Pacific Science (2002), vol. 56, no. 3:347-355© 2002 by University of Hawai'i PressAll rights reserved

THE HAWAIIAN ARCHIPELAGO presentsunique opportunities for the study of evo­lutionary biology because of its relative iso­lation, high levels of endemism, and theexplosive radiation of a number of taxa (Si­mon 1987, Wagner and Funk 1995). Forexample, the terrestrial invertebrate biota,which has been particularly well studied ingenera such as Drosophila flies, Tetragnathaspiders, and Achatinellinae tree snails, typi­cally exhibits extensive adaptive radiationscomprising species endemic to the archipel­ago (Roderick and Gillespie 1998, Thackerand Hadfield 2000). These patterns are

Two Genetically Distinct Populations of Bobtail Squid,Euprymna seolopes, Exist on the Island of O'ahu1

J. R. Kimbell,2 M. J. MeFall-Ngai,2 and G. K. Roderiek3

Abstract: Population structure of the endemic Hawaiian bobtail squid, Eu­prymna seolopes, was examined using both morphological and genetic data. Al­though allozyme polymorphism was negligible, measurements of eggs, juveniles,and adults, as well as genetic data sequences of mitochondrial cytochrome oxi­dase I, demonstrated highly significant population structuring between twopopulations found on the northeastern and southern coasts of the island ofO'ahu. These data suggest that extremely low levels of gene flow occur amongthese populations. Population subdivision of marine shallow-water invertebratesin Hawai'i is not expected based on earlier surveys, but may reflect a moregeneral pattern for organisms, both marine and terrestrial, that exhibit limiteddispersal. The subdivision also provides insight into the pathway through whichcoevolution between E. seolopes and its internal symbiont, Vibrio fiseheri, mayproceed.

thought to result from the extremely het­erogeneous nature of the terrestrial islandlandscape, which presents an abundance ofspatially limited, yet distinct, niches ripe foradaptive radiation (Gillespie et al. 2001).

Marine invertebrates, however, have muchlower rates of endemism than their terrestrialcounterparts, with endemism ranging from 2to 20% for nearshore gastropods, brachyurandecapods, and certain polychaetes and echi­noderms of the central Pacific (Scheltema1986, Kay and Palumbi 1987). These benthicmarine invertebrates appear instead to repre­sent attenuated Indo-Pacific fauna, and themarine endemic species that do exist are usu-ally distributed throughout all of the islands

1 This work was supported by NSF grants IBN 99- of the archipelago (Scheltema 1986). The04601 to M.M.-N. and E. G. Ruby and DEB 0096189 wide geographic distributions of these marineto R. G. Gillespie and G.K.R., NIH grant RRI0926 to invertebrate species indicate that fairly highE. G. Ruby and M.M.-N., NOAA National SeaGrant gene flow must be occurring between pop­RlFM-5 to G.K.R. and C. Smith, and NOAA HCRIgrant NA870A0381 to G.K.R. Manuscript accepted 28 ulations within species (Palumbi 1992). SuchNovember 2001. gene flow is presumably facilitated by long

2 Pacific ~iomedical Research Center, University of planktonic larval phases, which ensure high-----Hawai'i, 41 'Ahui Street,-Honolulu,-Hawai'i-96816..------disp~-rsat-Yet,within-sudrwidespre-adsp-ecies----

3 Corresponding author: Department of Environ-mental Science, Policy, and Management, University of some population structure may neverthelessCalifornia, 201 Wellman Hall MC 3112, Berkeley, Cali- persist (Barber et al. 2000).fornia 94708-3112 (phone: 510-642-3327; fax: 510-642- Although much is known about marine7428; E-mail: roderick@nature.berkeley.edu). invertebrate species with extended larval life

histories, less is understood about species thatdo not have broadly dispersing larval or juve­nile stages. One such species is the bobtailsquid, Euprymna seolopes, which is endemic to

347

348 PACIFIC SCIENCE· July 2002

tNiu Valley

IKaneohe Bay, .

