Russian Journal of Herpetology. No. 2 (2009) by Evgenii Blagovidov

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 83 – 87

FIRST RECORD OF Tropiocolotes persicus euphorbiacola MINTON, ANDERSON AND ANDERSON 1970 (SAURIA: GEKKONIDAE) FROM THE REPUBLIC OF INDIA, WITH NOTES ON ITS HABITAT AND NATURAL HISTORY Ishan Agarwal1 Submitted October 22, 2007. Tropiocolotes persicus euphorbiacola is reported for the first time from India, based on two specimens collected from the Thar Desert, in Jaisalmer District, Rajasthan, India. Morphometrics and morphological characters of the specimens, along with notes on habitat and natural history of this species are presented. Keywords: Sauria, Gekkonidae, Tropiocolotes persicus euphorbiacola, first record, habitat, natural history, Thar Desert, Jaisalmer District, Rajasthan, India.

INTRODUCTION Tropiocolotes Peters, 1880 is a genus of small, terrestrial geckos distributed from the western Sahara through Northern Africa, eastward through south-western Asia (Iran, Afghanistan, and Pakistan; Anderson, 1999). Currently, Tropiocolotes contains nine species including species of the genus Microgecko Nikolsky, 1903 (T. helenae, T. latifi, T. persicus; Uetz and Hallerman, 2007). The Persian Dwarf gecko, Tropiocolotes persicus (Nikolsky, 1903), has three subspecies: T. p. persicus (Nikolsky, 1903) distributed from Iran to western Pakistan (Anderson, 1999; Khan, 2006); T. p. bakhtiari Minton, Anderson and Anderson, 1970 known from Iran (Anderson, 1999); and T. p. euphorbiacola Minton, Anderson and Anderson, 1970 known from central and eastern Pakistan (Khan, 2006). During a recent study on lizard ecology in and around the Desert National Park, Jaisalmer District, Rajasthan (Agarwal, 2007), I came across individuals of a small terrestrial, banded gecko. To confirm specific identity, two specimens were collected. On examination the species was identified as Tropiocolotes persicus euphorbiacola. After consulting relevant literature and checklists of Indian lizards (e.g., Prakash, 1974; Das 1996, 1997, 2002, 2007; Sharma, 1996, 2002; Das and Rathore, 2004), I found that these specimens represent the first record of this species and genus from within the 1

Wildlife Institute of India, Chandrabani, Dehradun 248001, India. Present address: 81/A Maker Kundan Gardens, Santacruz (W), Mumbai 400054, India. E-mail: ishan_a@rediffmail.com.

political boundaries of India. In this paper are presented morphological and morphometric details of the voucher specimens, as well as data on habitat and natural history. MATERIAL AND METHODS All searches involved two observers, and were carried out in the morning, afternoon (in winter), evening, and a few hours after sunset. The area was surveyed in winter (January 13 – 17, 2007), and early summer (March 6 – 10, 2007). In summer (April 12 – 17, 2007), systematic sampling was carried out, with three 1 ha plots sampled twice each [for a detailed description of sampling methods see Agarwal (2007)]. Two specimens of Tropiocolotes persicus euphorbiacola were hand collected from the rocky hills around Nabh Dongar (26°47¢11¢¢ N 70°41¢38¢¢ E) on April 15, 2007, and briefly kept for photography. Subsequently they were euthanized, preserved in 10% formalin, later transferred to 70% alcohol, and deposited in the collection of the Bombay Natural History Society (BNHS 1808 and 1809). Tissue samples were taken by preserving tail tips in 95% ethanol before fixation in formalin. The specimens were examined using a stereo microscope and measurements taken with a digital caliper (to the nearest 0.1 mm). Other individuals were observed in the field without disturbing them. EXTERNAL CHARACTERISTICS The specimens have the following characters diagnostic of Tropiocolotes: (after Minton, 1966; Minton et

Ishan Agarwal

Fig. 1. Tropiocolotes persicus euphorbiacola in life (BNHS 1808) from Jaisalmer District, Rajasthan, India.

Fig. 2. Tropiocolotes persicus euphorbiacola in life (BNHS 1809) from Jaisalmer District, Rajasthan, India.

al, 1970; Anderson, 1999; Khan, 2006) pupil vertical; dorsal scales without enlarged tubercles; no lateral fold; toes weakly angulate between last and next to last phalanx; digits not dilated, fringed, webbed, or ornamented above; males without preanal or femoral pores. The geckos were identified as T. persicus based on the following characters (after Minton, 1966; Minton et al., 1970; Anderson, 1999; Khan, 2006): dorsals, ven-

trals and lamellae smooth; 72 â&#x20AC;&#x201C; 76 scales along dorsal midline from axilla to groin; two pairs of postmentals; internasals enlarged, followed by a pair of enlarged scales; five scales (first labial, rostral, internasal and two postnasals) bordering nostril; dark crossbars on tail distinct. The subspecies T. p. euphorbiacola is distinguished by the dark bands on the body as wide or slightly narrower than the interspaces (as against wider in T. p. bakhtiari, or less than half the width in T. p. persicus); and 62 â&#x20AC;&#x201C; 76 scales along the dorsal midline from axilla to groin (Minton et al., 1970; Leviton and Anderson, 1972). The specimens at hand matched these descriptions well. Selected mensural and meristic data are listed in Table 1. Coloration in life (Figs. 1 and 2). Dorsum light brown; body with five dark bands, first between forelimbs and last between hindlimbs; darker portion of bands about as wide as interspaces; eight bands on the unregenerated tail of BNHS 1809 in life, regenerated portion of tail of BNHS 1808 without bands; limbs uniform pinkish, appear translucent; head uniform above; band on either side of the head beginning from internasals through eye, continuing behind to join first trunk band; labials white, finely speckled with black; venter uniform white; underside of tail faint yellow. Coloration in preservative faded, pattern similar; though dark portion of bars prominent only along posterior margin.

TABLE 1. Mensural and Meristic Data for Tropiocolotes persicus euphorbiacola from Jaisalmer District, Rajasthan Character

1808

1809

Snout to vent length Axilla to groin length Body width Head length Head width Head depth Eye diameter Ear diameter Eye to nostril distance Eye to ear distance Eye to snout distance Interorbital distance Internarial distance Subdigital lamellae (right/left): finger 1 finger 4 toe 1 toe 4 supralabials infralabials

28.7 12.7 5.5 8.4 5.2 3.8 1.9 0.7 2.7 2.4 3.1 2.5 1.1

26.5 12.2 5.4 8.2 4.7 3.5 1.7 0.6 2.5 2.2 3.0 2.3 1.0

6/7 7/6 10/12 10/11 7/7 8/8 15/16 16/14 9/8 8/9 8/7 9/8 Note. All mensural data in mm. Tail incomplete in both specimens.

HABITAT Nabh Dongar and the associated hills lie in Jaisalmer District, Rajasthan, about 25 km southwest of Jaisalmer city (Fig. 3), and are classified as rocky and

First Record of Tropiocolotes persicus euphorbiacola from India

Fig. 4. View of general habitat of Tropiocolotes persicus euphorbiacola from Nabh Dongar showing hill range. Fig. 3. Map of India and surrounding countries showing two locality records of T. persicus euphorbiacola: 1, present record from Nabh Dongar, Jaisalmer District, Rajasthan, India; 2, nearest previous locality, Nabisar, Thar Parkar District, Pakistan.

gravelly pediments (Kar, 1989). Jaisalmer District is in the Thar Desert, with average annual temperature a minimum of 7.9°C and maximum of 23.6°C in January, to a minimum of 25.8°C and a maximum of 41.6°C in May (Meena, 2000). Annual precipitation is about 164 mm (Gupta, 1986). The habitat in which the geckos were sighted consists of undulating to moderately steep rocky hills that do not rise above 290 m (Figs. 4 and 5). The terrain is broken, with rocky soil and low vegetation cover (9 – 23%). Trees are largely restricted to drainages and depressions, while Euphorbia caducifolia clumps are scattered across most areas but are concentrated on slopes and in drainages, with Euphorbia cover as high as 20%. Grasses include Aristida ssp., Cenchrus pennisetiformis, and Dactyloctenium scindicum. Dominant shrubs are Aerva ssp. and Fagonia cretica, while trees include Acacia senegal, Capparis decidua, Grewia tenax, and Salvadora oleoides. A more detailed account of this habitat as “rocky hills” can be found in Agarwal (2007). NATURAL HISTORY All nine sightings of T. p. euphorbiacola were in summer, from one to three hours after sunset (sampling not carried out after this period). Eight of these sightings were during 20.7 man-hours of night-time systematic sampling, and the additional sighting was opportunistic. This species was recorded on two of three 1 ha plots sampled. Vegetation cover was lowest in the plot in

Fig. 5. View of habitat of Tropiocolotes persicus euphorbiacola at Nabh Dongar showing an area with high Euphorbia cover where a total of six individuals were sighted in 1 ha.

which there were no sightings, and no gekkonids were recorded from that plot. Of the two plots in which Tropiocolotes was spotted, we recorded two and zero individuals in the first plot (on the first and second nighttime sampling sessions), and five and one in the second plot. The opportunistic sighting was adjacent to the first plot. Using the highest number sighted during any sampling session as a lower bound of abundance (Shenbrot and Krasnov, 1997; McNair, 2003), the abundance translates to 2.3 ind per 1 ha (for all three plots combined). The encounter rate was one sighting for every 2.6 man-hours of search effort. There were no sightings of this species in winter or early summer. No specimens were located in the day during any season, even with active searches under

Ishan Agarwal

rocks and in mounds of dead Euphorbia (Minton et al., 1970; Khan, 2006 found this species in such situations). Average air temperatures during the three sampling periods are shown in Table 2. The average air temperature recorded during night sampling in summer was 34.9°C (unrecorded in winter and early summer). These geckos were observed to be nocturnal and terrestrial, moving agilely among rocks and rubble. This species had a typical tail-waving behavior, both at rest and while active, with the tail constantly being sinuously waved back and forth horizontally. Sympatric geckos include Crossobamon orientalis, Cyrtopodion scabrum, Hemidactylus flaviviridis, and Hemidactylus sp. Sympatric lizards are Calotes versicolor (visual identification), Ophiomorus raithmai (only tracks recorded, no direct sightings), Ophisops jerdoni, and Trapelus agilis. The only snakes observed in the area were Echis carinatus sochureki (some authors consider this a valid species; e.g., Kochar et al., 2007) and Oligodon taeniolatus (color form I; Smith, 1943). DISCUSSION This record represents the first record of the genus Tropiocolotes and the species T. persicus euphorbiacola from within the political boundaries of India. The range extension is approximately 215 km northeast from the closest previously known locality in Nabisar, Thar Parkar District (reported by Leviton and Anderson, 1972), in the Thar Desert of Pakistan. This is also now the known easternmost limit for this genus. The occurrence of this species in India is not significant from a biogeographic point of view, as areas from which the species has been previously reported in Pakistan and the current localities are contiguous, forming part of the same biogeographic province — the Thar Desert (Udvardy, 1975), and are thus similar in aspects of terrain and climate. In fact, this finding merely reflects how little is known of the herpetofauna of this area, and more generally many parts of India. It is very likely that this species will be found in similar rocky habitats in the region, pos-

TABLE 2. Average Air Temperatures (°C) Recorded During the Three Sampling Periods Sampling period

Min

Max

Winter (January 13 – 17) Early summer (March 6 – 10) Summer (April 12 – 17)

6.4 15.4 24.5

25.2 30.5 41.3

Note. Data from Central Arid Zone Research Institute (C.A.Z.R.I.) field station about 30 km from Nabh Dongar.

sibly in other parts of Jaisalmer District, as well as Barmer District, Rajasthan and parts of Gujarat adjacent to Thar Parkar District, Pakistan. Acknowledgments. This study was conducted as part of the M. Sc. program at the Wildlife Institute of India. I thank my supervisors, S. P. Goyal and Q. Qureshi for all their help; Dr.H. C. Bohra, C.A.Z.R.I.; the Rajasthan forest department for permission to carry out this study: the PCCF R. N. Mehrotra, the Deputy Director, DNP, R. Jugtawat; the staff at Sam Chowki; Baba Anand Maharaj and Damodar at Nabh Dongar; my field assistant Tarun for all his help as well as for spotting the first gecko, and the Khichi family for all their support. A. Captain and N. D’Silva provided inputs on the draft and F. Tillack and S. Pawar sent vital references. I especially thank V. B. Giri (BNHS) for his help with the manuscript, examination of specimens, and his encouragement.

REFERENCES Agarwal I. (2007), Habitat Relationships and Resource Partitioning in a Lizard Community of the Thar Desert. M. Sc. Thesis, Saurashtra University, Rajkot, India. Anderson S. C. (1999), The Lizards of Iran, Society for the Study of Amphibians and Reptiles, Ithaca, NY. Das I. (1996), Biogeography of the Reptiles of South Asia, Krieger Publ. Co., Malabar, FL. Das I. (1997), “Checklist of the reptiles of India with English common names,” Hamadryad, 22, 32 – 45. Das I. (2002), A Photographic Guide to Snakes and Other Reptiles of India, New Holland publishers (UK) Ltd. Das S. K. (2007), “Checklist and distribution of saurian fauna in the Thar Desert of Rajasthan,” Tigerpapers, 34(2), 20 – 23. Das S. K. and Rathore N. S. (2004), “Herpetofauna of the Desert National Park, Rajasthan,” Zoosprint, 19(9), 1626 – 1627. Gupta R. K. (1986), “The Thar Desert,” in: Evenari M., NoyMeir I., and D. W. Goodall (eds.), Hot Deserts and Arid Shrublands, Elsevier Sci. Publishers B.V., Amsterdam, pp. 55 – 92. Kar A. (1989), “Terrain characteristics of Jaisalmer district,” Geogr. Rev. India, 51, 48 – 59. Khan M. S. (2006), Amphibians and Reptiles of Pakistan, Krieger Publ. Co., Malabar, FL. Kochar D. K., Tanwar P. D., Norris R. L., Sabir M., Nayak K. C., Agrawal T. D., Purohit V. P., Kochar A., and Simpson I. D. (2007), “Rediscovery of severe saw-scaled viper (Echis sochureki) envenoming in the Thar Desert region of Rajasthan, India,” Wilderness Environ. Med., 18(2), 75 – 85. Leviton A. E. and Anderson S. C. (1972), “Description of a new species of Tropiocolotes (Reptilia: Gekkonidae) with a revised key to the genus,” Occ. Pap. Calif. Acad. Sci., 96, 1 – 7.

First Record of Tropiocolotes persicus euphorbiacola from India McNair D. B. (2003), “Population estimate, habitat associations, and conservation of the St. Croix Ground Lizard Ameiva polops at Protestant Cay, United States Virgin Islands,” Carrib. J. Sci., 39(1), 94 – 99. Meena V. P. (2000), Desert Ecology, Pointer publishers, Jaipur. Minton S. A. (1966), “A contribution to the herpetology of West Pakistan,” Bull. Am. Mus. Nat. Hist., 134, 27 – 184. Minton S. A. Jr., Anderson S. C., and Anderson J. A. (1970), “Remarks on some geckos from southwest Asia, with descriptions of three new forms and a key to the genus Tropiocolotes,” Proc. Calif. Acad. Sci., 37, 333 – 362. Prakash I. (1974), “The ecology of vertebrates of the Indian Desert,” in: Mani M. S. (ed.), Ecology and Biogeography in India, Dr. W. Junk B.V., Publishers, The Hague, pp. 369 – 429 Sharma R. C. (1996), “Herpetology of the Thar Desert,” in: Ghosh A. K., Baqri Q. H., and Prakash I. (eds.), Faunal

Diversity in the Thar Desert: Gaps in Research, Scientific Publishers, Jodphur, pp. 297 – 306. Sharma R. C. (2002), Fauna of India and Adjacent Countries. Reptilia. Vol. II. Sauria, Published by Director, Zoological Survey of India, Kolkata. Shenbrot G. and Krasnov B. (1997), “Habitat relationships of the lizard fauna in the Ramon erosion cirque, Negev highland (Israel),” J. Zool., 241, 429 – 440. Smith M. A. (1943), The Fauna of British India, Ceylon and Burma, including the whole of the Indo-Chinese subregion. Reptilia and Amphibia. Vol. III. Serpentes, Taylor and Francis, London. Udvardy M. D. F. (1975), “A classification of the biogeographical provinces of the world,” IUCN Occasional Paper No. 18, Morges, Switzerland. Uetz P. and Hallerman J. (2007), The New Reptile Database, http://www.reptile-database.org, accessed June 10, 2007.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 88 – 94

ON THE BIOACOUSTIC BEHAVIOR OF MALE Homopholis fasciata

Dieter Gramentz1 Submitted January 2, 2008. The distress and threat calls of male Homopholis fasciata were examined. The lengths, call structures, frequencies and sound intensities along with associated behavior were described and analyzed. The calls are shown in oscillo- and audiospectrograms as well as three-dimensional images. Keywords: Homopholis fasciata, vocalization, distress call, threat call, interactive behavior.

INTRODUCTION Vocalization in geckos plays an important role in intraspecific communication and certain calls are used in male – male and male – female interactions. Some call types such as the advertisement call of Gekko gecko have specific meaning in courtship behavior (Gramentz, in press). Unfortunately until recently sound production and its importance for the behavioral ecology in gecko communication has only received minor attention despite the fact that first anecdotal information on vocalization in reptiles were presented by Mertens back in 1946. In certain unpleasant or potentially dangerous situations a number of gecko species are known to produce a distress call which is certainly the most described call in geckos (e.g., Morgue, 1913; Marcellini, 1974; Frankenberg, 1973, 1975, 1978; Werner et al., 1978; Szczerbak, 1981; Brown, 1984/85; Nettmann and Rykena, 1985; Kreuzer and Grossmann, 2003; Gramentz and Barts, 2004; Gramentz, 2005d, 2005c, 2007b; Barts, 2002, 2006). This type of call can either be produced in intraspecific interactions or when in contact with a potential predator. Contrary to the distress call threat calls have been far less documented in geckos (Marcellini, 1977; Rieppel, 1973; Zimmermann 1980; Gramentz, 2005b). The genus Homopholis is known to have a voice as it was mentioned by Cott (1934) who reported a weak but long lasting call of H. wahlbergii. That Homopholis fasciata has a voice was already mentioned by Barts (2004) who reported that adults produce a sound when they feel molested by maintenance works in the terrar1

Földerichstraße 7, D-13595 Berlin, Germany; E-mail: liteblu@gmx.de.

ium. This study is thought to give the first detailed description of the sound repertoire of H. fasciata. MATERIAL UND METHODS Both specimens used in this study were adult males obtained at a reptile fair in December of 2006. More than eight months both geckos were kept separately and isolated in two terrariums. In September 2007 one male was placed into the container with the other male and the acoustic interactions between them were recorded and digitalized. The recording equipment is the same described in Gramentz (2005a, 2005b). The sound card used was Creative Soundblaster Audigy 2 ZS Platinum Pro with a sample rate of 44100 Hz, 16 bit. As sound analysis software avisoft-SASLab was used. The containers in which the recordings were taken had a size of 25 ´ 40 ´ 30 cm (length ´ width ´ height). For sound isolation and avoidance of reflections the side walls were made of wood and covered with cork on the inside. The distance of the geckos and the microphone varied between approximately 15 and 30 cm. The air temperatures during the recordings ranged from 23.6 – 24.5°C. Analysis of the distress call was hindered to a certain degree as many of these calls were produced in the course of aggressive interactions with biting and chasing each other causing a number of noises which disturbed the recording. However, four distress call could be analyzed completely and another one partly. The red colored areas in the 3D figures below about 700 Hz result from working noises of the recorder.

On the Bioacoustic Behavior of Male Homopholis fasciata

Fig. 1. Oscillogram of a distress call of a male of Homopholis fasciata with a length of 0.110 sec.

Fig. 2. Oscillogram of a distress call of a male of Homopholis fasciata with a length of 0.160 sec.

RESULTS Males of the species possess at least two different call types. One call type (distress call) is emitted on physical contact and the other call type (threat call) before that. DISTRESS CALL Fig. 3. Audiospectrogram of the same distress call as in Fig. 2.

The distress call of H. fasciata is a very short call and it is produced when a gecko is bitten by another gecko. A call consists of about 7 – 12 pulses with a fairly strong amplitude in the main part of the call (Figs. 1 – 4). Occasionally distress calls show some kind of “tail” consisting of only a few pulses with very weak amplitudes. Details on frequency, sound intensity and length of the call are presented in Table 1. THREAT CALL The threat call is emitted by a male H. fasciata which is approached by another male. At a distance of only a few centimeters the usually passively standing male calls, while the other actively walks towards it. A threat call is always produced without or before physical contact. Within the three quarters of an hour after the geckos were put together about 10 distress calls were produced when they started to chase and bite each other. After that

Fig. 4. Audiospectrogram of a distress call of a male of Homopholis fasciata with a length of 0.183 sec.

biting ceased and for another 2.5 h 35 threat calls were emitted. Altogether 37 threat calls were recorded and analyzed.

TABLE 1. Various Bioacoustic Parameters of the Distress Call of Male Homopholis fasciata Parameter Length, sec Maximum frequency, Hz Minimum frequency, Hz Frequency at max. sound intensity, Hz Maximum sound intensity, dB

S.D.

Range

0.162 15148 232 6532 73.6

0.004 1030.03 93 2938.9 4.11

0.110 – 0.205 14453 – 16676 185 – 371 4262 – 10747 9.9 – 79.1

5 4 4 4 4

Dieter Gramentz

Fig. 5. Oscillogram of a single threat call of a male of Homopholis fasciata with a length of 1.887 sec. Fig. 7. Audiospectrogram of a short threat call of a male of Homopholis fasciata with a length of 1.028 sec.

Fig. 6. Audiospectrogram of a long threat call of a male of Homopholis fasciata with a length of 2.078 sec.

During an encounter between two males most frequently just one single threat call (Figs. 5 – 9) was emitted (45%). Two calls and three calls (Figs. 10 – 12) in a row were recorded 3 times each (15%). Very rarely five calls (Figs. 13 and 14) were produced in a row (10%). Finally three times (15%) some kind of double threat

call (Figs. 15 – 18) was emitted consisting of two single calls with only an extremely short gap in between or without a time gap at all. The duration of a call sequence has no discernable effect on the length of the threat calls in this sequence. However, in a multi-call sequence, not always but generally the time gap between threat calls increases with the length of the call sequence. The sequences with five threat calls in a row were produced by a male which had to pass the other male which was standing motionless but in the visual field of each other. As can be seen in Table 2 the threat call of H. fasciata is a rather high pitched call with a maximum frequency usually around 17 and 18 kHz. However, the fre-

Fig. 8. Three-dimensional semi logarithmic image of a long single threat call (the same as in Fig. 6) of a male of Homopholis fasciata.

On the Bioacoustic Behavior of Male Homopholis fasciata

Fig. 9. Three dimensional semi logarithmic image of a long single threat call of a male of Homopholis fasciata with a length of 1.934 sec.

quency were the maximum intensity of the call was noted is far lower. The maximum frequency recorded in the threat call of H. fasciata was 18159 kHz and the longest single call had duration of 2.750 sec. Although very high frequencies in a threat call are reached Figs. 8, 9 and 17 show that the main intensity is around 5420 Hz. Figure 17 also shows that in some calls frequencies above 8610 Hz are very much reduced, while in others, as can be seen in Fig. 12, the main intensity lies around 13 kHz.

Fig. 10. Oscillogram of three single threat calls in a row of a male Homopholis fasciata. Length of call sequence 6.258 sec, length of first call 1.324 sec, second call 1.502 sec, and third call 0.647 sec. Time gab between first and second call 1.187 sec, between second and third call 1.598 sec.

DISCUSSION The distress call of H. fasciata is similar in structure to the one of Cosymbotus platyurus (Gramentz, 2007a), Cyrtopodion scaber (Frankenberg, 1975), Hemidactylus frenatus (Marcellini, 1974), H. turcicus (Frankenberg, 1975), Pachydactylus rugosus (Gramentz and Barts, 2004), Stenodactylus stenurus (Gramentz, 2004), S. sthenodactylus (Frankenberg, 1975), Thecadactylus rapicauda (Gramentz, 2007b) which had a shorter distress call, and Hemidactylus angulatus (Gramentz, 2005) of which the distress call is slightly longer. In all these species the distress call consists usually of only a few pulses and is rather short in duration.

Fig. 11. Audiospectrogram of three single threat calls in a row of a male Homopholis fasciata. Length of call sequence 11.403 sec, length of first call 1.497 sec, second call 1.352 sec and third call 1.369 sec. Time gap between first and second call 4.496 sec, between second and third call 2.689 sec.

In practically all geckos in which a distress call was found it was also present in the sound repertoire of females which can also be assumed to be the case in H. fasciata. However, this should be verified.

Dieter Gramentz

Fig. 12. Three-dimensional semi logarithmic image of three single threat calls of a male of Homopholis fasciata with a sequence length of 14.977 sec. Length of first cal is 1.676 sec, second call 1.542 sec and third 1.855 sec. Time gap between first and second call 4.511 sec, between second and third call 5.393 sec.

Fig. 13. Oscillogram of five single threat calls in a row of male Homopholis fasciata. Length of call sequence 18.763 sec. Length of first call 2.095 sec, second call 1.957 sec, third call 1.213 sec, fourth call 1.792 sec, and fifth call 1.076. Time gap between first and second call 2.052 sec, between second and third call 2.278 sec, third and fourth call, 2.709 sec, fourth and fifth call 3.591 sec.

Fig. 15. Oscillogram of a double threat call of a male of Homopholis fasciata with a total length of 1.934 sec. Length of first part 1.242 sec, length of second part 0.692 sec.

The threat call of H. fasciata of this study resembles the call of Geckonia chazaliae [designated by me incorrectly as distress call (Gramentz, 2005b)] as well as in

Fig. 14. Audiospectrogram of five single threat calls in a row of male Homopholis fasciata. Length of call sequence 18.033 sec. Length of first call 1.876 sec, second call 2.750 sec, third call 1.516 sec, fourth call 1.182 sec and fifth call 1.311 sec. Time gap between first and second call 2.890 sec, between second and third call 2.010 sec, third and fourth call 2.067 sec, fourth and fifth call 2.431 sec.

Fig. 16. Audiospectrogram of a double threat call of a male of Homopholis fasciata with a total length of 2.297 sec. Length of first part 1.247 sec, length of second part 1.050 sec.

the context of call related behavior and also in sound characteristics. Some similarities in the threat calls be-

On the Bioacoustic Behavior of Male Homopholis fasciata

Fig. 17. Three-dimensional semi logarithmic image of a double threat call of a male of Homopholis fasciata with a total length of 1.762 sec. Length of first part 1.109 sec, length of second part 0.653 sec.

tween the two African species G. chazaliae and H. fasciata are in fact striking. The average length of a threat call in G. chazaliae was 1.844 sec in comparison to 1.672 sec in H. fasciata. The threat call of G. chazaliae is also very high pitched reaching frequencies around 17 and 22 kHz while 17 and 18 kHz were reached by H. fasciata. Furthermore G. chazaliae produces single threat calls but also at least two calls in a row. However, the similarity in the threat call of both species is most certainly not due to phylogenetic reasons as they are probably 120 million years divergent (Bauer, personal communication). Beside the similarities in these two species, there are, on the other hand, marked differences in the threat call structure between both geckos. While the threat call of G. chazaliae begins abruptly and loudly followed by a

Fig. 18. Oscillogram of two double threat calls of a male of Homopholis fasciata with a total length of 7.672 sec. Length of first double call 1.804 sec, length of second double call 1.596 sec. Time gap between both calls 4.272 sec.

gradual decline towards its end, in H. fasciata the start of the call is also abrupt however it begins with a lower intensity. From the beginning of the call the intensity increases gradually to its maximum still in the anterior part of the call. It then decreases gradually not unlike

TABLE 2. Various Bioacoustic Parameters of the Threat Call of Male Homopholis fasciata Parameter Length, sec Maximum frequency, Hz Minimum frequency, Hz Frequency at max. sound intensity, Hz Maximum sound intensity, dB

S.D.

Range

1.672 15788 1436 6798 84.0

0.468 2048.0 92.6 2366.4 2.6

0.999 – 2.750 9134 – 18159 1297 – 1668 5003 – 10932 75.5 – 88.3

33 35 36 29 34

94 the call of G. chazaliae. Another difference between the calls of the two species is that the maximum amplitude in the threat calls of H. fasciata is located in the anterior part (Figs. 5 and 10) but not at the very beginning as is the case in G. chazaliae. When comparing distress and threat call of H. fasciata both reach similar maximum frequencies, but sound intensity, length and minimum frequency are larger in the threat call than in the distress call. Most probably both calls function to prevent agonistic interactions which might result in injuries. What causes a male to emit a single call or a sequence of threat calls remains yet unclear. A possible cause may be the continued presence of the “aggressor” gecko in the visual field of the calling gecko. Probably as well, both call types have their meaning in the context of antipredator behavior. A distress call is most likely not only produced when a gecko is bitten by a conspecific but also during an attack of a potential predator. As was observed by Barts (2004) a threat call is directed towards the human hand which is certainly presumed to be of danger or a predator. Acknowledgment. I am very grateful to Aaron Bauer for providing information on the phylogenetic relationship between Geckonia and Homopholis.

REFERENCES Barts M. (2002), “Die Dickfingergeckos des südlichen Afrikas. Teil II. Die Haltung und Vermehrung des Gebänderten Dickfingergeckos, Pachydactylus fasciatus Boulenger, 1888,” Sauria, 24(1), 3 – 8. Barts M. (2004), “Der gebänderte Samtgecko und Wahlbergs Gecko Homopholis fasciata und Homopholis wahlbergii,” in: Art für Art, Natur und Tier – Verlag, Münster. Barts M. (2006), “Pachydactylus haackei Haacke’s Dickfingergecko,” Sauria, 28(1), 54. Brown A. M. (1984/85), “Ultrasound in gecko distress calls (Reptilia: Gekkonidae),” Israel J. Zool., 33, 95 – 101. Cott H. B. (1934), “The Zoological Society’s Expedition to the Zambezi, 1927: No. 5. On a collection of lizards, mainly from Portuguese East Africa, with description of new species of Zonurus, Monopeltis and Chirinda,” Proc. Zool. Soc. London, 1934, 145 – 172. Frankenberg E. (1973), “Vocalizations of the fan-toed gecko, Ptyodactylus hasselquistii,” Israel J. Zool., 22, 205. Frankenberg E. (1975), “Distress calls of gekkonid lizards from Israel and Sinai,” Israel J. Zool., 24, 43 – 53. Frankenberg E. (1978), “Calls of male and female tree geckos, Cyrtodactylus kotschyi,” Israel J. Zool., 27, 53 – 56. Gramentz D. (2004), “Das Antiprädationsverhalten von Pachydactylus turneri (Gray, 1864),” Sauria, 26(2), 37 – 41.

Dieter Gramentz Gramentz D. (2005a), “Zur intraspezifischen bioakustischen Kommunikation von Hemidactylus platycephalus Peters, 1854 (Reptilia: Sauria: Gekkonidae),” Gekkota, 5, 155 – 154. Gramentz D. (2005b), “Zum Defensivverhalten und Schrecklaut von Geckonia chazaliae Mocquard, 1895,” Sauria, 27(3), 23 – 27. Gramentz D. (2005c), “Der Schreckruf von Haemodracon riebeckii Peters, 1882 (Reptilia: Sauria: Gekkonidae),” Gekkota, 5, 170 – 178. Gramentz D. (2005d), “Zur intraspezifischen bioakustischen Kommunikation von Hemidactylus brookii angulatus Hallowell, 1852,” Sauria, 27(4), 41 – 46. Gramentz D. (2007a), “Zur akustischen und visuellen Kommunikation von Cosymbotus platyurus (Schneider, 1792),” Sauria, 29(2), 13 – 20. Gramentz D. (2007b), “Zum bioakustischen Verhalten männlicher Thecadactylus rapicauda Houttuyn, 1782,” Sauria, 29(3), 13 – 18. Gramentz D. (in press), “Zur Stimme des Tokeh, Gekko gecko Linné, 1758. Studien am Tokeh: 3. Der Anzeigeruf und der Schreckruf von Gekko gecko (Linnaeus, 1758) (Sauria: Gekkonidae),” Gekkota, 6. Gramentz D. and Barts M. (2004), “Der Schrecklaut von Pachydactylus rugosus A. Smith, 1849,” Sauria, 26(1), 23 – 26. Kreuzer M. and Grossmann W. (2003), “Beobachtungen an Gekko ulikovskii Darewski & Orlow, 1994 und Gekko grossmanni Günther, 1994 im Terrarium,” Sauria, 25(3), 3 – 11. Marcellini D. (1974), “Acoustic behavior of the gekkonid lizard, Hemidactylus frenatus,” Herpetologica, 30(1), 44 – 52. Marcellini D. (1977), “Acoustic and visual display behavior in gekkonid lizards,” Am. Zool., 17, 251 – 260. Mertens R. (1946), “Die Warn- und Drohreaktionen der Reptilien,” Abh. Senckenberg. Naturf. Ges., 471, 1 – 108. Morgue M. (1913), “Étude sur le Phyllodactylus d’Europe, ‘Phyllodactylus europaeus‘ Gené,” Bull. Soc. Linn. Marseille, 1, 45 – 51. Nettmann H.-K. and Rykena S. (1985), “Verhaltens- und fortpflanzungsbiologische Notizen über kanarische und nordafrikanische Tarentola-Arten,” Bonn. Zool. Beitr., 36(3/4), 287 – 305. Rieppel O. (1973), “Zur Kenntnis von Geckonia chazaliae,” Aquarien Terrarien Leipzig, 7, 230 – 233. Szczerbak N. (1981), “Cyrtodactylus russowii (Strauch 1887) — Transkaspischer Bogenfingergecko,” in: H. Böhme (ed.), Handbuch der Reptilien und Amphibien Europas, Akademische Verlagsgesellschaft, Wiesbaden, pp. 75 – 83. Werner Y. L., Frankenberg E., and Adar O. (1978), “Further observations on the distinctive vocal repertoire of Ptyodactylus hasselquistii cf. hasselquistii (Reptilia: Gekkonidae),” Israel J. Zool., 27, 176 – 188. Zimmermann H. (1980), “Durch Nachzucht erhalten: Faltengeckos,” Aquarien Mag., 14(7), 346 – 349.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 95 – 100

NEW RECORDS OF THE GECKONID LIZARD, Cyrtopodion heterocercum heterocercum (SAURIA: GEKKONIDAE) FROM ISFAHAN PROVINCE, CENTRAL IRAN, WITH AN EXTENDED DESCRIPTION AND NOTES ON DISTRIBUTION

Nasrullah Rastegar-Pouyani,1 Hamzeh Oraei,1 and Morteza Johari1

Submitted September 8, 2007. Based on extensive study and field work in various regions of the central Iranian Plateau, eight specimens of the geckonid lizard, Cyrtopodion heterocercum, were collected from Khansar, Kashan and Semirom in Isfahan Province, central Iran. The collected specimens were examined based on metric, meristic and pholidosis characters, as well as color pattern, and compared with 12 specimens of the same species, belonging to the Zoological Museum of Razi University, collected from Kermanshah and Hamedan Provinces. These comparisons were not indicative of distinct morphological differences between specimens occurring in the central Iranian Plateau (the new records) with those occurring in western regions of the Iranian Plateau (Kermanshah and Hamedan Provinces). Keywords: Gekkonidae; Cyrtopodion heterocercum heterocercum; new records; morphology; Isfahan Province; Iranian Plateau; distribution.

