Diversity and distribution of mitochondrial DNA

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Diversity and distribution of mitochondrial DNA lineages among humpback whales, Megaptera novaeangliae, in the Mexican Pacific Ocean Luis Medrano-Gonzalez, Anelio Aguayo-Lobo, Jorge UrbBn-Ramirez, and Charles Scott Baker

Abstract: We investigated the mitochondrial DNA (mtDNA) diversity of humpback whales, Megaptera novaeangliae, wintering off the Mexican Pacific coast and the Revillagigedo Islands. We amplified and sequenced a variable fragment of the mtDNA control region from skin samples of 65 whales and compared these with published sequences from whales in other regional habitats. Among the Mexican humpback whales, we distinguished eight haplotypes differing by 0.31 -3.75% along a consensus sequence length of 320 base pairs. A diagnostic restriction site outside the consensus sequence identified a ninth common haplotype. A phylogenetic reconstruction of the control region sequences revealed two main groupings: an AE group, which is common throughout the North Pacific, and a CF group, which is closely related to haplotypes from the southern hemisphere. We found a significant degree of geographic subdivision in the wintering grounds of the eastern North Pacific. Within Mexico, whales off the Revillagigedo Islands are weakly but significantly differentiated from those of the Mexican Pacific coast. Our data also suggest that mtDNA haplotypes are clinally distributed along the American Pacific coast and we hypothesize that the present distribution of these lineages among humpback whales in the eastern North Pacific is probably associated with weather changes after the last glaciation. RCsumC : Nous avons dtudid la diversitk gdndtique de I'ADN mitochondrial (ADNmt) chez le Rorqual a bosse, Megaptera novaeangliae, qui passe l'hiver prks de la c6te mdxicaine du Pacifique et des iles Revillagigedo. Nous avons procddd a l'amplification et au sdquen~aged'un fragment variable de la rdgion contrdle de 1'ADNmt provenant de biopsies de la peau de 65 rorquals et avons compard nos rdsultats des skquences connues obtenues en d'autres habitats rkgionaux. Chez les rorquals mkxicains, nous avons distingud huit haplotypes accusant des diffdrences de 0.31 -3.75% le long d'une sdquence consensus comportant 320 paires de base. Un sikge de restriction distinctif situd en dehors de la sdquence a donnd lieu a un neuvikme haplotype rkpandu. Une reconstruction phylogdndtique des sdquences de la rdgion contr6le a ddmontrd que les haplotypes foment deux groupes principaux, un groupe AE, commun dans le Pacifique Nord, et un groupe CF, trks apparent6 aux haplotypes de l'hkmisphkre austral. Nous avons trouvk une sdparation gdographique significative entre les zones d'hivernation du nord-est du Pacifique. Au Mdxique, les rorquals des iles Revillagigedo different lkgkrement mais significativement des rorquals de la c6te mCxicaine du Pacifique. Nos donnkes indiquent aussi que les haplotypes d'ADNmt suivent un gradient le long de la c6te americaine du Pacifique et nous croyons que la rdpartition des ligndes de Rorquals a bosse dans l'est du Pacifique Nord est probablement associde a des changements climatiques depuis la fin de la dernikre glaciation.

Introduction

whales, Mega~tera novaeangziae, in the North Pacific are thought to be divided into three stocks, based on Received November 15, 1994. Accepted May 16, 1995.

L. Medrano-Gonzalez and A. Aguayo-Lobo. Facultad de Ciencias, Universidad Nacional Autdnoma de Mdxico, Apartado Postal 70-572, Mdxico, DF 045 10, Mexico. J. Urban-Rarnirez. Departamento de Biologia Marina, Universidad Autdnoma de Baja California Sur, Apartado Postal 19-B, La Paz, BCS, 23081, Mexico. C.S. Baker. School of Biological Sciences, University of Auckland, Private Bag 920 19, Auckland, New Zealand. Can. J. Zool. 73: 1735- 1743 (1995). Printed in Canada 1 Imprime au Canada

