a harp seal × hooded seal hybrid

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MARINE MAMMAL SCIENCE, 13(3):460-468 (July 1997) 0 1997 by the Society for Marine Mammalogy A HARP SEAL X HOODED SEAL HYBRID KIT M. KOVACS’ Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl CHRISTIAN LYDERSEN Norwegian Polar Institute, Box 399, N-9001, TromsG, Norway MIKE 0. HAMMILL Institut Maurice LaMontagne, C. P. 1000, Mont-Joli, Quebec, Canada G5H 324 BRADLEY N. WHITE PAUL J. WILSON SOBIA MALIK Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4L8 ABSTRACT In this study we report the first documentation of mating between harp (Phoca groenlandica) and hooded seals (Cystophora cristata). The production of this hybrid was quite unusual, being the result of a cross between parents of different genera which are morphologically dissimilar and have quite different mating behavior and dramatically different body sizes. The molecular tech- niques (mtDNA and macrosatellite nuclear DNA banding patterns) used in this study will undoubtedly be applied widely to many different taxa in the near future, allowing us to re-examine many suspected cases of hybridization among marine mammals and, in a larger context, the meaning of the species concept. Key words: harp seal, Phoca groenlandica, hooded seal, Cystophora cristata, hy- bridization, mitochondrial DNA, macrosatellite DNA, species-specific se- quences. Hybridization among pinniped species has been suggested numerous ,times in the literature. Most suspected crosses in the wild have been reported among otariid seals. Suspicion of hybridization has been based on observations of animals that appeared to possess external characteristics representative of two species, skull measurements of specimens that were intermediate between two species, the lack of distinct separation of breeding seasons of closely related species in some localities, and observations of harems of individual males that ’ Present address: UNIS, N-9170, Longyearbyen, Svalbard, Norway. 460

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MARINE MAMMAL SCIENCE, 13(3):460-468 (July 1997) 0 1997 by the Society for Marine Mammalogy

A HARP SEAL X HOODED SEAL HYBRID KIT M. KOVACS’

Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl

CHRISTIAN LYDERSEN

Norwegian Polar Institute, Box 399, N-9001, TromsG, Norway

MIKE 0. HAMMILL

Institut Maurice LaMontagne, C. P. 1000, Mont-Joli, Quebec, Canada G5H 324

BRADLEY N. WHITE PAUL J. WILSON

SOBIA MALIK

Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4L8

ABSTRACT

In this study we report the first documentation of mating between harp (Phoca groenlandica) and hooded seals (Cystophora cristata). The production of this hybrid was quite unusual, being the result of a cross between parents of different genera which are morphologically dissimilar and have quite different mating behavior and dramatically different body sizes. The molecular tech- niques (mtDNA and macrosatellite nuclear DNA banding patterns) used in this study will undoubtedly be applied widely to many different taxa in the near future, allowing us to re-examine many suspected cases of hybridization among marine mammals and, in a larger context, the meaning of the species concept.

Key words: harp seal, Phoca groenlandica, hooded seal, Cystophora cristata, hy- bridization, mitochondrial DNA, macrosatellite DNA, species-specific se- quences.

Hybridization among pinniped species has been suggested numerous ,times in the literature. Most suspected crosses in the wild have been reported among otariid seals. Suspicion of hybridization has been based on observations of animals that appeared to possess external characteristics representative of two species, skull measurements of specimens that were intermediate between two species, the lack of distinct separation of breeding seasons of closely related species in some localities, and observations of harems of individual males that

’ Present address: UNIS, N-9170, Longyearbyen, Svalbard, Norway.

460

KOVACS ETAL.: HYBRID SEAL 461

contained females of two species (Condy 1978, Kerley 1983, Kerley and Rob- inson 1987, Shaughnessy and Fletcher 1987, Boness et al. 1993). Additionally, interspecific mating behavior has been observed between several combinations of otariid species (DeLong 1982, Stewart et al. 1987).

Among phocid seals, gray seals (Halichoerus grypus) have been seen copulat- ing with harbor seals (Phoca vitulina; Boness et al. 1993). Additionally, a cross- family mating situation was documented when an aberrant male southern elephant seal (Mirounga leonina) copulated with numerous Cape fur seal (Arc- tocephalus pusillus) females, crushing them to death in the process (Best et al. 1981).

None of the mating combinations between different genera, or higher tax- onomic units, have been reported to produce hybrid offspring. In this report we document the birth of a hybrid pup produced by a hooded seal female and a harp seal male.

MATERIAU AND METHODS

On 13 March 1994 we found a pup in the hooded seal herd in the Gulf of St. Lawrence that bore a combination of hooded seal and harp seal features (Fig. 1). The pup was only a few hours old when found. It was closely attended and defended by a hooded seal female. A male hooded seal rested about 5 m from the mother and pup, Both members of the mother-pup pair were cap- tured, weighed, and tagged with standard, individually numbered, domestic animal roto-tags placed through the webbing of a hind flipper (for details see Kovacs and Lavigne 1992). The skin plugs produced during the tagging pro- cedures were collected. Additionally, blood was taken in lo-ml Vacutainers (containing EDTA) from the hind flipper plexus of both animals. Blood and skin samples were frozen at -2O’C until they were analyzed.

