© 2005 International Council for the Exploration of the Sea
Genetic evidence that the northern calamary, Sepioteuthis lessoniana, is a species complex in Australian waters
Evolutionary Biology Unit South Australian Museum, North Terrace, Adelaide, SA 5000, Australia
*Correspondence to L. Triantafillos: Current contact address: Falkland Islands Fisheries Department, PO Box 598, FIPASS, Stanley, Falkland Islands; tel: +500 27260; fax: +500 27265. e-mail: ltriantafillos{at}fisheries.gov.fk.
Allozyme electrophoresis was used to investigate the taxonomic status of northern calamary Sepioteuthis lessoniana (Lesson 1830) from two sites in Shark Bay, Western Australia. Of the 40 squid examined at 38 presumptive loci, four individuals from the oceanic site were clearly differentiated from the rest by fixed allelic differences at four loci (Acp, Got2, Idh2, and PepD) and near-fixed differences at another three (Est, Ocdh, and 6Pgd). The genetic distances between these two groups of individuals (13% Fixed Differences and Nei, D (1978) = 0.178) were roughly twofold greater than those between the two cryptic taxa in the southern calamary S. australis, but are considerably smaller than those between the northern and southern calamary. The most likely explanation for these data is that S. lessoniana comprises two "cryptic" biological species in this region. Further studies are needed to delineate the total number of species found throughout Australasia for this important loliginid squid.
Keywords: allozyme electrophoresis, cryptic species, squid
Received 8 October 2004; accepted 14 June 2005.
 |
Introduction |
|---|
 Top Introduction Material and methodsResultsDiscussionConcluding remarksReferences |
|---|
Cryptic or sibling species are those not recognized a priori on morphological grounds and are a relatively common phenomenon among marine invertebrate groups (Knowlton, 1993; Thorpe et al., 2000). While ignorance of the real biodiversity in a group represents an intellectual irritant, cryptic species also become a major economic issue when the group in question is subject to commercial exploitation. In such situations, the undetected presence of cryptic species can invalidate a significant proportion of the biological information on individual "species" (Thorpe et al., 2000; Triantafillos, 2004) and will often render any fishery management plan inappropriate (Carvalho and Hauser, 1994). One such group is the cephalopods, which represents a major fishery both worldwide and in Australia (FAO, 1998). Here, as in other marine groups, molecular genetic techniques have revealed that cryptic species are a common occurrence (Knowlton, 1993). Thus far, cryptic speciation has been documented in cuttlefish (Kassahn et al., 2003), octopus (Levy et al., 1988; Soller et al., 2000), and both oegopsid (Smith et al., 1981; Brierley et al., 1993) and myopsid squid (Augustyn and Grant, 1988; Izuka et al., 1994; Yeatman and Benzie, 1994). In this light, it seems obvious that every important cephalopod fishery should be investigated for the presence of cryptic species, preferably before commencing any significant within-species study.
Little is known about the biology and ecology of cryptic species among commercially exploited squid. However, a recent study of Sepioteuthis australis has established that two different cryptic taxa and their hybrids display clear differences in several biological parameters of major importance to fisheries biologists (Triantafillos, 2004). Differences of the magnitude found in this study, combined with the frequent existence of cryptic species and their unknown contribution to respective fisheries, complicate markedly the construction of stock assessment models and provisions of subsequent fishery advice (Beddington et al., 1990).
The incidence of cryptic speciation does vary among cephalopods, particularly among squid. Of the 20 or so oegopsid squid examined using genetic techniques, cryptic speciation events have been found in Martialia hyadesi (Brierley et al., 1993) and Berryteuthis magister (Katugin, 2000). Cryptic speciation is also characteristic of (or present in) myopsid squid. Nowhere is this highlighted better than in Australian waters, where cryptic species have been revealed in all four "species" of loliginids examined so far (Photololigo chinensis and P. edulis, Yeatman and Benzie, 1994; Sepioteuthis australis, Triantafillos and Adams, 2001; Loliolus noctiluca, Citroen, 2001).
