Assessment of genetic structure in wild populations of Panulirus homarus (Linnaeus, 1758) across the south coast of Sri Lanka inferred from mitochondrial DNA sequences
SenevirathnaJ.D.M1, MunasingheD.H.N.2* and Peter B. Mather3
1, 2. Department of Zoology, Faculty of Science, University of Ruhuna, Matara, Sri Lanka.
3.School of Earth, Environmental and Biological Sciences, Science and Engineering Faculty, Queensland University of Technology, Australia.
*Corresponding author email:
Abstract
Scalloped spiny lobster (Panulirus homarus, Palinuridae) is an economically and ecologically important food species. Population structure of the scalloped spiny lobster across the southern coast of Sri Lanka (SCSL)(in Indian Ocean) was investigated using mitochondrial DNA (mtDNA) markers. Four sites belong two regions were sampled (Hikkaduwa, Weligama in the southwest and Godawaya, Kirinda in the southeast) and DNA was isolated from tissues of pereiopod and used for PCR amplification. Genetic variation in DNA sequences were examined for two mtDNA gene regions; Cytochrome Oxidase I (COI) and Cytochrome B (CytB). High levels of haplotype diversity and nucleotide diversity were detected in all populations andestimated average fixation index (-0.026285 (P < 0.01)) indicated essential homogeneity among population.Results ofAMOVA analysis confirmed an absence of significant population structure among sites. Tests of neutral evolution and analysis of mismatch distribution suggest that P. homarus populations across the SCSL may have experienced a recent population expansion. Information emerged from the current study could be utilized when design appropriate management strategies for conserving this vital wild marine resource.
Keywords;mtDNA, COI, CytB, Population genetics, Spiny lobster, Indian Ocean
1 Introduction
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 COIgene 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.
2 Materials and Methods
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 °C in 60 μL of 0.5 EDTA (pH 8), 250 µL of nucleic lysis solution (Promega kit wizard) and 3 μL of 20mg/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 01. 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 96oC for 40s, annealing at 50oC for 15s and extension at 60oC for 30s. 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).
3 Results
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).
Number of haplotypes (Nh), number of polymorphic locations (Np), haplotype diversity (Hd), and nucleotide diversity (Nd) estimates 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 observedin 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. 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.
Pairwise fixation index FST values were estimated to assess the degree of gene flow among four sampling sites in two geographical regions and it was evident that there was no significant genetic differentiation among sites eitherfor CytB or COI gene fragments (Table 4).
Results of the SSD en in the Table 5 and indicated that mismatch distributions were essentially unimodal for individual and overall locations at each locus. Raggedness (Rg) values measured the smoothness of mismatch distribution and wereranged from 0.0893 to 0.4178 for the COI gene fragment (0.0341 to 0.0633) and CytB (0.0602 to 0.1454) indicating characteristics of stable populations.
4 Discussion
MtDNA COI and CytB markers have not been previously examined forP. homarus populations in Sri Lanka. Information on genetic variation and population differentiation of P. homarus is not only crucial for developing appropriate conservation and management strategies for this species but may also could be used to reduce their over exploitation,which is happening across Asia (Babbucci et al., 2010).
Comparatively high haplotype and nucleotide diversity (high genetic diversity) observed in all populations suggest that populations were large historically. When initial population sizes are high, frequencies of ancestral haplotypes often decline as new mutants enter the population (Fernández et al., 2012). In fact, this has the effect of neutralizing the impacts of genetic drift by increasing the number of haplotypes and lineages. Over time stochastic loss of haplotypes and, thus lineages (lineage sorting) results in high haplotype diversity (Avise et al., 1984). This together with high original population sizes may explain the lack of prominent high frequency ancestral haplotypes in the sampled population in addition to occurrence of many unique haplotypes. High genetic diversity and a lack of population differentiation represent high levels of ongoing gene flow (Fernández et al., 2012). High genetic diversity evident in P. homarus populations in the current study is likely a combination of an expanding population with high initial population sizes and with ongoing gene flow (McMillen-Jackson & Bert, 2004).
Large decapods often exhibit high mtDNA diversity, with some shrimp taxa showing the highest values among them (McMillen-Jackson & Bert, 2004). Although high intra population indices were evident in the present study, results are consistent with studies conducted for other spiny lobster species(Tolley et al., 2005; Gopal et al., 2006).
High levels of genetic diversity of P. homarus across its wide distribution range potentially can be explained by the metabolic rate hypothesis (Da Silva et al., 2011). According to this hypothesis, a strong correlation exists between nucleotide substitution and metabolic rate, suggesting that increased growth rate leads to higher rate of DNA mutations (Da Silva et al., 2011). Temperature is a key factor influencing metabolic rate in P. homarus (Kemp & Britz, 2008). High intraspecific genetic variability often indicates a high level of population structuring (Da Silva et al., 2011). Significantly high levels of genetic and to a lesser extent, larval morphological diversity have been recognized in the widely distributed Indo-West pacific P. homarus (George, 2005; Naro-Maciel et al., 2011).
Presence of shared haplotypes among sites for both COI and CytB gene fragments and low FST and results of AMOVA analysis designate a single population of P. homarus distributed across the SCSL. Planktonic phyllosoma larvae of P. homarus are passively transport by oceanic currents and results of this study are supported by seasonal water current pattern across the SCSL. MtDNA markers have been successfully used in elucidating population genetic structure for a number of lobster species notably; P. argus (Sarver et al., 1998), Panulirus gilchristi (Tolley et al., 2005), P. delagoae (Gopal et al., 2006) and Panulirus elephas (Palero et al., 2011; Babucci et al., 2010).
In the present study, based on high haplotype diversity and negative Tajima’s D values for all populations, a large expanding population of P. homarus is the most reasonable inference from the aforementioned statistical results. The pattern of genetic diversity is suggestive of recent population expansions in P. homarus across this region after a period of low effective population size with extensive ongoing gene flow (Avise, 2000). Similar findings of only limited population structure with a history of recent population expansions have been reported in many other marine crustaceans (Benzie et al., 2002; McMillen-Jackson & Bert, 2004).
Limited population structure has also been observed recently in many other spiny lobster species (Perez-Enriquez et al., 2002; Tolley et al., 2005; Cannas et al., 2006; Garcı´a-Rodriguez and Perez-Enriquez, 2006; Gopal et al., 2006; Inoue et al., 2007; Palero et al., 2011) and these results are consistent with regional biophysical dispersal models (Roberts, 1997; Butler et al., 2008), especially since the exchange of only a few migrants per generation is sufficient to essentially maintain genetic panmixia (Crow & Aoki, 1984).
Absence of population structure in Panulirus lobster species is expected because all species have long pelagic larval stages that typically last well over 6 months, and the larvae dwell in open ocean habitats where their distribution would be greatly influenced by physical ocean processes. Under the hypothesis of neutrality, the results here show a signal of a recent demographic expansion for P. homarus in the south coast of Sri Lanka.
Author’s contributions
Senevirathna JDM conducted the study under the supervision of Munasinghe DHN. Peter B. Mather guided the study and support to enhance the quality of the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was supported by grant TURIS - RU/DVC/Pro 45 offered from University of Ruhuna and OSTP/2012/28 grant offered from the National Science Foundation (NSF), Sri Lanka. We wish to thank the Director and the team of Paul Herbert Centre for DNA Barcoding and Biodiversity studies, Aurangabad, India for technical support and guidance provided.
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