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

Mark S Springer; Christian F Guerrero-Juarez; Matthias Huelsmann; Matthew A Collin; Kerri Danil; Michael R McGowen; Ji Won Oh; Raul Ramos; Michael Hiller; Maksim V Plikus; John Gatesy

doi:10.1016/j.cub.2021.02.057

. Author manuscript; available in PMC: 2022 May 24.

Published in final edited form as: Curr Biol. 2021 Apr 1;31(10):2124–2139.e3. doi: 10.1016/j.cub.2021.02.057

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

Mark S Springer ^1,^#,^*, Christian F Guerrero-Juarez ^2,^3,^4,^5,^#, Matthias Huelsmann ^6,^7,^8,^#,^†,^‡, Matthew A Collin ^1,⁹, Kerri Danil ¹⁰, Michael R McGowen ¹¹, Ji Won Oh ^12,^13,¹⁴, Raul Ramos ^3,^4,⁵, Michael Hiller ^6,^7,^8,^15,^16,^17,^*, Maksim V Plikus ^3,^4,^5,^*, John Gatesy ^18,^*

¹Department of Evolution, Ecology, and Organismal Biology, University of California, Riverside, CA 92521, USA

²Department of Mathematics, University of California, Irvine, Irvine, CA 92697, USA

³NSF-Simons Center for Multiscale Cell Fate Research, University of California, Irvine, Irvine, CA 92697, USA

⁴Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697, USA

⁵Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, CA 92697, USA

⁶Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany

⁷Max Planck Institute for the Physics of Complex Systems, 01187 Dresden, Germany

⁸Center for Systems Biology Dresden, 01307 Dresden, Germany

⁹Department of Botany & Plant Sciences, University of California, Riverside, CA 92521, USA

¹⁰Southwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, La Jolla, CA 92037 USA

¹¹Department of Vertebrate Zoology, Smithsonian Museum of Natural History, 10th & Constitution Ave. NW, Washington DC 20560, USA

¹²Department of Anatomy, School of Medicine, Kyungpook National University, Daegu, Korea

¹³Biomedical Research Institute, Kyungpook National University Hospital, Daegu, Korea

¹⁴Hair Transplantation Center, Kyungpook National University Hospital, Daegu, Korea

¹⁵LOEWE Centre for Translational Biodiversity Genomics, 60325 Frankfurt, Germany

¹⁶Senckenberg Research Institute, 60325 Frankfurt, Germany

¹⁷Goethe-University, Faculty of Biosciences, 60438 Frankfurt, Germany

¹⁸Division of Vertebrate Zoology and Sackler Institute for Comparative Genomics, American Museum of Natural History, New York, NY 10024, USA

^†

Present address: Department of Environmental Microbiology, Eawag, 8600 Dübendorf, Switzerland

^‡

Present address: Department of Environmental Systems Science, ETH Zurich, 8092 Zürich, Switzerland

Co-first authors

AUTHOR CONTRIBUTIONS

Conceptualization, J.G., Mi.H., M.V.P., M.S.S.; Methodology, C.F.G.-J., M.V.P., J.O.H., Ma.H., Mi.H., M.A.C., M.S.S., J.G.; Investigation, M.S.S., C.F.G.-J., Ma.H., M.A.C., J.W.O., R.R., Mi.H., M.V.P., J.H.; Resources, K.D., M.R.M.; Writing – Original Draft, M.S.S., J.G., M.V.P., Mi.H., C.F.G.-J.; Writing – Review & Editing, M.S.S., J.G., C.F.G.-J., Ma.H., M.A.C., K.D., M.R.M., J.W.O., R.R., Ma.H., M.V.P.; Funding Acquisition, J.G., M.S.S., M.H., M.V.P.

Corresponding authors: MSS springer@ucr.edu (Lead Contact), MH michael.hiller@senckenberg.de, MVP plikus@uci.edu, JG jgatesy@amnh.org

PMCID: PMC8154672 NIHMSID: NIHMS1681163 PMID: 33798433

SUMMARY

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, 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 million years 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.

Keywords: Cetacea, epidermis, gene loss, Hippopotamidae, histology, skin

Graphical Abstract

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INTRODUCTION

The evolutionary history of Mammalia is mostly one of deployment and adaptation to different terrestrial habitats. Nevertheless, numerous mammalian clades have returned to aquatic habitats, either on a part-time basis or with full-time commitment to the aquatic realm. Fully aquatic clades include Cetacea and Sirenia, and in both cases there are extinct taxa that document the macroevolutionary transition from land to water [1,2]. Semiaquatic clades are more numerous and examples are found in a wide range of mammalian orders, e.g., Monotremata (platypuses), Rodentia (beavers, capybaras), Carnivora (pinnipeds, otters), and Cetartiodactyla (hippos). Adaptations to aquatic habitats are most extreme in fully aquatic forms where virtually every organ system has been highly modified. Locomotory adaptations in cetaceans include hind limb loss, modification of the front limbs into flippers, and conversion of the tail into a powerful fluke [1]; sensory system modifications include highly modified eyes with reduction or loss of color vision [3], olfactory structures that are highly reduced or absent [4,5], and ultrasonic hearing in odontocetes (toothed whales) [6].

Cetaceans also show changes to their integument. The skin of fully aquatic mammals constantly interacts with dense, viscous, and thermally conductive water, which poses unique physical challenges to its outer surface. The cetacean epidermis is exceptionally thick and undergoes constant cellular renewal [7–13]. Even with its increased thickness there are only three distinct histological layers – a basal layer (stratum basale), an intermediate layer (stratum spinosum), and an outer layer (stratum corneum) [14]. The stratum granulosum, which in land mammals lies beneath the stratum corneum, is ill-defined or absent [10,15]. The thick epidermis provides mechanical and thermal protection, and its fast rate of sloughing and extensive epidermal-dermal interdigitations guard against potential damage from locomotory shear stress [16]. The stratum basale forms deep root-like projections (= rete ridges) that extend into the underlying dermis [17–19]. Extensive cytoplasmic lipid vacuoles are present in the stratum spinosum and stratum corneum keratinocytes [20–22], and may play metabolic and/or thermo-insulating roles [22,23]. Unlike terrestrial mammals, cells of the stratum corneum retain nuclei and do not become fully keratinized in cetaceans [14], possibly because fully aquatic mammals do not need a functional epidermal barrier to a dry environment [24]. The cetacean dermis is thickened and consists of an upper papillary layer that interdigitates with the epidermis and a lower reticular layer that gradually transitions into the underlying blubber [10,11,18,25,26].

Cetaceans have reduced their ectodermal appendages including hair follicles and skin glands [27]. They show no evidence of pelage (i.e., fur) hair follicle formation during embryonic development although vibrissa follicles (i.e., whiskers) form on the head [28–32]. Mysticetes and some odontocetes cyclically grow whiskers as adults, but most adult odontocetes lose their whiskers and convert vibrissa follicles into degenerated pits that may perform sensory functions [30,33,34]. Cetaceans lack oil-secreting sebaceous glands [31,32,35] and sweat glands [1,14,27].

