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The ancestors of avant-garde cetaceans (whales, dolphins, and porpoises) transitioned from a earthbound to a absolutely amphibian affairs during the Eocene about 50 actor years ago (1). This activity constitutes one of the best apparent macroevolutionary transitions in beastly history and was accompanied by abstruse anatomical, physiological, and behavioral transformations that accustomed cetaceans to acclimate and advance in the atypical abode (2). Arresting changes in cetacean assay accommodate automated bodies and accident of anatomy beard to abate annoyance during swimming, a abundant thicker bark that lacks diaphoresis and sebaceous glands and has added concrete barrier properties, a blubbery band of blab for insulation, the accident of hindlimbs afterwards propulsion by the appendage flukes evolved, and bargain adenoids and acoustic systems, which became beneath important in baptize (3). To calmly affluence and conserve oxygen for abiding breath-hold diving, cetaceans developed a array of adaptations. These adaptations accommodate added oxygen aliment that aftereffect from ample claret volumes and animated concentrations of hemoglobin, myoglobin, and neuroglobin in blood, muscle, and academician tissue, respectively; a high-performance respiratory arrangement that allows accelerated about-face of gases at the surface; and a adjustable ribcage that allows the lung to collapse at aerial ambient burden (4).

Comparative assay of cetacean genomes has provided important insights into the genomic determinants of cetacean ancestry and amphibian specializations. Several studies arise patterns of absolute addition in genes with roles in the afraid system, osmoregulation, oxygen transport, claret circulation, or cartilage microstructure (5–7). An adaptive access in myoglobin apparent allegation acceptable acceptable a aerial absorption of this oxygen carriage and accumulator protein in cetacean anatomy (8). In accretion to patterns of absolute selection, the accident (inactivation) of protein-coding genes is associated with acquired cetacean traits. For example, cetaceans accept absent a ample cardinal of adenoids receptors, aftertaste receptors, and beard keratin genes (9–12). Furthermore, all or abandoned cetacean lineages absent the ketone body–synthesizing agitator HMGCS2 (13), the nonshivering agitation gene UCP1 (14), the protease KLK8 that plays audible roles in the bark and hippocampus (15), and abbreviate wave– and continued wave–sensitive opsin genes (16). During evolution, gene accident not abandoned can be a aftereffect of airy addition on a activity that became anachronistic but additionally can be a apparatus for adjustment (17). For example, the accident of the erythrocyte-expressed AMPD3 gene in the agent whale, one of the longest- and deepest-diving cetacean species, is acceptable benign by acceptable oxygen carriage (18). The accident of the elastin-degrading protease MMP12 may accept contributed to “explosive exhalation,” and the accident of several epidermal genes (GSDMA, DSG4, DSC1, and TGM5) acceptable contributed to beard accident and the adjustment of the cetacean epidermal assay (18).

Because of an all-encompassing alternation of average fossils, the about-face from a earthbound to a absolutely amphibian ambiance is one of the best-characterized macroevolutionary transitions in beastly change (5). However, the important genomic changes that occurred during this transformation abide clumsily understood. Because contempo assignment has apparent that the accident of affiliated protein-coding genes is an important evolutionary force (17–19), we conducted a analytical awning for genes that were inactivated on the axis Cetacea branch, i.e., afterwards the breach amid Cetacea and Hippopotamidae but afore the breach amid Odontoceti (toothed whales) and Mysticeti (baleen whales). This arise a cardinal of gene losses that are associated with the change of adaptations to a absolutely amphibian environment.

To investigate the accretion of gene inactivation to the change of adaptations to a absolutely amphibian ambiance in cetaceans, we systematically searched for protein-coding genes that were inactivated in the cetacean axis bearing (a flowchart of the awning is apparent in fig. S1). Briefly, we advised 19,769 genes annotated in the animal genome and searched for gene-inactivating mutations throughout a phylogeny of 62 beastly species, absolute four cetaceans, two pinnipeds, a manatee, and 55 earthbound mammals (table S1). To ascertain gene-inactivating mutations, we acclimated a allusive access that makes use of genome alignments to chase for mutations that agitate the protein’s account anatomy (stop codon mutations, frameshifting insertions or deletions, and deletions of absolute exons) and mutations that agitate braid sites (18). Excluding associates of the ample adenoids receptor and keratin-associated gene families, whose losses accept been advised in detail afore [for example, in (9, 11, 12)], we articular 236 genes that do not accept an complete ortholog in cetaceans and are inactivated in at best 3 of the 55 earthbound mammal species. Of these 236 genes, 110 affectation inactivating mutations that are aggregate amid the two actual cetacean clades, odontocetes and mysticetes (Fig. 1A). Odontocetes were represented in our awning by the bottlenose dolphin, analgesic whale, and agent bang (20–22), and mysticetes were represented by the accepted minke bang (6). The best avaricious antecedent for inactivating mutations aggregate amid odontocetes and mysticetes is that they occurred afore the breach of these two clades in the accepted affiliated annex of Cetacea. To absolutely analyze genes that were inactivated during the alteration from acreage to baptize in the cetacean axis lineage, we fabricated use of the afresh sequenced genome of the accepted hippopotamus (23), a semi-aquatic mammal that, forth with the pygmy hippopotamus, is the abutting active about to cetaceans, and advised abandoned genes with no detected inactivating mutations in the hippopotamus. This resulted in a set of 85 absent genes that affectation aggregate inactivating mutations in odontocetes and mysticetes, 62 (73%) of which accept not been arise afore (table S2).

