Genetic evaluation of the three captive mexican wolf lineages

The Mexican gray wolf appears to be extinct in the wild and exists now only in captivity. There are three captive putative Mexican wolf lineages: Certified, Ghost Ranch, and Aragón lineages. From pedigree analysis, the Certified lineage has the lowest level of inbreeding and has retained the most founder alleles. In contrast, the Ghost Ranch has a high level of inbreeding and fewest founder alleles. From molecular genetics data, particularly from microsatellite loci, it appears that all of the lineages consist of Mexican wolves, and none of them appear to have ancestry from dogs or coyotes. Further, the molecular data are consistent with the Certified lineage having three founders instead of the four previously assumed. From these findings, we recommend that the three lineages be combined to increase the number of founders and to postpone any inbreeding depression. The combination of pedigree analysis and molecular genetic data allowed us to resolve several long-standing concerns in Mexican wolves. Zoo Biol 16:47–69, 1997. © 1997 Wiley-Liss, Inc.     

Captive breeding and the reintroduction of Mexican and red wolves

Mexican and red wolves were both faced with extinction in the wild until captive populations were established more than two decades ago. These captive populations have been successfully managed genetically to minimize mean kinship and retain genetic variation. Descendants of these animals were subsequently used to start reintroduced populations, which now number about 40-50 Mexican wolves in Arizona and New Mexico and about 100 red wolves in North Carolina. The original captive Mexican wolf population was descended from three founders. Merging this lineage with two other captive lineages, each with two founders, has been successfully carried out in the captive population and is in progress in the reintroduced population. This effort has resulted in increased fitness of cross-lineage wolves, or genetic rescue, in both the captive and reintroduced populations. A number of coyote-red wolf hybrid litters were observed in the late 1990s in the reintroduced red wolf population. Intensive identification and management efforts appear to have resulted in the elimination of this threat. However, population reintroductions of both Mexican and red wolves appear to have reached numbers well below the generally recommended number for recovery and there is no current effort to re-establish other populations.     

Molecular genetics of the most endangered canid: the Ethiopian wolf Canis simensis

The world's most endangered canid is the Ethiopian wolf Canis simensis , which is found in six isolated areas of the Ethiopian highlands with a total population of no more than 500 individuals. Ethiopian wolf populations are declining due to habitat loss and extermination by humans. Moreover, in at least one population, Ethiopian wolves are sympatric with domestic dogs, which may hybridize with them, compete for food, and act as disease vectors. Using molecular techniques, we address four questions concerning Ethiopian wolves that have conservation implications. First, we determine the relationships of Ethiopian wolves to other wolf-like canids by phylogenetic analysis of 2001 base pairs of mitochondrial DNA (mtDNA) sequence. Our results suggest that the Ethiopian wolf is a distinct species more closely related to gray wolves and coyotes than to any African canid. The mtDNA sequence similarity with gray wolves implies that the Ethiopian wolf may hybridize with domestic dogs, a recent derivative of the gray wolf. We examine this possibility through mtDNA restriction fragment analysis and analysis of nine microsatellite loci in populations of Ethiopian wolves. The results imply that hybridization has occurred between female Ethiopian wolves and male domestic dogs in one population. Finally, we assess levels of variability within and between two Ethiopian wolf populations. Although these closely situated populations are not differentiated, the level of variability in both is low, suggesting long-term effective population sizes of less than a few hundred individuals. We recommend immediate captive breeding of Ethiopian wolves to protect their gene pool from dilution and further loss of genetic variability.     

