Ana R. Amaral

Species tree of a recent radiation : the subfamily Delphininae (Cetacea, Mammalia)

Lineages undergoing rapid radiations provide exceptional opportunities for studying speciation and adaptation, but also represent a challenge for molecular systematics because retention of ancestral polymorphisms and the occurrence of hybridization can obscure relationships among lineages. Dolphins in the subfamily Delphininae are one such case. Non-monophyly, rapid speciation events, and discordance between morphological and molecular characters have made the inference of phylogenetic relationships within this subfamily very difficult. Here we approach this problem by applying multiple methods intended to estimate species trees using a multi-gene dataset for the Delphininae (Sousa, Sotalia, Stenella, Tursiops, Delphinus and Lagenodelphis). Incongruent gene trees obtained indicate that incomplete lineage sorting and possibly hybridization are confounding the inference of species history in this group. Nonetheless, using coalescent-based methods, we have been able to extract an underlying species-tree signal from divergent histories of independent genes. This is the first time a molecular study provides support for such relationships. This study further illustrates how methods of species-tree inference can be very sensitive both to the characteristics of the dataset and the evolutionary processes affecting the evolution of the group under study.

Influences of past climatic changes on historical population structure and demography of a cosmopolitan marine predator, the common dolphin (genus Delphinus)

Climatic oscillations during the Pleistocene have greatly influenced the distribution and connectivity of many organisms, leading to extinctions but also generating biodiversity. While the effects of such changes have been extensively studied in the terrestrial environment, studies focusing on the marine realm are still scarce. Here we used sequence data from one mitochondrial and five nuclear loci to assess the potential influence of Pleistocene climatic changes on the phylogeography and demographic history of a cosmopolitan marine predator, the common dolphin (genus Delphinus). Population samples representing the three major morphotypes of Delphinus were obtained from 10 oceanic regions. Our results suggest that short‐beaked common dolphins are likely to have originated in the eastern Indo‐Pacific Ocean during the Pleistocene and expanded into the Atlantic Ocean through the Indian Ocean. On the other hand, long‐beaked common dolphins appear to have evolved more recently and independently in several oceans. Our results also suggest that short‐beaked common dolphins had recurrent demographic expansions concomitant with changes in sea surface temperature during the Pleistocene and its associated increases in resource availability, which differed between the North Atlantic and Pacific Ocean basins. By proposing how past environmental changes had an effect on the demography and speciation of a widely distributed marine mammal, we highlight the impacts that climate change may have on the distribution and abundance of marine predators and its ecological consequences for marine ecosystems.

Hybrid Speciation in a Marine Mammal: The Clymene Dolphin (Stenella clymene)

Natural hybridization may result in the exchange of genetic material between divergent lineages and even the formation of new taxa. Many of the Neo-Darwinian architects argued that, particularly for animal clades, natural hybridization was maladaptive. Recent evidence, however, has falsified this hypothesis, instead indicating that this process may lead to increased biodiversity through the formation of new species. Although such cases of hybrid speciation have been described in plants, fish and insects, they are considered exceptionally rare in mammals. Here we present evidence for a marine mammal, Stenella clymene, arising through natural hybridization. We found phylogenetic discordance between mitochondrial and nuclear markers, which, coupled with a pattern of transgressive segregation seen in the morphometric variation of some characters, support a case of hybrid speciation. S. clymene is currently genetically differentiated from its putative parental species, Stenella coerueloalba and Stenella longisrostris, although low levels of introgressive hybridization may be occurring. Although non-reticulate forms of evolution, such as incomplete lineage sorting, could explain our genetic results, we consider that the genetic and morphological evidence taken together argue more convincingly towards a case of hybrid speciation. We anticipate that our study will bring attention to this important aspect of reticulate evolution in non-model mammal species. The study of speciation through hybridization is an excellent opportunity to understand the mechanisms leading to speciation in the context of gene flow.

