Nuclear Internal Transcribed Spacer Sequences Research Articles

Carpinus austrobalcanica – A new highly polyploid species from the Balkan Peninsula closely related to European hornbeam

The Balkan Peninsula was the primary glacial refugium for many temperate tree species and contributed overproportionally to the postglacial recolonisation of central and eastern Europe. This is also the case for Carpinus betulus, the European hornbeam, whose main glacial refugium was in the Balkans. During our fieldwork in the southern Balkans, we discovered trees with a morphology similar to that of C. betulus, but differing in their rough, highly rigged bark and the shape of their fruit involucre. The aim of this study was to investigate the evolutionary origin and differentiation of these morphologically distinct populations of C. betulus from the North Pindus Mountain range in northern Greece and southern Albania using an integrative approach. Our study combined phylogenetic analyses of plastid and nuclear internal transcribed spacer sequences, amplified fragment length polymorphism fingerprinting, relative genome size estimation, and multivariate morphometric analyses. After establishing the genetic and morphological divergence of the aforementioned populations, we described them as a new species, Carpinus austrobalcanica D.Lakušić, Kuzmanović, Stevanoski, Schönsw. & Frajman, sp. nov. We provide diagnosis, description, geographical distribution, and conservation status of this enigmatic newly described tree species locally endemic to the southern Balkans.

Astragalicola ephedrae sp. nov., isolated from the stem of Ephedra gerardiana in Ladakh, India.

An endophytic fungus designated as EIT4T (MCC 9756T) was isolated from the asymptomatic stem tissue of Ephedra gerardiana collected from the Kargil district of Ladakh Union territory, India. Phylogenetic analysis based on concatenated nuclear ribosomal ITS (internal transcribed spacer) and LSU (large ribosomal subunit) sequence datasets revealed its placement within the genus Astragalicola. However, it formed a separate clade exhibiting strong bootstrap support value (80%). The highest nrITS sequence similarity between EIT4T and species of Astragalicola was 95.19% (A. vasilyevae) and 94.26% (A. amorpha), while nrLSU sequence similarity was 99.27% (A. amorpha). Morphologically, EIT4T differs from the other species of Astragalicola in having larger sub-globose to pyriform conidiomata, smaller and mostly unbranched conidiophores, and polymorphic translucent conidia with two terminal guttules. Based on combined cultural, micromorphological, molecular, and phylogenetic analyses, EIT4T represents a novel species in the genus Astragalicola proposed here as Astragalicola ephedrae sp. nov. Detailed description and illustrations of the novel species are provided. The type strain is EIT4T (= MCC 9756T = MN29T).

Plastome-based phylogeny and biogeography of Lactuca L. (Asteraceae) support revised lettuce gene pool categories.

This study generated and analyzed complete plastome and internal transcribed spacer (ITS) data of 46 Lactuca species, 13 African endemic (AE) Lactuca species, and 15 species from eight related genera in Lactucinae. The new plastome and nuclear ITS sequences were then used to reconstruct the phylogenetic relationships of Lactuca species. The whole-plastome data were used to estimate divergence time and ancestral area reconstruction of the identified major Lactuca lineages. The results showed that Lactuca species are generally similar in plastome size, Guanine and Cytosine (GC) content, gene structure, and categories, although crop lettuce (Lactuca sativa L.) and its gene pool relatives were found to have one unique pseudogene (ψ ndhF), and accD, atpF, cemA, clpP, and rpl22 showed signs of positive selection. Our phylogenomic analysis demonstrated that Lactuca is monophyletic after excluding Lactuca alatipes Collett and Hemsl and AE Lactuca species. AE Lactuca species are morphologically distinct from core Lactuca lineage and need to be excluded from Lactua. The core Lactuca species most likely originated from Asia-Temperate W ~6.82 Mya and then dispersed globally and formed nine clades. Finally, the lettuce gene pool concept was amended according to the phylogenetic and historical biogeographic analyses. This study revised the circumscription of Lactuca, revealed robust phylogenetic relationships within the genus, and provided insights into Lactucinae phylogeny. The lettuce gene pool species could be used as potential genetic resources for lettuce breeding.

