Tetrapod biodiversity in sambaquis from southern Brazil.

Fishermen-hunter-gatherers of sambaquis (Brazilian shell mounds) had an intimate affinity with marine-coastal environments, where they exploited a great variety of fish and mollusks that comprise the best documented fauna from sambaquis. However, other groups of animals as mammals, birds, reptiles, and amphibians are also present in these sites, but are relatively less studied. This paper is the first one focused exclusively on the Tetrapoda biodiversity of sambaquis and aimed to identify tetrapods of ten sites from southern Brazil. We present a faunal inventory and data regarding animal capture and environmental exploitation. We identified the specimens anatomically and taxonomically, analyzed them concerning fragmentation, and quantified the data for the number of identified specimens (NISP) and minimum number of individuals (MNI). Despite the high degree of fragmentation of remains, we identified 46 taxa. As expected, most were from marine animals: cetaceans (total NISP = 2,568 and MNI = 27), otariids (total NISP = 248 and MNI = 32), and seabirds (total NISP = 65 and MNI = 23), indicating great relevance of marine tetrapod fauna as a resource for sambaqui builders (79.39% of NISP). We thus document the close bond between fishermen-hunter-gatherers of sambaquis and the marine tetrapods in southern Brazil.

Phylogenomic Resolution of the Cetacean Tree of Life Using Target Sequence Capture.

The evolution of cetaceans, from their early transition to an aquatic lifestyle to their subsequent diversification, has been the subject of numerous studies. However, although the higher-level relationships among cetacean families have been largely settled, several aspects of the systematics within these groups remain unresolved. Problematic clades include the oceanic dolphins (37 spp.), which have experienced a recent rapid radiation, and the beaked whales (22 spp.), which have not been investigated in detail using nuclear loci. The combined application of high-throughput sequencing with techniques that target specific genomic sequences provide a powerful means of rapidly generating large volumes of orthologous sequence data for use in phylogenomic studies. To elucidate the phylogenetic relationships within the Cetacea, we combined sequence capture with Illumina sequencing to generate data for $\sim $3200 protein-coding genes for 68 cetacean species and their close relatives including the pygmy hippopotamus. By combining data from $>$38,000 exons with existing sequences from 11 cetaceans and seven outgroup taxa, we produced the first comprehensive comparative genomic data set for cetaceans, spanning 6,527,596 aligned base pairs (bp) and 89 taxa. Phylogenetic trees reconstructed with maximum likelihood and Bayesian inference of concatenated loci, as well as with coalescence analyses of individual gene trees, produced mostly concordant and well-supported trees. Our results completely resolve the relationships among beaked whales as well as the contentious relationships among oceanic dolphins, especially the problematic subfamily Delphinidae. We carried out Bayesian estimation of species divergence times using MCMCTree and compared our complete data set to a subset of clocklike genes. Analyses using the complete data set consistently showed less variance in divergence times than the reduced data set. In addition, integration of new fossils (e.g., Mystacodon selenensis) indicates that the diversification of Crown Cetacea began before the Late Eocene and the divergence of Crown Delphinidae as early as the Middle Miocene. [Cetaceans; phylogenomics; Delphinidae; Ziphiidae; dolphins; whales.].

Genetic identification of Anisakis spp. (Nematoda: Anisakidae) from cetaceans of the Southwestern Atlantic Ocean: ecological and zoogeographical implications.