A first approach to the usefulness of cytochrome c oxidase I barcodes in the identification of closely related delphinid cetacean species

The DNA barcode initiative has gained particular popularity as a promising tool to assist in species identification by using a single mitochondrial gene, cytochrome c oxidase I (COI). In some animal groups, COI barcodes have proved efficient in separating closely related taxa. However, several issues remain for discussion, namely how efficient this tool will be in animal groups with an unresolved taxonomy. Here, we examined COI sequences in delphinid cetaceans, a group where taxonomic uncertainty still exists. We analysed species belonging to the genera Stenella, Tursiops and Delphinus in the North-east Atlantic using cytochrome b gene sequences for comparison. We obtained values of COI interspecific genetic divergence ranging from 1.47% to 2.45%, which suggests a recent separation of the analysed taxa. S. coeruleoalba and D. delphis were the most similar species, with COI phylogenetic trees failing to separate them. On the other hand, the phylogenetic tree obtained with cytochrome b sequences correctly clustered species with high bootstrap support values. We thus consider that the application of COI barcodes in delphinid cetaceans should be done with caution; not only has the cytochrome b gene been shown to be phylogenetically more informative, but relying only on mitochondrial DNA genes alone may be problematic.

The World's Most Isolated and Distinct Whale Population? Humpback Whales of the Arabian Sea

A clear understanding of population structure is essential for assessing conservation status and implementing management strategies. A small, non-migratory population of humpback whales in the Arabian Sea is classified as “Endangered” on the IUCN Red List of Threatened Species, an assessment constrained by a lack of data, including limited understanding of its relationship to other populations. We analysed 11 microsatellite markers and mitochondrial DNA sequences extracted from 67 Arabian Sea humpback whale tissue samples and compared them to equivalent datasets from the Southern Hemisphere and North Pacific. Results show that the Arabian Sea population is highly distinct; estimates of gene flow and divergence times suggest a Southern Indian Ocean origin but indicate that it has been isolated for approximately 70,000 years, remarkable for a species that is typically highly migratory. Genetic diversity values are significantly lower than those obtained for Southern Hemisphere populations and signatures of ancient and recent genetic bottlenecks were identified. Our findings suggest this is the worlds most isolated humpback whale population, which, when combined with low population abundance estimates and anthropogenic threats, raises concern for its survival. We recommend an amendment of the status of the population to “Critically Endangered” on the IUCN Red List.

Cranial shape differentiation in three closely related delphinid cetacean species: Insights into evolutionary history

The present study investigates the pattern of differentiation of cranial shape in three closely related delphinid cetacean species of the complex Delphinus-Stenella-Tursiops: Delphinus delphis, Stenella coeruleoalba and Tursiops truncatus. Dorsal and ventral aspects of the cranium were analysed using landmark-based geometric morphometric methods. While there was no evidence of sexual dimorphism for shape or size, multivariate statistical analyses showed that there were interspecific differences in skull morphology. Skull shape differences between the three studied species were related with cranial width and differences in the length of the rostrum relative to the cranial portion of the skull. D. delphis and S. coeruleoalba showed high cranial shape similarity, which is indicative of their evolutionary proximity when compared with T. truncatus. Phenetic clusters based on cranial shape similarities were found to be concordant with the molecular phylogenetic clades obtained from mitochondrial DNA genes. Geometric morphometric methods can thus be an exceptionally useful tool for the study of differentiation of delphinid cetacean species and therefore provide some insights into their evolutionary history.

Physiopathologic mechanisms involved in mare endometrosis.

