Journal of Heredity Advance Access published online on June 4, 2007
Journal of Heredity, doi:10.1093/jhered/esm023
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Brief Communication |
Sexing Pinnipeds with ZFX and ZFY Loci
From the Department of Biology SCA 110, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620 (Curtis and Karl); the Hubbs-SeaWorld Research Institute, 2595 Ingraham Street, San Diego, CA (Stewart); and the Hawaii Institute of Marine Biology, University of Hawaii, Manoa, PO Box 1346, Kaneohe, Hawaii, 96744 (Karl)
Address correspondence to Stephen A. Karl at the address above, or e-mail: skarl{at}hawaii.edu.
We developed and tested a protocol for determining the sex of individual pinnipeds using the sex-chromosome–specific genes ZFX and ZFY. We screened a total of 368 seals (168 crabeater, Lobodon carcinophaga; 159 Weddell, Leptonychotes weddellii; and 41 Ross, Ommatophoca rossii) of known or unknown sex and compared the molecular sex to the sex assigned at the time of biopsy sample collection in the Ross and Amundsen seas, Antarctica. We also screened 6 captive northern elephant seals (Mirounga angustirostris) and 2 captive California sea lions (Zalophus californianus) of known sex. The assigned sex and genetic sex agreed for virtually all seals. Indeed, discrepancies ranged from 0.0% to 6.7% among species. It is not clear, however, if the few mis-assignments of sex occurred in situ or in the laboratory. The assigned morphological and molecular sex might both be correct with the discrepancies owing perhaps to developmental effects of environmental pollution. A subset of individuals sequenced at both loci revealed no intraspecific sequence variation. There was, however, sequence variation among species at both loci, which allowed them to be uniquely identified with as few as 2 and as many as 31 nucleotides.
The ability to accurately and reliably identify the sex of free-ranging animals is essential for estimating sex ratios, categorizing behavioral observations, and understanding almost every aspect of an species' life history. For many species, it may be relatively easy to distinguish between adult males and females if they are sexually dimorphic in size or color. Distinguishing subadult or juvenile males from females, however, can often be challenging. Even adult sex can be difficult to determine in animals like the phocid pinnipeds that live in pack-ice or fast-ice habitats of the Antarctic and exhibit little to no sexual dimorphism. This difficulty is even more pronounced when individuals are viewed from a distance with no direct physical examination. In phocid pinnipeds, male genitalia are inguinal (i.e., internal). Consequently, the only clue to sex in otherwise sexually monomorphic species is the presence (male) or absence (female) of a ventral penile opening, which is located just behind of the umbilical scar. For animals with chromosomal sex–determining mechanisms, molecular sex determination has the potential to unequivocally determine the genetic sex of individuals. This approach can circumvent many difficulties in identifying sex of animals in the field and is particularly useful in secretive species where only traces of the individual such as blood, hair, or scat are available.
Two sex-chromosome–specific genes, ZFX and ZFY, are zinc-finger homologues located on the X and Y chromosomes, respectively (Pecon-Slattery and O'Brien 1998). Because it is typically located outside the pseudoautosomal region of the Y chromosome in eutherian mammals (Page et al. 1987; Mardon and Page 1989), ZFY only rarely recombines with ZFX (but see Pecon-Slattery et al. 2000), and together, these genes have proved to be useful as a molecular method for determining sex in many mammals (e.g., canids, Lucchini et al. 2002; sea otters, Hattori et al. 2003; rodents, Marchal et al. 2003; pinnipeds, ungulates, and ursids, Shaw et al. 2003; prosimians and humans, Fredsted and Villesen 2004; cetaceans Morin et al. 2005; and felids, Pilgrim et al. 2005). Shaw et al. (2003) developed a polymerase chain reaction (PCR)–based ZFY/ZFX assay applicable in a variety of mammals, including one pinniped (the harbor seal, Phoca vitulina), by using a single generic primer pair to simultaneously amplify both homologues in a single PCR, and then verifying the presence or absence of the amplicons on an agarose gel. This approach relies, however, on differences in the size of the X- and Y-specific regions that may be subjected to PCR competition and leads to allele dropout of the larger allele producing incorrect sex assignment. Although we have no empirical indication that this is happening with the primers of Shaw et al. (2003), it is nonetheless a well-documented phenomenon and one we wished to avoid (Piyamongkol et al. 2003; Sefc et al. 2003; Buchan et al. 2005). We expanded on the work by Shaw et al. (2003) and created primers specifically targeted to separately amplify a portion of the ZFX or ZFY gene in pinnipeds. This allowed us to genotype each seal to determine sex and to directly sequence one or both of these genes that could be used for forensic or species identification purposes.
