Population structure in a continuously distributed coastal marine species, the harbor porpoise, based on microhaplotypes derived from poor-quality samples.

Harbor porpoise in the North Pacific are found in coastal waters from southern California to Japan, but population structure is poorly known outside of a few local areas. We used multiplexed amplicon sequencing of 292 loci and genotyped clusters of single nucleotide polymoirphisms as microhaplotypes (N = 271 samples) in addition to mitochondrial (mtDNA) sequence data (N = 413 samples) to examine the genetic structure from samples collected along the Pacific coast and inland waterways from California to southern British Columbia. We confirmed an overall pattern of strong isolation-by-distance, suggesting that individual dispersal is restricted. We also found evidence of regions where genetic differences are larger than expected based on geographical distance alone, implying current or historical barriers to gene flow. In particular, the southernmost population in California is genetically distinct (FST  = 0.02 [microhaplotypes]; 0.31 [mtDNA]), with both reduced genetic variability and high frequency of an otherwise rare mtDNA haplotype. At the northern end of our study range, we found significant genetic differentiation of samples from the Strait of Georgia, previously identified as a potential biogeographical boundary or secondary contact zone between harbor porpoise populations. Association of microhaplotypes with remotely sensed environmental variables indicated potential local adaptation, especially at the southern end of the species' range. These results inform conservation and management for this nearshore species, illustrate the value of genomic methods for detecting patterns of genetic structure within a continuously distributed marine species, and highlight the power of microhaplotype genotyping for detecting genetic structure in harbor porpoises despite reliance on poor-quality samples.

Modulation in Persistent Organic Pollutant Concentration and Profile by Prey Availability and Reproductive Status in Southern Resident Killer Whale Scat Samples.

Persistent organic pollutants (POPs), specifically PCBs, PBDEs, and DDTs, in the marine environment are well documented, however accumulation and mobilization patterns at the top of the food-web are poorly understood. This study broadens the understanding of POPs in the endangered Southern Resident killer whale population by addressing modulation by prey availability and reproductive status, along with endocrine disrupting effects. A total of 140 killer whale scat samples collected from 54 unique whales across a 4 year sampling period (2010-2013) were analyzed for concentrations of POPs. Toxicant measures were linked to pod, age, and birth order in genotyped individuals, prey abundance using open-source test fishery data, and pregnancy status based on hormone indices from the same sample. Toxicant concentrations were highest and had the greatest potential for toxicity when prey abundance was the lowest. In addition, these toxicants were likely from endogenous lipid stores. Bioaccumulation of POPs increased with age, with the exception of presumed nulliparous females. The exceptional pattern may be explained by females experiencing unobserved neonatal loss. Transfer of POPs through mobilization of endogenous lipid stores during lactation was highest for first-borns with diminished transfer to subsequent calves. Contrary to expectation, POP concentrations did not demonstrate an associated disruption of thyroid hormone, although this association may have been masked by impacts of prey abundance on thyroid hormone concentrations. The noninvasive method for measuring POP concentrations in killer whales through scat employed in this study may improve toxicant monitoring in the marine environment and promote conservation efforts.

Persistent Organic Pollutant Determination in Killer Whale Scat Samples: Optimization of a Gas Chromatography/Mass Spectrometry Method and Application to Field Samples.

Biologic sample collection in wild cetacean populations is challenging. Most information on toxicant levels is obtained from blubber biopsy samples; however, sample collection is invasive and strictly regulated under permit, thus limiting sample numbers. Methods are needed to monitor toxicant levels that increase temporal and repeat sampling of individuals for population health and recovery models. The objective of this study was to optimize measuring trace levels (parts per billion) of persistent organic pollutants (POPs), namely polychlorinated-biphenyls (PCBs), polybrominated-diphenyl-ethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs), and hexachlorocyclobenzene, in killer whale scat (fecal) samples. Archival scat samples, initially collected, lyophilized, and extracted with 70 % ethanol for hormone analyses, were used to analyze POP concentrations. The residual pellet was extracted and analyzed using gas chromatography coupled with mass spectrometry. Method detection limits ranged from 11 to 125 ng/g dry weight. The described method is suitable for p,p'-DDE, PCBs-138, 153, 180, and 187, and PBDEs-47 and 100; other POPs were below the limit of detection. We applied this method to 126 scat samples collected from Southern Resident killer whales. Scat samples from 22 adult whales also had known POP concentrations in blubber and demonstrated significant correlations (p < 0.01) between matrices across target analytes. Overall, the scat toxicant measures matched previously reported patterns from blubber samples of decreased levels in reproductive-age females and a decreased p,p'-DDE/∑PCB ratio in J-pod. Measuring toxicants in scat samples provides an unprecedented opportunity to noninvasively evaluate contaminant levels in wild cetacean populations; these data have the prospect to provide meaningful information for vital management decisions.