Y-chromosome microsatellite variation in Italian wolves: A contribution to the study of wolf-dog hybridization patterns

One major concern in wolf (Canis lupus) conservation is the risk of genetic contamination due to crossbreeding with domestic dogs. Although genetic monitoring of wolf populations has become widely used, the behavioural mechanisms involved in wolf-dog hybridization and the detrimental effects of genetic introgression are poorly known. In this study we analysed Y-chromosome microsatellite variation in the recovering Italian wolf population and detected strikingly different allele frequencies between wolves and dogs. Four Y haplotypes were found in 74 analysed male wolves, and all of them were present in a focus wolf population in the Apennines. On the other hand, only 1 haplotype was found in the recolonizing wolf population from the Western Alps. The most common haplotype in a sample of domestic dogs, was also found in 5 wolves, 2 of which revealing a signature of recent hybridization. Moreover, another suspect hybrid carried a private haplotype of possible canine origin. These results give support to the idea that female wolves can breed with male stray dogs in the wild. The Y-chromosome variation in Italian wolves contrasts with the previously observed lack of mitochondrial variation. Further investigations are needed to clarify at what extent historical or recent wolf-dog hybridization events may have contributed to the observed haplotype diversity. In conclusion, the two molecular markers employed in this study represent effective means to trace directional genetic introgression into the wolves male lineage and have the noteworthy advantage of being suitable for analyses on low-quality DNA samples.     

Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species

Captive breeding has become an important tool in species conservation programmes. Current management strategies for ex situ populations are based on theoretical models, which have mainly been tested in model species or assessed using studbook data. During recent years an increasing number of molecular genetic studies have been published on captive populations of several endangered species. However, a comprehensive analysis of these studies is still outstanding. Here, we present a review of the published literature on ex situ conservation genetics with a focus on molecular studies. We analysed 188 publications which either presented empirical studies using molecular markers (105), studbook analyses (26), theoretical work (38), or tested the genetic effects of management strategies using model species (19). The results show that inbreeding can be minimized by a thorough management of captive populations. There seems to be a minimum number of founders (15) and a minimum size of a captive population (100) necessary in order to minimize a loss of genetic diversity. Optimally, the founders should be unrelated and new founders should be integrated into the captive population successively. We recommend that genetic analyses should generally precede and accompany ex situ conservation projects in order to avoid inbreeding and outbreeding depression. Furthermore, many of the published studies do not provide all the relevant parameters (founder size, captive population size, H_o, H_e, inbreeding coefficients). We, therefore, propose that a general standard for the presentation of genetic studies should be established, which would allow integration of the data into a global database.     

Genetic evidence for multiple events of hybridization between wolves and domestic dogs in the Iberian Peninsula

Hybridization between wild species and their domestic counterparts may represent a major threat to natural populations. However, high genetic similarity between the hybridizing taxa makes the detection of hybrids a difficult task and may hinder attempts to assess the impact of hybridization in conservation biology. In this work, we used a combination of 42 autosomal microsatellites together with Y-chromosome microsatellite-defined haplotypes and mtDNA sequences to investigate the occurrence and dynamics of wolf-dog hybridization in the Iberian Peninsula. To do this, we applied a variety of Bayesian analyses and a parallel set of simulation studies to evaluate (i) the differences between Iberian wolves and dogs, (ii) the frequency and geographical distribution of hybridization and (iii) the directionality of hybridization. First, we show that Iberian wolves and dogs form two well-differentiated genetic entities, suggesting that introgressive hybridization is not a widespread phenomenon shaping both gene pools. Second, we found evidence for the existence of hybridization that is apparently restricted to more peripheral and recently expanded wolf populations. Third, we describe compelling evidence suggesting that the dynamics of hybridization in wolf populations is mediated by crosses between male dogs and female wolves. More importantly, the observation of a population showing the occurrence of a continuum of hybrid classes forming mixed packs may indicate that we have underestimated hybridization. If future studies confirm this pattern, then an intriguing avenue of research is to investigate how introgression from free-ranging domestic dogs is enabling wolf populations to adapt to the highly humanized habitats of southern Europe while still maintaining their genetic differentiation.     

