Mitochondrial genetic diversity, phylogeny and population structure of Hydropotes inermis in South Korea

ABSTRACT

The water deer (Hydropotes inermis) is one of the rarest species of deer in the family Cervidae. Only two subspecies exist in East Asia, and few studies have examined the genetic characteristics of the species. Here, we investigated the genetic diversity, phylogeny and population differentiation of the Korean subspecies (H. inermis argyropus). Seventeen mitochondrial D-loop haplotypes (822 bp) were detected and analyzed from 107 individual samples, together with a Chinese subspecies (H. inermis inermis) haplotype. The genetic diversity of the Korean subspecies is lower (π = 0.756%, h = 0.867) than that of the Chinese subspecies estimated in a previous study. This low genetic diversity may result from historical anthropogenic disturbances and/or a founder effect during the glacial period. The phylogenetic tree and median-joining network showed no location-specific distribution of D-loop haplotypes, but revealed two major lineages, A and B, of water deer. The A and B lineages were separated from each other at the beginning of the Pleistocene era (2.1–1.3 million years ago), with a genetic divergence of 1.332 ± 0.340%. The genetic divergence within lineages A and B was 0.525 ± 0.167% and 0.264 ± 0.113%, respectively. This suggests that climate change affected the division of the two lineages. Water deer sampled from the three Korean regions (26 locations) were slightly distinct in their genetic structure (AMOVA: F_ST = 0.28416, P < 0.00001; Φ_ST = 0.19239, P < 0.00001). Such slight population differentiation may be derived from differential dispersal ability in males and females. The use of genetic markers, such as nuclear microsatellite and Y-linked DNA markers, and samples collected from various localities in East Asia should improve our understanding of the water deer’s genetic characteristics.

INTRODUCTION

The water deer (Hydropotes inermis) is one of the most primitive members of the family Cervidae. While both the males and females lack antlers, the males exhibit a pair of long, sharp canine teeth similar to Old World deer (Flerov, 1952). This rare, small ruminant exists only in two countries in East Asia. The Chinese subspecies (H. inermis inermis Swinhoe, 1870) inhabits the Chinese mainland and islands, and the Korean subspecies (H. inermis argyropus Heude, 1884) lives throughout the Korean peninsula (Cooke and Farrell, 1998; Geist, 1998; Kim et al., 2011a).

The Chinese subspecies was once widely distributed and highly abundant in the central and eastern parts of China. However, its population has dramatically declined because of illegal hunting and habitat loss. Therefore, a small number of water deer now live only in four fragmented areas (Sheng, 1992; Wang, 1998; Xu et al., 1998; Zhang and Guo, 2000; Guo and Zhang, 2002, 2005). Recently, the subspecies was classified as a vulnerable species in the China Red Data Book of Endangered Animals (Wang, 1998) and the IUCN Red List (Harris and Duckworth, 2008), and its total population is about 10,000 (Butzler, 1990). The Korean subspecies was previously found along the ranges of Mt. Taebaek and Mt. Nangrim, distributed extensively throughout the Korean peninsula (Won and Smith, 1999). Like the Chinese subspecies, its abundance and distribution declined severely due to excessive harvesting and habitat destruction in the 1950s. Although the current population seems to be abundant and widely distributed in local habitats (Woo et al., 1990, Kim et al., 2011a), the total population size of the Korean subspecies has not yet been surveyed.

