Integrating technologies provides insight into the subsurface foraging behaviour of a humpback whale (Megaptera novaeangliae) feeding on walleye pollock (Gadus chalcogrammus) in Juan de Fuca Strait, Canada.

Subsurface foraging is an important proportion of the activity budget of rorqual whales, yet information on their behaviour underwater remains challenging to obtain. Rorquals are assumed to feed throughout the water column and to select prey as a function of depth, availability and density, but there remain limitations in the precise identification of targeted prey. Current data on rorqual foraging in western Canadian waters have thus been limited to observations of prey species amenable to surface feeding, such as euphausiids and Pacific herring (Clupea pallasii), with no information on deeper alternative prey sources. We measured the foraging behaviour of a humpback whale (Megaptera novaeangliae) in Juan de Fuca Strait, British Columbia, using three complimentary methods: whale-borne tag data, acoustic prey mapping, and fecal sub-sampling. Acoustically detected prey layers were near the seafloor and consistent with dense schools of walleye pollock (Gadus chalcogrammus) distributed above more diffuse aggregations of pollock. Analysis of a fecal sample from the tagged whale confirmed that it had been feeding on pollock. Integrating the dive profile with the prey data revealed that the whale's foraging effort followed the general pattern of areal prey density, wherein the whale had a higher lunge-feeding rate at the highest prey abundance and stopped feeding when prey became limited. Our findings of a humpback whale feeding on seasonally energy-dense fish like walleye pollock, which are potentially abundant in British Columbia, suggests that pollock may be an important prey source for this rapidly growing whale population. This result is informative when assessing regional fishing activities for semi-pelagic species as well as the whales' vulnerability to fishing gear entanglements and feeding disturbances during a narrow window of prey acquisition.

Impacts of Seawater pH Buffering on the Larval Microbiome and Carry-Over Effects on Later-Life Disease Susceptibility in Pacific Oysters.

Ocean acidification upwelling events and the resulting lowered aragonite saturation state of seawater have been linked to high mortality of marine bivalve larvae in hatcheries. Major oyster seed producers along North America's west coast have mitigated impacts via seawater pH buffering (e.g., addition of soda ash). However, little consideration has been given to whether such practice may impact the larval microbiome, with potential carry-over effects on immune competency and disease susceptibility in later-life stages. To investigate possible impacts, Pacific oysters (Crassostrea gigas) were reared under soda ash pH buffered or ambient pH seawater conditions for the first 24 h of development. Both treatment groups were then reared under ambient pH conditions for the remainder of the developmental period. Larval microbiome, immune status (via gene expression), growth, and survival were assessed throughout the developmental period. Juveniles and adults arising from the larval run were then subjected to laboratory-based disease challenges to investigate carry-over effects. Larvae reared under buffered conditions showed an altered microbiome, which was still evident in juvenile animals. Moreover, reduced survival was observed in both juveniles and adults of the buffered group under a simulated marine heatwave and Vibrio exposure compared with those reared under ambient conditions. Results suggest that soda ash pH buffering during early development may compromise later-life stages under stressor conditions, and illustrate the importance of a long-view approach with regard to hatchery husbandry practices and climate change mitigation. IMPORTANCE Shellfish industries are threatened worldwide by recurrent summer mortality events. Such incidences are often associated with Vibrio disease outbreaks, and thus, it is critical that animals are able to mount sufficient immune responses. The oyster immune system is linked to the microbiome which is laid down during early developmental stages. Consequently, shellfish hatcheries play a key role with regard to shaping the immune competency of later-life stages. This study represents the first in-depth examination of whether the adoption of seawater pH buffering practice by hatcheries for mitigation of ocean acidification may alter the larval microbiome, and thus, have repercussions for adult susceptibility to summer mortality events. Findings demonstrate that even minimal buffering results in a changed microbiome which is paralleled by increased mortality of later-life stages under Vibrio and temperature stressors, highlighting the importance of the hatchery environment with regard to shaping resilience to summer mortality events.

Genotyping-in-thousands by sequencing (GT-seq) of noninvasive faecal and degraded samples: A new panel to enable ongoing monitoring of Canadian polar bear populations.

