sorolepidium in polystichum (dryopteridaceae): evidence

Chinese Science Bulletin

© 2007 Science in China Press

Springer-Verlag

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Inclusion of the Eastern Asia endemic genus Sorolepidium in Polystichum (Dryopteridaceae): Evidence from the chloroplast rbcL gene and morphological characteristics

LIU HongMei1,2, ZHANG XianChun1†, CHEN ZhiDuan1 & QIU YinLong3

1 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China;

2 Graduate University of the Chinese Academy of Sciences, Beijing 100039, China; 3 Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA

The taxonomic status of the Eastern Asia endemic Sorolepidium is controversial. Some authors accept it as member of the large diverse genus Polystichum, whereas others suggest that it is an independent genus separated from the later by the exindusiate sorus and the absence of aristate teeth at the pinnae margins. Here we infer phylogenetic relationship of Sorolepidium using DNA sequences of the chloro-plast rbcL gene. Phylogenies were inferred using maximum-parsimony, maximum-likelihood, and Bayesian inference methods. Molecular data establish that Sorolepidium is deeply nested within the large genus Polystichum and has a close relationship with P. duthiei and P. lachenense in the model-based analyses. The Kimura 2-parameter distances of the rbcL sequences between S. glaciale and P. duthiei and P. lachenense were 0.1 and 0.2%, respectively. Furthermore, S. glaciale differed from P. duthiei by a single nucleotide in their rbcL sequences. Close relationships between S. glaciale and P. duthiei and P. lachenense are also supported by the shared spore ornamentation with echinate fenes-trate folds.

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Sorolepidium, Polystichum, Dryopteridaceae, phylogeny, rbcL, SEM

Sorolepidium Christ emend. Ching (Dryopteridaceae) is an oligogenus of one or two species[1,2], although four to five have been described from China alone[3,4]. The type of the genus, Sorolepidium glaciale Christ, has a dis-junct distribution from the East Himalayas to Taiwan, with its centers in Yunnan, Sichuan, Xizang and Gansu, China[2] (Figure 1). S. ovale Y. T. Hsieh, another species accepted in the Chinese version flora of China, has a restricted distribution in NW Yunnan, differing from the former only in minute morphology possibly caused by ecological variation.

Sorolepidium is endemic to the East Himalayas, has a disjunct distribution to Taiwan, and is sporadically dis-tributed in rigid, cold alpine regions, growing in cal-

careous rock crevices up to the snow line or by glaciers from 2600 to 4700 m[2]. The rhizomes are short, erect, and densely covered with reddish brown, ovate or broad-lanceolate scales. Leaves are arranged in a rosette, laminae are linear-lanceolate, simply pinnate or slightly bipinnate, and usually obtuse teeth on the edge of the pinnae are cartilaginous and reflexed. The large scales on the lower surface of leaves are densely imbricate and turn pale-white as the growing season advances. Veins are free and pinnate. Sori, at the apex of the veinlets, Received September 9, 2006; accepted December 26, 2006 doi: 10.1007/s11434-007-0115-2 †Corresponding author (email: [email protected]) Supported by the National Natural Science Foundation of China (Grant No. 30228004) and Wong Kuan Chung Education Foundation of Hong Kong

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are circular, exindusiate and eventually confluent, in a single row along the midrib (Figure 2).

Figure 1 The distribution map of Sorolepidium glaciale Christ.

Figure 2 Sorolepidium glaciale Christ. a, Habit; b, pinnule, dorsal re-view; c, pinnule, ventral review, illustration redrawn by Sun Y. B.

The genus Sorolepidium was established by Christ in 1911 based on specimens collected from Yunnan, Chi-na[1]. Although Sorolepidium has been known as an Eastern Asia endemic taxa of Dryopteridaceae[1―4], it has been controversial whether it is distinct from or merged with the large genus Polystichum. Sorolepidium glaciale is very similar to Polystichum duthiei (Hope) C. Chr. in gross morphology and habitat; the former was even re-garded as a synonym of the latter by some pteridolo-gists[5,6]. After a critical study, Ching[3] reestablished the genus and made some corrections to its morphological characteristics. Although Sorolepidium was accepted in all modern Chinese fern literature[2 ― 4,7,8], both Dai-

gobo[9] and Fraser-Jenkins[10] recognized S. glaciale in the section Sorolepidium (Christ) Tagawa in mono-graphs on Polystichum. Pichi Sermolli[11], Tryon and Tryon[12] and Kramer[13] all treated Sorolepidium as a synonym of Polystichum.

