Molecular Evidence for Relaxed Selection on the Enamel Genes of Toothed Whales (Odontoceti) with Degenerative Enamel Phenotypes.

Different species of toothed whales (Odontoceti) exhibit a variety of tooth forms and enamel types. Some odontocetes have highly prismatic enamel with Hunter-Schreger bands, whereas enamel is vestigial or entirely lacking in other species. Different tooth forms and enamel types are associated with alternate feeding strategies that range from biting and grasping prey with teeth in most oceanic and river dolphins to the suction feeding of softer prey items without the use of teeth in many beaked whales. At the molecular level, previous studies have documented inactivating mutations in the enamel-specific genes of some odontocete species that lack complex enamel. At a broader scale, however, it is unclear whether enamel complexity across the full diversity of extant Odontoceti correlates with the relative strength of purifying selection on enamel-specific genes. Here, we employ sequence alignments for seven enamel-specific genes (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) in 62 odontocete species that are representative of all extant families. The sequences for 33 odontocete species were obtained from databases, and sequences for the remaining 29 species were newly generated for this study. We screened these alignments for inactivating mutations (e.g., frameshift indels) and provide a comprehensive catalog of these mutations in species with one or more inactivated enamel genes. Inactivating mutations are rare in Delphinidae (oceanic dolphins) and Platanistidae/Inioidea (river dolphins) that have higher enamel complexity scores. By contrast, mutations are much more numerous in clades such as Monodontidae (narwhal, beluga), Ziphiidae (beaked whales), Physeteroidea (sperm whales), and Phocoenidae (porpoises) that are characterized by simpler enamel or even enamelless teeth. Further, several higher-level taxa (e.g., Hyperoodon, Kogiidae, Monodontidae) possess shared inactivating mutations in one or more enamel genes, which suggests loss of function of these genes in the common ancestor of each clade. We also performed selection (dN/dS) analyses on a concatenation of these genes and used linear regression and Spearman's rank-order correlation to test for correlations between enamel complexity and two different measures of selection intensity (# of inactivating mutations per million years, dN/dS values). Selection analyses revealed that relaxed purifying selection is especially prominent in physeteroids, monodontids, and phocoenids. Linear regressions and correlation analyses revealed a strong negative correlation between selective pressure (dN/dS values) and enamel complexity. Stronger purifying selection (low dN/dS) is found on branches with more complex enamel and weaker purifying selection (higher dN/dS) occurs on branches with less complex enamel or enamelless teeth. As odontocetes diversified into a variety of feeding modes, in particular, the suction capture of prey, a reduced reliance on the dentition for prey capture resulted in the relaxed selection of genes that are critical to enamel development.

Three Blind Moles: Molecular Evolutionary Insights on the Tempo and Mode of Convergent Eye Degeneration in Notoryctes typhlops (Southern Marsupial Mole) and Two Chrysochlorids (Golden Moles).

Golden moles (Chrysochloridae) and marsupial moles (Notoryctidae) are textbook examples of convergent evolution. Both taxa are highly adapted to subterranean lifestyles and have powerful limbs for digging through the soil/sand, ears that are adapted for low-frequency hearing, vestigial eyes that are covered by skin and fur, and the absence of optic nerve connections between the eyes and the brain. The eyes of marsupial moles also lack a lens as well as retinal rods and cones. Two hypotheses have been proposed to account for the greater degeneracy of the eyes of marsupial moles than golden moles. First, marsupial moles may have had more time to adapt to their underground habitat than other moles. Second, the eyes of marsupial moles may have been rapidly and recently vestigialized to (1) reduce the injurious effects of sand getting into the eyes and (2) accommodate the enlargement of lacrimal glands that keep the nasal cavity moist and prevent the entry of sand into the nasal passages during burrowing. Here, we employ molecular evolutionary methods on DNA sequences for 38 eye genes, most of which are eye-specific, to investigate the timing of relaxed selection (=neutral evolution) for different groups of eye-specific genes that serve as proxies for distinct functional components of the eye (rod phototransduction, cone phototransduction, lens/cornea). Our taxon sampling included 12 afrothere species, of which two are golden moles (Amblysomus hottentotus, Chrysochloris asiatica), and 28 marsupial species including two individuals of the southern marsupial mole (Notoryctes typhlops). Most of the sequences were mined from databases, but we also provide new genome data for A. hottentotus and one of the two N. typhlops individuals. Even though the eyes of golden moles are less degenerate than the eyes of marsupial moles, there are more inactivating mutations (e.g., frameshift indels, premature stop codons) in their cone phototransduction and lens/cornea genes than in orthologous genes of the marsupial mole. We estimate that cone phototransduction recovery genes were inactivated first in each group, followed by lens/cornea genes and then cone phototransduction activation genes. All three groups of genes were inactivated earlier in golden moles than in marsupial moles. For the latter, we estimate that lens/cornea genes were inactivated ~17.8 million years ago (MYA) when stem notoryctids were burrowing in the soft soils of Australian rainforests. Selection on phototransduction activation genes was relaxed much later (5.38 MYA), during the early stages of Australia's aridification that produced coastal sand plains and eventually sand dunes. Unlike cone phototransduction activation genes, rod phototransduction activation genes are intact in both golden moles and one of the two individuals of N. typhlops. A second marsupial mole individual has just a single inactivating mutation in one of the rod phototransduction activation genes (PDE6B). One explanation for this result is that some rod phototransduction activation genes are pleiotropic and are expressed in extraocular tissues, possibly in conjunction with sperm thermotaxis.

