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Fisheries Research 84 (2007) 87–95

Variations of the mitochondrial control region sequence in whalesharks (Rhincodon typus) from the Gulf of California, Mexico

Denı Ramırez-Macıas a,b,!, Ricardo Vazquez-Juarez b,Felipe Galvan-Magana a, Adrian Munguıa-Vega b

a Department of Fisheries, Centro Interdisciplinario de Ciencias Marinas, Av. Instituto Politecnico Nacional,s/n Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23096, Mexico

b Laboratory of Biotechnology of Marine Organisms, Centro de Investigaciones Biologicas del Noroeste, Mar Bermejo 195,Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico

Abstract

A highly variable fragment of the mtDNA control region of the whale shark was sequenced to investigate genetic population structure at threelocalities in the Gulf of California. We found high levels of variation with 14 haplotypes among 36 individuals (h = 0.90, ! = 0.005). AMOVAanalysis did not detect significant structuring among Gulf of California whale sharks (P > 0.12, "ST 0.029), which indicates a single, highly mobilepopulation. Genetic analysis, along with field observations, suggests natal philopatry of female whale sharks in the Gulf of California.© 2007 Published by Elsevier B.V.

Keywords: Whale shark; Control region; Genetic structure; Gulf of California

1. Introduction

The whale shark (Rhincodon typus) is epipelagic with cir-cumtropical distribution in tropical and warm temperate seas(Compagno, 1984; Colman, 1997a,b; Gunn et al., 1999; Eckertand Stewart, 2001). In the Indo-Pacific Ocean, there is little com-mercial fishing of the whale shark (Compagno, 1984; Colman,1997a,b; Norman, 2004), while their attraction for tourism hasgrown worldwide (Clark and Nelson, 1997; Colman, 1997a,b;Norman, 2004).

The whale shark has a K-selected life history that makes itvulnerable to exploitation (CITES, 2002), such as large size,slow growth, late maturation, extended longevity, long intervalbetween pregnancies, and probably small populations (Colman,1997a,b; CITES, 2002). It is a highly migratory organism (Eckertand Stewart, 2001; Norman, 2004), and sustainable use ofthis threatened species depends on international collaboration(Norman, 2004). Our knowledge of its biology, ecology, andbehavior, is limited (Wen-Been et al., 1997; Colman, 1997a,b;Eckert and Stewart, 2001) and hampers our ability to implementappropriate protection for conservation (Norman, 2004).

! Corresponding author. Tel.: +52 612 123 8484; fax: +52 612 125 3625.E-mail address: tiburonballena@gmail.com (D. Ramırez-Macıas).

In 2000, the whale shark was listed as vulnerable in theWorld Conservation Union (IUCN) red list (Norman, 2000)and included on the red list in Mexico in 2001 (DOF, 2002)because of declining populations (IUCN, 2002). Thus, it is nec-essary to obtain population data to determine the vulnerabilityof populations.

The whale shark is known to aggregate seasonally in threeareas in the Gulf of California, Mexico: Bahıa de Los Angeles,Bahıa de La Paz, and Banco Gorda, but little is known aboutwhale shark populations. Eckert and Stewart (2001) conducteda telemetry study to document movements of whale sharks in theGulf of California and the northern Pacific Ocean, suggestingthat there were no inter-regional or inter-oceanic genetic differ-ences among whale shark populations. Therefore, it is necessaryto investigate genetic relationships among whale shark popula-tions in the Gulf of California and define population structure.This knowledge has implications for global conservation of thespecies (Eckert and Stewart, 2001; Norman, 2004).