To date, a few genetic studies have been conducted on water deer using mitochondrial DNA markers (e.g., cytochrome b and the control region) (Hu et al., 2006; Koh et al., 2009) and nuclear microsatellite markers (Hu et al., 2007). Hu et al. (2006) ascertained that the overall Chinese subspecies displays no clear phylogenetic relationship between different regions of origin, even though a relatively higher genetic diversity in the mitochondrial D-loop region was found compared to that in other rare Cervidae. However, an analysis of molecular variance (AMOVA) showed a significant differentiation between the Zhoushan archipelago population and the mainland population, which indicated the existence of a geographic barrier to gene flow between the two populations (Hu et al., 2006). Furthermore, Hu et al. (2007) found that the Chinese water deer has a relatively high level of genetic diversity among microsatellite markers (n = 7) compared to other rare Cervidae, and strong differentiation between different regions of origin (mainland and Zhoushan archipelago). More recently, Koh et al. (2009) reported that the Chinese and Korean subspecies seem to be very closely related, displaying low levels of genetic distance for cytochrome b (1.3%) and the control region (2.1%), which contrasts with previous expectations that the two subspecies would have distinct characteristics based on the pelage color of the deer. This previous study only focused on the taxonomic status of the two water deer subspecies (Koh et al., 2009). Detailed genetic information about the Korean subspecies, such as the genetic diversity and population structure among various regions or localities of Korea, is scarce. Therefore, further studies are essential to improve our understanding of the genetic characteristics of the Korean subspecies.

The focus of this study was to examine in detail the mitochondrial genetic characteristics of the Korean subspecies. To this end, the genetic diversity, phylogeny and degree of genetic differentiation of the Korean subspecies were investigated for Korean water deer sampled across geographic localities from various regions in South Korea. Anthropogenic interference and climate change as possible causal effects to explain the present status of mitochondrial diversity of water deer in Korea are also discussed.

MATERIALS AND METHODS

Sampling sites and DNA samples

A total of 107 individual samples of water deer were studied in the wilds of the Korean peninsula (Table 1). Three different habitats were targeted for tissue and hair sampling: the mid-western region (Gyeonggi Province: 12 locations), the mid-eastern region (Gangwon Province: 5 locations), and the southwestern region (Jeolla Province: 9 locations) (Fig. 1). All tissue samples were collected in 2008 and 2009, and were frozen at −30℃ until they were used for DNA extraction.

Table 1. Geographical region, sample size and type, and GenBank accession number for each sample of water deer used in this study

No.	Region (Province)	Sample size	Sample type	Species name	GenBank no.
1	Gangwon, Korea	39	Tissue, hair	H. inermis argyropus	KC581917–KC581933
2	Jeolla, Korea	33	Tissue	H. inermis argyropus
3	Gyeonggi, Korea	35	Tissue, hair	H. inermis argyropus
1	China	1	No information	H. inermis inermis	EU315254
2	China	1	No information	H. inermis inermis	NC_011821
1	Russia	outgroup		Capreolus pygargus	Z70317

Fig. 1.

Map of Gyeonggi (squares, 12 sampling locations), Gangwon (triangles, 5 locations), and Jeolla (circles, 9 locations) Provinces in South Korea. More detailed information on sampling locations is provided in MATERIALS AND METHODS.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from tissue samples (e.g., muscle, tongue, skin, liver, kidney and spleen) using a LaboPass Tissue kit (Cosmo Genetech, Seoul, Korea). Additionally, a chelex method was used for DNA extraction from hair samples (Walsh et al., 1991). Two sequential PCR assays were carried out for the mitochondrial D-loop region in this study. One pair of primers, L15775 and H651, was first used to amplify a complete D-loop fragment (Kocher et al., 1989). A second pair of newly designed primers, BJWDDLOOP_F (5′-GCT CCA TAA AAA CCA AGA AC-3′) and BJWDDLOOP_R (5′-TTG AGT ACA AAA AAT GGC GC-3′), was then used to amplify a partial fragment from the first PCR products. PCR was carried out in a 20-μl reaction that contained 1 μl of DNA template, 1X PCR buffer (iNtRON Biotechnology, Seongnam, Korea), 2 mM MgCl₂ (iNtRON), 0.2 mM of each dNTP (iNtRON), 0.1 μM of each primer, 2.5 μg of BSA (Promega, Fitchburg, WI, USA) and 1.25 U of i-Star Taq polymerase (iNtRON).