Quantification of congruence among gene trees with polytomies using overall success of resolution for phylogenomic coalescent analyses.

Gene-tree-inference error can cause species-tree-inference artefacts in summary phylogenomic coalescent analyses. Here we integrate two ways of accommodating these inference errors: collapsing arbitrarily or dubiously resolved gene-tree branches, and subsampling gene trees based on their pairwise congruence. We tested the effect of collapsing gene-tree branches with 0% approximate-likelihood-ratio-test (SH-like aLRT) support in likelihood analyses and strict consensus trees for parsimony, and then subsampled those partially resolved trees based on congruence measures that do not penalize polytomies. For this purpose we developed a new TNT script for congruence sorting (congsort), and used it to calculate topological incongruence for eight phylogenomic datasets using three distance measures: standard Robinson-Foulds (RF) distances; overall success of resolution (OSR), which is based on counting both matching and contradicting clades; and RF contradictions, which only counts contradictory clades. As expected, we found that gene-tree incongruence was often concentrated in clades that are arbitrarily or dubiously resolved and that there was greater congruence between the partially collapsed gene trees and the coalescent and concatenation topologies inferred from those genes. Coalescent branch lengths typically increased as the most incongruent gene trees were excluded, although branch supports typically did not. We investigated two successful and complementary approaches to prioritizing genes for investigation of alignment or homology errors. Coalescent-tree clades that contradicted concatenation-tree clades were generally less robust to gene-tree subsampling than congruent clades. Our preferred approach to collapsing likelihood gene-tree clades (0% SH-like aLRT support) and subsampling those trees (OSR) generally outperformed competing approaches for a large fungal dataset with respect to branch lengths, support and congruence. We recommend widespread application of this approach (and strict consensus trees for parsimony-based analyses) for improving quantification of gene-tree congruence/conflict, estimating coalescent branch lengths, testing robustness of coalescent analyses to gene-tree-estimation error, and improving topological robustness of summary coalescent analyses. This approach is quick and easy to implement, even for huge datasets.

A genomic timescale for placental mammal evolution.

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signals using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes, and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic time trees reveal an accumulation of cladogenic events before and immediately after the Cretaceous-Paleogene (K-Pg) boundary, implying important roles for Cretaceous continental vicariance and the K-Pg extinction in the placental radiation.

Phylogenomic Coalescent Analyses of Avian Retroelements Infer Zero-Length Branches at the Base of Neoaves, Emergent Support for Controversial Clades, and Ancient Introgressive Hybridization in Afroaves.

