Genetic Diversity and Genetic Heterogeneity of Bigfin Reef Squid “Sepioteuthis lessoniana” Species Complex in Northwestern Pacific Ocean

The bigfin reef squid Sepioteuthis lessoniana Ferussac, 1831 in Lesson (1830–1831) is widely distributed in the Indo-Pacific, where it is a very valuable fishery resource (Dunning, 1998). Thus, a lot of ecological research of this species were reported (e.g. Ikeda, 1933; Choe & Ohshima, 1961; Segawa, 1987; Ueta, 2003; Ikeda et al., 2009). Segawa et al. (1993a; 1993b) showed that within Sepioteuthis lessoninana have diferrences of egg chracteristics and reproductive trait in Ishigakijima Island. Izuka et al. (1994) reported an allozyme analysis found so-called S. lessoniana around Ishigakijima in Okinawa Prefecture, Japan, includes at least three biological species (Figure 1 & 2). Local fishers call the three species “aka-ika,” which has a red body, “shiro-ika” or “aori-ika,” which has a white body, and “kua-ika,” which is smaller than the other two. Of these, the range of “shiro-ika” extends to the coast of the main Japanese islands. This is the extent of its taxonomic classification thus far. This is due in part to the limited number of distinguishing morphological characters but also because the type specimens is no longer available and type locality has not been disignated (Lu et al., 1995; Jereb & Roper, 2006). This makes it difficult to determain whether genetically recognized species are undescraibed species or one of 13 known synonymies (Young, 2002). In this study, we treated “aka-ika” as Sepioteuthis sp. 1, “shiro-ika” as Sepioteuthis sp. 2, and “kua-ika” as Sepioteuthis sp. 3.