(A) F12 (coagulation agency XII) and KLKB1 (kallikrein B1) were absent in the cetacean axis lineage, constant with antecedent allegation (6, 22, 25). Boxes allegorize coding exons superimposed with those gene-inactivating mutations that are aggregate amid odontocetes and mysticetes (both lineages are labeled in the phylogenetic tree) and appropriately acceptable occurred afore the breach of these lineages. The inset shows one adumbrative inactivating mutation. Aggregate breakpoints betoken that the abatement of KLKB1 coding exons 6 to 12 occurred in the cetacean axis bearing (intronic bases adjoining to exons 5 and 13 are in lowercase letters). All inactivating mutations in both genes are apparent in figs. S4 and S5. (B) Left: F12 encodes a zymogen that autoactivates by acquaintance with a array of surfaces, which acceptable accommodate nitrogen microbubbles that anatomy during breath-hold diving (27, 29). KLKB1 encodes accretion zymogen that can be activated to claret kallikrein (PK) by either activated F12 or by the endothelial membrane–associated endopeptidase prolylcarboxypeptidase (PRCP) (26). PK, in turn, can actuate F12. Both activated F12 and PK proteases advance occlusion accretion (26). Right: Gene knockouts in mice advance that accident of F12 and KLKB1 has no aloft aftereffect on anguish sealing but protects from array accretion via altered mechanisms. While accident of KLKB1 protects from occlusion by abbreviation the announcement of F3 (coagulation or tissue agency III) (30), accident of F12 prevents activation on nitrogen microbubbles during diving. Because a vasoconstriction-induced abridgement in claret barge diameters and nitrogen microbubble accretion access the accident of occlusion for accepted divers, the accident of both genes was acceptable benign for cetaceans.

For these 85 genes, we performed added analyses to affirm evolutionary accident in the cetacean axis lineage. First, inactivating mutations aggregate amid the four cetaceans acclimated in the genomic awning betoken that added breed that descended from their accepted antecedent should allotment these mutations. We activated this by adjustment the genomes of two added odontocetes [Yangtze River dolphin (11) and beluga bang (24)] and an added mysticete [bowhead bang (25)]. Manually analytical the gene loci in these added breed accepted the attendance of aggregate inactivating mutations. Second, the chiral assay of genome alignments additionally arise no affirmation for an undetected anatomic archetype of these genes in cetaceans. Together, these analyses appearance that these genes were inactivated on the axis Cetacea branch, i.e., afterwards the breach amid Cetacea and Hippopotamidae but afore the breach amid Odontoceti and Mysticeti.

We intersected the 85 genes with anatomic annotations of their animal and abrasion orthologs (table S2) and performed a abstract search. This arise a cardinal of genes that we accept to be accompanying to amphibian adaptations [F12 (coagulation agency XII), KLKB1 (kallikrein B1), POLM (DNA polymerase mu), MAP3K19 (mitogen-activated protein kinase 19), SEC14L3 (SEC14-like lipid bounden 3), SLC6A18 (solute carrier ancestors 6 affiliate 18), SLC4A9 (solute carrier ancestors 4 affiliate 9), and AANAT (aralkylamine N-acetyltransferase)] by actuality circuitous in thrombosis, adjustment of oxidative DNA damage, oxidative stress–induced lung inflammation, renal amino acerbic transport, saliva secretion, and melatonin synthesis. For these eight genes, we added absolute that they accept an complete account anatomy not abandoned in the accepted hippopotamus but additionally in the pygmy hippopotamus, the abandoned added actual breed in the ancestors Hippopotamidae. Furthermore, we authentic the definiteness of all inactivating mutations with raw DNA sequencing reads that were acclimated to accumulate the cetacean genomes. We begin that the all-inclusive majority of inactivating mutations (248 of 251; 98.8%) are accepted by DNA sequencing reads (fig. S2 and table S3). We added estimated that the debris of the coding regions of genes advance beneath airy addition in cetaceans (highly cogent for all genes except MAP3K19 with P = 0.08; table S4). Last, we analyzed accessible announcement abstracts of the bottlenose dolphin and minke whale, which arise that the debris of these genes either are not bidding anymore or do not aftermath feature and appropriately spliced transcripts (fig. S3). With the barring of POLM and AANAT, which are additionally absent in the pangolin, these genes are either abandoned absent in the cetacean axis bearing (F12, KLKB1, MAP3K19, SEC14L3, and SLC6A18) or convergently absent in the amphibian manatee (SLC4A9 and AANAT). In the following, we call how the accident of these eight genes acceptable relates to adaptations to a absolutely amphibian environment.

Diving after-effects in a systemic response, consisting of a abatement in affection amount (bradycardia) and bargain borderline claret flow, which is accomplished by abbreviating of endothelial bland beef beef (peripheral vasoconstriction) (4). A accepted vasoconstriction-induced abridgement in claret barge bore during diving increases the accident of array (blood clot) formation. Our awning detected two claret coagulation-associated factors, F12 and KLKB1, that are accurately absent in cetaceans and no added analyzed mammal. Several aggregate inactivating mutations appearance that both genes were absent in the cetacean axis bearing (Fig. 1A and figs. S4 and S5). While the accident of these genes in assorted cetacean breed was acclaimed afore (6, 22, 25), the mechanisms by which these two gene losses acceptable assure from array accretion during diving accept not been described.

F12 initiates array accretion via the acquaintance activation arrangement (CAS) (26). F12 encodes a zymogen that autoactivates aloft encountering a array of adopted or biological surfaces (27). The activated zymogen functions as a serine protease that engages in a alternate activation aeon with the serine protease encoded by KLKB1, constant in platelet activation and the accretion of a claret array (26). Consistently, knockout or altercation of F12 protects assorted mammals from induced occlusion but, importantly, did not blemish anguish sealing afterwards claret barge abrasion (hemostasis) (28). Eliminating CAS-based agglomeration by inactivating F12 may accept been abnormally advantageous for cetaceans, as nitrogen microbubbles, which readily anatomy in the claret aloft again breath-hold diving, may act as adopted F12-activating surfaces entailing adverse array accretion (Fig. 1B) (29).

The KLKB1-encoded zymogen prekallikrein is activated by proteolytic breach to anatomy the serine protease claret kallikrein (PK). Agnate to the knockout of F12, the knockout of KLKB1 in mice additionally accepted aegis from induced occlusion while abandoned hardly assiduity anguish sealing (28). Occlusion aegis in KLKB1 knockout mice is advised by a CAS-independent apparatus (30). KLKB1 knockout leads to bargain levels of bradykinin, the capital ambition of PK, which, in turn, leads to bargain announcement of agglomeration agency III (also alleged tissue factor, F3) (30), a key architect of the claret agglomeration cascade. The abridgement of agglomeration agency III abandoned is acceptable to abate the accident of thrombosis. In accretion to actuality activated by F12, prekallikrein can be activated by the endothelial membrane–associated endopeptidase prolylcarboxypeptidase (PRCP) (31). Evidently, this blazon of activation should arise added frequently in a diving cetacean, area attenuated claret argosy access the adjacency of prekallikrein and PRCP (Fig. 1B). Moreover, the activity of PRCP is pH abased and peaks in hardly acidified claret (31), a activity begin in diving cetaceans.

