Use of microsatellite data and pedigree information in the genetic management of two long-term salmon conservation programs

In this paper, we describe the utility of microsatellite data and genetic pedigree information to guide the genetic management of two long-term conservation programs for endangered populations of salmon: Snake River Sockeye Salmon, Oncorhynchus nerka, and inner Bay of Fundy Atlantic Salmon, Salmo salar. Both programs are captive broodstock (live gene banking) programs for endangered populations of salmon. In order for these programs to be successful for recovery efforts, genetic change, including accumulation of inbreeding, loss of genetic variation, and adaptation to captivity, must be minimized. We provide an overview of each program, describe broodstock selection and pairing for spawning, and discuss how pedigree data are being used to evaluate different management practices. While there are inherent species and programmatic differences, both of these programs use widely accepted genetic conservation strategies (minimize mean kinship, reduce variance in family size, minimize inbreeding in the next generation, maintain large census and effective population size) to potentially mitigate some unintended side-effects associated with the rearing of small populations in captivity. These case studies highlight the benefits and practical limitations of applying these strategies in the genetic management of salmon, and may be used to inform other conservation programs.     

Evaluation of the genetic management of the endangered black‐footed ferret (Mustela nigripes)

Empirical support for the genetic management strategies employed by captive breeding and reintroduction programs is scarce. We evaluated the genetic management plan for the highly endangered black‐footed ferret (Mustela nigripes) developed by the American Zoo and Aquarium Associations (AZA) as a part of the species survival plan (SSP). We contrasted data collected from five microsatellite loci to predictions from a pedigree‐based kinship matrix analysis of the captive black‐footed ferret population. We compared genetic diversity among captive populations managed for continued captive breeding or reintroduction, and among wild‐born individuals from two reintroduced populations. Microsatellite data gave an accurate but only moderately precise estimate of heterozygosity. Genetic diversity was similar in captive populations maintained for breeding and release, and it appears that the recovery program will achieve its goal of maintaining 80% of the genetic diversity of the founder population over 25 years. Wild‐born individuals from reintroduced populations maintained genetic diversity and avoided close inbreeding. We detected small but measurable genetic differentiation between the reintroduced populations. The model of random mating predicted only slightly lower levels of heterozygosity retention compared to the SSP strategy. The random mating strategy may be a viable alternative for managing large, stable, captive populations such as that of the black‐footed ferret. Zoo Biol 22:287–298, 2003. © 2003 Wiley‐Liss, Inc.     

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.     

Partitioning kin groups of broodstock in the critically endangered Chinese sturgeon,Acipenser sinensis Gray 1835

Pedigrees of broodstock with unknown relationship of the critically endangered Chinese sturgeon, Acipenser sinensis, was evaluated using microsatellite markers to facilitate genetic management in restocking programs with small broodstock size. We characterized the distributions of relatedness values to reconstruct kin groups in four hatchery families with known pedigrees using microsatellites. The distributions of relatedness values for kin classes were used for partitioning full sibling groups of wild A. sinensis broodstock kept in two hatcheries, resulted in 13 full sibling clusters, four of which containing 62% of all the wild individuals. This indicates high probability of choosing close related breeder pairs in random mating, thus selective breeding is necessary to minimize inbreeding and maintain genetic diversity. This study provides a useful tool for genetic management in conservation programs of A. sinensis in aim of preserving self‐sustained wild populations.     

LANDSCAPE GENOMICS IN ATLANTIC SALMON (SALMO SALAR): SEARCHING FOR GENE–ENVIRONMENT INTERACTIONS DRIVING LOCAL ADAPTATION

