Journal of Heredity 2004:95(4):291-300
© 2004 The American Genetic Association
Low Genetic Variability in the Highly Endangered Mediterranean Monk Seal
From the Department of Animal Biology, University of Barcelona, Diagonal 645, 08028 Barcelona, Spain (Pastor and Aguilar); Department of Integrative Biology, University of California, Berkeley, CA 94720-3160 (Garza); Southwest Fisheries Science Center, Santa Cruz Laboratory, 110 Shaffer Rd., Santa Cruz, CA 95060 (Garza); and Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK (Allen and Amos).
Address correspondence to Teresa Pastor at the address above, or e-mail: teresa_pastor{at}ub.edu.
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Abstract |
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Genetic variability is an important component in the ability of populations to adapt in the face of environmental change. Here we report the first description of nuclear genetic variability in the only remaining sizable colony of the Mediterranean monk seal (Monachus monachus), located at Cap Blanc (Western Sahara, Mauritania), whose estimated size during the study period (1994–May 1997) was about 320 individuals. We tested 42 microsatellite loci isolated from five pinniped species in a sample of 52 pups. Three loci failed to give any product, and of the remaining 39, only 15 were polymorphic, with a maximum of 3 alleles detected. Three loci appeared to be X-linked. No departures from Hardy-Weinberg equilibrium were detected and no genetic structure was found between the two nursing caves currently occupied by the seals. Several analytical methods show that, as a consequence of a severe bottleneck, the population has suffered a decrease in genetic variability over the last few centuries.
The Mediterranean monk seal (Monachus monachus) is one of the world's most endangered mammals (IUCN 1996). Historically the species was apparently fairly common and its range included both the Mediterranean and Black seas, islands in the Atlantic Ocean, and the coast of northwest Africa south to Senegal. Today the world population is estimated at only 300–400 individuals, divided into three main geographic areas: the Greek islands, Cyprus, and the Turkish coast (200–250 individuals, scattered in small groups); the Mediterranean coastline of Morocco and western Algeria (10–20 individuals); and the Atlantic Ocean, including the Madeira archipelago and the northwest coast of Africa (100–130 individuals, after a mass mortality event in June 1997) (Aguilar 1999).
The species' decline began due to intense hunting by the early Greek, Roman, and Byzantine civilizations in the Mediterranean region (Johnson and Lavigne 1999) and by the action of Spanish and Portuguese sealers during the 15th–18th centuries in the Atlantic Ocean (Marchessaux 1989). The few surviving colonies have been further reduced by other human-related pressures, such as habitat destruction, reduction of fishing stocks, pollution, and deliberate aggression by fishermen (Aguilar 1999). The largest colony remaining today, located on the peninsula of Cap Blanc, on the west coast of Africa (Western Sahara, Mauritania), was subject to a mass mortality event in 1997 in which 80% of the adult population died (Forcada et al. 1999). The severity of that mortality event, coupled with the low reproductive rate of females in the colony (Gazo et al. 1999), raises the question of whether genetic effects related of small population size, such as inbreeding depression, are operating.
Since 1973 the species has been the focus of numerous studies and recovery plans aimed at preventing further declines (González 1996). However, none of these plans have incorporated genetic information, primarily because of the lack of adequate data, which is largely due to the inaccessibility of monk seal haul-out sites. For example, at Cap Blanc, almost all seals haul out in one of two caves with access only from the sea, which makes the collection of samples very difficult. There has been only one previous genetic study of the species, which examined sequence variation in the normally highly variable mitochondrial DNA (mtDNA) control region (350–444 bp) from skin and hair of carcasses (Harwood et al. 1996; Stanley and Harwood 1997). In their work (n = 18 sequences), only one mtDNA genotype was detected. However, it is difficult to draw general conclusions based solely on this finding, because mtDNA is maternally inherited and has a smaller effective population size than nuclear markers. Small populations may therefore lose mitochondrial variability while retaining significant nuclear diversity (Avise et al. 1985).
Here we examine levels of nuclear genetic variability in the Mediterranean monk seal using a set of 42 microsatellite loci assayed in 52 animals from the Cap Blanc population. We compare this variation with that found in other pinniped species and discuss possible causes for the low levels of variation observed.
