Biochemical Systematics and Ecology 38 (2010) 897–905

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Biochemical Systematics and Ecology

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Molecular phylogenetics and population structure of Sousa chinensis inChinese waters inferred from mitochondrial control region sequences

Lian Chen a,b, Susana Caballero c, Kaiya Zhou a, Guang Yang a,*

a Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, ChinabNanjing Institute of Environmental Sciences, Ministry of Environmental Protection of China, Nanjing 210042, Chinac Laboratorio de Ecologia Molecular de Vertebrados Acuáticos LEMVA, Departamento de Ciencias Biológicas, Universidad de los Andes, Carrera 1 No. 18A-10,Bogotá, Colombia

a r t i c l e i n f o

Article history:Received 1 June 2010Accepted 24 September 2010Available online 10 November 2010

Keywords:SousaGenetic variationPopulation structuremtDNA

* Corresponding author. Fax: þ86 25 85891163.E-mail address: gyang@njnu.edu.cn (G. Yang).

0305-1978/$ – see front matter � 2010 Elsevier Ltddoi:10.1016/j.bse.2010.09.009

a b s t r a c t

Phylogenetic analyses of the genus Sousawere carried out across their geographic range andfocused especially on the genetic variation patterns and population structure of Sousa chi-nensis in Chinese waters. The analyses were based on 287-bp of the mitochondrial controlregion from 122 Indo-Pacific humpback dolphins (Sousa chinensis) and two Atlantic hump-backdolphins (Sousa teuszii). All specimens fromChina grouped together in awell-supportedclade with high bootstrap (BS) and Bayesian posterior probability values (BPP), stronglysuggesting that only one species of Sousa exists in China. Six haplotypeswere identified in 65individuals from Xiamen and Pearl River Estuary in China. The extremely low level ofdetectedmtDNA genetic diversity strongly suggests a high priority in conservation of Sousa.The highly significant genetic differentiation between the two populations in Xiamen andPearl River Estuary supports the designation of these two populations as separatemanagement units (MUs) when designing management programs for this species.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Although the humpback dolphin genus Sousa has an extensive geographic range in the shallow, near- shore waters of thetropical Atlantic, Indian, and Western Pacific Oceans, its taxonomy and systematics have remained highly controversial. Todate, five species have been described for this genus: Sousa chinensis (Osbeck, 1765), Sousa plumbea (Cuvier, 1829),S. lentigenosa (Owen, 1866), Sousa teuszii (Kükenthal, 1892) and S. borneensis (Lydekker, 1901), but by the mid-2000s, mostscientific and taxonomic authorities accepted two species-the Atlantic humpback dolphin S. teuszii, ranging fromMauritaniasouth to Angola, and the Indo-Pacific humpback dolphin S. chinensis (grouping S. plumbea, S. lentigenosa and S. borneensis),ranging from southern China and north Australia in the east, along the coasts of the Indian andwestern Pacific Ocean, to SouthAfrica in the west (Jefferson and Karczmarski, 2001; Reeves et al., 2003). However, Rice (1998) identified three valid speciesfor this genus in his exhaustive review of marine mammal species classification, i.e. S. chinensis (Indo-Pacific humpbackdolphin or Chinese white dolphin), found in the eastern Indian Ocean and Pacific; S. plumbea (Indian humpback dolphin),found in the western Indian Ocean; and S. teuszii (Atlantic humpback dolphin), found in the Atlantic Ocean off West Africa.A study on skull morphology of over 200 humpback dolphins originating from much of the range of this genus supportedevidence for three groups (Atlantic Ocean/West Africa, West Indian Ocean, Eastern Indian Ocean/Pacific Ocean respectively),corresponding to the ‘teuszii’, ‘plumbea’, and ‘chinensis’ forms (Jefferson and Van Waerebeek, 2004). However, the externalmorphological characters that have been traditionally used to differentiate these forms may be plesiomorphic (ancestral) or

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the result of convergent evolution, and as such, may not be phylogenetically informative. Recently, a mitochondrial DNAanalysis (Frère et al., 2008) surprisingly found that humpback dolphins from South Africa and China (Hong Kong) formeda strongly-supported clade with the Atlantic S. teuszii to the exclusion of animals from Australia, which suggested thatAustralian humpback dolphins are not S. chinensis but may represent a distinct species in their own right. These resultsindicate that a revision of humpback dolphin (Sousa spp.) taxonomy and systematics is urgently needed.

