Genetic Characteristics of the Japanese Serow Capricornis

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Genetic Characteristics of the Japanese Serow Capricornis crispus in the Kii Mountain Range, Central Japan Authors: Arisa Iwahori, Jyun-ichi Kitamura, and Kouichi Kawamura Source: Zoological Science, 36(4) : 306-315 Published By: Zoological Society of Japan

URL: https://doi.org/10.2108/zs180187

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Downloaded From: https://bioone.org/journals/Zoological-Science on 07 Aug 2019 Terms of Use: https://bioone.org/terms-of-use Access provided by Zoological Society of Japan ZOOLOGICAL306 SCIENCE 36: 306–315 (2019) A. Iwahori et al. © 2019 Zoological Society of Japan

Genetic Characteristics of the Japanese Serow Capricornis crispus in the Kii Mountain Range, Central Japan

Arisa Iwahori1, Jyun-ichi Kitamura2, and Kouichi Kawamura1*

1Faculty of Bioresources, Mie University, Kurimamachiya 1577, Tsu, Mie 514-8507, Japan 2Mie Prefectural Museum, 3060 Isshinden-kouzubeta, Tsu, Mie 514-0061, Japan

The Japanese serow, Capricornis crispus, is an indigenous bovid species exclusively inhabiting mountain regions in the main Japanese islands, excepting Hokkaido. It had decreased in abundance to its lowest level due to overhunting and deforestation, with its distribution severely fragmented from the middle of the 20th century, many populations of C. crispus currently facing the risk of extinction. The Kii Mountain Range (KM) on Honshu is one such location that has seen a drastic population decline of C. crispus. In this study, we examined genetic characteristics of C. crispus in KM and neighboring regions of the Chubu district, using mtDNA and microsatellite markers, in order to devise strategies for its conservation. Results for mtDNA were characterized by low nucleotide diversity with five endemic and two dominant haplotypes shared by individuals in neighboring regions. A Bayesian skyline plot indicated a gradual increase after the last glacial maximum. For microsatellites, the genetic diversity of C. crispus in KM was comparable to Shi- zuoka and higher than Shikoku. Recent genetic bottlenecks were strongly suggested in C. crispus in KM. Bayesian clustering showed a genetic cline between KM and neighboring regions, where multivariate analysis suggested three local populations. A Mantel test indicated male-biased dispersal. These results indicate that C. crispus in KM and neighboring regions constitute multiple local populations, connected through restricted gene flow. For the conservation of C. crispus, it is important to define small-scale conservation units, among which genetic connectivity should be facilitated to prevent further loss of genetic diversity.

Key words: Capricornis crispus, conservation unit, dispersal, genetic bottleneck, genetic cline

decrease of C. crispus to the lowest levels (Ochiai, 2016). In INTRODUCTION 1955 the species was designated as a “Special National The Japanese serow, Capricornis crispus, is an indige- Monument of Japan” by the Japanese Government. Owing nous bovid species in Japan. Capricornis species are widely to the subsequent strict protection, some populations of C. distributed in East and Middle Asia, ranging from the tropical crispus on Honshu showed signs of recovery. However, fol- to the subarctic zone (Ochiai, 2016). The genus was formerly lowing increases problems associated with damage to agri- considered to be primitive in the subfamily Caprinae, belong- cultural and forestry sites emerged in some prefectures, ing to the tribe Rupicaprini (Simpson, 1945). However, recent such as Gifu and Aichi in central Honshu (Ochiai, 2016). molecular studies suggest that Capricornis is not a member Consequently, in 1979 the Japanese government desig- of the Rupicaprini, but genetically related to species of the nated legally protected areas for C. crispus while allowing tribe Ovibovini (Ropiquet and Hassanin, 2009). The genus culling for population control in other regions under special Capricornis comprises three species, C. sumatraensis, C. license (MEGJ, 2010). Populations in Shikoku and Kyushu swinhoei and C. crispus (Novak, 1999). Although C. swinhoei have remained small and fragmented, and continue to face in Taiwan had long been considered to be closely related to the risk of extinction (Okumura, 2003). C. crispus, recent studies using mtDNA have shown it to be The Kii Mountain Range (KM) in central Honshu is one a sister species of C. sumatraensis (Min et al., 2004). of the principal habitats of C. crispus. In this region, compre- Formerly, C. crispus was widely distributed in mountain- hensive research on C. crispus has been periodically con- ous regions of the main Japanese islands, excepting ducted since 1986 by the collaboration of Mie, Nara and Hokkaido (Ochiai, 2016). However, hunting and deforesta- Wakayama Prefectures to elucidate the ecology, distribution tion resulted in its decline, resulting not only the fragmenta- and population dynamics of the species (PBEMNW, 2010). tion of populations but also their disappearance from Based on the results of the first tranche of research in 1986– Chugoku, Fukuoka and Izu districts. Indiscriminate hunting 1987, KM was registered as a protected area for C. crispus in the 19th and 20th centuries in particular drove the in 1989. However, the fourth phase of research on C. crispus in the KM protected area (FSRCPK, conducted in 2008– * Corresponding author. E-mail: [email protected] 2009) reported a decrease in numbers and the fragmenta- doi:10.2108/zs180187 tion of habitat, despite an increase in the distribution of C.