Among semiaquatic forms, hippos are the largest herbivores [36]. Both extant species (Hippopotamus amphibius [river hippo], Choeropsis liberiensis [pygmy hippo]) spend their days in the water and emerge at dusk to feed on grasses and other vegetation. H. amphibius has a thick epidermis and its stratum basale forms distinct projections into the papillary dermis [15]. H. amphibius shows elevated levels of epidermal lipid storage, although unlike cetaceans the most prominent lipid deposits occur in intercellular locations within the stratum corneum [23]. Hippos have bristle-like whiskers on their muzzle, and pelage hairs that are sparsely distributed across most of the body [15]. Sebaceous glands have not been reported, but previous histological studies have only examined limited regions of the body [15,37–39]. Hippo skin contains anatomically complex sweat glands [37,38] that secrete a red-orange pigmented sweat that may have sunscreen and/or antimicrobial properties [40,41].

The traditional view based on morphology is that cetaceans are excluded from a monophyletic Artiodactyla (even-toed hoofed mammals) [42,43] and that hippopotamids are the sister taxon to pigs (Suidae) and/or peccaries (Tayassuidae) [44–48]. However, molecular studies challenged this view and eventually provided conclusive evidence that cetaceans are nested within Artiodactyla [49,50] as the sister to Hippopotamidae [51–54].

Given that cetaceans and hippopotamids share a variety of morphological and behavioral characters that may be related to aquatic habitats (hairless or nearly hairless body, lack of sebaceous glands, lack of scrotal testes, underwater parturition and nursing, underwater detection of sound directionality), the most parsimonious hypothesis is that these characters evolved in the common ancestor of these two clades (= Cetancodonta) and that this common ancestor was semiaquatic [51,52,55,56] (Figure 1A). O’Leary and Gatesy [57] favored the common origin hypothesis based on ancestral reconstructions of underwater hearing and other aquatic characters. Gatesy et al. [1] favored the aquatic ancestry hypothesis, specifically in freshwater, based on oxygen isotope values in pakicetid cetaceans and the presence of dense, osteosclerotic bones in both hippos and basal stem cetaceans (raoellids, pakicetids). Alternatively, semiaquatic adaptations evolved convergently in hippos and cetaceans [36] (Figure 1B). In part, support for these two competing hypotheses turns on the phylogenetic placement of various ‘Anthracotheriidae’, which collectively are the paraphyletic stem group to Hippopotamidae. Some anthracotheres in the subfamilies Anthrocotheriinae (e.g., Anthracotherium) and Microbunodontinae (e.g., Microbunodon) are inferred to have been terrestrial based on oxygen isotope values, but members of Bothriodontinae (e.g., Bothriogenys, Merycopotamus) have values that are consistent with a semiaquatic lifestyle [58–62]. Cooper et al. [62] performed ancestral reconstructions on bone microanatomy for a data set that included both anthracotheres and stem cetaceans (“archaeocetes”) and concluded that the most recent common ancestor of Cetancodonta was probably semiaquatic. Soe et al. [63] reported skeletal material for the Eocene anthracotheriid Siamotherium, which they recovered as the most basal genus of Anthracotheriidae in their phylogenetic analysis, and concluded there is no evidence of clear-cut aquatic adaptations in Siamotherium.

Figure 1. — (A) Evolution of shared aquatic features in the common ancestor of Hippopotamidae and Cetacea, and (B) independent evolution of aquatic features on the cetacean stem lineage and also on the hippopotamid stem lineage. Encircled red stars mark the initial evolution of behavioral, physiological, and anatomical characteristics associated with adaptation to aquatic environments. Aquatic (blue) and terrestrial (brown) specializations of extant taxa (squares) and ancestral nodes (circles) are indicated in each scenario.

At the molecular level, positive selection analyses provide minimal support for shared aquatic adaptations in the ancestry of Cetancodonta [64]. However, branch-site selection analyses can have weak statistical power. A more promising approach may be to search for skin genes that have been inactivated in the common ancestor of cetaceans and hippos. Previous authors have employed this approach to identify skin genes that were inactivated in the common ancestor of cetaceans [16,24,65–68]. However, only two candidate gene studies [69–70] have searched for skin genes that are inactivated in cetaceans and hippos. It remains to be determined if a comprehensive genomic screen [sensu 67] will reveal additional skin genes that have been inactivated in both cetaceans and hippos.

Here, we present morphological and molecular evidence to evaluate competing hypotheses that aquatic skin adaptations evolved in the common ancestor of cetaceans and hippos versus independently in these two groups (Figure 1). First, we provide histological data for different regions of the integument in two cetacean species and both extant hippos. Next, we perform a comprehensive genomic screen on representative cetaceans and hippopotamids for protein-coding genes that have been inactivated in both of these clades and assess the timing of pseudogenization for these genes. Finally, we discuss our results in the context of the rich fossil record for stem cetaceans (“archaeocetes”) and stem hippos (“anthracotheres”).

RESULTS AND DISCUSSION

Comparative Skin Histology Between Terrestrial and Aquatic Cetartiodactyla

We analyzed skin samples from the face, eyelid, ear, dorsum, ventrum, and tail in both hippopotamid species, and from the facial region of one odontocete (Tursiops truncatus [bottlenose dolphin]) and one mysticete (Eschrichtius robustus [gray whale]). Table 1 summarizes features of the skin in these taxa and also includes data from the literature for terrestrial mammals including humans and two cetartiodactyls (cow, pig) that are close relatives of Cetancodonta [71–79].

Table 1. Anatomical features of the skin.

Anatomical features of the skin in human, cow, pig, hippo, and two cetaceans.

Cellular/anatomical feature	Human	Pig	Cow	Hippo	Grey whale (facial skin only)	Common bottlenose dolphin (facial skin only)
Epidermal thickness	29.6 to 96.5 μm^a	45.8 to 91.7 μm^b	34.9 to 55.6 μm^c	River hippo: 216 to 1,134 μm^d (adult trunk); 42 +/− 7 μm (neonatal, facial skin) Pygmy hippo: 44 +/− 12 μm (neonatal, facial skin	6,334 +/− 872 μm	945 +/− 267 μm
Epidermal rete ridges	Medium-size rete ridges that differ in size across body	Medium-size rete ridges that differ in size across body	Absence of definitive rete ridges	Shallow rete ridges that differ in size across body	Prominent, branched rete ridges	Prominent, branched rete ridges
Cytoplasmic lipid vacuoles in epidermal living layers	Absent	N/A	N/A	Extensive cytoplasmic lipid vacuoles in all epidermal layers	Extensive cytoplasmic lipid vacuoles in all epidermal layers	Extensive cytoplasmic lipid vacuoles in all suprabasal epidermal layers
Lipid distribution in stratum corneum	Inter-cellular	N/A	N/A	Mostly intercellular	Mostly intracellular	Mostly intracellular
Stratum basale	Present	Present	Present	Present	Present	Present
Stratum spinosum	Present, thin	Present, medium	Present	Present, medium	Present, very thick	Present, very thick
Stratum granulosum	Present	Present	Present	Poorly defined	Absent	Absent
Stratum corneum	Present, variable thickness	Present, variable thickness	Present, thin	Present, medium	Present, very thick	Present, very thick
Epidermal barrier	Intact	Intact	Intact	Intact	Disrupted	Disrupted
Pelage hair follicles	Present, variable hair density across the body	Present, variable hair density across the body	Present, high hair density	Present, variable hair density; sparse over most of the body	Absent	Absent
Vibrissa hair follicles	Absent	Present	Present	Present	Present, limited to cranial skin	Present, limited to cranial skin
Ability of hair follicles to cycle	Able to cycle	Able to cycle	Able to cycle	Able to cycle	Able to cycle	Hair follicles cycle only once in neonates and degenerate in adults to become pits
Hair follicle-associated sebaceous gland	Present, variable in size and often prominent	Present, rudimentary	Present	Absent	Absent	Absent
Meibomian glands	Present	Present	Present	Absent	Absent	Absent
Sweat glands	Apocrine and eccrine glands	Apocrine glands only	Apocrine glands only	Eccrine glands^d; possibly apocrine glands	Absent	Absent
Dermal adipose tissue	Prominent	Prominent	Poorly defined	Absent	Abundant adipocyte clusters throughout the dermis	Abundant adipocyte clusters throughout the dermis

