The use of marker‐based relationship information to estimate the heritability of body weight in a natural population: a cautionary tale

A number of procedures have been developed that allow the genetic parameters of natural populations to be estimated using relationship information inferred from marker data rather than known pedigrees. Three published approaches are available; the regression, pair‐wise likelihood and Markov Chain Monte Carlo (MCMC) sib‐ship reconstruction methods. These were applied to body weight and molecular data collected from the Soay sheep population of St. Kilda, which has a previously determined pedigree. The regression and pair‐wise likelihood approaches do not specify an exact pedigree and yielded unreliable heritability estimates, that were sensitive to alteration of the fixed effects. The MCMC method, which specifies a pedigree prior to heritability estimation, yielded results closer to those determined using the known pedigree. In populations of low average relationship, such as the Soay sheep population, determination of a reliable pedigree is more useful than indirect approaches that do not specify a pedigree.     

Pedigree reconstruction in wild cichlid fish populations

It is common practice to use microsatellites to detect parents and their offspring in wild and captive populations, in order to reconstruct a pedigree. However, correct inference is often constrained by a number of factors, including the absence of demographic data and ignorance regarding the completeness of parental sampling. Here we present a new Bayesian estimator that simultaneously estimates the pedigree and the size of the unsampled population. The method is robust to genotyping error, and can estimate pedigrees in the absence of demographic data. Using a large-scale microsatellite assay in four wild cichlid fish populations of Lake Tanganyika (1000 individuals in total), we assess the performance of the Bayesian estimator against the most popular assignment program, Cervus. We found small but significant pedigrees in each of the tested populations using the Bayesian procedure, but Cervus had very high type I error rates when the size of the unsampled population was assumed to be lower than what it was. The need of pedigree relationships to infer adaptive processes in natural populations places strong constraints on sampling design and identification of multigenerational pedigrees in natural populations.     

Indirect genetic estimates of breeding population size in the polyploid green sturgeon (Acipenser medirostris)

The utility of genetic measures for kinship reconstruction in polysomic species is not well evaluated. We developed a framework to test hypotheses about estimating breeding population size indirectly from collections of outmigrating green sturgeon juveniles. We evaluated a polysomic dataset, in allelic frequency and phenotypic formats, from green sturgeon to describe the relationship among known progeny from experimental families. The distributions of relatedness values for kin classes were used for reconstructing green sturgeon pedigrees from juveniles of unknown relationship. We compared three rarefaction functions that described the relationship between the number of kin groups and number of samples in a pedigree to estimate the annual abundance of spawners contributing to the threatened green sturgeon Southern Distinct Population Segment in the upper Sacramento River. Results suggested the estimated abundance of breeding green sturgeon remained roughly constant in the upper Sacramento River over a 5‐year period, ranging from 10 to 28 individuals depending on the year and rarefaction method. These results demonstrate an empirical understanding for the distribution of relatedness values among individuals is a benefit for assessing pedigree reconstruction methods and identifying misclassification rates. Monitoring of rare species using these indirect methods is feasible and can provide insight into breeding and ontogenetic behaviour. While this framework was developed for specific application to studying fish populations in a riverscape, the framework could be advanced to improve genetic estimation of breeding population size and to identify important breeding habitats of rare species when combined with finer‐scaled sampling of offspring.     

Pedigree analysis for the genetic management of group‐living species

Captive breeding programs are an important tool for the conservation of endangered species. These programs are commonly managed using pedigrees containing information about the history of each individual's family, such as breeding pairs and parentage. However, there are some species that are kept in groups where it is hard to distinguish between particular individuals within the group, making it very difficult to record any information at an individual level. Currently, software and methods commonly used for registering and analyzing pedigrees to help manage populations at an individual level are not adequate for managing these group‐living species. Therefore, there is a need to further develop these tools and methodologies for pedigree analysis to better manage group‐living species. PMx is a program used for the management of ex situ populations in zoos and aquariums. We adapted the pedigree analysis method implemented in PMx to analyze pedigrees (records of descendant lineages) of group‐living species. In addition, we developed a group pedigree data entry sheet and group2PMx, a converter program that enables group datasets to be imported into PMx. We show how pedigree analysis of a group‐living species can be used for population management using the studbook of the endangered Texas blind cave salamander Eurycea rathbuni. Such analyses of the pedigree of groups can improve the management of group‐living species in ex situ breeding programs. Firstly, it enables better management decisions based on more accurate genetic measures between groups, allowing for greater control of inbreeding. Secondly, it can improve the conditions in which group‐living species are held by adapting husbandry practices to better reflect conditions of these species living in the wild. The use of the spreadsheet and group2PMx extends the application of PMx_, allowing conservation managers and other institutions outside the zoo and aquarium community to easily import and analyze their pedigree data.     

