Phylogenetic analysis of questionable tetraploid species in Roegneria and Pseudoroegneria (Poaceae: Triticeae) inferred from a gene encoding plastid acety1-CoA carboxylase

To evaluate the phylogenetic relationships of questionable tetraploid species Roegneria alashanica Keng, Roegneria magnicaespes (D.F. Cui) L.B. Cai, Roegneria elytrigioides C. Yen et J.L. Yang, Roegneria grandis Keng and Pseudoroegneria geniculata (Trin.) Á. Löve, the single copy sequences of the plastid acetyl-CoA carboxylase gene (Acc1) were analyzed among the five species and the related diploid and tetraploid species. The results indicated that: (a) R. alashanica contained one set of modified St genome which was closely related to the E^e genome, and the other set of genome was closely related to the P genome; (b) R. magnicaespes contained one set of St genome, the other set of genome might be closely related to the P genome. There are close affinities between R. magnicaespes and R. alashanica; (c) R. elytrigioides contained two sets of St genomes, and it is reasonable to be treated as Pseudoreogneria elytrigioides (C. Yen et J.L. Yang) B.R. Lu; (d) the genome of R. grandis should be designed as St^gY. The St^g genome was a differentiated form of the St genome in Pseudoroegneria and was homoeologous with the Y genome in Roegneria; (e) the genomic constitution of P. geniculata was similar to that of R. magnicaespes and R. alashanica and distinctly related to P. geniculata ssp. scythica (E^eSt). They should be treated as different species in different genera; and (f) the Y genome was possibly originated from the St genome, and was sister to the St, E^e, E^b and W genomes.     

Phylogeny and maternal donors of the tetraploid species with St genome (Poaceae: Triticeae) inferred from CoxII and ITS sequences

Mitochondrial CoxII and ITS sequences of 29 tetraploid species with St genome were compared with their related genera and species of Pseudoroegneria (Nevski) Á. Löve (St), Hordeum L. (H), Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (E^b), Lophopyrum elongatum (Host) Á. Löve (E^e), Agropyron Gaertner (P), Australopyrum (Tzvelev) Löve (W) and Psathyrostachys Nevski (Ns). The results indicate that: (1) the maternal donors of North American and Eurasian StStHH tetraploid species may have acquired their St genome from distinct Pseudoroegneria gene pools, with Pse. spicata (Pursh) Á. Löve potentially the maternal donor of North American species; (2) Lophopyrum is the maternal donor of StStE^eE^e tetraploid species, hence, Pse. geniculata ssp. scythica (Nevski) Á. Löve, Elytrigia caespitosa (K. Koch) Nevski and El. caespitosa ssp. nodosa (Nevski) Tzvelev should be identified as species of Trichopyrum Á. Löve; (3) tetraploid species with the same maternal donors were more closely related to each other than those with different maternal donors.     

Molecular phylogeny of RNA polymerase II gene reveals the relationships of tetraploid species with St genome (Triticeae: Poaceae)

To evaluate phylogeny of tetraploid with St genome, phylogenetic analyses of RNA polymerase II (RPB2), a member of the nuclear gene family encoding the second largest subunit, were performed. Our results showed that: (1) Roegneria magnicaespes and Roegneria alashanica are related to Pseudoroegneria. (2) Roegneria elytrigioides has StStStSt genomes and should therefore be classified as Pseudoroegneria elytrigioides. (3) Pseudoroegneria tauri and Pseudoroegneria deweyi which have StStPP genomes should be transferred to Douglasdeweya and be renamed as Douglasdeweya wangii and Douglasdeweya deweyi, respectively. (4) Pseudoroegneria geniculata ssp. scythica is related to Pseudoroegneria and Lophyrum, and hence should be identified as a species of Trichopyrum. (5) Pseudoroegneria libanotica might be a parental donor for Elytrigia caespitosa rather than Elytrigia caespitosa ssp. nodosa. It is unreasonable to recognize El. caespitosa ssp. nodosa as a subspecies of El. caespitosa. (6) Interspecific and intergeneric variations are detected in St genome of these tetraploid species.     