INTRODUCTION The bent-toed geckonid lizards of the genus Cyrtopodion Fitzinger, 1843 consist of about 82 species (www.zipcodezoo.com), of which about 15 species are documented from Iran (Anderson, 1999). The genus is distributed from countries of the eastern Mediterranean (Egypt, the Balkans) east through southwest Asia across the north Arabian Desert, through Pakistan to northern India and the flanks of the Himalayas, north to the southern republics of Central Asia (Anderson, 1999). Cyrtopodion heterocercum (Blanford, 1874) is known as rough-scaled gecko or Asia minor thin–toed gecko. Blanford in 1874 distinguished and described it as Gymnodactylus heterocercus. The type locality of the Blanford species is given as Hamedan province, western Iran (Blanford, 1974). Later on, Anderson (1974:33) used the name Cyrtodactylus heterocercus for this lizard. Thereafter, Szczerbak and Golubev (1984:54, 1986:182, 1996:180) established the name Teniodactylus heterocercus for this species. 1

Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran; E-mail: nasrullah.r@gmail.com

Some years later, the senior author, for the first time, collected this gecko from Kermanshah Province (Rastegar-Pouyani, 1991). Apparently Cyrtopodion heterocercum heterocercum is known from only four specimens, all from Hamadan and Kermanshah Provinces (e.g., Blanford, 1874; Rastegar-Pouyani, 1991; Anderson, 1999), but recently a new record of this species was found from Markazi Province (Mozaffari, 2007). Another subspecies, Cyrtopodion heterocercum mardinensis (Mertens, 1924) is known from Gaziantep, Nisi, Mardin and Siirt, all in southern Turkey as well as northern Iraq (Nader and Jawadt, 1976; Baran and Gruber, 1982; Szczerbak and Golubev, 1986; Leviton et al., 1992; Anderson, 1999; Ugurtas et al, 2007). Szczerbak and Golubev (1986) discussed the possible occurrence of C. heterocercum in northern Syria. Some years later, Moravec and Modry (1994:53 – 56) collected four specimens from Halab, Syria. This record is about 100 km west of the westernmost published locality for Cyrtopodion heterocercum mardinensis (Gaziantep in Turkey). So far, there is no definite record for the occurrence of Cyrtopodion heterocercum heterocercum in central regions of the Iranian Plateau.

Nasrullah Rastegar-Pouyani et al.

Kashan

In this paper we report Cyrtopodion heterocercum heterocercum for the first time from the central Iranian Plateau based on material collected from different regions of Isfahan Province.

Khansar

SOURCE OF MATERIAL Semirom

Fig. 1. Location of Isfahan Province on the Iranian Plateau and the three new localities in which Cyrtopodion heterocercum heterocercum specimens were collected.

TABLE 1. The Main Metric and Meristic Characters of the Adult Cyrtopodion heterocercum Specimens Used in This Study Characters

Definition

During long-term excursions and field work in central Iranian Plateau in 2005 – 2007, eight specimens (four adults and four juveniles) of Cyrtopodion heterocercum heterocercum were collected from three different localities (Qamsar area, about 30 km southwest of Kashan as well as Khansar and Semirom) in Isfahan Province, central Iran (Fig. 1). Also, since 2000 and during extensive field work in various regions of the Zagros Mountains and neighboring areas, 12 specimens of C. h. heterocercum from Kermanshah and Hamedan Provinces were collected and deposited in the Razi University Zoological Museum (RUZM). Furthermore, for comparison, one specimen of C. h. mardinensis, two specimens of C. russowi and five specimens of the closely related species C. kotchyi from the GNHM (Gothenburg Natural History Museum, Gothenburg, Sweden) were studied (see material examined).

SVL

Length of snout to vent (from tip of snout to anterior edge of cloaca)

Length of tail (from posterior edge of cloaca to tip of tail)

Length of head (from tip of snout to anterior edge of tympanum)

Width of head (greatest distance between ear openings)

Height of head

SDLT

Subdigital lamellae under the fourth toe (total number of lamellae under the right fourth toe)

SDLF

Subdigital lamellae under the fourth finger.

EYD

Eye diameter (from upper corner to lower corner of eye). Greatest diameter

NED

Nostril-eye distance (from anterior corner of eye to posterior edge of nostril)

EED

Eye-ear distance (from the posterior corner of eye to anterior edge of tympanum)

Number of supralabials

Number of infralabials

IOR

Interorbital distance (between anterior or posterior corner of orbits)

RESULTS

Neck length (from the posterior edge of tympanum to anterior edge of shoulder)

ORD

Orbit diameter

Snout width (between nostrils)

LFE

Length of femur

Length of leg

Main morphological characters of the studied specimens of Cyrtopodion heterocercum heterocercum and their basic data are presented in Tables 1 – 2. Description of Cyrtopodion heterocercum heterocercum. A species belonging to the genus Cyrtopodion Fitzinger 1843; 5 – 7 pairs of postmentals which is apparently unique in this character among the Iranian species of Cyrtopodion; the first and second pairs in full contact with each other; also the first pair is the largest, about twice the size of the second pair; the third pair about 1/2 size of the second and widely separated from each other by 4 – 6 enlarged granules; furthermore there are 4 – 5 extra pairs of postmentals in continuation of

Length of arm

LFO

Length of forearm

DHF

Distance between hindlimbs and forelimbs

NDS

Number of dorsal scales around body

NVS

Number of ventral scales (from mental to anterior edge of cloaca)

RVS

Row of ventral scales (in longitudinal rows)

DISTRIBUTION Cyrtopodion heterocercum has a limited and littleknown distribution in Iran. As mentioned before, prior to this study, a few records from Kermanshah and Hamadan are the only known records of distribution of this lizard in Iran. Now, and based on our new data, the distribution of this taxon is extended toward the central Iranian Plateau in Isfahan Province.

New Records of the Geckonid Lizard, Cyrtopodion heterocercum heterocercum from Central Iran the first pair, and in contact with lower labials on each side, each pair is widely separated from one another by 6 – 15 granules or by second and third pairs of postmentals (this may be a unique arrangement). There are 128 – 132 scales, in a single longitudinal row, from symphysis of chin shields to vent; gular scales smooth, granular, juxtaposed (not imbricate); ventral scales smooth, larger (about 2 – 3 times the size of gulars), mostly cycloid, juxtaposed and sometimes relatively subimbricate; about 25 – 28 ventrals in a single transverse row across belly (on the widest area); scales of lower parts of hind limb plate-like and mostly subimbricate, smooth or sometimes very weakly keeled under the surface of tibia; fourth toe the longest, covered below by 23 – 25 compressed and weakly keeled lamellae; third and fourth fingers almost the same size, 17 – 19 lamellae under the fourth finger (less compressed and very weakly keeled); lower surfaces of hind legs covered by flat, juxtaposed, smooth scales, those of forearm covered by imbricate, slightly keeled plates; there are 14 – 17 granules across gular at the level of last (7th) postmental; mental large, lozenge–shaped, pointed backward and in contact with first sublabial as well as first pair of postmentals; rostral plate broad, in contact with first supralabial on

each side as well as nasal and 4 – 6 small scales (internasals); nostril bordered by rostral, first supralabial and one or two nasals; there are 6 – 8 pairs of enlarged and swollen scales (in median region) between nasal region and anterior margin of eyes; 20 – 24 granules, in a single longitudinal row, from behind rostral to posterior edge of eyes (in the median region); upper head scales mostly granular, but there are some enlarged tubercle-like scales intermixed (and slightly keeled); upper surfaces of forelimbs covered by large, plate-like, keeled and imbricate scales (almost without tubercles); dorsal scales granular, intermixed with trihedral, weakly to moderately keeled tubercles, the size of which being less than the interspaces, more intense on the paravertebral region, less intense on the vertebral region; 12 – 15 tubercles across widest part of dorsum; there are 21 – 24 tubercles from occiput to the level of vent (on the paravertebral region); 80 – 85 scales in a single longitudinal row from occiput to a point just above vent (in vertebral region); 31 – 35 scales (including tubercles) across widest part of dorsum; upper surface of hind limbs covered by keeled and large scales, intermixed with large trihedral and keeled, pointed tubercles; tail covered dorsally by large, keeled and pointed tubercles, 6 – 8 in the

TABLE 2. The Basic Statistics of Morphological Characters of the Adult Cyrtopodion heterocercum Specimens Used in this Study Characters SVL TL SL IL IO SA TP SAM SM SLU HL HB SNL OS ED EYD SVL/TL HL/HB ED/EYD TP/SVL LS LS/SNL CD

Kermanshah and Hamedan (n = 12) min

mean

max

Khansar (n = 1)

24.54 19.74 7.00 6.00 12.00 23.00 20.00 22.00 92.00 13.00 6.86 5.21 3.26 2.59 0.50 1.56 0.78 1.15 0.26 0.55 8.00 1.78 2.49

34.01 36.43 8.25 7.08 13.83 29.09 22.63 25.50 99.33 18.66 8.57 6.59 4.01 3.21 0.08 1.96 0.91 1.30 0.41 0.67 8.81 2.18 3.26

41.61 45.56 10.00 8.00 16.00 37.00 26.00 30.00 110.00 23.00 10.16 7.99 4.78 3.97 1.54 2.56 1.33 1.43 0.06 0.09 11.00 2.63 4.20

39.55 — 6.00 6.00 15.00 25.00 25.00 25.00 114.00 19.00 9.70 7.68 4.98 5.26 1.13 2.46 — 1.26 0.45 0.63 9.00 1.81 3.37

Kashan (n = 2) min

mean

max

Semirom (n = 1)

36.91 46.43 7.00 9.00 13.00 29.00 22.00 23.00 102.00 20.00 7.72 6.14 3.76 3.25 0.55 1.86 0.87 1.13 0.20 0.54 8.00 2.12 2.39

38.67 46.43 6.50 9.00 13.50 29.50 22.00 24.00 105.00 21.50 8.15 6.84 3.76 3.39 0.55 2.26 0.87 1.13 0.20 0.54 8.50 2.25 2.82

40.43 46.43 6.00 9.00 14.00 30.00 22.00 25.00 108.00 23.00 8.58 7.54 3.77 3.53 0.55 2.66 0.87 1.25 0.25 0.59 9.00 2.39 3.25

35.90 48.12 13.00 9.00 14.00 31.00 27.00 25.00 120.00 24.00 9.10 6.94 4.07 4.00 0.68 1.86 0.74 1.31 0.40 0.75 10.00 2.45 2.68

Nasrullah Rastegar-Pouyani et al.

Fig. 2. Two of Cyrtopodion heterocercum heterocercum specimens (RUZM, GC.10.2 and GC.10.3) collected from the Ghamsar area, about 30 km southwest of Kashan, Isfahan Province, central Iran.

Fig. 4. Habitat of Cyrtopodion heterocercum heterocercum, an old building in the suburbs of the city of Khansar, Isfahan Province, central Iran (about 2250 m elevation).

Fig. 3. Habitat of Cyrtopodion heterocercum heterocercum, about 30 km southwest of the city of Kashan, Isfahan Province, central Iran (more that 2000 m elevation).

dorsolateral regions; first dark cross-bar on occiput, the last one on sacrum; upper surfaces of limbs an intermixture of dark bars and reticulation; all of the ventral surfaces whitish except the mental and post mental areas that are yellowish-cream. Biological and ecological observations. Cyrtopodion heterocercum heterocercum in central Iranian Plateau has evolved certain differences in niche specificity. It means that it occurs both in the rocky areas on boulders or under stones as well as on the walls of ruined, old buildings close to human populations (Figs. 3 and 4). Two specimens from the Ghamsar area, 30 km southwest of Kashan were collected in a semi-mountainous region on boulders (Fig. 3) at an elevation of about 2000m, where they were syntopic with the other geckonid lizard Tropiocolotes latifii. These two specimens were collected during the day when they were relatively active. On the other hand the specimens from Khansar (about 2250m elevation), and Semirom (2400 m elevation), were collected during the night on the walls, or ceiling, of old buildings (Fig. 4) where they were active, looking for insects. The senior author collected C. heterocercum heterocercum and C. scabrum at the same place in suburbs of Kermanshah city, western Iran on the walls of buildings. Both species were active during the night as sit-and wait predators for capturing insects. Specimens collected from Khansar had two enlarged nodules behind their head and also around their eyes as well as on each side of the neck region. These nodules were also observed almost on the same areas of body in Cyrtopodion scabrum. With more careful examination,

upper surface of the first segment of intermixed with smaller scales, lower surface of tail covered by imbricate, keeled scales. Coloration and color pattern. The coloration and color pattern of this species is distinctive among all the Iranian species of the genus Cyrtopodion (Fig. 2); upper head region with brownâ&#x20AC;&#x201C;gray intermixed, four dark bars on occipital region direction of which being towards the head, these longitudinal bars are posteriorly connected to a transverse dark bar that is the first of the 8 â&#x20AC;&#x201C; 9 dark cross-bars presenting on dorsum. The dorsal dark crossbars are relatively irregular and cross each other frequently, making up a reticulate pattern, especially on

New Records of the Geckonid Lizard, Cyrtopodion heterocercum heterocercum from Central Iran it was found that these nodules are masses of small mites infecting the lizard. Further study on these parasites is now being carried out. DISCUSSION Based on statistics, color pattern and other pertinent characters, the newly collected specimens of C. heterocercum heterocercum are similar to those already collected from the Zagros area in Kermanshah and Hamedan provinces. It means that all the population of this taxon in the Zagros area as well as in the central Iranian Plateau are morphologically homogenous and belong to a single taxonomic entity. This may be indicative of a recent invasion by this lizard from the western Zagros to the central Iranian Plateau. Prior to this study, most workers considered the Zagros range as the main distribution area for Cyrtopodion heterocercum, as it was, apparently, restricted in distribution to this region with two subspecies (Anderson, 19968, 1999; Baran and Gruber, 1982; Leviton et al., 1992; Mertens, 1924b, 1956; Rastegar-Pouyani, 1991; Szczerbak and Golubev, 1986; Ugurtas et al., 2007). Further, these authors speculated that the Zagros Mountains may even serve as a geographic barrier to further eastward distribution of this lizard. Now, by collecting C. heterocercum from the central Iranian Plateau, we postulate that, perhaps, the main geographic barriers for more eastern distribution of this taxon are the great Iranian Deserts (Kavir and Lut Deserts). Some herpetologists (e.g., Mertens, 1924, 1956; Anderson, 1968, 1974, 1999; Rastegar-Pouyani, 1991; Kluge 1991, 1993) have been interested in taxonomy and biogeography of the southwest and central Asian geckonid lizards of the genus Cyrtopodion. Of all the Iranian Plateau species of Cyrtopodion, most likely, C. heterocercum is the least known taxon and, as mentioned before, prior to this study only a few records of its occurrence in Iran were documented. Collecting of C. heterocercum heterocercum in the central Iranian Plateau is indicative of a relatively wide distribution of this taxon in Iran. Golubev and Szczerbak (1986, 1996) divided all the Cyrtopodion species into four groups. Of these, C. heterocercum occurs within the Mediodactylus group (Szczerbak and Golubev, 1977b: 130). This species group encompasses: Cyrtopodion amictopholis (Hoofien), C. heterocercum (Blanford), C. kotschyi (Steindachner), C. rossowii (Strauch), C. sagittifer (Nikolsky), and C. spinicauda (Strauch). According to the another scenario (e.g., Anderson, 1999) C. heterocercum belongs to the “kotschi” group. This arrangement is different from that of Golubev and Szczerbak

(see above). The “kotschi” group in this view is composed of Cyrtopodion kotschyi, C. russowii, C. heterocercum, C. sagittifer, and C. spinicauda. With regards to the above-mentioned scenarios, it is clear that taxonomic status of some of the Iranian species of Cyrtopodion (including C. heterocercum) is still not resolved. Further study and more material from the areas of distribution of these lizards may shed more light on their systematics and phylogeny. Material examined Cyrtopodion heterocercum heterocercum (n = 2) (RUZM, GC.10.2 and GC.10.3) collected from Ghamsar about 30 km southwest of the city of Kashan, in the Karkas Mountains, Isfahan Province, central Iran (May 2006). Cyrtopodion heterocercum heterocercum (n = 1) (RUZM, GC.10.1) collected from Khansar city, Isfahan Province, central Iran (July 2006). Cyrtopodion heterocercum heterocercum (n = 5, one adult, 4 juveniles) (RUZM, GC.10.4 – 8) collected from Semirom, Isfahan Province, central Iran (July 2005). Cyrtopodion heterocercum heterocercum (n = 12): RUZM, GC.69 and GC. 70: collected from around the city of Hamedan, on the walls of a workshop (May 2003); RUZM, GC.78, GC.81 and GC.82, collected from Kangavar city, about 85km east of Kermanshah city, Kermanshah Province, western Iran. RUZM, GC.15, GC.20, GC.26, GC.30, GC.34, GC.38, and GC.59: collected from suburbs of Kermanshah city, Kermanshah province, western Iran. C. heterocercus mardinensis (n = 1): GNHM Re.ex. 6158: (Mertens, 1924) Karacay-Tekenpinar, 11 km SW Antakya, Province Antakya, Turkey. C. kotschyi ciliensis (n = 1): GNHM Re.ex. 6154 (Baran and Gruber, 1982), 20 km north of Tarsus, Adana, Turkey (26-4-82). C. kotschyi colchicus (n = 1): GNHM Re.ex. 6157 (Nikolsky, 1902), 36 km SSW Artvin, Province Artvin, Turkey (t-0262). C. kotschyi ciliciensis (n = 1): GNHM Re.ex. 6155 (Baran and Gruber 1982), Gulek Bogaz, Province Mersin, Turkey (t-0260). C. kotschyi syriacus (n = 1): GNHM Re.ex. 6156: (Stepanek, 1937), Yilankule Province, Adana, Turkey (t-0261). C. kotschyi danilewskii (n = 1): GNHM Re.ex. 6153 (Strauch, 1887), Termessos, Province Antalya, Turkey (t-0159). C. russowi (n = 2): GNHM Re.ex. 10633 and 10636 (Strauch), Eastern Kazakhstan, around Ili River, near Kerbulak Village.

Abbreviations. GNHM Re.ex., Gothenburg Natural History Museum, Reptilia Exotica, Gothenburg, Sweden; RUZM GC., Razi University Zoological Museum, Gekkonidae/Cyrtopodion.

100 Acknowledgments. We are grateful to the Razi University authorities for financial support during field work in western and central Iran. We also wish to thank Professor Göran Nilson (Gothenburg Natural History Museum, Gothenburg, Sweden) for providing the senior author the time and space to examine the material from his museum.

REFERENCES Anderson S. C. (1974), “Preliminary key to the turtles, lizards and amphisbaenians of Iran,” Fieldiana Zool., 65(4), 27 – 44. Anderson S. C. (1968), “Zoogeographic analysis of the lizard fauna of Iran. Chapter 10,” in: W. B. Fisher (ed.), The Land of Iran. The Cambridge History of Iran. Vol. 1, Cambridge Univ. Press, Cambridge, UK, pp. 305 – 371. Anderson S. C. (1999), The Lizards of Iran, Soc. for the Study of Amphibians and Reptiles. Baran I. and Gruber U. F. (1982), “Taxonomische Untersuchangen an türkischen Gekkoniden,” Spixiana, 5(2), 109 – 138. Blanford W. T. (1874), “Description of new lizards from Persia and Baluchistan,” Ann. Mag. Nat. Hist. Ser. 4, 13(78), 453 – 455. Kluge A. G. (1991), “Checklist of gekkonid lizards,” Smithsonian Herpetol. Inf. Serv., 85, 1 – 35. Kluge A. G. (1993), Gekkonid Lizard Taxonomy, Int. Gecko Soc., San Diego. Leviton A. E., Anderson S. C., Adler K. K., and Minton S. A. (1992), Handbook to Middle East Amphibians and Reptiles. Contributions in Herpetology, No. 8, Soc. for the study of Amphibians and Reptiles. Mertens R. F. W. (1924), “Ein neuer Gecko aus Mesopotamia,” Senckenberg. Biol., 6, 84.

Nasrullah Rastegar-Pouyani et al. Mertens R. F. W. (1956), “Amphibien und Reptilien aus S. O. Iran, 1954,” Jahr. Vereins vaterländ. Naturkunde Würtemberg, 111(1), 90 – 97. Moravec J. and Modry D. (1994), “On the occurrence of the Cyrtopodion heterocercum mardinensis and Pseudocerastes persicus fieldi in Syria,” Zool. Middle East, 10, 53 – 56. Mozaffari O. (2007), “On the occurrence of Cyrtopodion heterocercum heterocercum from Markazi (Arak) Province, Center of Iran,” Herpetol. Rev., 38(3), 2007. Nader I. A. and Jawdat S. Z. (1976), “Taxonomic study of the Geckos of Iraq (Reptilia: Gekkonidae),” Bull. Biol. Res. Center. Univ. Baghdad, (5), 1 – 41. Rastegar-Pouyani N. (1991), Biosystematics of Lizards of Kermanshah Province, Western Iran, MSc. Thesis, Tehran University. Szczerbak N. N. and Golubev M. L. (1977), “[Systematics of the Palearctic geckos (genera Gymnodactylus, Bunopus, Alsophylax )],” Gerpetol. Sb. Trudy Zool. Inst. AN SSSR, 74, 120 – 133 [in Russian with English summary]. Szczerbak N. N. and Golubev M. L. (1984), “[On generic assignment and generic structure of the Palearctic Cyrtodactylus lizard species (Reptilia, Gekkonidae, Tenuidactylus gen. n.)],” Vestnik Zool., 1984(2), 50 – 56 [in Russian]. Szczerbak N. N. and Golubev M. L. (1986), [The Gecko fauna of the USSR and Adjacent Regions], Nauka Dumka, Kiev [in Russian]. Szczerbak N. N. and Golubev M. L. (1996), The Gecko Fauna of the USSR and Adjacent Regions [English Edition, translated from the Russian by Mikhail L. Golubev and Sasha A. Malinsky; Alan E. Leviton and George R. Zug, eds.], Soc. for the study of Amphibians and Reptiles. Ugurtas I. H., Yildirimhan H. S., and Sevinc M. (2007), “Distribution of Gekkonidae in southeast Anatolia, Turkey, and new localities,” Tur. J. Zool., 31, 137 – 141.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 101 – 106

MORPHOLOGY OF PERIPHERAL BLOOD CELLS FROM SOME LACERTID LIZARDS FROM TURKEY

Hüseyin Arýkan,1 Bayram Göçmen,1 Mehmet Zülfü Yildiz,1,2 Çetin Ilgaz,3 and Yusuf Kumlutaº3 Submitted February 23, 2008. The present study is on the morphologies and sizes of peripheral blood cells (erythrocytes, leukocytes, and thrombocytes) on blood smears, stained with Wright’s stain, in some lacertid lizards species [Apathya cappadocica (Werner, 1902), Acanthodactylus boskianus (Daudin, 1802), Acanthodactylus harranensis Baran et al. 2005, Anatolacerta danfordi (Günther, 1876), Darevskia praticola (Eversmann, 1834), D. uzzelli (Darevsky and Danielyan, 1977), D. valentini (Boettger, 1892), Parvilacerta parva (Boulenger, 1887), Lacerta pamphylica Schmidtler, 1975, L. trilineata Bedriaga, 1886, L. viridis (Laurenti, 1768), Ophisops elegans Menetries, 1832, Mesalina brevirostris Blanford, 1876, Podarcis muralis (Laurenti, 1768), P. sicula (Rafinesque-Schmaltz, 1810), Timon princeps (Blanford, 1874)] from Turkey. As a result of our survey, it was determined that the blood cells of the investigated species are shows significant variations in sizes and of leukocytes, agranulocytic leukocytes (lymphocytes and monocytes) are present as predominant cells. Moreover, of granulocytes, neutrophils were no observed in A. danfordi, D. praticola, D. uzzelli, and P. parva. Keywords: Lacertidae; blood cell morphology; blood smears; Wright’s stain.

INTRODUCTION

MATERIAL AND METHODS

The first investigations about hematology of reptiles consist of comparing blood cells of reptiles with other vertebrates’ ones (Gulliver, 1840, 1842). The later works include sexual and seasonal variations on cell counts and sizes (Gulliver, 1875; Werzberg, 1910; Leowenthal, 1928, 1930; Wintrobe, 1933; Jordan, 1938; Ryerson, 1949; Altman and Dittmer, 1961; Hartman and Lessler, 1964; Hutchison and Szarski, 1965; Szarski and Czopek, 1966; Saint Girons and Saint Girons, 1969; Saint Girons, 1970; Duguy, 1970; Canfield and Shea, 1988; Sevinç and Uðurtaþ, 2001; Atatür et al. , 2001). In present study, the blood cell morphologies and sizes of 16 lacertid lizard species known from Turkey were investigated comparatively and obtained data discussed in literature.

Specimens of 16 species in family Lacertidae were collected different date and various localities from Turkey. The blood samples were obtained according to MacLean et al. (1973), from postorbital sinuses of the specimens via heparinized glass capillaries (MacLean et al., 1973). Blood smears were prepared and stained with Wright’s stain to facilitate the measurements of morphological and size parameters of the blood cells. The cells were measured under a light microscope, using a MOB-1-15x micrometrical ocular. From each blood smear, 40 erythrocytes were randomly chosen for the measurements of their lengths (L), widths (W), nuclear lengths (NL) and nuclear widths (NW). The sizes of the erythrocytes (ES) and their nuclei (NS) were computed from ES = LWð/4 and NS = NLNWð/4. Comparisons of cell and nuclear shapes were done from L/W and NL/NW ratios and that of nucleus/cytoplasm from NS/ES ratio. From the blood smears of each species, measurements of leukocytes (lymphocytes, monocytes, neutrophils, eosinophils, basophils) and thrombocytes (TL, TW) were also taken to determine their sizes. One

Zoology Section, Department of Biology, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey. Harran University, Faculty of Art and Science, Department of Biology, Zoology Section, Osmanbey Campus, Sanliurfa, Turkey. Department of Biology, Faculty of Education, Dokuz Eylül University, 35160 Buca, Izmir, Turkey.

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Fig. 1. Photomicrographs of peripheral blood cells of various lizards from Turkey, Wright’s stain: A, normal erythrocytes (Apathya cappadocica); B, small lymphocyte (Lacerta trilineata); C, large lymphocyte (Anatolacerta dandordi); D, monocyte (Mesalina brevirostris); E, neutrophil (Ophisops elegans); F, eosinophile (Lacerta pamphylica); G, basophil (A. cappadocica); H, almost nearly spherical shaped thrombocytes (O. elegans); I, a group of spindle shaped thrombocytes (M. brevirostris). Scale bar is 15 ìm.

way ANOVA test was utilized in the comparisons of the obtained data, á = 0.05 in all of the analyses. The photomicrographs of the blood cells were taken with Olympus BX51-Altra 20 Soft Imaging System. RESULTS The characteristic shape of erythrocytes in lacertid lizard is oval and with nuclei just like those of all other vertebrates except of mammals. Nuclei are also ellipsoidal, more or less as regular shape uniformly localized in a central place of the cell (Fig. 1A). On smears stained with Wright’s stain, the cytoplasms were light yellowish pink, the chromophilic nuclei were dark purplish blue.

The erythrocyte measurements (lengths and widths), sizes and L/W ratios are given in Table 1; nuclear measurements of the erythrocytes and nucleocytoplasmic ratios (NS/ES) are given in Table 2. Regarding the erythrocyte length, width and size, it was detected significant interspecific, in some cases even intraspecific variations. A statistical comparison of the species showed that the longest erythrocytes were found in L. pamphylica (F15,624 = 30.463, P = 0.000), the shortest, the narrowest and the smallest in O. elegans, the widest Anatolacerta danfordi (F15,624 = 27.001, P = 0.000) and the largest in A. harranensis (F15,624 = 29.210, P = 0.000). Regarding the L/W ratio, the most strongly ellipsoidal erythrocytes were observed

Blood Cells from Various Turkish Lizards

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TABLE 1. The Erythrocyte Measurements in the Peripheral Blood Cells of 16 Lacertid Lizards Species from Turkey Species A. cappadocica A. boskianus A. harranensis A. danfordi D. praticola D. uzzelli D. valentini P. parva L. pamphylica L. trilineata L. viridis O. elegans M. brevirostris P. muralis P. sicula T. princeps

Erythrocytes L, ìm

W, ìm

L/W

ES, ìm2

13.42 ± 0.90 12.00 – 15.75 14.22 ± 0.98 12.25 – 16.50 15.46 ± 1.24 12.50 – 18.00 14.14 ± 1.17 11.50 – 16.50 13.08 ± 0.97 11.50 – 15.25 13.65 ± 0.96 11.50 – 15.50 13.32 ± 0.93 12.00 – 16.25 13.63 ± 0.86 11.75 – 15.50 15.61 ± 1.00 13.25 – 18.00 14.39 ± 1.01 12.00 – 17.00 14.94 ± 1.04 12.50 – 17.50 12.43 ± 0.65 11.25 – 14.00 14.06 ± 0.91 12.50 – 16.50 13.93 ± 0.95 11.75 – 16.25 13.89 ± 0.94 12.25 – 16.00 14.98 ± 1.14 12.00 – 17.75

7.94 ± 0.47 6.50 – 8.75 7.92 ± 0.41 7.00 – 8.50 8.56 ± 0.59 7.25 – 10.00 9.09 ± 0.51 7.75 – 10.00 8.01 ± 0.38 7.25 – 9.00 7.84 ± 0.48 7.00 – 8.75 7.73 ± 0.57 6.50 – 9.00 8.01 ± 0.44 7.25 – 9.00 7.89 ± 0.52 6.75 – 9.00 7.63 ± 0.49 6.75 – 9.00 8.16 ± 0.56 7.25 – 9.25 7.51 ± 0.25 7.00 – 8.00 8.07 ± 0.42 6.75 – 8.75 8.43 ± 0.59 7.00 – 9.50 8.1 ± 0.35 7.25 – 8.75 8.43 ± 0.47 7.50 – 9.25

1.69 ± 0.14 1.45 – 2.12 1.80 ± 0.14 1.58 – 2.03 1.81 ± 0.13 1.47 – 2.07 1.56 ± 0.11 1.35 – 1.85 1.64 ± 0.13 1.36 – 1.97 1.74 ± 0.12 1.48 – 2.00 1.73 ± 0.14 1.47 – 2.04 1.70 ± 0.12 1.42 – 1.93 1.99 ± 0.16 1.64 – 2.30 1.89 ± 0.12 1.56 – 2.18 1.83 ± 0.1 1.58 – 2.12 1.66 ± 0.09 1.52 – 2.00 1.75 ± 0.14 1.52 – 2.10 1.66 ± 0.11 1.47 – 1.96 1.74 ± 0.12 1.50 – 2.00 1.78 ± 0.14 1.45 – 2.15

83.73 ± 8.13 70.16 – 102.00 88.45 ± 8.37 74.53 – 110.10 104.22 ± 13.53 73.59 – 137.38 101.13 ± 12.10 69.96 – 122.46 82.34 ± 7.78 67.71 – 101.76 84.22 ± 9.33 64.57 – 100.09 80.97 ± 9.47 63.78 – 102.05 85.80 ± 8.39 73.59 – 107.74 96.77 ± 9.91 75.51 – 127.17 86.31 ± 10.22 68.88 – 116.77 96.03 ± 11.78 71.14 – 118.00 73.27 ± 4.88 63.19 – 83.21 89.09 ± 7.52 106.86 – 118.00 92.46 ± 11.32 65.94 – 114.81 87.41 ± 7.85 72.12 – 109.90 99.27 ± 10.83 77.72 – 120.20

Note. L, erythrocyte length; W, erythrocyte width; ES, erythrocyte size.

in L. pamphylica, the least ellipsoidal in A. danfordi (Table 1). In nuclear measurements, the largest nuclei were found in L. trilineata (F15,624 = 11.947, P = 0.000), the shortest in D. uzzelli, the widest and the largest in A. danfordi (F15,624 = 90.886, P = 0.000; F15,624 = 21.825, P = 0.000), the narrowest in O. elegans and M. brevirostris, the smallest in T. princeps. Regarding NL/NW, the most strongly ellipsoidal nuclei were observed in L. trilineata, the least ellipsoidal in D. uzzelli (F15,624 = 24.704; P = 0.000). Nucleocytoplasmical ratio (NS/ES) was the highest in O. elegans, the lowest in T. princeps (F15,624 = 28.336, P = 0.000; Table 2). On blood smears belonging to investigated species, lymphocytes as predominant cell amongst the different types of leukocytes were observed both small and large types. In small lymphocytes, fairly chromophilic nuclei almost filled the whole cell (Fig. 1B). Cytoplasm was

pushed to a side as a small peripheral zone. In small lymphocytes diameter was ranged between 7.19 – 7.96 ìm (Table 3). Large lymphocytes had a relatively wider zone of cytoplasm. Cytoplasm was stained a pale blue, nuclei purplish blue with Wright’s stain (Fig. 1C). In large lymphocytes diameter was ranged from 10.07 to 13.04 ìm (Table 3). Of the agranulocytes, monocytes were similar to large lymphocytes in size but could be easily differentiated by their kidney shaped nuclei. Cytoplasm was stained a light gray, the nuclei dark purplish blue with Wright’s stain. In monocytes, diameter was ranged from 10.07 to 13.39 ìm (Table 3). Of granulocytes, the neutrophils or heterophils are spheroidal cells; their nuclei were consisted of 2 – 4 lobes (Fig. 1E). No neutrophils were observed in A. danfordi, D. praticola, D. uzzelli, P. parva, and P. sicula. In

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TABLE 2. The Erythrocyte Nuclei Measurements in the Peripheral Bloods of 16 Lacertid Lizards Species from Turkey Species A. cappadocica A. boskianus A. harranensis A. danfordi D. praticola D. uzzelli D. valentini P. parva L. pamphylica L. trilineata L. viridis O. elegans M. brevirostris P. muralis P. sicula T. princeps

NL, ìm

NW, ìm

NL/NW

NS, ìm2

NS/ES

6.33 ± 0.46 5.50 – 7.00 6.24 ± 0.55 5.25 – 7.50 6.59 ± 0.50 5.50 – 7.75 6.73 ± 0.61 5.50 – 8.50 6.19 ± 0.43 5.25 – 7.00 5.98 ± 0.36 5.50 – 6.75 6.13 ± 0.48 5.25 – 7.25 6.12 ± 0.48 5.25 – 7.25 6.33 ± 0.41 5.75 – 7.25 6.93 ± 0.52 6.00 – 8.25 6.64 ± 0.52 5.75 – 7.75 6.51 ± 0.34 5.75 – 750 6.46 ± 0.49 5.50 – 7.75 6.36 ± 0.54 5.00 – 7.50 6.59 ± 0.51 5.75 – 7.75 6.04 ± 0.53 5.00 – 7.50

4.11 ± 0.13 4.00 – 4.25 4.02 ± 0.12 3.75 – 4.25 4.07 ± 0.13 3.75 – 4.25 4.41 ± 0.13 4.25 – 4.75 4.31 ± 0.12 4.00 – 4.50 4.34 ± 0.14 4.00 – 4.50 4.28 ± 0.16 4.00 – 4.50 3.98 ± 0.10 3.75 – 4.25 4.23 ± 0.11 4.00 – 4.50 3.93 ± 0.11 3.75 – 4.00 4.36 ± 0.13 4.25 – 4.50 3.84 ± 0.15 3.50 – 4.25 3.84 ± 0.15 3.50 – 4.25 4.35 ± 0.12 4.25 – 4.50 4.19 ± 0.12 4.00 – 4.50 3.99 ± 0.11 3.75 – 4.25