The influence of nonrandom extra‐pair paternity on heritability estimates derived from wild pedigrees

Quantitative genetic analysis is often fundamental for understanding evolutionary processes in wild populations. Avian populations provide a model system due to the relative ease of inferring relatedness among individuals through observation. However, extra‐pair paternity (EPP) creates erroneous links within the social pedigree. Previous work has suggested this causes minor underestimation of heritability if paternal misassignment is random and hence not influenced by the trait being studied. Nevertheless, much literature suggests numerous traits are associated with EPP and the accuracy of heritability estimates for such traits remains unexplored. We show analytically how nonrandom pedigree errors can influence heritability estimates. Then, combining empirical data from a large great tit (Parus major) pedigree with simulations, we assess how heritability estimates derived from social pedigrees change depending on the mode of the relationship between EPP and the focal trait. We show that the magnitude of the underestimation is typically small (<15%). Hence, our analyses suggest that quantitative genetic inference from pedigrees derived from observations of social relationships is relatively robust; our approach also provides a widely applicable method for assessing the consequences of nonrandom EPP.     

Large‐scale parentage analysis reveals reproductive patterns and heritability of spawn timing in a hatchery population of steelhead (Oncorhynchus mykiss)

Understanding life history traits is an important first step in formulating effective conservation and management strategies. The use of artificial propagation and supplementation as such a strategy can have numerous effects on the supplemented natural populations and minimizing life history divergence is crucial in minimizing these effects. Here, we use single nucleotide polymorphism (SNP) genotypes for large‐scale parentage analysis and pedigree reconstruction in a hatchery population of steelhead, the anadromous form of rainbow trout. Nearly complete sampling of the broodstock for several consecutive years in two hatchery programmes allowed inference about multiple aspects of life history. Reconstruction of cohort age distribution revealed a strong component of fish that spawn at 2 years of age, in contrast to programme goals and distinct from naturally spawning steelhead in the region, which raises a significant conservation concern. The first estimates of variance in family size for steelhead in this region can be used to calculate effective population size and probabilities of inbreeding, and estimation of iteroparity rate indicates that it is reduced by hatchery production. Finally, correlations between family members in the day of spawning revealed for the first time a strongly heritable component to this important life history trait in steelhead and demonstrated the potential for selection to alter life history traits rapidly in response to changes in environmental conditions. Taken together, these results demonstrate the extraordinary promise of SNP‐based pedigree reconstruction for providing biological inference in high‐fecundity organisms that is not easily achievable with traditional physical tags.     

Reconstructing sibling relationships in wild populations

Reconstruction of sibling relationships from genetic data is an important component of many biological applications. In particular, the growing application of molecular markers (microsatellites) to study wild populations of plant and animals has created the need for new computational methods of establishing pedigree relationships, such as sibgroups, among individuals in these populations. Most current methods for sibship reconstruction from microsatellite data use statistical and heuristic techniques that rely on a priori knowledge about various parameter distributions. Moreover, these methods are designed for data with large number of sampled loci and small family groups, both of which typically do not hold for wild populations. We present a deterministic technique that parsimoniously reconstructs sibling groups using only Mendelian laws of inheritance. We validate our approach using both simulated and real biological data and compare it to other methods. Our method is highly accurate on real data and compares favorably with other methods on simulated data with few loci and large family groups. It is the only method that does not rely on a priori knowledge about the population under study. Thus, our method is particularly appropriate for reconstructing sibling groups in wild populations.     

spip 1.0: a program for simulating pedigrees and genetic data in age‐structured populations

We present the program spip for simulating multilocus genetic data on individuals in age‐structured populations. In addition to genetic data on sampled individuals, the pedigree connecting all individuals in the population is recorded. This allows investigation of the relationship between family structure and population parameters. We foresee that spip will be useful for evaluating multilocus estimators of pairwise relatedness and population structure, and for simulating the distribution of relatedness in populations with varying demographies. It also provides a method for simulating genetic drift in complex populations.     

Are shed hair genomes the most effective noninvasive resource for estimating relationships in the wild?