Genetic monitoring using noninvasive samples provides a complement or alternative to traditional population monitoring methods. However, next-generation sequencing approaches to monitoring typically require high quality DNA and the use of noninvasive samples (e.g., scat) is often challenged by poor DNA quality and contamination by nontarget species. One promising solution is a highly multiplexed sequencing approach called genotyping-in-thousands by sequencing (GT-seq), which can enable cost-efficient genomics-based monitoring for populations based on noninvasively collected samples. Here, we develop and validate a GT-seq panel of 324 single nucleotide polymorphisms (SNPs) optimized for genotyping of polar bears based on DNA from noninvasively collected faecal samples. We demonstrate (1) successful GT-seq genotyping of DNA from a range of sample sources, including successful genotyping (>50% loci) of 62.9% of noninvasively collected faecal samples determined to contain polar bear DNA; and (2) that we can reliably differentiate individuals, ascertain sex, assess relatedness, and resolve population structure of Canadian polar bear subpopulations based on a GT-seq panel of 324 SNPs. Our GT-seq data reveal spatial-genetic patterns similar to previous polar bear studies but at lesser cost per sample and through use of noninvasively collected samples, indicating the potential of this approach for population monitoring. This GT-seq panel provides the foundation for a noninvasive toolkit for polar bear monitoring and can contribute to community-based programmes - a framework which may serve as a model for wildlife conservation and management for species worldwide.

Fine-scale diversity of prey detected in humpback whale feces.

Predator diets are largely influenced by prey availability and abundance. Yet, in heterogenous marine environments, identifying the prey species consumed by diving mammals remains a fundamental challenge. For rorqual whales, the energetic constraints of prey engulfment require that the whales seek areas of high prey abundance and execute discrete lunge feeding events on patches of high-density prey. Prey occurrences in feces should therefore provide meaningful insight into the dominant taxa in food patches selected by the animal. We investigated the prey consumed by humpback whales in three regions in southern British Columbia (BC), Canada, using opportunistic fecal sampling, microscopy, and DNA metabarcoding of 14 fecal samples. Fish including Pacific herring (Clupea pallasii), hake (Merluccius productus), and eulachon (Thaleichthys pacificus) were the most common fish species potentially targeted by humpback whales in two regions. The krill Euphausia pacifica was the most prevalent invertebrate DNA detected in all three regions, while sergestid and mysid shrimp may also be important. High DNA read abundances from walleye pollock (Gadus chalcogrammus) and sablefish (Anoplopoma fimbria) were also recovered in one sample each, suggesting that juveniles of these semi-pelagic species may occasionally be targeted. In general, we observed heavily digested fecal material that drove substantial dissimilarities in taxonomic resolution between polymerase chain reaction-based and morphological analyses of the feces. Pacific herring and walleye pollock were the only prey species confirmed by both methods. Our results highlight that molecular and visual analyses of fecal samples provide a complementary approach to diet analysis, with each method providing unique insight into prey diversity.

Cytonuclear discordance in the crowned-sparrows, Zonotrichia atricapilla and Zonotrichia leucophrys.

The golden-crowned (Zonotrichia atricapilla) and white-crowned (Z. leucophrys) sparrows have been presented as a compelling case for rapid speciation. They display divergence in song and plumage with overlap in their breeding ranges implying reproductive isolation, but have almost identical mitochondrial genomes. Previous research proposed hybridization and subsequent mitochondrial introgression as an alternate explanation, but lacked robust nuclear gene trees to distinguish between introgression and incomplete lineage sorting. We test for signatures of these processes between Z. atricapilla and Z. leucophrys, and investigate the relationships among Z. leucophrys subspecies, using mitochondrial sequencing and a reduced representation nuclear genomic dataset. Contrary to the paraphyly evident in mitochondrial gene trees, we confirmed the reciprocal monophyly of Z. atricapilla and Z. leucophrys using large panels of single nucleotide polymorphisms (SNPs). The pattern of cytonuclear discordance is consistent with limited, historical hybridization and mitochondrial introgression, rather than a recent origin and incomplete lineage sorting between recent sister species. We found evidence of nuclear phylogeographic structure within Z. leucophrys with two distinct clades. Altogether, our results indicate deeper divergences between Z. atricapilla and Z. leucophrys than inferred using mitochondrial markers. Our results demonstrate the limitations of relying solely on mitochondrial DNA for taxonomy, and raise questions about the possibility of selection on the mitochondrial genome during temperature oscillations (e.g. during the Pleistocene). Historical mitochondrial introgression facilitated by past environmental changes could cause erroneous dating of lineage splitting in other taxa when based on mitochondrial DNA alone.

Skull diversity and evolution in miniaturized amphibians, genus Brachycephalus (Anura: Brachycephalidae).

