bystropogon

Molecular Ecology (2005) 14, 1177–1189

doi: 10.1111/j.1365-294X.2005.02487.x

Molecular phylogenetics of the Macaronesian-endemic genus Bystropogon (Lamiaceae): palaeo-islands, ecological shifts and interisland colonizations

Blackwell Publishing, Ltd.

J E N N I F E R L . T R U S T Y ,*¶ R I C H A R D G . O L M S T E A D ,† A R N O L D O S A N T O S - G U E R R A ,‡ S U S A N A S Á - F O N T I N H A ,§ and J A V I E R F R A N C I S C O - O R T E G A * *Department of Biological Sciences, Florida International University, University Park Campus, Miami, FL 33199, USA †Department of Biology, University of Washington, PO Box 355325, Seattle, WA 98195, USA, ‡Jardín de Aclimatación de La Orotava, Calle Retama Num. 2, Puerto de La Cruz, E-38400, Tenerife, Canary Islands, Spain, §Parque Natural da Madeira/CEM, Caminho do Meio, Bom Sucesso, PT-9050-251, Funchal, Madeira, Portugal, ¶Fairchild Tropical Botanic Garden, 11935 Old Cutler Road, Coral Gables, FL 33156, USA

Abstract A molecular phylogenetic study of Bystropogon L’Hèr. (Lamiaceae) is presented. We performed a cladistic analysis of nucleotide sequences of the internal transcribed spacers (ITS), of the nuclear ribosomal DNA, and of the trnL gene and trnL-trnF intergenic spacer of the chloroplast DNA. Bystropogon odoratissimus is the only species endemic to the Canary Islands that occurs in the three palaeo-islands of Tenerife. This species is not part of an early diverging lineage of Bystropogon and we suggest that it has a recent origin. This phylogenetic pattern is followed by most of the species endemic to the palaeo-islands of Tenerife. The two sections currently recognized in Bystropogon form two monophyletic groups. Taxa belonging to the section Bystropogon clade show interisland colonization limited to the Canary Islands with ecological shifts among three ecological zones. Taxa from the section Canariense clade show interisland colonization both within the Canary Islands and between the Canary Islands and Madeira. Speciation events within this clade are mostly limited to the laurel forest. The genus has followed a colonization route from the Canaries towards Madeira. This route has also been followed by at least five other plant genera with species endemic to Macaronesia. Major incongruences were found between the current infrasectional classification and the molecular phylogeny, because the varieties of Bystropogon origanifolius and Bystropogon canariensis do not form two monophyletic groups. The widespread B. origanifolius appears as progenitor of the other species in section Bystropogon with a more restricted distribution.

Keywords: adaptive radiation, biogeography, evolution, molecular phylogenetics, oceanic islands, quantum speciation Received 20 October 2004; revision received 6 January 2005; accepted 6 January 2005

Introduction The Macaronesian Islands comprise the Atlantic archipelagos of the Azores, Madeira, Selvagens, Canaries, and Cape Verde. In the last 10 years, many evolutionary biology studies have focused on this region, and these island systems have played an important role in the understanding of Correspondence: Javier Francisco-Ortega, Fax: 1305 3481986; E-mail: ortegaj@fiu.edu © 2005 Blackwell Publishing Ltd

plant speciation processes in archipelagos worldwide (Baldwin et al. 1998; Juan et al. 2000; Emerson 2002; Silvertown 2004; Valido et al. 2004). Research into the interisland relationships of Macaronesian endemics gives insight into the relative roles of dispersal and ecological adaptation as evolutionary avenues (Francisco-Ortega et al. 1996, 2002; Panero et al. 1999). However, none of these studies have focused on interpreting phylogenetic patterns in the framework of the geological history of Macaronesia. The island of Tenerife (Canary Islands) has a complex geological history

1178 J . L . T R U S T Y E T A L .

Fig. 1 Geographical distribution of Bystropogon in the Canary and the Madeira Islands.

The palaeo-islands of Adeje, Anaga, and Teno are indicated by hatched shading.

and is a good model system to investigate these kind of questions. Three of the mountain systems of this island, Anaga, Adeje, and Teno, are considered ‘palaeo-islands’ (Ancochea et al. 1990; Marrero & Francisco-Ortega 2001; Marrero 2004; Fig. 1). These regions are estimated to be between 4 (Anaga) and 12 (Adeje) million years (Myr) old; Teno is approximately 6 Myr old (Thirlwall et al.

2000; Guillou et al. 2004). Volcanic activity during the late Tertiary and early Quaternary led to the merger of these three palaeo-islands, which eventually formed the current island of Tenerife approximately 1 million years ago (Ma) (Ancochea et al. 1990). These three regions have a high number of endemics (Bramwell & Bramwell 1974; SantosGuerra & Fernández-Galván 1983; Martín et al. 1999), and some authors have suggested that they have some of the oldest plant lineages of the archipelago (reviewed by Marrero & Francisco-Ortega 2001; Marrero 2004).

The Macaronesian-endemic genus Bystropogon L’Hèr.

(Lamiaceae) presents an opportunity to address the role played by the geologically old island areas in the diversification of island endemics. Bystropogon has seven species and four varieties (La Serna-Ramos 1984; Fig. 1). Two of the species are restricted to the island of Madeira (Bystropogon maderensis and Bystropogon punctatus), while the rest of the species are endemic to the Canary Islands. Among the Canary Islands, only Tenerife and La Palma harbour single-island endemics of this genus. Bystropogon odoratissimus and Bystropogon plumosus are restricted to Tenerife, whereas Bystropogon wildpretii is endemic to La Palma. All the species of Bystropogon are abundant with the exception of B. odoratissimus and B. wildpretti.

