Synchronized Expansion and Contraction of Olfactory, Vomeronasal, and Taste Receptor Gene Families in Hystricomorph Rodents.

Chemical senses, including olfaction, pheromones, and taste, are crucial for the survival of most animals. There has long been a debate about whether different types of senses might influence each other. For instance, primates with a strong sense of vision are thought to have weakened olfactory abilities, although the oversimplified trade-off theory is now being questioned. It is uncertain whether such interactions between different chemical senses occur during evolution. To address this question, we examined four receptor gene families related to olfaction, pheromones, and taste: olfactory receptor (OR), vomeronasal receptor type 1 and type 2 (V1R and V2R), and bitter taste receptor (T2R) genes in Hystricomorpha, which is morphologically and ecologically the most diverse group of rodents. We also sequenced and assembled the genome of the grasscutter, Thryonomys swinderianus. By examining 16 available genome assemblies alongside the grasscutter genome, we identified orthologous gene groups among hystricomorph rodents for these gene families to separate the gene gain and loss events in each phylogenetic branch of the Hystricomorpha evolutionary tree. Our analysis revealed that the expansion or contraction of the four gene families occurred synchronously, indicating that when one chemical sense develops or deteriorates, the others follow suit. The results also showed that V1R/V2R genes underwent the fastest evolution, followed by OR genes, and T2R genes were the most evolutionarily stable. This variation likely reflects the difference in ligands of V1R/V2Rs, ORs, and T2Rs: species-specific pheromones, environment-based scents, and toxic substances common to many animals, respectively.

Localized Expression of Olfactory Receptor Genes in the Olfactory Organ of Common Minke Whales.

Baleen whales (Mysticeti) possess the necessary anatomical structures and genetic elements for olfaction. Nevertheless, the olfactory receptor gene (OR) repertoire has undergone substantial degeneration in the cetacean lineage following the divergence of the Artiodactyla and Cetacea. The functionality of highly degenerated mysticete ORs within their olfactory epithelium remains unknown. In this study, we extracted total RNA from the nasal mucosae of common minke whales (Balaenoptera acutorostrata) to investigate ORs' localized expression. All three sections of the mucosae examined in the nasal chamber displayed comparable histological structure. However, the posterior portion of the frontoturbinal region exhibited notably high OR expression. Neither the olfactory bulb nor the external skin exhibited the expression of these genes. Although this species possesses four intact non-class-2 ORs, all the ORs expressed in the nasal mucosae belong to class-2, implying the loss of aversion to specific odorants. These anatomical and genomic analyses suggest that ORs are still responsible for olfaction within the nasal region of baleen whales, enabling them to detect desirable scents such as prey and potential mating partners.

Unstable environmental conditions constrain the fine-tune between opsin sensitivity and underwater light in an Amazon forest stream fish.

Visual adaptations can stem from variations in amino acid composition, chromophore utilization, and differential opsin gene expression levels, enabling individuals to adjust their light sensitivity to environmental lighting conditions. In stable environments, adaptations often involve amino acid substitutions, whereas in unstable conditions, differential gene expression may be a more relevant mechanism. Amazon forest streams present diverse underwater lighting conditions and experience short-term water colour fluctuations. In these environments, it is less likely for genetic and amino acid sequences to undergo modifications that tailor opsin proteins to the prevailing lighting conditions, particularly in species having several copies of the same gene. The sailfin tetra, Crenuchus spilurus, inhabits black and clear water Amazon forest streams. The long-wavelength sensitivity (LWS) is an important component for foraging and courtship. Here, we investigated LWS opsin genes in the sailfin tetra. Three copies of LWS1 and two copies of LWS2 genes were found. The maximum absorbance wavelength (λmax) estimated from the amino acid sequences of LWS1 genes exhibited variation among the different copies. In contrast, the copies of LWS2 genes showed identical expected λmax values. Although the amino acid positions affecting λmax varied among LWS genes, they remained consistent among populations living in different water colours. The relative expression levels of LWS genes differed between gene copies. While not formally tested, our results suggest that in fluctuating environments, visual adaptations may primarily stem from alterations in gene expression profiles and/or chromophore usage rather than precise genetic tuning of protein light sensitivity to environmental lighting conditions.

Genomics reveals a genetically isolated population of the Pacific white-sided dolphin (Lagenorhynchus obliquidens) distributed in the Sea of Japan.