Hybridisation,genomic constitution and generic delimitation inElymus s. l. (Poaceae: Triticeae)

Intergeneric crosses were made between representatives of the genomically-defined generaElymus, Agropyron, Elytrigia, Pseudoroegneria, andThinopyrum. The genomic constitution ofElytrigia repens, the type species ofElytrigia, is shown to be SSH, a genomic combination otherwise found only inElymus. The S genome ofPseudoroegneria has almost always a dominant influence on the morphology of the taxa of which it is a component.Wang (1989) showed that the J genome inThinopyrum and the S genome have considerable homoeology, with a mean c-value of 0.35 in diploid SJ hybrids. A genetic coherence from S to SJ^e, J^e, J^eJ^b, and J^b can be expected, agreeing with the continuous morphologic variation pattern observed. Because of the absence of morphological discontinuities between the taxa,Pseudoroegneria (S),Elymus (SH, SY, sometimes with additional genomes),Elytrigia (SSH, SSHX), andThinopyrum (SJ, SJJ, J) are best treated as a single genus,Elymus, following the generic concept ofMelderis in Flora Europaea and Flora of Turkey. The basic genomic constituents ofElymus will then be the S and/or J genomes.Agropyron, with diploids, tetraploids, and hexaploids based on the P genome is morphologically distinct from other genera inTriticeae. In a few species ofElymus andPseudoroegneria, a P genome is an additional constituent. In these cases the P genome has a negligible morphological influence. Therefore, it seems reasonable to maintainAgropyron as a separate genus.     

Phylogenetic relationships among Leymus and related diploid genera (Triticeae: Poaceae) based on chloroplast trnQ–rps16 sequences

To investigate the phylogenetic relationships among Leymus and related diploid genera, the genome donor of Leymus, and the evolutionary history of polyploid Leymus species, chloroplast trnQ–rps16 sequences were analyzed for 36 accessions of Leymus representing 25 species, together with 11 diploid taxa from six monogenomic genera. The phylogenetic analyses (Neighbor‐Joining and MJ network) supported three major clades (Ns, St and Xm). Sequence diversity and genealogical analysis suggested that 1) Leymus species from the same areas or neighboring geographic regions are closely related; 2) most of the Eurasian Leymus species are closely related to Psathyrostachys: P. juncea might serve as the Ns genome donor of polyploid Eurasian Leymus species; 3) the Xm genome may originate from ancestral lineages of Pseudoroegneria (St), Lophopyrum (E^e), Australopyrum (W) and Agropyron (P); 4) the trnQ–rps16 sequences of Leymus are evolutionarily distinct, and may clarify parental lineages and phylogenetic relationships in Leymus.     

Genome constitutions of Pseudoroegneriageniculata, P. geniculata ssp. scythica and P. geniculata ssp. pruinifera (Poaceae: Triticeae) revealed by genomic in situ hybridization

Genomic constitutions of three taxa of Pseudoroegneria á. L?ve, P. geniculata, P. geniculata ssp. scythica, and P. geniculata ssp. pruinifera, were characterized using genomic in situ hybridization (GISH). The results indicated that ploidy level and genomic constitution in the three Pseudoroegneria taxa studied were as follows: P. geniculata (2n = 4x = 28; genomic formula StSt ^g ), P. geniculata ssp. scythica (2n = 4x = 28; genomic formula ESt), and P. geniculata ssp. pruinifera (2n = 6x = 42; genomic formula EESt). P. geniculata ssp. scythica and P. geniculata ssp. pruinifera should be transferred to Trichopyrum á. L?ve following L?ve’s principles and treated as T. scythicum and T. pruiniferum, respectively.     

Genome constitutions of Roegneriaalashanica,R. elytrigioides,R. magnicaespes and R. grandis (Poaceae: Triticeae) via genomic in‐situ hybridization

The genomic constitutions of Roegneria alashanica, R. elytrigioides, R. magnicaespes and R. grandis were studied using GISH. DNA of Pseudoroegneria spicata (St), P. libanotica (St), P. stipifolia (St), R. ciliaris (StY), Lopophyllum elongatum (E^e), Agropyron cristatum (P) and Hordeum bogdanii (H) were used for probing, respectively. The results indicated that: 1) R. alashanica and R. magnicaespes contained one St genome, the other genome was unidentified, however, it was not an E, P, H or Y genome; 2) R. elytrigioides contained two St genomes and should on this basis be included in Pseudoroegneria; 3) R. grandis contained an St and an St^g genome. The St^g genome is suggested to be a modified form of the St genome of Pseudoroegneria and to be homoeologous with the Y genome. It might be an intermediate type between the St and Y genomes. Therefore, R. elytrigioides should be treated as Pseudoroegneriaelytrigioides.Roegneria alashanica, R. magnicaespes and R. grandis does apparently not belong in the genus Roegneria but further studies are needed to establish their correct taxonomic position.     

