Taxonomic significance of leaf anatomy of Aniselytron (Poaceae) as ...

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Plant ResLife Sciences23610.1007/s10265-005-0236-0

J Plant Res (2005) 118:401–414 Digital Object Identifier (DOI) 10.1007/s10265-005-0236-0

© The Botanical Society of Japan and Springer-Verlag Tokyo 2005

REGULAR PAPER

Hai-Ying Ma • Hua Peng • De-Zhu Li

Taxonomic significance of leaf anatomy of Aniselytron (Poaceae) as an evidence to support its generic validity against Calamagrostis s. l.

Received: March 30, 2005 / Accepted: August 17, 2005 / Published online: October 28, 2005 The Botanical Society of Japan and Springer-Verlag 2005

Abstract A comparative study of leaf anatomy on Aniselytron Merr. and Calamagrostis Adans. s. l. was conducted to review the systematic status of Aniselytron Merr. Calamagrostis s. l. exhibits wide variation in many features, but basic leaf structures of the genus remain constant: absence of a midrib-keel; median and large vascular bundles are central, with double sheaths, accompanied by girders both adaxially and abaxially; prickles have a bulbous base and are not sunken; the abaxial epidermal cells are short and wide and relatively thick-walled. Aniselytron differs from Calamagrostis s. l. in: midrib-keel is present, consisting of a large central bundle with small ones on either side; all vascular bundles are abaxially situated, with abaxial girders only, parenchyma takes the place of the adaxial sclerenchyma; the bases of the prickles are sunken and are not bulbous; the abaxial epidermal cells are tall and thinwalled. These distinct anatomical features, in combination with the differences in spikelet structure and habitat, suggest that Aniselytron should be generically separated from and not merged with Calamagrostis s. l. Due to the adaxial parenchyma in the midrib which has never been found in Pooideae, Aniselytron might have a relationship with some other subfamily. Key words Aniselytron · Calamagrostis · Deyeuxia · Leaf anatomy · Poaceae

Introduction The genus Aniselytron Merr., with only two species A. treutleri and A. agrostoides, is distributed in the Philippines, H.-Y. Ma · H. Peng (*) · D.-Z. Li Laboratory of Plant Biogeography and Biodiversity, Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, 650204, People’s Republic of China e-mail: [email protected] H.-Y. Ma Graduate School of the Chinese Academy of Sciences, Beijing, People’s Republic of China

Malaysia, Japan, North Vietnam, China (including Taiwan), Myanmar, Bhutan and Sikkim (Hackel 1907; Merrill 1910; Keng 1959; Korthof and Veldkamp 1984; Lu 1987; Noltie 2000). The genus was established by Hackel (1907) and Merrill (1910) independently, using the name Aulacolepis and Aniselytron respectively. Later the monotypic genus Aniselytron was combined into Aulacolepis (Ohwi 1935). It was only recently realized that Aulacolepis was a homonym of a fossil fir in Paleobotany, thus the generic name Aniselytron was resurrected (Bennett and Raizada 1981). A recent revision by Korthof and Veldkamp (1984) affirmed that there are only two species in the genus. The genus was accepted in all major accounts for the countries in which the species occur, except for Bhutan (Noltie 2000) where it is included in Calamagrostis s. l. (sensu Clayton and Renvoize 1986). Although the nomenclature of the genus Aniselytron and taxonomy of its two species seem clear, the systematic position of the genus in Pooideae remains uncertain because of its unusual spikelet structure. In their revision, Korthof and Veldkamp (1984) noted that Aniselytron closely resembles four other genera: Deyeuxia Beauv., in the induration of the lemma; Poa L., in the glabrous rachilla process and the unusual absence of an awn; Arctagrostis Griseb. and Simplicia Kirk in the reduction of glume size. Overall, they considered it closest to Deyeuxia. Following them, Clayton and Renvoize (1986) placed Aniselytron in subtribe Alopecurinae of Tribe Aveneae, subfamily Pooideae, and treated it as a part of Calamagrostis Adans. s. l., therefore Calamagrostis s. l. (sensu Clayton and Renvoize 1986) includes Calamagrostis s. str., Deyeuxia and Aniselytron. However, they noted that this delimitation of the genus Calamagrostis s. l. is rather artificial. As no further evidence has been put forward, some later researchers adopted this treatment: Shukla (1996) transferred species of Aniselytron to Calamagrostis s. l. and this treatment was also adopted by another author (Noltie 2000). Privately, some agrostologists are still skeptical, questioning its affinity with Deyeuxia or Calamagrostis s. l. (S.M. Phillips and R.J. Soreng, personal communication).

402

Leaf anatomy has been the focus of a lot of attention since Prat (1936) demonstrated its potential for taxonomy in Poaceae and the delimitation of subfamilies is now firmly based upon differences in leaf anatomy (Clayton and Renvoize 1986; Ellis 1986). Prat (1936) and Metcalfe (1960) both indicated that Calamagrostis has a Festucoid type of leaf anatomy. Türpe (1962) and Escalona (1991) studied leaf anatomy of 13 and 14 species of Deyeuxia respectively from South America. No anatomical work on Aniselytron has been performed before. The present study investigates the similarities and differences between Aniselytron and Calamagrostis s. l. in order to reveal the systematic status of Aniselytron. To make this comparison, an intensive study on Calamagrostis s. l. has also been conducted. For this study the generic names Calamagrostis s. str. and Deyeuxia have been used because both are recognized in China.

