A comparison of genomic diversity and demographic history of the North Atlantic and Southwest Atlantic southern right whales.

Right whales (genus Eubalaena) were among the first, and most extensively pursued, targets of commercial whaling. However, understanding the impacts of this persecution requires knowledge of the demographic histories of these species prior to exploitation. We used deep whole genome sequencing (~40×) of 12 North Atlantic (E. glacialis) and 10 Southwest Atlantic southern (E. australis) right whales to quantify contemporary levels of genetic diversity and infer their demographic histories over time. Using coalescent- and identity-by-descent-based modelling to estimate ancestral effective population sizes from genomic data, we demonstrate that North Atlantic right whales have lived with smaller effective population sizes (Ne ) than southern right whales in the Southwest Atlantic since their divergence and describe the decline in both populations around the time of whaling. North Atlantic right whales exhibit reduced genetic diversity and longer runs of homozygosity leading to higher inbreeding coefficients compared to the sampled population of southern right whales. This study represents the first comprehensive assessment of genome-wide diversity of right whales in the western Atlantic and underscores the benefits of high coverage, genome-wide datasets to help resolve long-standing questions about how historical changes in effective population size over different time scales shape contemporary diversity estimates. This knowledge is crucial to improve our understanding of the right whales' history and inform our approaches to address contemporary conservation issues. Understanding and quantifying the cumulative impact of long-term small Ne , low levels of diversity and recent inbreeding on North Atlantic right whale recovery will be important next steps.

Genetic Diversity and Connectivity of Southern Right Whales (Eubalaena australis) Found in the Brazil and Chile-Peru Wintering Grounds and the South Georgia (Islas Georgias del Sur) Feeding Ground.

As species recover from exploitation, continued assessments of connectivity and population structure are warranted to provide information for conservation and management. This is particularly true in species with high dispersal capacity, such as migratory whales, where patterns of connectivity could change rapidly. Here we build on a previous long-term, large-scale collaboration on southern right whales (Eubalaena australis) to combine new (nnew) and published (npub) mitochondrial (mtDNA) and microsatellite genetic data from all major wintering grounds and, uniquely, the South Georgia (Islas Georgias del Sur: SG) feeding grounds. Specifically, we include data from Argentina (npub mtDNA/microsatellite = 208/46), Brazil (nnew mtDNA/microsatellite = 50/50), South Africa (nnew mtDNA/microsatellite = 66/77, npub mtDNA/microsatellite = 350/47), Chile-Peru (nnew mtDNA/microsatellite = 1/1), the Indo-Pacific (npub mtDNA/microsatellite = 769/126), and SG (npub mtDNA/microsatellite = 8/0, nnew mtDNA/microsatellite = 3/11) to investigate the position of previously unstudied habitats in the migratory network: Brazil, SG, and Chile-Peru. These new genetic data show connectivity between Brazil and Argentina, exemplified by weak genetic differentiation and the movement of 1 genetically identified individual between the South American grounds. The single sample from Chile-Peru had an mtDNA haplotype previously only observed in the Indo-Pacific and had a nuclear genotype that appeared admixed between the Indo-Pacific and South Atlantic, based on genetic clustering and assignment algorithms. The SG samples were clearly South Atlantic and were more similar to the South American than the South African wintering grounds. This study highlights how international collaborations are critical to provide context for emerging or recovering regions, like the SG feeding ground, as well as those that remain critically endangered, such as Chile-Peru.

Cultural Hitchhiking in the Matrilineal Whales.

Five species of whale with matrilineal social systems (daughters remain with mothers) have remarkably low levels of mitochondrial DNA diversity. Non-heritable matriline-level demography could reduce genetic diversity but the required conditions are not consistent with the natural histories of the matrilineal whales. The diversity of nuclear microsatellites is little reduced in the matrilineal whales arguing against bottlenecks. Selective sweeps of the mitochondrial genome are feasible causes but it is not clear why these only occurred in the matrilineal species. Cultural hitchhiking (cultural selection reducing diversity at neutral genetic loci transmitted in parallel to the culture) is supported in sperm whales which possess suitable matrilineal socio-cultural groups (coda clans). Killer whales are delineated into ecotypes which likely originated culturally. Culture, bottlenecks and selection, as well as their interactions, operating between- or within-ecotypes, may have reduced their mitochondrial diversity. The societies, cultures and genetics of false killer and two pilot whale species are insufficiently known to assess drivers of low mitochondrial diversity.