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Materials and Methods |
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Sample Collection and DNA Extraction
From 1994 to May 1997, we collected skin samples from 52 pups (37 while tagging and 15 from carcasses) of the population inhabiting the peninsula of Cap Blanc (Western Sahara, Mauritania). This is the only sizeable colony of the species remaining today (Figure 1) since monk seals in the Mediterranean live in small groups, but no longer form colonies. Most pups were sampled in the two main breeding caves of the colony, known as cave 1 (n = 15) and cave 3 (n = 20) (Gazo et al. 1999). Gender was determined by observation of the genital area or by coloration pattern, which is sexually dimorphic (Badosa et al. 1998). Samples were collected before the mass mortality event that occurred in June 1997 (see below). Capture-recapture estimates of the size of the colony during the sample collection period indicate that it was composed of approximately 320 individuals, including adults and juveniles (Forcada et al. 1999).
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Three female-fetus pairs collected during the mass mortality event were also analyzed to confirm Mendelian segregation and to check for the presence of null alleles, which are known to segregate in other mammalian populations (Callen et al. 1993; Pemberton et al. 1995). These three adult females, as well as other adults sampled during the study period, were not included in the analysis of population genetics to restrict the survey to samples from a single generation.
Tissue samples were preserved in 20% dimethyl sulfoxide (DMSO) saturated with salt and, once in the laboratory, frozen until analysis. DNA was extracted using a protocol involving proteinase K digestion, followed by phenol/chloroform extractions and a final precipitation with ethanol (Sambrook et al. 1989).
Microsatellite Markers
We tested a panel of 42 pairs of microsatellite primers (Table 1), originally isolated in the grey seal (Halichoerus grypus), harbor seal (Phoca vitulina), northern elephant seal (Mirounga angustirostris), southern elephant seal (Mirounga leonina), and South American fur seal (Arctocephalus australis). Primers were initially tested on samples from 10 Mediterranean monk seals. Loci that revealed polymorphism in this limited set were used to genotype the entire sample. Polymerase chain reactions (PCRs) were carried out using end-labeled primers following the protocol described in Garza et al. (1995). PCR products were separated by electrophoresis on 6% acrylamide gels and visualized by autoradiography. Alleles were sized by comparison with either grey or harbor seal samples of known genotype or with a standard M13 sequence.
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Genetic Variation Analysis
Some of the amplified loci are known to be sex linked in other pinnipeds. Consequently attention was paid to the patterns of variability in the two sexes. Loci in which all males (n = 23) were homozygous while at least 5 of the 29 females were heterozygous show a significant deviation from random expectations and were considered sex linked. Genetic variability was assessed as the proportion of polymorphic loci and the mean number of alleles per locus (allelic diversity). Observed heterozygosity, Ho, was compared with the unbiased estimate of heterozygosity expected under Hardy-Weinberg assumptions, He (Nei 1978). Possible departures from Hardy-Weinberg equilibrium were examined by calculating exact significance probabilities following the procedure described by Louis and Dempster (1987). Complete enumeration could be used because a maximum of three alleles was detected at any locus. Males were excluded from these calculations for the loci presumed to be X-linked. Linkage disequilibria were evaluated using an exact test, with sequential Bonferroni correction for multiple comparisons. For detection of genic and genotypic differences between pups born in the two caves, exact tests were carried out following Raymond and Rousset (1995a). The above analyses were performed using the GenePop software package (Raymond and Rousset 1995b).
Bottleneck Detection
We assessed the genetic consequences of population size reduction (or bottlenecks) using three methods. The first is a graphical examination of allele frequencies described by Luikart et al. (1998). In a population of constant size, many microsatellite alleles should be rare. In contrast, a recently bottlenecked population is expected to show fewer rare alleles, as they are lost most quickly. A histogram of the proportion of alleles in a dataset at different frequencies should thus reveal a deficit of rare alleles, or a "mode shift." Although this qualitative method is not a proper statistical test, it can potentially identify bottlenecked populations (Luikart et al. 1998).
We also employed two quantitative methods to detect the occurrence of a bottleneck. The first is the heterozygosity excess method described by Cornuet and Luikart (1996) and implemented in the Bottleneck software package (Piry et al. 1997). This method exploits the fact that allelic diversity is reduced faster than heterozygosity during a bottleneck, because rare alleles are lost rapidly and have little effect on heterozygosity, thus producing a transient excess in heterozygosity relative to that expected in a population of constant size with the same number of alleles (Cornuet and Luikart 1996). Several statistical tests have been proposed to evaluate such differences, and we applied the Wilcoxon signed-rank test, as it does not require a large number of polymorphic loci, which are scarce in a population with low variability. Moreover, in a recent comparative analysis of statistical methods, the Wilcoxon signed-rank test performed better in identifying bottlenecked populations (Maudet et al. 2002). As is appropriate for microsatellites, we carried out 5000 replicates and assumed that all loci follow the two-phase mutation model (TPM), in which most mutations are one-step, but a small percentage (5–10%) are multistep (Di Rienzo et al. 1994).