Materials and methods Leaves of 64 samples comprising 2 species of Aniselytron and 33 species and 1 variety of Calamagrostis s. l. from China, Japan, Germany, Russia and USA were examined. Some of the materials were collected in the field by the first author while the others were gathered from herbarium specimens in The Institute of Botany (PE) and Kunming Institute of Botany (KUN) of The Chinese Academy of Sciences, The Kochi Prefectural Makino Botanical Garden (MBK) in Japan and National Museum of Natural History, Smithsonian Institution (US) in USA. The materials used are listed in Table 1. Voucher specimens are housed in KUN, PE, MBK and US. Leaves from living plants were fixed in FAA in the field. Leaves from herbarium specimens were boiled in water for 10 min or more until fully expanded, and then treated with FAA for at least 1 day before use. The middle part of the mature leaf was used for sectioning. The materials were first treated in 10% hydrofluoric acid for 24–48 h to remove silica, a method introduced by Metcalfe (1960). They were then dehydrated through an alcohol series and embedded in paraffin. Sections were prepared at a thickness of 12 µm and stained with safranin and fast green. Samples were observed and photographed with a LM Olympus BX51 microscope. For classification of the species, we followed Lu et al. (manuscript for Flora of China, English edition) for Calamagrostis s. str. and Deyeuxia respectively for Chinese species. For European and American species, we followed Tsvelev (1983). For the description of the anatomical features, we use the nomenclature of Metcalfe (1960) and Ellis (1976).

Results Anatomical description of Aniselytron treutleri Thickness of the leaf blade is about 400 µm thick in the median and 100 µm in the marginal parts (Figs. 1a, 2a–e).

Transverse section outline is expanded as a whole and Vshaped in the middle (Fig. 1a); the projecting keel gives the appearance of the V-shape (Fig. 2a). Slight, shallow furrows (f) are present on adaxial surface, between two vascular bundles; ribs (r) are obtuse, situated over the vascular bundles; no ribs or furrows are present on the abaxial surface (Fig. 2c, d). All types of vascular bundles1 are present; large vascular bundles are of basic type (Fig. 2b–d); small bundles are not conspicuously angular in outline (Fig. 2d, e); there are five first-order vascular bundles in the half lamina, nine in entire blade, progressively fewer first- and more thirdorder vascular bundles toward the margins. A keel is present, rounded, projecting abaxially, and composed of three vascular bundles: the median one and two small ones (Fig. 2a). Median vascular bundle is abaxially positioned (Fig. 2a, b), associated with a small abaxial inverted triangular girder2 (gr) but adaxially only a small strand (str); large parenchyma cells (pc) are present between the bundle and the strand (Fig. 2a, b). Large vascular bundles are usually associated with very narrow girder abaxially and relatively wide girders or strands adaxially; third-order vascular bundles (tvb) are usually without sclerenchyma (Fig. 2c–e). Sclerenchyma cells are absent or very sparse in leaf margins (Fig. 2e); no sclerenchyma cells are present between vascular bundles (Fig. 2c). Sclerenchyma cells are not heavily thickened (Fig. 2a, b). Vascular bundle sheaths are double, in large vascular bundles the inner sheath (is) complete and the outer sheath (os) interrupted by abaxial girders, in small vascular bundles both sheaths complete (Fig. 2a–d). Chlorenchyma (cl) is irregular, arranged loosely and lacunae are present in mesophyll, only the layer connecting the abaxial epidermis seems regular (Fig. 2a–d). Epidermal cells (ec) in transverse section bulliform cells (bc) are present, large and fan-shaped (Fig. 2a, b); stomata (st) are present on adaxial surface only (Fig. 2b); prickles (pr) are infrequent and present on adaxial surface only; prickles are sunken at base, not bulbous (Fig. 2b–d); all epidermal cells have thin walls, the width of cell is shorter than the height (Fig. 2a, b).

1 Vascular bundles are classified into three types according to the size and structure. The first-order bundles are large, with conspicuous metaxylem vessels and exhibiting varying degrees of sclerosis of the phloem. The second-order bundles refer to those with one or two metaxylem vessels. The third-order bundles are small bundles containing no conspicuously large metaxylem vessels, and xylem and phloem are not easy to distinguish from one another in transverse section. There is no essential difference between the first and the second order vascular bundles and they are often referred as large vascular bundles, and the third-order bundles are referred as small vascular bundles. 2 There are various terms to describe sclerenchyma. Girder is used when the sclerenchyma extends from the epidermis on its outer surface to the bundle-sheath on its inner surface. Strand is applied to sclerenchyma of which the outer surface rests against the epidermis but does not penetrate sufficiently deeply to make contact with the bundle-sheath on its inner surface. Hypodermal strand is to describe the sclerenchyma which is situated between two vascular bundles and makes contact with epidermal cells on one surface. Sclerenchyma cells in leaf margin have no special term.

403 Table 1. Materials examined Taxon

Collector and specimen nos.

Location and altitude

Herbarium

Aniselytron treutleri (Kuntze) Soják Aniselytron treutleri (Kuntze) Soják Aniselytron treutleri (Kuntze) Soják

H.Y. Ma 46 H. Peng 4001 T. Makino 144957

kun kun mbk

Aniselytron agrostoides Merr. Deyeuxia sinelatior Keng D. neglecta (Ehrh.) Kunth D. neglecta (Ehrh.) Kunth

J. Ohwi 3623 K.M. Liou 5288 Sh.H. Gong no number H.Y. Ma 240

D. hakonensis (Franchet & Savatier) Keng D. lapponica (Wahlenb.) Kunth D. sichuanensis (J.L. Yang) S.M. Phillips & W.L. Chen D. purpurea (Trin.) Kunth D. purpurea (Trin.) Kunth D. korotkyi (Litv.) S.M. Phillips & W.L. Chen D. pyramidalis (Host) Veldkamp D. pyramidalis (Host) Veldkamp D. pyramidalis (Host) Veldkamp D. pyramidalis (Host) Veldkamp D. pyramidalis (Host) Veldkamp D. effusiflora Rendle D. effusiflora Rendle D. himalaica L. Liu ex W.L. Chen D. nyingchiensis P.C. Kuo et S.L. Lu D. scabrescens (Griseb.) Munro ex Duthie D. pulchella J.D. Hooker D. pulchella J.D. Hooker D. pulchella J.D. Hooker D. rosea Bor. D. rosea Bor. D. flavens Keng D. flavens Keng D. flavens keng D. nivicola Hook. f.