PCR was performed in a PTC-100 PCR Thermal Cycler (MJ Research, Waltham, MA, USA) under the following conditions: initial denaturation for 4 min at 94℃ followed by 40 cycles of [94℃ for 60 s, 50℃ or 57℃ (for the first and second PCR, respectively) for 45 s, and 72℃ for 60 s], with a final extension for 5 min at 72℃. The PCR products were resolved by electrophoresis on a 2% agarose gel, stained with ethidium bromide, and visualized under a UV transilluminator. All the PCR products were purified from the gel using a Zymoclean Gel DNA Recovery kit (Zymo Research, Orange, CA, USA). The purified PCR products were bidirectionally sequenced using forward (BJWDDLOOP_F) and reverse (BJWDDLOOP_R) primers with an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Data analysis

Partial sequences of the mitochondrial D-loop region were aligned using the AlignIR program, version 2.1 (LI-COR, Lincoln, NE, USA). These sequences were manually edited at least five times on the alignment program. Any sequence that varied by at least one nucleotide was considered to be a different haplotype. The number of polymorphic sites, parsimonious informative sites, singletons and haplotypes (A), and the amount of haplotype diversity (h) and nucleotide diversity (π), were calculated using DNASP version 4.10.9 (Rozas et al., 2003). Sequence divergences (p_s) at each region and all regions, and pairwise sequence divergences among regions and clades were calculated using the Kimura 2 parameter (K2P; Kimura, 1980) with MEGA version 4.0 (Kumar et al., 2004).

The phylogenetic relationships between the haplotypes of the Korean and Chinese subspecies (accession no. EU315254 and NC011821) were reconstructed using the neighbor joining (NJ) method under the K2P model and the maximum parsimony (MP) method (Saitou and Nei, 1987). The maximum likelihood (ML) phylogenetic tree was also constructed. To select the best-fit model, ML analysis was subjected to the Akaike Information Criterion (Akaike, 1974) using jModelTest version 0.1 (Posada and Crandall, 1998). The HKY+I model was determined to be most appropriate for our DNA sequences with the following parameters. Both MP and ML heuristic searches were conducted using a procedure with a tree-bisection-reconnection branch swapping algorithm with 100 random addition sequence replications. The mitochondrial control region sequence of the Siberian roe deer (Capreolus pygargus) was chosen as an outgroup to root the phylogenetic trees, and retrieved from GenBank (accession no. Z70317) (Douzery and Randi, 1997). Confidence in the estimated relationship was determined using the bootstrap approach and obtained through 1,000 (for NJ) or 100 (for MP and ML) replications incorporating the same model as detailed above (Felsenstein, 1985). Bootstrap analysis and phylogeny reconstruction were both conducted using PAUP version 4b10 (Swofford, 2001). Both NJ and MP were used, but the ML tree was not used due to its poor resolution with relatively low bootstrap values (< 50%). In addition, a median-joining network was created by combining the haplotypes of the water deer using the program Network version 4.0 (Bandelt et al., 1999).

Fisher’s exact test was performed with GENEPOP DOS version 4.0.10 (Raymond and Rousset, 1995). In addition, an AMOVA was conducted to investigate population differentiation (both F_ST and Φ_ST), using the program ARLEQUIN version 3.0 (Excoffier et al., 2005, Weir and Cockerham, 1984). Pairwise Φ_ST values were calculated in the analysis with 1,000 permutations of haplotypes between the three regions, which consisted of 26 locations under the hypothesis of panmixia. The software was further used to examine the demographic histories of the populations for regions and lineages. The distribution of the pairwise differences (mismatch distribution) was constructed to distinguish stable populations over time with regard to recent expansion or reduction. Tajima’s D and Fu’s F statistics were also computed.

