Phylogeographic Relationships of Scotophilus Kuhlii Between Hainan Island and Mainland China

Mammal Study 37: 139–146 (2012) © The Mammal Society of Japan

Phylogeographic relationships of Scotophilus kuhlii between Hainan Island and mainland China

Wenhua Yu1,3, Zhong Chen3, Yuchun Li2,* and Yi Wu1,* 1 College of Life Science, Guangzhou University, Guangzhou 510006, China 2 Marine College, Shandong University at Weihai, Weihai 264209, China 3 Department of Biology, Hainan Normal University, Haikou 571158, China

Abstract. Phylogeographic analysis of Scotophilus kuhlii between Hainan Island and Guangdong Province was conducted to validate the existence of two subspecies separated by the Qiongzhou Strait. A total of 37 individuals from 3 assumed populations (8 roosts) were examined using 617 bp mtDNA control region segments. Nineteen individuals from cross-strait populations of Hainan Island and the mainland shared 3 of 13 haplotypes. Overall nucleotides diversity was 0.003, a low genetic variation level compared to previous reports for other chiropteran species. The neutral test, mismatch distribution, and star-like TCS network suggested a bottleneck effect occurring at 21,000 years BP during the last glacial maximum and a subsequent population expansion at 7,000 years BP, corresponding with a global warming period. AMOVA analysis, intertwined haplotypes in the TCS network, and rejection of “Isolation by Distance” by the Mantel test indicated no distinct population structure among these populations. Three populations seem to have been derived from a single panmictic unit and the Qiongzhou Strait did not hinder gene flow. Our result, a striking difference in population structure compared with other sympatric chiropteran phylogeographic reports, may indicate variations in the barrier effect of the strait in different species. Based on these results, we suggest that the cross-strait population should be classified as one subspecies, namely S. k. consobrinus, instead of two traditional subspecies.

Key words: Hainan Island, lesser yellow bat, phylogeography, Scotophilus kuhlii, subspecies.

Islands have been described as valuable natural laborato- graphic barrier of the Qiongzhou Strait, comprising 22% ries in the macroevolution and speciation of organisms of chiropteran species of Hainan (Wang 2003). However, (e.g., Darwin 1859; Wallace 1880; MacArthur and most of these subspecies lack validation from phylogeo- Wilson 1967; Diamond 1975). Indeed, island popula- graphic or metrical evidence. Several phylogeographic tions may be less affected by gene flow from continental reports have examined the barrier effect of the Qiong- populations due to the barrier effect of a strait, and zhou Strait in cross-strait populations, such as Grey- rapidly evolve into new subspecies or new species cheeked fulvetta (Alcippe morrisonia) (Zou et al. 2007; under the effective and sufficient time scale of geograph- Song et al. 2009), spiny tree fern (Alsophila spinulosa) ic segregation, genetic drift and mutations (Phillimore (Su et al. 2005), Japanese Pipistrelle (Pipistrellus abra- et al. 2008). mus) (Wei et al. 2010), and intermediate horseshoe bat Hainan Island is the second largest island in China, (Rhinolophus affinis) (Mao et al. 2010). However, these with an area of 34,000 km2, separated from mainland species are all of low dispersal capability; a different China by the Qiongzhou Strait, which is 18–33.5 km in outcome might be expected in examining the genetic width (Yan 2008). Hainan Island is one of the valuable structure of species with strong dispersal capability. tropical areas of China, with the highest abundance of The lesser Asiatic yellow bat (Scotophilus kuhlii), a chiropteran species (Li et al. 2005). Traditionally, 7 vespertilionid species occupying Hainan Island and chiropteran species in Hainan Island have been described southern China (Zhang et al. 1997; Wang 2003), is of as geographic subspecies due to the presumed biogeo- high wing loading and moderately high aspect ratio,