DNA was extracted from the blood and skin samples according to Guglich et al. (1994). Mitochondrial DNA was used to confirm the maternal species of the seal pup. Primers (5’- ccatccaacatcteagcatgatgaaa-3’ and 5’-ccctcagaat- gatatttgtccta-3’) were used to amplify and sequence a 285 b-p. region of the cytochrome b gene. The following reaction conditions were used to amplify this region of the cytochrome b gene: 100 ng of template DNA, 1.5 mM MgCl,, 2 mM KCl, 10 mM tris-HCl (pH 8.3), 0.2 mM dNTPs, 1.5 U Taq polymerase (Perkin-Elmer-Cetus), 10 pmol of each primer. The reactions were processed through 30 cycles at 95°C for 30 set, 55°C for 15 set, and 72°C for 30 sec. PCR products were sequenced with a Model 373A DNA Sequencing System (MOBIX Facilities, McMaster University). Cytochrome b sequences were obtained from the adult female attending the pup, the seal pup, two control samples from harp seals, and two control samples from hooded seals.

Highly repetitive satellite DNA, i.e., macrosatellite DNA, was also exam- ined in these samples to identify species-specific patterns in the nuclear DNA to determine any contribution of genetic material from more than one seal species to the pup. Macrosatellite DNA is localized in the centromeric regions of chromosomes in tandemly repeated units and has been observed to dem-

462 MARINE MAMMAL SCIENCE, VOL. 13, NO. 3, 1997

Figure 1. A 4-d-old hooded seal blueback (top), the hybrid at 5 d of age (middle),and a 25-d-old harp seal beater (bottom).

KOVACS ETAL.: HYBRID SEAL 463

onstrate various levels of chromosome specificity (Willard 1989). As a result of concerted evolution (Dover 1982), satellite DNA subunits demonstrate dif- ferences in the size and sequence of tandemly repeated units between species and demonstrate homogenized sequences and unit sizes within a species. In- terspecific sequence variants can be detected by restriction enzyme analyses, which provide a diagnostic indication of satellite DNA subunits within a species. Restriction enzyme analysis and gel electrophoresis were performed according to Guglich et al. (1994). Digested DNA from the adult female attending the pup, the seal pup, and harp and hooded seal controls was ana- lyzed by Southern Blotting using radioactively labeled genomic harp seal DNA (50 ng) (Feinburg and Vogelstein 1983, Westneat et al. 1988, Quinn et al. 1992). Random primer extension with 50 pCi alpha 3*P {dCTP] (Du- pont/NEN, Inc.) produced specific activities of > 1.0 X lo8 cpm/kg. Hy- bridization was carried out at 65°C for 15-20 h. Blots were washed in 0.05 X SSC, 0.5% SDS; twice for 15 min, and once for 30 min. Blots were exposed to Cronex x-ray film using one intensifying screen at -70°C for 24-48 h.

All animal handling procedures were conducted in accordance with the principles and guidelines of the Canadian Council for Animal Care.

RESULTS

The mother appeared to be a normal adult hooded seal in good condition. She weighed 225 kg. The pup weighed 9 kg when it was first weighed. It was partially covered in greyish white lanugo, but the front half of its body had molted. The exposed pelt was spotted and looked like that of a harp seal beater (Fig. l), although the background color was lighter grey than that of harp seal pups when they have shed their first coat. The lanugo that had been shed prior to birth was silver in color and was associated with the placenta in the form of tight disks. The disks were lighter in color than those of normal hooded seal pups. The pup’s head was flattened in appearance and it had very large eyes, like a hooded seal blueback. It performed fright paralysis behavior when handled.

The mother attended and fed the pup for five days; on the morning of 18 March it was found alone at its birth site. The pup weighed only 18.5 kg at weaning but appeared fat for its length. Once weaned by its mother the pup remained near its birth site for three days before it left the area.

The sequence of the cytochrome b gene in the seal pup confirmed that the maternally inherited mtDNA was from a hooded seal when it was compared to published sequences (Arnason et al. 1995, Perry et al. 1995). Nuclear DNA analysis using macrosatellite DNA markers demonstrated restriction-hybrid- ization banding patterns that distinguished between hooded seals and harp seals, i.e., species diagnostic variants (Fig. 2). The attending mother demon- strated a pattern identical to two hooded seal control samples. The pup pro- duced DNA banding patterns which contained the diagnostic patterns of both hooded and harp seals, strongly suggesting a genetic contribution from both of these two species. The additive evidence of DNA markers identifying harp

464 MARINE MAMMAL SCIENCE, VOL. 13, NO. 3, 1997

Figure 2. Species-specific banding patterns of repetitive DNA with EcoRI digestedDNA from hooded seal controls, the mother of the pup, the hybrid pup, and harp sealcontrols. Visualization of the ethidium-stained DNA samples confirmed an equalamount of DNA was loaded for each sample. Restriction-hybridization banding pat-terns distinguished harp seal DNA and hooded seal DNA. Two species-specific frag-ments (A and B) were observed in the EcoRI digested harp seal DNA and the seal pupthat were absent in the mother and the hooded seal controls. Two additional enzymes,HindIII and MspI, demonstrated a total of four harp seal specific fragments which werepresent in the pup and absent in the hooded seal samples. A total of four fragmentswere observed to be diagnostic for hooded seals, with the three enzymes. All of thesefragments were observed in the pup. The additive evidence strongly suggests that thematernal species of the pup was hooded seal and the paternal species was harp seal.

and hooded seals as species which were consistently present in the seal pupwith three restriction enzymes is consistent with an F1 hybrid pup resultingfrom a maternal hooded seal and a paternal harp seal.