Another loliginid species subjected to significant commercial exploitation is the northern calamary, Sepioteuthis lessoniana Lesson 1830. This large squid is common throughout the shallow coastal waters of the Indo-West Pacific region and, in Australia, displays a continuous distribution from central Western Australia around to southern Queensland. Compared with most squid, the northern calamary has been well studied, particularly with respect to growth (Jackson, 1989, 1993; Jackson and Moltschaniwskyj, 2002) and aquaculture (Segawa, 1993; Forsythe et al., 2001). Some of the above-mentioned growth studies have revealed local and geographical heterogeneity in growth and reproductive biology, more than might be expected for a single biological species.
Although cryptic species may be uncovered using a variety of non-morphological data, they are most commonly detected using molecular genetic techniques (Knowlton, 1993; Thorpe et al., 2000). The only previous genetic investigations undertaken on S. lessoniana have already demonstrated three taxa in the waters around Japan, based on their allozyme profiles (Izuka et al., 1994, 1996). These three taxa displayed non-random distributions, with one found only in the northeast, another mainly in the southwest, and the third occurring throughout Japanese waters. Clearly, there is a need to extend these initial genetic studies to populations throughout the extensive geographic range of this species, both to determine which of the three above-mentioned taxa are widespread and to determine whether additional cryptic forms are present elsewhere.
This study represents a first look at the northern calamary of Australian waters using a genetic technique. Our technique of first choice remains allozyme electrophoresis, both for its demonstrated utility in the initial investigation of species boundaries (Richardson et al., 1986; Avise, 1994; Hillis et al., 1996) and for its historical significance in diagnosing marine sibling species in general (Knowlton, 1993) and cephalopods in particular. Allozyme data have also provided important insights into within-species population structure in a number of economically important cephalopod species (e.g. Triantafillos and Adams, 2001; Kassahn et al., 2003). Herein, we have used allozyme data to demonstrate that the northern calamary in the waters around Shark Bay, Western Australia, comprise at least two cryptic biological species.
|
Material and methods |
|---|
|
TopIntroductionMaterial and methodsResultsDiscussionConcluding remarksReferences |
|---|
Sample collection
Samples of Sepioteuthis lessoniana were collected from two sites in Shark Bay, Western Australia. A squid jig was used to collect 11 individuals from the Denham jetty (site 1) on 27 September 1996. After 12 h, the same gear was used on a boat to collect another nine animals near Dirt Hartog Island (site 2). The distance between these two sites was 18 km (Figure 1). In addition, another 20 individuals were collected from site 1, 20 months after the original collection date.
|
Within 15 min of collection of all squid, a piece of tentacle tissue was removed from each individual and frozen in liquid nitrogen. Tissue samples were returned to the laboratory and stored at –80°C, pending analysis. All tissues received identical treatment leading up to allozyme analysis, thus ruling out treatment-related, post-translational modification as a potential source of electrophoretic variability.
Allozyme electrophoresis
Allozyme electrophoresis was undertaken on cellulose acetate gels (CELLOGEL©, M.A.L.T.A., Milan) following the principles and procedures of Richardson et al. (1986). Muscle homogenates were prepared as outlined by Triantafillos and Adams (2001). A total of 35 enzymes displayed banding patterns of sufficient activity and resolution to permit allozymic interpretation. Apart from diaphorase (DIA, EC 1.6.99) and octopine dehydrogenase (OCDH, EC 1.5.1.11
[EC]
), the details of which enzymes correspond to the locus abbreviations used herein are presented in Triantafillos et al. (2004). The nomenclature used to refer to loci and allozymes follows Adams et al. (1987). Representative tissues of the "central" and "peripheral" forms of the southern calamary Sepioteuthis australis were also included in the study for comparative purposes. All ten specimens of S. australis (five centrals and five peripherals) used were collected by Triantafillos and Adams (2001) from Albany, Western Australia, in September 1996. The tissues of these individuals were included on the same gels as S. lessoniana gels, allowing for direct interspecific comparisons of allelic frequencies.