In China, the distribution of the humpback dolphin appears to be discontinuous due to habitat fragmentation, from themouth of the Yangtze River in the north along the coastline to the Beibuwan Gulf adjacent to Vietnam border in the South(Jefferson, 2000; Jefferson andHung, 2004; Zhou, 2004). Five Sousa populations have been confirmed occurring in the South andEast China Seas: southern Fujian (Xiamen/Chinmen), Hong Kong and the Pearl River Estuary, Leizhou Bay, western coast ofTaiwan, BeibuwanGulf (Jefferson, 2000; Liu and Huang, 2000; Zhou et al., 2003, 2007;Wang et al., 2004; Chen et al., 2008a), butonly very preliminary investigations of genetic diversity and population structure have been conducted for these populations,primarily based on mitochondrial DNA control region sequences. The finding that Indo-Pacific humpback dolphins from northand south of Lantau Island inHong Kong are genetically isolated (Porter,1998)was regarded as highly unlikely, for itwas inferredfrom a small sample size of only 10 individuals and contradicted with the results of photo-identification results and fieldobservations (Jefferson, 2000). Jefferson (2000) provided a weak suggestion of population genetic subdivision between HongKong and Xiamen. Recently, Chen et al. (2008b) conducted a comparative analysis of mitochondrial DNA control region andcytochrome b (cyt b) sequences of Indo-Pacific humpback dolphins from the Pearl River Estuary and Xiamen, which suggestedthe possibility for genetic exchange between both populations, and that the Xiamen population might represent a uniquematernal lineage. However, considering that only a very small sample size (n ¼ 12) was examined, this result requires furtherconfirmation before we make applicable suggestions for conservation and management programs. Moreover, Zhou (2004) andZhou et al. (2003) suggested that Sousa in the BeibuwanGulfwas distinct from S. chinensis in other Chinesewaters and should beidentified as S. plumbea. However, so far there is no convincing evidence to support the existence of S. plumbea in Chinesewaterswith only occasional and equivocal sighting records and only one sample available from the Beibuwan Gulf.

Despite the endangered status of Sousa chinensis in China, little is known about its population genetics so far. The absenceof detailed population genetic information has led conservation agencies to develop management strategies based onavailable morphological evidence and descriptions, but relevant information about population structure is missing in theirdecision making. Effective conservation of endangered species requires determining which taxawithin closely related groupsare different species, as well as identification of evolutionarily significant units (ESUs) within species and management units(MUs) (Moritz, 1994) within ESUs so that evolutionarily important groups are preserved and conservation actions may beimplemented. An understanding of the genetic diversity and the spatial structure of populations is also important forestablishing the appropriate scale and subunits for conservationmanagement and tominimize genetic erosion (Moritz,1999).

To better understand the genetic diversity and population structure of Indo-Pacific humpback dolphins in Chinese waters,we examined variation at a highly variable portion of the mtDNA control region across the entire known range of this speciesin China, especially for the Xiamen population and the Pearl River Estuary population. A phylogenetic survey of Sousa chi-nensis in Chinese waters was conducted based on sequences from a total of 122 Indo-Pacific humpback dolphins and twoAtlantic humpback dolphins. We also intended to identify management units for conservation of this endangered species.

2. Materials and methods

2.1. Sampling, DNA extraction, PCR amplification, and sequencing

Genomic DNAwas isolated from13 Sousa samples of Chinesewaters (see detailed information in Table 1) using the DNeasyTissue Extraction Kit (QIAGEN) following the manufacturer’s instructions. Amplification of the mtDNA control region was

Table 1Summary of samples used in this study. Numbers in the parentheses refer to sample sizes.

