Phylogenetic position of the genus Bryhnia Kaurin (Brachytheciaceae,Bryophyta) inferred from chloroplast (rpl16, trnG,trnL–F) and nuclear (ITS) sequence data

Abstract

The systematic position of the genus Bryhnia Kaurin has been problematic and remains unsettled. A phylogenetic analysis of Bryhnia and closely related genera, based on the nuclear marker ITS1–5·8S–ITS2 and the plastid markers rpl16, trnG, and trnL–F, was carried out to clarify their relationships and systematic positions. The phylogenetic trees generated from Maximum parsimony and Bayesian analyses show that Bryhnia s.s., represented by B. novae-angliae (Sull. & Lesq.) Grout, B. hultenii E.B.Bartram, and B. scabrida (Lindb.) Kaurin, forms a maximally supported clade nested within Brachythecium Schimp. ‘Bryhnia scabrida’ sensu is Bryhnia brachycladula Cardot, and together they form a monophyletic group with Eurhynchiadelphus eustegia (Besch.) Ignatov & Huttunen and Myuroclada maximowiczii (G.G.Borshch.) Steere & W.B.Schofield. We propose the inclusion of the species currently recognized in Bryhnia in Brachythecium, with the transfer of Bryhnia brachycladula to the genus Myuroclada Besch. Bryhnia brachycladula is further treated as a synonym of the new combination, Myuroclada longiramea (Müll.Hal.) M.Li, Y.F.Wang, M.S.Ignatov & S.Huttunen.     

Molecular phylogenetic relationships reveal taxonomic and biogeographic clades in Dianella (flax lilies; Asphodelaceae,Hemerocallidoideae)

A phylogeny of Dianella is presented based on Bayesian and maximum parsimony analyses of a combined molecular data set using three chloroplast markers (trnQ^UUG–5'rps16, 3'rps16–5'trnK^(UUU) and rpl14–rps8–infA–rpl36) and two nuclear markers (ITS and ETS). Accessions included most Dianella species, including all species from Australia, the centre of diversity for the genus, and related outgroup genera Eccremis, Stypandra, Thelionema and Herpolirion. The phylogeny showed Stypandra sister to Herpolirion + Thelionema, and confirmed the monophyly of Dianella. Within Dianella, a number of clades were resolved that revealed biogeographic relationships. Accessions from south-western Australia (extending into South Australia) formed the earliest diverging clade, followed by D. serrulata from New Guinea, sister to all other clades of Dianella from Australia and other regions. Tropical North Queensland species, including the D. pavopennacea complex, were related to a clade of accessions from New Caledonia and the Hawaiian Islands in the Pacific, and a clade that included samples of D. carolinensis (Caroline Islands) and the widespread D. ensifolia from South-East Asia and across the Indian Ocean to Mauritius and Madagascar. However, D. ensifolia is not monophyletic, with accessions from Japan and Taiwan related to a clade of Queensland samples that are part of the D. revoluta complex. Three New Zealand species (diploid, 2n?=?16) were found to be related to Norfolk Island D. intermedia (type locality; octoploid, 2n?=?64). In contrast ‘D. intermedia’ from Lord Howe Island was resolved as sister to the eastern Australian D. caerulea complex. The phylogenetic results indicate the need for taxonomic revision, particularly revision of the species ‘complexes’ D. longifolia and D. caerulea in Australia, and recognition of more than one species within D. ensifolia and within D. sandwicensis on the Hawaiian Islands.     

Phylogeny and taxonomy of Halimeda incrassata,including descriptions of H. kanaloana and H. heteromorpha spp. nov. (Bryopsidales,Chlorophyta)

The tropical green algal genus Halimeda is one of the best studied examples of pseudo-cryptic diversity within the algae. Previous molecular and morphometric studies revealed that within Halimeda section Rhipsalis, Halimeda incrassata included three pseudo-cryptic entities and that the morphological boundaries between H. incrassata and Halimeda melanesica were ill-defined. In this paper, the taxonomy of H. incrassata is revised: two pseudo-cryptic entities are described as new species, Halimeda kanaloana and Halimed heteromorpha, while H. incrassata is redefined to encompass a single, monophyletic entity. Similarities and differences between the three species and H. melanesica are discussed. Monophyly of H. heteromorpha, which was questioned in a former study, is reinvestigated using sets of 32 ITS1–ITS2 and 21 plastid rps3 sequences and various alignment and inference methods. The phylogenetic relationships within Halimeda section Rhipsalis are inferred from nuclear 18S–ITS1–5.8S–ITS2 and concatenated plastid sequences (tufA & rpl5–rps8–infA) and interpreted in a biogeographic context.     