Phylogenetic relationships between Leymus (Poaceae,Triticeae) and related diploid Triticeae species based on isozyme and genome-specific random amplified polymorphic DNA (RAPD) markers

Abstract

To investigate the phylogenetic relationships between Leymus and related diploid species of the Triticeae tribe, the esterase isozyme (EST), superoxide dismutase (SOD) isozymes, and genome-specific random amplified polymorphic DNA (RAPD) markers were used to analyze for 14 Leymus species, together with two Psathyrostachys species (Ns), three Pseudoroegneria species (St), two Hordeum species (H), Lophopyrum elongatum (E^e), Australopyrum retrofractum (W), and Agropyron cristatum (P). The data were used to construct dendrograms by means of UPGMA in the NTSYS-pc computer program. The results suggested that (1) isozyme analysis can be used in the systematic studies of these perennial Triticeae; (2) there is a close relationship between Leymus, Psathyrostachys juncea, three Pseudoroegneria species, and Lophopyrum elongatum; (3) the Ns genome-specific RAPD marker was present in all 14 polyploid species of Leymus, while the E^e and P genome-specific RAPD markers were absent in 14 polyploid species of Leymus; the St, W and H genome-specific RAPD markers were present in some species of Leymus; (4) Leymus species have multiple origins, and different Leymus species derived their genomes from different donors.     

Phylogenetic relationships among the species of <Emphasis Type="Italic">Elymus</Emphasis> sensu lato in Triticeae (Poaceae) based on nuclear rDNA ITS sequences

The genus Elymus L. sensu lato includes Roegneria, Elymus, Hystrix, Sitanion and Kengyilia, and they are very important group in the tribe Triticeae. However, the phylogenetic relationships and taxonomic status of them are still in dispute. The ITS sequences were obtained and analyzed for their phylogenetic relationships by using Maximum Parsimony (MP) and Bayesian Inference (BI) methods. The main results were as follows: (1) Most species in Roegneria, Elymus and Sitanion were clustered in the St clade with diploid St genome species, and it was difficult to distinguish the species in Roegneria and Elymus; (2) The polyploid species with St genomes in the St clade were divided into three groups, which suggests that there exists differentiation of St genome in polyploids; (3) Most species of Kengyilia have only P-type of clone and clustered with diploid Agropyron species, which may suggest that Kengyilia is a valid genus; (4) Hy. patula, the type species of Hystrix was clustered with species of Elymus, while Hy. duthiei ssp. duthiei, Hy. duthiei ssp. longearistata, Hy. coreana and Hy. komarovii were grouped with diploid Psathyrostachys species. It indicated that Hy. patula is distinct related to other Hystrix species, and it is reasonable to treat Hystrix patula as Elymus hystrix and other species in Hystrix should be transferred to Leymus; (5) The “clones bias” in ITS sequences are widespread in the allopolyploid species. The article is published in the original.     

Cytogenetic and genomic relationships ofThinopyrum elongatum with twoPsathyrostachys species and withLeymus secalinus (Poaceae)

Intergeneric hybridizations were made betweenT. elongatum, and twoPsathyrostachys and fiveLeymus species. The seed set obtained onT. elongatum ×Leymus hybrids ranged from 5.65% to 20.00%, depending onLeymus species. The seed set obtained onT. elongatum ×Psathyrostachys hybrids ranged from 16.07% to 19.70%. Meiotic pairing at metaphase-I in JN diploid hybrids ofT. elongatum ×Psathyrostachys species revealed a very low level homology between the basic J and N genomes, and further demonstrated that the two genomes are quite diverged. Chromosome pairing in theT. elongatum ×Leymus secalinus hybrid averaged 15.19 univalents + 2.62 rod bivalents + 0.26 ring bivalents + 0.02 trivalents, suggesting that the partial J^e chromosomes ofT. elongatum has homology withLeymus secalinus genomes.L. secalinus might have 3–4 chromosomes originating from J^e genome.     