Small Ne of the Isolated and Unmanaged Horse Population on Sable Island.

For small, isolated populations 2 common conservation concerns relate to genetic threats: inbreeding and negative consequences associated with loss of genetic diversity due to drift. Mitigating these threats often involves conservation actions that can be controversial, such as translocations or captive breeding programs. Although such actions have been successful in some situations, in others they have had undesirable outcomes. Here, we estimated the effective population size (N e ) of the Sable Island horses to assess the risk to this population of these genetic threats. We found surprising consistency of N e estimates across the 5 different methods used, with a mean of 48 effective individuals. This estimate falls below the 50 criterion of the "50/500 rule," below which inbreeding depression is a concern for population viability. However, simulations and knowledge of population history indicate that this population is still in its early stages of approaching equilibrium between mutation, drift, and genetic diversity; and no negative consequences have been identified that could be associated with inbreeding depression. Therefore, we do not recommend taking management action (such as translocations) at this stage. Rather, we propose continued monitoring of genetic diversity and fitness over time so that trends and any substantial changes can be detected. This represents one of the few unmanaged horse populations in the world, and therefore these data will not only alert us to serious concerns regarding their conservation status, but will also provide a wealth of information about how natural processes drive patterns of reproduction, mortality, and population growth over time.

Effects of inbreeding on reproductive success in endangered North Atlantic right whales.

Only approximately 356 North Atlantic right whales (Eubalaena glacialis) remain. With extremely low levels of genetic diversity, limited options for mates, and variation in reproductive success across females, there is concern regarding the potential for genetic limitations of population growth from inbreeding depression. In this study, we quantified reproductive success of female North Atlantic right whales with a modified de-lifing approach using reproductive history information collected over decades of field observations. We used double-digest restriction site-associated sequencing to sequence approximately 2% of the genome of 105 female North Atlantic right whales and combined genomic inbreeding estimates with individual fecundity values to assess evidence of inbreeding depression. Inbreeding depression could not explain the variance in reproductive success of females, however we present evidence that inbreeding depression may be affecting the viability of inbred fetuses-potentially lowering the reproductive success of the species as a whole. Combined, these results allay some concerns that genetic factors are impacting species survival as genetic diversity is being retained through selection against inbred fetuses. While still far fewer calves are being born each year than expected, the small role of genetics underlying variance in female fecundity suggests that variance may be explained by external factors that can potentially be mitigated through protection measures designed to reduce serious injury and mortality from human activities.

An example of DNA methylation as a means to quantify stress in wildlife using killer whales.

The cumulative effects of non-lethal stressors on the health of biodiversity are a primary concern for conservation, yet difficulties remain regarding their quantification. In mammals, many stressors are processed through a common stress-response pathway, and therefore epigenetic changes in genes of this pathway may provide a powerful tool for quantifying cumulative effects. As a preliminary assessment of this approach, we investigated epigenetic manifestations of stress in two killer whale populations with different levels of exposure to anthropogenic stressors. We used bisulfite amplicon sequencing to compare patterns of DNA methylation at 25 CpG sites found in three genes involved in stress response and identified large differences in the level of methylation at two sites consistent with differential stress exposure between Northern and Southern Resident killer whale populations. DNA methylation patterns could therefore represent a useful method to assess the cumulative effects of non-lethal stressors in wildlife.

Increased efficiency of genetic profiling through quantity and quality assessment of fluorescently labeled oligonucleotide primers.

Optimizing the amount of primer to use in PCR amplification is one of the most important steps when developing protocols for genetic profiling, where subtle changes in primer concentration result in major impacts on the amount of desired product that is amplified. However; there are frequently discrepancies between the reported and actual quantity of primers delivered by suppliers, resulting in a need for re-optimization of conditions between primer orders and limiting the ability to standardize conditions between laboratories. To increase the consistency of genetic profiling protocols, we have developed a simple method to assess the quantity and quality of fluorescently labeled primers and therefore standardize reaction conditions through time and across laboratories. The method is based on analysis by electrophoresis with an automated fluorescent DNA analyzer.

