DOI: 10.5061/dryad.b8gtht787
title: Genetic delimitation of Oreocharis species from Hainan Island

description: 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.
description: <b><i>Materials collection and DNA
 extraction</i></b> Twelve geographic populations of
 <i>Oreocharis</i> 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 <i>O. dasyantha</i>, populations QX (Qixian), WZA (Wuzhishan A) and WZB (Wuzhishan B) from <i>O. flavida</i>, and populations YG (Yingge) and LM (Limu) from unidentified <i>Oreocharis</i> 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 <i>Oreocharis</i> 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). <b><i>PCR amplification and sequencing</i></b> One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising spacer 1, the 5.8S gene and spacer 2 (White<i> </i>et al. 1990) and two chloroplast DNA (cpDNA) intron–spacer region <i>trn</i>L<i>-trn</i>F (Taberlet et al. 1991) and <i>ycf</i>1b<i> </i>(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 ddH<sub>2</sub>O, 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 <i>trn</i>L<i>-trn</i>F 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 <i>ycf</i>1b 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.