Distinct origin of the Y and St genome in Elymus species: evidence from the analysis of a large sample of St genome species using two nuclear genes

Background

Previous cytological and single copy nuclear genes data suggested the St and Y genome in the StY-genomic Elymus species originated from different donors: the St from a diploid species in Pseudoroegneria and the Y from an unknown diploid species, which are now extinct or undiscovered. However, ITS data suggested that the Y and St genome shared the same progenitor although rather few St genome species were studied. In a recent analysis of many samples of St genome species Pseudoroegneria spicata (Pursh) À. Löve suggested that one accession of P. spicata species was the most likely donor of the Y genome. The present study tested whether intraspecific variation during sampling could affect the outcome of analyses to determining the origin of Y genome in allotetraploid StY species. We also explored the evolutionary dynamics of these species.

Methodology/Principal Findings

Two single copy nuclear genes, the second largest subunit of RNA polymerase II (RPB2) and the translation elongation factor G (EF-G) sequences from 58 accessions of Pseudoroegneria and Elymus species, together with those from Hordeum (H), Agropyron (P), Australopyrum (W), Lophopyrum (E^e), Thinopyrum (E^a), Thinopyrum (E^b), and Dasypyrum (V) were analyzed using maximum parsimony, maximum likelihood and Bayesian methods. Sequence comparisons among all these genomes revealed that the St and Y genomes are relatively dissimilar. Extensive sequence variations have been detected not only between the sequences from St and Y genome, but also among the sequences from diploid St genome species. Phylogenetic analyses separated the Y sequences from the St sequences.

Conclusions/Significance

Our results confirmed that St and Y genome in Elymus species have originated from different donors, and demonstrated that intraspecific variation does not affect the identification of genome origin in polyploids. Moreover, sequence data showed evidence to support the suggestion of the genome convergent evolution in allopolyploid StY genome species.     

Meiotic pairing behaviour reveals differences in genomic constitution between Hystrix patula and other species of the genus Hystrix Moench (Poaceae, Triticeae)

Interspecific and intergeneric hybridizations were carried out to evaluate the genomic relationships among species of Hystrix Moench and to study the relationships between Hystrix species and Psathyrostachys huashanica Keng (2n=2x=14, Ns^h). Meiotic pairing in hybrids of Hystrix duthiei ssp. duthiei × P. huashanica (2n=3x=21), Hystrix duthiei ssp. longearistata × P. huashanica (2n=3x=21) and H. patula × P. huashanica (2n=3x=21) averaged 5.18, 5.11 and 0.29 bivalents per cell, while H. patula × H. duthiei ssp. longearistata (2n=4x=28) averaged 25.36 univalents and 1.32 bivalents per cell, respectively. The results indicate that (i) H. duthiei ssp. duthiei and H. duthiei ssp. longearistata have one set of Ns genome from Psathyrostachys; (ii) H. patula has no Ns genome; (iii) genomes of H. duthiei ssp. duthiei and H. duthiei ssp. longearistata are non-homologous to those of H. patula. The genomic relationships between H. patula and other Hystrix species are also discussed.     

Phylogenetic analysis of <Emphasis Type="Italic">Kengyilia</Emphasis> species based on nuclear ribosomal DNA internal transcribed spacer sequences

Phylogenetic analysis was conducted based on sequences of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA in 17 species of Kengyilia, together with those of 18 species from Pseudoroegneria, Agropyron, Roegneria and Douglasdeweya by the maximum parsimony, maximum likelihood and neighbor-joining distance methods. The results indicate that species of Kengyilia had close affinities to species of Douglasdeweya and Agropyron. The species in Kengyilia was identified as two subgroups with regard to geographic distribution, indicating that species from the same distribution had a closer phylogenetic relationship. The genus Kengyilia was found as a ligament-group between Roegneria and Agropyron. The ITS sequence is a useful tool for studying the phylogeny of closely related species.     