What factors shape genetic diversity in cetaceans?

Understanding what factors drive patterns of genetic diversity is a central aspect of many biological questions, ranging from the inference of historical demography to assessing the evolutionary potential of a species. However, as a larger number of datasets have become available, it is becoming clear that the relationship between the characteristics of a species and its genetic diversity is more complex than previously assumed. This may be particularly true for cetaceans, due to their relatively long lifespans, long generation times, complex social structures, and extensive ranges. In this study, we used microsatellite and mitochondrial DNA data from a systematic literature review to produce estimates of diversity for both markers across 42 cetacean species. Factors relating to demography, distribution, classification, biology, and behavior were then tested using phylogenetic methods and linear models to assess their relative influence on the genetic diversity of both marker types. The results show that while relative nuclear diversity is correlated with population size, mitochondrial diversity is not. This is particularly relevant given the widespread use of mitochondrial DNA to infer historical demography. Instead, mitochondrial diversity was mostly influenced by the range and social structure of the species. In addition to population size, habitat type (neritic vs. oceanic) had a significant correlation with relative nuclear diversity. Combined, these results show that many often-unconsidered factors are likely influencing patterns of genetic diversity in cetaceans, with implications regarding how to interpret, and what can be inferred from, existing patterns of diversity.

Kinship influences sperm whale social organization within, but generally not among, social units.

Sperm whales have a multi-level social structure based upon long-term, cooperative social units. What role kinship plays in structuring this society is poorly understood. We combined extensive association data (518 days, during 2005-2016) and genetic data (18 microsatellites and 346 bp mitochondrial DNA (mtDNA) control region sequences) for 65 individuals from 12 social units from the Eastern Caribbean to examine patterns of kinship and social behaviour. Social units were clearly matrilineally based, evidenced by greater relatedness within social units (mean r = 0.14) than between them (mean r = 0.00) and uniform mtDNA haplotypes within social units. Additionally, most individuals (82.5%) had a first-degree relative in their social unit, while we found no first-degree relatives between social units. Generally and within social units, individuals associated more with their closer relatives (matrix correlations: 0.18-0.25). However, excepting a highly related pair of social units that merged over the study period, associations between social units were not correlated with kinship (p > 0.1). These results are the first to robustly demonstrate kinship's contribution to social unit composition and association preferences, though they also reveal variability in association preferences that is unexplained by kinship. Comparisons with other matrilineal species highlight the range of possible matrilineal societies and how they can vary between and even within species.

Gamete compatibility genes in mammals: candidates, applications and a potential path forward.

Fertilization represents a critical stage in biology, where successful alleles of a previous generation are shuffled into new arrangements and subjected to the forces of selection in the next generation. Although much research has been conducted on how variation in morphological and behavioural traits lead to variation in fertilization patterns, surprisingly little is known about fertilization at a molecular level, and specifically about how genes expressed on the sperm and egg themselves influence fertilization patterns. In mammals, several genes have been identified whose products are expressed on either the sperm or the egg, and which influence the fertilization process, but the specific mechanisms are not yet known. Additionally, in 2014 an interacting pair of proteins was identified: 'Izumo' on the sperm, and 'Juno' on the egg. With the identification of these genes comes the first opportunity to understand the molecular aspects of fertilization in mammals, and to identify how the genetic characteristics of these genes influence fertilization patterns. Here, we review recent progress in our understanding of fertilization and gamete compatibility in mammals, which should provide a helpful guide to researchers interested in untangling the molecular mechanisms of fertilization and the resulting impacts on population biology and evolutionary processes.

Conservation genetics of the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi): River valleys are critical features for snakes at northern range limits.

