Multiple lines of evidence supports the two varieties of Halenia elliptica (Gentianaceae) as two species.

Delimiting species requires multiple sources of evidence. Here, we delimited two varieties of Halenia elliptica (Gentianaceae) using several lines of evidence, including morphological traits and mating system in a sympatric population, phylogenetic relationships based on nrITS and cpDNA (rpl16) data, and complete chloroplast genome sequences. Comparative analysis of 21 morphological traits clearly separates the two varieties of H. elliptica. Examination of the flowering process and pollination treatments indicate that H. elliptica var. grandiflora produces seeds via outcrossing, whereas H. elliptica var. elliptica produces seeds via mixed mating. Furthermore, hand-pollinated hybridization of the two varieties produced no seeds. Observations of pollinators showed that when bees began a pollination bout on H. elliptica var. grandiflora they preferred to continue pollinating this variety; however, when they began a pollination bout on H. elliptica var. elliptica, they showed no preference for either variety. Phylogenetic analysis confirmed the monophyly of H. elliptica, which was further divided into two monophyletic clades corresponding to the two varieties. A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H. elliptica. We recommend that the two varieties of H. elliptica should be revised as two species (H. elliptica and H. grandiflora). Our findings indicate that H. elliptica varieties may have split into two separate species due to a shift in mating system, changes in flowering phenology and/or post-pollination reproductive isolation.

Evolutionary history of a desert perennial Arnebia szechenyi (Boraginaceae): Intraspecific divergence, regional expansion and asymmetric gene flow.

The complex interactions of historical, geological and climatic events on plant evolution have been an important research focus for many years. However, the role of desert formation and expansion in shaping the genetic structures and demographic histories of plants occurring in arid areas has not been well explored. In the present study, we investigated the phylogeography of Arnebia szechenyi, a desert herb showing a near-circular distribution surrounding the Tengger Desert in Northwest China. We measured genetic diversity of populations using three maternally inherited chloroplast DNA (cpDNA) fragments and seven bi-paternally inherited nuclear DNA (nDNA) loci that were sequenced from individuals collected from 16 natural populations across its range and modelled current and historical potential habitats of the species. Our data indicated a considerably high level of genetic variation within A. szechenyi and noteworthy asymmetry in historical migration from the east to the west. Moreover, two nuclear genetic groups of populations were revealed, corresponding to the two geographic regions separated by the Tengger Desert. However, analysis of cpDNA data did not show significant geographic structure. The most plausible explanation for the discrepancy between our findings based on cpDNA and nDNA data is that A. szechenyi populations experienced long periods of geographic isolation followed by range expansion, which would have promoted generalized recombination of the nuclear genome. Our findings further highlight the important role that the Tengger Desert, together with the Helan Mountains, has played in the evolution of desert plants and the preservation of biodiversity in arid Northwest China.

Climatic Changes and Orogeneses in the Late Miocene of Eurasia: The Main Triggers of an Expansion at a Continental Scale?

Migrations from the Qinghai-Tibetan Plateau (QTP) to other temperate regions represent one of the main biogeographical patterns for the Northern Hemisphere. However, the ages and routes of these migrations are largely not known. We aimed to reconstruct a well-resolved and dated phylogeny of Hippophae L. (Elaeagnaceae) and test hypothesis of a westward migration of this plant out of the QTP across Eurasian mountains in the Miocene. We produced two data matrices of five chloroplast DNA (cpDNA) and five nuclear DNA markers for all distinct taxa of Hippophae. These matrices were used to reconstruct phylogenetic relationships in the genus. In dating analyses, we first estimated the stem node age of Elaeagnaceae using five fossil records evenly distributed across a tree of Rosales. We used this estimate and two fossil records to calibrate the cpDNA and nDNA phylogenies of Hippophae. The same phylogenies were used to reconstruct ancestral areas within the genus. The monophyly of Hippophae, all five species, and most of subspecies was strongly supported by both plastid and nuclear data sets. Diversification of Hippophae likely started in central Himalayas/southern Tibet in the early Miocene and all extant distinct species had probably originated by the middle Miocene. Diversification of Hippophae rhamnoides likely started in the late Miocene east of the QTP from where this species rapidly expanded to central and western Eurasia. Our findings highlight the impact of different stages in uplift of the QTP and Eurasian mountains and climatic changes in the Neogene on diversification and range shifts in the highland flora on the continent. The results provide support to the idea of an immigration route for some European highland plants from their ancestral areas on the QTP across central and western mountain ranges of Eurasia in the late Miocene.