1.54 ± 0.13 1.29 – 1.75 1.56 ± 0.15 1.24 – 1.88 1.62 ± 0.15 1.29 – 1.94 1.53 ± 0.14 1.28 – 1.89 1.44 ± 0.02 1.17 – 1.69 1.38 ± 0.11 1.22 – 1.69 1.43 ± 0.10 1.24 – 1.61 1.54 ± 0.12 1.31 – 1.75 1.49 ± 0.09 1.28 – 1.71 1.77 ± 0.16 1.50 – 2.20 1.53 ± 0.14 1.28 – 1.76 1.70 ± 0.11 1.41 – 1.93 1.69 ± 0.17 1.38 – 2.07 1.46 ± 0.12 1.18 – 1.76 1.57 ± 0.13 1.35 – 1.82 1.52 ± 0.15 1.25 – 2.00

20.42 ± 1.58 17.27 – 22.52 19.69 ± 1.70 16.49 – 23.55 21.02 ± 1.49 18.06 – 25.02 23.32 ± 2.30 18.35 – 30.03 20.93 ± 1.46 18.35 – 23.84 20.36 ± 1.22 18.35 – 23.84 20.63 ± 2.03 17.27 – 25.61 19.15 ± 1.67 16.19 – 24.19 21.01 ± 1.61 18.06 – 25.61 21.38 ± 1.56 17.66 – 25.12 22.68 ± 1.73 19.18 – 27.38 19.63 ± 1.22 16.93 – 23.55 19.48 ± 1.34 16.93 – 22.81 21.74 ± 2.06 16.68 – 25.61 21.69 ± 1.82 18.84 – 25.86 18.89 ± 1.67 15.70 – 22.08

0.25 ± 0.02 0.21 – 0.30 0.23 ± 0.02 0.19 – 0.31 0.21 ± 0.02 0.16 – 0.26 0.23 ± 0.03 0.19 – 0.36 0.26 ± 0.02 0.21 – 0.32 0.25 ± 0.03 0.20 – 0.33 0.26 ± 0.03 0.20 – 0.33 0.22 ± 0.02 0.18 – 0.28 0.22 ± 0.02 0.18 – 0.27 0.25 ± 0.02 0.21 – 0.31 0.24 ± 0.02 0.19 – 0.31 0.27 ± 0.02 0.23 – 0.30 0.22 ± 0.02 0.18 – 0.29 0.24 ± 0.02 0.19 – 0.31 0.25 ± 0.02 0.21 – 0.32 0.19 ± 0.02 0.15 – 0.24

Note. NL, nucleus length; NW, nucleus width; NS, nucleus size; NS/ES, nucleocytoplasmic ratio

TABLE 3. The Mean Measurement Values of Agranulocytic Leukocytes, Granulocytic Leukocytes, and Thrombocytes in the Peripheral Bloods of 16 Lacertid Lizards Species from Turkey, Together with Their Standard Deviation Species

Lymphocyte (small), ìm

Lymphocyte (large), ìm

Monocyte, ìm

Neutrophil, ìm

Eosinophil, ìm

Basophil, ìm

TL, ìm

TW, ìm

A. cappadocica A. boskianus A. harranensis A. danfordi D. praticola D. uzzelli D. valentini P. parva L. pamphylica L. trilineata L. viridis O. elegans M. brevirostris P. muralis P. sicula T. princeps

7.73 ± 0.26 7.96 ± 0.34 7.65 ± 0.24 7.90 ± 0.58 7.48 ± 0.19 7.42 ± 0.18 7.33 ± 0.29 7.47 ± 0.40 7.50 ± 0.19 7.31 ± 0.26 7.54 ± 0.23 7.19 ± 0.37 7.50 ± 0.37 7.75 ± 0.31 7.90 ± 0.36 7.42 ± 0.42

10.68 ± 0.72 13.04 ± 1.16 10.86 ± 0.71 10.57 ± 0.82 11.22 ± 0.51 10.34 ± 0.67 10.07 ± 0.86 10.86 ± 0.77 10.31 ± 0.95 10.18 ± 1.52 11.15 ± 0.71 10.03 ± 0.67 11.03 ± 1.26 10.78 ± 1.03 12.19 ± 1.27 10.61 ± 0.41

11.34 ± 0.55 13.39 ± 1.14 11.25 ± 0.57 12.33 ± 1.09 11.63 ± 0.55 11.00 ± 0.45 11.28 ± 1.00 11.59 ± 0.79 11.20 ± 0.78 10.07 ± 0.47 11.68 ± 0.61 10.70 ± 0.45 12.30 ± 0.93 12.48 ± 1.13 12.27 ± 0.71 10.73 ± 0.62

10.40 ± 0.29 13.38 ± 0.97 11.13 ± 0.32 — — — 9.42 ± 0.14 — 10.15 ± 0.14 9.83 ± 0.38 10.95 ± 0.41 10.05 ± 0.31 11.55 ± 0.48 10.38 ± 0.14 — 10.37 ± 0.32

10.12 ± 0.30 10.20 ± 0.21 9.95 ± 0.21 10.38 ± 0.14 9.80 ± 0.21 9.54 ± 0.17 9.88 ± 0.14 10.04 ± 0.19 9.75 ± 0.20 9.95 ± 0.21 10.50 ± 0.43 9.38 ± 0.75 10.25 ± 0.39 10.15 ± 0.29 9.96 ± 0.19 9.88 ± 0.14

9.70 ± 0.33 9.95 ± 0.21 9.33 ± 0.13 10.56 ± 0.13 9.20 ± 0.21 8.93 ± 0.31 9.40 ± 0.38 9.46 ± 0.23 9.95 ± 0.19 9.50 ± 0.20 9.25 ± 0.25 8.75 ± 0.25 9.36 ± 0.18 10.00 ± 0.40 9.25 ± 0.46 9.50 ± 0.31

6.35 ± 0.32 7.68 ± 0.53 7.08 ± 0.45 6.80 ± 0.33 6.28 ± 0.53 6.70 ± 0.37 6.40 ± 0.39 8.19 ± 0.47 6.95 ± 0.44 7.66 ± 0.63 7.28 ± 0.42 6.30 ± 0.35 7.29 ± 0.44 7.18 ± 0.46 8.42 ± 0.25 7.46 ± 0.42

5.33 ± 0.21 5.10 ± 0.32 4.96 ± 0.23 5.30 ± 0.20 4.69 ± 0.26 4.89 ± 0.30 4.69 ± 0.24 5.07 ± 0.25 4.90 ± 0.38 5.00 ± 0.46 4.88 ± 0.21 4.50 ± 0.20 5.14 ± 0.33 5.20 ± 0.39 5.31 ± 0.21 5.35 ± 0.24

Note. TL, thrombocyte length; TW, thrombocyte width.

Blood Cells from Various Turkish Lizards neutrophils, diameter was ranged from 10.05 to 13.38 ìm (Table 3). Eosinophils were easily distinguished with large bright red granules in their cytoplasm. The darkly stained nuclei were masked by the cytoplasmic granules so that their nuclei shape was not readily distinguishable (Fig. 1F). In eosinophils, diameter was ranged from 9.38 to 10.50 ìm (Table 3). Basophils were rarely seen on blood smears. Purplish black granules in their cytoplasm were characteristic for these cells. Nuclei were masked by the cytoplasmic granules as being in eosinophils nuclei (Fig. 1G). Basophils diameter was ranged from 8.75 to 10.56 ìm (Table 3). In some species, thrombocytes were observed nearly spheroidal while they were spindle shaped in some species. Quite chromophilic nuclei were large as well as almost filling the whole cell (Fig. 1H). Nucleoplasmic ratio in spindle shaped thrombocytes was very high. On blood smears, they were observed in groups of two or more cells (Fig. 1I). The length (diameter) and width of thrombocytes was ranged from 6.28 to 8.42 ìm and from 4.50 to 5.35 ìm, respectively (Table 3). DISCUSSION AND CONCLUSION Among vertebrates, the largest erythrocytes were found in urodeles (Hartman and Lessler, 1964; Foxon, 1964). Reptiles constitute a heterogeneous group among vertebrates, regarding their blood cell morphologies (Hartman and Lessler, 1964; Szarski and Czopek, 1966; Szarski, 1968; Saint Girons, 1970). According to Saint Girons and Saint Girons (1969), the largest erythrocytes are found in Sphenodon punctatus, member of old group, the later in turtles and crocodiles; the smallest in lacertid lizards. The numbers of erythrocytes in reptiles were lower than aves and mammals; lizards have the more high numbers than turtles in reptiles. Consequently, there is a negative correlation between the number of erythrocytes and the body sizes (Duguy, 1970). According to various researchers (Hartman and Lessler, 1964; Szarski and Czopek, 1966; Saint Girons and Saint Girons, 1970; Sevinç et al., 2000; Atatür et al., 2001), sizes of erythrocytes in lizards varied not only amongst the families, even sometimes amongst different species in same family. In the present study, it is determined that the size of the erythrocytes varied not only amongst species, but also on the different blood smears of the same species in investigated 16 lacertid lizards coinciding the present literature. The largest erythrocytes were observed in A. harranensis, the smallest in O. ele-

105 gans. We believe that these differentiations were stemmed from the possible different activation levels or environmental factors. Regarding L/W ratios, the more ellipsoidal erythrocytes were found in L. pamphylica, the least ellipsoidal or nearly spheroidal ones in A. danfordi. Atatür et al. (2001) reported that the erythrocytic nuclei were showed some interspecific variation in scincid lizards, and except of Eumeces schneiderii their nuclei were generally regular and more or less spherical in shape, and also there were a positive correlation between the sizes of the erythrocytes and their nuclei. The present work on blood smears established variations between the sizes of erythrocyte nuclei of 16 species which belong to the family Lacertidae. It was also determined that the nuclei were more ore less regular in shape and regarding NL/NW ratio, except some species (L. trilineata, O. elegans, and M. brevirostris) were established nearly spherical shape. The present study also established that the size of leukocytes varied between the species; in all of the investigated lizards agranulocytes were dominant cells; of granulocytic leukocytes, the nuclei of eosinophils and basophils were not easily seen because of the dense granulations of their cytoplasms. This finding supports Saint Girons (1970), Arýkan et al. (2004) and Arýkan et al. (in press). Of granulocytic leukocytes, no neutrophils were also observed in blood smears of A. danfordi, D. praticola, D. uzzelli, and P. parva. These results agree with the observations of Cannon et al. (1996) on some lizards, of Arýkan et al. (2004) on some viperids and of Arýkan et al. (in press) in one elapid species, Walterinnesia aegyptia. Thrombocytes have been described as small, ovoidal cells with centrally localized extremely chromophilic nuclei by various Authors (Taylor and Kaplan, 1961; Saint Girons, 1970; Camfield and Shea, 1988; Sevinç and Uðurtaþ, 2001; Arýkan, et al., 2004). The present study established the presence of both small, ovoidal cells and nearly spheroidal thrombocytes. REFERENCES Altman P. L. and Dittmer D. S. (1961), Blood and Other Body Fluids, Fed. Am. Soc. Exp. Biol., Washington. Arýkan H., Kumlutaº Y., Türkozan O., Baran Ý., and Ilgaz Ç. (2004), “The morphology and size of blood cells of some viperid snakes from Turkey,” Amphibia–Reptilia, 25, 465 – 470. Arýkan H., Göçmen B., Atatür M. K., Kumlutaº Y., and Çiçek K. (in press), “Morphology of peripheral blood cells from various Turkish snakes,” Biologia.

106 Atatür K. M., Arýkan H., Çevik Ý. E., and Mermer A. (2001), “Erythrocyte measurements of some scincids from Turkey,” Tur. J. Zool., 25, 149 – 152. Canfield P. J. and Shea G. M. (1988), “Morphological observations on the erythrocytes leukocytes and thrombocytes of blue tongue lizards (Lacertilia: Scincidae: Tiliqua),” Anat. Histol. Embryol., 17, 328 – 342. Cannon M. S., Freed D. A., and Freed P. S. (1996), “The leukocytes of the roughtail gecko Cryptopodion: a bright-field and phase-contrast study,” Anat. Histol. Embryol., 25, 11 – 14. Duguy R. (1970), “Numbers of blood cells and their variation,” in: C. Gans and T. S. Parsons (eds.), Biology of Reptilia. Vol. 3, Acad. Press, New York, pp. 93 – 109. Foxon G. E. H. (1964), “Blood and Respiration,” in: J. Moore (ed.), A Physiology of the Amphibia, Acad. Press, New York, pp. 151 – 209. Gulliver G. (1840), “On the blood corpuscles of the Crocodilia,” Proc. Zool. Soc. London, 8, 131 – 133. Gulliver G. (1842), “On the blood corpuscles of the British ophidians reptiles and other oviparous vertebrates,” Proc. Zool. Soc. London, 10, 108 – 111. Gulliver G. (1875), “Observations on the sizes and shapes of the red corpuscles of the blood of the vertebrates with drawings of them to a uniform scale and extended and revised tables of measurements,” Proc. Zool. Soc. London, 43, 474 – 495. Hartman F. A. and Lessler M. A. (1964), “Erythrocyte measurements in fishes, amphibians and reptiles,” Biol. Bull., 126, 83 – 88. Hutchison V. H. and Szarski H. (1965), “Number of erythrocytes in some amphibians and reptiles,” Copeia, 1965(3), 373 – 375. Jordan H. E. (1938), “Comparative hematology (Reptilia),” in: H. Downey (ed.), Handbook of Hematology, Hoeber, New York, pp. 776 – 788. Leowenthal N. (1928), “Etude sur les globules blanes du sang dans la séries des Vertébres. Reptiles,” Arc. Anat., 8, 255 – 273.

Hüseyin Arýkan et al. Leowenthal N. (1930), “Nouvelles observations sur les globules blanes du sang chez les animaux Vertébres. Reptiles,” Arc. Anat., 11, 283 – 279. MacLean G. S., Lee S. K., and Wilson K. F. (1973), “A simple method of obtaining blood from lizards,” Copeia, 1973(2), 338 – 339. Ryerson D. L. (1949), “A preliminary survey of reptilian blood,” J. Ent. Zool., 41, 49 – 55. Saint Girons M. C. and Saint Girons H. (1969), “Contribution à la morphologie comparée des érythrocytes chez les reptiles,” Br. J. Herpetol., 4, 67 – 82. Saint Girons M. C. (1970), “Morphology of the circulating blood cells,” in: C. Gans and T. S. Parsons (eds.), Biology of Reptilia. Vol. 3. Morphology C, Acad. Press, pp. 73 – 91. Sevinç M., Uðurtaþ Ý. H., and Yýldýrýmhan H. S. (2000), “Erythrocyte measurements in Lacerta rudis (Reptilia Lacertidae),” Tur. J. Zool., 24, 207 – 209. Sevinç M. and Uðurtaþ Ý. H. (2001), “The morphology and size of blood cells of Lacerta rudis bithynica (Squamata: Reptilia) Turkey,” Asiatic Herpetol. Res., 9, 122 – 129. Szarski H. and Czopek G. (1966), “Erythrocyte diameter in some amphibians and reptiles,” Bull. Acad. Pol. Sci. Cl. II Sér. Sci. Biol., 14, 433 – 437. Szarski H. (1968), “Evolution of cell size in lower vertebrates,,” in: A. S. Romer (ed.), Current Problems of Lower Vertebrate Phylogeny, Interscience (Wiley), pp. 445 – 453. Taylor K. and Kaplan H. M. (1961), “Light microscopy of the blood cells of pseudemyd turtles,” Herpetologica, 17, 186 – 196. Werzberg A. (1910), “Über Blutplättchen und Thrombocyten ihre Beziehungen zu Erthrocyten und Lymphozyten nebst einem Anhang Über die Erythrogenese,” Folia Haematol., 10(2), 301. Wintrobe M. M. (1933), “Variations in the size and haemoglobin concentration of erythrocytes in the blood of various vertebrates,” Folia Haematol., 51, 32 – 49.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 107 â&#x20AC;&#x201C; 118

ARE TOAD-HEADED LIZARDS Phrynocephalus przewalskii AND P. frontalis (FAMILY AGAMIDAE) THE SAME SPECIES? DEFINING SPECIES BOUNDARIES WITH MORPHOLOGICAL AND MOLECULAR DATA Agnes Gozdzik1 and Jinzhong Fu1,2 Submitted February 26, 2008. Toad-headed lizards of the Phrynocephalus przewalskii complex provide a challenging case for delimiting species boundaries. We tested the species status of P. przewalskii and P. frontalis using mitochondrial DNA (mtDNA) sequence and morphological data. A phylogenetic analysis was applied to the mtDNA data and principal component analysis (PCA) was applied to the morphological data. Furthermore, Mantel tests were used to test congruence between the patristic distance matrix derived from the phylogenetic tree and Euclidean distance matrix derived from PCA. The phylogenetic tree presented deeply diverged discreet clades that largely correspond to the two putative species. Nevertheless, PCA revealed no distinct clustering of individuals. Unique maternal inheritance might explain the discrete mtDNA variations while nuclear gene based morphological variations were continuous. Mantal tests suggested the mtDNA and morphology diverged in concordance; both had evidence of a west to east clinal variation. We conclude that P. frontalis is a synonym of P. przewalskii. Furthermore, the Mantel test is a useful method to compare mtDNA data with morphological data, but insufficient to delimit species boundaries. Keywords: species validity, Mantel test, mtDNA sequence, phylogeny, PCA, multivariate morphometrics, Phrynocephalus

INTRODUCTION Toad-headed lizards of the Phrynocephalus przewalskii complex are the dominant reptiles in the desert of northern China and adjacent Mongolia. The species group traditionally includes three nominal species, P. frontalis, P. przewalskii, and P. versicolor (e.g., Strauch, 1876; Zhao et al., 1999). There is substantial color variation within and between species, which may have caused the taxonomic confusion within this group. For example, over 20 names have been applied to these populations (Barabanov and Ananjeva, 2007). A recent phylogenetic study (Wang and Fu, 2004) of the complex using mitochondrial ND2 gene sequences revealed no clear distinction between the three nominal species and suggested that they were possibly one species (P. przewalskii). This is directly in conflict with several previous morphology based studies. Both Pope (1935) and 1

Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Address correspondence and reprint requests to Dr. Jinzhong Fu, Department of Integrative Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada; Phone: +1 (519) 824-4120 ext. 52715; Fax: +1 (519) 767-1656; E-mail: jfu@uoguelph.ca.

Zhao et al. (1999) distinguished the three species by morphological traits, although both heavily relied on color patterns on the hind limbs and tails to diagnose species. A study by Wang and Wang (1993) on populations that were traditionally identified as P. przewalskii and P. frontalis found that the two groups differed significantly based on multivariate morphometric analysis of metric (mensural) and meristic traits. The conflicting conclusions from mitochondrial DNA (mtDNA) phylogeny and morphology not only indicates that detailed scrutiny is needed to resolve the species status within this complex, but also presents an excellent case study for comparing mtDNA data with morphology data in delineating species boundaries. Morphological traits have been used as the primary evidence for species diagnosis for more than two centuries. More recently statistical analyses, such as multivariate methods that summarize information from several traits, continue to be used as methods to delimit species (e.g., Masters and Bragg, 2000; Montanucci, 2004). The principal component analysis (PCA) is a commonly used multivariate morphometric method. It is capable of summarizing variation in a number of traits simultaneously, and individual specimens do not need to be as-

1026-2296/2009/1602-0107 ÂŠ 2009 Folium Publishing Company

108 signed to groups prior to analysis. Therefore, PCA allows species delineation to be investigated without the bias of a priori taxonomic designations and is capable of identifying cryptic taxa (Masters and Bragg, 2000). On the other hand, mtDNA information is mostly the product of the last 20 years and is accumulating at an exponential rate along with the rapid improvement in DNA technology. The construction of mtDNA gene trees has become a standard method for testing traditional morphology-based taxonomy, as well as a method for species discovery in its own right (Avise and Ball, 1990; Graybeal, 1995; Olmstead, 1995; Templeton, 2001; Hebert et al., 2004). Among many advantages of mtDNA, the lack of reticulation and rapid lineage coalescence make it most suitable for inferring evolutionary history of closely related species and species complexes. Newly formed species often are more distinct in their mtDNA trees than in morphology. The objective of this study is to test the species status of two nominal species of the complex, P. przewalskii and P. frontalis, and explore methods of comparing mtDNA with morphological data. Wang and Fu (2004) hypothesized that P. frontalis is a junior synonym of P. przewalskii. The distribution ranges of the two species are next to each other while P. przewalskii occurs at the west and P. frontalis occurs at the east. Helan Mountains and Yellow River are located in the middle. We employed a phylogenetic analysis for mtDNA data and a principal component analysis for the morphological data. To make the comparison between the molecular and morphological data more reliable, we collected both types of data from the same specimens. METHODS Sampling Our sampling effort was concentrated on a 500 km west to east transect, including the Tengger Desert and the Ordos Dessert, which are separated by the Yellow River and the Helan Mountains. The Tengger Desert is the type locality of P. przewalskii, and most Tengger populations were previously identified as P. przewalskii. The Ordos Desert is the type locality for P. frontalis and the Ordos populations were predominantly identified as P. frontalis. A total of 313 specimens from 25 localities (population 1 – 25) were examined for morphological data. Among them, representatives from all populations, which included a total of 195 specimens, were sequenced. An additional 22 sequences of the P. przewalskii complex from Wang and Fu (2004) were obtained from GenBank. Figure 1 presents a map of northern

Agnes Gozdzik and Jinzhong Fu China and the sample sites, and Appendix I provides a list of specimens used in this study including their localities and voucher numbers. Phrynocephalus guttatus, P. versicolor, and an unnamed species (as defined in Wang and Fu, 2004) were selected as outgroup for the phylogenetic analysis based on the current understanding of the phylogeny of the genus Phrynocephalus (Arnold, 1999; Pang et al., 2003; Wang and Fu, 2004). Six sequences of the outgroup species were obtained from Genbank and one sequence was obtained in this study. The locality data of these samples are also presented in Appendix I. Molecular Data Collection and Phylogenetic Analyses Total genomic DNA was extracted from frozen muscle tissues using a Promega Wizard® Genomic DNA kit. The mitochondrial NADH dehydrogenase subunit 2 (ND2) gene was chosen for this study because of its high variability. Amplification of the ND2 gene was performed using the primers L4447 5¢-AAG CAG TTG GGC CCA TGC CCC AAA AAC GG-3¢ and H56222 5¢-TAT TTT AAT TAA AAT ATC TGA GTT GCA-3¢ (Wang and Fu, 2004). Standard PCR reactions were performed on a PTC-200 thermal cycler with an annealing temperature of 50°C. PCR products were purified using QiaQuick PCR purification kit (Qiagen) and were subsequently sequenced on an ABI 377 automatic sequencer using the Big Dye Terminator sequencing kit (Perkin Elmer). Initially, three to five randomly selected specimens per population of the focal species were selected for sequencing. However, all of the available specimens of a population were sequenced if phylogenetic analysis of the randomly chosen samples in a single population indicated the presence of haplotypes that belonged to more than one major cluster. A minimum of three specimens were also sequenced from the adjacent populations. Sequence editing for all sequences was performed with Sequencher (version 3.1.1, Gene Codes Corp.). Sequence alignment was performed in MacClade (version 4.06; Maddison and Maddison, 2003). Identical haplotypes were merged for phylogenetic analyses. Maximum parsimony (MP) method was used to infer the ND2 gene tree topology. All trees were rooted with the outgroup members. A model based Bayesian analysis was also conducted to verify the MP topology. The MP phylogenetic analyses were performed in PAUP* (version 4.0b10; Swofford, 2002). The large number of highly similar haplotypes generated many equally parsimonious trees for which branch swapping

Delineating Species Boundaries in Toad-Headed Lizards

109

Fig. 1. Map of northern China, showing geographic location of sample sites. The exact location of each sample site is given in Appendix I. The outlined areas represent the approximate distributions of the mitochdondrial haplotype clades for populations of the P. przewalskii complex.

would have been computationally impractical. We derived an approximation searching method in PAUP. The number of random addition replicates was set to 1000 and no more than 1000 trees of score greater than or equal to the shortest tree from a preliminary search were saved in each replicate. Two additional searches were conducted using the shortest tree score from the previous search to limit the 1000 trees for each replicate. All characters were unordered and weighed equally. All parsimony uninformative characters were excluded from the analysis. Heuristic searches were performed, with tree-bisection reconnection (TBR) branch swapping and 1000 random step-wise addition replicates. Bootstrap proportions (BSP; Felsenstein, 1985) were used to establish nodal support and were determined using a heuristic search strategy with 100 pseudoreplicates. Ten random addition replicates were conducted per bootstrap pseudoreplicate and a maximum of 1 Â´ 109 tree rearrangements was limited in each random addition replicate to reduce the search times. We do not expect

this reduction in search effort will significantly bias the bootstrap values. Bayesian analyses were conducted with MrBayes (version 3.1.2; Ronquist and Huelsenbeck, 2003). The parameters of the evolutionary model were selected by the likelihood ratio test performed in MrModeltest (version 2.1; Nylander, 2004). A GTR + I + G (nst = 6) model was found to be the best-fit model, and was implemented in the Bayesian analysis. Four Markov chains were employed for the MCMC simulations and 1 Â´ 107 generations were run to allow for convergence of log likelihood scores. Trees were sampled every 100 generations and only the last 10,000 trees were used to estimate the 50% majority rule consensus tree. Two independent searches, including four runs, were conducted to test the convergence of tree topologies. Morphological Data Collection and Analyses The traits used for morphometric analysis included 12 metric (mensural), 7 meristic, and 3 qualitative present/absent traits (Appendix II). These three qualitative

110 traits have been used as diagnostic traits to differentiate between P. frontalis and P. przewalskii (Zhao et al., 1999). Qualitative traits were binary coded. All specimens were sexed, but only individuals of snout to vent length (SVL) greater than 45 mm were considered sexually mature; all others were considered to be juveniles (Wang and Wang, 1993). Metric measurements were collected using a Mitutoyo Absolute Digimatric Digital Caliper, and recorded to two decimal places. All measurements were performed on the left side of the individuals, unless the body was damaged. An analysis of variance (ANOVA) was used to test if characters have significant differences among populations. Multivariate morphometric methods were employed using correlation based principal component analysis (PCA) on untransformed data. Metric and meristic traits were analyzed in separate PCA due to the lack of correlation between these two trait types. Additionally, ANOVA was carried out on both the metric and meristic data sets to test for sexual dimorphism. Statistically significant differences were found (p < 0.05) between males, females and juveniles in the metric dataset. As a result, metric data collected from juvenile specimens was not included in the PCA, and data collected from male and female specimens were analyzed in separate PCA. In the meristic dataset, none of the traits showed sexual dimorphism or adult-juvenile differences, hence, all individuals, including juveniles, were included in a single PCA analysis. All morphometric analyses were conducted with SPSS 11 (SPSS Inc., Chicago, USA). Mantel Tests Pairwise and partial matrix Mantel tests were used to examine the relationship between morphological and mtDNA variation (Daltry et al., 1996; Puorto et al., 2001). Between-specimen Euclidean distance matrices were constructed to summarize patterns of meristic and morphometric variation. MtDNA variation was represented by patristic distances calculated from the phylogenetic tree topology. Patristic distance is defined as the sum of the branch length between two taxa via their most recent common ancestor. Patristic distance encompasses both phylogenetic relationship and genetic similarity (Puorto et al., 2001), and therefore, is better than genetic distance. We used the tree derived from the MP analysis because a patristic distance matrix can be readily generated by PAUP. The male and female metric data were pooled for this test, following suggestions of Puorto et al. (2001). Distance matrices were also constructed to test the effects of sex of specimens, presence of putative diag-

Agnes Gozdzik and Jinzhong Fu nostic traits and geographic distances between localities on the morphological and molecular variation in specimens of the P. przewalskii complex. Three matrices were created for the following diagnostic traits: red axillary patch, black gular patch and black abdominal patch. A geographic distance dissimilarity matrix was calculated using distances between sample sites calculated from GPS coordinates of the localities. Both simple pairwise and partial Mantel tests were implemented to test for potential associations. First, correlations between morphology and mtDNA patristic distances, geographic distance and sex were examined with simple pairwise Mantel tests. Second, for testing the significance of the relationship between patristic distances and morphological variation among specimens, while eliminating the confounding effects of geographical distance, partial Mantel tests were performed. The meristic and metric data sets were used in a separate series of partial Mantel tests since there were no correlations between these data in both the pairwise Mantel tests and the initial correlation analyses prior to PCA. Third, potential associations between variations in patristic distances with variation in traditional diagnostic traits used to identify P. frontalis and P. przewalskii were examined by both simple and partial Mantel tests. Distance matrices for each of the diagnostic traits were first correlated with patristic distance in simple Mantel tests. If any relationship was observed, additional partial Mantel tests were performed to test for effects of geographic distances on this relationship. Each Mantel test was conducted with 10,000 randomizations using R package software (Le Progiciel R). RESULTS Phylogenetic Hypothesis of mtDNA Haplotypes A total of up to 850 base pairs of the ND2 gene were successfully sequenced for 196 individuals (195 ingroup and one outgroup). Combining with the 28 sequences from GenBank (22 ingroup and six outgroup), a total of 117 unique haplotypes were identified (110 ingroup and seven outgroup) and the alignment did not produce any gaps. Sequences were properly translated into protein using vertebrate mitochondrial codes, and therefore, we assumed that the sequences were genuine mitochondrial DNA, not pseudogenes. Of the 850 aligned base pairs, 252 sites were variable, 201 of which were parsimony informative. Excluding the parsimony-uninformative characters, the heuristic search under the parsimony criterion revealed more than 77,000 multiple equally parsimonious trees

Delineating Species Boundaries in Toad-Headed Lizards 89

111

[1] [11] [11]

100 83

B1 B2

1 change

Fig. 2. A maximum parsimony tree derived from 850 base pairs of mitochondrial ND2 gene sequences. Numbers above the branches are bootstrap proportions greater than 70%. Haplotype clades are denoted by letters. Dashed lines indicate unresolved nodes on the strict consensus tree. Clade A is traditional designated as P. frontalis and Clade B is traditional designated as P. przewalskii. Numbers in brackets correspond to population numbers for which exact localities can be found in Appendix I and Fig. 1.

100

(MPTs), with tree length of 381, CI of 0.6037 and RI of 0.9314. A strict consensus tree revealed that the unresolved nodes were primarily the tip nodes, most likely due to the large number of similar haplotypes. The differences of the patristic distances provided by different MPTs should be minimum. Therefore, a most parsimonious tree was arbitrarily chosen for constructing the patristic distance data matrix. Figure 2 presents the tree used for constructing the patristic distance matrix, with all unresolved nodes among the MPTs marked on the tree. The bootstrap proportions greater than 70 were mapped on the tree. The Bayesian analysis revealed a nearly identical topology as the parsimony analysis (tree not shown). The branching patterns of the basal nodes were identical and the branch lengths were similar. Two major lineages were clearly defined and these lineages largely correspond to the traditional taxonomic groupings of P. frontalis and P. przewalskii. Generally, clade A corresponds to populations of P. frontalis in the east including the type locality, population [32], and clade B corresponds to populations of P. przewalskii in the west including the type locality, population [6]. However, the two mitochondrial lineages did not show

[1] [11,12] [13] [1] [8,13,14,16] 73 [11] [13] [8] [8] [8] [15] 79 [8,13,15] [11,12,13,14,15,18,19,20,25] [13] [13,15,17] [13] [15] [15] 79 [19] 79 [19] 93 [19] [24,25] [20] [20] [21] [21] [21] [12] 87 [20,21,22,23] [22] 73 [30] [32] [20] 73 85 [20] [20,21,23] 70 99 [21] [21] 94 [31] [31] [18] [18] [18] [19] 87 [19] [19]

[1] [11] [2,8] [2] [8] [4] [2,9] [2] [10] [16] [16] [13,15,16,17] [15] [12] [13] [17] [17] [17] [15,17] [15] [17] [16] [16] [16] [17] [16,17] [17] [7,12] [9,10,12] [17] [2] [2] [5] [5] [7] [9]

[3] [4] [6] 87 [6] [33,34] [8] [9] 77 [10] [10] [9] [9,10] [10] [10] [10] [10] [10] [10]

geographic exclusivity. All populations along the Helan Shan Mountain ([1], [8], [11] â&#x20AC;&#x201C; [13], [15] â&#x20AC;&#x201C; [17]) were found to have members of both clades A and B (Fig. 1). Population [14] was an exception with all individuals belonging to clade A, but this is likely due to the small sample size of three specimens. Considering its geographical location (Fig. 1), we treated the population [14] as in the contact zone. Additionally, a population located north of the Yin Shan Mountain [30] had members in both clade A and clade B. The two lineages, A and B, were highly diverged with pairwise sequences differences ranging from 3.6 to 7.3%. Within each of the two major lineages, haplotypes demonstrated substantial variation. Within clade A, two deeply diverged branches, A1 and A2, were defined. The pairwise sequence difference between the two branches ranged from 2.0 to 2.8%. Members of populations [18] and [19], which were traditionally considered as P. versicolor (but included in P. przewalskii by Wang and Fu, 2004) and located at the northern end of the Helan Mountain Chain, had members from both A1 and A2. Within clade B, two diverged lineages were shown (Fig. 2). The pairwise sequence difference between the

112

Agnes Gozdzik and Jinzhong Fu

two lineages ranged from 2.6 to 4.9%. Clade B2 is composed of populations from the northeast, which were traditionally identified as P. versicolor, but Wang and Fu (2004) considered them as P. przewalskii. We did not have voucher specimens from these populations, therefore, no morphological analysis was performed on them. Morphometric Analyses The statistical null hypothesis of equal means between populations with one-way ANOVA was rejected for all 22 metric/meristic characters (p < 0.05). Taxonomic identities, based on mtDNA clade designations were assigned to individuals following the analyses to determine if clusters corresponded to genetically distinct or geographically recognized groups. The results of PCA of metric morphology from male and female lizards were very similar to each other. Three principal components account for 85% of the observed variation in males (Table 1). In females, three principal components account for 84% of the variation (Table 1). Based on factor loadings, PC1 can be interpreted as a measure of body size and PC2 appears to be a measure of midbody width for both males and females. Figure 3A, B show plots of PC1 and PC2 scores for male and female individuals, respectively. Members of putative P. frontalis (clade A) and P. przewalskii (clade B) appear to have considerable overlap in morphology, particularly in populations where individuals from both clades A and B co-exist. We further analyzed the data by excluding populations from the contact zone [population

1, 8, 11 – 17]. When only individuals from the “pure” populations, which were composed of individuals from only one clade, were included, the overlap in morphology was reduced but remained substantial (Fig. 3C, D). Furthermore, when longitude was plotted against PC1 scores (Fig. 3E, F), a clinal geographical pattern became evident for females; females at the west appear larger than those at the east. Four principal components account for 82% of the total variation in the meristic PCA (Table 2). All factor loading scores were high on PC1, particularly eyelid festoons (DEFN and VEFN) and longest hindlimb digit lamellae number (HLN). PC2 can be interpreted as the number of dorsal eyelid festoons (DEFN), while PC3 can be interpreted as a combination of parietal-nasal scale number (PNSN) and lower labial scale number (LLSN). From Fig. 4A, it is clear that, with regards to these traits, populations of putative P. przewalskii and P. frontalis show little differentiation. When individuals from only the “pure” populations were plotted in Fig. 4B, clustering becomes noticeable but overlap remains substantial. When plotted against longitude, PC1 scores for the meristic traits also demonstrate clinal differentiation from west to east (Fig. 4C). Mantel Tests A total of 165 individuals belonging to populations of P. frontalis and P. przewalskii had complete morphological data and patristic distance data estimated from the mtDNA phylogeny. Therefore, correlation examina-

TABLE 1. Principal Component Analysis Using a Correlation Matrix of Metric Traits Measured from Male and Female Specimens Traits

Males PCA1

1. SVL 0.91 2. TL 0.85 3. PNL 0.91 4. PL 0.84 5. CL 0.92 6. SL 0.88 7. BL 0.77 8. AML 0.93 9. HW 0.92 10. HL 0.89 11. MBW 0.77 12. TW 0.71 Eigenvalues 8.90 Percent variation 74 % Cumulative variation 74 Note. Factor loading scores above 0.5 are in explaining total variation.