Introduction
The bigfin reef squid Sepioteuthis lessoniana Férussac, 1831in Lesson (1830-1831) is widely distributed in the Indo-Pacific, where it is a very valuable fishery resource (Dunning, 1998).Thus, a lot of ecological research of this species were reported (e.g.Ikeda, 1933;Choe & Ohshima, 1961;Segawa, 1987;Ueta, 2003;Ikeda et al., 2009).Segawa et al. (1993a;1993b) showed that within Sepioteuthis lessoninana have diferrences of egg chracteristics and reproductive trait in Ishigakijima Island.Izuka et al. (1994) reported an allozyme analysis found so-called S. lessoniana around Ishigakijima in Okinawa Prefecture, Japan, includes at least three biological species (Figure 1 & 2).Local fishers call the three species "aka-ika," which has a red body, "shiro-ika" or "aori-ika," which has a white body, and "kua-ika," which is smaller than the other two.Of these, the range of "shiro-ika" extends to the coast of the main Japanese islands.This is the extent of its taxonomic classification thus far.This is due in part to the limited number of distinguishing morphological characters but also because the type specimens is no longer available and type locality has not been disignated (Lu et al., 1995;Jereb & Roper, 2006).This makes it difficult to determain whether genetically recognized species are undescraibed species or one of 13 known synonymies (Young, 2002).In this study, we treated "aka-ika" as Sepioteuthis sp. 1, "shiro-ika" as Sepioteuthis sp. 2, and "kua-ika" as Sepioteuthis sp. 3.
A previous population genetics study found significant differences in the genetic heterogeneity of Sepioteuthis sp. 2 between Pacific Ocean and Japan Sea populations using allozyme analysis (Yokogawa & Ueta, 2000).Yokogawa and Ueta (2000) did not include the Okinawan Sepioteuthis sp. 2 population in their study.In addition, Pratoomchat et al. (2001) found no significant genetic heterogeneity between Japanese and Thai Sepioteuthis sp. 2 populations, while our present study tried significant differences in the genetic heterogeneity of the Japanese and Vietnumese Sepioteuthis sp. 2 populations.Recently, Aoki et al. (2008a) reported significant genetic heterogeneity between Japanese and Vietnumese populations of Sepioteuthis sp. 2 using DNA sequencing analysis of the mitochondrial noncoding region.
Therefore, this study examined the genetic diversity (i.e., the average heterogeneity) and gene flow among Sepioteuthis sp. 2 populations using allozyme analysis and among populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 using mitochondrial DNA noncoding region sequencing of populations from Japanese, Taiwanese, and Vietnamese waters.
specimens were fresh and immediately sent to a refrigerator in the laboratory.The buccal bulb muscle was removed and kept frozen at -40°C until the allozyme analysis.Small pieces of liver and skeletal muscle were dissected from selected specimens and minced individually in an equal volume of distilled water on ice.Electrophoresis was conducted in a glass box with ice on top of it.The box was in a refrigerator at a constant voltage (250 V) until the Amido Black 10B marker moved seven cm from the origin.The allozymes were tested using 12.5% horizontal starch-gel electrophoresis and the two buffer systems described by Clayton and Tretiak (1972) and modified by Numachi (1989): citric acid N-(3-aminopropyl) diethanolamin e ( C A E A , p H 7 ) a n d c i t r ic acid N-(3-aminopropyl) morpholine (CAPM, pH 6).Each gel was sliced into six 1-mm-thick sheets with a wire gel cutter (Numachi, 1981) and stained for the enzymes aspartate aminotransferase (AAT), isocitrate dehydrogenase (IDHP), lactate dehydrogenase (LDH), phosphoglucomutase (PGM), and phosphogluconate dehydrogenase (6PGD) according to Shaw and Prasad (1970), Numachi (1970a, b), and Taniguchi and Numachi (1978).The locus and gene nomenclature followed Shaklee et al. (1990).Polymorphisms involving several alleles with frequencies of more than 5% were tested at a significance level of 0.05 to determine whether they were consistent with Hardy-Weinberg equilibrium.The average heterozygosity H (Nei, 1978) was calculated as a measure of genetic diversity.The χ 2 homogeneity test of allele frequency among samples was also performed.30), and Ishigakijima (30).Crude DNA was extracted by TNES 8M Urea buffer and proteinase K digestion followed by a phenolchloroform isoamyl method described Imai et al. (2004).
A GeneAmp 9700 (Applied Biosystems, USA) thermal cycler was used with the following setting: 94°C for 120 s, followed by 30 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s.The PCR products were purified using a PCR Product Pre-sequencing Kit (USB, USA).The nucleotide sequences were determined using ABI 3700 (Applied Biosystems, USA) genetic analyzers.All sequences were initially aligned using ClustalX ver.1.83.1 (Thompson et al., 1997) and then edited manually using MacClade4 ver.4.08 (Maddison and Maddison, 2005).
For Sepioteuthis sp. 3, AMOVA could not be performed because there were fewer than three localities.Therefore, homogeneity was tested using the chi-square randomization method (Monte Carlo simulation) with 100,000 randomizations of the data (Roff and Benzen, 1989).Significance thresholds were Bonferroni-corrected for multiple pairwise comparisons.Relationships of haplotypes were assessed using a minimum spanning tree created via the Minspanet algorithm in Arlequin and drawn by hand.