their winter distribution and from observations of migratory movement by naturally marked individuals (Perry et al. 1988, 1990; Rice 1974) An Asian stock winters near Taiwan and around the Ryukyu Islands and Osagawara Islands south of Japan. This stock is presumed to summer primarily in the Sea of Okhotsk, around the Kamchatka Peninsula and the Aleutian Islands (Nishiwaki 1966). A central stock winters near the main islands of Hawaii and summers off the Alaska coast (Baker et al. 1986; Darling and McSweeney 1985). An American stock winters in Mexican and Central American Pacific waters from the Baja California peninsula to Costa Rica, including the Revillagigedo Islands, and is known to summer mainly around the coast of California (Fig. 1). Humpback whales have been sighted in the summer months

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Can. J. Zool. Vol. 73, 1995


Fig. 1. Migratory movements and seasonal distribution of humpback whales in the North Pacific. Broken lines denote sightings of whales in different reproductive regions, usually in different years.

feeding in the Sea of CortCs (Calambokidis et al. 1990; Gendron and Urban 1993; Rice 1974; Steiger et al. 1991; Urban and Aguayo 1987). It has been estimated that commercial exploitation of humpback whales in the North Pacific during the 20th century decreased the population from its original size of 15 000 - 20 000 individuals to a few hundreds in 1965, when hunting was banned by the International Whaling Commission (Rice 1974, 1978). At present, mark -recapture estimates of photoidentified animals indicate that around 1400 .2000 animals winter in Hawaii (Baker and Herman 1987; Darling and Morowitz 1986; Perry et al. 1988, 1990). Recent estimates suggest that 2200 -2800 humpback whales winter in the whole Mexican Pacific Ocean, including the Revillagigedo Islands (Urban et a1. 1994). Photoidentification (Baker et al. 1987; Darling and Cerchio 1993; Perry et al. 1988, 1990) and the distribution of mitochondrial DNA (mtDNA) haplotypes (Baker et al. 1990, 1993, 1994) have shown that humpback whales in the North Pacific consistently assort in a particular feeding region but may mix on the breeding grounds. Despite stocks intermingling in wintering areas, there is significant genetic divergence of mtDNA, a maternally inherited genetic marker, J.R. Urbin, A.L. Jaramillo, M.Z. Salinas, J . Jacobsen, K. Balcomb, P.P. Ladr6n de Guevara, and A.L. Aguayo. 1994. Population size of the humpback whale (Megaptera novaeangliae) in the Mexican Pacific. Report of the International Whaling Commission No. SC/46/np4.

between the Hawaii-Alaska and Mexico - central California stocks (Baker et al. 1993, 1994). It is thought that this geographic segregation of mtDNA haplotypes is influenced by maternally directed fidelity to migratory destinations (Baker et al. 1990). These maternal traditions are presumably learned during the first year of life, when the calf accompanies its mother on a round-trip migration between seasonal habitats (Baker et al. 1986, 1987). Other studies on mtDNA variation in humpback whales in the Atlantic Ocean also suggest this influence of philopatric behaviour on the population structure at an evolutionary scale (Palsboll et al. 1995). Here we analyze the variability in the mitochondrial control region or D-loop of humpback whales in the Revillagigedo Islands and along the Pacific coast of Mexico. The analysis of mitochondrial control region sequences has shown that at least six haplotypes are found in the North Pacific; two are common in the Hawaii-Alaska stock and six in the central California - Mexican Pacific stock, where greater genetic diversity is found (Baker et al. 1993, 1994). The Mexican Pacific wintering grounds are of particular interest because of apparent heterogeneity of habitat use by different age - sex classes (Campos 1989; Ladr6n de Guevara 1995; Medrano 1993; Medrano et al. 1994; Salas 1993) and possible segregation between coastal and offshore regions. Photoidentification studies indicate a low resighting rate between the Revillagigedo Islands and the mainland coast (Alvarez et al. 1990; Urban et al. 1994),' as well as a low rate between the whole Mexican Pacific and Hawaiian breeding grounds (Perry et al. 1990). Unlike those of whales found

Medrano-Gonzalez et al.


in Hawaii and coastal Mexico, the primary feeding grounds of Revillagigedo Islands whales have not been determined. These facts suggest that the latter could be a separate subpopulation (Alvarez et al. 1990; Perry et al. 1988, 1990; Urban and Aguayo 1987; Urban et al. 1994, see footnote I). The results of the present study extend the previous analysis of the mtDNA variation in humpback whales from Baja California, Hawaii, California, and southeastern Alaska (Baker et al. 1990, 1993, 1994) and provide further insight into the genetic structure and historical demography of the humpback whales in the North Pacific.