Geographic and temporal dynamics of a global radiation and diversification in the killer whale.

Global climate change during the Late Pleistocene periodically encroached and then released habitat during the glacial cycles, causing range expansions and contractions in some species. These dynamics have played a major role in geographic radiations, diversification and speciation. We investigate these dynamics in the most widely distributed of marine mammals, the killer whale (Orcinus orca), using a global data set of over 450 samples. This marine top predator inhabits coastal and pelagic ecosystems ranging from the ice edge to the tropics, often exhibiting ecological, behavioural and morphological variation suggestive of local adaptation accompanied by reproductive isolation. Results suggest a rapid global radiation occurred over the last 350 000 years. Based on habitat models, we estimated there was only a 15% global contraction of core suitable habitat during the last glacial maximum, and the resources appeared to sustain a constant global effective female population size throughout the Late Pleistocene. Reconstruction of the ancestral phylogeography highlighted the high mobility of this species, identifying 22 strongly supported long-range dispersal events including interoceanic and interhemispheric movement. Despite this propensity for geographic dispersal, the increased sampling of this study uncovered very few potential examples of ancestral dispersal among ecotypes. Concordance of nuclear and mitochondrial data further confirms genetic cohesiveness, with little or no current gene flow among sympatric ecotypes. Taken as a whole, our data suggest that the glacial cycles influenced local populations in different ways, with no clear global pattern, but with secondary contact among lineages following long-range dispersal as a potential mechanism driving ecological diversification.

Revised taxonomy of eastern North Pacific killer whales (Orcinus orca): Bigg's and resident ecotypes deserve species status.

Killer whales (Orcinus orca) are currently recognized as a single ecologically and morphologically diverse, globally distributed species. Multiple morphotypes or ecotypes have been described, often associated with feeding specialization, and several studies have suggested taxonomic revision to include multiple subspecies or species in the genus. We review the ecological, morphological and genetic data for the well-studied 'resident' and Bigg's (aka 'transient') ecotypes in the eastern North Pacific and use quantitative taxonomic guidelines and standards to determine whether the taxonomic status of these killer whale ecotypes should be revised. Our review and new analyses indicate that species-level status is justified in both cases, and we conclude that eastern North Pacific Bigg's killer whales should be recognized as Orcinus rectipinnus (Cope in Scammon, 1869) and resident killer whales should be recognized as Orcinus ater (Cope in Scammon, 1869).

Geographic patterns of genetic differentiation among killer whales in the northern North Pacific.

The difficulties associated with detecting population boundaries have long constrained the conservation and management of highly mobile, wide-ranging marine species, such as killer whales (Orcinus orca). In this study, we use data from 26 nuclear microsatellite loci and mitochondrial DNA sequences (988bp) to test a priori hypotheses about population subdivisions generated from a decade of killer whale surveys across the northern North Pacific. A total of 462 remote skin biopsies were collected from wild killer whales primarily between 2001 and 2010 from the northern Gulf of Alaska to the Sea of Okhotsk, representing both the piscivorous "resident" and the mammal-eating "transient" (or Bigg's) killer whales. Divergence of the 2 ecotypes was supported by both mtDNA and microsatellites. Geographic patterns of genetic differentiation were supported by significant regions of genetic discontinuity, providing evidence of population structuring within both ecotypes and corroborating direct observations of restricted movements of individual whales. In the Aleutian Islands (Alaska), subpopulations, or groups with significantly different mtDNA and microsatellite allele frequencies, were largely delimited by major oceanographic boundaries for resident killer whales. Although Amchitka Pass represented a major subdivision for transient killer whales between the central and western Aleutian Islands, several smaller subpopulations were evident throughout the eastern Aleutians and Bering Sea. Support for seasonally sympatric transient subpopulations around Unimak Island suggests isolating mechanisms other than geographic distance within this highly mobile top predator.

Inbreeding depression explains killer whale population dynamics.

Understanding the factors that cause endangered populations to either grow or decline is crucial for preserving biodiversity. Conservation efforts often address extrinsic threats, such as environmental degradation and overexploitation, that can limit the recovery of endangered populations. Genetic factors such as inbreeding depression can also affect population dynamics but these effects are rarely measured in the wild and thus often neglected in conservation efforts. Here we show that inbreeding depression strongly influences the population dynamics of an endangered killer whale population, despite genomic signatures of purging of deleterious alleles via natural selection. We find that the 'Southern Residents', which are currently endangered despite nearly 50 years of conservation efforts, exhibit strong inbreeding depression for survival. Our population models suggest that this inbreeding depression limits population growth and predict further decline if the population remains genetically isolated and typical environmental conditions continue. The Southern Residents also had more inferred homozygous deleterious alleles than three other, growing, populations, further suggesting that inbreeding depression affects population fitness. These results demonstrate that inbreeding depression can substantially limit the recovery of endangered populations. Conservation actions focused only on extrinsic threats may therefore fail to account for key intrinsic genetic factors that also limit population growth.

