Highly abundant core taxa in the blow within and across captive bottlenose dolphins provide evidence for a temporally stable airway microbiota.

BACKGROUND: The analysis of blow microbiota has been proposed as a biomarker for respiratory health analysis in cetaceans. Yet, we lack crucial knowledge on the long-term stability of the blow microbiota and its potential changes during disease. Research in humans and mice have provided evidence that respiratory disease is accompanied by a shift in microbial communities of the airways. We investigate here the stability of the community composition of the blow microbiota for 13 captive bottlenose dolphins over eight months including both sick and healthy individuals. We used barcoded tag sequencing of the bacterial 16S rRNA gene. Four of the dolphins experienced distinct medical conditions and received systemic antimicrobial treatment during the study. RESULTS: We showed that each dolphin harboured a unique community of zero-radius operational taxonomic units (zOTUs) that was present throughout the entire sampling period ('intra-core'). Although for most dolphins there was significant variation over time, overall the intra-core accounted for an average of 73% of relative abundance of the blow microbiota. In addition, the dolphins shared between 8 and 66 zOTUs on any of the sampling occasions ('inter-core'), accounting for a relative abundance between 17 and 41% of any dolphin's airway microbiota. The majority of the intra-core and all of the inter-core zOTUs in this study are commonly found in captive and free-ranging dolphins and have previously been reported from several different body sites. While we did not find a clear effect of microbial treatment on blow microbiota, age and sex of the dolphins did have such an effect. CONCLUSIONS: The airways of dolphins were colonized by an individual intra-core 'signature' that varied in abundance relative to more temporary bacteria. We speculate that the intra-core bacteria interact with the immune response of the respiratory tract and support its function. This study provides the first evidence of individual-specific airway microbiota in cetaceans that is stable over eight months.

Postglacial Colonization of Northern Coastal Habitat by Bottlenose Dolphins: A Marine Leading-Edge Expansion?

Oscillations in the Earth's temperature and the subsequent retreating and advancing of ice-sheets around the polar regions are thought to have played an important role in shaping the distribution and genetic structuring of contemporary high-latitude populations. After the Last Glacial Maximum (LGM), retreating of the ice-sheets would have enabled early colonizers to rapidly occupy suitable niches to the exclusion of other conspecifics, thereby reducing genetic diversity at the leading-edge. Bottlenose dolphins (genus Tursiops) form distinct coastal and pelagic ecotypes, with finer-scale genetic structuring observed within each ecotype. We reconstruct the postglacial colonization of the Northeast Atlantic (NEA) by bottlenose dolphins using habitat modeling and phylogenetics. The AquaMaps model hindcasted suitable habitat for the LGM in the Atlantic lower latitude waters and parts of the Mediterranean Sea. The time-calibrated phylogeny, constructed with 86 complete mitochondrial genomes including 30 generated for this study and created using a multispecies coalescent model, suggests that the expansion to the available coastal habitat in the NEA happened via founder events starting ~15 000 years ago (95% highest posterior density interval: 4 900-26 400). The founders of the 2 distinct coastal NEA populations comprised as few as 2 maternal lineages that originated from the pelagic population. The low effective population size and genetic diversity estimated for the shared ancestral coastal population subsequent to divergence from the pelagic source population are consistent with leading-edge expansion. These findings highlight the legacy of the Late Pleistocene glacial cycles on the genetic structuring and diversity of contemporary populations.

A cerebellar substrate for cognition evolved multiple times independently in mammals.

Given that complex behavior evolved multiple times independently in different lineages, a crucial question is whether these independent evolutionary events coincided with modifications to common neural systems. To test this question in mammals, we investigate the lateral cerebellum, a neurobiological system that is novel to mammals, and is associated with higher cognitive functions. We map the evolutionary diversification of the mammalian cerebellum and find that relative volumetric changes of the lateral cerebellar hemispheres (independent of cerebellar size) are correlated with measures of domain-general cognition in primates, and are characterized by a combination of parallel and convergent shifts towards similar levels of expansion in distantly related mammalian lineages. Results suggest that multiple independent evolutionary occurrences of increased behavioral complexity in mammals may at least partly be explained by selection on a common neural system, the cerebellum, which may have been subject to multiple independent neurodevelopmental remodeling events during mammalian evolution.

Cryptic lineage differentiation among Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the northwest Indian Ocean.

