Spiny lobsters are marine cryptic, benthic crustaceans with a unique lifestyle that includes a very long-lived planktonic phyllosoma larval stage (Booth and Phillips, 1994; Sekiguchi and Inoue, 2002; Dixon et al., 2003). Typically, spiny lobsters with a long Planktonic Larval Duration (PLD) have the potential to disperse and settle over vast geographical distances (Briones-Fourzan et al., 2008; Butler et al., 2011) promoting high levels of gene flow. Dispersal capacity of long lived-larvae is largely enhanced by dominant ocean currents and winds but could potentially be limited by physical, chemical or biological barriers to natural gene flow (Gopal et al., 2006; von der Heyden et al., 2007).

 

Among commercial spiny lobster species which were listed by the Food and Agriculture Organization of the United Nations (FAO), the scalloped spiny lobster is the most common commercially fished species in the Indo-West Pacific region (Holthius, 1991) and this species is also the most commercially important marine lobster species found in Sri Lanka. In spite of its relative economic importance, knowledge about its biology in Sri Lankan waters is currently limited. However, information on reproductive activity, size ranges, population dynamics and sex composition, is known from various studies (De Bruin, 1969; Jayakody, 1989, 1991, 1993; Jayawickrema, 1991; George, 1997) that has been used in implementation of management practices. More exhaustive studies of wild population structure are required to improve the management options for lobster fishing industry and stock sustainability (Amarakoon et al., 2006 a, b; Gunawardane et al., 2006 a, b; Koralagama et al., 2007). Improved management strategies for exploited fishery resources should be based on recognition of natural population boundaries. If the boundary of the fishery does not encompass a stock, appropriate management may be affected because different growth and mortality characteristics of discrete populations will not be addressed appropriately (Skillman, 1989). Understanding wild population genetic structure is therefore, an important first step for effective conservation and management of any species (Thorpe et al., 2000; Massimiliano et al., 2010).

 

To date, population genetic studies of P. homarus have been conducted applying allozyme markers (Garcia-Rodriguez and Perez-Enriquez, 2006) and mitochondrial DNA (mtDNA) fragment length polymorphisms (Sarver et al., 1998; García-Rodríguez and Perez-Enriquez, 2006; Dharani et al., 2009). Mitochondrial DNA has many attributes that make it particularly suitable for population genetic studies, including a rapid rate of evolution, lack of recombination, and maternal inheritance (Hoelzel et al., 1991). To resolve population genetic structure, the mitochondrial COI gene region has been investigated in a wide range of crustacean species (Presa et al., 2002; David and Brian, 2012) including marine lobsters (Seinen et al., 2011; David and Brian, 2012). The CytB gene contains both slowly and rapidly evolving regions as well as more conservative and more variable regions or domains overall and this marker has also been used widely for population genetic studies on a number of crustacean taxa (Pfeiler et al., 2005).

 

The aim of the current study was to assess the extant population genetic diversity patterns of wild populations of P. homarus across the SCSL employing partial sequence analysis of the mtDNA COI and CytB genes and to discuss its implications in long-term conservation and sustainable management of these important aquatic resources.

 

Adult individuals of P. homarus were collected from four sites across the SCSL; Kirinda (KIR), Godawaya (GOD), Weligama (WEL) and Hikkaduwa (HIK) using scuba diving and nets (Figure 1). Upon sampling specimens were frozen in ice immediately and were transferred into 100% ethanol at the Research Laboratory of the Department of Zoology, University of Ruhuna, Matara, Sri Lanka where the reference samples have been stored. Molecular analyses were conducted at the Paul Herbert Centre for DNA Barcoding and Biodiversity Studies, Aurangabad, India.

Genomic DNA was extracted from leg muscle of scalloped spiny lobsters. Approximately 25 mg of muscle tissue per individual was digested overnight at 55? in 60 μL of 0.5 EDTA (pH 8), 250 µL of nucleic lysis solution (Promega kit wizard) and 3 μL of 20 mg/mL proteinase K. DNA was collected following brief centrifugation and the samples were washed in 300 μL isopropanol and twice with 75% ethanol before being air-dried, and then dissolved in TE buffer.