Retroelement insertions (RIs) are low-homoplasy characters that are ideal data for addressing deep evolutionary radiations, where gene tree reconstruction errors can severely hinder phylogenetic inference with DNA and protein sequence data. Phylogenomic studies of Neoaves, a large clade of birds (>9000 species) that first diversified near the Cretaceous−Paleogene boundary, have yielded an array of robustly supported, contradictory relationships among deep lineages. Here, we reanalyzed a large RI matrix for birds using recently proposed quartet-based coalescent methods that enable inference of large species trees including branch lengths in coalescent units, clade-support, statistical tests for gene flow, and combined analysis with DNA-sequence-based gene trees. Genome-scale coalescent analyses revealed extremely short branches at the base of Neoaves, meager branch support, and limited congruence with previous work at the most challenging nodes. Despite widespread topological conflicts with DNA-sequence-based trees, combined analyses of RIs with thousands of gene trees show emergent support for multiple higher-level clades (Columbea, Passerea, Columbimorphae, Otidimorphae, Phaethoquornithes). RIs express asymmetrical support for deep relationships within the subclade Afroaves that hints at ancient gene flow involving the owl lineage (Strigiformes). Because DNA-sequence data are challenged by gene tree-reconstruction error, analysis of RIs represents one approach for improving gene tree-based methods when divergences are deep, internodes are short, terminal branches are long, and introgressive hybridization further confounds species−tree inference.

Contradictory Phylogenetic Signals in the Laurasiatheria Anomaly Zone.

Relationships among laurasiatherian clades represent one of the most highly disputed topics in mammalian phylogeny. In this study, we attempt to disentangle laurasiatherian interordinal relationships using two independent genome-level approaches: (1) quantifying retrotransposon presence/absence patterns, and (2) comparisons of exon datasets at the levels of nucleotides and amino acids. The two approaches revealed contradictory phylogenetic signals, possibly due to a high level of ancestral incomplete lineage sorting. The positions of Eulipotyphla and Chiroptera as the first and second earliest divergences were consistent across the approaches. However, the phylogenetic relationships of Perissodactyla, Cetartiodactyla, and Ferae, were contradictory. While retrotransposon insertion analyses suggest a clade with Cetartiodactyla and Ferae, the exon dataset favoured Cetartiodactyla and Perissodactyla. Future analyses of hitherto unsampled laurasiatherian lineages and synergistic analyses of retrotransposon insertions, exon and conserved intron/intergenic sequences might unravel the conflicting patterns of relationships in this major mammalian clade.

Molecular evolutionary analyses of tooth genes support sequential loss of enamel and teeth in baleen whales (Mysticeti).

The loss of teeth and evolution of baleen racks in Mysticeti was a profound transformation that permitted baleen whales to radiate and diversify into a previously underutilized ecological niche of bulk filter-feeding on zooplankton and other small prey. Ancestral state reconstructions suggest that postnatal teeth were lost in the common ancestor of crown Mysticeti. Genomic studies provide some support for this hypothesis and suggest that the genetic toolkit for enamel production was inactivated in the common ancestor of living baleen whales. However, molecular studies to date have not provided direct evidence for the complete loss of teeth, including their dentin component, on the stem mysticete branch. Given these results, several questions remain unanswered: (1) Were teeth lost in a single step or did enamel loss precede dentin loss? (2) Was enamel lost early or late on the stem mysticete branch? (3) If enamel and dentin/tooth loss were decoupled in the ancestry of baleen whales, did dentin loss occur on the stem mysticete branch or independently in different crown mysticete lineages? To address these outstanding questions, we compiled and analyzed complete protein-coding sequences for nine tooth-related genes from cetaceans with available genome data. Seven of these genes are associated with enamel formation (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) whereas two other genes are either dentin-specific (DSPP) or tooth-specific (ODAPH) but not enamel-specific. Molecular evolutionary analyses indicate that all seven enamel-specific genes have inactivating mutations that are scattered across branches of the mysticete tree. Three of the enamel genes (ACP4, KLK4, MMP20) have inactivating mutations that are shared by all mysticetes. The two genes that are dentin-specific (DSPP) or tooth-specific (ODAPH) do not have any inactivating mutations that are shared by all mysticetes, but there are shared mutations in Balaenidae as well as in Plicogulae (Neobalaenidae + Balaenopteroidea). These shared mutations suggest that teeth were lost at most two times. Shared inactivating mutations and dN/dS analyses, in combination with cetacean divergence times, were used to estimate inactivation times of genes and by proxy enamel and tooth phenotypes at ancestral nodes. The results of these analyses are most compatible with a two-step model for the loss of teeth in the ancestry of living baleen whales: enamel was lost very early on the stem Mysticeti branch followed by the independent loss of dentin (and teeth) in the common ancestors of Balaenidae and Plicogulae, respectively. These results imply that some stem mysticetes, and even early crown mysticetes, may have had vestigial teeth comprised of dentin with no enamel. Our results also demonstrate that all odontocete species (in our study) with absent or degenerative enamel have inactivating mutations in one or more of their enamel genes.

