Tracking Five Millennia of Horse Management with Extensive Ancient Genome Time Series.

Horse domestication revolutionized warfare and accelerated travel, trade, and the geographic expansion of languages. Here, we present the largest DNA time series for a non-human organism to date, including genome-scale data from 149 ancient animals and 129 ancient genomes (≥1-fold coverage), 87 of which are new. This extensive dataset allows us to assess the modern legacy of past equestrian civilizations. We find that two extinct horse lineages existed during early domestication, one at the far western (Iberia) and the other at the far eastern range (Siberia) of Eurasia. None of these contributed significantly to modern diversity. We show that the influence of Persian-related horse lineages increased following the Islamic conquests in Europe and Asia. Multiple alleles associated with elite-racing, including at the MSTN "speed gene," only rose in popularity within the last millennium. Finally, the development of modern breeding impacted genetic diversity more dramatically than the previous millennia of human management.

Million-year-old DNA sheds light on the genomic history of mammoths.

Temporal genomic data hold great potential for studying evolutionary processes such as speciation. However, sampling across speciation events would, in many cases, require genomic time series that stretch well back into the Early Pleistocene subepoch. Although theoretical models suggest that DNA should survive on this timescale1, the oldest genomic data recovered so far are from a horse specimen dated to 780-560 thousand years ago2. Here we report the recovery of genome-wide data from three mammoth specimens dating to the Early and Middle Pleistocene subepochs, two of which are more than one million years old. We find that two distinct mammoth lineages were present in eastern Siberia during the Early Pleistocene. One of these lineages gave rise to the woolly mammoth and the other represents a previously unrecognized lineage that was ancestral to the first mammoths to colonize North America. Our analyses reveal that the Columbian mammoth of North America traces its ancestry to a Middle Pleistocene hybridization between these two lineages, with roughly equal admixture proportions. Finally, we show that the majority of protein-coding changes associated with cold adaptation in woolly mammoths were already present one million years ago. These findings highlight the potential of deep-time palaeogenomics to expand our understanding of speciation and long-term adaptive evolution.

Complex patterns of gene flow and convergence in the evolutionary history of the spiral-horned antelopes (Tragelaphini).

The Tragelaphini, also known as spiral-horned antelope, is a phenotypically diverse mammalian tribe comprising a single genus, Tragelaphus. The evolutionary history of this tribe has attracted the attention of taxonomists and molecular geneticists for decades because its diversity is characterised by conflicts between morphological and molecular data as well as between mitochondrial, nuclear and chromosomal DNA. These inconsistencies point to a complex history of ecological diversification, coupled by either phenotypic convergence or introgression. Therefore, to unravel the phylogenetic relationships among spiral-horned antelopes, and to further investigate the role of divergence and gene flow in trait evolution, we sequenced genomes for all nine accepted species of the genus Tragelaphus, including a genome each for the highly divergent bushbuck lineages (T. s. scriptus and T. s. sylvaticus). We successfully reconstructed the Tragelaphus species tree, providing genome-level support for the early Pliocene divergence and monophyly of the nyala (T. angasii) and lesser kudu (T. imberbis), the monophyly of the two eland species (T. oryx and T. derbianus) and, importantly, the monophyly of kéwel (T. s. scriptus) and imbabala (T. s. sylvaticus) bushbuck. We found strong evidence for gene flow in at least four of eight nodes on the species tree. Among the six phenotypic traits assessed here, only habitat type mapped onto the species tree without homoplasy, showing that trait evolution was the result of complex patterns of divergence, introgression and convergent evolution.

Wide-spread dispersal in a deep-sea brooding polychaete: the role of natural history collections in assessing the distribution in quill worms (Onuphidae, Annelida).

