Qingbo Gao

Phylogeography of Spiraea alpina (Rosaceae) in the Qinghai–Tibetan Plateau inferred from chloroplast DNA sequence variations

Abstract  The aim of the present study was to investigate the phylogeographic patterns of Spiraea alpina (Rosaceae) and clarify its response to past climatic changes in the climate‐sensitive Qinghai‐Tibetan Plateau (QTP). We sequenced a chloroplast DNA fragment (trnL–trnF) from 528 individuals representing 43 populations. We identified 10 haplotypes, which were tentatively divided into three groups. These haplotypes or groups were distributed in the different regions of the QTP. Only half the populations were fixed by a single haplotype, whereas the others contained two or more. In the central and eastern regions, adjacent populations at the local scale shared the same haplotype. Our phylogeographic analyses suggest that this alpine shrub survived in multiple refugia during the Last Glacial Maximum and that earlier glaciations may have trigged deep intraspecific divergences. Post‐glacial expansions occurred only within populations or across multiple populations within a local range. The findings of the present study together with previous phylogeographic reports suggest that evolutionary histories of plants in the QTP are complex and variable depending on the species investigated.

Phylogeny and speciation in Saxifraga sect. Ciliatae (Saxifragaceae): Evidence from psbA-trnH, trnL-F and ITS sequences

Nearly half of the species in the large genus Saxifraga belong to Saxifraga sect. Ciliatae, a largely Sino-Himalayan taxon. We report here that evidence from chloroplast DNA sequences (psbA-trnH, trnL-F) and from nuclear sequences (ITS) indicates that this section is monophyletic and composed of at least three main lineages, corresponding to (1) a clade made up of species from S. subsect. Gemmiparae, subsect. Cinerascentes, subsect. Flagellares and subsect. Hemisphaericae, in which the last three subsections are nested in the first; (2) a clade of species belonging to S. subsect. Rosulares (including S. subsect. Serpyllifoliae); and (3) a clade of species belonging to S. subsect. Hirculoideae. Species relationships in S. subsect. Rosulares and subsect. Hirculoideae are not well resolved. A molecular clock analysis indicates that the diversification of S. sect. Ciliatae into its three lineages dates from ca. 9.48 Ma, coinciding with orogenic events associated with one of the most important phases of uplift of the Qinghai-Tibet Plateau. Extensive diversifications within S. subsect. Rosulares and subsect. Hirculoideae have been more recent (ca. 4.51 Ma and 2.12 Ma, respectively), again correlated with Qinghai-Tibet Plateau uplift events and, in the case of S. subsect. Hirculoideae, have occurred at a rate comparable to that seen in the radiation of Hawaiian fruit flies.

Phylogenetic analyses of Spiraea (Rosaceae) distributed in the Qinghai-Tibetan Plateau and adjacent regions: insights from molecular data

Abstract The Qinghai-Tibetan Plateau (QTP) and adjacent regions comprise an excellent mountainous system to study plant diversification and speciation within East Asia. The uplift and eco-environmental processes of QTP have had an obvious effect on evolution of organisms in this region. The present study intends to test the potential correlation between evolutionary events (such as speciation and diversification) and orogenetic events (such as the intense uplift of QTP). Sequence data from five plastid DNA regions (trnL–trnF, rpl20–rpl12, rps15–ycf1, psbA–trnH, and trnS–trnG) and one nuclear ribosomal internal transcribed spacer of 19 species of the genus Spiraea L. were used in the study. Maximum parsimony and maximum likelihood trees were constructed in PAUP*, while divergence time was estimated with BEAST v1.7.5. Phylogenetic reconstruction revealed that these species form a single clade and can be divided into three sections. Diversification of Spiraea species began in middle Miocene (ca. 13.38 million years ago) during the first stage of uplifting at QTP. Diversification of Spiraea was further triggered and accelerated during the second stage of QTP uplifting in late Pliocene (ca. Last four million years). The estimated divergences time indicate that this rapid diversification was most likely triggered by the uplifting of QTP in early Pliocene, and accelerated during the Quaternary climatic oscillations.

Isolation of 16 Microsatellite Markers for Spiraea alpina and S. mongolica (Rosaceae) of the Qinghai–Tibet Plateau

Premise of the study: A set of microsatellite markers were developed to characterize the level of genetic diversity and gene flow in two plant species endemic to the Qinghai–Tibet Plateau, Spiraea alpina and S. mongolica. Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) method, 16 microsatellite loci showed polymorphisms in both species. In two populations of each species, the number of alleles per locus ranged from three to 18 in S. alpina and from four to 30 in S. mongolica. Conclusions: These microsatellite markers provide an efficient tool for population genetic studies and will be used to assess the genetic diversity and spatial genetic structure of S. alpina and S. mongolica.