Bystropogon odoratissimus is restricted to the three palaeoislands of Tenerife. This species has a disjunct distribution and is found only in Teno, Anaga, and Adeje. This unique © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

B Y S T R O P O G O N P H Y L O G E N Y 1179 biogeographical pattern is not known for any other plant species of Tenerife and has led some authors to consider B. odoratissimus to be part of an ancient lineage of the genus (Hernández & García 1996).

Two widespread Bystropogon species, B. canariensis and B. origanifolius, are highly polymorphic and form two species complexes. Bystropogon origanifolius has three varieties restricted to Gran Canaria (var. canariae), El Hierro (var. ferrensis), and La Palma (var. palmensis). Bystropogon origanifolius var. origanifolius is endemic to the islands of La Gomera and Tenerife. Bystropogon canariensis has two varieties. The type variety canariensis occurs on the islands of Gran Canaria, La Palma, and Tenerife, while B. canariensis var. smithianus occurs on four islands, La Gomera, El Hierro, La Palma, and Tenerife.

The latest taxonomic revision of Bystropogon produced

by La Serna-Ramos (1984) recognized two sections: Bystropogon section Bystropogon and Bystropogon section Canariense La Serna. Bystropogon canariensis and the two Madeiran species, B. maderensis and B. punctatus, were placed in Bystropogon section Canariense. The remaining four species comprise Bystropogon section Bystropogon.

The three species of Bystropogon section Canariense are primarily restricted to the laurel forest ecological zone (Pruno hixae-Lauretea novocanariensis) (La Serna-Ramos 1984; Capelo et al. 1999; Jardim & Francisco 2000). This vegetation zone is found on the northern slopes of the islands which are under the direct influence of the humid and cool northeastern trade winds and receive approximately 1000 mm of average rainfall per year (Fernández-Palacios 1999).

Two species of Bystropogon section Bystropogon (i.e. B. origanifolius and B. wildpretii) are confined mostly to the pine forest ecological zone (Chamaecytiso-Pinetea canariensis) (La Serna-Ramos 1984). This ecological zone only occurs in the Canary Islands and is situated above the laurel forest (on the northern slopes of the islands) or above the lowland scrub (on the southern slopes). Rainfall in this forest ranges between 400 and 800 mm per year (Fernández-Palacios 1999).

The two other species of this section, B. odoratissimus and B. plumosus, are primarily found in the lowland scrub (Rhamno crenulatae-Oleetea cerasiformis) (La Serna-Ramos 1984; Hernández & García 1996). The lowland scrub is situated above the coastal xerophytic belt (Kleinio-Euphorbietea canariensis) and receives up to 550 m of rainfall per year (Fernández-Palacios 1999).

Bystropogon origanifolius var. origanifolius has the widest ecological amplitude of any of Bystropogon species. This taxon is reported in the few natural pine forests of La Gomera (Fernández-Galván 1983; Del Arco-Aguilar et al.

1990); however, it is much more abundant in the lowland scrub vegetation zone and in sunny areas of the laurel forest of this island (La Serna-Ramos 1984). B. origanifolius var. origanifolius has a different ecology in Tenerife, where it is © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

an extremely common understory species in the pine forest belt. Occasionally, this species can be found in the lowland scrub and laurel forests areas of Tenerife.

In this study we present a molecular phylogenetic study of the genus Bystropogon based on nucleotide sequences of the nuclear and chloroplast genomes. The three major objectives of our study were (i) to determine if B. odoratissimus is part of a early diverging lineage of Bystropogon and investigate the biogeographical role played by the three palaeo-islands of Tenerife; (ii) to discuss the main ecological and geographical avenues followed by the genus during its evolutionary history in the context of other Macaronesian-endemic groups; and (iii) to determine if the molecular phylogeny of the genus is concordant with the latest taxonomic classification of Bystropogon.

Materials and methods Plant materials The ingroup consisted of 17 taxa representing all the species and varieties of Bystropogon in Macaronesia from the two sections currently recognized by La Serna-Ramos (1984).

When a species was found on multiple islands, a single representative from each island was included in the analysis.

Mentha L., Minthostachys (Benth.) Spach, Pycnanthemum Michx., and Ziziphora L. were chosen as the outgroups. The outgroup was selected based on a previous phylogenetic study of the tribe Mentheae (Trusty et al. 2004). Details of the plant material, including voucher information, accession provenance, and nucleotide sequence identification, are listed in Table 1.

DNA extraction, PCR amplification, and sequencing DNA was extracted from either fresh or silica-gel-dried material using the DNeasy protocol (QIAGEN). Both strands of the two nuclear ribosomal DNA internal transcribed spacer (ITS) regions including the 5.8S gene were amplified using primers ITS4 (White et al. 1990) and ITS5 (Downie & Katz-Downie 1996). Polymerase chain reaction (PCR) amplification conditions are described in Kim & Jansen (1994). Difficulty in amplifying the ITS region of some taxa was ameliorated through the use of the Ready-To-Go PCR Bead Kit (Amersham/Pharmacia Biotech).

The chloroplast trnL gene and trnL-trnF intergenic spacer (trnL/F) were amplified using the ‘C’ and ‘F’ primers according to the protocol described by Taberlet et al. (1991).