,//"(\.

/ .....•.........•.........

N

i

FIGURE 1. Collection sites on O'ahu, Hawaiian Archi­pelago, for specimens examined in this smdy.

windward O'ahu (Figure 1). Specimens weremaintained in running seawater tables at 24°Cat the Kewalo Marine Laboratory, Universityof Hawai'i.

the Hawaiian Islands (Singley 1983). FemaleE. seolopes lay clutches of eggs on hard sub­strates in the shallow waters of the back reef.No true larval stages occur in this species (i.e.,the juveniles hatch with a morphology andbehavior very similar to those of the adult[Arnold et al. 1972]). Specifically, after a briefplanktonic stage of hours to a few days, thejuvenile squid take on the diel pattern of be­havior characteristic of the species (i.e., theybury in the sand during the day and forage inthe water column at night).

Previously, it was observed that E. seolopesis notable for its habitat loyalty, and ithas been suggested that two reproductivelyisolated populations exist on northeasternand southern coasts of the island of O'ahu(Singley 1983). The E. seolopes-Vibrio fiseheriassociation has become a model for animal­bacterial symbiosis and in particular the per­sistent colonization of animal epithelia byextracellular bacteria (McFall-Ngai and Ruby1998). Recent studies of the squid-vibriosymbioses at the level of species have pro­vided strong evidence for coevolution of thehost and symbiont (Nishiguchi et al. 1998).Whether population subdivision of E. seolopesexists is critical to understanding the co­evolution of this species with its bacteriapartners-how isolated are populations of E.seolopes and at what scale would population­specific strains of V. fiseheri be expected toevolve? In this study we tested the hypothesisof population subdivision of E. seolopes withboth morphological and genetic data. Ourresults indicate that populations of E. seolopeson O'ahu are indeed distinct. Further, theresults demonstrate that at least some speciesof Hawaiian marine invertebrates are moresubdivided than previously realized, likely asa consequence of a life history lacking dis­persive egg or larval stages.

Morphometric Data

Measurements of morphological characterswere performed to determine whether signif­icant phenotypic differences occur betweenKane'ohe Bay and Niu Valley animals. Threecharacters were measured on specimens fromeach of these two sites: female mantle length,egg diameter, and hatchling mantle length.The latter two measurements were madeusing a dissection microscope (Wild M5) atlOx with an ocular micrometer. Eggs weremeasured on the day that they were laid. Thehatchlings were first anesthetized in a 50: 50solution of 0.37 M MgClz:seawater beforethe mantle-length measurements were taken.

...... .-- ..---------------- - -------:A:pproximately-75--specimenswere-examirredfor each character per locality.

MATERIALS AND METHODS

Capture and Maintenance ofSpecimens

Adult Euprymna seolopes were collected in1997 and 1998 from two sites, the shallowsand flats offNiu Valley along leeward O'ahuand along the shores of Kane'ohe Bay on

Allozymes: Cellulose Acetate Gel Electrophoresis

Variation at allozyme loci was examinedthrough cellulose acetate gel electrophoresis,a method that requires very little tissue(Richardson et al. 1986, Hebert and Beaton

Two Genetically Distinct Populations of Bobtail Squid on O'ahu . Kimbell et at. 349