BACKGROUND: Modern integrative taxonomy-based annelid species descriptions are detailed combining morphological data and, since the last decades, also molecular information. Historic species descriptions are often comparatively brief lacking such detail. Adoptions of species names from western literature in the past led to the assumption of cosmopolitan ranges for many species, which, in many cases, were later found to include cryptic or pseudocryptic lineages with subtle morphological differences. Natural history collections and databases can aid in assessing the geographic ranges of species but depend on correct species identification. Obtaining DNA sequence information from wet-collection museum specimens of marine annelids is often impeded by the use of  formaldehyde and/or long-term storage in ethanol resulting in DNA degradation and cross-linking. RESULTS: The application of ancient DNA extraction methodology in combination with single-stranded DNA library preparation and target gene capture resulted in successful sequencing of a 110-year-old collection specimen of quill worms. Furthermore, a 40-year-old specimen of quill worms was successfully sequenced using a standard extraction protocol for modern samples, PCR and Sanger sequencing. Our study presents the first molecular analysis of Hyalinoecia species including the previously known species Hyalinoecia robusta, H. tubicloa, H. artifex, and H. longibranchiata, and a potentially undescribed species from equatorial western Africa morphologically indistinguishable from H. tubicola. The study also investigates the distribution of these five Hyalinoecia species. Reassessing the distribution of H. robusta reveals a geographical range covering both the Atlantic and the Indian Oceans as indicated by molecular data obtained from recent and historical specimens. CONCLUSION: Our results represent an example of a very wide geographical distribution of a brooding deep-sea annelid with a complex reproduction strategy and seemingly very limited dispersal capacity of its offspring, and highlights the importance of molecular information from museum specimens for integrative annelid taxonomy and biogeography.

Blue Turns to Gray: Paleogenomic Insights into the Evolutionary History and Extinction of the Blue Antelope (Hippotragus leucophaeus).

The blue antelope (Hippotragus leucophaeus) is the only large African mammal species to have become extinct in historical times, yet no nuclear genomic information is available for this species. A recent study showed that many alleged blue antelope museum specimens are either roan (Hippotragus equinus) or sable (Hippotragus niger) antelopes, further reducing the possibilities for obtaining genomic information for this extinct species. While the blue antelope has a rich fossil record from South Africa, climatic conditions in the region are generally unfavorable to the preservation of ancient DNA. Nevertheless, we recovered two blue antelope draft genomes, one at 3.4× mean coverage from a historical specimen (∼200 years old) and one at 2.1× mean coverage from a fossil specimen dating to 9,800-9,300 cal years BP, making it currently the oldest paleogenome from Africa. Phylogenomic analyses show that blue and sable antelope are sister species, confirming previous mitogenomic results, and demonstrate ancient gene flow from roan into blue antelope. We show that blue antelope genomic diversity was much lower than in roan and sable antelope, indicative of a low population size since at least the early Holocene. This supports observations from the fossil record documenting major decreases in the abundance of blue antelope after the Pleistocene-Holocene transition. Finally, the persistence of this species throughout the Holocene despite low population size suggests that colonial-era human impact was likely the decisive factor in the blue antelope's extinction.

Genome Sequencing of up to 6,000-Year-Old Citrullus Seeds Reveals Use of a Bitter-Fleshed Species Prior to Watermelon Domestication.

Iconographic evidence from Egypt suggests that watermelon pulp was consumed there as a dessert by 4,360 BP. Earlier archaeobotanical evidence comes from seeds from Neolithic settlements in Libya, but whether these were watermelons with sweet pulp or other forms is unknown. We generated genome sequences from 6,000- and 3,300-year-old seeds from Libya and Sudan, and from worldwide herbarium collections made between 1824 and 2019, and analyzed these data together with resequenced genomes from important germplasm collections for a total of 131 accessions. Phylogenomic and population-genomic analyses reveal that (1) much of the nuclear genome of both ancient seeds is traceable to West African seed-use "egusi-type" watermelon (Citrullus mucosospermus) rather than domesticated pulp-use watermelon (Citrullus lanatus ssp. vulgaris); (2) the 6,000-year-old watermelon likely had bitter pulp and greenish-white flesh as today found in C. mucosospermus, given alleles in the bitterness regulators ClBT and in the red color marker LYCB; and (3) both ancient genomes showed admixture from C. mucosospermus, C. lanatus ssp. cordophanus, C. lanatus ssp. vulgaris, and even South African Citrullus amarus, and evident introgression between the Libyan seed (UMB-6) and populations of C. lanatus. An unexpected new insight is that Citrullus appears to have initially been collected or cultivated for its seeds, not its flesh, consistent with seed damage patterns induced by human teeth in the oldest Libyan material.

Is it inappropriate to ask for your age? Evaluating parameter impact on tree dating in a challenging clade (Macroscelidea).

