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Genetic delimitation of Oreocharis species from Hainan Island

Citation

Ling, Shao-Jun et al. (2021), Genetic delimitation of Oreocharis species from Hainan Island, Dryad, Dataset, https://doi.org/10.5061/dryad.b8gtht787

Abstract

Hainan Island harbours an extraordinary diversity of Gesneriaceae with 14 genera and 23 species, among which two species and one variety are recognized in the genus Oreocharis. These three Oreocharis taxa are all Hainan-endemics and show complex geographical distribution pattern with considerable morphological intermixtures. In this study, we combined DNA (nuclear ITS sequences and cpDNA trnL-trnF and ycf1b) to evaluate genetic delimitation for 12 Oreocharis populations from the island, together with morphological similarity analysis using 16 morphological traits. The results showed Hainan Oreocharis taxa was monophyly with relative low genetic diversity within populations, highly significant genetic differentiation among populations and a significant phylogeographical structure. The 12 populations formed three genetically distinct groups, roughly correspondent to the currently recognized two species and one unknown lineage. The PCA analyses of morphological traits indicate three distinctive groups, differing mainly in petal color and corolla shape and types. The roles of river isolation in the origin and distribution of these three lineages are discussed.

Methods

Materials collection and DNA extraction

Twelve geographic populations of Oreocharis taxa covering all the suitable habitat of the genus on the island were collected, including populations DW (Dongwu in Bawangling), DE (Donger in Bawangling), FT (Futou in Bawangling), NG (Nangao), HM (Houmi), JF (Jianfeng) and CH (Chahe) from O. dasyantha, populations QX (Qixian), WZA (Wuzhishan A) and WZB (Wuzhishan B) from O. flavida, and populations YG (Yingge) and LM (Limu) from unidentified Oreocharis sp. (Table 1, Fig. 1). Fresh leaves were collected and stored dried in silica gel from the south-central mountains in Hainan Island in 2015, 2016 and 2017. Totally 238 leaf samples from 12 populations that represented the whole geographical range of Oreocharis taxa on Hainan Island were collected (Fig.1).

Total genomic DNA for each individual was extracted using CTAB methods (Doyle and Doyle 1987) and served as template for the polymerase chain reaction. AL2000 DNA maker (Aidlab Biotechnologies Co., Ltd) was used to detect DNA quality and quantity on 0.8% agarose gels stained with 2.5 μL Goldview (Aidlab Biotechnologies Co., Ltd) in DTU-48 spectrophotometer (Hangzhou Miu Instruments Co., Ltd, China).

PCR amplification and sequencing

One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (White et al. 1990) and two chloroplast DNA (cpDNA) intron–spacer region trnL-trnF (Taberlet et al. 1991) and ycf1b (Dong et al. 2015) were used in this study (Table 2). PCR reactions were set up in a volume of 25 μL consisting of 20 μL ddH2O, 2.5 μL 10×Buffer, 0.5 uL μ10 mM dNTPs, 0.5 μL each 5 μM primer, 0.5 μL DNA template and 0.5 μL 5 U/μL Taq polymerase (Aidlab Biotechnologies Co., Ltd). PCR was conducted in a 2720 Thermal cycler (Applied Biosystems by Life Technologies, made in Singapore) and Veriti® 96-Well Thermal Cycler (Applied Biosystems by Life Technologies, made in Singapore). PCR program for nrDNA and trnL-trnF was designed to an initial denaturation at 94℃ 5 min, followed by 35 cycles of 1 min at 94℃, 1 min at 55℃, 1 min at 72℃, and with a final extension of 10 min at 72℃. Amplification of ycf1b used the following protocol: 4 min at 94℃, 35 cycles of 30 s at 94℃, 40 s at 58℃, and 1 min at 72℃, ending with 10 min at 72℃. All the PCR products were checked by electrophoresis. Then purification and sequencing of PCR products were finally sequenced by an ABI 3730 DNA Analyzer based on the BigDye Terminator Cycle Sequencing Ready Kit (Applied Biosystems, Foster City, CA) in BGI (Beijing Genomics institution), the chemistry and primers were used above in BGI.