DNA, 0.4 !J,M of each primer, 5 ~l of 8 mMdNTPs, and 0.12 ~l ofTaq polymerase (Per­kin Elmer, Norwalk, California). Amplifica­tions were performed using a thermocycler(GeneAmp PCR 9600). The PCR conditionswere as follows: 35 cycles of 94°C for 30 sec,47°C for 30 sec, and noc for 60 sec. Theprimers used to amplify COl were CI-J-I718(26 mer, 5'-GGAGGATTTGGAAATT­GATTAGTTCC-3') and CI-N-2191 (alias"Nancy" 26 mer, 5'-CCCGGTAAAAT­T AAAATATAAACTTC-3') (primer desig­nations given in Simon et al. [1994]). Theresulting PCR products were purified with aGeneClean Kit (Bio 101 Inc., La Jolla, Cali­fornia). Seven microliters of the resultantDNA and 4 ~l of 1 !J,M CI-J-1718 primerwere used for cycle sequencing using thePRISM Ready Reaction Terminator CycleSequencing Kit with an ABI Model No. 377automated sequencer (PE Applied Biosys­terns, Foster City, California). A total of 18COl sequences was obtained from 10 indi­viduals from Kane'ohe Bay and 8 individualsfrom Niu Valley. All DNA sequences wereexamined and aligned using Sequencher 3.1(GeneCodes, Ann Arbor, Michigan).

1989). Squid tissue was frozen in liquid ni­trogen and stored at -80°C until used inanalyses. Mantle tissue, because it is of lowyield, and light organs, because they containsymbionts, were removed before homogeni­zation. Tissue samples (0.5 g) were placed onice and homogenized in an equal volume ofdistilled water. The homogenates were cen­trifuged for 5 min at 12,000x g at 4°C topellet tissue debris. Supernatant fluids wereplaced in microcentrifuge tubes on ice untiluse. Allozyme electrophoresis was run on cel­lulose acetate gels in electrophoresis cham­bers (Zip-zone, Helena Laboratories Inc.,Beaumont, Texas). The histochemical stain­ing recipes used were given in Hebert andBeaton (1989). Two buffer systems resulted inthe best resolution: "tris-glycine"-0.4 MTris with 2 M glycine, pH 8.5, with gellbufferdilution 1: 9; and "tris-maleate"-0.1 M tris­maleate, pH 7.8. Of the 18 presumed en­zymes assayed 7 yielded acceptable enzymeactivity and clear resolution: isocitrate de­hydrogenase (IDH; E.c. 1.1.1.42), malatedehydrogenase (MDH; E.C. 1.1.1.37), malatedehydrogenase NADP+ (ME; E.C. 1.1.1.40),glucose-6-phosphate isomerase (GPI; E.C.5.3.1.9), mannose-6-phosphate isomerase(MPI; E.C. 5.3.1.8), phosphoglucomutase(PGM; E.C. 5.4.2.2), and aspartate aminotransferase (AAT; E.C. 2.6.1.1). The tris­glycine buffer was used for PGM and ME,and the remaining loci were run with tris­maleate. Horizontal gels were run at 180 Vand 3 rnA for 45 min at 4°C. Approximately30 alleles were scored at each locus for 20individuals from each locality.

Phylogeog;raphic Analysis

The evolutionary history of the alleles at alocus can be represented in a phylogenetictree, which can then be interpreted in light ofthe geographical distribution of those alleles;this approach is termed phylogeography (Avise2000). The interpretation of such an analysiscan be complicated for nuclear loci becauseof recombination (Begun and Aquadro 1992,

Mitochondrial DNA: Cytochrome Oxidase I Hudson 1994) and for both nuclear and mi­tochondrialloci because of the persistence of

Squid DNA was extracted from frozen testis ancestral alleles (Templeton and Sing 1993).tissue (25 mg) with a Qiagen QIAamp Tissue There is no evidence that mitochondrial

-----Rir-tc-at:-n-o-:-z9t(4):-1'lre-rsuhrt-eLl-g-enomlc---sequences--m-dUs species recombine, yetDNA was reprecipitated with 95% ethanol, persistence of ancestral alleles is an issue.washed with 70% ethanol, and resuspended Templeton et al. (1992) and Templeton andin TE buffer (10 mM Tris-HCl and 1 mM Sing (1993) provided a method for examiningEDTA, pH 8.0). A polymerase chain reaction the recent history of alleles at a locus. The(PCR) was used to produce DNA templates method focuses phylogenetic reconstructionfor the sequencing of the mitochondrial cy- on the most recent substitutions before at­tochrome oxidase I (COl) gene. Each 50-~l tempting to resolve deeper nodes. AssumingPCR reaction contained 4 ~g of template that more recent changes are less likely to in-

350 PACIFIC SCIENCE· July 2002

RESULTS

dude homoplasy or recombination, sequencesseparated by one substitution are linked first,the resulting groups form the basis for linkingalleles separated by two substitutions, andso on. Details of the method are providedby Templeton and Sing (1993), Templeton(1998), Davies et al. (1999), and Posada andCrandall (2001).