Polystichum, comprising 180―230[13] or as many as 300 species[8], is nearly cosmopolitan and mainly dis-tributed in temperate and subtropical montane regions, with a center of diversity in southwest China[4,8]. A re-cent phylogenetic analysis of Polystichum using se-quences of the rbcL gene has shown that Polystichum is paraphyletic with respect to some putative segregates such as Cyrtomium. The analysis also indicated that the sister groups of Polystichum sensu lato (s.l.) are Phan-erophlebia and Polystichopsis[14].

Many studies have employed phylogenetic analysis of DNA sequences of the gene encoding the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) to assess phylogenetic relationships of ferns at various taxonomic rank[14―22]. A large amount of Polys-tichum rbcL sequences are now available[14,19,21], permit-ting analyses across a diverse array of Polystichum.

In clarifying systematic relationship at various levels in pteridophytes, especially at the low level of species, varieties and hybrids, spore ornamentation is a valuable character[23,24]. Mitui[25] recognized 8 spore types in 40 Polystichum species that range from echinate to folded and suggested the echinate type was the fundamental type. Tryon and Lugardon[24] recognized three main types and suggested that the outer perispore fold was usually fenestrate, echinulate and echinate based on 43 species in Polystichum. Based on observations on 79 species and one variety in Polystichum from Yunnan, China, 16 spore types were suggested, in which four types are most frequent; they are fenestrate, winged, coarse tubercles and granulate[26].

Since Sorolepidium was not included in any of those analyses, its systematic status is still uncertain and de-serving of a new phylogenetic investigation. We con-ducted molecular analyses using nucleotide sequence data of the chloroplast gene rbcL from S. glaciale and a suite of Polystichum species. Additionally, we used a scanning electron microscope (SEM) to study the spore morphology of S. glaciale in comparison to Polystichum species.

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as outgroups based on previous molecular studies[14,19,21]. Eighteen sequences were obtained from GenBank (Table 1). Vouchers are deposited in the Herbarium (PE), Insti-tute of Botany, the Chinese Academy of Sciences, Bei-jing. The generic and sectional circumscriptions and taxonomic names of Polystichum and Sorolepidium fol-lowed the treatment of Kung et al.[8].

1 Materials and methods

1.1 Taxon sampling

Sequences of 35 accessions representing Sorolepidium, Polystichum, Cyrtomium, and outgroup taxa were ana-lyzed in this study (Table 1). Our sampling scheme in-cludes a basic spectrum of morphological variation and sectional divisions in Polystichum. Since Cyrtomium is considered to either have a close relationship with or be included in Polystichum[13,14,19], six representatives of Cyrtomium were included in the present analysis. Spe-cies of Phanerophlebia and Polystichopsis were selected

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1.2 DNA extraction, PCR amplification and sequencing

Total genomic DNA was extracted from silica-gel- dried leaves using the modified CTAB procedure of Doyle

Table 1 Taxa used in this study, with voucher information, and GenBank accession numbers Taxon Voucher informationa) Source localities GenBank Accession No.b)

Polystichum acutidens X.C. Zhang 3404 Hubei, China EF394244

Polystichum anomalum H.M. Liu A379 Jiangxi, China EF394248

Polystichum atkinsonii X.C. Zhang 3305 Hubei, China EF394250

Polystichum attenuatum Lu SG/A30 Yunnan, China AY545503 b)

Polystichum brachypterum Lu SG/B9 Yunnan, China AY545501 b)

Polystichum capillipes X.C. Zhang 3304 Hubei, China EF394249

Polystichum christii Lu SG/H13 Yunnan, China AY545486 b)

Polystichum craspedosorum M. Kato s.n. Japan AF537238 b)

Polystichum crinigerum LJM 062 Yunnan, China AY694813 b)

Polystichum duthiei Z.D. Fang F009 Yunnan, China EF394252

Polystichum grandifrons Lu SG/I31 Yunnan, China AY545484 b)

Polystichum hancockii H.M. Liu A366 Jiangxi, China EF394243

Polystichum lachenense Z.D. Fang F004 Yunnan, China EF394251

Polystichum lentum NYBG 474/77 US AF537246 b)