PCR products were cleaned using the QIAquick silica columns (QIAGEN) following the manufacturer’s instructions. The purified PCR products were cycle-sequenced in both directions using the ABI Prism BigDye, Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Applied

1180 J . L . T R U S T Y E T A L .

Table 1 List of plant material used in this study GenBank Accession nos ITS/trnL-F

Taxon

Island

Voucher

Bystropogon canariensis (L.) L′ Hèr. var. canariensis

Gran Canaria La Palma Tenerife La Gomera El Hierro La Palma Tenerife Madeira Tenerife Tenerife La Gomera Gran Canaria

AY04590/AY704605 AY04583/AY704607 AY506634/AY506597 ------------/AY704603 AY704592/AY704595 AY704593/AY704606 AY706475/AY704594 AY506633/AY506596 AY704589/AY704596 AY506635/AY506598 AY704591/AY704604 AY704587/AY704600

El Hierro

Los Marteles; Marrero, Caujapé & Francisco-Ortega (LPA) Sobre Tagoja; Santos-Guerra 6–1999 (ORT) Pedro Alvarez; Santos-Guerra 7-6-1999 (ORT) Chorros de Epina; Santos-Guerra 10-3-2000 (ORT) El Golfo; Santos-Guerra 8-2-2000 (ORT) Breña Baja, Santos-Guerra 6-10-2002 (ORT) Miradores de la Cumbre; Santos-Guerra 8-8-1999 (ORT) Ribeiro Frio; Fontinha and Roberto s. n. (MADJ) Punta Hidalgo, Tenerife; Santos-Guerra 6-20-1999 (ORT) Montaña de Los Poleos; Santos-Guerra 8-6-1999 (ORT) Vallehermoso; Santos-Guerra 5-3-1997 (ORT) Riscos de Tamadaba; Marrero, Caujapé and Francisco-Ortega (LPA) Isora; Santos Guerra 8-2-2000 (ORT)

La Palma

Roque de los Muchachos; Santos Guerra 7–1999 (ORT)

AY704588/AY704599

Tenerife Madeira La Palma

Articosa; Santos-Guerra 8-31-2000 (ORT) Folhadal; Fontinha and Roberto s. n. (MADJ) Tijarafe; Santos-Guerra 6–1999 (ORT)

AY704586/AY704598 AY704582/AY704602 AY704584/AY704601

Wagstaff 88–026 (BHO) Tulcan, Ecuador; Thompson and Rawlins 942 (BHO)

AY506645/AY506610 AY506638/AY506601

Bloomington, Indiana; Olmstead 90–06 (WTU)

AY506640/AY506604

Madrid, Spain; Sánchez-Mata and Gavilán 100 (MSC)

AF369166/AY506595

Bystropogon canariensis (L.) L′ Hèr. var. smithianus Christ

Bystropogon maderensis Webb Bystropogon odoratissimus C. Bolle Bystropogon origanifolius L′ Hèr. var. origanifolius Bystropogon origanifolius L′ Hèr. var. canariae I. La-Serna Bystropogon origanifolius L′ Hèr. var. ferrensis (Ceb. and Ort.) I. La-Serna Bystropogon origanifolius L′ Hèr. var. palmensis L′ Hèr.

Bystropogon plumosus (L. f.) L′ Hèr.

Bystropogon punctatus L′ Hèr.

Bystropogon wildpretii I. La-Serna Outgroup species: Mentha rotundifolia (L.) Huds.

Minthostachys mollis (Kunth) Griseb.

Pycnanthemum incanum (L.).

Michx.

Ziziphora hispanica L.

AY704585/AY704597

Herbaria (Holmgren et al. 1990; www.nybg.org/bsci/ih/ih.html) where vouchers are deposited are indicated in brackets.

Biosystems Division) with AmpliTaq DNA polymerase. The sequencing reactions were conducted using the same primers that were used for the PCR amplifications.

Dye-terminator reactions were carried out in 10-µL reactions, diluted 50:50 using AmpliTaq FS buffer (Perkin-Elmer, Applied Biosystems Division) and amplified according to manufacturer’s protocol. Cycle sequencing products were separated on an ABI 377 automated sequencer at the Florida International University (FIU) DNA Core Facility.

MFOLD

analysis

Thermodynamic stability of the ITS sequences were analysed using mfold version 3.1 (Zuker 2003). The parameters were set with the folding temperature of 37 °C and the sodium concentration of 1 m. Comparisons of free energy estimates (dG values) among ITS sequences allowed us to identify putative nonfunctional paralogue copies (pseudogenes) which were consequently excluded from the phylogenetic analyses.

Data analysis Sequences were assembled, edited, and aligned using sequencher 4.1 (Genecodes) and clustal x (Thompson et al. 1997), respectively. Minor adjustments were made manually to the final alignment using se-al version 2a08 (A. Rambaut, University of Oxford, UK). Fewer than 0.1% of data matrix cells were scored as missing. Gaps were coded as binary characters following the ‘simple indel coding’ procedure of Simmons & Ochoterena (2000). The aligned data matrices are deposited in the treebase database (Accession no. SN1952).

Heuristic phylogenetic parsimony analyses were performed using paup* (Swofford 2002). Heuristic searches with 1000 random taxon addition replicates (using the TBR and MulPars options of paup*) to look for multiple optimal tree islands (Maddison 1991). Phylogenetic support for each clade was evaluated through bootstrap analysis (Felsenstein 1985) of 1000 replicates with one random sequence addition per replicate and the TBR and MulPars © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

B Y S T R O P O G O N P H Y L O G E N Y 1181 options (DeBry & Olmstead 2000). In the trnL/F bootstrap analysis, the protocol was modified to save only 20 trees per replicate because of computer memory limitations.