*To whom correspondence should be addressed. E-mail: [email protected] or [email protected] 140 Mammal Study 37 (2012) which implies their capability for long distance and Our purposes were: 1) to examine the population genetic steady flight (Norberg and Rayner 1987; Kitchener et al. structure and evolutionary history in cross-strait popula- 1990; Kunz and Fenton 2003; Pottie et al. 2005). This tions of S. kuhlii; 2) to certify the validity of subspecies species could be a good indicator for the examination of differentiation of S. kuhlii on both sides of the Qiong- the variations of the strait barrier effect compared to bats zhou Strait by phylogeographic perspective. with low dispersal capability. Furthermore, their subspe- cies taxonomy is still the subject of debate. Wang (2003) Materials and methods suggested that there are 3 subspecies occupying southern areas of China: Huanan subspecies (S. k. swinhoei Blyth, Samples and DNA extraction 1860), distributed in Guangdong, Guangxi, Fujian, and A total of 37 specimens of S. kuhlii were collected in Hong Kong; Hainan subspecies (S. k. consobrinus Allen, Guangdong and Hainan from 1999 to 2007 (Fig. 1). All 1906), distributed in Hainan (type locality) and Taiwan samples were preserved in 70–100% ethanol for use. For Island; and Thailand subspecies (S. k. gairdneri Kloss, the mainland specimens, we assigned specimens from 1917), distributed in the southern part of Yunnan. How- Guangzhou, Huizhou, Zhongshan, and Longmen to one ever, Smith and Xie (2008) argue that there is only one population, namely the Pearl River Delta population subspecies (S. k. consobrinus) in China. (abbreviated as PRD) because of close distances <200 In this study, we used the control region of mitochon- km among these sites, while specimens from Gaozhou drial DNA (mtDNA) to assess the genetic variation were assigned to Gaozhou population (GZ). Specimens between the populations of Hainan Island and the nearby from Hainan Island were assigned to Hainan population Chinese mainland separated by the Qiongzhou Strait. (HN).

Fig. 1. Sampling locales of Scotophilus kuhlii. The specimen number is showed for each locale, and the number of female individual of each locale is showed in parentheses. Yu et al., Phylogeography of S. kuhlii in China 141