crispus. Results also suggested that KM supported the low- prior to DNA extraction. DNA was extracted using DNeasy Blood est population density of C. crispus among the protected and Tissue Kit (Qiagen, USA). areas of C. crispus in Japan, along with the Southern Ou- Sex of collected individuals was primarily judged from external Mountain Range (PBEMNW, 2010). genital features. For sexually ambiguous individuals, PCR-based sex identification was employed as described below. Most collected In an analysis of the cytochrome b region of the mtDNA individuals were adult, and only three individuals (two females and of C. crispus (sequence data not published), PBEMNW one male) in KM were young (less than three years), judging from (2010) reported that the dominant haplotype of C. crispus in horn size (Kishimoto, 1988). KM was found in individuals from Gifu, Shizuoka and Yamagata Prefectures, although some haplotypes were PCR-based Sex identification endemic to KM. These findings suggested that C. crispus in For sexually ambiguous individuals (n = 30) PCR-based sex KM and that of neighboring regions were not genetically identification was used. In mammals, the amelogenin gene is separated, but were connected through a small number of widely used for sex identification, because it has a locus on both occasional migrants. Yamashiro and Yamashiro (2012) com- sex chromosomes (X and Y) and respective base sequences (Pfei- pared the genetic diversity of C. crispus in Shikoku, KM ffer and Brenig, 2005). This gene was found to be effective for the sex identification of C. crispus (Nishimura et al., 2010). We ampli- (including the Shiga population) and Shizuoka, using five fied the amelogenin gene via PCR, using the primer pairs: SE47 microsatellite markers (genotype data not published). They and SE38 and PCR protocols described by Nishimura et al. (2010). reported that the Shikoku population expressed the lowest The PCR products were electrophoresed on 2% agarose gel with genetic diversity, while the other two populations showed TAE buffer. The gel was stained by GelStar nucleic acid stain (FMC approximately the same level of genetic diversity. In the pre- Fig. 1 IwahoriBioproducts, et al. USA) and visualized under UV illumination to identify ceding studies of C. crispus (e.g., PBEMNW, 2010; Nishimura et al., 2011; Yamashiro and Yamashiro, 2012), genetic information was exclusively restricted to one kind of genetic N marker, which is insufficient for the elucidation of the genetic 36°N characteristics of the species (Gompert et al., 2006). Hokkaido HM In conservation management, it is important to clarify Japan the genetic characteristics of the targeted species, because it enables inference of their genetic structure, genetic rela- Yamagata Gifu tionships and demographic history. Such information is especially indispensable for the identification of conserva- Honshu tion units, which are a requisite for the design of a rational Shizuoka conservation plan (Allendorf et al., 2013). In the estimation of Shikoku conservation units of endangered species, the use of multi- Kyushu Kii Peninsula SM Aichi ple genetic markers with different modes of inheritance, Siga 35°N such as mtDNA and microsatellite markers, is strongly recommended (Gompert et al., 2006). Discordance of conser- Ise vation units between markers, mainly due to the differences Mie Bay in effective population size and gene flow, is reported from many taxa (Allendorf et al., 2013). Such discordance is mainly caused by hybridization of different clades, non- Nara Pacific monophyly, and differences in genealogy between different Ocean genes. We therefore sought to collect genetic information on KM C. crispus in KM and neighboring regions using mtDNA and microsatellite markers to inform the formulation of a conser- Kumano-nada 34°N Sea vation strategy for the species. At present, many wild mammals, including C. crispus, face the risk of extinction in female Japan (Ohdachi et al., 2009). The utility of approaches male adopted in this study are also discussed in the broader con- Wakayama sex unknown text of the conservation in wild mammals. MATERIALS AND METHDOS 136°E 137°E Fig. 1. Approximate sample locations of C. crispus (n = 57) used in Sample collection and DNA extraction this study. Solid and open circles, and grey triangles show male, We used tissue samples (n = 57) of C. crispus preserved at female and sex unknown respectively. For region code, see Table 1. Mie Prefectural Museum. They were collected from three regions of KM (n = 45); the Suzuka Mountains (SM) (n = 3) and the Hida Mountains (HM) (n = 9) in the Chubu district in 2004–2012 (Fig. 1, Table 1. Samples of C. crispus used in this study (See Fig. 1). Table 1). As C. crispus is designated as a special natural monu- Region Abbreviation Female Male Unknown Total ment by the Japanese Government, and as a near-threatened species by Mie and Wakayama Prefectures (WPG, 2012; MPG, 2015), Hida Mountains HM 5 3 1 9 its hunting is strictly prohibited. Our samples, collected in FSRCPK Suzuka Mountains SM 2 1 0 3 (PBEMNW, 2010), are derived from individuals that died from natu- Kii Mountainland KM 16 27 2 45 ral causes or that were killed in road traffic accidents. All samples had ears surgically removed, and the tissue fixed in 99% ethanol Total 23 31 3 57

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the sex based on the band patterns of PCR products. rected using Benjamini and Yekutieli’s (2001) method (B-Y correction) (see Narum, 2006). Genetic diversity parameters within indi- mtDNA analysis viduals for each region were estimated using GENEPOP and For analysis of mtDNA variability, a complete sequence (1,140 FSTAT 2.9.3.2 (Goudet, 2001): expected (HE: Nei’s unbiased esti- bp) of the cytochrome b region (Cyt b) was amplified via PCR, using mates; Nei, 1978) and observed (HO) heterozygosities, and the the following primer pairs: Capri-GLUL (5′-TGATATGAAAAAT- inbreeding coefficient within individuals in each region (FIS). We CATCGTTG-3′) and Capri-CB6THRH (5′-TCTCCTTCTCTGGTT- also used MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004) to TACAAG-3′) and PCR protocols described by Kawamura et al. check for evidence of null alleles, allelic dropouts or mis-scoring. (2014). After post-PCR cleaning with ExoSAP-IT (USB, USA), the To determine whether any isolated populations carried the PCR products were sequenced by the ABI PRISM BigDye Terminator molecular signature of a recent genetic bottleneck, Wilcoxon’s het- Cycle Sequencing kit 1.1 standard protocol (using the same primer) erozygosity excess test (Piry et al., 1999) and allele frequency dis- with ABI PRISM 3730 DNA Analyzer (Applied Biosystems, USA). tribution Mode shift indicator (Luikart et al., 1998) were performed Sequence alignment of edited sequences of mtDNA was per- using BOTTLENECK version 1.2.02 (Piry et al., 1999). For the Wil- formed with the computer software DNASYS version 3.1 (Hitachi coxon’s test, data were examined using both the stepwise mutation Software, Japan). Haplotype sequences were deposited in DDBJ (SMM) and two-phase (TPM) models, considered most appropriate (LC316120-LC316130). Within-population genetic diversity was for microsatellite data (Piry et al., 1999). For the TPM, a model of qualified by the number of haplotypes N( H), the number of private 90% single-step mutations and 10% multiple mutations was haplotypes (NPH), haplotype diversity (h) and nucleotide diversity adopted with a variance of 10 (Garza and Williamson, 2001). The (π), using ARLEQUIN 3.5.2.2 (Excoffier and Lischer, 2010). Median- average pairwise relatedness (rXY: Lynch and Ritland, 1999) was joining (MJ) networks (Bandelt et al., 1999) among mtDNA haplo- calculated among individuals using GenAlEx 6.503 (Peakall and types including two haplotypes published on DDBJ (AP003429 and Smouse, 2012). We calculated this parameter per sex and 95% D32191) were constructed using NETWORK 5.0.0.1 (http://www. confidence intervals (CI) using 9,999 permutations and 9,999 boot- fluxus-engineering.com). straps. In these analyses, individuals of HM and SM were excluded, The demographic history of C. crispus was tested by mismatch because of their small sample sizes (n < 14) (Luikart et al., 1998). distribution and Bayesian skyline plot (BSP) of the Cyt b gene. To assess the genetic structure of C. crispus in KM, we per- Analyses were performed in individuals of KM and all individuals, formed a principal component analysis (PCA) with Adegenet R-pack- respectively. Mismatch distribution was first carried out with ARLE- age (Jombart, 2008; Jombart et al., 2009). PCA was performed on QUIN. The observed distribution was compared with the expected microsatellite data on sexual groups for each region. The spatial pat- distribution under a model of sudden population expansion, and terns of genetic variability of C. crispus in KM and neighboring any deviation from this model was evaluated by calculating regions were explored by performing a spatial principal component Harpending’s (1994) raggedness index (Hrag) and the sum of analysis (sPCA) with Adegenet (Jombart et al., 2009). sPCA is a mul- squared difference (SSD) of Rogers (1995); the significance of Hrag tivariate analysis that uses the same information as PCA, but and SSD was assessed with 10,000 parametric bootstraps. In addi- includes geographical information. We used a distance-based con- tion, Fu’s test of neutrality (Fs) was performed to test for recent nection network (minimum distance between neighbors = 0 m, max- population growth or selection (Fu, 1997). The significance of Fu’s imum Euclidian distance = 41,000 m) based upon the approximate test was calculated from 10,000 data bootstraps using ARLEQUIN. mean dispersal distance of the mountain goat, Oreamnos america- Second, historical population trends were assessed by BSP nus, in the USA from Festa-Bianchet and Côté (2012) to define (Drummond et al., 2005) in BEAST v1.10.2 (Suchard et al., 2018). neighbors for the calculation of Moran’s I. Dispersal distance for O. This approach was found to be more sensitive to recent demo- americanus, which has a similar ecology to C. crispus, was used graphic bottlenecks than classical bottleneck tests (Hoffman et al., because of a lack of information for C. crispus (Festa-Bianchet and 2011). MtDNA data were partitioned into codon positions 1 + 2, and Côté, 2012). sPCA defines synthetic components that optimize the 3 (as recommended by Shapiro et al., 2006) and analyzed using a product of the variance and Moran’s I, summarizing spatial patterns GTR + I + G substitution model, 10 skyline groups and a strict of genetic structure. These components are separated into positive molecular clock. A substitution model was determined by AIC crite- (global) and negative (local) eigenvalues. Global scores can identify rion in jModelTest 2.1.1 (Darriba et al., 2012) and mutation rate was genetically distinguishable groups, clines in allele frequency and assumed to be 2% per million years (Rezaei et al., 2010). Analyses intermediate individuals, whereas local scores can detect differentia- were conducted with 100 million MCMC (Markov chain Monte Carlo tion between neighboring individuals. In addition, to test for isolation method) steps, sampling every 5,000 steps after discarding the first by geographica1 distance (IBD), the independence between geo- 10% as burn-in. Four independent replicate analyses were combined graphical and genetic distances was tested per sex by permuting the with LogCombiner v1.10.2 and then visualized with Tracer v1.6.0. Genetic diversity of C. crispus in KM and neighboring Microsatellite analysis Table 2. For analyzing microsatellite variability, 13 microsatellite loci regions at mtDNA Cyt b region and 12 microsatellite loci. For region were surveyed: FS33, FS50, FS58, FS81, FS90, FS152, FS175 code, see Table 1. (Chang et al., 2012), TGLA53 (Brezinsky et al., 1993), BM203, mtDNA Microsatellite BM6438, BM3628 (Bishop et al., 1994) and SY84, SY84B (An et al., Code 2005), using the fluorescent labelling protocol of Schuelke (2000) n NH NPH h π (%) n A NPA HE HO FIS and the PCR conditions described in the respective papers for each HM 9 4 3 0.750 0.210 9 3.000 3 0.509 0.472 0.073 primer set. PCR fragments were analyzed on ABI PRISM 3730 DNA SM 3 2 1 0.667 0.059 3 2.083 0 0.396 0.417 −0.053 Analyzer, according to the manufacturer’s recommendations. Allele KM 43 7 5 0.627 0.113 45 4.167 13 0.493 0.464 0.060 sizes were determined with GeneMapper 4.1 (Applied Biosystems). Total 55 11 – 0.702 0.137 57 4.417 – 0.507 0.463 0.087 Gametic disequilibrium (GD: Allendorf et al., 2013) for all pairs of loci, and genotype frequency to Hardy-Weinberg equilibrium n, number of samples; NH, number of haplotypes; NPH, number of (HWE) for each locus and for individuals in each region were tested private haplotypes; h, haplotype diversity; π, percent nucleotide using GENEPOP v4.2 (Raymond and Rousset, 1995). Levels of sig- diversity; A, number of alleles (averaged on seven loci); NPA, num- nificance for multiple tests in GD and HWE were determined by ber of private alleles; HE, expected heterozygosity; HO, observed sampling regions. Significance levels for all multiple tests were cor- heterozygosity; FIS, fixation index within region.