On the North American Great Plains, several snake species reach their northern range limit where they rely on sparsely distributed hibernacula located in major river valleys. Independent colonization histories for the river valleys and barriers to gene flow caused by the lack of suitable habitat between them may have produced genetically differentiated snake populations. To test this hypothesis, we used 10 microsatellite loci to examine the population structure of two species of conservation concern in Canada: the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi) in 3 major river valleys in southern Saskatchewan. Fixation indices (FST) showed that populations in river valleys were significantly differentiated for both species (racers, FST = 0.096, P = 0.001; bullsnakes FST = 0.045-0.157, P = 0.001). Bayesian assignment (STRUCTURE) and ordination (DAPC) strongly supported genetically differentiated groups in the geographically distinct river valleys. Finer-scale subdivision of populations within river valleys was not apparent based on our data, but is a topic that should be investigated further. Our findings highlight the importance of major river valleys for snakes at the northern extent of their ranges, and raise the possibility that populations in each river valley may warrant separate management strategies.

A note on the use of multiple linear regression in molecular ecology.

Multiple linear regression analyses (also often referred to as generalized linear models--GLMs, or generalized linear mixed models--GLMMs) are widely used in the analysis of data in molecular ecology, often to assess the relative effects of genetic characteristics on individual fitness or traits, or how environmental characteristics influence patterns of genetic differentiation. However, the coefficients resulting from multiple regression analyses are sometimes misinterpreted, which can lead to incorrect interpretations and conclusions within individual studies, and can propagate to wider-spread errors in the general understanding of a topic. The primary issue revolves around the interpretation of coefficients for independent variables when interaction terms are also included in the analyses. In this scenario, the coefficients associated with each independent variable are often interpreted as the independent effect of each predictor variable on the predicted variable. However, this interpretation is incorrect. The correct interpretation is that these coefficients represent the effect of each predictor variable on the predicted variable when all other predictor variables are zero. This difference may sound subtle, but the ramifications cannot be overstated. Here, my goals are to raise awareness of this issue, to demonstrate and emphasize the problems that can result and to provide alternative approaches for obtaining the desired information.

related: an R package for analysing pairwise relatedness from codominant molecular markers.

Analyses of pairwise relatedness represent a key component to addressing many topics in biology. However, such analyses have been limited because most available programs provide a means to estimate relatedness based on only a single estimator, making comparison across estimators difficult. Second, all programs to date have been platform specific, working only on a specific operating system. This has the undesirable outcome of making choice of relatedness estimator limited by operating system preference, rather than being based on scientific rationale. Here, we present a new R package, called related, that can calculate relatedness based on seven estimators, can account for genotyping errors, missing data and inbreeding, and can estimate 95% confidence intervals. Moreover, simulation functions are provided that allow for easy comparison of the performance of different estimators and for analyses of how much resolution to expect from a given data set. Because this package works in R, it is platform independent. Combined, this functionality should allow for more appropriate analyses and interpretation of pairwise relatedness and will also allow for the integration of relatedness data into larger R workflows.

Population Genetic Structure Within and among Seasonal Site Types in the Little Brown Bat (Myotis lucifugus) and the Northern Long-Eared Bat (M. septentrionalis).

During late summer and early autumn, temperate bats migrate from their summering sites to swarming sites, where mating likely occurs. However, the extent to which individuals of a single summering site migrate to the same swarming site, and vice versa, is not known. We examined the migratory connectivity between summering and swarming sites in two temperate, North American, bat species, the little brown bat (Myotis lucifugus) and the northern long-eared bat (Myotis septentrionalis). Using mitochondrial and microsatellite DNA markers, we examined population structuring within and among summering and swarming sites. Both species exhibited moderate degrees of mitochondrial DNA differentiation (little brown bat: FST(SUMMER) = 0.093, FST(SWARMING) = 0.052; northern long-eared bat: FST(SUMMER) = 0.117, FST(SWARMING) = 0.043) and little microsatellite DNA differentiation among summering and among swarming sites[corrected]. Haplotype diversity was significantly higher at swarming sites than summering sites, supporting the idea that swarming sites are comprised of individuals from various summering sites. Further, pairwise analyses suggest that swarming sites are not necessarily comprised of only individuals from the most proximal summering colonies.

Assessment of the extirpated Maritimes walrus using morphological and ancient DNA analysis.

