Ancestral chromosomal signatures of Paenungulata (Afroteria) reveal the karyotype of Amazonian manatee (Trichechus inunguis, Sirenia: Trichechidae) as the oldest among American manatees.

BACKGROUND: Chromosomal painting in manatees has clarified questions about the rapid evolution of sirenians within the Paenungulata clade. Further cytogenetic studies in Afrotherian species may provide information about their evolutionary dynamics, revealing important insights into the ancestral karyotype in the clade representatives. The karyotype of Trichechus inunguis (TIN, Amazonian manatee) was investigated by chromosome painting, using probes from Trichechus manatus latirostris (TML, Florida manatee) to analyze the homeologies between these sirenians. RESULTS: A high similarity was found between these species, with 31 homologous segments in TIN, nineteen of which are whole autosomes, besides the X and Y sex chromosomes. Four chromosomes from TML (4, 6, 8, and 9) resulted in two hybridization signals, totaling eight acrocentrics in the TIN karyotype. This study confirmed in TIN the chromosomal associations of Homo sapiens (HSA) shared in Afrotheria, such as the 5/21 synteny, and in the Paenungulata clade with the syntenies HSA 2/3, 8/22, and 18/19, in addition to the absence of HSA 4/8 common in eutherian ancestral karyotype (EAK). CONCLUSIONS: TIN shares more conserved chromosomal signals with the Paenungulata Ancestral Karyotype (APK, 2n = 58) than Procavia capensis (Hyracoidea), Loxodonta africana (Proboscidea) and TML (Sirenia), where TML presents less conserved signals with APK, demonstrating that its karyotype is the most derived among the representatives of Paenungulata. The chromosomal changes that evolved from APK to the T. manatus and T. inunguis karyotypes (7 and 4 changes, respectively) are more substantial within the Trichechus genus compared to other paenungulates. Among these species, T. inunguis presents conserved traits of APK in the American manatee genus. Consequently, the karyotype of T. manatus is more derived than that of T. inunguis.

Spectral tuning and deactivation kinetics of marine mammal melanopsins.

In mammals, the photopigment melanopsin (Opn4) is found in a subset of retinal ganglion cells that serve light detection for circadian photoentrainment and pupil constriction (i.e., mydriasis). For a given species, the efficiency of photoentrainment and length of time that mydriasis occurs is determined by the spectral sensitivity and deactivation kinetics of melanopsin, respectively, and to date, neither of these properties have been described in marine mammals. Previous work has indicated that the absorbance maxima (λmax) of marine mammal rhodopsins (Rh1) have diversified to match the available light spectra at foraging depths. However, similar to the melanopsin λmax of terrestrial mammals (~480 nm), the melanopsins of marine mammals may be conserved, with λmax values tuned to the spectrum of solar irradiance at the water's surface. Here, we investigated the Opn4 pigments of 17 marine mammal species inhabiting diverse photic environments including the Infraorder Cetacea, as well as the Orders Sirenia and Carnivora. Both genomic and cDNA sequences were used to deduce amino acid sequences to identify substitutions most likely involved in spectral tuning and deactivation kinetics of the Opn4 pigments. Our results show that there appears to be no amino acid substitutions in marine mammal Opn4 opsins that would result in any significant change in λmax values relative to their terrestrial counterparts. We also found some marine mammal species to lack several phosphorylation sites in the carboxyl terminal domain of their Opn4 pigments that result in significantly slower deactivation kinetics, and thus longer mydriasis, compared to terrestrial controls. This finding was restricted to cetacean species previously found to lack cone photoreceptor opsins, a condition known as rod monochromacy. These results suggest that the rod monochromat whales rely on extended pupillary constriction to prevent photobleaching of the highly photosensitive all-rod retina when moving between photopic and scotopic conditions.

Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia.

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Differential Evolution of the Epidermal Keratin Cytoskeleton in Terrestrial and Aquatic Mammals.