Regions Populations Sampling sites and sample sizes Reference

China Xiamen (XM, 24) Xiamen (9a), FujianProvince This studyXiamen (5), FujianProvince Chen et al. (2008b)Xiamen (10), FujianProvince Jefferson (2000)

Pearl River Estuary (41) Zhuhai (ZH, 6), GuangdongProvince Chen et al. (2008b)Hong Kong (HK, 19) Frère et al. (2008)Hong Kong (HK, 16) Jefferson (2000)

Others (4) Yueqing (YQ, 1, NNU0380), ZhejiangProvince This studyNantong (NT, 1, NNU0216), JiangsuProvinceLeizhou (LZ, 1, NNU0553), GuangdongProvinceBeihai (BH, 1, NNU0475), GuangxiProvince

South Africa South Africa (SA, 23) Frère et al. (2008)Australia Australia (AUS, 28) Frère et al. (2008)India India (IND, 2) DQ364693, EF061406West Africa (Sousa teuszii) Mauritania (MTN, 2) Frère et al. (2008)

a Voucher numbers for these nine samples are NNU(Nanjing Normal University) 0472, 0474, 0502–5, 0537–8, and 0568.

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carried out using the primers Strobeck (50-TAATATACTGGTCTTGTAAACC-30) (Murray et al., 1995) and H00034 (50-TAC-CAAATGTATGAAACCTCAG-30) (Rosel et al., 1995). PCR amplifications were conducted in a total volume of 30-mL containing thefollowing components: 50 ng of DNA, Ex Taq premix buffer 15 mL (TaKaRa), 1.0 pm of each primer. PCR was performed usinga thermal cycler with the following protocol: 5min at 95 �C followed by 35 cycles of 30 s at 95 �C, 30 s at 50 �C and 40 s at 72 �Cwith a final extension time of 10min at 72 �C. PCR products were purifiedwith the QIAquick PCR Purification Kit (QIAGEN). AllPCR products were sequenced on an ABI Prism 3100 automated sequencer using ABI’s Big Dye Terminator Kit (AppliedBiosysterms).

2.2. Data analyses

2.2.1. Phylogenetic reconstructionsAnalyses included sequences from the 13 Sousa samples analyzed in this study combined with 111 sequences previously

published in GenBank or previously used in other published papers. Therefore, a total of 124 available Sousa samplesincluding 69 from China, 28 from Australia, 23 from South Africa, two from India, and two from West Africa. Samplinginformation for Australia, South Africa, and West Africa is available in Frère et al. (2008), sampling information for India isavailable in Jayasankar et al. (2008), whereas sampling information for China (including 13 new ones added in the presentstudy, 19 samples examined in Frère et al. (2008), and another 37 ones used in other published papers) is shown in Fig. 1 andTable 1. A total of 124 Sequences and sequences only for Xiamen and Pearl River Estuary were aligned using CLUSTAL Walgorithm implemented in MEGA 3.0 software respectively (Kumar et al., 2004). Alignments were checked and edited byhand. Segments not present in all sequences were excluded from relevant data analyses.

Phylogenetic relationships among Sousa mtDNA haplotypes were reconstructed by maximum likelihood (ML) imple-mented in PAUP version 4.0 (Swofford, 2003) and Bayesian analyses as implemented in MRBAYES 3.0 (Huelsenbeck andRonquist, 2001). The most appropriate model of sequence evolution for the likelihood analysis was selected using theAkaike Information Criterion (AIC) (Akaike, 1973) using Modeltest version 3.7 (Posada and Crandall, 1998). ML analyses wereperformed using the K81ufþG model with gamma (G) shape parameter (a) ¼ 0.2591. Bootstrapping (500 replications withrandom number seed; groups with a frequency of >50% retained) was used to determine the robustness and support of

Fig. 1. Source populations of humpback dolphin samples (Sousa spp.) in Chinese waters available for the present study. Numbers indicate sample size in eachlocation.

