Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

A Preliminary Molecular Phylogeny of the Rhynchosporeae (Cyperaceae)

The tribe Rhynchosporeae comprises the genera Rhynchospora and Pleurostachys and has never been studied using molecular techniques. The objective of this study was to use an analysis of trnL-F sequences to evaluate the hypothesized taxonomic divisions within the Rhynchosporeae including the monophyly of the genera and the soundness of the subgenera and sections. A total of 44 ingroup species were studied, 41 of Rhynchospora representing 22 of Kükenthal’s 28 sections, and three of Pleurostachys. Five outgroup species from other genera were also included. The cladistic analysis of 50 trnL intron and trnL-F intergenic spacer sequences resulted in 16 trees and a strict consensus tree. The Rhynchosporeae form two well-supported primary clades with several well-supported smaller clades, many of which agree with previously hypothesized sections. Pleurostachys is embedded within the second primary clade.     

Comparison of molecular and morphological data on St Helena: Elaphoglossum

The endemic elaphoglossoid ferns, Elaphoglossum dimorphum, E. nervosum and Microstaphyla furcata of St Helena, form a closely related group within section Lepidoglossa when analysed phylogenetically using sequences from the chloroplast trnL intron (partial) and trnL-F intergenic spacer. Microstaphyla furcata, traditionally placed in its own genus, is clearly shown to belong to Elaphoglossum confirming the previous transfer of this species to Elaphoglossum as E. bifurcatum. There is hardly any trnL-F sequence divergence between the species, in fact sequences of E. nervosum and E. dimorphum are identical. These results are consistent with the possible origin of E. dimorphum as a hybrid between E. bifurcatum and E. nervosum or with the view that the three species are the result of a recent radiation. The potential conflict between phylogenetic and morphological distinctness in determining species conservation priorities is discussed.     

两栖蓼的分子系统学研究

两栖蓼是一种水陆两栖植物,植株在不同生态环境下外部形态差异较大,同时两栖蓼的系统位置存在争议,被归入春蓼组(sect.Persicaria)或提升为两栖蓼组(sect.Amphibium)。该文选取两栖蓼及春蓼组植物12种,以及刺蓼组、头状蓼组、神血宁组、拳参组、萹蓄组和外类群掌叶大黄共23种植物进行研究。植物总DNA的提取采用改进的CTAB法,所测序列以及从Genbank数据库下载的序列,以掌叶大黄为外类群,采用最大简约法和贝叶斯法对核糖体ITS序列和叶绿体trn L-F序列进行了系统发育分析。ITS序列对位排列的长度为735 bp,包括489个可变位点,272个位点是信息位点。简约法得到9个简约树,步长为1 084,CI指数为0.680,RI指数为0.614。trn L-F序列对位排列的长度为1 121 bp,包括427个可变位点,239个位点是信息位点。简约法寻找到9个简约树,步长为551(CI=0.911,RI=0.910)。贝叶斯法和简约法得到的树基本一致。分子序列分析结果显示,trn L-F序列树类似于ITS序列树。ITS序列构建的发育树上,两栖蓼与刺蓼组植物、春蓼组其他植物形成3个并列的分支;在trn L-F序列树上,两栖蓼则与其他春蓼组植物形成两个并列的分支。由此可见,两栖蓼与春蓼组其他植物的亲缘关系较远,成一独立的分支。两个分子证据支持将两栖蓼提升为两栖蓼组的处理意见。此外,两栖蓼的花粉具散沟,与典型的春蓼组的具散孔花粉不一致。再加上两栖蓼水陆两栖的特性,因此支持把两栖蓼提升为两栖蓼组的观点。两栖蓼组的界定为多年生草本,水陆两栖,根状茎横生,生于水中茎漂浮,叶长圆形或椭圆形,生于陆地茎直立,叶披针形或长圆状披针形,托叶鞘为筒状、薄膜质,总状花序穗状,瘦果近圆形,花粉具散沟。     

