"Type D" killer whale genomes reveal long-term small population size and low genetic diversity.

Genome sequences can reveal the extent of inbreeding in small populations. Here, we present the first genomic characterization of type D killer whales, a distinctive eco/morphotype with a circumpolar, subantarctic distribution. Effective population size is the lowest estimated from any killer whale genome and indicates a severe population bottleneck. Consequently, type D genomes show among the highest level of inbreeding reported for any mammalian species (FROH ≥ 0.65). Detected recombination cross-over events of different haplotypes are up to an order of magnitude rarer than in other killer whale genomes studied to date. Comparison of genomic data from a museum specimen of a type D killer whale that stranded in New Zealand in 1955, with 3 modern genomes from the Cape Horn area, reveals high covariance and identity-by-state of alleles, suggesting these genomic characteristics and demographic history are shared among geographically dispersed social groups within this morphotype. Limitations to the insights gained in this study stem from the nonindependence of the 3 closely related modern genomes, the recent coalescence time of most variation within the genomes, and the nonequilibrium population history which violates the assumptions of many model-based methods. Long-range linkage disequilibrium and extensive runs of homozygosity found in type D genomes provide the potential basis for both the distinctive morphology, and the coupling of genetic barriers to gene flow with other killer whale populations.

Complete mitochondrial genome phylogeographic analysis of killer whales (Orcinus orca) indicates multiple species.

Killer whales (Orcinus orca) currently comprise a single, cosmopolitan species with a diverse diet. However, studies over the last 30 yr have revealed populations of sympatric "ecotypes" with discrete prey preferences, morphology, and behaviors. Although these ecotypes avoid social interactions and are not known to interbreed, genetic studies to date have found extremely low levels of diversity in the mitochondrial control region, and few clear phylogeographic patterns worldwide. This low level of diversity is likely due to low mitochondrial mutation rates that are common to cetaceans. Using killer whales as a case study, we have developed a method to readily sequence, assemble, and analyze complete mitochondrial genomes from large numbers of samples to more accurately assess phylogeography and estimate divergence times. This represents an important tool for wildlife management, not only for killer whales but for many marine taxa. We used high-throughput sequencing to survey whole mitochondrial genome variation of 139 samples from the North Pacific, North Atlantic, and southern oceans. Phylogenetic analysis indicated that each of the known ecotypes represents a strongly supported clade with divergence times ranging from approximately 150,000 to 700,000 yr ago. We recommend that three named ecotypes be elevated to full species, and that the remaining types be recognized as subspecies pending additional data. Establishing appropriate taxonomic designations will greatly aid in understanding the ecological impacts and conservation needs of these important marine predators. We predict that phylogeographic mitogenomics will become an important tool for improved statistical phylogeography and more precise estimates of divergence times.

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.

Positive selection on the killer whale mitogenome.

Mitochondria produce up to 95 per cent of the eukaryotic cell's energy. The coding genes of the mitochondrial DNA may therefore evolve under selection owing to metabolic requirements. The killer whale, Orcinus orca, is polymorphic, has a global distribution and occupies a range of ecological niches. It is therefore a suitable organism for testing this hypothesis. We compared a global dataset of the complete mitochondrial genomes of 139 individuals for amino acid changes that were associated with radical physico-chemical property changes and were influenced by positive selection. Two such selected non-synonymous amino acid changes were found; one in each of two ecotypes that inhabit the Antarctic pack ice. Both substitutions were associated with changes in local polarity, increased steric constraints and α-helical tendencies that could influence overall metabolic performance, suggesting a functional change.

The world's smallest whale population?

The North Pacific right whale (Eubalaena japonica) was heavily exploited by both nineteenth century whaling and recent (1960s) illegal Soviet catches. Today, the species remains extremely rare especially in the eastern North Pacific. Here, we use photographic and genotype data to calculate the first mark-recapture estimates of abundance for right whales in the Bering Sea and Aleutian Islands. The estimates were very similar: photographic = 31 (95% CL 23-54), genotyping = 28 (95% CL 24-42). We also estimated the population contains eight females (95% CL 7-18) and 20 males (95% CL 17-37). Although these estimates may relate to a Bering Sea subpopulation, other data suggest that the total eastern North Pacific population is unlikely to be much larger. Its precarious status today-the world's smallest whale population for which an abundance estimate exists-is a direct consequence of uncontrolled and illegal whaling, and highlights the past failure of international management to prevent such abuses.