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topologies obtained for the likelihood trees. Bayesian posterior probabilities (BPP) were calculated using a Metropolis-coupled, Markov chain Monte Carlo (MCMC) sampling approach with four simultaneous Markov chains run for 1,000,000generations and trees sampled every 100 generations. The Indo Pacific bottlenose dolphin Tursiops aduncus (AF459520) andshort-beaked common dolphin Delphinus delphis (FM211538) were used as outgroups in all analyses. A median-joiningnetwork of all unique haplotypes was constructed to visualize phylogeographic relationships among haplotypes and pop-ulations using NETWORK version 4.2.0.1 (http://www.fluxus-engineering.com, Bandelt et al., 1999).

2.2.2. Genetic diversity and population structure of Sousa chinensis in ChinaDNASP 4.0 (Rozas et al., 2003) was used to estimate haplotype diversity (h, the probability that two randomly chosen

mtDNA lineages are different in the sample) and nucleotide diversity (p per nucleotide site, the probability that two randomlychosen homologous nucleotides are different in the sample), especially for Xiamen and Peal River Estuary populations.

The amount of genetic variability partitioned within and among populations was assessed by an analysis of molecularvariance (AMOVA; Excoffier et al., 1992). Significance of estimated FST values was tested using 10 000 permutations.We testedfor past demographic history of dolphins in Xiamen and Peal River Estuary using several methods. Tajima’s D (Tajima, 1989)and Fu’s Fs (Fu, 1997) tests conducted through ARLEQUIN 3.1(Excoffier et al., 2005) were carried out to examine deviationsfrom neutrality (as would be expected under population expansion). Tajima’s test is widely used as a test for neutrality, andFu’s Fs test outperforms other tests in detecting population growth for large sample sizes (Ramos-Onsins and Rozas, 2002).We also examined the distribution of pairwise differences (mismatch distributions) for each population to look for evidenceof past expansion (Rogers and Harpending, 1992). In general, populations undergoing recent and sudden expansion exhibita Poisson-shaped mismatch distribution while populations in equilibrium tend to have ragged distributions (Slatkin andHudson, 1991).

3. Results

3.1. Genetic diversity

Sequences generated in this study have been deposited in GenBank under the accession numbers HQ200193-5 andHQ221868-80. Forty-one variable sites defining 15 unique haplotypes were identified in the 287-base pair (bp) fragment ofmtDNA control region sequences obtained from 124 Sousa samples available for the present study (Table 2). No haplotypeswere shared between major sampling regions (i.e. China, Australia, India, South Africa, West Africa), and each region wasdominated by one or two different haplotypes. In China, five haplotypes were identified in seven sampling locations, whiletwo haplotypes (HK017 and HK020) were only found in the Hong Kong population. Haplotype CH01 was dominant (62.3%)and distributed over a vast region of the dolphins’ geographic distribution in China. However, when considering a 335-bpsequence only for Xiamen and Pearl River Estuary (Zhuhai and Hong Kong) populations, nine variable sites were detectedamong the 65 individual samples examined, defining six unique haplotypes. Of these, two haplotypes (HK017 and HK020)were unique to Pearl River Estuary and two haplotpyes were found only in Xiamen (CH02 and CH03). The Xiamen populationexhibited relatively higher haplotype diversity than Pearl River Estuary population, whereas nucleotide diversity was similarfor both populations (Table 3).

Table 2Polymorphic sites found among the 15 mtDNA control region haplotypes identified in 124 humpback dolphins based (287-bp fragment). Dots indicateidentity to the top sequence. Dashes represent insertion–deletion events. Number represents frequency of each haplotype in particular geographic regions.See Table 1 for the localities abbreviations of YQ, XM, HK, NT, ZH, LZ, BH, SA, AUS, IND, and MTN.

Table 3Haplotype (h) and nucleotide diversity (p) � standard error (s.e.) in Xiamen and Pearl River Estuary populations. n represents the frequency of eachhaplotype based on 335 mtDNA control region fragments.