Z. S. Yue 3664

Lushui, Yunnan, 2,780 m, China Fanjingshan, Guizhou, 2,200 m, China Okinoyama, Yato-cho, Yaze-gun, Tottori, Japan Mt. Arisan, Taiwan Lushi, Henan, China Inner Mongolia, 980 m, China Zhegushan, Ma’erkang, Sichuan, 3,500 m, China Wugongshan, Jiangxi, 1,600 m, China

N.R. Cui 820541 H. Y. Ma 239 Ch.P. Wang 271 G.Sh. Zhou et al. 32 R.C. Ching s. n. H.Y. Ma 071 H.Y. Ma 073 H.Y. Ma 138 Bijiang Exped. 1542 M.X. Nie & S.S. Lai 5187 P.H. Yu 1055 H.Y. Ma 257 H.Y. Ma 94 H.Y. Ma 223 H.Y. Ma 066 H.Y. Ma 82 H.Y. Ma 86 H.Y. Ma 95 G.X. Fu 846 H.Y. Ma 243 F.B. Wang 83225 Y.C. Yang 1355 H.Y. Ma 250 H.Y. Ma 56

us pe pe kun kun

Halasi Lake, XinJiang, China Zhegushan, Ma’erkang, Sichuan, 3,000 m, China Wumeng, Inner Mongolia, China Jingpo Lake, 350 m, China Qinghe, Xinjiang, 1,800 m, China

pe kun

Xiangcheng, Sichuan, 3,500 m, China Daocheng, Sichuan, 3,550 m, China Heqing, Yunnan, 3,000 m, China Lushui, Yunnan, 2,100 m, China Dexing, Jiangxi, China Zhenxiong, Yunnan, 1,850 m, China Baoxing, Sichuan, 2,700 m, China Xiangcheng, Sichuan, 4,390 m, China Linzhi, Tibet, 3,500 m, China Laojunshan, Jianchuan, Yunnan, 2,900 m, China Daocheng, Sichuan, 4,000 m, China Daocheng, Sichuan, 4,000 m, China Xiangcheng, Sichuan, 3,500 m, China Jiangzi Farm, Tibet, 3,900 m, China Zhegushan, Hongyuan, Sichuan, China Cui’ershan, Sichuan, China Anqian, Qinghai, 4,300, China Hongyuan, Sichuan, 3,800 m, China Laojunshan, Jianchuan, Yunnan, 3,900 m, China Daxueshan, Zhongdian, Yunnan, 4,410 m, China Russia Ruo Qiang, Xinjiang, China Shinaihai, Qinghai, 3,200 m, China Bange, Tibet, 4,650 m, China

kun kun kun kun kun kun kun kun kun kun

pe pe kun pe

Maduo, Qinghai, 4,500 m, China

kun pe pe pe kun

kun kun kun kun kun kun kun kun pe pe kun

D. nivicola Hook. f.

H.Y. Ma 63

D. anthoxanthoides Munro ex Hook. f. D. tianschanica (Rupr.) Bor D. kokonorica Keng D. tibetica var. przevalskyi (Tzvel.) P.C. Kuo et S.L Lu D. tibetica var. przevalskyi (Tzvel.) P.C. Kuo et S.L Lu D. holciformis (Jaub. et Spach) Bor D. holciformis (Jaub. et Spach) Bor Deyeuxia debilis (Hook. f.)Veldk. D. moupinensis (Franch.) Pilger

Unclear K. Guo & D. Zheng 12316 H.Y. Ma 233 Naqu Branch, Qinghai & Tibet Expedition 10611 H.Y. Ma 235 K.Y. Lang 9851 K.Y. Lang 9869 CAS 14929 H.Y. Ma256

D. diffusa Keng

H.Y. Ma 148

D. diffusa Keng D. petelotii (Hitchcock) S.M. Phillips & W.L. Chen D. yanyuanensis (J.L. Yang) L. Liu D. mazzettii Veldk. D. mazzettii Veldk. D. mazzettii Veldk. D. mazzettii Veldk. D. mazzettii Veldk. Calamagrostis epigeios (L.) Roth C. epigeios (L.) Roth C. epigeios (L.) Roth

H.Y. Ma 136 H.Y. Ma 001

Shuanghu, Tibet, 4,800 m, China Shuanghu, Tibet, 4,900 m, China Mt. Gonggala, Bomi, Tibet, China Ganyanggou, Baoxing,Sichuan, 2,700 m, China Mt. Cangshan, Dali, Yunnan, 3,000 m, China Heqing, Yunnan, 3,000 m, China Kunming, Yunnan, 2,000 m, China

H.Y. Ma 262 H.Y. Ma 74 H.Y. Ma 101 H.Y. Ma 115 H.Y. Ma 127 H.Y. Ma 134 Hexi Exped. 1309 R.C. Ching 6854 S.S. L 3806

Yanyuan, Sichuan, 2,600 m, China Daocheng, Sichuan, China Xiangcheng, Sichuan, China Zhongdian, Yunnan, China Mt. Yulong, Lijiang, Yunnan, China Heqing, Yunnan, 3,000 m, China Gansu, 2,900 m, China Unclear, China Mt. Dahai, Jiangxi, China

pe pe pe

kun kun kun pe kun pe kun kun kun kun

kun

404 Table 1. (Continued) Taxon

Collector and specimen nos.

Location and altitude

Herbarium

C. epigeios (L.) Roth C. varia (Schrader) Host.

S.Z. Wang 1014 German–Chinese Exped. 36791

kun kun

C. canadensis (Michx.) P. Beauv. C. pseudophragmites (Hall. f.) Koel. C. pseudophragmites (Hall. f.) Koel. C. hedinii Pilger C. macrolepis Litv. C. macrolepis Litv C. emodensis Griseb. C. emodensis Griseb.

