Palaeoworld 16 (2007) 294–300

Available online at www.sciencedirect.com

Research paper

Phylogeny and biogeography of Chinese and AustralasianPolystichum ferns as inferred from chloroplast trnL-F

and rps4-trnS sequence data

Chun-Xiang Li a,∗, Shu-Gang Lu b, Qun Yang a

a State Key Laboratory of Palaeobiology and Stratigraphy of Nanjing Institute of Geology and Palaeontology,The Chinese Academy of Science, Nanjing 210008, People’s Republic of China

b Institute of Ecology and Geobotany, Yunnan University, Kunming 650091, People’s Republic of China

Received 20 July 2006; received in revised form 11 April 2007; accepted 10 July 2007Available online 17 July 2007

Abstract

Polystichum, one of the largest genera of ferns, occurs worldwide with the greatest diversity in southwest China and adjacentregions. Although there have been studies of Chinese Polystichum on its traditional classification, geographic distributions, andeven a few on its molecular systematics, its relationships to other species outside China remain little known. Here, we investigatedthe phylogeny and biogeography of the Polystichum species from China and Australasia. The evolutionary relationships among42 Polystichum species found in China (29 taxa) and Australasia (13 taxa) were inferred from phylogenetic analyses of twochloroplast DNA sequence data sets: rps4-trnS and trnL-F intergenic spacers. The divergence time between Chinese and AustralasianPolystichum was estimated. The results indicated that the Australasian species comprise a monophyletic group that is nested withinthe Chinese diversity, and that the New Zealand species are likewise a monophyletic group nested within the Australasian species.The divergence time estimates suggested that Chinese Polystichum migrated into Australasia from around 40 Ma ago, and from

there to New Zealand from about 14 Ma. The diversification of the New Zealand Polystichum species began about 10 Ma. Theseresults indicated that Polystichum probably originated in eastern Asia and migrated into Australasia: first into Australia and theninto New Zealand.© 2007 Published by Elsevier Ltd on behalf of Nanjing Institute of Geology and Palaeontology, CAS.

; Diver

Keywords: Polystichum; rps4-trnS; trnL-F; Phylogeny; Biogeography

1. Introduction

Polystichum Roth, one of the largest genera of ferns,

comprises about 300 species (Wu and Ching, 1991). Thisgenus is nearly cosmopolitan in distribution, but is abun-dant in southwest China and adjacent regions with 168

∗ Corresponding author. Tel.: +86 25 8328 2286;fax: +86 25 8335 7026.

E-mail address: cxli@nigpas.ac.cn (C.-X. Li).

1871-174X/$ – see front matter © 2007 Published by Elsevier Ltd on behalf odoi:10.1016/j.palwor.2007.07.003

gence time

species (Kung et al., 2001). At present, traditional clas-sification, geographic distributions, and a few molecularsystematic studies of Chinese Polystichum have beenconducted (Kramer and Green, 1990; Kung et al., 2001;Little and Barrington, 2003; Li et al., 2004); however, itsrelationships to those species out of China are obscure.

Recent phylogenetic studies have shed light on the

evolutionary relationships and historical biogeographyof Polystichum and allied genera. Little and Barrington(2003) used rbcL sequences and morphology, and Liet al. (2004) examined rbcL sequences exclusively,

f Nanjing Institute of Geology and Palaeontology, CAS.

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ielding basic insights into the phylogeny of the genus.hese works have identified the sister group ofolystichum; defined a monophyletic Polystichum s.l.;nd provided evidence that the genus originated inastern Asia. However, these studies focused on thehylogeny of Polystichum from throughout the world;nly a few species from southwest China (a major cen-er of diversity for Polystichum) were included in thenalyses. In addition, the rbcL sequence data did not pro-ide enough informative characters to allow resolutionf species relationships from China and other regions.ntergenic spacer sequences, such as trnL-F and rps4-rnS, have proven more effective than coding regions foresolving recent divergence events in the ferns (Smallt al., 2005). For instance, trnL-F has proved useful intudies of Cyrtomium, an allied genus of PolystichumLu et al., 2005 in combination with rbcL sequences).nother intergenic spacer, rps4-trnS, has only recentlyeen applied to infrageneric phylogenetic work in theerns (Hymenophyllum, Hennequin et al., 2003 andlaphoglossum, Rouhan et al., 2004; Skog et al., 2004).

