金鼎的論文更正版(壓縮檔) by li shai fu

國立嘉義大學生物資源學系碩士學位論文

台灣產卷柏屬植物葉片解剖與葉綠體多樣性之研究 (Structural Study on Leaf Anatomy and the Diversity of Chloroplasts in Selaginella from Taiwan)

指導教授：許秋容 Chiou-rong Sheue 研究生：吳金鼎 Chin-ting Wu 中華民國一○一年六月

Acknowledgements First, I acknowledge my advisor, Dr. Chiou-Rong Sheue (許秋容) and Dr. Chiung-Ru Yang (楊瓊儒) for their great help, guidance, advice, and patience throughout the past two and a half years. Thanks are due to Show-Chin Chang (張秀琴), Edward Wu(吳業樺) and Win-Li Hwang (黃雯莉), who are the members of Plant Reservation Laboratory in the Department of Biological Resources, National Chaiyi University. I feel lucky study together with 黃子軒,張宇甄,陳淑芬 in the Nematode Laboratory in the Department of Biological Resources, National Chaiyi University. I sincerely thank Miss Sue (蘇碧華) in the Electron Microscopy Laboratory, National Chaiyi University, for her help over the days I prepared the samples and used Transmission Electron Microscope. Appreciation is extended to Dr. Chi-Jou Tsai (蔡奇助) and Miss Jade Lin (林文粦) (Kaohsiung District Agricultural Research and Extension Station) for the use of a Confocal Laser Scanning Microscope. I amvery grateful to Yushan National Park Headquarters, who approved the certificate of collecting Selaginella species in the restricted area. In addition, thanks are given to Dr. Kou (郭城孟) for his identification of Selaginella species. Finally, I would like to thank my wife, Jen-Win Wu (吳靜雯), and my daughter, Isabella Wu (吳胤瑄). I finish this work with their love and support.

摘要通常葉片是扁平的器官，專為光合作用。在維管植物，其組成為表皮組織、葉肉組織和葉脈。葉肉組織由含有葉綠體的薄壁細胞所組成，是一種同化組織及光合作用的最初所在。大部份維管束植物的表皮細胞缺乏葉綠體，然而，有數種水生植物及陰性植物是例外，包括卷柏屬。葉片解剖顯示出的特徵是上表皮細胞富含葉綠體，本研究中的 12 種台灣產卷柏，其葉肉組織大致上分為兩群，一是葉肉組織稀疏，例如緣毛卷柏、全緣卷柏；另一則是葉肉組織密集，例如異葉卷柏、擬密葉卷柏和萬年松等，前者又可分為缺乏葉肉組織，只有上下表皮細胞，以及葉肉組織疏鬆，例如生根卷柏和玉山卷柏等。後者又可分為沒有柵狀組織，只有海棉組織，例如異葉卷柏，以及具有明顯分化出柵狀組織和海棉組織，例如擬密葉卷柏和萬年松。疏葉卷柏的氣孔罕見地在葉緣和被觀察到，可能因為其棲地是潮濕的林蔭下，要排出植物體內多餘的水份，具有泌水孔的作用。日本卷柏的葉片呈一層顯著的角質層表皮細胞。本研究的目的在探討近軸腹小葉表皮細胞葉綠體的特性。使用共軛焦雷射顯微鏡觀察 12 種原產於台灣的卷柏屬物種腹小葉近軸表皮細胞，腹小葉近軸側表皮細胞從縱切面看呈現圓形和長方形。在表皮細胞發現 2 種葉綠體的分佈模式，即中間型和週邊型。清楚地顯出台灣產卷柏腹小葉近軸表皮細胞屬在大小上具有很大的多樣性(c. 4- 18 µm)和數量上的多樣性(每個細胞的葉綠體從 1 個到 17 個)。全緣卷柏的生鮮徒手切片小葉的解剖構造包括上下表皮細胞和海綿組織的葉肉。值得注意的是其缺少柵狀組織。上表皮細胞富含綠色的葉綠體。姬卷柏在被報導出為第二種具有雙區葉綠體的維管束植物。它出現在被其他植物密集遮蔽處(PAR 4– 39μ mol/m2s at mid-day: 0.2-0.3% full sun)，棲地是適度潮溼的岩石具有薄薄的土壤層。在姬卷柏中發現的二區葉綠體在超微構造上相似於 Selaginella erythropus。但是姬卷柏的二區葉綠體具有一種不同的型式，相似於捕手的手套來捕捉光(而非球形)。具有一種凹陷的結構，吾人推測它靈活的變化型式乃是為了因應優勢的光環境。這兩種同屬的植物，分別生長在舊大陸的台灣及新大陸的巴西，相隔數千公里的太平洋，其葉綠體卻演化出相類似的超微構造，具有趨同演化的重大意義，本研究也推測，未來在卷柏屬中將陸續發現具有二區葉綠體構造的物種，這是值得進一步的探討與研究的課題。

關鍵字：葉綠體、卷柏、二區葉綠體、趨同演化

Abstract A leaf is typicallyflat (laminar) plant organspecializedfor photosynthesis, and consists of epidermis, mesophyll and vein in vascular plants. Mesophyll consisted of parenchyma with chloroplasts is an assimilationtissue and the primarylocation of photosynthesis. The epidermal cells of most vascular plants are devoid of chloroplasts, however, several aquatic or shade plants are exceptions, including Selaginella. This paper describes remarkable leaf anatomy and chloroplast ultrastructure in Selaginella species in Taiwan. Physiological leaf anatomy and chloroplast morphology of the adaxial ventral microphylls in twelve species of the genus Selaginella fromTaiwan–S. bonenisis, S. ciliaris, S. delicatula, S. doederleinii, S. heterostachys, S. labordei, S. nipponica, S. remotifolia, S. repanda, S. moellendorffii,S. stauntoniana and S. tamariscina –the last three species are recurrection plants observed inthe habitat. Twelve species were investigated by using light microscopy, confocal laser scanningmicroscopy (CLSM) andtransmissionelectron microscopy. Four distinct morphological types of mesophyll inthe leaf and distinguished diversity of chloroplasts inthe upper epidermal cells are identified. The features of leaf anatomy showthat the upper epidermal cells have conspicuous chloroplasts, and the mesophyll lacks a palisade layer, but which in Selaginella stauntoniana and Selaginella tamariscina is differentiated into palisade tissue and spongy tissue. Stomata are observed rarely onthe leaf margin as well as on the lower epidermis of S. remotifolia. S. nipponicaleaf obviously shows a single layer of cuticularized epiderml cells. Mesophyll consisted of parenchyma with chloroplasts is anassimilation tissue and the primarylocation of photosynthesis. The epidermal cells of most vascular plants are devoid of chloroplasts, however, several aquatic or shade plants are exceptions, including Selaginella.This paper describes remarkable leaf anatomy and chloroplast ultrastructure in Selaginella species in Taiwan. Physiological leaf anatomy and chloroplast morphology of the adaxial ventral microphylls in twelve species of the genus Selaginella fromTaiwan–S. bonenisis, S. ciliaris, S. delicatula, S. doederleinii, S. heterostachys, S. labordei, S. nipponica, S. remotifolia, S. repanda, S. moellendorffii,S. stauntoniana and S. tamariscina –the last three species are recurrection plants observed inthe habitat. Twelve species were investigated by using light microscopy, confocal laser scanningmicroscopy (CLSM) andtransmissionelectron microscopy. Four distinct morphological types of mesophyll in the leaf and distinguished diversity of chloroplasts in the upper epidermal cells are identified The aim of this studyis toinvestigate the features of chloroplast in adaxial ventral epidermal cell. The adaxial epidermal cells of the ventral microphylls of 12 species of 4

Selaginella native inTaiwan were observed by confocal laser scanning microscopy. The epidermal cells at adaxial side of ventral microphylls appear as circular or rectangular in shape fromlongitudinal section. Two distribution patterns of chloroplast in epidermal cells were found, namelymiddle and margin patterns. It clearly demonstrated that the chloroplasts in adaxial ventral epidermal cells of Selaginella in Taiwan posses great diversityin size (c. 4- 18µm) and number (from one to 17 per cell). The anatomical structure of the microphyll from fresh hand-sections of Selaginella delicatulal contains both the upper and the lower epidermis andspongy mesophyll. Note that palisade mesophyll is absent. The upper epidermal cells have abundant green chloroplasts. Selaginella heterostachys is here reportedas the second species of vascular plant with bizonoplasts. It occurs densely shaded by other plants (PAR4–39 μmol/m2s at mid-day: 0.2-0.3% full sun) in moderately damp rocky habitat with shallowsoil layers. The bizonoplast found in Selaginella heterostachys is similar to that of Selaginella erythropus in ultrastructure. However, the bizonocloroplasts of S. heterostachys have a different shape and are similar to a catcher’s mitt (for photons instead of balls), with a lobed structure, which we suppose is flexible, changing form tothe prevailing light conditions. The features of leaf anatomy show that the upper epidermal cells have conspicuous chloroplasts, and the mesophyll lacks a palisade layer, but which in Selaginella stauntoniana and Selaginella tamariscina is differentiated into palisade tissue and spongy tissue.

Key words: bizonocloroplast, confocal microscopy, epidermis, Selaginella, ventral microphyll

Contents Acknowledgements .......................................................................................................... 2 ć&#x2018;&#x2DC;čŚ .................................................................................................................................. 3 Abstract ............................................................................................................................. 4 List of Tables .................................................................................................................... 8 List of Figures ................................................................................................................... 9 Chapter 1 Introduction .................................................................................................... 20 Chapter 2 Materials and methods ................................................................................... 26 2.1. Plant materials .................................................................................................. 26 2.2. Free hand-sections of fresh plant specimens and light microscopy ................. 40 2.3. Semi-thin sections and light microscopy ......................................................... 40 2.4. Confocal scanning light microscopy ................................................................ 41 2.5. Ultra-thin sections and transmission electron microscopy (TEM)................... 41 Chapter 3 Results ............................................................................................................ 42 3. 1. The mesophyll of S. ciliaris is poorly developed. ........................................... 42 3. 2. One giant chloroplast is found in the adaxial ventral epidermis of S. delicatula. ................................................................................................................................. 47 3. 4. Abizonoplast was found in the ventral epidermal cell of S. heterostachys. .... 63 There are two giant chloroplasts per adaxial ventral epidermal cell of S. labordei. 73 3. 6. The mesophyll cells are dense in S. moellendorffii ......................................... 77 3.7. S. nipponica leaf obviously shows a single layer of cuticularized epidermaial cells.......................................................................................................................... 82 3.8. Stomata are observed rarely on the leaf margin as well as on the ventral epidermis of S. remotifolia. ..................................................................................... 88 3.9. There is normally one giant chloroplast per adaxial ventral epidermal cell of S. repanda.................................................................................................................... 94 6

3. 10. The mesophyll is differentiated into typical palisade tissue and spongy tissue in S. stauntoniana. ................................................................................................. 103 3. 11. The mesophyll is differentiated into seudo-palisade layers and spongy layers in S. tamariscina.................................................................................................... 111 Chapter 4 Discussion and Conclusion .......................................................................... 121 4.1 Two group microphyll types of Selaginella in this study ............................... 121 4.2 . The importance of abundant intercellulal space inmesophyll ....................... 121 4.3. Novel marginal stomata in S. remotifolia....................................................... 123 4.4. The diversity of chloroplsast in number and size of Selaginella ................... 124 4.5 Selaginella hetrostachys: the second species with bizonoplasts similar to S. erythropus.............................................................................................................. 125 References .................................................................................................................... 127

List of Tables TABLE 2-1 LIST OF SELAGINELLA SPECIES COLLECTED FROM TAIWAN IN THIS STUDY. 26

TABLE 3 1 COMPARING IN THE FEATURE OF CHLOROPLAST OF VENTRAL MICROPHYLL 64

BETWEEN S.HETEROSTACHYS AND S ERYTHROPUS TABLE 3-2 C OMPARISON OF ANATOMICAL FEATURES OF ADAXIAL MICROPHYLL

AMONG 12 SPECIES OF SELAGINELLA COLLECTED FROM TAIWAN IN THIS S TUDY. A TOTAL OF 108 CELLS WERE MEASURED FROM3 INDIVIDUALS PER SPECIES (MEAN ± 118

SE). TABLE 3 3 THE CHLOROPLAST SIZE, NUMBER P ER CELL A ND THE D ISTRIBUTION

PATTERN IN A DAXIAL EP IDERM AL C ELLS OF VENTRAL MICROPHY LLS OF SELAG 119

INE LLA IN TAIWA N OF THIS STUDY.

TABLE 3-4 THE CHARACTERISTICS OF CHLOROPLAST IN AD AXIA L EPIDERMA L CELLS OF VENTRA L MICROP HYLLS OF S ELAG INE LLA IN TAIWA N OBSERV ED IN LASER CONFOC AL MICROSC OPES OF THIS STUDY (N= 31).

120

List of Figures FIGURE 1 THE CHLOROPLAST ADAPTATION SCHEME PRESENTED THE SUN-TYPE CHLOROPLASTS POSSES AN ULTRASTRUCTURE, PIGMENT COMPOSITION AND PHOTOSYNTHETIC RATE WHICH ARE QUITE DIFFERENT FROM THOSE SHADE-TYPE CHLOROPLASTS. P, PLOLASTOGLOBULI (LICHTENTHALER AND BURKART, 1999). .... 21 FIGURE 1-2 GENERALLY THE EPIDERMAL CELLS OF MOST VASCULAR PLANTS ARE DEVOID OF CHLOROPLASTS. MESOPHYLL CONTAIN THE VAST MAJORITY OF THE CHLOROPLASTS. LIGHT MICROGRAPHS SHOWING CROSS SECTIONS OF THE FRESH LEAVES OF (A) IXERIS CHINENSIS 19 (THUND.) NAKAI (B) BOMBOX MALABARICA DC., AND (C) CINNAMOMUN CAMPHORA (L.) NEES & EBERM. ......................................... 22 FIGURE 1-3 CROSS OF A FRESH SELAGINELLA LEAF.(A) LIGHT MICROGRAPH SHOWING CROSS SECTION OF A VENTRAL MICROPHYLL OF S. DOEDERLEINII HERON,.(B) AN ENLARGED VIEW OF A SHOWING CHLOROPLASTS IN DORSAL EPIDERMAL CELLS AND MESOPHYLL CELLS............................................................................................................. 23 FIGURE 1-4 THIS DECONVOLUTED IMAGE IN PSEUDOCOLOR WAS RECONSTRUCTED FROM 3D OF BIZONOPLASTS (SHEUE ET AL., 2007). ............................................................. 24 FIGURE 1-5 SIMPLIFIED PHYLOGENETIC TREE SHOWING THE MINIMUM STRATIGRAPHIC RANGES OF SELECTED GROUPS BASED ON MEGAFOSSILS (THICK BARS) AND THEIR MINIMUM IMPLIED RANGE EXTENSIONS (THIN LINES) (KENRICK AND CRANE, 1997). ........................................................................................................................................................... 25