A growing number of studies are examining the factors driving historical and contemporary evolution in wild populations. By combining surveys of genomic variation with a comprehensive assessment of environmental parameters, such studies can increase our understanding of the genomic and geographical extent of local adaptation in wild populations. We used a large‐scale landscape genomics approach to examine adaptive and neutral differentiation across 54 North American populations of Atlantic salmon representing seven previously defined genetically distinct regional groups. Over 5500 genome‐wide single nucleotide polymorphisms were genotyped in 641 individuals and 28 bulk assays of 25 pooled individuals each. Genome scans, linkage map, and 49 environmental variables were combined to conduct an innovative landscape genomic analysis. Our results provide valuable insight into the links between environmental variation and both neutral and potentially adaptive genetic divergence. In particular, we identified markers potentially under divergent selection, as well as associated selective environmental factors and biological functions with the observed adaptive divergence. Multivariate landscape genetic analysis revealed strong associations of both genetic and environmental structures. We found an enrichment of growth‐related functions among outlier markers. Climate (temperature–precipitation) and geological characteristics were significantly associated with both potentially adaptive and neutral genetic divergence and should be considered as candidate loci involved in adaptation at the regional scale in Atlantic salmon. Hence, this study significantly contributes to the improvement of tools used in modern conservation and management schemes of Atlantic salmon wild populations.     

Strategies for maintaining genetic diversity in captive populations through reproductive technology

The maintenance of genetic diversity in captive populations is a primary goal of captive breeding plans, and it is becoming increasingly apparent that reproductive technology has much to offer captive breeding programs in attaining this goal. Reproductive technology can best assist captive breeding programs in this task by developing strategies that effectively increase the genetic contribution of new wild founders to a population as well as increase the reproductive life span of existing founders and their close descendents. This will act to reduce genetic drift and inbreeding effects in the population and thereby minimize the loss of genetic diversity. Considering only one aspect of reproductive technology, semen collection, this paper examines some of the genetic considerations that might be used for choosing which males in a population to collect semen from, assuming the goal of the captive breeding program is the preservation of genetic diversity. It is shown that semen collection and preservation, with future intent of artificial insemination, can make significant contributions to the maintenance of genetic diversity if careful consideration is given to the selection of donor males. Finally, the pedigree of the captive population of Asian lions (Panthera leo persica) is used to illustrate some of these genetic concepts that might be important in selecting males as semen donors.     

Genetic variability,management, and conservation implications of the critically endangered Brazilian pitviper Bothrops insularis

Information on demographic, genetic, and environmental parameters of wild and captive animal populations has proven to be crucial to conservation programs and strategies. Genetic approaches in conservation programs of Brazilian snakes remain scarce despite their importance for critically endangered species, such as Bothrops insularis, the golden lancehead, which is endemic to Ilha da Queimada Grande, coast of São Paulo State, Brazil. This study aims to (a) characterize the genetic diversity of ex situ and in situ populations of B. insularis using heterologous microsatellites; (b) investigate genetic structure among and within these populations; and (c) provide data for the conservation program of the species. Twelve informative microsatellites obtained from three species of the B. neuwiedi group were used to access genetic diversity indexes of ex situ and in situ populations. Low‐to‐medium genetic diversity parameters were found. Both populations showed low—albeit significant—values of system of mating inbreeding coefficient, whereas only the in situ population showed a significant value of pedigree inbreeding coefficient. Significant values of genetic differentiation indexes suggest a small differentiation between the two populations. Discriminant analysis of principal components (DAPC) recovered five clusters. No geographic relationship was found in the island, suggesting the occurrence of gene flow. Also, our data allowed the establishment of six preferential breeding couples, aiming to minimize inbreeding and elucidate uncertain parental relationships in the captive population. In a conservation perspective, continuous monitoring of both populations is demanded: it involves the incorporation of new individuals from the island into the captive population to avoid inbreeding and to achieve the recommended allelic similarity between the two populations. At last, we recommend that the genetic data support researches as a base to maintain a viable and healthy captive population, highly genetically similar to the in situ one, which is crucial for considering a reintroduction process into the island.     

Contrasting patterns of gene diversity between microsatellites and mitochondrial SNPs in farm and wild Atlantic salmon