Molecular and morphological analyses to improve taxonomic classification ofMetridia lucens/pacificain the North Pacific

AbstractMolecular and morphological analyses were used to resolve the taxonomy of Metridia lucens/pacifica in the North Pacific. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunit I (mtCOI) revealed two lineages of M. lucens and M. pacifica with an average sequence difference of 13.0%, which were supported by variations in nuclear internal transcribed spacer sequences. Metridia pseudopacifica in the eastern Pacific, mentioned by Mackas and Galbraith (2002), was included in M. lucens. The presence of M. lucens and M. pacifica was confirmed across the North Pacific, and different genetic population structures were suggested between the two species in the North Pacific based on mtCOI sequences. The morphological identification character of length of setae on the fifth pair of legs was not useful for classifying adult female M. pacifica and M. lucens. There were regional variations in prosome length and head angle; these two characters successfully classified >98.2% of M. pacifica and M. lucens specimens from the North Pacific. As M. lucens and M. pacifica are key copepods in the North Pacific, accurate classification of individuals should be made in future studies to understand their relative contributions to marine ecosystems and improve detection of ecosystem change.

Diversification of Cynoglossinae genera (Cynoglosseae-Boraginaceae) in the western Irano–Turanian bioregion

The Irano–Turanian (I–T) bioregion harbours one of the Old World’s greatest repositories of botanical diversity; however, the diversification patterns and the phenotypic evolution of its flora are sorely understudied. The subtribe Cynoglossinae is characteristic of the western I–T bioregion, species-rich in both the desertic lowlands and the more mesic highlands of the Iranian plateau. About 70 species of Cynoglossinae are present in the Iranian plateau, 47 of which are endemic to the plateau. Herein, nuclear internal transcribed spacer sequences as well as cpDNA rpl32-trnL and trnH–psbA sequences were used to investigate the molecular phylogeny, historical biogeography, and ancestral character states of Cynoglossinae. Molecular dating and ancestral range reconstruction analyses indicated that the subtribe Cynoglossinae has initiated its diversification from the eastern part of the western I–T during the mid-Miocene, concomitantly with the uplift of the Pamir and Hindu Kush mountains. Moreover, from the Pliocene onwards, the Afghan–India collision and extensive deformation of the Arabia–Eurasia convergence likely promoted allopatric speciation in Cynoglossinae via mostly vicariance events. Evolution of annuals with small nutlets from perennials with large nutlets was accompanied by mesic to desert habitat shifts. Herein, to explain distribution of Cynoglossinae in the western I–T, the congruence between cladogenetic, geological, and palaeoclimatic events was investigated.

Plastome phylogeny and lineage diversification of Salicaceae with focus on poplars and willows.

Phylogenetic relationships and lineage diversification of the family Salicaceae sensu lato (s.l.) remain poorly understood. In this study, we examined phylogenetic relationships between 42 species from six genera based on the complete plastomes. Phylogenetic analyses of 77 protein coding genes of the plastomes produced good resolution of the interrelationships among most sampled species and the recovered clades. Of the sampled genera from the family, Flacourtia was identified as the most basal and the successive clades comprised both Itoa and Poliothyrsis, Idesia, two genera of the Salicaceae sensu stricto (s.s.) (Populus and Salix). Five major subclades were recovered within the Populus clade. These subclades and their interrelationships are largely inconsistent with morphological classifications and molecular phylogeny based on nuclear internal transcribed spacer sequence variations. Two major subclades were identified for the Salix clade. Molecular dating suggested that species diversification of the major subclades in the Populus and Salix clades occurred mainly within the recent Pliocene. In addition, we found that the rpl32 gene was lost and the rps7 gene evolved into a pseudogene multiple times in the sampled genera of the Salicaceae s.l. Compared with previous studies, our results provide a well‐resolved phylogeny from the perspective of the plastomes.

Molecular phylogeny of the genus Corchorus (Grewioideae, Malvaceae s.l.) based on nuclear rDNA ITS sequences