Polystichum lonchitis J. Macoun s.n. Alaska, US AF537247 b)

Polystichum luctuosum X.C. Zhang 2421 Sichuan, China EF394245

Polystichum makinoi X.C. Zhang 3365 Hubei, China EF394247

Polystichum neolobatum X.C. Zhang 3330 Hubei, China EF394246

Polystichum nepalense Lu SG/C59 Yunnan, China AY545499 b)

Polystichum omeiense Lu SG/D5 Yunnan, China AY545497 b)

Polystichum stenophyllum D. Boufford 27327 Sichuan, China AF537256 b)

Polystichum subacutidens Lu SG/D8 Yunnan, China AY545488 b)

Polystichum tripteron Hasebe et al.1995 Japan U30832 b)

Polystichum xiphophyllum LJM038 Unkown DQ508788 b)

Polystichum yunnanense Lu SG/28 Yunnan, China AY545504 b)

Polysticum yuanum Lu SG/H11 Yunnan, China AY545487 b)

Sorolepidium glaciale X.C. Zhang 2772 Yunnan, China EF394253

Cyrtomium falcatum X.C. Zhang 2397 Seoul, Korea EF394238

Cyrtomium fortunei X.C. Zhang 2700 Chongqing, China EF394237

Cyrtomium macrophyllum H.M. Liu A249 Jiangxi, China EF394239

Cyrtomium nephrolepioides X.C. Zhang 2595 Chongqing, China EF394242

Cyrtomium pachyphyllum X.C. Zhang 2594 Chongqing, China EF394241

Cyrtomium urophyllum Z.Y. Liu 2022824 Yunnan, China EF394240

Phanerophlebia nobilis G. Yatskievych et al. 85-211 Mexico AF537231 b)

Polystichopsis chaerophylloides D.S.Barrington 140 Puerto Rico AF537234 b)

a) X. C. Zhang, Zhang XianChun; H. M. Liu, Liu HongMei; Z.D. Fang, Fang ZhenDong; Z.Y. Liu, Liu ZhengYu. b) Sequences from GenBank.

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and Doyle[27]. The selected DNA regions were amplified with standard polymerase chain reaction (PCR). The amplification of the rbcL gene used primers 1F[28] and 1351R[22]; an additional sequencing primer ， 440F (5′-GGT AAT GTT TTT GGA TTT AAG GC-3′)[22], was also used in this study. The PCR protocol used to am-plify the rbcL gene was identical to that described in Li et al.[29]. The PCR products were purified using a GFXTM PCR DNA and Gel Band Purification Kit, then directly sequenced. Sequencing reactions were con-ducted using the DYEnamicTM ET Dye Terminator Cy-cle Sequencing Kit. Sequences were analyzed using MegaBACETM1000 DNA Analysis Systems, following the manufacture’s protocol.

1.3 Phylogenetic analyses

Sequences were aligned using CLUSTAL X[30] and re-fined manually by eye. Phylogenetic analyses were per-formed with maximum parsimony (MP), maximum like-lihood (ML), and Bayesian inference (BI) methods, re-spectively. For MP analysis, heuristic searches were performed in PAUP* 4.0b10[31] with 1000 random taxa-addition replicates, one tree held at each step during stepwise addition, tree-bisection-reconnection (TBR) branch swapping, MulTrees in effect, and steepest de-scent off. All characters were unordered and equally weighted. To assess node support, bootstrap analyses[32] were performed using 1000 heuristic search replicates as described above. For ML analyses, an optimal model of nucleotide evolution was selected using the Akaike In-formation Criterion (AIC) as implemented in Modeltest 3.06[33]. ML analyses of the rbcL sequences were per-formed in PHYML[34] using the general time reversible model (GTR) with invariant sites and additional among site rate variation modeled as a discrete gamma point with the appropriate parameters. ML bootstrap analyses were carried out with 1000 replicates. BI was conducted using MrBayes version 3.0b4[35]. The posterior probabil-ity (PP) was estimated with four chains; each chain was initiated with a random tree, run for 1000000 genera-tions, and sampled every 1000 generations. For all analyses, the first 50 samples from each run were dis-carded as burn-in to ensure that the chains reached sta-tionarity. Sequence divergence among taxa was esti-mated using the Kimura 2-parameter distance for all pairs of sequences in PAUP.