Aggregations of whale sharks in the Gulf of Californiarepresent an opportunity to study aspects of whale shark biol-ogy, providing more detailed history and population structureusing molecular techniques. Quantification of inter- and intra-specific sequence variations within the mitochondrial (mtDNA)genome is a powerful tool for examining questions of populationgenetic structure, gene flow, and migratory movements within

0165-7836/$ – see front matter © 2007 Published by Elsevier B.V.doi:10.1016/j.fishres.2006.11.038

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and among different populations of fish and sharks (Heist et al.,1996; Rosel and Block, 1996; Haig, 1998). It is important toselect a locus or loci with a relative high mutation rate to detectsufficient polymorphism for population level studies (Heist etal., 1996). Various studies have shown that the rate of mutationis greater in mtDNA than in coding regions of nuclear DNA(Heist et al., 1996; Parker et al., 1998). The mtDNA genomealso includes a small non-coding region known as the controlregion or displacement loop (D-loop), which serves as the originof replication for the mitochondrial genome and is usually morevariable than other coding genes (Parker et al., 1998; Avise,1994). As maternal inheritance and haploid condition, mito-chondrial genes have an effective size one-fourth that of nucleargenes, Nf = 1/4Ne (Randi, 2000), and therefore, mtDNA vari-ability is sensitive to random drift in small populations and anideal marker for assessing genetic structure of recently divergedor closely related populations or species (Avise, 1994; Randi,2000).

Sequencing of the mitochondrial control region has been sug-gested as a useful tool to evaluate genetic structure in sharks(Heist et al., 1996; Keeney et al., 2003). Pardini et al. (2001) ana-lyzed the control region in white shark Carcharodon carcharias,which revealed significant differences between Australian/NewZealand and South African populations. Keeney et al. (2003)analyzed the entire control region sequence of black tip sharkCarcharhinus limbatus and detected significant partitioning ofhaplotypes between Gulf and Atlantic nurseries.

We developed new primer pairs for a 650 bp fragment fromthe control region of the whale shark, and analyzed the sequencevariation among juveniles (and a small number of adults) sam-pled at three separate locations in the Gulf of California. We alsocollected data on sex and total length to distinguish young andmature sharks, and photo-identified individual sharks.

2. Materials and methods

2.1. Study area

The whale shark is known to aggregate seasonally in threeareas of the Gulf of California, Mexico: Bahıa de Los Angelesoff the north central Baja California Peninsula, Bahıa de La Pazoff the southeastern coast of the peninsula, and Banco Gorda,several sea mounts off the tip of the peninsula (Fig. 1). Weobserved and obtained skin tissue from whale sharks in autumn2002–2004 in Bahıa de La Paz, in summer over Banco Gorda,and in autumn 2003 and 2004 in Bahıa de Los Angeles.

2.2. Sample collection

Skin tissue was obtained from sharks using a pole spear witha core modified from those used for taking biopsies of marinemammals. The skin tissues were preserved in ethanol (98%) andstored at "20 #C. Each individual was identified with photos ofthe pattern of lateral markings behind the five gills slits on theleft side (Taylor, 1994; Iniguez and Enrıquez, 2004; Norman,2004), and scars and other markings (Taylor, 1994; Clark andNelson, 1997). Sex was determined by the presence or absence of

Fig. 1. Whale shark collecting areas near the Baja California Peninsula in theGulf of California (Bahıa de Los Angeles = BLA, Bahıa de La Paz = BLP, andBanco Gorda = BG).

claspers. Total length was measured while swimming and usinga metric band or was estimated from repeated observations of thesharks next to the 5–7 m tracking boat (Ketchum, unpublished).Total length was used as a proxy of sexual maturity based on thecriteria that adult whale shark reach sexual maturity at a lengthof about 9 m (Taylor, 1994; Colman, 1997a,b).

2.3. DNA extraction, PCR amplification, and sequencing

Total genomic DNA was isolated from approximately20–50 mg of skin tissue that was minced and digested overnightat 60 #C in 500 !l of extraction buffer [10 mM Tris at pH 8.0,2 mM EDTA, 10 mM NaCl, 1% sodium dodecyl sulfate (SDS),8 mg ml"1 dithiothreitol (DTT), and 0.2 mg ml"1 proteinase K](Rosel and Block, 1996). Samples were then extracted twice withphenol and once with chloroform (Hoelzel, 2001), followed byethanol precipitation.