Levels of genetic variability at 12 microsatellite loci and 19 single nucleotide polymorphisms in mitochondrial DNA were studied in four farm strains and four wild populations of Atlantic salmon. Within populations, the farm strains showed significantly lower allelic richness and expected heterozygosity than wild populations at the 12 microsatellite loci, but a significantly higher genetic variability with respect to observed number of haplotypes and haplotype diversity in mtDNA. Significant differences in allele- and haplotype-frequencies were observed between farm strains and wild populations, as well as between different farm strains and between different wild populations. The large genetic differentiation at mitochondrial DNA between wild populations (F_ST = 0.24), suggests that the farm strains attained a high mitochondrial genetic variability when created from different wild populations seven generations ago. A large proportion of this variability remains despite an expected lower effective population size for mitochondrial than nuclear DNA. This is best explained by the particular mating schemes in the breeding programmes, with 2–4 females per male. Our observations suggest that for some genetic polymorphisms farm populations might currently hold equal or higher genetic variability than wild populations, but lower overall genetic variability. In the short-term, genetic interactions between escaped farm salmon and wild salmon might increase genetic variability in wild populations, for some, but not most, genetic polymorphisms. In the long term, further losses of genetic variability in farm populations are expected for all genetic polymorphisms, and genetic variability in wild populations will be reduced if escapes of farm salmon continue.     

Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program

The main goal of ex situ conservation programs is to improve the chances of long term survival of natural populations by founding and managing captive colonies that can serve as a source of individuals for future reintroductions or to reinforce existing populations. The degree in which a captive breeding program has captured the genetic diversity existing in the source wild population has seldom been evaluated. In this study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison. Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indicating that no substantial loss of diversity has occurred in the captive-breeding program. The effective number of founders of the program was estimated as ∼ ∼16, suggesting that all founders in the studbook pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to distinguish wolves, dogs and their hybrids.     

Development and testing of a genetic marker‐based pedigree reconstruction system ‘PR‐genie’ incorporating size‐class data

For wildlife populations, it is often difficult to determine biological parameters that indicate breeding patterns and population mixing, but knowledge of these parameters is essential for effective management. A pedigree encodes the relationship between individuals and can provide insight into the dynamics of a population over its recent history. Here, we present a method for the reconstruction of pedigrees for wild populations of animals that live long enough to breed multiple times over their lifetime and that have complex or unknown generational structures. Reconstruction was based on microsatellite genotype data along with ancillary biological information: sex and observed body size class as an indicator of relative age of individuals within the population. Using body size‐class data to infer relative age has not been considered previously in wildlife genealogy and provides a marked improvement in accuracy of pedigree reconstruction. Body size‐class data are particularly useful for wild populations because it is much easier to collect noninvasively than absolute age data. This new pedigree reconstruction system, PR‐genie, performs reconstruction using maximum likelihood with optimization driven by the cross‐entropy method. We demonstrated pedigree reconstruction performance on simulated populations (comparing reconstructed pedigrees to known true pedigrees) over a wide range of population parameters and under assortative and intergenerational mating schema. Reconstruction accuracy increased with the presence of size‐class data and as the amount and quality of genetic data increased. We provide recommendations as to the amount and quality of data necessary to provide insight into detailed familial relationships in a wildlife population using this pedigree reconstruction technique.     

Inbreeding, heterozygosity and fitness in a reintroduced population of endangered African wild dogs (Lycaon pictus)

It is crucial to understand the genetic health and implications of inbreeding in wildlife populations, especially of vulnerable species. Using extensive demographic and genetic data, we investigated the relationships among pedigree inbreeding coefficients, metrics of molecular heterozygosity and fitness for a large population of endangered African wild dogs (Lycaon pictus) in South Africa. Molecular metrics based on 19 microsatellite loci were significantly, but modestly correlated to inbreeding coefficients in this population. Inbred wild dogs with inbreeding coefficients of ??0.25 and subordinate individuals had shorter lifespans than outbred and dominant contemporaries, suggesting some deleterious effects of inbreeding. However, this trend was confounded by pack-specific effects as many inbred individuals originated from a single large pack. Despite wild dogs being endangered and existing in small populations, findings within our sample population indicated that molecular metrics were not robust predictors in models of fitness based on breeding pack formation, dominance, reproductive success or lifespan of individuals. Nonetheless, our approach has generated a vital database for future comparative studies to examine these relationships over longer periods of time. Such detailed assessments are essential given knowledge that wild canids can be highly vulnerable to inbreeding effects over a few short generations.     

