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.     

Phylogenetics of Chinese Dryopteris (Dryopteridaceae) based on the chloroplast rps4-trnS sequence data

Dryopteris is one of the largest and most taxonomically complex fern genera in the Dryopteridaceae, with 127 species occurring throughout temperate, sub-temperate, subtropical, and tropical China. Investigations of the evolutionary relationships of a subset of these Chinese Dryopteris species, using DNA sequence-based methods, specifically tested the monophyly of the genus and the validity of the previous subgeneric classifications. Sixty species of Dryopteris, four closely related non-Dryopteris and three species of Arachniodes, were used as outgroup taxa. The rps4-trnS region of the chloroplast genome was sequenced in these species for the first time. Both maximum parsimony (MP) and neighbor-joining (NJ) analyses identified six polyphyletic clades that contained Dryopteris species. These results were supported by a Bayesian analysis of the same data set. The phylogenetic patterns strongly suggest the polyphyletic status of Dryopteris; the monophyletic groupings of the species do not correspond with either Fraser-Jenkins [In: Bull Brit Mus (Nat Hist) Bot 14(3):183–218, 1986} or Wu (In: Flora Reipublica Popularis Sinicae Tomus 5 (1) pp 1–241, 2000] subgeneric classification of Dryopteris, except in a few specific cases. This work represents the first molecular systematic analyses of Chinese Dryopteris, and we propose the next steps necessary to recognize new subgenera of the genus.     

Biogeography of temperate Australasian Polystichum ferns as inferred from chloroplast sequence and AFLP

Aim and location New Zealand began to separate from Gondwana c. 85 Ma, and has been isolated from the nearest large landmass, Australia, by some 2000 km of the Tasman Sea since c. 60 Ma. Given New Zealand's long geographical isolation, there has been considerable interest in explaining the origins of its different biotic elements. Here we investigate the biogeography of the fern genus Polystichum from temperate Australasia. Six species are found in New Zealand, four in Australia, and two on Lord Howe Island. Methods The evolutionary relationships between the twelve Polystichum species found in temperate Australasia were inferred from phylogenetic analyses of two molecular data sets: DNA sequence from the chloroplast rps4–trnS spacer locus; and AFLP DNA‐fingerprinting. The timing of the separation between Australian and New Zealand Polystichum was estimated by using the fossil record to temporally calibrate the rbcL sequence differentiation between representative species from these regions. Results Species of Polystichum from New Zealand appear to comprise a monophyletic group. This suggests that Polystichum crossed the Tasman only once. Temporal calibration of the rbcL sequence differentiation between Australian and New Zealand Polystichum indicates that a vicariant explanation for their separation can be rejected in favour of trans‐oceanic dispersal. Main conclusions The extant diversity within New Zealand Polystichum appears to have been derived from a single, trans‐oceanic dispersal event (within the last c. 20 Myr), followed by a relatively extensive in situ ecological radiation.     

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.     

Molecular phylogeny of the fern genus Elaphoglossum (Elaphoglossaceae) based on chloroplast non-coding DNA sequences: contributions of species from the Indian Ocean area

We performed a phylogenetic analysis of the fern genus Elaphoglossum using two non-coding chloroplast spacers: trnL-trnF and rps4-trnS. The sampling includes 123 species, of which 80 have not been previously sequenced, and for the first time includes species from Africa and the Indian Ocean area. The results of this expanded study largely agree with an earlier molecular study based on a smaller group of neotropical species and with the morphology-based classification of Mickel and Atehortua. We found, however, that some infrageneric groups such as section Elaphoglossum are not monophyletic. Besides section Elaphoglossum pro parte, we recognize six sections: two new monospecific, unnamed sections, and the previously established sections Lepidoglossa, Squamipedia, Amygdalifolia, and "Subulate-scaled clade." We divide the subulate-scaled clade into subsection Setosa (hydathodes present) and Polytrichia (hydathodes absent), and section Elaphoglossum is divided into subsections Platyglossa and Pachyglossa, two groups that do not appear to be supported by any single morphological character. In general, however, the main clades are supported by morphology. Finally, we discuss the species of the Indian Ocean region and their affinities with the neotropical ones. Out of the 11 species pairs postulated by Moran and Smith on the basis of morphology, two are well supported (E. eximium-E. aubertii; E. piloselloides-E. spatulatum) and three are not supported (E. ciliatum-E. humbertii; E. muscosum-E. poolii; E. paleaceum-E. deckenii), and two remain unresolved (E. erinaceum-E. hybridum; E. glabellum-E. acrostichoides) because our molecular markers were not variable enough. Four species pairs could not be tested because specimens were lacking. Unsupported species pairs are best interpreted as morphological convergences. Two additional species pairs are proposed: E. cuspidatum-E. succisaefolium; E. doanense-E. hornei. Placement of the species from the Indian Ocean suggests that at least 13 long-distance dispersal events occurred between the Neotropics and the Indian Ocean-Africa.     