RESULTS

Genetic diversity

An 822-bp mitochondrial D-loop region sequence was successfully determined for 107 water deer from three different regions (26 locations) (Table 2). There were no insertions or deletions in the sequenced D-loop region. A total of 17 haplotypes was defined using 18 polymorphic sites (18 transitions and no transversions), which consisted of two singleton sites and 16 parsimony informative sites in the Korean populations (A = 8: H1, 3, 5, 6, 7, 9, 11 and 12 for Gyeonggi Province; A = 13: H1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 14, 15 and 16 for Gangwon Province; and A = 7: H1, 3, 5, 6, 8, 13 and 17 for Jeolla Province) (Supplementary Fig. S1). Among the 17 haplotypes, six (H1, 3, 5, 6, 9 and 11), four (H1, 3, 5 and 6) and five (H1, 3, 5, 6 and 8) were shared between Gyeonggi and Gangwon, Gyeonggi and Jeolla, and Gangwon and Jeolla Provinces, respectively. Two (H7 and 12), six (H2, 4, 10, 14, 15 and 16) and two (H13 and 17) were detected only in Gyeonggi, Gangwon and Jeolla Provinces, respectively (Supplementary Fig. S1). The most dominant haplotype was H5 (n = 26 individuals), followed by H1 (n = 20), H6 (n = 15) and H11 (n = 14). The mean sequence divergence (p_s) within Gyeonggi, Gangwon, Jeolla and all populations was 0.848%, 0.897%, 0.890% and 0.883%, respectively (Table 3). The nucleotide diversity (π) and haplotype diversity (h) for the entire populations were 0.756% (0.549–0.801%) and 0.867 (0.598–0.865), respectively (Table 3).

Table 2. Summary of haplotype definition of all obtained mitochondrial control region sequences of water deer individuals showing the polymorphic sites only. The numbers in the columns refer to variable nucleotide positions in the 822-bp DNA sequences

	Polymorphic site																					Lineage	N
Haplotype	4 7	1 0 5	1 5 2	1 6 3	1 8 6	1 9 1	1 9 2	1 9 3	2 0 2	2 0 7	2 0 8	2 2 6	2 4 4	2 5 4	2 5 6	2 6 0	2 7 9	2 9 9	3 0 1	3 3 1	7 1 9
																								CWDH	G	C	A	C	C	G	T	C	T	A	T	A	G	T	T	C	C	G	C	T	C	A	2
																								H1	.	.	.	.	T	A	.	.	C	G	.	G	A	.	.	T	T	A	.	.	T	A	20
H2	.	T	.	.	T	A	.	.	.	G	.	G	A	.	.	T	T	A	.	.	T	A	1
H3	.	.	.	.	.	A	.	.	C	.	.	.	A	C	.	T	T	A	T	.	T	A	5
H4	.	.	.	.	.	A	.	.	C	.	C	G	A	C	.	T	T	A	T	.	T	A	4
H5	.	.	.	.	.	A	.	.	C	.	.	G	A	C	.	T	T	A	T	.	T	A	26
H6	A	.	.	T	T	.	.	T	.	.	.	.	A	C	.	T	T	A	.	C	T	B	15
H7	A	.	.	T	T	.	.	T	.	.	.	.	A	C	C	T	T	A	.	C	T	B	5
H8	A	.	G	T	T	.	.	T	.	.	.	.	A	C	C	T	.	A	.	C	T	B	2
H9	A	.	.	T	T	.	.	T	.	.	.	.	A	C	C	T	.	A	.	C	T	B	6
H10	A	T	.	T	T	.	.	T	.	.	.	.	A	C	C	T	.	A	.	C	T	B	2
H11	A	T	.	T	T	.	.	T	.	.	.	.	A	C	C	T	T	A	.	C	T	B	14
H12	.	T	.	.	T	A	.	.	C	C	.	C	A	.	.	T	T	.	.	.	T	A	4
H13	.	.	.	.	T	A	.	T	C	.	.	C	A	.	.	T	T	.	.	.	T	A	1
H14	A	T	.	T	T	.	.	T	C	.	.	.	A	C	C	T	T	A	.	C	T	B	1
H15	.	.	.	.	.	A	.	.	C	.	C	.	A	C	.	T	T	A	T	.	T	A	1
H16	A	T	.	T	T	.	C	T	.	.	.	.	A	C	C	T	.	A	.	C	T	B	1
H17	A	.	G	T	T	.	.	T	.	.	.	.	A	C	C	T	T	A	.	C	T	B	1

H: haplotype; CWDH: Chinese water deer haplotype.