A phylogeny of Cariniana (Lecythidaceae) based on morphological and anatomical data

Cariniana as previously circumscribed is a genus of 16 species restricted to neotropical forest habitats on well-drained sites. A phylogenetic analysis of the genus based on 33 morphological and anatomical characters was undertaken. The results show that Cariniana consists of two clades: the Allantoma/Cariniana decandra clade includes Allantoma lineata and seven species of actinomorphic-flowered Cariniana and is characterized by 5-merous flowers, carnose petals, incurved petal apex, scarcely lobed calyces, eucamptodromous secondary veins, dichotomizing venation, and poorly developed areolation; the C. legalis clade is made up of nine species and is characterized by an obliquely zygomorphic androecium, reticulate tertiary venation, and anomocytic stomata. The actinomorphic-flowered Cariniana are more closely related to the monotypic Allantoma lineata than they are to the species of the C. legalis clade. In order to reflect these relationships, Cariniana is divided into two genera: species in the C. legalis clade, which includes the generic type C. legalis, remain as Cariniana while species of Cariniana in the Allantoma/Cariniana decandra clade are transferred to Allantoma. The following new combinations are proposed: Allantoma decandra, A. integrifolia, A. kuhlmannii, A. pluriflora (a nomen novum for Cariniana multiflora because Allantoma multiflora is a synonym of Couratari multiflora), A. pachyantha, A. pauciramosa, and A. uaupensis.     

Molecular diversity and phylogenetic analyses of y-type high-molecular-weight glutenin promoters from different genomes in Triticeae

Sequence polymorphisms and phylogenetic relationships from different genomes of 25 diploid species in Triticeae (Poaceae) were evaluated by using the sequences of y-type high-molecular-weight glutenin promoter (y-HGP). The length of the amplified y-HGP sequences ranged from 845 to 915 base pairs (bp) in the 25 species of Triticeae. Multiple sequence alignment showed conserved and variable parts in the y-HGP sequences. Higher sequence conservation was detected in the regulatory elements of y-HGP. An 85-bp deletion was found in eight species of Triticum, Aegilops, and Hordeum. Several species-specific indels were identified in the y-HGP from Psathyrostachys, Hordeum, and Pseudoroegneria. Maximum parsimony (MP) and Bayesian analyses defined an Aegilops/Triticum group consisting of closely related species. A close relationship between Pseudoroegneria and the clade of Australopyrum, Dasypyrum, and Agropyron was also strongly supported in the topologies of MP and Bayesian trees. As y-HGP has sufficient amounts of genetic variation and is a single-copy region in diploid Triticeae, it is useful in phylogenetic analyses of this group.     

Morphological plasticity following species-specific recognition and competition in two perennial grasses

Morphological characteristics and biomass allocation of two perennial grasses, Pseudoroegneria spicata (Pursh) A. Löve ssp. spicata (bluebunch wheatgrass) and Agropyron desertorum (Fisch. ex Link) Schult. (crested wheatgrass), were compared under different competition and nutrient treatments. The competitive responses of two plants grown in containers under field conditions were assessed in monocultures and mixtures in two experiments using different scales of nutrient application. In the Small-Scale Experiment, a localized fertilization was applied in the rooting zone between two plants; in the Large-Scale Experiment the entire container was supplied with nutrients. Agropyron responded more vigorously to fertilization than did Pseudoroegneria, but based on the relative performance of Agropyron in monoculture and mixture, it was not superior to Pseudoroegneria in resource competition. Pseudoroegneria was apparently able to recognize neighboring plants as either conspecifics or individuals of the other species. The responses included changes in shoot architecture, root morphology, and allocation between roots and shoots. Agropyron generally did not exhibit such morphological flexibility. In field plot plantings of 4-yr-old tussocks similar shoot differences were seen in Pseudoroegneria. There was, however, no indication of superior resource competition for Agropyron. Thus, any early advantage of Agropyron in vigorous growth of young plants in response to nutrients was apparently lost by the time the plants had reached this stage of development. Morphological and allocation flexibility of Pseudoroegneria may have compensated for slower, less vigorous growth. If species-specific recognition and morphological plasticity are common in nature, this complicates our attempts to understand mechanisms of competition.     