A molecular phylogenetic analysis of the genus Corchorus (Grewioideae, Malvaceae s.l.) is presented, based on sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 144 accessions representing 47 species. Several other genera from the subfamily Grewioideae, namely Pseudocorchorus, Triumfetta, Sparrmannia, Entelea, and Grewia, were included as outgroups. The monophyly of the genus was well supported by all phylogenetic analyses (maximum likelihood, Bayesian approaches, and parsimony), and Corchorus was divided into four major clades. The majority of African species formed a statistically highly supported and distinct clade separated from the other pantropically distributed species. Several endemic species from Australia, New Caledonia, and tropical America were nested within this distinct clade, indicating dispersal from Africa to the rest of the pantropics. Based on the taxa included in this study, the two cultivated species (C. olitorius and C. capsularis) shared a common ancestry with wild species of C. africanus, C. brevicornatus, C. pseudocapsularis, C. pseudo-olitorius, C. urticifolius, C. pilosus, C. orinocensis, and C. cunninghamii. Pseudocorchorus, previously considered an accepted genus, was nested within the genus Corchorus and shared a common ancestry especially with C. depressus and C. siliquosus. Based on morphological and anatomical similarity as well as the results of the present molecular findings, inclusion of the six Pseudocorchorus species into Corchorus is proposed, with Pseudocorchorus as a synonym of Corchorus. Of the included outgroup taxa, Triumfetta is the closest sister to Corchorus, while the common ancestor of Corchorus/Pseudocorchorus, Triumfetta, Sparrmannia, and Entelea is Grewia. A further phylogenetic study with more taxa mainly from Australia, together with additional molecular markers and morphological investigation, would help to test the hypothesis on the biogeography and growth form evolution of the genus Corchorus.

Development of novel simple sequence repeat markers from ramie (Boehmeria nivea L. Gaudich) and analysis of genetic diversity in its genetic resources

Boehmeria nivea (ramie), a subshrub of the Urticaceae family and an important fiber crop, has been grown in Asian countries, including Korea, for many centuries. Traditionally, in some regions of Korea, ramie leaves are also used as a major ingredient in ‘Songpyeon’ rice cakes. Despite its economic importance, the molecular genetics of ramie have not yet been studied in detail. Researchers from Yeong-Gwang Agricultural Technology Center collected genetic resources of ramie from a variety of local sites in Korea. Nuclear internal transcribed spacer sequences amplified from 90 genetic resources showed no variation in size or sequence between them or B. nivea reference samples, indicating that all the genetic resources could be taxonomically classified as B. nivea. To systematically and efficiently manage these genetic resources, we developed simple sequence repeat (SSR) markers for ramie using the magnetic bead hybridization selection method. Finally, we selected 17 SSR markers that showed possible polymorphism among the genetic resources and analyzed the genetic diversity of ramie collection. These 17 markers detected a total of 140 alleles, ranging from 3 to 17 alleles per accession. The average genetic diversity value was 0.61, ranging from 0.37 to 0.84. Average polymorphism information content was 0.56, ranging from 0.34 to 0.82. Genetic cluster analysis based on SSR data revealed that the genetic collection of ramie plants could be classified into four distinct clusters. A purity test on several of the genetic resources using polymorphic SSR markers revealed that some resources were impure. These newly developed SSR markers might be useful for diverse genetic analysis including the management of genetic collections, establishment of core collections, and cultivar identification.

Natural hybridization between Torreya jackii and T. grandis (Taxaceae) in southeast China

AbstractThe recognition of natural hybridization can help to understand the origin and maintenance of biodiversity. Based on intermediate morphology and overlapped distribution, Torreya grandis var. jiulongshanensis Z. Y. Li, Z. C. Tang & N. Kang was regarded as a putative natural hybrid between T. jackii Chun and T. grandis Fort. ex Lindl. in southeast China. This study tested the hybridization origin of T. grandis var. jiulongshanensis using nuclear internal transcribed spacer (ITS) and cytoplasm (chloroplast rbcL and rpl16 and mitochondrial rps3) DNA sequences. The results showed that most heterozygous sites of nuclear ITS sequences in T. grandis var. jiulongshanensis showed additivity between T. jackii and T. grandis. Cloned ITS sequences of T. grandis var. jiulongshanensis separately clustered with T. jackii and T. grandis in the phylogenetic analyses. Meanwhile, all of chloroplast and mitochondrial haplotypes of T. grandis var. jiulongshanensis were identical to those of T. jackii, not T. grandis. On the chloroplast DNA phylogenetic trees, T. grandis var. jiulongshanensis were grouped into the clade of T. jackii. These results suggest that T. grandis var. jiulongshanensis is a natural hybrid between T. jackii (maternal parent) and T. grandis (paternal parent). This study also implies that T. grandis should be cultivated with caution near the sites of T. jackii, because the endangered status of the latter might be deteriorated due to hybridization.