Afrotheria.

Elephants and sea cows and tenrecs; hyraxes and aardvarks and sengis and golden moles. What do these very divergent and different looking mammals have in common? They are each other's closest living relatives, and all belong to the placental mammal clade Afrotheria ('African beasts'), which is one of the four major clades of placental mammals along with Xenarthra (anteaters, sloths, armadillos), Euarchontoglires (e.g. rodents, rabbits, primates), and Laurasiatheria (e.g. bats, carnivorans, odd-toed and even-toed ungulates) (Figure 1). Unlike many animal groups that were recognized and named long ago based on anatomical features, the Afrotheria emerged as a natural clade only in the 1990s when molecular techniques were applied to the problem of placental mammal classification. The recognition of Afrotheria represents a triumph of molecular phylogenetics and brings together a fantastically diverse assemblage of placental mammals with widely disparate ecological and morphological adaptations. Although Afrotheria was not previously proposed based on studies of anatomical characters, additional support for the monophyly of this clade comes from geography and the fossil record. Specifically, the six extant orders in Afrotheria share with each other early fossil representatives that are known from Africa or along the margins of the ancient Tethys Sea, hence Afrotheria.

Gene-tree misrooting drives conflicts in phylogenomic coalescent analyses of palaeognath birds.

Phylogenomic analyses of ancient rapid radiations can produce conflicting results that are driven by differential sampling of taxa and characters as well as the limitations of alternative analytical methods. We re-examine basal relationships of palaeognath birds (ratites and tinamous) using recently published datasets of nucleotide characters from 20,850 loci as well as 4301 retroelement insertions. The original studies attributed conflicting resolutions of rheas in their inferred coalescent and concatenation trees to concatenation failing in the anomaly zone. By contrast, we find that the coalescent-based resolution of rheas is premised upon extensive gene-tree estimation errors. Furthermore, retroelement insertions contain much more conflict than originally reported and multiple insertion loci support the basal position of rheas found in concatenation trees, while none were reported in the original publication. We demonstrate how even remarkable congruence in phylogenomic studies may be driven by long-branch misplacement of a divergent outgroup, highly incongruent gene trees, differential taxon sampling that can result in gene-tree misrooting errors that bias species-tree inference, and gross homology errors. What was previously interpreted as broad, robustly supported corroboration for a single resolution in coalescent analyses may instead indicate a common bias that taints phylogenomic results across multiple genome-scale datasets. The updated retroelement dataset now supports a species tree with branch lengths that suggest an ancient anomaly zone, and both concatenation and coalescent analyses of the huge nucleotide datasets fail to yield coherent, reliable results in this challenging phylogenetic context.

Theoretical and Practical Considerations when using Retroelement Insertions to Estimate Species Trees in the Anomaly Zone.

A potential shortcoming of concatenation methods for species tree estimation is their failure to account for incomplete lineage sorting. Coalescent methods address this problem but make various assumptions that, if violated, can result in worse performance than concatenation. Given the challenges of analyzing DNA sequences with both concatenation and coalescent methods, retroelement insertions (RIs) have emerged as powerful phylogenomic markers for species tree estimation. Here, we show that two recently proposed quartet-based methods, SDPquartets and ASTRAL_BP, are statistically consistent estimators of the unrooted species tree topology under the coalescent when RIs follow a neutral infinite-sites model of mutation and the expected number of new RIs per generation is constant across the species tree. The accuracy of these (and other) methods for inferring species trees from RIs has yet to be assessed on simulated data sets, where the true species tree topology is known. Therefore, we evaluated eight methods given RIs simulated from four model species trees, all of which have short branches and at least three of which are in the anomaly zone. In our simulation study, ASTRAL_BP and SDPquartets always recovered the correct species tree topology when given a sufficiently large number of RIs, as predicted. A distance-based method (ASTRID_BP) and Dollo parsimony also performed well in recovering the species tree topology. In contrast, unordered, polymorphism, and Camin-Sokal parsimony (as well as an approach based on MDC) typically fail to recover the correct species tree topology in anomaly zone situations with more than four ingroup taxa. Of the methods studied, only ASTRAL_BP automatically estimates internal branch lengths (in coalescent units) and support values (i.e., local posterior probabilities). We examined the accuracy of branch length estimation, finding that estimated lengths were accurate for short branches but upwardly biased otherwise. This led us to derive the maximum likelihood (branch length) estimate for when RIs are given as input instead of binary gene trees; this corrected formula produced accurate estimates of branch lengths in our simulation study provided that a sufficiently large number of RIs were given as input. Lastly, we evaluated the impact of data quantity on species tree estimation by repeating the above experiments with input sizes varying from 100 to 100,000 parsimony-informative RIs. We found that, when given just 1000 parsimony-informative RIs as input, ASTRAL_BP successfully reconstructed major clades (i.e., clades separated by branches $>0.3$ coalescent units) with high support and identified rapid radiations (i.e., shorter connected branches), although not their precise branching order. The local posterior probability was effective for controlling false positive branches in these scenarios. [Coalescence; incomplete lineage sorting; Laurasiatheria; Palaeognathae; parsimony; polymorphism parsimony; retroelement insertions; species trees; transposon.].