Keratins are the main intermediate filament proteins of epithelial cells. In keratinocytes of the mammalian epidermis they form a cytoskeleton that resists mechanical stress and thereby are essential for the function of the skin as a barrier against the environment. Here, we performed a comparative genomics study of epidermal keratin genes in terrestrial and fully aquatic mammals to determine adaptations of the epidermal keratin cytoskeleton to different environments. We show that keratins K5 and K14 of the innermost (basal), proliferation-competent layer of the epidermis are conserved in all mammals investigated. In contrast, K1 and K10, which form the main part of the cytoskeleton in the outer (suprabasal) layers of the epidermis of terrestrial mammals, have been lost in whales and dolphins (cetaceans) and in the manatee. Whereas in terrestrial mammalian epidermis K6 and K17 are expressed only upon stress-induced epidermal thickening, high levels of K6 and K17 are consistently present in dolphin skin, indicating constitutive expression and substitution of K1 and K10. K2 and K9, which are expressed in a body site-restricted manner in human and mouse suprabasal epidermis, have been lost not only in cetaceans and manatee but also in some terrestrial mammals. The evolution of alternative splicing of K10 and differentiation-dependent upregulation of K23 have increased the complexity of keratin expression in the epidermis of terrestrial mammals. Taken together, these results reveal evolutionary diversification of the epidermal cytoskeleton in mammals and suggest a complete replacement of the quantitatively predominant epidermal proteins of terrestrial mammals by originally stress-inducible keratins in cetaceans.

Hundreds of Genes Experienced Convergent Shifts in Selective Pressure in Marine Mammals.

Mammal species have made the transition to the marine environment several times, and their lineages represent one of the classical examples of convergent evolution in morphological and physiological traits. Nevertheless, the genetic mechanisms of their phenotypic transition are poorly understood, and investigations into convergence at the molecular level have been inconclusive. While past studies have searched for convergent changes at specific amino acid sites, we propose an alternative strategy to identify those genes that experienced convergent changes in their selective pressures, visible as changes in evolutionary rate specifically in the marine lineages. We present evidence of widespread convergence at the gene level by identifying parallel shifts in evolutionary rate during three independent episodes of mammalian adaptation to the marine environment. Hundreds of genes accelerated their evolutionary rates in all three marine mammal lineages during their transition to aquatic life. These marine-accelerated genes are highly enriched for pathways that control recognized functional adaptations in marine mammals, including muscle physiology, lipid-metabolism, sensory systems, and skin and connective tissue. The accelerations resulted from both adaptive evolution as seen in skin and lung genes, and loss of function as in gustatory and olfactory genes. In regard to sensory systems, this finding provides further evidence that reduced senses of taste and smell are ubiquitous in marine mammals. Our analysis demonstrates the feasibility of identifying genes underlying convergent organism-level characteristics on a genome-wide scale and without prior knowledge of adaptations, and provides a powerful approach for investigating the physiological functions of mammalian genes.

Interordinal gene capture, the phylogenetic position of Steller's sea cow based on molecular and morphological data, and the macroevolutionary history of Sirenia.

The recently extinct (ca. 1768) Steller's sea cow (Hydrodamalis gigas) was a large, edentulous North Pacific sirenian. The phylogenetic affinities of this taxon to other members of this clade, living and extinct, are uncertain based on previous morphological and molecular studies. We employed hybridization capture methods and second generation sequencing technology to obtain >30kb of exon sequences from 26 nuclear genes for both H. gigas and Dugong dugon. We also obtained complete coding sequences for the tooth-related enamelin (ENAM) gene. Hybridization probes designed using dugong and manatee sequences were both highly effective in retrieving sequences from H. gigas (mean=98.8% coverage), as were more divergent probes for regions of ENAM (99.0% coverage) that were designed exclusively from a proboscidean (African elephant) and a hyracoid (Cape hyrax). New sequences were combined with available sequences for representatives of all other afrotherian orders. We also expanded a previously published morphological matrix for living and fossil Sirenia by adding both new taxa and nine new postcranial characters. Maximum likelihood and parsimony analyses of the molecular data provide robust support for an association of H. gigas and D. dugon to the exclusion of living trichechids (manatees). Parsimony analyses of the morphological data also support the inclusion of H. gigas in Dugongidae with D. dugon and fossil dugongids. Timetree analyses based on calibration density approaches with hard- and soft-bounded constraints suggest that H. gigas and D. dugon diverged in the Oligocene and that crown sirenians last shared a common ancestor in the Eocene. The coding sequence for the ENAM gene in H. gigas does not contain frameshift mutations or stop codons, but there is a transversion mutation (AG to CG) in the acceptor splice site of intron 2. This disruption in the edentulous Steller's sea cow is consistent with previous studies that have documented inactivating mutations in tooth-specific loci of a variety of edentulous and enamelless vertebrates including birds, turtles, aardvarks, pangolins, xenarthrans, and baleen whales. Further, branch-site dN/dS analyses provide evidence for positive selection in ENAM on the stem dugongid branch where extensive tooth reduction occurred, followed by neutral evolution on the Hydrodamalis branch. Finally, we present a synthetic evolutionary tree for living and fossil sirenians showing several key innovations in the history of this clade including character state changes that parallel those that occurred in the evolutionary history of cetaceans.