Population h p Haplotypes n

Xiamen 0.649 � 0.050 0.0045 � 0.0004 CH01 9CH02 11CH03 2CH04 2

Pearl River Estuary 0.383 � 0.091 0.0029 � 0.0008 CH01 32CH04 2HK017 3HK020 4

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3.2. Phylogenetic position of Sousa chinensis in Chinese waters

Phylogenetic reconstruction, based on maximum likelihood (ML) using the K81ufþG model of evolution as the mostappropriatemodel and Bayesian inference (BI), generated similar topologies (Fig. 2). All Sousa haplotypes grouped together tothe exclusion of sequences from outgroup species (i.e. Delphinus delphis and Tursiops aduncus) confirming the monophyly ofmtDNA lineages for this genus. The humpback dolphins were divided into four major clades corresponding to dolphins fromAustralia, South Africa and India, and West Africa (S. teuszii) respectively. The dolphins from South Africa, India, West Africa,and China formed a monophyletic clade, having sister relationship with dolphins from Australia supported by high bootstrapvalues and high Bayesian posterior probability scores. However, phylogenetic relationships among South Africa þ India,China, and West Africa were not well resolved. The median-joining network (Fig. 3) showed a similar pattern of relationshipamong haplotypes as revealed in the phylogenetic reconstructions.

3.3. Population structure and demographic history of Sousa chinensis in China

The analysis of molecular variance (AMOVA) found significant genetic structure between dolphins from Xiamen and PearlRiver Estuary populations. The differences between populations accounted for 27.36% of the total variance, i.e. FST ¼ 0.274，P < 0.001.

Fig. 2. Maximum likelihood (ML) reconstruction of relationships among Sousa spp. based on mtDNA control region haplotypes (287-bp) from five regions: China(Xiamen, Hong Kong and other regions), South Africa (Natal), Australia (Queensland and other regions), India, and West Africa (Mauritania). ML bootstrap scoresare shown above internal nodes and Bayesian posterior probability scores are shown below. ML and Bayesian methods converged on the same tree. Terminalnodes are labeled with haplotype codes as in Table 2. Sequences from Delphinus delphis and Tursiops aduncus were used as outgroups.

Fig. 3. Median-joining network showing the genetic relationships and genetic distance among 15 haplotypes of Sousa. Colours indicate the proportion ofindividuals sampled in different geographical regions within the study area. Red dots represent putative mutational steps between haplotypes. The size of thecircle is proportional to the frequency of that haplotype.

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Tests of population expansion revealed no clear pattern of expansion or contraction in these populations. Tajima’s D valuewas positive in Xiamen population (D ¼ 2.170, P > 0.1) but was negative in Pearl River Estuary population (D ¼ �0.428,P > 0.1). Fu’s Fs values were positive and none of them were significant for either population (Xiamen: Fs ¼ 1.5008, P > 0.1;Pearl River Estuary: Fs ¼ 1.6323, P > 0.1). Distributions of pairwise differences (mismatch distributions) suggested noexpansion in these populations (Fig. 4).

4. Discussion

4.1. Phylogenetic position of Sousa chinensis in Chinese waters

In the present study, some newcontrol region sequences of humpback dolphins from China and Indiawere combinedwithpreviously published sequences in order to do a more comprehensive analysis of the phylogenetic position of Sousa chinensisin Chinese waters. In the phylogenetic reconstructions, all samples coming from different localities in China grouped togetherin a well-supported clade (Fig. 2). As of particular interest, a sample from Beihai (the Beibuwan Gulf) shared the samehaplotype (CH02) with dolphins in Xiamen. The species status S. plumbea assigned for the dolphins in the Beibuwan Gulf byZhou et al. (2003) and Zhou (2004) was not supported by the present analysis of mitochondrial control region sequences.In another words, the present finding suggests that only one species of Sousa exists in Chinese waters, although the samplesizes for most localities in China except for Xiamen and Pear River Estuary make it necessary for further confirmation withmore samples in future especially when this finding is practically applied in designing of conservation programme for this

Fig. 4. Mismatch distribution analyses for mtDNA haplotypes from (a) Xiamen, and (b) Pearl River Estuary populations. Bars represent observed distribution ofpairwise differences among samples, while the line shows the distribution modeled for sudden population growth.