Population Analysis

Population structure was determined throughthe analysis of molecular variation (AMOVA)method of Excoffier et al. (1992) as imple­mented by the computer program Arlequin2.0 (Schneider et al. 2000). Indices of popu­lation subdivision analogous to standard Fstatistics (Wright 1951,1965) were calculatedwith significance assessed using permutationprocedures that randomly redistribute indi­viduals among populations. This general ap­proach was used to estimate F statistics basedupon (1) allele frequencies only, and (2) thegenetic distance among alleles as well as theirfrequencies. The F statistics used were (1) ()(Weir and Cockerham 1984, see Excoffier etal. 1992), and (2) <1>, calculated using the av­erage Kimura 2-parameter distance observedbetween alleles. Exact tests were performedbased on the distribution among populationsof (1) alleles and (2) phylogenetically derivedallele groups (TFPGA, Miller 1998). In thelatter case, alleles in the same one-step dadeof the Templeton networks were pooled.

plications of differing egg and female bodysizes among these populations have not yetbeen examined.

The seven enzyme stains resolved nineenzyme-coding loci (lDH and AAT eachstained for two loci). AlI loci examined weremonomorphic. Though surprising, this resultis not unexpected for squid-other intra!interspecific studies of squid population struc­ture have yielded negligible allozyme poly­morphism and in some cases monomorphism(Brierley et al. 1993, 1995, lzuka et al. 1996).However, other allozyme studies have dis­tinguished among closely related squid spe­cies (lzuka et al. 1994) and seasonal cohorts(Kang et al. 1996). Although allozyme datacollected here contribute no information con­cerning the hypothesis of distinct populations,they do raise a question for future work: towhat extent does selection contribute to thelack of variation in squid electrophoreticproteins?

Partial Cal sequences of approximately430 base pairs from both Niu Valley andKane'ohe Bay animals were obtained. Tensequences from Kane'ohe Bay and eight fromNiu Valley were resolved. Measures of genediversity, mean number of pairwise differ­ences, and base composition were very similarin the two populations (Table 1). Our resultsyielded six distinct haplotypes distinguishedby seven polymorphic sites, with only onehaplotype being shared among the two pop­ulations (Figure 3).

Statistical analysis revealed significant pop­ulation structure between the populations.Both frequency- and distance-based F statis­

Significant differences were found among tics were higWy significant, with the distance­populations for both morphological and ge- based estimates, <I> (0.60), being considerablynetic characters. Niu Valley females were larger than the frequency-based () (0.49) (Ta­higWy significantly larger and produced higWy ble 2), illustrating that the sequence data dosignificantly larger eggs and hatchlings than provide additional information with regard to

----Rarre'uhe-B-ay-femates-tpigure-2-)-;-suppurtirrg----population-structuringin-this-species~----­

Singley's (1983) earlier observations. Al- The values of both F statistics are extremelythough phenotypic differences between the high, especially considering the small distancetwo populations do not provide direct evi- between populations. These data, coupleddence of genetic, or inherited, differences with the observation of only one shared hap­between the two populations, such data are lotype between the populations (frequency ofcertainly consistent with this hypothesis of approximately 10% in each population), sug­genetic differentiation. The life history im- gest that gene flow is minimal between these

Two Genetically Distinct Populations of Bobtail Squid on O'ahu . Kimbell et al. 351