Adult Anisakis Dujardin, 1845 were found in two specimens of killer whale Orcinus orca and one specimen of franciscana Pontoporia blainvillei stranded from off the coast of Buenos Aires Province, Argentina. Genetic identification of the nematodes (N = 144) was performed by sequence analysis of the mitochondrial (mtDNA cox2) and the nuclear (nas 10 nDNA) gene loci. Anisakis pegreffii and Anisakis berlandi were detected in the two individuals of O. orca, while Anisakis typica and A. pegreffii were identified in P. blainvillei. Morphological and morphometric analysis also carried out on adult specimens of A. pegreffii and A. berlandi has allowed to underlining the usefulness of genetic/molecular markers in their recognition. This represents the first record of A. pegreffii in O. orca and P. blainvillei and of A. berlandi in O. orca. This is also the first sympatric and syntopic occurrence, as adults, of A. pegreffii and A. berlandi from the Austral Region of the Atlantic Ocean waters. These results provide insights into the knowledge of the host ranges and geographical distribution of these parasites in the basin waters of the region. Pontoporia blainvillei showed low abundance values of infection with Anisakis spp., which is the general pattern for coastal dolphins in the area, whereas O. orca harboured higher abundance of Anisakis spp. than those previously recorded among cetacean species in the Argentine Sea. Differences in the Anisakis spp. distribution and their parasitic loads, observed among the three host specimens, are discussed in relation to the oceanographic parameters, as well as to the host ecology. The usefulness of genetic/molecular markers in the recognition of adults of the sibling species A. pegreffii and A. berlandi with considerable overlapping in morphometric and morphological characters was underlined. The distribution of Anisakis species from Southwestern Atlantic waters is discussed in relation to their value as indicators for studies on the zoogeography of their hosts at a regional-scale level.

Characterizing lineage-specific evolution and the processes driving genomic diversification in chordates.

BACKGROUND: Understanding the origins of genome content has long been a goal of molecular evolution and comparative genomics. By examining genome evolution through the guise of lineage-specific evolution, it is possible to make inferences about the evolutionary events that have given rise to species-specific diversification. Here we characterize the evolutionary trends found in chordate species using The Adaptive Evolution Database (TAED). TAED is a database of phylogenetically indexed gene families designed to detect episodes of directional or diversifying selection across chordates. Gene families within the database have been assessed for lineage-specific estimates of dN/dS and have been reconciled to the chordate species to identify retained duplicates. Gene families have also been mapped to the functional pathways and amino acid changes which occurred on high dN/dS lineages have been mapped to protein structures. RESULTS: An analysis of this exhaustive database has enabled a characterization of the processes of lineage-specific diversification in chordates. A pathway level enrichment analysis of TAED determined that pathways most commonly found to have elevated rates of evolution included those involved in metabolism, immunity, and cell signaling. An analysis of protein fold presence on proteins, after normalizing for frequency in the database, found common folds such as Rossmann folds, Jelly Roll folds, and TIM barrels were overrepresented on proteins most likely to undergo directional selection. A set of gene families which experience increased numbers of duplications within short evolutionary times are associated with pathways involved in metabolism, olfactory reception, and signaling. An analysis of protein secondary structure indicated more relaxed constraint in ß-sheets and stronger constraint on alpha Helices, amidst a general preference for substitutions at exposed sites. Lastly a detailed analysis of the ornithine decarboxylase gene family, a key enzyme in the pathway for polyamine synthesis, revealed lineage-specific evolution along the lineage leading to Cetacea through rapid sequence evolution in a duplicate gene with amino acid substitutions causing active site rearrangement. CONCLUSION: Episodes of lineage-specific evolution are frequent throughout chordate species. Both duplication and directional selection have played large roles in the evolution of the phylum. TAED is a powerful tool for facilitating this understanding of lineage-specific evolution.

Ignoring stratigraphic age uncertainty leads to erroneous estimates of species divergence times under the fossilized birth-death process.

Fossil information is essential for estimating species divergence times, and can be integrated into Bayesian phylogenetic inference using the fossilized birth-death (FBD) process. An important aspect of palaeontological data is the uncertainty surrounding specimen ages, which can be handled in different ways during inference. The most common approach is to fix fossil ages to a point estimate within the known age interval. Alternatively, age uncertainty can be incorporated by using priors, and fossil ages are then directly sampled as part of the inference. This study presents a comparison of alternative approaches for handling fossil age uncertainty in analysis using the FBD process. Based on simulations, we find that fixing fossil ages to the midpoint or a random point drawn from within the stratigraphic age range leads to biases in divergence time estimates, while sampling fossil ages leads to estimates that are similar to inferences that employ the correct ages of fossils. Second, we show a comparison using an empirical dataset of extant and fossil cetaceans, which confirms that different methods of handling fossil age uncertainty lead to large differences in estimated node ages. Stratigraphic age uncertainty should thus not be ignored in divergence time estimation and instead should be incorporated explicitly.