The processes by which animals become genetically isolated in an open environment such as the ocean have not yet been fully elucidated. Morphologically different populations of Pacific white-sided dolphin Lagenorhynchus obliquidens are observed sympatrically in the Sea of Japan. However, genetic studies that exclusively used limited mitochondrial loci or microsatellite DNA have failed to demonstrate the existence of genetically distinct populations. Here, to reveal the population structure, we analysed genome-wide population genetic data using single nucleotide polymorphisms (SNPs) gathered in 2018-2021 from all domestic captive individuals in aquaria, the majority of which originated from the wild, as well as from some stranded individuals, together covering a wide area of coastal water around Japan (n = 123). Multiplexed intersimple sequence repeat genotyping-by-sequencing analysis was performed to obtain the SNP data. Principal coordinate analysis and the clustering method structure indicated that two genetically-distinct populations exist, with little interpopulation gene flow revealed. In addition, the genotypic segregation was reflected in differences in external morphotype. Furthermore, a population demographic analysis based on the whole-genome sequences of an individual from each population indicated that sea-level changes during the Last Glacial Period probably led to allopatric divergence of this species in a limited area of the Sea of Japan, with that group subsequently sharing a distribution area with the other population. These findings yield insights into the formation of genetically isolated sympatric populations in the ocean.

Olfactory epithelium and ontogeny of the nasal chambers in the bowhead whale (Balaena mysticetus).

In a species of baleen whale, we identify olfactory epithelium that suggests a functional sense of smell and document the ontogeny of the surrounding olfactory anatomy. Whales must surface to breathe, thereby providing an opportunity to detect airborne odorants. Although many toothed whales (odontocetes) lack olfactory anatomy, baleen whales (mysticetes) have retained theirs. Here, we investigate fetal and postnatal specimens of bowhead whales (Balaena mysticetus). Computed tomography (CT) reveals the presence of nasal passages and nasal chambers with simple ethmoturbinates through ontogeny. Additionally, we describe the dorsal nasal meatuses and olfactory bulb chambers. The cribriform plate has foramina that communicate with the nasal chambers. We show this anatomy within the context of the whole prenatal and postnatal skull. We document the tunnel for the ethmoidal nerve (ethmoid foramen) and the rostrolateral recess of the nasal chamber, which appears postnatally. Bilateral symmetry was apparent in the postnatal nasal chambers. No such symmetry was found prenatally, possibly due to tissue deformation. No nasal air sacs were found in fetal development. Olfactory epithelium, identified histologically, covers at least part of the ethmoturbinates. We identify olfactory epithelium using six explicit criteria of mammalian olfactory epithelium. Immunohistochemistry revealed the presence of olfactory marker protein (OMP), which is only found in mature olfactory sensory neurons. Although it seems that these neurons are scarce in bowhead whales compared to typical terrestrial mammals, our results suggest that bowhead whales have a functional sense of smell, which they may use to find prey.

Olfaction of aquatic amniotes.

Amniotes originated on land, but aquatic/amphibious groups emerged multiple times independently in amniotes. On becoming aquatic, species with different phylogenetic backgrounds and body plans have to adapt themselves to handle similar problems inflicted by their new environment, and this makes aquatic adaptation of amniotes one of the greatest natural experiments. Particularly, evolution of the sense of smell upon aquatic adaptation is of great interest because receptors required for underwater olfaction differ remarkably from those for terrestrial olfaction. Here, I review the olfactory capabilities of aquatic/amphibious amniotes, especially those of cetaceans and sea snakes. Most aquatic/amphibious amniotes show reduced olfactory organs, receptor gene repertoires, and olfactory capabilities. Remarkably, cetaceans and sea snakes show extreme examples: cetaceans have lost the vomeronasal system, and furthermore, toothed whales have lost all of their olfactory nervous systems. Baleen whales can smell in the air, but their olfactory capability is limited. Fully aquatic sea snakes have lost the main olfactory system but they retain the vomeronasal system for sensing underwater. Amphibious species show an intermediate status between terrestrial and aquatic species, implying their importance on understanding the process of aquatic adaptation. The olfactory capabilities of aquatic amniotes are diverse, reflecting their diverse phylogenetic backgrounds and ecology.

Population history and genomic admixture of sea snakes of the genus Laticauda in the West Pacific.