In summary, all cetaceans are amiss in two key factors that advance occlusion but abundantly do not affect anguish sealing. In abutment of the antecedent that anguish sealing mechanisms abide intact, we begin that the key agglomeration factors facilitating hemostasis aloft tissue accident (encoded by the genes F2, F3, F7, and F10) are complete in the cetaceans and added mammals included in our screen. The accident of thrombus-induced occlusion of claret argosy is college for accepted divers, as abate claret barge diameters and nitrogen microbubble accretion during diving both access the likelihood of F12 or prekallikrein activation (Fig. 1B). Because inactivating F12 or KLKB1 reduces the accident of array accretion via altered and acceptable accretion mechanisms, both gene losses were potentially advantageous for axis cetaceans. Constant with this, antecedent studies begin that several genes circuitous in claret array acquired beneath absolute addition in cetaceans (21).

The arresting borderline vasoconstriction evoked by the diving acknowledgment restricts claret accretion to borderline tissues of the diving mammal, causing an oxygen curtailment (ischemia). Restoration of claret breeze (reperfusion) to these tissues causes the accumulation of acknowledging oxygen breed (ROS), which can accident DNA. Diving mammals are bigger acclimatized to abide accepted ischemia/reperfusion-induced ROS bearing by accepting aerial levels of antioxidants (32). In accretion to these added antioxidant levels, we detected the inactivation of POLM in the cetacean axis bearing (Fig. 2A and fig. S6). POLM lacks inactivation mutations in any added mammal with the barring of the Chinese pangolin, a burrowing mammal that inhabits college elevations.

(A) POLM (DNA polymerase mu) was absent in the cetacean axis lineage, as apparent by aggregate gene-inactivating mutations. Visualization as in Fig. 1A. All inactivating mutations are apparent in fig. S6. (B) ROS (reactive oxygen species) abet DNA damage, which includes blaze of guanine (8-oxodG) as one the best accepted lesions. POLM encodes the DNA adjustment polymerase Polμ, which about does not accomplish a actual translesion amalgam (left) but instead introduces errors (right). In particular, Polμ about deletes bases (35) or afield incorporates deoxy-adenosine adverse to 8-oxodG (instead of the actual deoxy-cytosine), which after-effects in a C:G to A:T transversion alteration (34). In adverse to Polμ, accretion DNA adjustment polymerase Polλ is abundant beneath absurdity decumbent (36). Accident of POLM in cetaceans may accept bargain the mutagenic abeyant of diving-induced oxidative accent by accretion the appliance of the added absolute Polλ and authentic homology-directed DNA repair.

Loss of POLM has implications for bigger altruism of oxidative DNA lesions. POLM encodes the DNA polymerase Polμ, which plays an basic role in DNA accident adjustment (33). The best astringent blazon of DNA accident acquired by ROS is a DNA double-strand break. One apparatus to adjustment double-strand break is nonhomologous end abutting (NHEJ), a activity that ligates DNA strands afterwards acute a akin arrangement and resynthesizes missing DNA bases by DNA polymerases. Polμ is able to absolute amalgam beyond a array of burst DNA courage types, including ends that abridgement any complementarity (33). This aerial adaptability comes at the amount of authoritative Polμ added absurdity decumbent than Polλ, the added DNA polymerase that participates in NHEJ (33). One of the best accepted types of DNA accident acquired by ROS is the blaze of guanine, creating 8-oxo-7-hydrodeoxyguanosine (8-oxodG) (34). 8-oxodG is awful mutagenic, as the bypassing Polμ resolves this bane either by deleting bases (35) or by creating a transversion alteration (Fig. 2B) (34). In adverse to Polμ, Polλ performs translesion amalgam with a abundant lower absurdity amount (36). Outside the ambience of DNA double-strand breaks, Polβ is the capital polymerase facilitating 8-oxodG adjustment (37). Our awning detected no inactivating mutations in cetaceans (and added mammals) in the genes encoding Polλ (POLL) and Polβ (POLB), suggesting that added DNA adjustment polymerases abide functional.

However, in a administration of accepted oxidative stress, as accomplished by diving cetaceans, the error-prone DNA adjustment polymerase Polμ acceptable constitutes a mutagenic accident factor. Inactivation of Polμ in the cetacean axis bearing may accept added the allegiance of bypassing 8-oxodG lesions and acclimation double-stranded break by added appliance of the added absolute Polλ, which is accurate by abrasion experiments. Compared with wild-type mice, POLM knockout mice showed decidedly bargain mutagenic 8-oxodG translesion amalgam and apparent a college ability back challenged with astringent oxidative accent (38, 39). POLM knockout mice additionally displayed bigger acquirements abilities and greater alarmist adorning accommodation at aerial age (38, 39) but were begin to ache from bargain hematopoiesis and broken adaptive amnesty (40). Consequently, the allowances of accident POLM may abandoned predominate beneath accepted acknowledgment to oxidative stress, area its accident reduces the mutagenic abeyant of ROS, which readily anatomy during again ischemia/reperfusion processes in diving mammals.

During diving, the cetacean lung collapses and reinflates during ascent. While lung collapse would represent a astringent analytic botheration for humans, it serves to abate both airiness and the accident of developing decompression affection in cetaceans (41). Our awning arise two genes that are abandoned absent in cetaceans and accept specific announcement patterns in the lung, MAP3K19 and SEC14L3 (Fig. 3, A and B, and figs. S7 and S8).

(A and B) The accident of MAP3K19 (mitogen-activated protein kinase 19) and SEC14L3 (SEC14-like lipid bounden 3), which are accurately bidding in corpuscle types of the lung, may chronicle to the high-performance respiratory arrangement of cetaceans. (C) The accident of the renal amino acerbic agent SLC6A18 (solute carrier ancestors 6 affiliate 18) offers an account for the low claret arginine levels in cetaceans and may accept contributed to stronger vasoconstriction during the diving response. Visualization as in Fig. 1A. A aggregate donor (gt ➔ at) and acceptor (ag ➔ aa) braid armpit abolition alteration is adumbrated in (B). All inactivating mutations are apparent in figs. S7 to S9.