1.4 Spore morphology

Spores were removed from herbarium sheets. The spores

were mounted on aluminum stubs and coated with gold-palladium before direct observation using a Hitachi S-800 scanning electron microscope (SEM) to collect micrographs. The terminology of spore surfaces corre-sponded to that used by Tryon and Lugardon[24].

2 Results

2.1 Molecular data and phylogenetic analyses

Seventeen rbcL sequences were newly obtained in this study. The alignment of the rbcL gene in 35 accessions produced a data set of 1204 characters, with 162 vari-able sites (13.5%), 89 of which were phylogenetically informative (7.4%). Parsimony analysis of the rbcL data set resulted in 32622 most parsimonious trees (MPTs) with a total length of 246 steps, a consistency index (CI) of 0.691 and a retention index (RI) of 0.775.

Selection criteria implemented in Modeltest sug-gested that the general time reversible model (GTR) best fit the rbcL data set. The maximum-likelihood tree is shown in Figure 3, with Bayesian posterior probabilities and ML bootstrap values indicated on the ML tree. The BI and ML analyses yielded trees with nearly identical topologies, with a few minor differences at the species level (not shown, Figure 3). Within the context of Cyr-tomium and all section representatives of Polystichum examined, three main clades were resolved from both ML and BI analyses: clades A, B and Cyrtomium (PP=0.80, 1.0 and 1.0 and BPML=72%, 87% and 99%, respectively), with clade B as the basal group among them. The sister relationship between clade A and Cyr-tomium was moderate-highly supported (PP=0.94, BPML=82%). Within clade A, two subclades A1 and A2 were also recognized, although the subclade A2 received only weak supports (PP, BPP

ML<50%). S. glaciale was deeply nested within clade A, forming a group with P. duthiei and P. lachenense (PP=0.73, BPML=62%).

The MP trees were identical to the ML and BI trees in recognizing monophyletic Cyrtomium and B clades, but clade A had unresolved polytomies and lower support values. The strict consensus tree is shown in Figure 4, with bootstrap values shown above the branches. S. gla-ciale was clearly nested within the genus of Polystichum, although its relative(s) was not identified in the MP analysis.

2.2 Sequence divergence

The sequence divergence of the rbcL gene among


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Figure 3 Phylogram resulting from the maximum-likelihood analysis of the chloroplast rbcL data. The Bayesian topology was similar to the ML topology and their branch-support measures are mapped to the ML phylo-gram. Numbers above branches present posterior probabilities (>50%) obtained in the Bayesian inference. Numbers below branches represent ML bootstrap values above 50%. Three main clades (A, B and Cyrtomium) are labed on the tree. Shaded boxes denote the generic and taxonomical sections of Polystichum followed the treatment of Kung et al.[8]. Soro-lepidium glaciale is in bold. taxa was estimated using the Kimura 2-parameter dis-tance for all pairs of sequences in PAUP, but only diver-gence values among Sorolepidium and Polystichum are shown in Table 2. In the rbcL gene, sequence divergence within Polystichum ranged from 0.3% (between P. luc-tuosum and P. xiphophyllum, and between P. makinoi and P. yunnanense, with the rbcL sequences of P. omeiense and P. christii were identical) to 3.6% (be-tween P. tripteron and P. lentum). The divergence be-tween Sorolepidium and Polystichum ranged from 0.1% (between S. glaciale and P. duthiei) to 2.8% (between S. glaciale and P. tripteron). S. glaciale differs from P. duthiei by a single nucleotide, and two nucleotide varia-tions are found between S. glaciale and P. lachenense in their rbcL sequences.

2.3 Spore morphology

Under the SEM, spores of Sorolepidium glaciale and eleven species of Polystichum examined showed three

Figure 4 The strict consensus tree of the 32622 equally most- parsimonious trees in the maximum parsimony analyses of the rbcL data set (tree with length of 246 steps, a consistency index of 0.691, a retention index (RI) of 0.775). Numbers above branches correspond to bootstrap values (>50%) obtained in the most parsimonious analysis. The generic and taxonomical sections of Polystichum followed the treatment of Kung et al.[8]. Sorolepidium glaciale is in bold. ornamentation types (Figure 5), i.e. fenestrate or echi-nate fenestrate folds (Figure 5(a)―(f)), winged folds (Figure 5(g) and (h)), and coarse tubercles (Figure 5(i)); the first is the most common type in Polystichum. S. glaciale has spores sculptured with echinate fenestrate folds, a type also found in P. duthiei, P. lachenense, P. moupinense and P. sinense (Figure 5(b) and (d)―(f)).