DNA sequencing and data analysis The goodness-of-fit test based on the sum of squared Genomic DNA was isolated from approximate 20 mg deviations (SSD) was performed to test for the signifi- of muscle tissue using the Universal Genomic DNA cance of fit of distribution by parametric bootstrapping Extraction Kit (TAKARA). A partial segment of mtDNA (10,000 replicates). For smooth and unimodal distribu- control region was amplified by polymerase chain reac- tion where an expansion model could not be rejected, we tion (PCR) using the primers C (5'-TGA ATT GGA further estimated the time of the expansion from the rela- GGA CAA CCA GT-3') and primer E (5'-CCT GAA tionship τ = 2ut, where u is the mutation rate per locus GTA GGA ACC AGA TG-3') (Wilkinson and Chapman per generation. We used a divergence rate of 20% per 1991). This portion of the control region spans the million years (Ma), which had been widely used in phy- hypervariable domain (HVI), which is of proven impor- logeographic analyses of other bats (Petit et al. 1999; tance in the study of intraspecific variation (Vigilant et Salgueiro et al. 2004; Chen et al. 2006; Mao et al. 2010), al. 1991; Wilkinson and Fleming 1996). PCRs were and a generation time of 1 year based on recapture exper- carried out in a final volume of 50 μl, containing iment and field observations of S. kuhlii in Guangdong approximately 5.0–50 ng DNA, 0.2 mM of each dNTP, from 2003 to 2005. This calibrated rate is consistent 0.4 mM of each primer, 1.5 mM MgCl2 and 2.0 U Taq with several other studies in mammalian species (see polymerase (TAKARA) in the manufacturer’s buffer. Petit et al. 1999) and should provide a rough estimate of Amplification was performed using a MyCycler Thermal divergence time even though it is not so unique at some Cycler (BioRad) with the following profile: 94°C 4 min; extent. Second, we derived Tajima’s D (Tajima 1989) 37 cycles of 94°C for 30 sec, 50°C for 30 sec, 72°C for and Fu’s Fs (Fu 1997) statistics in ARLEQUIN 3.01 and 1 min; 72°C for 5 min. DNA sequencing was performed assessed their significance by simulation (10,000). on an ABI PRISM 3700 DNA Analyser (Applied Bio- Third, we used Bayesian MCMC approach implemented systems). The chromatograms were edited with BIO- in BEAST 1.43 (Drummond and Rambaut 2007) to esti- EDIT (Hall 1999) and aligned using CLUSTAL_X mate the time to the most recent common ancestor (Thompson et al. 1997). (TMRCA) of the whole population. We applied an The number of haplotypes, haplotype diversity (h), HKY model of evolution based on MODELTEST 3.06 and nucleotide diversity (π) for the 3 populations were (Posada and Crandall 1998), and a relaxed-clock model calculated using ARLEQUIN 3.01 (Excoffier et al. with an uncorrelated lognormal distribution for the sub- 2005). In order to estimate the partitioning of genetic stitution rate. We performed two independent runs for variation among 3 different populations and between 2 10 million generations, each with a burn-in of 1 million subspecies, a hierarchical analysis of molecular variation generations, and sampled every 1,000 steps. Both results (AMOVA) (Excoffier et al. 1992) was performed with were then combined in TRACER 1.4 (Rambaut and 1,000 permutations in ARLEQUIN (Excoffier et al. Drummond 2007). To convert the estimates scaled by 2005). Additionally, we used SAMOVA (Dupanloup et mutation rate to calendar years, we used the divergence al. 2002) to determine the maximum partitioning of rate of 20% Ma as applied in the above time estimation. genetic variation. We also calculated pairwise FST and Nm values across all populations using DNASP 4.10 Results (Rozas et al. 2003), and tested for genetic isolation by distance among 7 sampling locales using the Mantel test Sequence characteristics and genetic diversity as implemented in ARLEQUIN (Excoffier et al. 2005). In total, we identified 13 unique haplotypes from 37 The gene genealogy was examined by constructing a par- Scotophilus kuhlii individuals, based on 617 base pairs of simony haplotype network using the TCS 1.21 software the mitochondrial control region. The most frequent (Clement et al. 2000). Because no significant difference haplotype was H2, which was shared by 13 individuals in genetic structure occurred in the whole population, we from PRD and HN populations (Table 1). The values of inferred the demographic history of the whole population nucleotide diversity (π) of the 3 populations were low, instead of making inferences for each population. Three ranging from 0.002 to 0.003 (Table 1). Nine polymor- methods were used for inferring the demographic his- phic sites were recorded, and no insertions or deletions tory. First, a mismatch distribution was determined were observed. No trend for finding more shared haplo- according to the sudden expansion model (Rogers and types among the closer locales was observed. Further- Harpending 1992) in DNASP 4.10 (Rozas et al. 2003). more, haplotypes H2, H8, and H9 were found in HN and 142 Mammal Study 37 (2012)

Table 1. Haplotype diversity, nucleotide diversity and haplotypes in different populations of Scotophilus kuhlii

Number of Haplotype diversity Nucleotide diversity Population N NFa Haplotype (number of individuals) Haplotypes (h ± SD) (π ± SD) Pearl River Delta 21 12 9 0.852 ± 0.055 0.003 ± 0.002 H01 (1), H02 (7), H03 (1), H04 (3), H05 (4), H08 (2), H09 (1), H10 (1), H11 (1) Gaozhou 6 5 3 0.600 ± 0.215 0.002 ± 0.002 H06 (1), H07 (4), H08 (1) Hainan 10 6 5 0.667 ± 0.163 0.002 ± 0.002 H02 (6), H08 (1), H09 (1), H12 (1), H13 (1) a The number of female specimen used in this study.

