nrDNA Sequence Data Research Articles

Integrative species delimitation of the Selaginella sanguinolenta (Selaginellaceae) group with description of a new species, S. baodongii

AbstractSelaginella sanguinolenta group is widely distributed from the Himalayas to Siberia and the Russian Far East, including four to nine taxa. Despite progress in recent phylogenetic studies of the S. sanguinolenta group, the problem of species delimitation in this group remains. We revise the taxonomy of the S. sanguinolenta group based on an integrative approach considering morphological, molecular, and geographical evidence. The sampling for molecular analysis based on rbcL, psbA, and 26S nrDNA sequence data involved 71 individuals, covering the geographical range of this group. A total of 21 morphological traits from 103 specimens were explored using principal component analysis and linear discriminant analysis (LDA). The LDA results indicate that the combination of ventral leaf cilium length and ventral/dorsal leaf carinate characteristics enable a success rate of more than 90% in distinguishing between S. aitchisonii (western Himalayas to northern China), S. jacquemontii (western Himalayas to southwest China), and S. sanguinolenta (Russian Far East regions to northeast China). The phylogenetic analysis results of nuclear and plastid DNA markers further disclose the clear molecular differentiation. Selaginella nummularifolia, S. rossii, and S. sajanensis are well‐defined species based on morphological and molecular evidence with narrow distribution ranges respectively. Based on molecular and morphological evidence, in combination with distribution information, we finally recognize seven species in the S. sanguinolenta group, including a new species, S. baodongii sp. nov. from the bordering regions of Russia and China in northeast Asia.

Revised Taxonomy of theArctotisAnnual Clade (Arctotideae, Asteraceae) from Southern Africa: Integration of Molecular Phylogenetic and Morphological Evidence

Abstract—Previous phylogenetic analysis of ITS nrDNA sequence data for Arctotidinae species resolved a strongly supported clade containing all but one of the showy annualArctotisspecies (informally designated theArctotisAnnual Clade). In the present study, phylogenetic relationships in theArctotisAnnual Clade were investigated by Bayesian inference and maximum parsimony analyses of cpDNA (trnT-trnL-trnFandtrnH-psbA) and nrDNA (ITS) sequence data. The cpDNA and nrDNA phylogenies were notably incongruent.Arctotis venustaand a putative unnamed species (A.‘sp. B’) were strongly supported as monophyletic by both data sets. The monophyly ofA. leiocarpawas strongly supported by the ITS data set, whereas the remaining accessions formed a poorly resolved complex (the ‘A. fastuosacomplex’). Within theA. fastuosacomplex,A. hirsutawas monophyletic with strong support in the ITS phylogeny. A statistical parsimony-derived cpDNA haplotype network resolved five broad groups of haplotypes and showed no consistent geographical structure, but species-specific haplotype lineages forA. venustaandA.sp. B were resolved.Arctotis fastuosaaccessions were distributed among four haplotype groups. Incongruence between the data sets and poor resolution within theA. fastuosacomplex may reflect reticulate evolution, ancestral polymorphism, and incomplete lineage sorting, in tandem with the low information content of the data sets. The greatest phenotypic diversification in the clade is in cypsela morphology. Comparison of cypsela morphology with the phylogenies suggests a general trend for reduction in the sizes of the cypsela, abaxial wings, pappus scales, and loss of pubescence during diversification. A revised taxonomy, integrating currently available evidence, accompanied by full descriptive accounts and a key to the taxa are presented. Eight species are recognized, including the nomenclatural noveltiesArctotis chrysantha(sp. nov.) andArctotis namibiensis(sp. nov.). The namesArctotis karasmontana,Venidium fugax, andVenidium macrocephalumare lectotypified.