Procedures

Skin samples were collected from humpback whales of different social groupings by biopsy darting, using the systems described by Medrano (1993) and Weinrich et al. (1991), in the following regions (Fig. 4): 21 samples from Bahia Banderas, Nayarit-Jalisco (20°40'N, 105"301W), in winters 1990- 1992; 23 samples from the Revillagigedo Islands around Isla Socorro (18"401N, 111"00'W) in winters 19911992; 21 samples from the surroundings of Los Cabos, Baja California Sur (23 "OO'N, 109"301W), during 1991 (Baker et al. 1993, 1994); 26 samples near the main islands of Hawaii (20°30'N, 156"001W): 16 in 1989 (Baker et al. 1990) and 10 in 1992. Biopsied material was preserved in 20% dimethylsulfoxide in saturated salt solution or 70% ethanol at -75°C for up to 30 months before analysis. Photoidentification (Katona and Whitehead 1981) was conducted in combination with biopsy sampling, so that cases of duplicate sampling could be eliminated from the genetic analysis. For example, two nursing females with the rare haplotype E2 sampled in February 1992 at Bahia Banderas were confirmed to be unique individuals after the inspection of the photographs. The sex of the animals was identified by molecular analysis following the method of Palsboll et al. (1992) as described elsewhere (Medrano et a1. 1994). Total genomic DNA was extracted using standard methods (Sambrook et al. 1989) as modified by Baker et al. (199 1). Homogenized skin tissue was digested with proteinase K and DNA was extracted with phenol-chloroform, precipitated in ethanol, and resuspended in Tris-EDTA buffer. A 462 base pair (bp) fragment comprising the most variable region of the niitochondrial D-loop was amplified by the polymerase chain reaction (PCR), using the primers DlplO (biotinylated) and Dlp5 (Baker et al. 1993) in a standard composition of polymerase, buffer, and 2-deoxynucleosidetriphosphates (Palumbi et al. 1991b; Saiki 1990). Complementary mtDNA strands from the amplified double-stranded DNA were isolated by attaching 40 pL of PCR product with the biotinylated primer to streptavidin-coated magnetic beads (Hultman et al. 1989). Both DNA strands between positions 95 and 414 of the mitochondrial D-loop, in reference to the fin whale ( ~ r n a s o n 1991),2 were sequenced by the dideoxy chain termination method (Sanger et al. 1977; Sanger 1981), using the Sequenase 2.0 protocol (U.S . Biochemicals, Cleveland, Ohio). Additional analysis of D-loop variation was conducted by sequencing representative individuals of each haplotype, -

U. Arnason. 1991. Sequence composition of the D-loop in mitochondrial DNA of the fin, blue and humpback whales. Report to the International Whaling Commission No. SC/F91 /F3 1 .