Miniaturized amphibians of the genus Brachycephalus are phenotypically diverse. The species of Brachycephalus have bufoniform or leptodactyliform Baupläne and any of three skeletal states: nonhyperossified, hyperossified without dorsal shield, and hyperossified with dorsal shield. We integrate high-resolution microcomputed tomography, geometric morphometrics, and an estimate of molecular phylogenetic relationships to investigate skull diversity in shape and size-shape space in selected species of Brachycephalus. Skull diversity amongst species of Brachycephalus can be partitioned into shape and size-shape space according to the four conditions of skeletal states-Baupläne, namely, nonhyperossified leptodactyliform, nonhyperossified bufoniform, hyperossified bufoniform without dorsal shield, and hyperossified bufoniform with dorsal shield. Skull diversity in shape and size-shape space in nonhyperossified leptodactyliform species of Brachycephalus is markedly larger, when compared to skull diversity in species of the three other conditions of skeletal states-Baupläne. Variation in skull shape scales with size across Brachycephalus and, therefore, can be explained by allometry. Skull diversity, Baupläne, and skeletal states covary to a large extent with monophyletic lineages of Brachycephalus, as revealed by a mitochondrial DNA species tree. Nonhyperossified bufoniform species and hyperossified bufoniform species with or without dorsal shield are monophyletic lineages, as inferred from a mitochondrial DNA species tree. Nonhyperossified leptodactyliform species of Brachycephalus do not share, however, a most recent common ancestor. The nonhyperossified leptodactyliform species of Brachycephalus, due to their marked skull diversity and lack of monophyly, emerge as evolutionarily complex. Therefore, further sampling of the nonhyperossified leptodactyliform condition of skeletal states-Baupläne will be necessary to further understand the evolutionary history of Brachycephalus.

Os anfíbios miniaturizados do gênero Brachycephalus são fenotipicamente diversos. As espécies de Brachycephalus têm o plano corporal bufoniforme ou leptodactyliforme e três estados esqueléticos: não-hiperossificado, hiperossificado sem placa dorsal e hiperossificado com placa dorsal. Neste trabalho nós integramos tomografia micro-computadorizada de alta resolução, morfometria geométrica e uma estimativa de relações filogenéticas moleculares para investigar diversidade craniana nos espaços de forma e tamanho-forma em determinadas espécies de Brachycephalus. A diversidade craniana entre espécies de Brachycephalus pode ser dividida no espaço de forma e tamanho-forma segundo as quatro condições de plano corporal-esqueleto, a saber, leptodactiliforme não-hiperossificado, bufoniforme não-hiperossificado, bufoniforme hiperossificado sem placa dorsal e bufoniforme hiperossificado com placa dorsal. A diversidade craniana nos espaços de forma e tamanho-forma nas espécies de Brachycephalus leptodactiliformes não-hiperossificadas é pronunciadamente maior quando comparada àquela das espécies nas outras trcs condições de plano corporal-esqueleto. A variação na forma craniana aumenta com o tamanho craniano em Brachycephalus e, portanto, pode ser explicada por alometria. Diversidade craniana, plano corporal e estados esqueléticos covariam consideravelmente com as linhagens monofiléticas de Brachycephalus, como estimado pela filogenia mitocondrial. As espécies de Brachycephalus leptodactiliformes não-hiperossificadas e bufoniformes hiperossificadas com ou sem placa dorsal são linhagens monofiléticas, como estimado pela filogenia mitocondrial. As espécies leptodactiliformes não-hiperossificadas não compartilham, todavia, um ancestral comum mais recente. As espécies de Brachycephalus leptodactiliformes não-hiperossificadas devido a sua pronunciada diversidade e não-monofilia emergem claramente como entidades evolutivamente complexas. Por conseguinte, a amostragem adicional de populações leptodactiliformes não-hiperossificadas será necessária para uma melhor compreensão da história evolutiva do gênero Brachycephalus.

Comparing fish prey diversity for a critically endangered aquatic mammal in a reserve and the wild using eDNA metabarcoding.

Using environmental DNA (eDNA) metabarcoding, we compared fish diversity in two distinct water bodies within the Yangtze River Basin with known populations of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis; YFP): the Tian-e-Zhou Reserve and Poyang Lake. We aimed to create a fish surveying tool for use in the Yangtze River Basin, while also gaining a better understanding of the prey distribution and diversity within two of the remaining strongholds of YFP. 16S rRNA universal primers were developed to amplify fish eDNA. After high-throughput sequencing and stringent data filtering, we identified a total of 75 fish species (6 orders, 9 families, 57 genera) across seasons and regions. Nine of the 75 fish species were among the 28 known YFP prey species, three of which were detected in all water samples. Our eDNA metabarcoding identified many species that had been previously captured using traditional netting practices, but also numerous species not previously collected in these water bodies. Fish diversity was higher in Poyang Lake than in Tian-e-Zhou Reserve, as well as higher in the spring than in summer. These methods provide a broadly applicable tool to quantify fish diversity and distributions throughout the Yangtze River Basin, and to inform conservation strategies of YFP.