一个可用于复杂大群体的可视化系谱绘制和分析软件

对于在遗传研究和家系研究中大的系谱结构图还很难分析。系谱的绘制通常是遗传性状的分析研究的第一步。系图可以反映整个群体的结构、每个个体之间的相互关系以及基因流的走向,便于理解遗传性状的本质。因为所用家系数目的增大和复杂性的增加,绘制1个清晰的系谱有时变得十分困难。因此开发了1种名为Pedigraph软件,可以解决这个问题。Pedigraph能够完成对于大的复杂的群体的系谱绘制工作,并能进行相应的系谱分析。初步的测试表明这个软件在研究动植物的遗传育种中是1个有用的工具,同时它也可以用于人类的群体和历史等方面的研究。     

Genetic management of captive prosimian populations,a report of experience withLoris tardigradus

Extinction of small, closed populations in captivity as well as in the wild is believed to be nearly inevitable, because inbreeding will adversely effect reproductive success, mortality, sex ratio and also the susceptibility to epidemic diseases and environmental stress. An ever increasing number of primate species exist only in small isolated populations, which contain only a part of the original genetic variability. In captive breeding programs research about genetic management strategies is, therefore, of essential importance. In 1980 we imported 9Loris tardigrdus nordicus (4 females, 5 males) from NE-Sri Lanka. The founders came from one natural breeding population. All sexual mature females are breeding. Up to now the colony contains 36 living individuals. The main goal of our long-term genetic management plan was to minimize inbreeding and to preserve the genetic diversity. Therefore, we try to pass the founder bottleneck rapidly by enlarging the population to a desired minimum population size of 25 pairs and to equalize the founder representation within any generation. The need to control the spread of sublethal genes, introduced by one of the founders, conflicts directly with the aim of equalizing the founder representation. A solution of this problem is discussed. To produce a sufficiently large population we intend to give animals to other institutions and to build up an exchange-system for offspring individuals, which should lead to an international studbook.     

Effects of species, culture history, size and residency on relative competitive ability of salmonids

The relative competitive ability of juvenile farm and wild salmonids was investigated to provide insight into the potential effects of introduction of cultured salmon on wild Pacific salmonid ( Oncorhynchus ) species. Aquarium experiments involving equal contests ( i.e. size matched, simultaneously introduced individuals) indicated that two wild coho salmon Oncorhynchus kisutch populations were competitively equal to a farm coho salmon population. In equal contests between farm Atlantic salmon Salmo salar (Mowi strain) and these wild coho salmon populations or coastal cutthroat trout Oncorhynchus clarki clarki , Atlantic salmon were subordinate in all cases. When Atlantic salmon were given a residence advantage, however, they were competitively equal to both wild coho salmon populations, but remained subordinate to coastal cutthroat trout. When Atlantic salmon were given a 10–30% length advantage, they were competitively equal to one wild coho salmon population but remained subordinate to the other. In equal contests in semi-natural stream channels, both wild coho and farm Atlantic salmon grew significantly more in the presence of the other species than when alone. It appears that coho salmon obtain additional food ration by out competing Atlantic salmon, whereas Atlantic salmon were stimulated to feed more in the presence of coho salmon competitors. These results suggest that wild coho salmon and cutthroat trout should out compete farm Atlantic salmon of a similar size in nature. As the relative competitive ability of Atlantic salmon improves when they have a size and residence advantage, should feral populations become established, they may exist on a more equal competitive footing owing to the long freshwater residence of Atlantic salmon.     

Pedigrees and microsatellites among endangered ungulates: what do they tell us?

Relationships between pedigree coefficients of inbreeding and molecular metrics are generally weak, suggesting that measures of heterozygosity estimated using microsatellites may be poor surrogates of genome-wide inbreeding. We compare three endangered species of gazelles ( Gazella ) with different degrees of threat in their natural habitats, for which captive breeding programmes exist. For G. dorcas, the species with the largest founding population, the highest and most recent number of founding events, the correlation between pedigree coefficient of inbreeding and molecular metrics was higher than for outbred populations of mammals, probably because it has both higher mean f and variance. For the two species with smaller founding populations, conventional assumptions about founders, i.e. outbred and unrelated, are unrealistic. When realistic assumptions about the founders were made, clear relationships between pedigree coefficients of inbreeding and molecular metrics were revealed for G. cuvieri. This population had a small founding population, but it did experience admixture years later; thus, the relationship between inbreeding and molecular metrics in G. cuvieri is very similar to the expected values but lower than in G. dorcas . In contrast, no relationship was found for G. dama mhorr which had a much smaller founding population than had been previously assumed, which probably had high levels of inbreeding and low levels of genetic variability, and no admixture. In conclusion, the strength of the association between pedigree coefficient of inbreeding and molecular metrics among endangered species depends on the level of inbreeding and genetic variability present in the founding population, its size and its history.     