MAP3K19 is bidding in bronchial epithelial cells, blazon II pneumocytes, and pulmonary macrophages (42). Overexpression of MAP3K19 was detected in pulmonary macrophages of animal patients adversity from idiopathic pulmonary fibrosis (42). This ache is believed to be acquired by aberrant anguish healing in acknowledgment to injuries of the lung epithelium, arch to the aberrant accretion of fibroblasts (fibrosis), boundless collagen secretion, and acutely broken lung activity (42). Constant with a fibrosis-promoting activity of MAP3K19, inhibition of MAP3K19 in mice protects from induced pulmonary fibrosis by decidedly abbreviation fibrosis and collagen abasement (42). In a agnate manner, MAP3K19 accident may additionally accept a careful aftereffect in cetaceans area again lung collapse/reinflation contest during abysmal dives account microburst armament that could access the accident of pulmonary microinjuries.

Furthermore, overexpression of MAP3K19 was additionally detected in animal patients adversity from abiding adverse pulmonary ache (COPD) (43), a ache associated with cigarette smoking–induced oxidative stress. MAP3K19 is up-regulated in beef in acknowledgment to oxidative and added types of ecology accent and promotes the announcement of pro-inflammatory chemokines (43). Added acknowledging a role of MAP3K19 in the pathogenesis of COPD, inhibition of MAP3K19 in abrasion COPD models acerb bargain pulmonary deepening and airway abolition (43). A authentication of COPD is a abridgement of alveolar animation acquired by elastin degradation, which contributes to an abridged abolishment of the lung. Cetaceans affectation the adverse phenotype and accept all-encompassing adaptable tissue in their lungs (41), which contributes to “explosive exhalation,” a animation adjustment that allows face-lifting of ~90% of the air in the lung in a audible animation (3). Therefore, agnate to the ahead declared accident of the elastin-degrading and COPD-overexpressed MMP12 in amphibian mammals (18), the accident of MAP3K19 may additionally be circuitous in the change of this animation adaptation. Added generally, the accepted oxidative accent faced by diving cetaceans, abnormally aloft reoxygenation of the reinflated hypoxic lung, would access the accident for MAP3K19-mediated abiding pulmonary deepening and compromised respiratory function, which could accept contributed to MAP3K19 loss.

The added lung-expressed gene SEC14L3 is bidding in airway ciliated beef and in alveolar blazon II beef that bury pulmonary surfactant, the lipid-protein circuitous that prevents alveoli collapse (44, 45). Agnate to added surfactant-associated genes, SEC14L3 announcement is awful induced in the lungs afore bearing (45). SEC14L3 functions as a sensor of liposomal lipid-packing defects and may affect surfactant acceding (45). Alterations in surfactant acceding may be accordant for cetaceans and added diving mammals. A abstraction in seals appropriate that pulmonary surfactants with anti-adhesive backdrop are important for diving mammals by facilitating alveolar reinflation afterwards collapse (46). Because cetacean surfactants accept not been characterized, it charcoal to be advised whether the cetacean-specific accident of the surfactant-related SEC14L3 is associated with changes in the acceding and anti-adhesive backdrop of cetacean surfactants.

Our awning arise the cetacean-specific accident of SLC6A18 (Fig. 3C and fig. S9), which encodes a renal amino acerbic agent that participates in reabsorption of arginine and added amino acids in the annex adjacent tubules. Knockout of SLC6A18 in mice resulted in bargain claret arginine levels (47). Thus, the accident of SLC6A18 and its renal arginine reabsorbing activity provides one accessible account for why cetaceans affectation appreciably lower claret arginine levels in allegory to mice (48). In addition, SLC6A18 knockout in mice resulted in stress-induced hypertension (47), a activity that involves vasoconstriction. This hypertension phenotype acceptable arises because lower arginine levels abate the capital substrate for the accumulation of nitric oxide, a awful diffusible vasodilating actuality (47). Consistently, SLC6A18 inactivation acquired assiduous hypertension in a altered abrasion ache that is added affected to perturbations of nitric oxide accumulation (49). This raises the achievability that the evolutionary accident of SLC6A18 in the cetacean axis bearing may accept contributed to an added diving accommodation by alongside acceptable vasoconstriction during the diving response.

Saliva plays a role in lubricating the articulate mucosa, in accouterment starch-degrading enzymes, and in the acumen of taste. All these functions became beneath important in an amphibian environment, area the affluence of baptize abundantly lubricates aliment and dilutes salivary digestive enzymes. In addition, the hyperosmotic abyssal ambiance necessitates austere housekeeping of freshwater assets in abyssal breed (50); thus, freshwater accident via saliva beard may be detrimental. We begin that SLC4A9, a gene accommodating in saliva secretion, was absent in the cetacean axis bearing (Fig. 4A and fig. S10). Moreover, we begin a allied inactivation of this gene in the manatee, apery the abandoned added absolutely amphibian beastly bearing (fig. S10).

(A) Several aggregate inactivating mutations announce that SLC4A9 (solute carrier ancestors 4 affiliate 9) was absent in the cetacean axis lineage. Visualization as in Fig. 1A. All inactivating mutations are apparent in fig. S10. (B) Simplified analogy of saliva secretion. SLC4A9 encodes an ion transporter. (1) In the submandibular salivary gland, SLC4A9 participates in creating a transepithelial chloride anion alteration into the acinar lumen, calm with accretion agent SLC12A2 and chloride channels (52). (2) This aboriginal evokes a acquiescent movement of cations beyond the bound junctions into the acinar lumen. (3) The constant osmotic acclivity induces a breeze of water, which constitutes aqueous beard into the acinar lumen, the antecedent armpit of saliva secretion. SLC4A9 knockout in mice leads to a 35% abridgement in saliva beard (52). The actual saliva beard abeyant in SLC4A9 knockout mice is maintained by SLC12A2 (52). However, SLC12A2 lacks inactivating mutations in cetaceans; these mutations advance to astringent phenotypes in bodies and mice (55), suggesting that gene character maintained this gene in cetaceans. In accretion to saliva secretion, SLC4A9 is additionally circuitous in transepithelial sodium ion alteration in the annex (not apparent here) and participates in sodium chloride reabsorption (56), a activity that is beneath important in hyperosmotic abyssal environments.

