nrDNA ITS Research Articles

Post‐Oligocene diversification in Australia: Phylogeography, systematics and new species of Gonocarpus (Haloragaceae)

AbstractThe angiosperm genus Gonocarpus (Haloragaceae) is an enigmatic group of herbs and shrubs that occurs primarily in Australia (36/41 spp.) with four species ranging into New Zealand and/or Asia. Much of Gonocarpus diversity is found in the Southwest Australian Floristic Region (SWAFR), an international biodiversity hotspot, where several species are endemic and recognized as rare or of priority concern. The diversification of Gonocarpus throughout Australia started in the early Miocene, which makes it a strong candidate for examining phylogenetic patterns related to post‐Oligocene aridity events. The most recent taxonomic treatment for the genus was based on morphology alone and included relatively few specimens from some taxa, as collections were limited. This genus is modest in floral displays, but its striking intricate floral and fruit morphology have been critical to help define the species. Here, we use a molecular phylogenetic approach with DNA sequence data collected from the nrDNA ITS and cpDNA regions to construct phylogenetic hypotheses to examine species limits, determine times of divergence, and reconstruct ancestral distribution. Most of the SWAFR diversity originates from a most recent common ancestor (MRCA) ≈8 Ma with most diversification occurring over the late Miocene. There were two other diversification events into Southwest Australia from a geographically widespread clade in the genus also from a MRCA ≈8 Ma. Diversification in the southeast temperate region occurred during the Pliocene/Pleistocene. Late Miocene aridification is likely important for most of the patterns of historical biogeography found in the genus, rather than extreme climate fluctuation of the Pleistocene. Two new species are described for the genus.

Mimosa brevicalyx (Leguminosae-Caesalpinioideae): a new species based on molecular, anatomical, and morphological data

Phylogenetic, taxonomic, and biogeographic studies of Mimosa sect. Mimosa ser. Mimosa subser. Polycephalae revealed a new species, Mimosa brevicalyx T.P.Mendes & M.J.Silva (sp. nova), endemic to the savannas of central Goias State, Brazil. The species is described here, illustrated, compared with related taxa (Mimosa hypoglauca var. hypoglauca, M. hypoglauca var. dawsonii, M. papposa var. papposa, and M. papposa var. segrex), its geographic distribution is mapped, its conservation status, flowering and fruiting times and systematic position is discussed, and images are provided. Additionally, the new species and its related congeners have their phylogenetic positions elucidated and discussed based on molecular phylogenies derived from the union of ITS (nrDNA) and trnD-T (cpDNA) markers and are compared using leaf-anatomical data, including the micromorphology of its trichomes. Trichomes are of extreme importance for determining the taxa considered here, as they show stable and discontinuous differences related to the environments occupied by the species and are therefore all of likely ecological relevance.

Aster sanqingshanica (Asteraceae, Astereae), a new species from Jiangxi, China

Aster sanqingshanica J.W. Xiao & W.P. Li, a new species of Asteraceae from Jiangxi, China, is described, illustrated and compared with related taxa based on evidence from morphology and molecular phylogeny. Morphological observations have shown that A. sanqingshanica is most similar to A. tianmenshanensis, but readily differs by its ascending stem (versus erect), marcescent basal leaves (versus deciduous), ciliate basal leaf margins (versus glabrous), 4–5‐seriate phyllaries (versus 2–3‐seriate), purple phyllaries (apex) (versus green), flat receptacle (versus convex) and broader lamina of the ray florets (3–4 mm versus 2–3 mm). Phylogenetic analysis based on nrDNA ITS and ETS sequence data show that A. sanqingshanica is closely related to A. tonglingensis.

First report of powdery mildew of rainforest spinach (Elatostema reticulatum), native to Australia, caused by Podosphaera xanthii

Powdery mildew-infected rainforest spinach (Elatostema reticulatum) plants were found in Queensland, Australia. Based on morphology and nrDNA ITS sequence analyses, the pathogen was identified as Podosphaera xanthii, a powdery mildew species with a worldwide distribution and a broad host range. This is the first record of P. xanthii infecting E. reticulatum, and the first powdery mildew infection recorded for this native Australian plant.

Genetic diversity of Oxytropis section Xerobia (Fabaceae) in one of the centres of speciation.

