Genetic Analysis of Cuttlefish Sepia Pharaonis (Ehrenberg, 1831) Populations in Persian Gulf with Microsatellite Markers

Journal of Shellﬁsh Research, Vol. 34, No. 2, 1–5, 2015.

GENETIC ANALYSIS OF CUTTLEFISH SEPIA PHARAONIS (EHRENBERG, 1831) POPULATIONS IN PERSIAN GULF WITH MICROSATELLITE MARKERS

HEDAYAT Y. MOGHADAM,1 HOSSEIN ZOLGHARNEIN,1* MOHAMAD A. S. ALIABADI,1 SAEED KEYVANSHOKOOH2 AND MOHAMAD MODARRESI3 1Department of Marine Biology, College of Marine Science, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran; 2Department of Fisheries, College of Marine Natural Resources, Khorramshahr University of Marine Science and Technology, Khorramshahr, Khouzestan, Iran; 3Persian Gulf Research and Studies Center, Persian Gulf University, Bushehr, Iran

AU1 ABSTRACT The pharaoh cuttlefish Sepia pharaonis (Ehrenberg, 1831) is an abundant cephalopod in Persian Gulf and Oman Sea. Despite the commercial and conservation importance of this species, information on population structure and diversity at the molecular level in S. pharaonis is scarce. Seventeen microsatellite loci from other species were analyzed to study genetic variation in populations of S. pharaonis from Iranian waters of the northern Persian Gulf coasts (Bandarabass, Lengeh, and Bushehr). Eleven of the seventeen primer pairs failed to amplify, whereas six primer pairs successfully amplified: four pairs were polymorphic and two pairs were monomorphic. All loci were significantly deviated from Hardy–Weinberg equilibrium (P < 0.01).

In the three samples of Persian Gulf analyzed, the four polymorphic microsatellite loci exhibited a mean allele number (Na) of 7.5, mean expected heterozygosity (He) of 0.802 and mean observed heterozygosity (Ho) of 0.541.Weak genetic differentiation was present among the populations with FST values ranging from 0.020 to 0.31.This investigation represents the ﬁrst microsatellite study of population genetics of this species in Iran, and the reported results could be of interest for management and conservation programmers of this species.

KEY WORDS: genetic diversity, cuttleﬁsh Sepia pharaonis, microsatellite, Persian Gulf

INTRODUCTION is known about the phylogeography and population genetics of this species (Anderson et al. 2007). The aim of the present study The pharaoh cuttlefish Sepia pharaonis (Ehrenberg, 1831) is was to assess the genetic relationships among S. pharaonis a broadly distributed species with substantial fisheries impor- specimens from various locations of its distribution range in tance found from east Africa to southern Japan (Roper et al. 1984) Iranian waters of the northern coasts of the Persian Gulf, using that inhabits in shallow coastal waters to 100 m depth (Norman & microsatellite markers. Reid 2000). This species is the most common species of cuttlefish trapped in the Persian Gulf and Oman Sea and also an important MATERIALS AND METHODS fishery in this area. For commercial mariculture, S. pharaonis, with features such as short life span, fast growth rates, tolerance Sampling to crowding and handling, resistance to disease, and feeding AU2 habits, is an excellent candidate (Minton et al. 2002). In recent By trawling, Sepia pharaonis samples were collected from years, pharaoh cuttlefish is listed among the endangered species three localities from northern coasts of the Persian Gulf in many regions of its distribution range and needs conservation including Bandarabass (BA, n ¼ 21), Lengeh (LE, n ¼ 30), (IUCN 2012). and Bushehr (BU, n ¼ 30) (Fig. 1). About 0.5 g of tentacle F1 Genetic diversity is important for maintaining the adaptive tissue (arm tips) was dissected using a sterile cutter and stored potential of populations and the ability to deal with changing in absolute ethanol at 4°C. environmental conditions (Hauser et al. 2002). Microsatellite markers have shown widespread utility for assaying genetic DNA Extraction and Microsatellite Analysis population structure, particularly in organisms showing low variability visible by other techniques (Estoup et al. 1998). Approximately 50 mg of tissue was cut into small pieces Codominant, multiallelic, and highly polymorphic microsatel- with scissors. Total genomic DNA was extracted using a CTAB lite markers are suitable tools for examination of genetic method modified from Winnepenninckx et al. (1993). The variation and population structure. Due to low levels of genetic quality and concentration of DNA were assessed by agarose variability observed in cephalopods, microsatellite markers are 1.0% gel electrophoresis and stored at –20°C until use. We useful for understanding the genetic population structure screened and assessed variation at 17 microsatellite loci from (Doubleday et al. 2009). In many species, semelparous life cycle three species of the cuttlefish genus of Sepia: Sepia officinalis, with unpredictable juvenile causes decreased population size Sof1, Sof2, Sof6 (Shaw & Perez-Losada 2000); Sepia apama, (Boyle & Boletzky 1996). Therefore, it is necessary to construct Sap19, Sap65, Sap31, Sap22b, Sap66 (Shaw 2003); and Sepia a correct picture of genetic structure of cephalopods for rational esculenta, Secu 3, Secu 15, Secu17, Secu 25, Secu 42, Secu 44, management of exploitation (Shaw 2003). Despite the high Secu 47, Secu 57, and Secu 77 (Wang et al. 2010). economic and commercial importance of Sepia pharaonis, little Polymerase chain reaction was performed in a 25 ml reaction volume containing 100 ng of template DNA, 10 pmol *Corresponding author. E-mail: [email protected] of each primer, 200 mM each of the dNTPs, 1 U of Taq DNA DOI: 10.2983/035.034.0200 polymerase (Cinnagen, Tehran, Iran), 1.5–2.5 mM MgCl2 and