PCA2

Females PCA3

0.16 –0.04 –0.22 0.11 0.21 –0.05 –0.35 –0.15 –0.19 0.07 –0.18 0.02 –0.12 0.04 –0.24 –0.07 0.23 –0.14 0.07 –0.22 0.54 –0.10 0.14 0.67 0.75 0.57 6 5 80 85 bold. Eigenvalue scores and

PCA4

PCA 1

PCA2

PCA3

0.01 0.92 0.14 0.09 –0.16 0.82 –0.20 –0.16 0.04 0.89 0.14 0.06 –0.18 0.84 –0.33 –0.27 –0.13 0.85 –0.30 –0.06 0.13 0.88 –0.14 0.14 0.60 0.75 –0.32 0.55 –0.10 0.88 –0.19 –0.18 0.00 0.90 0.25 –0.03 0.00 0.91 0.08 –0.07 –0.10 0.76 0.50 0.09 –0.06 0.78 0.37 –0.09 0.48 8.68 0.88 0.49 4 72 7 4 89 72 80 84 percent variation indicate relative contribution of the four

PCA4 –0.07 –0.17 –0.27 0.04 0.20 0.12 0.04 0.05 –0.15 –0.17 0.08 0.38 0.38 3 87 PC axes in

Delineating Species Boundaries in Toad-Headed Lizards

113 4

0 –1 –2

PC2 scores

–3 –3

–2

4 3 2 1 0 –1 –2 –3 –4 D –5 –2.5 –1.5

–1

0 1 PC1 scores

2.5

3.5

0 –1 –2

B –3

–2

–1

0 1 PC1 scores

1 0 –1

103

104

105 106 107 Longitude, °E

108

109

–2

–1

103

104

0 1 PC1 scores

108

109

1 0 –1 –2

–3 102

–3 –3

–2 –0.5 0.5 1.5 PC1 scores

PC2 scores

4 3 2 1 0 –1 –2 –3 –4 –5

PC2 scores

–3 102

105 106 107 Longitude, °E

Fig. 3. Morphometric analyses of the 12 metric traits. Symbols correspond to the mtDNA haplotype clade of each individual ( , Clade A; , Clade B). A – D: Projection of PC1 and PC2 scores from PCA analysis. A: All male specimens. PC1 explains 74% and PC2 explains 6% of the total variance. B: All female specimens. PC1 explains 72% and PC2 explains 7% of the total variance. C: Male specimens from populations that belong to only one clade. D: Female specimens from populations that belong to only one clade. E: Plot of the first principal component scores against longitude for male specimens. F: Plot of the first principal component scores against longitude for female specimens.

tions were based on data collected from these 165 specimens. Table 3 presents the results from simple pairwise Mantel tests that examined correlations between morphology and patristic distance, geography and sex. Table 3 presents the results of the partial Mantel tests that examined associations between morphology and patristic distances while holding the geographic distance matrix constant. Several significant associations were identified. The metric morphological traits revealed significant association with sex (r = 0.163, p = 0.0001) from the

simple Mantel test. These results are not surprising given that PCA analysis of metric traits and ANOVA had revealed strong sexual dimorphism. The metric data showed no correlation with geographic distance, which is surprising given that the PCA analysis revealed some patterns in clinal variation (Fig. 3C – E). Both simple and partial Mantel tests found no correlation between the metric data and patristic distance (Table 3). However, meristic morphological traits revealed a significant association with patristic distance, geographical distance and sex in the simple Mantel tests (Table 3). The

TABLE 2. Principal Component Analysis of Meristic Traits Measured from Male, Female, and Juvenile Specimens Using a Correlation Matrix Traits

PCA 1

PCA2

PCA3

PCA4

1. DEFN 0.73 –0.51 –0.23 0.02 2. VEFN 0.71 –0.46 –0.38 –0.03 3. PNSN 0.58 –0.03 0.62 0.39 4. LLSN 0.62 –0.22 0.51 –0.20 5. HFN 0.61 0.45 –0.30 0.41 6. HLN 0.72 0.43 –0.10 0.00 7. FLNP 0.65 0.42 0.04 –0.52 Eigenvalues 3.06 1.076 0.95 0.64 Percent variation 44 15 14 9 % Cumulative variation 44 59 73 82 Note. Factor loading scores above 0.5 are in bold. Eigenvalue scores and percent variation indicate relative contribution of the four PC axes to explaining total variation.

Agnes Gozdzik and Jinzhong Fu 3

0 –1 –2 –3

0 –1

–2

–1

0 1 PC1 scores

–4 –4

1 0 –1

–2 –3

–4 –3

PC2 scores

114

–2

B –2

0 2 PC1 scores

–3 102

103

104

105 106 107 Longitude, °E

108

109

Fig. 4. Morphometric analyses of the 7 meristic traits. Symbols indicate the mtDNA haplotype clade of each individual ( , Clade A; , Clade B). A: Projection of PC1 and PC2 scores from PCA analysis of all specimens. PC1 explains 44% and PC2 explains 15% of the total variance. B: Projection of PC1 and PC2 scores from PCA analysis of specimens from populations that belong to only one clade. C: Plot of the first principal component scores against longitude.

significant correlation between the meristic morphology and patristic distance was further confirmed by the partial Mantel test, when the geographical distance was controlled (Table 3). Only one of the three traditional diagnostic characters used for identification of P. frontalis and P. przewalskii, the presence of a red axillary patch, was significantly correlated with the patristic distances both in simple and partial Mantel tests (Table 3). However, both the red axillary spot and the black abdominal spot showed significant associations with geographic distance and all three revealed significant association with sex (Table 3). DISCUSSION Phrynocephalus frontalis Is a Synonym of P. przewalskii All data suggest that the populations of P. frontalis and P. przewalskii are one species, as Wang and Fu (2004) proposed. The principal component analysis,

which incorporated a total of 12 metric and 7 meristic morphological characters from 313 specimens, failed to reveal any distinct clustering. Even when the populations from the putative contact zone were excluded, characters of the two putative species remained largely overlapped (Figs. 3 and 4). With all specimens pre-identified by morphology, Wang and Fu (2004) found the two nominal species did not form respective monophyletic groups on the mtDNA tree. In this study, we did not assign specimens to species prior to the analysis; rather, we allowed pattern, if any, to reveal itself along with the analysis. MtDNA sequence data revealed discrete groups and a deep divergence between the eastern and western populations (Fig. 2), while nuclear gene based morphology suggested all populations as one unit. This is likely due to the unique maternal inheritance of mtDNA. Maternal inheritance produces no reticulation, and therefore, a bifurcating tree is always applicable to depict the relationships among the mtDNA haplotypes. Naturally, on any

TABLE 3. Correlation Coefficients (Above) and p-Values (below) from Simple and Partial Mantel Tests Holding the Geographic Distance Matrix Constant Two matrix Mantel patristic distance Metric morphology

geographic distance

Three matrix Mantel sex

patristic distance

–0.001352 –0.035570 0.163084 0.005417 0.387600 0.190000 0.000100 0.244700 Meristic morphology 0.043640 0.053310 0.043038 0.037902 0.000300 0.098900 0.031700 0.001300 Red axillary patch 0.084362 0.230750 0.048138 0.060243 0.000100 0.000100 0.038600 0.000300 Black gular patch –0.003270 0.027034 0.028988 –0.06373 0.459200 0.020200 0.027800 0.13500 Black abdominal patch –0.005900 –0.001132 0.065548 –0.005809 0.258500 0.509000 0.009800 0.250400 Note. Significant associations are in bold. A separated simple Mantel test of patristic distance and geographic distance also yielded significant values (r = 0.113204, p = 0.000100).

Delineating Species Boundaries in Toad-Headed Lizards tree, there are discrete groups (clades). Furthermore, maternal inheritance also reduces effective population size for mitochondrial evolution to a quarter of any nuclear genes, and therefore, a four times faster coalescence process. With this fast evolutionary rate, mtDNA is unlikely to maintain continuous variation across a large geographical area without breaking into segments. Numerous studies have demonstrated that mtDNA is much more likely to display geographical structure than any nuclear gene (Funk and Omland, 2003). Furthermore, behavioral differences between males and females may enhance the discrepancy between mitochondrial and nuclear genome evolution. Male-mediated gene flow, such as in the case of Green sea turtle (Roberts et al., 2004), would also create deeply diverged mtDNA lineages while nuclear genes remain homogenous. This study also illustrates the danger of solely relying on mtDNA for species delineation. MtDNA sequence data can serve as excellent precursory indicator, but are insufficient to define species boundaries if used alone (Fu and Zeng, 2008). Although the morphological variation is continuous and the mtDNA has breaks, variation of the two types of data responses to geography in a similar fashion. Mantel test revealed an significant correlation between the patristic distance matrix derived from the mtDNA and the Euclidean distance matrix derived from morphology (Table 3). Much of the variation is likely due to geography. On the mtDNA tree, specimens from the same geographical proximity were generally clustered together (Fig. 2); the meristic morphology revealed a significant correlation with geographical distance (Table 3). Part of the variation is also clinal; the meristic morphology had a clear west to east transition (Fig. 4) and clade A and B on the mtDNA tree were primarily made of eastern and western populations respectively. The red axillary patch used by Pope (1935) and Zhao et al. (1997) for species diagnosis also showed a west to east clinal variation (Table 3). Most individuals from the eastern populations, who are also the primary members of clade A, have the patch, while most individuals from the west, who are the primary members of clade B, do not have the patch. However, among the central populations, individuals with and without the patch are present in the same location. Wang and Wang (1993) found populations of P. przewalskii and P. frontalis differed significantly based on multivariate morphometric analysis. The different conclusions between our study and Wang and Wang’s are likely due to differences in sample size and methods of analysis. Wang and Wang used a smaller sample size and analyzed fewer morphological charac-

115 ters. More importantly, they used populations as analytical units and identified specimens to species prior to analysis. The multivariate analysis only confirmed the pre-defined groups, but did not distinguish intra-specific geographical variation from inter-specific variation. The Mantel Test Puorto et al. (2001) proposed the Mantel test as a means of examining congruence between morphological and mtDNA sequence data, and advertised the method for delineating species boundaries. They argued that the presence of two species should be characterized both by the possession of different mtDNA clades, and by patterns in morphological variation that are congruent with species identity as revealed by mtDNA sequence variation. Under this criterion, P. frontalis and P. przewalskii would be recognized as two valid species. The method undoubtedly represents an important attempt to bring morphology together with mtDNA data (Sites and Marshall, 2004). However, it only tests the congruence between the two types of data without addressing a crucial question: at what level of differentiation do diverged populations become different species? Many current methods have been proposed to address this question (e.g., Wiens and Penkrot, 2002; Hebert et al., 2004; Knowles and Carstens, 2007; Fu and Zeng, 2008), but all have severe limitations (Sites and Marshall, 2004). Much work is needed to reach a consensus. Acknowledgments. This project was supported by a NSERC (Canada) discovery grant to JF. We would like to thank Y. Wang, Z. Liu, Y. Zheng, V. Collins, C. Weadick, and A. Holliss for field or lab assistance. Thanks also to D. Noble and D. Melnikov for their comments on this manuscript. Drs. B. Robinson and T. Crease provided invaluable help for the completion of this project.

REFERENCES Arnold E. N. (1999), “Phylogenetic relationships of toadheaded lizards (Phynocephalus, Agamidae) based on morphology,” Bull. Nat. Mus. Lond. Zool., 65, 1 – 13. Avise J. C. and Ball R. J., Jr. (1990), “Principles of genealogical concordance in species concepts and biological taxonomy,” Oxford Surveys Evol. Biol., 7, 45 – 67. Barabanov A. V. and Ananjeva N. B. (2007), “Catalogue of the available scientific species-group names for lizards of the genus Phrynocephalus Kaup, 1825 (Reptilia, Sauria, Agamidae),” Zootaxa, 1399, 1 – 56. Daltry J. C., Wuster W., and Thorpe R. S. (1996), “Diet and snake venom evolution,” Nature, 379, 537 – 540. Felsenstein J. (1985), “Confidence limits on phylogenies — An approach using the bootstrap,” Evolution, 39, 783 – 791.

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Fu J. and Zeng X. (2008), “How many species are in the genus Batrachuperus? A phylogeographical analysis of the stream salamanders (family Hynobiidae) from southwestern China,” Mol. Ecol., 17, 1469 – 1488. Funk D. J. and Omland K. E. (2003), “Species-level paraphyly and polyphyly: Frequency, causes, and consequences, with insights from animal mitochondrial DNA,” Ann. Rev. Ecol. Evol. Syst., 34, 397 – 423. Graybeal A. (1995), “Naming Species,” Syst. Biol., 44, 237 – 250. Hebert P. D. N., Penton E. H., Burns J. M., Janzen D. H., and Hallwachs W. (2004), “Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator,” Proc. Natl. Acad. Sci. USA, 101, 14812 – 14817. Knowles L. L. and Carstens B. (2007), “Delimiting species without monophyletic gene trees,” Syst. Biol., 56, 887 – 895. Maddison W. P. and Maddison D. R. (2003), MacClade: Analysis of Phylogeny and Character Evolution, Sinauer Associates, Sunderland. Masters J. C. and Bragg N. P. (2000), “Morphological correlates of speciation in bush babies,” Int. J. Primatol., 21, 793 – 813. Montanucci R. R. (2004), “Geographic variation in Phrynosoma coronatum (Lacertilia, Phrynosomatidae): Further evidence for a Peninsular Archipelago,” Herpetologica, 60, 117 – 139. Nylander J. A. A. (2004), MrModeltest version 2.1 (Computer program distributed by the author), Uppsala University, Uppsala. Olmstead R. G. (1995), “Species concepts and plesiomorphic species,” Syst. Bot., 20, 623 – 630. Pang J., Wang Y., Zhong Y., Hoelzel A. R., Papenfuss T. J., Zeng X., Ananjeva N. B., and Zhang Y. P. (2003), “A phylogeny of Chinese species in the genus Phrynocephalus (Agamidae) inferred from mitochondrial DNA sequences,” Mol. Phylogen. Evol., 27, 398 – 409. Pope C. H. (1935), The Reptiles of China, Am. Mus. Nat. Hist., New York.

Puorto G., Salomao M. D., Theakston R. D. G., Thorpe R. S., Warrell D. A., and Wüster W. (2001), “Combining mitochondrial DNA sequences and morphological data to infer species boundaries: Phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae),” J. Evol. Biol., 14, 527 – 538. Ronquist F. and Huelsenbeck J. P. (2003), “MRBAYES 3: Bayesian phylogenetic inference under mixed models,” Bioinformatics, 19, 1572 – 1574. Roberts M. A., Schwartz T. S., and Karl S. A. (2004), “Global population genetic structure and male-mediated gene flow in the green sea turtle (Chelonia mydas): Analysis of microsatellite loci,” Genetics, 166, 1857 – 1870. Sites J. W., Jr. and Marshall J. C. (2004), “Operational criteria for delimiting species,” Ann. Rev. Ecol. Evol. Syst., 35, 199 – 227. Strauch A. (1876), “Part III: Reptilia and Amphibia,” in: Przehevalsky N. M. (ed.), Mongolia and the Rangut County. Vol. 2, Russian Imperial Geographic Society, St. Petersburg [in Russian]. Swofford D. L. (2002), PAUP*: Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4.0b10, Sinauer Associates, Sunderland. Templeton A. R. (2001), “Using phylogeographic analyses of gene trees to test species status and processes,” Mol. Ecol. 10, 779 – 791. Wang Y. and Fu J. (2004), “Cladogenesis and vicariance patterns in the toad-headed lizard Phrynocepahlus versicolor species complex,” Copeia, 2004, 199 – 206. Wang Y. and Wang H. (1993), “Geographic variation and diversity in three species of Phrynocephalus in the Tengger Desert, Western China,” Asiatic Herpetol. Res., 5, 65 – 73. Wiens J. J. and Penkrot T. A. (2002), “Delimiting species using DNA and morphological variation and discordant species limits in spiny lizards (Sceloporus),” Syst. Biol., 51, 69 – 91. Zhao E., Zhao K., and Zhou K. (1999), Fauna Sinica. Reptilia Vol. 2. Squamata, Lacertilia, Science Press, Beijing [in Chinese].

APPENDIX I. Localities and Voucher Numbers/GenBank Accession Numbers of Specimens Examined Population

Locality

Latitude

Longitude

Voucher numbers/ Genbank accession No.

Phrynocephalus przewalskii complex (n = 328) 1

Zhongwei Co., Ningxia

37°30.688¢ N

105°27.672¢ E

UG-JF789 – UGJF796

Zhongwei Co., Ningxia

37°26.750¢ N

104°32.007¢ E

UG-JF797 – UGJF808

Zhongwei Co., Ningxia

37°26.191¢ N

104°21.208¢ E

UG-JF809 – UGJF814

Jingtai Co., Gansu

37°22.446¢ N

104°09.934¢ E

UG-JF815 – UGJF823

Gulang Co, Gansu

37°31.115¢N

103°23.597¢ E

UG-JF824 – UGJF838

Wuwei Co., Gansu

38°06.298¢ N

102°42.803¢ E

UG-JF839 – UGJF853

Wuwei Co., Gansu

37°53.063’N

102°56.190¢ E

UG-JF854 – UGJF865

Zhongwei Co., Ningxia

37°27.270¢ N

104°59.534¢ E

UG-JF866 – UGJF879

Delineating Species Boundaries in Toad-Headed Lizards

Population

Locality

Zhongwei Co., Ningxia

Alxa Zouqi, Inner Mongolia

117

Voucher numbers/ Genbank accession No.

Latitude

Longitude

37°35.140¢ N

105°01.256¢ E

UG-JF880 – UGJF887

104°58.150¢ E

UG-JF888 – UGJF902; UG-JF904

37°49.609¢ N

Alxa Zouqi, Inner Mongolia

37°37.691¢ N

105°21.780¢ E

UG-JF906 – UGJF912

Alxa Zouqi, Inner Mongolia

37°50.389¢ N

105°24.191¢ E

UG-JF923 – UGJF931; UG-JF933

Alxa Zouqi, Inner Mongolia

38°21.124¢ N

105°42.030¢ E

UG-JF934 – UGJF946; UG-JF948 – UG-JF952

38°32.722¢ N

105°38.849¢ E

UG-JF953 – UGJF955 UG-JF956 – UGJF979

Alxa Zouqi, Inner Mongolia

38°47.317¢ N

105°40.359¢ E

Alxa Zouqi, Inner Mongolia

39°02.824¢ N

105°39.534¢ E

UG-JF980 – UGJF992

Alxa Zouqi, Inner Mongolia

39°01.318¢ N

105°52.651¢ E

UG-JF993 – UGJF1015

Alxa Zouqi, Inner Mongolia

39°22.076¢ N

106°26.193¢ E

UG-JF1016 – UGJF1020; UG-JF1022

Wuhai, Inner Mongolia

39°26.687¢ N

106°43.192¢ E

UG-JF1024 – UGJF1027; UG-JF1029 – UG-JF1032; UG-JF1034;UG-JF1035

Otog Qi, Inner Mongolia

39°13.716¢ N

107°10.340¢ E

UG-JF1038 – UGJF1043; UG-JF1045 – UG-JF1048

Otog Qi, Inner Mongolia

39°08.841¢ N

107°53.763¢ E

UG-JF1051 – UG-JF1056; UG-JF1058 – UG-JF1071

Otog Qi, Inner Mongolia

39°00.187¢ N

108°09.676¢ E

UG-JF1072 – UGJF1091

Otog Qi, Inner Mongolia

38°55.481¢ N

107°34.146¢ E

UG-JF1092 – UGJF1109

Otog Qianqi, Inner Mongolia

38°38.876¢ N

107°19.772¢ E

UG-JF1110 – UGJF1123

Otog Qianqi, Inner Mongolia

38°08.359¢ N

107°31.097¢ E

UG-JF1124 – UGJF1135

Abag Qi, Inner Mongolia

43°56.19¢ N

114°33.24¢ E

AY396576, AY396577

Sonid Zuoqi, Inner Mongolia

43°47.86¢ N

113°36.63¢ E

AY396579 AY396574, AY396575

Erenhot, Inner Mongolia

43°20.46¢ N

112°11.12¢ E

Sonid Youqi, Inner Mongolia

43°07.47¢ N

112°25.20¢ E

AY396578

Bailingmiao, Inner Mongolia

41°31.64¢ N

110°32.89¢ E

AY396582, AY396583, AY396581, AY396580

Dongsheng City, Inner Mongolia

39°21.47¢ N

109°49.75¢ E

AY396591, AY396592

Yulin, Shaanxi Province

38°21.32¢ N

109°41.50¢ E

AY396595

Jinchang, Gansu Province

38°41¢ N

102°06¢ E

AY396586

Jinchang, Gansu Province

38°23.60’N

102°05.60’E

CIB- W0239

39°42¢ N

98°11¢ E

AY396596, UG-JF1393

41°37.19¢ N

95°14.31¢ E

AY396602

Outgroup Phrynocephalus sp. 35

Jiayuguan, Gansu

Xingxingxia, Gansu

Ejin Qi, Inner Mongolia

Hami, Xinjiang

Kuytun, Xinjiang

Burqin, Xinjiang

Phrynocephalus versicolor (n = 2) 41°58¢ N

101°06¢ E

AY396605

43°04.19¢ N

93°34.93¢ E

AY396613

Phrynocephalus guttatus (n = 3) 44°24.96¢ N 47°18.70¢ N

84°47.12¢ E

AY396571

86°46.11¢ E

AY396573

Note. UG, University of Guelph; CIB, Chengdu Institude of Biology. Only specimens from population 1 – 25 were used for morphological examination.

118

Agnes Gozdzik and Jinzhong Fu

APPENDIX II. The Morphological Traits Used for Morphometric Analysis and Their Acronyms Acronym

Description of morphological trait

SVL TL PNS PL CL SL BL AML HW HL MBW TW LLSN HLN FLNP PNSN DEFN VEFN HFN RAB BGP BAP

Snout to vent length Tail length Parietal nasal length Pes length Crus length Shank length Brachium length Antebrachium-manus length Head width Head length Midbody width Tail width lower labial scale number Number of subdigital lamellae on the longest digit of foot Number of subdigital lamellae on the first digit of the manus Number of scales from parietal eye to left nostril Number of dorsal eyelid festoons Number of ventral eyelid festoons Number of fringes on longest toe Presence of red axillary patch Presence of black gular patch Presence of black adominal patch

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 119 – 125

BLOOD CELL COUNTS AND SIZES OF SOME ANURANS FROM TURKEY1 Çiðdem Gül2 and Cemal Varol Tok2 Submitted February 13, 2008. We determined blood cell counts and sizes of Rana ridibunda, Rana dalmatina, Bufo viridis, Bufo bufo, Hyla arborea, and Pelobates syriacus, examining 72 specimens (33 males, 39 females) of species collected from Çanakkale Province of Turkey. For the blood cell counts and sizes, a sexual dimorphism was lacking in the species studied. The lowest erythrocytes count was found in R. ridibunda, the largest one in B. viridis. The smallest erythrocytes were found in P. syriacus and the largest ones in R. ridibunda. Semiaquatic and hydrophilous species had lower erythrocyte count in comparison with terrestrial ones. Keywords: Rana ridibunda, Rana dalmatina, Bufo viridis, Bufo bufo, Hyla arborea, Pelobates syriacus, Anura, Blood cells.

INTRODUCTION Most studies on hematology in various species of Anura are limited to single species or only erythrocyte measurements (Hutchison and Szarski, 1965; Carmena et al., 1980; Sinha, 1983; Arýkan, 1989; Atatür et al., 1999; Arýkan et al., 2001; Arýkan et al., 2003; Wojtaszek and Adamowicz, 2003). Various factors (habitat conditions, sex, season, body mass, age) are known to affect the blood cell counts and sizes in Amphibia species (Alder and Huber, 1923; Klieneberger, 1927; Arvy, 1947; Kaplan, 1951, 1952; Foxon, 1964; Hartman and Lessler, 1964; Hutchison and Szarski, 1965; Szarski and Czopek, 1966; Rouf, 1969; Carmena et al., 1980; Kuramoto, 1981; Sinha, 1983; Arýkan, 1989; Atatür et al., 1999; Arýkan et al., 2001; Wojtaszek and Adamowicz, 2003). Atatür with coauthors (1999) examined erythrocyte cell sizes in some anurans from Turkey (Rana ridibunda, Bufo bufo, Bufo viridis, Pelobates syriacus, Bombina bombina, and Hyla arborea) and found the largest erythrocytes in Rana ridibunda, the widest in Bombina bombina, and the smallest in Pelobates syriacus. In addition, the blood cell counts and sizes of Rana ridibunda and Pelodytes caucasicus were given in detail by Arýkan (1989) and Arýkan with coauthors (2003). Information about blood cell counts and sizes of Rana dalmatina is lacking. 1 2

This study is a master thesis. Çanakkale Onsekiz Mart University, Faculty of Science and Arts, Department of Biology, Terzioðlu Campus, 17100 Çanakkale, Turkey; E-mail: gulcigdem@comu.edu.tr

The aim of this paper was to study blood cell counts and sizes in some Anura species from Turkey. MATERIAL AND METHODS The specimens of anuran species studied (Rana ridibunda, Rana dalmatina, Bufo viridis, Bufo bufo, Hyla arborea, and Pelobates syriacus) were collected from various localities in Çanakkale Province of Turkey. All specimens except of Rana dalmatina were caught from Çanakkale Center (western Anatolia) whereas Rana dalmatina was collected from Gelibolu Peninsula (Thrace). The taxonomical statuses of the anuran species examined in our study zone are as follows: Rana ridibunda complex (Alpagut Keskin, 2000), Rana dalmatina, Bufo viridis viridis (Yýlmaz, 1984; Tosunoðlu, 1999), Bufo bufo spinosus (Yýlmaz, 1984; Tok, 1995; Yýlmaz and Kumlutaþ, 1995), Hyla arborea arborea (Yýlmaz, 1984; Tok, 1995; Schneider, 2001), and Pelobates syriacus syriacus (Uðurtaþ, 1995). 72 sexually mature specimens (33 males, 39 females) were brought alive in a laboratory. Blood samples from these specimens were obtained within one day and were taken from etherized toads by means of cardiac (ventriculus) capillaries with heparinized hematocrit (Arýkan et al., 2003). Then these frogs were stored in Çanakkale Onsekiz Mart University collection connected to Zoology Department Ege University (Leviton et al., 1985).

120

Çiðdem Gül and Cemal Varol Tok

d e

Rana ridibunda

Rana dalmatina

Bufo viridis

b b

Bufo bufo

Hyla arborea

Pelobates syriacus

20 ìm

Fig. 1. The erythrocytes and nuclei of Rana ridibunda, Rana dalmatina, Bufo viridis, Bufo bufo, Hyla arborea, and Pelobates syriacus. a, Neutrophyl; b, lymphocyte; c, monocyte; d, eosinophyl; e, nuclei of erythrocytes on stained blood smears with Wright’s stain method.

The erythrocyte counts were done utilizing a Neubauer Hemocytometer as diluting solutions (the number of erythrocyte and leucocytes present in 1 mm3 of blood). The standard Hayem’s solution was used for erythrocytes, and the Turck’s solution was applied for leucocytes. Stained blood smears with Wright’s stain method were made for measurements of erythrocytes, leucocytes, and thrombocytes sizes, as well as erythrocyte nucleus sizes. Blood cell measurements were done under a microscope with Olympus 1-15x ocular micrometer. On each blood smear 40 erythrocytes were randomly chosen. An erythrocyte length (EL) and width (EW), nucleus length (NL) and width (NW) were measured. Dry erythrocyte cell (CA) and nucleus (NA) areas were computed by formulas: CA = EL ´ EW ´ ð/4 and

NA = NL ´ NW ´ ð/4. Cell and nucleus diameters are given in micrometers, and areas in ìm2. The photographs of blood cells were obtained with use of Olympus CX31 microscope (40´ magnify). Parametric descriptive statistics were analyzed by SPSS 10.0 (for Windows Student Version). RESULTS Significant differences between males and females were not found in the counts and sizes of the blood cells, therefore sexes were evaluated together. Erythrocytes were oval cells and contained nuclei for all species examined. Nuclei were centrally located and regular

TABLE 1. Blood Cell Data for Six Anurans Species Inhabiting of Turkey Character

Erythrocyte number Leucocyte number Erythrocyte length (EL) Erythrocyte width (EW) Erythrocyte sizes (CA) Nucleus length (NL)

10 6 10 10 10 10

Min – max Rana ridibunda 200,000 – 650,000 800 – 4520 21.59 – 24.09 12.40 – 12.81 210.8 – 242 7.71 – 9.00

Mean

320,500 2536 22.88 12.60 226.7 8.39

153,818.2 1447.22 0.70 0.13 9.13 0.46

48,641.2 590.82 0.220 0.041 2.888 0.144

Blood Cells Count and Size

121

TABLE 1 (continued) Character

Min – max

Mean

Nucleus width (NW) Nucleus sizes (NA) EL/EW NL/NW CA/NA Lymphocyte (large) diameter Lymphocyte (small) diameter Monocyte diameter Neutrophyl diameter Eosinophyl diameter Basophyl diameter Thrombocyte length Thrombocyte width

10 10 10 10 10 10 10 10 10 10 10 10 10

3.95 – 4.93 24.94 – 34.83 1.73 – 1.88 1.83 – 2.10 7.01 – 8.67 11.06 – 12.87 8.12 – 9.37 14.37 – 15.62 12.50 – 13.75 12.12 – 13.37 5.21 – 9.12 12.09 – 14.87 3.31 – 5.37

4.38 29.11 1.81 1.96 8.14 12.09 8.72 14.90 13.26 12.66 8.29 13.35 4.70

0.28 3.22 0.04 0.09 0.59 0.63 0.36 0.37 0.39 0.35 1.16 0.87 0.57

0.09 1.02 0.012 0.03 0.187 0.198 0.112 0.117 0.124 0.109 0.366 0.276 0.182

415,750 2683 21.80 12.57 215.41 8.53 3.84 25.92 1.73 2.24 8.60 11.82 8.87 14.51 13.24 12.24 8.74 10.36 5.27

74,115.2 865.83 0.96 0.37 10.21 0.44 0.31 3.05 0.10 0.14 0.68 1.51 1.22 0.45 0.53 0.48 0.90 0.97 0.43

26,203.7 353.47 0.34 0.132 3.611 0.155 0.109 1.078 0.036 0.049 0.24 0.533 0.432 0.169 0.187 0.16 0.319 0.343 0.153

721,750 1646 19.66 12.76 197.32 7.69 3.82 23.18 1.54 2.04 8.74 11.71 8.33 14.55 12.55 12.64 8.47 11.52 5.20

140,585.20 443.84 0.57 0.50 12.67 0.48 0.28 2.25 0.042 0.20 0.39 0.68 0.28 1.83 0.56 1.49 0.53 0.94 0.83

31,435.80 104.61 0.13 0.11 2.90 0.11 0.066 0.51 0.096 0.046 0.09 0.15 0.065 0.42 0.12 0.34 0.12 0.21 0.19

Rana dalmatina Erythrocyte number Leucocyte number Erythrocyte length (EL) Erythrocyte width (EW) Erythrocyte sizes (CA) Nucleus length (NL) Nucleus width (NW) Nucleus sizes (NA) EL/EW NL/NW CA/NA Lymphocyte (large) diameter Lymphocyte (small) diameter Monocyte diameter Neutrophyl diameter Eosinophyl diameter Basophyl diameter Thrombocyte length Thrombocyte width

8 6 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

310,000 – 550,000 2000 – 4100 20.75 – 23.56 12.12 – 13.31 208.02 – 237.52 7.87 – 9.28 3.57 – 4.56 23.07 – 32.13 1.56 – 1.87 1.99 – 2.43 7.28 – 9.37 10.56 – 15.31 8.25 – 11.87 13.87 – 15.37 12.37 – 13.87 11.62 – 13.06 7.87 – 10.75 9.12 – 11.93 4.81 – 6.06

Erythrocyte number Leucocyte number Erythrocyte length (EL) Erythrocyte width (EW) Erythrocyte sizes (CA) Nucleus length (NL) Nucleus width (NW) Nucleus sizes (NA) EL/EW NL/NW CA/NA Lymphocyte (large) diameter Lymphocyte (small) diameter Monocyte diameter Neutrophyl diameter Eosinophyl diameter Basophyl diameter Thrombocyte length Thrombocyte width

20 18 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19

450,000 – 900,000 900 – 2500 18.21 – 20.62 12.03 – 14.00 176.16 – 227.28 6.46 – 8.48 3.43 – 4.59 18.93 – 29.40 1.48 – 1.61 1.74 – 2.38 7.93 – 9.49 10.81 – 12.87 7.81 – 8.93 11.87 – 20.93 10.75 – 13.50 11.56 – 18.62 7.50 – 9.50 10.12 – 13.18 4.31 – 8.12

Bufo viridis

122

Çiðdem Gül and Cemal Varol Tok

TABLE 1 (continued) Character

Min – max

Mean

9 8 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11

Bufo bufo 453,000 – 703,000 1100 – 3800 20.37 – 22.78 12.09 – 13.31 193.55 – 231.42 7.40 – 9.18 3.82 – 4.92 22.64 – 35.32 1.60 – 1.76 1.60 – 2.15 6.56 – 9.67 10.87 – 13.06 7.93 – 8.62 14.12 – 16.12 11.56 – 14.37 10.12 – 14.00 7.75 – 9.25 11.56 – 13.12 4.81 – 6.06

534,722 2325 21.53 12.82 217.71 8.14 4.43 28.45 1.67 1.86 7.91 11.87 8.34 15.21 13.37 12.84 8.55 12.48 5.31

26,391.43 343.17 0.64 0.32 10.12 0.59 0.46 4.55 0.045 0.15 1.18 0.79 0.22 0.66 0.81 1.05 0.43 0.57 0.36

79,174.30 970.640 0.19 0.097 3.05 0.18 0.13 1.37 0.013 0.045 0.35 0.24 0.069 0.20 0.24 0.31 0.13 0.17 0.10

12 10 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14

Hyla arborea 405,000 – 703,000 460 – 4900 18.71 – 20.62 11.78 – 12.71 174.50 – 205.74 7.34 – 8.50 3.28 – 3.68 19.67 – 24.39 1.51 – 1.67 2.00 – 2.41 8.48 – 10.07 10.68 – 12.18 8.00 – 8.75 13.62 – 15.62 11.12 – 13.12 11.18 – 12.87 7.50 – 9.25 10.37 – 12.43 3.87 – 5.15

579,583 2980 19.62 12.26 189.16 7.64 3.49 21.15 1.59 2.18 9.07 11.33 8.35 14.61 12.34 12.16 8.41 11.52 4.70

81,980.5 1416.5 0.61 0.29 8.52 0.29 0.14 1.19 0.052 0.15 0.50 0.35 0.20 0.48 0.63 0.58 0.53 0.57 0.33

23,665.7 447.9 0.16 0.079 2.27 0.079 0.038 0.31 0.013 0.040 0.13 0.094 0.055 0.13 0.16 0.15 0.14 0.15 0.089

Erythrocyte number Leucocyte number Erythrocyte length (EL) Erythrocyte width (EW) Erythrocyte sizes (CA) Nucleus length (NL) Nucleus width (NW) Nucleus sizes (NA) EL/EW NL/NW

10 10 10 10 10 10 10 10 10 10

Pelobates syriacus 543,000 – 793,000 600 – 2500 18.07 – 20.37 10.03 – 11.60 143.01 – 185.77 6.21 – 7.28 3.68 – 4.31 18.52 – 24.65 1.69 – 1.84 1.63 – 1.80

657,100 1216 18.78 10.63 157.65 6.78 3.95 21.15 1.77 1.72

95,368.81 597.43 0.68 0.52 13.03 0.33 0.20 2.04 0.05 0.06

30,158.26 188.926 0.216 0.165 4.123 0.106 0.065 0.648 0.016 0.017

Blood Cells Count and Size

123

TABLE 1 (continued) Character

Min – max

Mean

10 6.51 – 8.88 7.65 0.63 0.20 CA/NA Lymphocyte (large) diameter 10 11.31 – 12.43 11.74 0.37 0.118 Lymphocyte (small) diameter 10 8.18 – 9.12 8.57 0.27 0.087 Monocyte diameter 10 13.37 – 14.68 14.27 0.42 0.133 Neutrophyl diameter 10 11.31 – 12.75 12.17 0.46 0.148 Eosinophyl diameter 10 11.25 – 12.62 12.10 0.42 0.134 Basophyl diameter 10 8.37 – 9.00 8.60 0.18 0.059 Thrombocyte length 10 10.65 – 12.03 11.22 0.38 0.123 Thrombocyte width 10 4.62 – 5.87 5.13 0.36 0.116 Note. N, the number of cells studied for each specimens; min, minimal value; max, maximum value; SD, standard deviation; SE, standard error of mean.