Table 2. AMOVA of genetic variation in Scotophilus kuhlii populations

Structure Source of variation Variation (%) Fixation indices P Hainan group vs Guangdong group Among regions –22.75 –0.228 0.66 (including PRD & GZ) Among populations/within regions 34.60 0.282 <0.05* Within populations 88.16 0.118 <0.05* No group Among populations 17.79 0.178 <0.01** Within populations 82.21 Gaozhou population vs other populations Among regions 22.69 0.227 0.35 (including PRD & HN) Among populations/within regions 5.00 0.065 0.07 Within populations 72.32 0.277 <0.01** * Statistically significant difference (P < 0.05). ** Statistically significant difference (P < 0.01).

Table 3. FST (below diagonal) and corresponding gene flow (Nm, In summary, AMOVA results did not support the divi- above diagonal) correspond to pairwise comparisons between Scotophilus sion of two unique subspecies on each side of the strait, kuhlii populations and further revealed a genetic pattern which is not con- Pearl River Delta Gaozhou Hainan sistent with the current geographic distribution. population population population (PRD) (GZ) (HN) Pairwise results of the genetic differentiation index PRD 0.600 3.410 (FST) and the gene flow values (Nm) of populations were in accordance with the AMOVA results (Table 3). High GZ 0.294* 0.570 and significant pairwise FST values were emerged under HN 0.068 0.305* the comparisons between GZ and other two populations * Statistically significant difference (P < 0.05). (0.294 for GZ vs PRD at P < 0.05 and 0.305 for GZ vs HN at P < 0.05), while the comparisons between PRD PRD populations, which are separated by the Qiongzhou and HN, which are geographically the furthest popula- Strait with a distance more than 700 km. tions in our study, showed low nonsignificant FST values (0.068, P > 0.05), and high gene flow values (Nm = Analysis of population structure 3.410). Results of the Mantel test, which rejected the The hierarchical AMOVA test under different group hypothesis of isolation by distance, indicated a non- settings revealed that high proportions of the total vari- significant correlation between geographic distances and ance were attributable to the variance within populations genetic distance of 8 sample locales (r = 0.202, P = (Table 2). Under the group setting according to the tradi- 0.214). tional distributions of two subspecies and no group A 95% statistical parsimony haplotype network setting, the proportions of variances within populations yielded a star-like and intertwined cladogram, in which 1 were 88.16% (P < 0.05) and 82.21%, respectively. or 2 mutation steps connected all haplotypes with each These findings indicated that the genetic differentia- other parsimoniously (Fig. 2). The most frequent haplo- tion between Hainan subspecies (Scotophilus kuhlii type H02 was the central one in the cladogram, and all consobrinus Allen, 1906) and Huanan subspecies (S. other haplotypes were derived from it. The pattern of the k. swinhoei Blyth, 1860) was low and nonsignificant. network was not in accord with their geographical pat- Yu et al., Phylogeography of S. kuhlii in China 143

smooth and unimodal mismatch distribution (Fig. 3), a significantly negative value of Fu’s Fs value (Fs = –6.723, P < 0.001), and a negative value of Tajima’s D (D = –1.634, 0.05 < P < 0.1) suggested that the entire population has experienced population expansion in the past, and the estimated time of the expansion was ap- proximate 7,000 years BP (95% CI, 3,800–10,000 years BP). Bayesian estimate of TMRCA provided reliable estimates with the ESS > 100. The inferred TMRCA for all sequences was approximate 21,000 years BP (95% CI, 7,500–38, 000 years BP).