10种棘豆属植物的trnL-F序列及其分子系统学研究

选择内蒙古27个样地采集的10种棘豆属植物54个单株,提取样品的基因组DNA,对其叶绿体trnL-F序列进行扩增、测序,所得序列利用ClustalX软件进行对位排列,并用MEGA5.0软件采用最大似然法构建系统发育树,以探讨棘豆属的种间关系与系统进化.结果显示:(1)10种棘豆属trnL-F的变异位点54个,信息位点46个,种间碱基差异百分率为1.9％,GC含量变化范围在30.69％～31.50％之间.(2)棘豆属与黄芪属各为一支,自展支持率达99％,支持棘豆属植物为单系起源.(3)系统树中小花棘豆的样本自成一支,为相对独立进化;多叶棘豆、砂珍棘豆和黄毛棘豆的样本相互混杂,表明亲缘关系很近,从而支持《内蒙古植物志》将三者归入真棘豆亚属轮叶棘豆组的观点.(4)刺叶柄棘豆的样本不同样地形成2个分支,对其亚属水平上的分类需进一步探讨.(5)缘毛棘豆与阴山棘豆的样本聚成一支,支持将二者归入矮生棘豆组.研究表明,trnL-F序列可为棘豆属下种间系统发育关系研究提供分子证据.     

Postglacial population expansion of Dacrydium pectinatum (Podocarpaceae) in Hainan, southem China, based on cpDNA trnL-F nocoding sequence data

This study determined the sequences of chloroplast DNA (cpDNA) trnL-F non-coding regions of individuals of a tropical coniferous species, Dacrydium pectinatum, collected from 12 natural populations located in Hainan Province, southern China. Sequence length varied from 868 bp to 876 bp, indicating length polymorphism. Base composition in the sequences was high in A+T content between 64.17% and 64.95%, and no recombination event occurred (Rm = 0). Thirty haplotypes were identified based on statistical parsimony algorithm by running the TCS program. Populations of D. pectinatum in Hainan were lacking genetic differentiation. Such a deduction was supported by the observed F _ST values (0.00), AMOVA (24.17% of molecular variance attributed to difference among populations, P>0.05), high values of Nm (ranging from 1.92 to 2.50) and the branching structure in neighbor-joining (NJ) tree constructed from haplotypes. A ‘star-like’ pattern was exhibited in the TCS network of trnL-F haplotypes, and majority of the haplotypes coalesced near the tips in NJ tree. Gene genealogies of cpDNA haplotypes proposed a recent population expansion of D. pectinatum in Hainan, which was further supported by the results from Tajima’s D test and mismatch distribution analysis. Our data, in conjunction with geological and palynological evidences, showed that in the Holocene, due to global warming, refugee populations of D. pectinatum in Hainan might experience a range expansion. __________ Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2005, 44(5): 70–74 [译自: 中山大学学报 (自然科学版), 2005, 44(5): 70–74]     

Molecular markers reveal a strong genetic differentiation between two European relic tree species: Zelkova abelicea (Lam.) Boissier and Z. sicula Di Pasquale, Garfì & Quézel (Ulmaceae)

Chloroplast (trnL) and ribosomal (ITS2)sequences and chloroplast DNA (PCR-RFLP andSSR) markers were analysed in two relicUlmaceae tree species: Zelkova abelicea,from Crete, and Z. sicula, from Sicily.The analysis of the plastidial trnLintron and of ITS2 ribosomal sequences revealedtheir divergence from the related speciesZ. carpinifolia, widespread in the Caucasianregion; one base substitution in the trnLintron was detected between the twoMediterranean species, thus suggesting theirrecent separation. Molecular markers(plastidial PCR-RFLP and SSR) showed an evidentgenetic differentiation between Z. siculaand Z. abelicea, the two species beingcharacterised by different haplotypes. Nowithin population variation was detected usingdifferent chloroplast markers inZ. abelicea and Z. sicula. Paleobotanicaldata proved that the genus Zelkova wasabundant and widespread in central Italy untilit became extinct in the continental part ofEurope during last glaciation events andsurvived only in two Mediterranean islands. Thesegregation of the two Mediterranean relicspecies might have occurred as a consequence ofthe strong reduction of their distribution andthe following geographic isolation. Geneticdrift may have determined the drastic reductionof within stand diversity as observed in othersmall, peripheral and geographically isolatedplant populations. The priorities forconservation programs are discussed in thelight of the different genetic resourcesrepresented by the two taxa.     

Zameioscirpus, a new genus of Cyperaceae from South America

Systematic studies of scirpoid species in the Andes showed the necessity to exclude one species each from Scirpus and Carex. They are combined in a new genus Zameioscirpus and a third new species is described. The autonomous generic position of Zameioscirpus within the Scirpeae is supported by a phylogenetic analysis based on rbcL and trnL-F sequencing data and by conspicuous morphological similarities.     