Genomic Organization and Phylogeny of MHC Class II Loci in Cetaceans.

Cetaceans are a suborder of secondarily adapted aquatic mammals with an enigmatic history involving a transition from land to sea approximately 55 Mya. During the transition period, cetaceans would have faced many new pathogen challenges, but limited information is available about the adaptive immune system of these mammals. The major histocompatibility complex (MHC) family plays a key role in antigen recognition and presentation in adaptive immunity, which is believed to have evolved in response to pathogens. In the present study, MHC class II loci were characterized in 7 published cetacean genome assemblies and the genomic organization of cetaceans was compared with that of their terrestrial relatives, the cow, sheep, and pig. A total of 9 MHC class II loci were identified in the cetacean genomes: DRA, DRB, DQA, DQB, DPB, DOA, DOB, DMA, and DMB. Sequences from 8 of the 9 genes included intact coding regions and were presumably functional. The organization of the MHC class II loci was conserved across the examined mammalian species, whereas the orientation and number of the alpha and beta genes varied among the species. The phylogenetic reconstruction of all MHC genes from Cetartiodactyla suggested that alpha and beta genes had different topologies. Additionally, based on a phylogenetic reconstruction of the multi-locus DRB, 2 (DRB1 and DRB2) of the 4 putative gene copies were hypothesized to have duplicated and evolved during the radiation of cetaceans.

Time-Dependent-Asymmetric-Linear-Parsimonious Ancestral State Reconstruction.

The time-dependent-asymmetric-linear parsimony is an ancestral state reconstruction method which extends the standard linear parsimony (a.k.a. Wagner parsimony) approach by taking into account both branch lengths and asymmetric evolutionary costs for reconstructing quantitative characters (asymmetric costs amount to assuming an evolutionary trend toward the direction with the lowest cost). A formal study of the influence of the asymmetry parameter shows that the time-dependent-asymmetric-linear parsimony infers states which are all taken among the known states, except for some degenerate cases corresponding to special values of the asymmetry parameter. This remarkable property holds in particular for the Wagner parsimony. This study leads to a polynomial algorithm which determines, and provides a compact representation of, the parametric reconstruction of a phylogenetic tree, that is for all the unknown nodes, the set of all the possible reconstructed states associated with the asymmetry parameters leading to them. The time-dependent-asymmetric-linear parsimony is finally illustrated with the parametric reconstruction of the body size of cetaceans.

Molecular identification of cetaceans from the West Atlantic using the E3-I5 region of COI.

Molecular identification is very useful in cases where morphology-based species identification is not possible. Examples for its application in cetaceans include the identification of carcasses of stranded animals in advanced state of decomposition and body parts that are illegally traded. One DNA region that is often used for molecular identification is the Folmer region of the mitochondrial gene cytochrome c oxidase subunit I (COI) (locus 48 to 705 bp). This locus has been used for the identification of several animal species, including whales and dolphins. The goal of the present study was to evaluate the usefulness of another region of COI, the E3-I5 (locus 685 to locus 1179; 495 bp) as a marker for identification of cetaceans from northeastern Canada and northeastern Brazil. The identification markers were successfully obtained for seven cetacean species after performing percent identity and Basic Local Alignment Search Tool analyses. The obtained markers are now publicly available and are useful for the identification of the endangered blue whale (Balaenoptera musculus), common minke whale (B. acutorostrata), vulnerable sperm whale (Physeter macrocephalus), harbor porpoise (Phocoena phocoena), common bottlenose dolphin (Tursiops truncatus), Guiana dolphin (Sotalia guianensis), and melon-headed whale (Peponocephala electra).

Hippopotamus and whale phylogeny.

Thewissen et al. describe new fossils from India that apparently support a phylogeny that places Cetacea (that is, whales, dolphins, porpoises) as the sister group to the extinct family Raoellidae, and Hippopotamidae as more closely related to pigs and peccaries (that is, Suina) than to cetaceans. However, our reanalysis of a modified version of the data set they used differs in retaining molecular characters and demonstrates that Hippopotamidae is the closest extant family to Cetacea and that raoellids are the closest extinct group, consistent with previous phylogenetic studies. This topology supports the view that the aquatic adaptations in hippopotamids and cetaceans are inherited from their common ancestor.