Phylogeography can provide insight into the potential for speciation and identify geographic regions and evolutionary processes associated with species richness and evolutionary endemism. In the marine environment, highly mobile species sometimes show structured patterns of diversity, but the processes isolating populations and promoting differentiation are often unclear. The Delphinidae (oceanic dolphins) are a striking case in point and, in particular, bottlenose dolphins (Tursiops spp.). Understanding the radiation of species in this genus is likely to provide broader inference about the processes that determine patterns of biogeography and speciation, because both fine-scale structure over a range of kilometers and relative panmixia over an oceanic range are known for Tursiops populations. In our study, novel Tursiops spp. sequences from the northwest Indian Ocean (including mitogenomes and two nuDNA loci) are included in a worldwide Tursiops spp. phylogeographic analysis. We discover a new 'aduncus' type lineage in the Arabian Sea (off India, Pakistan and Oman) that diverged from the Australasian lineage ∼261 Ka. Effective management of coastal dolphins in the region will need to consider this new lineage as an evolutionarily significant unit. We propose that the establishment of this lineage could have been in response to climate change during the Pleistocene and show data supporting hypotheses for multiple divergence events, including vicariance across the Indo-Pacific barrier and in the northwest Indian Ocean. These data provide valuable transferable inference on the potential mechanisms for population and species differentiation across this geographic range.

Categorizing click trains to increase taxonomic precision in echolocation click loggers.

Passive acoustic monitoring is an efficient way to study acoustically active animals but species identification remains a major challenge. C-PODs are popular logging devices that automatically detect odontocete echolocation clicks. However, the accompanying analysis software does not distinguish between delphinid species. Click train features logged by C-PODs were compared to frequency spectra from adjacently deployed continuous recorders. A generalized additive model was then used to categorize C-POD click trains into three groups: broadband click trains, produced by bottlenose dolphin (Tursiops truncatus) or common dolphin (Delphinus delphis), frequency-banded click trains, produced by Risso's (Grampus griseus) or white beaked dolphins (Lagenorhynchus albirostris), and unknown click trains. Incorrect categorization rates for broadband and frequency banded clicks were 0.02 (SD 0.01), but only 30% of the click trains met the categorization threshold. To increase the proportion of categorized click trains, model predictions were pooled within acoustic encounters and a likelihood ratio threshold was used to categorize encounters. This increased the proportion of the click trains meeting either the broadband or frequency banded categorization threshold to 98%. Predicted species distribution at the 30 study sites matched well to visual sighting records from the region.

Neural representation of the self-heard biosonar click in bottlenose dolphins (Tursiops truncatus).

The neural representation of the dolphin broadband biosonar click was investigated by measuring auditory brainstem responses (ABRs) to "self-heard" clicks masked with noise bursts having various high-pass cutoff frequencies. Narrowband ABRs were obtained by sequentially subtracting responses obtained with noise having lower high-pass cutoff frequencies from those obtained with noise having higher cutoff frequencies. For comparison to the biosonar data, ABRs were also measured in a passive listening experiment, where external clicks and masking noise were presented to the dolphins and narrowband ABRs were again derived using the subtractive high-pass noise technique. The results showed little change in the peak latencies of the ABR to the self-heard click from 28 to 113 kHz; i.e., the high-frequency neural responses to the self-heard click were delayed relative to those of an external, spectrally "pink" click. The neural representation of the self-heard click is thus highly synchronous across the echolocation frequencies and does not strongly resemble that of a frequency modulated downsweep (i.e., decreasing-frequency chirp). Longer ABR latencies at higher frequencies are hypothesized to arise from spectral differences between self-heard clicks and external clicks, forward masking from previously emitted biosonar clicks, or neural inhibition accompanying the emission of clicks.

The effects of click rate on the auditory brainstem response of bottlenose dolphins.

Rate manipulations can be used to study adaptation processes in the auditory nerve and brainstem. For this reason, rate effects on the click-evoked auditory brainstem response (ABR) have been investigated in many mammals, including humans. In this study, click-evoked ABRs were obtained in eight bottlenose dolphins (Tursiops truncatus) while varying stimulus rate using both conventional averaging and maximum length sequences (MLSs), which allow disentangling ABRs that overlap in time and thus permit the study of adaptation at high rates. Dolphins varied in age and upper cutoff frequency of hearing. Conventional stimulation rates were 25, 50, and 100 Hz and average MLS rates were approximately 50, 100, 250, 500, 1000, 2500, and 5000 Hz. Click peak-equivalent sound pressure levels for all conditions were 135 dB re 1 µPa. ABRs were observed in all dolphins, at all stimulus rates. With increasing rate, peak latencies increased and peak amplitudes decreased. There was a trend for an increase in the interwave intervals with increasing rate, which appeared more robust for the dolphins with a full range of hearing. For those rates where ABRs were obtained for both conventional and MLS approaches, the latencies of the mean data were in good agreement.