Range and niche expansion through multiple interspecific hybridization: a genotyping by sequencing analysis of Cherleria (Caryophyllaceae)

BackgroundCherleria (Caryophyllaceae) is a circumboreal genus that also occurs in the high mountains of the northern hemisphere. In this study, we focus on a clade that diversified in the European High Mountains, which was identified using nuclear ribosomal (nrDNA) sequence data in a previous study. With the nrDNA data, all but one species was monophyletic, with little sequence variation within most species. Here, we use genotyping by sequencing (GBS) data to determine whether the nrDNA data showed the full picture of the evolution in the genomes of these species.ResultsThe overall relationships found with the GBS data were congruent with those from the nrDNA study. Most of the species were still monophyletic and many of the same subclades were recovered, including a clade of three narrow endemic species from Greece and a clade of largely calcifuge species. The GBS data provided additional resolution within the two species with the best sampling, C. langii and C. laricifolia, with structure that was congruent with geography. In addition, the GBS data showed significant hybridization between several species, including species whose ranges did not currently overlap.ConclusionsThe hybridization led us to hypothesize that lineages came in contact on the Balkan Peninsula after they diverged, even when those lineages are no longer present on the Balkan Peninsula. Hybridization may also have helped lineages expand their niches to colonize new substrates and different areas. Not only do genome-wide data provide increased phylogenetic resolution of difficult nodes, they also give evidence for a more complex evolutionary history than what can be depicted by a simple, branching phylogeny.

Aster chuanshanensis (Asteraceae), a New Species from Shanxi and Sichuan, China

Aster chuanshanensis W.P. Li, a new species of Asteraceae from Shanxi and Sichuan, China, is described and illustrated based on morphological, micromorphological, molecular and cytological evidence. It is similar to A. indicus and A. shimadae, but differs from them in rhizome type, leaf size and texture, receptacle shape, floral tube and pappi lengths, and in the ratio of stigmatic line to the style arm appendage. Phylogenetic analysis based on nrDNA (ITS and ETS) sequence data supported A. chuanshanensis as a distinct species belonging to sect. Asteromoea. It is diploid with a karyotype 2n = 2x = 18m, which distinguishes it from the morphologically similar species.

Molecular phylogeny based on its sequences of nrDNA of some species belonging to dodder (Cuscuta L.) genus from various ecological sites of Turkey

Nuclear ribosomal DNA (nrDNA) sequence data of the Cuscuta genus, which have been considered as one of the most popular sequences for phylogenetic inference in plants, have been studied from a phylogenetic perspective in agricultural and non-agricultural lands of Turkey. The samples of Cuscuta spp. were collected from different geographical regions of Turkey between the years of 2013-2015. Some other species, not available locally, were taken from the herbarium samples of some research units. In order to study the phylogenetic relations of collected species, DNA isolations were made from body tissue samples. Conserved regions on ribosomal DNA (rDNA) were amplified by universal primers via PCR method and cloned into a proper cloning vector. The cloned DNA fragments were sequenced and analysed by web-based and computer programs. DNA sequences of certain species were recorded to the National Center for Biotechnology Information (NCBI) database. Based on the morphological examination and molecular analyses of fresh and the herbarium specimen, 8 species were identified. The identified species were C. hyalina (Gene bank accession no. KY020420), C. monogyna (KY020421), C. europaea (KY020422), C. palaestina (KY020423), C. approximata (KY020424), C. kurdica (KY020427), C. kotschyana (KY020430) and C. babylonica (KY020431). The ITS (Internal Transcribed Spacer) region contains several indels in identified Cuscuta species with the length varying from 668 to 730 bp. Sequence divergence ranges from 1.00% to 8.00% within Cuscuta spp. Based on our findings, the ITS sequences provided phylogenetically informative results in combination with the secondary structures.

Genetic diversity of Ixora macrophylla Bartl. and Ixora auriculata Elmer (Rubiaceae) inferred from nrDNA (ITS) and cpDNA (trnL-f) sequence data