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Data from references [130] and [131]

Data from reference [77].

Data from reference [132].

Glands are specialized and produce “colored” sweat with proposed sunblock and/or antimicrobial properties [41].

Cetaceans and hippos have prominent differences in the thickness and organization of the epidermis (Figure 2). Consistent with previous reports [17–19], the facial epidermis in adult Eschrichtius robustus (Figure 2E) and neonatal Tursiops truncatus (Figure 2F-G) is very thick, with a wide stratum spinosum and an undulated stratum basale with deep root-like rete ridges. By contrast, the epidermis in neonatal pygmy hippo is thin, with only shallow rete ridges (Figure 2A-D).

Hippo skin contains hair follicles of both pelage and vibrissa morphology. Prominent vibrissa follicles that contain collagen capsules and ringwulst (ring-like dermal structure that surrounds the follicle) occur in the upper lip skin (Figure 2A; Figure S1C). Some tail-tip hair follicles display collagen capsules (Figure 2B; Figure S1E), suggesting that the tail might contain both pelage and vibrissa hair types. Hair follicles in ear and eyelid skin have typical pelage morphology, and meibomian glands are absent (Figure 2D; Figure S1A-B, S2C). All hippo hair follicles lack sebaceous glands (Figure 2; Figure S1). The facial skin of both cetaceans has prominent vibrissa hair follicles (Figure 2E-G), but their structure differs from hippo vibrissae in lacking collagen capsules and ringwulst. In both cetaceans, mesenchymal dermal papillae and the epithelial hair matrix, two defining structures of actively growing hair follicles, are present. However, the hair matrix in dolphin vibrissae is uncharacteristically thin. There is no evidence of vibrissa-associated sebaceous glands in either cetacean species.

Prominent sweat glands occur in hippo skin in several locations, including the upper lip, dorsum, and ventrum (Figure 2C-D; Figure S2). The structure of the dermis is different in cetaceans and hippos. There are abundant clusters of adipocytes throughout the dermis in dolphin (Figure 2G), but we found no evidence for mature adipocytes in hippos. Hippos exhibit prominent differences in dermal thickness across body sites, with tail and ear dermis being the thinnest.

In conclusion, cetacean skin has a distinctly thick and highly undulated epidermis, a thick and adipocyte-rich dermis, and morphologically-specialized facial vibrissa hair follicles as the only skin appendage. Hippo skin is characterized by a much thinner epidermis, shallow rete ridges, a dermis of variable thickness without adipocytes, highly specialized sweat glands, and both pelage and vibrissa hair follicles. Cetaceans and hippos both lack sebaceous glands (Table 1). Our histological analyses for each species are limited to just one time point. Additional histological features, not captured in our studies, are expected for both hippos and cetaceans. For example, the epidermis undergoes annual molting in some cetaceans including belugas [80] and the Okhotsk population of bowheads [81]. During the spring molt the bowhead epidermis is at its thinnest and contains a highly vacuolated stratum spinosum that is not present in the fall [81,82].

Genomic Screens and Patterns of Gene Inactivation in Cetaceans and Hippos

We evaluated the shared versus independent aquatic ancestry hypotheses using existing and newly-generated genomic data. Changes in both protein-coding sequences and cis-regulatory elements could contribute to the evolution of skin phenotypes, but we focused on the former because the timing of gene inactivation can be estimated from protein-coding sequences [83]. We screened genomic alignments with 63 mammalian taxa for protein-coding genes that are inactivated in Hippopotamidae and Cetacea but not in terrestrial cetartiodactyls. Our screen included Hippopotamus amphibius, one baleen whale (Balaenoptera acutorostrata [common minke whale]), and three toothed whales (Physeter macrocephalus [sperm whale], Orcinus orca [killer whale], Tursiops truncatus). We identified 38 genes that have inactivating mutations (frameshift indels, premature stop codons, splice site mutations, deleted exons) or are completely deleted in these taxa (Table S1) including ten genes that have primary or sole functions related to skin and its ectodermal appendages (ALOX15, AWAT1, KPRP, KRT2, KRT26, KRT77, KRTAP6–2, KRTAP6–3, KRTAP7–1, TCHHL1). Two genes (KRTAP6–2, KRTAP6–3) were excluded because of ambiguous orthology relationships, leaving eight genes for a detailed analysis. ABCC11, which our screen found to be inactivated only in Cetacea, was added to our list because ABCC11 is a candidate gene of interest that is expressed in axillary sweat glands in humans [84,85].

Base errors in genome assemblies can mimic gene-inactivating mutations [86], so we used additional genomic data to confirm the validity of inactivating mutations and thus the loss of gene function. Our investigation of ten additional cetacean genomes revealed that all nine genes have mutations shared between at least two species (as exemplified in Figure 3A-B), which makes base errors highly unlikely. Three of these genes (AWAT1, KRTAP7–1, ABCC11) exhibit inactivating mutations shared between odontocetes and mysticetes, indicating gene loss on the stem cetacean branch (Figure 3C). Three genes exhibit large or entire gene deletions in odontocetes (KRT2, KRT26) or mysticetes (KRT77), and gene loss dating (below) indicates that KRT26 and KRT77 losses happened on the stem cetacean branch. The distribution of mutations in ALOX15 suggests that it was independently inactivated on three different cetacean branches (Figure 3C). Finally, two genes are absent from all cetacean (KPRP and TCHHL1) genomes. However, large deletions and rearrangements in these loci obscure reconstructions of when these genes were lost because there are no obvious breakpoints that are shared by odontocetes and mysticetes.