DNA methylation-based biomarkers for ageing long-lived cetaceans.

Epigenetic approaches for estimating the age of living organisms are revolutionizing studies of long-lived species. Molecular biomarkers that allow age estimates from small tissue biopsies promise to enhance studies of long-lived whales, addressing a fundamental and challenging parameter in wildlife management. DNA methylation (DNAm) can affect gene expression, and strong correlations between DNAm patterns and age have been documented in humans and nonhuman vertebrates and used to construct "epigenetic clocks". We present several epigenetic clocks for skin samples from two of the longest-lived cetaceans, killer whales and bowhead whales. Applying the mammalian methylation array to genomic DNA from skin samples we validate four different clocks with median errors of 2.3-3.7 years. These epigenetic clocks demonstrate the validity of using cytosine methylation data to estimate the age of long-lived cetaceans and have broad applications supporting the conservation and management of long-lived cetaceans using genomic DNA from remote tissue biopsies.

Signal and noise in metabarcoding data.

Metabarcoding is a powerful molecular tool for simultaneously surveying hundreds to thousands of species from a single sample, underpinning microbiome and environmental DNA (eDNA) methods. Deriving quantitative estimates of underlying biological communities from metabarcoding is critical for enhancing the utility of such approaches for health and conservation. Recent work has demonstrated that correcting for amplification biases in genetic metabarcoding data can yield quantitative estimates of template DNA concentrations. However, a major source of uncertainty in metabarcoding data stems from non-detections across technical PCR replicates where one replicate fails to detect a species observed in other replicates. Such non-detections are a special case of variability among technical replicates in metabarcoding data. While many sampling and amplification processes underlie observed variation in metabarcoding data, understanding the causes of non-detections is an important step in distinguishing signal from noise in metabarcoding studies. Here, we use both simulated and empirical data to 1) suggest how non-detections may arise in metabarcoding data, 2) outline steps to recognize uninformative data in practice, and 3) identify the conditions under which amplicon sequence data can reliably detect underlying biological signals. We show with both simulations and empirical data that, for a given species, the rate of non-detections among technical replicates is a function of both the template DNA concentration and species-specific amplification efficiency. Consequently, we conclude metabarcoding datasets are strongly affected by (1) deterministic amplification biases during PCR and (2) stochastic sampling of amplicons during sequencing-both of which we can model-but also by (3) stochastic sampling of rare molecules prior to PCR, which remains a frontier for quantitative metabarcoding. Our results highlight the importance of estimating species-specific amplification efficiencies and critically evaluating patterns of non-detection in metabarcoding datasets to better distinguish environmental signal from the noise inherent in molecular detections of rare targets.

Inferred Paternity and Male Reproductive Success in a Killer Whale (Orcinus orca) Population.

We used data from 78 individuals at 26 microsatellite loci to infer parental and sibling relationships within a community of fish-eating ("resident") eastern North Pacific killer whales (Orcinus orca). Paternity analysis involving 15 mother/calf pairs and 8 potential fathers and whole-pedigree analysis of the entire sample produced consistent results. The variance in male reproductive success was greater than expected by chance and similar to that of other aquatic mammals. Although the number of confirmed paternities was small, reproductive success appeared to increase with male age and size. We found no evidence that males from outside this small population sired any of the sampled individuals. In contrast to previous results in a different population, many offspring were the result of matings within the same "pod" (long-term social group). Despite this pattern of breeding within social groups, we found no evidence of offspring produced by matings between close relatives, and the average internal relatedness of individuals was significantly less than expected if mating were random. The population's estimated effective size was <30 or about 1/3 of the current census size. Patterns of allele frequency variation were consistent with a population bottleneck.

Skin microbiome of beluga whales: spatial, temporal, and health-related dynamics.

BACKGROUND: Host-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples. RESULTS: We sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health. CONCLUSIONS: We provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

Water, water everywhere: environmental DNA can unlock population structure in elusive marine species.