FIGURE 2-1 LOCATION MAP OF SELAGINELLA SPECIES COLLECTED FROM TAIWAN IN THIS STUDY. ................................................................................................................................... 27 FIGURE 2-2 HABITAT AND MORPHOLOGY OF S. BONINENSIS.. (A)PLANTS GROWING ON FOREST FLOORS IN TONGYUN, PINTUNG. THE INSET SHOWS WHERE MEASURED AT 1806.7 µMOL M-2S-1 OF FULL SUNLIGHT AND 37.11 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS LONG CREEPING, BEARING MANY SHORT LEAFY BRANCHES VERY SMALL. ........................................................................................................... 28 FIGURE 2-3 HABITAT AND MORPHOLOGY OF S. CILIARIS. (A)PLANTS GROWING ON EXPOSED GRASSY BANKS IN SUN-MOON LAKE. THE INSET SHOWS WHERE MEASURED AT 1854.8 µMOL M-2S-1 OF FULL SUNLIGHT AND 58.10 µMOL M-2S-1 OF HABITAT RADIATION. (B) PLANTS ARE VERY SMALL. MAIN STEMS CREEPING.TO SEMI-ERECT. ................................................................................................................................... 29 FIGURE 2-4 HABITAT AND MORPHOLOGY OF S. DELICATULA. (A) PLANTS GROWING ON THE FORESTFLOORS IN SITOU. THE INSET SHOWS WHERE MEASURED AT 1888.9 µMOL M-2S-1 OF FULL SUNLIGHTAND 23.41 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS ERECT. .................................................................................................................... 30 9

FIGURE 2-5 HABITAT AND MORPHOLOGY OF S. DOEDERLEINII. (A) PLANTS GROWING ON THE FORESTFLOORS IN SITOU. THE INSET SHOWS WHERE MEASURED AT 1957.7 µMOL M-2S-1 OF FULL SUNLIGHTAND 2.08 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS ERECT, BEARING LONGRHIZOPORES. ............................................................ 31 FIGURE 2-6 HABITAT AND MORPHOLOGY OF S. HETEROSTACHYS. (A) PLANTS GROWING ON THE FORESTFLOORS UNDER SHADE ENVIRONMENTS. THE INSET SHOWS WHERE MEASURED AT 1787.6 µMOLM-2S-1 OF FULL SUNLIGHT AND 4.12 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMSCREEPING TO SEMI-ERECT. ............................... 32 FIGURE 2-7 HABITAT AND MORPHOLOGY OF S. LABORDEI. (A) PLANTS GROWING ON THE FOREST FLOORS OF HIGH MOUNTAINS. THE INSET SHOWS WHERE MEASURED AT 1825.0 µMOL M-2S-1 OF FULL SUNLIGHT AND 43.41 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS ERECT. ...................................................................................... 33 FIGURE 2-8 HABITAT AND MORPHOLOGY OF S. MOELLENDORFFII. (A) PLANTS GROWING IN SHADES OFVALLEYS IN LIENHUACHIH. THE INSET SHOWS WHERE MEASURED AT 1855.7 µMOL M-2S-1 OF FULLSUNLIGHT AND 157.19 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS ERECT. ...................................................................................... 34 FIGURE 2-9 HABITAT AND MORPHOLOGY OF S. NIPPONICA. (A) PLANTS GROWING ON MOSSY BANKS IN SHIHTZULU. THE INSET SHOWS WHERE MEASURED AT 1803.5 µMOL M-2S-1 OF FULL SUNLIGHT AND 60.52 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS CREEPING. ....................................................................................................... 35 FIGURE 2-10 HABITAT AND MORPHOLOGY OF S. REMOTIFOLIA. (A) PLANTS GROWING IN MONTANEFORESTS IN ALISHAN. THE INSET SHOWS WHERE MEASURED AT 1868.5 µMOL M-2S-1 OF FULLSUNLIGHT AND 7.36 µMOL M-2S-1 OF HABITAT RADIATION. (B) PLANTS WIDELY CREEPING. ...................................................................................................... 36 FIGURE 2-11 HABITAT AND MORPHOLOGY OF S. REPANDA. (A) PLANTS GROWING ON EXPOSEDHILLSIDES IN LANTAN. THE INSET SHOWS WHERE MEASURED AT 1849.7 µMOL M-2S-1 OF FULLSUNLIGHT AND 95.53 µMOL M-2S-1 OF HABITAT RADIATION. (B) MAIN STEMS ERECT. .................................................................................................................... 37 FIGURE 2-12HABITAT AND MORPHOLOGY OF S. STAUNTONIANA. (A) PLANTS GROWING ON EXPOSEDROCKS IN SOUTHERN CROSS-ISLAND HIGHWAY. THE INSET SHOWS WHERE MEASURED AT 1976.2µMOL M-2S-1 OF FULL SUNLIGHT AND 536.9 µMOL M-2S-1 OF HABITAT RADIATION. (B) S.STAUNTONIANA IS A RESURRESCTION PLANT. ........................................................................................................................................................... 38 FIGURE 2-13 HABITAT AND MORPHOLOGY OF S. TAMARISCINA. (A) PLANTS GROWING ON EXPOSED ROCKS UNDER HIGHER-LIGHT ENVIRONMENTS IN KUKUAN. THE INSET SHOWS WHERE MEASURED AT 1923.6 µMOL M-2S-1 OF FULL SUNLIGHT AND 485.1 µMOL M-2S-1 OF HABITAT RADIATION. (B) S. TAMARISCINA IS A NOVELTY RESURRESCTION PLANT. THE INSET SHOWING THAT S. TAMARISCINA CURLS UP INTO A TIGHT BALL DURING A DROUGHT. ...................................................................................... 39 10

FIGURE 3-1 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. CILIARIS. (A) THE ENTIRE VIEW SHOWS THAT THE EPIDERMIS IS A SINGLE PERIPHERAL LAYER OF CELLS. (B) THE TRANSVERSE SECTION SHOWING A OPEN STOMA IN THE ABAXIAL LEAF SURFACE. (C) CHLOROPLASTS ARE FOUND IN BOTH THE ADAXIAL AND ABAXIAL EPIDERMAL CELLS. NOTE THAT MESOPHYLL IS POORLY DEVELOPED. ..... 43 FIGURE 3-2 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A) – (D) SHOW 3~4 CHLOROPLASTS IN THE ADAXIAL VENTRAL EPIDERMAL CELLS OF S. CILIARIS. ...... 43 FIGURE 3-3 THE TEMMICROGRAPHS OF S. CILIARIS. (A) THE CHLOROPLASTS ARE ELONGATED IN DORSAL EPIDERMAL CELLS, WHICH DISTRIBUTED ALONG THE ANTICLINAL WALLS, X 4,000. (B) THE CHLOROPLAST SHOWS THE LARGE ABUNDANT GRANA STACKS AND THYLAKOID STROMA LAMELLAE FACING THE CELL WALL, X 20,000. (C) THE STACKED GRANA CONTAINS 8 – 12 LAYERS OF THYLAKOID MEMBRANES, X 30,000. (D) THE PLASTOGLOBULI WERE FOUND IN THE STROMA BETWEEN THE INTERNAL MEMBRANES, X 50,000. ............................................................... 44 FIGURE 3-4 THE TEMMICROGRAPHS OF S. CILIARIS SHOWED (A) PLASTOGLOBULES ARE OFTEN ASSOCIATED WITH THYLAKOID MEMBRANES. (B) STARCH GRAINS AND WELL-DEVELOPED GRANA SYSTEM ARE VISIBLE. ............................................................. 45 FIGURE 3-5 THE TEMMICROGRAPHS OF S. CILIARIS SHOWED (A) THE CHLOROPLASTS, IN MESOPHYLL CELLS, ARE CUP-SHAPED AND OVAL TO LENTICULAR IN SHAPE. (B) THE CUP-SHAPED CHLOROPLAST IS VISIBLE AT HIGHER MAGNIFACTION. (C) THE CUP-SHAPED CHLOROPLAST HAS TWO STARCH GRAINS AND MANY UNSTACKED THYLAKOIDS. (D) THE CHLOROPLASTS HAVE MANY INTERCONNECTED THYLAKOID MEMBRNES AS WELL AS 17-21 LAYERS OF THYLAKOIDS PER GRANA STACKS, AND THERE IS SOME PLASTOGLOBULI. ........................................................................................... 46 FIGURE 3-6 THE TEM MICROGRAPH OF S. CILIARIS CLEARLY SHOWED THE OPEN PORE AND THE GUARD CELLS.............................................................................................................. 46 FIGURE 3-7 FRESH FREE-HAND SECTIONS OF THE VENTRAL MICROPHYLL FROM S. DELICATULA. (A) NUMEROUS CHLOROPLASTS ARE CLEARLY OBSERVED ON THE DORSAL AND THE VENTRAL EPIDERMIS AND MESOPHYLL, (B) THERE ARE LARGE INTERCELLULAR AIR SPACES ARE NEAR THE VEIN. (C) THE CUTICLE IS SEEN. ......... 48 FIGURE 3-8 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. DELICATULA. (A) THE LAMINA CONSISTS SOLELY OF EPIDERMAL CELLS AND THE MESOPHYLL COMPRISES ONE OR TWO LAYERS OF IRREGULARLY-SHAPED CELLS. (B) NOTE THE VARIOUS SIZES OF CHLOROPLASTS IN THE ADAXIAL CONICAL EPIDERMAL CELLS, MESOPHYLL CELLS, AND VENTRAL ELONGATED EPIDERMAL CELLS. (C) THE CHLOROPLASTS IN MESOPHYLL CELLS ARE GREATER THAN THOSE IN EPIDERMAL CELLS. .............................................................................................................................................. 49 11

FIGURE 3-9 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)~(D) ILLUSTRATE THERE IS ONLY ONE GIANT CHLOROPLAST PER VENTRAL EPIDERMAL CELLS IN S. DELICATULA FROM TOP VIEW. ................................................................................................. 50 FIGURE 3-10 THE TEMMICROGRAMSHOWS THE OVERVIEW OF S. DELICATULA. NOTE THERE IS ONE GIANT CHLOROPLAST OCCUPIED IN THE DORSAL EPIDERMAL CELL, X 1,000. ............................................................................................................................................. 50 FIGURE 3 11 THE TEM MICROGRAPHS OF S. DELICATULA. (A) THE ADAXIAL VENTRAL EPIDERMIAL CELL IS MAMMRAY-LIKE AND CONTAINS ONE CHLOROPLAST,. (B) THE PART OF THE CHLOROPLAST IS AT HIGHER MAGNIFICATION. ........................................ 51 FIGURE 3-12 THE TEMMICROGRAPHS OF S. DELICATULA. (A) A GIANT CHLOROPLAST IS ACCUMULATED PLASTOGLOBULI AND MANY THYLAKOIDS, X 3,500. (B) AGIANT CHLOROPLAST IN THE DORSAL EPIDERMAL CELL, CONTAINS GRANA AND STROMA THYLAKOIDS AND DENSE PLASTOGLIBULI, X 3,500. (C) AGIANT CHLOROPLAST AND A NUCLEUS WITH NUCLEOLUS ARE PRESENTED IN THE EPIDERMAL CEL, X 5,000. ... 51 FIGURE 3-13 THE TEM MICROGRAPHS OF S. DELICATULA. (A) THE MESOPHYLL CELL CONTAINS A CIRCULAR CHLOROPLAST. (B) THE PART OF THE CHLOROPLAST IS AT HIGHER MAGIFICATION. ............................................................................................................. 52 FIGURE 3-14 THE TEM MICROGRAPHS OF S. DELICATULA. (A) THE VENTRAL EPIDERMAL CELL SHOWS ONE CIRCULAR CHLOROPLAST. (B) THE CIRCULAR CHLOROPLAST IN THE VENTRAL EPIDERMAL CELL ARE SHOWN HERE AT HIGHER MAGNIFICATION. THE CHLOROPLAST CONTAINS LARGELY UNSTACKED THYLAKOIDS AND A MASS OF PLASTOGLOBULI. (C) A CHLOROPLAST AND A NUCLEUS WITH NUCLEOLUS ARE VISIBLE IN VENTRAL EPIDERMAL CELL. (D) THE VENTRAL EPIDERMAL CELL WALL IS MORE THICKER. ........................................................................................................................ 53 FIGURE 3-15 PHOTOGRAPHS OF FRESH FREE-HAND SECTIONS FROM S. DOEDERLEINII. (A) THE DORSAL EPIDERMAL CELLS DIFFERENTIATE AS CON-SHAPED CELLS WITH LARGE CHLOROPLAST FILLING THE BASE OF THE CELL. (B) THERE ARE THREE OR FOUR LAYERS OF MESOPHYLL ADJACENT TO THE VEIN . (C) THE MESOPHYLL TISSUE IS LESS WELL DEVELOPED GRADING TO THE MARGIN. ...................................... 55 FIGURE 3-16 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. DOEDERLEINII. (A) THE TRANSVERSE SECTION SHOWING THE LEAF S. DOEDERLEINII IS BROADER THAN OTHER SELAGINELLA SPECIES IN TAIWAN. (B) THE CHLOROPLASTS ARE MOST CLEARLY REVEALED IN EPIDERMAL CELLS. (C) THE VARIOUS SIZES OF CHLOROPLASTS IN THE DORSAL EPIDERMAL CELLS, MESOPHYLL CELLS, AND VENTRAL EPIDERMAL CELLS. (D) THE LEAF INTERIOR HAS IRREGULARLY SHAPED SPONGY CELLS WITH ABUNDANT INTERCELLULAR SPACES. (E) THE MESOPHYLL TISSUE IS LESS WELL DEVELOPED GRADING TO ONE LAYER NEAR THE MARGIN. ... 57 FIGURE 3-17 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)~(D) ILLUSTRATE THERE IS ONLY ONE GIANT CHLOROPLAST PER VENTRAL EPIDERMAL CELLS IN S. 12

DOEDERLEINII FROMTOP VIEW. ............................................................................................... 58 FIGURE 3-18 THE TEMMICROGRAPHS OF S. DOEDERLEINII. (A) ALOW MAGNIFACTION OVERVIEW, IT IS CHARACTERIZED BY ONE GIANT CHLOROPLAST PER DORSAL EPIDERMAL CELL. (B) THE CHLOROPLAST IN THE DORSAL EPIDERMAL CELL CONTAINS MORE STARCH GRAINS AND PLASTOGLOBULI. (C) THE MESOPHYLL HAS MANY IRREGULAR CELLS. ......................................................................................................... 59 FIGURE 3-19 THE TEMMICROGRAPHS OF S. DOEDERLEINII. (A) THE CHLOROPLAST IS WELL DEVELOPED WITH MANY GRANA STACKS AS WELL AS NUMEROUS STARCH GRAINS, AND THERE IS ACCUMULATION OF PLASTOGLOBULI. (B) THE GIANT CHLOROPLAST COMPOSES OF NUMEROUS STARCH GRAINS BETWEEN THE THYLAKOIDS AND PLASTOGLOBULI. (C) THE GIANT CHLOROPLAST HAS MANY DENSELY PLASTOGLOBULI IN CLUSTERS. ............................................................................. 60 FIGURE 3-20 THE TEM MICROGRAPHS OF S. DOEDERLEINII. (A) THE GIANT CHLOROPLAST IS SHOWN MANY GRANA WITH SOME STARCH GRAINS AND PLASTOGLOBULI IN CLUSTERS, (B) THE GIANT CUP-SHAPED CHLOROPLAST IS SHOWN MANY GRANA WITH SOME STARCH GRAINS AND PLASTOGLOBULI IN CLUSTERS. .............................. 61 FIGURE 3-21 THE TEM MICROGRAPHS OF S. DOEDERLEINII. (A) THE MESOPHYLL CHLOROPLASTS SHOWING THAT GRANA CONTAIN VERY NUMEROUS THYLAKOID MEMBRANES, X 3,000. (B) THE PART OF THE CHLOROPLAST IS AT HIGHER MAGNIFICATION. .......................................................................................................................... 61 FIGURE 3-22 THE TEM MICROGRAPHS OF S. DOEDERLEINII. (A) THE VENTRAL EPIDERMAL CELL CONTAINS A CHLOROPLAST, WHICH POSSESS NUMEROUS GRANA AND STARCH GRAINS,. (B) PART OF THE VENTRAL EPIDERMAL CELL, THE ABUNDANT GRANA STACKS IS SHOWN IN THE CHLOROPLAST AT HIGHER MAGNIFICATION. .......................................................................................................................... 62 FIGURE 3 23 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. HETEROSTACHYS. (A) THE DORSAL EPIDERMAL CELLS CLEARLY SHOW MUCH CHLOROPLASTS.(B) A THIN CUTICLE WHICH OFTEN BECAME DETACHED DURING THE EMBEDDING PROCESS, OVERLIES THE DORSAL EPIDERMAL CELLS. (B) STAMATA ARE PRESENT ON THE ABAXIAL SURFACE OF THE MICROPHYLL.(C) STAMATA ARE LOCALIZED NEAR THE VEIN. (D) THE CHLOROPLASTS IN THE DORSAL EPIDERMIS ARE AGGREGATED IN THE BASE OF THE CELLS. .......................................................................... 65 FIGURE 3 24 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)~(D) SHOW 4 CHLOROPLASTS IN THE VENTRAL EPIDERMAL CELLS OF S. HETEROSTACHYS. ........... 66 FIGURE 3 25 THE CONFOCAL MICROGRAPHS (A)~(B) SHOW ONE CHLOROPLAST IN THE VENTRAL EPIDERMAL CELLS OF S. HETEROSTACHYS. WITH DEEP LOBE ON LATERAL SECTION SIDE VIEW(PHTOED BY SHEUE ET AL.,2010). ........................................................ 67 FIGURE 3-26 (A) THE TEM MICROGAM SHOWS THE OVERVIEW OF S. HETEROSTACHYS, X 2,000. (B) ALOW MAGNIFACTION OVERVIEW. IT IS CHARACTERIZED BY PROMINENT 13