Figure 3. — Inactivating mutations in *KRTAP7–1* (A) and *ABCC11* (B). Genes are shown with exons represented by green rectangles proportional to their size and introns represented by horizontal lines. Inactivating mutations are premature stop codons (vertical black line and corresponding triplet), insertions (red arrowhead and corresponding insertion size), deletions (vertical red line and corresponding deletion size, or red rectangle for completely deleted exon), and splice site donor or acceptor mutations (red letters at the end or beginning of an exon, respectively). Insets show the DNA sequence context of representative mutations. Alignment files for inactivating mutations in all genes are available at https://doi.org/10.6084/m9.figshare.13549070.v1. (C) The phylogenetic pattern of skin gene inactivations mapped onto a timetree for Cetancodonta (Hippopotamidae + Cetacea). Independent inactivations of skin genes (red triangles) are marked on branches of the tree; inactivation times are average estimates for each locus based on dN/dS analyses. The mean dates of skin gene knockouts for six loci that were inactivated in the common ancestor of Cetacea and for eight loci that were inactivated in the common ancestor of Hippopotamidae are indicated on the stem lineages to these clades (encircled red stars). The timetree for extant lineages is based on the molecular clock analysis of McGowen et al. [129]. Extinct lineages for stem cetaceans (*Indohyus*, *Ambulocetus*, *Georgiacetus*) and stem hippopotamids (*Elomeryx*, *Libycosaurus*) are approximately positioned relative to the geological time scale based on earliest occurrence in the fossil record for each genus and phylogenetic hypotheses based on morphological characters. See also Table S2 and Table S3.

Genomic data for Choeropsis liberiensis are required to investigate whether mutations are shared between both hippopotamid species. Therefore, we generated ~40X coverage of Illumina whole-genome shotgun data for C. liberiensis and mapped these reads to the Hippopotamus amphibius assembly to obtain orthologous sequences. Eight of nine genes (ALOX15, AWAT1, KPRP, KRT2, KRT26, KRT77, KRTAP7–1, TCHHL1) exhibit shared inactivating mutations in both hippopotamid species (Figure 3). ABCC11 is intact in H. amphibius, but a two-bp frameshift deletion in exon 14 of C. liberiensis suggests a recent loss in pygmy hippo (Figure 3B).

Next we analyzed whether inactivating mutations in any of these nine skin-related genes are shared by cetaceans and hippos, which would indicate gene inactivation in the common ancestor of Cetancodonta. We did not find any shared inactivating mutations. Instead, all nine genes have different inactivating mutations that indicate independent gene loss in cetaceans and hippos (Figure 3, Table S2). Lopes-Marques et al. [70] reported the convergent loss of both AWAT1 and MOGAT3 in Cetacea and Hippopotamus amphibius. Our results confirm the independent inactivation of AWAT1 and further suggest that AWAT1 was inactivated in the common ancestor of H. amphibius and Choeropsis liberiensis. However, our investigation suggests that MOGAT3 underwent a tandem duplication, most likely in the common ancestor of the two extant hippopotamids (Figure 4), and whereas one copy is pseudogenized, a functional copy of MOGAT3 is present ~15.7 kb upstream (NCBI contig PVJP02910399).

Figure 4. — The gene tree for MOGAT3 shows inferred gene duplication event in Hippopotamidae (yellow circle) and parallel gene inactivations (red lineages). Seven gene knockouts are inferred, including pseudogenization of the *MOGAT3* paralog that was derived from a duplication event on the stem hippopotamid branch. Lineages with functional *MOGAT3* are gray, and lineages with inactivated *MOGAT3* are colored red. Branches where inactivation events (frameshift indels, premature stop codons) were inferred by parsimony optimization of indels are gray and red, indicating the transition from functional to non-functional. The dashed red lineage for *Choeropsis liberiensis* represents hypothesized deletion of the *MOGAT3* paralog in the genome of this species. Branch lengths are in expected numbers of substitutions per site.

Comparison of Inactivated Skin Genes and Epidermal Phenotypes in Cetaceans and Hippos

The cetacean epidermis is very thick and renews rapidly, yet does not fully differentiate into the highly keratinized stratum corneum of terrestrial mammals that confers barrier functions. These modifications are associated with loss of function of numerous genes that belong to the Epidermal Differentiation Complex (EDC) including S100 fused-type protein genes (CRNN, FLG, FLG2, HRNR, RPTN, TCHH, TCHHL1, TCHHL2) [27,65] and suprabasal epidermal keratin genes (KRT1, KRT2, KRT9, KRT10, KRT77, KRT23, KRT24) [16]. Our analyses confirm these findings (Figure 5).

Cetaceans and hippos share features such as reduced differentiation of the epidermis, but there are also differences. In particular, the hippo epidermis is much thinner, has shallower rete ridges, and partially preserves epidermal barrier function as hippos spend substantial time on land. These differences may explain why only one EDC gene, TCHHL1 (trichohyalin-like 1), is convergently inactivated in cetaceans and hippos (Figure 5). TCHHL1 is predominantly expressed in the stratum basale and its specific function in keratinocyte differentiation remains unclear [87,88]. Among suprabasal keratins, only KRT2 and KRT77 are independently inactivated in hippos (Figure 5), and the overall degree of suprabasal keratin inactivation in hippos is less than in obligately-aquatic manatees, which are more similar to cetaceans [16]. Other important epidermal genes that were reported as inactivated in cetaceans are intact in hippos. These include terminal keratinocyte differentiation-associated caspase CASP14 [65], PSORS1C2 (psoriasis susceptibility 1 candidate 2) [89], desmosome proteins DSG4 and DSC1, transglutaminase TGM5, and the atypical lipoxygenase ALOXE3 [90]. In summary, cetaceans and hippos both have inactivated copies of EDC and suprabasal keratin genes, but many more genes are knocked out in cetaceans.

Our genome-wide screen identified two new epidermal genes, KPRP and ALOX15, that are independently inactivated in cetaceans and hippos (Figure 5). KPRP (keratinocyte proline-rich protein) is an epidermal terminal differentiation-associated protein, part of the EDC, that is normally expressed in the stratum granulosum [91,92]. This epidermal layer is poorly defined in hippos [15] and absent in cetaceans [10]. A single-nucleotide polymorphism in human KPRP is associated with atopic dermatitis, a condition diagnosed by disrupted epidermal barrier function [93]. ALOX15 (arachidonate 15-lipoxygenase) belongs to the lipoxygenase family of enzymes that catalyze bioactive lipids synthesis, including resolvins that regulate resolution of excessive inflammatory responses [94,95]. ALOX15 is highly expressed in mouse epidermis and Axol15−^/− mutant mouse studies suggest a role in epidermal barrier function [96].