Mantel’s non-parametric test (Mantel, 1967) as implemented in regressed against the matrix of straight-line geographical distance Fig. 2 Iwahori et al. ARLEQUIN. The matrix of pairwise individual-by-individual genetic (in kilometers) between sampling locations (Rousset, 1997). P-values difference (Maguire et al., 2002), calculated using GenAlEx, was for the association of geographical and genetic distances were estimated by 1000 random permutation using ARLEQUIN. Assignment tests were also performed to investigate the mt10(1) genetic structure of C. crispus in KM and neighboring regions. The program STRUCTURE 2.3 (Falush et al., 2003) was used to assign mt1(17) mt11(1) individuals to putative clusters, based on their microsatellite pro-

mt9(1) files. The program uses a Bayesian clustering method to assign each multilocus genotype to one of k probable source populations. mt2(25) MCMC was used to estimate posterior probability distributions for mt4(4) each parameter integrated over all the other parameters. STRUC- TURE was run with 10 repetitions of 106 iterations following a burn- in period of 105 iterations, and with values of k from 1 to 10 under mt5(1) mt7(1) the “admixture model” and the “F model” (correlated allele frequen- mt8(2) cies) without prior information regarding the region from which an AP003429 individual was sampled. We selected the value of k showing the

mt3(1) optimal subdivisions of the data, using the ∆k method of Evanno et al. (2005) as implemented in STRUCTURE HARVESTER (Earl and mt6(1) vonHoldt, 2012).

HM RESULTS D32191 SM KM Genetic diversity in mtDNA and microsatellites The gender of 27 of 30 sexually ambiguous individuals Fig. 2. Median-joining (MJ) network of mtDNA Cyt b region of C. was identified using an amelogenin gene PCR-based DNA crispus. Each haplotype is represented by a pie chart, the size of Fig. 4 Iwahori et al. which is proportional to its frequency. Each line in the network repre- sex test. Although three individuals could not be sexually sents a single mutation step. Solid black circles indicate intermediate Fig. 3 haplotypesIwahori et al.that were not found in samples. The numbers in paren- (A) KM theses refer to the number of individuals representing the given hap- 1E8 lotype. The geographical regions of C. crispus are indicated by different colors. Blank triangles represent registered haplotypes in DDBJ.

1E7 (A) KM 0.5 Observed

Simulated Ne 1E6 0.4

0.3 P (SSD) = 0.088 1E5 P (Hrag) = 0.045 0.2 P (Fs) = 0.240

1E4 0.1 0 1000 2000 3000 4000 5000 6000 (B) All individuals 0 1E7 01234567

equen cy (B) All inidividuals 0.5 Fr

1E6 0.4

Ne 0.3 P (SSD) = 0.055 P (Hrag) = 0.014 1E5 0.2 P (Fs) = 0.034

0.1

0 1E4 01234567 0 2500 5000 7500 10000 12500 Pairwise difference Years before present Fig. 3. Mismatch distribution of mtDNA Cyt b region of C. crispus. Fig. 4. Bayesian skyline plots of mtDNA Cyt b region of C. crispus. Lines with open triangles indicate distribution expected under a The gray lines depict the median estimate of effective population model of sudden expansion: those with closed circles indicate the size (Ne) and shaded areas represent the 95% highest posterior observed distribution. density.