In a preliminary analysis, PCR was used to selectivelyamplify the entire mitochondrial control region of two whalesharks using primers located at the 3$ end of cytochromeB (primer Cytb-1040 5$-GGNCAACCMGTYGAACAACC-3$)and 5$ end of the ARNt Phe gene (primer 12 rev 326 5$-ACTCGTATAACGCGGTGGCT-3$) (Sandoval-Castillo et al.,2004). PCR amplification was conducted under the followingassay conditions: 3 mM MgCl2, 200 !M each dNTP, 50–100 ngDNA, 2 !M of each primer, and 0.5 U taq DNA polymerase insufficient 1% taq buffer (50 mM KCl, 10 mM Tris–HCl, 0.1%Triton X-100 at pH 9.0) to a total volume of 25 !l. Amplifica-

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tion was performed in a PE 9700 thermocycler, consisting ofan initial denaturation for 1 min at 94 #C, followed by 1 min at60 #C and 1 min at 72 #C for 35 cycles, and a final extensiontime of 10 min at 72 #C. We obtained an unspecific productsof 1220 and 1228 bp, and then extracted and purified the PCRproduct in agarose gel in spin columns (Qiagen PCR purifica-tion columns). PCR products were cloned in a PCR 2.1 cloningvector and transferred to E. coli competent cells (Invitrogen).We isolated plasmid DNA from positive colonies by standardmini-prep procedures (Sambrock et al., 1989). Insert DNA wassequenced in both directions using M13 primers on an ABI377automatic sequencer.

Upon examination of the variability across the entire con-trol region of the two individuals, we designed a new set ofprimers that encompassed the region of highest variability. Theresult was a shorter fragment (&650 bp) from the first half ofthe control region. Primers were designed by visual inspec-tion and then proven with PrimerSelect (DNAStar) software.PCR was used to amplify 37 whale sharks using novel primersTB31F-5$-TGCATGGTTTTATGTACGTC-3$ and RC-TB3R5$-GGCAGGTGTCGGAGCTT-3$. This product approximatelycorresponds to positions 15674–16403 from the control regionof the dog shark (Heterodontus francisci) (GenBank acces-sion no. AJ310141). PCR conditions were as above, except for1.5 mM of MgCl2. Amplification consisted of an initial denat-uration for 45 s at 94 #C, followed by 45 s at 61 #C and 45 s at72 #C for 35 cycles, and a final extension time of 10 min at 72 #C.PCR products were purified and sequenced as described, usingboth TB31F and RC-TB3R primers.

2.4. Analysis of the mitochondrial DNA control region

The sequences of the control region were edited with the com-puter programs Chromas and seqMan (DNAStar), Alignmentswere made by Clustal method, as implemented in MegAlign(DNAStar) software, and finally adjusted by visual inspection.

Summary statistics like haplotype frequencies, number ofpolymorphic sites, number of transitions, and transversions andnucleotide composition were calculated with the Arlequin 2.0program (Scheneider et al., 2000). Haplotype diversity, the prob-ability that two randomly selected homologous nucleotides inthe same sample are different, and the nucleotide sequence diver-sity were determined with the Arlequin 2.0 according to themethod of Nei (1987) (Scheneider et al., 2000).

We examined correlations between genetic distances andgeographic locations in the Gulf of California, using analysisof molecular variance (AMOVA) (Excoffier et al., 1992). InAMOVA analysis, individuals are assigned to populations andpopulations are nested into groups based on a non-genetic crite-rion, in this case, sampling location. We designated one group:Gulf of California with three populations mentioned above. TheAMOVA calculated genetic variance components and the hier-archical F-statistics analog ("ST), and tested the significanceof the observed variance using the permutation method. Anal-yses were determined with Arlequin 2.0 by comparing valuesof a null distribution of 1000 samples generated by permutatinghaplotypes between populations.