1. Knowledge of relationships in wild populations is critical for better understanding mating systems and inbreeding scenarios to inform conservation strategies for endangered species. To delineate pedigrees in wild populations, study genetic connectivity, study genotype‐phenotype associations, trace individuals, or track wildlife trade, many identified individuals need to be genotyped at thousands of loci, mostly from noninvasive samples. This requires us to (a) identify the most common noninvasive sample available from identified individuals, (b) assess the ability to acquire genome‐wide data from such samples, and (c) evaluate the quality of such genome‐wide data, and its ability to reconstruct relationships between animals within a population.
2. We followed identified individuals from a wild endangered tiger population and found that shed hair samples were the most common compared to scat samples, opportunistically found carcasses, and opportunistic invasive samples. We extracted DNA from these samples, prepared whole genome sequencing libraries, and sequenced genomes from these.
3. Whole genome sequencing methods resulted in between 25%–98% of the genome sequenced for five such samples. Exploratory population genetic analyses revealed that these data were free of holistic biases and could recover expected population structure and relatedness. Mitochondrial genomes recovered matrilineages in accordance with long‐term monitoring data. Even with just five samples, we were able to uncover the matrilineage for three individuals with unknown ancestry.
4. In summary, we demonstrated that noninvasive shed hair samples yield adequate quality and quantity of DNA in conjunction with sensitive library preparation methods, and provide reliable data from hundreds of thousands of SNPs across the genome. This makes shed hair an ideal noninvasive resource for studying individual‐based genetics of elusive endangered species in the wild.

     

RAD‐sequencing for estimating genomic relatedness matrix‐based heritability in the wild: A case study in roe deer

Estimating the evolutionary potential of quantitative traits and reliably predicting responses to selection in wild populations are important challenges in evolutionary biology. The genomic revolution has opened up opportunities for measuring relatedness among individuals with precision, enabling pedigree‐free estimation of trait heritabilities in wild populations. However, until now, most quantitative genetic studies based on a genomic relatedness matrix (GRM) have focused on long‐term monitored populations for which traditional pedigrees were also available, and have often had access to knowledge of genome sequence and variability. Here, we investigated the potential of RAD‐sequencing for estimating heritability in a free‐ranging roe deer (Capreolous capreolus) population for which no prior genomic resources were available. We propose a step‐by‐step analytical framework to optimize the quality and quantity of the genomic data and explore the impact of the single nucleotide polymorphism (SNP) calling and filtering processes on the GRM structure and GRM‐based heritability estimates. As expected, our results show that sequence coverage strongly affects the number of recovered loci, the genotyping error rate and the amount of missing data. Ultimately, this had little effect on heritability estimates and their standard errors, provided that the GRM was built from a minimum number of loci (above 7,000). Genomic relatedness matrix‐based heritability estimates thus appear robust to a moderate level of genotyping errors in the SNP data set. We also showed that quality filters, such as the removal of low‐frequency variants, affect the relatedness structure of the GRM, generating lower h² estimates. Our work illustrates the huge potential of RAD‐sequencing for estimating GRM‐based heritability in virtually any natural population.     

Estimating population size using single‐nucleotide polymorphism‐based pedigree data

Reliable population estimates are an important aspect of sustainable wildlife management and conservation but can be difficult to obtain for rare and elusive species. Here, we test a new census method based on pedigree reconstruction recently developed by Creel and Rosenblatt (2013). Using a panel of 96 single‐nucleotide polymorphisms (SNPs), we genotyped fecal samples from two Swedish brown bear populations for pedigree reconstruction. Based on 433 genotypes from central Sweden (CS) and 265 from northern Sweden (NS), the population estimates (N = 630 for CS, N = 408 for NS) fell within the 95% CI of the official estimates. The precision and accuracy improved with increasing sampling intensity. Like genetic capture–mark–recapture methods, this method can be applied to data from a single sampling session. Pedigree reconstruction combined with noninvasive genetic sampling may thus augment population estimates, particularly for rare and elusive species for which sampling may be challenging.     

Molecular and pedigree measures of relatedness provide similar estimates of inbreeding depression in a bottlenecked population