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identified due to PCR failure, samples that were successfully sion (Fig. 3A). Although the variance test (SSD) was not sig- amplified in PCR of mtDNA or microsatellites were used in nificant P( = 0.088), the raggedness index test (Hrag) sug- the subsequent analyses. gested that the curve significantly differed from the Sequence comparison of mtDNA Cyt b region in 55 distribution expected under a sudden expansion model (P = samples revealed 11 distinct haplotypes (mt1–11) defined by 0.045). The distribution produced was not entirely smooth, 11 polymorphic sites with ten transitions and one transver- with a single peak as expected. In addition, Fu’s test of neu- sion (no indels). The number of observed haplotypes was trality (FS) was not significantly indicative of expansion P( = four in HM, two in SM and seven in KM (Table 2). Haplotypes 0.240). The mismatch distribution of all individuals approxi- of mt1 and mt2 dominant in KM (86%) was observed in other mately showed the same pattern as that of KM (Fig. 3B). KM regions (67% of mt1 in SM and 33% of mt2 in HM). Nine and all individuals showed almost the same demographic haplotypes were region-specific (five in KM, one in SM and pattern in BSP (Fig. 4). BSP indicated a gradual increase three in HM). In a MJ network, region-specific clades were after the retreat of Wisconsin glaciers (~12 kya). not recognized (Fig. 2). For mtDNA, genetic diversity was lowest in KM (h = 0.627, π = 0. 00113), while it was highest Genetic differentiation among habitats in HM (h = 0.750, π = 0.00210). Based on PCA, males tended to be genetically more For microsatellites, unambiguously scorable amplifica- heterogeneous than females in all regions (Fig. 5). Females tion products were obtained for all 13 loci but with BM6438 in KM was totally encompassed by males in KM. SM (males monomorphic across all the samples (n = 57). Therefore, and females) was also encompassed in KM. While partially the remaining 12 loci were used in the analysis. Although no overlapping with KM, HM (males and females) differed from loci showed significant deviation from HWE in all regions, KM. two loci of FS90 and BM203 significantly deviated from Based on sPCA, the Monte-Carlo global test detected a HWE in total (B-Y corrected α = 0.016) (Supplementary clear spatial pattern of allele frequencies (max(t) = 0.070, Table S1). Significant GD (B-Y correctedα = 0.010) was not P = 0.001), while the Monte-Carlo local test was not signifi- observed in any pairs of loci in any other regions. MICRO- cant (max(t) = 0.032, P = 0.413), indicating that close indi- CHECKER suggested no evidence of null alleles or scoring viduals tended to be similar (positive spatial correlation). errors due to stuttering bands at any locus. At the sampling Three scores were obtained using sPCA (Fig. 6). The PC1 region level, FS33 in HM, FS90 in SM, BM203 in of SM and (I = 0.594) separated HM from KM and SM (Fig. 6A). PC2 KM, and SY84B in HM and SM were monomorphic. A total of (I = 0.560) approximately separated individuals from Mie 53 alleles (on average, 4.417 alleles per locus) were scored across all loci. The level of within-region genetic diversity for microsatellites ranged from 2.083 (SM) to 4.167 (KM) in number of alleles (on average), and from 0.396 (SM) to 0.509 (HM) in HE (Table 2). There was no significant difference among regions in genetic diversity parameters (Kruskall-Wallis test, P > 0.05). A total of 16 private alleles (PA) were identified in two regions (n = 3 in HM and n = 13 in KM). In FIS, HM and KM were not significantly positive, while SM was not significantly negative (P > 0.05). Significant heterozygosity excess consistent with a genetic bottleneck was detected in KM and all individuals with Wilcoxon’s test (P < 0.01) under the TPM and SMM models of mutation (Supplemen- tary Table S2). The Mode shift indicator also showed evidence of recent genetic bottlenecks in both groups. In KM, average pairwise relatedness (rXY) among individuals was − 0.009 in females and 0.006 in males. There was no significant difference in rXY between sexes (P > 0.05) and no sign of inbreeding was observed in either sex (P > 0.05).

Demographic pattern The mismatch distribution of haplotype Fig. 5. Result of principal component analysis (PCA) of C. crispus in KM and neigh- sequences for the Cyt b gene in KM was not boring regions. Individuals (dots) and regions (ovals) are discriminated using the first consistent with a model of population expan- two principal component axes. Ovals are 95% inertia eclipses.

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Conservation genetics of Japanese serow 311

A B C

PC1 PC2 PC3

-1.25 -0.75 -0.25 0.25 -0.25 0.25 0.75 1.25 -0.3 -0.1 0.1 0.3 0.5

Fig. 6. Results of the spatial principal component analysis (sPCA) of C. crispus in KM and neighboring regions. Projection of the individual lagged scores of each spatial principal component was positioned by its spatial coordinates. First (a), second (b) and third (c) principal components of sPCA. The color of squares (black and white) corresponds with the size of the score and the region is proportional to the absolute value of the score. Therefore, large black square individuals are well differentiated from large white square individuals.

Prefecture from those in other regions (Fig. 6B). PC3 (I = the last glacial maximum (LGM) (Tamate, 2013). 0.220) weakly distinguished individuals along the coastal Contrary to evidence for an absence of regional genetic region of the Kumano-nada Sea from those from inland structure in mtDNA, sPCA based on microsatellite data sug- regions (Fig. 6C). gested that C. crispus in KM and neighboring regions is sub- The results of a Mantel test with pairwise individual-by- structured, comprising three local populations (Fig. 6). PCA individual genetic distance, IBD was not suggested in males and admixture analysis showed the possibility that C. (P = 0.048) (Fig. 7B). However, it was significantly supported crispus from KM and neighboring regions are genetically in females (P = 0.02) and the null hypothesis of no correla- connected, forming a genetic cline (Figs. 5 and 8). In addition between geographical and genetic distances was tion, an IBD plot showed that dispersal potential is signifi- rejected (Fig. 7A). cantly higher in males than in females (Fig. 7). These results mean that C. crispus in KM and neighboring regions Among-population genetic structure comprises multiple local populations, which are genetically The posterior probability of microsatellite data, esti- connected by male-biased dispersal (Ochiai, 2016). Male- mated using STRUCTURE, showed the values of Ln P(D) biased dispersal is recognized in other taxa, such as Ursus drastically decreased from 1 to 2 k, indicating that the num- thibetanus (Ishibashi and Saitoh, 2004). ber of genetic clusters of C. crispus in KM and neighboring regions corresponds to one (Supplementary Figure S1). In k Population demography of C. crispus in the Kii Moun- = 2–4, however, the admixture ratio of clusters was not sta- tain Range ble among individuals (Fig. 8). Especially, in k = 2, dominant Capricornis crispus exclusively inhabits deciduous clusters differed between HM and KM. This pattern gradu- broad-leaved forests in temperate regions (Ochiai, 2016). In ally changed from northeast to southwest at the individual the LGM, it is presumed that forests in Japan contracted level, although the admixture ratio of clusters fluctuated substantially and acted as refugia for wild mammals (Tamate, among individuals. 2013). The effects of the LGM on the fauna and flora are considered to have been particularly large in eastern Japan DISCUSSION (Dobson, 1994), where genetic diversity is generally low in Genetic structure of C. crispus in the Kii Mountain Range many mammal species (Tamate, 2013). After the LGM, For mtDNA, haplotypes of mt1 and mt2 dominant in KM range and population expansions occurred in many taxa were also observed in HM and SM. PBEMNW (2010) reports (Dobson, 1994), which is conspicuous in mammal species of that the dominant haplotype of the Cyt b gene in KM was eastern Japan (Oshida et al., 2009; Nunome et al., 2010). In found in Gifu, Shizuoka and Yamagata Prefectures. These this study, BSP showed a gradual increase of C. crispus in findings suggest that C. crispus has a large mtDNA clade KM and neighboring regions after the LGM (~12 kya) (Fig. 4). distributed from the Tohoku district to the Kii Mountain With the wide distribution of a specific mtDNA haplotype in Range of Honshu. The same phenomena are observed in eastern Japan (PBEMNW, 2010), this finding suggests that other mammals, such as Cervus nippon (Tamate et al., C. crispus also experienced range and population expan- 1998), Macaca fuscata (Kawamoto et al., 2007) and Lepus sions after the LGM. brachyurus (Nunome et al., 2010). This pattern is generally In recent history, C. crispus decreased to its lowest considered to be due to a large-scale range expansion after abundance, with its distribution severely fragmented around