This study aims to assess the intraspecific genetic diversity of Ixora macrophylla, a widespread species of Philippine Ixora recorded from several islands of the Philippines, and I. auriculata, an endemic species that has yet to be included in molecular studies of the Philippine Ixora. The number of haplotypes, haplotype diversity, and nucleotide diversity of 19 trnL-F and 17 ITS sequences of I. macrophylla, as well as 4 trnL-F and 4 ITS sequences of I. auriculata were obtained using DNAsp 5.10.1 software, and pairwise distances were calculated using the nucleotide Kimura 2-parameter using MEGA 6.06. Two haplotypes of trnL-F and nine haplotypes of ITS were identified in six populations of I. macrophylla. One of the two haplotypes of trnL-F was unique to the Mindoro population. One of the nine haplotypes of ITS was common among seven individuals from four populations. Intraspecific pairwise distances ranged from 0 to 0.1% for trnL-F and 0 to 0.9% for ITS. Mantel test showed weak correlations between the genetic and geographic distances for both trnL-F (r = -0.0380) and ITS (r = 0.0980) sequences. For the genetic diversity of I. auriculata, two haplotypes of trnL-F and four haplotypes of ITS were identified, with intraspecific pairwise distances ranging from 0 to 0.1% in trnL-F and 0.3 to 3.5% in ITS. The results for genetic diversity may be used to better understand the population genetics of the Philippine Ixora and provide insights for conservation.

Mycena section Sacchariferae: three new species with basal discs from China

This current study presents three novel Mycena species that grow exclusively on specific substrata in southern China, including Castanea mollissima fruits, Fagaceae leaves and Lauraceae twigs or leaves. Mycena castaneicola belongs to Mycena section Sacchariferae stirps Amparoina, because its stipe has a prominent basal disc and it has cherocytes, acanthocysts and caulocystidia covered with spinules. The two other new Mycena species belong to M. section Sacchariferae stirps Adscendens, because of their basal disc characteristics, lack of cherocytes on the pileal surface and the presence of smooth caulocystidia. Mycena hyalinostipitata is characterised by a hyaline stipe, two types of acanthocysts and fusiform caulocystidia. Mycena substylobates can be easily distinguished by its obovoid acanthocysts and irregularly shaped caulocystidia. Detailed descriptions, line drawings and a discussion of morphological characteristics are provided herein. In addition to morphological comparisons with related taxa, phylogenetic analyses of internal transcribed spacer (ITS) nrDNA sequence data were conducted. Maximum likelihood and Bayesian inference analyses of the ITS dataset confirmed the morphology-based classification.

Phylogeny and evolution of achenial trichomes in theLucilia‐group (Asteraceae: Gnaphalieae) and their systematic significance

AbstractThe Gnaphalieae (Asteraceae) are a cosmopolitan tribe with around 185 genera and 2000 species. The New World is one of the centers of diversity of the tribe with 24 genera and over 100 species, most of which form a clade called theLucilia‐group with 21 genera. However, the generic classification of theLucilia‐group has been controversial with no agreement on delimitation or circumscription of genera. Especially controversial has been the taxonomic value of achenial trichomes and molecular studies have shown equivocal results so far. The major aims of this paper are to provide a nearly complete phylogeny of theLucilia‐group at generic level and to discuss the evolutionary trends and taxonomic significance of achenial trichome morphology. We conducted a phylogenetic analysis of the New World Gnaphalieae with nrDNA (ETS, ITS) sequence data from a sampling of 18 genera of theLucilia‐group and utilized these results to examine morphological evolution of achenial trichome types and presence of apical myxogenic cells. Seven well‐supported subclades can be recognized within theLucilia‐group (L1–L7). These results support Brazilian and AndeanBerroa,Facelis,Lucilia, andMicropsisforming a clade (L1), the inclusion of ChileanLuciliaunderBelloa(L2), the monophyly ofStuckertiella+Gamochaeta+Gamochaetopsis(L3),Chevreulia+Cuatrecasasiella(L4) andAntennaria(L5) excludingAntennaria linearifolia, which is resolved in a monophyletic group together withJalcophila,LoricariaandMniodes(L6), and the recognition ofGnaphaliothamnus(L7) removed from Brazilian taxa ofChionolaena(L2). Ancestral character state reconstruction of achenial trichome morphology suggests that clades are homogeneous in terms of trichome type, but with exceptions that make it highly homoplastic. Conversely, our results suggest that the presence of myxogenic apical cells is less homoplastic and that closely related species tend to resemble each other more than expected under random variation.

Revising the Schizoparmaceae: Coniella and its synonyms Pilidiella and Schizoparme.