TABLE 2. Leucocyte Formules (%) Species

Lymphocyte

Monocyte

Neutrophyl

Eosinophyl

Basophyl

R. ridibunda

10 (5xx + 5}})

40.9

16.4

15.1

7.6

R. dalmatina

8 (2xx + 6}})

37.5

19.25

19.85

15.25

10.25 8.21

B. viridis

19 (10xx + 9}})

35.27

18.52

19.15

18.26

B. bufo

11 (6xx + 5}})

37.36

16.72

22.72

15.72

7.45

H. arborea

14 (10xx + 4}})

37.28

17.28

19.84

19.14

7.42

P. syriacus

10 (9xx + 1})

40.2

23.1

15.3

7.4

shaped (Fig. 1). Blood cell counts and sizes are given in Table 1. The most erythrocyte count was found in Bufo viridis (mean 721.750), and the lowest one in Rana ridibunda (320.500). The erythrocyte count was decreased in hydrophilous (Rana dalmatina and Hyla arborea) and, especially, semiaquatic (Rana ridibunda) species and increased in most terrestrial Pelobates syriacus and Bufo viridis (Table 1). The most leucocyte count was found in Hyla arborea (2980) and the lowest one in Pelobates syriacus (1216). The largest erythrocyte cell and nucleus sizes were found in Rana ridibunda and the smallest one in Hyla arborea and Pelobates syriacus (Table 1). At leucocyte formulas, lymphocytes were the most abundant, and basophils were the most scarce (Table 2). DISCUSSION Some authors (Alder and Huber, 1923; Hutchison and Szarski, 1965; Klieneberger, 1927) studied individual variation of various Rana species in regard to blood cell counts and sizes. Sexual differences in the erythrocyte count were reported by Arvy (1947) for Rana tem-

poraria, Kaplan (1951) for Rana pipiens and Sinha (1983) for Rana esculenta. In contrast, we found no any significant sexual differences in specimens studied. Similarly, Arýkan (1989) reported that sexual dimorphism was not observed for erythrocyte count in Rana ridibunda. According to our data, the least erythrocyte count was found in Rana ridibunda. On the other hand, the leucocyte count of Rana ridibunda was similar with other anuran species studied. It is accorded with Arýkan’s (1989) findings in leucocyte count of Rana ridibunda. Furthermore, we did not find any differences in the leucocyte counts compared to the result of the previous study for Rana ridibunda (Arýkan, 1989). The erythrocyte count of Rana dalmatina was very close to that of Rana ridibunda. Nevertheless, erythrocyte and nucleus sizes of these species significantly differed. Then again, the leucocyte count was similar to those of other species in this study (Table 3). Bufo viridis was the species with the highest erythrocyte count among species studied. The erythrocyte cell and nucleus sizes of Bufo viridis presented by Atatür with coauthors (1999) is in agreement with our findings (Table 3).

124

Çiðdem Gül and Cemal Varol Tok

Alder and Huber (1923) studied erythrocyte counts in Hyla arborea. We found no significant differences in comparison with our data (Table 3). Compared with other Anura species, Hyla arborea possessed the highest leucocyte count (Table 3). However, our leucocyte count was taken in different conditions from previous studies (Arýkan, 1989). The smallest nucleus sizes of erythrocyte cell were encountered in Pelobates syriacus and Hyla arborea (Table 3). Among species studied, Pelobates syriacus succeeded to Bufo viridis with the highest erythrocyte count. Moreover, the erythrocyte count of Pelobates syriacus was very close to that of Pelodytes caucasicus (Arýkan et al. 2003). Compared with the leucocyte counts of other anurans mentioned in previous studies, Pelobates syriacus had the lowest amount. We found that erythrocyte cell and nucleus sizes were in inverse proportion with erythrocyte count. For instance, among the species studied, Rana ridibunda had the lowest erythrocyte count (320.500) but the highest erythrocyte size (226.7). On the another hand, Pelobates syriacus has one from the highest erythrocyte counts (657.100) but the lowest erythrocyte size (157.7). The erythrocyte size increased from Pelobates syriacus to Hyla arborea, Bufo viridis, Rana dalmatina, Bufo bufo, and Rana ridibunda, respectively.

Acknowledgments. I would like to thanks Asist. Prof. Dr. Murat Tosunoðlu for helping during studies.

REFERENCES Alder A. and Huber E. (1923), “Untersuchungen über Blutzellen und Zellbildung bei Amphibien und Reptilien,” Folia Haematol., 29, 1 – 22. Alpagut Keskin N. (2000), Güneybatý Anadolu Rana ridibunda Palas, 1771 (Anura: Ranidae) Populasyonlarýnda Sitogenetik ve Ýzoenzimik Özelliklerin Karþýlaþtýrmalý Ýncelenmesi [Comparative Studies on Cytogenetics and Isoenzyme Features of Southwest Anatolian Rana ridibunda Pallas 1771 (Anura, Ranidae) Populations]. Postgraduate Thesis, Ege University, Faculty of Science [in Turkish]. Arýkan H. (1989), “Anadolu’daki Rana ridibunda (Anura: Ranidae) populasyonlarýnýn kan hücrelerinin sayýsý bakýmýndan incelenmesi [Investigation on the blood cell counts of Rana ridibunda (Anura, Ranidae) populations in Anatolica],” Doða Tu Zooloji D, 13(2), 54 – 59 [in Turkish]. Arýkan H., Çevik Ý. E., Kaya U., and Mermer A. (2001), “Anadolu Dað Kurbaðalarýnda Eritrosit Ölçümleri [Erythrocyte measurements in Anatolian Mountain Frogs],” Anadolu Üniversitesi Bilim ve Teknoloji Dergisi, 2(2), 387 – 391 [in Turkish]. Arýkan H., Atatür M. K., and Tosunoðlu M. (2003), “A study on the blood cells of the Caucasus Frog, Pelodytes caucasicus,” Zool. Middle East, 30, 43 – 47.

TABLE 3. Sizes of Blood Cells in Different Anura Species Referred by Various Authors Researchers

Species

Present Study

R. ridibunda 320,500 R. dalmatina 415,750 B. viridis 721,750 B. bufo 499,954 H. arborea 579,583 P. syriacus 657,100 H. arborea 674,000 R. ridibunda 326,620 R. ridibunda — B. bufo — B. viridis — P. syriacus — B. bombina — H. arborea — R. holtzi — R. macrocnemis — R. camerani — B. bombina 340,000 x 290,000 } P. caucasicus 776,000

Alder and Huber, 1923 Arýkan, 1989 Atatür et al., 1999

Arýkan et al., 2001

Wojtaszek and Adamowicz, 2003 Arýkan et al., 2003

2536 2683 1646 2663 2980 1216 29,000 3142 — — — — — — — — —

22.88 21.80 19.66 21.53 19.62 18.78 — — 24.36 20.85 17.86 17.56 21.80 19.80 19.10 20.55 19.81 22.08

12.60 12.57 12.76 12.82 12.26 10.63 — — 14.46 13.45 12.71 11.70 15.05 12.89 12.80 13.46 12.78 14.74

226.7 215.41 197.32 217.71 189.16 157.65 — — 276.62 221.22 179.18 161.85 258.14 200.33 192.81 217.68 198.85 255.82

8.39 8.53 7.69 8.14 7.64 6.78 — — — — — — — — 7.84 8.66 8.45 10.77

4.38 3.84 3.82 4.43 7.64 3.95 — — — — — — — — 4.13 4.14 3.94 7.11

29.11 25.92 23.18 28.45 21.15 21.15 — — — — — — — — 25.46 28.03 26.09 —

15.29

9.68

116.42

6.21

3.81

18.71

9734 x 7030 } 2560

Blood Cells Count and Size Arvy L. (1947), “Le dimorphisme sexual sanguine chez Rana temporaria L. et Bufo vulgaris Laur.,” Compt. Rend. Soc. Biol., 141, 457 – 459. Atatür M. K., Arýkan H., and Çevik I. E. (1999), “Erythrocyte sizes of some anurans from Turkey,” Tur. J. Zool., 23, 111 – 114. Carmena A. S., Siret J. P., Callejas J., and Carmena D. A. (1980), “Blood volume in male Hyla septentrionalis (Tree frog) and Rana catesbeiana (Bullfrog),” Comp. Biochem. Physiol., 67A, 187 – 189. Foxon G. E. H. (1964), “Blood and respiration,” in: Moore J. A. (ed.), Physiology of the Amphibia, Acad. Press, New York, pp. 151 – 209. Hartman F. A. and Lessler M. A. (1964), “Erythrocyte measurements in fisches, amphibia and reptiles,” Biol. Bull., 126, 83 – 88. Hutchison H. V. and Szarski H. (1965), “Number of erythrocytes in some amphibians and reptiles,” Copeia, 1965(3), 373 – 375. Kaplan H. M. (1951), “A study of frog blood in red leg disease,” Trans. Ill. State Acad. Sci., 44, 209 – 215. Kaplan H. M. (1952), “Variations in white blood cells between normal and red leg frogs,” Trans. Ill. State Acad. Sci., 45, 170 – 176. Klieneberger C. (1927), Die Blutmorphologie der Laboratoriumstiere, Barth, Leipzig. Kuramoto M. (1981), “Relationships between number size and shape of red blood cells in Amphibians,” Comp. Physiol., 69, 771 – 775. Leviton A. E., Gibbs R. H., Jr., Heal E., and Dawson C. E. (1985), “Standard in herpetology and ichthyology: Part I. Standard symbolic codes for institional resource collections in herpetology and ichthyology,” Copeia, 1985(3), 802 – 832. Rouf M. A. (1969), “Hematology of the leopard frog, Rana pipiens,” Copeia, 1969, 682 – 687. Schneider H. (2001), “The distribution of Hyla arborea and H. savignyi on the South Coast of Turkey (Amphibia: Anura),” Zool. Middle East, 23, 61 – 69.

125 Sinha R. C. (1983), “Haematological studies on the prewintering and wintering frog, Rana esculenta,” Comp. Biochem. Physiol., 74(2), 311 – 314. Szarski H. and Czopek G. (1966), “Erythrocyte diameter in some amphibians and reptiles,” Bull. Acad. Pol. Sci. Cl. II. Ser. Sci. Biol., 14(6), 433 – 437. Tok C. V. (1995), “Reþadiye (Datça) Yarýmadasýnýn Herpetofaunasý [Herpetofauna of Reþadiye (Datça) Peninsula],” Tur. J. Zool., 19, 119 – 121 [in Turkish]. Tosunoðlu M. (1999), “Türkiye Bufo viridis (Anura: Bufonidae) Populasyonlarý Üzerinde Morfolojik, Osteolojik ve Serolojik Araþtýrmalar [Morphological, osteological, and serological investigations on Bufo viridis (Anura: Bufonidae) populations in Turkey],” Tur. J. Zool., 23, 849 – 871 [in Turkish]. Uðurtaþ Ý. H. (1995), “Türkiye’deki Pelobates syriacus Boettger 1889 (Anura, Pelobatidae)’un Taksonomi, Biyoloji ve Daðýlýþý Üzerine Araþtýrmalar [Taxonomical, biological, and distributional investigations on Pelobates syriacus Boettger 1889 (Anura, Pelobatidae) in Turkey],” Tur. J. Zool., 19, 123 – 145 [in Turkish]. Wojtaszek J. and Adamowicz A. (2003), “Haematology of the fire-bellied toad, Bombina bombina L.,” Comp. Clin. Pathol., 12, 129 – 134. Yýlmaz Ý. (1984), “Trakya Kuyruksuz Kurbaðalarý Üzerine Morfolojik ve Taksonomik bir Araþtýrma (Anura: Discoglossidae, Pelobatidae, Bufonidae, Hylidae, Ranidae) [A morphological and taxonomical investigation of Thracian anura (Anura, Discoglossidae, Pelobatidae, Bufonidae, Hylidae, Ranidae)],” Doða Bilim Dergisi, 8(2), 244 – 264 [in Turkish]. Yýlmaz Ý. and Kumlutaþ Y. (1995), “Türkiye’de yaþayan Bufo bufo (Linnaeus) 1758’nun Daðýlýþý ve Taksonomik Durumu Hakkýnda Bir Ýnceleme [Distribution of Bufo bufo (Linnaeus) 1758 living in Turkey and study on it’s taxonomic status],” Tur. J. Zool., 19, 277 – 284 [in Turkish].

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 126 – 130

MORPHOLOGY OF SOUTHEAST ASIAN TADPOLES: Hoplobatrachus chinensis (DICROGLOSSIDAE), Leptolalax pelodytoides (MEGOPHRYIDAE), AND OTHER MEGOPHRYIDS Ronald Altig,1 Amy Lathrop,2 and Robert W. Murphy2,3 Submitted April 25, 2008. The structure and development of the large, spike-like, biserial teeth of the tadpole of Hoplobatrachus chinensis are unique. The presently erupted, conical tooth has at least 5 replacement teeth of decreasing sizes stacked inside, and the whole assembly projects only slightly into the oral epithelium. The ventrolateral abdominal wall of Leptolalax pelodytoides has well delineated, lymphatic spaces that presumably aid in stabilizing the tadpole among rocky substrates. The external narial apertures of megophryid tadpoles have a diversity of ornamentations that range from a simple rim, a lobate rim or a distinct tube. Keywords: tadpole, morphology, mouthparts, Hoplobatrachus, Leptolalax, Megophryidae.

A largely common structure and developmental mode of the labial teeth of most tadpoles is overlain by a large range of poorly understood interspecific and ontogenetic variations (e.g., Altig and Pace, 1974; Hosoi et al., 1995). Typically several replacement teeth occur in progressive stages of development embedded deeply within the tissue of the tooth ridge, and the head of each tooth of the series is interdigitated in the sheath of the next peripheral tooth (Altig, 2007). One tooth row per ridge is the norm, and most cases of biserial or multiserial tooth rows occur in “primitive frogs.” Chou and Lin (1997), Grosjean et al. (2004), and Khan (2004) reported 5/6 biserial tooth rows in the tadpoles of the dicroglossid Hoplobatrachus chinensis, and our examinations revealed the form and method of growth and shedding of these unusual teeth. Amiet (1971) did not discuss the anatomy or suggest a function for the prominent “lateral sacs,” presumably large lymph sinuses, along the flanks of the muscular, elongate tadpoles of Nyctibates corrugatus. Similar but 1

Department of Biological Sciences, Mississippi State University, Mississippi State, Mississippi 39762, USA; E-mail: raltig@biology.msstate.edu Department of Natural History, Royal Ontario Museum, 100 Queen’s Park, Toronto, Ontario, M5S 2C6, Canada; E-mail: amyl@rom.on.ca; drbob@zoo.utoronto.ca. Department of Ecology & Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario, M5S 3B2, Canada; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan, 650223, P. R. China.

lesser developed structures occur in a probable ecological analogue, the tadpole of the megophryid Leptolalax pelodytoides. The external nares of tadpoles commonly are rounded apertures flush with the local surface. Variations in aperture shape and ornamentation around the aperture and position relative to surrounding surfaces are known. These variations were surveyed among megophryid tadpoles. The range of these morphological features of Asian tadpoles provides a better understanding of the diversity of tadpole morphology. MATERIAL AND METHODS Intact oral discs of Hoplobatrachus chinensis (N = 10; Gosner, 1960, stages 30 – 34; Cat Tien National Park, Dong Nai Province, Vietnam; ROM 37847) were examined at 8-50X after staining with Crystal Violet to increase contrast of translucent tissues. Histological sections (7ì, hemotoxylin and eosin, upper labium, stage 34) revealed the attachment mode of the teeth to the tissue of the tooth ridge, and SEM photomicrographs showed the structure of the teeth and their growth patterns. The right abdominal wall of a specimen of Leptolalax pelodytoides (stage 34; 53.8 mm TL; Quang Thanh, Cao Bang Province, Vietnam; ROM 35225) was sectioned as outlined above, and narial ornaments were surveyed for a number of megophryid taxa.

Asian Tadpole Morphology

127

Fig. 1. Hoplobatrachus chinensis: A, spike-like, biserial teeth in right side of row A-1 (scale line is 20 ìm; white arrow, tip of outer tooth sloughed); B, broken tooth with arrows showing five successive teeth in a stack (scale line is 20 ìm) from outermost at top to innermost at bottom (electronically darkened for emphasis); C, histological longitudinal section of part of lower labium (scale line is 100 ìm; tadpole facing right (notations: CJ, cleft between lower jaw sheath and lower labium; CR, cleft proximal to LTR P-2; LJ, lower jaw sheath; P-1 and P-2, first and second posterior tooth rows); D, comparisons with two teeth in a tooth series removed from the tooth ridge tissue of Hyla chrysoscelis, presently erupted tooth uppermost (notations: vertical black line, approximate plane of tooth eruption above tooth ridge tissue; C, cusps on head of replacement tooth; S-1, sheath of erupted tooth; S-2, sheath of replacement tooth); E, labial teeth of Hyla gratiosa showing (arrow) typical form of breakage of an old tooth (compare with pane A).

RESULTS Each of the large teeth of Hoplobatrachus chinensis (Fig. 1A) is a broadly curved, oblate cone that is rigidly attached (Fig. 1B, tooth broken without disrupting the base) to the oral epithelium. We know little about tooth function, and the mode of tooth attachment versus function (i.e., relative flexion of a tooth or tooth series) has never been evaluated. Evaluations with a probe suggest that the teeth of Hoplobatrachus (i.e., attached as a series of stacked cones to the surface of a short, broad tooth ridge) are much more solidly fastened than are the interdigitated teeth in a tooth series. This oral armament is impressive, but it remains unknown whether tadpole

teeth function primarily as scarifying/tearing structures versus stabilizers for the oral disc during a bite. The teeth are widely spaced, and the surface of each tooth is smooth. A typical large tooth is ca. 58 ìm long in a straight line measurement, 23 ìm in long diameter at the base, and 20 ìm in the short diameter. The squamous epithelium of the oral disc (Fig. 1C) typically is about three cell layers thick but increases to a rounded pocket of about nine cell layers beneath a tooth stack that extends about six layers into the epithelium. The lack of birefringence in transmitted polarized light suggests that connective tissue and muscles are absent. Each tooth is formed from a small hillock of mitotic tissue at the base of the tooth stack, and there is an abrupt inter-row valley immediately proximal to each row

128

Fig. 2. Abdominal lymph sac of Leptolalax pelodytoides: A, ventral view of intact tadpole, anterior to left, showing the lower margin of the left lymphatic sac (notations: white arrow, spiracle; black arrow, ventral margin of lymph sac; white line, approximate orientation of histological section in B); B, histological cross-section of the abdominal, lymphatic sac (notations: extent of the sac shown by black arrows at far left [dorsal] and far right [ventral]; AM, abdominal musculature; IN, intestine; LLS, lateral lymph sac). C – H, Representative narial ornamentations of megophryid tadpoles: C, Leptobrachium cf. xanthospilum; D, Scutiger glandulatus; E, Oreolalax major; F, Leptolalax oshanensis; G, L. pelodytoides; H, Ophryophryne sp.

(Fig. 1C). All replacement teeth are stacked entirely within the presently erupted tooth, and thus all teeth are above the surface of the epithelium. The broken tooth (Fig. 1B) shows five teeth in a stack, and the arrows indicate layers 1 (outermost, presently erupted tooth) through 5 (tooth that will grow and erupt after the previous four have sloughed, electronically darkened for better visibility). Compare this arrangement with the more typical one (Hyla chrysoscelis where only the head of the replacement tooth inserts loosely into the sheath of the erupted tooth, and the sheath of the erupted tooth and all replacement teeth are embedded in the soft tissue of the tooth ridge (Fig. 1D, right of vertical black line). In such a

Ronald Altig et al. case, either the head of the erupted tooth breaks off to expose the head of the next tooth (Fig. 1E, arrow) or the entire tooth falls out. Tooth replacement in Hoplobatrachus is accomplished by an initial breakage (Fig. 1A, white arrow) of part of the outer tooth, and then progressively larger pieces break off to uncover the next tooth inside. No teeth with appreciable wear were noticed, so we suspect that growth of the inner teeth constantly pushes the outermost tooth free at reasonably short intervals. Smaller teeth adjacent to larger ones likely indicate breakage of a layer(s) of a given stack so that a younger tooth was functional at that site when the tadpole was preserved. What appears to be cellular debris between successive layers of the broken tooth is likely the result of apoptosis that provides separation of the teeth in a stack so that they can grow and eventually shed independently. The ventrolateral areas of the body of the tadpole of Leptolalax pelodytoides are visibly delimited from the adjacent areas and have a slightly different surface texture (Fig. 2A, black arrow). The lymph sac lies external to the abdominal musculature (Fig. 2B). Narial ornamentations of megophryid taxa vary as follows: simple rim, may vary in height or aperture round versus crudely oval (e.g., Vibrissaphora boringiae, Leptobrachium xanthospilum, and Scutiger glandulatus; Fig. 2C, D), rim with 1 – 3 indistinct papillae (e.g., Oreolalax major, O. omeimontis; Fig. 2E) or 2 – 5 distinct papillae (Leptobrachium chapaensis, Leptolalax pelodytoides, L. oshanensis, L. pluvialis, and Oreolalax major; Fig. 2F, G), or aperture with tubular extension, usually with marginal papillae (e.g., Megophrys ssp., Ophryophryne ssp., Xenophrys brachykolos, and X. omeimontis; Fig. 2H). In Leptolalax ssp. the papillate rim is stiff, white and often with deeply positioned melanophores, and this was observed in L. pelodytoides at stage 42. DISCUSSION Hoplobatrachus tadpoles are large, active and voracious carnivores in temporary pools. Individuals less than 1 cm TL turned vertically upwards to attack floating microhylid egg films from the bottom soon after the eggs were deposited. All eggs in a film were sometimes eaten by daybreak, and tadpoles of various taxa were either eaten or badly wounded if transported to the laboratory with Hoplobatrachus tadpoles. The teeth of the majority of tadpoles develop by a similar pattern and have a grossly similar form, and the known cases of unusual teeth likely involve a histologi-

Asian Tadpole Morphology cal structure and growth pattern similar to that described above. The bromeliad-dwelling, hylid tadpole of Phyllodytes gyrinaethes (Peixoto et al., 2003) has many unusual characteristics, including flattened, spade-shaped teeth that lie at a low angle to the surrounding tissue (R. Altig, unpublished data). The structures and the three rows of straight, upright spikes on the lower labium of Mantidactylus lugubris (Altig and McDiarmid, 2006) likely have a similar structure and mode of formation as the teeth of Hoplobatrachus. While assuming the systematics of the taxa are correct, the occurrences of novel teeth within genera in which other members usually have typical teeth is odd. Are the novel teeth in these genera homologous with typical labial teeth and if so, which form might be ancestral? Comparable cases of profound modifications of jaw sheaths seem to involve opposite responses — reduction of the sheath until only derivatives of the serrations remain (e.g., hatchlings of Heleophryne, Visser, 1985; tadpoles of Lepidobatrachus, Cei, 1968) or a hypertrophy of the serrations (e.g., Mantidactylus lugubris, Altig and McDiarmid, 2006; Otophryne pyburni, Pyburn, 1980). Other than Hoplobatrachus, the normal presence of bi- or multiserial teeth is restricted to alytids, bombinatorids, and leiopelmatids. One sometimes finds short sections of bi- or triserial teeth in taxa that typically have uniserial teeth, and these presumed atavisms indicate that biseriality might appear in any tadpole. No ideas have been presented to explain the conditions that produce such rows. Judged by the phylogenetic analysis of Cannatella (1985), the ancestral tadpole might have resembled that of Bombina (Maglia et al., 2001), which would suggest that biserial rows are the primitive condition. If so, did uniserial teeth arise by fusion of the rows in a biserial condition or loss of one line of the biserial state? Histological examinations of embryos during tooth formation may reveal a likely scenario. Recent studies present a relevance to the tadpole stage in the evolution of lineages instead of relegating the larva to being a hapless follower. Grosjean et al. (2004) hypothesized that the occurrence of a large, fast developing (e.g., ca. 70 h from fertilization to stage 25 at ca. 26.2°C, about half the time required for Fejervarya limnocharis; Pan and Liang, 1990), carnivorous larva in temporary water was the driving force in the success and geographic expansion of the genus Hoplobatrachus. Also, the detection of correlative changes among adults and larvae within lineages from different faunas (Bossuyt and Milinkovitch, 2000) suggest common, though limited and largely unknown, ranges of morphospace among lineages. Viewing amphibian biology as a series of life cycles instead of either adults or larvae (e.g., Ar-

129 thur, 2004), and not presenting eggs and development as separate pieces, will surely produce a more coherent view than considering these pieces as independent units. Modifications of the body wall of tadpoles are uncommon but sometimes elaborate (e.g., dorsal head flap of Schismaderma carens, Bufonidae, Wager, 1965; abdominal sucker, New World bufonid, Chaunus chrysophorus, McCranie et al., 1989 and Old World ranids, Amolops, Inger and Gritis, 1983; abdominal flaps, Thoropa petropolitana, Cyclorhamphidae, Altig and McDiarmid, 1999; snout flap of Chacophrys pierottii, Ceratophryidae, Quinzio et al., 2006). Most such structures function in some way for position maintenance, but the head flap of Schismaderma may serve a respiratory function (e.g., grows larger when tadpoles occur in hypoxic water; Channing, 2001), and the function of the snout flap of Chacophrys is unknown. Homology is unlikely among all such cases, but convergence, especially in the abdominal suckers, is profound. The elongate, muscular tadpole of Nyctibates corrugatus (Amiet, 1971) has prominent “lateral sacs.” It is sometimes noted that the skin of a tadpole seems far removed from underlying structures (e.g., Grosjean et al., 2007), and similar lymph sinuses surely occur in every tadpole (Viertel and Richter, 1999:101). Noteworthy cases include those where certain elongate, stream-dwelling tadpoles have the sacs strongly delimited from adjacent areas by connective tissue (e.g., Amiet, 1971; Huang and Fei, 1981). We suggest that these sacs aid in position maintenance in tadpoles that live among cobble, although this supposition implies that at least the turgidity of these lymphatic cavities can be willfully modified. Reconstructions of the lymphatic system from histological sections accompanied by behavior studies would be needed to verify the anatomy and ecological function. Although the sacs appear empty in preserved specimens, they are not filled with air as mentioned by Huang and Fei (1981; Nanorana conaensis). Configurations of the external nares are typically mentioned in descriptions of tadpoles, but the range of variations in these structure have not been summarized (see myobatrachid genera: Dziminski and Anstis, 2004) or discussed in any detail. The position, size, and shape of the aperture is variable, but most taxa lack narial ornamentations. Potential relationships of external narial ornaments and internal buccal papillae have not been detected, but some function associated with modulating water flow or inhibiting materials from entering the naris should be considered. No function is ascribed to the unusual contrasty color and apparent stiffness of the narial ornaments of Leptolalax pelodytoides. The megophryid taxa with umbelliform mouthparts all have tubu-

130 lar nares, and benthic forms have a simple rim or a rim with slight lobes. Acknowledgments. Histological preparations were made at the MSU College of Veterinary Medicine, G. Thibaudeau and W. A. Monroe of the MSU Electron Microscopy Center provided SEM assistance, and N. E. Karraker supplied the tadpoles of Xenophrys. Fieldwork in Vietnam was supported by grants from the Royal Ontario Museum (Foundation and Members’ Volunteer Committee), private donations, and by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to RWM. All fieldwork was conducted following approved Animal Use Protocols. The Institute of Ecology and Biological Resources facilitated export permits; special thanks to the late Cao Van Sung for his assistance.

REFERENCES Altig R. (2007), “A primer for the morphology of anuran tadpoles,” Herpetol. Cons. Biol., 2, 73 – 76. Altig R. and McDiarmid R. W. (1999), “Body plan: development and morphology,” in: R. W. McDiarmid and R. Altig (eds.), Tadpoles: The Biology of Anuran Larvae, Univ. Chicago Press, Chicago, pp. 24 – 51. Altig R. and McDiarmid R. W. (2006), “Descriptions and biological notes on three unusual mantellid tadpoles (Amphibia: Anura: Mantellidae) from southeastern Madagascar,” Proc. Biol. Soc. Wash. (USA), 19, 279 – 286. Altig R. and Pace W. L. (1974), “Scanning electron photomicrographs of tadpole labial teeth,” J. Herpetol., 8, 247 – 251. Amiet J.-L. (1971), “Le têtard d’Astylosternus corrugatus Boulenger (Amphibien Anoure),” Ann. Fac. Sci. Cameroun, 1971, 85 – 98. Arthur W. (2004), Biased Embryos and Evolution, Cambridge Univ. Press, Cambridge, UK. Bossuyt F. and Milinkovitch M. C. (2000), “Convergent adaptive radiations in Madagascar and Asian ranid frogs reveal covariation between larval and adults traits,” Proc. Natl. Acad. Sci. USA, 97, 6586 – 6590. Cannatella D. C. (1985), A Phylogeny of Primitive Frogs (Archaeobatrachians). Unpublished Ph.D. Dissertation, Univ. of Kansas, Lawrence. Cei J. M. (1968), “Notes on the tadpoles and breeding ecology of Lepidobatrachus,” Herpetologica, 24, 141 – 146. Channing A. (2001), Amphibians of Central and Southern Africa, Comstock Publ. Ass., Ithaca (NY). Chou W.-H. and Lin J.-Y. (1997), “Tadpoles of Taiwan,” Spec. Publ. Natl. Mus. Taiwan, 7, 1 – 98. Dziminski M. A. and Anstis M. 2004, “Embryonic and larval development of the sunset frog, Spicospina flammocaerulea (Anura: Myobatrachidae), from southwestern Australia,” Copeia, 2004, 896 – 902.

Ronald Altig et al. Gosner K. L. (1960), “A simplified table for staging anuran embryos and larvae with notes on identification,” Herpetologica, 16, 183 – 190. Grosjean S., Glos J., Teschke M., Glaw F., and Vences M. (2007), “Comparative larval morphology of Madagascar toadlets of the genus Scaphiophryne: phylogenetic and taxonomic inferences,” Zool. J. Linn. Soc., 151, 555 – 576. Grosjean S., Vences M., and Dubois A. (2004), “Evolutionary significance of oral morphology in the carnivorous tadpoles of tiger frogs genus Hoplobatrachus (Ranidae),” Biol. J. Linn. Soc., 81, 171 – 181. Hosoi M., Niida S., Yoshiko Y., Suemune S., and Maeda N. (1995), “Scanning electron microscopy of horny teeth in the anuran tadpole Rhacophoridae, Rhacophorus arboreus and Rhacophorus schlegelii,” J. Electron Microsc., 44, 351 – 357. Huang Y.-Z. and Fei L. (1981), “Two new species of amphibians in Tibet,” Acta Zootaxon. Sinica, 6, 211 – 215. Inger R. F. and Gritis P. A. (1983), “Variation in Bornean frogs of the Amolops jerboa species group, with descriptions of two new species,” Fieldiana Zool. New Ser., 19, 1 – 13. Khan M. S. (2004), “Riparian tadpoles: Hoplobatrachus tigerinus (Daudin, 1802) with notes on breeding habits and feeding ecology,” Bull. Chicago Herpetol. Soc., 39, 101 – 104. Maglia A. M., Pugener L. A., and Trueb L. (2001), “Comparative development of anurans: using phylogeny to understand ontogeny,” Am. Zool., 41, 538 – 551. McCranie J. R., Jr., Wilson L. D., and Williams K. L. (1989), “A new genus and species of toad (Anura: Bufonidae) with an extraordinary stream-adapted tadpole from northern Honduras,” Occ. Pap. Mus. Nat. Hist. Univ. Kansas, 129, 1 – 18. Pan J. and Liang D. (1990), “Studies of the early embryonic development of Rana rugulosa Wiegmann,” Asiatic Herpetol. Res., 3, 85 – 100. Peixoto O. L., Caramaschi U., and Freire E. M. X. (2003), “Two new species of Phyllodytes (Anura: Hylidae) from the state of Alagoas, northeastern Brazil,” Herpetologica, 59, 235 – 246. Pyburn W. F. (1980), “An unusual anuran larvae from the Vaupés region of southeastern Colombia,” Pap. Avulsos Zool., 33, 231 – 238. Quinzio S. I., Fabrezi M., and Faivovich J. (2006), “Redescription of the tadpole of Chacophrys pierottii (Vellard, 1948)(Anura: Ceratophryidae),” South Am. J. Herpetol., 1, 202 – 209. Viertel B. and Richter S. (1999), “Anatomy: viscera and endocrines,” in: R. W. McDiarmid and R. Altig (eds.), Tadpoles: The Biology of Anuran Larvae, Univ. Chicago Press, Chicago, pp. 92 – 148. Visser J. (1985), “The fang-like teeth of the early larvae of some Heleophryne,” South Afr. J. Sci., 81, 200 – 202. Wager V. A. (1965), The Frogs of South Africa, Purnell and Sons, Cape Town.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 131 – 133

A NEW LOCALITY OF THE SOUTHERN CRESTED NEWT, Triturus karelinii (STRAUCH, 1870) (URODELA: SALAMANDRIDAE), FROM AFYON PROVINCE IN TURKEY Mehmet Öz,1* Mustafa Yavuz,1 Mehmet Rýzvan Tunç,1 and Ali Erdoðan1 Submitted March 1, 2008. In this study, we described 46 specimens of Triturus karelinii which were collected from Büyük Kalecik-District Gölcük Lake in Afyon Province. The specimens and habitat characteristics are described. This is the first record of T. karelinii from Afyon Province and, therefore, the species distributional range is being extend to the west of Central Anatolian in Turkey. Keywords: Triturus karelinii, Crested Newt, Distribution, Afyon, Turkey.