We next focused on genes that are associated with hair follicles, sebaceous glands, and sweat glands. We confirm previous findings that some or all cetaceans have inactivated hair inner root sheath keratins (KRT25, KRT26, KRT27, KRT28, KRT71, KRT72, KRT73, KRT74), hair, nail and tongue papillae keratins (KRT32, KRT33A, KRT33B, KRT34, KRT35, KRT38, KRT39, KRT40, KRT82, KRT83, KRT84), as well as keratins KRT3 and KRT6B [16,97]. Of these, hippos have independently inactivated only KRT26. The retention of functional hair and nail keratins in hippos is consistent with the presence of prominent keratinized hoofs [98] and both pelage and vibrissa hairs. Brush-like hairs on the tail aid in spreading feces during defecation, a behavior used by hippos for marking territory [99]. Our analyses validate that cetaceans have inactivating mutations in genes (AWAT1, DGAT2L6, FABP9, ELOVL3, MOGAT3, MC5R) associated with sebaceous gland function [69,70]. AWAT1 is inactivated in both hippos, but in contradiction to Lopes-Marques et al. [70] we show that these species retain an intact copy of MOGAT3 (Figure 4). Finally, we found that ABCC11, which is associated with axillary sweat gland function in humans, is inactivated in all cetaceans and independently in pygmy hippo, which like river hippo has active sweat glands that produce pigmented secretion. Therefore, ABCC11 function does not appear to be critical for pygmy hippos’ sweat gland biology.

The Timing of Gene Inactivations in Hippopotamidae and Cetacea

To understand when gene losses occurred, we performed dN/dS analyses with the coding remnants of inactivated genes and equations from Meredith et al. [83]. To obtain robust inactivation dates, we calculated dates using eight different combinations of codon frequency model (CF1, CF2), fixed versus estimated ω values for the pseudogenic branch category, and one versus two rates for synonymous substitutions (Table S3). Mean inactivation dates based on these estimates are shown in Figure 3 and Table 2. Mean estimates for eight genes (ALOX15, AWAT1, KPRP, KRT2, KRT26, KRT77, KRTAP7–1, TCHHL1) that were inactivated on the stem Hippopotamidae branch range from 53.92 to 5.42 Ma. These inactivation dates suggest that derived changes in hippopotamid skin, including the loss of sebaceous glands, have a long history that encompasses the entirety of the stem hippopotamid branch. The mean date for inactivation of these eight genes is ~30.5 Ma, which is near the midpoint of the stem hippopotamid branch. In addition, ABCC11 appears to have been inactivated very recently in Choeropsis liberiensis (Figure 3; Table 2).

Table 2. Inactivation dates for pseudogenized skin genes.

Mean inactivation dates for skin-related genes in cetaceans and hippos based on eight different combinations of codon frequency model (CF1, CF2), fixed versus estimated dN/dS values for the pseudogenic branch category, and one versus two rates for synonymous substitutions. Estimates based on individual analyses are provided in Table S3.

Gene	Hippopotamidae	Choeropsis	Cetacea	Mysticeti	Balaenoptera	Delphinida	Physeteroidea
ABCC11		0	40.54
ALOX15	23.54				15.74	34.13	21.44
AWAT1	53.92		48.23
KPRP	39.01		CDS deleted
KRT2	26.41			33.64^a
KRT26	15.98		48.93^b
KRT77	45.75		53.92^c
KRTAP 7–1	34.18		40.94
MOGAT3			46.37
TCHHL1	5.42		CDS deleted

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CDS (partial) only preset in Mysticeti.

CDS only present in Delphinida.

CDS only present in Mysticti.

For Cetacea, estimated inactivation dates for four genes (ABCC11, AWAT1, KRTAP7–1, MOGAT3) with evidence of pseudogenization on the stem cetacean branch based on shared inactivating mutations range from 48.23 to 40.54 Ma (mean = 44.02 Ma). Three additional genes are inactivated in all cetaceans, but are completely absent from either odontocetes (KRT2, KRT77) or mysticetes (KRT26). As complete gene deletion could have erased smaller mutations that inactivated these genes earlier in evolution, we used the remnants of these genes to estimate whether loss may have occurred on the stem Cetacea branch. Inactivation dates suggest that two of these genes were pseudogenized on the cetacean stem branch (53.92 Ma [KRT77], 48.93 Ma [KRT26]) whereas the date for KRT2 [33.64 Ma] is slightly younger than the most recent common ancestor of crown Cetacea at ~36.72 Ma. The mean date for the six genes (ABCC11, AWAT1, KRT26, KRT77, KRTAP7–1, MOGAT3) inactivated on the stem cetacean branch is ~46.5 Ma, which is near the midpoint of the stem cetacean branch. Overall, the mean inactivation date for these six genes in cetaceans is ~16.0 million years older than the mean inactivation date for eight genes that were inactivated on the stem hippo branch. Among four overlapping genes (AWAT1, KRT26, KRT77, KRTAP7–1), the mean date for inactivation on the stem Cetacea branch (48.00 Ma) is 10.54 Ma older than the mean date for inactivation on the stem hippo branch (37.46 Ma). Two additional genes (KPRP, TCHHL1) are completely absent in all examined cetacean genomes, so the timing of these gene losses on the stem cetacean branch could not be estimated. Finally, ALOX15 was independently inactivated in the common ancestor of Delphinida (34.13 Ma), on the branch leading to Physeter (21.44 Ma), and in the common ancestor of Balaenoptera acutorostrata and B. bonaerensis (15.74 Ma).

Integration of Molecular, Histological, and Paleontological Data

Our analyses shed light on a key question pertaining to the evolution of Cetancodonta: Did shared features associated with a (semi)aquatic lifestyle evolve in the common ancestor of this clade or independently in Cetacea and Hippopotamidae (Figure 1)? If aquatic features of hippos represent an intermediate condition in the transition from land to sea in the ancestry of Cetacea [1], then features of extant hippos might provide insights into the behavior and morphology of the earliest cetaceans from the Eocene [51]. Shared morphological features include the loss of sebaceous glands and the general reduction or complete loss of pelage hairs that cover the body. Previous histological studies that reported the absence of sebaceous glands in river hippo were limited to skin from the trunk, neck, and limbs that has few to no hairs [15]. However, sebaceous glands and hair follicles comprise an anatomically connected pilosebaceous unit. Therefore, we thoroughly investigated several regions of hair-bearing skin and provide more definitive evidence for the absence of sebaceous glands in both extant hippo species. Meibomian glands, which are modified sebaceous glands in the eyelids, are also absent in both hippos (also see [39]). Further, vibrissa follicles in newborn dolphin and adult gray whale lack sebaceous glands. These findings suggest the complete body-wide absence of sebaceous glands in hippopotamids and cetaceans. The most parsimonious explanation is that these glands were lost in the common ancestor of Cetancodonta. Similarly, the most parsimonious explanation for body hair reduction (hippos) or loss (cetaceans) is that pelage density started to decrease in the common ancestor of Cetancodonta. By contrast, the distribution of lipids is intracellular in the stratum corneum of cetaceans whereas hippos have intercellular stratum corneum lipids, which suggests parallel evolution of the highly modified epidermis in these taxa.

If sebaceous gland loss and pelage reduction occurred in the common ancestor of Cetancodonta, then we should find evidence of shared inactivating mutations in one or more skin-specific genes in hippos and cetaceans. By contrast, the independent origins hypothesis predicts convergent gene inactivations in cetaceans and hippos. Our genomic screens identified several skin-specific genes that are inactivated in hippos and cetaceans. Strikingly, while our genomic screens identified several skin-specific genes that are inactivated in hippos and cetaceans, none of these genes have shared inactivating mutations, suggesting that pelage reduction and the loss of sebaceous glands occurred independently in Hippopotamidae and Cetacea.