Discussion

The mitochondrial control region, a rapid evolving Fig. 2. TCS network of haplotypes on the sequences of control segment, is useful for chiropteran intraspecies phylogeo- region of Scotophilus kuhlii. graphic study because it contains a great deal of genetic information (Worthington et al. 1999; Petit et al. 2000; Ruedi and Castella 2003; Russell et al. 2005; Chen et al. 2006; Ruedi et al. 2008; Mao et al. 2010). Nucleotide diversity (π) of this segment is an important index for the evaluation of intraspecies genetic diversity. In this study, our results revealed a lower genetic variability (ranging from 0.002 to 0.003) in Scotophilus kuhlii populations than other reported chiropteran species, such as Curaçaoan long-nosed bat (Leptonycteris curasoae) (0.006, Wilkinson and Fleming 1996), ghost bat (Macro- derma gigas) (0.007, Worthington et al. 1999), New Zealand lesser short-tailed bat (Mystacina tuberculata) (0.036, Lloyd 2003), least horseshoe bat (Rhinolophus monoceros) (0.013, Chen et al. 2006), intermediate horseshoe bat (R. affinis) (0.041, Mao et al. 2010), Bra- Fig. 3. The mismatch distribution of Scotophilus kuhlii. The black bars are the observational frequencies; the gray bars are the expecta- zilian free-tailed bat (Tadarida brasiliensis mexicana) tion frequencies. (0.045, Russell et al. 2005), noctule (Nyctalus noctula) (HVII) (0.011, Petit et al. 2000), and greater mouse- eared bat (Myotis myotis) (HVII) (0.006, Ruedi and tern of populations or sampling locales. Furthermore, Castella 2003). The low nucleotide diversity and mod- although the results of AMOVA and population genetic erate haplotypes diversity detected may be indicative of differentiation indicated a greater genetic differentiation a bottleneck effect to their ancestral population, great between GZ and the other 2 populations, the pattern of genetic flow among the locales or a short evolutionary GZ haplotypes did not show any evidence of separated history of a small effective population. structure. The haplotypes from different populations The Bayesian estimate of TMRCA for all sequences were intertwined. The results suggested that the 3 S. was 21,000 years BP, corresponding broadly to a period kuhlii populations did not have a distinct population of the last glacial maximum (LGM) in Holocene. During structure, and all groups might be derived from one this period, the eastern Asiatic zone experienced a cooler ancestral panmictic population and/or behave as a pan- and drier climate than today, leading to the displacement mictic unit. of moist forest by xerophytic vegetation (Chen 1984; Our mismatch distribution was unable to reject a Huang and Zhang 2007; Clark et al. 2009). According to sudden expansion model (SSD value P > 0.05). A our findings of low genetic diversity and unimodal mis- 144 Mammal Study 37 (2012) match distribution (Slatkin and Hudson 1991; Rogers S. kuhlii in Guangzhou and Longmen during 2003–2007 and Harpending 1992), the common ancestral population also found that S. kuhlii in these areas left their summer of the 3 populations seems to have suffered a bottleneck habitats in autumn and returned the next spring. It is due to the climate oscillations in Holocene, especially reasonable to assume that they spent their winter in a the cold impact of the LGM on them and/or indirectly warmer place, such as Hainan Island. Moreover, in our through their habitat resources. After deglaciation, cli- feeding experiences we observed that the captured mate returned to a warmer and moister state at around females which were subsequently fed separately were all 7,500–10,000 years BP (Dawson 1992; Zhao and Wang pregnant, which indicates that they copulated with males 2007), and this provided an important chance for post- before their returning to summer habitat from wintering glacial Scotophilus kuhlii to recover and expand due to areas or hibernaculums. These habits are often found in the availability of better food resources and an improved seasonal migratory chiropteran species, such as Schreiber’s habitat than in the LGM. The expansion time, which long-fingered bat (Miniopterus schreibersii), Curaçaoan occurred about 7,000 years BP, broadly corresponds with long-nosed bat (Leptonycteris curasoae), and Brazilian this global warming period. free-tailed bat (Tadarida brasiliensis) (Kunz and Fenton Although the pairwise FST values revealed a significant 2003), and suggest that S. kuhlii should be one kind of genetic differentiation of GZ population from the rest of migratory species. In fact, as this study revealed, the two populations (Table 3), no separated population struc- lack of distinct genetic population structures among the 3 ture was found from our TCS network (Fig. 