Allozyme analysis of Sepioteuthis sp. 2
Regarding the eight loci for the six enzymes analyzed, the five loci Aat-1*, Idhp-1*, Ldh-1*, Mdh-1*, and Mdh-3* showed no differences among and within localities, and no genetic polymorphism was recognized.Two Mdh-2* heterozygotes were found in Vietnam, although the frequency was 0.017; therefore, it was not considered a polymorphic allozyme locus (Table 1).Genetic polymorphism was detected within a locality for Pgm* and 6pgd*.
Table 1.Allele frequencies at eight loci and indices of genetic heterozygosities within six localities of Sepioteuthis sp. 2.
No allele frequency gap was observed among different localities for the polymorphic allozyme loci 6pgd* allele frequency.In contrast, a significant difference was detected between Pgm* in the Japanese and Vietnamese localities (Table 2).This result differed greatly from that of Pratoomchat et al. (2001) (2000) is repeatable, the difference in a highly polymorphic marker among populations would not always be detected.For example, regarding Theragra chalcogramma, a restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA and microsatellite DNA could not identify the difference among populations seen in the allele frequency of the superoxide dismutase (SOD) allozyme marker (Iwata, 1975;Mulligan et al., 1992;Bailey et al., 1999;Chow, 2001).Our finding of gene flow between Taiwanese and Japanese localities detected in the allozyme analysis of Sepioteuthis sp. 2 was not consistent with the sequencing analysis of the mitochondrial noncoding region by Aoki et al. (2008a), perhaps because of the low level of polymorphic loci for the allozyme analysis.
In addition, noncoding regions such as the mitochondrial control region accumulate more variation than allozyme markers.The relative smallness of the effective population size (female) with nuclear DNA made it easier to detection interpopulation genetic differentiation by genetic drift (Williams et al., 2002).Therefore, Aoki et al. (2008a) used the mitochondrial noncoding region and found low genetic diversity in Japanese waters.Furthermore, Aoki et al. (2008a) revealed the independence of gene flow within the populations in Japanese waters from others.

Mitochondrial non-coding region of Sepioteuthis sp. 1
We sequenced 552 base pairs (bp) of the NC2 sequence for 116 Sepioteuthis sp. 2 specimens from three localities.From a total of 35 haplotypes, 23 variable sites were identified (Table 3).One haplotype was shared among three localities, and the remaining 31 haplotypes were each specific to a single locality.Among the populations, 23.3% of the samples belonged to haplotype no. 1, which was the major haplotype in all Japanese localities.In contrast, haplotype no. 3 was the major haplotype in Taiwan (Figure 3).
The haplotype diversity (h) ranged from 0.7994 for Ishigakijima to 0.8665 for Okinawajima, and the nucleotide diversity (π) varied from 0.0035 for Ishigakijima to 0.0052 for Okinawajima (Table 4).Among the three Sepioteuthis sp. 1 localities, the level of genetic diversity did not differ much.The AMOVA indicated that the genetic variation over all of the Japanese localities was 34.87%, whereas the within-locality variation was 65.13% (p<0.01 [Table 5]).The estimated pairwise Fst values for the three pairs of three localities ranged from 0.0538 to 0.5329.All combinations of locality samples had significant pairwise Fst values (p<0.05[Table 6]).Therefore, each locality had an independent population with restricted gene flow, concurring with Aoki et al. (2008a).Sepioteuthis sp. 2 had gene flow within the territorial waters of Japan and showed genetic homogeneity.The relationships among the haplotypes Fig. 3. Pie chart representation of the haplotype frequencies of Sepioteuthis sp. 1 of the three localities.
Table 5. Analysis of Molecular Variance on pairwaise differences and P-value of Sepioteuthis sp. 1 among three localities is the probability of a more extreme variance component.were represented on a minimum spanning tree, and the shape indicated that the population had long-term stability (Figure 4).

Mitochondrial non-coding region of Sepioteuthis sp. 3
We sequenced 557 bp NC2 sequences for 60 Sepioteuthis sp. 3 specimens from two localities.From a total of 15 haplotypes, 13 variable sites were identified (Table 7).Seven haplotypes were shared between the two localities, and the remaining eight were specific to a single locality.Overall, 31.6% of the samples belonged to haplotype no. 1, which was not the major haplotype in both localities.Haplotype no. 2 was the major haplotype in Okinawajima (Figure 5).The haplotype diversity (h) ranged from 0.7103 for Ishigakijima to 0.8828 for Okinawajima, and the nucleotide diversity (π) varied from 0.0037 for Ishigakijima to 0.0044 for Okinawajima (Table 8).The level of genetic diversity was similar to that of Sepioteuthis sp. 1.
Significant heterogeneity was observed between the Okinawajima and Ishigakijima populations (χ 2 =23.89, p<0.01).Therefore, these two populations could be distinguished by the haplotype frequency.Izuka et al. (1996) reported that each population was genetically independent based on the allozyme analysis for Ishigakijima and the Ogasawara Islands.These results suggest that Sepioteuthis sp. 3 does not experience larval dispersal, but completes its life history within coral reefs.The relationships among the haplotypes were represented on a minimum spanning tree, and the shape indicated that haplotypes could not be divided into clusters (Figure 6).

