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.

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.

Click reception in the harbor porpoise (Phocoena phocoena): Effects of electrode and contact transducer location on the auditory brainstem response.

Unlike terrestrial mammals that have unambiguous aerial sound transmission pathways via the outer ear and tympanum, sound reception pathways in most odontocetes are not well understood. Recent studies have used auditory brainstem response (ABR) measurements to examine sound reception pathways. This study sought to determine how sound source placements, recording electrode arrangements, and ABR peak analyses affect interpretations of sound reception in the harbor porpoise (Phocoena phocoena). Click stimuli were delivered in air from a contact transducer ("jawphone"). Early ABR peaks (representing auditory nerve responses), and later peaks reflecting higher brainstem activity, were analyzed across jawphone and recording electrode positions. Auditory nerve responses were similar for jawphone placements from the ipsilateral posterior mandible to the tip of the rostrum. Later peaks, however, suggested a possible region of highest sensitivity midway between the posterior mandible and the rostrum tip. These findings are generally similar to previous data for porpoises. In contrast to auditory nerve responses that were largest when recorded near the ipsilateral meatus, later ABR peaks were largest when recorded with a contralateral (opposing) electrode. These results provide information on the processes underlying peaks of the ABR, and inform stimulus delivery and ABR recording parameters in odontocete sound reception studies.

Extensive Core Microbiome in Drone-Captured Whale Blow Supports a Framework for Health Monitoring.

The pulmonary system is a common site for bacterial infections in cetaceans, but very little is known about their respiratory microbiome. We used a small, unmanned hexacopter to collect exhaled breath condensate (blow) from two geographically distinct populations of apparently healthy humpback whales (Megaptera novaeangliae), sampled in the Massachusetts coastal waters off Cape Cod (n = 17) and coastal waters around Vancouver Island (n = 9). Bacterial and archaeal small-subunit rRNA genes were amplified and sequenced from blow samples, including many of sparse volume, as well as seawater and other controls, to characterize the associated microbial community. The blow microbiomes were distinct from the seawater microbiomes and included 25 phylogenetically diverse bacteria common to all sampled whales. This core assemblage comprised on average 36% of the microbiome, making it one of the more consistent animal microbiomes studied to date. The closest phylogenetic relatives of 20 of these core microbes were previously detected in marine mammals, suggesting that this core microbiome assemblage is specialized for marine mammals and may indicate a healthy, noninfected pulmonary system. Pathogen screening was conducted on the microbiomes at the genus level, which showed that all blow and few seawater microbiomes contained relatives of bacterial pathogens; no known cetacean respiratory pathogens were detected in the blow. Overall, the discovery of a shared large core microbiome in humpback whales is an important advancement for health and disease monitoring of this species and of other large whales. IMPORTANCE The conservation and management of large whales rely in part upon health monitoring of individuals and populations, and methods generally necessitate invasive sampling. Here, we used a small, unmanned hexacopter drone to noninvasively fly above humpback whales from two populations, capture their exhaled breath (blow), and examine the associated microbiome. In the first extensive examination of the large-whale blow microbiome, we present surprising results about the discovery of a large core microbiome that was shared across individual whales from geographically separated populations in two ocean basins. We suggest that this core microbiome, in addition to other microbiome characteristics, could be a useful feature for health monitoring of large whales worldwide.

Physical constraints of cultural evolution of dialects in killer whales.

Odontocete sounds are produced by two pairs of phonic lips situated in soft nares below the blowhole; the right pair is larger and is more likely to produce clicks, while the left pair is more likely to produce whistles. This has important implications for the cultural evolution of delphinid sounds: the greater the physical constraints, the greater the probability of random convergence. In this paper the authors examine the call structure of eight killer whale populations to identify structural constraints and to determine if they are consistent among all populations. Constraints were especially pronounced in two-voiced calls. In the calls of all eight populations, the lower component of two-voiced (biphonic) calls was typically centered below 4 kHz, while the upper component was typically above that value. The lower component of two-voiced calls had a narrower frequency range than single-voiced calls in all populations. This may be because some single-voiced calls are homologous to the lower component, while others are homologous to the higher component of two-voiced calls. Physical constraints on the call structure reduce the possible variation and increase the probability of random convergence, producing similar calls in different populations.

Hybridization in the Cetacea: widespread occurrence and associated morphological, behavioral, and ecological factors.

Hybridization has been documented in a many different pairs of cetacean species both in captivity and in the wild. The widespread occurrence of hybridization indicates that postmating barriers to interbreeding are incomplete within the order Cetacea, and therefore raises questions about how species integrity is maintained in the face of interspecific (and often intergeneric) gene flow. We examined hybridization across the order Cetacea (oceanic species included: N = 78; species with 44 chromosomes included: N = 52) to test for associations between the occurrence of hybridization and similarity across 13 ecological, morphological and behavioral traits in hybridizing vs. non-hybridizing species pairs. We found that species pairs that share a greater number of traits had a higher propensity to hybridize than pairs of species that did not. This trend was driven by behavioral and morphological traits such as vocalization frequency and body size. Together our findings suggest the importance of divergent selection on morphological and behavioral traits within sympatric species in constraining opportunities for hybridization and preventing the collapse of parental species.

Killer whale call frequency is similar across the oceans, but varies across sympatric ecotypes.

