Hai-Fei Yan

Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants

A two-marker combination of plastid rbcL and matK has previously been recommended as the core plant barcode, to be supplemented with additional markers such as plastid trnH–psbA and nuclear ribosomal internal transcribed spacer (ITS). To assess the effectiveness and universality of these barcode markers in seed plants, we sampled 6,286 individuals representing 1,757 species in 141 genera of 75 families (42 orders) by using four different methods of data analysis. These analyses indicate that (i) the three plastid markers showed high levels of universality (87.1–92.7%), whereas ITS performed relatively well (79%) in angiosperms but not so well in gymnosperms; (ii) in taxonomic groups for which direct sequencing of the marker is possible, ITS showed the highest discriminatory power of the four markers, and a combination of ITS and any plastid DNA marker was able to discriminate 69.9–79.1% of species, compared with only 49.7% with rbcL + matK; and (iii) where multiple individuals of a single species were tested, ascriptions based on ITS and plastid DNA barcodes were incongruent in some samples for 45.2% of the sampled genera (for genera with more than one species sampled). This finding highlights the importance of both sampling multiple individuals and using markers with different modes of inheritance. In cases where it is difficult to amplify and directly sequence ITS in its entirety, just using ITS2 is a useful backup because it is easier to amplify and sequence this subset of the marker. We therefore propose that ITS/ITS2 should be incorporated into the core barcode for seed plants.

Testing DNA barcoding in closely related groups of Lysimachia L. (Myrsinaceae)

It has been suggested that rbcL and matK are the core barcodes in plants, but they are not powerful enough to distinguish between closely related plant groups. Additional barcodes need to be evaluated to improve the level of discrimination between plant species. Because of their well‐studied taxonomy and extreme diversity, we used Chinese Lysimachia (Myrsinaceae) species to test the performance of core barcodes (rbcL and matK) and two additional candidate barcodes (trnH‐psbA and the nuclear ribosomal ITS); 97 accessions from four subgenus representing 34 putative Lysimachia species were included in this study. And many closely related species pairs in subgen. Lysimachia were covered to detect their discriminatory power. The inefficiency of rbcL and matK alone or combined in closely related plant groups was validated in this study. TrnH‐psbA combined with rbcL + matK did not yet perform well in Lysimachia groups. In contrast, ITS, alone or combined with rbcL and/or matK, revealed high resolving ability in Lysimachia. We support ITS as a supplementary barcode on the basis of core barcode rbcL and matK. Besides, this study also illustrates several mistakes or underlying evolutionary events in Lysimachia detected by DNA barcoding.

Comparative transcriptome analysis and marker development of two closely related Primrose species (Primula poissonii and Primula wilsonii)

BackgroundPrimula species are important early spring garden plants with a centre of diversity and speciation in the East Himalaya-Hengduan Mountains in Western China. Studies on population genetics, speciation and phylogeny of Primula have been impeded by a lack of genomic resources. In the present study, we sequenced the transcriptomes of two closely related primrose species, Primula poissonii and Primula wilsonii, using short reads on the Illumina Genome Analyzer platform.ResultsWe obtained 55,284 and 55,011 contigs with N50 values of 938 and 1,085 for P. poissonii and P. wilsonii, respectively, and 6,654 pairs of putative orthologs were identified between the two species. Estimations of non-synonymous/synonymous substitution rate ratios for these orthologs indicated that 877 of the pairs may be under positive selection (Ka/Ks > 0.5), and functional enrichment analysis revealed that significant proportions of the orthologs were in the categories DNA repair, stress resistance, which may provide some hints as to how the two closely related Primula species adapted differentially to extreme environments, such as habitats characterized by aridity, high altitude and high levels of ionizing radiation. It was possible for the first time to estimate the divergence time between the radiated species pair, P. poissonii and P. wilsonii; this was found to be approximately 0.90 ± 0.57 Mya, which falls between the Donau and Gunz glaciation in the Middle Pleistocene. Primers based on 54 pairs of orthologous SSR-containing sequences between the two Primula species were designed and verified. About half of these pairs successfully amplified for both species. Of the 959 single copy nuclear genes shared by four model plants (known as APVO genes), 111 single copy nuclear genes were verified as being present in both Primula species and exon-anchored and intron-spanned primers were designed for use.ConclusionWe characterized the transcriptomes for the two Primula species, and produced an unprecedented amount of genomic resources for these important garden plants. Evolutionary analysis of these two Primula species not only revealed a more precise divergence time, but also provided some novel insights into how differential adaptations occurred in extreme habitats. Furthermore, we developed two sets of genetic markers, single copy nuclear genes and nuclear microsatellites (EST-SSR). Both these sets of markers will facilitate studies on the genetic improvement, population genetics and phylogenetics of this rapidly adapting taxon.