3 Discussion

3.1 The taxonomic position for Sorolepidium

A previous molecular study by Little and Barrington[14] recognizes Cyrtomium and several other Polystichum allies as segregate genera and accepts a more narrowly circumscribed Polystichum. In our analyses, all repre-sentatives of Cyrtomium formed a well supported mo-nophyletic group apart from members of Polystichum.

Our molecular data indicate that Sorolepidium is deeply nested within the genus Polystichum and forms a group with P. duthiei and P. lachenense (Figure 3). Al-


Figure 5 SEM photographs of spores in Sorolepidium glaciale and several Polystichum. (a) S. glaciale Christ; (b) P. duthiei (Hope) C. Chr.; (c) P. anomalum (Hook. & Arn.) J. Sm.; (d) P. moupinense (Franch.) Bedd.; (e) P. sinense Christ; (f) P. lachenense (Hook.) Bedd.; (g) P. herbaceum Ching & Z.Y. Liu; (h) P. stimulans (Kunze & Mett.) Bedd.; (i) P. craspedosorum (Maxim.) Diels. Vouchers: (a) H-M, Liu YN285; (b), (d), (g)―(i), X-C, Zhang 2915, 3442, 3329, 3398, 3408, respectively; (c) Wu-zhi Shan fern Exp. HN182; (e) B-Q, Zhong 5233; (f) Tibet Exp. 7825; all vouchers were deposited in PE. Scale bars: =15 μm ((a), (e) and (g)); =17.6 μm ((b) and (f)); =12.0 μm ((c) and (h)); =23.1 μm (d); =22.9 μm (i). Table 2 Sequence divergence values in rbcL for Sorolepidium and Polystichum Taxon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

1 - 12 22 27 29 35 35 35 35 35 35 36 35 38 40 28 36 40 33 41 38 37 37 38 34 31 322 1 - 18 23 23 29 29 31 31 34 33 33 29 33 35 25 30 34 29 35 34 34 33 34 30 27 283 1.9 1.5 - 11 13 20 21 27 27 26 27 27 25 29 31 21 26 30 25 31 28 28 27 28 24 21 224 2.3 1.9 0.9 - 14 21 22 24 24 25 24 26 26 26 28 21 25 29 24 32 27 25 26 27 23 20 215 2.5 2.0 1.1 1.2 - 21 22 28 27 29 29 29 24 27 30 22 25 29 24 32 29 29 27 28 25 22 236 3.1 2.5 1.7 1.8 1.9 - 0 30 30 33 32 31 30 33 35 24 29 35 30 38 35 34 34 35 31 28 297 3.0 2.5 1.8 1.9 1.9 0 - 30 30 33 32 32 30 34 36 24 29 35 30 38 35 34 34 35 31 28 298 3.0 2.6 2.3 2.0 2.4 2.6 2.6 - 4 15 14 22 18 22 24 15 19 21 18 26 23 23 22 23 21 20 199 3.0 2.6 2.3 2.0 2.4 2.6 2.6 0.3 - 13 12 20 18 20 22 14 17 19 16 24 21 21 20 21 19 18 17

