Molecular Phylogenetics and Evolution 85 (2015) 238–246 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Repeated range expansions and inter-/postglacial recolonization routes of Sargentodoxa cuneata (Oliv.) Rehd. et Wils. (Lardizabalaceae) in subtropical China revealed by chloroplast phylogeography Shuang Tian a,b,c,1, Shu-Qing Lei b,1, Wan Hu a,b,1, Ling-Li Deng b,BoLib, Qing-Lin Meng b, ⇑ ⇑ Douglas E. Soltis d,e, Pamela S. Soltis e, Deng-Mei Fan b, , Zhi-Yong Zhang b, a College of Forestry, Jiangxi Agricultural University, 330045 Nanchang, Jiangxi, China b Laboratory of Subtropical Biodiversity, Jiangxi Agricultural University, 330045 Nanchang, Jiangxi, China c Jiangdezhen College, 333000 Jingdezhen, Jiangxi, China d Department of Biology, University of Florida, Gainesville, FL 17 32611, USA e Florida Museum of Natural History, University of Florida, Gainesville, FL 17 32611, USA article info abstract Article history: Most plant phylogeographic studies in subtropical China have stressed the importance of multiple refugia Received 18 December 2014 and limited admixture among refugia. Little attention has been paid to range expansion and recoloniza- Revised 15 February 2015 tion routes in this region. In this study, we implemented a phylogeographic survey on Sargentodoxa Accepted 18 February 2015 cuneata, a widespread woody deciduous climber in subtropical China to determine if it conforms to Available online 27 February 2015 the expansion–contraction (EC) model during the Pleistocene. Sequence variation of two chloroplast intergenic spacers (IGSs) in 369 individuals from 54 populations of S. cuneata was examined. Twenty- Keywords: six chloroplast haplotypes were recovered. One of these (H5) occurred across the range of S. cuneata Range expansion and was absent from only 13 populations. Sixteen of the 26 haplotypes were connected to H5 by one Recolonization Chloroplast mutation and displayed a star-like pattern in the haplotype network. All chloroplast haplotypes clustered Phylogeography into two lineages (A and B) in a Bayesian tree, and most haplotypes (18 out of 26) originated during the mid-Pleistocene (0.63–1.07 Ma). Demographic analyses detected a recent range expansion that occurred at 95.98 ka (CI: 61.7–112.53 ka) for Lineage A. The genetic signature of an ancient range expansion after the Middle Pleistocene Transition (MPT) was also evident. Three recolonization routes were identified in subtropical China. The results suggest that temperate plants in subtropical China may conform to the EC model to some extent. However, the genetic signature from multiple historical processes may complicate the phylogeographic patterns of organisms in the region due to the mild Pleistocene climate. This study provides a new perspective for understanding the evolutionary history of temperate plants in subtropical China. Ó 2015 Elsevier Inc. All rights reserved. 1. Introduction areas of both continents uninhabitable (reviewed by Hewitt, 1996, 2000; Shafer et al., 2010; Soltis et al., 2006). During glacial periods, Most extant plant species from the Northern Hemisphere have temperate species of these regions were driven southward but persisted through glacial–interglacial cycles over the past 2.5 then expanded their distributions northward during warmer million years. Range shifts are the most conspicuous response of interglacial/postglacial periods. However, the range shift of plant taxa to Pleistocene climatic fluctuations (Davis and Shaw, temperate plants in subtropical China is much more complex, only 2001), especially for temperate species in Europe and North several cases found regional expansions (e.g., Lei et al., 2012; Qi America, where the development of massive ice sheets made large et al., 2012; Sun et al., 2014) but few conform to a range-wide expansion–contraction (EC) model that is typical for many species from Europe and North America (Harrison et al., 2001; Liu et al., ⇑ Corresponding authors at: No. 1101, Zhimin Road, Nanchang, Jiangxi 330045, 2012; Ni et al., 2010; Qiu et al., 2011). PR China. Fax: +86 791 83813047. The hilly mid-elevation area of eastern China between the E-mail addresses: [email protected] (D.-M. Fan), [email protected] Qinling Mts. Huai River line (at C. 34°N) and the tropical South (Z.-Y. Zhang). 1 6 These authors contributed equally. ( 22°N), and bordered by the Qinghai-Tibetan Plateau (105°E) in http://dx.doi.org/10.1016/j.ympev.2015.02.016 1055-7903/Ó 2015 Elsevier Inc. All rights reserved. S. Tian et al. / Molecular Phylogenetics and Evolution 85 (2015) 238–246 239 the west and the coastline in the east, is generally referred to as extant member, S. cuneata (Oliv.) Rehd. et Wils., is a deciduous ‘subtropical China’ (Zhao, 1986). This area now harbors warm- woody climber, mostly confined to subtropical China with occa- temperate evergreen forests (i.e., subtropical evergreen broad- sional occurrences in Laos and northern Vietnam (Chen and leaved forests) which are interspersed with warm-temperate Tatemi, 2001). Plants of S. cuneata frequently grow in different deciduous