Results ITS phylogenetic analyses The ITS sequence from Bystropogon canariensis var. smithianus from La Gomera had an unusually high number of changes when compared with the other sequences. In addition, alignment of this sequence with the rest of the sequences was difficult to achieve. Uncorrected ‘p ’ distances (Swofford 2002) were determined to estimate levels of nucleotide divergence among sequences. Distance p values between outgroup and ingroup varied between 0.06 and 0.09 for sequences with dG values lower than −50. The unusual sequence of B. canariensis var. smithianus from La Gomera had dG values of −35.9 (for the ITS1 region) and −28.1 (for the ITS2 region). The sequence diverged from the two other complete ITS Bystropogon sequences of the same variety by 0.13 and 0.17. Pairwise P values within the ingroup (excluding this sequence from La Gomera) varied between 0.02 and 0.07. This unusual ITS sequence was therefore identified as a putative nonfunctional paralogue and was subsequently excluded from the phylogenetic analysis. We were able to successfully sequence only the ITS2 region for B. canariensis var. smithianus from La Palma. The dG value (−64.7) for this ITS2 sequence indicated that it belonged to a functional copy. This ITS2 sequence was included in the phylogenetic analyses.

The final aligned ITS matrix was 648 nucleotide positions in length and included only one informative coded gap.

There were 140 variable characters, with 39% (55) of these being parsimony informative. The search yielded nine equally most-parsimonious trees [length = 184 steps; consistency index (CI) = 0.859; retention index (RI) = 0.865].

One of the nine most-parsimonious trees (equivalent to the strict consensus tree) is shown in Fig. 2. When the phylogenetic analysis was conducted using only the ITS2 data, 21 trees were found. The strict consensus of the ITS2 tree was consistent with the strict consensus of the complete ITS, except for reduced resolution within the two major clades.

The ITS strict consensus tree supported a basal split of Bystropogon into two major clades (Fig. 2). One of these major clades (98% bootstrap value) contained all the members of Bystropogon section Canariense. B. canariensis var. smithianus from La Palma was sister to the rest of this clade. This basal split was also found in the ITS2 topology.

The rest of the taxa of this section formed a monophyletic assemblage weakly supported by a bootstrap value of 69%.

The second major clade (99% bootstrap value) contained all the members of Bystropogon section Bystropogon. Two major lineages were identified at the base of this second major © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

Fig. 2 Nuclear and chloroplast DNA phylogenies of Bystropogon. (a) One of the nine most parsimonious trees from the ITS analysis (184 steps; CI = 0.859; RI = 0.865). This tree was identical to the strict consensus tree. (b) One of the 28 most-parsimonious trees from the trnL/F analysis analysis [68 steps; CI = 0.897; RI = 0.829).

Branches which collapse in the strict consensus tree are indicated by dashed lines. Bootstrap values are below branches. Branch lengths are above branches.

clade. One was moderately supported with a 72% bootstrap value and contains Bystropogon wildpretti, Bystropogon origanifolius var. ferrensis and Bystropogon origanifolius var. palmensis. The second was strongly supported (95%) and contains the rest of the taxa of the section, including the palaeo-island-endemic Bystropogon odoratissimus.

trnL/F phylogenetic analyses The final aligned matrix used for parsimony analyses of trnL/F was of 832 DNA characters in length and included five parsimony informative gaps. There were 60 variable characters, with 30% (18) of these being parsimony informative. Parsimony analysis yielded 28 most-parsimonious trees (length = 68 steps each; CI = 0.897; RI = 0.829). One of these 28 trees and the strict consensus tree are shown in Fig. 2.

The trnL/F strict consensus tree identified three major clades. One of these clades (68% bootstrap value) comprised the outgroup genera Minthostachys and Pycnanthemum.

1182 J . L . T R U S T Y E T A L .

Fig. 3 One of the 48 most-parsimonious trees from the combined analyses of ITS and trnL/F data (257 steps; CI = 0.852; RI = 0.837). Branches which collapse in the strict consensus tree are indicated by dashed lines. Bootstrap values are below branches.

Branch lengths are above branches. Open circles indicate taxa found in the pine forest, closed circles indicate taxa found to the laurel forest, and closed squares indicate taxa found in the lowland scrub.

Another clade consisted of Bystropogon section Canariense; this clade had low bootstrap support below 50%. The third major clade comprised all of the members of Bystropogon section Bystropogon (61% bootstrap value). Two major lineages were distinguished in this clade, the first lineage had only the Gran Canaria endemic B. origanifolius var. canariae.

The remaining six taxa of Bystropogon sect. Bystropogon, including B. odoratissimus, formed the second lineage with no resolution among them.

Combined analyses The final aligned matrix obtained from the combination of ITS and trnL/F data was 1480 positions in length and had six phylogenetically informative gaps. This matrix did

not include B. canariensis var. smithianus from La Gomera because we were unable to obtain ITS sequences for this accession. There were 190 variable characters, with 38% (73) of these being parsimony informative. Forty-eight equally most-parsimonious trees were obtained (length = 257 steps, CI = 0.852; RI = 0.837). One of the most-parsimonious trees and the strict consensus of all trees are shown in Fig. 3. The combined ITS – trnL/F strict consensus tree also showed the two major clades obtained after the phylogenetic analyses of ITS data.