Molecular systematics of southern African monkey orange Strychnos L. (Loganiaceae)

Strychnos is the largest genus of the Loganiaceae with about 200 species distributed across Africa (including Madagascar), the Americas, Australia and Asia. Recent molecular phylogenetic effort at elucidating relationships globally provided a useful overview for the genus, based on the internal transcribed spacer (ITS) data. However, an understanding of evolutionary and ecological patterns at regional levels is better served by fine scale phylogenetic analysis to resolve species complexes for conservation and allied reasons. In this study, we use plastid (trnL-trnF, trnS-trnG) and nuclear ribosomal ITS sequence data to infer phylogenetic patterns among members of southern Africa Strychnos. We also evaluate sectional validity of the current classification for African members with ITS sequence data. Our findings support the monophyly of Strychnos, although several of the sections are not monophyletic, thus raising the need for sectional reappraisal of the genus. S. xantha is sister to our expanded representation of southern African taxa, while S. aculeata is sister to all African taxa. The uncertain relationships among S. innocua, S. madagascariensis and S. gerrardii were partly resolved in the phylogenetic analysis of combined datasets. S. innocua is sister to the other two species in a well-supported clade. S. gerrardii and S. madagascariensis are also sister taxa that are not yet reciprocally monophyletic, but possess other features to distinguish them. The trnS-trnG marker revealed a relatively large indel event of potential taxonomic value in section Densiflorae. There is also a clear ecological signal in both the plastid and nuclear datasets, as both consistently placed forest species at the base of the phylogeny, and savanna species in more derived positions. We addressed the adaptive significance of such signals as they relate to fruit and growth forms, and submit proposals towards a comprehensive sectional revision of African Strychnos.

Phylotranscriptomic analyses in plants using Betulaceae as an example

AbstractWith the advance of next‐generation sequencing techniques, phylogenetic analyses based on genomic data are becoming frequent in plants. However, the efficacy of different methods used to retrieve orthologs for phylogenetic studies based on transcriptome sequences is rarely compared and examined for closely related species with low genetic differentiation. In this study, we used both low‐copy gene‐based approaches and mapping‐based approaches to construct a phylogeny of Betulaceae. We found that both approaches showed no distinct differences in phylogeny construction and associated bootstrap support, although mapping‐based methods seemed to be superior to the former in all analyses particularly when the summed length of orthologs was not long enough. Phylogenetic relationships within the family, largely consistent with those previously based on chloroplast DNA and nuclear internal transcribed spacer sequence variations, received high support for all clades and subclades. However, we found that support values for the sister relationship between two polyploid genera were lower than those between diploid groups if only a few orthologs were sampled. This may result from ortholog misidentification due to genome duplications.

Phylogenetic relationships among Elymus and related diploid genera (Triticeae: Poaceae) based on nuclear rDNA ITS sequences

AbstractTo investigate the phylogenetic relationships among Elymus and related diploid genera, the genome donor of Elymus, and the evolutionary history of polyploid Elymus species, nuclear ribosomal internal transcribed spacer (ITS) sequences were analyzed for 10 Elymus species, together with 17 diploid taxa from 5 monogenomic genera. The phylogenetic analyses (Neighbor-Joining) supported two major clades (St and H). Sequence diversity and genealogical analysis suggested that (1) Elymus species were unambiguously closely related to Pseudoroegeria; (2) Pse. stipifolia might be serve as the St genome donor of polyploid Elymus species; (3) the Y genome might be originated from ancestral lineage of Pseudoroegneria (St); (4) the ITS sequences of Elymus were evolutionarily distinct and may clarify parental lineages and phylogenetic relationships in Elymus.

Molecular phylogenetic analysis ofTulipa(Liliaceae) based on noncoding plastid and nuclear DNA sequences with an emphasis on Turkey

We investigated the phylogenetic relationships in Tulipa in Turkey using DNA sequences from the plastid trnL-trnF region and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. We generated trnL-trnF and nuclear ITS sequences for 11 Tulipa spp. from Turkey and compared the utility of trnL-trnF and ITS sequences for phylogenetic analysis. Neighbor-joining, Bayesian and maximum parsimony methods were implemented using the same matrices. Our study of Tulipa based on molecular data revealed congruent results with previous studies. Despite the relatively lower resolution of trnL-trnF than that of ITS, both sequence matrices generated similar results. Three clades were clearly distinguished, corresponding to subgenera Tulipa, Eriostemones and Orithyia. It is not fully resolved whether Clusianae should be recognized as a separate section of subgenus Tulipa or a distinct subgenus. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 270–279.