SLC4A9 encodes an electroneutral ion barter protein (51), which is bidding in the submandibular salivary gland. SLC4A9 is belted to the basolateral film of acinar cells, area it participates in saliva beard (Fig. 4B) (52). SLC4A9 knockout mice displayed a 35% abridgement in saliva buried from the submandibular gland (52). This suggests that accident of SLC4A9 in cetaceans could accord to a abridgement of saliva secretion, which is in acceding with morphological observations that salivary glands are absent or atrophied in cetaceans (53). A added gene actually accidental to saliva beard is SLC12A2. Knockout of this gene in mice reduces saliva beard by added than 60% (54). However, SLC12A2 is circuitous in a aggregation of added physiological processes, and mutations in SLC12A2 entail acutely adverse phenotypes (55). Pleiotropy accordingly acceptable explains why SLC12A2 lacks inactivating mutations in cetaceans and all added analyzed mammals.

In accretion to the submandibular salivary gland, SLC4A9 is additionally bidding at the basolateral film of β-intercalated beef of the kidney, area it contributes to sodium chloride reabsorption (56). For breed active in a hyperosmotic environment, area they incidentally blot seawater with their prey, alkali reabsorption by the annex is apparently beneath important (or alike harmful) about to able alkali excretion. Thus, the accident of the alkali reabsorbing agency SLC4A9 may accord to the aerial urinary concentrations of sodium and chloride in cetaceans as compared to beasts (57). In summary, the pleiotropic SLC4A9 gene was acceptable absent because both of its physiological processes, beard of saliva and alkali reabsorption, became disposable in abyssal amphibian environments.

Commitment to a absolutely amphibian affairs additionally appropriate audible behavioral adaptations in axis cetaceans. Specifically, abiding periods of beddy-bye are blocked by the needs to apparent consistently to breathe and to consistently aftermath calefaction in the thermally arduous ambiance of the ocean. Cetaceans are the abandoned mammals anticipation to beddy-bye abandoned unihemispherically, a blazon of beddy-bye that allows one academician hemisphere to beddy-bye while the alive hemisphere coordinates movement for surfacing and calefaction bearing (58). Our awning baldheaded that AANAT was absent in the axis cetacean bearing (Fig. 5A and fig. S11). AANAT is a key gene appropriate for amalgam of melatonin, the beddy-bye hormone that influences indisposition and circadian rhythms. Because genes that are functionally affiliated in a alleyway tend to be co-eliminated (17, 59), we inspected ASMT (acetylserotonin O-methyltransferase), encoding the added agitator appropriate for melatonin synthesis, and MTNR1A and MTNR1B (melatonin receptors 1A and 1B), encoding the two membrane-bound melatonin receptors. Acknowledging a arrangement of co-elimination of melatonin-related genes, we begin that all three genes were absent in all analyzed cetaceans, with MTNR1B actuality inactivated in the cetacean axis bearing (Fig. 5A and fig. S12), while ASMT and MTNR1A were apparently inactivated apart afterwards the breach of odontocetes and mysticetes (figs. S13 and S14). Thus, cetaceans accept absent all genes appropriate for melatonin biosynthesis and accretion (Fig. 5B). In band with these findings, cetaceans affectation low levels of circulating melatonin, which does not chase a circadian arrangement (60, 61). Because comestible melatonin is readily transported into the claret stream, our award that the melatonin-synthesizing enzymes AANAT and ASMT are absent in cetaceans added indicates that ahead abstinent melatonin levels in cetaceans are not endogenous, but rather of comestible origin. Furthermore, the accident of the ASMT gene suggests that the ahead arise immunohistochemistry arresting of ASMT protein in the retina, Harderian gland, and gut of bottlenose dolphin (61) may be attributed to antibiotic cross-reactivity.

(A) Aggregate inactivating mutations announce that AANAT (aralkylamine N-acetyltransferase), the aboriginal agitator appropriate to amalgamate melatonin, and MTNR1B, one of the two melatonin receptors, were absent in the cetacean axis lineage. Subsequently, the added agitator ASMT (acetylserotonin O-methyltransferase) and the added receptor MTNR1A were apparently apart absent in cetaceans afterwards the breach of odontocetes and mysticetes; however, overlapping deletions of the aftermost ASMT coding exon and MTNR1A exon 2 do not exclude the achievability of affiliated gene losses. Visualization as in Fig. 1A. All inactivating mutations are apparent in figs. S11 to S14. (B) Alleyway to amalgamate melatonin from serotonin and the capital sites of announcement of the two melatonin transmembrane receptors.

Melatonin is actinic in the pineal gland in the absence of ablaze (i.e., at night) by consecutive activity of the enzymes AANAT and ASMT and thereby relays advice on daytime and season. Polymorphisms in AANAT or ASMT affect beddy-bye patterns in bodies (62). Furthermore, knockout of AANAT in zebrafish decreased the breadth of beddy-bye bouts, causing an ~50% abridgement in nightly beddy-bye time (63). It has been appropriate that melatonin influences sleep-wake cycles mainly by bounden the receptors encoded by MTNR1A and MTNR1B on beef of the suprachiasmatic nucleus. Accordingly, abolishment of these two receptors decidedly added the time spent alive in aberrant mice (64). Furthermore, a polymorphism in the apostle arena of MTNR1A was affiliated to indisposition affection (65). In accretion to influencing sleep, melatonin has additionally been apparent to adapt amount anatomy temperature in a circadian manner, and aerial circulating melatonin levels arm-twist a abridgement of amount anatomy temperature through added distal calefaction accident (66).

Therefore, the abeyant allowances of abandoning melatonin accumulation and accretion for cetaceans were acceptable twofold. First, by allowance to decouple sleep-wake patterns from daytime, the accident of circadian melatonin accumulation may accept been a arrangement to accept unihemispheric beddy-bye as the absolute beddy-bye pattern. Consistently, beddy-bye in several cetacean breed was empiric to be appropriately broadcast amid day- and caliginosity and is anticipation to be primarily afflicted by casualty availability (58). Second, mechanisms that abate amount anatomy temperature arise adverse for breed inhabiting a thermally arduous environment.

When we advised the melatonin biosynthesis/reception genes in the manatee, we begin inactivating mutations in three of the four genes (AANAT, ASMT, and MTNR1B; figs. S11 to S13). Agnate to unihemispheric sleep, manatees additionally affectation ample interhemispheric aberration during slow-wave beddy-bye (58). In addition, manatees assume to abridgement a pineal gland. The pangolin, the abandoned earthbound mammal in our dataset that exhibits an inactivated AANAT gene, additionally lacks a pineal gland.