Speciation in the open ocean has long been studied, but it remains largely elusive what factors promote or inhibit speciation in such an open environment. Marine amniotes, which evolved from terrestrial ancestors, provide valuable opportunities for studying speciation in the ocean because of their evident aquatic origins. Sea snakes are phylogenetically related to terrestrial elapid snakes and consist of two monophyletic groups (Hydrophiini and Laticaudini). These two groups migrated from land to water almost at the same time, but species diversities are remarkably different: there are approx. 60 species in 16 genera described for hydrophiins, whereas only eight species in the genus Laticauda are described for laticaudins. Here, we provide a high-quality reference genome assembly of a laticaudin L. colubrina with a scaffold N50 value of 40 Mbp, and focused on laticaudins to consider why they have seldom speciated. We performed whole-genome shotgun sequencing of several species of laticaudins sampled in their southmost (Vanuatu) and northmost (Ryukyu) habitats. Demographic histories of Vanuatu and Ryukyu populations suggest that populations of broadly distributed major species are geographically structured. Each species is genetically clearly distinguished, but there is a considerable amount of gene flow between two sibling species distributed sympatrically in Vanuatu. In addition, inter-species genomic admixture is ubiquitously observed among laticaudins even between phylogenetically distant species. Broad distribution of major species combined with such genetic mixability might have prevented laticaudins from genetic isolation and speciation.

Antioxidative Potency of Dolphin Serum Albumin Is Stronger Than That of Human Serum Albumin Irrespective of Substitution of 34Cysteine With Serine.

Serum albumin (SA), the most abundant protein in circulation, functions as a carrier protein, osmoregulator, and antioxidant. Generally, SA exerts its antioxidative effects by scavenging reactive oxygen species. Because marine mammals are superior divers, they are intermittently exposed to oxidative stress induced by rapid reperfusion of oxygen to ischemic tissues after the dive. Although several antioxidants in marine mammals have been described, SA activity remains largely uncharacterized. In this study, we investigated the antioxidative activity of SA in marine mammals by comparing features of the primary and steric structures, biochemical properties, and antioxidative activities of common bottlenose dolphin SA (DSA) and human SA (HSA). Our results revealed that DSA lacked free cysteine at position 34 that is important for the antioxidative activity of HSA; however, the antioxidative capacity and thiol activity of DSA were stronger than those of HSA. Circular dichroism spectra showed different patterns in DSA and HSA. Ultraviolet fluorescence intensities of DSA were higher than those of HSA, suggesting lower surface hydrophobicity of DSA. Additionally, DSA showed higher excess heat capacity than HSA. We then compared a homology model of DSA with a 3D model of HSA. Our results indicate that DSA was more unstable than HSA at least in the body-temperature range, probably due to the mode of molecules involved in the disulfide bonds and/or the lower surface hydrophobicity, and it may be related to the equivalent or stronger antioxidant potency of DSA. These data show that DSA is an effective antioxidant in the circulation of the dolphin.

Visual adaptation of opsin genes to the aquatic environment in sea snakes.

BACKGROUND: Evolutionary transitions from terrestrial to aquatic life history cause drastic changes in sensory systems. Indeed, the drastic changes in vision have been reported in many aquatic amniotes, convergently. Recently, the opsin genes of the full-aquatic sea snakes have been reported. However, those of the amphibious sea snakes have not been examined in detail. RESULTS: Here, we investigated opsin genes and visual pigments of sea snakes. We determined the sequences of SWS1, LWS, and RH1 genes from one terrestrial, three amphibious and four fully-aquatic elapids. Amino acid replacements at four and one spectra-tuning positions were found in LWS and RH1, respectively. We measured or predicted absorption of LWS and RH1 pigments with A1-derived retinal. During their evolution, blue shifts of LWS pigments have occurred stepwise in amphibious sea snakes and convergently in both amphibious and fully-aquatic species. CONCLUSIONS: Blue shifted LWS pigments may have adapted to deep water or open water environments dominated by blue light. The evolution of opsins differs between marine mammals (cetaceans and pinnipeds) and sea snakes in two fundamental ways: (1) pseudogenization of opsins in marine mammals; and (2) large blue shifts of LWS pigments in sea snakes. It may be possible to explain these two differences at the level of photoreceptor cell composition given that cone and rod cells both exist in mammals whereas only cone cells exist in fully-aquatic sea snakes. We hypothesize that the differences in photoreceptor cell compositions may have differentially affected the evolution of opsins in divergent amniote lineages.

Phylogenomics of the genus Tursiops and closely related Delphininae reveals extensive reticulation among lineages and provides inference about eco-evolutionary drivers.