Killer whale populations may differ in genetics, morphology, ecology, and behavior. In the North Pacific, two sympatric populations ("resident" and "transient") specialize on different prey (fish and marine mammals) and retain reproductive isolation. In the eastern North Atlantic, whales from the same populations have been observed feeding on both fish and marine mammals. Fish-eating North Pacific "residents" are more genetically related to eastern North Atlantic killer whales than to sympatric mammal-eating "transients." In this paper, a comparison of frequency variables in killer whale calls recorded from four North Pacific resident, two North Pacific transient, and two eastern North Atlantic populations is reported to assess which factors drive the large-scale changes in call structure. Both low-frequency and high-frequency components of North Pacific transient killer whale calls have significantly lower frequencies than those of the North Pacific resident and North Atlantic populations. The difference in frequencies could be related to ecological specialization or to the phylogenetic history of these populations. North Pacific transient killer whales may have genetically inherited predisposition toward lower frequencies that may shape their learned repertoires.

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.

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.

Ultrasonic whistles of killer whales (Orcinus orca) recorded in the North Pacific (L).

Ultrasonic whistles were previously found in North Atlantic killer whales and were suggested to occur in eastern North Pacific killer whales based on the data from autonomous recorders. In this study ultrasonic whistles were found in the recordings from two encounters with the eastern North Pacific offshore ecotype killer whales and one encounter with the western North Pacific killer whales of unknown ecotype. All ultrasonic whistles were highly stereotyped and all but two had downsweep contours. These results demonstrate that specific sound categories can be shared by killer whales from different ocean basins.

Estimation of southern resident killer whale exposure to exhaust emissions from whale-watching vessels and potential adverse health effects and toxicity thresholds.

Southern resident killer whales in British Columbia and Washington are exposed to heavy vessel traffic. This study investigates their exposure to exhaust gases from whale-watching vessels by using a simple dispersion model incorporating data on whale and vessel behavior, atmospheric conditions, and output of airborne pollutants from the whale-watching fleet based on emissions data from regulatory agencies. Our findings suggest that current whale-watching guidelines are usually effective in limiting pollutant exposure to levels at or just below those at which measurable adverse health effects would be expected in killer whales. However, safe pollutant levels are exceeded under worst-case conditions and certain average-case conditions. To reduce killer whale exposure to exhaust we recommend: vessels position on the downwind side of whales, a maximum of 20 whale-watching vessels should be within 800 m at any given time, viewing periods should be limited, and current whale-watch guidelines and laws should be enforced.

The structure of stereotyped calls reflects kinship and social affiliation in resident killer whales (Orcinus orca).

A few species of mammals produce group-specific vocalisations that are passed on by learning, but the function of learned vocal variation remains poorly understood. Resident killer whales live in stable matrilineal groups with repertoires of seven to 17 stereotyped call types. Some types are shared among matrilines, but their structure typically shows matriline-specific differences. Our objective was to analyse calls of nine killer whale matrilines in British Columbia to test whether call similarity primarily reflects social or genetic relationships. Recordings were made in 1985-1995 in the presence of focal matrilines that were either alone or with groups with non-overlapping repertoires. We used neural network discrimination performance to measure the similarity of call types produced by different matrilines and determined matriline association rates from 757 encounters with one or more focal matrilines. Relatedness was measured by comparing variation at 11 microsatellite loci for the oldest female in each group. Call similarity was positively correlated with association rates for two of the three call types analysed. Similarity of the N4 call type was also correlated with matriarch relatedness. No relationship between relatedness and association frequency was detected. These results show that call structure reflects relatedness and social affiliation, but not because related groups spend more time together. Instead, call structure appears to play a role in kin recognition and shapes the association behaviour of killer whale groups. Our results therefore support the hypothesis that increasing social complexity plays a role in the evolution of learned vocalisations in some mammalian species.

Allonursing in captive belugas (Delphinapterus leucas).

This study describes allonursing (females nursing offspring that are not their own) in captive belugas (Delphinapterus leucas). In addition to the calf's mother, two females that were not pregnant or nursing at the time of the calf's birth spontaneously lactated and nursed the male calf intermittently throughout 34 months of his life at the Vancouver Aquarium Marine Science Center. These observations suggest that allonursing may also take place in their wild counterparts and add to existing evidence of alloparental care in this species. Alloparental care, the care of nonoffspring, has been observed in every major mammalian taxon; the protection of calves through alloparental care may be a strong evolutionary benefit.

PBDEs, PBBs, and PCNs in three communities of free-ranging killer whales (Orcinus orca) from the northeastern Pacific Ocean.

Polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls (PBBs), and polychlorinated naphthalenes (PCNs) were quantified in blubber biopsy samples collected from free-ranging male and female killer whales (Orcinus orca) belonging to three distinct communities (southern residents, northern residents, and transients) from the northeastern Pacific Ocean. High concentrations of sigmaPBDE were observed in male southern residents (942+/-582 ng/g Iw), male and female transients (1015+/-605 and 885+/-706 ng/g Iw, respectively), and male and female northern residents (203+/-116 and 415+/-676 ng/g Iw, respectively). Because of large variation within sample groups, sigmaPBDE levels generally did not differ statistically with the exception of male northern residents, which had lower sigmaPBDE concentrations than male southern residents, male transients, and female transients, perhaps reflecting the consumption of less contaminated prey items. Male transient killer whales, which consume high trophic level prey including other cetaceans and occasionally spend time near populated areas, had sigmaPBDE concentrations approximately equal to southern residents. No significant age-related relationships were observed for sigmaPBDE concentrations. sigmaPBDE concentrations were approximately 1-3 orders of magnitude greater than those of sigmaPBB (3.0-31 ng/g Iw) and sigmaPCN (20-167 ng/g Iw) measured in a subset of samples, suggesting that PBDEs may represent a contaminant class of concern in these marine mammals.