Rodrigues fruit bats (<Emphasis Type="Italic">Pteropus rodricensis</Emphasis>, Megachiroptera: Pteropodidae) retain genetic diversity despite population declines and founder events

Fruit bats of the genus Pteropus are important contributors to ecosystem maintenance on islands through their roles as pollinators and seed dispersers. However, island faunas are the most prone to extinction and there is a real need to assess the possible genetic implications of population reductions in terms of extinction risk. An effective method of ameliorating extinction risk in endangered species is the establishment of captive populations ex situ. The effectiveness of captive breeding programmes may be assessed by comparing the genetic variability of captive colonies to that of wild counterparts. Here, we use polymorphic microsatellite loci to assess genetic variability in wild, critically endangered Rodrigues fruit bats (Pteropus rodricensis, Dobson 1878) and we compare this variability to that in a captive colony. We document remarkable conservation of genetic variability in both the wild and captive populations, despite population declines and founder events. Our results demonstrate that the wild population has withstood the negative effects of population reductions and that captive breeding programmes can fulfil the goals of retaining genetic diversity and limiting inbreeding.     

Using microsatellite diversity in wild Anegada iguanas (<Emphasis Type="Italic">Cyclura pinguis</Emphasis>) to establish relatedness in a captive breeding group of this critically endangered species

Awareness of the genealogical relationships between founder animals in captive breeding programs is essential for the selection of mating pairs that maintain genetic diversity. If captive founder relationships are unknown they can be inferred using genetic data from wild populations. Here, we report the results of such an analysis for six Cyclura pinguis (Sauria: Iguanidae) acquired as adults in 1999 by the San Diego Zoo Institute for Conservation Research to begin a captive breeding program for this critically endangered species. The six founder animals were reportedly hatched in captivity from eggs collected on Anegada in 1985. No records exist, however, as to where on Anegada the eggs were collected or from how many nests they originated. To assist determination of genealogical relationships, we genotyped the six captive founders, their first six offspring, and 33 wild adult iguanas from Anegada at 23 informative microsatellite loci. With these data, we estimated allele frequencies among the wild samples and then estimated the relatedness of the captive population. Using likelihood inference, we determined that three closely related pairs exist among the six captive founders and that each pair is not closely related to the other two. In addition, we were able to assign parentage for all six of the founders’ offspring tested, one of which had been previously misdiagnosed. Using the assigned parentage and inferred relatedness of the six founders, we calculated mean kinship for each of the six founders and their five living offspring. Finally, based on the allelic diversity of the wild iguanas sampled, we conclude that the C. pinguis population on Anegada is not excessively inbred; however, further investigation is warranted.     

Effects of inbreeding on reproductive success,performance, litter size,and survival in captive red wolves (Canis rufus)

Captive‐breeding programs have been widely used in the conservation of imperiled species, but the effects of inbreeding, frequently expressed in traits related to fitness, are nearly unavoidable in small populations with few founders. Following its planned extirpation in the wild, the endangered red wolf (Canis rufus) was preserved in captivity with just 14 founders. In this study, we evaluated the captive red wolf population for relationships between inbreeding and reproductive performance and fitness. Over 30 years of managed breeding, the level of inbreeding in the captive population has increased, and litter size has declined. Inbreeding levels were lower in sire and dam wolves that reproduced than in those that did not reproduce. However, there was no difference in the inbreeding level of actual litters and predicted litters. Litter size was negatively affected by offspring and paternal levels of inbreeding, but the effect of inbreeding on offspring survival was restricted to a positive influence. There was no apparent relationship between inbreeding and method of rearing offspring. The observable effects of inbreeding in the captive red wolf population currently do not appear to be a limiting factor in the conservation of the red wolf population. Additional studies exploring the extent of the effects of inbreeding will be required as inbreeding levels increase in the captive population. Zoo Biol 29:36–49, 2010. © 2009 Wiley‐Liss, Inc.     

Genetic Variability of the Grey Wolf Canis lupus in the Caucasus in Comparison with Europe and the Middle East: Distinct or Intermediary Population?