SILICA BODIES ON THE DORSAL EPIDERMAL CELLS. (C) THE TEMMICROGRAPH OF S. HETEROSTACHYS SHOWING THE CHLOROPLASTS ARE ELONAGED IN SHAPE, IN DORSAL EPIDERMAL CELLS, ARE DISTRIBUTED ALONG THE ANTICLINAL WALLS. (D) AHIGHER MAGNIFACTION OVERVIEW. THE MITOCHONDRON AND THYLOKOIDS WERE OBSERVED IN THE THE DORSAL EPIDERMAL CELLS. ............................................. 68 FIGURE 3-27 (A) THE ADAXIAL CELL HAS HAS A GREAT CHLOROPLAST WITH A MITOCONDRON ANA NUCLEUS.. (B) THE TEM MICROGRAPH SHOWS THE MITOCONDRIA ARE GATHERED NEAR THE CHLOROPLAST. (C) THE TEM MICROGRAPH SHOWS THAT THE MITICONDRON IS CLOSE TO THE CHLOROPLAST. AND THE CHLOROPLAST SHOWS LONG STRANDS OF THYLAKOIDS INSTEAD OF THE NORMAL GRANA AND FRET MEMBRANES,.(D) THE CHLOROPLAST SHOS THE UNSTACKED THYLAKOIDS. ....................................................................................................... 69 FIGURE 3-28 (A) ASURPRISING OBSERVATION IS THAT THYLAKOIDS GROUPPED INPARALLEL STRANDS EXTEND FROMONE SIDE OF THE PLASTID TO THE OTHER, .(B) ALARGE MAGNIFACTION OF THE CHLOROPLAST. THE CHLOROPLAST SHOWS LONG STRANDS OF THYLAKOIDS AND CLEAR MITOCHONDRIA. ................................................ 70 FIGURE 3-29 (A) IT WAS BSERVEDTHAT THERE ARE TWO CIRCULAR CHLOROPLAST IN THE MESOPHYLL CELL.(B) THE ORGANELLE IS COMPOSED OF CHLOROPLASTS AND ABUNDANT MITOCONDRIA........................................................................................................ 71 FIGURE 3-30 (A) THE STOMA SHOWING WITH MANY STRCH GRINS IN THE CLOSE CONDITION. (B) THE STOMA APPEARS TO THE NORMAL PHANEROGANIC TYPE, WITH TWO GUARD-CELLS, EACH PROVIDED WITH BIG EMPTY-LOOKING VACULES, AND THE CHLOROPLAST HAS A VARIABLE NUMBER OF STARCH GRAINS. ................. 72 FIGURE 3-31 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. LABORDEI. (A) THE EPIDERMIS IS COMPOSED OF A SINGLE LAYER, LACKING A HYPODERMIS. (B).THE OUTER SURFACE OF THE EPIDERMAL CELLS ARE OFTEN CONVEX, AND THE CHLOROPLASTS ARE TIGHTLY PACKED WITHIN THESE INNER REGION. (C) THE STOMA IS PRESENT DISTINCTIVELY WITH A LARGE SUBSTOMATIC CAVITY. (D) THE MESOPHYLL IS LOOSE AND THE LAMINA CONSISTS SOLELY OF EPIDERMAL CELLS. ........................................................................................................................................................... 74 FIGURE 3-32 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)~(D) SHOWING THE EPIDERMAL CELLS OF S. LABORDEI ARE SLIGHTLY DOMED IN SHAPE, ALTHOUGH APPEAR CIRCULAR LIKELY FLATTENED, WHICH CONTAIN 2 CHLOROPLASTS IN HEMISPHERE-SHAPED AND IN CIRCULAR.............................................................................. 75 FIGURE 3-33 THE TEM MICROGRAPHS OF VENTRAL MICROPHYLL OF S. LABORDEI. (A) THE EPIDERMIS HAVE LARGE CHLOROPLAST (B).THE LARGE CHLOROPLAST IS OCCUPIED THE BASE OF THE EPIDERMAL CELLL. (C) THE CHLOROPLAST HAS MUCH GPLASTOGLOBULI. ....................................................................................................................... 76 FIGURE 3-34 THE TEM MICROGRAPHS OF MESOPHYLL CELL IN S.LABORDEI. (A) THE 14

CHLOROPLAST STROMA WHIN WHICH STACKED GRANA THYLAKOIDS AND UNSTACKED STROMA THYLAKOIDS CAN BE RECONIZED. (B) A HIGH MEGNICATION OVERVIEW. GRANA ARE INTERNECTED BY MUTIPLE STROMA THYLAKOIDS. (C) FUTHER ENLARGEMENT OF THYLAKOIDS MEMBRANES. SHOWING STACKING OF THYLAKOIDS MEMBRANES. ...................................................................................................... 76 FIGURE 3-35 PHOTOGRAPHS OF FRESH, UNSTAINED HAND SECTIONS FROMS. MOELLENDORFFIII. (A) LARGE AMOUNT OF CHLOROPLAST IS CLEARLY PRESENTED IN THE DORSAL EPIDERMIS. (B) SPONGY MESOPHYLL CELLS ARE DENSE. (C) THE CUTICLE WAS COTING AROUND THE DORSAL AND VENTRAL EPIDERMIS. ................. 78 FIGURE 3-36 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. MOELLENDORFFII. (A) THE SPONGY CHLORENCHYMA LDENSELY IS ARRANGED IN THE LAMINA.(B) THE TRANSVERSE SECTION SHOWING A LARGE QUANTITY OF SPONGY MESOPHYLL CELLS ARE VARIOUSLY SHAPED IN THE MICROPHYLL. (C) THE MESOPHYLL CELLS CONTAIN MUCH CHLOROPLASTS, WITH NUMEROUS SMALL INTERCELLULAR SPACES. ........................................................................................................................................... 79 FIGURE 3-37 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) SHOW 3~4 CHLOROPLASTS ON THE MARGIN OF THE VENTRAL EPIDERMAL CELLS OF S. MOLLENDORFFI. ............................................................................................................................ 80 FIGURE 3 38 (A) ALOW MAGNICICATION OVERVIEW OF S. MOELLENDORFFII,.(B) THE TEM MICROGRAPH SHOWS THAT THE DORSAL EPIDERMAL CELL CONTAINS LARGE CHLOROPLASTS WITH A NUCLEUS. (C) THE DORSAL EPIDERMAL CELLS HAVE CHLOROPLASTS, A NUCLEUS, AND NUCLEOLUS, AS WELL AS MITOCHONDRIA, X 6,000. ................................................................................................................................................. 80 FIGURE 3-39 (A) AHIGHER MAGNICICATION OF S. MOELLENDORFFII. (B) THE CHLOROPLAST HAS ABUNDANT GRANA AS WELL AS STARCH GRAINS AND PLASTOGLUBI.(C) CHLOROPLAST TYPICALLY CONTAINS MANY THYLAKOIDS PER GRANUM. ........................................................................................................................................ 81 FIGURE 3-40 (A) ACCUMULATION OF MITOCHONDRIA WITH CHLOROPLASTS AND NUCLEUS IN THE EPIDERMAL CELL OF S. MOELLENDORFFII. MITOCHONDRIA ARE ACCUMULATED ALONG THE OUTER PERICLINAL WALL TOGATHER WITH CHLOROPLAS. (B) AHIGHER MAGNICICATION OF S. MOELLENDORFFI.(C) GRANA ARE INTERCONNECTED BY MULTIPLE STROMA THYLAKOIDS. ............................................... 81 FIGURE 3-41 (A) THE DISC-SHAPED CHLOROPLASTS IN THE MESOPHYLL CELLS HAVE MANY GRANA THYLAKOIDS AND STACH GRAINS. (B) DETAIL OF A DISC-SHAPED CHLOROPLAST IN THE MESOPHYLL CEL. (C) THE MESOPHYLL CELL CHLOROPLAST CONTAINS NUMEROUS STARCH GRAINS IN THE STROMA BETWEEN THE AGRANAL AND INTERNAL MEMBRANES.................................................................................................... 81 FIGURE 3-42 PHOTOGRAPHS OF FRESH FREE-HAND SECTIONS OF S. NIPPONICA. (A) A DISTINCT WAXY CUTICLE SURROUNDS BOTH THE EPIDERMIS. (B) THE SILIC BODIES 15

OCCUR BOTH IN THE EPIDERMIS. (C) THE SPONGY PARENCHYMA IS COMPOSED OF LOOSELY ARRANGED CHLORENCHYMA CELLS WITH PROMINENT INTERCELLULAR SPACES. (D) BOTH THE EPIDERMAL CELLS CONTAIN A VERY LARGE NUMBER OF CHLOROPLASTS. ........................................................................................................................... 84 FIGURE 3 43 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. NIPPONICA. (A) THE LAMINA IS COMPRISED OF A SINGLE LAYER EPIDERMIS AND A SINGLE LAYER MESOPHYLL. (B) THE CHLOROPLASTS IN THE VENTRAL EPIDERMAL CELLS ARE UNIFORMLY DISTRIBUTED AROUND THE CELL PERIPHERY. (C) THE SPONGY MESOPHYLL SHOWS NUMEROUS INTERCELLULAR SPACES AROUND THE VEIN. (D) AN EPIDERMAL CELL CLEARLY CONTAINS A SILICA BODY (CIRCULAR). .................... 85 FIGURE 3-44 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) SHOWING THE EPIDERMAL CELLS OF S. NIPPONICA ARE SLIGHTLY DOMED IN SHAPE, ALTHOUGH APPEAR CIRCULAR LIKELY FLATTENED, WHICH CONTAIN 5 CHLOROPLASTS. .......... 86 FIGURE 3-45 ( A) SILICA BODIES OCCUR IN BOTH DORSAL AND VENTRAL EPIDERMIS. (B) THE TEM MICROGRAPH SHOWS THERE ARE NUMEROUS SMALL THYLAKOID GRANA IN THE CHLOROPLAST.(C) SILICA BODIES ARE VISIBLE, USUALLY ONE, BUT OCCASIONALLY MORE (CIRCLE) TO AN EPIDERMAL CELL. ............................................. 86 FIGURE 3-46 THE CHLOROPLAST SHOWS CUP-SHAPED WITH DEEP LOBE. ............................ 87 FIGURE 3-47 THE VENTRAL EPIDERMAL CELL CONTAINS A CHLOROPLAST WITH INERGRANAL THYLAKOIDS AND PLASTOGLOBULI. ........................................................... 87 FIGURE 3-48 (A)THE DORSAL EPIDERMISAL CELLS AREFILLED WITH CHLOROPLASTS. (B) THE VEIN IS NEAR THE DORSAL EPIDERMIS AND BELOW THE VEIN IS LARGE INTERCELLULAR SPACE. (C)THE MESOPHYLL TISSUE IS LESS WELL DEVELOPED GRADING TO THE MARGIN. ........................................................................................................ 89 FIGURE 3-49 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. REMOTIFOLIA. (A) THE MESOPHYLL COMPRISES ONE OR TWO LAYERS OF IRREGULARLY-SHAPED CELLS. (B) CHLOROPLASTS IN MESOPHYLL ARE MORE THAN EPIDERMIS (C) THE SPONGY MESOPHYLL SHOWS LARGE INTERCELLULAR SPACES AROUND THE VEIN. (D) AN STRICT FEATURE IS A STOMATA WAS FOUND ON THE LEAF MARGIN. ............ 90 FIGURE 3-50 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) ILLUSTRATE THERE ARE 6 CHLOROPLASTS IN THE VENTRAL EPIDERMAL CELLS OF S. REMOTIFOLIA. .... 91 FIGURE 3-51 (A) THE CHLOROPLASTS ARE ARRANGED IN CUP-SHAPED SHOWING DISTINCT GRANA IN THE DORSAL EPIDERMAL CELL. (B) THE CHLOROPLASTS ARE LOCATED AT THE BASE OF DORSAL EPIDERMAL CELLS. (C) CHLOROPLASTS ARE ARRANGED ALONG THE CELL WALL. ..................................................................................... 92 FIGURE 3-52 THE CHLOROPLASTS ARE ARRANGED IN CUP-SHAPED SHOWING DISTINCT GRANA IN THE DORSAL EPIDERMAL CELL. (B) THE CHLOROPLASTS ARE LOCATED AT THE BASE OF DORSAL EPIDERMAL CELLS. (C) FOUR DISC-SHAPED CHLOROPLASTS ARE TYPICALLY FOUND IN THE SPONGY MESOPHYLL CELLS.(D) IN 16