Genetic assessment of a conservation breeding program of the houbara bustard (Chlamydotis undulata undulata) in Morocco,based on pedigree and molecular analyses

Protection and restoration of species in the wild may require conservation breeding programs under genetic management to minimize deleterious effects of genetic changes that occur in captivity, while preserving populations' genetic diversity and evolutionary resilience. Here, through interannual pedigree analyses, we first assessed the efficiency of a 21-year genetic management, including minimization of mean kinship, inbreeding avoidance, and regular addition of founders, of a conservation breeding program targeting on Houbara bustard (Chlamydotis undulata undulata) in Morocco. Secondly, we compared pedigree analyses, the classical way of assessing and managing genetic diversity in captivity, to molecular analyses based on seven microsatellites. Pedigree-based results indicated an efficient maintenance of the genetic diversity (99% of the initial genetic diversity retained) while molecular-based results indicated an increase in allelic richness and an increase in unbiased expected heterozygosity across time. The pedigree-based average inbreeding coefficient F remained low (between 0.0004 and 0.003 in 2017) while the proportion of highly inbred individuals (F > .1) decreased over time and reached 0.2% in 2017. Furthermore, pedigree-based F and molecular-based individual multilocus heterozygosity were weakly negatively correlated, (Pearson's r = −.061 when considering all genotyped individuals), suggesting that they cannot be considered as alternatives, but rather as complementary sources of information. These findings suggest that a strict genetic monitoring and management, based on both pedigree and molecular tools can help mitigate genetic changes and allow to preserve genetic diversity and evolutionary resilience in conservation breeding programs.     

Determinants of hierarchical genetic structure in Atlantic salmon populations: environmental factors vs. anthropogenic influences

Disentangling the effects of natural environmental features and anthropogenic factors on the genetic structure of endangered populations is an important challenge for conservation biology. Here, we investigated the combined influences of major environmental features and stocking with non‐native fish on the genetic structure and local adaptation of Atlantic salmon (Salmo salar) populations. We used 17 microsatellite loci to genotype 975 individuals originating from 34 French rivers. Bayesian analyses revealed a hierarchical genetic structure into five geographically distinct clusters. Coastal distance, geological substrate and river length were strong predictors of population structure. Gene flow was higher among rivers with similar geologies, suggesting local adaptation to geological substrate. The effect of river length was mainly owing to one highly differentiated population that has the farthest spawning grounds off the river mouth (up to 900 km) and the largest fish, suggesting local adaptation to river length. We detected high levels of admixture in stocked populations but also in neighbouring ones, implying large‐scale impacts of stocking through dispersal of non‐native individuals. However, we found relatively few admixed individuals suggesting a lower fitness of stocked fish and/or some reproductive isolation between wild and stocked individuals. When excluding stocked populations, genetic structure increased as did its correlation with environmental factors. This study overall indicates that geological substrate and river length are major environmental factors influencing gene flow and potential local adaptation among Atlantic salmon populations but that stocking with non‐native individuals may ultimately disrupt these natural patterns of gene flow among locally adapted populations.     

Alternative mating strategies in Atlantic salmon and brown trout

By screening variable number of tandem repeat (VNTR) loci, multiple paternity within clutches has been found in wild populations of southern European Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). For Atlantic salmon, we determined the relative contribution of alternative male phenotypes to the next generation. Individual males that are morphologically juvenile yet sexually mature fertilized a large proportion of eggs, and they thereby contributed to an increase of genetic variability in wild populations via (1) balancing the sex ratio, (2) increasing outbreeding, and (3) enlarging the effective population size, in part a consequence of (1) and (2). In addition, these precocious males ensured that interspecific spawns involving Atlantic salmon females and brown trout males (a fairly common occurrence in southern Europe where the two species are sympatric) resulted mostly in Atlantic salmon progeny. For brown trout, preliminary genetic results indicated that multiple paternity, when present, was not due to alternative mating strategies by males, but rather to successive fertilizations by adult suitors.