Abstract
BackgroundTetrastigma hemsleyanum is of great medicinal importance and used as a model system to address the evolutionary history of warm-temperate evergreen (WTE) forest biomes in East Asia over Neogene time scales. However, further studies on the neutral and adaptive divergence processes of T. hemsleyanum are currently impeded by a lack of genomic resources. In this study, we de novo assembled and annotated a reference transcriptome for two cpDNA lineages (Central-South-East vs. Southwest) of T. hemsleyanum. We further used comparative genomic and multilocus coalescent approaches to investigate the tempo and mode of lineage diversification in T. hemsleyanum.ResultsA total of 52,838 and 65,197 unigenes with an N50 of 1,667 and 1,841 bp for Central-South-East (CSE) and Southwest (SW) lineages, respectively, were recovered, and 6,692 putative orthologs were identified between the two lineages. Estimation of Ka/Ks ratios for these orthologs revealed that ten genes had Ka/Ks values significantly greater than 0.5 (P < 0.05), whereas 2,099 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. Based on three bioinformatic strategies, we identified a total of 1,018 single-copy nuclear genes (SCNGs) from the orthologs. We successfully designed eight nuclear gene primer pairs with high intraspecific variation (e.g. hT = 0.923, πT = 1.68×10-3), when surveyed across a subset of T. hemsleyanum individuals. Concordant with the previous cpDNA data, the haplotype networks constructed for most nuclear gene loci clearly identified the two lineages. A multilocus coalescence analysis suggested that the separation between the two lineages appears to have occurred during the mid-Pliocene. Despite their ancient divergence, both lineages experienced expansion at rather localized scales and have continued to exchange genes at a low rate.ConclusionsThis study demonstrated the utility of transcriptome sequencing as a basis for SCNG development in non-model species and the advantages of integrating multiple nuclear loci for phylogeographic and phylogenetic studies.
Highlights
Tetrastigma hemsleyanum is of great medicinal importance and used as a model system to address the evolutionary history of warm-temperate evergreen (WTE) forest biomes in East Asia over Neogene time scales
Since T. hemsleyanum is a typical component of warmtemperate evergreen (WTE) forest habitats in subtropical China, it has emerged as an excellent model species to address the evolutionary history of WTE forest biomes in eastern Asia over Neogene time scales
Prediction of protein-coding sequence (CDS) regions According to the BLAST search results against various protein databases, we identified and extracted the CDS regions of 49,915 unigenes in the CSE lineage and 54,373 in the SW lineage, which were translated into amino sequences with the standard codon table
Summary
Tetrastigma hemsleyanum Diels et Gilg (Vitaceae) is a diploid, perennial herb, with red berries dispersed by birds, bats and mammals, and is distinguished as the sole herbaceous climber of a genus that otherwise comprises only woody lianas [1,2,3,4] This species is widely distributed throughout subtropical China, and occurs rarely farther south on Hainan and Taiwan [5, 6]. The two major lineages likely diverged through climate/the uplift of QTP-induced vicariance of an ancestral southern range during the early Pliocene [6] Given that this climber of the WTE forest is primarily dispersed by frugivorous birds [12, 13] and shows a nearly continuous range at present, we presume that adaptive divergence processes may play an additional role in maintaining the separation of two cpDNA lineages of T. hemsleyanum inhabiting different floristic regions [6]. A paucity of genetic resources such as genomic and transcriptomic sequences has made further studies on the neutral and adaptive divergence processes of T. hemsleyanum and other non-model species a challenging task
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