Canadian polar bear population structure using genome-wide markers.

Predicting the consequences of environmental changes, including human-mediated climate change on species, requires that we quantify range-wide patterns of genetic diversity and identify the ecological, environmental, and historical factors that have contributed to it. Here, we generate baseline data on polar bear population structure across most Canadian subpopulations (n = 358) using 13,488 genome-wide single nucleotide polymorphisms (SNPs) identified with double-digest restriction site-associated DNA sequencing (ddRAD). Our ddRAD dataset showed three genetic clusters in the sampled Canadian range, congruent with previous studies based on microsatellites across the same regions; however, due to a lack of sampling in Norwegian Bay, we were unable to confirm the existence of a unique cluster in that subpopulation. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range.

Spatial distribution, prevalence and diversity of haemosporidians in the rufous-collared sparrow, Zonotrichia capensis.

BACKGROUND: Parasite prevalence and diversity are determined by the distribution of hosts and vectors and by the interplay among a suite of environmental factors. Distributions of parasite lineages vary based on host susceptibility and geographical barriers. Hemoparasites of the genera Haemoproteus and Plasmodium have wide distributions, and high prevalence and genetic diversity within perching birds (Order Passeriformes). The rufous-collared sparrow (Zonotrichia capensis) is widely distributed in Central and South America across an immense diversity of environments from sea level to more than 4000 meters above sea level. It therefore provides an excellent model to investigate whether altitudinal and latitudinal gradients influence the distribution, prevalence and diversity of haemosporidian parasites, their population structure and the biogeographical boundaries of distinct parasite lineages. RESULTS: We assembled samples from 1317 rufous-collared sparrows spanning 75 locales from across Central and South America (between 9.5°N and 54°S; 10-4655 meters above sea level). We used DNA sequence data from a fragment of the mitochondrial cytochrome b gene (cytb) of Haemoproteus and Plasmodium from 325 positive samples and found prevalences of 22 and 3%, respectively. Haemoproteus exhibited a higher prevalence than Plasmodium but with comparatively lower genetic diversity. We detected a relationship of Plasmodium and Haemoproteus prevalence with altitude and latitude; however, altitude and latitude did not influence parasite diversity. CONCLUSIONS: Parasite lineages showed a phylogeographical boundary coincident with the Andes Mountains, although we also observed a north-south disjunction in Peru for Haemoproteus. Haemosporidian distribution was not homogeneous but differed based on latitude and altitude. This is most probably due to environmental factors that have influenced both vector distribution and abundance, as well as parasite development. Our study provides key insights on the distribution of haemoparasite lineages and parasite dynamics within hosts.

Vocalizations, tadpole, and natural history of Crossodactylus werneri Pimenta, Cruz Caramaschi, 2014 (Anura: Hylodidae), with comments on distribution and intraspecific variation.

Crossodactylus werneri was described based on specimens collected in the 1970's at Parque Nacional do Itatiaia, being also reported for nearby localities. We collected specimens that we assigned to C. werneri, and recorded calls of the species during fieldworks at Serra das Cabras (Campinas, state of São Paulo). In this paper, we describe for the first time the vocalizations, tadpole, coloration in life, and comment on aspects of the natural history of C. werneri. Besides, the examination of specimens in zoological collections allowed us to extend the geographic range for this species. We also make remarks on morphological/chromatic variation and provide 16S rDNA sequences for the species. Adults were found along a slow-flowing streamlet with sandy/muddy bottom within a small fragment of secondary forest. Males called between sunset and first hours of the night. Advertisement call consisted of series of pulsed notes. Call duration lasted around 3 s, emitted at the highest rate of 17 calls per minute and six notes per second. Note duration lasted around 18 ms. Notes had poorly defined pulses (irregular and/or weak amplitude modulations along the note). The dominant frequency was about 3380 Hz. Territorial call had a long, well-defined pulsed portion followed by a higher-amplitude "squeak". The dominant frequency was around 3400 Hz. Tadpoles were essentially similar to those of other Crossodactylus species, except by not having nostril ornamentation. Our record of C. werneri in Serra das Cabras might be regarded a rediscovery of this species since C. werneri had not been recorded for more than 30 years until our first record of C. werneri in the field from 2011 and subsequent years. Our record is approximately 100 km west, and Mococa 200 km northwest, from Santo Antônio do Pinhal, the westernmost previous record for C. werneri up to date. Gene sequences (16S rRNA) give insights into the genetic divergence between C. werneri and some congeners.