Individual‐based estimates of the degree of inbreeding or parental relatedness from pedigrees provide a critical starting point for studies of inbreeding depression, but in practice wild pedigrees are difficult to obtain. Because inbreeding increases the proportion of genomewide loci that are identical by descent, inbreeding variation within populations has the potential to generate observable correlations between heterozygosity measured using molecular markers and a variety of fitness related traits. Termed heterozygosity‐fitness correlations (HFCs), these correlations have been observed in a wide variety of taxa. The difficulty of obtaining wild pedigree data, however, means that empirical investigations of how pedigree inbreeding influences HFCs are rare. Here, we assess evidence for inbreeding depression in three life‐history traits (hatching and fledging success and juvenile survival) in an isolated population of Stewart Island robins using both pedigree‐ and molecular‐derived measures of relatedness. We found results from the two measures were highly correlated and supported evidence for significant but weak inbreeding depression. However, standardized effect sizes for inbreeding depression based on the pedigree‐based kin coefficients (k) were greater and had smaller standard errors than those based on molecular genetic measures of relatedness (RI), particularly for hatching and fledging success. Nevertheless, the results presented here support the use of molecular‐based measures of relatedness in bottlenecked populations when information regarding inbreeding depression is desired but pedigree data on relatedness are unavailable.     

Historical Pedigree Reconstruction from Extant Populations Using PArtitioning of RElatives (PREPARE)

Recent technological improvements in the field of genetic data extraction give rise to the possibility of reconstructing the historical pedigrees of entire populations from the genotypes of individuals living today. Current methods are still not practical for real data scenarios as they have limited accuracy and assume unrealistic assumptions of monogamy and synchronized generations. In order to address these issues, we develop a new method for pedigree reconstruction, , which is based on formulations of the pedigree reconstruction problem as variants of graph coloring. The new formulation allows us to consider features that were overlooked by previous methods, resulting in a reconstruction of up to 5 generations back in time, with an order of magnitude improvement of false-negatives rates over the state of the art, while keeping a lower level of false positive rates. We demonstrate the accuracy of compared to previous approaches using simulation studies over a range of population sizes, including inbred and outbred populations, monogamous and polygamous mating patterns, as well as synchronous and asynchronous mating.     

Molecular marker-based pedigrees for animal conservation biologists

Pedigrees, depicting the genealogical relationships between individuals in a population, are of fundamental importance to several research areas including conservation biology. For example, they are useful for estimating inbreeding, heritability, selection, studying kin selection and for measuring gene flow between populations. Pedigrees constructed from direct observations of reproduction are usually unavailable for wild populations. Therefore, pedigrees for these populations are usually estimated using molecular marker data. Despite their obvious importance, and the fact that pedigrees are conceptually well understood, the methods, and limitations of marker-based pedigree inference are often less well understood. Here we introduce animal conservation biologists to molecular marker-based pedigrees. We briefly describe the history of pedigree inference research, before explaining the underlying theory and basic mechanics of pedigree construction using standard methods. We explain the assumptions and limitations that accompany many of these methods, before going on to explain methods that relax several of these assumptions. Finally, we look to future and discuss some recent exciting advances such as the use of single-nucleotide polymorphisms, inference of multigenerational pedigrees and incorporation of non-genetic data such as field observations into the calculations. We also provide some guidelines on efficient marker selection in order to maximize accuracy and power. Throughout we use examples from the field of animal conservation and refer readers to appropriate software where possible. It is our hope that this review will help animal conservation biologists to understand, choose, and use the methods and tools of this fast-moving field.     

Trawl selectivity‐induced evolution effects on age structure and size‐at‐age of largehead hairtail (Trichiurus lepturus) Linnaeus, 1758 in the East China Sea,China

Increasing evidence has demonstrated that the life history traits of fishes have changed in many exploited populations, caused principally by intense fishing mortality and size‐selectivity of the fishing gear. Broad and intensive trawl fishing over an extended period has the enormous potential to change the biological characters of exploited fish populations. An individual‐based model was developed to explore the interactions between trawl fishing and evolutionary changes in length‐at‐age and age structure of an exploited fish population. A perennial fish population was simulated with a multiple age structure in the model to examine the effects of long‐term trawl fishing on hairtail, Trichiurus lepturus, in the East China Sea. The results revealed that distribution of the body length‐at‐age and the age structure of the fish population were irreversibly changed under long‐term trawl fishing. The simulated results confirm that the length‐at‐age is increasing shorter, the younger individuals dominate, the influence of trawl selectivity on the biological traits of the fish population is highly significant, and that these changes have potentially evolutionary consequences on the fish body length‐at‐age.     