Fred H. Utech 79-356 H.Y. Ma 143 F.T. Wang 2229 H.Y. Ma 231 Xinjiang Expedition 652 Geobotany group 40 H.Y. Ma 133 Sichuan economical plant expedition 1523

Funing, Yunnan, 1,540 m, China Allgau Alps, Bavaria Swabóa, 1,050 m, Germany New York, 1,000 m, USA Lushui, Yunnan, 2,780 m, China Kunming, Yunnan 2,000 m, China Ge’ermu, Qinghai, 2,700 m, China Kashgar, Xinjiang, 1,420 m, China Ge’ermu farm, Qinghai, 4,300, China Lushui, Yunnan, 2,780 m, China Hongqilewu, Sichuan, 2,300 m, China

kun kun kun kun pe pe kun kun

PE Herbarium of Institute of Botany, The Chinese Academy of Sciences, KUN Herbarium of Kunming Institute of Botany, The Chinese Academy of Sciences, US Smithonian Institution, National Museum of Natural History of the United States of America, MBK The Kochi Prefectural Makino Botanical Garden, Japan

Anatomical features of Aniselytron agrostoides Due to the age, the leaf blade of the specimen had greatly degenerated and was not fully restored, causing poor quality sections. Fortunately the middle and marginal parts of the transverse section are still clear, exhibiting a similar anatomy to that of A. treutleri: median vascular bundle is abaxially positioned, associated with an abaxial girder and a small adaxial strand, with large parenchyma cells between the vascular bundle and the adaxial strand (Fig. 3k); there is a small abaxially positioned third-order vascular bundle on either side of the median vascular bundle (Fig. 3k); the epidermal cells have thin walls, the width of cell is shorter than the height (Fig. 3k); there is no sclerenchyma in the leaf margins. The rest of the section was not very clear. Anatomical features of Calamagrostis s. l. (including Deyeuxia and Calamagrostis s. str.) As found in previous studies (Metcalfe 1960; Türpe 1962; Escalona 1991), all the species examined exhibit a Festucoid type of leaf blade, with a wide range of variations. Each species has a specific leaf anatomy, and the structure of each species appears to be constant. The leaf anatomical features, from the materials we studied, are given in the descriptions below.

D. petelotii (Fig. 1c) have the thinnest leaf, about 100 µm thick, and D. scabrescens has the thickest one (Figs. 1c, 3a), about 250 µm. Most species have an even leaf thickness across the section of between 100 and 200 µm. Only D. effusiflora has different thickness throughout the leaf blade, with 400 µm thick in the middle but only 100 µm thick in the other ribs (Fig. 4b). Adaxial furrows and ribs Some species in Deyeuxia, such as D. scabrescens, D. himalaica, D. petelotii and D. pulchella (Fig. 3a–d), have shallow furrows. Other species, such as Deyeuxia hakonensis (Fig. 3e) and Deyeuxia nyingchiensis (Fig. 3f), have moderate furrows. Four of the five species of Calamagrostis s. str., such as Calamagrostis epigeios (Fig. 3i) and Calamagrostis hedinii (Fig. 3j) and some species in Deyeuxia, such as Deyeuxia kokonorica (Fig. 3g) and Deyeuxia tibetica var. przevalskyi (Fig. 3h), have deep narrow furrows. Adaxial furrows are present between all vascular bundles, except for D. tibetica var. przevalskyi, in which the adaxial furrows are situated between large vascular bundles and above third order vascular bundles (Fig. 3h). Adaxial ribs are situated over all vascular bundles, with obtuse, round, rectangular or triangular tops (Fig. 3a–j). Abaxial furrows and ribs

Transverse section outline The outline of the transverse section exhibits expanded, round, or reduced V- or U-shape (Fig. 1b–g). But this feature is not constant. Samples immediately fixed in the field tend to exhibit a more expanded outline, while those restored from dry leaves are more or less in-rolled.

No species examined have distinct abaxial ribs, only some species have slight abaxial protrusions as those in Calamagrostis epigeios (Fig. 3i) and C. hedinii (Fig. 3j), due to thickening of sclerenchyma, or undulation opposite the adaxial ribs similar to that of D. tibetica var. przevalskyi (Fig. 3h). Median vascular bundle or midrib

Leaf thickness The leaf thickness was measured with an average thickness of between 100 and 250 µm. Deyeuxia debilis, D. diffusa and

Median vascular bundles are present but mostly indistinguishable from other large vascular bundles, as that of Deyeuxia mazzettii (Fig. 4a), or only distinguishable from others

405 Fig. 1. Transverse sections of leaves of selected species. a Aniselytron treutleri, b Deyeuxia petelotii, c D. scabrescens, d D. himalaica, e D. tibetica var. przevalskyi, f D. kokonorica and g D. nivicola. The outline of the transverse section exhibit expanded, round or reduced V- or U-shape. Scale bar 200 µm

by size, as that of D. himalaica (Fig. 4c). Midrib is only present in D. effusiflora (Fig. 4b) and Deyeuxia moupinensis. None of the samples forms a keel in the median vascular bundle.

second-order vascular bundles are uncommon. The vascular bundles of the first and second order are usually circular or elliptical and third-order vascular bundles are inconspicuously angular.