The plants of the southern hemisphere have tra-itionally been considered as having a fundamentallyicariant history (Raven and Axelrod, 1972; Barron etl., 1981). The common trans-Pacific disjunctions oflants are usually explained by the sequential breakupf the supercontinent Gondwana during the last 165 mil-ion years, causing successive vicariance of a commonncestral biota (Brundin, 1966). However, recent bio-eographic studies, based on molecular estimates andore accurate paleogeographic reconstructions, indicate

hat dispersal may have been more important than tra-itionally assumed vicariant events (Winkworth et al.,002; Sanmartin and Ronquist, 2004). Such a dispersalvent was invoked to explain the disjunct distribution ofolystichum ferns between Australia and New Zealandy Perrie et al. (2003) based on chloroplast rps4-trnS andbcL sequences and AFLP data. In the present study,e newly sequenced two plastid intergeneic spacers

rps4-trnS and trnL-F) for representatives of the Chi-ese Polystichum species in addition to the previouslytudied species. Based on the phylogenetic analyses andhe estimates of separation time, we aimed to address theelationships of Polystichum species between China andustralasia.

. Materials and methods

.1. Plant material

In addition to the 14 species examined in Perrie etl. (2003), as listed in Table 1, we sequenced 28 species

16 (2007) 294–300 295

from China, representing 10 of the 13 sections in the clas-sification of Kung et al. (2001). Based on the previousstudies by Little and Barrington (2003), Cyrtomidictyumlepodocaulon, Dryopteris sinofibrillosa, and Dryopterischampionii were chosen as the outgroups. Material forthis study was collected in the wild or, in a few cases,from cultivated plants (see Table 2 for Voucher informa-tion).

2.2. DNA extraction

Total genomic DNA was extracted from 2 g offresh or 1 g of silica gel dried leaves using the CTAB(cetyltrimethylammonium bromide) procedure (Hillis etal., 1996), as modified by Shi et al. (1996). Leaveswere ground in 65 ◦C 2× CTAB buffer supplementedwith 2% PVP (polyvinylpyrrolidone), extracted twice inchloroform:isoamylalcohol, 24:1, precipitated in ethanolovernight at −20 ◦C, spun, washed in 70% ethanol,resuspended in Tris–EDTA (10 mmol/L Tris, 1 mmol/LEDTA, pH 8.0), and purified with glass powder whennecessary.

2.3. PCR amplification and DNA sequencing

Polymerase chain reaction (PCR) of the rps4-trnSand trnL-F regions were performed in 50 �L vol-umes containing 2.5 units DNA polymerase, 1× buffer,2.5 mmol/L MgCl2, 0.1 �mol/L dNTP, 0.1 �mol/L eachprimer, 5% BSA, and ∼100 ng sample DNA. Theprimers “e” and “f” of Taberlet et al. (1991) wereused for trnL-F region amplification, with a ther-mocycling profile of: initial denaturation at 94 ◦Cfor 2 min, 40 cycles at 94 ◦C for 1 min, 55 ◦C for1 min, 72 ◦C for 1 min, and final extension at 72 ◦Cfor 7 min. The primers 5′-ATG AAT T (A/G)T TAGTTG TTG AG-3′ (which we designed) and 5′-TACCGAGGG TTC GAA TC-3′ (Souza-Chies et al., 1997)were used to amplify the rps4-trnS spacer region,with a thermocycling profile of: initial denaturationat 94 ◦C for 2 min, 40 cycles at 94 ◦C for 1 min,56 ◦C for 1 min, 72 ◦C for 1 min, and final exten-sion at 72 ◦C for 7 min. The amplified products werepurified for sequencing by WizardR PCR preps DNAPurification System (Promega, USA) following the sup-plier’s specifications to remove the redundant smallmolecular fragments of primers and dNTPs. Purifieddouble-stranded DNAs were sequenced on an ABI

377 automated sequencer (Applied Biosystems, Califor-nia, USA) with the Bigdye cycle sequencing kit. Thesequences were determined in both directions using theamplification primers.