Speciation in the deep: genomics and morphology reveal a new species of beaked whale Mesoplodon eueu.

The deep sea has been described as the last major ecological frontier, as much of its biodiversity is yet to be discovered and described. Beaked whales (ziphiids) are among the most visible inhabitants of the deep sea, due to their large size and worldwide distribution, and their taxonomic diversity and much about their natural history remain poorly understood. We combine genomic and morphometric analyses to reveal a new Southern Hemisphere ziphiid species, Ramari's beaked whale, Mesoplodon eueu, whose name is linked to the Indigenous peoples of the lands from which the species holotype and paratypes were recovered. Mitogenome and ddRAD-derived phylogenies demonstrate reciprocally monophyletic divergence between M. eueu and True's beaked whale (M. mirus) from the North Atlantic, with which it was previously subsumed. Morphometric analyses of skulls also distinguish the two species. A time-calibrated mitogenome phylogeny and analysis of two nuclear genomes indicate divergence began circa 2 million years ago (Ma), with geneflow ceasing 0.35-0.55 Ma. This is an example of how deep sea biodiversity can be unravelled through increasing international collaboration and genome sequencing of archival specimens. Our consultation and involvement with Indigenous peoples offers a model for broadening the cultural scope of the scientific naming process.

The effects of fossil taxa, hypothetical predicted ancestors, and a molecular scaffold on pseudoextinction analyses of extant placental orders.

Pseudoextinction analyses, which simulate extinction in extant taxa, use molecular phylogenetics to assess the accuracy of morphological phylogenetics. Previous pseudoextinction analyses have shown a failure of morphological phylogenetics to place some individual placental orders in the correct superordinal clade. Recent work suggests that the inclusion of hypothetical ancestors of extant placental clades, estimated by ancestral state reconstructions of morphological characters, may increase the accuracy of morphological phylogenetic analyses. However, these studies reconstructed direct hypothetical ancestors for each extant taxon based on a well-corroborated molecular phylogeny, which is not possible for extinct taxa that lack molecular data. It remains to be determined if pseudoextinct taxa, and by proxy extinct taxa, can be accurately placed when their immediate hypothetical ancestors are unknown. To investigate this, we employed molecular scaffolds with the largest available morphological data set for placental mammals. Each placental order was sequentially treated as pseudoextinct by exempting it from the molecular scaffold and recoding soft morphological characters as missing for all its constituent species. For each pseudoextinct data set, we omitted the pseudoextinct taxon and performed a parsimony ancestral state reconstruction to obtain hypothetical predicted ancestors. Each pseudoextinct order was then evaluated in seven parsimony analyses that employed combinations of fossil taxa, hypothetical predicted ancestors, and a molecular scaffold. In treatments that included fossils, hypothetical predicted ancestors, and a molecular scaffold, only 8 of 19 pseudoextinct placental orders (42%) retained the same interordinal placement as on the molecular scaffold. In treatments that included hypothetical predicted ancestors but not fossils or a scaffold, only four placental orders (21%) were recovered in positions that are congruent with the scaffold. These results indicate that hypothetical predicted ancestors do not increase the accuracy of pseudoextinct taxon placement when the immediate hypothetical ancestor of the taxon is unknown. Hypothetical predicted ancestors are not a panacea for morphological phylogenetics.

Myoglobin primary structure reveals multiple convergent transitions to semi-aquatic life in the world's smallest mammalian divers.