Vascular bundle arrangement

Vascular bundle sheaths

Vascular bundles of all three orders are present, varying in the numbers and the arrangement. In some species the

Vascular bundles have double sheaths, and these are not always distinguishable in third order vascular bundles. The

406 Fig. 2. Transverse section of leaves of Aniselytron treutleri. a Structure of the keel, composed of a large middle vascular bundle and a very small vascular bundle on both sides. Bulliform cells are large, fan-shaped. Stomata are distributed on adaxial surface. Prickles are infrequent adaxially and absent abaxially; the base is sunken and not bulbous. All epidermal cells are thin-walled, the width of epidermal cells shorter than the height. b The middle vascular bundle, abaxially positioned, associated with an abaxial girder and a small adaxial strand, large parenchyma cells between the bundle and the adaxial strand. c Lamina halfway to margin, show furrows and ribs. d Furrows and ribs about halfway from middle to margin, show prickles and lacunae. e Margin, without sclerenchyma cells. bc bulliform cells, cl chlorenchyma, ec epidermal cells, f furrow, is inner sheath, lc lacunae, mvb median vascular bundle, os outer sheath, pr prickle, r rib, scl sclerenchyma, st stomata, str strand, tvb third vascular bundle, pc parenchyma cells. Scale bar 100 µm

407 Fig. 3. Transverse sections of leaves of Calamagrostis s. l. (a–j) and Aniselytron agrostoides (k) to show ribs and furrows. a Deyeuxia scabrescens, b D. himalaica, c D. petelotii, d D. pulchella, e D. hakonensis, f D. nyingchiensis, g D. kokonorica, h D. tibetica var. przevalskyi, i Calamagrostis epigeios, j C. hedinii and k Aniselytron agrostoides. Adaxial furrows are shallow in (a–d), while deep in (g–j) and moderate in (e–f). The median vascular bundle of A. agrostoides (k) is abaxially positioned, associated with an abaxial girder and a small adaxial strand, with parenchyma between the vascular bundle and the adaxial strand. tvb third-order vascular bundle, bc bulliform cells, f furrow, mvb median vascular bundle, pr prickle, r rib, scl sclerenchyma, st stomata, sbv small vascular bundle. Scale bar 100 µm

408 Fig. 4. Vascular bundles and sclerenchyma in Calamagrostis s. l. a Deyeuxia mazzettii, b D. effusiflora, c, d D. himalaica, e Calamagrostis canadensis, f C. hedinii, g D. scabrescens h, i D. mazzettii and j D. scabrescens. Median vascular bundle is not distinguishable from other large vascular bundles as in (a), or distinguishable as in (b) and (c). Vascular bundles are all central positioned in Calamagrostis s. l.; sclerenchyma occur in leaf crosssections as girders associated with the vascular bundles, varying in shape as show in (e–h), or as strands as show in (i), or as hypodermal strands between vascular bundles as show in (j); sclerenchyma cells also occur in the margins as show in (j). mvb median vascular bundle, bc bulliform cells, gr grinder, is inner sheath, os outer sheath, pr prickle, scl sclerenchyma, st stomata. Scale bars: 100 µm in (a–d), (j); 100 µm in (e–i) and 50 µm

inner sheath is always complete, and the inner tangential and radial walls of the cells are heavily thickened (Fig. 4e– h). The outer sheath is made up of parenchyma, well differentiated from the chlorenchyma (Fig. 4f–h). The outer sheaths are usually not complete: in large vascular bundles they are interrupted by both adaxial and abaxial girders (Fig. 4e–h); in small vascular bundles they are interrupted by both or either of abaxial and adaxial girders.

Sclerenchyma Sclerenchyma occurs in leaf cross-sections as girders and/or strands associated with the vascular bundles (Fig. 4e–i).

Girders vary in width, height and shape. T-shaped, reversed anchor-shaped, triangle-shaped and irregular rectangularshaped girders occur in this group (Fig. 4e–h). For median vascular bundles, the smallest girders are like those in Deyeuxia debilis and Calamagrostis canadensis (Fig. 4e), with only one to three layers of sclerenchyma, and the biggest is that in D. scabrescens (Fig. 4g), with five to eight layers of sclerenchyma. Usually the abaxial girder is wider than the adaxial one (Fig. 4g, h), while the reverse situation occurs in some species such as C. canadensis and C. hedinii (Fig. 4e, f). Hypodermal strands are only present in a few species, such as D. scabrescens (Fig. 4j), C. epigeios (Fig. 3i), C. hedinii (Fig. 3j) etc. Sclerenchyma in the leaf margin is usually present (Fig. 4j), but varies in size and cell wall thickness.

409

Mesophyll Chlorenchyma are not radiate, mostly closely arranged, without lacunae or airspace, some loosely arranged, with lacunae or airspace adjacent to the vascular bundles. However, this feature is only reliable in samples fixed directly from living leaves. In those from dried leaves, it seemed that some chlorenchyma cells have degenerated. There is no differentiation between palisade and spongy parenchyma.

Epidermal cells in transverse section Bulliform cells Usually five to eight fan-shaped bulliform cells fill the furrows on the adaxial surface. The cells uniformly have very thin walls and are conspicuously large or gradually larger than the rest of the epidermal cells (Fig. 3a–j). Thickness of cell walls Abaxial epidermal cells in most species have more or less thickened walls (Fig. 3a, b, d–j). Only a few species like Deyeuxia diffusa and D. petelotii (Fig. 3c) have thin-walled epidermal cells. Adaxial epidermal cells are normally thin-walled except those above the vascular bundles. Prickles These are present in almost every species; prickle cell bases are bulbous, not sunken. D. scabrescens (Fig. 3a), D. sichuanensis (Fig. 3b) and D. himalaica (Fig. 4c, d) have the densest prickles, making the epidermis coarse. Some species such as D. petelotii (Fig. 3c) have very few prickles thus the epidermis is smoother. Stomata No significant difference was noted in the morphology of the stomata in different species from the transverse section. Stomata are uniformly present on the adaxial surface. In some species, such as D. holciformis, D. tibetica var. przevalskyi, C. epigeios and C. hedinii (Fig. 3g–j), stomata occur on the abaxial surface as well.The anatomical features of all examined species are summarized in Table 2.