General discussion
In this research showed that genetic differentiation between Okinawajima and Ishigakijima population was identified for Sepioteuthis sp. 1 and Sepioteuthis sp. 3. The result showed that Sepioteuthis sp. 1 and sp. 3 prefer coast lines as habitat that limit periodic dispersal of larva and adult among islands.The result showed that there is no gene flow of Sepioteuthis sp1 between Ishigakijima and Taiwan, as Aoki et al. (2008a) showed for Sepioteuthis sp. 2. Geographical distance of these two areas is 300 km, which has no difference of the geographical distance between Okinawajima and Ishigakijima Island.However, genetic structure differentiation between Ishigakijima and Taiwan is bigger than that of Okinawajima and Ishigakijima.Thus, gene flow between Ishigakijima and Taiwan was disturbed for long period.Kuroshio Current possible is possibly disturbing the gene flow Kuroshio Current is warm current with the surface speed of 2m per second, strong flow that moves more than 50 million tons of water.The current axis starts from north equatorial countercurrent, go up towards north between Taiwan and Yonagunijima, through Tokara strait and flows into southern coast of main island Japan (Figure 7).The width between Taiwan and Yonagunijima is small.Kuroshio Current go up to the north along with continental shelf.The current split around Kyushu to Tsushima Current along with Japan Sea and main current that goes along with Pacific coast of main island, Japan.The current disperse when it goes to north, the flow of Kuroshio Currnet may take them to very northern part, that has lower temperature of ocean water.Geographical cal distribution of Panulirus longipes is another example in which Kuroshio Current is a barrier current between Taiwan and Ryukyu islands (Sekiguchi & Inoue, 2010).Accordingly, several marine organisms, Uca arcuata and Siganus guttatus shows different genetic structure between Ryukyu Archipelago and Taiwanese population showing no gene flow (Aoki et al., 2008b;Iwamoto et al., 2012).These genetic sturucture pattern may suggest the influence of Kuroshio Current.Especially squids has short longevity, some of the species has only one spawning season in a life.Drastic environmental change and some other accidental events may destroy population.In order to raise the fitness of squids, water temperature and appropriate environment of growth phase are essential (O'Dor & Coelho, 1993).Kuroshio Current may supply appropriate temperature and abundant feed resources for Sepioteuthis spp habitat.The comparative study of Sepioteuthis sp. 2 reported by Aoki et al. (2008a) and Sepioteuthis sp. 1 MST shape shows that these two species can belong to each clade of Japan and Taiwan.