The genetic diversity and phylogenetic relationships of Oxytropiscaespitosa, O.grandiflora, O.eriocarpa, O.mixotriche, O.nitens, O.peschkovae and O.triphylla, section Xerobia subgenus Oxytropis, in one of the main speciation centres of the genus Oxytropis (Baikal Siberia and adjacent territories of Northeastern Mongolia) were studied based on sequence analysis of the psbA-trnH, trnL-trnF and trnS-trnG intergenic spacers of cpDNA, as well as the ITS nrDNA. Most populations are characterized by a high level of chloroplast genetic diversity (h varied from 0.327 to 1.000 and π from 0.0001 to 0.0090) due to the ancient origin for some species and to hybridization and polyploidy for others. 67 haplotypes were identified, of which six were shared. Phylogenetic relationships among species could not be satisfactorily resolved. Only the haplotypes of O.triphylla formed a group with rather high support. Probably, O.caespitosa, O.grandiflora, O.mixotriche and O.nitens constitute a single genetic complex. As regards the ITS nrDNA polymorphism, we detected only two ribotypes (RX1, RX2). Both were found in O.caespitosa, O.eriocarpa, O.mixotriche and O.peschkovae, while RX1 was present in O.nitens and O.triphylla, RX2 in O.grandiflora. The absence of diagnostic species-specific variants for the markers studied, together with the sharing of cpDNA haplotypes and nrDNA ribotypes between species, and the resulting polytomies on the phylogenetic trees, confirm the hypothesis on the hybrid origin of some of them. Obviously, the reproductive barriers within the sect. Xerobia are weak. However, morphological differences between the species of the sect. Xerobia are clearly pronounced, even when they grow in sympatry.

An infraspecific dimension of bioindication? Comparison between genotypes and ecological distribution of Potamogeton coloratus

The role of within-species genetic variability and ecotypes has been poorly documented in research on water bioindication. Some studies already show that macrophytes could present genetic lineages with different optima for specific water parameters. In the present study, we tested this assumption on Potamogeton coloratus Hornem., a macrophyte described as characteristic of oligotrophic waters and threatened by freshwater eutrophication. Species population genetics, water parameters, and macrophyte and macroinvertebrate communities were analysed in 29 sites among three eco-regions of the eastern parts of France: Rhine Plain, Rhône Valley and Provence. The observed diversity of habitats can be divided into two main categories: the lotic hydrosystems such as springs and small watercourses often fed by groundwater, and the lentic waterbodies such as shallow pools or pond sides. Sampling sites were characterised by alkaline (mean pH 7.5 ± 0.4) and cool waters (mean 15.8 ± 4.1 °C) with low N-NH4+ concentration (median value of 20 μg/L), but large variation in other parameters (e.g. nitrates, phosphates). We found two main AFLP genotypic lineages, despite a poor genetic variability mainly distributed between populations, without regional structuring nor within-population diversity. We also sequenced only two ribotypes based on the nrDNA ITS. Genotypes, ribotypes and habitat categories were not correlated, arguing that the divergence of genotypic lineages is not due to ecotypic differentiation for P. coloratus. We highlight a putative event of long-distance dispersal, which illustrates the species ability for recolonization. Finally, we provide some suggestions for its conservation facing global changes.

Phylogenetic relationships and divergence time estimates in the genus Echinops L. (Asteraceae)

This study focuses on the phylogenetic relationships among taxa within the genus Echinops, in particular the taxa distributed in Turkey, since their systematic characterization and sectional taxonomy have not previously been fully determined. We used sequences of the plastid trnL‐trnF, rbcL, matK and nrDNA ITS gene regions obtained from this study and GenBank to reconstruct phylogenetic relationships among Echinops taxa based on Bayesian inference. In addition, divergence times of taxa within the genus were estimated by the Bayesian method using a relaxed clock model. In the phylogenetic tree, E. acantholepis, an annual species, was first split from all other echinops taxa and this split was estimated to have occurred approximately 19.99 Mya. Next, E. emiliae, a biannual species, separates from the other species, and finally forms a group with the perennial Echinops taxa. We were also to predict differences in the rate of speciation by comparing different gene regions. The results indicate that the number of sections of the genus Echinops distributed in Turkey should be increased from three to five by the addition of the sections Psectra and Acantholepis. Furthermore, it is indicated that some taxa currently treated as subspecies may deserve recognition at the species level. Our results also support previous studies suggesting that it is appropriate to incorporate the monotypic Acantholepis in Echinops and transfer A. orientalis to the genus Echinops under the name E. acantholepis. Consequently, it is concluded that the studied gene regions were significant and informative for resolving phylogenetic relationships among the taxa of Echinops.

<p><strong>Molecular and morphological evidence of <em>Onobrychis avanakensis</em>, a new species from Iran</strong></p>

Onobrychis avanakensis is described as a new species in sect. Onobrychis. The new species is restricted to the Alborz Mountains in the Qazvin province of Iran and distinguished from its closely related taxa in both morphological and molecular evidence based on nrDNA ITS sequences. O. avanakensis is well distinct from O. major and O. carduchorum by having different sizes of keel, wing and calyx. Based on the phylogenetic tree resulted from Bayesian inference, the new species is located in a separate branch. The distribution map and an illustration of the new species are provided.