The second quantitative method examines allele frequency distribution for gaps. In a population of constant size, most allele frequency distributions are expected to be more or less continuous, and the range in allele size, measured in repeat units, will be similar to the number of alleles. So in a population of constant size, the mean ratio M, the number of alleles/(range in allele size + 1), will be close to one (Garza and Williamson 2001). However, during a population size reduction, alleles are lost and, since they are not always the smallest or largest, gaps appear in the allele frequency distributions. As a result, the number of alleles decreases more rapidly than the range in allele size and M decreases (Garza and Williamson 2001). A simulation-based statistical test is used to evaluate the significance of the observed value through comparison with 10,000 simulated datasets from populations at equilibrium using the program M_P_Val (see Garza and Williamson 2001). When 15 variable loci are assayed and conservative assumptions about the mutation process are made, a value of M 
0.71 indicates that the population under study has experienced a recent reduction in size. M ratios were also calculated for the grey and harbor seal populations for comparative purposes.
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Results |
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Genetic Variation Analysis
Of the initial 42 microsatellite primer pairs tested in 10 samples (Table 1), only 3 (SGPv5, MA1.9, and TBPv1) failed to yield amplification products. For the other loci, one or two bands around the expected size were found. Of these 39 loci, 24 were monomorphic (61%), while 15 were polymorphic (39%) in the 10 samples. The number of alleles present for the polymorphic loci and their frequencies in the complete sample (N = 52) are represented graphically in Figure 2. Of the 15 polymorphic loci, 10 had two alleles and 5 had three alleles. Mean allelic diversity was 1.50 (±0.06) when all 39 loci were considered and 2.33 (±0.13) when only the 15 polymorphic loci were taken into account (Table 2). Three loci—SGPv17, Pvc63, and Pvc74—were found to be sex linked, with autosomal segregation rejected at P = 10–4, 10–14, 10–8, respectively. Two of these loci (SGPv17 and Pvc74) are known to be X-linked in other pinniped species (Coltman et al. 1996; Gemmell et al. 1997). The third locus, Pvc63, was surveyed by Coltman et al. (1996), but they did not have enough statistical power to detect sex linkage.
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For comparative purposes, the number of polymorphic loci and the allelic diversity in five other phocid species, for the same set of 24 microsatellite loci, are shown in Table 3. The estimated census size of the sampled populations for the five species is indicated. In general, allele sizes in the sample were similar to those described for the same loci in other pinniped species (Coltman et al. 1996). Moreover, some loci which were monomorphic in the source species (e.g., Pv9, Pv16, Pv17, and Pvc26) were polymorphic in the Mediterranean monk seal. However, the overall allelic diversity of the Mediterranean monk seal, together with that of the Hawaiian monk seal and that of the northern elephant seal, is among the lowest recorded (Table 3). However, despite the smallest census size, the Mediterranean monk seal has greater variability, both in terms of the number of polymorphic loci and allelic diversity, than either of these two species.
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Observed (Ho) and expected (He) heterozygosities are displayed in Table 2. Ho and He were highly correlated (r2 = 0.95), so we used the latter in our analyses, as it is considered to be less biased (Nei and Roychoudhury 1974). Estimates of He for the polymorphic loci ranged from 0.07 to 0.65, with a mean He per polymorphic locus of 0.41 (±0.05). Mean He for all 39 loci was 0.16 (±0.23). No deviation from Hardy-Weinberg proportions was detected for any of the loci (Table 2). No null alleles were detected at any locus in the three mother-fetus pairs analyzed. Linkage disequilibrium was detected between two pairs of loci: Hg3.6 and Pvc78 (P <.05) and Hg6.3 and MA11C (P <.05), which is less than the number of significant results expected by chance alone given the number of tests performed. No differences were detected for either genic (p = 0.75, SE = 0.002) or genotypic frequencies (p = 0.74, SE = 0.002) of pups born in the two different caves.
Bottleneck Detection
The graphical representation of the allele frequencies for all 15 polymorphic loci (Figure 3) shows a deficit of rare alleles (i.e., frequency less than 0.1) causing a mode-shift distortion, as is expected for recently bottlenecked populations, instead of an L-shaped distribution, typical of populations in equilibrium (Luikart et al. 1998). A statistically significant departure from mutation drift-equilibrium was detected with the heterozygosity excess method: 11 of 15 loci had an excess of heterozygosity compared with that of a population of the same size and same number of alleles at equilibrium (Wilcoxon signed-rank test: P =.00513; one tailed for heterozygosity excess) (Table 2).