Automatic detection of echolocation clicks based on a Gabor model of their waveform.

Prior research has shown that echolocation clicks of several species of terrestrial and marine fauna can be modelled as Gabor-like functions. Here, a system is proposed for the automatic detection of a variety of such signals. By means of mathematical formulation, it is shown that the output of the Teager-Kaiser Energy Operator (TKEO) applied to Gabor-like signals can be approximated by a Gaussian function. Based on the inferences, a detection algorithm involving the post-processing of the TKEO outputs is presented. The ratio of the outputs of two moving-average filters, a Gaussian and a rectangular filter, is shown to be an effective detection parameter. Detector performance is assessed using synthetic and real (taken from MobySound database) recordings. The detection method is shown to work readily with a variety of echolocation clicks and in various recording scenarios. The system exhibits low computational complexity and operates several times faster than real-time. Performance comparisons are made to other publicly available detectors including pamguard.

Acoustic detection of biosonar activity of deep diving odontocetes at Josephine Seamount High Seas Marine Protected Area.

The temporal occurrence of deep diving cetaceans in the Josephine Seamount High Seas Marine Protected Area (JSHSMPA), south-west Portugal, was monitored using a passive acoustic recorder. The recorder was deployed on 13 May 2010 at a depth of 814 m during the North Atlantic Treaty Organization Centre for Maritime Research and Experimentation cruise "Sirena10" and recovered on 6 June 2010. The recorder was programmed to record 40 s of data every 2 min. Acoustic data analysis, for the detection and classification of echolocation clicks, was performed using automatic detector/classification systems: M3R (Marine Mammal Monitoring on Navy Ranges), a custom matlab program, and an operator-supervised custom matlab program to assess the classification performance of the detector/classification systems. M3R CS-SVM algorithm contains templates to detect beaked whales, sperm whales, blackfish (pilot and false killer whales), and Risso's dolphins. The detections of each group of odontocetes was monitored as a function of time. Blackfish and Risso's dolphins were detected every day, while beaked whales and sperm whales were detected almost every day. The hourly distribution of detections reveals that blackfish and Risso's dolphins were more active at night, while beaked whales and sperm whales were more active during daylight hours.

The loss of taste genes in cetaceans.

BACKGROUND: Five basic taste modalities, sour, sweet, bitter, salt and umami, can be distinguished by humans and are fundamental for physical and ecological adaptations in mammals. Molecular genetic studies of the receptor genes for these tastes have been conducted in terrestrial mammals; however, little is known about the evolution and adaptation of these genes in marine mammals. RESULTS: Here, all five basic taste modalities, sour, sweet, bitter, salt and umami, were investigated in cetaceans. The sequence characteristics and evolutionary analyses of taste receptor genes suggested that nearly all cetaceans may have lost all taste modalities except for that of salt. CONCLUSIONS: This is the first study to comprehensively examine the five basic taste modalities in cetaceans with extensive taxa sampling. Our results suggest that cetaceans have lost four of the basic taste modalities including sour, sweet, umami, and most of the ability to sense bitter tastes. The integrity of the candidate salt taste receptor genes in all the cetaceans examined may be because of their function in Na(+) reabsorption, which is key to osmoregulation and aquatic adaptation.

Increased rate of hair keratin gene loss in the cetacean lineage.