Species biogeography is a result of complex events and factors associated with climate change, ecological interactions, anthropogenic impacts, physical geography, and evolution. To understand the contemporary biogeography of a species, it is necessary to understand its history. Specimens from areas of localized extinction are important, as extirpation of species from these areas may represent the loss of unique adaptations and a distinctive evolutionary trajectory. The walrus (Odobenus rosmarus) has a discontinuous circumpolar distribution in the arctic and subarctic that once included the southeastern Canadian Maritimes region. However, exploitation of the Maritimes population during the 16th-18th centuries led to extirpation, and the species has not inhabited areas south of 55°N for ∼250 years. We examined genetic and morphological characteristics of specimens from the Maritimes, Atlantic (O. r. rosmarus) and Pacific (O. r. divergens) populations to test the hypothesis that the first group was distinctive. Analysis of Atlantic and Maritimes specimens indicated that most skull and mandibular measurements were significantly different between the Maritimes and Atlantic groups and discriminant analysis of principal components confirmed them as distinctive groups, with complete isolation of skull features. The Maritimes walrus appear to have been larger animals, with larger and more robust tusks, skulls and mandibles. The mtDNA control region haplotypes identified in Maritimes specimens were unique to the region and a greater average number of nucleotide differences were found between the regions (Atlantic and Maritimes) than within either group. Levels of diversity (h and π) were lower in the Maritimes, consistent with other studies of species at range margins. Our data suggest that the Maritimes walrus was a morphologically and genetically distinctive group that was on a different evolutionary path from other walrus found in the north Atlantic.

Genetic connectivity among swarming sites in the wide ranging and recently declining little brown bat (Myotis lucifugus).

Characterizing movement dynamics and spatial aspects of gene flow within a species permits inference on population structuring. As patterns of structuring are products of historical and current demographics and gene flow, assessment of structure through time can yield an understanding of evolutionary dynamics acting on populations that are necessary to inform management. Recent dramatic population declines in hibernating bats in eastern North America from white-nose syndrome have prompted the need for information on movement dynamics for multiple bat species. We characterized population genetic structure of the little brown bat, Myotis lucifugus, at swarming sites in southeastern Canada using 9 nuclear microsatellites and a 292-bp region of the mitochondrial genome. Analyses of F ST, ΦST, and Bayesian clustering (STRUCTURE) found weak levels of genetic structure among swarming sites for the nuclear and mitochondrial genome (Global F ST = 0.001, P < 0.05, Global ΦST = 0.045, P < 0.01, STRUCTURE K = 1) suggesting high contemporary gene flow. Hierarchical AMOVA also suggests little structuring at a regional (provincial) level. Metrics of nuclear genetic structure were not found to differ between males and females suggesting weak asymmetries in gene flow between the sexes. However, a greater degree of mitochondrial structuring does support male-biased dispersal long term. Demographic analyses were consistent with past population growth and suggest a population expansion occurred from approximately 1250 to 12,500 BP, following Pleistocene deglaciation in the region. Our study suggests high gene flow and thus a high degree of connectivity among bats that visit swarming sites whereby mainland areas of the region may be best considered as one large gene pool for management and conservation.

STORM: software for testing hypotheses of relatedness and mating patterns.

Storm is a software package that allows users to test a variety of hypotheses regarding patterns of relatedness and patterns of mate choice and/or mate compatibility within a population. These functions are based on four main calculations that can be conducted either independently or in the hypothesis-testing framework: internal relatedness; homozygosity by loci; pairwise relatedness; and a new metric called allele inheritance, which calculates the proportion of loci at which an offspring inherits a paternal allele different from that inherited from its mother. STORM allows users to test four hypotheses based on these calculations and Monte Carlo simulations: (i) are individuals within observed associations or groupings more/less related than expected; (ii) do observed offspring have more/less genetic variability (based on internal relatedness or homozygosity by loci) than expected from the gene pool; (iii) are observed mating pairs more/less related than expected if mating is random with respect to relatedness; and (iv) do observed offspring inherit paternal alleles different from those inherited from the mother more/less often than expected based on Mendelian inheritance.