The speciose mammalian order Eulipotyphla (moles, shrews, hedgehogs, solenodons) combines an unusual diversity of semi-aquatic, semi-fossorial, and fossorial forms that arose from terrestrial forbearers. However, our understanding of the ecomorphological pathways leading to these lifestyles has been confounded by a fragmentary fossil record, unresolved phylogenetic relationships, and potential morphological convergence, calling for novel approaches. The net surface charge of the oxygen-storing muscle protein myoglobin (ZMb), which can be readily determined from its primary structure, provides an objective target to address this question due to mechanistic linkages with myoglobin concentration. Here, we generate a comprehensive 71 species molecular phylogeny that resolves previously intractable intra-family relationships and then ancestrally reconstruct ZMb evolution to identify ancient lifestyle transitions based on protein sequence alone. Our phylogenetically informed analyses confidently resolve fossorial habits having evolved twice in talpid moles and reveal five independent secondary aquatic transitions in the order housing the world's smallest endothermic divers.

The shrews, moles and hedgehogs that surround us all belong to the same large group of insect-eating mammals. While most members in this 'Eulipotyphla order' trot on land, some, like moles, have evolved to hunt their prey underground. A few species, such as the water shrews, have even ventured to adopt a semi-aquatic lifestyle, diving into ponds and streams to retrieve insects. These underwater foragers share unique challenges, burning a lot of energy and losing heat at a high rate while not being able to store much oxygen. It is still unclear how these semi-aquatic habits have come to be: the fossil record is fragmented and several species tend to display the same adaptations even though they have evolved separately. This makes it difficult to identify when and how many times the Eulipotyphla species started to inhabit water. The protein myoglobin, which gives muscles their red color, could help in this effort. This molecule helps muscles to capture oxygen from blood, a necessary step for cells to obtain energy. Penguins, seals and whales, which dive to get their food, often have much higher concentration of myoglobin so they can spend extended amount of time without having to surface for air. In addition, previous work has shown that eight groups of mammalian divers carry genetic changes that help newly synthetized myoglobin proteins to not stick to each other. This means that these animals can store more of the molecule in their muscles, increasing their oxygen intake and delivery. He et al. therefore speculated that all semi-aquatic Eulipotyphla species would carry genetic changes that made their myoglobin less likely to clump together; underground species, which also benefit from absorbing more oxygen, would display intermediate alterations. In addition, reconstructing the myoglobin sequences from the ancestors of living species would help to spot when the transition to aquatic life took place. A variety of approaches were harnessed to obtain myoglobin and other sequences from 55 eulipotyphlan mammals, which then were used to construct a strongly supported family tree for this group. The myoglobin results revealed that from terrestrial to subterranean to semi-aquatic species, genetic changes took place that would diminish the ability for the proteins to stick to each other. This pattern also showed that semi-aquatic lifestyles have independently evolved five separate times ­ twice in moles, three times in shrews. By retracing the evolutionary history of specific myoglobin properties, He et al. shed light on how one of the largest orders of mammals has come to be fantastically diverse.

Genomic and anatomical comparisons of skin support independent adaptation to life in water by cetaceans and hippos.

The macroevolutionary transition from terra firma to obligatory inhabitance of the marine hydrosphere has occurred twice in the history of Mammalia: Cetacea and Sirenia. In the case of Cetacea (whales, dolphins, and porpoises), molecular phylogenies provide unambiguous evidence that fully aquatic cetaceans and semiaquatic hippopotamids (hippos) are each other's closest living relatives. Ancestral reconstructions suggest that some adaptations to the aquatic realm evolved in the common ancestor of Cetancodonta (Cetacea + Hippopotamidae). An alternative hypothesis is that these adaptations evolved independently in cetaceans and hippos. Here, we focus on the integumentary system and evaluate these hypotheses by integrating new histological data for cetaceans and hippos, the first genome-scale data for pygmy hippopotamus, and comprehensive genomic screens and molecular evolutionary analyses for protein-coding genes that have been inactivated in hippos and cetaceans. We identified eight skin-related genes that are inactivated in both cetaceans and hippos, including genes that are related to sebaceous glands, hair follicles, and epidermal differentiation. However, none of these genes exhibit inactivating mutations that are shared by cetaceans and hippos. Mean dates for the inactivation of skin genes in these two clades serve as proxies for phenotypic changes and suggest that hair reduction/loss, the loss of sebaceous glands, and changes to the keratinization program occurred ∼16 Ma earlier in cetaceans (∼46.5 Ma) than in hippos (∼30.5 Ma). These results, together with histological differences in the integument and prior analyses of oxygen isotopes from stem hippopotamids ("anthracotheres"), support the hypothesis that aquatic skin adaptations evolved independently in hippos and cetaceans.