BACKGROUND: Hair represents an evolutionary innovation that appeared early on mammalian evolutionary history, and presumably contributed significantly to the rapid radiation of the group. An interesting event in hair evolution has been its secondary loss in some mammalian groups, such as cetaceans, whose hairless phenotype appears to be an adaptive response to better meet the environmental conditions. To determine whether different repertoire of keratin genes among mammals can potentially explain the phenotypic hair features of different lineages, we characterized the type I and II clusters of alpha keratins from eight mammalian species, including the hairless dolphin and minke whale representing the order Cetacea. RESULTS: We combined the available genomic information with phylogenetic analysis to conduct a comprehensive analysis of the evolutionary patterns of keratin gene clusters. We found that both type I and II gene clusters are fairly conserved among the terrestrial mammals included in this study, with lineage specific gene duplication and gene loss. Nevertheless, there is also evidence for an increased rate of pseudogenization in the cetacean lineage when compared to their terrestrial relatives, especially among the hair type keratins. CONCLUSIONS: Here we present a comprehensive characterization of alpha-keratin genes among mammals and elucidate the mechanisms involved in the evolution of this gene family. We identified lineage-specific gene duplications and gene loss among the Laurasiatherian and Euarchontoglires species included in the study. Interestingly, cetaceans present an increased loss of hair-type keratin genes when compared to other terrestrial mammals. As suggested by the 'less-is-more' hypothesis, we do not rule out the possibility that the gene loss of hair-type keratin genes in these species might be associated to the hairless phenotype and could have been adaptive in response to new selective pressures imposed by the colonization of a new habitat. Our study provides support for the idea that pseudogenes are not simply 'genomic fossils' but instead have adaptive roles during the evolutionary process.

Reconciling molecular phylogenies with the fossil record.

Historical patterns of species diversity inferred from phylogenies typically contradict the direct evidence found in the fossil record. According to the fossil record, species frequently go extinct, and many clades experience periods of dramatic diversity loss. However, most analyses of molecular phylogenies fail to identify any periods of declining diversity, and they typically infer low levels of extinction. This striking inconsistency between phylogenies and fossils limits our understanding of macroevolution, and it undermines our confidence in phylogenetic inference. Here, we show that realistic extinction rates and diversity trajectories can be inferred from molecular phylogenies. To make this inference, we derive an analytic expression for the likelihood of a phylogeny that accommodates scenarios of declining diversity, time-variable rates, and incomplete sampling; we show that this likelihood expression reliably detects periods of diversity loss using simulation. We then study the cetaceans (whales, dolphins, and porpoises), a group for which standard phylogenetic inferences are strikingly inconsistent with fossil data. When the cetacean phylogeny is considered as a whole, recently radiating clades, such as the Balaneopteridae, Delphinidae, Phocoenidae, and Ziphiidae, mask the signal of extinctions. However, when isolating these groups, we infer diversity dynamics that are consistent with the fossil record. These results reconcile molecular phylogenies with fossil data, and they suggest that most extant cetaceans arose from four recent radiations, with a few additional species arising from clades that have been in decline over the last ~10 Myr.

A pan-cetacean MHC amplicon sequencing panel developed and evaluated in combination with genome assemblies.

The major histocompatibility complex (MHC) is a highly polymorphic gene family that is crucial in immunity, and its diversity can be effectively used as a fitness marker for populations. Despite this, MHC remains poorly characterised in non-model species (e.g., cetaceans: whales, dolphins and porpoises) as high gene copy number variation, especially in the fast-evolving class I region, makes analyses of genomic sequences difficult. To date, only small sections of class I and IIa genes have been used to assess functional diversity in cetacean populations. Here, we undertook a systematic characterisation of the MHC class I and IIa regions in available cetacean genomes. We extracted full-length gene sequences to design pan-cetacean primers that amplified the complete exon 2 from MHC class I and IIa genes in one combined sequencing panel. We validated this panel in 19 cetacean species and described 354 alleles for both classes. Furthermore, we identified likely assembly artefacts for many MHC class I assemblies based on the presence of class I genes in the amplicon data compared to missing genes from genomes. Finally, we investigated MHC diversity using the panel in 25 humpback and 30 southern right whales, including four paternity trios for humpback whales. This revealed copy-number variable class I haplotypes in humpback whales, which is likely a common phenomenon across cetaceans. These MHC alleles will form the basis for a cetacean branch of the Immuno-Polymorphism Database (IPD-MHC), a curated resource intended to aid in the systematic compilation of MHC alleles across several species, to support conservation initiatives.

Automatic detection and classification of odontocete whistles.