Genetic distances were calculated as either %Fixed Differences (FD; Richardson et al., 1986) allowing a tolerance of 5% for shared alleles or Nei's genetic distance (D; Nei, 1978). Significance tests for Hardy–Weinberg expectations and heterogeneity of allele frequencies were carried out using GENEPOP version 3.1d (Raymond and Rousset, 1999), and adjusted for multiple tests using the sequential Bonferroni correction factor (Rice, 1989).
|
Results |
|---|
|
TopIntroductionMaterial and methodsResultsDiscussionConcluding remarksReferences |
|---|
In all, 38 putative loci were successfully screened in the allozyme study. Of these, the following 29 loci were invariant for the Sepioteuthis lessoniana examined: Acyc, Ada, Adh, Ak, Aldh, Argk, Dia, Enol, Fdp, Gapd, Glo, Got1, Gpd, Gpi, Idh1, Lap, Mdh, Mpi, Ndpk, Np, PepA1, PepA2, PepB, Pgam, Pgk, Pgm, Pk, Sordh, and Tpi. The remaining nine loci (Acp, Ald, Ap, Got2, Est, Idh2, Ocdh, PepD, and 6Pgd) were polymorphic, displaying between two and four alleles (Table 1). No sample exhibited electrophoretic patterns consistent with any of the non-genetic phenomena known to mimic allozyme variation (Richardson et al., 1986). Since there were no significant temporal differences in allele frequencies for the nine polymorphic loci at site 1, they were subsequently pooled and treated as a single sample.
|
Tests of observed genotype frequencies against the Hardy–Weinberg expectations for a panmictic population demonstrated a highly significant deficiency of heterozygotes at five of the nine polymorphic loci (p < 0.001 for Acp, Got2, Idh2, and Ocdh; p < 0.05 for Est). The reason for this deficiency became apparent upon examination of the multilocus genotypes, with two distinctive genetic groups immediately definable by their allelic profiles at four of the variable loci. The majority of animals (n = 36) belonged to genetic group A, which was homozygous for three loci (Acp, Got, and Idh) and polymorphic for a and b alleles at PepD. However, the remaining four animals (group B), all collected from site 2 (Figure 1), were homozygous for the alternate allele at Acp, Got, and Idh, plus possessed one or both of alleles c or d at PepD. Thus the two groups were characterized by fixed allelic differences, in sympatry, at these four loci.
Table 1 presents the allele frequencies at all variable loci for the two genetic groups. Apart from the four fixed differences outlined above, the two groups display "near-fixed" differences at an additional three loci (Est, Ocdh, 6Pgd; frequency of the shared allele ranges between 4 and 13%). Statistical tests indicated that the allele frequency differences between the two groups were highly significant at the seven key loci (all p values <0.001), despite the small sample sizes for group B. The genetic distance estimates are %FD = 13% and Nei D = 0.178 (Table 2). These genetic distances are roughly twofold greater than those between the two cryptic taxa in S. australis, but are considerably smaller than those that occur between the northern and southern calamary (Table 2; Figure 2).
|
|
|
Discussion |
|---|
|
TopIntroductionMaterial and methodsResultsDiscussionConcluding remarksReferences |
|---|
The present study has unequivocally demonstrated the presence of two readily diagnosable genetic groups among northern calamary from the Shark Bay region in Western Australia. Given the magnitude of their genetic distinctiveness, the conclusion that there are two taxa present among the animals sampled is justified, regardless of their geographic distributions. However, their co-occurrence at a single site, without evidence of hybridization or introgression, is sufficient to assign full biological species status to each taxon. This evidence, together with the observation that Sepioteuthis lessoniana appears also to be a species complex elsewhere in its distribution (Izuka et al., 1994, 1996), strongly argues for the need to carry out a detailed, Australia-wide genetic investigation of this important commercially harvested species.