Evolutionary and biogeographic patterns of the Badidae (Teleostei: Perciformes) inferred from mitochondrial and nuclear DNA sequence data

We reconstructed phylogenetic relationships of the family Badidae using both mitochondrial and nuclear nucleotide sequence data to address badid systematics and to evaluate the role of vicariant speciation on their evolution and current distribution. Phy-logenetic hypotheses were derived from complete cytochrome b (1,140 base pairs) sequences of 33 individuals representing 13 badid species, and using three species of Nandidae as outgroups. Additionally, we sequenced the nuclear RAG1 (1,473 base pairs) and Tmo-4C4 (511 base pairs) genes from each of the badid species and one representative of the outgroup. Our molecular data provide the first phylogenetic hypothesis of badid intrarelationships. Analysis of the mitochondrial and nuclear nucleotide sequence data sets resulted in well-supported trees, indicating a basal split between the genera Dario and Badis, and further supporting the division of the genus Badis into five species groups as suggested by a previous taxonomic revision of the Badidae. Within the genus Badis, mitochondrial and nuclear phylogenies differed in the relative position of B. kyar. We also used our molecular phylogeny to test a vicariant speciation hypothesis derived from geological evidence of large-scale changes in drainage patterns in the Miocene affecting the Irrawaddy- and Tsangpo-Brahmaputra drainages, in the southeastern Himalaya. Within both genera, Badis and Dario, we observed a divergence into Irrawaddy- and Tsangpo-Brahmaputra clades. Using a cytb substitution rate of 8.2 x 10(-9) (substitutions x base pair(-1) x year(-1), we tentatively date this vicariant event at the Oligocene-Miocene boundary (19-24Myr). It is concordant with a hypothesized paleo connection of the Tsangpo river with the Irrawaddy drainage that was most likely interrupted during Miocene orogenic events through tectonic uplifts in eastern Tibet. Our data, therefore, indicate a substantial role of vicariant-based speciation shaping the current distribution patterns of badids.     

Phylogeny of Caragana (Fabaceae) based on DNA sequence data from rbcL, trnS–trnG, and ITS

Phylogenetic relationships of 48 species of Caragana (Fabaceae: tribe Hedysareae) and one representative each of Astragalus, Calophaca, Halimodendron, and Hedysarum are estimated from DNA sequences of the rbcL gene, trnS–trnG intron and spacer, and ITS region. At least one representative of all five sections and 12 series within Caragana are included. Analyses yielded strongly supported clades corresponding to sections Caragana, Bracteolatae, and Frutescentes. The species of section Jubatae are distributed among three strongly supported clades, i.e., one with the species of section Bracteolatae, another with two species of section Spinosae, and a third as sister to section Frutescentes. All but the last of these six clades are corroborated by at least one unambiguously traced morphological character. The placement of the other four species of section Spinosae are not well supported and lack unambiguous morphological synapomorphies, and the samples of Calophaca and Halimodendron nest within Caragana with weak support.     

Phylogenetics,biogeography, and evolutionary trends of the Phalaenopsis sumatrana complex inferred from nuclear and chloroplast DNA

Phylogenetic trees inferred from the internal transcribed spacers 1 and 2 (ITS1 + ITS2) region from nuclear ribosomal DNA (nrDNA) and the intergenic spacer of atpB-rbcL from chloroplast DNA (cpDNA) were used to clarify the phylogenetics and evolutionary trends of the Phalaenopsis sumatrana complex. The P. sumatrana complex includes the two species P. sumatrana and Phalaenopsis corningiana as well as a problem species, Phalaenopsis zebrina, according to the concepts of Sweet (1980) and Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, the accessions of P. sumatrana cannot be separated from those of P. corningiana. In contrast, the accessions of P. zebrina can be separated from those of both P. sumatrana and P. corningiana. However, analyses of the sequences of the atpB-rbcL spacer apparently cannot discriminate among these three species of the P. sumatrana complex. An inspection of the morphological characters of the plants of the P. sumatrana complex and the floral fragrances of P. zebrina can be used to separate it from both P. sumatrana and P. corningiana. Based on the molecular data and floral fragrances, P. zebrina perhaps should not be treated as a synonym of P. sumatrana. On the evolutionary trend of the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex based on the phylogenetic tree and biogeography.     