Methods for the fully automatic detection and species classification of odontocete whistles are described. The detector applies a number of noise cancellation techniques to a spectrogram of sound data and then searches for connected regions of data which rise above a pre-determined threshold. When tested on a dataset of recordings which had been carefully annotated by a human operator, the detector was able to detect (recall) 79.6% of human identified sounds that had a signal-to-noise ratio above 10 dB, with 88% of the detections being valid. A significant problem with automatic detectors is that they tend to partially detect whistles or break whistles into several parts. A classifier has been developed specifically to work with fragmented whistle detections. By accumulating statistics over many whistle fragments, correct classification rates of over 94% have been achieved for four species. The success rate is, however, heavily dependent on the number of species included in the classifier mix, with the mean correct classification rate dropping to 58.5% when 12 species were included.

Classification of mysticete sounds using machine learning techniques.

Automatic classification of mysticete sounds has long been a challenging task in the bioacoustics field. The unknown statistical properties of the signals as well as the use of different recording apparatus and low signal-to-noise ratio conditions often lead to non-optimal systems. The goal of this paper is to design methods for the automatic classification of mysticete sounds using a restricted Boltzmann machine and a sparse auto-encoder that are widely used in the field of artificial intelligence. Experiments on five species of mysticetes are presented. The different methods are employed on the subset of species whose frequency range overlaps, as well as in all five species' calls. Moreover, results are offered with and without the use of a noise class. Overall, the systems are able to achieve an average classification accuracy of over 69% (with noise) and 80% (without noise) given the different architectures.

The effects of acoustic misclassification on cetacean species abundance estimation.

To estimate the density or abundance of a cetacean species using acoustic detection data, it is necessary to correctly identify the species that are detected. Developing an automated species classifier with 100% correct classification rate for any species is likely to stay out of reach. It is therefore necessary to consider the effect of misidentified detections on the number of observed data and consequently on abundance or density estimation, and develop methods to cope with these misidentifications. If misclassification rates are known, it is possible to estimate the true numbers of detected calls without bias. However, misclassification and uncertainties in the level of misclassification increase the variance of the estimates. If the true numbers of calls from different species are similar, then a small amount of misclassification between species and a small amount of uncertainty around the classification probabilities does not have an overly detrimental effect on the overall variance. However, if there is a difference in the encounter rate between species calls and/or a large amount of uncertainty in misclassification rates, then the variance of the estimates becomes very large and this dramatically increases the variance of the final abundance estimate.

Methodology and results of calibration of tonal click detectors for small odontocetes (C-PODs).

Static acoustic monitoring (SAM) is one major technology for observing small cetacean species. Automatic click loggers deployed for long time periods (>2 months) with a single hydrophone are a standard solution. Acoustic properties, like detection thresholds of these instruments, are essential for interpretation of results, but have nevertheless received little attention. A methodology for calibrating tonal click detectors in small tanks consisting of the determination of the horizontal directivity pattern and detection thresholds including a transfer function is presented. Two approaches were tested to determine detection thresholds by (a) determining the 50% detection threshold and (b) fitting a linear regression model to the recorded relative amplitudes. The tests were carried out on C-PODs (Cetacean PODs, tonal click detectors), the most commonly used instrument for SAM in Europe. Directivity and threshold were tested between 60 and 150 kHz. Directivity showed a maximum variation of 8.5 dB in the horizontal plane. Sensitivity is highest between 80 and 130 kHz and linear (± 3 dB) in this frequency range for most of the instruments tested. C-PODs have a detection threshold (calculated with the linear model) of 114.5 ± 1.2 (standard deviation) dB re 1 µPa peak-peak at 130 kHz.

Neglect of bandwidth of Odontocetes echo location clicks biases propagation loss and single hydrophone population estimates.

Passive acoustic monitoring with a single hydrophone has been suggested as a cost-effective method to monitor population density of echolocating marine mammals, by estimating the distance at which the hydrophone is able to intercept the echolocation clicks and distinguish these from the background. To avoid a bias in the estimated population density, this method relies on an unbiased estimate of the detection range and therefore of the propagation loss (PL). When applying this method, it is common practice to estimate PL at the center frequency of a broadband echolocation click and to assume this narrowband PL applies also to the broadband click. For a typical situation this narrowband approximation overestimates PL, underestimates the detection range and consequently overestimates the population density by an amount that for fixed center frequency increases with increasing pulse bandwidth and sonar figure of merit.