Sengis (order Macroscelidea) are small mammals endemic to Africa. The taxonomy and phylogeny of sengis has been difficult to resolve due to a lack of clear morphological apomorphies. Molecular phylogenies have already significantly revised sengi systematics, but until now no molecular phylogeny has included all 20 extant species. In addition, the age of origin of the sengi crown clade and the divergence age of its two extant families remain unclear. Two recently published studies based on different datasets and age-calibration parameters (DNA type, outgroup selection, fossil calibration points) proposed highly different divergent age estimates and evolutionary scenarios. We obtained nuclear and mitochondrial DNA from mainly museum specimens using target enrichment of single-stranded DNA libraries to generate the first phylogeny of all extant macroscelidean species. We then explored the effects of different parameters (type of DNA, ratio of ingroup to outgroup sampling, number and type of fossil calibration points) and their resulting impacts on age estimates for the origin and initial diversification of Macroscelidea. We show that, even after correcting for substitution saturation, both using mitochondrial DNA in conjunction with nuclear DNA or alone results in much older ages and different branch lengths than when using nuclear DNA alone. We further show that the former effect can be attributed to insufficient amounts of nuclear data. If multiple calibration points are included, the age of the sengi crown group fossil prior has minimal impact on the estimated time frame of sengi evolution. In contrast, the inclusion or exclusion of outgroup fossil priors has a major effect on the resulting node ages. We also find that a reduced sampling of ingroup species does not significantly affect overall age estimates and that terminal specific substitution rates can serve as a means to evaluate the biological likeliness of the produced temporal estimates. Our study demonstrates how commonly varied parameters in temporal calibration of phylogenies affect age estimates. Dated phylogenies should therefore always be seen in the context of the dataset which was used to produce them.

A genetic glimpse of the Chinese straight-tusked elephants.

Straight-tusked elephants (genus: Palaeoloxodon) including their island dwarf forms are extinct enigmatic members of the Pleistocene megafauna and the most common Pleistocene elephants after the mammoths. Their taxonomic placement has been revised several times. Using palaeogenomic evidence, previous studies suggested that the European P. antiquus has a hybrid origin, but no molecular data have been retrieved from their Asian counterparts, leaving a gap in our knowledge of the global phylogeography and population dynamics of Palaeoloxodon. Here, we captured a high-quality complete mitogenome from a Pleistocene Elephantidae molar (CADG841) from Northern China, which was previously morphologically assigned to the genus Elephas (Asian elephant), and partial mitochondrial sequences (838 bp) of another Palaeoloxodon sp. specimen (CADG1074) from Northeastern China. We found that both Chinese specimens cluster with a 244 000-year-old P. antiquus (specimen name: WE) from Western Europe, suggesting that this clade may represent a population with a large spatial span across Eurasia. Based on the fossil record and the molecular dating of both the divergences of different Palaeoloxodon mitochondrial clades and previously determined hybridization events, we propose that this Eurasian-wide WE clade provides evidence for an earlier migration and/or another hybridization event that happened in the evolutionary history of straight-tusked elephants.

Large variance in inbreeding within the Iberian wolf population.

The gray wolf (Canis lupus) population on the Iberian Peninsula was the largest in western and central Europe during most of the 20th century, with its size apparently never under a few hundred individuals. After partial legal protection in the 1970s in Spain, the northwest Iberian population increased to about 300-350 packs and then stabilized. In contrast to many current European wolf populations, which have been connected through gene flow, the Iberian wolf population has been isolated for decades. Here we measured changes on genomic diversity and inbreeding through the last decades in a geographic context. We find that the level of genomic diversity in Iberian wolves is low compared to other Eurasian wolf populations. Despite population expansion in the last 50 years, some modern wolves had very high inbreeding, especially in the recently recolonized and historical edge areas. These individuals contrast with others with low inbreeding within the same population. The high variance in inbreeding despite population expansion seems associated with small-scale fragmentation of the range that is revealed by the genetic similarity between modern and historical samples from close localities despite being separated by decades, remaining differentiated from other individuals that are just over 100 km away, a small distance for a species with great dispersal capacity inhabiting a continuous range. This illustrates that, despite its demographically stable condition, the population would probably benefit from favoring connectivity within the population as well as genetic exchange with other European wolf populations to avoid excessive fragmentation and local inbreeding depression.

Ecological Specialization and Evolutionary Reticulation in Extant Hyaenidae.