To estimate divergence among haplotypes, Tamura–Nei dis-tances were calculated with PAUP* (Swofford, 1998) and aneighbor-joining tree was constructed from divergence estimatesassuming different gamma values (0.5, 1). Maximum parsi-mony trees were generated, involving a heuristic search withstepwise addition and the tree-bisection-reconnection (TBR)setting. A maximum likelihood analysis with a heuristic search,as described was also conducted. On the neighbor-joining treeand maximum likelihood analysis, gaps were excluded fromthe analysis. Robustness of tree topology was tested with abootstrap analysis (1000 replicates). A control region sequencederived from H. francisci (GenBank accession no. AJ310141)was aligned by visual inspection and used as an out group.A median-joining network of all unique haplotypes was alsoconstructed using Netwok 4.1 (Bandelt et al., 1999).

3. Results

We obtained tissue samples from live juveniles (<9 m) in2002–2004 in the south end of Bahıa de La Paz (13 specimens:5 females, 6 males, 2 undetermined, sex ratio was 1 male to0.83 female) using a pole spear. From Bahıa de Los Angeles,we obtained tissue samples from live juveniles in 2003–2004(23 specimens: 9 females, 12 males, and 2 undetermined, sexratio was 1 male to 0.75 female). In 2002, samples were col-lected from a beached female at Bahıa de Los Angeles and in2001, a beached female from Punta Arenas (close to Bahıa deLa Paz). Of 36 samples of juveniles (15 females: 2 landed, 13from the two bays; 17 males from the 2 bays; and 4 undeter-mined specimens, 2 from each bay). The overall sex ratio was 1male to 0.88 female. In 2003 at the Banco Gorda, we found fivemature females (>9 m) that appeared to be pregnant because thebelly was distended. Comparison of pictures in the identificationcatalog showed four individuals were present in Bahıa de LosAngeles in 2003 and 2004. One whale shark sighted in Bahıade Los Angeles in 2004 was sighted in Bahıa de La Paz about1 month later (Fig. 2). Another whale shark sighted in Bahıade Los Angeles in 2003 was seen in Bahıa de La Paz in 2004(Fig. 3). Biopsies of re-sighted whale sharks were excluded afterthe analyses. Final sample size was 41 individuals (36 juveniles,5 adult females).

3.1. Sequence analysis

PCR products from the mitochondrial control region of36 individuals were obtained with the new whale shark spe-cific primers. These ranged from 600 to 619 bp (excludingprimers). The nucleotide composition of this fragment consistedof 18.55% cytosine, 41.42% thymine, 32.56%, adenine, and7.47% guanine. Altogether, 14 haplotypes were found in theGulf of California. Sequence variations were distributed among12 polymorphic sites; only one was a transversion, consistentwith the bias in favor of transitions characteristic of the controlregion in other animal species (Brown et al., 1982; Kocher et al.,1989). Also, two indels summing 33 bp (Table 1) were observed(GenBank accession numbers for the control region sequencesare DQ395261–DQ395274).

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Fig. 2. A whale shark, first sighted in Bahıa de Los Angeles in 2004, was sighted in Bahıa de La Paz 1 month later. This sighting demonstrates long migratorymovements (&850 km) in a short time.

Fig. 3. A whale shark, first sighted in Bahıa de Los Angeles in 2003, was sighted in Bahıa de La Paz in 2004.

Overall haplotype diversity and nucleotide sequence diver-sity for the three study areas were 0.90 and 0.005, respectively.Sample sizes, the number of haplotypes, haplotype diversity,and nucleotide diversity for each study area are listed in Table 2.The most common haplotype occurred in the three sampleareas (RC6). Five haplotypes occurred frequently in Bahıa deLos Angeles and Bahıa de La Paz, the highest frequency wasRC24 and RC13. Among the twelve haplotypes in Bahıa de LosAngeles specimens, there were six unique haplotypes. Among

the seven haplotypes in samples from Bahıa de La Paz, onehaplotype was unique. Among the four adult females at BancoGorda, one haplotype (RC7) was unique (Fig. 4).