using a primer designed to initiate amplification in the proline tRNA gene near the 5 ' end of the control region (Palumbi et al. 1991b). Alignment of this 550-bp region revealed a single nucleotide difference in an approximately 100-bp region of the D-loop among the common A haplotypes previously considered to be identical (Baker et al. 1990, 1993, 1994). The presence or absence of a Sau 961 site in the amplified fragment was used to assay this polymorphism in all other A-type samples. Amplified fragments were digested with 5 units of the enzyme Sau 961 following the manufacturer's recommendations (BRL Life Technologies Inc., Grand Island, N.Y.) and visualized in 3 % agarose gels following standard techniques (Sambrook et a1. 1989). Genetic divergence between whales from different regions was measured with the nucleotide diversity defined by Nei and Li (1979). The Gst statistic was used to estimate the fraction of genetic diversity due to differences between subpopulations (Nei 1973). The significance of genetic divisions was tested using the permutation procedure of Palumbi and Wilson (Palumbi et al. 1991a). In this method, each individual from the entire sample is randomly reassigned to a particular region, keeping constant the number of individuals in each region's subsample. The Gst statistic from this randomized data set is calculated and compared with the observed Gststatistic. This procedure is repeated 5000 times to generate a null distribution of the Gst statistics. This method allowed an estimate of the Type I error (a),i.e., the probability of incorrectly rejecting the null hypothesis of panmixis. For the management of endangered species, where sample sizes are often limited, it is also important to evaluate the Type I1 error (P), i.e., the probability of incorrec,tly accepting the null hypothesis. We estimated this probability following the procedure suggested by Dizon et al. (1994) .3 Briefly, the individuals included in the data set are randomly sampled with replacement and without changing their populational identity. We conducted 5000 samplings to generate an alternative Gst distribution. The Type I1 error was assigned to the fraction of the alternative distribution with Gst values lower than the observed value. The regional and sex-related heterogeneities of haplotype frequencies were tested using a randomized X 2 test of independence (Roff and Bentzen 1989). A permutation procedure with 5000 data shuffles was also used for testing the correlation between latitude and the ratio of haplotypes A and E in humpback whales of the Mexican Pacific Ocean.

Results A region of 320 bp was sequenced from the mitochondrial D-loop of 91 humpback whales from the Hawaiian and Mexican wintering grounds. Within this sequence set, we found 14 variable sites defining eight unique haplotypes referred to as A, AE, E l , E2, E3, E4, F l , and F2 following the arbitrary designations used previously (Baker et al. 1990, A.E. Dizon, G.M. O'Corry-Crowe, and B.L. Taylor. 1994. Why statistical power is necessary to link analyses of molecular variation to decisions about population structure. Workshop on the Analysis of Genetic Data to Address Problems of Stock Identity as Related to Management. South West Fisheries Science Center (GSID 3), La Jolla, Calif., September 26 -28.



Fig. 2. Sequence variations found in the mitochondria1 D-loop of humpback whales from the eastern North Pacific breeding grounds. Numbers indicate the position in the whole D-loop. Dots indicate base matches with the sequence of haplotype A + , which is used as a reference. The rest of the characters show the nucleotide differences in the remaining types. Variation at position 28 was analyzed as a restriction fragment length polymorphism using Sau 961 restriction enzyme to discriminate haplotypes A+ and A-. The identity of this site was analyzed only in some E and F types. 028 103 128 136 148 164 169 247 249 266 268 270 274 316 317

A+ A-

G A

C .

C

AE El E2 E3 E4 F1 F2

G

A

T

T

C

T

C

C

A

T

A

C

. T T

C

. G

A A

T

A

C A

T

. C

. T

C C

C C . .

. T

C

. . . .

C

T

G G

C C

T T

Fig. 3. Phylogenetic relationship of humpback whale D-loop haplotypes at Hawaii (HI), Isla Socorro (IS), Baja California (BC), and Bahia Banderas (BB) constructed by parsimony analysis using the computer program PAUP (Swofford 1993). The frequency of the haplotypes in each geographic region is shown adjacent to branch termini.

Total

1993, 1994). A Sau 961 site around position 15 927 with respect to the fin whale ( ~ r n a s o net al. 1991) further defined two haplotypes referred to as A + and A- (Fig. 2). These types were otherwise indistinguishable within the sequence set examined. Phylogenetic reconstructions indicated that the nine haplotypes group into two distinct clades, referred to as AE and CF (Baker et al. 1993; Fig. 3). The A , A - , AE, E l , E2, E3, and E4 types differ from each other by one to three substitutions within the sequence (0.3 1 -0.94 %). The F l and F2 types are distinguished from each other by 1 base