Kalinia, a New North American Genus for a Species Long Misplaced in Eragrostis (Poaceae, Chloridoideae)

Eragrostis obtusiflora (Poaceae, Chloridoideae), a species distributed from the southwestern United States to central Mexico, has long been recognized for exhibiting morphological and anatomical features atypical of Eragrostis. Phylogenetic analyses of nuclear internal transcribed spacer sequences and plastid sequences demonstrate that E. obtusiflora should be excluded from Eragrostis (Eragrostideae) and instead be placed in Cynodonteae, although its position within this tribe was unresolved. Additional data, including anatomical and micromorphological characters, suggest a close relationship with Distichlis. However, differences in spikelet and rhizome characters prevent its inclusion in Distichlis. Therefore, the species is transferred to a newly described genus, Kalinia, as K. obtusiflora.

Genetic diversity and phylogenetic analysis of genus Paeonia based on nuclear ribosomal DNA ITS sequence

The genus Paeonia belongs to the family Paeoniaceae having significant medicinal and ornamental importance. The present investigation was undertaken with an aim to understand phylogenetic relationships of three Paeonia species (P. lactiflora, P. obovata, and P. suffruticosa) that are widely distributed in China, Korea, and Japan, using nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) sequence and to compare the phylogeny results with investigations reported earlier using existed sequences of the same species. The size variation obtained among sequenced nrDNA ITS region was narrow and ranged from 722 to 726 bp. The highest interspecific genetic distance (GD) was found between P. lactiflora and P. suffruticosa or P. obovata. The phylogram obtained using our nrDNA ITS sequences showed non-congruence with previous hypothesis of the phylogeny between section Paeonia and section Moutan of genus Paeonia. This result was supported by the phylogenetic relations showed in the phylogram constructed with existed sequences in NCBI. The present study suggested that P. obovata belonging to section Paeonia was phylogenetically closer to P. suffruticosa representing section Moutan of genus Paeonia than P. lactiflora belonging to section Paeonia. The main reason of the paraphyly of section Paeonia is thought to be nucleotide additivity directly caused by origin hybridization. This study provides more sequence sources of genus Paeonia, and will help for further studies in intraspecies population, and their phylogentic analysis and molecular evolution.

Phylogeny of Korean Hornbeam ( Carpinus turczaninovii ) based on nuclear ribosomal ITS sequence

The genus Carpinus belonging to Coryloideae, Betulacea e, has significant economic and ornamental importance. This study was undertaken with the aim to understand the genetic diversity among eighteen isolates of Carpinus turczaninovii collected from different geographical regions of Korea, using ribosomal RNA (rRNA) internal transcribed spacer (ITS) sequences, to compare the infraspecificphylogenetic relationships among C. turczaninovii in Korea, and some known Carpinus plants. The size variation of sequenced rRNA ITS regions was not seen, with 215, 162, 222 bp of ITS1 region, 5.8S rRNA gene, ITS2 region, respectively. However, some certain nucleotide variations resulted in genetic diversity. In the genus Carpinus, C. turczaninovii closely genetic with Castanopsis kawakam ii, Carpinus orientalis, Carpinus monbeigiana , and Calyptranthes polynenra formed one monophyletic clade, while Carpinus betulus and Carpinus laxiflora , respectively formed one monophyletic clade. In the present ITS sequence analysis, Carpinus fangiana, Carpinus coreana, Carpinus japonica , and Carpinus caroliniana were considered as Out Group, compared to C. turczaninovii group. The highest intraspecific variation was found between C. caroliniana and C. japonica. The least intraspecific variation was obtained between C. turczaninovii and C. coreana . The results will help further understanding Carpinus infraspecies population and their phylogenetic analysis. Key words: Carpinus, Hornbeam, Carpinus turczaninovii, molecular identification, nuclear ribosomal RNA, internal transcribed spacer, infraspecific-phylogenetic relationship.