296 C.-X. Li et al. / Palaeoworld 16 (2007) 294–300

Table 1The samples and locality information for the new sequences in this study

Taxona Distribution Voucher or reference Genbank (trnL-F)b No. (rps4-trnS)

Cyrtomidictyum lepidocaulon (Hook.) Ching China Lu SG Q12 (PYU) DQ150392 DQ151855Dryopteris championii (Benth.) C. Chr. China Lu SG M3 (PYU) DQ150393 DQ151856Dryopteris sinofibrillosa Ching China Lu SG C60 (PYU) DQ150394 DQ151857Polystichum altum Ching ex L.B. Zhang et H.S. Kung China Lu SG EM3 (PYU) DQ150395 DQ151858Polystichum attenuatum Tagawa et Iwatsuki China Lu SG H10 (PYU) DQ150396 DQ151859Polystichum auriculum Ching China Lu SG 31 (PYU) DQ150397 DQ151860Polystichum australiense Tindale Australia Perrie et al. (2003) – AY164624Polystichum chingae Ching China Lu SG N7 (PYU) DQ150398 DQ151861Polystichum christii Ching China Lu SG H13 (PYU) DQ150399 DQ151862Polystichum cystostegia (Hook.) J.B. Armstr. New Zealand Perrie et al. (2003) – AY164630Polystichum dielsii H. Christ China Lu SG QC6 (PYU) DQ150400 DQ151863Polystichum discretum (Don) J. Sm. China Lu SG B41 (PYU) DQ150401 DQ151864Polystichum disjunctum Ching ex W.M. Chu et Z.R. He China Lu SG H15 (PYU) DQ150402 DQ151865Polystichum erosum Ching et Shing China Lu SG Z17 (PYU) DQ150403 DQ151866Polystichum fallax Tindale Australia Perrie et al. (2003) – AY164625Polystichum formosum Tindale Australia Perrie et al. (2003) – AY164626Polystichum gongboense Ching et S.K. Wu China Lu SG K43 (PYU) DQ150404 DQ151867Polystichum herbaceum Ching et Z.Y. Liu China Lu SG X18 (PYU) DQ150405 DQ151868Polystichum jizhushanense Ching China Lu SG 28 (PYU) DQ150406 DQ151869Polystichum lentum (Don) Moore Himalayas Perrie et al. (2003) – AY164637Polystichum longipinnulum Nair China Lu SG W14 (PYU) DQ150407 DQ151870Polystichum mayebarae Tagawa China Lu SG Z33 (PYU) DQ150408 DQ153079Polystichum moorei H. Christ Lord Howe Island Perrie et al. (2003) – AY164628Polystichum moupinense (Franch.) Bedd. China Lu SG Z18 (PYU) DQ150409 DQ151871Polystichum neozelandicum Fee subsp. neozelandicum New Zealand Perrie et al. (2003) – AY164631Polystichum neozelandicum Fee subsp. zerophyllum