Discussion and conclusion Calamagrostis s. l., including Deyeuxia but not Aniselytron, comprises of around 270 species (Clayton and Renvoize 1986). In the strictest sense Calamagrostis consists of only 15–20 species mainly from Eurasia. These have no or only rudimentary, glabrous rachilla extensions, callus hairs are longer than the thinly membranous lemma in length. Deyeuxia as generally treated in world floras, includes the remainder of species from temperate regions around the world. These exhibit well-developed pilose rachilla extensions, callus hairs are usually less than two-third length of the lemma, and lemmas are usually firmer than the glumes. In the broad sense the genus includes about 122 species endemic to the Americas and 45 endemic to Australia and

New Zealand. Some 80 species occur in Eurasia, about half of which occur in China, Japan and the Himalayas. Wasiljew (1960) divided Calamagrostis s. l. into five subgenera and a number of sections and series. It is an incomplete account, but it is the most recent and perhaps the only summary of the infrageneric taxonomy of the genus for the world: Calamagrostis subg. Calamagrostis, C. subg. Epigeios (Koch) Wassil., C. subg. Ankylanthera (Torges) Wassil. (=C. subg. Deyeuxia (Clarion ex P. Beauv.) Rchb.), C. subg. Paragrostis (Torges) Honda (monotypic, including the European C. villosa (Chaix.) Gmelin) and C. subg. Pararctagostis Wassil. [ditypic including the South American C. jamesonii Steud. and C. chrysostachya (E. Desv.) Kuntze]. For the flora of USSR Tsvelev (1983) recognized three sections in Calamagrostis s. l. He placed C. villosa in C. sect. Calamagrostis, and recognized C. sect. Deyeuxia and C. sect. Pseudophragmites (=C. subg. Epigeios). Neither Aniselytron nor its synonym Aulacolepis was included in Wasiljew’s account of Calamagrostis, since the genus does not occur in the former USSR. For Chinese Calamagrostis s. l. species, Keng (1959) and Lu (1987) recognized two genera: Calamagrostis (including C. subg. Epigeios) and Deyeuxia. Aniselytron has always been accepted as a separate genus in China (Keng 1959; Lu 1987). Our sample included representatives of the following subgenera, and mostly concentrates on Asian species: C. subg. Calamagrostis (15 sps., including 3 awnless sps.), C. subg. Epigeios (5 sps.), C. subg. Deyeuxia (13 sps.). We were not able to collect C. subg. Paragrostis and C. subg. Pararctagostis, which are each monotypic and ditypic, and do not represent the majority of Calamagrostis s. l. Three widely accepted subgenera, which represent Calamagrostis s. l. well, were all included in our study. Though abundant variations are found in Calamagrostis s. l., there is no substantial difference among species within it and no clear difference was found between Deyeuxia and Calamagrostis s. str. Basic leaf structures of the genus remain constant: large vascular bundles are central, with double sheaths, accompanied by both adaxial and abaxial girders; median vascular bundles do not form a keel, accompanied by both adaxial and abaxial girders; prickles are not sunken and have a bulbous base. Metcalfe (1960) reported a keel in C. epigeios, we found it to be a protrusion of the median vascular bundle, and it is not a keel according to the nomenclature of Ellis (1976). The variations found in South American species are also similar to our present study (Türpe 1962; Escalona 1991). In Aniselytron, the two species have very similar leaf anatomy. The anatomical features of A. agrostoides were not fully recognizable from our sections, but the most important features have been observed clearly. The common features of the leaf anatomy includes: a keel composed of a median vascular bundle, a third-order vascular bundle on either side; the median vascular bundles are both abaxially situated, associated with abaxial girder and a far adaxial strand only, large parenchyma cells are present between the median vascular bundles and the adaxial strands; the epidermal cells of both species are tall, narrow and thin-walled; no sclerenchyma is present in the leaf

400/100

220

Unclear

Slightly inrolled

Strongly inrolled

Strongly inrolled

Slightly inrolled

Slightly inrolled

Strongly inrolled

Slightly inrolled

Expanded 130

Slightly inrolled

Strongly inrolled

A. agrostoides

Calamagrostis epigeios

C. macrolepis

C. pseudophragmites

C. emodensis

C. hedinii

Deyeuxia hakonensis

D. sinelatior

D. lapponica

D. sichuanensis

D. neglecta

250

160

180

175

290

180

210

250

400/100

V-shaped

Aniselytron treutleri

Square alternate with triangular

Round alternate with triangular


Unclear

Obtuse

Adaxial rib tops

Round topped

Deep

Shallow

Rectangular alternate with triangular

Flat

Moderate Round alternate with triangular

Moderate Obtuse

Moderate Obtuse

Deep

Moderate Round topped

Deep

Deep

Deep

Shallow

Shallow

Leaf Adaxial thickness furrows (µm)

Outline

Taxon

Table 2. Leaf anatomical features of species examined

Smooth

Coarse by prickles

Smooth

Smooth

Smooth

Protrusion by abaxial girders

Smooth




Smooth

Smooth

Abaxial surface

Both adaxial and abaxial girders




Not distinguishable


Distinguishable Both adaxial and abaxial girders




Not distinguishable

+

–

–

+

+

+

+

+

–

–

+++

+++

+++

+++

+++

–

+

–

–

–

+

Abaxial girder – and adaxial parenchyma

–

Moderate, 5 cells

Large, 5 cells

Large, 5 cells

Large, 5 cells

Large

Large

Moderate

Moderate

Large

Large

Large, 5 cells

Large, 5 cells

Sclerenchyma Sclerenchyma Bulliform between vbs in margins cells

Abaxial girder – and adaxial parenchyma

Sclerenchyma associated with large vbs


Not distinguishable

Not distinguishable

Not distinguishable

Form a keel

Form a keel

Median vascular bundle or midrib

4

2

2

2

4

3.2

4.1

3

3.8

3

1

1

Stomata

Bulbous Both adaxial and abaxial

Bulbous Adaxial only







Bulbous Both adaxial and abaxial stomata


Sunken, Adaxial not only bulbous

Sunken, Adaxial not only bulbous

Thickness of Prickle epidermal base cell walls (µm)