Despite continuous historical distribution of the grey wolf (Canis lupus) throughout Eurasia, the species displays considerable morphological differentiation that resulted in delimitation of a number of subspecies. However, these morphological discontinuities are not always consistent with patterns of genetic differentiation. Here we assess genetic distinctiveness of grey wolves from the Caucasus (a region at the border between Europe and West Asia) that have been classified as a distinct subspecies C. l. cubanensis. We analysed their genetic variability based on mtDNA control region, microsatellite loci and genome-wide SNP genotypes (obtained for a subset of the samples), and found similar or higher levels of genetic diversity at all these types of loci as compared with other Eurasian populations. Although we found no evidence for a recent genetic bottleneck, genome-wide linkage disequilibrium patterns suggest a long-term demographic decline in the Caucasian population – a trend consistent with other Eurasian populations. Caucasian wolves share mtDNA haplotypes with both Eastern European and West Asian wolves, suggesting past or ongoing gene flow. Microsatellite data also suggest gene flow between the Caucasus and Eastern Europe. We found evidence for moderate admixture between the Caucasian wolves and domestic dogs, at a level comparable with other Eurasian populations. Taken together, our results show that Caucasian wolves are not genetically isolated from other Eurasian populations, share with them the same demographic trends, and are affected by similar conservation problems.     

The genetic legacy of extirpation and re-colonization in Vancouver Island wolves

Hybridization between wild and domestic species is of conservation concern because it can result in the loss of adaptations and/or disappearance of a distinct taxon. Wolves from Vancouver Island, British Columbia (Canada), have been subject to several eradication campaigns during the twentieth century and were considered virtually extirpated between 1950 and 1970. In this study, we use control region mitochondrial DNA sequences and 13 autosomal microsatellite loci to characterize Vancouver Island wolves as well as dogs from British Columbia. We observe a turnover in the haplotypes of wolves sampled before and after the 1950–1970 period, when there was no permanent wolf population on the island, supporting the probable local extinction of wolves on Vancouver Island during this time, followed by re-colonization of the island by wolves from mainland British Columbia. In addition, we report the presence of a domestic dog mtDNA haplotype in three individuals eliminated in 1986 that were morphologically identified as wolves. Here we show that Vancouver Island wolves were also identified as wolves based on autosomal microsatellite data. We attribute the hybridization event to the episodically small size of this population during the re-colonization event. Our results demonstrate that at least one female hybrid offspring, resulting from a cross of a male wolf and a female dog or a female hybrid pet with dog mtDNA, successfully introgressed into the wolf population. No dog mtDNA has been previously reported in a population of wild wolves. Genetic data show that Vancouver Island wolves are distinct from dogs and thus should be recognized as a population of wild wolves. We suggest that the introgression took place due to the Allee effect, specifically a lack of mates when population size was low. Our findings exemplify how small populations are at risk of hybridization.     

SNPs in ecological and conservation studies: a test in the Scandinavian wolf population

Single nucleotide polymorphisms (SNPs) have the potential to become the genetic marker of choice in studies of the ecology and conservation of natural populations because of their capacity to access variability across the genome. In this study, we provide one of the first demonstrations of SNP discovery in a wild population in order to address typical issues of importance in ecology and conservation in the recolonized Scandinavian and neighbouring Finnish wolf Canis lupus populations. Using end sequence from BAC (bacterial artificial chromosome) clones specific for dogs, we designed assays for 24 SNP loci, 20 sites of which had previously been shown to be polymorphic in domestic dogs and four sites were newly identified as polymorphic in wolves. Of the 24 assayed loci, 22 SNPs were found to be variable within the Scandinavian population and, importantly, these were able to distinguish individual wolves from one another (unbiased probability of identity of 4.33 x 10(-8)), providing equivalent results to that derived from 12 variable microsatellites genotyped in the same population. An assignment test shows differentiation between the Scandinavian and neighbouring Finnish wolf populations, although not all known immigrants are accurately identified. An exploration of the misclassification rates in the identification of relationships shows that neither 22 SNP nor 20 microsatellite loci are able to discriminate across single order relationships. Despite the remaining obstacle of SNP discovery in nonmodel organisms, the use of SNPs in ecological and conservation studies is encouraged by the advent of large scale screening methods. Furthermore, the ability to amplify extremely small fragments makes SNPs of particular use for population monitoring, where faecal and other noninvasive samples are routinely used.     