10 3.0 2.9 2.2 2.1 2.5 2.9 2.8 1.3 1.1 - 9 17 19 19 21 11 20 20 19 26 20 18 15 16 16 15 1411 3.0 2.8 2.3 2.0 2.5 2.8 2.7 1.2 1.0 0.8 - 16 18 18 20 11 19 21 18 26 19 17 16 17 15 14 1312 3.1 2.8 2.3 2.2 2.5 2.7 2.7 1.9 1.7 1.4 1.4 - 16 14 16 9 19 19 17 26 17 15 12 13 11 12 1113 3.0 2.5 2.1 2.2 2.1 2.6 2.5 1.5 1.5 1.6 1.5 1.3 - 14 16 10 15 14 13 22 14 14 14 15 13 12 1114 3.3 2.8 2.5 2.2 2.3 2.9 2.9 1.9 1.7 1.6 1.5 1.2 1.2 - 4 8 17 17 14 24 17 11 12 13 11 12 1115 3.5 3.0 2.6 2.4 3.6 3.1 3.1 2.0 1.9 1.8 1.7 1.3 1.3 0.3 - 10 19 19 16 26 19 13 14 15 13 14 1316 2.7 2.4 2.0 2.0 2.1 2.4 2.3 1.4 1.3 1.1 1.0 0.9 1.0 0.8 1 - 9 10 7 18 9 7 5 6 5 5 417 3.1 2.6 2.2 2.1 2.1 2.5 2.5 1.6 1.4 1.7 1.6 1.6 1.3 1.4 1.6 0.9 - 14 9 17 14 14 11 12 12 11 1018 3.5 2.9 2.6 2.5 2.5 3.1 3.0 1.8 1.6 1.7 1.8 1.6 1.1 1.4 1.6 0.9 1.2 - 9 17 14 14 13 14 12 11 1019 2.8 2.5 2.1 2.0 2.1 2.6 2.6 1.5 1.4 1.6 1.5 1.4 1.1 1.2 1.3 0.7 0.8 0.8 - 13 13 13 12 13 11 10 920 3.6 3.0 2.6 2.7 2.8 3.4 3.3 2.2 2.0 2.2 2.2 2.2 1.9 2.0 2.2 1.7 1.4 1.4 1.1 - 21 21 18 19 19 18 1721 3.3 2.9 2.4 2.3 2.5 3.1 3.0 1.9 1.8 1.7 1.6 1.4 1.1 1.4 1.6 0.9 1.2 1.2 1.1 1.8 - 10 11 12 10 9 822 3.2 2.9 2.4 2.1 2.5 3.0 3.0 1.9 1.8 1.5 1.4 1.3 1.2 0.9 1.1 0.7 1.2 1.2 1.1 1.8 0.8 - 9 10 8 7 623 3.2 2.8 2.3 2.2 2.3 3.0 2.9 1.9 1.7 1.3 1.3 1.0 1.2 1.0 1.2 0.5 0.9 1.1 1.0 1.5 0.9 0.8 - 1 5 6 524 3.3 2.9 2.4 2.3 2.4 3.1 3.0 1.9 1.8 1.3 1.4 1.1 1.3 1.1 1.3 0.6 1.0 1.2 1.1 1.6 1.0 0.8 0.1 - 6 7 625 2.9 2.6 2.0 1.9 2.1 2.7 2.6 1.8 1.6 1.3 1.3 0.9 1.1 0.9 1.1 0.5 1.0 1.0 0.9 1.6 0.8 0.7 0.4 0.5 - 3 226 2.7 2.2 1.8 1.7 1.9 2.5 2.4 1.7 1.5 1.3 1.2 1.0 1.0 1.0 1.2 0.4 0.9 0.9 0.8 1.6 0.8 0.6 0.5 0.6 0.3 - 127 2.8 2.4 1.9 1.8 2.0 2.5 2.5 1.6 1.4 1.2 1.1 0.9 0.9 0.9 1.1 0.4 0.8 0.8 0.8 1.4 0.7 0.5 0.4 0.5 0.2 0.1 -

a) Numbers above diagonal are absolute numbers of nucleotide differences; Numbers below diagonal are percentage differences (%). Taxon names are abbreviated as follows and are related to the data in Table 1. 1, Polystichum tripteron; 2, Polystichum hancockii; 3, Polystichum subacutidens; 4, Polysti-chum acutidens; 5, Polysticum yuanum; 6, Polystichum omeiense; 7, Polystichum christii; 8, Polystichum luctuosum; 9, Polystichum xiphophyllum; 10, Polystichum neolobatum; 11, Polystichum brachypterum; 12, Polystichum lonchitis; 13, Polystichum craspedosorum; 14, Polystichum yunnanense; 15, Polystichum makinoi; 16, Polystichum grandifrons; 17, Polystichum anomalum; 18, Polystichum nepalense; 19, Polystichum attenuatum; 20, Polystichum lentum; 21, Polystichum crinigerum; 22, Polystichum capillipes; 23, Polystichum atkinsonii; 24, Polystichum stenophyllum; 25, Polystichum lachenense; 26, Polystichum duthiei; 27, Sorolepidium glaciale.