The five Canary Islands where multiple taxa of Bystropogon occur have at least one taxon from each of the two major clades of the genus. Unfortunately we could not verify this result for the island of La Gomera, because functional ITS sequences of B. canariensis var. smithianus from this island © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

B Y S T R O P O G O N P H Y L O G E N Y 1183 were not available for the combined data set. However, based on the chloroplast DNA (cpDNA) phylogeny, this accession is part of the section Canariense clade. The clade comprising section Bystropogon does not have any species on the island of Madeira.

The clade comprising section Canariense was mostly limited to the laurel forest, and only one ecological shift was identified within this clade. In contrast section Bystropogon shows ecological shifts among three ecological zones: the laurel forest, the lowland scrub, and the pine forest.

The current species-level classification of B. origanifolius and B. canariensis is not supported in the combined analyses.

The four varieties of B. origanifolius did not form a monophyletic group. Varieties canariae and origanifolius formed a clade with B. odoratissimus and B. plumosus. The other varieties of B. origanifolius (i.e. var. ferrensis and var. palmensis) grouped with B. wildpretii. Together, the two varities of Bystropogon canariensis formed a paraphyletic group with respect to B. maderensis and B. punctatus.

Discussion The phylogenetic position of Bystropogon odoratissimus — phylogenetic patterns of plant species occurring in the three palaeo-islands of Tenerife Approximately 55 species are endemic to at least one of the three palaeo-islands of Tenerife (Sventenius 1948; SantosGuerra & Fernández-Galván 1983; Gómez-Campo 1996; Hernández & Bañares 1996; Beltrán-Tejera et al. 1999; Del Arco-Aguilar 2000; Varios Autores 2000). This number of endemic species represents approximately 10% of the flora endemic to the Canary Islands. Thirty-seven of these species have been included in molecular phylogenetic studies (Table 2), therefore there are phylogenies available for almost 67% of these palaeo-island endemics.

Although Bystropogon odoratissimus is restricted to the three palaeo-islands of Tenerife, our phylogenetic study does not support B. odoratissimus as an early diverging lineage of Bystropogon. This species is nested inside the section Bystropogon clade in a late branching position. The phylogenetic patterns of B. odoratissimus are not unique and 84% of the endemic species from the palaeo-islands follow similar patterns (Table 2).

Species restricted to Anaga/Adeje/Teno from the genera Aeonium Webb and Berthel., Argyranthemum Sch. Bip., Cheirolophus Cass., Crambe L., Echium L., Lotus L., Lugoa DC., Monanthes Haw., Sideritis L., Teline Medicus, and Tolpis Adanson are part of crown groups in their respective phylogenies, although in many cases the relevant nodes were supported by bootstrap values below 75%.

In contrast, species from only three genera, Lavatera L., the woody Sonchus alliance, and Vierea Webb and Berthel. are part of early diverging lineages. Lavatera has two © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

species endemic to Macaronesia. A phylogenetic study of this genus shows that the relatively common Canarian endemic L. acerifolia Cav. belongs to one of the crown groups of the Lavatera–Malva L. complex (Fuertes-Aguilar et al.

2002). The palaeo-island endemic Lavatera phoenicea is sister to the rest of the members of the complex.

The woody Sonchus alliance is the only taxonomic group with more than 20 species endemic to Macaronesia that has an early diverging species restricted to one of the palaeoislands of Tenerife. The ITS phylogeny of this alliance shows that the Teno-endemic S. tuberifer is part of an early diverging lineage of the woody Sonchus alliance. However most of the nodes of this phylogenetic tree are weakly supported with bootstrap values between 50% and 60% (Kim et al. 1996).

Finally, a molecular phylogenetic study of tribe Inuleae shows that Vierea and the North African genus Perralderia Coss. (three species) form an assemblage that is sister to a large clade of 10 genera with a predominantly Mediterranean distribution, however, this relationship is weakly supported (Francisco-Ortega et al. 2001b).

There are at least seven plant genera with species restricted to the old island of La Gomera and at least to one of the three palaeo-islands of Tenerife (Sventenius 1948; SantosGuerra and Fernández-Galván 1983; Gómez-Campo 1996; Hernández & Bañares 1996; Beltrán-Tejera et al. 1999; Del Arco-Aguilar 2000; Varios Autores 2000). La Gomera is approximately 9 Myr old (Guillou et al. 2004) and together with the three palaeo-islands of Tenerife formed a small archipelago located in the westernmost Canary Islands until the early Quaternary (Marrero & Francisco-Ortega 2001; Marrero 2004). It was only then that submarine volcanism lead to the formation of the islands of La Palma and El Hierro. Three of the species restricted to La Gomera and the palaeo-islands of Tenerife (i.e. Convolvulus volubilis, Dicheranthus plocamoides, Teline pallida) have been the subject of molecular phylogenetic studies (Table 2). Both D. plocamoides and T. pallida follow the pattern detected for most of the palaeo-island endemics and are part of terminal tips of their respective phylogenies (Oxelman et al. 2002; Percy & Cronk 2002; Smissen et al. 2002).

Convolvulus volubilis has a unique biogeographical pattern, in that it is the only Canarian species endemic to Adeje, Anaga, La Gomera, and Teno. This species represents the first branching lineage of endemic climbing species of Convolvulus and therefore is another example of an early diverging taxon with a palaeo-island (including La Gomera) distribution.