Whistle comparison of four delphinid species in Southeastern Brazil.

The present study evaluates variations in frequency and duration parameters of whistles of four dolphin species (Sotalia guianensis, Steno bredanensis, Stenella frontalis, and Tursiops truncatus), recorded in the Rio de Janeiro State Coast, Southeastern Brazil. A total of 487 whistles were analyzed. Acoustic parameters of the whistles were classified to species by discriminant function analysis. Overall classification score was 72.5%, with the highest classification score obtained for whistles of S. bredanensis and the lowest obtained for S. frontalis. Most differences were among S. bredanensis and S. guianensis, species that did not have their repertoires compared in other studies.

Echolocation parameters of Australian humpback dolphins (Sousa sahulensis) and Indo-Pacific bottlenose dolphins (Tursiops aduncus) in the wild.

Echolocation is a key sensory modality for toothed whale orientation, navigation, and foraging. However, a more comparative understanding of the biosonar properties of toothed whales is necessary to understand behavioral and evolutionary adaptions. To address this, two free-ranging sympatric delphinid species, Australian humpback dolphins (Sousa sahulensis) and Indo-Pacific bottlenose dolphins (Tursiops aduncus), were studied. Biosonar clicks from both species were recorded within the same stretch of coastal habitat in Exmouth Gulf, Western Australia, using a vertical seven element hydrophone array. S. sahulensis used biosonar clicks with a mean source level of 199 ± 3 dB re 1 µPa peak-peak (pp), mean centroid frequency of 106 ± 11 kHz, and emitted at interclick intervals (ICIs) of 79 ± 33 ms. These parameters were similar to click parameters of sympatric T. aduncus, characterized by mean source levels of 204 ± 4 dB re 1 µPa pp, centroid frequency of 112 ± 9 kHz, and ICIs of 73 ± 29 ms. These properties are comparable to those of other similar sized delphinids and suggest that biosonar parameters are independent of sympatric delphinids and possibly driven by body size. The dynamic biosonar behavior of these delphinids may have, consequently, allowed for adaptations to local environments through high levels of control over sonar beam properties.

Organochlorine contaminants in tissues of common bottlenose dolphins Tursiops truncatus from the northeastern part of the Adriatic Sea.

Levels of 24 organochlorine compounds, including toxic mono-ortho PCB congeners, were determined in the organs and tissues (blubber, kidney, lung, muscle, liver, heart) of 13 common bottlenose dolphins (Tursiops truncatus) stranded between 2000 and 2005 in the northern part of the Croatian territorial waters of the Adriatic Sea. Polychlorinated biphenyls (PCBs) were found at higher concentrations in comparison with organochlorine pesticides (OCPs) in all of the analyzed tissues. Sums of six indicator congeners (Σ6PCB) constituted around 50% of the total PCB amount, while PCB-153 and PCB-138 were found to have the highest concentrations. Among the seven investigated OCPs, p,p'-DDE was found at the highest concentrations. In blubber, mean values of 22,048 and 11,310ngg(-1) wet weight were determined for ΣPCB and ΣDDT, respectively. Much lower concentrations were found in muscle samples, followed by similar concentrations in kidneys, liver and heart, while the lowest levels of organochlorine contaminants were found in lungs. The results indicate that p,p'-DDT is still being introduced in the Mediterranean region. PCB concentrations are among the highest found in this region and toxicological assessments indicate that the health of this specie is at high risk.

Whistle source levels of free-ranging bottlenose dolphins and Atlantic spotted dolphins in the Gulf of Mexico.