Bucking the Trend in Wolf-Dog Hybridization: First Evidence from Europe of Hybridization between Female Dogs and Male Wolves

Studies on hybridization have proved critical for understanding key evolutionary processes such as speciation and adaptation. However, from the perspective of conservation, hybridization poses a concern, as it can threaten the integrity and fitness of many wild species, including canids. As a result of habitat fragmentation and extensive hunting pressure, gray wolf (Canis lupus) populations have declined dramatically in Europe and elsewhere during recent centuries. Small and fragmented populations have persisted, but often only in the presence of large numbers of dogs, which increase the potential for hybridization and introgression to deleteriously affect wolf populations. Here, we demonstrate hybridization between wolf and dog populations in Estonia and Latvia, and the role of both genders in the hybridization process, using combined analysis of maternal, paternal and biparental genetic markers. Eight animals exhibiting unusual external characteristics for wolves - six from Estonia and two from Latvia - proved to be wolf-dog hybrids. However, one of the hybridization events was extraordinary. Previous field observations and genetic studies have indicated that mating between wolves and dogs is sexually asymmetrical, occurring predominantly between female wolves and male dogs. While this was also the case among the Estonian hybrids, our data revealed the existence of dog mitochondrial genomes in the Latvian hybrids and, together with Y chromosome and autosomal microsatellite data, thus provided the first evidence from Europe of mating between male wolves and female dogs. We discuss patterns of sexual asymmetry in wolf-dog hybridization.     

Hybridization between wolf and domestic dog: First evidence from an endangered population in central Portugal

Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended.     

Genetic characterization of grey wolves (<Emphasis Type="Italic">Canis lupus</Emphasis> L. <Emphasis Type="Italic">1758</Emphasis>) from Bosnia and Herzegovina: implications for conservation

The grey wolves of Bosnia and Herzegovina form a subpopulation of the Dinaric-Balkan wolf population and represent one of Europe’s least studied wolf populations. Since the Dinaric-Balkan population is a valuable source of genetic diversity for neighboring populations, comprehensive assessments are warranted. We aimed to determine the genetic variability and structure of the grey wolf population from Bosnia and Herzegovina, as well as estimate levels of gene flow and inbreeding and evaluate genetic signals of a bottleneck. To do this, we analyzed the variability of eighteen microsatellite loci. We found moderately high genetic heterozygosity for wolves from Bosnia and Herzegovina, as described for other Dinaric-Balkan wolf populations. We reveal weak genetic structuring with two genetic clusters identified. Wolves from the eastern part of the region formed a relatively distinct cluster, whereas individuals in the second cluster overlapped quite considerably with admixed individuals. Despite the signal of genetic structure being weak, clustering of individuals from the eastern part of the country extended through all analyses. Thus, this cluster could be considered a separate management unit, perhaps requiring specific conservation attention.     