The allozyme data indicate that the A and B forms of S. lessoniana are closely related and, therefore, most likely represent genuine sibling species. This result is in stark contrast to the large genetic distances between the cryptic forms of S. lessoniana and those of S. australis, which display levels of genetic divergence typically associated with different genera (Thorpe, 1983). Nevertheless, it cannot be assumed that, as sibling species, S. lessoniana A and S. lessoniana B will necessarily have the same biology or ecology. Triantafillos (2004) has shown that the "central" and "peripheral" forms of S. australis vary markedly in their growth and reproductive biology, and yet our allozyme data suggest that these taxa are even more closely related than S. lessoniana A and S. lessoniana B.
Spatial and temporal heterogeneity for growth and life history features have been documented in S. lessoniana from northern Australia (Jackson and Moltschaniwskyj, 2002). Two very different life history modes were evident, namely a "hot growth" strategy of young, fast growth, and small body size shown in Townsville summer and Thailand equatorial populations; and a "cool growth" strategy of older, slower growth, and larger body size shown in Townsville winter and southern Queensland populations. Given that both modes were present in the waters around Townsville, one likely explanation for this finding is that each life history strategy is a fixed characteristic of different species, each with differing geographic distributions which, nevertheless, overlap (at least around Townsville). Several authors have implied that the high incidence of cryptic speciation among squid is due to inadequate taxonomy in a group with few hard parts or other obvious useful taxonomic characters (Thorpe et al., 2000). If this was true, then levels of speciation in ommastrephids should be comparable with those found in loliginids. They are not, as cryptic speciation has only been found in only 10% of the ommastrephids examined so far. An alternative explanation for the high levels of speciation observed in loliginids from Australia was put forward by Triantafillos et al. (2004), who proposed that the biological attributes of this group of squid, namely their neritic habitat, their nearshore benthic spawning habits, and their large hatchlings presumably provide them with less ability to disperse than ommastrephids and, therefore, greater opportunities to form localized, genetically isolated populations.
There is also some evidence to suggest that waterbodies with different oceanographic characteristics contribute to genetic divergence in squid. For example, an allozyme study of the squid Nototodarus gouldi around New Zealand revealed an allopatric sibling species, Nototodarus sloanii, the distributions of which were divided by the Substructuring Tropical Convergence Zone (Smith et al., 1981).
The Shark Bay region is characterized by salinoclines and three major waterbodies, namely oceanic, metahaline, and hypersaline. This distinct salinity pattern influences the distribution of marine flora and fauna within the bay (e.g. Pagrus auratus; Bastow et al., 2002), leading to three biotic zones. Affinities to these different waterbodies could account for the divergence between the two taxa of northern calamary. It is possible that under normal conditions the two Sepioteuthis lessoniana taxa may be largely allopatric, coming together in the Indian Ocean near Dirk Hartog Island only occasionally when prevailing oceanographic conditions allow.
|
Concluding remarks |
|---|
|
TopIntroductionMaterial and methodsResultsDiscussionConcluding remarksReferences |
|---|
The allozyme data demonstrate the presence of two species among the Sepioteuthis lessoniana of Shark Bay. This is at variance with current management perspectives on this important fisheries resource. Further studies are needed to delineate the number of taxa found within the Australasian region in order to successfully manage the northern calamary fishery in these waters. Without this information, this species would be vulnerable to population crashes, perhaps even local extinction, through overfishing. This risk is exacerbated by the general lack of overlapping generations, relatively low fecundity, and high interannual variability in recruitment of most loliginids (Boyle and Boletzky, 1996), and further compounded by the fact that most northern calamary are caught on spawning aggregations. The present study also casts doubts over the validity of all estimates of growth for this species, the results of which may not have been interpreted according to the true systematic complexity present. This is a serious problem, especially since Sepioteuthis lessoniana is fast becoming one of the most important loliginid squid species for modelling growth through the life cycle.
|
Acknowledgements |
|---|
This research was funded by the South Australian Marine Scalefish Integrated Management Committee and the Northern Territory University. We thank Terry Reardon for technical assistance and David Short and Gary Jackson for their tireless help in collecting many of the animals. Financial support was obtained by the senior author from a postgraduate scholarship from the Northern Territory University.
|
References |
|---|
|
TopIntroductionMaterial and methodsResultsDiscussionConcluding remarksReferences |
|---|
-
Adams M., Baverstock P.R., Watts C.H.S., Reardon T. (1987) Electrophoretic resolution of species boundaries in Australian Microchiroptera. I. Eptesicus (Chiroptera, Vespertilionidae). Australian Journal of Biological Sciences 40:143–162.