410

Expanded 120

Strongly inrolled

Strongly inrolled

V-shaped

Strongly inrolled

Slightly inrolled

Expanded 130

Expanded 200

Slightly inrolled

Expanded 140

Slightly inrolled

D. korotkyi

D. pyramidalis

D. effusiflora

D. himalaica

D. nyingchiensis

D. scabrescens

D. pulchella

D. rosea

D. nivicola

D. flavens

D. mazzettii

Flat






Not distinguishable


Form a midrib



Not distinguishable


Smooth

Smooth


Smooth

Protrusion by abaxial girders Both adaxial and abaxial girders


Not distinguishable



Not distinguishable


Not distinguishable


Protrusion Distinguishable Both adaxial by prickles and abaxial and abaxial girders girders

Coarse by prickles

Smooth

Coarse by prickles

Smooth

Round and Small sub-triangular protrusion with abaxial girders

Deep


Flat or rounded

Obtuse

Flat

Flat

Flat

D. tianschanica

Deep

Shallow

Deep

Shallow

Shallow

Shallow

Moderate Obtuse

Shallow

Moderate Obtuse


Expanded 225

Smooth

Smooth

Abaxial surface

Moderate Round or Smooth sub-triangular


Moderate Obtuse

Adaxial rib tops

D. Expanded 130 anthoxanthoides

175

150

200

120

350/150

400/100

140

130

Strongly inrolled

D. purpurea


Outline

Taxon

–

–

–

–

–

–

–

+

–

+

–

–

–

–

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Moderate

Large, 5 cells

Moderate

Large, 6 cells

Moderate, 5 cells

Moderate, 6–7 cells

Large, 5 cells

Large, 5 cells

Moderate

Large, 5 cells

Moderate, 5 cells

Large, 5 cells

Moderate, 5 cells

Moderate, 5 cells


4

2.5

3.6

2

3.5

1.4

1.5

5.5

2.5

4

2.5

2.5

2

2.5

Stomata

Bulbous Adaxial and sparsely abaxial














Thickness of Prickle base epidermal cell walls (µm)

411

U-shaped

Slightly inrolled

Slightly inrolled

Strongly inrolled

Expanded 100

Expanded 130

Expanded 120

V-shaped

Expanded 170

D. tibetica var. przevalskyi

D. holciformis

D. debilis

D. moupinensis

D. diffusa

D. petelotii

D. yanyuanensis

C. varia

C. canadensis

– absent, + present, +++ well-developed

130

100

100

270

230

270

Strongly inrolled

D. kokonorica



Most round, some triangular

Adaxial rib tops

Round ribs

Moderate Obtuse

Moderate Obtuse

Deep

Moderate Obtuse or flat



Moderate Obtuse

Deep

Deep

Deep


Outline

Taxon

Table 2. (Continued)

Smooth

Smooth

Smooth

Smooth

Smooth

Smooth

Smooth

Small protrusion with abaxial girders


Smooth

Abaxial surface









Not distinguishable



Not distinguishable

Not distinguishable

Not distinguishable


Not distinguishable


–

–

–

–

–

–

–

–

–

+

+

+

+

+

+

+

+

+

+

+

Moderate

Large, 5 cells

Moderate

Large, 5 cells

Moderate

Moderate

Moderate

Moderate

Moderate

Large, 8 cells


1.8

1.6

2

2

1.2

1.5

1.5

4

3

3

Stomata











Thickness of Prickle epidermal base cell walls (µm)

412

413

margins. These anatomical features are all different from those of Calamagrostis s. l. Some further distinct features are found in A. treutleri as the sections are clear in all parts. Firstly, the large vascular bundles are abaxially situated, close to the abaxial epidermis and far from adaxial one. Secondly, either the abaxial or adaxial girders, but never both, accompany the large vascular bundles; while in Calamgrostis s. l. both abaxial and adaxial girders appear. All these features are very distinct, and are not included within the variation range in Calamagrostis s. l. Above all, the differences between Aniselytron and Calamagrotis s. l. lie in four respects: the structure of median vascular bundle and the structures associated with it, the position of large vascular bundles in the transverse section, the morphology of prickles and the morphology of epidermal cells. The differences are listed in Table 3. Although very few comparative anatomical studies on Pooideae taxa have been conducted since 1960 (e.g., Connor 1960; Runemark and Heneen 1968; Aiken and Lefkovitch 1984; Aiken et al. 1985) and thus it is difficult to draw evolutionary lines or circumscribe tribes in this subfamily, leaf anatomy still might provide information to resolve systematic problems surrounding particular taxa (Macfarlane and Waston 1980; Ellis 1986). Many authors have pointed out that some characters are systematically useful and others are environmentally variable. Metcalfe (1960) pointed out that the degree of infolding or inrolling varies with environmental conditions and thus is not of much diagnostic value. Ellis (1986) remarked that characters such as the thickness of the leaf, the number and the arrangement of vascular bundles might be systematically useful, and characters such as the distribution of prickles, might be relatively stable or environmentally variable. Ellis (1976) particularly pointed out that the position of the vascular bundles in the blades appears to be a useful diagnostic character above the generic level that has been largely overlooked in the past. In the present study, there are no anatomical gaps among species within each group of Calamagrostis s. str. and Deyeuxia, variations within each of them being consecutive. These variations mostly include those addressed by Metcalfe (1960) and Ellis (1976) as environmentally related, such as the degree of infolding or inrolling, distribution of prickles, degree of development of sclerenchyma, etc. How-