A systematic study of North American Angelica species (Apiaceae) based on nrDNA ITS and cpDNA sequences and fruit morphology

Abstract Angelica is a taxonomically complex genus widespread throughout the North Temperate Zone. Previous phylogenetic studies of the genus have focused primarily on its East Asian species. The relationships among its North American members, the monophyly of these species, and the value of fruit morphology in circumscribing its taxa have yet to be examined. This study represents the most comprehensive sampling of Angelica to date (100 species) and includes all 26 species in North America. Relationships are inferred using Bayesian inference, maximum likelihood, and maximum parsimony analyses of ITS sequences and, for multiple accessions of each North American species, cpDNA ndhF‐rpl32, rpl32‐trnL, and psbM‐psbD sequences. The fruit morphological characters examined were those considered phylogenetically important in East Asian Angelica. The results revealed that the North American species fell into three major clades: North American Angelica clade, Archangelica clade, and the Eurasian Angelica clade. Angelica dawsonii has affinities with Lomatium brandegeei. Fourteen species within the North American Angelica clade were strongly supported as monophyletic. Two paraphyletic species resulted in new combinations in A. lineariloba and A. venenosa. Conflict between the ITS‐derived and cpDNA‐derived phylogenies and the lack of resolution in portions of the trees may be due to chloroplast capture and rapid species radiation. Fruit morphology supported some interspecific relationships based on molecular data, and relationships revealed by ITS and cpDNA data were roughly in accordance with fruit classification type and geographic distribution region, respectively. A diagnostic key based on fruit morphology is provided for the identification of the North American Angelica taxa.

<p>A new broad-leaved species of loquat from eastern Myanmar and its phylogenetic affinity in the genus <em>Eriobotrya </em>(Rosaceae)</p>

Eriobotrya shanense, a new species from the karst region of Shan State, eastern Myanmar, is described and illustrated. This evergreen tree is similar to the autumn and winter-flowering, large-leaved species E. malipoensis and E. japonica by having tomentose hairs on the abaxial leaf surface, but can be distinguished by its obovate, widely obelliptic or oval blade shape, leaf length-width ratio of 2:1 (vs. 3:1), and rounded or obtuse apex (vs. acute). The species also resembles less-known, undercollected Myanmar broad-leaved species E. wardii and E. platyphylla in leaf shape and length-width ratio, but can be easily differentiated by the presence of tomentose hairs on the leaves, and inflorescence. Phylogenetic analysis based on nrDNA ITS region supported its close affinity with E. malipoensis and E. japonica. Molecular data also generally grouped the 17 congeneric taxa accessions in congruence to their leaf morphology, with the entire Eriobotrya clade strongly supported to be monophyletic and separate from Rhaphiolepis.

Development of a taxon-discriminating molecular marker to trace and quantify a mycorrhizal inoculum in roots and soils of agroecosystems.

Crop inoculation with Glomus cubense isolate (INCAM-4, DAOM-241198) promotes yield in banana, cassava, forages, and others. Yield improvements range from 20 to 80% depending on crops, nutrient supply, and edaphoclimatic conditions. However, it is difficult to connect yield effects with G. cubense abundance in roots due to the lack of an adequate methodology to trace this taxon in the field. It is necessary to establish an accurate evaluation framework of its contribution to root colonization separated from native arbuscular mycorrhizal fungi (AMF). A taxon-discriminating primer set was designed based on the ITS nrDNA marker and two molecular approaches were optimized and validated (endpoint PCR and quantitative real-time PCR) to trace and quantify the G. cubense isolate in root and soil samples under greenhouse and environmental conditions. The detection limit and specificity assays were performed by both approaches. Different 18 AMF taxa were used for endpoint PCR specificity assay, showing that primers specifically amplified the INCAM-4 isolate yielding a 370bp-PCR product. In the greenhouse, Urochloa brizantha plants inoculated with three isolates (Rhizophagus irregularis, R. clarus, and G. cubense) and environmental root and soil samples were successfully traced and quantified by qPCR. The AMF root colonization reached 41-70% and the spore number 4-128 per g of soil. This study demonstrates for the first time the feasibility to trace and quantify the G. cubense isolate using a taxon-discriminating ITS marker in roots and soils. The validated approaches reveal their potential to be used for the quality control of other mycorrhizal inoculants and their relative quantification in agroecosystems.