Whistles of bottlenose dolphins (Tursiops truncatus) and Atlantic spotted dolphins (Stenella frontalis) in the eastern Gulf of Mexico were recorded and measured with a calibrated towed hydrophone array. Surveys encountered groups of both bottlenose (N = 10) and spotted dolphins (N = 5). Analysis of those data produced 1695 bottlenose dolphin whistles and 1273 spotted dolphin whistles with a high signal-to-noise ratio. Whistle frequency metrics were lower in bottlenose than spotted dolphins, while whistle duration was longer in spotted dolphins, data that may help inform automatic classification algorithms. Source levels were estimated by determining the range and bearing of an individual dolphin from the array and then adding the predicted transmission loss to the calculated received level. The median bottlenose dolphin source level was 138 dB re 1µPa at 1 m with a range of 114-163 dB re 1µPa at 1 m. The median spotted dolphin source level was 138 dB re 1µPa at 1 m with a range of 115-163 dB re 1µPa at 1 m. These source level measurements, in conjunction with estimates of vocalization rates and transmission loss models, can be used to improve passive acoustically determined dolphin abundance estimates in the Gulf of Mexico.

Combining whistle acoustic parameters to discriminate Mediterranean odontocetes during passive acoustic monitoring.

Acoustic observation can complement visual observation to more effectively monitor occurrence and distribution of marine mammals. For effective acoustic censuses, calibration methods must be determined by joint visual and acoustic studies. Research is still needed in the field of acoustic species identification, particularly for smaller odontocetes. From 1994 to 2012, whistles of four odontocete species were recorded in different areas of the Mediterranean Sea to determine how reliably these vocalizations can be classified to species. Recordings were attributed to species by simultaneous visual observation. The results of this study highlight that the frequency parameters, which are linked to physical features of animals, show lower variability than modulation parameters, which are likely to be more dependent on complex eco-ethological contexts. For all the studied species, minimum and maximum frequencies were linearly correlated with body size. DFA and Classification Tree Analysis (CART) show that these parameters were the most important for classifying species; however, both statistical methods highlighted the need for combining them with the number of contour minima and contour maxima for correct classification. Generally, DFA and CART results reflected both phylogenetic distance (especially for common and striped dolphins) and the size of the species.

Patterns of population structure for inshore bottlenose dolphins along the eastern United States.

Globally distributed, the bottlenose dolphin (Tursiops truncatus) is found in a range of offshore and coastal habitats. Using 15 microsatellite loci and mtDNA control region sequences, we investigated patterns of genetic differentiation among putative populations along the eastern US shoreline (the Indian River Lagoon, Florida, and Charleston Harbor, South Carolina) (microsatellite analyses: n = 125, mtDNA analyses: n = 132). We further utilized the mtDNA to compare these populations with those from the Northwest Atlantic, Gulf of Mexico, and Caribbean. Results showed strong differentiation among inshore, alongshore, and offshore habitats (ФST = 0.744). In addition, Bayesian clustering analyses revealed the presence of 2 genetic clusters (populations) within the 250 km Indian River Lagoon. Habitat heterogeneity is likely an important force diversifying bottlenose dolphin populations through its influence on social behavior and foraging strategy. We propose that the spatial pattern of genetic variation within the lagoon reflects both its steep longitudinal transition of climate and also its historical discontinuity and recent connection as part of Intracoastal Waterway development. These findings have important management implications as they emphasize the role of habitat and the consequence of its modification in shaping bottlenose dolphin population structure and highlight the possibility of multiple management units existing in discrete inshore habitats along the entire eastern US shoreline.

Developing genomic resources for the common bottlenose dolphin (Tursiops truncatus): isolation and characterization of 153 single nucleotide polymorphisms and 53 genotyping assays.

Although single nucleotide polymorphisms (SNPs) are commonly used in human genetics, they have only recently been incorporated into genetic studies of non-model organisms, including cetaceans. SNPs have several advantages over other molecular markers for studies of population genetics: they are quicker and more straightforward to score, cross-laboratory comparisons of data are less complicated, and they can be used successfully with low-quality DNA. We screened portions of the genome of one of the most abundant cetaceans in U.S. waters, the common bottlenose dolphin (Tursiops truncatus), and identified 153 SNPs resulting in an overall average of one SNP every 463 base pairs. Custom TaqMan(®) Assays were designed for 53 of these SNPs, and their performance was tested by genotyping a set of bottlenose dolphin samples, including some with low-quality DNA. We found that in 19% of the loci examined, the minor allele frequency (MAF) estimated during initial SNP ascertainment using a DNA pool of 10 individuals differed significantly from the final MAF after genotyping over 100 individuals, suggesting caution when making inferences about MAF values based on small data sets. For two assays, we also characterized the basis for unusual clustering patterns to determine whether their data could still be utilized for further genetic studies. Overall results support the use of these SNPs for accurate analysis of both poor and good-quality DNA. We report the first SNP markers and genotyping assays for use in population and conservation genetic studies of bottlenose dolphins.