Augustyn C.J. and Grant W.S. (1988) Biochemical and morphological systematics of Loligo vulgaris vulgaris Lamarck and Loligo vulgaris reynaudii d'Orbigny nov. comb. (Cephalopoda: Myopsida). Malacologia 29:215–233.[Web of Science]
Avise J.C. (1994) Molecular Markers, Natural History and Evolution(Chapman and Hall, New York) 511 pp.
Bastow T.P., Jackson G., Edmonds J.S. (2002) Elevated salinity and isotopic composition of fish otolith carbonate: stock delineation of pink snapper, Pagras auratus, in Shark Bay, Western Australia. Marine Biology 141:801–806.[CrossRef]
Beddington J.R., Rosenberg A.A., Crombie J.A., Kirkwood G.P. (1990) Stock assessment and the provision of management advice for the short fin squid fishery in Falkland Islands. Fisheries Research 8:351–365.[CrossRef][Web of Science]
Boyle P.R. and Boletzky S.v. (1996) Cephalopod populations: definition and dynamics. Philosophical Transactions of the Royal Society of London, Series B 351:985–1002.
Brierley A.S., Rodhouse P.G., Thorpe J.P., Clarke M.R. (1993) Genetic evidence of population heterogeneity and cryptic speciation in the ommastrephid squid Martiala hyadesi from the Patagonian Shelf and Antarctic Polar Frontal Zone. Marine Biology 116:593–602.[CrossRef]
Carvalho G.R. and Hauser L. (1994) Molecular markers and the stock concept in fisheries. Reviews in Fish Biology and Fisheries 4:326–350.[CrossRef][Web of Science]
Citroen R. (2001) Growth and genetic variation in the Australian squid Loliolus noctiluca. Honours thesis, University of Tasmania, Australia, 83 pp.
FAO. (1998) Fishery Statistics: Catches and Landings(FAO, Rome) vol. 87: 232 pp.
Forsythe J.W., Walsh L.S., Turk P.E., Lee P.G. (2001) Impact of temperature on juvenile growth and age at first egg-laying of the Pacific reef squid Sepioteuthis lessoniana reared in captivity. Marine Biology 138:103–112.[CrossRef]
Hillis D.M., Mabel B.K., Moritz C. (1996) Applications of molecular systematics. In Hillis D.M., Moritz C., Mabel B.K. (Eds.). Molecular Systematics(Sinauer Associates, Sunderland) pp. 515–543 655 pp.
Kassahn K.S., Donnellan S.C., Fowler A.J., Hall K.C., Adams M., Shaw P. (2003) Molecular and morphological analyses of the cuttlefish Sepia apama indicates a complex population structure. Marine Biology 143:947–962.[CrossRef]
Katugin O.N. (2000) A new subspecies of the schoolmaster gonate squid, Berryteuthis magister (Cephalopoda: Gonatidae), from the Japan Sea. The Veliger 43:82–97.
Knowlton N. (1993) Sibling species in the sea. Annual Review of Ecology and Systematics 124:189–216.
Levy J.A., Haimovici M., Conceição M. (1988) Genetic evidences for two species to the genus Eledone (Cephalopoda: Octopodidae) in south Brazil. Comparative Biochemistry and Physiology B 90:275–277.[CrossRef]
Izuka T., Segawa S., Okutani T. (1996) Biochemical study of the population heterogeneity and distribution of the oval squid Sepioteuthis lessoniana complex in southwestern Japan. American Malacological Bulletin 12:129–135.[Web of Science]
Izuka T., Segawa S., Okutani T., Numachi K. (1994) Evidence of the existence of three species in the oval squid Sepioteuthis lessoniana complex in Ishigaki Island, Okinawa, south western Japan by isozyme analyses. Japanese Journal of Malacology 53:217–228.