Phylogeny and biogeography of the Petaurista philippensis complex (Rodentia: Sciuridae), inter- and intraspecific relationships inferred from molecular and morphometric analysis

With modified DNA extraction and purification protocols, the complete cytochrome b gene sequences (1140 bp) were determined from degraded museum specimens. Molecular analysis and morphological examination of cranial characteristics of the giant flying squirrels of Petaurista philippensis complex (P. grandis, P. hainana, and P. yunanensis) and other Petaurista species yielded new insights into long-standing controversies in the Petaurista systematics. Patterns of genetic variations and morphological differences observed in this study indicate that P. hainana, P. albiventer, and P. yunanensis can be recognized as distinct species, and P. grandis and P. petaurista are conspecific populations. Phylogenetic relationships reconstructed by using parsimony, likelihood, and Bayesian methods reveal that, with P. leucogenys as the basal branch, all Petaurista groups formed two distinct clades. Petaurista philippensis, P. hainana, P. yunanensis, and P. albiventer are clustered in the same clade, while P. grandis shows a close relationship to P. petaurista. Deduced divergence times based on Bayesian analysis and the transversional substitution at the third codon suggest that the retreating of glaciers and upheavals or movements of tectonic plates in the Pliocene-Pleistocene were the major factors responsible for the present geographical distributions of Petaurista groups.     

Molecular phylogeny and biogeography of woolly flying squirrel (Rodentia: Sciuridae), inferred from mitochondrial cytochrome b gene sequences

To investigate the genetic diversity between the populations of woolly flying squirrels (Eupetaurus) from the eastern and western extremes of the Himalayas, partial mitochondrial cytochrome b gene sequences (390-810 bp) that were determined from the museum specimens were analyzed using maximum parsimony (MP) and maximum likelihood (ML) methods. The molecular data reveal that the two specimens that were collected in northwestern Yunnan (China) are members of the genus Eupetaurus. Reconstructed phylogenetic relationships show that the populations of Eupetaurus in the eastern and western extremes of the Himalayas are two distinct species with significant genetic differences (12%) and diverged about 10.8 million years ago. Eupetaurus is significantly different from Petaurista and Pteromys. The level of estimated pairwise-sequence divergence observed between Eupetaurus and Petaurista or Pteromys is greater than that observed between Eupetaurus and Trogopterus, Belomys, Glaucomys, or Hylopetes. Considering the divergence time of the two Eupetaurus groups, the glaciations and the uplift of the Himalayas and Qinghai-Tibet plateau during the Pliocene-Pleistocene period might be the major factors affecting the present distribution of Eupetaurus along the Himalayas.     

Phylogeography of Chrysanthemum indicum L. (Compositae) in China based on trnL-F sequences

Using chloroplast DNA (cpDNA) trnL-F sequences, we studied the phylogeographic pattern and demographic history of Chrysanthemum indicum with 81 individuals from 27 populations representing the partial geographic range in China. Fifteen haplotypes were identified based on nucleotide variation. Relatively high levels of haplotype diversity (Hd = 0.783) and low levels of nucleotide diversity (Pi = 0.00521) were detected in C. indicum. An analysis of molecular variance (AMOVA) indicated that most of the genetic variation (59.9%, P < 0.001) was partitioned within populations. Genetic differentiation among populations was high (F_ST = 0.35), suggesting that there is little gene flow among populations. Nested clade analysis showed that 15 haplotypes formed twelve 1-step clades, five 2-step clades and two 3-step clades, with four clades showing significant geographical association. We concluded that contiguous range expansion and restricted gene flow/dispersal but with some long distance dispersal seemed to have shaped the present-day distribution of the haplotypes of C. indicum in China.     