+

HI

IS

BC

BB

26

23

21

21

Total

91

substitution and differ from the AE types by 9- 12 substitutions (2.81 - 3.75 %). A previous analysis has shown that F types are most closely related to the C types common among southern hemisphere humpback whales (Baker et al. 1990, 1993). The average divergence between a fin whale ( ~ r n a s o n1991, see footnote 2) and the North Pacific humpback whales is 5.07-7.58%. Greater nucleotide diversity was found for the Mexican breeding grounds, 0.895 % on average, than for Hawaii, 0.628%. Net genetic distances among the three regions of


1739


Fig. 4. Distribution of the mtDNA clades A, E, and F in humpback whales on their wintering grounds in Hawaii and the Mexican Pacific Ocean. Data on the haplotypic composition of whales in feeding areas off the Alaska and California coasts are taken from Baker et al. (1993, 1994).

Bahia Banderas

I s l a Socorro

Table 1. Nucleotide divergence (%) within (boldface type) and between humpback whales on the breeding grounds of the eastern North Pacific.

Hawaii (HI) Isla Socorro (IS) BajaCalifornia(BC) Bahia Banderas (BB)

HI

IS

BC

BB

0.628 0.779 0.735 0.845

0.042 0.846 0.846 1.223

0.011 0.012 0.821 0.932

0.022 0.009 0.012 1.019

Note: Distance values corrected for within-population diversity are shown above the diagonal.

the Mexican breeding grounds, 0.009 -0.01 2 % (average 0.01 1%), are lower than between these regions and Hawaii, which average 0.025% (Table 1). Overall, whales on the wintering grounds of the central and eastern North Pacific have a nucleotide diversity of 0.857% and exhibit a significant degree of population subdivision, as shown by X 2 and randomized G,, tests. Approximately 11% of the total diversity can be attributed to the separation of the four wintering regions examined: Hawaii, Baja California, Bahia Banderas, and Isla Socorro (Table 2). Previous analyses have demonstrated significant differences in the composition of mtDNA haplotypes between the Hawaiian and Mexican wintering grounds and between Hawaii-Alaska and MexicoCalifornia (Baker et al. 1993, 1994; Medrano 1993). Of the Mexican regions, the tests showed a significant genetic difference between the Revillagigedo Islands and the wintering grounds of the Mexican Pacific coast. Neither test supported a significant difference between Bahia Banderas and Baja California (Table 2).

Table 2. G,,, Type I error (tested with X2 (ac) and null G,, (a) analyses) and Type I1 error (P) (tested with alternative G,, analysis) of mtDNA variation among humpback whales from the breeding grounds of the eastern North Pacific, and among males (BB,) and females (BB,) of this species in Bahia Banderas.

HI, IS, BC, BB IS, (BC BB) BC, BB BB,, BBf

+

Gst

a c

a

P

0.11 0.11 0.01 0.05

a > 0.13). However, a X2 test indicated that the proportion of males to females in the sample from the Mexican Pacific is significantly higher than the ratio of 1: 1 ( a < 0.01) generally accepted for humpback whales at birth (Chittleborough 1958, 1965; Clapharn and Mayo 1987). Therefore, we tested the genetic differences between the sexes, separating the haplotypes completely and applying the analyses only to Bahia Banderas, where the ratio of males to females is close to 1:1. X2 and G,, analyses showed that the mtDNA haplotypes of males and females from Bahia Banderas are not significantly different, although the Type I1 error indicates marginally that males and females may not be fully identical (Table 2).

Discussion Humpback whales on the wintering grounds of the central and eastern North Pacific do not seem to form a single, freely intermingling population or stock. Significant differences in mtDNA haplotype frequencies are found between Hawaii and Mexico, as well as between the waters off the Revillagigedo Islands and the coastal regions of Baja California and Bahia Banderas. These differences are in agreement with observations of low levels of migratory movement between these regions by naturally marked individuals (Alvarez et al. 1990; Perry et al. 1990; Urban et al. 1994, see footnote 1). It is worth noting that humpback whales from the Revillagigedo Islands seem to form a distinct stock, as is evident from a low resighting rate in other breeding regions and a characteristic composition of mtDNA haplotypes. The primary feeding ground of the Revillagigedo whales has not been identified despite resighting of a few animals near Vancouver and in the western Gulf of Alaska. The small absolute genetic distance between whales in the Revillagigedo Islands and those in other breeding areas in the Mexican Pacific suggests that this stock has recently diverged from the American subpopulation or maintains low levels of ongoing interchange. Based on observation of individual whales, those on the