基于5个基因片段的野胡麻属系统位置研究

对单种属野胡麻属(原隶属于广义玄参科)及其可能近缘类群广泛取样,选择合适外类群,通过对4个叶绿体基因联合数据(trnL-F、rps16、rbcL、rps2)、核基因ITS片段、叶绿体基因与核基因ITS片段联合数据,进行最大简约法和贝叶斯法分析,探讨野胡麻属在科级系统位置及其与近缘类群系统发育关系。结果表明,所有系统树都支持野胡麻属先和肉果草属构成姐妹群关系,叶绿体基因联合系统树支持率分别为97%[Bootstrap(BS)]和100%[Posterior probability(PP)];ITS系统树支持率分别为99%(BS)和100%(PP);叶绿体基因和ITS联合系统树支持率均为100%(BS和PP)。野胡麻属、肉果草属与通泉草属一起构成一单系群,叶绿体基因联合系统树、ITS系统树、叶绿体基因和ITS联合系统树的支持率均为100%(BS和PP)。形态学证据也支持野胡麻属、肉果草属和通泉草属这3个属的近缘关系。本研究结果支持单种属野胡麻属隶属于透骨草科的通泉草亚科,与肉果草属亲缘关系最近,这两个属进而与通泉草属近缘,可能隶属于通泉草属内,与岩白翠亲缘关系较近,也可能与通泉草属互为姐妹群关系。     

Mesoptychia chinensis Bakalin,Vilnet & Xiong sp. nov. (Jungermanniaceae,Marchantiophyta) and comments on the distribution of Mesoptychia south of the boreal zone in Asia

Abstract

Morphologically unique specimens of Mesoptychia (Lindb.) A.Evans recently collected in Guizhou Province, China were sequenced and analysed using a combined trnL-F+trnG-intron cpDNA dataset from 10 known taxa of Mesoptychia. Using a combination of morphological and molecular data, Mesoptychia chinensis Bakalin, Vilnet & Xiong sp. nov. is described as new to science. Mesoptychia polymorpha subsp. pakistanica Bakalin & Vilnet subsp. nov. is established based on morphological data only. Mesoptychia ussuriensis is newly recorded for China. New molecular data for species delimitation within Mesoptychia distributed in southern and south-east Asia are provided.     

Lack of intraspecific variation in cpDNA in Trichocolea tomentella

Abstract

The Asian genus Struckia Müll.Hal. is reduced to a synonym of Plagiothecium Bruch &; Schimp. according to a phylogenetic analysis involving S. argentata (Mitt.) Müll.Hal., S. enervis (Broth.) Ignatov, T.J.Kop. &; D.G.Long and 13 representative boreal species of Plagiothecium. Two nuclear regions, ITS and partial gapC, and five chloroplast regions, rbcL, rps4-trnS, psaB, trnG and trnL-F, were utilized to reconstruct the phylogenetic relationship between Struckia and Plagiothecium. Bayesian, maximum parsimony, and maximum likelihood analyses resulted in a strongly supported clade including Struckia and Plagiothecium. Plagiothecium handelii Broth. and P. paleaceum (Mitt.) A.Jaeger, which share similar geographical ranges with Struckia and intermediate morphological traits between Struckia and Plagiothecium, grouped with the Chinese Struckia, and all of them appeared as sister to the ‘core’ of Plagiothecium. The accurate position of P. piliferum (Sw.) Schimp. remains unclear due to moderate support from all analyses, and we suggest that it is retained in Plagiothecium until further evidence is available.     