Breaking constraint: axial patterning in Trichechus (Mammalia: Sirenia).

Meristic variation is often limited in serially homologous systems with high internal differentiation and high developmental modularity. The mammalian neck, an extreme example, has a fixed (at seven) count of diversely specialized segments. Imposition of the mammalian cervical constraint has been tentatively linked to the origin of the diaphragm, which is muscularized by cells that migrate from cervical somites during development. With six cervical vertebrae, the genus Trichechus (manatee) has apparently broken this constraint, although the mechanism of constraint escape is unknown. Hypotheses for the developmental origin of Trichechus cervical morphology include cervical rib 7 repatterning, a primaxial/abaxial patterning shift, and local homeosis at the cervical/thoracic boundary. We tested predictions of these hypotheses by documenting vertebral morphology, axial ossification patterns, regionalization of the postcranial skeleton, and the relationship of thoracic ribs to sternal subunits in a large data set of fetal and adult Trichechus and Dugong specimens. These observations forced rejection of all three hypotheses. We propose alternatively that a global slowing of the rate of somitogenesis reduced somite count and disrupted alignment of Hox-generated anatomical markers relative to somite (and vertebral) boundaries throughout the Trichechus column. This hypothesis is consistent with observations of the full range of traditional cervical morphologies in the six cervical vertebrae, conserved postcranial proportions, and column-wide reduction in count relative to its sister taxon, Dugong. It also suggests that the origin of the mammalian cervical constraint lies in patterning, not in count, and that Trichechus and the tree sloths have broken the constraint using different developmental mechanisms.

The chromosomes of Afrotheria and their bearing on mammalian genome evolution.

Afrotheria is the clade of placental mammals that, together with Xenarthra, Euarchontoglires and Laurasiatheria, represents 1 of the 4 main recognized supraordinal eutherian clades. It reunites 6 orders of African origin: Proboscidea, Sirenia, Hyracoidea, Macroscelidea, Afrosoricida and Tubulidentata. The apparently unlikely relationship among such disparate morphological taxa and their possible basal position at the base of the eutherian phylogenetic tree led to a great deal of attention and research on the group. The use of biomolecular data was pivotal in Afrotheria studies, as they were the basis for the recognition of this clade. Although morphological evidence is still scarce, a plethora of molecular data firmly attests to the phylogenetic relationship among these mammals of African origin. Modern cytogenetic techniques also gave a significant contribution to the study of Afrotheria, revealing chromosome signatures for the group as a whole, as well as for some of its internal relationships. The associations of human chromosomes HSA1/19 and 5/21 were found to be chromosome signatures for the group and provided further support for Afrotheria. Additional chromosome synapomorphies were also identified linking elephants and manatees in Tethytheria (the associations HSA2/3, 3/13, 8/22, 18/19 and the lack of HSA4/8) and elephant shrews with the aardvark (HSA2/8, 3/20 and 10/17). Herein, we review the current knowledge on Afrotheria chromosomes and genome evolution. The already available data on the group suggests that further work on this apparently bizarre assemblage of mammals will provide important data to a better understanding on mammalian genome evolution.

Osteological associations with unique tooth development in manatees (Trichechidae, Sirenia): a detailed look at modern Trichechus and a review of the fossil record.

Modern manatees have a unique type of tooth development, continually forming identical new molars in the posterior end of each quadrant of their mouths, and then progressively moving teeth anteriorly, only to reabsorb roots and spit out worn crowns. This process is not only developmentally complex, but requires space in the oral cavity that imposes its own limitations on other uses of that space. To gain a clearer understanding of the anatomical constraints on the evolution of this unique developmental process, we identified the specialized craniodental features in modern Trichechus that permit this specialization using visual observation and CT. Furthermore, to better understand the evolution of these traits, we review the fossil record of trichechids for these traits, including CT analysis of the skull of Miosiren kocki, a possible early member of the family from the Early Miocene of Belgium.

Marine mammals through time: when less is more in studying palaeodiversity.