Metabolic differences of two Ferula species as potential sources of galbanum: An NMR-based metabolomics study.

Ferula gummosa Boiss. and Ferula galbaniflua Boiss. & Buhse (Apiaceae) are two important Iranian plants that are considered as potential sources of galbanum (barijeh). Galbanum is traditionally used for treating different diseases including flatulence and memory impairment. According to a phylogenetic analysis of the nrDNA ITS sequence and the Flora Iranica, F. gummosa has been considered as a synonym of F. galbaniflua. However, F. galbaniflua and F. gummosa grow in two different geographical locations and have different metabolic patterns. Some researchers believe that F. gummosa and F. galbaniflua are two distinct species. To discriminate these species, we compared metabolic profiles of F. gummosa and F. galbaniflua samples. 1 H-NMR-based metabolomics analysis was used for classification of F. gummosa and F. galbaniflua samples collected from northeast Iran. The acquired data were analyzed using hierarchical cluster analysis (HCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). The result showed a clear separation between the two species that may be related to the quantity and diversity of their metabolites. Ferula gummosa had higher mogoltacin levels, while F. galbaniflua had higher feselol levels. Ligupersin A and conferdione were significantly detected in F. gummosa, whereas sterol compounds were significantly detected in F. galbaniflua. Our findings indicate that clear metabolomics discrimination of F. gummosa and F. galbaniflua makes their chemotaxonomic classification possible.

<p><strong>Molecular phylogeny and systematic evaluation of the <em>Caragana opulens</em> species complex (Fabaceae, Papilionoideae) based on the molecular and morphological data</strong></p>

In this study, we explored the evolutionary history and taxonomic treatment of the Caragana opulens complex taking information from morphological and molecular data. The complex consists of three species, C. opulens, C. licentiana and C. kansuensis. Moreover, the morphological characters currently used to differentiate the species present in the complex have been found insignificant and inconsistent and do not help diagnose the species. For the present study, we investigated its range and sampled 139 accessions from the different populations of the genus Caragana and 17 accessions of the complex. DNA sequence data from one nrDNA ITS and one cpDNA trnH-psbA loci were sequenced and analyzed using Maximum Likelihood and Bayesian methods. The resulting phylogenies were congruent in topologies. Based on morphological and molecular data, it is concluded that all three species of the complex are one of the same with significant morphological variations. Hence C. opulens is accepted as the correct name along with C. licentiana and C. kansuensis as synonyms.

Identification and Genetic Relationship Analysis of <i>Torreya</i> Based on nrDNA ITS Sequence

In order to evaluate the identification ability of nrDNA ITS sequences on Torreya , this study used ITS sequence and four different analysis methods (BLAST, K2P genetic distance, SNP analysis, NJ tree) to identify species of Torreya , and established phylogenetic trees to discuss their phylogenetic relationships. The results showed that the length of 48 ITS sequences was 1 095 bp~1 105 bp, the average intraspecies genetic distance was 0.001 6 and the average interspecies genetic distance was 0.015 0. At the species level, BLAST alignment and NJ tree had the highest efficiency in Torreya . The SNP loci analysis could effectively identify T. grandis cv . ‘ Merrillii ’ and T. grandis . The analysis of genetic relationship showed that all the other species of Torreya were single-line branches, but the quince tree of T. yunnanensis and T. fargesii were clustered into a cluster in the ML tree, indicating that the two were closely related. This provided evidence for the nuclear genome sequence proposed the incorporation of T. yunnanensis and T. fargesii . This study showed that ITS sequence could be used as a DNA barcode for the identification of Torreya , and providing reference for the identification and systematic relationship of Torreya.

Eriobotrya fusca sp. nov. (Rosaceae) from Yunnan province, China

Eriobotrya fusca Kuan ex Gao & Idrees, belonging to the family Rosaceae, a new species from Yunnan Province, China is described and illustrated based on both morphological and molecular data. The new species resembles closely morphologically to E. salwinensis Hand.-Mazz., but differs from it by having elliptic-oblong or oblong-ovate leaves, fewer pairs of lateral veins (12 - 15), lanceolate bracteoles, absent of pedicels, peduncles ca. 10 mm long, both densely brown tomentose, suborbicular petals with emarginate apices, triangular sepals with acute apices, and styles that are connate at their base. The phylogenetic analysis based on nrDNA ITS sequences confirmed the distinctiveness of this new species.