Mean dates for the inactivation of skin genes in these two clades serve as a proxy for phenotypic changes. These dates suggest that pelage reduction/loss, the loss of sebaceous glands, and changes to the keratinization program occurred 15.95 million years earlier in Cetacea (46.48 Ma) than Hippopotamidae (30.53 Ma) based on all estimates for gene inactivations or 10.54 million years earlier in Cetacea (48.00 Ma) than Hippopotamidae (37.46 Ma) based on an overlapping set of four genes (Figure 3, Table 2). The mean date of ~48.0–46.5 Ma for Cetacea is older than the first obligately aquatic cetaceans in the family Basilosauridae (e.g., Basilosaurus at ~41 Ma) and instead corresponds with the oldest protocetids (e.g., Rodhocetus) from the Lutetian (47.8–41.3 Ma), which may have utilized both land and water as sea lions do today [100]. The mean inactivation date (~37.5–30.5 Ma) for hippopotamid skin genes, in turn, is in the range of early bothriodontine anthracotheres (37.2–33.9 Ma) that are inferred to be the oldest semiaquatic members of Anthracotheriidae [36]. It is also noteworthy that far more skin-related genes have been inactivated in Cetacea than in Hippopotamidae (Figure 5), which is consistent with the more complete reorganization of the epidermis and its derivatives in cetaceans than hippopotamids (Table 1; Figures 2 and 5). Finally, our estimates of individual gene inactivations span tens of millions of years for both stem + crown cetaceans and stem + crown hippos (Figure 3, Table 2), suggesting that macroevolutionary changes to aquatic and semiaquatic habitats in these clades have long, stepwise histories. Indeed, AWAT1 inactivation on the stem hippopotamid branch and ABCC11 inactivation on the Choeropsis liberiensis branch have estimated pseudogenization dates that are separated by ~54 million years.

Paleontological evidence also provides an opportunity to evaluate competing hypotheses that (semi)aquatic features of cetaceans and hippos were acquired independently rather than in the common ancestor of this clade (Figure 1). If the initial shift to a semiaquatic lifestyle occurred in the common ancestor of Cetancodonta, then we might expect to find morphological and/or geochemical evidence for this transition in (1) stem cetancodontans that are close to the crown group, (2) the earliest stem cetaceans, and (3) the earliest stem hippopotamids. For stem cetaceans, the most primitive and earliest branching clade is Raoellidae. Thewissen et al. [101] inferred that the raoellid Indohyus was already semiaquatic based on a thickened medial wall in the auditory bulla (= involucrum) that is associated with underwater hearing. Dense limb bones (for ballast) and oxygen isotopic signatures of its teeth also suggest that Indohyus was semiaquatic [101]. Evidence for semiaquatic specializations in stem cetancodontans and stem hippopotamids is less forthcoming. Definitive stem members of Cetancodonta have been difficult to identify owing to the uncertainty of cladistic analyses. Possible stem cetancodontans have included various anthracotheres (e.g., Anthracotherium, Siamotherium [56]; Elomeryx, Heptacodon, Microbunodon [1]), an entelodontid (Brachyhyops [56], a cebochoerid (Cebochoerus [1,102]), and the enigmatic Andrewsarchus [56]. We are not aware of compelling evidence for aquatic adaptations in any of these taxa. Also, there is an emerging consensus that anthracotheres comprise the paraphyletic stem group that gave rise to Hippopotamidae [36,63,102]. Many anthracotheres (e.g., Anthracotherium, Siamotherium) appear to have been terrestrial based on oxygen isotope values whereas taxa in the subfamily Bothriodontinae have values that are consistent with a semiaquatic lifestyle [58,59,61,62]. However, the phylogenetic placement of presumed terrestrial anthracotheres as basal, stem Hippopotamidae suggests that specializations for an aquatic/semiaquatic lifestyle evolved independently in hippopotamids and cetaceans [36,63]. Thus, available paleontological evidence is largely aligned with distinct inactivating mutations in skin-related genes that favor the independent origins hypothesis. Additional testing of this hypothesis will benefit from more comprehensive taxonomic sampling of anthracotheres in both phylogenetic analyses and oxygen isotope studies.

When sister taxa share the same anatomical, physiological, or behavioral features, as is the case for aquatic features in Hippopotamidae and Cetacea, the simplest hypothesis is that these features evolved in their common ancestor. However, our results suggest otherwise and support the independent evolution of features that are related to the skin in hippos and cetaceans. Given that most synapomorphies for Cetancodonta are aquatic traits [1], morphological and behavioral support is very limited for this group if these traits evolved in parallel. Along the same lines, pinnipeds have lost their sweet and umami taste receptors. However, loss of these receptors occurred independently in the ancestors of the reciprocally monophyletic Phocidae (seals) and Otaroidea (sea lions, fur seals, walruses) rather than in the common ancestor of Pinnipedia [103]. Numerous morphological features related to raptorial feeding and hydrodynamic locomotion also evolved independently within Pinnipedia [104]. In Cetacea, multiple cranial and postcranial specializations for an aquatic lifestyle evolved convergently in odontocetes and mysticetes [1,105,106]. Morphological characters preserved in fossils and pseudogenic remnants of formerly functional genes provide complementary sources of evidence for elucidating such cases of convergent or parallel evolution.

Finally, gene inactivation dates have implications for understanding the physiology, behavior, and appearance of extinct organisms [1,107]. In the case of cetaceans, inactivation dates for AWAT1 and MOGAT3 are older than the inactivation date for ABCC11, which suggests sebaceous glands were lost before sweat glands on the stem cetacean branch. Given the timing of gene inactivations for these three genes, extinct protocetid whales may have retained sweat glands but not sebaceous glands. Similarly, the hair inner root sheath keratin KRT26 was lost relatively early on the stem cetacean branch (~49 Ma), suggesting that the program for generating body pelage hair had already been compromised at this early stage in cetacean evolution. Protocetids, which comprise a paraphyletic grade, were probably the earliest transoceanic cetaceans, but also spent time on land where they may have given birth and nursed their young [100,108,109]. These forms would certainly have looked different than modern whales due to their more prominent hindlimbs and primitive cranial morphology, but in other respects, such as having a largely hairless body, may have been similar to modern cetaceans.

Pelage reduction and other shared morphological features of the skin in Cetancodonta are not obligatory or exclusive features of a (semi)-aquatic lifestyle. For example, pinnipeds and beavers maintain dense hair despite being semi-aquatic. By contrast, sparse hair evolved in some fully terrestrial mammals (e.g., elephants, rhinoceroses) to enhance heat loss in savanna habitats [110]. Selection for efficient thermoregulation is also hypothesized to have contributed to hair reduction in hominins when our ancestors occupied the Africana savanna [110,111].