(A) Female k = 2 25

20 k = 3 obab ili t y r P 15 ss i gnmen t A k = 4 Genetic distance 10

5 HM SM KM r = 0.350, P = 0.002 Fig. 8. Admixture analysis of C. crispus in KM and neighboring regions with microsatellite data, calculated in STRUCTURE with k 0 50 100 150 200 250 300 = 2–4 under the F model. Each individual is represented as a verti- (B) Male cal bar partitioned into k segments whose length is proportional to the estimated membership in k clusters. Individuals were approximately arranged based on sampling locations from north-east to south-west. For region code, see Table 1.

Like C. crispus, Ce. nippon has also experienced pro- nounced population decline and habitat fragmentation resulting from indiscriminate overhunting from the mid-19th century combined with severe winters in 1879 and 1903 (Goodman et al., 2001). Owing to the restriction on hunting mammals beginning in the mid-20th Century, however, Ce.

Genetic distance nippon drastically recovered in numbers in many districts of 10 15 20 25 Japan towards the end of the 20th Century (Kaji, 2013). In contrast, the recovery of C. crispus is less obvious, except for some prefectures such as Aichi and Gifu (Ochiai, 2016). 5 The difference in recovery between the two species is attrib- r = 0.199, P = 0.048 uted to differences in behavior, ecology and reproduction. In 0 50 100 150 200 250 300 particular, C. crispus shows lower rates of increase than Ce. Geographical distance (km) nippon (Ochiai, 2006). It has recently been proposed that interspecific competition, arising from an overlap in the eco- Fig. 7. Measures of a pairwise individual-by-individual genetic dif- logical niches of the two species, may also negatively affect ference in C. crispus in KM and neighboring regions as a function the demography and distribution of C. crispus (Koganezawa, of geographical distances between sampling locations. Closed cir- 1999; Yamashiro et al., 2017). The same may also be the cles indicate comparisons in females and open circles indicate those in males. case in KM (PBEMNW, 2010).

Implications for conservation the beginning of the 1940s, mainly due to overhunting Based on mtDNA, individuals of C. crispus could not be (MEGJ, 2010; Ochiai, 2016). PBEMNW (2010) reports that distinguished among regions in a haplotype network, owing C. crispus in KM has continued to suffer a population decline to the sharing of dominant haplotypes (Fig. 2). However, on even after the imposition of strict protection beginning in the the basis of microsatellites, genetic heterogeneity among 1950s. In this study, recent genetic bottlenecks were sug- regions was shown by PCA and assignment tests (Figs. 5 gested by microsatellites (Supplementary Table S2). This and 8). In addition, sPCA suggested that C. crispus in KM finding seems to reflect the population decline in the 20th separates into three local populations in eastern (north to century. On the basis of microsatellites, C. crispus in KM middle regions in Mie), southern (Kumano region in Mie) and showed the same level of genetic diversity as the Shizuoka western regions (Wakayama and Nara regions) of KM, population, while the Shikoku population was the lowest respectively (Fig. 6). From the viewpoint of conservation, the (Supplementary Table S3). It is known that genetic diversity local populations in sPCA seem equivalent to Management in microsatellites reliably reflects effective population size Units (MUs: Moriz, 1994). At present, the prevailing conser- (Garza and Williamson, 2001). The low genetic diversity vation units are the Evolutionary Significant Unit (ESU) with observed in the present study thus strongly suggests that an emphasis of reciprocally monophyletic lineages seen in many populations of C. crispus in Japan continue to experi- mtDNA (Ryder, 1986), with MU a category subordinate to ence population decline. ESU, with an emphasis on differences in allele frequencies