THE MESOPHYLL CELLS, THE CHLOROPLASTS ARE ORIENTED FROMTHE CONSISTENT DIRECTION. ........................................................................................................... 93 FIGURE 3-53 (A) THE MESOPHYLL TISSUE IS LESS WELL DEVELOPED GRADING TO THE MARGIN (B) THEREARETHREEOR FOUR LAYERS OF MESOPHYLL ADJACENT TO THE VEIN. (C) THEDORSAL EPIDERMAL CELLS HAVE ABUNDANT CHLOROPLASTS. .......... 95 FIGURE 3-54 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S.REPANDA. (A) THE TRANSVERSE CSECTION SHOWING A SINGLE EPIDERMAL LAYER AND MORE LOOSE MESOPHYLL. (B) THE DORSAL EPIDERMIS IS THICKER THAN THE VENTRAL EPIDERMIS. (C) THE LAMINA CONSISTS OF FIVE LAYERS OF IRREGULAR SPONGY MESOPHYLL CELLS NEAR THE VEIN.(D) THE MESOPHYLL CELLS ARE FILLED WITH CHLOROPLASTS. ........................................................................................................................... 96 FIGURE 3-55 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) SHOW 3-4 CHLOROPLASTS IN THE DORSAL EPIDERMAL CELLS OF S. REPANDA. ........................... 97 FIGURE 3-56 THE TEM MICROGRAPH SHOWS THE OVERVIEW OF S. REPANDA IN LOWER MAGNICATION. .............................................................................................................................. 98 FIGURE 3-57 THE DORSAL EPIDERMAL CELLS HAVE THICK CELL WALLS AND CHLOROPLASTS ARRANGED IN CUP-SHAPED. ...................................................................... 98 FIGURE 3-58 THE TEM MICROGRAPH SHOWS THE EPIDERMAL CELL HAS CHLOROPLASTS WITH MANY MITOCHONDRIA AND A GIANT VACUOLE. .................................................... 99 FIGURE 3-59 THE CHLOROPLASTS ARRANGED ALONG THE BASE OF THE CELL. ................ 99 FIGURE 3-60 AMASS OF PLASTOGLOBULI GATHER IN THE STROMA..................................... 100 FIGURE 3-61 THE MESOPHYLL CELL HAS TWO CHLOROPLASTS AND A GIANT NUCLEUS. ......................................................................................................................................................... 100 FIGURE 3-62 THE TEM MICROGRAPH SHOWS A MESOPHYLL CELL AT HIGHER MAGNICATION. ............................................................................................................................ 101 FIGURE 3-63 THE MESOPHYLL CELL HAS CLEAR GRANA AND PLASTOGLOBULI IN GROUP. ......................................................................................................................................................... 101 FIGURE 3-64 THE OVERVIEW OF A MESOPHYLL CELL. .............................................................. 102 FIGURE 3-65 FRESH FREE-HAND SECTIONS OF S. STAUNTONIANA. (A) THE MICROPHYLL IS DIFFERENTIATED INTO FOUR DISTINCT TISSUE LAYERS: DORSAL MULTIPLE EPIDERMIS, A MULTIPLE LAYERS PALISADE PARENCHYMA, SPONGY MESOPHYLL, AND VENTRALL EPIDERMIS, X 400. (B) THE CELLS OF THE DORSAL AND VENTRAL EPIDERMIS CONTAIN NO CHLOROPLASTS. THESE ARE VISIBLE, HOWEVER, IN CELLS OF THE PALISADE LAYER OF COLUMNAR CELLS AND THE SPONGY MESOPHYLL. . 104 FIGURE 3-66 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. STAUNTONIANA. (A) THE MESOPHYLL IS DIFFERENTIATED INTO PALISADE AND SPONGY AREAS,(B) THE MESOPHYLL CONSISTS OF THE TIGHTLY-PACKED, ELONGATE CELLS OF THE PALISADE, THE SPONGY TISSUE SHOWS EXTENSIVE INTERCELLULAR SPACES, (C) THE MICROPHYLL SHOWING MORE COMPACT MESOPHYLL IN WHICH THE 17

INTERCELLULAR SPACES ARE REDUCED. THE MULTIPLE EPIDERMIS ARE PRESENTED, AND NO CHLOROPLAST IS FOUND THERE. .................................................. 105 FIGURE 3-67 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) SHOW 4-5 CHLOROPLASTS ARE DISTRIBUTED ALONG THE ANTICLINAL WALLS IN THE DORSAL EPIDERMAL CELLS OF S. STAUNTONIANA. ............................................................ 106 FIGURE 3-68 THE TEM MICROGRAPHS OF THE MICROPHYLL OF S. STAUNTONIANA.(A) MESOPHYLL TISSUE IS DIFFERENTIATED INTO COLUMNAR PALISADE CELLS AND IRREGULATED SPONGY CELLS.(B). THE CELLS OF THE PALISADE LAYER CONTAIN SEVERAL SMALL CHLOROPLASTS, WHICH ARE DISTRIBUTED ALONG THE ANTICLINAL WALLS OR ORIENTED WITH THEIR SURFACES FACING THE VACUOLE (C) TEM MICROGRAPH DEMONSTRATES THE COLUMNAR PALISADE CELLS CONTAIN SEVERAL CHLOROPLASTS, WHICH HAVE SOME STARCH GRAINS. ............................... 107 FIGURE 3-69 THE TEMMICROGRAPHS OF THE MICROPHYLL OF S. STAUNTONIANA.(A),(B),(C) THE CELLS OF THE PALISADE LAYER CONTAIN SEVERAL SMALL CHLOROPLASTS. ......................................................................................................................................................... 108 FIGURE 3-70 THE TEM MICROGRAPHS OF THE MICROPHYLL OF S.STAUNTONIANA.(A),(B),THE CELL WALLS ARE THICK. ................................................... 109 FIGURE 3-71 THE TEM MICROGRAPHS OF THE MICROPHYLL OF S..STAUNTONIANA. (A).THE PALISADE CELLS HAVE MANY CHLOROPLASTS. (B) THE CHLOROPLAST HAS MANY LAYERS THYLAKOIDS MEMBRANES AND MANY PLASTOGLOBULI. ............................ 110 FIGURE 3-72 FRESH FREE-HAND SECTIOS OF S. TAMARISCINA. (A) MESOPHYLL DIFFERENTIED INTO UPPER PALISADE PARENCHYMA AND LOWER SPONGY PARENCHYMA. (B) THE INTERCELLULAR SPACES BETWEEN THE IRREGULARLY SHAPED SPONGY MESOPHYLL CELLS WITHIN LEAF PERMIT FREE DIFFUSION OF GASES. ........................................................................................................................................... 112 FIGURE 3-73 LIGHT MICROGRAPHS OF VENTRAL MICROPHYLL OF S. TAMARISCINA. (A) A MUCH HIGHER PROPORTION OF SPONGY MESOPHYLL THAN PALISADE MESOPHYLL. (B) STAMATA ARE PRESENT ON THE ABAXIAL SURFACE OF THE LEAF AND ARE LOCALIZED NEAR THE VEIN. (C) THE MULTIPLE EPIDERMIS ARE PRESENTED, AND NO CHLOROPLAST IS FOUND THERE. THE SPONGY MESOPHYLL CELLS ARE COMPACT, WITH NUMEROUS SMALL INTERCELLULAR SPACES. .................................. 113 FIGURE 3 74 THE SEQUENCE OF CONFOCAL MICROGRAPHS (A)-(D) SHOW ABOUT 17 CHLOROPLASTS IN THE DORSAL EPIDERMAL CELLS OF S. TAMARISCINA. .................. 114 FIGURE 3 75 (A) (B) THE TEM MICROGRAPHS OF MICROPHYLL OF S. TAMARISCINA SHOW THICK CELL WALL. (C)THE CHLOROPLASTS IN THE PALISADE ARE ARRANGED ALONG THE CELL WALL. .......................................................................................................... 115 FIGURE 3-76 THE CHLOROPLAST HAS PLASTOGLOBULI IN GROUPS. .................................... 116 FIGURE 3-77 THE MESOPHYLL HAS MANY IRREGULAR CELLS. .............................................. 116 FIGURE 3-78 THE STRUCTURE OF MICROPHYLL OF 12 SPECIES OF SELAGINELLA FROM 18

TAIWANIN THIS STUDY ............................................................................................................. 126

FIGURE 4 1 SCHEMATIC DRAWING DEPICTING THE WILLSÄ TTTER AND STOLL THEORY ON THE PATHWAY OF LIGHT THROUGH LEAVES (SINCLAIR ET AL., 1973). ................. 122

Chapter 1 Introduction Photosynthesis is the process of a plant taking energy from the Sun and creating sugars. Light is essential to photosynthesis. Plants make their own food by converting light energy into chemical energy. The light environment within forests presents complex patterns of brightness and spectral distribution (Fedder and Tanner, 1996; Shashar et al., 1998). Irradiance is highly dynamic in many plant canopies (Valladares et al., 1997; Leakey et al., 2005). Light intensity is greatly reduced when passing through the highest canopy layer, and then gradually decreases further in the understorey. Light availability varies considerably within multi-layered forest canopies and therefore with each tree’sposition in this vertical profile (Keeling and Phillips, 2007). In many forests with closed canopies, only a small fraction (0.5-5%) of the solarradiation incident above the canopy reaches the understory (Chazdon and Pearcy, 1984). Light extinction can be as high as 94% over the first 5 m (Bazzaz and Pickett, 1980). Mean light intensity levels of 0.1-1.9% of full sunlight have been reported at ground level, where sun flecks contribute to the highest mean value (Chazdon and Fetcher, 1984; Théry, 2001) Leaf structure is closely associated with its photosynthetic function. Plants develop ‘sun’ or ‘shade’ leaves when acclimating to different irradiance levels. The anatomical and physiological differences between sun and shade leaves have been studied extensively (Yano and Terashima, 2001). Typically, sun leaves of laminar-leaved plants are smaller and/or more deeply lobed, thicker, and lighter incolor compared to those of shade plants. Also, sun leaves are commonly more amphistomatous with well-developed palisade layers, while shade leaves are typicallythinner, primarily hypostomatous, and without palisade layers (Cui et al., 1991; Johnson et al., 2005). Yano and Tamashita (2004) reported that sun and shade leaves are formed in highand low light environments, respectively. The size of chloroplasts is smaller and numbers are many in the high light environment. By contrast, chloroplasts are bigger and few in low light environment. Sun leaves have sun-type chloroplasts with less appressed thylakoid membranes or grana stacking, while shade leaves have shade-type chloroplasts having more appresses thylakoid membranes. Functionally, the sun leaves have higher rate of photosynthesis per unit leaf area, higher Chl a/b ratio, higher amounts of ribulose bisphosphate carboxylaseand oxygenase, cytochromes, and PSⅠ and PSⅡ core complexes than the shade leaves, on leaf area basis (Figure 1) (Anderson, et al., 1973; Lichtenthaler and Burkart, 1999; Dickison, 2000; Yano and Terashima, 2001; Yano and Terashima, 2004; Schulze et al., 2005). In addition, most shade plants do not have palisade mesophyll. In stead, they have large chloroplasts with numerous thylakoids per granum positioned at the base of conical chlorenchyma 20

(Nasrulhaq-Boyce and Duckett, 1991; Sheue et al., 2007).

Figure 1 The chloroplast adaptation scheme presented the sun-type chloroplasts posses an ultrastructure, pigment composition and photosynthetic rate which are quite different from those shade-type chloroplasts. P, plolastoglobuli (Lichtenthaler and Burkart, 1999). Mesophyll cells of leaves are specialized for photosynthesis. These cells usually in the middle of the leaf contain many chloroplasts, the organelles that perform photosynthesis. Generally each mesophyll cell often contains 50 or more chloroplasts (Gates et al., 1965; Whitmarsh and Govindjee, 1999). At maturity chloroplasts are usually oval to lenticular and 5-10 Îźm in length (Bowes, 1997; Whitmarsh and Govindjee, 1999). Jagels (1970) showed that such large cup-shaped chloroplasts in the 21

adaxial epidermal cells of Selaginella apoda (L.) Spring, S. martensii Spring, S. seperpens (Desv.) Spring, and in the subepidermal cells of Selaginella kraussiana (Kunze) A. Braun. Chloroplasts are semi-autonomous organelles comprised of two envelope membranes, an aqueous matrix known as stroma, and internal membranes called thylakoids. The stroma is the site of the biochemical carbon reduction reactions of photosynthesis that generate triose phosphate from carbon dioxide. The principal functions of thylakoids are the trapping of light energy and the transduction of this energy into the chemical energy forms (KutĂk, 1985; TichĂĄ, 1985; Brian et al., 1996; Staehelin and Staay, 1996; Larcher, 2003).

Figure 1-2 Generally the epidermal cells of most vascular plants are devoid of chloroplasts. Mesophyll contain the vast majority of the chloroplasts. Light micrographs showing cross sections of the fresh leaves of (A) Ixeris chinensis 19 (Thund.) Nakai (B) Bombox malabarica DC., and (C) Cinnamomun camphora (L.) Nees & Eberm.

Figure 1-3 Cross of a fresh Selaginella leaf.(A) Light micrograph showing cross section of a ventral microphyll of S. doederleinii Heron,.(B) An enlarged view of A showing chloroplasts in dorsal epidermal cells and mesophyll cells. Generally the epidermal cells of most vascular plants are devoid of chloroplasts (figure 1-2) , however, several aquatic or shade plants are exceptions, including Selaginella (figure 1-3). Haberlandt (1888) might have been the first to report the large cup-shaped chloroplasts in the funnel-shaped photosynthetic cells of several species of Selaginella, for example Selaginella martensiiSpring and S. grandisMoore. Priestley and Irving (1907) reported that Selaginella has large chloroplasts, up to 0.02 mm. Jagels (1970) showed that such large cup-shaped chloroplasts occur in the adaxial epidermal cells of Selaginella apoda (L.) Spring, S.martensii Spring, S. seperpens (Desv.) Spring, and in the subepidermal cells of S. kraussiana (Kunze) A. Braun. In addition, the occurrence of chloroplasts in Selaginella is not confined in mesophyll as most of vascular plants. For example, S. caulescens Spring (Bold et al., 1987) and S. doederleinii (Wu et al., 2009?) (Fig. 4) have chloroplasts located in both of dorsal and ventral epidermal cells, and mesophyll cells. Sheue et al. (2007) reported five types of chloroplasts, bases on size and number, can be recognized from the dorsal and ventral microphylls of the deep-shade-adapted plant Selaginella erythropus (Mart.) Spring: (1) disk-shaped chloroplasts in the mesophyll; (2) elongated or beadlike chloroplastsarranged as a chain in the elongated, adaxial epidermal cells of the ventral microphylls; (3) trichome chloroplasts; (4) stomatal chloroplasts; and (5) a new type of chloroplast in this plant, a single giant cup-shaped chloroplast, termed a bizonoplast (Figure 1-4).(Sheue et al., 2007). 23

Figure 1-4 This deconvoluted image in pseudocolor was reconstructed from 3D of bizonoplasts (Sheue et al., 2007). Selaginella, a lycophyte plant, is one of the most primitive vascular plants. A family of a single genus, with some 750 species distributed mainly in the tropical zones of the world, with a few species reaching the arctic-alpine zones in both hemispheres (Jermy, 1990). Dimorphic microphylls are characteristic of the dorsiventral species, with two rows of dorsal microphylls and another two rows of ventral microphylls (Bold et al.,1987). Kenrick and Crane (1997) suggested that clubmosses emerge from a poorly resolved grade of extinct Zosterophyllum-like plants. Within clubmosses, early leafy herbaceous fossils such as Baragwanathia and Asteroxylon are basal, and living Lycopodiaceae are resolved as sister group to a calde that comprises the extinct herbaceous Protolepidodendrales, living Selaginella and the predominantly arborescent carboniferous lepidodendrids, including living Isoetes (Figure1-6). (Delete)

Figure 1-5 Simplified phylogenetic tree showing the minimum stratigraphic ranges of selected groups based on megafossils (thick bars) and their minimum implied range extensions (thin lines) (Kenrick and Crane, 1997). In both editions of Flora of Taiwan, 14 species of Selaginella were recorded native to Taiwan (DeVol, 1979; Tsai and Shieh, 1994). They are Selaginella bonininensis Bak., S. ciliaris (Retz.) Spring, S. delicatula (Desv.) Alston, S. doederleinii Heron, S. heterostachys Bak., S. involvens (Sw.) Spring, S. labordei Heron, S. leptophylla Bak., S. moellendorffii Heron, S. nipponica Fr. & Sav., S. remotifolia Spring, S. repanda (Desv. Ex Poir) Spring, S. stauntoniana Spring, and S. tamariscina (Beauv.) Spring. More recently, Chang et al. (2009) reported a new recorded species, S. lutchuensis Koidz., collected from eastern Taiwan. 16 species ofSelaginella were studied in Chao’s “Leaf Morphology of Selaginella P. Beauv. and its 22 Taxonomic Significance in Taiwan”(2007). The aims of this study are: (1) to examine microphyll anatomy in Selaginella species with different habitats from Taiwan, (2) to investigate the diversity and ultrastructure of chloroplasts of Selaginella species from Taiwan with a view (3) to understanding environmental associations of these plant traits. In addition, we also aim to (4) know wether the unique chloroplast, bizonoplast, also occur in any other Selaginella native to Taiwan.