The records of Triturus karelinii in Turkey are Sarayiçi, Uzunköprü, Keþan (Edirne Province); Babeski, Lüleburgaz, Demirköy (Kýrklareli Province); Çorlu, Saray (Tekirdað Province); Yassýviran, Habibler Village, Altýnþehir, Þile, Yakacýk, Halkali (Ýstanbul Province); Arifiye, Karasu, Mekece, Ormanköy, Adapazari (Adapazarý Province); Çaycuma (Bartýn Province); Abant Lake, Taþlýyayla (Bolu Province); Karabük; Korgan, Ulubey (Ordu Province); Yomra (Trabzon Province); Yalova, Biga (Çanakkale Province); Karacabey, Uludað, Ýznik (Bursa Province); Ovacýk Village-Dikili, Bayraklý, 1

Akdeniz University, Faculty of Arts and Sciences, Department of Biology, Campus, 07058 Antalya, Turkey; Tel: +90 (242) 227-8900 Ext. 2235 or 2347, Fax: +90 (242) 227-89411

Bornova, Cumaovasý, Karagöl, Belevi (Ýzmir Province); Bozdað-Ödemiþ (Ýzmir-Manisa Province), Kula (Manisa Province); Efes (Aydýn Province); Reþadiye (Tokat Province); and Þerefiye (Sivas Province) (Baþoðlu et al., 1994; Kumlutaþ et al., 1998, 2004; Litvinchuk et al., 1999; Olgun et al., 2001, 2005; Sparreboom and Arntzen, 1987). The southern limits of distribution of T. karelinii in Turkey is Mersinbeleni (Aydýn Province) that was reported by Üzüm et al. (2004) in Western Anatolia, and the easternmost one is Gorgan (Iran) on the southeastern Caspian Sea (Haji, 1997). The records of T. karelinii in Afyon Province and Central Anatolia Region of Turkey are lacking.

* Address correspondence and/or reprint requests to: Mustafa Yavuz, Akdeniz University, Faculty of Arts and Sciences, Department of Biology, Campus, 07058 Antalya, Turkey; E-mail: myavuz@akdeniz.edu.tr, myavuz2002plus@yahoo.com.

Fig. 1. The new locality of Triturus karelinii collected from Büyük Kalecik-District Gölcük Lake in Afyon Province (Turkey).

Fig. 2. The habitat of Triturus karelinii in Büyük Kalecik-District Gölcük Lake in Afyon Province of Turkey.

132

Mehmet Öz et al.

16 males, 14 females, and 16 subadult specimens were collected on March 13, 2007, in Büyük KalecikDistrict Gölcük Lake (Fig. 1) in Afyon Province (1670 m), Central Anatolia Region (the western part of Central Anatolia Region) in Turkey (38°41¢21.48¢¢ N 30°27¢30.24¢¢ E), collectors M. R. Tunç, M. Yavuz,

M. Öz, and A. Erdoðan; collection is kept in the Faculty of Arts and Sciences of Department of Biology of Akdeniz University. The newts were found in the lake edged by graminea vegetation. The average air temperature in vicinity of the lake was 3°C, and the water temperature in the lake was

Fig. 3. Male (a) and female (b) of Triturus karelinii: dorsal (a) and ventral (b) views.

TABLE 1. Some Measurements (mm)1 and Indexes for 14 Males, 16 Females, and 16 Subadult Specimens of T. karelinii from Büyük Kalecik-District Gölcük Lake in Afyon Province (for abbreviations see the text) Character TL

Females mean ± SD (min – max)

Males SE

mean ± SD (min – max)

123.1 ± 26.0 (79.9 – 166.5)

6.9

LCD

60.4 ± 13.7 (37.4 – 82.6)

3.7

50.3 ± 11.6 (28.9 – 72.5)

13.7 ± 2.8 (9.4 – 20.7)

0.8

11.7 ± 2.5 (8.8 – 18.8)

LTC

11.1 ± 1.8 (7.7 – 13.9)

0.5

9.8 ± 1.6 (7.2 – 13.1)

Subadults SE

102.1 ± 23.8 (67.7 – 151.2) 5.9

mean ± SD (min – max)

Total SE

mean ± SD (min – max)

72.5 ± 15.1 (37.4 – 94.1)

3.8

98.2 ± 29.9 (37.4 – 166.5)

4.3

2.9

39.3 ± 4.0 (33.7 – 47.6)

1.1

50.1 ± 13.4 (28.9 – 82.6)

2.0

0.6

9.2 ± 0.7 (8.4 – 10.9)

0.2

11.4 ± 2.8 (8.4 – 20.7)

0.4

8.8 ± 0.5 (8.2 – 9.9)

0.1

9.8 ± 1.6 (7.2 – 13.9)

0.2

2.9 ± 0.6 (2.1 – 4.7)

0.2

2.6 ± 0.5 (1.7 – 3.7)

0.1

2.3 ± 0.2 (2.0 – 2.7)

0.1

2.6 ± 0.5 (1.7 – 4.7)

0.1

NED

3.4 ± 0.4 (2.7 – 4.3)

0.1

3.2 ± 0.4 (2.6 – 3.9)

0.1

2.9 ± 0.3 (2.2 – 3.4)

0.1

3.2 ± 0.4 (2.2 – 4.3)

0.1

3.3 ± 0.4 (2.5 – 4.1)

0.1

2.8 ± 0.5 (2.1 – 3.9)

0.1

2.6 ± 0.2 (2.3 – 2.9)

0.1

2.9 ± 0.5 (2.1 – 4.1)

0.1

22.4 ± 4.3 (13.9 – 28.6)

1.1

19.8 ± 5.7 (13.1 – 33.6)

1.4

14.8 ± 0.8 (13.6 – 16.7)

0.2

18.8 ± 5.2 (13.1 – 33.6)

0.8

23.6 ± 4.2 (14.4 – 27.2)

1.1

21.3 ± 5.7 (14.6 – 33.7)

1.4

15.5 ± 0.9 (14.5 – 17.9)

0.2

20.0 ± 5.3 (14.4 – 33.7)

0.8

LIE

35.9 ± 8.1 (21.3 – 48.8)

2.2

29.1 ± 7.4 (18.1 – 43.4)

1.9

20.7 ± 3.4 (16.5 – 29.7)

0.8

28.2 ± 8.9 (16.5 – 48.8)

1.3

SVL

80.1 ± 16.1 (53.5 – 105.5)

4.3

68.0 ± 13.9 (45.3 – 97.3)

3.5

49.7 ± 14.2 (14.8 – 64.4)

3.5

65.3 ± 19.1 (14.8 – 105.5)

2.8

LTC/LC

0.81 ± 0.05 (0.67 – 0.87)

0.01

0.85 ± 0.09 (0.70 – 1.02)

0.021

0.96 ± 0.03 (0.90 – 1.01)

0.008

0.88 ± 0.09 (0.67 – 1.02)

0.013

LTC/SVL

0.14 ± 0.01 (0.13 – 0.18)

0.004

0.15 ± 0.01 (0.13 – 0.17)

0.003

0.21 ± 0.13 (0.15 – 0.56)

0.034

0.17 ± 0.08 (0.13 – 0.56)

0.013

LC/SVL

0.17 ± 0.02 (0.16 – 0.21)

0.005

0.17 ± 0.02 (0.15 – 0.23)

0.005

0.22 ± 0.15 (0.16 – 0.61)

0.037

0.19 ± 0.09 (0.15 – 0.61)

0.013

LCD/SVL

0.75 ± 0.029 (0.69 – 0.78)

0.008

0.76 ± 0.03 (0.64 – 0.76)

0.008

0.63 ± 0.25 (0.11 – 0.74)

0.061

0.70 ± 0.15 (0.41 – 0.78)

0.023

LCD/TL

0.49 ± 0.03 (0.42 – 0.52)

0.008

0.49 ± 0.02 (0.43 – 0.53)

0.006

0.45 ± 0.17 (0.28 – 0.53)

0.044

0.48 ± 0.11 (0.24 – 0.53)

0.016

PA/SVL

0.28 ± 0.02 (0.25 – 0.33)

0.006

0.29 ± 0.03 (0.24 – 0.35)

0.008

0.36 ± 0.23 (0.25 – 0.97)

0.056

0.31 ± 0.14 (0.24 – 0.97)

0.020

PA/PP

0.95 ± 0.05 (0.89 – 1.08)

0.013

0.93 ± 0.03 (0.86 – 1.00)

0.009

0.95 ± 0.04 (0.90 – 1.01)

0.009

0.94 ± 0.04 (0.86 – 1.08)

0.006

PP/SVL

0.30 ± 0.04 (0.25 – 0.37)

0.010

0.31 ± 0.03 (0.27 – 0.36)

0.008

0.37 ± 0.24 (0.27 – 1.01)

0.061

0.33 ± 0.15 (0.25 – 1.01)

0.022

PP/LIE

0.67 ± 0.09 (0.56 – 0.84)

0.024

0.73 ± 0.04 (0.63 – 0.81)

0.011

0.76 ± 0.10 (0.51 – 0.94)

0.024

0.72 ± 0.09 (0.51 – 0.94)

0.013

PA/LIE

0.63 ± 0.07 (0.53 – 0.78)

0.019

0.68 ± 0.05 (0.61 – 0.78)

0.012

0.73 ± 0.09 (0.48 – 0.85)

0.023

0.68 ± 0.08 (0.48 – 0.85)

0.012

SVL, snout-vent length; TL, total length; LCD, tail length; LC, head length; LTC, head width; ND, nostril distance; NED, nostril-eye distance; DE, diameter of eye; PA, forelimb length; PP, hindlimb length; LIE, interlimbs distance; SD, standard deviation; SE, standard error.

A New Locality of the Southern Crested Newt in Turkey 1°C. Bottom and edge of the lake covered by algae (intensive eutrophication). Such plants as Prunus domestica, Pyrus communis, Crataegus sp., and Populus sp. were observed as well (Fig. 2). In addition, green frog Rana ridibunda caralitana was observed here. In all specimens examined, the ground coloration of the dorsum was various tones of brownish, greenish to grayish with more or less distinct black spots. The flanks with few small white stippling. Females with a thin yellow or brown vertebral stripe. Venter yellow or orange red, with black or bluish maculations (Fig. 3). The some morphological measurements and ratio values of specimens were given in Table 1. According to our results, the morphometry, color and pattern characteristics of specimens of Triturus karelinii were in agreement with data published previously by Baþoðlu et al. (1994) and Baran and Atatür (1998). Acknowledgments. This study was supported by Akdeniz University Research Foundation and thanks to Levent Turan from Hacettepe University, Sciences Department.

REFERENCES Baran I. and Atatür M. K. A. (1998), Turkish Herpetofauna (Amphibians and Reptiles), The Republic of Turkish Ministry of Environment Publications, Ankara. Baþoðlu M., Özeti N., and Yýlmaz Ý. (1994), Türkiye Amfibileri [Turkish Amphibians], Second Edition, Ege Üniv., Fen Fak., Kitaplar Ser., No. 151 [in Turkish].

133 Haji G. K. (1997), “Rediscovery of the southern crested newt, Triturus (cristatus) karelinii (Salamandridae), from its easternmost locality in Iran,” Zool. Middle East, 15, 5 – 8. Kumlutaº Y., Tok V., and Türkozan O. (1998), “The Herpetofauna of the Ordu-Giresun Region,” Tur. J. Zool., 22, 199 – 201. Kumlutaþ Y., Özdemir A., Ilgaz Ç., and Tosunoðlu M. (2004), “The amphibian and reptile species of Bozdað (Ödemiþ),” Tur. J. Zool., 28, 317 – 319. Litvinchuk S. N., Borkin L. J., D ukiæ G., Kaleziæ M. L., Khalturin M. D., and Rosanov Y. M. (1999), “Taxonomic status of Triturus karelinii on the Balkans, with some comments about other crested newt taxa,” Russ. J. Herpetol., 6(2), 153 – 163. Olgun K., Baran Ý., and Tok C. V. (2001), “Comparative morphology of Triturus karelinii populations from western and central Turkey (Amphibia: Urodela),” Zool. Middle East, 22, 57 – 65. Olgun K., Üzüm N., Avcý A., and Miaud C. (2005), “Age, size and growth of the southern crested newt Triturus karelinii (Strauch 1870) in a population from Bozdað (Western Turkey),” Amphibia–Reptilia, 26, 223 – 230. Sparreboom M. and Arntzen P. (1987), “Über die Amphibien in der Umgebung von Adapazari, Türkei,” Herpetofauna, 9(50)á 27 – 34. Üzüm N. T., Avcý A., Paksuz E. P. B., and Olgun K. (2004), “Türkiye’de Yaþayan Triturus karelinii (Strauch 1870) Ýçin Yeni Bir Populasyon: Mersinbeleni (Aydýn) Populasyonu. XVII [A new populations of Triturus karelinii (Strauch 1870) at Turkey: Mersinbeleni (Aydin) population. XVII],” in: Proc. of the Ulusal Biyoloji Kongresi, June 23, 2004. Abstract Book, Özet Kitabý [in Turkish].

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 134 – 138

NOTES ON THE NATURAL HISTORY OF Pseudocerastes urarachnoides (SQUAMATA: VIPERIDAE) Behzad Fathinia,1 Steven C. Anderson,2 Nasrullah Rastegar-Pouyani,3 Hasan Jahani,4 and Hosien Mohamadi5 Submitted September 15, 2008. An Iranian viper, Pseudocerastes urarachnoides, was collected alive during fieldwork in April 2008. The viper was transferred to the lab where many observations were made of its biology, behavior, feeding, habitat and distribution. During this survey we verified the previous speculation that its tail ornament (knob-like structure) is used as a lure to attract prey. Distribution was extended from Kermanshah Province to Khuzestan Province. Keywords: Pseudocerastes, Pseudocerastes urarachnoides, tail ornament, caudal luring, Squamata, Iran, Viperidae, behavior, feeding, distribution.

INTRODUCTION Pseudocerastes urarachnoides was first described by Bostanchi et al. (2006) from Ilam and Kermanshah Provinces, western Iran. The authors speculated that its elaborate caudal ornament, which resembles an arachnid, such as a spider or solpugid was probably used in caudal luring. The paratype specimen contained a bird in its stomach and the describers suggested that the snake might be a specialized feeder. The collection of a living specimen offered the opportunity to test these speculations and to make other observations on habitat, morphology, distribution, and behavior. The genus Pseudocerastes occurs across the North Arabian Desert from Sinai and southern Israel, Jordan, Iraq, southwestern Iran east to Afghanistan, and Pakistan west of the Indus River; an outlying population occurs in northern Oman (Bostanchi et al., 2006:444). Habitat. Ilam province is located in western Iran from 31°58¢ to 34°15¢ N and from 45°24¢ to 48°10¢ E. More than 78% of the province is covered with forests, meadows and arid lands. The province is located in three 1

School of Veterinary, Ilam University, Ilam, Iran; E-mail: bfathinia@gmail.com. Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA; E-mail: asaccus@aol.com. Department of Biological Sciences, Razi University, Kermanshah, Iran; E-mail: nasrullah.r@gmail.com. General office of Ilam Environment, Ilam, Iran; E-mail: jahanihasan@yahoo.com. Department of the Environment, Tehran, Iran.

geographic regions including the Zagros Mountains, western hills of the Zagros, and Khuzestan plain (Fathinia, 2006:13). Chakar, Bina, and Bijar, which is located from 33°22 to 33°46.08¢ N and from 45°48¢ to 46°05¢ E, is one of several No-Hunting Areas in Ilam province. The area of this region is almost 2% of the province area. The region is bounded to the northwest by Kermanshah province, to south and east by Mehran Township, to the northeast by Ilam Township and to the west by the Iraq border (Fig. 1). There are two types of climate in Ilam province; (A) Mediterranean and (B) dry and semidry climate, which is found in southern and western parts of the province [climatic types IV (III) and IV1 of Walter and Lieth (1960); see also Bobek, 1952: Fig. 6; Anderson, 1968:

Fig. 1. Location of No-hunting Areas in Ilam Province, Iran.

Notes on the Natural History of Pseudocerastes urarachnoides

135

Fig. 3. Habitat of Pseudocerastes urarachnoides in study area.

Fig. 2. Climate types of Walter and Lieth (from Anderson, 1968).

Fig. 94] and is in contrast to the desert climate in the westernmost lowland borders. The temperature of the study area is maximum in July and August (up to 50°C) and minimum in January (Rahnamaei, 1996a). (Fig. 2). Three types of plant associations occur in this area: Astragalus – Euphorbia; B) Bromus – Aegilops – Stipa; and C) Stipa – Bromus – Heteranthelium. Tree and bush species also are found in this region. See Rahnamaei (1996b) for details. Rahnamaei (1966b) and Fathinia (2006) have given accounts of the vertebrate fauna of this region. The immediate habitat of the viper contains hill and high grounds mostly composed of gypsum. This animal prefers deep cracks and holes in the gypsum where moisture and coolness are available during warm months of summer. There are some bushes (members of Polygonaceae and other bushes mentioned above) near their burrows and they can lie in ambush under them in cooler hours (morning and evening hours) of the day (Fig. 3). MATERIAL AND METHODS During fieldwork in April 2008 in Chakar, Bina, and Bijar No-Hunting Area, two specimens of Pseudocerastes urarachnoides were observed. One was a juvenile that formed a coil in the cool hours of the evening (17:00) at the opening of its burrow. We tried to catch it, but it escaped into the burrow. A torch was used to investigate cracks. Torchlight shown into a crack at twilight immediately elicited a loud hissing sound. The crack

was cleaved by crowbar and a specimen was caught by pinning its neck with a forked stick. The viper was put in a cloth bag and transported to the lab. A suitable environment similar to its natural habitat, including cracked gypsum was constructed. The viper was released in the environment and a closed circuit movie recording was set to study different aspects of its life including behavior and feeding. RESULTS Comments on morphology. This specimen, Razi University Zoological Museum (RUZMVP) 20.1, male, agrees with the original description in all essential characters (Table 1). Scale counts: 17 scales between horns, 20 scales around eye, 3 series of scales between eye and labials, 2 between nasal and rostral, 12 upper labials, 13 lower labials, 4 in contact with chin shields; 23 scales at midbody; ventrals 144, anal entire, subcaudals 15 pairs. Total length (TL) 840 mm, tail (T) 80 mm, TL/T = 10.5. Caudal ornament as described for holotype (Figs. 4 and 5). Anterior half of tongue is whiter and narrower than posterior half (Fig. 6). This is the first adult male specimen collected. The scales of the viper are more prominently rugose than in any other snakes found in Iran. This feature made the viper’s skin very rough. Because of the prominent scales along with more prominent appendages of the tail, local people who are familiar with it have named it Mar-e-pardar (= feathered snake) or Mare-gatch (= gypsum snake) (Fig. 7). The scales appear much more swollen and prominent in the living animal than in the preserved specimens illustrated by Bostanchi et al. (2006). The reason for this is that the snake inflates

136

Behzad Fathinia et al.

Fig. 4. Ventral surface of caudal lure.

Fig. 5. Dorsal surface of caudal lure.

its body or perhaps, uses dermal muscles, causing the scales to separate and stand out from one another. Contrast the relaxed lateral scales of Fig. 8 with their appearance in Fig. 5. Distribution. During the senior author’s recent visit to the Poisonous Animal Section of Razi Institute, Karaj, Iran, a preserved specimen of Pseudocerastes urarachnoides which had been collected from Khuzestan province and misidentified as Cerastes cerastes was seen.

This specimen extends the known range considerably to the south. According to the locality of the holotype (70 km SW of Ilam), paratype (25 km south of Qasr-eShirin), and the similarity of environments of the western borderland, which are mostly composed of gypsum sediments, we expect that this Iranian viper must be distributed from Qasr-e-Shirin to Khuzestan (Fig. 9). It should also be expected in the contiguous areas of Iraq to the northwest having similar habitat. Previous to the fieldwork, we had received from friends of Hasan Jahani a photograph of a specimen from the No-Hunting Area of Kooleg, a region just to the south of Chakar, Bina, and Bijar No-Hunting Area (Fig. 7). Feeding. This species eats birds (perhaps not exclusively), as feathers were observed in its excrement (Figs. 10 and 11). It regurgitated remains of a lark, apparently Galerida cristata (Fig. 12). This is in accord with the observation of the original describers, who found a bird in the stomach of the paratype. Observations in captivity support the notion that it uses caudal luring to attract birds. Behavior. All of the results which we discuss were observed in the lab and we can not generalize all of them to the natural habitat. When it exited alive from its burrow for the first time and was collected, it defecated twice on the collector’s arm. The viper uses both lateral and direct movements. It uses lateral movement when escaping and uses slow direct movement when coming out of its burrow. When alarmed, it gives a hissing sound and prepares to attack, raising head and neck from the ground and abruptly striking at its target without warning. Camouflage in its natural habitat is extremely effective because it perfectly resembles its environment (burned gypsum under sunlight) and one can hardly see

TABLE 1. Counts and Measurements of All Known Museum Specimens of Pseudocerastes urarachnoides

Character

Total length, mm Tail length, mm Total length/Tail Ventrals Subcaudals, number of pairs Midbody scale rows Scales between horns

RUZMVP 20.1 male 840 80 10.5 144 15 23 17

Scales around eye

Scales between eye and labials, number of series Scales between nasal and rostral

3 2

Upper labials Lower labials Labials in contact with chin shields

12 13 4

FMNH 170292 female holotype

ZMGU 1300 juv. male (?)

531 55 9.65 145 15 21 Head damaged Head damaged

432 46 9.39 146 15 23 16

3 Head damaged ~8 13/12 Head damaged

18/17

3 2 11/12 13/12 3–5

Notes on the Natural History of Pseudocerastes urarachnoides

Fig. 6. Protruding (RUZMVP 20.1).

tongue

Pseudocerastes

urarachnoides

137

Fig. 8. Lateral scales of Pseudocerastes urarachnoides (RUZMVP 20.1) in relaxed position.

Fig. 7. Defensive posture of Pseudocerastes urarachnoides in Kooleg No-hunting Area. Fig. 9. Estimated distribution of Pseudocerastes urarachnoides in Iran.

it until it moves. Speed of the strike is very fast and Ulead® software analysis of film revealed that it can complete its strike in less than 1 sec. We were able to observe and film the caudal luring originally suggested by the snake’s describers. It was very attractive and looked exactly like a spider moving rapidly. A video of this tail movement can be seen on Anderson’s web-site: http://swasiazoology.tripod.com. An experimental observation was planned and a chick placed in its proximity. After approximately half an hour the chick went toward the tail and pecked the knob-like structure. The viper pulled the tail structure toward itself, struck and bit the chick in less than 0.5 sec. The chick died after 1 h. In another examination a male sparrow was released in its environment, and then the viper

Fig. 10. Fecal mass of captive Pseudocerastes urarachnoides (RUZMVP 20.1).

138

Behzad Fathinia et al. in nature and lures for prey; some birds and even reptiles or small mammals, such as shrews, may be attracted toward this knob-like structure and when close enough they are struck. Many local people believe that the viper can climb into trees such as Pistacia atlantica and wait for prey.

Fig. 11. Dissected fecal mass showing feathers.

Acknowledgments. The senior author thanks his brothers, Fat’hollah and Behrooz and paternal uncle, Saeid, and Mr. Shanazar Mahmoodi who helped him to collect the viper; he thanks Hamid-Reza Goudarzi, Poisonous Animal Section, Razi Institute, Karaj, Iran, who assisted during his visit to the poisonous animal museum; and Mehrdad Kohzadian, General office of Natural Resources, Ilam, Iran, who gave us his photograph of the viper (Fig. 7).

REFERENCES

Fig. 12. Regurgitated bird remains.

slowly came out of its ambush, moved toward a corner and formed a coil, while putting its knob-like structure in front and contact with its mouth. Another sparrow offered to the snake was struck and it died within one minute. It is assumed that it keeps this position under bushes

Anderson S. C. (1968), “Zoogeographic analysis of the lizard fauna of Iran,” in: W. B. Fisher (ed.), The Land of Iran. Vol. 1. The Cambridge History of Iran. Chapter 10, Cambridge Univ. Press, London, pp. 305 – 371, 749 – 752. Anderson S. C., Web-site with video: http://swasiazoology.tripod.com. Bobek H. (1952), “Beiträge zur Klima-ökologische Gliederung Irans,” Erdkunde, 6(2 – 3), 65 – 84. Bostanchi H., Anderson S. C., Haji Gholi Kami, and Papenfuss Th. J. (2006), “A new species of Pseudocerastes with elaborate tail ornamentation from western Iran (Squamata: Viperidae),” Proc. Calif. Acad. Sci. Ser. 4, 57(14), 443 – 450. Fathinia B. (2006), Biosystematic Study of Lizards of Ilam Province. M.Sc. Thesis, University of Lorestan, Khoramabad, Iran. Rahnamaei M. T. (1996a), Effects of Development on Ilam Province. 2nd Stage, General office of Ilam Environment [in Farsi]. Rahnamaei M. T. (1996b), Effects of Development on Ilam Province. 1st Stage, General office of Ilam Environment [in Farsi]. Walter H. and Lieth H. (1960), “Klimadiagram,” in: Weltatlas, Gustav Fischer, Jena.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 139 – 142

COLOR VARIATION OF COMMON TOAD (Bufo bufo) LARVAE

Galina S. Surova1 Submitted May 18, 2008. In the natural pond (Moscow Oblast’) common toad tadpoles with unusual olive-red color have been found and registered three times for six year investigation. This color is similar to the color of yearlings soon after metamorphosis. “Red” tadpoles can be found at various stages of development but they always appeared after the beginning of metamorphosis of the most part of the population. Their occurrence is not affected neither by seasonal temperature, nor related with distinctions in number (density) of tadpoles between years. We suggest that this phenomenon could be explained by heterochrony. Keywords: Bufo bufo, development, color variation, common toad, tadpoles.

Identification of anuran larvae is mainly based on topology of morphological characteristics such as location of opercular and anal openings, height and shape of fin fold, and structure of mouthparts (Terentjev and Chernov, 1949; Bannikov et al., 1971; Kuznetsov, 1974). Only common toad larvae have such remarkable distinctive characteristic as black body color (Bannikov et al., 1977; Ananjeva et al., 1998; Kuzmin, 1999). However this feature cannot be considered as universal. In 2002, during our studies, we have found common toad tadpoles with red-olive color. During the following years we have continued the study to determine if this is casual or regular phenomenon.

nets were suspended to floats on the fishing-lines and placed to the depth of 0.5 – 0.7 m. The nets were checked daily for three days at a week interval. The temperature and the weather parameters were registered daily from 12 a.m. to 3 p.m. The stages of tadpole development were identified using the tables of normal development for B. bufo (Cambar and Gipoloux, 1956). Since the time of the second half of larval development we daily surveyed the pond visually and by net-sucks in searching the color tadpole variations.

MATERIAL AND METHODS

The red-olive tadpoles of B. bufo are greatly distinguished from common black tadpoles (Fig. 1). In water they were red-olive, and under good illumination, the tadpoles and yearlings looked as golden-red (Figs. 1 and 2). The other features such as the number of denticle rows, the disposition of oral tentacles and opercula opening and anus, the body shape and size and the fin fold were typical of common toad larvae. Under a microscope, dark (or “black”) tadpoles and metamorphosed individuals have the same black pigmentation with rare very fine golden spangles on the back. In the “red” individuals, spangles aggregates in spots under which black pigmentation become almost invisible. Such color is very similar to that of yearlings which appear several days later after metamorphosis when the skin gets red-brown color common for B. bufo in this area.

The study was carried out in summer 2002 – 2006 (occasionally in 2007), at Zvenigorod biological research station of Moscow State University (Odintsovo Rayyon, Moscow Oblast’, Russia). The common toad tadpoles (Bufo bufo L.) inhabit a forest pond formed from ravine. The pond size is 850 m2, perimeter is 145 m, and the average depth is 1 m (max — 1.8 m). The pond is fed by springs, well illuminated by the sun in the afternoon. The bottom on shallow sites is partly sandy and partly covered with a thick layer of tree waste. The number of tadpoles was estimated by round nettraps (0.5 m in diameter), with a cone 0.5 m high. The 1

Biological Faculty, Moscow State University, Leninskiye gory, 1, bldg. 12, Moscow 119992, Russia; E-mail: surova@hotbox.ru

RESULTS AND DISCUSSION

140

Galina S. Surova

Fig. 2. Red-olive (or “red”) color variation yearlings (left) and common color (or “black”) yearlings (right).

Fig. 1. Red-olive (or “red”) color variation tadpole (down left) and common color (or “black”) (right up).

For six years of studies, such tadpoles have been found during three years what excludes the occasional character of this phenomenon. Interestingly that individual tadpoles with such color appeared only after the beginning of metamorphosis of the most part of population. Along time scale their frequency in samples increased insignificantly but they always represent the minority of the pond population. Besides tadpoles could be both at early (IV2 — oval limb bud of hind feet), and late stages of development (IV11 — the elongated limbs with formed and separated fingers of the hind feet). In 2005 we carried out the following experiment. The “black” tadpoles at the stages preceding metamorphosis (IV11 – IV12 stages) were placed on the light background and the “red” tadpoles at the same stages — on the dark background. Metamorphosis occurred 6 days later in the “black” tadpoles and 9 days later — in

the “red” ones. Background has no effect on the color of metamorphosed individuals so they retain their own coloration. The question arises whether conditions of development causes the color variation. As shown in Table 1, the “red” tadpoles appeared in the pond either when yearlings came out to the land (2004) or much later (2002, 2005). The spawning of common toad occurs quite simultaneously, within 1 – 2 days, the duration of spawning usually does not exceed a week, and metamorphosis and coming out to the land extends until several weeks. Thus the “red forms” are individuals strongly extended development. It was shown, that duration of tadpole development is greatly affected by temperature and population density. The temperature dynamics along year scale is given in Fig. 3. Tadpoles of a common toad disperse from the aggregations all over the whole pond as late as in the end of May. Therefore we give the temperature data from the beginning of June. It is clear, that appearing of red color forms is not affected by temperature conditions of a year. Although 2003, 2004, and 2005 were colder than 2002 and 2006, “red” individuals appeared both in cold 2005 and 2004, and in warm 2002. Furthermore, they were absent both in warm 2006, and in cold 2003 (Table 1). The number of tadpoles in the pond varied from several thousands to 1 million individuals (Table 1). As the area and depth of the pond are

Color Variation of Common Toad (Bufo bufo) Larvae

2002 2003 2004 2005 2006

35 30

Temperature, °C

constant, tadpole abundance directly related to the density of population. In all the studied years with high density (2002, 2004, and 2005) the appearing of “red” color variations was not caused by temperature. In the years when the number of population was an order less than above (2003 and 2007), in the pond were no “red” tadpoles. However the “red” individuals did not also appear in 2006 when the population density was high enough. Therefore density does not affect the “red” form appearance. The presence of “red” forms does not also related to the total duration of metamorphosis (Table 1). Probably, toad tadpoles produce aggregations (shoals) of thousand individuals, and it buffer negative effects of temperature and density on duration of larvae development (Surova, 2006). For certain we only argue that “red” tadpoles which make up only the minor part of population always appear after the most part of yearlings have come out to the land. Therefore we assume that these individuals delaying in development due to slowing down morpho-physiological reconstructions, begin transformations with integuments change (i.e., they start reconstruction with the external rather than internal systems). In many Anura species the changes in developmental conditions influence the order of different system reconstructions. Such changes usually involve shape, body size, fin-folds, structure of the mouth parts what allow individuals to be more viable under different environmental changes (Laurila et. al., 2002; Vences, 2002; Gilbert, 2004). As a rule such changes are expected to appear in the whole population but in our case we observe color modifications only in separate individuals. The phenomenon what was observable by us (change of coloration) cannot be treated simply as modification change with extended ontogeny. In our laboratory experiments where common toad tadpoles were raised at a high density conditions we

141

25 20 15 10 5

II June

III

II July Date

III

II III August

Fig. 3. Ambient temperature during larvae development (average by decades).

have never observed such color variations (Surova, personal observation). However, Hephny (1991) found that adding of exogenous thyroxin initiates changes in integument of Rana temporaria larvae at later stages of development (prometamorhosis). Such tadpoles look like uneven-aged chimera. In our study, some tadpoles were characterized by “red” color typical for older stages of development — yearlings. Changes in the rate or timing of organ development are referred as “heterochrony” (Schmal’gauzen, 1969). Therefore we suggest that there are enough evidences to consider this phenomenon as heterochrony. Thus, we first found that in some common toad tadpoles integument develops at a faster rate than the other organs do, and it occurred regardless environmental factors. Acknowledgment. The study was supported by the Russian Foundation of Basic Research (05-04-48701-a) and the Program of support of leading scientific schools (8045.2006.4).

TABLE 1. Characteristics of Development of Common Toad (Bufo bufo) Duration Date of appearof coming out ance the color to the land of variation yearlings (days)

Year

Time (peak of spawning)

Larvae abundance: I — June; II — July

Time (peak of coming out to the land of yearlings)

2002

04/29 – 05/03 (04/29)

07/09 — 09/24 (07/24 – 07/25)

08/03

2003

05/08 – 05/12 (05/11)

07/18 – 08/09 (07/24)

2004 2005 2006

05/06 – 05/09 (05/07) 05/06 – 05/08 05/07 05/06 – 05/12 (05/07 – 05/09)

07/23 – 08/07 (07/25 – 07/26) 07/10 – 08/10 (07/15 – 07/16) 07/11 – 08/25 (07/14)

16 32 46

07/26 08/01 No

2007

05/01 – 05/09 (05/02)

I — 430,000; II — 170,000 I — 8000; II — 8000 II — 965,000 I — 800,000 I — 130,000; II — 106,000 Several thousands

07/25 – 08/02 (07/01 – 07/05)

142 REFERENCES Ananjeva N. B., Borkin L. J., Darevsky I. S., and Orlov N. L. (1998), Zemnovodnyye i Presmykayushchiyesya [Amphibians and Reptiles]. The Encyclopedia of Nature of Russia, ABF, Moscow [in Russian]. Bannikov A. G., Darevsky I. S., Ishchenko, V. G., and Rustamov A. K. (1971), Zemnovodnyye i Presmykayushchiyesya SSSR [Amphibians and Reptiles of the USSR], Mysl’, Moscow [in Russian]. Bannikov A. G., Darevsky I. S., Ishchenko, V. G., Rustamov A. K., and Szczerbak N. N. (1977), Opredelitel‘ Zemnovodnykh i Presmykayushchikhsya Fauny SSSR [Guide to Amphibians and Reptiles of the USSR Fauna], Prosveshchenie, Moscow [in Russian]. Cambar R. and Gipouloux J. (1956), “Table cronologiqiue du development embryonnair et larvaire du crapaud common Bufo bufo L.,” Bull. Biol. Fr. Belg., XC F2. Gilbert S. F. (2004), “Ecological biology of development — biology of development in the real world,” Ontogenez, 35(6), 425 – 438 [in Russian]. Hefni H. (1991), Development of Limbs of Amphibians in Norm and at Experimental Influences. Author‘s Abstract of Candidate‘s Thesis, Moscow State University [in Russian].