Phylogeny of the Madagascan endemic family Didiereaceae

A molecular phylogeny of the Didiereaceae was produced through parsimony analysis of chloroplastrpl16 intron andtrnL-trnF andtrnT-trnL intergenic spacer sequences of all eleven species of the Didiereaceae and several outgroup taxa from the Portulacaceae. Results indicated that: 1) the Didiereaceae were embedded within the Portulacaceae, withCalyptrotheca as the sister group of the family; 2) present generic limits were supported; 3)Alluaudiopsis was the most basal lineage; 4) at least two separate episodes of polyploidization within the genusAlluaudia had occurred, and 5) unusually low amounts of variation were present in rapidly evolving noncoding plastid sequences.     

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.     

Phylogenetic relationships in Blumea (Asteraceae: Inuleae) as evidenced by molecular and morphological data

The present study, based on sequences of cpDNA (trnL-F & psbA-trnH) and nrDNA (ITS) and morphology, examined the evolutionary relationships in Blumea and its position among related genera. The results confirmed that the closest relatives of Blumea are Caesulia, Duhaldea and Pentanema p.p., and showed that the monotypic genera Blumeopsis and Merrittia are nested within Blumea. In Blumea s.l., two major, well-supported clades were recognised and a single species, the widespread Blumea balsamifera, that could not be placed with certainty relative to the two main clades. The two main clades differ in habit, ecology and distribution. The Blumea densiflora clade contains shrubs and subshrubs of evergreen forests, distributed from continental Asia to New Guinea and Polynesia, whereas the Blumea lacera clade is a widespread paleotropical group that comprises mostly annual, weedy herbs of open forests and fields.     

TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.     

Phylogeography of two East Asian species in Croomia (Stemonaceae) inferred from chloroplast DNA and ISSR fingerprinting variation

The genus Croomia (Stemonaceae) comprises three herbaceous perennial species that are distributed in temperate-deciduous forests in Southeastern North America (C. pauciflora) and East Asia (C. japonica, C. heterosepala). The two Asian species have abutting ranges in South Japan, but C. japonica also occurs disjunctively on the adjacent Asiatic mainland in East China. In our phylogenetic analysis of Croomia, based on chloroplast (cp) DNA sequence variation of the trnL-F region, and rooted with Stemona spp., the two Asian species are identified as sister that likely diverged in the Mid-to-Late Pleistocene (0.84–0.13 mya), whereas the divergence of C. pauciflora dates back to the Late Plio-/Pleistocene (<2.6 mya). Phylogeographical analysis of the two East Asian species detected seven cpDNA (trnL-F) haplotypes across 16 populations surveyed, and all of those were fixed for a particular cpDNA haplotype (H_E = 0.0, G_ST = 1). A survey of inter-simple sequence repeats (ISSRs) markers also detected remarkably low levels of within-population diversity (C. japonica: H_E = 0.085; C. heterosepala: H_E = 0.125), and high levels of inter-population differentiation (C. japonica: Φ_ST = 0.736; C. heterosepala: Φ_ST = 0.550), at least partly due to pronounced regional genetic substructure within both species. Non-overlapping distributions of cpDNA haplotypes and strong genetic (cpDNA/ISSR) differentiation among populations and/or regions accord with findings of a nested clade analysis, which inferred allopatric fragmentation as the major process influencing the spatial haplotype distribution of the two species. Based on mismatch distribution analysis and neutrality tests, we do not find evidence of population expansion in both species. Overall, we conclude that components of temperate-deciduous forest types in South Japan and East China are particularly sensitive to range fragmentation, isolation, and enhanced (incipient) species formation through climate-induced expansions of other forest types over glacial and interglacial periods of the (Late) Quaternary.     

A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.     

Using progesterone 5β-reductase, a gene encoding a key enzyme in the cardenolide biosynthesis, to infer the phylogeny of the genus Digitalis

Summary The progesterone 5β-reductase (5β-POR), a key enzyme in the cardenolide biosynthesis, was sequenced for 21 species of Digitalis and Isoplexis to infer phylogenetic and biogeographic relationships. This new secondary metabolism molecular marker was compared to the previously applied nuclear ITS and plastid trnL-F sequences. The results from separate analyses show high congruence within the genus Digitalis and support the conclusion that all species of Isoplexis have a common origin and are embedded in Digitalis. The genus Isoplexis therefore should be reduced to sectional rank within the genus Digitalis. The sequence analyses give further evidence that additional sequence data increase support for relationships. It demonstrates that poorly supported relationships in smaller data sets may lead to erroneous conclusions about the evolution of the investigated taxa.     