The paucity of ancient, or early diverging groups (16%, Table 2) restricted to the palaeo-islands of Tenerife supports the view that several of the speciation events in the Canary Islands are relatively recent and that most of the species which are endemic to these palaeo-islands are not the result of ancient phylogenetic splits. Indeed, most of these

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Table 2 Molecular phylogenetic studies of species endemic in the palaeo-islands of Adeje, Anaga, or Teno Geographical distribution Species

Adeje

Aeonium ciliatum Webb and Berthel. (Crassulaceae) Aeonium haworthii (Webb and Berthel.) Webb and Berthel. (Crassulaceae) Aeonium mascaense Bramwell (Crassulaceae) Aeonium pseudourbicum Bañares (Crassulaceae) Aeonium volkeri H. Hernández and Bañares (Crassulaceae) Argyranthemum coronopifolium (Willd.) Humphries (Asteraceae) Argyranthemum foeniculaceum (Willd.) Sch. Bip. (Asteraceae) Argyranthemum lemsii Humphries (Asteraceae) Argyranthemum sundingii L. Borgen (Asteraceae) Bystropogon odoratissimus Bolle (Lamiaceae) Cheirolophus burchardii Susanna (Asteraceae) Cheirolophus canariensis (Willd.) Holub (Asteraceae) Cheirolophus tagananensis (Svent.) Holub (Asteraceae) Convolvulus volubilis Link (Convolvulaceae) Crambe laevigata Christ (Brassicaceae) Dicheranthus plocamoides Webb (Caryophyllaceae) Echium leuchophaeum Sprague and Hutch. (Boraginaceae) Echium simplex DC. (Boraginaceae) Lavatera phoenicea Vent. (Malvaceae) Hypochaeris oligocephala (Bramwell) Lack (Asteraceae) Lotus dumetorum Murr. (Fabaceae) Lotus mascaensis Burch. (Fabaceae) Lugoa revoluta DC. (Asteraceae) Monanthes anagensis Praeger (Crassulaceae) Monanthes minima (Bolle) Christ (Crassulaceae) Sideritis brevicaulis Mend.-Heuer (Lamiaceae) Sideritis cystosiphon Svent. (Lamiaceae) Sideritis infernalis Bolle (Lamiaceae) Sideritis macrostachya Poir. (Lamiaceae) Sideritis nervosa Poir. (Lamiaceae) Sonchus fauces-orci Knoche (Asteraceae) Sonchus tuberifer Svent. (Asteraceae) Teline salsoloides Arco and Acebes (Fabaceae) Teline pallida Arco and Acebes (Fabaceae) Tolpis crassiuscula Svent. (Asteraceae) Tolpis glabrescens Kämmer. (Asteraceae) Vierea laevigata Webb (Asteraceae)

Anaga

Teno

+ + + + + +

+

+ + +

+

+ +

+ + + +

+ +

+ + + + + +

+ + + +

+ + + + + + + +

+ +

+ + + + + +

+ +

Reference for phylogenetic study Mort et al. (2002) Mort et al. (2002) Mort et al. (2002) Mort et al. (2002) Mort et al. (2002) Francisco-Ortega et al. (1996)*† Francisco-Ortega et al. (1996)*† Francisco-Ortega et al. (1996)* Francisco-Ortega et al. (1996)*† Present study Susanna et al. (1999)*† Susanna et al. (1999)*† Susanna et al. (1999)*† Carine et al. (2004)*¶ Francisco-Ortega et al. (2002)*† Oxelman et al. (2001); Smissen et al. (2002)*†¶ Böhle et al. (1996)*† Böhle et al. (1996)*† Fuertes-Aguilar et al. (2002) Cerbah et al. (1998)* Allan et al. (2004)*† Allan et al. (2004)*† Francisco-Ortega et al. (2001a)*† Mort et al. (2002) Mort et al. (2002) Barber et al. (2000)*† Barber et al. (2000)*† Barber et al. (2000)*† Barber et al. (2000)*† Barber et al. (2000)*† Kim et al. (1996)*†§ Kim et al. (1996)*† Percy & Cronk (2002)*† Percy & Cronk (2002)*¶ Moore et al. (2002)*‡ Moore et al. (2002)* Francisco-Ortega et al. (2001b)*†

Geographical distribution compiled from Sventenius (1948), Santos-Guerra & Fernández-Galván (1983), Gómez-Campo (1996), Hernández & Bañares (1996), Beltrán-Tejera et al. (1999), Del Arco-Aguilar (2000), and Varios Autores (2000). Species in bold represent early diverging lineages in phylogenetic studies. *Phylogenetic study based on DNA data from one genomic compartment only. †Phylogenies show low bootstrap support (< 75%) for relevant nodes. ‡The presence of Tolpis crassiuscula in Adeje (Gómez-Campo 1996) needs further confirmation. §The presence of Sonchus fauces-orci outside Adeje and Teno (Gómez-Campo 1996) needs further confirmation. ¶Species is also present in La Gomera.

palaeo-island endemics appear to have a relatively recent origin and are terminal tips of their respective phylogenies.

Finally, Hypochaeris oligocephala has a unique pattern, is the only species of this genus endemic to one of the palaeoislands of Tenerife and its sister species, Hypochaeris cretensis (L.) Chaub. and Bory, is distributed in the eastern Medi-

terranean (Cerbah et al. 1998). Hypochaeris provides the only known phylogenetic evidence for a floristic connection between Macaronesian and the east Mediterranean.

The unusual phylogenetic patterns detected for Hypochaeris, Lavatera, Sonchus, and Vierea suggest that the three palaeoislands of Tenerife harbour some old endemic lineages © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

B Y S T R O P O G O N P H Y L O G E N Y 1185 and also provide evidence of floristic connections between Macaronesia and distant regions of the Mediterranean.

Interisland colonizations and ecological shifts The clades formed by sections Bystropogon and Canariense differ concerning their patterns of interisland colonization and ecological shifts. Speciation events within the section Canariense clade are mostly restricted to the laurel forest.