Conclusion
We analyzed the genetic heterogeneity of populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 in Okinawan waters using DNA analysis.The genetic diversity of Sepioteuthis sp. 1 and Sepioteuthis sp. 3 was higher than that of Sepioteuthis sp. 2 from Japanese waters.Moreover, the genetic heterogeneity of the populations differed significantly.The difference in genetic heterogeneity means that Sepioteuthis sp. 1 and Sepioteuthis sp. 3 do not have a large gene pool.We postulate that the reason for the genetic differentiation is that these two species prefer coastal habitats.Our results indicate that the Japanese populations of Sepioteuthis sp. 2 have very low genetic diversity compared to those of Taiwan and Vietnam.The minimum spanning tree showed that the Japanese populations were of the radiation type, implying that the Japanese populations had experienced founder effects.
The genetic heterogeneity in the Japanese populations was slightly different using AMOVA.This suggests that the mitochondrial noncoding region of the Japanese population lacks sufficient genetic diversity to assess the genetic heterogeneity in the Japanese populations.Moreover, our results suggest that only limited gene flow has occurred between the Ishigakijima and Taiwan populations of Sepioteuthis sp. 1 and Sepioteuthis sp. 2, implying the presence of barriers to gene flow.Kuroshio Current, a prominent current in this area, which moves at a rate of nearly 50 million m 3 /s, may prevent dispersal from Taiwan to Ishigakijima.
Lastly, it should be noted that Prof. Segawa's contribute to ecological research for Sepioteuthis lessoniana complex.Further study should focus on resolute species complex as soon as possible to develop ecological research of Sepioteuthis spp.It is necessary to identify species identification marker, however, frequently used allozyme marker needs fresh samples.Finding DNA marker that can be used for ethanol sample will be useful.DNA marker development should use allozyme marker of Izuka et al. (1994) and Triantafillos and Adams (2005) as the standard specimens.Allozyme analysis is the method to detect nuclear DNA polymorphism by the detection of enzyme molecule polymorphism.It cannot show nucleotide base-substitution mutation when it does not have amino-acid sequence variation.The different condition of electrophoresis buffer may produce different results, even though it is worth noting that it is a reliable tool to find cryptic species (Imai, 2006).The first author of this paper, Imai, H is currently working on development of species identification using DNA marker with some other researchers.
, who found the same gene pool in Thailand and Nagasaki, while our result supported the result ofAoki et al. (2008).Pratoomchat et al. (2001) used Pgm* and 6pgd*, but the results might have been influenced by differences in the electrophoresis buffer.No difference in allele frequency was observed among localities in Japan.Yokogawa and Ueta (2000) showed replacement of Ldh-4* between the main island Japan Sea and Pacific sides, although the allozyme band pattern shown in that paper may have been manipulated, and we find the results suspect.Therefore, we examined Ldh-4* with a fresh sample following the advice of Dr. Yokogawa, and we did not find it.Pratoomchat et al. (2001) citedYokogawa and Ueta (2000), but did not detect Ldh-4*.When Ldh-4* was eliminated, no difference existed between the Japan Sea and Pacific sides.Aoki et Okinawajima al. (2008a) could not show a difference between the Japan Sea and the main island Pacific side, even on analyzing the mitochondrial noncoding region sequence.If Ldh-4* of Yokogawa and Ueta

Fig. 5 .
Fig. 5. Pie chart representation of the haplotype frequencies of Sepioteuthis sp. 3 of the two localities.

Fig. 7 .
Fig. 7.The location of the main pathway of Kuroshio Current and sampling sites.

Sepioteuthis sp. 1 and sp. 3
part of the mantle muscles was kept in 90% ethanol and DNA was extracted with TNES 8M-Urea buffer.For Sepioteuthis sp. 3, 60 samples were collected between October 2005 and July 2006 from Nago, Okinawajima (

Table 4 .
The genetic diversity of Sepioteuthis sp. 1 was similar to that of Haplotype diversity and nucleotide diversity of Sepioteuthis sp. 1 among three localities.
www.intechopen.comGeneticDiversityand Genetic Heterogeneity of Bigfin Reef Squid "Sepioteuthis lessoniana" Species Complex in Northwestern Pacific Ocean 157 Table3.Haplotype distribution and variable sites of mitochondrial NC2 region of Sepioteuthis sp. 1 among three localities.

Table 6 .
Pairwise Fst and associated probability (P) of Sepioteuthis sp. 1 among three localities.values are below the diagonal and corresponding P values are above the diagonal.Bonfferroni correction P<0.05.

Table 7
. Haplotype distribution and variable sites of mitochondrial NC2 region of Sepioteuthis sp. 3 between Okinawajima and Ishigakijima Island.Table 8. Haplotype diversity and nucleotide diversity of Sepioteuthis sp. 3 between Okinawajima and Ishigakijima Island.