Chapter 2 Materials and methods 2.1. Plant materials Twelve species of Selaginella were studied in this investigation (Table 2-1). They were collected in the fields from Taiwan (Figure 2-1). The photographs were captured by Nikon D200 camera with Nikon AF-S VR MICRO NIKKOR 105mm 1:28 G ED lens (Figure 2-2A~2-13B). S. ciliaris grows on exposed grassy banks under shady grassy environments (at 58.10 μmol m-2s-1). S. boninensis, S.,delicatula, S. doedelleinii, S. heterostachys, S. labordei, S. moellendorffii, and S.remotifolia grow on the forest floors for photosynthesis and survival of those plants at extremely low quantum flux densities (at 7.36~157.15 μmol m-2s-1). S. nipponica was collected on mossy banks under lower levels of light environments (at 60.62 μmol m-2s-1). S. repanda was collected on the exposed hillside under shady trees (at 95.53 μmol m-2s-1). S. stauntoniana grows on drying rocky cliffs under rocky shadow (at 536.9 μmol m-2s-1), S. tamariscina growing on exposed rocks under higher-light environments (at 485.1 μmol m-2s-1) (Figures 2-9A~19B, Table 2-1). Table 2-1 List of Selaginella species collected from Taiwan in this study. No

Figure 2-1 Location map of Selaginella species collected from Taiwan in this study.

A A

B Figure 2-2 Habitat and morphology of S. boninensis.. (A)Plants growing on forest floors in Tongyun, Pintung. The inset shows where measured at 1806.7 Âľmol m-2s-1 of full sunlight and 37.11 Âľmol m-2s-1 of habitat radiation. (B) Main stems long creeping, bearing many short leafy branches very small. 28

B Figure 2-3 Habitat and morphology of S. ciliaris. (A)Plants growing on exposed grassy banks in Sun-Moon Lake. The inset shows where measured at 1854.8 Âľmol m-2s-1 of full sunlight and 58.10 Âľmol m-2s-1 of habitat radiation. (B) Plants are very small. Main stems creeping.to semi-erect.

B Figure 2-4 Habitat and morphology of S. delicatula. (A) Plants growing on the forestfloors in Sitou. The inset shows where measured at 1888.9 Âľmol m-2s-1 of full sunlightand 23.41 Âľmol m-2s-1 of habitat radiation. (B) Main stems erect.

B Figure 2-5 Habitat and morphology of S. doederleinii. (A) Plants growing on the forestfloors in Sitou. The inset shows where measured at 1957.7 Âľmol m-2s-1 of full sunlightand 2.08 Âľmol m-2s-1 of habitat radiation. (B) Main stems erect, bearing longrhizopores.

B Figure 2-6 Habitat and morphology of S. heterostachys. (A) Plants growing on the forestfloors under shade environments. The inset shows where measured at 1787.6 Âľmolm-2s-1 of full sunlight and 4.12 Âľmol m-2s-1 of habitat radiation. (B) Main stemscreeping to semi-erect.

B Figure 2-7 Habitat and morphology of S. labordei. (A) Plants growing on the forest floors of high mountains. The inset shows where measured at 1825.0 Âľmol m-2s-1 of full sunlight and 43.41 Âľmol m-2s-1 of habitat radiation. (B) Main stems erect.

B Figure 2-8 Habitat and morphology of S. moellendorffii. (A) Plants growing in shades ofvalleys in Lienhuachih. The inset shows where measured at 1855.7 Âľmol m-2s-1 of fullsunlight and 157.19 Âľmol m-2s-1 of habitat radiation. (B) Main stems erect.

B Figure 2-9 Habitat and morphology of S. nipponica. (A) Plants growing on mossy banks in Shihtzulu. The inset shows where measured at 1803.5 Âľmol m-2s-1 of full sunlight and 60.52 Âľmol m-2s-1 of habitat radiation. (B) Main stems creeping.

B Figure 2-10 Habitat and morphology of S. remotifolia. (A) Plants growing in montaneforests in Alishan. The inset shows where measured at 1868.5 Âľmol m-2s-1 of fullsunlight and 7.36 Âľmol m-2s-1 of habitat radiation. (B) Plants widely creeping.

B Figure 2-11 Habitat and morphology of S. repanda. (A) Plants growing on exposedhillsides in Lantan. The inset shows where measured at 1849.7 Âľmol m-2s-1 of fullsunlight and 95.53 Âľmol m-2s-1 of habitat radiation. (B) Main stems erect.

B Figure 2-12Habitat and morphology of S. stauntoniana. (A) Plants growing on exposedrocks in Southern Cross-Island Highway. The inset shows where measured at 1976.2Âľmol m-2s-1 of full sunlight and 536.9 Âľmol m-2s-1 of habitat radiation. (B) S.stauntoniana is a resurresction plant.

B Figure 2-13 Habitat and morphology of S. tamariscina. (A) Plants growing on exposed rocks under higher-light environments in Kukuan. The inset shows where measured at 1923.6 Âľmol m-2s-1 of full sunlight and 485.1 Âľmol m-2s-1 of habitat radiation. (B) S. tamariscina is a novelty resurresction plant. The inset showing that S. tamariscina curls up into a tight ball during a drought.

2.2. Free hand-sections of fresh plant specimens and light microscopy Immediately after collection, fresh free-hand sections were made with razor blade into 1 mm wide segments and viewed unstained. Living specimens were mounted in water onto a microscope slide and photographed (unstained) with a light microscope (Olympus BX51 and DP21 associated software, Tokyo, Japan).

2.3. Semi-thin sections and light microscopy Three dorsal and three ventral microphylls from each of three individual plants were sampled for anatomical and chloroplast ultrastructure investigation. Aerial branches, harvested after 24 hours, were cut into small pieces (2.0×2.0×0.5 mm) and fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3) for 4 hours at room temperature. After three washings in buffer for 30 minutes each, the specimens were postfixed in 1% OsO4 in the same buffer for 4 hours. After dehydration through an ethanol series, the material was infiltrated for 3 days and embedded in Spurr’s resin (Spurr, 1969). The embedded material was then polymerized in an oven at 70 ℃ for 12 hours. For light microscopy, semi-thin sections (0.5 μm) were cut on glass knives with an Ultracut E Microtome (Reichert-Jung, Wien, Austria) and stained with 1% toluidine blue (Feder and O’Brien, 1968) for examination with a light microscope (Olympus BX51, Tokyo, Japan). Imaged were digitally captured using Olympus BX51 and DP21 associated software (Tokyo, Japan).

Figue 2-1 The graph shows the ventral and dorsal microphyll of S. remotifolia.

2.4. Confocal scanning light microscopy Three ventral microphylls from each of three individual plants were sampled for microphyll structure and chloroplast investigation. Aerial branches, harvested after 24 hours, were cut into small pieces (2.0 × 2.0 × 0.5 mm3) for observation. Chloroplast size and number per cell was estimated from 99 adaxial ventral epidermal cells by a confocal laser scanning microscope (Leica TSC-SP5, Wetzlar, Germany). Theexcitation wavelength was 633 nm UV405 and the emission wavelength was 649–719 nm for observation with using a 40x oil immersion objective.

2.5. Ultra-thin sections and transmission electron microscopy (TEM) Leaf samples prepared for light microscopy were also used for transmission electron microscopy (TEM). Ultra-thin sections (70 nm) were cut on glass knives with an an Ultracut E Microtome (Reichert-Jung, Wien, Austria). Picked up onto 200 mesh formvar coated copper grids. Sections were post stained with uranyl acetate (5% in 50% methanol) followed by lead citrate (1% in water) (Reynolds, 1963) for examination with Hitachi H7100 (Tokyo, Japan) Transmission Electron Microscope (TEM) at 75.0 kV and associated software Amt V542 Image Capture Engine.

Chapter 3 Results The major results are presented in Figures 3-1~3-74; the more general relationships and implications are considered briefly below. 3. 1. The mesophyll of S. ciliaris is poorly developed. The lamina is 51.08±1.49 μm thick. It consists of a single peripheral layer of cells. And chloroplasts are found in both the adaxial and abaxial epidermal cells (figure3-2). The light micrographs of ventral microphyll of S. ciliaris show the mesophyll is poorly developed; there are one layer of mesophyll cell adjacent to the vein, grading to one layer or air space near the margin (figure 3-2). Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, range from 16~4.2 μm. The stomata are obviously found on the ventral epidermis (Figure 3-2B). Confocal laser scanning micrographs showing in the dorsal epidermal cells of S. cilairis are 3~4 chloroplasts, with mean size of 7.76±1.26 x 2.92±0.38 μm (length x width), which are arranged in close to margin (Figures 3-2). The TEM micrographs of S.ciliaris showing the chloroplasts are elonaged in shape, in dorsal epidermal cells, are distributed along the anticlinal walls (Figure 3-3). The chloroplasts are well developed with large abundant stacked granal and unstacked stromal thylakoids facing the cell wall. Some starch grains and plastoglobuli were found in the stroma between the internal membranes (Figure 3-23). The chloroplasts in mesophyll cells are cup-shaped and oval to lenticular in shape. The chloroplasts have many thylakoids as well as some distinct grana stacks, and there is some lipid drops (Figure 3-4). The grana contains 8~12 layers of thylakoid membranes per stack, 0.47–0.9 μm in size (n = 11). The ventral epidermis showed a open stoma containing chloroplasts and a large vacuoles (Figure 3-5).

Figure 3-1 Light micrographs of ventral microphyll of S. ciliaris. (A) The entire view shows that the epidermis is a single peripheral layer of cells. (B) The transverse section showing a open stoma in the abaxial leaf surface. (C) Chloroplasts are found in both the adaxial and abaxial epidermal cells. Note that mesophyll is poorly developed.

Figure 3-2 The sequence of confocal micrographs (A) â&#x20AC;&#x201C; (D) show 3~4 chloroplasts in 43

the adaxial ventral epidermal cells of S. ciliaris.

Figure 3-3 The TEMmicrographs of S. ciliaris. (A) The chloroplasts are elongated in dorsal epidermal cells, which distributed along the anticlinal walls, X 4,000. (B) The chloroplast shows the large abundant grana stacks and thylakoid stroma lamellae facing the cell wall, X 20,000. (C) The stacked grana contains 8 â&#x20AC;&#x201C; 12 layers of thylakoid membranes, X 30,000. (D) The plastoglobuli were found in the stroma between the internal membranes, X 50,000.

Figure 3-4 The TEMmicrographs of S. ciliaris showed (A) Plastoglobules are often associated with thylakoid membranes. (B) Starch grains and well-developed grana system are visible. 45

Figure 3-5 The TEMmicrographs of S. ciliaris showed (A) the chloroplasts, in mesophyll cells, are cup-shaped and oval to lenticular in shape. (B) The cup-shaped chloroplast is visible at higher magnifaction. (C) The cup-shaped chloroplast has two starch grains and many unstacked thylakoids. (D) The chloroplasts have many interconnected thylakoid membrnes as well as 17-21 layers of thylakoids per grana stacks, and there is some plastoglobuli.

Figure 3-6 The TEM micrograph of S. ciliaris clearly showed the open pore and the guard cells.

3. 2. One giant chloroplast is found in the adaxial ventral epidermis of S. delicatula. The anatomical structure of the microphyll from fresh hand-sections of S. delicatulal contains chloroplasts in boththe dorsal and the ventral epidermis and spongy mesophyll. Note that palisade mesophyll is absent. The dorsal epidermal cells have abundant green chloroplasts (Figure 3-7). There are large intercellular air spaces are near the vein (Figures 3-8). The lamina is 74.30±1.72 μm thick. The light micrographs of ventral microphyll of S. delicatula showing the the lamina consists solely of epidermal cells andthe mesophyll comprises one or two layers of irregularly-shaped cells with intercellular air spaces. Note the various sizes of chloroplasts in the dorsal conical epidermal cells, mesophyll cells, and ventral elongated epidermal cells (Figure 3-8). Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 6.07±0.32 µm. The epidermal cell curvature (θ) is 35.15∘±0.86. The stomata are obviouslyfound on the ventral epidermis (Figure 3-8). In addition, beneaththe stoma is a large substomatic cavity. Confocal laser scanning micrographs show S. delicatula has only one giant chloroplast, with mean size of 14.56±0.73 x 13.96±0.58 µm (length x width), which are arranged in middle within per adaxial epidermal cell. The chloroplast is almost giant enoughto fill the whole epidermal cell (Figure 3-9). The TEMmicrographs showing one giant chloroplast inthe dorsal epidermal cell, contains grana and stroma thylakoids and dense plastoglibuli, and a nucleus with nucleolus are presented (Figures 3-10, 3-11, 3-12). The mesophyll cell contains a circular chloroplast with manythylakoids as well as some distinct grana stacks, andthere is some lipid drops (Figure 3-13). The ventral epidermal cell shows one circular chloroplast also (Figure 3-14).

Figure 3-7 Fresh free-hand sections of the ventral microphyll from S. delicatula. (A) Numerous chloroplasts are clearly observed on the dorsal and the ventral epidermis and mesophyll, (B) There are large intercellular air spaces are near the vein. (C) The cuticle is seen.

Figure 3-8 Light micrographs of ventral microphyll of S. delicatula. (A) The lamina consists solely of epidermal cells and the mesophyll comprises one or two layers of irregularly-shaped cells. (B) Note the various sizes of chloroplasts in the adaxial conical epidermal cells, mesophyll cells, and ventral elongated epidermal cells. (C) The chloroplasts in mesophyll cells are greater than those in epidermal cells.

Figure 3-9 The sequence of confocal micrographs (A)~(D) illustrate there is only one giant chloroplast per ventral epidermal cells in S. delicatula from top view.

Figure 3-10 The TEMmicrogramshows the overview of S. delicatula. Note there is one giant chloroplast occupied in the Dorsal epidermal cell, X 1,000.

Figure 3 11 The TEM micrographs of S. delicatula. (A) The adaxial ventral epidermial cell is mammray-like and contains one chloroplast,. (B) The part of the chloroplast is at higher magnification.

Figure 3-12 The TEMmicrographs of S. delicatula. (A) A giant chloroplast is accumulated plastoglobuli and many thylakoids, X 3,500. (B) Agiant chloroplast in the dorsal epidermal cell, contains grana and stroma thylakoids and dense plastoglibuli, X 3,500. (C) Agiant chloroplast and a nucleus with nucleolus are presented in the epidermal cel, X 5,000. 51

Figure 3-13 The TEM micrographs of S. delicatula. (A) The mesophyll cell contains a circular chloroplast. (B) The part of the chloroplast is at higher magification.