In the trees that were generated, the 14 haplotypes appeared tobe consistently distributed among three major clades that showedmoderately significant bootstrap support. Only the neighbor-joining tree, based on Tamura–Nei distances, is shown (Fig. 5).Each clade represents different haplotype lineages, referred to asA–C (Fig. 5). Clade A consists of TB4, RC6, RC15, and RC21,

Table 1Variable nucleotide positions defining the mtDNA control region haplotype from each of 36 sampled whale sharks sampled.Total number of individuals of each haplotype (N). Sequence identity to reference sequence in top row (rc11)

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Table 2Statistics on sample of 36 whale sharks. BLA = Bahıa de Los Angeles,BLP = Bahıa de La Paz, BG = Banco Gorda, No. hap. = number of haplotypesfound, ! = nucleotide sequence diversity, and h = haplotype diversity for eacharea

Locality N No. hap. ! h

BLA 19 12 0.02 0.953BLP 13 7 0.02 0.897BG 4 2 0.01 0.5

representing the basal and oldest lineage, which is followed byclades B and C. On the maximum likelihood and maximumparsimony trees, clades B and C appeared switched, contrast-ing with the neighbor-joining tree (data not shown). Networkanalysis also grouped haplotypes among three different clusterscomposed of the same individual haplotypes present as cladesA–C in the phylogenetic analysis (Fig. 6). Cluster A on networkincluded the most common and widespread haplotype RC6 andthree derived haplotypes (RC15, RC21, and TB4), which dif-fer by a single nucleotide substitution. Cluster B starts withhaplotypes RC7 and TB3, differing by three substitutions, andfollowed by three haplotypes (RC24, RC16, and RC41) thatshare a common indel of 10 bp. Cluster C begins with haplotypeRC33 and RC11, and the network reticulates towards RC28,

Fig. 4. Haplotype frequency at the three localities. RC6 was the most widespreadhaplotype found in the three sampling areas. RC6 was also the most frequenthaplotype over all. Five haplotypes were common to Bahıa de Los Angeles(BLA) and Bahıa de La Paz (BLP). Among BLA specimens, 12 haplotypeswere found, of which 6 were exclusive to that area. Among BLP specimens,seven haplotypes were found and only one was unique to that area. Amongadult female specimens sampled at Banco Gorda (BG), only the RC7 haplotypewas unique to this area, and the common RC6 haplotype was also found.

Fig. 5. Phylogenetic tree of unique whale shark control region sequences usingthe neighbor-joining algorithm with gamma distances, based on the Tamura–Neimodel and a sequence from Heterodontus francisci as the outgroup. Bootstrapvalues (based on 1000 iterations) greater than 50% are shown. Haplotypes aregrouped among three distinct clades or lineages, referred to as A–C, respectively.

Table 3Analysis of variance of pairwise genetic distances among Gulf of Californiawhale shark in mtDNA control region sequences

Source of variation Variance % total P "ST

Among populations 0.01361 2.96 0.144 0.029Within populations 0.44587 97.04

RC20, and RC13, which share a common 17-bp indel (Table 1,Fig. 6). Network analysis did not reveal any obvious relation-ship between mitochondrial lineages and geographic locations.For example, clusters A and B included haplotypes from thethree study areas. Network analysis also suggested three miss-ing, intermediate haplotypes (Fig. 6). Analysis of molecularvariance (Table 3) showed most of the mtDNA control regionvariation (97%) was distributed within the three localities, andonly 3% among locations. This implies a lack of genetic structurebetween the three study areas (P > 0.12, "ST 0.029).