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endangered species in China. Furthermore, with more samples included in this study, it is further confirmed that humpbackdolphins from South Africa and China formed a supported clade with the Atlantic S. teuszii, whereas chinensis dolphins fromChina and Australia did not have direct affinity (Fig. 2).

4.2. Genetic diversity and population structure of Sousa chinensis in China

Although five populations of humpback dolphins in China have been confirmed (Chen et al., 2009) and abundant infor-mation has been gathered for some populations so far (Jefferson, 2000; Zhou et al., 2007; Chen et al., 2008a, 2009;Wang et al.,2004), the patterns of genetic variation, especially the level of genetic differentiation or gene flow among these populations,are still unclear. Different research groups are producing contradicting results, probably due to sampling and methodologicallimitations. For example, Jefferson (2000) identified three distinct haplotypes when analyzing 446-bp mtDNA control regionsequences from specimens in Hong Kong and Xiamen waters and provided some indication of population subdivisionbetween Hong Kong and Xiamen. By contrast, a recent study conducted by Chen et al. (2008b) found dolphins from the PearlRiver Estuary and Xiamen sharing a number of haplotypes. These authors suggested possible gene flow occurring betweenboth populations despite the long distance between them. However, both studies may be biased since only small sample sizeswere available (n¼ 26 for Jefferson, 2000, and n¼ 11 for Chen et al., 2008b, with one sample excluded due to uncertainty of itssequence) and only some preliminary analyses could be conducted. In the present study, 28 new samples (n ¼ 19 for the PearRiver Estuary and n ¼ 9 for Xiamen) were added, increasing the total sample size to 65. With a relatively larger sample size,a significant level of genetic structuring with high statistical support was found between Xiamen population and Pearl RiverEstuary population. Both populations have some unique haplotypes, i.e. CH02 and CH03 for Xiamen population and HK017and HK020 for the Pear River Estuary population. Even though two haplotypes (haplotype CH01 and CH04), were sharedbetween Xiamen and Pearl River Estuary populations, their frequencies were different. Most samples from the Pearl RiverEstuary (78.0%, 32/41) were identified as having the haplotype CH01, whereas this haplotype was found in 37.5% (9/24)specimens from the Xiamen population. These results imply genetic isolation and no genetic interchanging between thesetwo populations. These shared haplotypes could be alternatively explained as representation of shared and ancestral char-acters (symplesiomorphies).

Compared with the significant genetic structure between populations, within-population genetic diversities were rela-tively low. When all samples from Chinese waters were combined and treated as a single population, haplotype diversity andnucleotide diversity were h ¼ 0.574 � 0.059 and p ¼ 0.0082 � 0.0007, respectively, both comparable to those from Australia(h¼ 0.505� 0.094, p¼ 0.0090� 0.0015) and South Africa (h¼ 0.561�0.051, p¼ 0.0024� 0.0006).When Xiamen populationand Pearl River Estuary population were considered separately, the former exhibited relatively higher haplotype diversitythan the latter (Table 3), whereas nucleotide diversity was similar in both populations. These estimates for control regionvariability were lower than those reported for many other cetacean populations, e.g. Atlantic spotted dolphins (Stenellafrontalis) (430-bp, h ¼ 0.901 � 0.012, p ¼ 1.47% � 0.008) (Adams and Rosel, 2006), dusky dolphins (Lagenorhynchus obscurus)(591-bp, h ¼ 0.97 � 0.01, p ¼ 1.63% � 0.83) (Cassens et al., 2005), finless porpoises (Neophocoena phocaenoides) (345-bp,h¼ 0.79� 0.01, p¼ 0.44%) (Yang et al., 2008). Although the discrepancies in sampling strategy, sample size, and the short sizeof the gene segment examined would affect the validity and reliability of such a direct comparison, this preliminary analysisprovides valuable clues indicating that this species is characterized by a low level of mitochondrial control region variability inChinese waters. It also suggests that all Sousa populations in the world show low genetic diversity when comparing withother cetaceans.

