Genetic Structure and Demographic History of Cycas Chenii (Cycadaceae), an Endangered Species with Extremely Small Populations

Accepted Manuscript Genetic structure and demographic history of Cycas chenii (Cycadaceae), an endangered species with extremely small populations Rui Yang, Xiu-yan Feng, Xun Gong PII: S2468-2659(16)30024-5 DOI: 10.1016/j.pld.2016.11.003 Reference: PLD 42 To appear in: Plant Diversity Received Date: 9 May 2016 Revised Date: 14 November 2016 Accepted Date: 14 November 2016 Please cite this article as: Yang, R., Feng, X.-y., Gong, X., Genetic structure and demographic history of Cycas chenii (Cycadaceae), an endangered species with extremely small populations, Plant Diversity (2017), doi: 10.1016/j.pld.2016.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 2 Genetic structure and demographic history of Cycas chenii (Cycadaceae), an endangered species with 3 extremely small populations 4 5 ABSTRACT 6 Geological activities and climate oscillations during the Quaternary period profoundly impacted the distribution of 7 species in Southwest China. Some plant species may be harbored in refugia, such as the dry-hot valleys of Southwest 8 China. Cycas chenii X. Gong & W. Zhou, a critically endangered cycad species, which grows under the canopy in 9 subtropical evergreen broad-leaved forests along the upstream drainage area of the Red River, is endemic to this 10 refugium. In this study, 60 individuals of C.chenii collected from six populations were analyzed by sequencing two 11 chloroplast intergenic spacers (cpDNA: psb A-trn H and trn L-trn F) and two nuclear genes ( PHYP and RBP-1). Results 12 showed high genetic diversity at the species level, but low within-population genetic diversity and high 13 interpopulation genetic differentiation. A Bayesian phylogenetic tree based on cpDNA showed that five chloroplast 14 haplotypes were clustered into two clades, which corresponds to the division of the western and eastern bank of the 15 Red River. These data indicate a possible role for the Red River as a geographic barrier to gene flow in C. chenii . 16 Based on our findings, we propose appropriate in situ and ex situ conservation strategies for C. chenii . 17 18 Keywords: Cycas chenii ; Genetic variation; Phylogeography; Conservation 19 20 Rui Yang 1,2,3 , Xiu-yan Feng 1,2,3 , Xun Gong 1,2,4, * 21 22 1Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy 23 of Sciences, Kunming 650201, China 24 25 2Key Laboratory of Economic Plants and Biotechnology, KunmingMANUSCRIPT Institute of Botany, Chinese Academy of Sciences, 26 Kunming 650201, China 27 28 3University of Chinese Academy of Sciences, Beijing 100049, China 29 30 4Yunnan Key Laboratory for Wild Plant Resources, Kunming 650201, China 31 32 Corresponding author’s e-mail address: [email protected] 33 34 1. Introduction 35 Southwest China experienced glacial-interglacial cycles in the Pleistocene approximately 2.4-0.01 million years ago 36 (MYA) (Zhou et al., 2006; Royden et al., 2008). Therefore, the dry-hot valleys of Southwest China, such as Red River 37 valley, are recognized as potential refugia for some plant species (Guan and Zhou, 1996; Wang et al., 1996). 38 Extant cycads areACCEPTED composed of the two families (Cycadaceae and Zamiaceae) with ten genera, and are mainly 39 distributed in Asia, Australia, South and Central America and Africa. Approximately 200 (62%) cycads are threatened 40 with extinction (Jian et al., 2006; Hoffmann et al., 2010). There are about 25 Cycas species (21%) distributed in 41 China (Calonje et al., 2016). The range of these species is often limited by habitat destruction and fragmentation, 42 commonly attributed to planting economic crops along with over-collection for food, medicine and ornamentals 43 (Wang et al., 1996). The Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora 44 (WFF) gave all cycads ‘First Grade’ conservation status in China (Xiao et al., 2004). China harbors abundant Cycas 45 diversity, especially in the drainage areas of the Red River, which is considered a secondary diversification center of - 1 - ACCEPTED MANUSCRIPT 46 Cycas (Hill, 2008). 47 There are more than 14 Cycas species, of which 10 are endemic to the basin region of the Red River (Hill, 2008). 