4. Discussion

The control region has proven useful for population studies ofthe whale shark. The specific primers that we designed encom-pass the highly polymorphic fragment of the control region,permitting identification of 14 haplotypes from 36 individu-als. The study demonstrated genetic homogeneity among whalesharks in the Gulf of California.

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Fig. 6. Network analyses of unique mtDNA haplotypes for 36 whale sharks from the Gulf of California. Circle area is proximately proportional to the number ofsequences found with that haplotype. Transitions are denoted by bars, transversions by crosses. Bahıa de Los Angeles (BLA) is shown in white, Bahıa de La Paz(BLP) is shown with crosses, and Banco Gorda (BG) is shown with lines.

Using genetic analysis supported by hierarchical AMOVA(Excoffier et al., 1992) to analyze population genetic struc-ture, we determined that whale sharks in the Gulf of Californiaare a single population. This analysis included calculation ofstandard variance components and a series of haplotype corre-lation measures, referred to as "ST, which were used to inferthe degree of population subdivision. Analysis of the whaleshark mtDNA control region haplotypes revealed no significantdegree of genetic differentiation among animals in the three sam-ple areas (Table 3); hence a single genetic stock. These resultsare preliminary, since we have only a few samples from BancoGorda. Still, finding the most common haplotype in three of thefour whale sharks supports this (Fig. 4). The other female atBanco Gorda showed a unique haplotype (RC7), closely relatedto haplotypes in clade B (Fig. 5).

Eckert and Stewart (2001) suggested that there are no inter-regional or inter-oceanic genetic differences among populationsof the world’s whale sharks. Perhaps there is a meta-populationstructure which divides them into subpopulations, with geneticflow carried by mature males. Samples from other parts of theworld, particularly from localities in the Eastern Pacific, areneeded to prove this hypothesis.

At the three sampling sites, the most common haplotype,RC6, occurred in eight individuals (22.2%), including threeout of four pregnant females. According to coalescent theory(Posada and Crandall, 2001), the common haplotype most likelyrepresents the oldest mitochondrial lineage. The basal positionof RC6 at ancestral clade A in the neighbor-joining tree (Fig. 5)and the star shape of the network that radiates from cluster A(Fig. 6) strongly support this view.

Overall haplotypic diversity was high (h = 0.90), mainlyfrom the presence of many haplotypes, of which seven (50%)were rare and represented by single individuals. Of the threelocations, Bahıa de Los Angeles had the highest haplotypicdiversity from 12 haplotypes, six exclusive to this location.These values are considered tentative since haplotype diversityat the three localities could be biased by the relatively smallsample size (Table 2). Certainly, a larger sampling effort isneeded to find a more reliable estimate of haplotype frequen-cies. Network analysis (Fig. 6) suggests at least three haplotypes(or missing intermediates) were not sampled. This may be aconsequence of insufficient sampling or extinction of the hap-lotypes. The sampling bias could also explain, in part, highhaplotypic diversity (h = 0.90) found in whale sharks from theGulf of California (Table 2), compared to the entire controlregion (h = 0.371–0.796) of blacktip sharks C. limbatus alongthe United States Atlantic coast and Gulf of Mexico (Keeneyet al., 2003), and RFLP’s analysis (h = 0.507–0.909) of shortfinmako Isurus oxyrinchus from the Atlantic and Pacific Oceans(Heist et al., 1996).

Overall, nucleotide diversity was low (0.005), reflecting lowdivergence between haplotypes, as suggested by the short branchlengths on the neighbor-joining tree (Fig. 5). Analysis suggestedhaplotypes belong to three major lineages, and tree topology alsoindicated haplotypes could have arisen from a single commonancestor. Network analysis showed that most haplotypes couldhave arisen by single events of nucleotide substitution, insertion,or deletion (Fig. 6).