Table 3. Summary of sample size (N), number of haplotypes (A), haplotype diversity (h), mean sequence divergence (p_s) and nucleotide diversity (π) for the Korean water deer populations (Gyeonggi, Gangwon, Jeolla and total)

	Region
	Gyeonggi	Gangwon	Jeolla	Total
N	35	39	33	107
A	8	13	7	17
h	0.825	0.865	0.598	0.867
ps (%)	0.848	0.897	0.890	0.883
π (%)	0.701	0.801	0.549	0.756

Phylogeny and demographic history

NJ and MP trees were reconstructed with 17 Korean water deer haplotypes (accession no. KC581917–KC581933), one Chinese water deer haplotype (two reference sequences: accession no. EU315254 and NC_011821) and one Siberian roe deer haplotype (Z70317) (Fig. 2). Both the NJ tree and the MP tree showed identical topologies, with two major clades of Korean populations. Between the two clades (lineage A and B), one subclade (A1) was grouped with the other subclade (A2) that consisted of two Chinese water deer (Fig. 2). There was no clear location-specific distribution pattern in the Korean populations (Fig. 2 and Supplementary Fig. S1). The median-joining network also showed the same pattern as that of the phylogenetic trees (Fig. 3). Tajima’s D and Fu’s F test indicated that historical demography has had an important effect on the pattern of genetic variation of mitochondrial DNA and genetic diversity for lineage B (P < 0.05 in Fu’s F) (Supplementary Table S1). In addition, the mismatch distribution of the mitochondrial DNA lineage exhibited a clear, smooth, and unimodal shape (or bell shape) for lineage B (Supplementary Fig. S2), which suggests that a certain level of population expansion occurred for lineage B, although the raggedness index (P_RAG) was not supportive of the population expansion of the lineage (Supplementary Table S1).

Fig. 2.

Phylogenetic tree of mitochondrial control region haplotypes of the Korean subspecies of water deer. The tree was rooted using a Siberian roe deer sequence as the outgroup. The numbers of changes over the whole sequence are indicated by the scale. H: haplotype; A, B: lineage or clade; A1 and A2: sub-lineage or subclade. The first and second numbers on branches are bootstrap values obtained from NJ and MP analyses, respectively. Only values over 50% are indicated; those under 50% are shown as ‘–’.

Fig. 3.

Median-joining network of 18 control region haplotypes of water deer including Chinese subspecies. Branch lengths are scaled to the number of nucleotide substitutions. The size of the circles is proportional to the haplotype frequencies. H: haplotype; A and B: lineage or clade; A1 and A2: sublineage or subclade; CWDH: Chinese water deer haplotype.

Population differentiation

The null hypothesis that “the haplotype distribution is identical across three regions (Gyeonggi, Gangwon and Jeolla Provinces)” was rejected for the three regions comprising 26 locations in South Korea (Fisher’s exact test: P < 0.001; data not shown). Moreover, the result of AMOVA showed a significant genetic barrier among the three regions (F_ST = 0.28416, P < 0.00001; Φ_ST = 0.19239, P < 0.00001; Table 4). In particular, population differentiation was observed between Jeolla and Gyeonggi (Φ_ST = 0.31) and between Jeolla and Gangwon Provinces (Φ_ST = 0.23) (Table 5). Tajima’s D and Fu’s F test for three regions was not supportive of population fluctuation (Supplementary Table S1).