Cold call: the acoustic repertoire of Ross Sea killer whales (Orcinus orca, Type C) in McMurdo Sound, Antarctica.

Killer whales (Orcinus orca) are top marine predators occurring globally. In Antarctic waters, five ecotypes have been described, with Type C being the smallest form of killer whale known. Acoustic recordings of nine encounters of Type C killer whales were collected in 2012 and 2013 in McMurdo Sound, Ross Sea. In a combined 3.5 h of recordings, 6386 killer whale vocalizations were detected and graded based on their signal-to-noise ratio. Spectrograms of the highest-quality calls were examined for characteristic patterns yielding a catalogue of 28 call types (comprising 1250 calls). Acoustic parameters of each call were measured and summarized by call type. Type C killer whales produced complex calls, consisting of multiple frequency-modulated, amplitude-modulated and pulsed components. Often, two components occurred simultaneously, forming a biphonation; although the biphonic components did not necessarily start and end together, with one component lasting over several others. The addition and deletion of components yielded call subtypes. Call complexity appears stable over time and may be related to feeding ecology. Characterization of the Type C acoustic repertoire is an important step for the development of passive acoustic monitoring of the diverse assemblage of killer whale ecotypes in Antarctica's rapidly changing marine ecosystems.

Whale and dolphin behavioural responses to dead conspecifics.

The scientific study of death across animal taxa-comparative thanatology-investigates how animals respond behaviourally, physiologically and psychologically to dead conspecifics, and the processes behind such responses. Several species of cetaceans have been long known to care for, attend to, be aroused by, or show interest in dead or dying individuals. We investigated patterns and variation in cetacean responses to dead conspecifics across cetacean taxa based on a comprehensive literature review. We analysed 78 records reported between 1970 and 2016, involving 20 of the 88 extant cetacean species. We adopted a weighted comparative approach to take observation effort into account and found that odontocetes (toothed cetaceans) were much more likely than mysticetes (baleen whales) to attend to dead conspecifics. Dolphins (Delphinidae) had the greatest occurrence of attentive behaviour (92.3% of all records), with a weighed attendance index 18 times greater than the average of all other cetacean families. Two dolphin genera, Sousa and Tursiops, constituted 55.1% of all cetacean records (N=43) and showed the highest incidence of attentive behaviour. Results of analyses intended to investigate the reasons behind these differences suggested that encephalisation may be an important predictor, consistent with the "social brain" hypothesis. Among attending individuals or groups of known sex (N=28), the majority (75.0%) were adult females with dead calves or juveniles (possibly their own offspring, with exceptions), consistent with the strong mother-calf bond, or, in a few cases, with the bond between mothers and other females in the group. The remaining records (25.0%) involved males either showing sexual interest in a dead adult or subadult, or carrying a dead calf in the presence of females. Because an inanimate individual is potentially rescuable, responses to dead conspecifics-especially by females-can be explained at least in part by attempts to revive and protect, having a clear adaptive value. In some cases such responses are followed by apparently maladaptive behaviour such as the long-term carrying of, or standing by, a decomposed carcass, similar to observations of certain terrestrial mammals. Among the possible explanations for the observed cetacean behavioural responses to dead conspecifics are strong attachment resulting in a difficulty of "letting go"-possibly related to grieving-or perhaps individuals failing to recognise or accept that an offspring or companion has died. Our current understanding is challenged by small sample size, incomplete descriptions, and lack of information on the physiology and neural processes underpinning the observed behaviour. We provide research recommendations that would improve such understanding.

Phylogenetics and biogeography of the two-wing flyingfish (Exocoetidae: Exocoetus).