Identification and characterization of plastid trnF(GAA) pseudogenes in four species of Solanum (Solanaceae)

Non-functional trnF pseudogenes that rarely occur in embryophytes have been found in Solanaceae. We have sequenced the trnL-F intergenic spacer of four species of Solanum, and found duplicated regions of the original trnF gene. These repeats were 94–260 bp long causing large length variation in the trnL-F intergenic spacer resulting from differences in pseudogene copy number (2–4). The duplicated trnF regions are comprised of several highly structured motifs, which were partial residues, or entire parts of the Anticodon, T- and D-domains of the original gene, but all lacked the acceptor stems at the 5′- or 3′-end. Pseudogenes included several transitions and transversions in their sequences compared to the original trnF gene. Among pseudogene copies, T-domains were more frequent and fragmented than D-domain elements. Our results demonstrate that although chloroplast evolution is uniform such structural duplications in the sequences used for phylogenetic reconstructions should be treated with great caution.     

Biogeography and evolution of flower color in Veratrum (Melanthiaceae) through inference of a phylogeny based on multiple DNA markers

Veratrum (Melanthiaceae) comprises ca. 27 species with highly variable morphology. This study aims to construct the molecular phylogeny of this genus to infer its floral evolution and historical biogeography, which have not been examined in detail before. Maximum parsimony, maximum likelihood, and Bayesian analyses were performed on the separate and combined ITS, trnL-F, and atpB-rbcL sequences to reconstruct the phylogenetic tree of the genus. All Veratrum taxa formed a monophyletic group, within which two distinct clades were distinguished: species with white-to-green perianth formed one highly supported clade, and the species with black-purple perianth constituted another highly supported clade. Phylogenetic inference on flower color evolution suggested that white-to-green perianth was a plesiomorphic state and black-purple perianth was apomorphic for Veratrum. When species distribution areas were traced as a multi-state character, parsimonious optimization inferred that Veratrum possibly originated in East Asia. Our study confirmed previous phylogenetic and taxonomic suggestions on this genus and provided a typical example of plant radiation across the Northern Hemisphere.     

Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.     

Pollination by fungus gnats (Diptera: Mycetophilidae) and self-recognition sites in Tolmiea menziesii (Saxifragaceae)

Coleeae (Bignoniaceae) are a tribe almost entirely restricted to Madagascar. Coleeae have previously been placed in neotropical Crescentieae due to species with indehiscent fruits, a character otherwise unusual in Bignoniaceae. A phylogeny based on three chloroplast regions (ndhF, trnT-L spacer, trnL-F spacer) identifies a monophyletic Coleeae that is endemic to Madagascar and surrounding islands of the Indian Ocean (Seychelles, Comores and Mascarenes). African Kigelia is not a member of Coleeae, rather it is more closely related to a subset of African and Southeast Asian species of Tecomeae. The molecular phylogeny indicates that indehiscent fruit have arisen repeatedly in Bignoniaceae: in Coleeae, Kigelia and Crescentieae. The characteristic fleshy fruits of species of Coleeae likely arose autochthonously in Madagascar. Within Coleeae Colea and Ophiocolea are sisters, Phyllarthron is sister to Colea + Ophiocolea, and Rhodocolea is sister to the rest of the tribe.     

Phylogenetic relationships in Peniocereus (Cactaceae) inferred from plastid DNA sequence data

The phylogenetic relationships of Peniocereus (Cactaceae) species were studied using parsimony analyses of DNA sequence data. The plastid rpl16 and trnL-F regions were sequenced for 98 taxa including 17 species of Peniocereus, representatives from all genera of tribe Pachycereeae, four genera of tribe Hylocereeae, as well as from three additional outgroup genera of tribes Calymmantheae, Notocacteae, and Trichocereeae. Phylogenetic analyses support neither the monophyly of Peniocereus as currently circumscribed, nor the monophyly of tribe Pachycereeae since species of Peniocereus subgenus Pseudoacanthocereus are embedded within tribe Hylocereeae. Furthermore, these results show that the eight species of Peniocereus subgenus Peniocereus (Peniocereus sensu stricto) form a well-supported clade within subtribe Pachycereinae; P. serpentinus is also a member of this subtribe, but is sister to Bergerocactus. Moreover, Nyctocereus should be resurrected as a monotypic genus. Species of Peniocereus subgenus Pseudoacanthocereus are positioned among species of Acanthocereus within tribe Hylocereeae, indicating that they may be better classified within that genus. A number of morphological and anatomical characters, especially related to the presence or absence of dimorphic branches, are discussed to support these relationships.