Galina S. Surova Kuzmin S. L. (1999), Zemnovodnyye Byvshego SSSR [The Amphibians of the Former Soviet Union], KMK, Moscow [in Russian]. Kuznetsov B. A. (1974), Opredelitel’ Pozvonochnykh Fauny SSSR. Chast‘ 1 [The Guide of Vertebrates of Fauna of the USSR. Part 1], Prosveshcheniye, Moscow [in Russian]. Laurila A., Pakkasmaa S., Crochet P.-A., and Merila J. (2002), “Predator-induced plasticity in early life history and morphology in two anuran amphibians,” Population Ecol., 132, 524 – 530. Schmal’gausen I. I. (1969), Problemy Darvinizma [Problems of Darwinism], Nauka, Leningrad [in Russian]. Surova G. S. (2006), “Motor activity of amphibian larva — from schools to shoals ,” in: Vences M., Kohler J., Ziegler T., and Böhme W. (eds.), Herpetologia Bonnesis. II. Proc. of the 13th Congr. Soc. Eur. Herpetol., pp. 183 – 186. Terentjev P. V. and Chernov S. A. (1949), Opredelitel’ Presmykayushchikhsya i Zemnovodnykh [Guide-Book of Reptiles and Amphibians]. 3rd edition, Sovetskaya Nauka, Moscow – Leningrad [in Russian]. Vences M., Puente M., Nieto S., and Vieites D. (2002), “Phenotypic plasticity of anuran larvae: environmental variables influence body shape and oral morphology in Rana temporaria tadpoles,” J. Zool. Lond., 257, 155 – 162.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 143 – 145

A GIANT TADPOLE RECORD OF Rana esculenta IN NORTHWESTERN RUSSIA

Konstantin D. Milto1 Submitted November 19, 2007. This report describes the first record of giant neotenic tadpole of Rana esculenta from Northwestern Russia. Keywords: giant larva, Rana esculenta, neoteny.

Giant tadpoles are known for some species of anurans, including green frogs of the Rana esculenta complex. Larvae with total body length of more than 100 mm were described from France, England, Switzerland, Czechoslovakia, Romania, Germany, Denmark, Sweden, Poland, Latvia, Kazakhstan, Kyrgyzstan, and Uzbekistan. Known giant tadpoles were assigned to Rana ridibunda, except records from Poland and Latvia (Borkin et al., 1981, 1984; Covaciu-Marcov et al., 2003). On the territory of Russia the giant larvae of green frogs never were found. This paper describes the first record of giant tadpoles of Rana esculenta from Northwestern Russia. Three giant tadpoles of Rana esculenta were captured in a temporary water reservoir near Chernovo Village, Sebezh Rayon, Pskov Oblast’, in 24 – 28 September 2004. One larva was provided to Department of Herpetology in Zoological Institute, Russian Academy of Sciences, St. Petersburg (ZISP 7627). This larva was assigned to hybridogenic Rana esculenta because only this species was identified by DNA flow cytometry early, in 1998 (unpublished data) from this water reservoir. Description of a giant larva: L. — 63 mm, L.cd. — 105 mm, distance between nostrils — 9.8 mm, right eye is absent, hind limb length — 4 mm (Figs. 1 and 2). Tadpole was at the stage 34, according Gosner (1960) table and at the stage 44, according Dabagyan and Sleptsova (1975). It had well developed paired ovaries with eggs (Figs. 3 and 4). The water reservoir where giant tadpoles were collected was inhabited by Rana esculenta and is placed 1

Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia; E-mail: coluber@zin.ru.

near Sebezhskoye Lake on the territory of Sebezh landscape. This landscape is branching of Bezhanitsy Morainic Hills. Elongate morainic ridges and hills, sandy sites, lacustrine and marsh depressions and river valleys are characteristic for this landscape type. Forest land covers not more than 10 %, whereas ploughed land part is up to

Fig. 1. A giant tadpole of Rana esculenta.

Fig. 2. Giant tadpole, lateral view.

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Fig. 3. Ovaries with eggs.

Fig. 4. Ovaries with eggs.

40 %. The period with a daily mean temperature of above +10°C is equal to 130 days. The temperature sum for this period is 1800°C. In the north Rana esculenta is distributed in Estonia and Belarus’ and reaches Pskov Oblast’ in the northwestern Russia. All known records of Rana esculenta in the Pskov Oblast’ are concentrated only in the west (Pechory and Pytalovo) and south-west (Sebezh) parts. It inhabits different types of ponds and water reservoirs. Moreover, Rana esculenta inhabits coastal zone of big lakes, as for instance Osyno Lake and Beloye Lake in the Sebezh vicinities. In the Pechory Rayon this species is recorded on the south coast of Pskovskoye Lake. In the Pskov Oblast’ this species is rare and represented only in the mixed populations with Rana lessonae (LE-population system). “Pure” populations of Rana esculenta in the Northwestern Russia is unknown. Pa-

rental species Rana lessonae is very common in the Pskov Oblast’ and is represented by both pure and mixed populations types (L- and LE-types). Rana esculenta reaches the northern limit of its range in the Pskov region and exists here in severe climatic condition. In comparison with Rana lessonae, premetamorphosis development of Rana esculenta is prolonged, and tadpoles metamorphosed later. Postmetamorphic froglets can be found at first decade of October. Normally, the frogs migrate to hibernation sites in the middle September. Traditionally, the phenomenon of development retardation and tadpoles overwintering is named as a partial neoteny (Bannikov and Denisova, 1956). It is considered that innate thyroid glands or hypophysis disfunctions and prolactin hormone oversecretion lead to inability of metamorphosis and larval gigantism (Borkin, 1981). Non-metamorphosing anuran larvae continue to grow and develop into giant tadpoles individuals. For example, giant Xenopus laevis larvae lack thyroid glands and thyroid hormone that stimulates tail resorption and metamorphosis. As known, the differentiation of gonads in anurans is not dependent on thyroid hormone. Athyroid giant tadpoles may come to being neotenic (Rot-Nikcevic and Wassersug, 2003). Giant tadpoles of Rana ridibunda from Latvia also had normally developed gonads. Nine giant larvae were collected in 1952 – 1954 in Liepaja district. Three of nine them had testicles and one had oviducts with eggs (Lusis and Zaune, 1984). Based on degree of gonad development, the authors supposed that this female tadpole might reproduce next spring. This new record of giant tadpoles supports a Lusis and Zaune (1984) hypothesis about fertility of giant larvae of green frogs. In that way giant larvae after successful hibernation probably reach sexual maturity and can reproduce. Acknowledgments. Author is grateful to S. A. Fetisov (St. Petersburg University) provided to Zoological Institute with specimen of giant tadpole. This work was supported by the grant of Scientific Program of St. Petersburg Scientific Center Russian Academy of Sciences in 2007.

REFERENCES Bannikov A. G. and Denisova M. N. (1956), Essays on Biology of Amphibians, Uchpedgiz, Moscow [in Russian]. Borkin L. J. (1984), “On giant tadpoles of the marsh frog, Rana ridibunda, and a record of the common spadefoot, Pelobates fuscus, in Kirghizia,” in: Proc. Zool. Inst. Vol. 124. Ecology and Faunistic of Amphibians and Rep-

A Giant Tadpole Record of Rana esculenta in Northwestern Russia tiles of the USSR and the Adjacent Countries, Leningrad, pp. 140 – 141 [in Russian]. Borkin L. J., Berger L., and Günther R. (1981), “On giant tadpoles of the green frogs of the Rana esculenta complex,” in: Ananjeva N. B. and Borkin L. J. (eds.), Proc. Zool. Inst. Vol. 101. The Fauna and Ecology of Amphibians and Reptiles of the Palaearctic Asia, Leningrad, pp. 29 – 47 [in Russian]. Borkin L. J., Berger L., and Günther R. (1984), “Giant tadpoles of water frogs within Rana esculenta complex,” Zool. Poloniae, 29(1 – 2) for 1982, 103 – 127. Covaciu-Marcov S.-D., Ghira I., Ardeleanu A., and Cogalniceanu D. (2003), “Studies on the influence of thermal

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water from Western Romania upon Amphibians,” Biota, 4(1 – 2), 9 – 20. Dabagyan N. B. and Sleptsova L. A. (1975). “Common frog Rana temporaria L.,” in: Objects of Developmental Biology, Nauka, Moscow, pp. 442 – 462 [in Russian]. Gosner K. L. (1960), “A simplified table for staging anuran embryos and larvae with notes on identification,” Herpetologica, 16(3), 183 – 190. Lusis J. J. and Zaune I. A. (1984), “Giant tadpoles of green frogs in Latvia,” in: Faunistika, Dzîvnieku Ekoloìija, Rîga, pp. 117 – 128 [in Russian]. Rot-Nikcevic I. and Wassersug R. J. (2003), “Tissue sensitivity to thyroid hormone in athyroid Xenopus laevis larvae,” Dev. Growth. Diff., 45, 321 – 325.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 146 – 154

A NEW SPECIES OF THE GENUS Calamaria (SQUAMATA: OPHIDIA: COLUBRIDAE) FROM THE CENTRAL HIGHLANDS (NGOC LINH NATURE RESERVE, NGOC LINH MOUNTAIN, KON TUM PROVINCE), VIETNAM

Nikolai L. Orlov1 Submitted October 12, 2008. A new species of Calamaria is described from the Central Highlands, Ngoc Linh Nature Reserve, Ngoc Linh Mountain, Kon Tum Province, Vietnam. This species is characterized by a black body with blazing yelloworange spots on the ventral part of the body. Total body length (holotype) 482 mm; tip of a thick tapering tail is slightly sharpened, tail length 34 mm; 13 rows of dorsal scales around the midbody; 11 rows of dorsal scales on the level of a single anal plate; 159 – 174 ventral scales, 20 – 26 divided subcaudals; four supralabials (second and third entering orbit), five infralabials; rostral large, triangular, its height equal to width, in contact with nasal, prefrontal and first supralabial shield, visible from above; paraparietal surrounded by six shields and scales; one preocular scale; eight modified maxillary teeth in holotype. The new taxon is known only from two specimens (holotype and paratype) collected in the tropical rainforest. It is the eighth species of Calamaria recorded from Vietnam. Keywords: Central Highlands, Ngoc Linh Nature Reserve, Ngoc Linh Mountain, Kon Tum Province, Vietnam, Squamata, Ophidia, Colubridae, Calamaria sp. nov., morphology, taxonomy.

INTRODUCTION The genus Calamaria, with over 60 species, occupies a prominent place in the Oriental tropics and in the world ophidiofauna and, together with other species-rich genera, such as Oligodon, Lycodon, Elaphe sensu lato, Sinonatrix, Xenochrophis, Amphiesma, Rhabdophis, Dinodon, Boiga, and Dendrelaphis, represents the majority of biodiversity of the South Asian ophidiofauna. The results of the most significant study of the taxonomy and evolution of this genus were published in the monograph by Inger and Marx (1965), which recognized 50 species. The genus Calamaria is distributed from southern China and the Ryukyu Islands (south Japan) southward through the East Indies to the Philippines and Sulawesi. These highly specialized, burrowing, forest-dwelling snakes are easily recognized as members of a single genus. According to Inger and Marx (1965), the principal centre of evolution and dispersal of Calamaria was the Great Sunda Archipelago, namely the Borneo – Sumatra region that contains the majority (more than 60%) 1

Zoological Institute, Russian Academy of Sciences, Universitetskaya nab., 1, St. Petersburg 199034, Russia. E-mail: azemiops@zin.ru

of the Calamaria species. The most detailed analysis of taxonomy and distribution of the Calamaria species of Vietnam was made by Ziegler and Quyet Le Khac (2005), with description of a new Calamaria species from Annamite mountain range. A total of 7 species was registered in Vietnam: C. buchi Marx et Inger, 1955; C. lovii ingermarxorum Darevsky et Orlov, 1992; C. pavimentata Duméril et Bibron, 1854; C. septentrionalis Boulenger, 1890; C. thanhi Ziegler et Quyet Le Khac, 2005; C. sp. nov. Nguyen Quang Truong, Koch et Ziegler, in press; C. gialaiensis Ziegler, Nguyen Van Sang and Nguyen Quang Truong, 2008 (Darevsky and Orlov, 1992; Nguyen Van Sang and Ho, 1996, 2005; Orlov et al., 2000, 2003; Orlov, 2005; Ziegler, 2002; Ziegler et al., 2004; Ziegler and Quyet Le Khac, 2005; Nguyen Quang Truong et al., in press; Ziegler et al., 2008c). Three species of Calamaria: C. pavimentata, C. septentrionalis, and C. yunnanensis Chernov, 1962 are distributed in the southern regions of continental China adjacent to Vietnam, Taiwan and Hainan islands. In addition to C. pavimentata and C. septentrionalis, the following species are also recorded in the western and southern regions of Indochina: C. lumbricoidea Boie, 1827 (Thailand, western Malaysia); C. albiventer Gray, 1834

A New Species of the Genus Calamaria from the Central Highlands, Vietnam (western Malaysia); C. lovii gimleti Boulenger, 1890 (western Malaysia); C. schlegeli Duméril et Bibron, 1854 (western Malaysia); C. prakkei Lidth de Jeude, 1891 (Singapore); and C. ingeri Grismer, Kaiser et Yaakob, 2004 (Pahang, western Malaysia) (Inger and Marx, 1965; Darevsky and Orlov, 1992; Zhao and Adler, 1993; Zao et al., 1998; Nguyen and Ho, 1996; Grismer et al., 2004; Ziegler and Quyet Le Khac, 2005; Nguyen Quang Truong et al., in press). One cannot but agree with Ziegler et al. (2005) on the necessity of making descriptions of such rare secretive (fossorial or semifossorial) snakes even when only a single, poorly preserved, specimen is available, because the chance of finding another specimen in the foreseeable future could be extremely low. Such descriptions are of considerable value for the evaluation of real herpetodiversity. MATERIAL AND METHODS The new species is described from 2 specimens (female and juvenile) preserved first in 70% ethanol. The comparative material from herpetological collections of IEBR (Institute of Ecology and Biological Resources, Vietnam), ROM (Royal Ontario Museum, Canada), FMNH (Field Museum of Natural History, USA), ZMH (Zoological Museum, Hamburg University, Germany), ZMB (Zoological Museum, Berlin University, Germany), MVZ (Museum of Vertebrate Zoology, Berkeley, USA), CAS (California Academy of Sciences, USA), CIB (Chengdu Institute of Biology, China), KIZ (Kunming Institute of Zoology, China) and ZISP (Zoological Institute, St. Petersburg, Russia) was examined. The literature data on the records and their localities were analyzed. The following abbreviations for standard morphological characters were used: SVL, snout-vent length (in mm); Lcd, tail length from vent to tip (in mm); TL, total length; BD, body diameter (in mm); HL, head length from anterior part of rostral shield to posterior part of lower jaw (in mm); HW, head width at the widest point (in mm); ED, eye horizontal diameter (in mm); EN, eye to nostril distance from anterior corner of eye to posterior edge of nostril (in mm); SL, snout length from tip of snout to anterior edge of eye (in mm); IO, interorbital distance; SO, number of supraoculars; PrO, number of preoculars;

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PtO, number of postoculars; SubO, number of suboculars; PF, prefrontal; F, frontal; P, parietal; R, rostral; N, nasal; L, loreal; T, number of temporals [Ta, anterior temporal; Tp, posterior temporal]; IN, internasal; M, mental or symphysial; G, genials [Ga, anterior genials or anterior chin shields; Gp, posterior genials or posterior chin shields]; Supralab, number of supralabials [Spl-r, on the right; Spl-l, on the left]; Infralab, number of infralabials [Ifl-r, on the right, Ifl-l, on the left]; V, number of ventrals; Scd, number of subcaudals; Sq1, Sq2, Sq3, number of dorsal scale rows at body [1, at the level of the 15th ventral shield from the head; 2, at midbody; 3, at the level of the 15th ventral shield from the anal plate]; A, number of anal plate; SVL/Lcd, ratio of snout-vent length/tail length from vent to tip; SVL/HL, ratio of snout-vent length/head length from anterior part of rostral shield to posterior part of the lower jaw; SVL/BD, ratio of snout-vent length/body diameter; All measurements were taken with a caliper to the nearest 0.1 mm. The main measurements were made on a Leica MZ 8 stereomicroscope; the specimens were scanned on an Epson Twain 5 scanner; photographs were made with a Nikon D 200 camera using a Nikkormicro 105 mm lens. SPECIES DESCRIPTION Calamaria abramovi sp. nov. Holotype. ZISP 25569 adult female from above Mang Xang Village, Ngoc Linh Mountain, Dac Glei District, Kon Tum Province, Vietnam (15°05¢ N 107°57¢ E, elevation 1700 m), collected 15 September 1998 by Nikolai L. Orlov (Figs. 1 and 2). Paratype. IEBR A907, juvenile male from above Mang Xang Village, Ngoc Linh Mountain, Dac Glei District, Kon Tum Province, Vietnam (15°05¢ N 107°57¢ E, elevation 1900 m), on 25 March 2004 by Alexei V. Abramov. (Figs. 3 and 4).

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Fig. 1. Holotype ZISP 25569 of Calamaria abramovi sp. nov., general view 1.

Fig. 2. Holotype ZISP 25569 of Calamaria abramovi sp. nov., general view 2.

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Fig. 3. Paratype IEBR A907 of Calamaria abramovi sp. nov., dorsal view.

Fig. 4. Paratype IEBR A907 of Calamaria abramovi sp. nov., ventral view.

Diagnosis. Calamaria abramovi sp. nov. is small, robust colubrid snake, with round body in cross-section, total length 482 mm (snout-vent length 448 mm, tail length from vent to tip 34 mm), with calamaria-shaped fossorial morphotype. Small head rounded, not distinguished from the body, slightly depressed dorso-laterally, covered with large regular symmetric shields. Maxillary teeth modified. Eyes small, pupil round. No loreal shield. Rostral large, triangular, its height equal to width, in contact with nasals, prefrontals and first supralabials, clearly visible from above; tongue groove clearly visible on its ventral side; prefrontals shorter than frontal, touching first two supralabials; single nasal shield very small, triangular, located between first supralabial, prefrontal and rostral, oval nostril occupies greater part of shield; eye diameter smaller than eyemouth distance; Ratios of parietal shield length to lengths of supraocular, prefrontal and frontal shields 2.52, 2.05, and 1.56, respectively. Dorsal scales rhomboid, overlap

in roof-tile fashion, strongly smooth. All scales of equal size, number of dorsal scale rows at body Sq1 â&#x20AC;&#x201D; 12, Sq2 â&#x20AC;&#x201D; 13, Sq3 â&#x20AC;&#x201D; 13; single (1) anal plate; tail thick, short, tapering gradually from base, abruptly tapering at tip to a sharp point; dorsal scales reduced in number to four rows on tail opposite twenty to twenty six subcaudals anterior to terminal scute. Five shields around eye (second and third supralabials, one preocular, one supraocular, one postocular); paraparietal surrounded by six shields and scales; five supralabials, second and third touching eye; fourth supralabial longest and in broad contact with parietal; six infralabials, three touching anterior genial shields; mental not touching anterior genial shields; only anterior genial shields in contact along midline; two gular scales in midline between posterior genial shields and first ventral, the first one inserted between posterior genial shields, ending just short of anterior genial shields. Body black, strongly iridescent, with blazing yellow-orange spots on ventral side of body, dorsal side of body without spots [opaque]. Blazing yel-

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Fig. 5. Head of holotype ZISP 25569 of Calamaria abramovi sp. nov., dorsal view.

Fig. 6. Head of holotype ZISP 25569 of Calamaria abramovi sp. nov., ventral view.

low-orange spots on dorsal side arranged in 36 – 37 groups of 1 – 3 square spots; 2 – 3 groups of spots on ventral side of tail. Description of holotype. Adult female. Body robust, round in cross-section, total length 482 mm (snout-vent length 448 mm), tail length from vent to tip 34 mm). Body diameter 9.96 mm; ratio of body length to tail length 13.18; ratio of body length to body diameter 44.98; eight modified maxillary teeth; ventrals 174; subcaudals 20 pairs; dorsal scales rhomboid, overlap in roof-tile fashion, strongly smooth, without keel and tubercles, scales of nearly equal size, number of dorsal scale rows at body: Sq1 — 12, Sq2 — 13, Sq3 — 13, Sq on the level of anus 11; dorsal row of scales not enlarged; anal plate single; tail thick, short, tapering gradually from base, abruptly tapering at tip to sharp point; dorsal scales reduced in number to four rows on tail opposite twenty subcaudals anterior to terminal scute. Head small, roundish, short, covered by large regular symmetrical shields, not distinct from neck. Eyes small with round pupils, 5 shields around eye, horizontal eye diameter slightly smaller than distance from eye to mouth edge. Snout broad and short, HL/SL 3.18. Rostral large, triangular, its height equal to width, in contact with nasals, prefrontals and first supralabials, visible from above; single nasal shield very small, triangular, inserted between first supralabial, prefrontal and rostral, oval nostril occupies greater part of shield. Head length from anterior part of rostral shield to posterior part of lower jaw 13.05 mm; head width at widest point 7.23 mm; horizontal eye diameter 1.68 mm; snout length from snout tip to anterior edge of eye 4.10 mm; eye to nostril distance from anterior corner of eye to posterior edge of nostril 2.12 mm; interorbital distance 4.17 mm; no loreal; number of supraoculars 1 (large and wide, adjacent to frontal, prefrontal, preocular, postocu-

lar and parietal); preocular 1; postocular 1 on left and 2 on right; internasals absent; prefrontals paired, very large, hexagonal-shaped, slightly wider than long; length 2.41 mm, width 2.72 mm; frontal very large (length 3.17 mm), hexagonal in shape; paired parietals extremely large (length 4.95 mm), divided by suture. Small scale inserted between parietals in their lower part; suture between parietals 2.23 longer than that between prefrontals; paraparietal surrounded by six shields and scales. Mental shield triangular, distinct, not concealed in mental groove, touching anterior genial shields; anterior genial shields large and wide, touching 1st, 2nd, and 3th infralabials, posterior genials shields large, in contact with 3th and 4th infralabials; both pairs of genials shields meeting in midline; 2 gular scutes in midline between posterior genial shields and first ventral; first gular scute inserted between posterior genial shields and divides them almost to top. Supralabials 4 – 4, first supralabial smallest, fourth supralabial largest; first supralabial in contact with rostral, nasal and prefrontal shields; first one in contact with rostral, nasal and prefrontal; second one in contact with prefrontal, preocular and eye; third one in contact with postocular and eye. Infralabials 5 – 5, 4th and 5th infralabials largest (Figs. 5 and 6). Coloration. Body black, strongly iridescent, with blazing yellow-orange spots on ventral body side, dorsal body side immaculate. Blazing yellow-orange spots on dorsal side arranged in 36 – 37 groups of 1 – 3 square spots; 2 – 3 groups of spots are situated on ventral side of tail. Pointed tip of tail light-yellow. Measurements of holotype. TL 482 mm; SVL 448 mm; Lcd 34 mm; BD 9.96 mm; HL 13.05 mm; HW 7.23 mm; ED 1.68 mm; EN 2.12 mm; SL 4.10 mm; IO 4.17 mm; SVL/Lcd 13.18; SVL/HL 36.93; SVL/BD 44.98; V 174; Scd 20 pairs; A 1; Sq1 — 12,

A New Species of the Genus Calamaria from the Central Highlands, Vietnam Sq2 — 13, Sq3 — 13; SO — 1, its length 1.96 mm; PrO 1; PtO 1 on the left and 2 on the right; Supralab 4 – 4; Infralab 5 – 5; 5 shields around eye; PF length 2.41 mm, PF width 2.72 mm; F length 3.17 mm; P length 4.95 mm; P/SO 2.52; P/PF 2.05; P/F 1.56. Description of paratype. Juvenile male. Body robust, round in cross-section, total length 139.23 mm (snout-vent length 128.81 mm, tail length from vent to tip 10.42 mm); tail thick, short, tapering, with pointed tip. Body diameter 3.95 mm; body length/tail length ratio 12.36; ratio of body length to its diameter 32.61; phlolidosis identical to that of holotype: ventrals 159; subcaudals 26 pairs; dorsal scales rhomboid, tileshaped, strongly smooth, without keels or tubercles, scales of nearly equal size, number of rows of dorsal scales at body: Sq1 — 12, Sq2 — 13, Sq3 — 13, 11 Sq on level of anus; anal plate single. Head small, round, short, covered by large symmetric regular shields, not distinct from the neck. Snout broad and short, rostral large, triangular, with height equal to width, in contact with nasals, prefrontals and first supralabials, clearly visible from above; single nasal shield very small, triangular, inserted between first supralabial, prefrontal and rostral, large round nostril cut in nasal shield closer to anterior edge; no loreal; number of supraoculars 1 (large and wide, bordering frontal, prefrontal, preocular, postocular and parietal); preocular 1; postocular 1; internasals absent; prefrontals paired, very large, of hexagonal shape, slightly wider than long; frontal very large, of hexagonal shape; paired parietals extremely large (see measurements); paraparietal surrounded by six shields and scales. Mental shield triangular, distinct, not concealed in mental groove; anterior genial shields large and wide, in contact with 1st, 2nd and 3th infralabials, posterior genial shields large, in contact with 3th and 4th infralabials; small scale inserted from below between posterior genial shields divides them almost up to top. There are 4 – 4 supralabials, first of them smallest and fourth largest; first supralabial shield in contact with rostral, nasal and prefrontal shields; second one in contact with prefrontal, preocular and eye; third supralabial in contact with postocular and eye. Infralabials 5 – 5, fourth and fifth largest. Coloration. Body black, strongly iridescent, with blazing yellow-orange spots on ventral body side, dorsal body side without spots [opaque]. Blazing yellow-orange spots on dorsal side arranged in 36 groups of 1 – 3 square spots; 3 groups of spots on ventral side of tail. Pointed tip of tail light-yellow. Measurements of paratype. TL 142.48 mm; SVL 123.5 mm; Lcd 18.98 mm; BD 3.95 mm; SVL/Lcd

100°

151 105°

110° E

CHINA Hanoi Gulf of

20° E

To n k i n Hainan

THAILAND 15°

CAMBODIA

Gulf of

Ho Chi Minh City

10°

Thailand

Fig. 7. Map of type locality (yellow star) of Calamaria abramovi sp. nov.

12.36; SVL/BD 32.61; V 159; Scd 26 pairs; A 1; Sq1 — 12, Sq2 — 13, Sq3 — 13; SO 1; PrO 1; PtO 1; Supralab 4 – 4; Infralab 5 – 5. Etymology. The specific epithet abramovi is given after Dr. Alexey V. Abramov (Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia) in recognition of his many contributions to the study of Vietnam and many records of rare and new species of animals, in particular amphibians and reptiles. The second specimen (paratype) of a new species was collected by Dr. Alexey V. Abramov and kindly provided to me for study and description. Distribution and natural history. The species is known only from the type locality (Fig. 7). Both specimens were collected on a forested slope of Ngoc Linh Mount, elevation 1550 – 1700 m. The adult female was collected during the rain in September on a night excursion from the leaf litter in the flood plain of a forest stream (Fig. 8). The second specimen (juvenile male) was collected in the end of a dry season in March on a leaf litter covering the forest path. Comparisons and discussion. Calamaria abramovi sp. nov. differs from all known species of the genus by a combination of pholidosis characters and color-

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Fig. 8. Habitat of Calamaria abramovi sp. nov. in the type locality, above Mang Xang village, Ngoc Linh Mountain, Gac Glei district, Kon Tum province, Vietnam.

ation. Seven species are known in Vietnam ophidiofauna: C. buchi Marx et Inger, 1955; C. lovii ingermarxorum Darevsky et Orlov, 1992; C. pavimentata Duméril et Bibron, 1854; C. septentrionalis Boulenger, 1890; C. thanhi Ziegler et Quyet Le Khac, 2005; C. sp. nov. Nguyen Quang Truong, Koch et Ziegler, in press; C. gialaiensis sp. nov. Ziegler, Nguyen Van Sang, and Nguyen Quang Truong, 2008 (Darevsky and Orlov, 1992; Nguyen and Ho, 1996; Orlov et al., 2000, 2003; Orlov, 2005; Ziegler, 2002; Ziegler et al., 2004; Ziegler and Quyet Le Khac, 2005; Nguyen Quang Truong et al., in press; Ziegler et al., 2008c). In addition to the species listed above the following species are also recorded from the mainland part of Oriental province: C. yunnanensis Chernov, 1962 (Yunnan, China); C. lumbricoidea Boie, 1827; C. albiventer Gray, 1834; C. lovii gimleti Boulenger, 1890); C. schlegeli schlegeli Duméril et Bibron, 1854; C. prakkei Lidth de Jeude, 1891; and C. ingeri Grismer, Kaiser et Yaakob, 2004 (Thailand, western Malaysia, Singapore) (Inger and Marx, 1965; Darevsky and Orlov, 1992; Zhao and Adler, 1993; Nguyen and Ho, 1996; Grismer et al., 2004; Ziegler and Quyet, 2005).

Calamaria abramovi sp. nov. differs from C. buchi by a considerably smaller number of ventrals (V 159 – 174 vs. 221 – 236 in C. buchi — according to Inger and Marx, 1965); the new species is distinguished from C. lovii gimleti and C. lovii ingermarxorum by the presence of preoculars, smaller number of ventrals and higher number of subcaudals (especially in comparison with females of C. lovii gimleti), also by the ratio of the length of prefrontal and frontal shields (preocular 1, V 174, Scd 20 pairs, F > PF vs. no preocular, V 215 – 249, Scd 10 – 12 pairs, PF > F in C. lovii gimleti — according to Inger and Marx, 1965; and preocular 1, V 159 – 174, F > PF vs. no preocular, V 205, PF > F in C. lovii ingermarxorum, according to Darevsky and Orlov, 1992). The new species is distinguished from C. pavimentata by a completely different color pattern (body black, strongly iridescent, with blazing yellow-orange spots on ventral side of body, dorsal body side without spots [opaque] vs. dorsum with narrow, dark, longitudinal stripes, and with solid black color immediately behind neck in C. pavimentata — according to Inger and Marx, 1965; Ziegler and Quyet Le Khac, 2005); from

A New Species of the Genus Calamaria from the Central Highlands, Vietnam C. septentrionalis, the new species differs by a considerably higher number of subcaudals (Scd of female 20 vs. Scd 6, 11 in females of C. septentrionalis — according to Inger and Marx, 1965); from C. thanhi, by the presence of preocular and coloration patterns, in particular on the back (PrO present, dorsum black and immaculate vs. PrO absent, dorsum dark, with 4 – 6 light body bands in C. thanhi — according to Ziegler and Quyet Le Khac, 2005); from C. sp. Nguyen Quang Truong, Koch et Ziegler, in press — by a higher number of subcaudals, smaller number of ventrals and color pattern of ventral side (Scd 20 – 26, V 159 – 174, ventral side black with orange spots, 3 groups of spots on ventral side of tail vs. Scd19, V 2+190, ventral side cream, with dark transverse bands and a dark longitudinal stripe below the tail in C. sp. Nguyen Quang Truong, Koch et Ziegler, in press); from C. gialaiensis, by a smaller number of ventrals and higher number of shields and scales around paraparietal (V 159 – 174, paraparietal surrounded by six shields and scales vs. V 191 and paraparietal surrounded by five shields and scales in C. gialaiensis — according to Ziegler et al., 2008c); from C. yunnanensis, by the presence of preocular (PrO present vs. PrO absent in C. yunnanensis, according to Chernov, 1962 and Zhao et al., 1998); from C. lumbricoidea, by a higher number of shields and scales around paraparietal and another pattern of coloration (paraparietal surrounded by six shields and scales, ventral black with orange spots vs. paraparietal surrounded by 4 – 5 shields and scales, belly yellow with black crossbars wider than width of one ventral in C. lumbricoidea — according to Inger and Marx, 1965); from C. albiventer, by a smaller number of supralabials, higher number of shields and scales around paraparietal, pattern of supralabial shields entering orbit (Supralab 4, 6 shields and scales around paraparietal, second and third supralab entering orbit vs. 5, 5, third and fourth in C. albiventer — according to Inger and Marx, 1965). The new species differs from C. schlegeli schlegeli by a larger body size, smaller number of supralabials (TL of females 482 mm, 4 supralabials vs. TL of females 125 – 391 mm, 5 supralabials in C. schlegeli schlegeli, according to Inger and Marx, 1965); from C. prakkei — by a higher number of ventrals, smaller number of subcaudals, higher number of shields and scales around paraparietal, pattern of supralabial shields entering orbit and larger body size (V 159 – 174, Scd 20 – 26, 6 shields and scales around paraparietal, second and third supralab entering orbit, TL 482 mm vs. V 126 – 144, Scd 24 – 32, 5, third and fourth supralabials entering orbit, TL 172 – 256 mm, in C. prakkei — according to Inger and Marx, 1965); from C. ingeri, by a number of su-

153

pralabials and pattern of supralabial shields entering orbit (Supralab 4, second and third supralabial entering orbit vs. Supralab 5, third and fourth Supralab entering the orbit in C. ingeri — according to Grismer et al., 2004). It is important to note that Calamaria abramovi sp. nov. is characterized by a completely different color pattern from all the species under comparison and species of Sunda Archipelago (by Inger and Marx, 1965). Keys to the species of Calamaria genus are given by Inger and Marx (1965), Darevsky and Orlov (1992), Grismer et al. (2004); Ziegler and Quyet Le Khac (2005), Ziegler et al. (2008c). The description of 4 new species of Calamaria from central regions of Vietnam in a relatively short period (from 1992 to 2009) reflects the intensity of herpetological research in this region and in Indochina as a whole. Among them are snakes, including secretive and rarely recorded species such as fossorial and small water colubrids. Besides Calamaria we need to mention discoveries of a number of new species of colubrids and numerous records, which continually expand our understanding of the diversity and distribution of these snakes in Indochina (Fimbrios, Opistothropis, Parahelicops, Paratapinophis, Amphiesma) (David et al., 2007; Murphy et al., 2008; Orlov, 1995, 2005; Orlov et al., 2000, 2003; Szyndlar and Nguyen Van Sang, 1996; Stuart and Chuaynkern, 2007; Tillack et al., 2004; Ziegler and Herrmann, 2000; Ziegler and Le Khac Quyet, 2006; Ziegler et al., 2004, 2008a, 2008b, 2008c). Acknowledgments. We are very grateful to Vietnam’s Institute of Ecology and Biological Resources (IEBR) for the organization of the fieldwork in Vietnam, Director of IEBR, Prof. Le Xuan Canh and Director of Dept. of Vertebrate Zoology, Dr. Nguyen Xuan Dang. Many thanks to Natalia Ananjeva and Roman Khalikov for their kind assistance in the preparation of the manuscript. Our research was partially supported by VolkswagenStiftung and RFBR 08-04-0004. We are indebted to Harold Voris, Robert Inger and Alan Resetar (FMNH), Robert Murphy (ROM), Ted Papenfuss (MVZ), Jens Vindum (CAS), Alain Dubois and Patrick David (MNHN), Rainer Günther (ZMB), Nguyen Van Sang and Nguyen Quang Truong (IEBR), Zhao Ermi, Yuezhao Wang and Sun Erhu (CIB), Rao-Ding-qi (KIZ) for permitting us to examine snake specimens.

REFERENCES Darevsky I. S. and Orlov N. L. (1992), “A new subspecies of the dwarf snake Calamaria lowi ingermarxi ssp. nov. (Serpentes, Colubridae) from southern Vietnam,” Asiatic Herpetol. Res., 4, 13 – 17.