Development of SSR Markers for a Tibetan Medicinal Plant, Lancea tibetica (Phrymaceae), Based on RAD Sequencing

Premise of the study: Lancea tibetica (Phrymaceae), a Tibetan medicinal plant, is endemic to the Qinghai–Tibet Plateau. The over-exploitation of wild L. tibetica has led to the destruction of many populations. To enhance protection and management, biological research, especially population genetic studies, should be carried out on L. tibetica. Simple sequence repeat (SSR) markers of L. tibetica were developed to analyze population diversity. Methods and Results: Four thousand four hundred and forty-one SSR loci were identified for L. tibetica based on restriction-site associated DNA (RAD) sequencing on the Illumina HiSeq platform. One hundred SSR loci were arbitrarily selected for primer design, and 38 of them were successfully amplified. These markers were tested on 56 individuals from three populations of L. tibetica, and 10 markers displayed polymorphisms. The total number of alleles per locus ranged from three to eight, and observed and expected heterozygosities ranged from 0.200 to 1.000 and 0.683 to 0.879, respectively. We tested for cross-amplification of these 10 markers in the related species L. hirsuta and found that nine could be successfully amplified. Conclusions: The SSR markers characterized here are the first to be developed and tested in L. tibetica. They will be useful for future population genetic studies on L. tibetica and closely related species.

Development and characterization of polymorphic microsatellite loci for Saxifraga egregia (Saxifragaceae).

Premise of the study: Saxifraga egregia (Saxifragaceae) is a perennial herb that is endemic to the Qinghai–Tibet Plateau. We developed 12 polymorphic microsatellite loci for S. egregia to investigate its population genetics. Methods and Results: Forty-eight pairs of microsatellite primers (including 36 monomorphic loci) were isolated and characterized by magnetic bead enrichment. Twelve of these markers showed polymorphism, and the number of alleles per locus ranged from four to 14 across 50 individuals from three populations of S. egregia. No linkage disequilibrium was detected in any pair of loci. Conclusions: These polymorphic markers are expected to be helpful in further studies on the systematics and phylogeography of S. egregia in the Qinghai–Tibet Plateau.

Population genetic differentiation and taxonomy of three closely related species of saxifraga (Saxifragaceae) from southern tibet and the Hengduan mountains

The effects of rapid, recent uplift of the Hengduan Mountains on evolution and diversification of young floristic lineages still remain unclear. Here, we investigate diversification of three closely related Saxifraga species with a distribution restricted to the Hengduan Mountains (HM) and southern Tibet, and comment on their taxonomy based on molecular evidence. Three chloroplast DNA fragments (rbcL, trnL-F, trnS-G) and the nuclear ribosomal DNA internal transcribed spacer (ITS) were employed to study genetic structure across 104 individuals from 12 populations of Saxifraga umbellulata, S. pasumensis, and S. banmaensis. Chloroplast DNA (cpDNA) phylogenies revealed two well supported clades, corresponding to S. umbellulata and S. pasumensis plus S. banmaensis. Topology of the ITS phylogeny was largely congruent with that generated from cpDNA haplotypes, but with minor conflicts which might be caused by incomplete lineage sorting. Analyses of molecular variance of both cpDNA and ITS datasets revealed that most variation was held between S. pasumensis s.l. (with S. banmaensis) and S. umbellulata (92.31% for cpDNA; 69.78% for ITS), suggesting a high degree of genetic divergence between them. Molecular clock analysis based on ITS dataset suggested that the divergence between S. pasumensis s.l. and S. umbellulata can be dated to 8.50 Ma, probably a result of vicariant allopatric diversification associated with the uplift events of the HM. Vicariance associated with HM uplifts may also have been responsible for infraspecific differentiation in S. pasumensis. In contrast, infraspecific differentiation in S. umbellulata was most likely triggered by Quaternary glaciations. The much lower levels of gene diversity within populations of S. pasumensis compared with S. umbellulata could have resulted from both range contractions and human collection on account of its putative medicinal properties. Combining evidence from morphology, geographical distributions and molecular phylogenetic data, we recommend that S. banmaensis should be treated as a synonym of S. pasumensis which in turn, and based on the same sources of evidence, should be treated as a separate species rather than as a variety of S. umbellulata.