During the Miocene, Hyaenidae was a highly diverse family of Carnivora that has since been severely reduced to four species: the bone-cracking spotted, striped, and brown hyenas, and the specialized insectivorous aardwolf. Previous studies investigated the evolutionary histories of the spotted and brown hyenas, but little is known about the remaining two species. Moreover, the genomic underpinnings of scavenging and insectivory, defining traits of the extant species, remain elusive. Here, we generated an aardwolf genome and analyzed it together with the remaining three species to reveal their evolutionary relationships, genomic underpinnings of their scavenging and insectivorous lifestyles, and their respective genetic diversities and demographic histories. High levels of phylogenetic discordance suggest gene flow between the aardwolf lineage and the ancestral brown/striped hyena lineage. Genes related to immunity and digestion in the bone-cracking hyenas and craniofacial development in the aardwolf showed the strongest signals of selection, suggesting putative key adaptations to carrion and termite feeding, respectively. A family-wide expansion in olfactory receptor genes suggests that an acute sense of smell was a key early adaptation. Finally, we report very low levels of genetic diversity within the brown and striped hyenas despite no signs of inbreeding, putatively linked to their similarly slow decline in effective population size over the last ∼2 million years. High levels of genetic diversity and more stable population sizes through time are seen in the spotted hyena and aardwolf. Taken together, our findings highlight how ecological specialization can impact the evolutionary history, demographics, and adaptive genetic changes of an evolutionary lineage.

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication.

The date palm, Phoenix dactylifera, has been a cornerstone of Middle Eastern and North African agriculture for millennia. It was first domesticated in the Persian Gulf, and its evolution appears to have been influenced by gene flow from two wild relatives, P. theophrasti, currently restricted to Crete and Turkey, and P. sylvestris, widespread from Bangladesh to the West Himalayas. Genomes of ancient date palm seeds show that gene flow from P. theophrasti to P. dactylifera may have occurred by ∼2,200 years ago, but traces of P. sylvestris could not be detected. We here integrate archeogenomics of a ∼2,100-year-old P. dactylifera leaf from Saqqara (Egypt), molecular-clock dating, and coalescence approaches with population genomic tests, to probe the hybridization between the date palm and its two closest relatives and provide minimum and maximum timestamps for its reticulated evolution. The Saqqara date palm shares a close genetic affinity with North African date palm populations, and we find clear genomic admixture from both P. theophrasti, and P. sylvestris, indicating that both had contributed to the date palm genome by 2,100 years ago. Molecular-clocks placed the divergence of P. theophrasti from P. dactylifera/P. sylvestris and that of P. dactylifera from P. sylvestris in the Upper Miocene, but strongly supported, conflicting topologies point to older gene flow between P. theophrasti and P. dactylifera, and P. sylvestris and P. dactylifera. Our work highlights the ancient hybrid origin of the date palms, and prompts the investigation of the functional significance of genetic material introgressed from both close relatives, which in turn could prove useful for modern date palm breeding.

Ancient mitochondrial genomes from Chinese cave hyenas provide insights into the evolutionary history of the genus Crocuta.

Cave hyenas (genus Crocuta) are extinct bone-cracking carnivores from the family Hyaenidae and are generally split into two taxa that correspond to a European/Eurasian and an (East) Asian lineage. They are close relatives of the extant African spotted hyenas, the only extant member of the genus Crocuta. Cave hyenas inhabited a wide range across Eurasia during the Pleistocene, but became extinct at the end of the Late Pleistocene. Using genetic and genomic datasets, previous studies have proposed different scenarios about the evolutionary history of Crocuta. However, causes of the extinction of cave hyenas are widely speculative and samples from China are severely understudied. In this study, we assembled near-complete mitochondrial genomes from two cave hyenas from northeastern China dating to 20 240 and 20 253 calBP, representing the youngest directly dated fossils of Crocuta in Asia. Phylogenetic analyses suggest a monophyletic clade of these two samples within a deeply diverging mitochondrial haplogroup of Crocuta. Bayesian analyses suggest that the split of this Asian cave hyena mitochondrial lineage from their European and African relatives occurred approximately 1.85 Ma (95% CI 1.62-2.09 Ma), which is broadly concordant with the earliest Eurasian Crocuta fossil dating to approximately 2 Ma. Comparisons of mean genetic distance indicate that cave hyenas harboured higher genetic diversity than extant spotted hyenas, brown hyenas and aardwolves, but this is probably at least partially due to the fact that their mitochondrial lineages do not represent a monophyletic group, although this is also true for extant spotted hyenas. Moreover, the joint female effective population size of Crocuta (both cave hyenas and extant spotted hyenas) has sustained two declines during the Late Pleistocene. Combining this mitochondrial phylogeny, previous nuclear findings and fossil records, we discuss the possible relationship of fossil Crocuta in China and the extinction of cave hyenas.

A sliver of the past: The decimation of the genetic diversity of the Mexican wolf.