Table 4. Hierarchical analysis of molecular variance based on partial mitochondrial control region sequences. Percentage of variation explained by different levels of population structure, variance components, and F- and Φ-statistics

Source of variation	df	Variance components	Percent of variation	Fixation indices	P-value
Among regions	2	–0.02200 Va	–4.39	FCT = –0.04390	0.92571
		0.08333 Va	2.58	ΦCT = 0.02580	0.00293
Among populations within regions	23	0.16437 Vb	32.81	FSC = 0.31427	< 0.00001
		0.53818 Vb	16.66	ΦSC = 0.17101	< 0.00001
Within populations	81	0.35866 Vc	71.58	FST = 0.28416	< 0.00001
		2.60890 Vc	80.76	ΦST = 0.19239	< 0.00001
Total	106	3.23041
		3.02763

Table 5. Pairwise sequence divergence (%; above diagonal) and Φ_ST values (below diagonal) of the Korean water deer from three regions comprising 26 locations in South Korea

	Gyeonggi	Gangwon	Jeolla
Gyeonggi		0.801	0.780
Gangwon	0.06495*		0.844
Jeolla	0.31246***	0.23187***

*P < 0.001, ***P < 0.00001.

DISCUSSION

Of all the small ruminant species in East Asia, China and Korea, the water deer is one of the rarest, only inhabiting two countries. Despite this biogeographical status, the Korean subspecies has not been protected due to its high population density in South Korea, while the Chinese subspecies is protected in China. Recently, a restoration project for the Chinese subspecies began in Shanghai and the subspecies has been continuously studied in China. However, studies on water deer in Korea have rarely been conducted, regarding its ecology (e.g., population size, distribution and density) and genetics (e.g., genetic diversity, taxonomic status and population differentiation) (Kim et al., 2011a). In particular, the present status of the genetic diversity, phylogeny and population structure of the Korean subspecies is an important issue that needs to be examined to obtain a better understanding of its genetic characteristics and management.

Genetic diversity

From the present study, 17 haplotypes were found among 107 individuals. Their nucleotide diversity (π) and haplotype diversity (h) were 0.756% and 0.867, respectively (Table 3). These results indicate that the Korean subspecies has a relatively lower genetic diversity than the Chinese subspecies (Hu et al., 2006). A recent study of Korean water deer also showed that the Korean population has fewer haplotypes (Koh et al. 2009) (A = 21 from 28 samples for Chinese populations; A = 14 from 42 samples for Korean populations). In the present study, the mean sequence divergence (p_s) among all individuals was 0.883% (0.848–0.897%), which is within the range of the previous result (p_s = 0.5–2.1%) by Koh et al. (2009).

Hu et al. (2006) previously reported the genetic characteristics of the Chinese subspecies based on mitochondrial D-loop sequence (403 bp) variation. The subspecies showed 18 different haplotypes from 40 individuals, which represented slightly higher or similar genetic diversity (π = 1.318% and h = 0.923) compared to other Cervidae such as the sika deer (Cervus nippon) (π = 1.06% and h = 0.932; Wu et al., 2004), Eld’s deer (C. eldi) (π = 1.4–2.4% and h = 0.81–0.89; Balakrishnan et al., 2003) and black muntjac (Muntiacus muntja) (π = 0.562% and h = 0.862; Wu and Fang, 2005). The mean sequence divergence of all of the water deer was 1.7% (0.2–3.5%) (Hu et al., 2006).

