| The Qinghai-Tibetan Plateau (QTP) is known as "the roof to the world" and "the third pole of the earth". The QTP and its adjacent regions together comprise one of the centers for global biodiversity and a major hotspot for species delimitation, speciation, and biodiversity, supporting numerous endemic species. Orinus Hitchcock is an alpine endemic genus to the QTP and its adjacent regions in the grass family (Poaceae). This genus is the key or unique taxa growing under extremely sandy habitats in these regions. Orinus is of the great importance in the ecological restoration and potential usages in breeding new genetic resources. Since Orinus was established, six species were described for this genus, i. e.O. thoroldii, O. anomala, O. kokonorica, O. tibeticus, O. alticulmus, and O. longiglumis. However, the boundary and taxonomic treatments of these species remain disputed and their genetic variations between and within species have never been reported. So it provides a good model to study the species delimitation and speciation of new species under the extremely sandy and alpine habitats. On the other hand, the study of genetic diversity, genetic structure and intra-specific genetic differentiation for Orinus is till blank. Hereby, in this study, we conduct an extensive field exploration for the total genus and then delimitate species boundary based on studies in morphological statistics and DNA barcoding markers at the population level to answer these unsolved questions. We aim to provide a good and typical example for studying the species delimitation and speciation in the QTP regions. Simultaneously, genetic diversity, genetic structure and intra-specific genetic differentiation of this genus were surveyed using AFLP molecular markers, and scientific foundations were provided for the protection and utilization of Orinus germplasm resources. The main results and conclusions are summarized as follows.(1) Morphological variations and species delimitation within this genusThe presence/absence of hairs covering lemmas, glumes, leaf sheaths and leaf blades, spikelet color, and presence/absence of rhizome scales were widely viewed as the main morphological characters for interspecific delimitation of Orinus species. These above external morphological characters vary greatly among and within populations of the three previously recognized Orinus species including O. anomala, O. kokonorica and O. alticulmus. Equally, the external morphological ones also vary greatly among and within populations of the tentative O. thoroldii, O. tibeticus and O. longiglumis species. However, those characters are stable between these two groups, A total of 673 individuals from 90 geographical populations of the six previously recognised Orinus species described before were sampled for morphologically statistic analysis. A principle component analysis (PCA) based on the thirteen parameters of external morphological characters with larger classified value, i. e. plant height, ratio of leaf length and leaf width, presence/absence of hairs covering leaf blades and sheaths, panicle length, spikelet length and color, floret number per spikelet of the almost whole inflorescence, upper glume length and presence/absence of hairs covering upper glumes, the lowest lemma length and presence/absence of lemma hairs, presence/absence of rhizome scales, identified three distinct groups. The first group consisted of 362 individuals from 41 populations of the three species (O. anomala, O. kokonorica, and O. alticulmus). Within this group, the sampled individuals were plotted as a continuous distribution without distinct subgroups corresponding to any species or geographical populations. The second group was made up of 216 individuals from 33 populations of the three previously recognized species including O. thoroldii, O. tibeticus and O. longiglumis. No subgroups form within this group just like the first group. And the third group, being isolated distinctly from the above two groups by having scaleless rhizomes and 5-9 florets per spikelet, comprised 95 individuals from 16 populations of O. anomala and O. thoroldii distributed in the southeast of the QTP. Based on these results, we contended that the six previously recognized species should be treated as only three species, O. thoroldii, O. anomala and O. intermedia. Namely, O. kokonorica and O. alticulmus should be reduced to O. anomala, and they are regarded as the synonymy of O. anomala. Similarly, O. tibeticus, O. longiglumis and O. thoroldii should belong to the same species, and O. tibeticus and O. longiglumis can be reduced to synonymy of O. thoroldii. Especially, the third groups should be treated as a separate new species (O. intermedia).