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Allele size distributions were not continuous (Figure 2). While several loci had alleles that were separated by a single repeat unit, most had alleles that were separated by several repeats. M ratios are presented in Table 2. Mean ratios for current grey and harbor seal populations were close to 1 (0.89 for both), as predicted for populations that have not suffered a recent bottleneck. In contrast, the average ratio for the Mediterranean monk seal was much lower (0.66). This value falls below the critical value (0.71) that indicates that a population has suffered a recent reduction in size.
Mean estimated He is 0.16 or 0.41, if calculated with all loci or only polymorphic ones, respectively (Table 2). If it is assumed that the Mediterranean monk seal initially had a mean heterozygosity of 0.75, as found in other pinniped populations with no evidence of a bottleneck, such as the grey seal from North Rona (He = 0.75 for 8 polymorphic loci) (Allen et al. 1995) or the Atlantic walrus (He = 0.76 for 10 polymorphic loci) (Buchanan et al. 1998), the observed value then suggests that the Mediterranean monk seal has suffered a heterozygosity loss of approximately 53%. This is probably an underestimate, as it considers only polymorphic loci, and the proportion of monomorphic loci is much higher in the monk seal.
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Discussion |
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We present here the first study of nuclear genetic variability in the Mediterranean monk seal. All but 3 of 42 microsatellite primer sets (93%), isolated from five other pinniped species, produced specific PCR products. This high level of conservation of primer sites is consistent with that found in previous studies of related species, including pinnipeds (Coltman et al. 1996; Gemmell et al. 1997), and is most likely due to the monophyletic origin and relatively recent divergence of the major pinniped taxa (Arnason et al. 1995).
However, the level of microsatellite variation (maximum of three alleles) is among the lowest reported for any mammal. Similar levels have only been found in a few severely bottlenecked species, such as the northern hairy-nosed wombat (Lasiorhinus krefftii) (Taylor et al. 1994) or the Kangaroo Island population of koalas (Phascolarctos cinereus) (Houlden et al. 1996). Among pinnipeds, the Mediterranean monk seal also has among the lowest variability, together with the Hawaiian monk seal (Gemmell et al. 1997; Kretzmann et al. 2001) and the northern elephant seal (Garza 1998), two species that have gone through well-documented reductions in population size. But despite having the smallest population size, the amount of variability both in terms of number of polymorphic loci and allelic diversity found in Mediterranean monk seals is still higher than in the other two species.
Cross-species comparisons should be interpreted with caution because ascertainment bias may potentially produce higher values of polymorphism in source species (Ellegren et al. 1999). However, this effect should be negligible in our study because we did not use only those loci with high variability in the source species. We assayed all available loci, including those that were monomorphic in the source species, which in some cases proved to be polymorphic in the Mediterranean monk seal. Moreover, other nonsource pinniped species analyzed with the same loci had higher levels of polymorphism than the Mediterranean monk seal, and sometimes than the source species, even when fewer animals were examined (Garza 1998; Gemmell et al. 1997). Furthermore, the low M ratios (e.g., gaps in allele frequency distribution) also suggest that ascertainment bias is not the cause of the low variation, as they indicate that the assayed loci have recently lost variation, not that they are inherently low in variation due to slow evolution and/or small effective sizes.
Moreover, microsatellite mutation rates are generally of the same order of magnitude among mammals (Dallas 1992; Ellegren 1995; Weber and Wong 1993). The observed differences are due to properties of the particular loci sampled and not different mechanisms affecting the entire genome. In the Mediterranean monk seal, the finding that variability is low at many independent nuclear loci (present study), as well as in the mitochondrial genome (Harwood et al. 1996), also contradicts the idea that the species is inherently low in variation due to low genome-wide mutation rates.
Another possible explanation is small effective size in the absence of a bottleneck, which can result from strong population subdivision, high variance in reproductive success for one or both sexes, and/or low absolute numbers (Hartl and Clark 1989). Population subdivision might exist, for example, if animals always return to reproduce in the caves where they were born. However, we did not detect any genetic difference between pups from cave 1 and cave 3, the two main breeding sites. This is consistent with our observation of the same females giving birth in alternate years in both caves, which are located only 1.1 km apart. High variance in reproductive success might be due to a strongly polygynous mating system, as observed in other pinnipeds (Riedman 1990), which can greatly reduce the number of males contributing to reproduction. However, despite the uncertainties about the mating system of the Mediterranean monk seal, preliminary analysis of genetic relatedness indicates that reproduction is not dominated by a single male or a small number of males (Pastor T, et al., unpublished data).