of codominant markers, such as microsatellites and allo- SUPPLEMENTARY MATERIALS zymes (Moriz, 1994). Accordingly, it seems that C. crispus in KM and neighboring regions is composed of multiple MUs Supplementary materials for this article are available online. based on microsatellites, while it corresponds to a single (URL: https://bioone.org/journals/supplementalcontent/10.2108/ ESU based on mtDNA. zs180187/10.2108.zsj.36.306.s1.pdf). Supplementary Figure S1. Posterior probability of the data Ln In the present study, the difference in genetic structures P(D) against the number of k clusters, computed using STRUC- of C. crispus populations identified using microsatellites and TURE for k = 1–10 for microsatellite data from three regions of C. mtDNA seems to have arisen from different mutation rates crispus in KM and neighboring regions. between the genetic markers. The mutation rates of micro- Supplementary Table S1. Summary statistics for 12 microsat- satellites are generally higher than mtDNA and could drive ellite loci of C. crispus. genetic differentiation among populations in a short period Supplementary Table S2. Results of Wilcoxon’s heterozygos- (Avise, 2004). The same pattern to that seen with C. crispus ity excess test and Mode shift indicator for a genetic bottleneck by in this study was observed in Males anakuma (Tashima et BOTTLENECK (Piry et al., 1999) in C. crispus from KM and neigh- al., 2010). Tashima et al. (2010) consider that genetic differ- boring regions. entiation of M. anakuma in Japan occurred over a relatively Supplementary Table S3. Comparison of genetic diversity of four populations of C. crispus in Japan. short evolutionary time-scale (7 kya). Sato (2017) inferred that the most recent common ancestor of C. crispus in REFERENCES Japan could be coalesced in the late Pleistocene (90 kya), Allendorf FW, Luikart G, Aitken SN (2013) Conservation and the based upon the nucleotide diversity of the Cyt b gene. With Genetics of Populations, 2nd edn. Wiley–Blackwell, West Sus- no regional genetic structure in mtDNA, these findings sug- sex gest that genetic differentiation among local populations of An J, Min MS, Sommer J, Louis E Jr, Brenneman R, Kwon SW, et al. C. crispus in KM and neighboring regions is likely to have (2005) Isolation and characterization of 15 microsatellite loci in occurred in a short period after the LGM. the Korean goral (Nemorhaedus caudatus). Mol Ecol Notes 5: Legally protected areas for C. crispus have been cre- 421–423 ated in 13 areas in Honshu. Additional protected areas are Avise JC (2004) Molecular Markers, Natural History, and Evolution. Sinauer Associates, Sunderland, Massachusetts now being prepared in Shikoku and Kyushu (MEGJ, 2010). Bandelt H, Forster P, Rhöl A (1999) Median-joing networks for infer- The main goals of protected areas are the harmonious com- ring intraspecific phylogenies. Mol Biol Evol 16: 37–48 bination of the conservation of C. crispus and the reduction Benjamini Y, Yekutieli D (2001) The control of the false discovery of agricultural and forestry damage. Protected areas cov- rate in multiple testing under dependency. Ann Stat 29: 1165– ered 20–30% of the distribution of C. crispus in 1983. 1188 However, at present there have been critiques of the effec- Bishop MD, Kappes SM, Keele JW, Stone RT, Sunden S, Hawkins tiveness of these protected areas, due to the failure to moni- GA, et al. (1994) A genetic linkage map for cattle. Genetics tor them adequately and their discordance with the main 136: 619–639 habitats of C. crispus (Ochiai, 2016). The results of this Brezinsky L, Kemp SJ, Teale AJ (1993) ILSTS005: a polymorphic study highlight the necessity of an evaluation of local popu- bovine microsatellite. Anim Genet 24: 73 Chang YY, Chao MC, Ding ST, Lin EC, Tsao HS, Yuan HW, et al. lations as MUs in the conservation of C. crispus. The same (2012) Development of microsatellite markers in an ungulate also may apply to other mammals with small home ranges or mammal, the Formosan serow (Capricornis swinhoei). Con- low mobility. In addition, it will be important to maintain serv Genet Resour 4: 755–757 genetic connectivity among local populations by preserving Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: the continuity of habitats to limit the further loss of genetic more models, new heuristics and parallel computing. Nat diversity and maximize the potential for adaptability Methods 9: 772 (PBEMNW, 2010; Allendorf et al., 2013). Dobson M (1994) Patterns of distribution in Japanese land mammals. Mammal Rev 24: 91–111 ACKNOWLEDGMENTS Drummond AJ, Rambaut A, Shapiro B, Pybus OG (2005) Bayesian We would like to thank the staff of Mie Prefectural Museum for coalescent inference of past population dynamics from molec- providing tissue samples of C. crispus. S. Miura and A. Yamashiro ular sequences. Mol Biol Evol 22: 1185–1192 kindly gave us valuable information on the genetic and ecological Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a web- characteristics of C. crispus in Japan. We are also grateful to C. site and program for visualizing STRUCTURE output and Smith (University of St. Andrews, UK) for comments and English implementing the Evanno method. Conserv Genet Resour 4: correction. We express sincere thanks to three anonymous review- 359–361 ers for critical comments and useful suggestions that have helped Evanno G, Regnaut S, Goudet J (2005) Detecting the number of us improve our paper considerably. clusters of individuals using the software STRUCTURE: a sim- ulation study. Mol Ecol 14: 2611–2620 COMPETING INTERESTS Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under The authors have no competing interests to declare. Linux and Windows. Mol Ecol Resour 10: 564–567 AUTHOR CONTRIBUTIONS Falush D, Stephens M, Pritchard JK (2003) Inference of population structure using multilocus genotype data: linked loci and cor- KK and JK designed the research project and obtained funding related allele frequencies. Genetics 164: 1567–1587 for this study. JK collected tissue samples of C. crispus. AI and KK Festa-Bianchet M, Côté SD (2012) Mountain goats: ecology, behav- conducted genetic analyses and wrote the paper. ior, and conservation of an alpine ungulate. Island Press, Washington Fu YX (1997) Statistical tests of neutrality of mutations against pop-

Downloaded From: https://bioone.org/journals/Zoological-Science on 07 Aug 2019 Terms of Use: https://bioone.org/terms-of-use Access provided by Zoological Society of Japan 314 A. Iwahori et al.