Phylogeny and divergence times of some racerunner lizards (Lacertidae: Eremias) inferred from mitochondrial 16S rRNA gene segments

Eremias, or racerunners, is a widespread lacertid genus occurring in China, Mongolia, Korea, Central Asia, Southwest Asia and Southeast Europe. It has been through a series of taxonomic revisions, but the phylogenetic relationships among the species and subgenera remain unclear. In this study, a frequently studied region of the mitochondrial 16S rRNA was used to (i) reassess the phylogenetic relationships of some Eremias species, (ii) test if the viviparous species form a monophyletic group, and (iii) estimate divergence time among lineages using a Bayesian relaxed molecular-clock approach. The resulting phylogeny supports monophyly of Eremias sensu Szczerbak and a clade comprising Eremias, Acanthodactylus and Latastia. An earlier finding demonstrating monophyly of the subgenus Pareremias is corroborated, with Eremias argus being the sister taxon to Eremias brenchleyi. We present the first evidence that viviparous species form a monophyletic group. In addition, Eremias przewalskii is nested within Eremias multiocellata, suggesting that the latter is likely a paraphyletic species or a species complex. Eremias acutirostris and Eremias persica form a clade that is closely related to the subgenus Pareremias. However, the subgenera Aspidorhinus, Scapteira, and Rhabderemias seem not to be monophyletic, respectively. The Bayesian divergence-time estimation suggests that Eremias originated at about 9.9 million years ago (with the 95% confidence interval ranging from 7.6 to 12 Ma), and diversified from Late Miocene to Pleistocene. Specifically, the divergence time of the subgenus Pareremias was dated to about 6.3 million years ago (with the 95% confidence interval ranging from 5.3 to 8.5 Ma), which suggests that the diversification of this subgenus might be correlated with the evolution of an East Asian monsoon climate triggered by the rapid uplift of the Tibetan Plateau approximately 8 Ma.     

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.     

The chloroplast generps 4 as a tool for the study ofPoaceae phylogeny

Phylogenetic analyses of 28Poaceae species based on the chloroplastrps 4 gene are presented using parsimony and distance methods. Two monocots from other families were used as outgroups. The chloroplast generps 4 was amplified, cloned, and sequenced for each species. The inferred phylogenetic trees were compared to recent classifications and are shown to fit their general features. There is a dichotomy in our tree between the pooid group and the other grasses. This is in contradiction with other molecular phylogenies, where the bamboos appear first within the family. This result led us to discuss some hypotheses about the relationships of the bambusoids with the other groups of grasses, and also about the relative position of rice and bamboo, which are found close to each other in our trees.     

Phylogeny and classification of the Grimmiaceae/Ptychomitriaceae complex (Bryophyta) inferred from cpDNA

Phylogenetic relationships within the Grimmiaceae/Ptychomitriaceae were studied using a plastid tRNA cluster, including four tRNAs (trnS, trnT, trnL, trnF), a fast evolving gene (rps4), four spacers separating the coding regions, as well as one group I intron. Secondary structure analyses of the spacers as well as the trnL intron P8 domain identified several homoplastic inversions. Tracing the structural evolution of P8 we were able to identify lineage specific modifications that are mainly explained by inversions often in combination with large indel events. Phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian methods indicate that Jaffueliobryum and Indusiella are closely related to Ptychomitrium and form the Ptychomitriaceae s. str. As Campylostelium is neither resolved within Ptychomitriaceae s. str. nor Grimmiaceae s. str., we prefer to treat it in its own family, Campylosteliaceae De Not. The systematic position of Glyphomitrium, as also found by other authors, should be considered in a broader analysis of haplolepidous mosses as our analyses indicate that it is not part of Campylosteliaceae, Grimmiaceae, or Ptychomitriaceae. Within Grimmiaceae s. str., Racomitrium is recognized as a monophyletic group sister to a clade including Dryptodon, Grimmia, and Schistidium. Coscinodon species appear disperse in Grimmia s. str. next to species sharing the same gametophyte morphology, and thus the genus is synonymized with Grimmia. Finally, Schistidium is resolved monophyletic with high statistical support, and seems to represent a rapidly evolving group of species. Our results are not fully congruent with recently published treatments splitting Grimmiaceae in a fairly high number of genera, neither with a comprehensive Grimmia including Dryptodon and Grimmia s. str.     

Taxonomic implications of Liassic ferns Cladophlebis Brongniart and Todites Seward from Hungary

Ferns are diverse and abundant in the Lower Liassic flora of the Mecsek Mountains in south Hungary. The morphogenus Cladophlebis Brongniart, 1849, and the genus Todites Seward, 1900, show a high diversity. In the examined material, most of the characteristics used in taxonomy vary, showing transitional forms that cast doubt on the separation of species established on the grounds of either a low number of specimens, a low number of features or uncharacteristic differences.