A new dolphin species, the Burrunan Dolphin Tursiops australis sp. nov., endemic to southern Australian coastal waters.

Small coastal dolphins endemic to south-eastern Australia have variously been assigned to described species Tursiops truncatus, T. aduncus or T. maugeanus; however the specific affinities of these animals is controversial and have recently been questioned. Historically 'the southern Australian Tursiops' was identified as unique and was formally named Tursiops maugeanus but was later synonymised with T. truncatus. Morphologically, these coastal dolphins share some characters with both aforementioned recognised Tursiops species, but they also possess unique characters not found in either. Recent mtDNA and microsatellite genetic evidence indicates deep evolutionary divergence between this dolphin and the two currently recognised Tursiops species. However, in accordance with the recommendations of the Workshop on Cetacean Systematics, and the Unified Species Concept the use of molecular evidence alone is inadequate for describing new species. Here we describe the macro-morphological, colouration and cranial characters of these animals, assess the available and new genetic data, and conclude that multiple lines of evidence clearly indicate a new species of dolphin. We demonstrate that the syntype material of T. maugeanus comprises two different species, one of which is the historical 'southern form of Tursiops' most similar to T. truncatus, and the other is representative of the new species and requires formal classification. These dolphins are here described as Tursiops australis sp. nov., with the common name of 'Burrunan Dolphin' following Australian aboriginal narrative. The recognition of T. australis sp. nov. is particularly significant given the endemism of this new species to a small geographic region of southern and south-eastern Australia, where only two small resident populations in close proximity to a major urban and agricultural centre are known, giving them a high conservation value and making them susceptible to numerous anthropogenic threats.

The use of carcasses for the analysis of cetacean population genetic structure: a comparative study in two dolphin species.

Advances in molecular techniques have enabled the study of genetic diversity and population structure in many different contexts. Studies that assess the genetic structure of cetacean populations often use biopsy samples from free-ranging individuals and tissue samples from stranded animals or individuals that became entangled in fishery or aquaculture equipment. This leads to the question of how representative the location of a stranded or entangled animal is with respect to its natural range, and whether similar results would be obtained when comparing carcass samples with samples from free-ranging individuals in studies of population structure. Here we use tissue samples from carcasses of dolphins that stranded or died as a result of bycatch in South Australia to investigate spatial population structure in two species: coastal bottlenose (Tursiops sp.) and short-beaked common dolphins (Delphinus delphis). We compare these results with those previously obtained from biopsy sampled free-ranging dolphins in the same area to test whether carcass samples yield similar patterns of genetic variability and population structure. Data from dolphin carcasses were gathered using seven microsatellite markers and a fragment of the mitochondrial DNA control region. Analyses based on carcass samples alone failed to detect genetic structure in Tursiops sp., a species previously shown to exhibit restricted dispersal and moderate genetic differentiation across a small spatial scale in this region. However, genetic structure was correctly inferred in D. delphis, a species previously shown to have reduced genetic structure over a similar geographic area. We propose that in the absence of corroborating data, and when population structure is assessed over relatively small spatial scales, the sole use of carcasses may lead to an underestimate of genetic differentiation. This can lead to a failure in identifying management units for conservation. Therefore, this risk should be carefully assessed when planning population genetic studies of cetaceans.

Gaussian mixture model classification of odontocetes in the Southern California Bight and the Gulf of California.

A method for the automatic classification of free-ranging delphinid vocalizations is presented. The vocalizations of short-beaked and long-beaked common (Delphinus delphis and Delphinus capensis), Pacific white-sided (Lagenorhynchus obliquidens), and bottlenose (Tursiops truncatus) dolphins were recorded in a pelagic environment of the Southern California Bight and the Gulf of California over a period of 4 years. Cepstral feature vectors are extracted from call data which contain simultaneous overlapping whistles, burst-pulses, and clicks from a single species. These features are grouped into multisecond segments. A portion of the data is used to train Gaussian mixture models of varying orders for each species. The remaining call data are used to test the performance of the models. Species are predicted based upon probabilistic measures of model similarity with test segment groups having durations between 1 and 25 s. For this data set, 256 mixture Gaussian mixture models and segments of at least 10 s of call data resulted in the best classification results. The classifier predicts the species of groups with 67%-75% accuracy depending upon the partitioning of the training and test data.