ever, great anatomical gaps are found between Calamagrostis s. l. and Aniselytron. Especially a distinct feature of Aniselytron is that the vascular bundles are abaxially situated throughout the leaf blade while those of Calamagrostis s. l. are central. According to Ellis (1976), this could have sufficiently proved that Aniselytron is a genus different from Calamagrostis s. l., without the other differences. Beyond all the above, the most remarkable feature of Aniselytron is the presence of adaxial parenchyma in keel. This has never been found in Pooideae and Arundinoideae, but is common in Panicoideae and rare in Bambusoideae and Chloridoideae (Ellis 1986). Aniselytron is surely a genus in Pooideae, not a group in Bambusoideae, Panicoideae or Chloridoideae, since Bambusoideae is characterized by complex vasculature, and both Panicoideae and Chloridoideae are characterized by Kranz structure. This very rare feature in Pooideae demonstrated its uniqueness not only in Calamagrostis s. l., but also in Pooideae, therefore there is no doubt regarding it as a genus separate from Calamagrostis s. l. This also suggests that it would probably have some relationship with another subfamily. The distribution and habitat of Aniselytron are very different from those of Calamagrostis s. l. Calamagrostis s. l. is mainly distributed in the cool temperate and boreal zones of the world and mainly grows in comparatively wet areas, but can also be found in drier areas. Those few species of Calamagrostis s. l. do extend into subtropical areas, such as D. diffusa and D. petelotii in our study, have typical Calamagrostis leaf anatomy in the genus. Therefore Calamagrostis s. l. is probably derived in cold regions. Aniselytron is, however, distributed in East Asia only, and grows on tropical and subtropical mountains and always in very moist places, usually beside streams. Considering its unique leaf anatomy, it might be derived from tropical areas. Looking back to the spikelet structure, Calamagrostis s. l. is characterized by equal glumes, longer than lemma. Aniselytron is aberrant when included in Calamagrostis s. l.: its two glumes are unequal and much shorter than the lemma; its rachilla extension is reduced and glabrous. A few species of Deyeuxia, including the type species, have a glabrous and reduced rachilla extension like Aniselytron. Some workers have considered the naked and somewhat reduced rachilla extension and sparse minute callus hairs of Aniselytron as a link to Deyeuxia, because some other species

Table 3. Comparison of leaf anatomy of Aniselytron and Calamagrostis s. l. Character

Aniselytron

Calamagrostis s. l.

Median vascular bundle Position of median vascular bundle Cells associated with median vascular bundle Girders accompanying large vascular bundles Large vascular bundles

Forms a keel Abaxial Abaxial girder only; adaxially large parenchyma and a small adaxial strand Either adaxial or abaxial girders

Does not form a keel Central Both adaxial and abaxial girders; with no adaxial parenchyma cells Both adaxial and abaxial girders

Abaxial (in A. agrostoides only the median vascular bundle observed) Sunken based, not bulbous Tall, thin-walled

Central

Prickles Epidermal cells

Not sunken, bulbous based Short and wide, relatively thick-walled

414 Table 4. Comparison of glume length (mm) in Aniselytron and Deyeuxia petelotii Taxon

Lower glume

Upper glume

Lemma

Aniselytron treutleri Aniselytron agrostoides Deyeuxia petelotii

0.5–2.5 0.2–0.7 2.2

2–3.5 1–2.7 2.7

2.5–5 2.5–4 2–2.5

have slightly unequal glumes or of those slightly shorter than the lemma (Korthof and Veldkamp 1984). Lacking any additional corroborative evidence of Aniselytron, the main arguments for or against recognizing Aniselytron have revolved around whether these spikelet features are derived independently from Deyeuxia. Our present study offers evidence against this speculation. We included one species with unequal glumes in our present study: D. petelotii. This species was described as new several times (Hitchcock 1934; Handel-Mazzetti 1936; Keng 1958) in different genera because of its unusual spikelet structure and was once placed in Aniselytron (Hitchcock 1934). Korthof and Veldkamp (1984) regarded it as one of the linking species (though they mistakenly combined it with D. abnormis Hooker). In our study it has a leaf anatomy that is similar to other species of Deyeuxia while dissimilar to that of Aniselytron. This has resulted in a reconsideration of the spikelet structure of Aniselytron. We made a comparison of the glume length in two species of Aniselytron and D. petelotii in Table 4. It is clear that the two glumes of D. petelotii are subequal to the lemma, unlike that of Aniselytron whose glumes are very unequal to the lemma. We believe that the most characteristic feature of glumes in Aniselytron is the greater reduction of lower glume, which is reduced to 0.5 and 0.2 mm in A. treutleri and A. agrostoides respectively, and this is not the case in Deyeuxia. As there is such a great difference between Deyeuxia and Aniselytron in glume length, the unequal glumes in Aniselytron must have come from another origin, not the one derived from Deyeuxia. As a very unusual group, Aniselytron received a lot of attention recently. We have been informed recently that phylogenetic analysis based on cpDNA sequencing demonstrated that Aniselytron is far from Calamagrostis s. l. and much closer to Poa (Davis, unpublished data). Therefore it is increasingly clear that Aniselytron is separate from Calamagrostis s. l. Acknowledgements The financial support of NSFC (30070051 and 40332021), NSFYN (2000C0069 M) and MOST (2003CB415103) are gratefully acknowledged. Our thanks are also to Prof. Hang Sun and Mr. Lisong Wang of Kunming Institute of Botany (KUN) for their help in the fieldwork. We are also indebted to curators of PE, MBK and US for loaning their specimens, and to Dr. Hiroyuki Motomura of Tohoku University of Japan and Dr. Wenli Chen of PE for providing their literature collections. Special thanks go to Dr. Robert J. Soreng of Smithsonian Institution for improvements to the manuscript and to Mr. Martyn Dickson of the Royal Botanic Garden Edinburgh for editing

English. We are also very grateful to two anonymous reviewers for constructive comments.

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