Figure 3-14 The TEM micrographs of S. delicatula. (A) The ventral epidermal cell shows one circular chloroplast. (B) The circular chloroplast in the ventral epidermal cell are shown here at higher magnification. The chloroplast contains largely unstacked thylakoids and a mass of plastoglobuli. (C) A chloroplast and a nucleus with nucleolus are visible in ventral epidermal cell. (D) The ventral epidermal cell wall is more thicker.

3. 3. There is normally one giant chloroplast per ventral ventral epidermal cell of S. doederleinii. Photographs of freshfree-hand sections from S. doederleinii showingthe dorsal epidermal cells differentiate as con-shaped cells with large chloroplast fillingthe base of the cell. There are three or four layers of mesophyll adjacent tothe vein. The mesophyll tissue is less well developed gradingto the margin (Figure 3-16). The lamina is 122.55±3.91 μm thick. Light micrographs of ventral microphyll of S. doederleinii showing the microphyll is broader than other Selaginella species in Taiwan. The chloroplasts are most clearly revealed inepidermal cells. The microphyll interior has irregularly shaped spongy cells with abundant intercellular spaces. The mesophyll tissue is less well developed grading to one layer near the margin(Figure 3-17). Note that palisade mesophyll is absent. Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 8.03±0.51µm. The epidermal cell curvature (θ) is 40.55∘± 3.17. The stomata are obviously found on the ventral epidermis (Figure 3-18). Confocal laser scanning micrographs show there is only one giant chloroplast per ventral epidermal cells in S. doederleinii, with diameters of up to 22µm(22.95±1.68 × 21.14±2.45). The single chloroplast is so big and occupies most of the ventral epidermal cell (Figure 3-19). The TEM micrographs of S. doederleinii showthat It is characterized by one giant chloroplast per dorsal epidermal cell. The chloroplast in the dorsal epidermal cell contains more starch grains and plastoglobuli (Figure 3-20,3-21,3-22 ). The mesophyll has many irregular cells, in which the chloroplasts are circular or oval in shape, with very numerous thylakoidmembranes (Figure 3-21). The ventral epidermal cell contains a chloroplast, which possess numerous grana and starch grains (Figure 3-22).

Figure 3-15 Photographs of fresh free-hand sections from S. doederleinii. (A) The dorsal epidermal cells differentiate as con-shaped cells with large chloroplast filling the base of the cell. (B) There are three or four layers of mesophyll adjacent to the vein . (C) The mesophyll tissue is less well developed grading to the margin.

Figure 3-16 Light micrographs of ventral microphyll of S. doederleinii. (A) The transverse section showing the leaf S. doederleinii is broader than other Selaginella species in Taiwan. (B) The chloroplasts are most clearly revealed in epidermal cells. (C) The various sizes of chloroplasts in the dorsal epidermal cells, mesophyll cells, and ventral epidermal cells. (D) The leaf interior has irregularly shaped spongy cells with abundant intercellular spaces. (E) The mesophyll tissue is less well developed grading to one layer near the margin.

Figure 3-17 The sequence of confocal micrographs (A)~(D) illustrate there is only one giant chloroplast per ventral epidermal cells in S. doederleinii fromtop view.

Dorsall epidermis Dorsall epidermis

Ventral epidermis Figure 3-18 The TEMmicrographs of S. doederleinii. (A) Alow magnifaction overview, It is characterized by one giant chloroplast per dorsal epidermal cell. (B) The chloroplast in the dorsal epidermal cell contains more starch grains and plastoglobuli. (C) The mesophyll has many irregular cells.

Figure 3-19 The TEMmicrographs of S. doederleinii. (A) The chloroplast is well developed with many grana stacks as well as numerous starch grains, and there is accumulation of plastoglobuli. (B) The giant chloroplast composes of numerous starch grains between the thylakoids and plastoglobuli. (C) The giant chloroplast has many densely plastoglobuli in clusters.

Figure 3-20 The TEM micrographs of S. doederleinii. (A) The giant chloroplast is shown many grana with some starch grains and plastoglobuli in clusters, (B) The giant cup-shaped chloroplast is shown many grana with some starch grains and plastoglobuli in clusters.

Figure 3-21 The TEM micrographs of S. doederleinii. (A) The mesophyll chloroplasts showing that grana contain very numerous thylakoid membranes, X 3,000. (B) The part of the chloroplast is at higher magnification.

Ventral epidermis

Figure 3-22 The TEM micrographs of S. doederleinii. (A) The ventral epidermal cell contains a chloroplast, which possess numerous grana and starch grains,. (B) Part of the ventral epidermal cell, the abundant grana stacks is shown in the chloroplast at higher magnification.

3. 4. Abizonoplast was found in the ventral epidermal cell of S. heterostachys. The lamina is 123.95 ± 2.25μm thick. The light micrographs of ventral microphyll of S. heterostachys showing that It consists of a single peripheral layer of cells. The dorsal epidermal cells clearly showmuch chloroplasts, which are aggregatedin the base of the cells. Athin cuticle which often became detachedduring the embedding process, overlies the dorsal epidermal cells(Figure 3-15). The mesophyll tissue is 3~4 layers of spongy cells near the vein, grading to one layer or air space near the margin, and lacks of palisade cells(Figure 3-15). Radius of curvature measurements, taken from invididual cells in cross-sections of microphyll, is 6.51 ± 1.26 µm. The epidermal cell curvature (θ) is 38.05∘± 3.84. Stamata are present on the dorsal surface of the microphyll and are localized near the vein(Figure 3-15). Confocal laser scanningmicrographs showS. heterostachys has four chloroplasts on top view, with mean size of 16.32 ± 1.64 × 8.52 ± 1.86 µm (length x width) (Figure 3-21).But the confocal laser scanning migraphs showS. heterostachys has one chloroplast with deep lobe on lateral section view(Figure 3-20). The TEMmicrograph of S. heterostachys showingthe chloroplasts are elonaged in shape, in dorsal epidermal cells, are distributed along the anticlinal walls (Figure 3-22). The chloroplasts are well developed with large abundant stacked granal and unstacked stromal thylakoids facing the cell wall (Figure 3-21). The chloroplasts in mesophyll cells are cIrcular. The chloroplasts have many thylakoids as well as some distinct grana stacks(Figure3-23). The stacked grana contains 8 – 12 layers of thylakoidmembranes, 0.47– 0.9 µm in size (n=11). The dorsal epidermis showed a open stoma containing chloroplasts and a large vacuoles (Figure 3-22). Unusual chloroplastsin terms of size and thylakoid membrane stacking have been described previously inseveral deep-shade plants. Inthis study, Asurprising observation is that thylakoids groupped inparallel strands extendfrom one side of the plastid tothe other (Figure 3-21).a singlegiant cup-shaped chloroplast, termed a bizonoplast, was found in the abaxial epidermal cells of the dorsal microphylls and the adaxial epidermal cells of the ventral microphylls in thedeep-shade spike moss Selaginella erythropus. Bizonoplasts aredimorphic in ultrastructure: the dorsal zone is occupied by numerous layers of 2–4stackedthylakoid membranes while the ventral zone contains both unstacked stromal thylakoids and thylakoidlamellae stacked in normal grana structure oriented in different directions. In contrast, other cell types in the microphyll scontain chloroplasts with typical structure. This unique chloroplast has not been reported from any other species(Sheue et al.,2007).

Table 3 1 Comparing in the feature of chloroplast of ventral microphyll between S.heterostachys and S erythropus

S erythropus

S.heterostachys

Chloroplast number

Chloroplast shape

Chloroplast size

Grana layers

Native from

One

Cup-shaped

19.6 x 13.4 3x 17.0µm

15-25

Brazil

one

Cup-shape with lobe

16.32 ± 1.64 × 8.52 ± 1.86 µm

8~12

Taiwan

Figure 3 23 Light micrographs of ventral microphyll of S. heterostachys. (A) The dorsal epidermal cells clearly show much chloroplasts.(B) A thin cuticle which often became detached during the embedding process, overlies the dorsal epidermal cells. (B) Stamata are present on the abaxial surface of the microphyll.(C) Stamata are localized near the vein. (D) The chloroplasts in the dorsal epidermis are aggregated in the base of the cells.

Figure 3 24 The sequence of confocal micrographs (A)~(D) show 4 chloroplasts in the ventral epidermal cells of S. heterostachys.

Figure 3 25 The confocal micrographs (A)~(B) show one chloroplast in the ventral epidermal cells of S. heterostachys. with deep lobe on lateral section side view(phtoed by Sheue et al.,2010).

Figure 3-26 (A) The TEM microgam shows the overview of S. heterostachys, X 2,000. (B) Alow magnifaction overview. It is characterized by prominent silica bodies on the dorsal epidermal cells. (C) The TEMmicrograph of S. heterostachys showing the chloroplasts are elonaged in shape, in dorsal epidermal cells, are distributed along the anticlinal walls. (D) Ahigher magnifaction overview. The mitochondron and thylokoids were observed in the the dorsal epidermal cells.

Figure 3-27 (A) The adaxial cell has has a great chloroplast with a mitocondron ana nucleus.. (B) The TEM micrograph shows the mitocondria are gathered near the chloroplast. (C) The TEM micrograph shows that the miticondron is close to the chloroplast. And the chloroplast shows long strands of thylakoids instead of the normal grana and fret membranes,.(D) The chloroplast shos the unstacked thylakoids.

Figure 3-28 (A) Asurprising observation is that thylakoids groupped inparallel strands extend fromone side of the plastid to the other, .(B) Alarge magnifaction of the chloroplast. The chloroplast shows long strands of thylakoids and clear mitochondria.

Figure 3-29 (A) It was bservedthat there are two circular chloroplast in the mesophyll cell.(B) The organelle is composed of chloroplasts and abundant mitocondria.

Figure 3-30 (A) The stoma showing with many strch grins in the close condition. (B) The stoma appears to the normal phaneroganic type, with two guard-cells, each provided with big empty-looking vacules, and the chloroplast has a variable number of starch grains.

There are two giant chloroplasts per adaxial ventral epidermal cell of S. labordei. The lamina is 86.34±2.57μm thick. Light micrographs of ventral microphyll of S. lbordei showingthe epidermis is composed of a single layers (Figure 3-23).The chloroplasts are most clearly revealed inepidermal cells. The outer surface of the epidermal cells are often convex, andthe chloroplasts are tightly packed within these inner region Figure 3-23). The stoma is present distinctively with a large substomatic cavity(Figure 3-23). The mesophyll is loose .The leaf interior has irregularly shaped spongy cells with abundant intercellular spaces. The mesophyll tissue is less well developed grading to one layer near the margin (Figure 3-23). Radius of curvature measurements, takenfrominvididual cells in cross-sections of leaf, is 5.37±1.08µm. The epidermal cell curvature (θ) is 41.09∘±2.95.The stomata are obviouslyfound on the ventral epidermis (Figures 3-23).Confocal laser scanning micrographs showthere is only one giant chloroplast per adaxial epidermal cells in S. labordei, with diameters of upto 22 µm (22.95±1.68 × 21.14±2.45). The single chloroplast is so bigand occupies most of the dorsal epidermal cell (Figure 3-24). The TEMmicrographs of S. labordei showthat It is characterized by one giant chloroplast per upper epidermal cell. The chloroplast inthe upper epidermal cell contains more starch grains and plastoglobuli (Figures 3-25). The mesophyll has many irregular cells, in which the chloroplasts are circular or oval in shape, with very numerous thylakoid membranes (Figure 3-26).The ventral epidermal cell contains a chloroplast, which possess numerous grana and starch grains (Figure 3-26).

Figure 3-31 Light micrographs of ventral microphyll of S. labordei. (A) The epidermis is composed of a single layer, lacking a hypodermis. (B).The outer surface of the epidermal cells are often convex, and the chloroplasts are tightly packed within these inner region. (C) The stoma is present distinctively with a large substomatic cavity. (D) The mesophyll is loose and the lamina consists solely of epidermal cells. 74

Figure 3-32 The sequence of confocal micrographs (A)~(D) showing the epidermal cells of S. labordei are slightly domed in shape, although appear circular likely flattened, which contain 2 chloroplasts in hemisphere-shaped and in circular.

Figure 3-33 The TEM micrographs of ventral microphyll of S. labordei. (A) The epidermis have large chloroplast (B).The large chloroplast is occupied the base of the epidermal celll. (C) The chloroplast has much gplastoglobuli.

Figure 3-34 The TEM micrographs of mesophyll cell in S.labordei. (A) The chloroplast stroma whin which stacked grana thylakoids and unstacked stroma thylakoids can be reconized. (B) A high megnication overview. Grana are internected by mutiple stroma thylakoids. (C) Futher enlargement of thylakoids membranes. Showing stacking of thylakoids membranes.

3. 6. The mesophyll cells are dense in S. moellendorffii Photographs of fresh, unstained hand sections from S. moellendorffiii. showing large amount of chloroplast is clearly presented inthe dorsal epidermis (figure39A).The spongy mesophyll cells are dense (Figure 3-27). The cuticle was coting around the dorsal and ventral epidermis.(Figure 3-27). The lamina is 122.55±3.91 μm thick. Light micrographs of ventral microphyll of S. moellendorffiii showing spongy mesophyll cells are dense(Figur 3-28). The transverse sectionshowing a large quantity of spongymesophyll cells are variously shaped in the leaf (Figure 3=32 C). The mesophyll cells contain much chloroplasts, with numerous small Intercellular spaces(Figure 3-28). Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 5.04 ± 0.60µm. The epidermal cell curvature (θ) is 27.96∘± 3.84. The stomata are not clearly.Confocal laser scanning micrographs showthere is only one giant chloroplast per darsal epidermal cells in S. moellendorffiii, with diameters of up to 22 µm (22.95±1.68 × 21.14±2.45). The single chloroplast is so big and occupies most of the ventral epidermal cell (Figure 3-29). The TEMmicrographs of S. moellendoffii showthat It is characterized by one giant chloroplast per dorsal epidermal cell. Mitochondria are accumulated alongthe outer periclinal wall togather with chloroplast (Figure 3-30,3-31). The chloroplast in the dorsal epidermal cell contains more starch grains and plastoglobuli (Figures 3-32). The mesophyll has many irregular cells, in which the chloroplasts are circular or oval in shape, which possess numerous grana with very numerous thylakoid membranes and starch grains (Figure 3-33).

Figure 3-35 Photographs of fresh, unstained hand sections fromS. moellendorffiii. (A) Large amount of chloroplast is clearly presented in the dorsal epidermis. (B) spongy mesophyll cells are dense. (C) The cuticle was coting around the dorsal and ventral epidermis.

Figure 3-36 Light micrographs of ventral microphyll of S. moellendorffii. (A) The spongy chlorenchyma ldensely is arranged in the lamina.(B) The transverse section showing a large quantity of spongy mesophyll cells are variously shaped in the microphyll. (C) The mesophyll cells contain much chloroplasts, with numerous small Intercellular spaces.

Figure 3-37 The sequence of confocal micrographs (A)-(D) show 3~4 chloroplasts on the margin of the ventral epidermal cells of S. mollendorffi. Dorsal epidermis Dorsal epidermis

Figure 3 38 (A) Alow magnicication overview of S. moellendorffii,.(B) The TEM micrograph shows that the dorsal epidermal cell contains large chloroplasts with a nucleus. (C) The dorsal epidermal cells have chloroplasts, a nucleus, and nucleolus, as well as mitochondria, X 6,000.