Kaneohe Bay

D Niu Valley

3.5 35

- 3.0 - 30E EE E- 2.5 - 25CD CD.~ ,~tJ) 2.0 tJ) 20.! .!.- asc:CD 1.5 E 15> CD~ -'- .:!:::'" 1.0 10en ~

en "Cw

0.5«

5

0.0 0Egg Hatchling Femalediameter mantle mantle

FIGURE 2. Morphological differences among individuals collected in Kane'ohe Bay and Niu Valley locations. Samplesizes were for eggs, hatchlings, and females (78, 62, and 30, respectively) for each population. All comparisons werehighly statistically significant (t-tests, P < 0.001). Female age was uncertain; females were measured the day after dyingin the laboratory.

two populations. Using the standard migra- DISCUSSION

tion estimator for mitochondrial data (Slatkin1994), migration between these two pop- The analysis of both morphological and ge­ulations is calculated as approximately one netic data indicates that populations of E.migrant exchanged between the populations seolopes on O'ahu are extremely subdivided,only every two to three generations, with a likely exchanging very few, if any, migrants,generation time of 3 to 4 months. This effec- each generation. The extent to which othertive rate of migration is very low, yet it is still populations of E. seolopes are so subdivided in

_. --sufficient-to-prevent-complete-divergence-be~---Hawai!.i-is-clearly-important-and-weH-worth---­tween the populations by genetic drift alone. investigating. Kane'ohe Bay is surrounded byIt should be noted that this estimate of gene the only true barrier reef in the Hawaiian Is-flow should be considered an upper estimate- lands (Kay and Palumbi 1987), and it may bethe two populations may also show some ge- that the well-studied Kane'ohe Bay popu­netic similarity, if they share a recent history lation is an anomaly. Yet, water does flow(see Bohonak 1999, Bohonak and Roderick actively in and out of the bay.2001). Subdivision of the E. seolopes populations

352 PACIFIC SCIENCE· July 2002

Kaneohe Bay

o NiuValley

FIGURE 3. Mitochondrial haplotype network and local­ities as estimated by the method described by Templeton(1998). Haplotype identities are noted within circles witha letter, and distances between haplotypes are propor­tional to the number of base pair differences (one or twosteps) between haplotypes. The size of the circles is pro­portional to the number of individuals (n = 18) sharing aparticular haplotype.

has implications beyond the species. If thelevel of subdivision observed here is foundelsewhere, one can also ask, at what level ofdivergence does one see a concomitant changein the symbiont Vibrio fiseheri? At the mecha­nistic level, under these very refined con-

ditions, one may be able to pinpoint thosetraits (likely to be cell surface determinantson the host or symbiont) that change as thepartners coevolve and become, as a unit, ge­netically distinct from their congeneric squid­bacteria counterparts. For example, symbiontsofJapanese squid E. morsei infect Hawaiian E.seolopes easily by themselves, but quickly losein competition with the native Hawaiianstrain (Nishiguchi et al. 1998). This compet­itive difference was easily determined becauseE. morsei strains of V fiseheri are brightly lu­minous in culture, whereas E. seolopes strainsare dim in culture. Thus, when symbioticorgans of such competition experiments areplated on agar, the ratio of the two strains iseasily determined. Unfortunately, all isolatedstrains of V fiseheri from Hawai'i are dim inculture and, therefore, to compete the sym­bionts of Hawaiian populations of E. seolopeswould require genetic engineering of Vfiseheri strains to introduce distinguishinggenetic markers.

The extent of population structure is alsoimportant in determining whether host isola­tion occurs before symbiont isolation or viceversa, or whether host and symbiont trackeach other closely. The numbers of V fiseheri

TABLE 1

Within- and Between-Population Comparisons of Mitochondrial COl DNA Sequences

Locality

Kane'oheBay (n = 10)

Niu Valley(n = 8)

Haplotype Data Frequency Frequency 101

20.436.027.316.2

20.735.827.416.1

A 0.700 0.000 GB 0.100 0.125C 0.100 0.000D 0.100 0.000

----E----------o~OOO---O;iSO------A

F 0.000 0.125Gene diversity 0.53 ± 0.18 0.46 ± 0.20Mean no. pairwise differences 1.32 ± 0.89 0.93 ± 0.71Nucleotide composition (%)CTAG

a Differences in base positions are shown relative to a reference haplotype (A), with identities shown by".".