In summary, the integration of new histological data with comprehensive analyses of inactivated protein-coding genes provides strong support for the hypothesis that aquatic adaptations of the skin evolved independently in cetaceans and hippos. Our study further illustrates the potential of genomic data and in particular remnants of once functional genes as dateable ‘molecular vestiges’ to complement morphological data in providing novel insights into ancestry and timing of key trait changes and macroevolutionary transitions [68,83,86,107,112–117].

STAR*METHODS

RESOURCE AVAILABILITY

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mark S. Springer (springer@ucr.edu).

Materials Availability

Gene alignments generated in this study have been deposited to FigShare [https://doi.org/10.6084/m9.figshare.13549070.v1].

Data and Code Availability

The Illumina sequencing data generated for this study are available under NCBI BioProject PRJNA694317.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Genomic DNA from Choeropsis liberiensis (pygmy hippopotamus) was provided by G. Amato (formerly at New York Zoological Society). The sources for skin samples used in histological analyses are as follows: Choeropsis liberiensis (pygmy hippopotamus) skin samples are from Smithsonian National Museum of Natural History – specimen number 395848 (unknown gender, neonatal); Hippopotamus amphibius (river hippopotamus) skin samples are from Smithsonian National Museum of Natural History – specimen number 254870 (male, neonatal); Tursiops truncatus (common bottlenose dolphin) skin samples are from Southwest Fisheries Science Center (NOAA) specimen numbers KKS0032 (unknown gender, neonatal) and KXD0206 (late term fetus); Eschrichtius robustus (gray whale) skin samples are from Southwest Fisheries Science Center (NOAA) Fisheries – specimen number NEB0083 (unknown gender, adult).

METHOD DETAILS

Histology

Formalin-fixed Choeropsis liberiensis and Hippopotamus amphibius skin samples were first hydrated and rinsed in 1X PBS. Samples were then dehydrated through an ethanol gradient (from 25% to 100%), processed through histoclear and embedded in paraffin. Each hydration and dehydration step lasted for 12 hours. Tissues were sectioned at a thickness of 10 μm with a microtome (Leica). Samples were stained with hematoxylin and eosin using standard methods with minor modifications. Tissue sections were mounted with Permount mounting media and visualized with Nikon Ti-E Upright microscope. Tissue whole-mounts were captured with a Nikon dissecting microscope. Individual fields of Eschrichtius robustus rostral skin were visualized and stitched together with Keyence microscope.

Quantification of Epidermal Thickness

Epithelial thickness of Choeropsis liberiensis, Hippopotamus amphibius, Tursiops truncatus and Eschrichtius robustus rostral skin was quantified using ImageJ (NIH). We included measurements ranging from: a) top to start of rete ridge; b) start to end of rete ridge; and c) entire epidermis including rete ridge. Up to 10 individual measurements were included per image field per species. Measurements are reported as average epidermal thickness (μm) ± standard deviation (μm) in Table 1.

Pygmy Hippopotamus Genome

Genomic DNA was sonicated at the University of California, Riverside (UCR), Genomics Core Facility into ~550 bp fragments. We constructed a genomic library using Illumina’s NeoPrep Library Prep System. Paired-end sequencing (150 bp) was performed at UCR. Raw Illumina sequence data have been deposited at NCBI (PRJNA694317).

Genomic Screens for Inactivated Genes

We used a genome alignment of placental mammals with the human hg38 assembly as the reference [118]. Our gene loss detection method [68,119] was used to screen for genes that exhibit inactivating mutations in the genomes of bottlenose dolphin, killer whale, sperm whale, common minke whale, and river hippo. We started with 19,769 genes annotated by Ensembl (http://www.ensembl.org) version 90 [120] in the human genome and considered 18,363 genes that are present in the assemblies of at least 31 of 63 placental mammals. To identify genes that were potentially inactivated on the branch leading to hippos and cetaceans, we further extracted genes that are inactivated in all cetaceans and the river hippo. We excluded genes that are intact in less than three of six terrestrial outgroup artiodactyls included in the screen (Bos taurus [cow], Capra hircus [goat], Camelus ferus [wild Bactrian camel], Pantholops hodgsonii [Tibetan antilope], Bison bison [bison], Vicugna pacos [alpaca]). Finally, we used a more recent assembly of the river hippo genome (GCA_004027065.2) to exclude instances where assembly errors mistakenly led to genes classified as inactivated in the river hippo. This resulted in a final list of 38 genes (Table S1).

BLAST Searches and Alignments

Genomic sequences encoding ten genes of interest (ABCC11, ALOX15, AWAT1, KPRP, KRT2, KRT26, KRT77, KRTAP7–1, MOGAT3, TCHHL1) were downloaded from NCBI for Homo sapiens (human), Bos taurus (cow), and Equus caballus (horse). Sequences for each gene were aligned and exon annotations in Bos and Equus were compared against those in Homo to ensure that orthologous regions were annotated. Protein-coding sequences and flanking introns from Bos and Equus were employed in BLAST searches against other cetartiodactyls and perissodactyls, respectively, in NCBI’s ‘RefSeq Genome’ and ‘Whole-genome shotgun contigs’ databases. Additional perissodactyls included Ceratotherium simum (white rhinoceros) and Dicerorhinus sumatrensis (Sumatran rhinoceros). Additional cetartiodactyls included two camelids (Camelus ferus [wild Bactrian camel], Vicugna pacos [alpaca]), one suid (Sus scrofa [pig]), two bovids (Bubalus bubalis [water buffalo], Capra hircus [goat]), two giraffids (Giraffa camelopardalis [giraffe], Okapia johnstoni [okapi]), two cervids (Axis porcinus [hog deer], Odocoileus virginianus [white-tailed deer]), one hippopotamid (Hippopotamus amphibius [river hippopotamus]), four mysticetes (Balaena mysticetus [bowhead, downloaded from http://www.bowhead-whale.org/], Balaenoptera acutorostrata [common minke whale], B. bonaerensis [Antarctic minke whale], Eschrichtius robustus [gray whale]), and ten odontocetes (Physeter macrocephalus [sperm whale], Lipotes vexillifer [baiji], Delphinapterus leucas [beluga], Phocoena phocoena [harbor porpoise], Neophocaena asiaorientalis [narrow-ridged finless porpoise], Orcinus orca [killer whale], Lagenorhynchus obliquidens [Pacific white-sided dolphin], Sousa chinensis [Indo-Pacific humpback dolphin], Tursiops aduncus [Indo-Pacific bottlenose dolphin], Tursiops truncatus [common bottlenose dolphin]). Additional searches were performed with other perissodactyls or cetartiodactyls when the initial searches with Equus and Bos were unsuccessful in retrieving complete orthologs. Megablast was employed for highly similar sequences and blastn for less similar sequences. Complete protein-coding sequences and intervening introns were imported into Geneious 11.1.5 [121] and aligned against reference sequences with MAFFT [122] with minor adjustments by eye. Aligned sequences were annotated for exons and inspected for splice site mutations. Illumina sequences for Choeropsis liberiensis were imported into Geneious and protein-coding sequences for the above-mentioned genes were obtained using a map to reference approach with probe sequences from the closely related Hippopotamus amphibius. We allowed for a maximum mismatch of 6% per read and required at least two reads for base calling with a consensus threshold of 65%. We also used MAFFT to align complete protein-coding sequences from all taxa for each gene.