(2) DNA barcoding and species delimitation within this genusBased on the different types of DNA barcoding fragments (rbcL, matK., trnH-psbA, nrITS), the Orinus species were delimitated in this article. The results showed whether any single chloroplast DNA barcoding fragment or three chloroplast DNA barcoding combined fragment always indicated low level of species discrimination rate for Orinus species. On the contrary, the nuclear ribosomal internal transcribed sequence (nrITS) always shows low level of species discrimination rate for Orinus species. Based on it, three Orinus species delimitated by morphology can completely be distinguished, which suggested that nrITS is a useful fragment as a core barcode to delimitate the closely-related species. The combination of any single cpDNA barcoding fragment and nrITS fragment had higher resolution for identifying Orinus species, especially for trnH-psbA+nrITS and rbcL+nrITS, whose discrimination rate can reach up to 100% and which strictly divides the Orinus species into three groups.Phylogenetic inference and network based on cpDNA and nrITS data indicated that all the Orinus species formed a well-supported monophyletic clade. Specifically, the six previously recognized Orinus species clustered into two stable and well-supported clades I & clade â…¡ on the basis of the combined cpDNA data. The clade I constituted by O. anomala, O. kokonorica, O. alticulmus distributed in the east of the QTP, and O. anomala, O. thoroldii distributed in the southeast of the QTP. The clade II consisted of O. thoroldii, O. tibeticus, and O. longiglumis distributed in the west of the QTP. In the same way, the six previously recognized Orinus species strictly clustered into four stable and well-supported clades I-IV based on nrITS data. Thereinto, the component of clade IV is completely same with clade II divided by cpDNA data. The clade I is made up of O. anomala, O. kokonorica, O. alticulmus distributed in the east of the QTP. The clade II was made up of O. anomala and O. thoroldii originated from the west of Sichuan province. The clade III was composed of two populations of O. thoroldii from Qamdo of Tibet. There were obvious superposition loci between clade â…¡ & â…¢ and clade â… & â…£. Particularly, there were seven shared mutant nucleotides between clade â…¡ & â…¢ and clade I, while there were only two shared mutant nucleotides between clade â…¡ & â…¢ and clade IV. Furthermore, five unique and shared mutant nucleotides existed between clade â…¢ and clade IV. So the clade II and clade III should be treated as a monophyletic, rather than paraphyletic taxa based on the above results and conclusions from morphological studies. Therefore, the results of species delimitation of Orinus species according to DNA barcoding are consistent with the conclusions obtained through the morphological classification.(3) Genetic diversity and genetic structure of this genusThe eight pairs of selective primers with high polymorphism were filtered from 64 pairs of AFLP primer combinations. These eight pairs of primers were employed to make the AFLP amplification of 98 individuals from 20 populations of O. thoroldii,94 individuals from 19 populations of O. anomala, and 39 individuals from 9 populaitons of O. intermedia, which were delimitated based on morphological characters and DNA barcodes, totaling 231 individuals from 48 populations. The results showed that among three Orinus species, total genetic diversity of O. intermedia is the highest, and that of O. thoroldii is the lowest, while that of O. anomala is between them. The FST value of genetic differentiation coefficient is 0.45657 between O. thoroldii and O. anomala, and that of genetic differentiation coefficient is 0.44090 between O. thoroldii and O. intermedia, while that of genetic differentiation coefficient is 0.34960 between O. anomala and O. intermedia. Thus, we thought that there was significant genetic differentiation among three species. Meanwhile, analysis of molecular variance (AMOVA) indicated that the genetic variation within the populations was higher than that among the populations. In detail, the genetic variation was 30,04% among three species, that among the populations was 13.31%, and that within the populations was 56.65%. Likewise, STRUCTURE analyses revealed that there was a significant genetic differentiation among the three Orinus species. In addition, UPGMA cluster analyses indicated that the 48 populations of Orinus species were divided into three clades when similarity coefficient of simple matching was equal to 0.776. These results further support the accuracy and precision of Orinus species delimitation based on morphology and DNA barcodes.
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