Figure 3-39 (A) Ahigher magnicication of S. moellendorffii. (B) The chloroplast has abundant grana as well as starch grains and plastoglubi.(C) Chloroplast typically contains many thylakoids per granum.

Figure 3-40 (A) Accumulation of mitochondria with chloroplasts and nucleus in the epidermal cell of S. moellendorffii. Mitochondria are accumulated along the outer periclinal wall togather with chloroplas. (B) Ahigher magnicication of S. moellendorffi.(C) Grana are interconnected by multiple stroma thylakoids.

Figure 3-41 (A) The disc-shaped chloroplasts in the mesophyll cells have many grana thylakoids and stach grains. (B) Detail of a disc-shaped chloroplast in the mesophyll cel. (C) The mesophyll cell chloroplast contains numerous starch grains in the stroma between the agranal and internal membranes.

3.7. S. nipponica leaf obviously shows a single layer of cuticularized epidermaial cells. Photographs of fresh free-hand sections of S. nipponica. Showing a distinct waxy cuticle surrounds both the epidermis (.figure 3-34) The silic bodies occur both in the epidermis (figure 3-34). The spongy parenchyma is composed of loosely arranged chlorenchyma cells with prominent intercellular spaces(figure 3-34). Both the epidermal cells contain a very large number of chloroplasts( figure 3-34). The lamina is 183,09±2.29 μm thick. The lamina is comprised of a single layer epidermis and a single layer mesophyll (figure 3-35A), The chloroplasts in the lower epidermal cells are uniformly distributed around the cell periphery. (figure 3-35B), The spongy mesophyll shows numerous intercellular spaces around the vein (Figure 3-35 C).and an epidermal cell clearlycontains a silica body ((figure 3-35 D). Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 6.10±0.76µm. The epidermal cell curvature (θ) is 32.3-35∘±2.55. Confocal laser scanning micrographs showthere is only one giant chloroplast per dorsal epidermal cells in S.nipponica, with diameters of upto 22 µm (22.95±1.68 × 21.14±2.45). The single chloroplast is so bigand occupies most of the dorsal epidermal cell (figure 3-36). The TEMmicrographs of S. npponicai showthat Silica bodies occur in boththe upper and lower epidermal cells.(Figure 3-37). Silica bodies are visible, usually one, but occasionally more to an epidermal cell,(Figure 3-37). The TEMmicrograph shows there are numerous small thylakoid grana in the chloroplast ( Figure 3-37). The chloroplast shows cup-shaped with deep lobe (Figure 3-38), The ventralr epidermal cell contains a chloroplast withinergranal thylakoids and plastoglobuli (Figure 3-43).

Figure 3-42 Photographs of fresh free-hand sections of S. nipponica. (A) A distinct waxy cuticle surrounds both the epidermis. (B) The silic bodies occur both in the epidermis. (C) The spongy parenchyma is composed of loosely arranged chlorenchyma cells with prominent intercellular spaces. (D) Both the epidermal cells contain a very large number of chloroplasts.

Figure 3 43 Light micrographs of ventral microphyll of S. nipponica. (A) The lamina is comprised of a single layer epidermis and a single layer mesophyll. (B) The chloroplasts in the ventral epidermal cells are uniformly distributed around the cell periphery. (C) The spongy mesophyll shows numerous intercellular spaces around the vein. (D) An epidermal cell clearly contains a silica body (circular). 85

Figure 3-44 The sequence of confocal micrographs (A)-(D) showing the epidermal cells of S. nipponica are slightly domed in shape, although appear circular likely flattened, which contain 5 chloroplasts.

Figure 3-45 ( A) Silica bodies occur in both dorsal and ventral epidermis. (B) The TEM micrograph shows there are numerous small thylakoid grana in the chloroplast.(C) Silica bodies are visible, usually one, but occasionally more (circle) to an epidermal cell.

Figure 3-46 The chloroplast shows cup-shaped with deep lobe.

Figure 3-47 The ventral epidermal cell contains a chloroplast with inergranal thylakoids and plastoglobuli. 87

3.8. Stomata are observed rarely on the leaf margin as well as on the ventral epidermis of S. remotifolia. Photographs of freshfree-hand sections from S. remotifolia showing the dorsal epidermal cells are filled with chloroplast (figure 3-4004 A). The vein is near the dorsal epidermis and belowthe vein is large intercellular space(figure 3-40B)..The mesophyll tissue is less well developed gradingto the margin (figure 3-40). The lamina is 122.55±3.91 μm thick. Light micrographs of ventral microphyll of S remotifolia showing the mesophyll comprises one or two layers of irregularly-shaped cells.(figure 3-41). Chloroplasts in mesophyll are more than epidermis (figure 3-41) The spongy mesophyll shows large intercellular spaces around the vein (figure 3-41). An strict feature is stomata was found on the leaf margin as well as on the ventral epidermis(figure 3-41)or has irregularly shaped spongy cells with abundant intercellular spaces. The mesophyll tissue is less well developed grading to one layer near the margin (figure 3-41). Radius of curvature. measurements, taken from invididual cells in cross-sections of leaf, is 8.16±0.26 µm. The epidermal cell curvature (θ) is 32.61∘±2.26. The stomata are obviouslyfound on the ventral epidermis (figures 3-41). Confocal laser scanning micrographs showthere is only one giant chloroplast per ventral epidermal cells in S. remotifolia, with diameters of upto 22 µm (22.95±1.68 × 21.14±2.45). The single chloroplast is so bigand occupies most of the ventral epidermal cell (figure 3-42). The TEMmicrographs of S.remotifoliai showthat The chloroplasts are arranged in cup-shaped showing distinct grana inthe dorsal epidermal cell The chloroplasts are located at the base of dorsal epidermal cells. Chloroplasts are arrangedalong the cell wall cells (figure 3-43). Four disc-shaped chloroplasts are typically found in the spongy mesophyll cells. In the mesophyll cells, the chloroplasts are oriented fromthe consistent direction. (figure 3-44).

Figure 3-48 (A)The dorsal epidermisal cells arefilled with chloroplasts. (B) The vein is near the dorsal epidermis and below the vein is large intercellular space. (C)The mesophyll tissue is less well developed grading to the margin.

Figure 3-49 Light micrographs of ventral microphyll of S. remotifolia. (A) The mesophyll comprises one or two layers of irregularly-shaped cells. (B) Chloroplasts in mesophyll are more than epidermis (C) The spongy mesophyll shows large intercellular spaces around the vein. (D) An strict feature is a stomata was found on the leaf margin.

Figure 3-50 The sequence of confocal micrographs (A)-(D) illustrate there are 6 chloroplasts in the ventral epidermal cells of S. remotifolia.

Dorsal epidermis

Dorsal epidermis cell

Figure 3-51 (A) The chloroplasts are arranged in cup-shaped showing distinct grana in the dorsal epidermal cell. (B) The chloroplasts are located at the base of dorsal epidermal cells. (C) Chloroplasts are arranged along the cell wall.

Dorsal epidermis cell

Figure 3-52 The chloroplasts are arranged in cup-shaped showing distinct grana in the dorsal epidermal cell. (B) The chloroplasts are located at the base of dorsal epidermal cells. (C) Four disc-shaped chloroplasts are typically found in the spongy mesophyll cells.(D) In the mesophyll cells, the chloroplasts are oriented fromthe consistent direction.

3.9. There is normally one giant chloroplast per adaxial ventral epidermal cell of S. repanda. Photographs of freshfree-hand sections from S. repanda showingthe dorsal epidermal cells differentiate as con-shaped cells with abundant chloroplasts fillingthe base of the cell. There are three or four layers of mesophyll adjacent to the vein. The mesophyll tissue is less well developed gradingto the margin . The lamina is 5±3.91 μm thick. Light micrographs of ventral microphyll of S. repanda showing the leaf is consists of five layers of irregular spongy mesophyll cells near the vein. The chloroplasts are most clearlyrevealed in epidermal cells. The microphyll interior has irregularlyshaped spongy cells with abundant intercellular spaces. The mesophyll tissue is less well developed grading to one layer near the margin(Figure 3-46). Radius of curvature measurements, taken from invididual cells in cross-sections of microphyll, is 8.03±0.51µm. The epidermal cell curvature (θ) is 40.55∘± 3.17. The stomata are obviouslyfound on the abaxial epidermis (Figures 3-46).Confocal laser scanning micrographs showthere are 4-5 chloroplast per dorsal epidermal cells in S. repanda, insize with 15.77±1.2.17x.58±1.10. (Figure 3-47`). The TEMmicrographs of S. repanda showthat It is characterized by two chloroplast locatedat the bottom of the dorsal epidermal cell.. The dorsal epidermal cells have thick cell walls and chloroplasts arranged in cup-shaped (figure 3-48). The TEM micrograph shows the epidermal cell has chloroplasts with many mitochondria and a giant vacuole(Figure 3-49). The chloroplasts arranged along the base of the cell because the high sunlight (figure 3-50).

Figure 3-53 (A) The mesophyll tissue is less well developed grading to the margin (B) Therearethreeor four layers of mesophyll adjacent to the vein. (C) Thedorsal epidermal cells have abundant chloroplasts.

Figure 3-54 Light micrographs of ventral microphyll of S.repanda. (A) The transverse csection showing a single epidermal layer and more loose mesophyll. (B) The dorsal epidermis is thicker than the ventral epidermis. (C) The lamina consists of five layers of irregular spongy mesophyll cells near the vein.(D) The mesophyll cells are filled with chloroplasts.

Figure 3-55 The sequence of confocal micrographs (A)-(D) show 3-4 chloroplasts in the dorsal epidermal cells of S. repanda.

Figure 3-56 The TEM micrograph shows the overview of S. repanda in lower magnication.

Figure 3-57 The dorsal epidermal cells have thick cell walls and chloroplasts arranged in cup-shaped. 98

Figure 3-58 The TEM micrograph shows the epidermal cell has chloroplasts with many mitochondria and a giant vacuole.

Figure 3-59 The chloroplasts arranged along the base of the cell.

Figure 3-60 Amass of plastoglobuli gather in the stroma.

Figure 3-61 The mesophyll cell has two chloroplasts and a giant nucleus.

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Figure 3-62 The TEM micrograph shows a mesophyll cell at higher magnication.

Figure 3-63 The mesophyll cell has clear grana and plastoglobuli in group.

101

Figure 3-64 The overview of a mesophyll cell.

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3. 10. The mesophyll is differentiated into typical palisade tissue and spongy tissue in S. stauntoniana. Photographs of freshfree-hand sections from S. stauntoniana showingthe dorsal epidermal cells differentiate as con-shaped cells with large chloroplast fillingthe base of the cell. There are three or four layers of mesophyll adjacent tothe vein. The mesophyll tissue is less well developed gradingto the margin (Figure 3-56). The lamina is 122.55±3.91 μm thick. Light micrographs of ventral microphyll of S. stauntonian showing the microphyll has thick cell wall. The microphyll interior has irregularly shaped spongy cells with abundant intercellular spaces. The mesophyll tissue is less well developed grading to one layer near the margin (Figure 3-56). The microphyll of S. stauntonianais well adaptedto dry conditions with a thick epidermis with stoma recessedintothe into the surface. Beneath the epidermis is the thick walled cells of the hypodermis which helps reduce water evaporation from the leaf. The pine leaf has an endodermis inside the mesophyll whichis not seenin either of the other leaf types observed. The outside surface of the epidermis tissues is usually covered witha waxy substance called cutin, which reduces water loss. In ferns and most fabaxialing plants it is dividedintotwo layers: a palisade layer of tightly packed, vertically elongated cells, one totwo cells thick, directly beneaththe dorsal epidermis. Its cells contain manymore chloroplasts thanthe spongy layer. These long cylindrical cells are regularly arrangedin one tofive rows. Cylindrical cells, withthe chloroplasts close to the walls of the cell, cantake optimal advantage of light. The slight separation of the cells provides maximal absorption of carbon dioxide. This separation must be minimal to afford capillary action for water distribution. In order to adapt to their different environment (such as sun or shade), plants hadto adapt this structure to obtain optimal result. Sun leaves have a multi-layered palisade layer, while shade leaves or older leaves closer tothe soil, are single-layered. Beneath the palisade layer is the spongylayer. The cells of the spongy layer are more rounded and not sotightly packed. There are large intercellular air spaces (substomatal chambers). These cells containless chloroplasts Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 8.03±0.51 µm. The epidermal cell curvature (θ) is 40.55∘± 3.17. The stomata are obviouslyfound on the ventral epidermis (figures3-57). Confocal laser scanning micrographs show4-5 chloroplast in the dorsal epidermal cells of S. stauntonian (figure 3-58) The TEM micrographs of S. stauntoniana showthat the cell walls are very thick (figure 3-59). The mesophyll has many irregular cells, in which the chloroplasts are circular or oval in shape, with very numerous thylakoidmembranes (Figure 3-59,figure 3-60). The palisade cells have many chloroplasts. The chloroplast has manylayers thylakoids membranes and many plastoglobuli (Figure 3-60). Note the well differentiated mesophyll with a palisade mesophyll onthe ventral surface andthe spongymesophyll below (Figutr 3-.59).

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A Palisade mesophyll

Spongy mesophyll

Vein

Figure 3-65 Fresh free-hand sections of S. stauntoniana. (A) The microphyll is differentiated into four distinct tissue layers: dorsal multiple epidermis, a multiple layers palisade parenchyma, spongy mesophyll, and ventrall epidermis, X 400. (B) The cells of the dorsal and ventral epidermis contain no chloroplasts. These are visible, however, in cells of the palisade layer of columnar cells and the spongy mesophyll.

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Figure 3-66 Light micrographs of ventral microphyll of S. stauntoniana. (A) The mesophyll is differentiated into palisade and spongy areas,(B) The mesophyll consists of the tightly-packed, elongate cells of the palisade, the spongy tissue shows extensive intercellular spaces, (C) The microphyll showing more compact mesophyll in which the intercellular spaces are reduced. The multiple epidermis are presented, and no chloroplast is found there. 105

Figure 3-67 The sequence of confocal micrographs (A)-(D) show 4-5 chloroplasts are distributed along the anticlinal walls in the dorsal epidermal cells of S. stauntoniana.

106

Figure 3-68 The TEM micrographs of the microphyll of S. stauntoniana.(A) Mesophyll tissue is differentiated into columnar palisade cells and irregulated spongy cells.(B). The cells of the palisade layer contain several small chloroplasts, which are distributed along the anticlinal walls or oriented with their surfaces facing the vacuole (C) TEM micrograph demonstrates the columnar palisade cells contain several chloroplasts, which have some starch grains.

107

Figure 3-69 The TEMmicrographs of the microphyll of S. stauntoniana.(A),(B),(C) The cells of the palisade layer contain several small chloroplasts.

108

Figure 3-70 The TEM micrographs of the microphyll of S.stauntoniana.(A),(B),The cell walls are thick.

109

Figure 3-71 The TEM micrographs of the microphyll of S..stauntoniana. (A).The palisade cells have many chloroplasts. (B) The chloroplast has many layers thylakoids membranes and many plastoglobuli.