Several hypotheses can be suggested to explain the relatively low genetic diversity of the Korean population. First, this low genetic diversity (number of haplotypes, sequence divergence, nucleotide diversity and haplotype diversity) may result from historical anthropogenic effects, such as excessive harvesting, poaching, and political upheavals such as war and famine. Historically, for more than a thousand years, the distribution of water deer in the Korean peninsula seems to have been quite different from their current distribution. Currently, water deer are found in all South Korean regions, including high-altitude mountainous areas (Woo et al., 1990). However, the historical range of water deer in the Korean peninsula was restricted mainly to the southwestern, lowland region of the peninsula. This was probably because water deer prefer lowland habitats near water sources, such as riverside wetlands. A high density of predator (tigers, leopards and wolves) and competing ungulate species (sika deer and roe deer) might also have prevented the expansion of their range further into the eastern mountainous areas of the peninsula. However, the situation changed during the last hundred years: most major predator species along with the major competing species became extinct in South Korea, and consequently water deer became the most abundant ungulate species in the whole of the country. Thus, the historical distribution of water deer had been restricted to the southwestern part of the Korean peninsula, which coincides with the most intensively developed agricultural area and the highest density of human population of the peninsula. Therefore, the historically localized water deer population may have been highly vulnerable to human activities like hunting, land reclamation and war. The South Korean water deer population has become more abundant during the last two decades, but more time will be required to recover from their lowered genetic diversity.

A second cause of this low genetic diversity may have been a founder effect during a glacial period, as historical evidence of climate change in the Korean peninsula suggests the existence of glaciers in high mountainous northern regions during the Pleistocene period (Kim, 1994; Kong and Watts, 1993). In contrast, no glaciers existed in the central or southern parts of the Korean peninsula despite the extreme cold. However, since southern coastal areas are presumed to have been comparatively warmer (Kong and Watts, 1993), a refuge may have formed somewhere along these warmer coastlines. The existence of a small founder population in such a refuge could be the cause of the comparatively lower genetic diversity. This hypothesis would concur with the population expansion in lineage B based on mismatch distribution.

Phylogeny

Water deer were previously considered to be a species consisting of two subspecies, H. inermis argyropus and H. inermis inermis (Cooke and Farrell, 1998; Geist, 1998). These two subspecies have very similar phenotypes, except for their coat colors (Tate, 1947). The Korean subspecies has a darker coat color and a much more reddish-colored head than the Chinese subspecies (Koh et al., 2009). Koh et al. (2009) reported two sympatric phylogroups and low levels of mitochondrial genetic distance between the two subspecies (Korean and Chinese) in both the cytochrome b gene (1.3%) and the D-loop region (2.1%). Therefore, they suggested that the validity of the subspecies designation amongst water deer needs to be re-evaluated by performing detailed statistical analyses of morphological characters, including coat color, as well as genetic characteristics.

Likewise, we found that there are two sympatric clades (A and B) in water deer (Fig. 2). The Chinese D-loop sequences (EU315254 and NC_011821) are grouped with one (A) of the two clades. The genetic distance between lineage A and lineage B was 1.332 ± 0.340% (data not shown). The standard equation d = 2Tu (where u is the substitution rate and T is time (Saitou and Nei, 1987)) and a conservative fossil record calibration of 25 million years ago (MYA) for the most recent common ancestor of Cervidae and Bovids (Vrba and Schaller, 2000) produced a substitution rate of approximately 0.39% (u = 0.00393) per site per million years (Ludt et al., 2004). These estimates concordantly suggest that the A and B lineages (2.1–1.3 MYA) separated at the beginning of the Pleistocene period. The genetic distances within lineage A and lineage B were 0.525 ± 0.167% and 0.264 ± 0.113%, respectively. This indicates that both lineages have a very close phylogenetic relationship in terms of mitochondrial D-loop haplotypes. The median-joining network showed that of the two location-nonspecific lineages, one sub-lineage (A1) was grouped with the Chinese sub-lineage (A2) (Fig. 3). This result also supports the possibility that the two water deer subspecies are from the same taxonomic group.