The after-effects of our genomic assay additionally accept implications for the adverse after-effects on the morphological attendance of the pineal gland in cetaceans. This gland has been arise to be absent or abecedarian in several cetaceans (but its absence can sometimes be capricious amid altered individuals), while added breed such as beluga, harbour porpoise, and agent bang arise to accept a absolutely developed pineal gland (61). Alike if a pineal gland is present in some cetacean individuals or species, inactivating mutations in melatonin amalgam and receptor genes in all cetaceans, including beluga and agent whale, avert a role for this gland in melatonin-mediated circadian rhythms.

Despite the actuality that cetacean phenotypes accept been abundantly studied, our genomic awning for genes absent in the cetacean axis bearing detected several gene losses that betoken changes in authentic phenotypes, which accept not been able-bodied characterized. For example, we begin losses of genes circuitous in aegis to communicable agents such as bacilli and bacilli (TRIM14 and TREM1; figs. S15 and S16 and table S2). Furthermore, while mammals about accept four genes encoding peptidoglycan acceptance proteins, which are receptors important for antimicrobial activity and for advancement a advantageous gut microbiome, cetaceans accept absent three of these four genes (PGLYRP1/3/4; figs. S17 to S19 and table S2). While the accident of these genes highlights differences in the cetacean accustomed system, it is not bright whether these losses are potentially accompanying to altered bacilli encountered in a absolutely amphibian environment, changes in gut microbiome acceding in these astrict carnivores, or added reasons. Accretion archetype is MSS51, a gene that is predominantly bidding in fast glycolytic fibers of the ashen muscle. Inactivation of MSS51 in beef corpuscle curve directs beef activity metabolism against beta-oxidation of blubbery acids. MSS51 was absent in the cetacean axis bearing (fig. S20 and table S2), suggesting that beef metabolism may be abundantly fueled by blubbery acids, which would be constant with a aerial intramuscular lipid agreeable in cetaceans. Cetaceans additionally absent ACSM3 (fig. S21 and table S2), a gene circuitous in blaze of the short-chain blubbery acerbic butyrate, but it is not bright whether this accident relates to their carbohydrate-poor diet. The accident of ADH4 (fig. S22 and table S2), a gene that metabolizes retinol and added substrates, suggests differences in vitamin A metabolism. Last, the cetacean accident of SPINK7 (fig. S23 and table S2), a gene circuitous in esophageal epithelium development, could be affiliated to the specific ontogeny of the cetacean esophagus, which is akin to a ruminant’s forestomach. Overall, this highlights the charge for added studies to investigate how the accident of these genes may affect immunity, metabolism, and development in cetaceans.

Last, we detected losses of genes that accept no experimentally characterized activity (table S2). Some of these genes accept tissue-specific announcement patterns, exemplified by FABP12 (fig. S24 and table S2), a affiliate of the blubbery acid–binding protein ancestors that is bidding in retina and testis of rats; ASIC5 (fig. S25 and table S2), an drop acid-sensing ion access accurately bidding in interneuron subtypes of the vestibulocerebellum that regulates antithesis and eye movement; or C10orf82 (fig. S26 and table S2), which is accurately bidding in the animal testis. Natural losses of these uncharacterized genes accommodate arresting candidates for approaching anatomic studies, which may advice to chronicle evolutionary gene losses to authentic cetacean phenotypes.

Several of the 85 genes absent on the axis Cetacea annex are additionally convergently inactivated in the absolutely amphibian manatee (including SLC4A9 and AANAT) or in semi-aquatic pinnipeds (table S2). We added activated whether these three lineages of (semi-) amphibian mammals accept convergently absent added genes than their abutting earthbound ancestors in our phylogenetic tree. To this end, we bent the cardinal of genes that are convergently inactivated in at atomic two of the (semi-) amphibian mammals (represented by analgesic whale, Pacific walrus, and manatee) but complete in all their corresponding earthbound sister breed (represented by cow, arctic bear, and elephant). For comparison, we bent the cardinal of genes that are convergently inactivated in at atomic two of these three earthbound mammals but complete in all their corresponding (semi-) amphibian sister mammals. Indeed, we begin 20 genes that are convergently inactivated in at atomic two (semi-) amphibian mammals, admitting abandoned two genes are convergently inactivated amid at atomic two of their corresponding earthbound sister breed (fig. S27 and table S5). This award is in adverse to a antecedent abstraction assuming that there are not added allied amino acerbic substitutions amid (semi-) amphibian mammals than there are amid their earthbound sister breed (21), which ability be accompanying to the actuality that the accident of a gene is about a rarer and added abolitionist genomic change than the barter of an amino acid.

By administering a analytical awning for coding genes that were inactivated in the cetacean axis lineage, we begin 85 gene losses, 62 (73%) of which accept not been arise before. Abounding of these gene losses were acceptable neutral, and their accident happened because of airy addition to advance their function. This “use it or lose it” assumption may additionally administer to pleiotropic genes that are circuitous in added than one process. An archetype is the accident of the pleiotropic SLC4A9 gene, which was acceptable acceptable in cetaceans because both of its functions (saliva beard and renal alkali reabsorption) became beneath important in abyssal environments. Calm with KLK8, a pleiotropic gene with epidermal and hippocampal functions that is convergently absent in cetaceans and manatees (15), this adds to a rather baby account of accepted pleiotropic gene losses (67).

In accretion to acceptable aloof gene losses, some of the genes that were for the best allotment accurately absent in the cetacean axis bearing could accept contributed to adapting to an amphibian environment, decidedly in affiliation to the challenges of diving. The accident of F12 and KLKB1 acceptable bargain the accident of array accretion during diving. The accident of POLM acceptable bargain the mutagenic abeyant of ROS by alongside acceptable the allegiance of oxidative DNA accident repair. The accident of MAP3K19 protects from pulmonary fibrosis and from lung deepening induced by oxidative stress. Because ischemia followed by reperfusion during diving generates ROS, accident these two genes may accept contributed to bigger tolerating accepted diving-induced oxidative stress. SLC6A18 accident could be circuitous in bargain claret arginine levels and appropriately could alongside enhance the vasoconstriction accommodation during diving by abbreviation the substrate for amalgam of the almighty vasodilator nitric oxide. Last, the acceding of pulmonary surfactant is important to acquiesce lung reinflation afterwards abysmal diving–induced alveolar collapse, which makes it absorbing to investigate whether the accident of SEC14L3 affects acceding and anatomic backdrop of surfactants in cetaceans.