1 2 MOGHADAM ET AL.

Figure 1. Map showing sampling locations of three populations of Sepia pharaonis: BU (1), LE (2), BA (3).

13 PCR buffer. The temperature profile consisted of 5-min Multilocus FST values between populations ranged from initial denaturation at 94°Cfollowedby30cyclesof30sat 0.020 to 0.031. Population differentiation value between 94°C, 30 s at annealing temperature, and 45 s at 72°C, ending BA and BU was the highest (0.031) and significant among with 7 min at 72°C. PCR products were separated on 8% the population pairs (P ¼ 0.001), with the lowest gene flow polyacrylamide gels stained with silver nitrate. (7.77). The lowest FST (0.020) with the highest gene flow

Data Analysis

Microsatellite polymorphism within samples was measured TABLE 1. with the mean number of alleles, and observed (Ho)and Genetic variation at four microsatellite loci in the Sepia expected (He) heterozygosities calculated using the Genetix pharaonis samples from the Persian Gulf. Number of in- 4.05 Software package (Belkhir et al. 2004). Deviations from dividuals (n), number of alleles (Na), unbiased expected Hardy–Weinberg equilibrium (HWE) were tested using the heterozygosity (He), and observed heterozygosity (Ho). Fishers exact test with the level of signiﬁcance determined P values of deviation from HWE are given. by a Markov chain method using GENEPOP 4.0.10 software (Raymond & Rousset 1995) as implemented for online use Sampling site (http://genepop.curtin.edu.au/). The genetic distance between $ $ $ any two populations was estimated from Nei standard genetic Locus BU (n 30) LE (n 30) BA (n 21) Mean distance and genetic similarity index (Nei 1972). Genetic Secu15

differentiation between populations was also evaluated by Na 7 6 7 0.667/6 the calculation of pairwise estimates of FST values. These Ho 0.433 0.300 0.619 0.451 calculations were conducted using the GENALEX version He 0.788 0.791 0.817 0.799 6.4 (Peakall & Smouse 2006). All loci were tested for possible P *** *** *** genotyping errors, associated with null alleles and mis-scoring Secu17 N 8 7 8 7.667 using MICRO-CHECKER version 2.2.3 (Van Oosterhout a H 0.467 0.300 0.571 0.446 et al. 2004). o He 0.832 0.704 0.797 0.778 P *** *** ** RESULTS Secu44 N 8 9 8 8.333 In this study, the ampliﬁcation of six primer pairs was a Ho 0.833 0.567 0.619 0.673 T1 successful: four pairs were polymorphic (Table 1), whereas two He 0.849 0.827 0.859 0.845 pairs (Sof2 and Secu3) were monomorphic. Allele numbers and P *** *** *** genetic variability at the microsatellite loci in the Persian Gulf Secu77

samples of Sepia pharaonis are shown in Table 1. Mean number Na 7 8 7 7.333 of alleles per population was 7.50. The mean observed and Ho 0.400 0.667 0.714 0.594 expected heterozygosities ranged from 0.458 to 0.631 (average He 0.682 0.851 0.824 0.786 overall loci 0.541) and 0.788 to 0.825 (average overall loci 0.802), P *** *** ** respectively (Table1). Mean Na 7.50 7.50 7.50 7.50 Mean H 0.533 0.458 0.631 0.541 Signiﬁcant deviations from HWE at the locus level are o Mean H 0.788 0.793 0.825 0.802 shown in Table 1. All of the loci were signiﬁcantly deviated e from HWE (P < 0.01). ** P < 0.01; *** P < 0.001. GENETIC ANALYSIS OF CUTTLEFISH, SEPIA PHARAONIS 3

TABLE 2. TABLE 3.

Multilocus Nm (above diagonal) and FST values (below di- Genetic distance (D) (above diagonal) and genetic similarity agonal) between pairs of Sepia pharaonis populations across (below diagonal) between pairs of Sepia pharaonis all loci. populations.