forests (Harrison et al., 2001; Wu, 1980). Although sub- types of forests with sufficient sun and in thickets at forest margins tropical China was probably not glaciated during the Pleistocene from 130 m to 2400 m above sea level (a.s.l., Ying et al., 1993). This (Shi, 1998), the region underwent profound climate changes species bears fleshy, dark blue berries (Chen and Tatemi, 2001), throughout the Pleistocene. At the Last Glacial Maximum (LGM), which may facilitate its dispersal by birds. Given the wide dis- for example, the climate of this region was cooler by c. 4–6 °C tribution of the species in subtropical China, its occurrence in and drier by c. 400–600 mm/yr (Qiu et al., 2011). Paleoecological diverse habitats, and efficient seed-dispersal, S. cuneata may repre- reconstructions based on fossil pollen showed that marked climate sent a good system to test if there are some temperate plants that change caused warm-temperate evergreen forests to retreat south- conform to the EC model in subtropical China. If so, this species ward c. 1000 km relative to today (Harrison et al., 2001; Ni et al., would provide a good opportunity to identify the recolonization 2010; Yu et al., 2000). However, the temperate deciduous forests routes in subtropical China following the Pleistocene glaciations. retreated to the continental mountains and were separated by We investigated the phylogeographic structure of S. cuneata in more cold-tolerant biomes in the lowlands, such as boreal, cool- subtropical China using chloroplast DNA sequence data. The main temperate coniferous, and mixed (boreal conifers with the most objectives were: (i) to examine the phylogeographic structure of S. cold-tolerant temperate taxa) forests. After the Holocene, warm- cuneata; (ii) to investigate signals of range expansion(s) and to temperate evergreen forests re-colonized the mid-latitude lowland attempt to peel through different layers of historical processes; areas up to 30/33°N. The expansion of evergreen forests displaced and (iii) to identify potential recolonization routes in subtropical temperate deciduous forests over the same area, resulting in cur- China for temperate plant species. rently isolated patches of temperate deciduous forest at both higher elevations and more northern latitudes (Harrison et al., 2. Materials and methods 2001; Ni et al., 2010; Yu et al., 2000). The history of temperate deciduous forests in subtropical China 2.1. Population sampling and experimental procedures indicates that temperate plant species in this region may have been isolated during both glacial and inter-/postglacial stages, We made extensive field surveys throughout subtropical China giving rise to an expected phylogeographic pattern of high diver- from 2010 to 2014 and collected leaf samples from 584 individuals sity among populations due to genetic drift and limited genetic in 54 populations (Fig. 1 and Supplementary Table 1). Because the admixture between presently disjunct populations. A few recent species is a very long vine (up to five meters or more), sampled studies (e.g., Gong et al., 2008; Lei et al., 2012; Qiu et al., 2009; individuals were spaced by ca. 20–100 m to avoid repeatedly col- Zhang et al., 2013) have suggested that the phylogeographic struc- lecting the same individual. All samples were desiccated in silica tures of temperate plants in subtropical China are indeed different gel and stored at À20 °C until being processed. Genomic DNA from those of their counterparts in North America and Europe. was extracted using a modified CTAB procedure. These findings match the predictions of high population differ- After a screening of a dozen of intergenic spacers (IGSs) of the entiation and limited genetic admixture due to long-term pop- chloroplast genome, two IGSs, atpI–atpH and trnS–trnG, had high ulation isolation (Harrison et al., 2001; Ni et al., 2010; Yu et al., sequence variation and were selected as molecular markers for this 2000). However, both paleoecological reconstructions and previous study. PCR amplification reactions using previously reported phylogeographic studies have limitations, which may lead to an primers (Shaw et al., 2007) were carried out in a volume of 20 ll incomplete understanding of the evolutionary history of temperate containing 10 ll2Â Taq PCR MasterMix (Tiangen, Shanghai, plants in subtropical China. First, paleoecological reconstructions China), 1 ll each forward and reverse primer (0.2 lM), 1 ll tem- of entire forest biomes cannot provide a detailed picture of past plate DNA (ca. 50–100 ng) and 7 ll ddH O. Amplification was car- fragmentation, admixture, and/or expansion of populations of par- 2 ried out in a Bioer XP cycler (Bioer, Hangzhou, China) programmed ticular species (Qian and Ricklefs, 2001). Second, fossil pollen for an initial 240 s at 94 °C, followed by 30 cycles of 60 s at 94 °C, records used for such biome reconstructions are largely restricted 60 s at 54 °C(atpI–atpH)or57°C(trnS–trnG), 60 s at 72 °C, and a to the most recent (LGM/Holocene) time periods and are limited final 600 s at 72 °C.