Populations of Bystropopon canariensis var. smithianus from Tenerife are limited to the southeastern sector of this island where they are linked both to the laurel and pine forest ecological zones. This clade shows extensive interisland colonizations; indeed this is the only clade that has reached the northern archipelago of Madeira. Our data suggest that B. canariensis var. smithianus which is endemic to La Palma is the sister taxon to the rest of this clade. These results imply that Bystropogon originated in the Canary Islands and dispersed to Madeira, probably as a single colonization event. Bystropogon provides the sixth known example of a colonization route from the Canaries toward Madeira.

The other five taxa that appear to have followed this dispersal route are the Aeonium alliance (Mort et al. 2002; Fairfield et al. 2004), Convolvulus L. (Convolvulaceae) (Carine et al. 2004); Crambe (Francisco-Ortega et al. 2002), Pericallis (Panero et al. 1999), and the woody Sonchus alliance (Kim et al. 1996).

Interisland dispersal is common in the section Canariense clade, since each of the Canarian taxa are distributed on more than one island and there has been at least one dispersal to Madeira. Previous phylogenetic studies of the Macaronesian species have demonstrated that interisland colonization between similar ecological zones is one of the modes of species diversification in the Canary Islands (Francisco-Ortega et al. 2001a). However, very few studies have found that this pattern also can involve species from the Canary and Madeira archipelagos. Indeed Bystropogon and Crambe are the only known cases where sister relationships between Canarian and Madeira taxa are confined to only one ecological zone (Francisco-Ortega et al. 2002).

Crambe sventeni Bramwell & Sunding from the island of Fuerteventura is sister to the Madeira-endemic Crambe fruticosa L. f. Both species thrive in the lowland scrub of their respective islands.

In contrast, speciation events within the section Bystropogon clade are defined by ecological shifts among three different ecosystems. This clade does not have any endemic species in Madeira; and each island of the Canaries, with the exception of La Gomera, has at least one singleisland endemic taxon. The section Bystropogon clade has followed an evolutionary route in which radiation into different ecological zones seems as important as interisland colonization. This mode of plant speciation is followed by most of the Canary Island species. Good examples are the Š 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177â&#x20AC;&#x201C;1189

Aeonium alliance, Crambe, Lotus, Pericallis, Sideritis, and the woody Sonchus alliance (Kim et al. 1996; Panero et al. 1999; Barber et al. 2000; Francisco-Ortega et al. 2002; Mort et al.

2002; Allan et al. 2004). These genera follow a common pattern in which the majority of their clades show both interisland colonization and several ecological shifts.

The two different modes of speciation shown by Bystropogon are not unique in Macaronesia. A phylogenetic study of the Canarian endemic Gonospermum Less. alliance (Asteraceae) showed that this group is also composed of two major clades (Francisco-Ortega et al. 2001a). One of these clades has three species restricted to Gran Canaria, and they have radiated into three different ecological zones. The second clade has two major lineages and does not have any representative in Gran Canaria; it shows interisland colonizations among La Gomera, El Hierro, Tenerife, and La Palma. One of the two lineages is restricted to the lowland scrub, the second lineage is confined to the pine forest.

Evolutionary implications and morphological differentiation Barriers to gene flow between the various species of Bystropogon appear to be eco-geographical rather than genetic. Several hybrids between the two sections of this genus have been found in the wild and formally described (La Serna-Ramos 1984). In addition, during our field studies we have also observed several morphological forms that seem to be hybrids between Bystropogon plumosus and Bystropogon origanifolius in Tenerife (A. Santos-Guerra, unpublished).

Both the ITS and trees are congruent in supporting the sectional classification of Bystropogon proposed by La Serna-Ramos (1984). It seems that gene flow between these two sections has not been a major factor in the evolutionary history of this genus, even though natural intersectional hybrids are known to occur. Despite the congruence in sectional classification, disagreements between taxonomy and species-level phylogeny were detected within each of the sections. Multiple accessions from the two varieties of B. canariensis did not form two separate clades. In addition, the two species from Madeira were nested within a clade formed by the six accessions of B. canariensis. A similar pattern was detected within the section Bystropogon clade, where the four varieties of B. origanifolius did not form a monophyletic group. The three other species of this section were nested inside the clade with the four varieties of B. origanifolius.

Molecular phylogenetic studies for other Macaronesian genera have found major incongruences between taxonomic classifications and phylogenetic patterns. These discrepancies have been considered to be the result of hybridization events. The two best examples are provided

1186 J . L . T R U S T Y E T A L .

by Argyranthemum and Sideritis (Francisco-Ortega et al.

1996; Barber et al. 2000). Natural interspecific hybrids are known to occur in these two genera and it has been suggested that hybridization may be the cause of incongruence between groups obtained from the taxonomic classification and those detected in the phylogenetic trees.

Morphological differences between the two Bystropogon sections are based on reproductive characters concerning calyx teeth, corolla shape, inflorescence architecture, and seed coat structure. In contrast, species boundaries within each section rely mostly on leaf characters such as leaf hairiness, shape, and plant fragrance. We believe that these few leaf traits are more prone to respond rapidly to selection and are likely to be highly homoplasious. We cannot rule out that these morphological traits are under simple genetic control as it has been proven for the Hawaiian endemic species of Tetramolopium Nees (Asteraceae).

Sex expression differences among island endemics of this genus are mostly controlled by two loci (Whitkus et al.

2000).