(Colenso) PerrieNew Zealand Perrie et al. (2003) – AY164632

Polystichum nudisorum Ching China Lu SG H8 (PYU) DQ150410 DQ151872Polystichum oculatum (Hook.) J.B. Armstr. New Zealand Perrie et al. (2003) A AY164633Polystichum parvifoliolatum W.M. Chu China Lu SG N9 (PYU) DQ150411 DQ151873Polystichum piceopaleaceum Tagawa China Lu SG K50 (PYU) DQ150412 DQ151874Polystichum proliferum (R.Br.) C. Presl Australia Perrie et al. (2003) A AY164627Polystichum punctiferum C. Chr. China Lu SG K45 (PYU) DQ150413 DQ151875Polystichum pycnopterum (H. Christ) Ching China Lu SG B11 (PYU) DQ150414 DQ151876Polystichum semifertile (Clarke) Ching China Lu SG B32 (PYU) DQ150415 DQ151877Polystichum silvaticum (Colenso) Diels New Zealand Perrie et al. (2003) – AY164634Polystichum sinotsus-simense Ching et Z.Y. Liu China Lu SG Z16 (PYU) DQ150416 DQ151878Polystichum stimulans (Kunze ex Mett.) Bedd. China Lu SG K48 (PYU) DQ150417 DQ151879Polystichum subacutidens Ching ex L.L. Xiang China Lu SG D8 (PYU) DQ150418 DQ151880Polystichum tsus-simense (Hook.) J. Sm. China Lu SG J24 (PYU) DQ150419 DQ151881Polystichum vestitum (G. Forst.) C. Presl New Zealand Perrie et al. (2003) A AY164635Polystichum wawranum (Szyszyl. in Wawra) Perrie New Zealand Perrie et al. (2003) – AY164636Polystichum whiteleggei Watts Lord Howe Island Perrie et al. (2003) – AY164629Polystichum xiphophyllum (Baker) Diels China Lu SG EM43 (PYU) DQ150420 DQ151882Polystichum yuanum Ching China Lu SG H11 (PYU) DQ150421 DQ151883

a pularisthose se

Taxonomic names follow the classification in Flora Reipubicae Pob “–” represents that the sequences are not available; “A” represents

Dr. Leon Perrie; they are available from the first author upon request.

2.4. Sequence analysis

The newly obtained sequences have been sub-mitted to GenBank (Table 1). The sequences werealigned using the Clustal X program (Thompson et al.,1997). Phylogenetic analyses were performed by the

Sinicae (Kung et al., 2001).quences without GenBank accession numbers, but we got them from

maximum parsimony (MP) and neighbor-joining (NJ)methods with Mega 2 (Kumar et al., 2001) software

packages. Parsimony analysis was performed via theclose-neighbor-interchange (CNI) searching algorithmwith uniformly (standard parsimony) weighting meth-ods, using all sites, and random addition trees. Character

C.-X. Li et al. / Palaeoworld 16 (2007) 294–300 297

Table 2Comparison of results generated with maximum parsimony analysis for different data sets, CI = consistency index, RI = retention index

Data set Number of variablecharacters (%)

Number of informativecharacters (%)

Number of trees Length of trees CI RI

trnL-F spacer 129 (27.51%) 65 (13.86%) 94 171 0.8070 0.8813rC

sgtmcptoui2shPwdn(runrpedfrtdw“su2

2

feLoR

ps4-trnS spacer 233 (44.89%) 125 (24.08%)ombined data 447 (43.95%) 186 (18.29%)

tates (nucleotides) were specified as unordered, andaps were treated as missing data. The genetic dis-ance used in NJ analysis was Kimura’s two-parameter

ethod (Kimura, 1980). Bootstrapping with 1000 repli-ates for NJ and 500 replicates for MP analysis wereerformed to estimate the support on each branch ofhe phylogenetic trees constructed. A combined data setf the two-chloroplast DNA sequences was analyzedsing the Bayesian inference (BI) method implementedn MrBayes version 3.1.2 (Ronquist and Huelsenbeck,003) and MP analysis. Before combining the two dataets, data congruence was assessed with the partitionomogeneity test (Farris et al., 1995) implemented withAUP*4.0b5 (Swofford, 2003). One thousand replicatesere performed and the resulting P-value was used toetermine that combining the data sets for phyloge-etic reconstruction was appropriate. An MCMCMCmetropolis-coupled Markov chain Monte Carlo) algo-ithm that runs four Markov chains simultaneously wassed to estimate the posterior probability of phyloge-etic trees. The Markov chains were started from aandom tree and run for 1,000,000 generations, sam-ling every 100 generations for a total of 10,000 samplesach run. The first 2500 samples from each run wereiscarded as burn-in. The standard deviation of splitrequencies was less than 0.01 and the potential scaleeduction factor (PSRF) was around 1.00 for all parame-ers at this point, suggesting convergence to a stationaryistribution had been reached. The remaining samplesere combined into a single file and analyzed using the

sumt” command in MrBayes version 3.1.2. MrBayesettings for the best-fit model (GTR + G) were selectedsing the AIC criterion in MrModeltest 2.0 (Nylander,004).