154 David P., Bain R., Nguyen Quang Truong, Orlov N., Vogel G., Vu Ngoc Thanh, and Ziegler T. (2007), “Another new species of the natricine snake genus Amphiesma from Central Vietnam (Squamata: Colubridae: Natricinae),” Zootaxa, 1462, 41 – 60. Inger R. F. and Marx H. (1965), “The systematics and evolution of the Oriental colubrid snakes of the genus Calamaria,” Fieldiana Zool., 49, 1 – 304. Grismer L. L., Kaiser R. H., and Yakob N. S. (2004), “A new species of reed snake of the genus Calamaria H. Boie, 1827, from Pulau Tioman, Pahang, West Malaysia,” Hamadryad, 28, 1 – 6. Murphy J. C., Tanya Chan-Ard, Sonchai Mekchai, Cota M., and Voris H. K. (2008), “The rediscovery of Angel’s Stream Snake, Paratapinophis praemaxillaris Angel, 1929 (Reptilia: Serpentes: Natricidae),” Nat. Hist. J. Chulalongkorn Univ., 8(2), 169 – 183. Nguyen Van Sang and Ho Thu Cuc (1996), Danh Luc Bo Sat Va Ech Nhai Viet Nam [A Checklist of Reptiles and Amphibians of Vietnam], Nha Xuat Ban Khoa Hoc Va Ky Thu At [Science and Technic Publishing House], Hanoi [in Vietnamese]. Nguyen Van Sang and Ho Thu Cuc (2005), A Checklist of Amphibians and Reptiles of Vietnam, Nha xuat ban nong nghiep [Agriculture Publishing House], Hanoi [in Vietnamese]. Nguyen Quang Truong, Koch A., and Ziegler T., “A new species of reed snake, Calamaria Boie, 1827 (Squamata: Colubridae), from central Vietnam,” Hamadryad, 34(1), in press. Orlov N. L. (1995), “Rare snakes of the mountainous forests of northern Indochina,” Russ. J. Herpetol., 2(2), 179 – 183. Orlov N. L. (2005), “New species of the genus Vibrissaphora Liu, 1945 (Anura: Megophryidae) from mount Ngoc Linh (Kon Tum Province) and analysis of the extent of species overlap in the fauna of amphibians and reptiles of the North-West of Vietnam and Central Highland,” Russ. J. Herpetol., 12(1), 10 – 31. Orlov N. L., Murphy R. W., and Papenfuss T. J. (2000), “List of Snakes of Tam-Dao Mountain Ridge (Tonkin, Vietnam),” Russ. J. Herpetol., 7(1), 69 – 80. Orlov N. L., Ryabov S. A., Nguyen Van Sang, and Nguyen Quang Truong (2003), “New records and data on the poorly known snakes of Vietnam,” Russ. J. Herpetol., 10(3), 217 – 240. Stuart Bryan L. and Yodchaiy Chuayankern (2007), “A new Opisthotropis (Serpentes: Colubridae: Natricinae) from Northeastern Thailand,” Curr. Herpetol., 26(1), 35 – 40.

Nikolai L. Orlov Szyndlar Z. and Nguyen Van Sang (1996), “Terrestrial snake fauna of Vietnam: distributional records,” The Snake, 27, 91 – 98. Tillack F., Ziegler T., and Le Khac Quyet (2004), “Eine neue Art der Gattung Boiga Fitzinger, 1826 (Serpentes: Colubridae: Colubrinae) aus dem zentralen Vietnam,” Sauria, 26, 3 – 12. Zhao E. and Adler K. (1993), Herpetology of China. Contribution to Herpetology. No. 10, Soc. Study Amphib. Reptiles. Zhao E.-M., Huang M.-H., Zong Y., et al. (1998), Fauna Sinica. Reptilia. Vol. 3. Squamata. Serpentes, Science Press, Beijing. Ziegler T. (2002), Die Amphibien and Reptilien eines Tieflandfeuuchtwald-Schutzgebietes in Vietnam, Natur und Tier-Verlag, Münster. Ziegler T. and Herrmann H.-W. (2000), “Preliminary list of the herpetofauna of the Phong Nha – Ke Bang area in Quang Binh province, Vietnam,” Biogeographica, 76, 49 – 62. Ziegler T. and Le K. Q. (2006) “A new natricine snake of the genus Amphiesma (Squamata: Colubridae: Natricinae) from the central Truong Son, Vietnam,” Zootaxa, 1225, 39 – 56. Ziegler T. and Quyet Le Khac (2005), “A new species of reed snake, Calamaria (Squamata: Colubridae), from the Central Truong Son (Annamite mountain range), Vietnam,” Zootaxa, 1042, 27 – 38. Ziegler T., David P., Miralles A., Doan Van Kien and Nguyen Quang Truong (2008a), “A new species of the snake genus Fimbrios from Phong Nha—Ke Bang National Park, Truong Son, central Vietnam (Squamata: Xenodermatidae),” Zootaxa, 1729, 37 – 48. Ziegler T., David P., and Vu Ngoc Thanh (2008b), “A new natricine snake of the genus Opisthotropis from Tam Dao, Vinh Phuc Province, northern Vietnam (Squamata, Colubridae),” Zoosyst. Evol., 84(2), 197 – 203. Ziegler T., Nguyen Van Sang, and Nguyen Quang Truong (2008c), “A New Reed Snake of the genus Calamaria Boie (Squamata: Colubridae) from Vietnam,” Curr. Herpetol., 27(2), 71 – 80. Ziegler T., Hendrix R., Vu N. T., Vogt M., Forster B., and Dang N. K. (2007), “The diversity of a snake community in a karst forest ecosystem in the central Truong Son, Vietnam, with an identification key,” Zootaxa, 1493, 1 – 40. Ziegler T., Herrmann H.-W., Vu Ngok Thanh, Le Khac Quyet, Nguyen Tan Hiep, Cao Xuan Chinh, Luu Minh Thank, and Dinh Huy Tri (2004), “The amphibians and reptiles of the Phong Nha-Ke Bang National Park, Quang Binh Province, Vietnam,” Hamadryad, 28(1 – 2), 19 – 42.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 155 – 158

A NEW LOCALITY FOR Vipera (Pelias) kaznakovi NIKOLSKY, 1909 (REPTILIA, VIPERIDAE) IN THE NORTH-EASTERN ANATOLIA Murat Afsar1* and Birgül Afsar1 Submitted June 12, 2008. In this study, Vipera (Pelias) kaznakovi specimens collected from Camili Biosphere Reserve (Borçka, Artvin province Turkey) were investigated in terms of morphological characters. Information on morphological, ecological features and population status of this species in the Biosphere Reserve is given. Furthermore, the distribution range of Vipera (Pelias) kaznakovi has been extended in Turkey. Keywords: Vipera (Pelias) kaznakovi, taxonomy, morphology, distribution, Biosphere Reserve.

INTRODUCTION

MATERIAL AND METHODS

Vipera (Pelias) kaznakovi, was first described from Tsebelda in Georgia by Nikolsky in 1909. According to the Orlov and Tuniyev 1990 and Tuniyev and Ostrovskikh, 2001 the distribution range of Vipera (Pelias) kaznakovi is vicinity of Tuapse on the north-west of Black Sea, then cross the Main Ridge to Goryachiy Klyuch and continues eastward to Belaya River on the northern slope of Western Caucasus. Specimen examined in the north-eastern Anatolia was first described as Vipera berus ornata by Baþoðlu (1947) from Hopa. However Mertens (1952) synonymized it with Vipera kaznakovi which was subsequently accepted by Kramer (1961), Kretz (1972), Baran (1976), and others. More recently Baþoðlu and Baran (1980), Nilson et al. (1988), Orlov and Tuniyev (1990), Nilson et al. (1994), Baran and Atatür (1998) have reported that V. (Pelias) kaznakovi inhabits only Hopa (Artvin, Turkey) region in Turkey, but Baran et al. (2005) have extended the distribution range of Vipera (Pelias) kaznakovi to Borçka, (Artvin, Turkey) (Figure 1). In this study, Vipera(Pelias) kaznakovi specimens collected from Camili (Artvin) were examined in terms of morphological characters, ecological features and compared with the related literature. Population status of this species in Camili Biosphere Reserve is also given.

A total of 4 (1 }, 2 xx s.ad., 1 x) specimens were collected from May to September 2003 from Camili Biosphere reserve. Camili region is the first Biosphere Reserve in Turkey appointed from UNESCO since 29 July 2005. The Camili basin is part of the Karçal Mountains important plant area, which is the only one of the 122 important plant area defined in Turkey. Camili basin is located in Borçka district of Artvin province and 52 km from Borçka city center (Fig. 1). The lowest points in the basin is Camili with 450 m and the highest point is an unnamed hill in Karçal Mountains with 3435 m. The area is 25274.58 ha. The specimens fixed in a mixture of 7% formalin and 7% ethanol, and later deposited 70% ethanol. The following meristic pholidolial characteristics were

Celal Bayar University, Faculty of Sciences & Literature, Department of Biology, Muradiye Campus, 45030 Manisa, Turkey. *Address correspondence and reprint requests to: Murat Afsar, E-mail: murat.afsar@bayar.edu.tr; Tel: +90 (236) 241-2151/404; Fax: +90 (236) 241-2158.

Fig. 1. The distribution of Vipera kaznakovi in Turkey: , old localities of Vipera kaznakovi; ¢, new locality of Vipera kaznakovi.

156

Murat Afsar and Birgül Afsar

taken: while taking the morphological measurements, digital calipers of 0.01 mm [for the rostral height (RH) and rostral weight (RW)] sensitivity and tape measure of 0.1 mm [for the total length (TL) and tail length (TaL)] sensitivity were used. The ventral plate count was conducted according to Dowling (1951). The specimens, which were given collection number at ZDEU (Zoology Department, Ege University), are now kept at the Zoology Department, Faculty of Literature and Science, Celal Bayar University (Table 1). RESULTS Pholidosis. Dorsally the head is covered with large scales. Supraocularia, parietalia and frontale are pronounced. Rostral reaches two apical scales on upper snout. Apicalia 2 in tree specimens, 1 in the fourth; canthalia 2; lorealia 7/7 in female specimen, 6/6, 4/5, 5/5 in the two subadult male specimens and one adult male specimen respectively; intercanthalia and intersupraocularia 22 in female specimen, 21, 17, 16 in the two subadult male specimens and one adult male specimen; supralabialia 10/9 in female specimen, 10/–, 9/9, 9/8 in the two subadult male specimens and one adult male specimen. Double row of scales is located between the row of orbital and supralabial plates in the tree specimens, a single row of scales is located between the rows of orbital and supralabial plates in the one specimen found from vicinity of Camili village. The number of small scales around the eyes 10/10 in female specimen, 13/13, 9/9, 8/8 in the two subadult male specimens and one adult male specimen respectively. Number of dorsal scales in the midbody 21 in the tree specimens, uncounted in the one specimen collected from vicinity of Baltacýk village because of damaged on its body. The number of ventralia 135 both specimens collected from Düzenli and Efeler, uncounted other specimens from Camili and Baltacýk village because of damaged on its body. The number of subcaudalia 26 in female specimen, 34, 35, 35 in the two subadult male specimens and one adult male specimen, respectively.

Measurements. Total length 435 mm in female specimen from Düzenli and 324 mm in subadult male specimen from Efeler, total length unmeasured Camili and Baltacýk village specimens because of damaged on its body. Tail length 45 mm in female specimen, 45, 50, 60 mm in the two subadult male specimens and one adult male specimen respectively. Rostral width 2.59 in the female specimen, 2.79, 2.68, 2.82 mm in the two subadult male specimens and one adult male specimen, respectively. Rostral height 3.61 mm in female specimen, 3.07, 3.13, 3.44 mm in the two subadult male specimens and one adult male specimen respectively. Rostral index 0.77 in female specimen, 0.90, 0.85, 0.82 mm in the two subadult male specimens and one adult male specimen, respectively. Color and pattern. Top of the head is black not any pattern present on the ground color. Black temporal band is pronounced, upper and lower labials are yellow. Black dorsal striped is present and zigzag shaped in all specimens along the body (Fig. 2). Ventrum is black with numerous a few light tiny spots. Ecology. Female specimen collected from Düzenli was found on a sunny day near the hazel grow on a good vegetated rocky hillside covered with forest. The altitude of the area was 563 m. The temperature was 17°C. Subadult male specimen which has damaged on its head collected from Efeler was found on a cloudy day near the big stones on a good vegetated rocky hillside covered with forest. The altitude of the area was 500 m. The temperature was 20°C. Subadult male specimen which has damaged on its body collected from Camili village was found near the road. The altitude of the area was 950 m. The temperature was 17°C. Male specimen which has damaged on its head and body collected from Baltacýk village was found on a cloudy day at an 1044 m. The temperature was 13°C. In the same habitat, amphibian and reptile specimens such as Bufo verrucosissimus, Hyla arborea, Mertensiella caucasica, Darevskia rudis, Anguis fragilis, Coronella austriaca, Natrix natrix, Zamenis longissimus were found and Fagus orientalia, Castanea sativa, Picea orientalia, Corylus avellana, Abies nordmanniana, Quercus hartwissiana, Quercus pontica groves species were also noted.

TABLE 1. Specimens Used in This Study Material

Number of Specimens

Locality

Collecting Date

ZDEU 129/2003

1 (x)

Baltacýk/Camili Artvin

05/18/2003

ZDEU 130/2003

1 (s.ad.x)

Efeler/Camili Artvin

06/19/2003

ZDEU 131/2003

1 (s.ad.x)

Camili/Camili Artvin

09/21/2003

ZDEU 132/2003

1 (})

Düzenli/Camili Artvin

09/23/2003

New Locality for Vipera (Pelias) kaznakovi in Anatolia

157

Fig. 2. Dorsal view and pattern characteristics of the “Vipera kaznakovi” collected from Camili Biosphere Reserve: a, female specimen from Düzenli; b, subadult male specimen from Efeler; c, subadult male specimen from Camili; d, male specimen from Baltacýk.

DISCUSSION The color pattern and pholidosis of four specimens were compared with literature data (Baþoðlu, 1947; Mertens, 1952; Kramer, 1961; Kretz, 1972; Baran, 1976; Nilson et al., 1995; Tuniyev and Ostrovskikh, 2001; Baran et al., 2005). Our sample from Camili are remarkable in being similar in head shape and color pattern to Vipera (Pelias) kaznakovi. According to Tuniyev and Ostrovskikh (2001), in Western Caucasus Vipera (Pelias) kaznakovi population have 5 – 12 (mean 8.88) numbers of intercanthals and intersupraoculars. However we found out that numbers of intercanthals and intersupraoculars of our samples were 16 – 22 (mean 19). From the view point of this character our material is almost identical with Hopa Vipera (Pelias) kaznakovi population given by Nilson et al. (1995). But Nilson et al. (1995) has reported that the number of loreal scales (counted as sum of the both sides) ranged between 5 and 12 (mean, 8.69) in Hopa population these values of our samples have a slightly higher range (9 – 14, mean 11.25). Tuniyev and Ostrovskikh (2001) have recorded

the circumocularia numbers (counted as sum of the both sides) as 17 – 26 (mean 20.56) in Western Caucasus Vipera (Pelias) kaznakovi population and Nilson et al. (1995) have recorded the circumocularia numbers as 16 – 23 (mean 20.2) in Sochi-Adler population, as 15 – 21 (mean 19.31) in Hopa population, coinciding with our data obtained. Ventrals, subcaudals, supralabials, sublabials, apicals, and mid-body scales counts of our material fall within the range of other Vipera (Pelias) kaznakovi population given in previous studies (Baþoðlu, 1947; Mertens, 1952; Kramer, 1961; Kretz, 1972; Baran, 1976; Nilson et al., 1995; Tuniyev and Ostrovskikh, 2001; Baran et al., 2005). When the measurements of the four specimens compared with literature data (Baþoðlu, 1947; Mertens, 1952; Kramer, 1961; Baran, 1976; Nilson et al., 1995; Tuniyev and Ostrovskikh, 2001; Baran et al., 2005) in the present study only rostral index was found lower than that previous studies. Baran (1976) and Nilson et al. (1995) have reported that rostral index values were ranged from 0.87 to 1.5 (mean 1.1) in Hopa population and from 1.0 to 1.27 (mean 1.1) in Sochi-Adler popula-

158 tion. However, we found out that rostral index of our specimens were from 0.72 to 0.91 (mean 0.82). other measurements of the four specimens correspond to those given on previous studies. Until recently, Vipera (Pelias) kaznakovi has been reported from only Hopa region in Turkey (Baþoðlu, 1947; Mertens, 1952; Kramer, 1961; Kretz, 1972; Baran, 1976; Baþoðlu and Baran, 1980; Nilson et al., 1988, 1994; Baran and Atatür, 1998) and Nilson et al. (1988), Höggren et al. (1993), and Baran and Atatür (1998) also have stated that this species is locally abundant in the Hopa region of Turkey. But Höggren et al. (1993) have claimed that in Turkey, Russia and Georgia, Vipera (Pelias) kaznakovi populations along the Black sea coast have been seriously affected by exploitation of habitats and Tuniyev and Tuniyev (2007) have stated that Vipera (Pelias) kaznakovi has disappeared from many localities because of threatened by continuous decline in habitat suitability and dense populations still exist in the Sochi National Park. In this study, great numbers of specimens have been observed during field trips in Artvin Camili complex Caucasian mild rainforest area but, because of dense vegetation population size is not estimated. Camili Biosphere Reserve due to geographical condition, strategic military interests and the immediate border zone has not been accessible to foreigners and this area offering a refuge for viable Vipera (Pelias) kaznakovi as other species (e.g., Tetreo mlokosiewiczi Caucasian black grause, Apis mellifera caucasica Caucasus bee race). As a result, this study extended the distribution range of Vipera (Pelias) kaznakovi to Camili, 52 km northeast of Borçka in Turkey. Acknowledgments. This study was supported by the Grant Agreement of Project “Biodiversity and Natural Resource Management” prepared by the Ministry of Forestry in collaboration with World Bank and financed by the Global Environment Facility (GEF) between 2000 and 2006. We would like to extend our gratitude to Sýtký Eraydýn, the responsible Forest Engineer for the Fauna Survey of PAMA-Camili, Özgür Alaçam and other co-workers of the project for their support and help in the field. We are also most grateful to G. Nilson for providing some references.

REFERENCES Baran Ý. (1976), “The taxonomic revision of Turkey snakes and their geographical distribution,” Tübýtak yayýnlarý Ankara, 309, 1 – 117.

Murat Afsar and Birgül Afsar Baran Ý. and Atatür M. K. (1998), Turkish Herpetofauna (Amphibians and Reptiles), Turkish Ministry of Environment, Ankara. Baran Ý., Tok C. V., Olgun K., Ýret F., and Avcý A. (2005), “On viperid (Serpentes: Sauria) specimens collected from Northeastern Anatolia,” Tur. J. Zool., 29, 225 – 228. Baþoðlu M. (1947), “On some varieties Vipera berus from the extreme northeastern Anatolia,” Rev. Fac. Sci. Univ. Istanbul. Ser. B, 12, 182 – 190. Baþoðlu M. and Baran Ý. (1980), “Türkiye Sürüngenleri. Kýsým II. Yýlanlar,” Ege Üniv. Fen Fak. Kitaplar Ser., No. 76, 1 – 217. Dowling H. G. (1951), “A proposed standard of counting ventrals in snakes,” Br. J. Herpetol., 1, 97 – 99. Höggren M., Nilson G., Andrén C., Orlov N., and Tuniyev B. (1993), ”Vipers of the Caucasus natural history and systematic review,” Herpetol. Nat. Hist., 1(2), 11 – 19. Kramer E. (1961), “Variation, Sexualdimorfismus, Wachstum und Taxonomie von Vipera ursinii (Bonaporte, 1835) und Vipera kaznakovi Nikolskij 1909,” Rev. Suisse Zool., 68(4), 627 – 725. Kretz J. (1972), “Über Vipera kaznakovi Nikolskij, 1909 aus Nordostanatolien (Reptilia, Viperidae),” Jb. Naturhist. Mus. Bern, 4, 125 – 134. Mertens R. (1952), “Amphibien und reptilien aus der Türkei,” Ýstanbul Üniv. Fen Fak. Mecmuasý Ser. B, 17, 41 – 75. Nikolsky A. M. (1909), “A new viper species from Caucasus, Vipera kaznakovi,” Byull. Kavkaz. Muz. Tiflis [in Russian], No. 3/4, 173 – 174. Nilson G., Andren C., and Flärdh B. (1988), “Die Vipern in der Türkei,” Salamandra, 24, 215 – 247. Nilson G., Höggren M., Tuniyev B., Orlov N., and Andren C. (1994), “Phylogeny of the Vipers of the Caucasus (Reptilia, Viperidae),” Zool. Scripta, 23(4), 353 – 360. Nilson G., Höggren M., Tuniyev B., Orlov N., and Andren C. (1995), “Systematics of the vipers of the Caucasus: Polymorphism or sibling species?” Asiatic. Herpetol. Res., 6, 1 – 26. Orlov N. and Tuniyev S. B. (1990), “Three species in the Vipera kaznakovi complex (Eurosiberian group) in the Caucasus: their present distribution, possible genesis, and phylogeny,” Asiatic Herpetol. Res., 3, 1 – 36. Tuniyev S. B. and Ostrovskikh S. V. (2001), “Two new species of vipers of ‘kaznakovi‘ complex (Ophidia, Viperinae) from the Western Caucasus,” Russ. J. Herpetol., 8, 117 – 126. Tuniyev B. and Tuniyev S. (2007), “Vipers of the Krasnodarsky Territory, Russia,” in: Abstrs. of the 2nd Biol. of the Vipers Conf., September 24 – 27, 2007, Portugal, p. 49.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 159 – 160

OCCURRENCE OF Enhydris sieboldii (SCHLEGEL, 1837) IN BHOPAL, MADHYA PRADESH STATE OF INDIA

Sanjay Thakur1 and Aparna Watve1 Submitted January 29, 2008. Enhydris sieboldii is reported from Bhopal in Madhya Pradesh state of India, with comments on its occurrence. Keywords: Enhydris sieboldii, Bhopal, Madhya Pradesh, India.

The genus Enhydris Sonnini et Latreille, 1802 is represented by four species in India. They are water snakes of the subfamily Homalopsinae, inhabiting permanent as well as temporary water bodies. Enhydris enhydris (common smooth-scaled water snake) is the most common species of this group distributed throughout central and eastern India. E. dussumieri (Dussumier’s Smooth-scaled Water Snake) is endemic and known from the southwestern coast of India (Kerala) only (Parameswaran, 1954; Murphy, 2007). In India, E. plumbea (Plumbeous Smooth-scaled Water Snake) has only been reported from Great Nicobar Island (Whitaker and Captain, 2004). The fourth species E. sieboldii is widely distributed but not common anywhere. In India, it is known from few scattered collections at some localities. Hence, it was thought necessary to report a collection of this species from an additional locality in Madhya Pradesh. In June 2005, a specimen (Fig. 1) was collected by local fishermen in the backwaters of the Kaliya Sot dam while fishing during evening time (Fig. 1). The local people suspected it to be a juvenile of Python molurus molurus (Indian Rock Python) and thought it a novelty to find a python living underwater. It was brought alive for identification and was confirmed as Enhydris sieboldii after counting its scales. It was about 60 cm long (total length) and 15 cm girth round the midbody. It fitted within the already described range for the taxon and did not show any aberrations. The snake was released in the same locality after photographing it. This is the first photographic record of the species from India. The earlier published photo1

Biome Conservation Foundation, 18, Silver Moon, S.No. 1/2A/2, Bavdhan Kh., Pune 411 021, India; E-mail: biome1@vsnl.net.

graphs of the species are from Nepal (Schleich and Kästle, 2002; Whitaker and Captain, 2004). The place of collection is part of Kaliya Sot dam backwaters and is located south of Bhopal city, about 7 km from the city center. The snake was found in fishing nets put in shallow water at about 0.5 m depth. The surrounding area is open scrub with several rocky boulders along the waterbody. It is locally used for grazing and small scale fishing by the local populace. Fishermen reported that this species is often found in fishing nets in the Bhoj wetland, a large lake near Bhopal city. The photograph of the snake was shown to snake rescuers and snake charmers around the Bhopal area. However they could not identify it and had never seen it. Thus, it appears that the snake does not ever enter areas with heavy human activity. The behavior of the snake was observed at the time of release and was also noted from previous experience of the fishermen. The snake is slow moving on land but fast in water. It is very aggressive, actively

Fig. 1

160 lunging and biting and hence is commonly killed if trapped in fishing nets. After the release, fishermen caught two more specimens within a month’s time from the same locality. A specimen of E. sieboldii has been previously collected only once from Madhya Pradesh (formerly included in the “Central Provinces”) by Dreckmann (1886) from Saugor (now spelt as Sagar), which was identified by Murray (1886). Sagar is situated about 150 km north of Bhopal. Smith (1943) and Sharma (2003) report it from localities in Uttar Pradesh, Delhi, Bihar, Madhya Pradesh, Maharashtra, Karnataka, Rajasthan, West Bengal, Assam and Kerala. Whitaker and Captain (2004) have added Uttaranchal (now named Uttarakhand) and Nagaland in its distribution but have mentioned that old records from Maharashtra (Mumbai) and Kerala need confirmation. Outside India, it is known from Bangladesh, Nepal, and Myanmar. A record of this species from West Malaysia was based on a specimen deposited in the British Museum, sent by Dr. Cantor from Penang (Boulenger, 1896). However, Cantor’s locality data are doubtful in many cases (Wall, 1923; Smith, 1931; Adler, 2007). No further specimen of E. sieboldii was collected since eastward from Myanmar. Smith (1943) did not list Malaysia for the distribution of this species and also Wall (1924) discredited Cantor’s record. During the CAMP assessment (1998) E. sieboldii has been included in the category of lower risk — near threatened based upon the available data. Wall (1898) had reported the aggressive behavior of this species based on live specimens collected from Jumna in Delhi. Apart from these reports, very little is known about the biology of the species such as feeding, behavior, population size and ecology. In view of this the species should actually be included as data deficient. The true status of the species in the wild can only be judged after an extensive survey of the potential habitats including stagnant as well as running water bodies. Local fishermen can provide reliable data on this based upon the accidental trapping records. This snake is misidentified in some cases as a juvenile Rock Python based on the coloration. It can however be easily distinguished from the python by having nostrils on the upper surface of the head and the absence of labial pits. The coloration of E. sieboldii is very distinct and cannot be confused with any other Enhydris species in India. It is hoped that the present note and photograph will be useful for researchers to identify this snake in the

Sanjay Thakur and Aparna Watve field and add to the present knowledge of this poorly known species. Acknowledgment. We thank Mr. Ashok Captain for reviewing the manuscript and giving important suggestions. We gratefully acknowledge the help from the reviewers of MS especially regarding the distribution data.

REFERENCES Adler K. (ed.) (2007), Contributions to the History of Herpetology. Vol. 2, Contributions to Herpetology. Vol. 21, Soc. for the Study of Amphibians and Reptiles, St. Louis. Anonymous (1998), CAMP Report: Reptiles of India, Zoo Outreach Organization and CBSG, Coimbatore (India). Boulenger G. A. (1896), Catalogue of the Snakes in the British Museum (Natural History). Vol. III. Colubridae (Opisthoglyphae and Proteroglyphae), Amblycephalidae, and Viperidae, British Museum (Natural History), London. Dreckmann F. (1886), “Note on an undescribed Homalopsida,” J. Bombay Nat. Hist. Soc., 1(1), 20 – 21. Gyi K. K. (1970), “A revision of Colubrid snakes of the subfamily Homalopsinae,” Univ. Kansas Publ. Mus. Nat. Hist. Lawrence, 20(2), 47 – 233. Murphy J. C. (2007), Homalopsid Snakes, Evolution in the Mud, Krieger Publ. Co., Malabar (FL). Murray J. A. (1886), “Note on the Homalopsidae in the Society’s collection,” J. Bombay Nat. Hist. Soc., 1(1), 219. Parameswaran K. N. (1954), “On the viviparous habit oft the fresh-water snake, Enhydris dussumieri,” Curr. Sci., 1, 27 – 28. Schleich H. H. and Kästle W. (2002), Amphibians and Reptiles of Nepal, A. R. G. Gantner Verlag KG, Ruggell. Sharma R. C. (2003), Indian Snakes. Handbook, Zoological Survey of India, Kolkata. Smith M. A. (1931), The fauna of British India, including Ceylon and Burma. Reptilia and Amphibia. Vol. I. Loricata. Testudines, Taylor and Francis, London. Smith M. A. (1943), The Fauna of British India, Ceylon and Burma, including the whole of the Indo-chinese sub-region. Reptilia and Amphibia. 3. Serpentes, London (reprinted 1961 and 1981). Wall F. (1898), “Notes on two specimens of Hypsirhina sieboldii,” J. Bombay Nat. Hist. Soc., 11, 732 – 734. Wall F. (1923), “A hand-list of the snakes of the Indian Empire,” J. Bombay Nat. Hist. Soc., 29(2), 345 – 361. Wall F. (1924), “A hand-list of the snakes of the Indian Empire. Part III,” J. Bombay Nat. Hist. Soc., 29(4), 864 – 878. Whitaker R. and Captain A. (2004), Snakes of India. The Field Guide, Draco Books, Chennai.

Russian Journal of Herpetology

Vol. 16, No. 2, 2009, pp. 161 – 162

ON THE NOMENCLATURAL STATUS OF Agama cristata MOCQUARD, 1905: A REPLY TO A. BARABANOV (2008) Philipp Wagner1 and Wolfgang Böhme1 Submitted June 12, 2008. Keywords: Squamata, Agamidae, Iguanidae sensu lato (Corythophanidae), Agama cristata, Agama maria nom. nov., synonymy.

In a recent note, Barabanov (2008) proposed to replace the species name of the West African agamid lizard Agama cristata Mocquard, 1905 by Agama maria nom. nov., because of primary homonymy of the former with Agama cristata Merrem, 1820 which is the original name of the Central American iguanid (sensu lato) Corythophanes cristatus. Merrem’s (1820) species was erroneously said to originate from Ceylon (= Sri Lanka) but its type locality was later restricted to Orizaba, Veracruz, Mexico by Smith and Taylor (1950). Agama cristata Merrem, 1820 was designated to be the type species of the genus Corythophanes Boie, 1827, which is now an American iguanid [or, according to Frost and Etheridge (1989), corythophanid] genus. This genus was first mentioned by Boie (in Schlegel, 1827:235) in 1826 with Agama cristata Merrem, 1820 as type species, but only later described in more detail (Boie, 1827). At first glance, Barabanov (2008) was correct to replace a junior primary homonym by a new name, according to Art. 57.2 and 60.1 of the Code (ICZN, 1999). There are, however, two problems which in our view invalidate his action: 1. According to Art. 60.1, a new name can only be given for a primary junior homonym when no other synonym is available. Here, Agama insularis Chabanaud, 1918, described as an endemic species of some minute offshore islands of Guinea, West Africa, would be available to replace the name of the Guinean mainland species A. cristata. The supposed synonymy of the latter with A. sankaranica Chabanaud, 1918 is only based on unfounded assumptions (see the references cited in Barabanov 2008) and certainly not justified. Our current studies on African Agama species will soon resolve these problems (Wagner et al., in preparation). 1

Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, D-53113 Bonn, Germany; E-mail: philipp.wagner.zfmk@unibonn.de, w.boehme.zfmk@uni-bonn.de

2. Art. 57.2 refers also to Art. 23.9. 5 which states that if “an author discovers that a species group name in use is a primary junior homonym of another species group name also in use, but the names apply to taxa not considered congeneric after 1899, the author must not automatically replace the junior homonym; the case should be referred to the Commission for a ruling under the plenary power and meanwhile prevailing usage of both names is to be maintained.” Because “Agama” cristata Merrem, 1820 was transferred to the iguanid genus Corythophanes already by Boie (1827), an action followed by Duméril and Bibron 1837 and Boulenger, 1885 [and more recently also by Smith and Taylor (1950) and Ortleb and Heatwole 1965]. Therefore, both nominal species were in different genera (and even in different families) already seven decades before 1899 and the same is true until today. A similar case in the same genus is the problem on the nomenclatural status of Agama armata Peters, 1855, which is a junior primary homonym of Agama armata Hardwicke and Gray, 1827, now known as Acanthosaura armata (Agamidae). Wermuth (1967) also proposed Agama mertensi as a nom. nov. for the former taxon but also failed to refer the case to the Commission because both species were not considered as congeneric since 1837. Therefore, like in the above mentioned case, Agama armata Peters, 1855 is still valid. From the above arguments we conclude that the creation of Agama maria Barabanov, 2008 to replace Agama cristata Mocquard, 1905 was premature as he did not consider A. insularis as a possible junior available synonymous replacement name, and as he finally did not refer his case to the Commission. Consequently, Agama cristata Mocquard, 1905 should stay in use for this species until the taxonomic problems will be resolved or the Commission will be approached with this case.

162 Acknowledgments. We thank Andreas Schmitz (Genève, Switzerland) and Aaron Bauer (Philadelphia, USA) for reviewing this manuscript.

REFERENCES Barabanov A. (2008), “A case of homonymy in the genus Agama (Reptilia: Sauria: Agamidae),” Russ. J. Herpetol., 15(3), 206. Boie F. (1827), “Bemerkungen über Merrem’s Versuch eines Systems der Amphibien. 1. Lieferung: Ophidier,” Isis van Oken Jena, 20, 508 – 566. Boulenger G. A. (1885), Catalogue of the Lizards in the British Museum (Natural History). Vol. 2. Second Edition, London. Duméril A. M. C. and Bibron G. (1837), Erpétologie Générale ou Histoire Naturelle Complete des Reptiles. Vol. 4, Libr. Encyclopédique Roret, Paris. Frost D. and Etheridge R. (1989), “A phylogenetic analysis and taxonomy of iguanian lizards (Reptilia: Squamata),” Univ. Kansas Mus. Nat. Hist. Misc. Publ., Issue 81. ICZN (International Commission of Zoological Nomenclature) (1999), International Code of Zoological Nomencla-

Philipp Wagner and Wolfgang Böhme ture. 4th Edition Adopted by the International Union of Biological Sciences, International Trust of Zoological Nomenclature, London. Merrem B. (1820), Versuch eines Systems der Amphibien I (Tentamen Systematis Amphibiorum), J. C. Kriegeri, Marburg. Schlegel H. (1827), “Notice sur l’Erpétologie de l’île de Java; Par M. Boïé,” Bull. Sci. Nat. Géol., 9, 233 – 240. Smith H. M. and Taylor E. H. (1950), “An annotated checklist and key to the reptiles of Mexico exclusive of the snakes,” U.S. Natl. Mus. Bull., 199, 1 – 253. Ortleb E. and Heatwole H. (1965), “Comments on some Panamanian lizards with a key to the species from Barro Colorado Island, C. Z. and vicinity,” Caribbean J. Sci., 5(3 – 4), 141 – 147. Wagner P., Ineich I., Leaché A., Wilms T., Trape S., Mane Y., Böhme W., and Schmitz A. (in preparation), “Studies on African Agama. V. On the status of Agama insularis Chabanaud, 1918 and Agama cristata Mocquard, 1905 (Sauria: Agamidae).” Wermuth H. (1967), “Liste der rezenten Amphibien und Reptilien. Agamidae,” Das Tierreich, 86.