DNA barcoding approach fails to discriminate Central European bladderworts (Utricularia,Lentibulariaceae), but provides insights concerning their evolution

Abstract

The main features to distinguish the seven native Utricularia species occurring in central Europe are found in flower shape, but being rarely flowering identification is often doubtful and uncertain. A recent morphometric work highlighted that there are no univocal reliable extra-floral morphological features allowing a safe identification at species level. Therefore, DNA barcoding approach is attempted here. Molecular analyses were performed to search for DNA barcodes using nuclear ITS (rDNA), plastid (cpDNA) trnL-trnF IGS and rps16 intron sequences. Generally, the barcoding approach failed to discriminate Utricularia species, although it could be of some help in the U. minor aggregate. With few exceptions, U. bremii shows peculiar DNA regions different from U. minor for both plastid markers investigated. However, interesting hypotheses could be derived from the obtained networks, including hybridization events to explain the rise of mostly sterile species, such as U. stygia. This species clusters with the other species of the U. intermedia aggregate in plastid phylogenetic graphs, while it is closely related to species of the U. minor aggregate in ITS phylogenetic graphs. Additionally to U. stygia, U. ochroleuca also shows some incongruences in the different markers, at least for some accessions, pointing to the possible occurrence of hybrids.     

基于ITS和trnL-F以及rps4基因片段的 粗疣藓属分类学地位研究

选择粗疣藓属及相关属样本作为内群,油藓属样本作为外群,选取核基因ITS片段以及叶绿体基因trnL-F和rps4片段进行PCR扩增、测序,运用贝叶斯推论(BI)、最大似然法(ML)和最大简约法(MP)构建分子系统发育树,以明确粗疣藓属的分类学地位。结果表明:(1)用于分子系统分析的基因序列共有1 744个位点,其中747个位点属于核基因片段,997个位点属于叶绿体基因片段。(2)3种系统发育树拓扑结构基本一致,都显示粗疣藓属与毛锦藓科成员聚成一支,支持粗疣藓属应隶属于毛锦藓科的观点。(3)小粗疣藓和粗疣藓的种间界限模糊。(4)小鼠尾藓属的3个种与棉藓属成员聚成一支,支持将其归入棉藓科的观点。     

Molecular phylogeny of the endemic fern genera Cyrtomidictyum and Cyrtogonellum (Dryopteridaceae) from East Asia

Cyrtomidictyum Ching and Cyrtogonellum Ching are two eastern Asian endemic genera whose taxonomic affinities and phylogenetic relationships have long been controversial. The main uncertainty surrounds the separation of the two genera from the species-rich genus Polystichum. Here we present a phylogenetic study focusing on the phylogenetic relationships of these polystichoid ferns. We reconstructed the relationships based on DNA sequence variation in four chloroplast genome regions, rbcL, atpB, and the intergenic spacers (IGS) rps4-trnS and trnL-trnF. Maximum likelihood and Bayesian inference analyses confirm earlier results that were based on less comprehensive taxon sampling and either only a single gene (rbcL) or two IGS (rps4-trnS and trnL-trnF). Cyrtomidictyum is the sister of the clade of polystichoid ferns that includes Cyrtogonellum, Cyrtomium subser. Balansana and three sections of Polystichum. Cyrtogonellum groups with several species of Polystichum, and constitutes the sister taxon to Polystichum sect. Sphaenopolystichum. We support the recognition of Cyrtomidictyum as circumscribed initially, rather than expansion of the genus to include either several Polystichum species or Cyrtogonellum, some Polystichum and Cyrtomium species. The monophyly of Cyrtomidictyum is supported by morphological characters such as once-pinnate leaves, free venation, prolongated leaf apices, and exindusiate sori. Two synapomorphic indels in the chloroplast genome, one 15-bp deletion in rps4-trnS, and one 3-bp insertion in trnL-trnF further differentiate Cyrtomidictyum from other polystichoid ferns. The close affinity of Cyrtogonellum to section Sphaenopolystichum of Polystichum s.s. is highly supported by molecular data. However, no shared morphological characters or molecular indels have been detected, although the distinctness of Cyrtogonellum is shown by a 13-bp insertion in the rps4-trnS alignment.     