Phytogeographic patterns and cryptic diversity in an aposematic toad from NW Argentina.

The Yungas Redbelly Toad, Melanophryniscus rubriventris, is patchily distributed in Argentina, confined to the upland portion (1000-2000m above sea level) of the montane forests of northern and central regions of Salta, and in central-eastern and south-eastern Jujuy. This species is known for its striking aposematic color variation across its geographic distribution, and was once treated as a complex of three subspecies based on distinctive color patterns. Here we assess the geographical genetic variation within M. rubriventris and quantify divergence in color and pattern among individuals sampled from Northwestern Argentina. We compare multi-gene phylogeography of M. rubriventris to patterns of dorsal and ventral coloration to test whether evolutionary affinities predict variation in warning color. Our results reveal two well-supported species lineages: one confined to the extreme northern portion of our sampling area, and the other extending over most of the Argentine portion of the species' range, within which there are two populations. However, these well-supported evolutionary relationships do not mirror the marked variation in warning coloration. This discordance between DNA genealogy and warning color variation may reflect selection brought about by differences in local predation pressures, potentially coupled with effects of sexual selection and thermoregulation.

Molecular phylogenetic relationships and phenotypic diversity in miniaturized toadlets, genus Brachycephalus (Amphibia: Anura: Brachycephalidae).

Toadlets of the genus Brachycephalus are endemic to the Atlantic rainforests of southeastern and southern Brazil. The 14 species currently described have snout-vent lengths less than 18 mm and are thought to have evolved through miniaturization: an evolutionary process leading to an extremely small adult body size. Here, we present the first comprehensive phylogenetic analysis for Brachycephalus, using a multilocus approach based on two nuclear (Rag-1 and Tyr) and three mitochondrial (Cyt b, 12S, and 16S rRNA) gene regions. Phylogenetic relationships were inferred using a partitioned Bayesian analysis of concatenated sequences and the hierarchical Bayesian method (BEST) that estimates species trees based on the multispecies coalescent model. Individual gene trees showed conflict and also varied in resolution. With the exception of the mitochondrial gene tree, no gene tree was completely resolved. The concatenated gene tree was completely resolved and is identical in topology and degree of statistical support to the individual mtDNA gene tree. On the other hand, the BEST species tree showed reduced significant node support relative to the concatenate tree and recovered a basal trichotomy, although some bipartitions were significantly supported at the tips of the species tree. Comparison of the log likelihoods for the concatenated and BEST trees suggests that the method implemented in BEST explains the multilocus data for Brachycephalus better than the Bayesian analysis of concatenated data. Landmark-based geometric morphometrics revealed marked variation in cranial shape between the species of Brachycephalus. In addition, a statistically significant association was demonstrated between variation in cranial shape and genetic distances estimated from the mtDNA and nuclear loci. Notably, B. ephippium and B. garbeana that are predicted to be sister-species in the individual and concatenated gene trees and the BEST species tree share an evolutionary novelty, the hyperossified dorsal plate.

Hyperossification in miniaturized toadlets of the genus Brachycephalus (Amphibia: Anura: Brachycephalidae): Microscopic structure and macroscopic patterns of variation.

Species of the genus Brachycephalus, have a snout-vent length of less than 18 mm and are believed to have evolved through miniaturization. Brachycephalus ephippium, is particularly interesting; because its entire skull is hyperossified, and the presacral vertebrae and transverse processes are covered by a dorsal shield. We demonstrate in this paper that, at the macroscopic level, a completely hyperossified skull and dorsal shield occur only in B. ephippium, but not in B. ferruginus, B. izechsohni, B. pernix, B. pombali, B. brunneus, B. didactylus, and B. hermogenesi. An intermediate condition, in which the skull is hyperossified but a dorsal shield is absent, occurs in B. vertebralis, B. nodoterga, B. pitanga, and B. alipioi. The microscopic structure of hyperossification was examined in skulls of B. ephippium and B. pitanga, revealing a complex organization involving the presence of Sharpey fibers, which in humans are characteristic of periodontal connections.