Overall, low sequence diversity (! = 0.005) among a rela-tively small number of polymorphic sites and the star shape of

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the phylogeny and network analyses are consistent with a popu-lation expansion after a bottleneck (Slatking and Hudson, 1991;Aris-Brosou and Excoffier, 1996; Tajima, 1989), which prob-ably occurred hundreds or thousands of years ago. This couldimply that the whale shark population in the Gulf of Californiawas founded by a relatively small population involving haplo-type RC6 that expanded rapidly (Figs. 5 and 6). In this case,most polymorphic sites could have emerged during a demo-graphic expansion that probably reached moderate populationsizes, which might explain the presence of 14 haplotypes (14)and three lineages. Also, the low levels of sequence divergenceobserved among individuals of other cosmopolitan species(such as humpback whales) can be explained by their greatermobility and genetic exchange among populations (Barker etal., 1993), thus supporting the highly migratory nature of thewhale shark (Eckert and Stewart, 2001; CITES, 2002; Norman,2004).

Our results demonstrate the utility of molecular studies, inconjunction with other observations of sex, length, and photo-identification of whale sharks. Without photo-identification, ourresults would be biased if we had analyzed samples more thanonce. Our observations of four recaptures from Bahıa de LosAngeles in 2003 and 2004 show seasonal residence in the areaand confirmed the conclusion of Eckert and Stewart (2001) thatwhale sharks are local residents. Taylor (1994) used photo-identification to demonstrate seasonal residence in WesternAustralia. Norman (2004) mentioned that many sharks weresighted repeatedly during a ‘season’ and between ‘seasons’.Also, two whale sharks that had been sampled Bahıa de LosAngeles and again in Bahıa de La Paz is direct evidence ofhigh mobility and local migration patterns within the Gulf ofCalifornia, and also support the conclusion that a single popu-lation of whale sharks inhabits the Gulf of California, as wassuggested by AMOVA analysis. One juvenile female migratedapproximately 850 km from Bahıa de Los Angeles (sighted20 October 2004), to Bahıa de La Paz (sighted 21 November2004). This record is consistent with others records of rapid andwidespread migration (Norman, 2004; CITES, 2002). Eckertand Stewart (2001) reported the longest known migration usingtagging and satellite tracking when a whale shark from Bahıade Los Angeles migrated 12,620 km. However, in that investiga-tion, none of the whale sharks tagged in Bahıa de Los Angelesmigrated to Bahıa de La Paz. The two recaptures does show thatwhale sharks move between these two bays. Migratory behav-ior must be taken into account for regional and internationalconservation.

Our data also confirm the observations of Eckert and Stewart(2001) that whale sharks in the Gulf of California segregatethemselves by size. At Banco Gorda, the whale sharks were adultfemales (>9 m) and appeared pregnant, whereas in the northernand central gulf, we found only juveniles (<9 m). Eckert andStewart (2001) also suggested segregation by sex; however, thiswas more evident at Banco Gorda.

Ketchum (unpublished) also found segregation of whalesharks at Bahıa de La Paz: juveniles were found near the coast(juvenile sex ratio of 1 male:0.67 female), along with veryyoung sharks (<2 m). Adults (mostly gravid females) were found

far offshore in Bahıa de La Paz (adult sex ratio of 1 male:5females). Segregation of juveniles and adults appeared to bestrongly related to environmental conditions and prey prefer-ences of the two age groups (Ketchum, unpublished). Theseobservations are consistent with our results. Banco Gorda is faroffshore. Juveniles occupied areas close to the coast. Addition-ally, Ketchum (unpublished) suggested that whale sharks use thedeep waters of Bahıa de La Paz as their primary nursery areaand the shallow coastal waters of the southern part of the gulfas a secondary nursery area. Our observations of whale sharksin near-shore locations included a small juvenile (&2 m) closeto the El Mogote sand bar in the most southern part of Bahıa deLa Paz and several juveniles (>3.5 m) in Bahıa de Los Angeles.The importance of near-shore areas as nurseries needs to beconsidered for conservation of whale sharks.