Crabeater (Lobodon carcinophaga), Ross (Ommatophoca rossii), and Weddell (Leptonychotes weddellii) seals are phocid pinnipeds that live almost exclusively in fast ice or pack-ice habitats around the Antarctic Continent (Reeves et al. 1992; Reeves and Stewart 2003). Males and females of each species are similar in size and color and are not easily distinguished most of the time without close inspection for the presence or absence of a ventral penile opening. Consequently, a genetic method for determining or verifying sex may be useful to a variety of ecological studies in these species.
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Materials and Methods |
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 Top Materials and Methods Results and DiscussionAppendixReferences |
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Skin biopsy samples were collected from 168 free-living crabeater seals (L. carcinophaga), 159 Weddell seals (L. weddellii), and 41 Ross seals (O. rossii) via remote darting or direct handling during the 2000 Antarctic Pack Ice Seal scientific cruise (Decker et al. 2002; Solls et al. 2005). Although all 3 species are circumpolar, most samples were collected from the pack-ice zone of the western Amundsen and eastern Ross seas, approximately 67°–78° S, 129°–180° W (Figure 1), and some Weddell seal samples came from McMurdo Sound in the western Ross Sea. Of the crabeater seals, there were 71 field identified males, 56 females, and 41 sex unknown. For Weddell seals, there were 90 males, 64 females, and 5 unknown, and for the Ross seals, there were 25 males and 16 females. We also assayed 4 male and 2 female northern elephant seals (Mirounga angustirostris) and one male and one female California sea lion (Zalophus californianus). Because samples from the latter 2 species were from captive animals, we are confident of their true morphological sex. The sampled Antarctic phocids were all free ranging, however, and many were sampled remotely by biopsy dart and not directly examined.
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We designed primers specifically targeting the last intron of the phocid ZFX and ZFY genes. To accomplish this, we used the 2 previously published sets of nested generic ZFX and ZFY felid primers (Pecon-Slattery and O'Brien 1998; Table 1) to simultaneously amplify both gene regions from a male crabeater seal. PCR products were cloned (Original TA Cloning Kit, Invitrogen, Inc., Carlsbad, CA) and sequenced on an ABI 3730XL automatic sequencer (Macrogen Inc., Seoul, Korea). Sequences were aligned using Sequencher software (GeneCodes Corp., Ann Arbor, MI) and compared with published ZFX and ZFY sequences in GenBank. From the aligned crabeater seal and GenBank sequences, locus-specific primers were designed to target the ZFX or ZFY loci separately (Table 1). A subset of male crabeater, Weddell, and Ross seals were amplified and sequenced at both loci. We also assayed ZFY and ZFX in one northern elephant seal and one California sea lion. To determine the sex of seals, we set up separate PCRs for the ZFX and ZFY genes for each seal and visualized the amplifications side by side on an agarose gel. There are 4 possible amplification patters. If both ZFX and ZFY or just ZFY alone amplified, the individual was assigned male. If the ZFX but not the ZFY amplified, the individual was assigned female. If neither locus amplified, the individual was classified as unresolved. It should also be noted that individuals that amplify for the ZFX locus and not the ZFY could be either females or nonamplifying males (i.e., false negative for ZFY). The inclusion of a second male-specific locus (e.g., SRY) can be helpful in confirming the results (Gilson et al. 1998).
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DNA was extracted from frozen tissue samples using standard phenol–chloroform techniques and/or using a DNeasy Tissue Kit (Qiagen, Valencia, CA). Amplification reactions generally were 25 µl and contained 0.5 µl total cell DNA, 1x reaction buffer (Promega, Madison, WI), 2.0 mM MgCl2, 0.2 mM of each dNTP, 10 pmol of each primer, 6 mg bovine serum albumin, and 1.25 U Taq polymerase (Promega). Thermocycling conditions were 95° C, 2 min; 35 cycles of 95° C, 1 min; annealing temperature (Table 1) 1 min; and 72° C, 1 min, followed by a final extension at 72° C for 7 min. PCR products were visualized on a 2% agarose gel with ethidium bromide to assess quantity and fidelity of amplification and then purified using either Microcon Centrifugal Filter Units (Millipore Corp., Billerica, MA) or QIAquick spin columns (Qiagen). Approximately 100 ng of purified PCR product was directly sequenced in both directions on an ABI 3730XL automatic sequencer (Macrogen Inc.).