The endangered Mexican wolf (Canis lupus baileyi) is known to carry exceedingly low levels of genetic diversity. This could be (i) the result of long-term evolutionary patterns as they exist at the southernmost limit of the species distribution at a relatively reduced effective size, or (ii) due to rapid population decline caused by human persecution over the last century. If the former, purifying selection is expected to have minimized the impact of inbreeding. If the latter, rapid and recent declines in genetic diversity may have resulted in severe fitness consequences. To differentiate these hypotheses, we conducted comparative whole-genome analyses of five historical Mexican wolves (1907-1917) and 18 contemporary Mexican and grey wolves from North America and Eurasia. Based on whole-genome data, historical and modern Mexican wolves together form a discrete unit. Moreover, we found that modern Mexican wolves have reduced genetic diversity and increased inbreeding relative to the historical population, which was widespread across the southwestern United States and not restricted to Mexico as previously assumed. Finally, although Mexican wolves have evolved in sympatry with coyotes (C. latrans), we observed lower introgression between historical Mexican wolves and coyotes than with modern Mexican wolves, despite similarities in body size. Taken together, our data show that recent population declines probably caused the reduced level of genetic diversity, but not the observed differentiation of the Mexican wolves from other North American wolves.

Phylotranscriptomic evidence for pervasive ancient hybridization among Old World salamanders.

Hybridization can leave genealogical signatures in an organism's genome, originating from the parental lineages and persisting over time. This potentially confounds phylogenetic inference methods that aim to represent evolution as a strictly bifurcating tree. We apply a phylotranscriptomic approach to study the evolutionary history of, and test for inter-lineage introgression in the Salamandridae, a Holarctic salamanders group of interest in studies of toxicity and aposematism, courtship behavior, and molecular evolution. Although the relationships between the 21 currently recognized salamandrid genera have been the subject of numerous molecular phylogenetic studies, some branches have remained controversial and sometimes affected by discordances between mitochondrial vs. nuclear trees. To resolve the phylogeny of this family, and understand the source of mito-nuclear discordance, we generated new transcriptomic (RNAseq) data for 20 salamandrids and used these along with published data, including 28 mitochondrial genomes, to obtain a comprehensive nuclear and mitochondrial perspective on salamandrid evolution. Our final phylotranscriptomic data set included 5455 gene alignments for 40 species representing 17 of the 21 salamandrid genera. Using concatenation and species-tree phylogenetic methods, we find (1) Salamandrina sister to the clade of the "True Salamanders" (consisting of Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), (2) Ichthyosaura sister to the Near Eastern genera Neurergus and Ommatotriton, (3) Triturus sister to Lissotriton, and (4) Cynops paraphyletic with respect to Paramesotriton and Pachytriton. Combining introgression tests and phylogenetic networks, we find evidence for introgression among taxa within the clades of "Modern Asian Newts" and "Modern European Newts". However, we could not unambiguously identify the number, position, and direction of introgressive events. Combining evidence from nuclear gene analysis with the observed mito-nuclear phylogenetic discordances, we hypothesize a scenario with hybridization and mitochondrial capture among ancestral lineages of (1) Lissotriton into Ichthyosaura and (2) Triturus into Calotriton, plus introgression of nuclear genes from Triturus into Lissotriton. Furthermore, both mitochondrial capture and nuclear introgression may have occurred among lineages assigned to Cynops. More comprehensive genomic data will, in the future, allow testing this against alternative scenarios involving hybridization with other, extinct lineages of newts.

Ancient proteins resolve the evolutionary history of Darwin's South American ungulates.

No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin, who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago, or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen α1- and α2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies. Toxodon and Macrauchenia form a monophyletic group whose sister taxon is not Afrotheria or any of its constituent clades as recently claimed, but instead crown Perissodactyla (horses, tapirs, and rhinoceroses). These results are consistent with the origin of at least some South American native ungulates from 'condylarths', a paraphyletic assembly of archaic placentals. With ongoing improvements in instrumentation and analytical procedures, proteomics may produce a revolution in systematics such as that achieved by genomics, but with the possibility of reaching much further back in time.

A comprehensive genomic history of extinct and living elephants.