Life‐history traits and geographical divergence in wild rice (Oryza rufipogon) gene pool in Indochina Peninsula region

Indochina Peninsula is the primary centre of diversity of rice and lies partly in the centre of origin of cultivated rice (Oryza sativa) where the wild ancestor (Oryza rufipogon) is still abundant. The wild gene pool is potentially endangered by urbanisation and the expansion of agriculture, and by introgression hybridisation with locally cultivated rice varieties. To determine genetic diversity and structure of the wild rice of the region we genotyped nearly 1000 individuals using 20 microsatellite loci. We found ecological differentiation in 48 populations, distinguishable by their life‐history traits and the country of origin. Geographical divergence was suggested by isolation of the perennial Myanmar populations from those of Cambodia, Laos and Thailand. The annual types would be most likely to have lost genetic variation because of genetic drift and inbreeding. The growing of cultivated and wild rice together, however, gives ample opportunities for hybridisation, which already shows signs of genetic mixing, and will ultimately lead to replacement of the original wild rice gene pool. For conservation we suggest that wild rice should be conserved ex situ in order to prevent introgression from cultivated rice, along with in situ conservation in individual countries for the recurrent evolutionary process through local adaptation, but with sufficient isolation from cultivated rice fields to preserve genetic integrity of the wild populations.     

Genetic structure of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens and its implications for the management of threatened island populations

The Red‐headed Wood Pigeon Columba janthina nitens is endemic to the Ogasawara Islands, an oceanic island chain located 1000 km south of the main islands of Japan. The subspecies is at high risk of extinction because of its small population size and restricted habitat range. We undertook genetic analyses of this pigeon using sequences of a portion of the mitochondrial control region and five microsatellite markers to estimate the genetic characteristics of two wild populations from the Bonin and Volcano Islands, as well as one captive breeding population. The genetic diversity of the wild individuals was exceptionally low in both the mitochondria (nucleotide diversity = 0.00105) and at the microsatellite (3.2 alleles per locus and H_E = 0.12) loci. Higher numbers of microsatellite genotypes were observed in the Volcano Islands population than in the Bonin Islands population, which may be because of the relatively low impact of human disturbance. The most common mitochondrial haplotypes and microsatellite alleles observed in the two wild populations were completely fixed in the captive population. Our results suggest that the genetic diversity of the captive population needs to be increased. However, introduction of a wild individual into a captive population can lead to a decreased genetic diversity in the wild population and therefore should be done with caution. The genetic differentiation between the Bonin and the Volcano island groups was low, and the populations of the two island groups should be regarded as a single evolutionarily significant unit. However, special consideration is required for habitat conservation in the Volcano Islands, which may be functioning as a sanctuary for the Red‐headed Wood Pigeon. For the long‐term conservation of threatened bird species that live on remote oceanic islands, determination of management units considering gene flow caused by their flying capacity and maintenance of genetically suitable wild and captive populations are essential.     

Looking back to go forward: genetics informs future management of captive and reintroduced populations of the black-footed rock-wallaby <Emphasis Type="Italic">Petrogale lateralis</Emphasis>

Active management is essential to the survival of many threatened species globally. Captive breeding programmes can play an important role in facilitating the supplementation, translocation and reintroduction of wild populations. However, understanding the genetic dynamics within and among wild and captive populations is crucial to the planning and implementation of ex situ management, as adaptive potential is, in part, driven by genetic diversity. Here, we use 14 microsatellite loci and mitochondrial Control Region sequence to examine the population genetics of both wild populations and captive colonies of the endangered warru (the MacDonnell Ranges race of the black-footed rock-wallaby Petrogale lateralis) in central Australia, to understand how historical evolutionary processes have shaped current diversity and ensure effective ex situ management. Whilst microsatellite data reveal significant contemporary differentiation amongst remnant warru populations, evidence of contemporary dispersal and relatively weak isolation by distance, as well as a lack of phylogeographic structure suggests historical connectivity. Genetic diversity within current captive populations is lower than in the wild source populations. Based on our genetic data and ecological observations, we predict outbreeding depression is unlikely and hence make the recommendation that captive populations be managed as one genetic group. This will increase genetic diversity within the captive population and as a result increase the adaptive potential of reintroduced populations. We also identify a new site in the Musgrave Ranges which contains unique alleles but also connectivity with a population 6 km away. This novel genetic diversity could be used as a future source for supplementation.