The asexual genera Coniella (1918) and Pilidiella (1927), including their sexual morphs in Schizoparme (1923), have a cosmopolitan distribution and are associated with foliar, fruit, leaf, stem and root diseases on a wide variety of hosts. Species of these genera sometimes occur as secondary invaders of plant tissues infected by other organisms or that are injured by other causes. Several studies published over the last few decades had conflicting ideas as to whether Coniella, Pilidiella and Schizoparme should be regarded as synonymous or as separate genera. The present study aims to resolve the generic classification of these genera through phylogenetic analyses of the concatenated alignment of partial LSU nrDNA, rpb2, ITS nrDNA and tef1 sequence data of 117 isolates, combined with their morphology. Results revealed that all strains cluster in a single well-supported clade. Conidial colour, traditionally the distinguishing character between Coniella and Pilidiella, evolved multiple times throughout the clade, and is not a good character at generic level in Schizoparmaceae. The three genera should therefore be regarded as synonymous, with the older name Coniella having priority. Furthermore, this study delineated 13 new species, and new combinations were proposed for a further 15 species.

Schoenus (Cyperaceae) is not monophyletic based on ITS nrDNA sequence data

We used nuclear rDNA-sequence data from the internal transcribed spacer (ITS) region to test the monophyly of Schoenus by using maximum parsimony and Bayesian inference. Schoenus is not monophyletic, with strong bootstrap support for most branches and congruence across analyses. nrITS does not resolve terminal taxa fully and, therefore, needs to be used in combination with other lines of evidence to address questions of species limits.

Clathrosporium retortum, a novel aeroaquatic fungus in the Sordariomycetidae (Ascomycota) from Brazil

Clathrosporium retortum sp. nov., collected on submerged mixed leaf litter samples at Ilha do Cardoso State Park, São Paulo state, Brazil, is described based on morphological and molecular data. The fungus is characterized by forming whitish, dense, subglobose to irregular propagules, hyaline to subhyaline when young, subhyaline to dark brown at maturity, that are formed by densely interwoven conidial filaments with each conidial cell repeatedly branching bilaterally or occasionally unilaterally. Phylogenetic analyses using partial LSU nrDNA sequence data suggest that C. retortum belongs in the Sordariomycetes (Ascomycota) where it forms a well-supported clade with Clohesia corticola in the Sordariomycetidae, but its ordinal or familial placement remains unresolved. Its phylogenetic placement confirms the polyphyletic nature of aeroaquatic fungi like Clathrosporium, as it was distantly related to one available sequence in GenBank named as C. intricatum, the type species, which is phylogenetically related to the Helotiales (Leotiomycetes). However, due to lack of authenticity of the identity of this sequence with the type specimen of C. intricatum, a broad concept of Clathrosporium is tentatively adopted here to accommodate the present fungus instead of introducing a new genus. Beverwykella clathrata, Helicoön septatissimum and Peyronelina glomerulata are recorded for the first time from Brazil. Cancellidium applanatum and Candelabrum brocchiatum are new records for the state of São Paulo.

Pseudodidymosphaeria gen. nov. in Massarinaceae

Didymosphaeria spartii was collected from dead branches of Spartium junceum in Italy. Multi-gene phylogenetic analyses of ITS, 18S and 28S nrDNA sequence data were carried out using maximum likelihood and Bayesian analysis. The resulting phylogenetic trees showed this to be a new genus in a well-supported clade in Massarinaceae. A new genus Pseudodidymosphaeria is therefore introduced to accommodate this species based on molecular phylogeny and morphology. A illustrated account is provided for the new genus with its asexual morph and the new taxon is compared with Massarina and Didymosphaeria.