coastal Mexican wintering grounds would be expected to be more genetically similar to .those in feeding regions near central California than to those near Alaska. Surprisingly, the frequency of mtDNA haplotypes of whales on the Mexican wintering grounds is nearly intermediate between that observed in southeastern Alaska and in central California. This suggests a rate of migratory interchange higher than that observed by means of photoidentification between individuals from the two described feeding grounds, or the presence of a substantial portion of the eastern North Pacific population that has not been accounted for on the two known feeding grounds. The unequal representation of the A and A- haplotypes in winter and summer distribution areas further supports this second option (C .S. Baker, unpublished data). We have previously suggested that age-sex classes of humpback whales in the Mexican Pacific may have specific patterns of temporal and spatial distribution (Campos 1989; Ladr6n de Guevara 1995; Medrano 1993; Medrano et al. 1994; Salas 1993), as is recognized for Hawaiian wintering grounds (Perry et al. 1990). Thus, it is necessary to consider the potential effects of sex-biased dispersal on the distribution of mtDNA lineages. Unfortunately, the small size of our sample and its unequal representation of males and females preclude a detailed analysis of this factor. For example, the apparent lack of F-type females on the Mexican wintering grounds (Table 3) may be due to the combination of a low frequency of F-type individuals and an unintentional bias against females in sampling. Although imperfect, our data do not show regional or global heterogeneities of mtDNA identities between the sexes, as might be expected if sex-biased dispersal affects .the distribution of mtDNA lineages. In mammals, males generally tend to disperse more than females, thereby contributing to uniformity of genetic variation among subpopulations (Avise 1994; Greenwood 1980). Because our samples consisted mostly of males, and geographic subdivision of mtDNA haplotypes is observed, the greater mobility of males, a factor, which is not well established, that accounts for the interchange between subpopulations of humpback whales (Perry et al. 1990), seems not to affect the distribution of such maternal lineages. The influence of possible sex-biased dispersal on the genetic structure of a population can be discounted when regional differences in haplotype frequencies, such as those between offshore and coastal Mexico, are significant. Where regional differences are not significant, sex-biased dispersal cannot be excluded as a confounding factor. For example, our analyses suggest that the nonsignificant genetic differences between whales in Baja California and Bahia Banderas may be accounted for by sex-related variability, as seen at Bahia Banderas (Table 2), where the sampling was less biased and genetic differences between males and females seem to be greater (Table 3), though not significantly so. Palsboll et al. (1995) also found no sex-related heterogeneities in the distribution of mtDNA lineages in whales in the Atlantic Ocean. Proper evaluation of the role of sex-biased dispersal in the genetic structure of humpback whale populations will require samples large enough to allow independent analysis of panmixis for males and females or the testing of sex biases for each haplotype. More important than increasing the sample size, however, is the need to obtain unbiased samples. This will require a bet-