Two-wing flyingfish (Exocoetus spp.) are widely distributed, epipelagic, mid-trophic organisms that feed on zooplankton and are preyed upon by numerous predators (e.g., tunas, dolphinfish, tropical seabirds), yet an understanding of their speciation and systematics is lacking. As a model of epipelagic fish speciation and to investigate mechanisms that increase biodiversity, we studied the phylogeny and biogeography of Exocoetus, a highly abundant holoepipelagic fish taxon of the tropical open ocean. Morphological and molecular data were used to evaluate the phylogenetic relationships, species boundaries, and biogeographic patterns of the five putative Exocoetus species. We show that the most widespread species (E. volitans) is sister to all other species, and we find no evidence for cryptic species in this taxon. Sister relationship between E. monocirrhus (Indo-Pacific) and E. obtusirostris (Atlantic) indicates the Isthmus of Panama and/or Benguela Barrier may have played a role in their divergence via allopatric speciation. The sister species E. peruvianus and E. gibbosus are found in different regions of the Pacific Ocean; however, our molecular results do not show a clear distinction between these species, indicating recent divergence or ongoing gene flow. Overall, our phylogeny reveals that the most spatially restricted species are more recently derived, suggesting that allopatric barriers may drive speciation, but subsequent dispersal and range expansion may affect the distributions of species.

Population Genetic Structure of the Tropical Two-Wing Flyingfish (Exocoetus volitans).

Delineating populations of pantropical marine fish is a difficult process, due to widespread geographic ranges and complex life history traits in most species. Exocoetus volitans, a species of two-winged flyingfish, is a good model for understanding large-scale patterns of epipelagic fish population structure because it has a circumtropical geographic range and completes its entire life cycle in the epipelagic zone. Buoyant pelagic eggs should dictate high local dispersal capacity in this species, although a brief larval phase, small body size, and short lifespan may limit the dispersal of individuals over large spatial scales. Based on these biological features, we hypothesized that E. volitans would exhibit statistically and biologically significant population structure defined by recognized oceanographic barriers. We tested this hypothesis by analyzing cytochrome b mtDNA sequence data (1106 bps) from specimens collected in the Pacific, Atlantic and Indian oceans (n = 266). AMOVA, Bayesian, and coalescent analytical approaches were used to assess and interpret population-level genetic variability. A parsimony-based haplotype network did not reveal population subdivision among ocean basins, but AMOVA revealed limited, statistically significant population structure between the Pacific and Atlantic Oceans (ΦST = 0.035, p<0.001). A spatially-unbiased Bayesian approach identified two circumtropical population clusters north and south of the Equator (ΦST = 0.026, p<0.001), a previously unknown dispersal barrier for an epipelagic fish. Bayesian demographic modeling suggested the effective population size of this species increased by at least an order of magnitude ~150,000 years ago, to more than 1 billion individuals currently. Thus, high levels of genetic similarity observed in E. volitans can be explained by high rates of gene flow, a dramatic and recent population expansion, as well as extensive and consistent dispersal throughout the geographic range of the species.

Octopaminergic modulation of synaptic transmission between an identified sensory afferent and flight motoneuron in the locust.

The role of the biogenic amine octopamine in modulating cholinergic synaptic transmission between the locust forewing stretch receptor neuron (fSR) and the first basalar motoneuron (BA1) was investigated. The amines 5-hydroxytryptamine (5-HT, serotonin) and dopamine were also studied. Bath application of octopamine, 5-HT, and dopamine at concentrations of 10(-4) M reversibly decreased the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in BA1 by electrically stimulating the fSR axon. These effects occurred without any detectable change in either input resistance or membrane potential of BA1. The amines also reversibly decreased the amplitude of responses to acetylcholine (ACh) pressure-applied to the soma of BA1. The muscarinic antagonist scopolamine (10(-6) M) had no significant effect on the octopamine-induced decrease in ACh responses. These observations suggest that these amines potentially could physiologically depress cholinergic transmission between fSR and BA1, at least in part, by altering nicotinic rather than muscarinic cholinergic receptor function. Although the octopaminergic agonists naphazoline and tolazoline both mimicked the actions of octopamine, the receptor responsible for octopamine-mediated modulation could not be characterized since amine receptor antagonists tested on the preparation had complex actions. Confocal immunocytochemistry revealed intense octopamine immunoreactivity in the anterior lateral association center, thus confirming the presence of octopamine in neuropil regions containing fSR/BA1 synapses and therefore supporting a role for this amine in the modulation of synaptic transmission between the fSR and BA1. 5-HT-immunoreactivity, conversely, was concentrated within the ventral association centers; very little staining was observed in the dorsal neuropil regions in which fSR/BA1 synapses are located.

Concordant preferences for opposite-sex signals? Human pheromones and facial characteristics.