Two Genetically Distinct Populations of Bobtail Squid on O'abu . Kimbell et al. 353

ACKNOWLEDGMENTS

TABLE 2

We thank two anonymous reviewers forhelpful and insightful comments.

Note: Statistics are presented that are based solely on the fre­quency of alleles (IJ) and that also consider the genetic distanceamong alleles (<1».

Wallace's line? Nature (Lond.) 406:692­693.

Begun, D.]., and C. F. Aquadro. 1992. Levelsof naturally occurring DNA polymorphismcorrelate with recombination rates in D.melanogaster. Nature (Lond.) 356:519-520.

Bohonak, A. J. 1999. Dispersal, gene flow,and population structure. Q. Rev. BioI.74:21-45.

Bohonak, A. ]., and G. K Roderick. 2001.Dispersal of invertebrates among tempo­rary ponds: Are genetic estimates accurate?1sr. J. Zoo1. (in press).

Brierley, A. S., P. G. Rodhouse,J. P. Thorpe,and M. R. Clarke. 1993. Genetic evidenceof population heterogeneity and crypticspeciation in the ommastrephid squid Mar­tialia hyadesi from the Patagonian Shelfand Antarctic Polar Frontal Zone. Mar.BioI. (Ber1.) 116:593-602.

Brierley, A. S., J. P. Thorpe, G. J. Pierce, M.R. Clarke, and P. R. Boyle. 1995. Geneticvariation in the neritic squid Laligo fobesi(Myopsida: Loliginidae) in the northeastAtlantic Ocean. Mar. BioI. (Ber1.) 122:79­86.

Davies, N., F. X. Villablanca, and G. K Ro­derick. 1999. Bioinvasions of the medfly,Ceratitis eapitata: Source estimation usingDNA sequences at multiple intron loci.Genetics 153:351-360.

Excoffier, L., P. Smouse, and J. Quattro.1992. Analysis of molecular variance in­ferred from metric distances among DNAhaplotypes: Application to human mito­chondrial DNA restriction data. Genetics131:479-491.

Gillespie, R. G., F. G. Howarth, and G. KRoderick. 2001. Adaptive radiation. Pages25-44 in S. A. Levin, ed. Encyclopedia ofbiodiversity. Academic Press, New York.

Hebert, P. D. N., and M. J. Beaton. 1989.MethOdOlogies for allozyme analysis usingcellulose acetate electrophoresis. HelenaLaboratories, Beaumont, Texas.

Hudson, R. R. 1994. How can the low levelsof DNA sequence variation in regions ofthe Drosophila genome with low recom­bination rates be explained. Proc. Natl.Acad. Sci. U.S.A. 91:6815-6818.

1zuka, T., S. Segawa, T. Okutani, and K-I.

p

<0.00001<0.00001

F

.492

.604

F Statistics and Exact Tests for PopulationDifferentiation

Frequency (0)Distance (<1»

Method

in the water are strongly dependent on thedensity of host squid in the environment (Leeand Ruby 1994). Thus, at the mouth of Ka­ne'ohe Bay, where there are few hosts, thereare very few V. fiseheri. There may also be littlegenetic exchange between symbiont popula­tions.

In sum, populations of E. sealapes on twosides of O'ahu are more distinct than pre­viously realized, suggesting that gene flowmay be substantially limited in some marinespecies in Hawai'i. This result likely reflectsthe lack of long-lived pelagic larvae. Further,the Hawai'i populations ofE. sealopes and theirsymbionts offer some interesting opportunitiesto gain insight into the coevolution of bacte­ria and their animal hosts. Evidence that thehost populations can be extremely subdividedis an intriguing start.

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