.5. Divergence time estimation

In the absence of a clear calibration point from theossil record, we used the published divergence time

stimation that Polystichum s.l. originated in Asia in theate Cretaceous (≈76 Ma) (Li et al., 2004) to infer thenset of diversification of other Polystichum lineages.elative-rate tests were conducted to assess homogene-

783 364 0.7720 0.80051161 646 0.8202 0.8316

ity of substitution rates among the lineages with RRTree(Robinson et al., 1998) and Mega2 (Kumar et al., 2001)software. The species or clades that failed to pass thetests were excluded from divergence time estimation.

3. Results and discussion

The three data sets subjected to phylogenetic anal-ysis were: (1) the trnL-F spacer data set containing 31taxa, 469 bp in length; (2) the rps4-trnS spacer data setcontaining 45 taxa, 519 bp; (3) the combined data setcontaining 45 taxa, 1017 bp. The strict consensus (notshown) of maximum-parsimony trees of the rps4-trnSand trnL-F spacer data analyzed yielded largely unre-solved topologies, though several clades were supportedby high bootstrap values. The parameters of the mostparsimonious trees obtained from the three data setsare presented in Table 2. The P-value resulting fromthe partition-homogeneity test allowed us to concludethat the rps4-trnS and trnL-F spacer data sets were con-gruent (P = 1.000), so phylogenetic analyses were thenconducted using the combined data (Farris et al., 1995).Though we were unable to sequence the trnL-F spac-ers of 13 Australasian taxa and one Himalayan taxon(Table 1), they were included in the analysis of the com-bined data.

The phylogenetic trees constructed by using BI, MPand NJ from the combined data set were similar (Fig. 1),but at almost all nodes, posterior probability values ofBI were greater than bootstrap values of the MP andNJ analyses, and the posterior probability values (above0.90) for all clades could be found in MP and NJ analyseswith high bootstrap support (above 50%).

The phylogenetic trees indicated that the earliestdivergence within Polystichum was between Polystichumerosum and the remainder of the genus, which com-prised three major well-supported clades. Two of thesecontained only Chinese species, while the Australasianspecies were nested within the third. The species from

New Zealand formed a clade within the Australasianspecies.

The evolutionary relationships presented here arelargely congruent with those found in recent world phy-

298 C.-X. Li et al. / Palaeoworld 16 (2007) 294–300

Fig. 1. The phylogenetic tree constructed based on trnL-F and rps4-trnS sequences of Polystichum with Mega2 and MrBayes version 3.1.2 software.replica

elow brais are s

Left: tree constructed from Mega2 software (Bootstrap values of 1000values of 500 replicates of maximum parsimony analysis are shown b(the posterior probability values (100×) of Bayesian inference analystimes are indicated, *76 Ma: the published time estimation.

logenetic studies of Polystichum based exclusively onrbcL (Li et al., 2004), rbcL and morphology (Littleand Barrington, 2003), and on Chinese Polystichum andallies based on pairs of chloroplast markers (rbcL andtrnL-F—Lu et al., 2005). However, unlike the resultsof the previous workers working with rbcL alone (Li etal., 2004), rbcL and morphology (Little and Barrington,2003), or rbcL and trnL-F (Lu et al., 2005), our sub-stantially larger sample size from China and Australasiashows a good resolution for the relationship betweenChinese and Australasian Polystichum (Fig. 1). Thespecies from Australasia appeared to comprise a mono-phyletic group and are obviously nested within the