Molecular ecological approaches to studying the evolutionary impact of selective harvesting in wildlife

Harvesting of wildlife populations by humans is usually targeted by sex, age or phenotypic criteria, and is therefore selective. Selective harvesting has the potential to elicit a genetic response from the target populations in several ways. First, selective harvesting may affect population demographic structure (age structure, sex ratio), which in turn may have consequences for effective population size and hence genetic diversity. Second, wildlife-harvesting regimes that use selective criteria based on phenotypic characteristics (e.g. minimum body size, horn length or antler size) have the potential to impose artificial selection on harvested populations. If there is heritable genetic variation for the target characteristic and harvesting occurs before the age of maturity, then an evolutionary response over time may ensue. Molecular ecological techniques offer ways to predict and detect genetic change in harvested populations, and therefore have great utility for effective wildlife management. Molecular markers can be used to assess the genetic structure of wildlife populations, and thereby assist in the prediction of genetic impacts by delineating evolutionarily meaningful management units. Genetic markers can be used for monitoring genetic diversity and changes in effective population size and breeding systems. Tracking evolutionary change at the phenotypic level in the wild through quantitative genetic analysis can be made possible by genetically determined pedigrees. Finally, advances in genome sequencing and bioinformatics offer the opportunity to study the molecular basis of phenotypic variation through trait mapping and candidate gene approaches. With this understanding, it could be possible to monitor the selective impacts of harvesting at a molecular level in the future. Effective wildlife management practice needs to consider more than the direct impact of harvesting on population dynamics. Programs that utilize molecular genetic tools will be better positioned to assess the long-term evolutionary impact of artificial selection on the evolutionary trajectory and viability of harvested populations.     

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.     

Age‐specific,population‐level pedigree of wild black bears provides insights into reproduction,paternity, and maternal effects on offspring apparent survival

Wildlife pedigrees provide insights into ecological and evolutionary processes. DNA obtained from noninvasively collected hair is often used to determine individual identities for pedigrees and other genetic analyses. However, detection rates associated with some noninvasive DNA studies can be relatively low, and genetic data do not provide information on individual birth year. Supplementing hair DNA stations with video cameras should increase the individual detection rate, assuming accurate identification of individuals via video data. Video data can also provide birth year information for individuals captured as young of the year, which can enrich population‐level pedigrees. We placed video cameras at hair stations and combined genetic and video data to reconstruct an age‐specific, population‐level pedigree of wild black bears during 2010–2020. Combining individual birth year with mother–offspring relatedness, we also estimated litter size, interlitter interval, primiparity, and fecundity. We used the Cormack‐Jolly‐Seber model in Program Mark to evaluate the effect of maternal identity on offspring apparent survival. We compared model rankings of apparent survival and parameter estimates based on combined genetic and video data with those based on only genetic data. We observed 42 mother–offspring relationships. Of these, 21 (50%) would not have been detected had we used hair DNA alone. Moreover, video data allowed for the cub and yearling age classes to be determined. Mean annual fecundity was 0.42 (95% CI: 0.27, 0.56). Maternal identity influenced offspring apparent survival, where offspring of one mother experienced significantly lower apparent survival (0.39; SE = 0.15) than that of offspring of four other mothers (0.89–1.00; SE = 0.00–0.06). We video‐documented cub abandonment by the mother whose offspring experienced low apparent survival, indicating individual behaviors (e.g., maternal care) may scale up to affect population‐level parameters (e.g., cub survival). Our findings provide insights into evolutionary processes and are broadly relevant to wildlife ecology and conservation.     

ROADTRIPS: Case-Control Association Testing with Partially or Completely Unknown Population and Pedigree Structure

Genome-wide association studies are routinely conducted to identify genetic variants that influence complex disorders. It is well known that failure to properly account for population or pedigree structure can lead to spurious association as well as reduced power. We propose a method, ROADTRIPS, for case-control association testing in samples with partially or completely unknown population and pedigree structure. ROADTRIPS uses a covariance matrix estimated from genome-screen data to correct for unknown population and pedigree structure while maintaining high power by taking advantage of known pedigree information when it is available. ROADTRIPS can incorporate data on arbitrary combinations of related and unrelated individuals and is computationally feasible for the analysis of genetic studies with millions of markers. In simulations with related individuals and population structure, including admixture, we demonstrate that ROADTRIPS provides a substantial improvement over existing methods in terms of power and type 1 error. The ROADTRIPS method can be used across a variety of study designs, ranging from studies that have a combination of unrelated individuals and small pedigrees to studies of isolated founder populations with partially known or completely unknown pedigrees. We apply the method to analyze two data sets: a study of rheumatoid arthritis in small UK pedigrees, from Genetic Analysis Workshop 15, and data from the Collaborative Study of the Genetics of Alcoholism on alcohol dependence in a sample of moderate-size pedigrees of European descent, from Genetic Analysis Workshop 14. We detect genome-wide significant association, after Bonferroni correction, in both studies.     

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.