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3. 11. The mesophyll is differentiated into seudo-palisade layers and spongy layers in S. tamariscina. Photographs of fresh free-hand sections from S. tamariscinai showing the dorsal has thick cell wall.The mesophyll tissue is less well developed intopalisade layer and spongy layer.(figure 3-69). The lamina is 122.55±3.91 μmthick. The chloroplasts are most clearly revealed in mesophyll cells. The microphyll interior has irregularly shaped spongy cells with abundant intercellular spaces.(Figure 3-69,Figure 3-70). Radius of curvature measurements, taken from invididual cells in cross-sections of leaf, is 8.03±0.51 µm. The epidermal cell curvature (θ) is 40.55∘± 3.17. The stomata are obviously found on the ventral epidermis (Figure 3-70). Confocal laser scanning micrographs show there are 17 chloroplasts per adaxial epidermal cells in S. tamariscina, in size the range among 5.93±1.08 × 4.01±0.76.(Figure 3-71). The TEM micrographs of S. tamariscinai show that It is characterized that the cell walls are thick and palisade tissue clearly appear. The chloroplast in the palisade cells contain more starch grains and plastoglobuli (figure 3-72, figure 3-73). The mesophyll has many irregular cells, in which the chloroplasts are arranged along the cell wall (figure 3-74).

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Figure 3-72 Fresh free-hand sectios of S. tamariscina. (A) Mesophyll differentied into upper palisade parenchyma and lower spongy parenchyma. (B) The intercellular spaces between the irregularly shaped spongy mesophyll cells within leaf permit free diffusion of gases.

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Figure 3-73 Light micrographs of ventral microphyll of S. tamariscina. (A) A much higher proportion of spongy mesophyll than palisade mesophyll. (B) Stamata are present on the abaxial surface of the leaf and are localized near the vein. (C) The multiple epidermis are presented, and no chloroplast is found there. The spongy mesophyll cells are compact, with numerous small intercellular spaces.

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Figure 3 74 The sequence of confocal micrographs (A)-(D) show about 17 chloroplasts in the dorsal epidermal cells of S. tamariscina.

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Figure 3 75 (A) (B) The TEM micrographs of microphyll of S. tamariscina show thick cell wall. (C)The chloroplasts in the palisade are arranged along the cell wall.

115

Figure 3-76 The chloroplast has plastoglobuli in groups.

Figure 3-77 The mesophyll has many irregular cells.

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Results are sumarried belowtables. Table 3-1. Comparison of anatomical features of adaxial microphyll among 12species of Selaginella collectedfromTaiwan in this study. Table 3-2. The characteristics of chloroplast in adaxial epidermal cells of ventral microphylls of Selaginella inTaiwan observed inlaser confocal microscopes of this study. Figure 3-74. The structure of microphyll of 12 species of Selaginella fromTaiwanin this study.

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Table 3-2 C omparison of anatomical features of adaxial microphyll among 12 species of Selaginella collected from Taiwan in this s tudy. A total of 108 cells were measured from3 individuals per species (Mean Âą SE).

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Table 3 3 The chloroplast size, number p er cell a nd the d istribution pattern in a daxial ep iderm al c ells of ventral microphy lls of Selag ine lla in Taiwa n of this study.

119

Table 3-4 The characteristics of chloroplast in ad axia l epiderma l cells of ventra l microp hylls of S elag ine lla in Taiwa n observ ed in laser confoc al microsc opes of this study (n= 31).

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Chapter 4 Discussion and Conclusion 4.1 Two group microphyll types of Selaginella in this study In this study, the mesophyll tissue is usually absent andthe mesophyll cells are somewhat asymmetrically distributedin the leaves of Selaginella species fromTaiwan. The mesophyll tissue is less well development in the microphylls; there are usually two or three layers of mesophyll adjacent to the vein, grading to one or two layers of cells and air spaces near the margin. Gibson(1897) describedthat the microphyll of Selaginellahas five types in anatomy:A) Martensii-type,which has the epidermis onthe ligular and aligular surfaces of the leaves dissimilar, and a mesophyll consiststing of reticulate parenchyma, B) Braunii-type,which has a distinct palisade-layer aswell as reticulate mesophyll. c) Galeottii-type, ,which stomataoccur on the aligular face only,the guard-cells of which are very frequently folded outwards intothe ajacent epidermal cells, D) Spinosa-type, which the margins of all the leaves are strongly cuticularized, E) Lyallii-type, which stomata occur onthe aligular face, close to and over the midrib, where the epidermal cells are much shorter. But,there were nocriteria in Gibsonâ&#x20AC;&#x2122;s describing(1897). In this study, 12 species of Selaginella from Taiwan were classifiedintotwo groups,one have loose mesophyll, e.g. S.ciliaris,another have dense mesophyll, e.g. S.moellendorffii.And the latter group can be divided into two group, one has no palisade,e.g. S.moellendorffii, another has palisade, e.g. S.tamariscina.(Figure 4-1). 4.2 . The importance of abundant intercellulal space inmesophyll Typically, sun leaves of laminar-leaved plants are smaller and/or more deeply lobed, thicker, andlighter incolor compared to shade plants. Also, sunleaves are commonlymore amphistomatous with well-developed palisade layers, while shade leaves are typically thinner, primarily hypostomatous, and without palisade layers (Allen et al., 1973; Mauseth, 1988; Cui et al., Dickison, 2000; Johnson et al, 2005; Schulze et al., 2005). The intercellular air spaces in the shade leaf are larger than those in the sun leaf (Allen et al., 1973). In contrast, the intercellular spaces are greatlyreduced, particularly in more xerophytic species (Cutler et al., 2008). microphyll that develop under conditions of lowirradiance are thinner, e.g. S. ciliaris, S. delicatula, S. doederleinii, S. labordei, S. heterostachys, S. mollendorffii, S. nipponica, S. remotifolia, and S. repanda they have a mesophyll region composed of no palisade cells and abundant intercellular spaces. Habitat connection can be add more. microphyll that develop under conditions of high irradiance (light intensity c. 95.3~536.9 Âľmol m-2s-1) are thicker, e.g. S. stauntoniana and S. tamariscina, they have more layers of palisade cells and smaller intercellular spaces. Intercellular air spaces play an important role in leaf reflectance (Gates et al., 1965; Allen et al., 1973; Sinclair et al., 1973). Evert et al. (2004) reportedthat light scattering 121

in leaves is largely determined bythe intercellulare air spaces. Because the intercellular spaces of leaves are critical to photosynthetic efficiency (Mauseth, 1988). Scattering arises from the jumpin refractive index (n) between air (n = 1) and cells (n = 1.33 to 1.45), which creates mirror-like reflections (Woolly, 1971). The path of light through a leaf can be thought of as a series of deflections between cells and air spaces. Total intercellular air spaces volume is important, but so is the three-dimensional geometry of that air space (Evert et al., 2004). The spongy mesophyll of leaves contains large intercellular spaces andcells with very irregular structure, and with cell walls oriented at virtually all angels. The pathway of the light inleaves as envisioned by Wills채ttter and Stoll theory (Figure 39, Sinclair et al., 1973). Brief description of theory needed

Figure 4 1 Schematic drawing depicting the Wills채ttter and Stoll theory on the pathway of light through leaves (Sinclair et al., 1973). 4.4 Two major types of mesophyll: The mesophyll is differentiatedinto obvious multi-layered palisade cells and multi-layeredspongy cells In this study, only two species with dense type? Mesophyll. The mesophyll is differentiated into distinctlymulti-layered palisade cells andcompact spongy cells with extensive intercellular spaces in S. tamariscina and S. tamariscina?microphyll (Figures 3-70). The palisade tissue has become specializedin such a way that the efficiency of photosynthesis has been increased. In mesophyll that can be clearly dividedinto palisade and spongy parenchyma and the large majority of the chloroplasts are found in the palisade cells. Because of the shape andarrangement of the palisade cells the chloroplasts can be placed so as to enable the maximum utilization of light (Fahn, 1982). Another important factor that increases photosynthetic efficiency is the presence of a well-developed system of intercellular spaces which is present inthe mesophyll and facilitates rapid gas exchange. Because of the cell arrangement inthe mesophyll large surface areas of the cells are exposed and so brought into contact withair (Fahn, 1982). The most obvious features of xeromorphic leaves are the increasingly developed palisade tissue andthe smaller intercellular spaces (Fahn, 1982).The palisade cells were 122

more densely arranged, andthe spongy mesophyll was more highly developedin the sunlit leaves (Sinclair et al., 1973, Cutler et al., 2008). In this study, two sun-adapted species sport that dense developed mesophyll are foundin their microphylls as previous reports (Sinclair et al., 1973, Cutler et al., 2008). The cells of typical palisade parenchyma are elongated, andin cross-sections of S. stauntioniana microphyll they are rod-shaped and appear tobe arranged inrows. The columnar-like palisade regionin the leaf of S. stauntoniana appears tofacilitate the exchange of carbon dioxide between mesophyll cells andintercellular air spaces and plays animportant role inthe distribution of light withinthe light. Because photosynthesis depends onthe balance between internal concentrations of both light and carbon dioxide, increasedleaf thickness and the development of palisade layers directlyinfluence this balance and optimize the rate of whole leaf photosynthesis (Dickison, 2000). To obtain higher efficiency of photosynthesis or to avoid photo-damage of chloroplasts, chloroplasts change their location along the cell surface adjacent to the cell wall. In the dark, chloroplasts become locatedalong anticlinal walls and/or at the bottom of the cells. In the daytime, chloroplasts move upto the periclinal walls of palisade cells (Kagawa and Wada,1993; 2002). When light passes through a forest canopy, green and especiallyfar red (FR) wavelengths are preferentially transmitted or reflected while red (R) and blue wavelengths are preferentially absorbed. Diffuse light in the understorey is strongly reduced in photosynthetically active radiation (PAR) and has a decrease inthe ratio of red tofar-red wavelengths (R/FR). Sunflecks pass through holes of the canopy producing a highly dynamic light environment. Thus, the obvious changes inlight quantity between understorey and open habitats are accompanied by differences in light quality and dynamics of the light environment (Tinoco-Ojanguren and Pearcy, 1995). Growth under verylowlight leads to a dramatic increase inlevels of PS 1, while very highlight leads to changes in photosystems Ⅱ(PSⅡ) organization including reductions in the levels of minor LHCcomponent (Bailey et al., 2001). Under conditions of natural shade where light reaching the plant is enrichedin far-red wavelengths preferentially absorbed by photosystems Ⅰ (PS Ⅰ ) (Walters, 2005). The effect of changes inlight quality on photosynthetic acclimationto shade. It is an improtant role of light quality on the the regulation of chloroplast membrane organizationand function. Chloroplasts of lowR/FRshade acclimated plants have a higher density of grana stacking and a decrease inintergranal lamellae. Since photosystems Ⅱ(PSⅡ) preferentially locatedin the appressed grana, lower chlorophyll a/b and PSⅠ/PSⅡratios are also found under FRenriched conditions (Melis, 1984; Glick et al., 1985; Chow, 1990). 4.3. Novel marginal stomata in S. remotifolia. Stomata are the principal conduits through which CO2 diffuses intothe leaf and 123

water vapor diffuses out (Givnish, 1988). In comparing the occurance of stomata in ventral microphylls of different species , it is interestingthat the distribution of the stomata shows great variation (table 2). In most of species stamata are found on theventral epidermis. However, besides stomata on ventral side, S. remotifolia uniquely has marginal stomata alongmicrophylls. It is highly possible that unnecessary water in the plant body is secreted by the marginal stomata, since this plant grows in very wet circumstances under shady forest. Menwhile, an evident intercellualar space (air chamber) is associated withthe position of this marginal stoma. These marginal stomata may play a role as hydathode onleaf margins of in flowering plants (Wood, 1970). 4.4. The diversity of chloroplsast in number and size of Selaginella Confocal laser scanning micrographs show2 chloroplasts in the epidermal cells of S.labordei , S heterostachy, 4 chloroplasts in ,3-4 chloroplasts in S. mollendorffii , 5 chloroplasts both in S. nipponica and S. repanda ,6 chloroplasts in S. remotifolia, and 4-5 chloroplasts in S. stauntoniana. The chloroplasts in dorsal epidermal cells are different in size (c. 8.0- 18.0 µm) (Table 3-2). Some species growinshade environments of forest (at 4.12 -95.53µmol m-2s-1 (able 2-1 ,tabel 3-3). S.bonesis, S. heterostachys, S.labordei, S. staunotiana and S. tamariscina. needmore obversionobserv.? Confocal laser scanning micrographs shownumerous small chloroplasts appearing as spherical or ovoid inthree species growingin sun and open places (at 485.1-1201.5 µmol m-2s-1, Table 2, Figures 6A, 16A). Selaginella ciliaris with diameters of c7.76±1.26 ╳. 2.92±0.38 µm(table 3-3), and S. tamariscina with diameters of c. 5.93±1.08╳4.01±0.76 µm(table 3-3). The results showed that the chloroplasts in dorsal epidermal cells of Selaginella in Taiwan posses great diversityin size and number, and it is species specific and habitat related. In this study this association of chloroplast features andenvironment is first pointed out. There is only one giant chloroplast per dorsal epidermal cells in both S. delicatula and S. doederleinii, ?S.het , which growin deep-shade environment. In contrast, there are numerous small chloroplasts per dorsal epidermal cell, epsecially in S. ciliaris and S. tamariscina, which growin high light and open environment. Relatedto previous study. In many cases chloroplasts have the power of changingtheir shape inresponse to external influences, particularly photonic stimulation. Haberlandt (1914) assumed advantages of numerous small chloroplasts in each photosynthetic element: enhance mobility of the whole apparatus, accelerate of the efflux of synthetic products and get a more even distribution of light, especially when illumination is feeble. large size implications?It is indeed noteworthythat chloroplasts of Selaginella showed such diversity in number, size and distribution pattern, which is related to different environments. It clearly demonstrated that the chloroplasts in dorsal epidermal cells of Selaginella 124

in Taiwan posses great diversityin size (c. 4- 22 µm) and number (from one to 17 per cell). The number of chloroplasts in dorsal epidermal cells varies in different species ranging from one to 17. 4.5 Selaginella hetrostachys: the second species with bizonoplasts similar to S. erythropus Note that the confocal microgrphs showtwo chloroplast on top viewof S. heterostachys, but showone chloroplast onlateral viewfrom microphyll transverse section of it. Selaginella heterostachys is here reported as the second species of vascular plant with bizonoplasts. It occurs densely shaded by other plants (PAR4–39 μmol/m2s at mid-day: 0.2-0.3%full sun) in moderately damprocky habitat with a shallowsoil layer. The bizonoplast foundin S. heterostachys is similar to that of S. erythropus in ultrastructure (Sheue et al., 2007). However, the bizonoplasts of S. heterostachys have a different shape and are similar to a catcher’s mitt (for photons instead of balls), with a lobed structure, which we conjecture is flexible changing form tothe prevailing light conditions. Compare upper zone? . Selaginella heterostachy and S. erythropus belong to different subgenera of Selaginella suggesting the potential of this chloroplast feature throughout the genus, given the right environmental conditions. However, giant chloroplasts were commonly found in deep shade Selaginella, but of the 12 species in this study, only S. heterostachys has bizonoplasts. Thus, the prerequisites for bizonoplasts could not be fully characterized. Functional trait analysis is current major thrust in plant ecology, but with very different traits from those considered here, and very different taxa. When wider taxonomic diversityis considered, rather different traits, including chloroplast traits may be needed. Herblandt (1914) statedthat in many cases chloroplasts have the ability of changing their shape inresponse to external influences—particularly photic stimulation. Among algae and mosses, the chloroplasts contract and become rounded or hemispherical in very intense sunlight (as well as after prolonged darking). Theconfocal micrographs showtwo chloroplast ontop viewof S. heterostachys, but show one cup-shape chloroplast on cross side viewof it. It must be needed to researched.

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Figure 3-78 The structure of microphyll of 12 species of Selaginella from Taiwanin this study

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