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though the placement of Sorolepidium as a sister clade to P. duthiei lacks strong support in our phylogenetic analyses, the number of substitutions per site in the ML analyses (Figure 3) and the number of character state changes in the MP analyses (not shown) indicate that the recovered relationships of Sorolepidium are not likely to be the result of long-branch attraction. The sequence divergence between Sorolepidium and Polystichum is lower than that within Polystichum (0.1%―2.8% < 0.3%―3.6%). The nearly identical rbcL sequences of S. glaciale and P. duthiei and P. lachenense support close relationships between S. glaciale and P. duthiei and P. lachenense. This molecular evidence suggests that it is inappropriate to consider Sorolepidium as a separate genus.

The genus Sorolepidium was established based mainly on the exindusiate sori, absence of aristate teeth at the pinnae margins and its characteristic large scales covering the plant body and even hiding the sori[1]. After a revision and some corrections to its morphological characteristics, Ching[3] emphasized its specific mor-phological and ecological characters and reestablished the genus. However, these morphological characters do not distinguish it well from Polystichum. (1) The indu-sium character is not a synapomorphy of Polystichum, for many Polystichum species are exindusiate (such as P. grandi frons and P . ru fopaleaceum ) or have ill-developed indusia (as in P. altum and P. parvifoli-olatum). The exindusiate condition of the sorus might be a secondary loss as has occurred in many other members of Dryopteridaceae[36]. The absence of indusium in S. glaciale emphasised by Christ[1] and Ching[3] to support the distinctness of Sorolepidium may be interpreted merely as retention of a phylogenetically uninformative homoplasy. (2) The pinnae of Sorolepidium are ob-tuse-crenate or entire, while most species of Polystichum have aristate teeth on the margin. However, pinnae in some species of Polystichum are either entire (P. moupinense), slightly pinnatifid or have obtuse teeth (P. duthiei and P. paramoupinense). Leaf shapes and pinnae margins are always different in varied environments and may show changes in the same species even on the same leafy organs. Thus, the loss of spinules in the pinnae might be a synapomorphy for Sorolepidium and is re-

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garded as a useful character at species or lower levels

but not at the generic level[10]. (3) The large, dense- cov-ering scales might be the result of adaptation to extreme of drought and cold at high altitudes. This character is also found in P. duthiei and some other high alpine spe-cies[12,37] and since it probably has adaptive significance it does not necessarily indicate affinity. Actually, except for the exindusiate sori, S. glaciale and P. duthiei are very similar in morphology and habit, often occur to-gether in the same environment in the eastern Himalayas, and can readily be misidentified as the same species. Therefore, the results of comparative morphology and the molecular phylogeny show that S. glaciale should be included in the large genus Polystichum.

3.2 The possible close relative(s) of S. glaciale within Polystichum

The molecular data support the placement of Soro-lepidium within Polystichum and suggest a close rela-tionship with P. duthiei and P. lachenense. In the model-based methods (Figure 3), S. glaciale is nested within the subclade A2 and forms a group with P. duthiei and P. lachenense (PP=0.73, BPML=62%). This relationship is also supported by the nearly identical se-quences between S. glaciale and P. duthiei, and between S. glaciale and P. lachenense.

In our observations of spores of S. glaciale and 11 Polystichum species under the SEM, three main types are recognized: fenestrate or echinate fenestrate folds, winged folds and coarse tubercles. The spore ornamen-tation of S. glaciale is identical to that of P. duthiei, P. lachenense, P. moupinense and P. sinense, in that they have fenestrate or echinate fenestrate folds. The shared spore ornamentation with echinate fenestrate folds also supports the close relationship between S. glaciale and P. duthiei and P. lachenense. Thus, the shared morpho-logical features and spore ornamentation as described above and the molecular phylogeny show that S. gla-ciale should be placed in Polystichum and has a close relationship with P. duthiei and P. lachenense, two al-pine species of Polystichum in eastern Himalayas.

The authors thank Fang ZhenDong and Liu ZhengYu for providing mate-rials; Huang ChaCha for lab assistance; Sun YingBao for drawing figure; Wang Wei and Liu Yang for much help and valuable discussion; George Yatskievych and David S. Barrington for comments on an earlier draft of this manuscript.


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sorolepidium in polystichum (dryopteridaceae): evidence

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