48 Cycas chenii X. Gong & W. Zhou (Section Stangerioides ) is a recently described species (Zhou et al., 2015). This 49 species is distributed in Chuxiong and Honghe of Yunnan Province, China, along the upstream drainage areas of the 50 Red River (also called Yuanjiang in China). It occurs on a range of substrates from limestone to shale or schist, which 51 is characteristic of steep slopes at altitudes ranging from 500 m to 1300 m (Zhou et al., 2015). Only six populations 52 have been discovered, four at the northeast side of the Red River and two at the southwest side. To date, the total 53 population size of C. chenii is estimated at less than 500 individuals across its geographic distribution, with all the 54 know populations of the species being far from any protected areas (Zhou et al., 2015). This species is threatened by 55 ongoing land-clearing and over-collection. Narrow range, small population sizes and presence of a potential barrier to 56 gene flow (the Red River) necessitates understanding extent and structure of genetic variation as a prerequisite for 57 working out the appropriate conservation strategy for this species. The organelle DNA of cycads is maternally 58 inherited and is transmitted only by seeds while their nuclear DNA is biparentally inherited and is transmitted via 59 both seeds and pollens (Huang et al., 2004; Zhong et al., 2011; Feng et al., 2014). Therefore, using these two genetic 60 markers together allows a greater understanding of the role that seed vs. pollen flow play in spatial structuring of 61 genetic variation. Thus, we employed both maternally inherited chloroplast DNA (cpDNA) and biparentally inherited 62 nuclear DNA (nDNA) markers to investigate (i) the extent and structure of genetic variation in C. chenii , and (ii) the 63 role of the Red River in shaping this structure. Based on our results, we propose suitable conservation strategy for the 64 species. 65 66 2. Materials and Methods 67 2.1. Study species and population sampling 68 All C. chenii populations have less than 100 individuals. All currently known populations of C. chenii were sampled 69 during August to September in 2012. Four populations (ZS, DT, WJ and SP) were sampled from the northeast of the 70 Red River (NE group) and the other two (ML and LH) were fromMANUSCRIPT the southwest of the Red River (SW group) (Fig. 1). 71 The distance between sampled individuals was at least 5 m, increasing the likelihood of sampling genetically 72 unrelated individuals. Fresh leaves were dried in silica gel after collection and stored at room temperature until DNA 73 extraction. Voucher specimens were stored in the herbarium of the Kunming Institute of Botany, Chinese Academy of 74 Sciences (KUN). 75 76 2.2. DNA extraction, PCR amplification and DNA sequencing 77 We extracted genomic DNA from dried leaves using the modified CTAB method (Doyle, 1991). After preliminary 78 screening of DNA fragments from universal chloroplast and nuclear primers, we chose two cpDNA intergenic spacers, 79 psb A-trnH and trn L-trn F (Taberlet et al., 1991; Shaw et al., 2005), and two nuclear genes, the phytochrome P gene 80 PHYP (Zhou et al., 2015) and the gene that encodes the largest subunit of RNA polymerase II, RBP-1 (Liu et al., 81 2015). PCR amplification was carried out in 30 µL reactions. For cpDNA, the PCR reactions contained 10 ng of 82 DNA, 3.0 µL of 10 × PCR buffer, 1.5 µL of dNTPs (10 mM), 1.5 µL of MgCl2 (25 mM), 0.45 µL of Taq DNA 83 Polymerase (5 U/ µL), ACCEPTED0.45 µL of each primer, 1.5 µL of DMSO (20 mg/mL) and 19.65 µL of double-distilled water. 84 For nDNA, the PCR reactions contained 20 ng DNA, 3.0 µL 10 × PCR buffer, 1.5 µL dNTPs (10 mM), 2.25 µL 85 MgCl2 (25 mM), 0.45 µL Taq DNA Polymerase (5 U/ µL), 0.525 µL of each primer, 0.75 µL DMSO (20 mg/mL) and 86 18 µL double-distilled water. PCR amplification for cpDNA included an initial denaturation stage for 5 min at 80 °C, 87 followed by 30 cycles of 1 min at 95 °C, 1 min annealing at 50 °C, extension for 1.5 min at 65 °C, and a final 88 extension at 65 °C for 10 min. For nDNA: an initial denaturation stage at 94 °C for 5min, was followed by 35 cycles 89 at 95 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2 min, and a final extension for 10 min at 90 72 °C. All PCR products were sequenced in both directions with the same primers for the amplification reactions, - 2 - ACCEPTED MANUSCRIPT 91 using an ABI 3770 automated sequencer at Shanghai Majorbio Bio-pharm Technology Company Ltd. 92 93 2.3. Data analysis 94 We edited and assembled sequences using SeqMan (Swindell and Plasterer, 1997). Multiple alignments of the DNA 95 sequences were performed in Clustal X, version 1.83 (Thompson et al., 1997), then the DNA sequences were adjusted 96 by Bioedit, version 7.0.4.1 (Hall, 1999).

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