Macromitrium maolanense Zeyou Zhang,D.D.Li,Jing Yu & S.L.Guo,a new species from China based on molecular and morphological evidence

Introduction. During a field excursion in Guizhou Province, China, we collected some interesting moss specimens with branch leaves subulate in the upper part, partially and variably bistratose laminae, and a Macromitrium-like epiphytic growth habit on tree trunks. We present morphological and phylogenetic arguments for recognising these plants as a new moss species in the genus Macromitrium Brid. (Orthotrichaceae).

Methods. We compared the morphology of the potential new species with closely related species of Macromitrium, and constructed a phylogenetic tree based on ITS2, trnL and trnG including sampling from 14 other morphologically similar species of Macromitrium.

Key results. The proposed new species belongs to the genus Macromitrium (Orthotrichaceae, Musci). It is closely related to M. gymnostomum Sull. & Lesq. in the phylogenetic tree and according to gametophytic morphological features, represents a hitherto undescribed species.

Conclusions. A new moss species, Macromitrium maolanense Zeyou Zhang, D.D.Li, Jing Yu & S.L.Guo, is described and illustrated. The new species can be distinguished from all congeners by the combination of the following features of the branch leaves: (1) oblong-lanceolate, lanceolate to linear-lanceolate, gradually narrowed to an easily broken subula; (2) rather obscure upper and medial cells, often with blackish stains among cells, densely pluripapillose; (3) variably and partially bistratose laminae in the upper 1/3 portion; (4) basal cells clear, hyaline and smooth, those near costae forming a ‘cancellina region’; and (5) with numerous brownish, clavate gemmae on upper portion. We also discuss the principal distinctive characters separating the new species from its nearest congeners.     

Chloroplastrps16 intron phylogeny of the tribeSileneae (Caryophyllaceae)

Intron sequences of the chloroplast generps16 from 46 species were used to examine phylogenetic relationships indicated by nrDNA ITS sequence variation in the tribeSileneae (Caryophyllaceae, Caryophylloideae). This region has previously not been utilized for phylogenetic purposes but the results presented here suggest that it is a consistent and valuable complement to the ITS sequences. Therps16 intron trees are largely congruent with the ITS trees. All the major hypotheses suggested by the ITS data are supported, often at similar bootstrap levels. The joint usage ofrps16 intron and ITS sequences provides a powerful tool for resolving many of the difficult taxonomic issues in the tribeSileneae. Dedicated to emer. Univ.-Prof. DrFriedrich Ehrendorfer on the occasion of his 70th birthday     

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.     

Origin and early evolution of Ceropegieae (Apocynaceae-Asclepiadoideae)

The first-branching lineages of the tribe Ceropegieae, Anisotominae, Leptadeniinae, and Heterostemminae, are the focus of the present study. In the ingroup we molecularly analysed 34 samples from southern and (north)eastern Africa and Asia for the nuclear internal transcribed spacer (ITS) region, and five plastid markers (trnT-L, trnL-F and trnH-psbA intergenic spacers, trnL, and rps16 introns). Maximum Parsimony and Bayesian analyses were conducted for phylogenetic reconstruction and Bayesian binary MCMC (BBM) analysis as implemented in RASP for testing biogeographic inference. Sister to the well-known hyperdiverse subtribe Stapeliinae (around 700 species) are the Anisotominae, an African subtribe of c. 30 species, with the tropical Neoschumannia in sister-group position to the remaining genera, Anisotoma, Emplectanthus, Riocreuxia, and Sisyranthus. Emplectanthus is sequenced in full for the first time, and its monophyly as sister to Anisotoma is documented. As in Sisyranthus, new sequences in Riocreuxia, especially in Riocreuxia torulosa s.l., reveal very low genetic variation pointing to an actively radiating and speciating group, possibly in adaptation to pollinator pressure. Molecular and morphological data necessitate the transfer of Brachystelma natalense to Anisotoma, and the new combination Anisotoma natalensis is proposed. Leptadeniinae are sister to the Stapeliinae–Anisotominae clade. The position of the strictly Asian, rheophytic Pentasachme could be resolved as sister to the remaining genera of Leptadeniinae, Conomitra, Leptadenia, and Orthanthera. The position of the S Asian/Australasian wet forest lianas of the genus Heterostemma as sister to the remaining Ceropegieae is confirmed, indicating a humid Asian origin for this tribe known for its arid-adapted African succulents.     