Phylogeographic analysis and environmental niche modeling of widespread shrub Rhododendron simsii in China reveals multiple glacial refugia during the last glacial maximum

Abstract  The phylogeography of common and widespread species can help us to understand the history of local flora and vegetation. Here, we study the semi‐evergreen shrub Rhododendron simsii Planch., which is found in most areas of current evergreen broad leaved forest in China. Two noncoding chloroplast DNA (cpDNA) regions (rpl20‐rps12 and trnL‐F) and three amplified fragment length polymorphism (AFLP) primer sets (E‐AAC/M‐CTA, E‐AGC/M‐CTA and E‐AGG/M‐CAT) were used to examine the phylogeographic pattern in relation to past (last glacial maximum) and present distributions based on ecological niche modeling. The cpDNA data revealed four phylogeographic groups (East, South, West, and North groups) corresponding to geographic regions. Molecular dating suggests that lineage diversification within species likely occurred during the mid‐to‐late Pleistocene. In contrast, the four main cpDNA phylogeographic groups were not supported by the AFLP dataset. The highest likelihood of the AFLP data was obtained when samples were clustered into three groups (K= 3). However, these groupings did not correspond to separate geographic regions supported by cpDNA data. Both mismatch distribution analysis and environmental niche modeling (ENM) indicated that multiple glacial refugia were maintained across the range of Rhododendron simsii during the last glacial maximum, contrary to the previous hypothesis that subtropical broad leaved evergreen forests were forced to retreat southward as far as 25° N. The discordance between the patterns revealed by cpDNA and AFLP data indicate that localized postglacial range expansions may facilitate extensive gene flow between the major glacial refugia.

Population Expanding with the Phalanx Model and Lineages Split by Environmental Heterogeneity: A Case Study of Primula obconica in Subtropical China

Background Current and historical events have both affected the current distribution patterns and intraspecific divergence of plants. While numerous studies have focused on the Qinghai-Tibetan Plateau (QTP), the impacts of such events on the flora of subtropical China remain poorly understood. Subtropical China is famous for its highly complex topography and the limited impact from glaciation during the Pleistocene; this may have resulted in a different genetic legacy for species in this region compared to fully glaciated areas. Methodology/Principal Findings We used plastid and nuclear DNA sequence data and distribution modeling to analyze the divergence patterns and demographic history of Primula obconica Hance, a widespread herbaceous montane species in subtropical China. The phylogenetic analysis revealed two major lineages (lineage A and lineage B), representing a west-east split into the Yunnan and Eastern groups, and the Sichuan and Central groups, respectively. The Eastern and Central groups comprised relatively new derived haplotypes. Nested Clade Analysis and Bayesian Skyline Plot analyses both indicated that P. obconica mainly experienced a gradual expansion of populations. In addition, the simulated distribution of P. obconica during the Last Glacial Maximum was slightly larger than its present-day distribution. Conclusion/Significance Our results are the first to identify a west-east migration of P. obconica. The gradual expansion pattern and a larger potential distribution range in cold periods detected for P. obconica indicate that the population expansion of this species is consistent with the phalanx model. In addition, the current patterns of genetic differentiation have persisted as a result of the extensive environmental heterogeneity that exists in subtropical China.

Chloroplast DNA phylogeography of Primula ovalifolia in central and adjacent southwestern China: Past gradual expansion and geographical isolation

Abstract  To investigate the mechanisms responsible for the high diversity in central and adjacent southwestern China, we inferred the phylogeographic history of Primula ovalifolia from chloroplast DNA data. One hundred and thirty five individuals from 13 natural populations (including one Primula tardiflora population) were analyzed. A total of 23 haplotypes were identified. Most of them were exclusive to a single population. Strong phylogeography structure was detected, with NST (0.936) significantly higher than GST (0.784). Phylogenetic analysis showed that all haplotypes were clustered into three lineages (clades I, II, and III). High genetic diversity was revealed, possibly due to the interglacial contraction and glacial expansion. The three identified lineages may have undergone different historical processes after the mid‐Pleistocene, due to their early divergent time. Multiple refugia were inferred for the three lineages. Pleistocene climatic oscillations, combined with the complex local topography, were responsible for the strong phylogeographic pattern of P. ovalifolia. Our analysis supports the hypothesis that the central and adjacent southwestern region of China was an important refugium for the survival, persistence, and further speciation of most East Asia flora, which has led to high species diversity in this region.