In conclusion, our allegation advance that gene losses in cetaceans not abandoned are associated with amphibian specializations but could accept been circuitous in adapting to a absolutely amphibian environment, which added supports that accident of affiliated genes can be a apparatus for phenotypic adjustment (17, 18). Added generally, our abstraction highlights important genomic changes that occurred during the alteration from acreage to baptize in the cetacean bearing and appropriately helps to accept the atomic determinants of their arresting adaptations.

We aboriginal searched for genes that affectation inactivating mutations in the bottlenose dolphin, analgesic whale, agent whale, and accepted minke bang application abstracts generated by a ahead developed gene accident apprehension access (18). Briefly, this access acclimated the animal Ensembl (www.ensembl.org) adaptation 90 gene comment and a genome alignment with the animal hg38 accumulation as the advertence (68) (all analyzed assemblies with their accretion numbers are listed in table S1) to ascertain stop codon mutations, frameshifting insertions or deletions, deletions of absolute exons or genes, and mutations that agitate braid sites (18). The access performs a alternation of clarify accomplish to abolish artifacts accompanying to genome accumulation or alignment and evolutionary exon-intron anatomy changes in conserved genes. These accomplish comprise excluding those deletions that overlap accumulation gaps in a concern genome, re-aligning all coding exons with CESAR (Codon Exon Anatomy Aware Realigner) to ascertain evolutionary braid armpit accouterment and to abstain affected frameshifts due to alignment ambiguities (69), and excluding alignments to paralogs or candy pseudogenes. Last, the access considers all arch or addition APPRIS isoforms (http://appris.bioinfo.cnio.es) of a gene and outputs abstracts for the isoform with the aboriginal cardinal of inactivating mutations.

To analyze genes absent on the axis Cetacea branch, our genomic awning comprised the afterward accomplish (summarized in fig. S1). Starting with 19,769 genes annotated in the animal genome, we advised the 18,363 genes, which are present in the genome assemblies of at atomic bisected (31) of the 62 placental mammals. Next, we extracted the 2472 genes that were not classified as complete in any of the four analyzed cetaceans. To access applicant genes whose accident may be circuitous in amphibian adaptations, we afar all genes that were inactivated in added than 5% (3 of 55) of the earthbound mammals, constant in 350 genes. These genes included 114 genes acceptance to the keratin-associated and adenoids receptor gene families. Because genome alignments accept lower accurateness in adjustment associates of these ample gene families and because losses of keratin-associated and adenoids receptor genes accept been advised in detail ahead (9, 11, 12), we focused on the 236 actual genes.

For these 236 genes, we manually advised whether they were inactivated afore the breach of odontocetes and mysticetes. To this end, we aboriginal extracted those genes with stop codon, frameshift, or braid armpit mutations that are aggregate amid breed from both clades. Second, we classified genes that are partially or absolutely deleted in odontocetes and mysticetes into two groups, those that accept aggregate abatement breakpoints amid at atomic one asperous and baleen bang and those area the abatement breakpoints are not aggregate amid both lineages. To appraise the abatement breakpoints up- and afterwards of the deleted genes, we manually inspected the pairwise genome alignment chains (70) amid animal and cetaceans in the University of California, Santa Cruz (UCSC) genome browser. We abandoned included genes that affectation aggregate breakpoints (such as KLKB1; see Fig. 1A) because the best avaricious account is a audible abatement accident in the cetacean axis lineage. However, it should be acclaimed that a audible affiliated abatement accident may accept been blocked by consecutive adulteration of the breakpoint regions in abandoned lineages; thus, gene deletions afterwards aggregate breakpoints, which are not included in this study, may accept additionally occurred in the cetacean axis lineage. Likewise, we additionally afar genes that affectation abate (stop codon, frameshift, and braid site) inactivating mutations in one clade and are deleted in the added clade, although the abatement may accept happened afterwards a audible gene inactivation accident occurred in the cetacean axis lineage. This assay resulted in 110 genes that were inactivated afore the breach of odontocetes and mysticetes.

To ascertain those genes that were inactivated during the alteration from acreage to baptize in the cetacean axis lineage, we abutting afar all genes that accept inactivating mutations in the hippopotamus lineage. To this end, we aboriginal accumbent the genome of the accepted hippopotamus (23) to the animal hg38 accumulation application lastz (parameters K = 2400, L = 3000, and absence scoring matrix), axtChain, chainCleaner, and chainNet (all with absence parameters) as done afore (13) and acclimated our access to ascertain inactivating mutations in the accepted hippopotamus. We acquired 85 genes that were inactivated in the cetacean axis lineage, acceptation afterwards the breach amid Cetacea and Hippopotamidae.

For the 11 genes discussed in detail (F12, KLKB1, POLM, MAP3K19, SEC14L3, SLC6A18, SLC4A9, AANAT, ASMT, MTNR1A, and MTNR1B), we added corroborated the abridgement of inactivating mutations in the hippopotamus lineage. To this end, we fabricated use of amiss sequencing reads of the pygmy hippopotamus (Choeropsis liberiensis). We mapped these reads to exonic sequences of the accepted hippopotamus, including ~60–base brace (bp) crabbed intron on anniversary ancillary application Geneious 11.1.5 (www.geneious.com). In cases area an exon was not present in the accepted hippopotamus because of an accumulation gap, we acclimated the orthologous exon from cow and BLASTed the Sequence Apprehend Archive (SRA) of the accepted hippopotamus (SRR5663647) to balance and accumulate the missing exons. Chiral assay accepted that all of the complete exons in the accepted hippopotamus additionally abridgement inactivating mutations in the pygmy hippopotamus.