What is Astragalus wiesneri? Disentangling a new species from its relatives in section Anthylloidei

Here, we describe and illustrate a new alpine species of Astragalus sect. Anthylloidei from Iran. Astragalus wiesneri sp. nov., an interesting and rare species, is only known from the type locality in the Bul Mountain (near Eghlid city) in the southern Zagros Mountains. The new species is compared with close relatives, including A. nigrohirsutus and A. lalesarensis, which are also endemic to Iran. In addition, we assembled a dataset of nrDNA ITS sequences of 21 species (22 accessions) representing A. sect. Anthylloidei and ten species of other related spiny-cushion forming sections. Maximum likelihood methods and Bayesian Inference retrieved A. wiesneri sister to A. nigrohirsutus, a species growing in the same area as the new species. The description of A. wiesneri is presented along with a distribution map, some ecological information, field images, conservation status, and pertinent notes.

Two new Bacidina species (Lecanorales, Ascomycota) from Pakistan


 
 
 Bacidina margallensis and B. iqbalii from Pakistan are described and illustrated. Phylogeny of ITS nrDNA region confirms their position within the genus Bacidina, and morphological data make them distinct from other known species of the genus. Based on molecular data, corticolous Bacidina margallensis appears to be a sister species to B. chloroticula, but morphologically, when dry, is the most similar to known from Europe B. mendax because of the granular and warted greenish grey thallus, whitish-cream to dark brown and often piebald apothecia. It differs from that species by shorter, wider and less septate ascospores; 1–3-septate in B. margallensis vs 3–5(6)-septate in B. mendax, and by unusual parrot-green colour of wet thallus. Saxicolous Bacidina iqbalii is closely related to B. neosquamulosa but differs in having crustaceous thallus, transculent when wet entirely pale apothecia, larger asci and less septate large ascospores.
 
 

Phylogenetic study of Silene sections Auriculatae , Spergulifoliae , Ampullatae , and Lasiocalycinae in Iran

Phylogenetic analyses of some sections of Silene (Caryophyllaceae) were presented by using nrDNA ITS and cpDNA rps16 intron sequences. Forty taxa, belonging to the sections Auriculatae, Spergulifoliae, Lasiocalycinae, and Ampullatae, were investigated. The molecular results showed that the species belonging to S. sect. Auriculatae, Spergulifoliae, and Ampullatae were strongly supported as one separated clade. Furthermore, species in S. sect. Lasiocalycinae remain closely related to those in the S. sect. Auriculatae. In this study, a comparative evaluation was performed among these four sections to determine which classification (local flora or recent molecular studies) can be proven. Furthermore, the phylogenetic position of some new species, which have been described and reported from Iran after Melzheimer investigation, were discussed. Some species were nested within S. sect. Auriculatae displayed great affinity.

<p><strong>A new species of <em>Usnea</em> (Parmeliaceae, lichenized Ascomycota) from Southwest China</strong></p>

During the study of Usnea from China, we identified and illustrated a new species, Usnea sulphuridiscoidea S. Y. Guo & L. F. Han from the Ailaoshan Mountain of Southwest China, based on morphological characteristics and nrDNA ITS sequence data. This new species can be distinguished morphologically from other apotheciate members of Usnea by the special lateral apothecia with sulphur disc, the inflated branches with glossy surface and numerous papillae as well as many fibrils, meanwhile lacking pseudocyphellae and soralia. The nrDNA ITS sequence data supported the recognition of the new species.

Species of <i>Odontia</i> and <i>Tomentella</i> (Thelephorales, Basidiomycota) new to Dagestan, Russia

Aim. To obtain new data on the species diversity, phylogenetic structure, and ecological characteristics of thelephoroid fungi (Thelephorales, Basidiomycota) in the Republic of Dagestan.Material and Methods. Both micromorphological and molecular analyses were used for studying of the fungal specimens collected by the authors in 2018-2019 in the Gunibsky and Magaramkentsky Districts of Dagestan. Additional specimens from the Mycological Herbarium of the Komarov Botanical Institute of the Russian Academy of Sciences (LE) were studied. The ITS region of nrDNA was amplified with two pairs of primers, ITS1F/ITS4 and ITS5/ITS4.Results. Sixteen ITS sequences belonging to eight species were obtained from the studied material. Of them, 14 sequences clustered in the Tomentella clade and two sequences nested within the Odontia clade. Four species – Odontia duemmeri, Tomentella lapida, T. radiosa, T. terrestris – were registered for the first time for Dagestan. Detailed information on the specimens studied is presented. Species identification of Odontia fibrosa, Tomentella badia, T. ferruginea, and T. stuposa was confirmed by ITS nrDNA analysis.Conclusion. Data on the species richness of the genera Odontia and Tomentella in Dagestan is updated, and the species T. lilacinogrisea is excluded from the regional funga. To date the genera Odontia and Tomentella in the Republic of Dagestan are represented by three and fifteen species, respectively.