Elephantids are the world's most iconic megafaunal family, yet there is no comprehensive genomic assessment of their relationships. We report a total of 14 genomes, including 2 from the American mastodon, which is an extinct elephantid relative, and 12 spanning all three extant and three extinct elephantid species including an ∼120,000-y-old straight-tusked elephant, a Columbian mammoth, and woolly mammoths. Earlier genetic studies modeled elephantid evolution via simple bifurcating trees, but here we show that interspecies hybridization has been a recurrent feature of elephantid evolution. We found that the genetic makeup of the straight-tusked elephant, previously placed as a sister group to African forest elephants based on lower coverage data, in fact comprises three major components. Most of the straight-tusked elephant's ancestry derives from a lineage related to the ancestor of African elephants while its remaining ancestry consists of a large contribution from a lineage related to forest elephants and another related to mammoths. Columbian and woolly mammoths also showed evidence of interbreeding, likely following a latitudinal cline across North America. While hybridization events have shaped elephantid history in profound ways, isolation also appears to have played an important role. Our data reveal nearly complete isolation between the ancestors of the African forest and savanna elephants for ∼500,000 y, providing compelling justification for the conservation of forest and savanna elephants as separate species.

Moose genomes reveal past glacial demography and the origin of modern lineages.

BACKGROUND: Numerous megafauna species from northern latitudes went extinct during the Pleistocene/Holocene transition as a result of climate-induced habitat changes. However, several ungulate species managed to successfully track their habitats during this period to eventually flourish and recolonise the holarctic regions. So far, the genomic impacts of these climate fluctuations on ungulates from high latitudes have been little explored. Here, we assemble a de-novo genome for the European moose (Alces alces) and analyse it together with re-sequenced nuclear genomes and ancient and modern mitogenomes from across the moose range in Eurasia and North America. RESULTS: We found that moose demographic history was greatly influenced by glacial cycles, with demographic responses to the Pleistocene/Holocene transition similar to other temperate ungulates. Our results further support that modern moose lineages trace their origin back to populations that inhabited distinct glacial refugia during the Last Glacial Maximum (LGM). Finally, we found that present day moose in Europe and North America show low to moderate inbreeding levels resulting from post-glacial bottlenecks and founder effects, but no evidence for recent inbreeding resulting from human-induced population declines. CONCLUSIONS: Taken together, our results highlight the dynamic recent evolutionary history of the moose and provide an important resource for further genomic studies.

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.

Novel Genes, Ancient Genes, and Gene Co-Option Contributed to the Genetic Basis of the Radula, a Molluscan Innovation.

The radula is the central foraging organ and apomorphy of the Mollusca. However, in contrast to other innovations, including the mollusk shell, genetic underpinnings of radula formation remain virtually unknown. Here, we present the first radula formative tissue transcriptome using the viviparous freshwater snail Tylomelania sarasinorum and compare it to foot tissue and the shell-building mantle of the same species. We combine differential expression, functional enrichment, and phylostratigraphic analyses to identify both specific and shared genetic underpinnings of the three tissues as well as their dominant functions and evolutionary origins. Gene expression of radula formative tissue is very distinct, but nevertheless more similar to mantle than to foot. Generally, the genetic bases of both radula and shell formation were shaped by novel orchestration of preexisting genes and continuous evolution of novel genes. A significantly increased proportion of radula-specific genes originated since the origin of stem-mollusks, indicating that novel genes were especially important for radula evolution. Genes with radula-specific expression in our study are frequently also expressed during the formation of other lophotrochozoan hard structures, like chaetae (hes1, arx), spicules (gbx), and shells of mollusks (gbx, heph) and brachiopods (heph), suggesting gene co-option for hard structure formation. Finally, a Lophotrochozoa-specific chitin synthase with a myosin motor domain (CS-MD), which is expressed during mollusk and brachiopod shell formation, had radula-specific expression in our study. CS-MD potentially facilitated the construction of complex chitinous structures and points at the potential of molecular novelties to promote the evolution of different morphological innovations.

Extended and Continuous Decline in Effective Population Size Results in Low Genomic Diversity in the World's Rarest Hyena Species, the Brown Hyena.

Hyenas (family Hyaenidae), as the sister group to cats (family Felidae), represent a deeply diverging branch within the cat-like carnivores (Feliformia). With an estimated population size of <10,000 individuals worldwide, the brown hyena (Parahyaena brunnea) represents the rarest of the four extant hyena species and has been listed as Near Threatened by the IUCN. Here, we report a high-coverage genome from a captive bred brown hyena and both mitochondrial and low-coverage nuclear genomes of 14 wild-caught brown hyena individuals from across southern Africa. We find that brown hyena harbor extremely low genetic diversity on both the mitochondrial and nuclear level, most likely resulting from a continuous and ongoing decline in effective population size that started ∼1 Ma and dramatically accelerated towards the end of the Pleistocene. Despite the strikingly low genetic diversity, we find no evidence of inbreeding within the captive bred individual and reveal phylogeographic structure, suggesting the existence of several potential subpopulations within the species.