Chinese Polystichum. The previous studies (Little andBarrington, 2003; Li et al., 2004) based on rbcL andmorphology revealed that all species of Polystichum s.l.(including Cyrtomium and Cyrtomidictyum) formed a

tes of neighbor-joining analysis are shown above branches, bootstrapnches). Right: tree constructed from MrBayes version 3.1.2 software

hown above branches). The Localities of Polystichum and divergence

monophyletic group. The basal split in Polystichum s.l.separated a clade with all Asian members from a cladecontaining other species from all over the world, soLi et al. (2004) suggested that Polystichum s.l. origi-nated in Asia. Here, the new rps4-trnS and trnL-F spacersequence data sets were applied to examine the relation-ships between Chinese and Australasian Polystichum.Our results indicated a close correlation between theirphylogenetic relationships and geographic distribution.The Australasian species are nested within Polystichums.s. from China (eastern Asia). This is congruent withthe “Asian origin of Polystichum” hypothesis and pro-vides more molecular evidence for the Asian origin for

Polystichum. The species from Australasia appeared tocomprise a monophyletic group and are obviously nestedwithin the Chinese Polystichum based our phylogenetictrees.

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It was found that no clades or taxa evolved at a sig-ificantly different rate after the relative-rate tests wereonducted for the rps4-trnS spacer sequences (only theps4-trnS spacer data set contained all of the sampledolystichum species). Therefore, the divergence timesould be estimated between the Polystichum lineages.

Using the published divergence time estimation thatolystichum s.l. originated in Asia in the Late Cre-

aceous (≈76 Ma) (Li et al., 2004) and the geneticistance among the analyzed Polystichum taxa in thistudy (0.0540 ± 0.0050, the genetic distance of theimura-2 parameters), an evolutionary rate was esti-ated for the rps4-trnS sequences. Based on this rate

nd the genetic distances between taxa, the last com-on ancestor (divergence time) of various groups could

e estimated as follows: Australasian and Chineseolystichum shared their last common ancestor (afterhich Chinese Polystichum began to migrate into Aus-

ralasia) at 40.1 ± 3.8 Ma (Fig. 1, node C), Australasianolystichum plants shared their last common ancestorafter which Australian Polystichum began to migratento New Zealand) at 14.1 ± 3.3 Ma (Fig. 1, node B), andhe New Zealand Polystichum shared their last commonncestor (after which New Zealand Polystichum begano diversify) at 9.9 ± 2.8 Ma (Fig. 1, node A).

The divergence time estimation indicated that Chi-ese Polystichum might migrate into Australasia atround 40.1 Ma ago and from there to New Zealand atbout 14.1 Ma. Molecular calibration of branching timen phylogenetic trees is controversial and problematicHillis et al., 1996; Sanderson, 1997), but when paleon-ological data are lacking, molecular estimates providehe only means of inferring the ages of lineage (Li,997; Yang, 2006). With only one reference point (Lit al., 2004), the molecular dating approach employedere is simplistic. However, our estimation of the diver-ence time of Polystichum lineages between Australiand New Zealand corresponds well with that estimationabout 13 Ma) made by Perrie et al. (2003) based on rbcLequences; we have used independent sequence data anddifferent calibration to get a very similar answer to thatf Perrie et al. (2003).

cknowledgements

We are grateful to Prof. Shi Su-hua (Sunyatsenniversity, Guangzhou) for discussion and techni-

al assistance. This study is supported by the Major

esearch Project of the Chinese Academy of Science

KZCX2–SW–130) and National Natural Science Foun-ation of China (40302003 and 30370116). We wouldike to thank Dr. Jianwei (Jerry) Li in Institute for

16 (2007) 294–300 299

Genomic Research at Rockville, MD, USA for criti-cal reading of the manuscript. We also thank the tworeviewers (Prof. Shi Su-hua and Dr. Leon Perrie) and theeditors for comments, which have greatly improved themanuscript.

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