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To better understand the responses of arid-adapted, alpine plants to Quaternary climatic oscillations, we investigated the genetic variation and phylogeographic history of Orinus, an endemic genus of Poaceae comprising three species from the dry grasslands of the Qinghai-Tibet Plateau (QTP) in China. We measured the genetic variation of 476 individuals from 88 populations using three maternally inherited plastid DNA markers (matK, rbcL and psbAtrnH), the biparentally inherited nuclear ribosomal internal transcribed spacer (nrITS) and amplified fragment length polymorphisms (AFLPs). We found that the plastid DNA, nrITS and AFLPs show considerable, recent differentiation among the species. We detected 14 plastid haplotypes (H1-H14), of which only three were shared among all species, and 30 nrITS ribotypes (S1-S30), of which one (S10) was shared between two species, O. kokonoricus and O. intermedius, but absent in O. thoroldii. The nrITS types formed clades that were inconsistent with species boundaries. Based on these data, we propose and illustrate a complex hypothesis for the evolutionary history of Orinus involving lineage sorting and introgression, the latter of which may explain the shared S10 nrITS type. The AFLP results showed clades corresponding to current species delineation and suggest that lineage sorting in the genus is probably complete. We estimated the crown age of Orinus to be 2.85 (95% highest posterior density: 0.58-12.45) Mya (late Pliocene), and subsequent divergence occurred in the Quaternary. Early divergences were allopatric. More recently, Orinus probably underwent regional expansions corresponding to Quaternary climatic changes, especially glaciation, which is consistent with our divergence time estimates. These climatic changes could have facilitated the S10 event and other hybridization events. Our data also suggest that species of this small genus of grasses survived the Quaternary glacial period in the extremely adverse habitats of the QTP. [ABSTRACT FROM AUTHOR]

* Background and Aims Accurate identification of species is essential for the majority of biological studies. However, defining species objectively and consistently remains a challenge, especially for plants distributed in remote regions where there is often a lack of sufficient previous specimens. In this study, multiple approaches and lines of evidence were used to determine species boundaries for plants occurring in the Qinghai-Tibet Plateau, using the genus Orinus (Poaceae) as a model system for an integrative approach to delimiting species. * Methods A total of 786 individuals from 102 populations of six previously recognized species were collected for niche, morphological and genetic analyses. Three plastid DNA regions (matK, rbcL and trnH-psbA) and one nuclear DNA region [internal transcribed space (ITS)] were sequenced. * Key Results Whereas six species had been previously recognized, statistical analyses based on character variation, molecular data and niche differentiation identified only two well-delimited clusters, together with a third possibly originating from relatively recent hybridization between, or historical introgression from, the other two. * Conclusions Based on a principle of integrative species delimitation to reconcile different sources of data, the results provide compelling evidence that the six previously recognized species of the genus Orinus that were examined should be reduced to two, with new circumscriptions, and a third, identified in this study, should be described as a new species. This empirical study highlights the value of applying genetic differentiation, morphometric statistics and ecological niche modelling in an integrative approach to re-circumscribing species boundaries. The results produce relatively objective, operational and unbiased taxonomic classifications of plants occurring in remote regions. [ABSTRACT FROM AUTHOR]