Genomic evidence for the parallel regression of melatonin synthesis and signaling pathways in placental mammals.

Background: The study of regressive evolution has yielded a wealth of examples where the underlying genes bear molecular signatures of trait degradation, such as pseudogenization or deletion. Typically, it appears that such disrupted genes are limited to the function of the regressed trait, whereas pleiotropic genes tend to be maintained by natural selection to support their myriad purposes. One such set of pleiotropic genes is involved in the synthesis ( AANAT, ASMT) and signaling ( MTNR1A, MTNR1B) of melatonin, a hormone secreted by the vertebrate pineal gland. Melatonin provides a signal of environmental darkness, thereby influencing the circadian and circannual rhythmicity of numerous physiological traits. Therefore, the complete loss of a pineal gland and the underlying melatonin pathway genes seems likely to be maladaptive, unless compensated by extrapineal sources of melatonin. Methods: We examined AANAT, ASMT, MTNR1A and MTNR1B in 123 vertebrate species, including pineal-less placental mammals and crocodylians. We searched for inactivating mutations and modelled selective pressures (dN/dS) to test whether the genes remain functionally intact. Results: We report that crocodylians retain intact melatonin genes and express AANAT and ASMT in their eyes, whereas all four genes have been repeatedly inactivated in the pineal-less xenarthrans, pangolins, sirenians, and whales. Furthermore, colugos have lost these genes, and several lineages of subterranean mammals have partial melatonin pathway dysfunction. These results are supported by the presence of shared inactivating mutations across clades and analyses of selection pressure based on the ratio of non-synonymous to synonymous substitutions (dN/dS), suggesting extended periods of relaxed selection on these genes. Conclusions: The losses of melatonin synthesis and signaling date to tens of millions of years ago in several lineages of placental mammals, raising questions about the evolutionary resilience of pleiotropic genes, and the causes and consequences of losing melatonin pathways in these species.

Phylogenomics and the Genetic Architecture of the Placental Mammal Radiation.

The genomes of placental mammals are being sequenced at an unprecedented rate. Alignments of hundreds, and one day thousands, of genomes spanning the rich living and extinct diversity of species offer unparalleled power to resolve phylogenetic controversies, identify genomic innovations of adaptation, and dissect the genetic architecture of reproductive isolation. We highlight outstanding questions about the earliest phases of placental mammal diversification and the promise of newer methods, as well as remaining challenges, toward using whole genome data to resolve placental mammal phylogeny. The next phase of mammalian comparative genomics will see the completion and application of finished-quality, gapless genome assemblies from many ordinal lineages and closely related species. Interspecific comparisons between the most hypervariable genomic loci will likely reveal large, but heretofore mostly underappreciated, effects on population divergence, morphological innovation, and the origin of new species.

ILS-Aware Analysis of Low-Homoplasy Retroelement Insertions: Inference of Species Trees and Introgression Using Quartets.

DNA sequence alignments have provided the majority of data for inferring phylogenetic relationships with both concatenation and coalescent methods. However, DNA sequences are susceptible to extensive homoplasy, especially for deep divergences in the Tree of Life. Retroelement insertions have emerged as a powerful alternative to sequences for deciphering evolutionary relationships because these data are nearly homoplasy-free. In addition, retroelement insertions satisfy the "no intralocus-recombination" assumption of summary coalescent methods because they are singular events and better approximate neutrality relative to DNA loci commonly sampled in phylogenomic studies. Retroelements have traditionally been analyzed with parsimony, distance, and network methods. Here, we analyze retroelement data sets for vertebrate clades (Placentalia, Laurasiatheria, Balaenopteroidea, Palaeognathae) with 2 ILS-aware methods that operate by extracting, weighting, and then assembling unrooted quartets into a species tree. The first approach constructs a species tree from retroelement bipartitions with ASTRAL, and the second method is based on split-decomposition with parsimony. We also develop a Quartet-Asymmetry test to detect hybridization using retroelements. Both ILS-aware methods recovered the same species-tree topology for each data set. The ASTRAL species trees for Laurasiatheria have consecutive short branch lengths in the anomaly zone whereas Palaeognathae is outside of this zone. For the Balaenopteroidea data set, which includes rorquals (Balaenopteridae) and gray whale (Eschrichtiidae), both ILS-aware methods resolved balaeonopterids as paraphyletic. Application of the Quartet-Asymmetry test to this data set detected 19 different quartets of species for which historical introgression may be inferred. Evidence for introgression was not detected in the other data sets.