 

Details of primers used to amplify COI and CYB gene regions, details of the PCR mixture and thermal cycle parameters are given in the Table 1. Di-deoxy chain-termination DNA sequencing was performed (Sanger et al., 1977) using a Sequencing kit (version 2.0, United States Biochemical with [a 35S]-dATP). DNA sequencing reactions were conducted using the following cycling parameters: 35 cycles of denaturation at 96 ? for 40 s, annealing at 50 ? for 15 s and extension at 60 ? for 30 s. Sequence runs were performed according to the manufacturer’s recommendations on an ABI 3130 Genetic analyzer capillary sequencer.

Sequences were edited and aligned using Codon Code (Codon Code Corporation 2012) and MEGA 5.0 software (Tamura et al., 2011). Relative diversities of the two mtDNA gene regions were evaluated by determining haplotype diversity (Hd) and nucleotide diversity (Nd) for each population.

 

To estimate hypothesized patterns of spatial genetic structure, hierarchical analysis of molecular variance (AMOVA) was used to estimate variances within and among populations. Isolation-by-distance effects on population genetic structure were estimated using pairwise FST statistics (Wright, 1965). The neutrality analysis (Tajima’s D test) and the shape of mismatch distributions were assessed by sum of squared deviations (SSD) and were used to deduce whether a population had potentially undergone recent sudden population expansions. All analyses were implemented in ARLEQUIN version 3.0 (Excoffier et al., 2005).

 

High quality sequences were obtained for COI and CytB gene regions. A total of 493-bp for COI from 27 individuals and 809-bp for CytB from 38 individuals were resolved for P. homarus from the four sampled sites. Derived sequences for three gene regions were deposited into the NCBI GenBank database (COI sequence accession numbers: from KF715528 to KF715555 and CytB sequence accession numbers: from KF738886 to KF738922, KF765484 respectively).

 

Estimated values for number of haplotypes (Nh), number of polymorphic sites (Np), haplotype diversity (Hd), and nucleotide diversity (Nd) for each population are presented in Table 2. COI sequences were characterized by low nucleotide and high haplotype diversity. Haplotype diversities for the COI gene region in each population were lower than those for the CytB gene region and ranged from 0.8667 in GOD to 1.0000 in HIK, KIR, while nucleotide diversities ranged from 0.0114 in WEL to 0.0162 in GOD. Overall, 20 unique COI haplotypes were detected and 31 polymorphic sites were observed in the 493-bp (6.288%) fragment of sequence. CytB sequences also showed low nucleotide and high haplotype diversity. Haplotype diversity in the CytB gene fragment was higher than for the COI gene fragment in the four sampled populations and ranged from 0.9778 in WEL and GOD to 1.0000 in KIR and HIK), while nucleotide diversities (p) ranged from 0.0106 in WEL to 0.0156 in HIK. CytB sequences yielded 34 discrete haplotypes with 77 polymorphic sites in the 809-bp (9.51%) sequence fragment. 

The distribution of haplotypes (represent by H) at each site is presented in Figure 1. Some COI gene and CytB haplotypes were shared between sites. For COI; H1 (7.4%) was shared between WEL and GOD, H2 (11.1%) between GOD and HIK, H8 (7.4%) between HIK and KIR and H9 (11.1%) between WEL and HIK were shared while the other 16 haplotypes were unique to specific populations. All four populations possessed a set of unique haplotypes. The KIR population had the highest number of unique haplotypes (6), followed by HIK (5) and WEL (3). The GOD population had the lowest number of unique haplotypes (2). For CytB; H3 was shared (15.8%) among all four populations, H1 (5.2%) between GOD and WEL, H25 (7.9%) between KIR and WEL were shared and the other 28 haplotypes were unique to specific populations. All four locations had unique CytB haplotypes. The HIK population had the highest number of unique haplotypes (8), followed by GOD (7), KIR (7) and WEL (6).

 

AMOVA results indicated the presence of variation within populations (Table 3), but very limited divergence was evident among populations for both gene fragments due to negative variations among populations. The average fixation index values also indicated only weak genetic structuring among the four sampled sites for each gene fragment. The genetic structure among the sampled sites was further investigated using FST analysis.

 

Table 3 AMOVA results for Panulirus homarus among four sites from the southern coast of Sri Lanka.

 

Pairwise fixation index FST values were estimated to assess the degree of gene flow among four sampling sites in two geographical regions. According to the results all positive values are less than 0.05 significant level and others are negative (Table 4).