ulation growth, hitchhiking and background selection. Genetics and management plans for the Japanese serrow. Available at 147: 915–925 https://www.env.go.jp/nature/choju/plan/plan3-2b/index.html Garza JC, Williamson EG (2001) Detection of reduction in popula- (In Japanese) tion size using data from microsatellite loci. Mol Ecol 10: 305– Moritz C (1994) Defining ‘evolutionarily significant units’ for conser- 318 vation. Trends Ecol Evol 9: 373–375 Gompert Z, Nice CC, Fordyce JA, Forister ML, Shapiro AM (2006) Narum SR (2006) Beyond Bonferroni: less conservative analyses Identifying units for conservation using molecular systematics: for conservation genetics. Conserv Genet 7: 783–787 the cautionary tale of the Karner blue butterfly. Mol Ecol 15: Nei M (1978) Estimation of average heterozygosity and genetic dis- 1759–1768 tance from a small number of individuals. Genetics 89: 583– Goodman SJ, Tamate HB, Wilson R, Nagata J, Tatsuzawa S, 590 Swanson GM, et al. (2001) Bottlenecks, drift and differentia- Nishimura T, Yamauchi K, Deguchi Y, Aoi T, Tsujimoto T, Matsubara tion: the population structure and demographic history of sika K (2011) Development of microsatellite marker assay for indi- deer (Cervus nippon) in the Japanese archipelago. Mol Ecol vidual identification in Japanese serows Capricornis( crispus). 10: 1357–1370 Jpn J Zoo Wildl Med 16: 75–78 Goudet J (2001) FSTAT, Version 2.9.3: a program to estimate and Nishimura T, Yamauchi K, Saitoh Y, Deguchi Y, Aoi T, Tsujimoto T, test gene diversities and fixation indices. Available at http:// et al. (2010) Sex determination of the Japanese serow www.unil.ch/izea/software/fstat.html (Capricornis crispus) by fecal DNA analysis. Jpn J Zoo Wildl Harpending H (1994) Signature of ancient population growth in a Med 15: 73–78 low-resolution mitochondrial DNA mismatch distribution. Hum Novak R (1999) Walker’s mammals of the world, 6th edn. Johns Biol: 591–600 Hopkins Univ Press, Baltimore and London Hoffman J, Grant S, Forcada J, Phillips C (2011) Bayesian inference Nunome M, Torii H, Matsuki R, Kinoshita G, Suzuki H (2010) The of a historical bottleneck in a heavily exploited marine mam- Influence of Pleistocene refugia on the evolutionary history of mal. Mol Ecol 20: 3989–4008 the Japanese hare, Lepus brachyurus. Zool Sci 27: 746–754 Ishibashi Y, Saitoh T (2004) Phylogenetic relationships among frag- Ochiai K (2016) The Japanese serrow: behavior and ecology. Tokyo mented Asian black bear (Ursus thibetanus) populations in Univ Press, Tokyo (In Japanese) western Japan. Conserv Genet 5: 311–323 Ohdachi SD, Ishibashi Y, Iwasa MA, Saitoh T (2009) The Wild Jombart T (2008) Adegenet: a R package for the multivariate analy- Mammals of Japan. Shoukadoh, Tokyo sis of genetic markers. Bioinformatics 24: 1403–1405 Okumura H (2003) Japanese serrow in the northern and southern Jombart T, Pontier D, Dufour A (2009) Genetic markers in the play- distribution limits. Shinrin Kagaku 38: 59–63 (In Japanese) ground of multivariate analysis. Heredity 102: 330 Oshida T, Masuda R, Ikeda K (2009) Phylogeography of the Kaji K (2013) Increase and population management of Cervus Japanese giant flying squirrel, Petaurista leucogenys nippon in Japan. Water Science 57: 2–11 (In Japanese) (Rodentia: Sciuridae): implication of glacial refugia in an arbo- Kawamoto Y, Shotake T, Nozawa K, Kawamoto S, Tomari K-i, Kawai real small mammal in the Japanese Islands. Biol J Linnean Soc S, et al. (2007) Postglacial population expansion of Japanese 98: 47–60 macaques (Macaca fuscata) inferred from mitochondrial DNA Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in phylogeography. Primates 48: 27–40 Excel. Population genetic software for teaching and research- Kawamura K, Ueda T, Arai R, Smith C (2014) Phylogenetic relation- an update. Bioinformatics 28: 2537–2539 ships of bitterling fishes (Teleostei: Cypriniformes: Acheilo- Pfeiffer I, Brenig B (2005) X- and Y-chromosome specific variants of gnathinae), inferred from mitochondrial cytochrome b the amelogenin gene allow sex determination in sheep (Ovis sequences. Zool Sci 31: 321–329 aries) and European red deer (Cervus elaphus). BMC Genet 6: Kishimoto R (1988) Age and sex determination of the Japanese 16 serow Capricornis crispus in the field study. J Mammal Soc Piry S, Luikart G, Cornuet JM (1999) BOTTLENECK: a computer Jpn 13: 51–58 program for detecting recent reductions in the effective size Koganezawa M (1999) Changes in the population dynamics of using allele frequency data. J Hered 90: 502–503 Japanese serow and sika deer as a result of competitive inter- Prefecural Boards of Education in Mie, Nara, and, Wakayama actions in the Ashio Mountains, central Japan. Biosphere con- (PBEMNW) (2010) Report of the fourth special research in the servation: for nature, wildlife, and humans 2: 35–44 protection habitat of Japanese serrow in the Kii Peninsula. Luikart G, Allendorf FW, Cornuet JM, Sherwin WB (1998) Distortion Available at http://www.bunka.pref.mie.lg.jp/common/content/ of allele frequency distributions provides a test for recent popu- 000146744.pdf (In Japanese) lation bottlenecks. J Hered 89: 238–247 Raymond M, Rousset F (1995) GENEPOP (Version 1.2): population Lynch M, Ritland K (1999) Estimation of pairwise relatedness with genetics software for exact tests and ecumenicism. J Hered molecular markers. Genetics 152: 1753–1766 86: 248–249 Maguire T, Peakall R, Saenger P (2002) Comparative analysis of Rezaei HR, Naderi S, Chintauan-Marquier IC, Taberlet P, Virk AT, genetic diversity in the mangrove species Avicennia marina Naghash HR, et al. (2010) Evolution and taxonomy of the wild (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs. species of the genus Ovis (Mammalia, Artiodactyla, Bovidae). Theor Appl Genet 104: 388–398 Mol Phylogenet Evol 54: 315–326 Mantel N (1967) The detection of disease clustering and a general- Rogers AR (1995) Genetic evidence for a Pleistocene population ized regression approach. Cancer Res 27: 209–220 explosion. Evolution: 608–615 Mie Prefectural Government (MPG) (2015) Mie red data book 2015: Ropiquet A, Li B, Hassanin A (2009) SuperTRI: A new approach endangered wildlife in Mie Prefecture. Mie Prefectural Govern- based on branch support analyses of multiple independent ment, Tsu (In Japanese) data sets for assessing reliability of phylogenetic inferences. C Min MS, Okumura H, Jo DJ, An JH, Kim KS, Kim CB, et al. (2004) R Biol 332: 832–847 Molecular phylogenetic status of the Korean goral and Japa- Rousset F (1997) Genetic differentiation and estimation of gene nese serow based on partial sequences of the mitochondrial flow from F-statistics under isolation by distance. Genetics cytochrome b gene. Mol Cells 17: 365–372 145: 1219–1228 Ministry of the Environment Government of Japan (MEGJ) (2010) Ryder O (1986) Species conservation and systematics: the dilemma Guideline for the design of the specified wildlife conservation of subspecies. Trends Ecol Evol 1: 9–10

Sato JJ (2017) A review of the processes of mammalian faunal anakuma), as revealed by microsatellite analysis. Mammal assembly in Japan: insight from molecular phylogenetics. In study 35: 221–226 “Species Diversity of Animals in Japan” Ed by M Motokawa, H van Oosterhout C, Hutchinson WF, Wills DP, Shipley P (2004) Kajihara, Springer Japan, Tokyo, pp 49–116 MICRO–CHECKER: software for identifying and correcting Schuelke M (2000) An economic method for the fluorescent label- genotyping errors in microsatellite data. Mol Ecol Notes 4: ing of PCR fragments. Nat Biotech 18: 233–234 535–538 Shapiro B, Rambaut A, Drummond AJ (2006) Choosing appropriate Wakayama Prefectural Government (WPG) (2012) Red list of substitution models for the phylogenetic analysis of protein- Wakayama Prefecture. Available at http://www.pref.wakayama. coding sequences. Mol Biol Evol 23: 7–9 lg.jp/prefg/032000/032500/reddate2012/ (In Japansese) Simpson GG (1945) The principles of classification and a classifica- Yamashiro A, Yamashiro T (2012) Analysis of Japanese serrow in tion of mammals. Bull Amer Mus Nat Hist 85: 1–350 the Shikoku Mountainlands using genetic markers. In “Report Suchard MA, Lemey P, Baele G, Ayres DL, Drummond AJ, Rambaut on Special Research of Japanese Serrow in the Shikoku A (2018) Bayesian phylogenetic and phylodynamic data inte- Mountainislands” Ed by Prefecural Boards of Education in gration using BEAST 1.10. Virsu Evolution 4: vey016 Tokushima and Kochi, and Scientific research center of natural Tamate HB (2013) Genetic diversity in the mammalian fauna of history in Shikoku, Tokushima, pp 60–83 Japan: past, present and future. Chikyu Kankyo 18: 159–167 Yamashiro A, Kaneshiro Y, Kawaguchi Y, Yamashiro T (2017) Spe- (In Japanese) cies, sex, and individual identification of Japanese serow Tamate HB, Tatsuzawa S, Suda K, Izawa M, Doi T, Sunagawa K, et (Capricornis crispus) and sika deer (Cervus nippon) in sympat- al. (1998) Mitochondrial DNA variations in local populations of ric region based on the fecal DNA samples. Conserv Genet the Japanese sika deer, Cervus nippon. J Mammal 79: 1396– Res 9: 333–338 1403 Tashima S, Kaneko Y, Anezaki T, Baba M, Yachimori S, Masuda R (Received November 25, 2018 / Accepted February 28, 2019) (2010) Genetic diversity within the Japanese badgers (Meles

Downloaded From: https://bioone.org/journals/Zoological-Science on 07 Aug 2019 Terms of Use: https://bioone.org/terms-of-use Access provided by Zoological Society of Japan Fig. S1. Posterior probability of the data Ln P(D) against the number of k clusters, computed using STRUCTURE for k = 1-10 for microsatellite data from three regions of C. crispus in KM and neighboring regions.