We have investigated whether preferences for masculine and feminine characteristics are correlated across two modalities, olfaction and vision. In study 1, subjects rated the pleasantness of putative male (4,16-androstadien-3-one; 5alpha-androst-16-en-3-one) and female (1,3,5 (10),16-estratetraen-3-ol) pheromones, and chose the most attractive face shape from a masculine-feminine continuum for a long- and a short-term relationship. Study 2 replicated study 1 and further explored the effects of relationship context on pheromone ratings. For long-term relationships, women's preferences for masculine face shapes correlated with ratings of 4,16-androstadien-3-one and men's preferences for feminine face shapes correlated with ratings of 1,3,5(10),16-estratetraen-3-ol. These studies link sex-specific preferences for putative human sex pheromones and sexually dimorphic facial characteristics. Our findings suggest that putative sex pheromones and sexually dimorphic facial characteristics convey common information about the quality of potential mates.

Mitochondrial sequence divergence among Antarctic killer whale ecotypes is consistent with multiple species.

Recently, three visually distinct forms of killer whales (Orcinus orca) were described from Antarctic waters and designated as types A, B and C. Based on consistent differences in prey selection and habitat preferences, morphological divergence and apparent lack of interbreeding among these broadly sympatric forms, it was suggested that they may represent separate species. To evaluate this hypothesis, we compared complete sequences of the mitochondrial control region from 81 Antarctic killer whale samples, including 9 type A, 18 type B, 47 type C and 7 type-undetermined individuals. We found three fixed differences that separated type A from B and C, and a single fixed difference that separated type C from A and B. These results are consistent with reproductive isolation among the different forms, although caution is needed in drawing further conclusions. Despite dramatic differences in morphology and ecology, the relatively low levels of sequence divergence in Antarctic killer whales indicate that these evolutionary changes occurred relatively rapidly and recently.

First human-caused extinction of a cetacean species?

The Yangtze River dolphin or baiji (Lipotes vexillifer), an obligate freshwater odontocete known only from the middle-lower Yangtze River system and neighbouring Qiantang River in eastern China, has long been recognized as one of the world's rarest and most threatened mammal species. The status of the baiji has not been investigated since the late 1990s, when the surviving population was estimated to be as low as 13 individuals. An intensive six-week multi-vessel visual and acoustic survey carried out in November-December 2006, covering the entire historical range of the baiji in the main Yangtze channel, failed to find any evidence that the species survives. We are forced to conclude that the baiji is now likely to be extinct, probably due to unsustainable by-catch in local fisheries. This represents the first global extinction of a large vertebrate for over 50 years, only the fourth disappearance of an entire mammal family since AD 1500, and the first cetacean species to be driven to extinction by human activity. Immediate and extreme measures may be necessary to prevent the extinction of other endangered cetaceans, including the sympatric Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis).

Modulation by 5-hydroxytryptamine of nicotinic acetylcholine responses recorded from an identified cockroach (Periplaneta americana) motoneuron.

Recordings from the soma of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) were made while acetylcholine (ACh) was regularly pressure-applied locally from a micropipette. The modulatory effects upon these nicotinic ACh responses of bath-applied 5-hydroxytryptamine (5-HT, serotonin), dopamine and octopamine were investigated under either current-clamp or voltage-clamp conditions. The biogenic amines reversibly suppressed, but never totally abolished, ACh responses, 5-HT being the most potent, with a threshold near 10(-6) m (EC50 = 5 x 10(-5) m). Occlusion experiments indicate that the amines share a common mechanism at the level of either receptors or second messenger pathways. The amines also modulated responses to nicotine or carbachol (each of which resists hydrolysis by acetylcholinesterases), indicating that the amines did not act by accelerating ACh degradation. Pharmacological antagonists were used in an attempt to characterize the receptor responsible for amine-mediated modulation. Although a number of antagonists mimicked the action of amines rather than producing blockade, the antagonistic actions of LSD and RS23597 pointed strongly to a receptor-mediated mechanism, but did not allow receptor identification. The magnitude of the modulatory effect of 5-HT was significantly reduced by intracellular guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S), indicating involvement of a G-protein. Intracellular injection of the calcium chelator BAPTA did not block the modulatory effect of 5-HT, showing that the amines do not operate through the calcium-dependent pathway by which muscarinic receptors act on nicotinic currents. The adenylate cyclase inhibitor dideoxyadenosine (DDA), on the other hand, did attenuate the action of 5-HT, suggesting involvement of cyclic AMP.