Hybridization between two oil-secreting orchids in South Africa

Natural hybrids among the specialized terrestrial oil-secreting orchids of South Africa are extremely rare even where multiple closely related species co-occur. We found putative hybrids between Pterygodium catholicum Sw. and P. acutifolium Lindl., two closely related oil-secreting orchids that lack morphological barriers to inter-breeding. The purpose of this study was to confirm the parentage of the putative hybrids using molecular data from one nuclear (ITS) and two plastid (matK-trnK and trnL/F introns) DNA regions. Phylogenetic analyses of sequences as well as nucleotide substitution patterns of the putative hybrids, putative parents, and their closest relatives were consistent with a hypothesis of hybridization. We suggest that the hybrids were the result of visits to both orchids by a single species of oil-collecting bee during a brief period of flowering overlap. These results suggest that the rarity of hybrids between these orchids is due to factors other than genetic incompatibility.     

MOLECULAR AND MORPHOMETRIC DATA PINPOINT SPECIES BOUNDARIES IN HALIMEDA SECTION RHIPSALIS (BRYOPSIDALES,CHLOROPHYTA)1

Molecular systematic studies have changed the face of algal taxonomy. Particularly at the species level, molecular phylogenetic research has revealed the inaccuracy of morphology‐based taxonomy: Cryptic and pseudo‐cryptic species were shown to exist within many morphologically conceived species. This study focused on section Rhipsalis of the green algal genus Halimeda. This section was known to contain cryptic diversity and to comprise species with overlapping morphological boundaries. In the present study, species diversity within the section and identity of individual specimens were assessed using ITS1–5.8S–ITS2 (nrDNA) and rps3 (cpDNA) sequence data. The sequences grouped in a number of clear‐cut genotypic clusters that were considered species. The same specimens were subjected to morphometric analysis of external morphological and anatomical structures. Morphological differences between the genotypic cluster species were assessed using discriminant analysis. It was shown that significant morphological differences exist between genetically delineated species and that allocation of specimens to species on the basis of morphometric variables is nearly perfect. Anatomical characters yielded better results than external morphological characters. Two approaches were offered to allow future morphological identifications: a probabilistic approach based on classification functions of discriminant analyses and the classical approach of an identification key.     

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.     

Mutational dynamics and phylogenetic utility of noncoding chloroplast DNA

Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference. The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats, short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability. A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential, the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.     

Natural Hybridization and Introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae,Senecioneae) in Four Different Locations

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5''rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.     

Phylogeny of Pyroleae (Ericaceae): implications for character evolution

Pyroleae (Ericaceae) consist of four genera, all of which are distributed widely in temperate coniferous or sometimes deciduous forests of the Northern Hemisphere. To investigate the phylogenetic relationships among these genera and to explore the evolution of the characteristics of the subfamily, we conducted maximum parsimony and Bayesian analyses with nrDNA ITS and three cpDNA intergenic spacers (atpB-rbcL, trnS-trnG and trnL-trnF). The results from cpDNA and combined cpDNA + ITS data sets strongly support the monophyly of Pyroleae as well as a sister relationship between Pyrola and Moneses–Chimaphila, with Orthilia as the basal lineage. The sister-group relationship between Moneses and Chimaphila is supported by a set of synapomorphies, e.g., single flower, colpate pollen, five bundles in the style, straight fruiting pedicel orientation, complete capsule dehiscence, and the basic chromosome number, x = 13. The Moneses–Chimaphila–Pyrola clade is supported by at least one homologous character of pollen in tetrads. Conflicts associated with the phylogenetic position of Orthilia may imply a hybrid origin for it, and therefore further study is needed.