Segregation by size in the Gulf of California and theabsence of adult males agrees with other observations. Norman(unpublished) mentioned that a seasonal and area populationsegregation occurs because aggregations of whale sharks are ofsimilar size and largely the same sex in a specific area. In otherareas, aggregations are of other age classes and dominated bythe other sex (CITES, 2002). These observations suggest thatage and sex groups have different migration patterns. The factthat adult males were conspicuously absent, raise the question ofthe location of adult males. Pardini et al. (2001) suggested thatmale white sharks migrate across ocean basins, while femalestend to remain in continental coastal waters. Keeney et al. (2003)demonstrated female philopatry in blacktip sharks. The presenceof the most common haplotype (RC6) in three of the five preg-nant females sampled suggest at least some females show fidelityto the area. This observation agrees with philopatry observed infemales of other shark species where adult males migrate toreproduce in other areas. However, these results should be con-sidered preliminary for several reasons. First, our data coveredonly three sampling locations with samples limited particularlyto adult individuals. That males migrate to other areas of theGulf of California is a strong possibility. Also, more samplingof adult females could confirm how the commonality of femalenatal philopatry. DNA analysis combined with telemetry tech-niques will give more information about the movements of thewhale shark in the Gulf of California.

Our observations lead to the following null hypotheses:

1. Whale sharks in the Gulf of California are born at far off-shore localities, such as Banco Gorda and Bahıa de La Paz.When they reach about 2 m, they migrate to protected shal-low coastal areas along the south shores of Bahıa de La Paz(El Mogote and Punta Prieta). They remain in the Gulf ofCalifornia and aggregate seasonally in coastal areas of highproductivity, such as Bahıa de Los Angeles and Bahıa de LaPaz.

2. When males reach adulthood, they migrate to other areas,where reproduction takes place.

3. Adult females remain close to the Gulf of California (orreturn) and use far offshore areas adjacent to Bahıa de LaPaz and Banco Gorda as primary nurseries, while coastalwaters of Bahıa de La Paz are used as secondary nurseries.

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Data from other parts of the world and analysis of nucleargenes must be obtained before these hypotheses can be fullyaccepted.

5. Conclusions

This study demonstrated high variability in the mitochondrialcontrol region among whale sharks in the Gulf of Califor-nia, but they comprise a single population, probably recentlyexpanded. Integration of genetic and photo-identification dataconfirms that juveniles are resident to the Gulf of California,and probably natal philopatry of female whale shark is preva-lent in the Gulf of California. Where adult males migrate to breedis the most interesting unanswered question. Given the highlymigratory nature of whale sharks, regional and international col-laborations are crucial to fill important gaps on whale sharkbiology, particularly migration patterns. Such information willassist in sustainable use, management, and conservation of thespecies.

Acknowledgements

Field work was provided by James Ketchum, MauricioHoyos, Abraham Vazquez, Ana Hacohen, Roberto Enriquez,Lucina Iniguez, Felipe Morales, Nezahualpilli Tovar, MarioSalinas, Francisco J. Vergara, Yoel Goldchein, Florencia Cerutti,Flavio Zepeda, Ciro Calderon, Gabriel Aguirre, Romeo Saldıvar,Lilia Gama, Liza Gomez, Mario Jaime, Ciro Arista, Calvillo,Nicolas Quinonez, Agnes Rocha, Rodrigo Rocha. Collection ofsamples of beached whale shark tissues was provided by JamesKetchum, Antonio Resendiz, and Jonathan Nelson. Hever Latis-nere, Dariel Tovar, and Neftalı Gutierrez helped with criticalsteps in the laboratory. Francisco Garcıa de Leon and ClarissaGalindo provided insightful observations about genetic analy-sis. Editing and related aspects of the English text were providedby the editor at CIBNOR. CONACYT and the Instituto Politec-nico Nacional provided PIFI scholarships to D.R.M. COFAAprovided a fellowship. Funding was provided by SEMARNAT-CONACYT (project no. FOSEMARNAT-2004-01-457).

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