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Results and Discussion |
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TopMaterials and MethodsResults and DiscussionAppendixReferences |
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All 5 species successfully amplified for both loci. The length of the crabeater seal ZFY fragment, including the primer sequences, was 931 nucleotides (nt). The other species produced DNA fragments of similar length. The length of the crabeater seal ZFX fragment, including the primer sequences, was 1045 nt, and the other species produced fragments of similar length. The ZFX fragment matches with 83% identity to the final ZFX intron of Bos taurus (Lawson and Hewitt 2002; GenBank accession no. AF241273 [GenBank] ), and our ZFY fragment was 79% identical to the final intron of the Amur leopard (GenBank accession no. AB211426 [GenBank] ). After removing segments for which reliable sequence data were not obtained form all individuals (usually at the beginning and end of the sequence), we were able to clearly resolve 851 and 956 nt of sequence for the ZFY and ZFX loci, respectively.
The genotypic sex of virtually all seals (95.8%) agreed with the sex assigned in the field (Table 2). Indeed, discrepancies between the field and laboratory assigned sex ranged from 0% to only 6.7%. Conflicts between the laboratory and field assigned sex of an individual can arise from either field-sexing errors or an unidentified laboratory artifact. We are most confident in the laboratory assessment when both ZFX and ZFY amplifications produce strong, clear bands because nonhomologous amplification of an appropriate-sized fragment is unlikely given the specificity of the primers. Furthermore, the homology of the fragment can be verified by DNA sequence or restriction fragment length polymorphism analysis. Any male that amplifies for the ZFX gene but fails to amplify for the ZFY gene would appear to be a female by our genetic tests. The amplification of the ZFX gene indicates that the failure of the ZFY gene to amplify was not due to poor template quality or other general problems of amplifications. Because we saw no intraspecific variation and as these primers generally worked well in all 3 species, we think that this type of locus-specific artifact is also unlikely. Nonetheless, we cannot definitively rule out that the 8 individuals that were field identified as male but failed to amplify for the ZFY locus may indeed be male. A field-identified female appearing to be male based on amplification of both the ZFY and ZFX loci is a likely candidate for field misidentification. Sex of individuals in the field was determined in one of 3 ways. First, many crabeater and Weddell seals were closely approached so that skin samples could be taken from the trailing edge of their rear flippers while they slept. At this time, individuals were visually examined for the presence of a ventral penile opening or for distinctive scarring around the neck and fore flippers (suggesting male in crabeater seals). Six of the incorrectly identified individuals in Table 2 were sampled this way, evenly split between the 2 possible discrepancies (i.e., field male but genetic female and field female but genetic male). Second, a number of seals (particularly crabeater seals that are more difficult to approach) were remotely sampled by biopsy darting either using a crossbow or a hand-held dart pole. The sex in these cases was assigned based on the quickly observed presence or absence of a penile opening or scarring on neck and near the fore flippers. Five of the total 15 incorrectly sexed individuals were sampled in this way, and all but one of these 5, a Weddell seal, were field males but genetic females. Finally, a smaller number of seals (13) were captured and anesthetized while samples were taken and telemetry instruments attached. These individuals were examined more closely and assigned a sex by an experienced field assistant. Only one individual field identified as male but failing to amplify the ZFY gene (a crabeater seal) was among the examined individuals. This may indicate that, although rare, false-negative amplification of ZFY has occurred.
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Interestingly, 3 of the examined individuals field identified as female (2 crabeater seals and 1 Ross seal) produced strong ZFY (and ZFX) amplifications. We think that false-positive amplification is unlikely. Instead, those seals may have been misidentified in the field or were errors in reporting data to recorders or in transcribing information to sample labels.
Although we cannot fully validate either the field or laboratory methods for sex identification, it is possible that the observed discrepancy of a field-identified female clearly being a genetic male does not involve errors in either method. One of us (B.S.S., personal observation) has observed 3 northern elephant seals (M. angustirostris) in California that clearly had secondary sexual characteristics of adult males and yet lack a penile opening leading to ambiguous sexual assignment. Unfortunately, we did not have access or samples from these individuals to determine genetic sex. Presumably, if we did, they would produce positive amplifications for ZFY.