Westwards and northwards dispersal of Triosteum himalayanum (Caprifoliaceae) from the Hengduan Mountains region based on chloroplast DNA phylogeography

The varying topography and environment that resulted from paleoorogeny and climate fluctuations of the Himalaya–Hengduan Mountains (HHM) areas had a considerable impact on the evolution of biota during the Quaternary. To understand the phylogeographic pattern and historical dynamics of Triosteum himalayanum (Caprifoliaceae), we sequenced three chloroplast DNA fragments (rbcL-accD, rps15-ycf1, and trnH-psbA) from 238 individuals representing 20 populations. Nineteen haplotypes (H1–H19) were identified based on 23 single-site mutations and eight indels. Most haplotypes were restricted to a single population or neighboring populations. Analysis of molecular variance revealed that variations among populations were much higher than that within populations for the overall gene pool, as well as for the East Himalayan group (EH group) and the North Hengduan group (NHM group), but not for the Hengduan Mountains group (HM group). Ecoregions representing relatively high genetic diversity or high frequencies of private haplotypes were discovered, suggesting that this alpine herbaceous plant underwent enhanced allopatric divergence in isolated and fragmented locations during the Quaternary glaciations. The current phylogeographic structure of T. himalayanum might be due to heterogeneous habitats and Quaternary climatic oscillations. Based on the phylogeographic structure of T. himalayanum populations, the phylogenetic relationship of identified haplotypes and palaeodistributional reconstruction, we postulated both westwards and northwards expansion from the HM group for this species. The westwards dispersal corridor could be long, narrow mountain areas and/or the Yarlung Zangbo Valley, while the northwards movement path could be south–north oriented mountains and low-elevation valleys.

The Complete Chloroplast Genomes of Two Lancea Species with Comparative Analysis

The genus Lancea is native to the Qinghai-Tibetan Plateau and consists of two species, Lancea tibetica Hook. f. et Thoms. and Lancea hirsuta Bonati. Here, we report the complete sequences of the chloroplast genomes of L. tibetica and L. hirsuta, which were 153,665 and 154,045 bp in length, respectively, and each included a pair of inverted repeated regions (25,624 and 25,838 bp in length, respectively) that were separated by a large single copy region (84,401 and 84,588 bp in length, respectively) and a smaller single copy region (18,016 and 17,781 bp in length, respectively). A total of 106 genes in L. tibetica and 105 in L. hirsuta comprised 79 protein-coding genes, and 4 ribosomal RNA (rRNA) genes, as well as 23 and 22 transfer RNA (tRNA) genes in L. tibetica and L. hirsuta, respectively. The gene order, content, and orientation of the two Lancea chloroplast genomes exhibited high similarity. A large number of informative repetitive sequences, including SSRs, were observed in both genomes. Comparisons of the genomes with those of three other Lamiales species revealed 12 highly divergent regions in the intergenic spacers and in the matK, rpoA, rps19, ndhF, ccsA, ndhD, and ycf1 coding regions. A phylogenomic analysis suggested that Lancea forms a monophyletic group that is closely related to the clade composed of the families Phrymaceae, Paulowniaceae, and Rehmanniaceae.

The Complete Plastome Sequences of Seven Species in Gentiana sect. Kudoa (Gentianaceae): Insights Into Plastid Gene Loss and Molecular Evolution

The chloroplast (cp) genome is useful in the study of phylogenomics, molecular dating, and molecular evolution. Gentiana sect. Kudoa is a predominantly alpine flowering plant that is valued for its contributions to medicine, ecology, and horticulture. Previous evolutionary studies showed that the plastid gene loss pattern and intra-sectional phylogenetics in sect. Kudoa are still unclear. In this study, we compared 11 Gentiana plastomes, including 7 newly sequenced plastomes from sect. Kudoa, to represent its three serious: ser. Ornatae, ser. Verticillatae, and ser. Monanthae. The cp genome sizes of the seven species ranged from 137,278 to 147,156 bp. The plastome size variation mainly occurred in the small single-copy and long single-copy regions rather than the inverted repeat regions. Compared with sect. Cruciata, the plastomes in ser. Ornatae and ser. Verticillatae had lost approximately 11 kb of sequences containing 11 ndh genes. Conversely, far fewer losses were observed in ser. Monanthae. The phylogenetic tree revealed that sect. Kudoa was not monophyletic and that ser. Monanthae was more closely related to other sections rather than sect. Kudoa. The molecular dating analysis indicated that ser. Monanthae and sect. Kudoa diverged around 8.23 Ma. In ser. Ornatae and ser. Verticillatae, the divergence occurred at around 0.07–1.78 Ma. The nucleotide diversity analysis indicated that the intergenic regions trnH-psbA, trnK-trnQ, ycf3-trnS and rpl32-trnL constituted divergence hotspots in both sect. Kudoa and Gentiana, and would be useful for future phylogenetic and population genetic studies.