Evolutionary Models for the Diversification of Placental Mammals Across the KPg Boundary.

Deciphering the timing of the placental mammal radiation is a longstanding problem in evolutionary biology, but consensus on the tempo and mode of placental diversification remains elusive. Nevertheless, an accurate timetree is essential for understanding the role of important events in Earth history (e.g., Cretaceous Terrestrial Revolution, KPg mass extinction) in promoting the taxonomic and ecomorphological diversification of Placentalia. Archibald and Deutschman described three competing models for the diversification of placental mammals, which are the Explosive, Long Fuse, and Short Fuse Models. More recently, the Soft Explosive Model and Trans-KPg Model have emerged as additional hypotheses for the placental radiation. Here, we review molecular and paleontological evidence for each of these five models including the identification of general problems that can negatively impact divergence time estimates. The Long Fuse Model has received more support from relaxed clock studies than any of the other models, but this model is not supported by morphological cladistic studies that position Cretaceous eutherians outside of crown Placentalia. At the same time, morphological cladistics has a poor track record of reconstructing higher-level relationships among the orders of placental mammals including the results of new pseudoextinction analyses that we performed on the largest available morphological data set for mammals (4,541 characters). We also examine the strengths and weaknesses of different timetree methods (node dating, tip dating, and fossilized birth-death dating) that may now be applied to estimate the timing of the placental radiation. While new methods such as tip dating are promising, they also have problems that must be addressed if these methods are to effectively discriminate among competing hypotheses for placental diversification. Finally, we discuss the complexities of timetree estimation when the signal of speciation times is impacted by incomplete lineage sorting (ILS) and hybridization. Not accounting for ILS results in dates that are older than speciation events. Hybridization, in turn, can result in dates than are younger or older than speciation dates. Disregarding this potential variation in "gene" history across the genome can distort phylogenetic branch lengths and divergence estimates when multiple unlinked genomic loci are combined together in a timetree analysis.

Genes lost during the transition from land to water in cetaceans highlight genomic changes associated with aquatic adaptations.

The transition from land to water in whales and dolphins (cetaceans) was accompanied by remarkable adaptations. To reveal genomic changes that occurred during this transition, we screened for protein-coding genes that were inactivated in the ancestral cetacean lineage. We found 85 gene losses. Some of these were likely beneficial for cetaceans, for example, by reducing the risk of thrombus formation during diving (F12 and KLKB1), erroneous DNA damage repair (POLM), and oxidative stress-induced lung inflammation (MAP3K19). Additional gene losses may reflect other diving-related adaptations, such as enhanced vasoconstriction during the diving response (mediated by SLC6A18) and altered pulmonary surfactant composition (SEC14L3), while loss of SLC4A9 relates to a reduced need for saliva. Last, loss of melatonin synthesis and receptor genes (AANAT, ASMT, and MTNR1A/B) may have been a precondition for adopting unihemispheric sleep. Our findings suggest that some genes lost in ancestral cetaceans were likely involved in adapting to a fully aquatic lifestyle.

How conflict shapes evolution in poeciliid fishes.

In live-bearing animal lineages, the evolution of the placenta is predicted to create an arena for genomic conflict during pregnancy, drive patterns of male sexual selection, and increase the rate of speciation. Here we test these predictions of the viviparity driven conflict hypothesis (VDCH) in live-bearing poecilid fishes, a group showing multiple independent origins of placentation and extreme variation in male sexually selected traits. As predicted, male sexually selected traits are only gained in lineages that lack placentas; while there is little or no influence of male traits on the evolution of placentas. Both results are consistent with the mode of female provisioning governing the evolution of male attributes. Moreover, it is the presence of male sexually selected traits (pre-copulatory), rather than placentation (post-copulatory), that are associated with higher rates of speciation. These results highlight a causal interaction between female reproductive mode, male sexual selection and the rate of speciation, suggesting a role for conflict in shaping diverse aspects of organismal biology.