Endometrosis is a degenerative chronic process, characterized by paramount fibrosis development in mare endometrium. This condition is one of the major causes of subfertility/infertility in mares. As in other organs, fibrosis might be a pathologic sequel of many chronic inflammatory diseases. However, aetiology and physiopathologic mechanisms involved in endometrial fibrosis are still controversial. This review presents new hypotheses based on our newest data. As the first line of innate immune defence, systemic neutrophils arrive in the uterus at mating or in the presence of pathogens. A novel paradigm is that neutrophils cast out their DNA in response to infectious stimuli and form neutrophil extracellular traps (NETs). We have shown that bacterial strains of Streptococcus zooepidemicus, Escherichia coli or Staphylococcus capitis, known to cause endometritis in mares were able to induce NETs release in vitro by equine PMN to different extents. An intriguing dilemma is the dual action of NETs. While NETs play a desirable role fighting micro-organisms in mare uterus, they may also contribute to endometrial fibrosis. A long-term in vitro exposure of mare endometrium explants to NETs components (myeloperoxidase, elastase and cathepsin G) up-regulated fibrosis markers TGFβ and Tissue inhibitor of metalloproteinase (TIMP-1). Also, pro-fibrotic cytokines regulated collagen deposition and fibrosis. Changes in expression of connective tissue growth factor (CTGF), interleukins (IL)1-α, IL-1β, IL-6 and receptors in endometrium with different degrees of fibrosis and/or inflammation were observed. A putative role of CTGF, IL and NETs components in endometrosis development should be considered. Additionally, we speculate that in sustained endometritis in mares, prostaglandins may not only cause early luteolysis or early pregnancy loss, but may also be related to endometrial fibrosis pathogenesis by stimulating collagen deposition.

Opposing Roles of Leptin and Ghrelin in the Equine Corpus Luteum Regulation: An In Vitro Study

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2 and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α (TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4 in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. The in vitro effect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

Historical Biogeography of Delphininae Dolphins and Related Taxa (Artiodactyla: Delphinidae)

Delphinine dolphins arose via a recent, rapid radiation, probably within the last four million years. Although molecular phylogenies are increasingly well resolved, patterns of morphology-ecology-geography are hard to link to phylogeny or to translate into taxonomy. Such problems might be tackled through understanding the drivers of the delphinine radiation. Here, we examine delphinine historical biogeography using the phylogeny of McGowen et al. (Mol Phylogenet Evol 53:891–906, 2009) as our working hypothesis. We used the “Spatial Analysis of Vicariance” method to delimit modern distribution patterns, including disjunctions involving sister nodes in the Delphininae. The analysis identified disjunct sister nodes, allowing some interpretation of Delphininae biogeography. The Central American Seaway was probably an important gateway for early delphinids, but the succeeding “hard” barrier of the Panama Isthmus had little influence. Southern African waters form the Atlantic-Indo-Pacific gateway, which is sometimes considered a “soft” barrier because of the variation in the Benguela and Agulhas currents, in turn driven by tectonic changes and/or Pleistocene glacial and interglacial cycles. The latter cycles probably fragmented coastal habitats, allowing allopatric speciation. Geological patterns of turnover in Southern Ocean diatoms, which link to physical oceanic change, closely match the main cluster of delphinine divergences. The Eastern Pacific Barrier, and perhaps the associated Humboldt Current and equatorial “cold tongue,” affect modern distributions, but cause and effect are poorly understood. Future research should involve molecular-morphological phylogenetics for all species, subspecies, and ecomorphs. Complete distributions must be known for all taxa to understand how vicariance and dispersal shaped the distribution of delphinines.

Evolution of 2 Reproductive Proteins, ZP3 and PKDREJ, in Cetaceans

The rapid evolution of proteins involved in reproduction has been documented in several animal taxa. This is thought to be the result of forces involved in sexual selection and is expected to be particularly strong in promiscuous mating systems. In this study, a range of cetacean species were used to analyze the patterns of evolution in 2 reproductive proteins involved in fertilization: the zona pellucida 3 (ZP3), present in the egg coat, and PKDREJ, localized in the sperm head. We targeted exons 6 and 7 of ZP3 and a part of the REJ domain in PKDREJ for a total of 958 bp in 18 species. We found very low levels of amino acid sequence divergence in both proteins, a very weak signal of positive selection in ZP3 and no signal in PKDREJ. These results were consistent with previous reports of a slow rate of molecular evolution in cetaceans but unexpected due to the existence of promiscuous mating systems in these species. The results raise questions about the evolution of reproductive isolation and species recognition in whales and dolphins.