Some aspects of the moss population development on the Svalbard glaciers

Glaciers are rather unusual habitat for mosses, but sometimes they can be suitable for some species due to presence of sufficient moisture and cryoconite substrate in the ablation zone. To date, moss populations were found only on a few glaciers in Alaska, Iceland and Svalbard. An origin and development of moss cushions on ice (so called "glacier mice") are still unclear. In this study, some aspects of the moss population development were explored on ice of the Svalbard glaciers – Bertilbreen (Billefjorden) and Austre Grnfjordbreen (Grnfjorden) in 2012 and 2013. On Bertilbreen, populations of Hygrohypnella polaris (Lindb.) Ignatov & Ignatova and Schistidium abrupticostatum (Bryhn) Ignatova & H.H. Blom were found for the first time. Due to putative morphological features, identification of S. abrupticostatum was confirmed by com-paring ITS1-2 nrDNA sequence data to BLAST searches (megablast). The results indicated a genetic heterogeneity of the population. Although visually moss-free, examination of cryoconite sediments revealed development of new individuals of S. abrupticostatum from filamentous structures consisting of caulonema and rhizoids. The developmental stages of young plants were revealed. Therefore, besides fragmen-tation of existing cushions, cryoconite sediments provided a source of new moss cush-ions in glacier populations. Additionally a few plants of Pohlia cf. wahlenbergii (F. Weber & D. Mohr) Andrews and a gametophyte fragment of Philonotis sp. were found in aggregation of cryoconite. Presence of Paludella squarrosa (Hedw.) Brid. reported for Bertilbreen has not been confirmed. On Austre Grnfjordbreen Bryum cryophilum Mrtensson, Sanionia uncinata (Hedw.) Loeske were found invading into some Hygrohypnella polaris cushions. Each moss polster on ice represents a separate mini-ecosystem that includes successive colonization events.

Multi-Locus Phylogeny Reveals the Sexual State ofTiarosporellain Botryosphaeriaceae

A new taxon of Botryosphaeriaceae associated with Arrhenatherum and Dactylis was collected in Italy. Single ascospore isolates were obtained and formed asexual morphs in culture. Multi-gene phylogenetic analysis was carried out using ITS, LSU nrDNA and EF1-α sequence data and the resulting phylogenetic tree showed that new species clustered in Botryosphaeriaceae together with Tiarosporella species with high bootstrap support. The new species is introduced as a sexual morph of Tiarosporella and detailed descriptions and illustrations are provided. A discussion of the asexual morphs of Tiarosporella is provided.

Elucidating the Ramularia eucalypti species complex.

The genus Ramularia includes numerous phytopathogenic species, several of which are economically important. Ramularia eucalypti is currently the only species of this genus known to infect Eucalyptus by causing severe leaf-spotting symptoms on this host. However, several isolates identified as R. eucalypti based on morphology and on nrDNA sequence data of the ITS region have recently been isolated from other plant hosts, from environmental samples and also from human clinical specimens. Identification of closely related species based on morphology is often difficult and the ITS region has previously been shown to be unreliable for species level identification in several genera. In this study we aimed to resolve this species-complex by applying a polyphasic approach involving morphology, multi-gene phylogeny and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Six partial genes (ITS, ACT, TEF1-α, HIS3, GAPDH and RPB2) were amplified and sequenced for a total of 44 isolates representing R. eucalypti s.lat. and closely related species. A multi-gene Bayesian phylogenetic analysis and parsimony analysis were performed, and both the resulting trees showed significant support for separation of seven species in R. eucalypti s.lat., including two previously described (R. eucalypti and R. miae), four novel species here described (R. haroldporteri, R. glennii, R. mali and R. plurivora) and one undescribed Ramularia species (sterile). Additionally, Mycosphaerella nyssicola is newly combined in Ramularia as R. nyssicola. Main mass spectra (MSPs) of several R. eucalypti s.lat. strains were generated using MALDI-TOF MS and were compared through a Principal Component Analysis (PCA) dendogram. The PCA dendrogram supported three clades containing R. plurivora, R. glenni/R. mali and R. eucalypti/R. miae. Although the dendrogram separation of species differed from the phylogenetic analysis, the clinically relevant strains were successfully identified by MALDI-TOF MS.

Diversity within the genus Elymus (Poaceae: Triticeae) as investigated by the analysis of the nr5S rDNA variation in species with St and H haplomes.

The genus Elymus ("Ryegrass") is a repository for a range of species with a variety of haplome contents; hence the pejorative name "dustbin" genus. We have analyzed 1,059 sequences from 128 accessions representing 24 species to investigate the relationships among the StH haplomes-containing species described by Yen and Yang (Genus Elymus Beijing 5:58-362, 2013). Sequences were assigned to "unit classes" of orthologous sequences and subjected to a suite of analyses including BLAST (Basic Local Alignment Search Tool) searches, phylogenetic analysis and population genetic analysis to estimate species diversity. Our results support the genome analyses in Yen and Yang (Genus Elymus Beijing 5:58-362, 2013), i.e., genomic constitution StStHH including variants restricted to Elymus. Population genetic analysis of the 5S nrDNA sequence data revealed that the within-species variance component is roughly ±89%; thus, we were unable to identify molecular markers capable to separate the 24 species analyzed. Separate phylogenetic analyses of the two unit classes and of all the data exhibit a trend only of the species to cluster on the phylograms. Finally, the analysis provides evidence for the multiple origins of American and Eurasian species.