Conservation of rDNA inPolystichum (Dryopteridaceae)

Restriction site variation in the nuclear 18S–25S ribosomal RNA genes (rDNA) was analyzed hierarchically in a species complex in the fern genusPolystichum. Two distinct rDNA repeat types were present in all individuals ofPolystichum examined. No variation was detected among individuals within a population ofP. munitum, among populations ofP. munitum orP. imbricans, or among the six diploid species ofPolystichum from North America, including the circumborealP. lonchitis. The identity of rDNA repeats across all six North American species ofPolystichum may reflect an overall similarity of the nuclear genomes of these species, an observation supported by isozyme data as well. However, this nuclear similarity contrasts sharply with the highly divergent chloroplast genomes of these six species. The conservative nature of the rDNA inPolystichum also is in contrast to the much more variable rDNAs of most angiosperms investigated. Perhaps the tempo and mode of evolution of rDNA in ferns differ from those of angiosperms; however, the data base for fern rDNA is very small. Furthermore, the number of repeat types per individual is consistent with a diploid, rather than polyploid, condition despite the high chromosome number (n = 41) of these plants, although homogenization of multiple, divergent rRNA genes cannot be disproven.     

Molecular verification and characterization of BYDV-resistant germ plasms derived from hybrids of wheat with Thinopyrum ponticum and Th. intermedium

Twenty-five partial amphiploids (2n=8x=56), which were derived from hybrids of wheat (Triticum aestivum L.) with either Thinopyrum ponticum (Podpera) Liu & Wang, Th. intermedium (Host) Barkworth & D. Dewey, or Th. junceum (L.) A. Löve, were assayed for resistance to BYDV serotype PAV by slot-blot hybridization with viral cDNA of a partial coat protein gene. Three immune lines were found among seven partial amphiploids involving Th. ponticum. Seven highly resistant lines were found in ten partial amphiploids involving Th. intermedium. None of eight partial amphiploids or 13 addition lines of Chinese Spring — Th. junceum were resistant to BYDV. Genomic in situ hybridization demonstrated that all of the resistant partial amphiploids, except TAF46, carried an alien genome most closely related to St, whether it was derived from Th. ponticum or Th. intermedium. The two partial amphiploids carrying an intact E genome of Th. ponticum are very susceptible to BYDV-PAV. In TAF46, which contains three pairs of St- and four pairs of E-genome chromo somes, the gene for BYDV resistance has been located to a modified 7 St chromosome in the addition line L1. This indicates that BYDV resistance in perennial polyploid parents, i.e., Th. ponticum and Th. intermedium, of these partial amphiploids is probably controlled by a gene(s) located on the St-genome chromosome(s).     

Molecular evidence for the taxonomic status of three species of the <Emphasis Type="Italic">Sphyraena obtusata</Emphasis> group (Perciformes: Sphyraenidae) from East Asia

Genetic differences among the three species of Sphyraena with two gill rakers from East Asia (S. iburiensis, S. obtusata, and S. pinguis, defined recently as the S. obtusata group), were investigated using 799-bp sequences of the mitochondrial cytochrome b gene. Pairwise sequence differences within each of the three species were 0.0–0.4% (S. iburiensis), 0.0–0.4% (S. obtusata), and 0.0–0.6% (S. pinguis), respectively, pairwise sequence differences among the three species being 18.3–18.6% (S. iburiensis vs. S. obtusata), 14.9–15.4% (S. iburiensis vs. S. pinguis), and 17.6–18.3% (S. obtusata vs. S. pinguis), respectively. The extent of the latter were comparable to those among four other congeneric species, S. africana, S. forsteri, S. helleri, and S. japonica (16.0–24.5%). Phylogenetic analysis using the maximum-likelihood method indicated reciprocal monophyly of the three species, each clade being supported by a bootstrap value of 100%. These findings fully supported the taxonomic status of these species, recently elucidated by detailed morphological comparisons. In addition, the S. obtusata group formed a strongly supported clade against the four other congeneric species.     

Phylogeny of Chinese <Emphasis Type="Italic">Polystichum</Emphasis> (Dryopteridaceae) based on chloroplast DNA sequence data (<Emphasis Type="Italic">trnL-F</Emphasis> and <Emphasis Type="Italic">rps4-trnS</Emphasis>)

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.