Phylogeography of a rare and endemic tree of Western Ghats reveals its ice age dynamics Rajasri Ray1,3, TV Ramachandra1 and Avik Ray2,3* 1 - Center for Ecological Sciences, Indian Institute of Science, Bangalore - 560012, Karnataka, India; 2 - Ashoka Trust for Research In Ecology and Environment (ATREE), Royal Enclave, Srirampu- ra, Jakkur Post, Bangalore - 560054, Karnataka, India; 3 - Center for studies in Ethnobiology, Biodiversity, and sustainability (CEiBa), B.G. Road, Mokdumpur, Malda - 732103, WB, India *[email protected] 1 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27043v1 | CC BY 4.0 Open Access | rec: 19 Jul 2018, publ: 19 Jul 2018 ABSTRACT: The climatic shifts in the Quaternary Period acted as an important driving force for evolution of the world's biodiversity. In this study, responses to the ice age of a rare and endangered tree of Indian Western Ghats, Syzygium travancoricum, was investigated through a combination of mo- lecular data and ecological niche modeling. Results indicate the moderate (nrDNA) to high (cpDNA) population structure, presence of pri- vate alleles, and absence of phylogeographic structure. Spatial distribution of diversity does not significantly support southern colonisation, nor the Palghat Gap as a geographical barrier based on the genetic structure. Combined insights from Ecological Niche Modelling and population history depicts a probable colonisation of the Western Ghats in the late Miocene or later fol- lowed by a split into separate southern and northern populations from the middle to late Pleisto- cene. The prevalence of favourable conditions in the post-Last interglacial period stimulated rap- id population expansion and a very recent decline. The absence of phylogeographic structure and overall climatic stability from Last Interglacial do not offer support to an existence of refugia in the Western Ghats. We conclude by discussing appropriate conservation measures for this rare and endangered tree based on our findings. Keywords:Quaternary period, Western Ghats, endemic tree, Syzygium travancoricum, glacial refugia, Myrtaceae Abbreviations: WG: Western Ghats; ENM: Ecological niche model; LIG: Last Interglacial peri- od; LGM: Last Glacial Maxima, nrDNA - nuclear DNA, ptDNA - chloroplast DNA 2 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27043v1 | CC BY 4.0 Open Access | rec: 19 Jul 2018, publ: 19 Jul 2018 Running title: Quaternary dynamics of a Western Ghats endemic tree Word count: 3672 Introduction The Quaternary Period is seen as a key driver of evolution of the world's biota resulting in the current geographic patterns (Hewitt, 2000; 2004 and references therein). A great deal of research on the biotic elements of Euro-America and China has yielded insights into the responses to this period. For example, the identification of glacial refugia or cryptic refugia, prediction of extinc- tion events, range contraction and expansion, colonisation of new landscapes, and divergence and speciation (Hewitt, 2004; Willis and Niklas, 2004; Provan and Bennett, 2007; Stewart et al., 2010). In the past, a few paleo-palynological studies have recreated a preliminary vegetation and cli- mate history of India during the Quaternary Period and have described the fate of the Western Ghats and surrounding regions (Singh et al., 1974; Sukumar et al. 1993; Rajagopalan et al., 1997; Meher-Homji and Gupta, 1999). Likewise, a number of studies have analysed pollen deposits from across various regions of India and elucidated local floral assemblages during similar time- scale (e.g. Sharma et al., 2006; Chauhan et al., 2015). A recent study by Bose et al. (2016) re- vealed a local persistence of most of the Western Ghats endemics in the Quaternary and pre- Quaternary Periods, which they attributed to climatic stability sheathing taxa from migration and/or extinction. Altogether, it suggests a serious lack of studies researching the Quaternary history of the Western Ghats biota. Integration of the existing palynological insights with 3 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27043v1 | CC BY 4.0 Open Access | rec: 19 Jul 2018, publ: 19 Jul 2018 knowledge from various disciplines, e.g., insights from genealogical history would help us to understand the persistence of taxa under past climatic events, and may also give clues to their biogeographic evolution. Syzygium travancoricum Gamble is a critically endangered and endemic tree of Western Ghats of family Myrtaceae,(IUCN 2015). This plant is mostly found in Myristica-dominated swamps or forest patches of Western Ghats of Kerala, Karnataka, and Goa states (Sasidharan, 1997; Chan- dran et al., 2008, 2010; Roby et al., 2013; Ray et al., 2014). Paleo niche modelling in a phyloge- ographic framework was