For all 85 genes listed in table S2, we advised whether aggregate inactivating mutations are present in the genomes of three added cetaceans that were not allotment of the whole-genome alignment (68), the Yangtze River dolphin, beluga whale, and bowhead bang (11, 24, 25). To this end, we accumbent these genomes to the hg38 accumulation as declared aloft and manually accepted the attendance of aggregate inactivation mutations. Furthermore, we acclimated our alignment chains, which are acute abundant to abduction abounding gene duplications that happened afore the alteration of mammals, to affirm that these 85 absent genes do not accept accretion complete archetype abroad in a cetacean genome due to a added contempo gene duplication event.

For the 11 genes discussed in detail (F12, KLKB1, POLM, MAP3K19, SEC14L3, SLC6A18, SLC4A9, AANAT, ASMT, MTNR1A, and MTNR1B), we authentic the definiteness of all 251 inactivating mutations that are present in the bottlenose dolphin, analgesic whale, agent whale, accepted minke whale, and manatee genome application raw sequencing reads. To this end, we extracted 100-bp sequences accoutrement the corresponding mutations and acclimated BLAST to retrieve alignments to the species’ raw sequencing reads deposited on the SRA (experiment IDs are listed in table S3). We again bent the cardinal of reads that abutment or do not abutment anniversary alteration (table S3).

For these 11 genes, we added advised whether they still aftermath spliced transcripts application accessible RNA sequencing (RNA-seq) abstracts of cetaceans. We downloaded RNA-seq abstracts of the bottlenose dolphin bark and claret (PRJNA385781, SRA study: SRP106690) and the minke bang brain, heart, kidney, liver, lung, and beef (PRJNA72723, SRA study: SRP025154) (6) from the National Center for Biotechnology Advice (NCBI) SRA. We candy all SRA apprehend files with fastq-dump application ambit for removing abstruse reads (skip technical), clarification (read-filter = pass) and removing tags (clip), agreeable paired-end reads into according files (split files), befitting apprehend identifiers (readids), and formatting abstracts into abject amplitude (dumpbase). Reads were again mapped to the genome accumulation of the bottlenose dolphin (turTru3) and the minke bang (balAcu1) application STAR [Spliced Transcripts Alignment to a Advertence (version 2.4.2a); https://github.com/alexdobin/STAR/releases/tag/STAR_2.4.2a). For bottlenose dolphin, genome indexes were generated with absence parameters. For minke whale, we adapted the cardinal of bins for the indexes according to the cardinal of absolute bases and scaffolds in the accumulation (genomeChrBinNbits = 18). The minke bang RNA-seq abstracts abide of paired-end reads, admitting the dolphin RNA-seq abstracts abide of single-end reads. During mapping, we defined ascribe files according to paired- or single-end apprehend data. All runs were mapped alone application ambit for removing reads that map to abounding altered locations (outFilterMultimapNmax = 20) and attached the cardinal of accustomed mismatches for mapped reads (outFilterMismatchNoverLmax = 0.04). We acclimated bedtools (https://bedtools.readthedocs.io/en/latest/) and bedGraphToBigWig (UCSC genome browser antecedent code) to anticipate the apprehend advantage in the UCSC genome browser. The dolphin RNA-seq abstracts accommodate 10 runs from claret samples and 25 runs from bark samples. We accumulated all runs from claret samples and all runs from bark samples application bigWigMerge. The apprehend advantage beyond the seven applicant gene losses was again inspected to appraise whether debris of the absent genes are still bidding and appropriately spliced in dolphin and minke whale.

We acclimated our gene accident apprehension access to investigate which of the genes that are absent in the cetacean axis bearing are additionally convergently absent in added amphibian (manatee) or semi-aquatic (pinnipeds) beastly lineages. Because pinnipeds were abandoned represented by the Weddell allowance (family Phocidae) and the walrus, we downloaded the genome of the Antarctic fur allowance (family Otariidae) (https://datadryad.org/resource/doi:10.5061/dryad.8kn8c.2), accumbent it to the hg38 accumulation as declared above, and activated our gene accident apprehension pipeline. All genes convergently absent amid cetaceans and manatees and/or pinnipeds are adumbrated in table S2.

We added buried for genes that are convergently absent amid any amphibian or semi-aquatic beastly lineages and compared the prevalence of these allied gene losses to allied losses amid their earthbound sister species. To this end, we aboriginal extracted from our dataset those genes that are classified as absent in one adumbrative of the three (semi-) amphibian lineages but that are classified as complete in their corresponding earthbound sister species, absorption on genes not acceptance to keratin-associated and adenoids receptor gene families. Specifically, we extracted genes absent in the analgesic bang but not in the cow, genes absent in the Pacific walrus but not in the arctic bear, and genes absent in the manatee but not in the elephant. Then, we asked how abounding of these genes are convergently absent in at atomic two (semi-) amphibian mammals (table S5). We compared this cardinal to the cardinal of allied losses detected back swapping the three (semi-) amphibian and three earthbound mammals. These abstracts are visualized as Venn diagrams in fig. S27.

Acknowledgments: We acknowledge the genomics association for sequencing and accumulating the genomes and the UCSC genome browser accumulation for accouterment software and genome annotations. We additionally acknowledge J. G. Roscito for advice with gene annotations, G. Amato (NYZS) for accouterment a tissue sample for pygmy adipose genome sequencing, M. Collin for advice with class work, and the Computer Service Facilities of the MPI-CBG and MPI-PKS for support. Funding: This assignment was accurate by the Max Planck Society, the German Research Foundation (HI1423/3-1), the Leibniz Association (SAW-2016-SGN-2), and the National Science Foundation (USA) admission (DEB-1457735). Author contributions: M.Hi., J.G., and M.S.S. conceived the study. M.Hu. performed the gene accident awning and analyzed the data. N.H. analyzed the data. M.S.S., J.G., V.S., and M.Hi. contributed to the abstracts assay and interpretation. M.Hu. and M.Hi. drafted the manuscript. All authors alarmingly revised the manuscript. Aggressive interests: The authors acknowledge that they accept no aggressive interests. Abstracts and abstracts availability: All abstracts bare to appraise the abstracts in the cardboard are present in the cardboard and/or the Supplementary Materials. Specifically, all analyzed genome assemblies (table S1) are about accessible on the UCSC genome browser and from NCBI. The account of 85 genes absent in the cetacean axis lineage, calm with anatomic annotations, is provided in table S2. The account of genes convergently inactivated in two or added (semi-) amphibian mammals is provided in table S5. Added abstracts accompanying to this cardboard may be requested from the authors.

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