2). Given to populations implies that S. kuhlii may migrate between these results, the significant FST values seems be due to Hainan Island and the mainland across the Qiongzhou our insufficient sample size from this locale or its special Strait. In other words, the groups of S. kuhlii summering geographic site where there may be a mix with western in Hainan Island and the mainland (PRD and GZ) would lineages rather than the distinct and separated lineage have gene flow when sharing the same or nearby winter- from PRD and HN populations. In fact, no distinct popu- ing areas or hibernaculums, which is a common habit of lation structure from TCS network, AMOVA or Mantel species that migrate seasonally (Kunz and Fenton 2003). test are consistent with the phylogeographic report of Therefore, we suggest that the populations examined in Scotophilus kuhlii in the Indonesian archipelago using this study should behave as a panmictic unit, and the allozyme as a genetic marker (Hisheh et al. 2004), and independent subspecies on each side of Qiongzhou Strait indicate that cross-strait populations should derive from are not valid and should be classified as the same sub- a single panmictic unit and/or behave as a panmictic species. Although the S. k. swinhoei Blyth, 1860 is a unit. We suggest that this result is due to the complex valid and early scientific name, Blyth’s description of paleogeographic history of Qiongzhou Strait and the Nycticejus swinhoei seems to be unidentifiable and the biological and behavioral traits of S. kuhlii. First, the type specimen appears to have been lost (Smith and Xie Qiongzhou Strait grew out from a normal low land, and 2008). The insistency of this subspecific name would Hainan Island was connected with the Leizhou Peninsula cause the inconvenience and difficulty in examining or several times due to cycles of glaciations. The most comparing the type specimen in the feature. Given that, recent connection was from 24,000 to 8,000 years BP we propose the name of S. k. consobrinus Allen, 1906 for due to a 100 meter decrease in the sea level (Zhao et al. them; however, their Chinese name should be modified 1999; Zhao and Wang 2007; Yao et al. 2009); therefore, from Hainan subspecies to Huanan subspecies to avoid the cross-strait populations of S. kuhlii should have been taxonomic confusion. able to have gene flow during this period. Second, S. To verify the efficiency of a biogeographic barrier, kuhlii has been described as a strong flier (Medway animal banding or radio telemetry tracing may be the 1978; Kitchener et al. 1990), with a high wing loading most straightforward methods, but they consume a lot of and a moderately high aspect ratio, which is often associ- time and resources, and are quite difficult in practice. ated with fast, long distance air flight because of better Recently, phylogeographic analysis has replaced these aerodynamic efficiency (Norberg and Rayner 1987; methods. The phylogeographic research of Grey-cheeked Pottie et al. 2005). Such features imply that S. kuhlii fulvetta (Alcippe morrisonia) revealed an independent should be able to fly across the Qiongzhou Strait, genetic population structure between populations on because the narrowest width of the strait is only 14 km. each side of Qiongzhou Strait (Zou et al. 2007; Song et Third, our field observations and banding research of al. 2009), and similar results were also observed in Japa- Yu et al., Phylogeography of S. kuhlii in China 145 nese Pipistrelle (Pipistrellus abramus) (Wei et al. 2010) Science (JSPS) (30811140092), NSFC research grants and intermediate horseshoe bat (Rhinolophus affinis) (30670277), JSPS AA Science Platform Program, Natural (Mao et al. 2010). These sympathetic phylogeographic Science Foundation of Guangdong (8151009101000005), reports revealing separated population structure indicate the Research Innovation Program for College Graduates a barrier effect of Qiongzhou Strait on these species with of Jiangsu Province (CXZZ11_0883) and Natural Sci- low dispersal capability. However, our study revealed a ence Foundation of Hainan Province of China (311049). strikingly different result, in which the cross-strait popu- lations were of low genetic variations and behaved as a References single panmictic unit. There are two alternative explana- tions for these findings. One is that our unique genetic Chen, S. F., Rossiter, S. R., Faulkes, C. G. and Jones, G. 2006. Popu- structure is attributed to the short time for cross-strait lation genetic structure and demographic history of the endemic populations to accumulate genetic variations after colori- Formosan lesser horseshoe bat (Rhinolophus monoceros). 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