Persistent organic pollutants (POPs) are nearly ubiquitous in the environment (Damstra et al. 2002), and many of them (e.g., dithiothreitol, polychlorinated biphenyls, and tributyltin) are endocrine-disrupting chemicals. In vertebrates, some POPs act as estrogen mimics or androgen antagonists resulting in genetically male individuals possessing female physical characteristics (as is seen here; Hayes et al. 2002; Ayaki et al. 2005; Penaz et al. 2005). Although the near absence of industrial development and its remoteness make the Antarctic appear to be an unlikely place for POPs, this is clearly not the case. While studying sediment cores in McMurdo Sound, Ross Sea, and Antarctica, Negri et al. (2004) documented detectable levels of butyltins (i.e., TBT, DBT, and MBT) at 6 of 8 surveyed sites. Butyltins are commonly used in antifouling paints on large boats including the icebreakers that visit McMurdo Sound. Negri et al. (2004) thought that butyltins might be introduced into the sediment from paint chips rubbed off of icebreakers. One of their sampling sites in McMurdo Sound, Cape Armitage, had extremely high levels of butyltin "...only exceeded in very busy harbours ..." (Negri et al. 2004). Goerke et al. (2004) also documented a 30- to 160-fold biomagnification of several POPs in Weddell seals and southern elephant seals (Mirounga leonina).
Although it is difficult to say at this time what, if any, effect Antarctic POPs are having on the health and sexual development of Antarctic pack-ice seals, there is a wealth of studies indicating that the presence of POPs in the environment can have long lasting and dire consequences for wildlife. They also may have been contributing factors resulting in field sex identifications not agreeing with our genetic sex assignments. Regardless, being able to genetically assess the sex of free-ranging seals can provide a backup method for testing the veracity of visual designations, allow sex determination of DNA samples when individuals are not handled or even sighted, and provide a key to the understanding of the impacts that POPs might have in marine ecosystems. Furthermore, to fully assess the potential effects that POPs may be having on natural populations, it is necessary to have both genetic and morphological information along with some understanding of the individual exposure to POPs.
We found no intraspecific ZFY variation after sequencing 12 crabeater (GenBank accession no. DQ493902
[GenBank]
), 10 Weddell (GenBank accession no. DQ493904
[GenBank]
), or 10 Ross seals (GenBank accession no. DQ493903). There also was no intraspecific variation in ZFX genes of those species after screening 4 (GenBank accession no. DQ811091), 2 (GenBank accession no. DQ811093), and 2 (GenBank accession no. DQ811092) individuals, respectively. In addition, we sequenced ZFY and ZFX from one each of northern elephant seals (ZFY, GenBank accession no. DQ493906
[GenBank]
; ZFX, GenBank accession no. DQ811095) and California sea lion (ZFY, GenBank accession no. DQ493905
[GenBank]
; ZFX, GenBank accession no. DQ811094). Among all 5 species, there was considerable interspecific variation at both the ZFY and ZFX genes (Appendixes A and B). At the ZFX locus, there were 50 variable nucleotide positions (
5%). Of those, 29 were transitions, 10 were transversions, and 3 were deletions (one 3 nt and two 4 nt). At ZFY, 61 of the nucleotide sites were variable (7%). Of those, 33 were transitions, 13 were transversions, one had both a transition and a transversion, and one had both a deletion and a transition. There was a single 4 nt deletion in the Weddell sequence and there were two 2 nt and two 3 nt deletions in the California sea lion sequence. It appears that all species can be uniquely identified from all others through DNA sequence by at least 2 (crabeater seal at ZFX) to at most 31 (California sea lion at ZFY) sites. Our assessment of intraspecific variation, however, was limited, and species designations should be made on multiple sites or a larger number of individuals from throughout the range of the species. Nonetheless, this level of variation is likely to be useful in identifying species from forensic samples and for phylogenetic analyses.
The results of this study have been 3-fold. First, we have found that the ZFX and ZFY loci are likely to be good nuclear markers for species identification of the California sea lion, and crabeater, Weddell, Ross, and northern elephant seals. Second, we have demonstrated the utility of new ZFX and ZFY markers for assigning the sex of pinnipeds especially when access to the animal is highly limited. Third, we uncovered several cases where the sex designated based on morphology appears to be in conflict with molecular markers. In some of these cases, it likely is simply owing to misidentification or errors in reporting or transcribing data in the field. Others, however, may be indicating that persistent organic pollutants are having significant impact on Antarctic fauna. Further targeted research, however, is needed before final conclusions about the likelihood and magnitude of the effect can be reached.
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Appendix |
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Acknowledgments |
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Phocid samples in this study were collected in Antarctica by B.S.S. during a multidisciplinary research cruise to the Ross and Amundsen seas in 2000. We thank Claudia Rocha for laboratory assistance and 3 anonymous reviewers for helpful comments on earlier drafts of the manuscript. Funding for this study was provided by an American Museum of Natural History, Lerner-Gray grant to C.C. and by the National Science Foundation grants OPP 98-16011 and OPP 98-16035 to B.S.S. and DEB 98-06905 and DEB 03-21924 to S.A.K.
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Footnotes |
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Corresponding Editor: C. Scott Baker
Received August 7, 2006
Accepted April 11, 2007
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References |
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