Ln P(D) (mean ± SD) Ln P (D)

Number of clusters (k) Supplementary Figure S1. Posterior probability of the data Ln P(D) against the number of k clusters, computed using STRUC- TURE for k = 1–10 for microsatellite data from three regions of C. crispus in KM and neighboring regions. Supplementary Table S1. Summary statistics for 12 microsatellite loci of C. crispus . For region code, see Table 1. HM SM KM All FS33 n 934456 Total number of alleles: 3 A 1233

P A 001-

Size range of allele: 211-215 bp H E 0.000 0.333 0.283 0.242

H O 0.000 0.333 0.273 0.232 HWE - - 0.187 0.149

F IS - - 0.035 0.042 FS50 n 934557 Total number of alleles: 7 A 4377

P A 002-

Size range of allele: 185-197 bp H E 0.729 0.750 0.707 0.749

H O 0.667 1.000 0.733 0.737 HWE 0.920 1.000 0.377 0.249

F IS 0.086 -0.333 -0.037 0.016 FS58 n 934557 Total number of alleles: 6 A 5356

P A 101-

Size range of allele: 162-180 bp H E 0.750 0.667 0.739 0.764

H O 0.778 1.000 0.644 0.684 HWE 0.570 1.000 0.156 0.131

F IS -0.037 -0.500 0.128 0.104 FS81 n 934456 Total number of alleles: 7 A 4477

P A 002-

Size range of allele: 204-218 bp H E 0.632 0.833 0.709 0.698

H O 0.667 0.667 0.727 0.714 HWE 1.000 0.606 0.857 0.810

F IS -0.055 0.200 -0.025 -0.024 FS90 n 934557 Total number of alleles: 4 A 3134

P A 101-

Size range of allele: 244-260 bp H E 0.528 0.000 0.296 0.325

H O 0.444 0.000 0.200 0.228 HWE 0.421 - 0.019 0.014*

F IS 0.158 - 0.323 0.298 FS152 n 934557 Total number of alleles: 5 A 3255

P A 001-

Size range of allele: 212-220 bp H E 0.625 0.333 0.765 0.763

H O 0.556 0.333 0.822 0.754 HWE 0.341 - 0.025 0.066

F IS 0.111 - -0.075 0.011 FS175 n 934557 Total number of alleles: 2 A 2222

P A 000-

Size range of allele: 218-221 bp H E 0.208 0.667 0.442 0.423

H O 0.222 0.333 0.378 0.351 HWE 1.000 1.000 0.492 0.221

F IS -0.067 0.500 0.146 0.170 TGLA53 n 934557 Total number of alleles: 4 A 3244

P A 001-

Size range of allele: 141-155 bp H E 0.611 0.333 0.518 0.529

H O 0.778 0.333 0.622 0.632 HWE 0.449 - 0.234 0.140

F IS -0.273 0.000 -0.200 -0.194 BM203 n 934557 Total number of alleles: 2 A 2112

P A 100-

Size range of allele: 219-224 bp H E 0.514 0.000 0.000 0.117

H O 0.333 0.000 0.000 0.053 HWE 0.492 - - 0.009*

F IS 0.351 - - 0.550 SY84 n 934557 Total number of alleles: 7 A 4277

P A 003-

Size range of allele: 190-202 bp H E 0.736 0.500 0.737 0.744

H O 0.778 0.667 0.578 0.614 HWE 1.000 1.000 0.026 0.115

F IS -0.057 -0.333 0.216 0.175 BM3628 n 934456 Total number of alleles: 4 A 4244

P A 000-

Size range of allele: 262-268 bp H E 0.778 0.333 0.524 0.564

H O 0.444 0.333 0.455 0.446 HWE 0.030 - 0.141 0.030

F IS 0.429 0.000 0.133 0.209 SY84B n 934557 Total number of alleles: 2 A 1122

P A 001-

Size range of allele: 220-222 bp H E 0.000 0.000 0.201 0.162

H O 0.000 0.000 0.133 0.105 HWE - - 0.073 0.047

F IS - - 0.335 0.350 Average for all samples A 3.000 2.083 4.167 4.417

H E 0.509 0.396 0.493 0.507

H O 0.472 0.417 0.464 0.463

F IS 0.073 -0.053 0.060 0.087 n , number of samples; A , number of alleles; P A, number of private alleles; H E, expected heterozygosity; H O, observed heterozygosity; F IS, fixation index within region. Locus BM6438 (Bishop et al., 1994) was monomorphic in all individuals. Significance of the tests for HWE was adjusted by regions, using B-Y corrections (Benjamini and Yekutieli, 2001). *p < 0.05 Supplementary Table S2. Results of Wilcoxon's heterozygosity excess test and Mode shift indicator for a genetic bottleneck by BOTTLENECK (Piry et al., 1999) in C. crispus from KM and neighboring regions. Region n Wilcoxon's test Mode shift test TPM SMM KM 45 0.00049*** 0.00098*** Yes All individual 57 0.00244** 0.00342** Yes n , number of samples ** p < 0.01, *** p < 0.001 Individuals of HM and SM were included in all individuals, owing to small sample size (n < 14) (Luikart et al., 1998). Supplementary Table S3. Comparison of genetic diversity of four populations of C. crispus in Japan. Microsatellite Shikoku (n = 46)*,** KM (n = 32)*,*** Shizuoka (n = 16)* KM (n = 45)**** locus A H E H O A H E H O A H E H O A H E H O TGLA53 2 0.487 0.361 3 0.405 0.406 4 0.558 0.438 4 0.518 0.622 SY84 5 0.706 0.694 7 0.747 0.844 6 0.783 0.813 7 0.737 0.578 SY84B 2 0.086 0.027 2 0.146 0.156 2 0.271 0.313 2 0.201 0.133 BM203 2 0.387 0.417 2 0.421 0.656 3 0.363 0.438 1 - - BM3628 3 0.459 0.111 4 0.600 0.219 3 0.658 0.188 4 0.524 0.455 BM6438 1- - 1- - 1- - 1- - Average 2.500 0.425 0.322 3.167 0.464 0.456 3.167 0.527 0.438 3.167 0.495 0.447 n , number of samples; A , number of alleles; H E, expected heterozygosity; H O, observed heterozygosity. *Yamashiro and Yamashiro (2012) **Populations of Tokushima and Kochi included ***Populations of Wakayama, Mie, Shiga and Nara included ****This study