Complete plastid genome sequence of Primula sinensis (Primulaceae): structure comparison, sequence variation and evidence for accD transfer to nucleus

Species-rich genus Primula L. is a typical plant group with which to understand genetic variance between species in different levels of relationships. Chloroplast genome sequences are used to be the information resource for quantifying this difference and reconstructing evolutionary history. In this study, we reported the complete chloroplast genome sequence of Primula sinensis and compared it with other related species. This genome of chloroplast showed a typical circular quadripartite structure with 150,859 bp in sequence length consisting of 37.2% GC base. Two inverted repeated regions (25,535 bp) were separated by a large single-copy region (82,064 bp) and a small single-copy region (17,725 bp). The genome consists of 112 genes, including 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Among them, seven coding genes, seven tRNA genes and four rRNA genes have two copies due to their locations in the IR regions. The accD and infA genes lacking intact open reading frames (ORF) were identified as pseudogenes. SSR and sequence variation analyses were also performed on the plastome of Primula sinensis, comparing with another available plastome of P. poissonii. The four most variable regions, rpl36–rps8, rps16–trnQ, trnH–psbA and ndhC–trnV, were identified. Phylogenetic relationship estimates using three sub-datasets extracted from a matrix of 57 protein-coding gene sequences showed the identical result that was consistent with previous studies. A transcript found from P. sinensis transcriptome showed a high similarity to plastid accD functional region and was identified as a putative plastid transit peptide at the N-terminal region. The result strongly suggested that plastid accD has been functionally transferred to the nucleus in P. sinensis.

DNA Barcoding Evaluation and Its Taxonomic Implications in the Species-Rich Genus Primula L. in China

The genus Primula is extremely diverse in the east Himalaya-Hengduan Mountains (HHM) in China as a result of rapid radiation. In order to overcome the difficulty of morphological classification of this genus, we surveyed three plastid regions (rbcL, matK, and trnH-psbA) and two nuclear markers (ITS and ITS2) from 227 accessions representing 66 Primula species across 18 sections, to assess their discriminatory power as barcodes. We found that ITS alone or combined with plastid regions showed the best discrimination across different infrageneric ranks and at species level. We suggest rbcL + matK + ITS as the first choice at present to barcode Primula plants. Although the present barcoding combination performed poorly in many closely related species of Primula, it still provided many new insights into current Primula taxonomy, such as the underlying presence of cryptic species, and several potential improper taxonomic treatments. DNA barcoding is one useful technique in the integrative taxonomy of the genus Primula, but it still requires further efforts to improve its effectiveness in some taxonomically challenging groups.

The Use of DNA Barcoding on Recently Diverged Species in the Genus Gentiana (Gentianaceae) in China.

DNA barcoding of plants poses particular challenges, especially in differentiating, recently diverged taxa. The genus Gentiana (Gentianaceae) is a species-rich plant group which rapidly radiated in the Himalaya-Hengduan Mountains in China. In this study, we tested the core plant barcode (rbcL + matK) and three promising complementary barcodes (trnH-psbA, ITS and ITS2) in 30 Gentiana species across 6 sections using three methods (the genetic distance-based method, Best Close Match and tree-based method). rbcL had the highest PCR efficiency and sequencing success (100%), while the lowest sequence recoverability was from ITS (68.35%). The presence of indels and inversions in trnH-psbA in Gentiana led to difficulties in sequence alignment. When using a single region for analysis, ITS exhibited the highest discriminatory power (60%-74.42%). Of the combinations, matK + ITS provided the highest discrimination success (71.43%-88.24%) and is recommended as the DNA barcode for the genus Gentiana. DNA barcoding proved effective in assigning most species to sections, though it performed poorly in some closely related species in sect. Cruciata because of hybridization events. Our analysis suggests that the status of G. pseudosquarrosa needs to be studied further. The utility of DNA barcoding was also verified in authenticating ‘Qin-Jiao’ Gentiana medicinal plants (G. macrophylla, G. crassicaulis, G. straminea, and G. dahurica), which can help ensure safe and correct usage of these well-known Chinese traditional medicinal herbs.

A Remarkable New Species of Liparis (Orchidaceae) from China and Its Phylogenetic Implications

In the present study, we formally describe Liparis pingxiangensis as a new species from Guangxi, China on the basis of morphological and molecular phylogenetic analyses. It is easily distinguished from closely related species by strongly curved column without column wings, and broadly rhombic-elliptic lip with 2 uncinate calli at the base. In particular, it differs most markedly from its congeners in possessing two pollinia attached by long and prominent caudicles (not stipes), to a distinct sticky disc. This type of pollinarium, as far as we know, is not found in any other species of Liparis, and is also unique among the orchids with waxy pollinia. We then proceeded to a phylogenetic analysis to ascertain the systematic position of this enigmatic species. Molecular study based on nuclear ribosomal ITS and plastid matK DNA sequence data supports L. pingxiangensis as a distinct species, which forms an independent lineage sister to L. nervosa and its allies (93% BS, 1.00 BPP). In the light of previous work, the findings have important implications for a better understanding of the well-supported pattern mainly based on vegetative features in Malaxideae.