The validity of biological explanations of patterns of palaeodiversity has been called into question owing to an apparent correlation of diversity with the amount of sedimentary rock preserved. However, this claim has largely been based on comprehensive estimates of global marine Phanerozoic diversity, thus raising the question of whether a similar bias applies to the records of smaller, well-defined taxonomic groups. Here, new data on European Caenozoic marine sedimentary rock outcrop area are presented and compared with European occurrences of three groups of marine mammals (cetaceans, pinnipedimorphs and sirenians). Limited evidence was found for a correlation of outcrop area with marine mammal palaeodiversity. In addition, similar patterns were identified in the cetacean and pinnipedimorph diversity data. This may point to the preservation of a genuine biological signal not overwhelmed by geological biases in the marine mammal diversity data, and opens the door to further analyses of both marine mammal evolution and geological bias in other small and well-defined groups of taxa.

Positive selection in the N-terminal extramembrane domain of lung surfactant protein C (SP-C) in marine mammals.

Maximum-likelihood models of codon and amino acid substitution were used to analyze the lung-specific surfactant protein C (SP-C) from terrestrial, semi-aquatic, and diving mammals to identify lineages and amino acid sites under positive selection. Site models used the nonsynonymous/synonymous rate ratio (omega) as an indicator of selection pressure. Mechanistic models used physicochemical distances between amino acid substitutions to specify nonsynonymous substitution rates. Site models strongly identified positive selection at different sites in the polar N-terminal extramembrane domain of SP-C in the three diving lineages: site 2 in the cetaceans (whales and dolphins), sites 7, 9, and 10 in the pinnipeds (seals and sea lions), and sites 2, 9, and 10 in the sirenians (dugongs and manatees). The only semi-aquatic contrast to indicate positive selection at site 10 was that including the polar bear, which had the largest body mass of the semi-aquatic species. Analysis of the biophysical properties that were influential in determining the amino acid substitutions showed that isoelectric point, chemical composition of the side chain, polarity, and hydrophobicity were the crucial determinants. Amino acid substitutions at these sites may lead to stronger binding of the N-terminal domain to the surfactant phospholipid film and to increased adsorption of the protein to the air-liquid interface. Both properties are advantageous for the repeated collapse and reinflation of the lung upon diving and resurfacing and may reflect adaptations to the high hydrostatic pressures experienced during diving.

A retroposon analysis of Afrotherian phylogeny.

Recent comprehensive studies of DNA sequences support the monophyly of Afrotheria, comprising elephants, sirenians (dugongs and manatees), hyraxes, tenrecs, golden moles, aardvarks, and elephant shrews, as well as that of Paenungulata, comprising elephants, sirenians, and hyraxes. However, phylogenetic relationships among paenungulates, as well as among nonpaenungulates, have remained ambiguous. Here we applied an extensive retroposon analysis to these problems to support the monophyly of aardvarks, tenrecs, and golden moles, with elephant shrews as their sister group. Regarding phylogenetic relationships in Paenungulata, we could characterize only one informative locus, although we could isolate many insertions specific to each of three lineages, namely, Proboscidea, Sirenia, and Hyracoidea. These data prompted us to reexamine phylogenetic relationships among Paenungulata using 19 nuclear gene sequences resulting in three different analyses, namely, short interspersed element (SINE) insertions, nuclear sequence analyses, and morphological cladistics, supporting different respective phylogenies. We concluded that these three lineages diverged very rapidly in a very short evolutionary period, with the consequence that ancestral polymorphism present in the last common ancestor of Paenungulata results in such incongruence. Our results suggest the rapid fixation of many large-scale morphological synapomorphies for Tethytheria; implications of this in relation to the morphological evolution in Paenungulata are discussed.

The developing renal, reproductive, and respiratory systems of the African elephant suggest an aquatic ancestry.

The early embryology of the elephant has never been studied before. We have obtained a rare series of African elephant (Loxodonta africana) embryos and fetuses ranging in weight from 0.04 to 18.5 g, estimated gestational ages 58-166 days (duration of gestation is approximately 660 days). Nephrostomes, a feature of aquatic vertebrates, were found in the mesonephric kidneys at all stages of development whereas they have never been recorded in the mesonephric kidneys of other viviparous mammals. The trunk was well developed even in the earliest fetus. The testes were intra-abdominal, and there was no evidence of a gubernaculum, pampiniform plexus, processus vaginalis, or a scrotum, confirming that the elephant, like the dugong, is one of the few primary testicond mammals. The palaeontological evidence suggests that the elephant's ancestors were aquatic, and recent immunological and molecular evidence shows an extremely close affinity between present-day elephants and the aquatic Sirenia (dugong and manatees). The evidence from our embryological study of the elephant also suggests that it evolved from an aquatic mammal.