Moreover, the results of the previous research in China and Korea and ours indicate that both lineages A and B appeared in Korea while only lineage A appeared in China (Hu et al., 2006; Koh et al., 2009). This suggests that lineage B originated in Korea. Furthermore, the two lineages were separated from each other in the early Pleistocene period when climatic fluctuations greatly affected the distribution of species. At the beginning of the Pleistocene, South Korea was connected with China, as a fresh-water lake or river existed where the Yellow Sea currently resides (Dobson, 1994; Keigwin and Gorbarenko, 1992; Kim, 1994; Nelson, 1993). The water deer may therefore have been distributed widely in both Korea and China without a specific barrier. At the start of the Ice Age (about 1.6 MYA), two refuges may have formed in comparatively warm areas. Their populations gradually became lineages A and B. Lineage B may have inhabited the southern coastline of Korea, and then population expansion toward the north and west during interglacial periods, and/or exchanges between the two lineage populations in China and Korea during relatively mild glacial periods, may have occurred. As some groups of lineage A migrated eastward, a complete separation of Korea and China, about 10,000 years ago, may have been the cause of the two dependent lineages existing in Korea (Kong and Watts, 1993). Although the two lineages also existed in China, the survival of only one lineage may have been due to a decrease in population because of human intervention (Koh et al., 2009). However, more detailed analyses, including DNA sequencing using fossils in China, would be required to test this hypothesis.

Population differentiation

The AMOVA test suggested the existence of distinct populations with slightly different genetic structures (F_ST = 0.28416, P < 0.00001; Φ_ST = 0.19239, P < 0.00001; Table 4). This indicates that there has been a barrier between the populations to prevent random gene flow among them. In particular, population isolation was observed between the Jeolla and Gyeonggi (pairwise Φ_ST = 0.31246, P < 0.00001) or Gangwon (pairwise Φ_ST = 0.23187, P < 0.00001) regions. Hu et al. (2006) also showed that a distinctive genetic barrier existed between the Zhoushan archipelago and mainland China (F_ST = 0.088, P < 0.001; Φ_ST = 0.075, P = 0.043). According to Hu et al. (2006), a geographical separation of island and continent caused population differentiation. However, population differentiation could also be derived from a different dispersal ability in males and females. Territory and dispersal of male water deer are generally wider than those of females (Kim et al., 2011b). The result of analysis of mtDNA, which is maternally inherited, indicated a slight population differentiation in this study. Further study is needed using other markers, such as a nuclear microsatellite markers, to show more precise regional differentiation.

This study confirmed the low genetic diversity and controversial phylogenetic status of the Korean water deer, and showed that there may be a certain level of differentiation among the populations at the regional scale. However, only water deer from restricted regions (n = 3) were analyzed. Furthermore, we did not analyze the genetic differentiation at finer scales, such as between water deer from the southern and northern parts of Gyeonggi Province (or the Han River), between the northern areas of Gyeonggi Province and western areas of Gangwon Province, and between the western and eastern sites of Gangwon Province (or the Taebaek Mountains). In addition, we did not include the other provinces in South Korea (e.g., Chungcheong and Gyeongsang Provinces), due to the insufficient number of samples collected from these regions. Future studies using more samples collected from various localities in China as well as Korea will provide better resolution of the genetic structure of water deer at regional and local scales. Our analyses were based only on matrilineal mitochondrial D-loop markers, and therefore do not reflect any patrilineal influence. Therefore, further use of other genetic markers (e.g., nuclear microsatellite DNA or Y-linked DNA markers) would help us to find unbiased and comprehensive information related to the genetic diversity, phylogeny and population differentiation of water deer in East Asia.

ACKNOWLEDGMENTS

This study was supported in part by the Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University and a Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) (No. 2009-0080227). This study was also partially supported by the Korea Ministry of Environment and the National Research Foundation of Korea (NRF 2009-83257). We would also like to express our extreme gratitude to Mr. Jong-In Choe (Ansan City Hall), Mr. Su-Ho Kim (The Korean Association for Bird Protection), Dr. Chang-Yong Choi (Oklahoma University & USGS), Dr. Tae-Young Choi (National Institute of Ecology), Dr. Young-Jun Kim (National Institute of Ecology), Mr. Han-Chan Park (Seoul National University) and Wildlife Rescue Center (Paju, Jeonnam, Gyeonggi) for providing us with water deer samples during this study period.

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