The most likely cause of the low levels of genetic variability observed is the demographic history and current small population size of the colony at Cap Blanc, estimated at about 320 during the sample period (1994–1997) (Forcada et al. 1999). Moreover, the Cap Blanc colony is effectively isolated from the animals in other remaining sites. The nearest Atlantic breeding group is located at the Desertas Islands (Madeira Archipelago), approximately 1300 km away (Figure 1). The most recent census (1998) at this site found only 16 animals (Neves and Pires 1998).
While historical population sizes are difficult to estimate, the fact that the Mediterranean monk seal was once heavily exploited for oil and pelts (Marchessaux 1989) suggests that the species was relatively abundant. Fifteenth century Portuguese hunters reported as many as 5000 animals in the Western Sahara region (Monod 1932). Although the accuracy of such estimates is uncertain, the current population size estimate (see above) suggests that the colony is about 3% of its historic size.
Much of the population size reduction probably occurred during sealing operations in the 15th and 16th centuries (Marchessaux 1989). Afterwards, despite several expeditions to the area, references to seals do not reappear until the beginning of the 20th century. These references indicate that during at least the last 80 years, the population has been very small and has been subject to various mortality factors such as human persecution (Marchessaux 1989), collapse of inhabited caves (Trotignon 1979), and a mass mortality event attributed to either a morbillivirus epizootic (Osterhaus et al. 1997) or a toxic algae bloom (Hernández et al. 1998). It is possible that similar unrecorded events during the past 500 years have also contributed to the reduction in a population already diminished by sealing operations.
The present study supports the idea that a decrease in population size is the main factor accounting for the low levels of variability observed. The three methods applied to detect recent population reductions—mode-shift distortion, heterozygosity excess, and M ratio—all indicate a recent reduction in effective population size. Genetic depletion due to a drastic decrease in population size greatly influences the amount of variation found within a population, independent of its current size, because the generation of genetic variability is slow, even for sequences with high mutation rates (Avise 1994). Thus species that have undergone a bottleneck in the past and have recovered their numbers, such as the northern elephant seal, may not have regained the levels of genetic variability that would be expected from their current population abundance (Bonnell and Selander 1974; Garza 1998).
Moreover, purifying selection associated with inbreeding depression can further contribute to lower population numbers and levels of genetic variability, one of the so-called vortices of extinction (Gilpin and Soulé 1986). We recorded the occurrence of several stillborn pups and other instances of perinatal mortality with no apparent external cause, suggesting that some females might be producing offspring with congenital problems. Nevertheless, we did not observe any deviation from Hardy-Weinberg proportions, which would indicate that mating between relatives is low or that there is not sufficient variability in our data to detect them. Another possibility would be that mating between relatives is so detrimental that the pups are not brought to term, and thus are not sampled or included in the calculations. Both possibilities are consistent with the low reproductive rate observed for females in this colony (Gazo et al. 1999).
Small populations are generally considered to be susceptible to a number of genetic problems that can compromise long-term survival. Inbreeding and low levels of genetic variability have been associated with low fitness in populations (Jimenez et al. 1994; Keller et al. 1994; Madsen et al. 1996). Genetic erosion has also been suggested to reduce the genetic resources of populations to overcome the effect of infectious or other disease agents (O'Brien and Evermann 1988). Given this context, it is possible that low genetic variability played a role in the severity of the mass mortality event that affected the Cap Blanc colony in 1997. Further research is needed to clarify the role of genetic effects on the future demographic trajectory of this colony. This work should include an evaluation of genetic distance between the Cap Blanc and other remnant monk seal colonies, with consideration of population translocation of individual seals. Finally, detailed information on parentage and survival would help to answer the question of whether inbreeding depression is already operating in this population.
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Acknowledgments |
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We thank all those who assisted in the field work. We are also indebted to researchers who donated primers: T. Bert, D. Coltman, N. Gemmell, and S. Goodman. J. Forcada provided useful comments to the manuscript. The European Commission LIFE projects B4-3200/94/0000/D2 and B4-3200/96/510 funded this study. T. Pastor had a grant from the Vicerectorat de Recerca of the University of Barcelona. P. Allen was funded by NERC (grant GR3/10084). The samples used for this study were supplied by the BMA (Banco Medio Ambiental) with the support of the Pew Fellows Program in Marine Conservation and Earthtrust.
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Footnotes |
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Corresponding Editor: C. Scott Baker
Received June 17, 2003
Accepted March 30, 2004
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