Population BA LE BU Population BA LE BU BA – 11.96 7.77 BA – 0.232 0.282 LE 0.020 – 8.17 LE 0.793 – 0.233 BU 0.031 0.030 – BU 0.754 0.792 –

(11.96) was, however, observed between BA and LE pop- coastward migrations of mature cuttlefish for reproduction are T2 ulations (Table 2). shown (Carvalhoetal 1992, Watanuki & Kawamura 1999). Genetic distance (D) and genetic similarity index (I)between Migration could cause the intermixing of individuals from T3 any two populations are shown in Table 3. The genetic distance different groups and might have caused observed departures was smallest (0.232) between BA and LE populations and largest from HWE in the collected samples (Garoia et al. 2004, Zheng (0.282) between BU and BA populations (Table 3). In this case, et al. 2009). Deviations from the HWE by an excess of the nature of the deviations is suggestive of null alleles, as homozygotes have been reported to be an ordinary event in indicated by the MICRO-CHECKER analysis. cephalopod populations (Perez-Losada et al. 2002, Garoia et al. 2004). For Persian Gulf cuttlefish, homozygote excesses DISCUSSION may also be related to null alleles as well as to sampling effects due to migration cohorts and overexploitation. Despite the commercial and ecological importance of The value of FST is a useful measure of genetic differenti- Sepia pharaonis in Iran, natural population stocks of this ation among populations, and different values mean different species have been declining in recent years because of over- degrees of divergence. Our microsatellite data provide the first exploitation and environmental conditions. Information evidence of genetic differentiation among populations of Sepia about S. pharaonis populations is critical for their conserva- pharaonis inthePersianGulf.Overall,thelevelofgenetic tion and sustainable use. Unfortunately, the knowledge of differentiation among the S. pharaonis populations was low molecular genetics and genetic structure of this species is (Table 2). The FST value ranged from 0.020 to 0.031. FST scarce (Anderson et al. 2007). Previous studies indicated that values observed in the present study are similar to that microsatellites markers have great potential for resolving reported in a congeneric species, Sepia officinalis, along the genetic population structure, and an understanding of genetic Iberian Atlantic coast (q ¼ 0.014–0.022, Perez-Losada et al. structure is vital for the management and conservation of 2002), in the Adriatic Sea populations (FST ¼ 0.018–0.022, cuttlefish resources (Zheng et al. 2007). In this study, we used Garoia et al. 2004), Sepia esculenta along the coast of Japan microsatellites from other species to assess the genetic vari- and China (0.009–0.037, Zheng et al. 2007), and Octopus ability and the population structure of cuttlefish, S. pharaonis, maorum (0.001–0.035, Doubleday et al. 2009). In the present in the Persian Gulf. study, the FST value between BA and BU was the highest The results of this study indicated that all of the loci were (0.031), and the lowest FST (0.020) was observed between BA significantly deviated from HWE (P < 0.01). Consistent to our and LE populations (Table 3). Therefore, the apparent findings, heterozygote deficiency relative to Hardy–Weinberg correlation observed between genetic and geographic dis- expectations was previously reported in cephalopod popula- tances among the populations suggests that the data conform tion (Perez-Losada et al. 2002, Garoia et al. 2004, Wolfram to an isolation-by-distance model of gene flow, and this model et al. 2006, Cabranes et al. 2008, Zheng et al. 2009). The (isolation-by-distance) is suitable for investigating marine heterozygote deficiency observed can be a result of factors such distribution (Palumbi 2003). Pelagic organisms in the open as the presence of null alleles (nonamplifying) or biological ocean usually have low levels of population differentiation factors such as inbreeding and Wahlund effect (mixing of (Zheng et al. 2005). In addition, water circulation affects populations) (Zheng et al. 2009, Doubleday et al. 2009). migration patterns of coastal cephalopod species (Semmens Deficiencies of heterozygotes have been broadly observed in et al. 2007, Zheng et al. 2009). Moreover, according to the cephalopod populations (Shaw & Perez-Losada 2000, Perez- pattern of water circulation in the Persian Gulf (Fig. 2), in F2 Losada et al. 2002, present study). Previous studies on which water moves from the Strait of Hormuz and adjacent to cephalopod population by microsatellite markers indicated the Iranian coast (moving from BA toward BU) (Reynolds that heterozygote deficits could be largely caused by null alleles 1993), we explain the gene flow, similarity, and differentiation (Perez-Losada et al. 2002, Wolfram et al. 2006). In Adriatic observed between Sepia pharaonis populations. cuttlefish (Sepia officinalis)andSepia esculenta (from coasts of In conclusion, microsatellite data provide the first evi- Japan and China), homozygote excesses were related to null dence on genetic differentiation among populations of Sepia alleles as well as to sampling effects due to migrating cohorts of pharaonis along the coast of the Persian Gulf. The data mature cuttlefish for reproduction (Garoiaetal 2004, Zheng generated in this study can be applied to future genetic et al. 2009) and therefore, homozygote excess can be the result improvement by selective breeding and to the design of of nonamplifying (null) alleles or Wahlund effect (mixing of suitable guidelines for managing genetic resources of this population) (Doubleday et al. 2009). In addition, the seasonal species. 4 MOGHADAM ET AL.

Figure 2. Schematic of surface currents and circulation processes in Persian Gulf (Reynolds 1993).

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