Phylogenetic Analysis Reveals Multiple Cases of Morphological Parallelism and Taxonomic Polyphyly in <I>Lomatium</I> (Apiaceae)

Abstract The genera Lomatium and Cymopterus, along with many others, form a group that has been referred to previously as the perennial endemic Apiaceae subfamily Apioideae of western North America. This group of ecologically important and widespread species has been the target of numerous systematic studies, but the evolutionary relationships among these species remain elusive. Here we show that this confusion is due to high levels of morphological parallelism and homoplasy in the characters that have traditionally been used to define them, a result that is concordant with previous studies of the group. We explore patterns of evolution in traditionally important morphological characters using Bayesian stochastic character mapping on a phylogeny constructed from novel nrDNA and cpDNA sequence data for 96 specific and infraspecific taxa of the estimated 200 species in the group. We consider the implications of these results for taxonomic classification, the evolution of morphologies, and the utility of the...

The olive goblet: Peziza oliviae, a new cup fungus growing underwater in Oregon

A new species, Peziza oliviae, is described from stream habitats in the Oregon Cascade Mountains (USA) based on morphology, habitat, and ITS and LSU nrDNA sequence data. This new member of the Ascomycota grows underwater on sticks and decomposing wood as well as near running water where the woody substrate is saturated.

<i>Deniquelata barringtoniae gen. et sp. nov.</i>, associated with leaf spots of <i>Barringtonia asiatica</i>

Deniquelata barringtoniae gen. et sp. nov. (Montagnulaceae) forms numerous ascomata on distinct zonate leaf spots of Barringtonia asiatica (Lecythidaceae). We isolated this taxon and sequenced the 18S and 28S nrDNA. The result of phylogenetic analysis based on 18S and 28S nrDNA sequence data indicate that the genus belongs in the family Montagnulaceae, Dothideomycetes, Ascomycota. The ascomata are immersed, dark brown to black, with bitunicate asci and brown, muriform ascospores. Deniquelata is distinguished from the other genera in Montagnulaceae based on its short, broad, furcate and pedicellate asci, verruculose ascospores with short narrow pseudoparaphyses with parasitic naturee and this is also supported by molecular data. A new genus and species is therefore introduced to accommodate this taxon. We used isolates of this species to show via pathogenicity testing that the taxon is able to cause leaf spots when leaves are pin pricked.

Resolving the Diaporthe species occurring on soybean in Croatia.

Diaporthe (anamorph = Phomopsis) species are plant pathogens and endophytes on a wide range of hosts including economically important crops. At least four Diaporthe taxa occur on soybean and they are responsible for serious diseases and significant yield losses. Although several studies have extensively described the culture and morphological characters of these pathogens, their taxonomy has not been fully resolved. Diaporthe and Phomopsis isolates were obtained from soybean and other plant hosts throughout Croatia. Phylogenetic relationships were determined through analyses of partial translation elongation factor 1-alpha (EF1-α) gene and ITS nrDNA sequence data. By combining morphological and molecular data, four species could be distinguished on soybeans in Croatia. Diaporthe phaseolorum is described in this study and its synonyms are discussed. Diaporthe phaseolorum var. caulivora is raised to species status and the name Diaporthe caulivora is introduced to accommodate it. A species previously known as Phomopsis sp. 9 from earlier studies on sunflower, grapevine, rooibos and hydrangea is reported for the first time on soybean, and is formally described as Diaporthe novem. The well-known soybean pathogen Phomopsis longicolla was also collected in the present study and was transferred to Diaporthe longicolla comb. nov. The presence of these species on herbaceous hosts raises once more the relevance of weeds as reservoirs for pathogens of economically important plants.