ГЕНЕТИКА, 2012, том 48, № 12, с. 1410-1417
ГЕНЕТИКА ЖИВОТНЫХ
УДК 575.17
POPULATION GENETIC STRUCTURE AND HISTORICAL DEMOGRAPHY OF Oratosquilla oratoria REVEALED BY MITOCHONDRIAL DNA SEQUENCES
© 2012 D. Zhang, Ge Ding, B. Ge, H. Zhang, B. Tang
Jiangsu Provincial Key Laboratory of Coastal Wetland Bioresources and Environmental Protection, Yancheng Teachers
University, Jiangsu Yancheng 224051 China e-mail: zhangdaizhen@yahoo.com.cn Received February 28, 2012
Genetic diversity, population genetic structure and molecular phylogeographic pattern of mantis shrimp Oratosquilla oratoria in Bohai Sea and South China Sea were analyzed by mitochondrial DNA sequences. Nucleotide and haplotype diversities were 0.00409—0.00669 and 0.894—0.953 respectively. Neighbor-Joining phylogenetic tree clustered two distinct lineages. Both phylogenetic tree and median-joining network showed the consistent genetic structure corresponding to geographical distribution. Mismatch distributions, negative neutral test and "star-like" network supported a sudden population expansion event. And the time was estimated about 44000 and 50000 years ago.
As the predatory marine organisms, mantis shrimps were characterized by the greatly developed second maxilliped modified as large powerful raptorial appendages [1]. In the growing seasons, these planktonic larvae dispersed by ocean currents and adults moved following their aquatic preys [1, 2], so high genetic homogeneity was likely characterized over broad spatial scale. But now, significant genetic differentiation has been discovered in some marine organisms, which were similar to mantis shrimps in habitat [3—6].
As the important species of stomatopod, Oratosquilla oratoria (Crustacea: Squillidae) occurred in a wide range of habitats from the shore down to coral reefs and level substrates [7]. It was an abundant species that had been regarded as the commercial valuable for its good taste and rich nutrition, especially in west-pacific countries [8—11]. In recent years, the natural resources of O. oratoria have been seriously damaged due to excessive exploiture [11]. However, the genetic diversity, population structure and demographic history are still not well understood. DNA markers have been widely used for population diversity analysis [12, 13].
The aim of the present study is to estimate the genetic diversity and genetic structure, also provide some genetic information for conservation and stock management of this species.
MATERIALS AND METHODS
Sample collection. A total of 73 sequences of O. oratoria were analyzed in the study, in which 48 were from Bohai Sea, and the other 25 sequences of South China Sea were retrieved from GenBank [14, 15] (Fig. 1, table). Samples and sequences identities were confirmed with the morphological features and blasted in GenBank.
DNA extraction, PCR amplification and sequencing. Total genomic DNA was extracted from maxilliped muscle using standard phenol-chloroform method. A partial fragment of mitochondrial cytochrome coxi-dase I (COI) was amplified with the primers of Folmer et al. [16]. Each 30 ^l amplification reaction was consisted of thermophilic buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 2.0 mM MgCl2, 10pmol of each primer, 0.2 mM dNTPs and 1 unit Taq DNA polymerase. Polymerase chain reaction (PCR) was performed on a GeneAmp® PCR System 9700 (ABI) or Mastercycler® gradient (Eppendorf) thermocycler with a temperature profile of 30 cycles at 94°C for 40 s, 50°C for 40 s and 72°C for 60 s. The PCR products were purified using a PCR purification kit, and cycle sequenced on an ABI Prism 3730 automated sequencer.
Data analysis. Sequences were aligned with Clustal 1.83 [17] using the multiple alignment default parameters, and corrected by hand. Phylogenetic analysis of Neighbor-Joining (NJ) was executed with boot-strap=1000 in MEGA 3.0 [18]. Nucleotide diversity (n) and haplotype diversity (h) were calculated to evaluate the level of genetic diversity using the program DnaSP version 4.0 [19]. The genetic divergence was computed with MEGA 3.0.
The divergence time was estimated according to molecular clock. Both Tajima's D [20] and Fu's Ftest [21] of selective neutrality were performed by DnaSP [19]. The mismatch distribution analysis about recent demographic change in populations was also addressed with DnaSP software. If a rapid expansion was observed in the demographic analysis, the relationship t = 2ut [22] was used to further estimate the time of expansion (t), where t was the mode of the mismatch distribution, expressed in units of evolutionary time, and u was the mutation rate for the whole sequence.