Inactivating Mutations

Final alignments for protein-coding sequences for each gene included 15–29 taxa given that some genes are deleted in one or more cetaceans. We inspected the final protein-coding alignment for each gene for inactivating mutations including exon deletions, frameshift insertions and deletions, altered start and stop codons, and premature stop codons (splice site mutations screened above). For each gene, parsimony optimizations with delayed transformation (deltran) were performed with PAUP* 4.0a150 [123] to map inactivating mutations to branches of the species tree (see below).

QUANTIFICATION AND STATISTICAL ANALYSIS

Phylogenetic Analyses

RAxML 8.2.11 [124] was run in Geneious to estimate maximum likelihood gene trees for each protein-coding alignment. Gene trees were inspected for suspicious relationships that conflict with the species tree (e.g., Camelus grouping with Sus instead of Vicugna) but none were found. Instead, gene tree incongruence was confined to conflicts that are readily explained by ILS such as Ruminantia grouping with Suoidea or Physeter grouping with Mysticeti [125]. Rapid bootstrap analysis (100 pseudoreplications) and a search for the best tree were performed in a single run. These analyses were performed with a GTR + Γ model of sequence evolution.

DN/dS Analyses

DN/dS analyses were performed with the codeml program of PAML 4.4 [126]. Analyses for each gene were performed with separate dN/dS categories for functional branches that lack inactivating mutations, fully pseudogenic branches that post-date the occurrence of an inactivating mutation on an earlier branch, and each transitional branch that records the first occurrence of an inactivating mutation (e.g., [83]). Analyses were performed with the codon frequency 1 (CF1) and codon frequency 2 (CF2) models of codeml. We also performed analyses with estimated and fixed (dN/dS =1.00) values for the fully pseudogenic branch category. We employed a species tree with higher level (interordinal, interfamilial) relationships from Meredith et al. [127] and intrafamilial relationships from Hassanin et al. [128] for terrestrial cetartiodactyls and McGowen et al. [129] for cetancodontans.

Estimation of Gene Inactivation Times

Equations from Meredith et al. [83] were used to estimate gene inactivation times in hippopotamids and cetaceans. We performed calculations using eight different combinations of codon model (CF1 or CF2), fixed (1.0) versus estimated values for dN/dS on fully pseudogenic branches, and equations that allow for one versus two synonymous substitution rates [83]. Mean inactivation dates for each gene are averages based on these eight different combinations. Divergence times for relevant nodes in these calculations were taken from McGowen et al. [129].

Supplementary Material

NIHMS1681163-supplement-1.pdf^{(1.9MB, pdf)}

Table S3: Estimated inactivation dates for pseudogenized skin genes, related to Figure 3.

NIHMS1681163-supplement-2.xlsx^{(34.9KB, xlsx)}

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Deposited Data
Choeropsis liberiensis Illumina whole-genome shotgun sequences	This paper	PRJNA694317
Nexus alignments of inactivated genes	This paper	https://doi.org/10.6084/m9.figshare.13549070.v1
Software and Algorithms
PAML	[126]	http://abacus.gene.ucl.ac.uk/software/paml.html
Geneious 11.1.5	[121]	https://www.geneious.com

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Springer et al. perform genomic and anatomical comparisons to determine if aquatic adaptations of the skin in hippos and cetaceans are shared derived or convergent features in these two clades. The results of these comparisons support the hypothesis that aquatic adaptions of the skin are convergent characters in hippos and cetaceans.

Cetaceans and hippos have differences in the thickness and organization of the skin
Genomic screens identified 8 skin genes that are inactivated in hippos and cetaceans
None of these 8 genes share inactivating mutations in hippos and cetaceans
Aquatic skin adaptations evolved independently in hippos and cetaceans

ACKNOWLEDGMENTS

This work was supported by NSF grant DEB-1457735 (J.G., M.S.S.), NIH grants U01-AR073159 and P30-AR075047 (M.V.P.), NSF grant DMS-1951144 (M.V.P.), and Pew Charitable Trust and LEO Foundation (M.V.P.). C.F.G-J. was supported by UC Irvine Chancellor’s ADVANCE Postdoctoral Fellowship Program, NSF-Simons Postdoctoral Fellowship, NSF grant (DMS1763272, to Qing Nie), Simons Foundation Grant (594598, to Qing Nie), and by a kind gift from the Howard Hughes Medical Institute Hanna H. Gray Postdoctoral Fellowship Program. M.H. was supported by the Max Planck Society and the LOEWE-Centre for Translational Biodiversity Genomics (TBG) funded by the Hessen State Ministry of Higher Education, Research and the Arts (HMWK). We thank the Smithsonian Institution for skin samples of hippos. C. Buell provided images of mammals. J. Geisler and four anonymous reviewers provided helpful comments on this manuscript.

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1681163-supplement-1.pdf^{(1.9MB, pdf)}

Table S3: Estimated inactivation dates for pseudogenized skin genes, related to Figure 3.

NIHMS1681163-supplement-2.xlsx^{(34.9KB, xlsx)}

Data Availability Statement

The Illumina sequencing data generated for this study are available under NCBI BioProject PRJNA694317.

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PERMALINK

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

Mark S Springer

Christian F Guerrero-Juarez

Matthias Huelsmann

Matthew A Collin

Kerri Danil

Michael R McGowen

Ji Won Oh

Raul Ramos

Michael Hiller

Maksim V Plikus

John Gatesy

SUMMARY

Graphical Abstract

INTRODUCTION

Figure 1. Two hypotheses for the evolution of aquatic adaptations.

RESULTS AND DISCUSSION

Comparative Skin Histology Between Terrestrial and Aquatic Cetartiodactyla

Table 1. Anatomical features of the skin.

Figure 2. Histological features of the skin in hippos and cetaceans.

Genomic Screens and Patterns of Gene Inactivation in Cetaceans and Hippos

Figure 3. Inactivating mutations in cetaceans and hippos.

Figure 4. Gene tree for MOGAT3.

Comparison of Inactivated Skin Genes and Epidermal Phenotypes in Cetaceans and Hippos

Figure 5. Skin structures and inactivated skin genes.

The Timing of Gene Inactivations in Hippopotamidae and Cetacea

Table 2. Inactivation dates for pseudogenized skin genes.

Integration of Molecular, Histological, and Paleontological Data

STAR*METHODS

RESOURCE AVAILABILITY

Lead Contact

Materials Availability

Data and Code Availability

EXPERIMENTAL MODEL AND SUBJECT DETAILS

METHOD DETAILS

Histology

Quantification of Epidermal Thickness

Pygmy Hippopotamus Genome

Genomic Screens for Inactivated Genes

BLAST Searches and Alignments

Inactivating Mutations

QUANTIFICATION AND STATISTICAL ANALYSIS

Phylogenetic Analyses

DN/dS Analyses

Estimation of Gene Inactivation Times

Supplementary Material

ACKNOWLEDGMENTS

Footnotes

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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