Determining management units for natural populations is critical for effective conservation and management. However, collecting the requisite tissue samples for population genetic analyses remains the primary limiting factor for a number of marine species. The harbour porpoise (Phocoena phocoena), one of the smallest cetaceans in the Northern Hemisphere, is a primary example. These elusive, highly mobile small animals confound traditional approaches of collecting tissue samples for genetic analyses, yet their nearshore habitat makes them highly vulnerable to fisheries by-catch and the effects of habitat degradation. By exploiting the naturally shed cellular material in seawater and the power of next-generation sequencing, we develop a novel approach for generating population-specific mitochondrial sequence data from environmental DNA (eDNA) using surface seawater samples. Indications of significant genetic differentiation within a currently recognized management stock highlights the need for dedicated eDNA sampling throughout the population's range in southeast Alaska. This indirect sampling tactic for characterizing stock structure of small and endangered marine mammals has the potential to revolutionize population assessment for otherwise inaccessible marine taxa.

Pre-oil spill baseline profiling for contaminants in Southern Resident killer whale fecal samples indicates possible exposure to vessel exhaust.

The Southern Resident killer whale population (Orcinus orca) was listed as endangered in 2005 and shows little sign of recovery. Exposure to contaminants and risk of an oil spill are identified threats. Previous studies on contaminants have largely focused on legacy pollutants. Here we measure polycyclic aromatic hydrocarbons (PAHs) in whale fecal (scat) samples. PAHs are a diverse group of hazardous compounds (e.g., carcinogenic, mutagenic), and are a component of crude and refined oil as well as motor exhaust. The central finding from this study indicates low concentrations of the measured PAHs (<10 ppb, wet weight), as expected; however, PAHs were as high as 104 ppb prior to implementation of guidelines mandating increased distance between vessels and whales. While causality is unclear, the potential PAH exposure from vessels warrants continued monitoring. Historical precedent similarly emphasizes the importance of having pre-oil spill exposure data available as baseline to guide remediation goals.

Physiological, morphological, and ecological tradeoffs influence vertical habitat use of deep-diving toothed-whales in the Bahamas.

Dive capacity among toothed whales (suborder: Odontoceti) has been shown to generally increase with body mass in a relationship closely linked to the allometric scaling of metabolic rates. However, two odontocete species tagged in this study, the Blainville's beaked whale Mesoplodon densirostris and the Cuvier's beaked whale Ziphius cavirostris, confounded expectations of a simple allometric relationship, with exceptionally long (mean: 46.1 min & 65.4 min) and deep dives (mean: 1129 m & 1179 m), and comparatively small body masses (med.: 842.9 kg & 1556.7 kg). These two species also exhibited exceptionally long recovery periods between successive deep dives, or inter-deep-dive intervals (M. densirostris: med. 62 min; Z. cavirostris: med. 68 min). We examined competing hypotheses to explain observed patterns of vertical habitat use based on body mass, oxygen binding protein concentrations, and inter-deep-dive intervals in an assemblage of five sympatric toothed whales species in the Bahamas. Hypotheses were evaluated using dive data from satellite tags attached to the two beaked whales (M. densirostris, n = 12; Z. cavirostris, n = 7), as well as melon-headed whales Peponocephala electra (n = 13), short-finned pilot whales Globicephala macrorhynchus (n = 15), and sperm whales Physeter macrocephalus (n = 27). Body mass and myoglobin concentration together explained only 36% of the variance in maximum dive durations. The inclusion of inter-deep-dive intervals, substantially improved model fits (R2 = 0.92). This finding supported a hypothesis that beaked whales extend foraging dives by exceeding aerobic dive limits, with the extension of inter-deep-dive intervals corresponding to metabolism of accumulated lactic acid. This inference points to intriguing tradeoffs between body size, access to prey in different depth strata, and time allocation within dive cycles. These tradeoffs and resulting differences in habitat use have important implications for spatial distribution patterns, and relative vulnerabilities to anthropogenic impacts.

Quantifying the influence of sociality on population structure in bottlenose dolphins.

1. The social structure of a population plays a key role in many aspects of its ecology and biology. It influences its genetic make-up, the way diseases spread through it and the way animals exploit their environment. However, the description of social structure in nonprimate animals is receiving little attention because of the difficulty in abstracting social structure from the description of association patterns between individuals. 2. Here we focus on recently developed analytical techniques that facilitate inference about social structure from association patterns. We apply them to the population of bottlenose dolphins residing along the Scottish east coast, to detect the presence of communities within this population and infer its social structure from the temporal variation in association patterns between individuals. 3. Using network analytical techniques, we show that the population is composed of two social units with restricted interactions. These two units seem to be related to known differences in the ranging pattern of individuals. By examining social structuring at different spatial scales, we confirm that the identification of these two units is the result of genuine social affiliation and is not an artefact of their spatial distribution. 4. We also show that the structure of this fission-fusion society relies principally on short-term casual acquaintances lasting a few days with a smaller proportion of associations lasting several years. These findings highlight how network analyses can be used to detect and understand the forces driving social organization of bottlenose dolphins and other social species.