Stably variable sites found in 33 haplotypes from different sea areas
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Stably variable sites
Haplotype 00011112222222233333345555 Number in South China Sea Number in Bohai Sea Sequences source
68944550013336822478831224 53569286920396136473646178
SC01 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 FJ229786 [15]
SC02 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621812 [14]
SC03 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621827 [14]
SC04 CTCTGCGCTGGGTCGTTCCGTCTGGT 0 FJ229782.83,87[15], HQ621813.23 [14]
SC05 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 FJ229784 [15]
SC06 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621822 [14]
SC07 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621814 [14]
SC08 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621817[14]
SC09 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 FJ229785 [15]
SC10 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621826 [14]
SC11 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621819 [14]
SC12 CTCTGCGCTGGGTCGTTCCGTCTGGT 0 HQ621815, 28 [14]
SC13 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621811 [14]
SC14 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621820 [14]
SC15 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621816 [14]
SC16 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621821 [14]
SC17 CTCTGCGCTGGGTCGTTCCGTCTGGT 3 0 HQ621818.25,29 [14]
SC18 CTCTGCGCTGGGTCGTTCCGTCTGGT 1 0 HQ621824 [14]
BH01 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH02 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH03 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH04 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH05 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH06 TCTCTTATCAAACGACCTTACTCATC 0 14 Present study
BH07 TCTCTTATCAAACGACCTTACTCATC 0 4 Present study
BH08 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH09 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH10 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH11 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH12 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
BH13 TCTCTTATCAAACGACCTTACTCATC 0 4 Present study
BH14 TCTCTTATCAAACGACCTTACTCATC 0 4 Present study
BH15 TCTCTTATCAAACGACCTTACTCATC 0 2 Present study
Total 25 48 73
Note: SC: haplotypes of South China Sea; BH: haplotypes of Bohai Sea; ]: number of reference.
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Fig. 1. Map showing samples localities of O. oratoria in this study. The unrooted radiation tree of all 33 haplotypes was corresponding to the geographical distribution. BH: haplotypes lineage of Bohai Sea. SC: haplotypes lineage of South China Sea. A: Bohai Sea area. B: the South China Sea area. Pi_n: divergence between BH and SC. Pbh: divergence of haplotypes in Bohai Sea. Psc: divergence of haplotypes in South China Sea.
And u was calculated using the formula u = ^k, where ^ was the mutation rate per nucleotide and k was the number of assayed nucleotides. A mutation rate of 2.33% per million years (Myr) was used as the evolutionary rate of mitochondrial DNA in Crustacea [23, 24]. To estimate phylogeographic structure, a median-joining network was generated with Network Program [25].
RESULTS
Genetic diversity
We identified 62 polymorphic sites in 580 aligned sites, and 33 haplotypes were defined. Haplotype BH06 was shared by 14 sequences of Bohai Sea. And SC04 was shared by 4 ones of South China Sea. 26 stably variable sites were found between Bohai and South China Sea, which could be regarded as the barcoding sites for identification. The number of parsimony information sites was 53. The average nucleotide diversity (n) was 0.03159. And average haplotype diversity (h) was 0.949. But nucleotide diversity of Bohai Sea and South China Sea was 0.00409 and 0.00669 respectively. Haplotype diversity was 0.894 and 0.953. No shared haplotype was found between Bohai and South China Sea.
Population structure
Neighbor-Joining phylogenetic analysis consistently separated two distinct lineages with high statistical support (Fig. 2). One lineage (named BH) was represented by all mantis shrimps of Bohai Sea. The other one (named SC) was South China Sea. The unrooted radiation tree showed significant population genetic structure corresponding to geographical distribution (Fig. 1). Lineages BH and SC were strict correspondence with the positions of Bohai Sea (A) and the South China Sea (B). The average genetic differentiation of Bohai and South China Sea was 0.0052 and 0.0075 respectively. But the mean value (0.0677) was about 10 times of the above values.
When a mutation rate of 2.33% per million years (Myr) was used, we concluded that the two lineages was diverged about 2900000 years ago. A median-joining network analysis separated the haplotypes into two independent networks, which was corresponding to the two phylogenetic lineages. But network BH was constituted with two related groups (Fig. 3). In the whole network, the central haplotypes SC4, BH4 and BH6 were the most common and ancestral haplotypes. Other "star-like" haplotypes were all located at the de-
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100
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SC4
— SC5
- SC2
- SC1 SC6
- SC3 SC7
L SC8 rSC15
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SC16 SC17 "T-SC18 SC10
SC11 ,— SC14 -SC13 SC12
SC9 _
-BH15 — BH13 —BH14 I-BH1 T-BH2 —BH5 -BH3 BH4 BH11
Eighteen haptypes of O. oratoria from South China Sea
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BH12 i—BH8 BH6 -BH10 -BH7
Fifteen haptypes of O. oratoria from Bohai Sea
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Oratosquillina interrupta (outgroup)
Fig. 2. Neighbor-Joining tree showing genetic divergence of Oratosquilla oratoria.
rived clades.
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