undertaken to explain its response and to test the following hypotheses: i) Syzygium travancoricum underwent a southward colonization in the Western Ghats, ii) the Palghat Gap acted as a geographic barrier for this species, and iii) a few areas of Western Ghats acted as glacial refugia to sustain climatic upheaval. Materials andMethods Sampling, DNA Extraction, PCR, and Sequencing Fresh leaf samples were collected from wild populations of Syzygium travancoricum between 2013-15 from various locations across its known distribution (Table 1, Figure 1a-b). We pro- cured necessary permission from Forest Departments for sample collection. We sampled one to ten individuals per population. Fresh leaves were dried and stored at - 20°C. For each sample, approximately 50 mg of leaves were used to extract total genomic DNA ac- cording to a modified CTAB procedure (Doyle and Doyle, 1987). We have amplified and se- quenced two ptDNA (trnH-psbA and rpl20-rps12) and two nrDNA fragments (ITS and ETS) using our designed primers SjITS and SjETS respectively (Table S1). The polymerase chain re- action (PCR) was performed in total volume of 25μL, which contained 30–60ng plant DNA, 4 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27043v1 | CC BY 4.0 Open Access | rec: 19 Jul 2018, publ: 19 Jul 2018 50mM Tris-HCl, 1.5mMMgCl2, 0.5mMdNTPs, 2mM of each primer and 0.75 unit of Taq poly- merase. All reactions were performed using the following temperature profile: 3 min at 94°C, 35 cycles of 1min at 94°C, 1 min of annealing at 58°C, and 2 min at 72°C, with a final 10 min ex- tension at 72°C. Sequencing was performed using an ABI Prism BigDye Terminator Cycle V3.1 Sequencing Kit (Applied Biosystems, Foster City, CA, USA) following the manufacturers’ protocols. All DNA sequences were edited, aligned with ClustalW as implemented in MEGA 5.2, double-checked manually, and insertions/deletions (indels) were excluded. Genetic diversity and Haplotype network construction Sequences were used to estimate the basic population genetic parameters, including the number of segregating sites (S), Watterson’s parameter (θw), nucleotide diversity (π), haplotype number (h), and diversity (He) in DnaSP version 5.00.04 (Librado and Rozas, 2009). For both chloro- plast and nuclear DNA, evolutionary relationships among the individuals were drawn employing median joining algorithm in NETWORK V4.5.1.6 (www.fluxus-engineering.com) (Bandelt et al., 1999). Population and Phylogeographic Structure In order to understand the extent of divergence, the FST value for nrDNA was estimated using an AMOVA as implemented in ARLEQUIN version 3.1.1 (Excoffier and Lischer, 2000). This was done by dividing the total distribution range, taking Palghat Gap into account, and by delimiting populations on either side of it, i.e. central and southern Western Ghats (henceforth CWG and SWG respectively) followed by a further division of CWG and SWG into smaller sub- populations (i.e. G, UK, TH, DK-NK, and CK, SK details of which is provided in table -1) thereby testing hierarchical genetic structure. This enabled us to test the hypothesis that the 5 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27043v1 | CC BY 4.0 Open Access | rec: 19 Jul 2018, publ: 19 Jul 2018 Palghat Gap has been acting as biogeographic barrier for this taxon. Additionally, to test for phy- logeographic structure and to identify the glacial refugia, hT and vT were estimated using PER- MUT (Pons and Petit 1996) using the nuclear markers. Inference and Simulation of Demographic Histories The population history of Syzygium travancoricum was investigated using an Approximate Bayesian Computation (ABC) framework in DIYABC 2.0.4 based on nrDNA sequences (Cor- nuet et al. 2014) (Figure 2) to understand the responses of the species to Quaternary period. The dataset was divided into two groups consisted of combined CWG + CK populations and SK. The rationale of the grouping was largely based on high pair-wise FST values between DK-NK and SK coupled with the minimal effect of Palghat Gap as barrier. We have performed several itera- tions to test the following scenarios: i) simple divergence; ii) divergence with a bottleneck; iii) divergence with expansion; and iv) single population (data not shown), where 1 × 106 data sets for each scenario were simulated (Table S2). The fitness of each scenario was determined stepwise by comparing with the real data sets by estimating errors and precision, best model selection, and finally quantification of the posterior largely following Shang et al. (2015): i) we selected the ‘pre-evaluation scenario-prior combina- tions’ option which was employed to check the similarity of the simulated data with the observed data; ii) type I and type II errors were computed to assess