+

ter understanding of the sex composition of social groupings, as well as their behavioral and seasonal patterns. Alternatively, the influence of sex-biased dispersal on long-term population structure may be addressed by studying nuclear genetic markers. Palumbi and Baker (1994) have recently shown that variation in an intron of the nuclear actin gene shows far less geographic subdivision than mtDNA among humpback whales in the eastern North Pacific. This contrasting pattern of population structure may be due to greater dispersal of males, in combination with the larger effective size of populations for nuclear genetic markers (Palumbi and Baker 1994). Our data also suggest that the influence of historic demography may still be apparent in the current distribution of mitochondria1 haplotypes along the American North Pacific coast. Prior to the retreat of the last ice age, 12 000 - 6000 years ago (Fairbridge 1960), the distribution of humpback whale feeding and wintering grounds may have been considerably more constrained than it is at present. With the end of the last glaciation, the distribution of humpback whales may have shifted northwards from wintering grounds along the Central American coast until it became the scattered and clinal distribution currently observed along the Mexican Pacific coast. An analysis of the distribution of coalescence times according to Avise et al. (1988) and a computer simulation of the F1 type propagation within the framework of humpback whale historical demography are consistent with this hypothesis. These analyses suggest that divergence events within clades E and F in the eastern North Pacific can be traced back 10 000 - 40 000 years, and that separation of the Revillagigedo Islands aggregation is associated with the end of last glaciation (Medrano 1993). The existence of breeding regions along the coast of Central America, which may have been the main reproductive areas in the past and are now relictual, would also have allowed exchange with southern hemisphere whales whose descendants are the present F types. The observed temporal and geographic distribution of F-type whales in Mexico suggests that animals of this maternal lineage may still migrate more to southerly regions such as Costa Rica, a known breeding ground for some whales summering near central California (Steiger et al. 1991). The F-type whales may travel, or even breed, along the Mexican coast, earlier, on average, than whales of the AE group. This testable hypothesis is suggested by several sightings of humpback whales off the Michoacin, Guerrero, and Oaxaca coasts to south of Bahia Banderas (Gallo et al. 1986) and by several observations made by the research group of the Facultad de Ciencias, Universidad Nacional Aut6noma de Mexico. Colonization of the Hawaiian wintering ground may have proceeded with the expansion of populations into feeding ranges to the north and west along the coast of Alaska and the isolation of a few maternal lineages. However, the effects of human activities, especially whaling, on distribution changes and occupation of new areas cannot be discounted. As discussed by Herman (1979), humpback whales may have begun wintering in the main Hawaiian Islands only during the last two centuries. At present, the slow rate of mutational change in mysticete mtDNA (Martin and Palumbi 1993) and the level of DNA sequence resolution presented here do not allow more precise dating of this colonization event.



Humpback whales in the Mexican Pacific Ocean do not have a simple and homogeneous population structure. Geographic isolation of the Revillagigedo Islands grounds, mixing with Hawaiian whales, potential heterogeneous migratory patterns, the genetic influence of whales from the southern hemisphere, the recent historical changes in distribution, and the generation of genetic diversity have contributed to forming a complex and dynamic system. Therefore, the details concerning the Mexican aggregations of humpback whales may be important for the recovery of the North Pacific population, in terms not only of abundance but also of genetic diversity and thus of evolutionary significance.

Acknowledgements We are greatly indebted to S.R. Palumbi and the staff of the Kewalo Marine Laboratory, University of Hawaii, for their kind hospitality and facilities for analyzing tissue samples, and to A. Perry, M. Betancourt, P. Miramontes, A. Lazcano, G. Cocho, 0 . Flores, M. Uribe, L. Eguiarte, P. Nicolas, J. Prieto, G. Rosas, R. Robles, M. Salinas, P. Ladr6n de Guevara, N. Vargas, I. Salas, C. Esquivel, J. Jacobsen, L. Rojas, H. Rosenbaum, T. Bosques, R. Ruiz, J. Patton, students and colleagues from the Universidad Nacional Aut6noma de Mkxico (UNAM) and the Universidad Aut6noma de Baja California Sur, J. Niebla, fishermen from Punta de Mita and I. Casillas for provision of laboratory facilities, technical assistance, collaboration in field activities, advice, and (or) comments. We thank two anonymous reviewers for their attention to this article. We also acknowledge H. Armada de Mkxico, Facultad de Medicina UNAM, and Centro de Estudios Tecnol6gicos del Mar 6 for logistical aid. This work was supported by grants from the Programa de Apoyo a las Divisiones de Estudio de Posgrado UNAM, Consejo Nacional de Ciencia y Tecnologia, and Sistema Nacional de Investigadores to A. Aguayo and L. Medrano, from the International Whaling Commission to C.S. Baker, and from the National Science Foundation to C.S. Baker and S.R. Palumbi. Samples were collected in United States waters in full compliance with the conditions of U.S. National Marine Fisheries Service Permit No. 675 and in Mexican waters according to specifications of the Secretaria de Desarrollo Urbano y Ecologia in documents 412 (1) 03735 and 412 (1) 05748.

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