DISCUSSION

The hybrid pup bore a combination of morphological and behavior char-acteristics of its two parental species. It had a birth mass typical of harp seals(Kovacs and Lavigne 1985). However, its lanugo was grey rather than white,

KOVACS ETAL.: HYBRID SEAL 465

and half of its body had shed the birth coat in zltero. The shed hair at the birth site was in tightly woven disks, typical of hooded seals (Oftedal et al. 1991). The exposed juvenile pelt was similar to that of a harp seal beater, although it was lighter in color. Facially, the pup more closely resembled a blueback hooded seal as opposed to a harp seal whitecoat; it had a flattened head with very large eyes. The pup performed paralysis behavior when touched, which is typical behavior for harp seal whitecoats (Lydersen and Ko- vats 1995). This behavior has never been observed in hooded seals.

The hybrid pup was fed for five days before being weaned by its mother. Hooded seals normally feed their young for less than four full days (Bowen et al. 1985, Kovacs and Lavigne 1992), while harp seals feed their young for an average of 12 d (Stewart and Lavigne 1980, Kovacs and Lavigne 1985). The small size of the hybrid at weaning is not typical of either harp or hooded seals, which are normally weaned when they are 35 and 45 kg, respectively. The short sedentary period displayed by the hybrid after it was weaned was normal behavior for a hooded seal pup but unlike that of harp seals, which remain quite sedentary for several weeks after weaning (Kovacs 1987, 1995).

The production of this intergeneric hybrid is unusual for a variety of reasons. Hooded seal females are approximately twice the body mass of male harp seals, and hooded seals are a much more aggressive species. Male hooded seals are usually 1.5-2 times the body mass of their female partners (Kovacs and La- vigne 1986). Thus, the father of the hybrid was approximately one-third the mass of a normal mating partner for a hooded seal female. During the lactation period, hooded seal females are normally guarded by a male, or series of males, that actively defend their attendance position (Kovacs 1990, McRae and Ko- vats 1994). Harp seals appear to be promiscuous and do not attend females on the ice-surface prior to mating (Kovacs 1995).

However, the breeding seasons of the two species do overlap, with the end of the harp season marking the start of the hooded seal season, and both species breed on the pack ice in a relatively restricted area in the Gulf of St. Lawrence. Additionally, they may be more closely related than previously thought. Perry et al. (1995) recognized harp and hooded seals as a tribal group within the subfamily Phocinae. The harp and hooded seal herds in the Gulf of St. Law- rence are usually 40+ km apart, but each year a few hooded seal females that give birth early in the season do so at the edges of the harp seal herd. These females are frequently not attended by male hooded seals. The mother of the hybrid may have been one of these females in 1993.

Hybrid crosses between these species are in all likelihood extremely rare. Although these species breed on offshore pack ice, they have had a very long history of intense human predation (Lavigne and Kovacs 1988), and if hybrids occurred with regularity they would almost certainly have been reported pre- viously in some descriptive form in sealing ships’ logs or by other scientific researchers.

Hybridization is likely to be confirmed with increasing frequency in the near future in a wide variety of taxa due to the rapid development of molecular techniques for dealing with species identification using various forms of DNA

466 MARINE MAMMAL SCIENCE, VOL. 13, NO. 3, 1997

(Hoelzel 1993). Species-specific repetitive DNA sequences have been identified in avian and mammalian species (Arnason et al. 1988, Quinn et al. 199%) and have been used to identify a hybrid of two cetacean species (Spillaert et al. 1991). In 1991, the first hybrid baleen whale cross was confirmed, between a fin whale female and a blue whale male (Spillaert et al. 1991). The fact that this hybrid had successfully been impregnated by a blue whale was also very interesting. The future survival and reproductive status of the hybrid pup documented here is, of course, unknown, but upon last sighting it was alert, responsive, and in good condition.

Other cases of suspected crosses that are thought to occur more commonly among animals that are more readily accessible than pack-ice-breeding pho- cids, or large pelagic cetaceans, will undoubtedly come under genetic scrutiny with increasing frequency. At the very least, the production of viable hybrids will serve to cause reflection on species isolating mechanisms, the species con- cept, and the role of natural hybridization in evolution (Arnold and Hodges

1995).

ACKNOWLEDGMENTS

This research was funded by the Natural Sciences and Engineering Council of Canada (NSERC), the Department of Fisheries and Oceans, Canada, the Norwegian Fisheries Research Council, and a NATO Collaborative Research Grant (#921216).

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Received: 29 April 1996 Accepted: 7 June 1996