Phylogenetic analysis of eastern Asian and eastern North American disjunct Lespedeza (Fabaceae) inferred from nuclear ribosomal ITS and plastid region sequences

Lespedeza (tribe Desmodieae, Fabaceae) follows a disjunct distribution in eastern Asia and eastern North America. Phylogenetic relationships among its species and related taxa were inferred from nuclear ribosomal internal transcribed spacer (ITS) and plastid sequences (trnH-psbA, psbK-psbI, trnK-matK and rpoC1). We examined 35 species of Lespedeza, two of Kummerowia and one of Campylotropis, the sole constituents of the Lespedeza group. An analysis of these data revealed that the genus Campylotropis is sister to the other two genera. However, we were unable to resolve the relationships between Kummerowia and Lespedeza in the strict consensus trees of parsimony analyses based on plastid and combined DNA data. In the genus Lespedeza, the Old World subgenus Macrolespedeza is monophyletic, whereas the transcontinental subgenus Lespedeza is paraphyletic. Monophyly of eastern Asian species and of North American species is strongly supported. Although inconsistent with the traditional classification, this phylogenetic finding is consistent with seedling morphology. Three subgroups recognized in subgenus Macrolespedeza were unresolved in our phylogenetic trees. An incongruence length difference (ILD) test indicated that the two partitions (nuclear ITS and plastid sequences) were significantly incongruent, perhaps because of hybridization between species in Lespedeza. Most of the primary clades of tribe Desmodieae are Asian, implying that the relatively few New World ones, such as those in Lespedeza, are more recently derived from Asia. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 164, 221–235.

Development of ITS sequence based molecular marker to distinguish, Tribulus terrestris L. (Zygophyllaceae) from its adulterants

Tribulus terrestris L. (Zygophyllaceae) is one of the highly traded raw drugs and also used as a stimulative food additive in Europe and USA. While, Ayurvedic Pharmacopoeia of India recognizes T. terrestris as Goksura, Tribulus lanuginosus and T. subramanyamii are also traded by the same name raising issues of quality control . The nuclear ribosomal RNA genes and ITS (internal transcribed spacer) sequence were used to develop species-specific DNA markers. The species-specific markers efficiently amplified 295 bp for T. terrestris (TT1F and TT1R), 300 bp for T. lanuginosus (TL1F and TL1R) and 214 bp for T. subramanyamii (TS1F and TS1R). These DNA markers can be used to distinguish T. terrestris from its adulterants.

Polymorphism hides cryptic species in Oerstedia dorsalis (Nemertea, Hoplonemertea)

The marine hoplonemertean Oerstedia dorsalis is considered to be a highly polymorphic species with extensive geographic distribution. We show, based on mitochondrial cytochrome c oxidase subunit I, 16S and nuclear internal transcribed spacer sequences, that there are genetic subsets withing this species. Seventy-one specimens of various colours from different geographic localities (in Europe) were sequenced and analysed using statistical parsimony and Bayesian analysis. Both analyses supported nine major clades. We conclude that O. dorsalis hides different species with geographic resolution. These species, however, appear to be polymorphic as well, and we find no diagnostic features in pigmentation or external characters to separate species within this complex. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 556–567.

Phylogenetic relationships of species in Pseudoroegneria (Poaceae: Triticeae) and related genera inferred from nuclear rDNA ITS (internal transcribed spacer) sequences

To evaluate the phylogenetic relationships of species in Pseudoroegneria and related genera, the nuclear ribosomal internal transcribed spacer (ITS) sequences were analyzed for eighteen Pseudoroegneria (St), two Elytrigia (EeSt), two Douglasdeweya (StP), three Lophopyrum (Ee and Eb), three Agropyron (P), two Hordeum (H), two Australopyrum (W) and two Psathyrostachys (Ns) accessions. The main results were: (i) Pseudoroegneria gracillima, P. stipifolia, P. cognata and P. strigosa (2x) were in one clade, while P. libanotica, P. tauri and P. spicata (2x) were in the other clade, indicating there are the differentiations of St genome among diploid Pseudoroegneria species; (ii) P. geniculata ssp. scythica, P. geniculata ssp. pruinifera, Elytriga caespitosa and Et. caespitosa ssp. nodosa formed the EeSt clade with 6-bp indel in ITS1 regions; and (iii) Douglasdeweya wangii, D. deweyi, Agropyron cristatum and A. puberulum comprised the P clade. It is unreasonable to treat P. geniculata ssp. scythica and P. geniculata ssp. pruinifera as the subspecies of P. geniculata, and they should be transferred to a new genus Trichopyrum, which consists of species with EeSt genomes. It is also suggested that one of the diploid donor of D. wangii and D. deweyi is derived from Agropyron species, and it is reasonable to treat tetraploid species with StP genomes into Douglasdeweya.