The microsatellite genotypes of the pup and three-candidate mothers at the four loci unambiguously identified CM2 as the mother; no allelic mismatches were observed between CM2 and the pup, whereas CM1 and CM3 were clearly excluded by allelic mismatches at three of the four loci (). Despite the collectively high allelic diversity and heterozygosity of these four markers in the source population (Chapman et al. 2004
), the pup was uniformly homozygous for one of its mother's alleles. The composite pup microsatellite genotype strongly supports the absence of paternal genetic contribution (i.e. asexual reproduction occurred) for two reasons. First, the pup had no unique (paternal) alleles at these four loci. Second, the probability of the observed homozygous genotype at all four loci assuming biparental reproduction is vanishingly small (p
) given the population rarity of alleles possessed by the pup at two of the loci (Sti
01 (allele 187, expected population homozygote frequency 0.0009), Sti
10 (allele 304, expected population homozygote frequency 0.002)). Furthermore, although the probability of biparental allelic inheritance is not theoretically eliminated (i.e. extremely small but not zero), none of the wild 119 S. tiburo
screened were homozygous at all four loci. In addition, any such theoretically possible individuals would be expected to exhibit homozygous combinations of the most common alleles in the source population, rather than some of the rare ones seen in this pup.
Table 1 Genotypes of the three S. tiburo candidate mothers (CM1–3) and pup at four microsatellite loci. (CM1 and CM3 are excluded as the mother by allelic mismatches at three of the four loci (non-bold) in each case. CM2 is the mother of the pup, as shown (more ...)
AFLP fingerprinting analysis also confirmed the identity of the CM2 as the mother because it shared a higher percentage of AFLP fragments with the pup (84%) than did the other two females (less than 69%; not shown). More importantly, all AFLP fragments observed in the pup were also found in CM2, with no evidence of any unique paternal bands. Finally, 16% of the bands observed in the mother were absent in the pup, which is consistent with the complete homozygosity observed in the pup's composite microsatellite genotype. Based on these observations, the alternative hypothesis that the pup's very unusual, all homozygous microsatellite composite genotype coupled with an absence of non-maternal AFLP fragments could have resulted from sexual reproduction is extremely improbable.
The pup's homozygosity at all four microsatellite loci and reduced number of AFLP fragments compared with its mother is consistent with an automictic rather than an apomictic parthenogenetic pathway. Automixis also produces homozygosity for sex chromosomes, and the documented cases in vertebrates (birds and reptiles) all have heterogametic females (ZW), and so only produce viable ZZ males and an equal proportion of inviable WW zygotes (Olsen 1975
; Schuett et al. 1997
). The contrasting heterogametic male system (XX females, XY males) should only produce viable females by automixis. The female sex of the S. tiburo
pup is therefore consistent with automixis and female homogamety (XX) in carcharhiniform sharks as proposed from karyotyping (Maddock & Schwartz 1996
With this discovery of parthenogenesis in a cartilaginous fish, asexual reproduction has now been demonstrated in all major jawed vertebrate lineages except mammals (Spurway 1953
; Olsen 1975
; Schuett et al. 1997
; this study), where its absence is due to genomic imprinting. The maternal and paternal genomes in the mammalian zygote are imprinted and differentially expressed, thus both genomes are required for normal foetal development (Kono 2006
). This imprinting is believed to have evolved in response to conflicts that develop between the embryonic maternal and paternal genomes with regard to maternal resource allocation in lineages where there is a direct maternal–embryonic connection, such as a placenta (Moore & Haig 1991
; Haig 2004
). The same intergenomic conflict and selection for imprinting could reasonably be hypothesized to operate in placental sharks with their long evolutionary history of this mode of development (Hamlett & Koob 1999
; Feldheim et al. 2004
). Our finding of successful parthenogenesis in the placentally viviparous S. tiburo
argues that genomic imprinting in this species is absent, or at least does not occur to the extent that development of a gynogenetic embryo is prevented. This observation raises questions about whether genomic imprinting is absent in sharks generally, despite relatively common placental viviparity in this lineage. Given the wide range of reproductive modes from oviparity to placental viviparity in elasmobranchs (Hamlett & Koob 1999
), further investigation into the occurrence of parthenogenesis across this lineage could provide valuable insights into the role of reproductive mode in the evolution of genome imprinting.
Parthenogenesis is difficult to detect in ordinarily sexually reproducing vertebrate species, and its prevalence and potential effects on population genetic diversity are poorly understood. Our results suggest that accumulating cases of female sharks producing healthy offspring in the absence of males (Castro et al. 1988
; Voss et al. 2001
; Heist 2004
) warrant genetic evaluation to determine how common asexual reproduction, especially automixis, is among these ancient fishes. In some of these cases, females have produced several viable offspring over multiple reproductive cycles (Castro et al. 1988
; D. Sweet 2005, Detroit Aquarium personal communication), suggesting that parthenogenesis may be facultative in situations where female sharks have difficulty encountering suitable mates (e.g. a possibility in the wild due to low population densities caused by overexploitation or in emerging captive breeding programmes for endangered sharks). A similar recent discovery of automictic parthenogenesis in Komodo dragons (Varanus komodoensishas
) raised concerns about the possible negative effects of this form of asexual reproduction on the genetic diversity in small natural or captive populations of this and other endangered reptiles (Watts et al. 2006
). Our finding for a shark extends the known evolutionary occurrence of automictic parthenogenesis to a major basal vertebrate lineage, indicating that these concerns about the conservation of genetic diversity could apply to threatened species over a much broader range of vertebrate taxa.