4.3. Implications for conservation

Coastal areas are among the marine habitats at higher risk from human activities (Moore, 1999). Therefore, the coastaldistribution of humpback dolphins renders them particularly susceptible to various anthropogenic threats. Genetic diversityis generally believed to be a prerequisite for the short- and long- term survival of an endangered species (Lande,1988). Loss ofgenetic diversity, therefore, could lead to a decline in a species’ ability to copewith a changing environment and demographicfluctuations both in the short and long term (Ellstrand and Elam, 1993; Milligan et al., 1994; Reisch et al., 2003). Molecularmeasures of genetic diversity will assist in deciding what constitutes a genetically viable population, and measures ofpopulation differentiation can contribute to placing value on the different ecological groupings. The extent of genetic vari-ability characteristic of the mtDNA control region has been widely used in studies of population structure and can be veryinformative in identifying meaningful population subdivisions (Moritz, 1994), an important prerequisite to effectiveconservation, management and monitoring programs. Such analyses of mitochondrial DNA sequences have already provedinvaluable in investigating population differentiation in a number of cetacean species (e.g. Adams and Rosel, 2006; Daleboutet al., 2005; Gaspari et al., 2007; Parsons et al., 2002; Yang et al., 2008).

According to Moritz’s (1994) criterion, management units (MUs) are identified by significant differences in allelefrequency distributions and significant divergence in mitochondrial or nuclear loci, whereas evolutionarily significant units(ESUs) are designated on the basis of populations that are reciprocally monophyletic for mtDNA haplotypes and showsignificant differentiation at nuclear loci. In the present study, there is no significant phylogeographic partitioning of mtDNAhaplotypes present in populations from Sousa chinensis in Chinese waters, which are clearly not reciprocal monophyleticgroups. Two haplotypes were shared between Xiamen population and Pearl River Estuary populations (CH01 and CH04).

L. Chen et al. / Biochemical Systematics and Ecology 38 (2010) 897–905904

Using Mortiz’s criterion, these populations do not correspond perfectly to two distinct ESUs. However, based on significantdifferences in frequencies of mtDNA haplotypes and especially significant FST value, the status of the Xiamen and the PearlRiver Estuary populations should stand as two distinct MUs, and should be separately managed to best preserve the geneticdiversity at species level. Very low genetic diversity in Sousa chinensis from Chinese waters might decrease the species’potential to adapt to diseases or environmental modifications. For this reason, the low genetic variation in Sousa chinensis ofChinese waters, along with continued risks of habitat loss, climate change and environmental pollution, require an urgentconservation and management plan by region.

It is noteworthy that a lack of variability in the mitochondrial control region might not necessarily reflect low levels ofgenetic heterozygosity in the nuclear genome (Rosel and Rojas-Bracho,1999). Although we have isolated a set of polymorphicmicrosatellite markers for the Indo-Pacific humpback dolphins (Chen and Yang, 2009), the difficulty in collecting additionalsamples hampered the application of these nuclear markers to further investigate genetic variation and population structureof Sousa chinensis in Chinese waters at present. Furthermore, except for Sousa chinensis in Xiamen and Pearl River Estuary,population genetics information is almost completely absent for other populations in China. A more comprehensive surveyand sampling, and especially adding samples for other populations will help us to better understand the extent of populationsubdivision and document the relative amount of genetic diversity remaining in the extant populations of Sousa chinensis inChinese waters. This will further help us to design a more scientifically based and effective conservation program for thisendangered species in China.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (NSFC) key project grant no. 30830016,NSFC grant nos 30670294 and 30470253, the Program for New Century Excellent Talents in University (NCET-07-0445), theMinistry of Education of China, the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP20060319002), the Ministry of Education of China, and the major project of the Natural Science Foundation of the JiangsuHigher Education Institutions of China (07KJA18016). S. Caballero is grateful to the SFWFS DNA and Tissue Archive, T. Jeffersonfrom the NMFSSouth West Fisheries Science Center and to the Agriculture, Fisheries and Conservation Department of HongKong for access to samples.

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