Development and characterization of novel EST-SSR markers for Speranskia tuberculata (Euphorbiaceae).

PREMISE OF THE STUDY: The first set of expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed and characterized for Speranskia tuberculata (Euphorbiaceae), a traditional medicinal plant endemic to northern China, to explore the effects of recent habitat fragmentation on the genetic diversity and structure of this species. METHODS AND RESULTS: In this study, a total of 18 novel polymorphic microsatellite (EST-SSR) markers were developed for S. tuberculata using high-throughput transcriptome sequencing. Analysis of 24 individuals of S. tuberculata from four natural populations revealed their robust polymorphic reliability. The number of alleles per locus ranged from two to 11, while the expected and observed heterozygosity per marker varied from 0.187 to 0.827 and 0.042 to 0.917, respectively. Of these markers, 13 showed good amplification results in the closely related species S. cantonensis. CONCLUSIONS: These newly generated SSR markers are expected to provide novel tools for genetic studies of S. tuberculata, which will contribute to the conservation and sustainable use of the species' wild genetic resources.

Changes of flowering phenology and flower size in rosaceous plants from a biodiversity hotspot in the past century.

Responses of plant traits to climate changes are complex, which could be mirrored by the investigations of herbarium specimens. By examining specimens of Rosa and Cotoneaster species collected since 1920s in Hengduan Mountains, we analyzed the changes of flowering phenology and flower size in the past century when climate changes were considered to be intensified. We found that flowering phenology of Rosa showed no significant change, but flowering phenology of Cotoneaster was delayed in recent years. Flower size of Rosa species showed a marginally significant decrease over the past century. The results suggested that responses of flowering time to global changes and pollinator mediated selection on floral traits might be more complex than what were expected. Our results indicated that future researches based on investigations of herbarium specimens should be carried out on multiple plant species with different flower structures and life histories to better understand the effects of climate changes on plant traits.

Out of the Qinghai-Tibet Plateau: evidence for the origin and dispersal of Eurasian temperate plants from a phylogeographic study of Hippophaë rhamnoides (Elaeagnaceae).

Numerous temperate plants now distributed across Eurasia are hypothesized to have originated and migrated from the Qinghai-Tibet Plateau (QTP) and adjacent regions. However, this hypothesis has never been tested through a phylogeographic analysis of a widely distributed species. Here, we use Hippophaë rhamnoides as a model to test this hypothesis. We collected 635 individuals from 63 populations of the nine subspecies of H. rhamnoides. We sequenced two maternally inherited chloroplast (cp) DNA fragments and also the bi-paternally inherited nuclear ribosomal ITS. We recovered five major clades in phylogenetic trees constructed from cpDNA and internal transcribed spacer (ITS) sequence variation. Most sampled individuals of six subspecies that are distributed in northern China, central Asia and Asia Minor/Europe, respectively, comprised monophyletic clades (or subclades) nested within those found in the QTP. Two subspecies in the QTP were paraphyletic, while the placement of another subspecies from the Mongolian Plateau differed between the ITS and cpDNA phylogenetic trees. Our phylogeographic analyses supported an 'out-of-QTP' hypothesis for H. rhamnoides followed by allopatric divergence, hybridization and introgression. These findings highlight the complexity of intraspecific evolutions and the importance of the QTP as a center of origin for many temperate plants.