Jackson G.D. (1989) Age and growth of the tropical nearshore loliginid squid Sepioteuthis lessoniana determined from statolith growth-ring analysis. Fishery Bulletin US 88:113–118.
Jackson G.D. (1993) Season variation in reproductive investment in the tropical loligind squid Loligo chinensis and the small tropical sepioid Idiosepius pygmaeus. Fishery Bulletin US 91:260–270.
Jackson G.D. and Moltschaniwskyj N.A. (2002) Spatial and temporal variation in growth rates and maturity in the Indo-Pacific squid Sepioteuthis lessoniana (Cephalopoda: Loliginidae). Marine Biology 140:747–754.[CrossRef]
Nei M. (1978) Estimation of average heterozygosity and genetic distance from a number of individuals. Genetics 89:583–590.
Raymond M. and Rousset F. (1995) GENEPOP (Version 3.1d): An updated version of GENEPOP (version 1.2) described in: Raymond, M, and Rousser, F. 1995. GENEPOP (version 1.2)–populations genetics software for exact tests and ecumenicism. Journal of Heredity 86:248–249.
Rice W.R. (1989) Analyzing tables of statistical tests. Evolution 43:223–225.[CrossRef][Web of Science]
Richardson B.J., Baverstock P.R., Adams M. (1986) Allozyme Electrophoresis: A Handbook of Animal Systematics and Population Studies(Academic Press, New York) 410 pp.
Segawa S. (1993) Field and laboratory feeding studies of the Japanese oval squid, Sepioteuthis lessoniana. In Okutani T., O'Dor R.K., Kubodera T. (Eds.). Recent Advances in Cephalopod Fisheries Biology(Tokai University Press, Tokyo) pp. 499–511 752 pp.
Smith P.J., Roberts P.E., Hurst R.J. (1981) Evidence for two species of arrow squid in the New Zealand fishery. New Zealand Journal of Marine and Freshwater Research 15:247–253.[Web of Science]
Soller R., Warnke K., Saint-Paul U., Blohm D. (2000) Sequence divergence of mitochondrial DNA indicates cryptic biodiversity in Octopus vulgaris and supports the taxonomic distinctiveness of Octopus mimus (Cephalopoda: Octopodidae). Marine Biology 136:29–35.[CrossRef]
Thorpe J.P. (1983) Enzyme variation, genetic distance and evolutionary divergence in relation to levels of taxonomic separation. In Oxford G.S. and Rollinson D. (Eds.). Protein Polymorphism: Adaptive and Taxonomic Significance(Academic Press, London) pp. 131–152 405 pp.
Thorpe J.P., Solé-Cava A.M., Watts P.C. (2000) Exploited marine invertebrates: genetics and fisheries. Hydrobiologia 420:165–184.[CrossRef][Web of Science]
Triantafillos L. (2004) Effects of genetic and environmental factors on growth of southern calamary, Sepioteuthis australis from southern Australia and northern New Zealand. Marine and Freshwater Research 55:439–446.[CrossRef][Web of Science]
Triantafillos L. and Adams M. (2001) Allozyme analysis reveals a complex population structure in the southern calamary Sepioteuthis australis from Australia and New Zealand. Marine Ecology Progress Series 212:193–209.[CrossRef][Web of Science]
Triantafillos L., Jackson G.D., Adams M., McGrath-Steer B. (2004) An allozyme investigation of the stock structure of arrow squid Nototodarus gouldi (Cephalopoda: Ommastrephidae) from Australia. ICES Journal of Marine Science 61:829–835.
Yeatman J. and Benzie J.A.H. (1994) Genetic structure and distribution of Photololigo spp. in Australia. Marine Biology 118:79–87.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||