La Serna-Ramos (1984) distinguished two morphological groups in Bystropogon origanifolius. The first of these groups has the two varieties from La Palma and El Hierro (i.e. var. ferrensis and palmensis), and they have spatulate leaves that are attenuated at their bases. The second group has the other three varieties; plants from this group have leaves which are neither spatulate nor attenuated at their bases.

Bystropogon section Bystropogon is split into two major clades which concord with the morphological groups suggested by La Serna-Ramos (1984). These clades had bootstrap support of 70% and 78%. However, there were other single-island endemics nested in each of these major clades. Bystropogon wildpretii from La Palma grouped with B. origanifolius var. ferrensis and B. origanifolius var. palmensis.

The leaves of Bystropogon wildpretii are also spatulate and slightly attenuated at their bases (I. La Serna-Ramos, personal communication). The second major clade has the remaining varieties of B. origanifolius together with B. odoratissimus and B. plumosus. Taxa from this clade do not have spatulate leaves, although plants of B. odoratissimus and B. plumosus have leaves which are sometimes slightly attenuated at their bases.

Our phylogenetic results show the two species with the most restricted distribution (B. odoratissimus and B. wildpretii) are terminal branches within the clade of the widespread and polymorphic species B. origanifolius. This suggests B. origanifolius as progenitor of the two other species. This phylogenetic pattern has been previously reported for continental taxa, and there are several examples from the California flora (Baldwin 2003; Gottlieb 2003). ‘Quantum speciation’(Grant 1981) is considered as the evolutionary mechanism behind these phylogenetic results (Rieseberg & Brouilet 1994). In this speciation mode

a particular species gives rise to other species leading to a progenitor-derivative pattern. As far as we are aware this speciation mode has not been reported previously in any other oceanic island group. Although, a recent molecular phylogeny for Macaronesian species of Aichryson Webb and Berthel. (Crassulaceae) also shows a common and highly polymorphic species (A. pachycaulon) forming a paraphyletic group with respect to three other taxa with more restricted distributions (Fairfield et al. 2004).

In Bystropogon peripheral populations of widespread species are prone to genetic and morphological changes through genetic drift and inbreeding. Most Bystropogon taxa are restricted to single islands, therefore these two evolutionary processes may act to accelerate speciation events.

Conclusions and future molecular perspectives With only seven species, Bystropogon is a small genus compared with other taxonomic groups with species endemic to Macaronesia. These groups include the Aeonium alliance (63 endemic species), Argyranthemum (26 endemic species), Echium (27 endemic species), Sideritis (26 endemic species), or the woody Sonchus alliance (21 endemic species) (Böhle et al. 1996; Kim et al. 1996; Santos-Guerra 2001; Mort et al.

2002; Marrero & Navarro 2003). These groups not only have large number of endemic species, but they also occur in many of the Macaronesian islands and show extensive radiations in all of the ecosystems of Macaronesia.

Despite its small size, Bystropogon provides unique insights into the evolutionary history of plants endemic to the Macaronesian islands, particularly on the phylogenetic position of taxa endemic to geologically ancient regions, and how interisland colonizations and ecological shifts shaped plant biodiversity in Macaronesia. Further population genetic studies are needed to understand better the potential occurrence of multiple dispersal events between islands, and to what extent populations of taxa occurring in the palaeo-islands of Tenerife have unique patterns of genetic diversity not found in the rest of the archipelago.

Acknowledgements We dedicate this paper to Henrique Miguel Costa Neves, pioneer of Biological Conservation in the archipelago of Madeira. Without his perseverance, enthusiasm, and tireless work the few remaining Macaronesian individuals of the Mediterranean monk seal would not have found a secure refuge in the Desertas Islands at Parque Natural da Madeira. Mark Carine, T. Collins, J. Geiger, C. Lewis, A. Marrero, M. Maunder, I. La Serna-Ramos and S. Zona critically read and provided valuable suggestions to an early draft of this paper. Irene La Serna-Ramos provided helpful discussions on the morphology, ecology, and evolutionary patterns of Bystropogon. Aguedo Marrero and J. Caujapé-Castells kindly helped JFO © 2005 Blackwell Publishing Ltd, Molecular Ecology, 14, 1177–1189

B Y S T R O P O G O N P H Y L O G E N Y 1187 during field studies in the island of Gran Canaria. Carl Lewis provided valuable insights on the interpretation of dG values. Bruce Baldwin guided us on relevant literature on quantum speciation and shared with us unpublished results from his research with Californian tarweeds. This work was supported by an FIU Presidential Fellowship, a United States Environmental Protection Agency STAR Fellowship, and a Tropical Biology Program (TBPFIU) research assistantship to JT. Additional support was provided by start-up funds from the Tropical Biology Program of FIU, research funds from Fairchild Tropical Botanic Garden (FTBG), an FIU Provost’s Office Summer Research Competition grant, and a Summer Research Position from FTBG to JFO. This is contribution number 045 of the TBP-FIU.

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Jennifer Trusty is a graduate student at FIU/FTBG, her research focuses on the origin, evolution, and conservation genetics of island plants, particularly from the Antilles, Cocos Island (Pacific Ocean), and Macaronesia. Richard Olmstead is Professor of Biology at University of Washington. His main research concerns plant systematics, particularly within the Lamiales and Solanales.

Arnoldo Santos-Guerra is Director of Research of ORT. His main research concerns plant biodiversity in Macaronesia. Susana Sa-Fontinha is Director of PNM, she specializes on conservation and taxonomy of Macaronesian plants. Javier Francisco-Ortega is Associate Professor at FIU, he is interested in island plant biodiversity and conservation.