Abstract
BACKGROUND: 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. 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. RESULTS: A 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 K(a)/K(s) ratios for these orthologs revealed that ten genes had K(a)/K(s) values significantly greater than 0.5 (P < 0.05), whereas 2,099 (K(a)/K(s) < 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. h(T) = 0.923, π(T) = 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. CONCLUSIONS: This 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.