Phylogeographic inference has provided extensive insight into the relative roles of geographical isolation and ecological processes during evolutionary radiations. However, the importance of cross-lineage admixture in facilitating adaptive radiations is increasingly being recognised, and suggested as a main cause of phylogenetic uncertainty. In this study, we used a double digest RADseq protocol to provide a high resolution (~4 Million bp) nuclear phylogeny of the Delphininae. Phylogenetic resolution of this group has been especially intractable, likely because it has experienced a recent species radiation. We carried out cross-lineage reticulation analyses, and tested for several sources of potential bias in determining phylogenies from genome sampling data. We assessed the divergence time and historical demography of T. truncatus and T. aduncus by sequencing the T. aduncus genome and comparing it with the T. truncatus reference genome. Our results suggest monophyly for the genus Tursiops, with the recently proposed T. australis species falling within the T. aduncus lineage. We also show the presence of extensive cross-lineage gene flow between pelagic and European coastal ecotypes of T. truncatus, as well as in the early stages of diversification between spotted (Stenella frontalis; Stenella attenuata), spinner (Stenella longirostris), striped (Stenella coeruleoalba), common (Delphinus delphis), and Fraser's (Lagenodelphis hosei) dolphins. Our study suggests that cross-lineage gene flow in this group has been more extensive and complex than previously thought. In the context of biogeography and local habitat dependence, these results improve our understanding of the evolutionary processes determining the history of this lineage.

Loss of olfaction in sea snakes provides new perspectives on the aquatic adaptation of amniotes.

Marine amniotes, a polyphyletic group, provide an excellent opportunity for studying convergent evolution. Their sense of smell tends to degenerate, but this process has not been explored by comparing fully aquatic species with their amphibious relatives in an evolutionary context. Here, we sequenced the genomes of fully aquatic and amphibious sea snakes and identified repertoires of chemosensory receptor genes involved in olfaction. Snakes possess large numbers of the olfactory receptor (OR) genes and the type-2 vomeronasal receptor (V2R) genes, and expression profiling in the olfactory tissues suggests that snakes use the ORs in the main olfactory system (MOS) and the V2Rs in the vomeronasal system (VNS). The number of OR genes has decreased in sea snakes, and fully aquatic species lost MOS which is responsible for detecting airborne odours. By contrast, sea snakes including fully aquatic species retain a number of V2R genes and a well-developed VNS for smelling underwater. This study suggests that the sense of smell also degenerated in sea snakes, particularly in fully aquatic species, but their residual olfactory capability is distinct from that of other fully aquatic amniotes. Amphibious species show an intermediate status between terrestrial and fully aquatic snakes, implying their importance in understanding the process of aquatic adaptation.

How Snakes Find Prey Underwater: Sea Snakes Use Visual and Chemical Cues for Foraging.

Fully aquatic adaptation generally leads amniotes to change sensory modalities drastically. Terrestrial snakes rely heavily on chemical cues to locate and recognize prey, but little is known about how sea snakes find prey fishes underwater. Sea snakes of the genus Hydrophis are fish-eating marine elapids which adapted from land to water approximately 5-10 million years ago. Here, using two species of captive Hydrophis snakes, we show that they can recognize and discriminate their preferred fish species solely by using olfactory cues. However, they locate places where their preferred fishes may hide without relying on chemical cues. These findings indicate that Hydrophis snakes find prey in water as follows: they use visual cues to locate a place where their prey fishes are likely to hide, and then use chemical cues to find and attack prey. As is the case for other aquatic amniotes, snakes also modified their sensory modalities upon becoming aquatic.

A Single Pheromone Receptor Gene Conserved across 400 My of Vertebrate Evolution.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island.

Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.

Evolution of the alternative AQP2 gene: Acquisition of a novel protein-coding sequence in dolphins.

Taxon-specific de novo protein-coding sequences are thought to be important for taxon-specific environmental adaptation. A recent study revealed that bottlenose dolphins acquired a novel isoform of aquaporin 2 generated by alternative splicing (alternative AQP2), which helps dolphins to live in hyperosmotic seawater. The AQP2 gene consists of four exons, but the alternative AQP2 gene lacks the fourth exon and instead has a longer third exon that includes the original third exon and a part of the original third intron. Here, we show that the latter half of the third exon of the alternative AQP2 arose from a non-protein-coding sequence. Intact ORF of this de novo sequence is shared not by all cetaceans, but only by delphinoids. However, this sequence is conservative in all modern cetaceans, implying that this de novo sequence potentially plays important roles for marine adaptation in cetaceans.

Aquatic adaptation and the evolution of smell and taste in whales.

INTRODUCTION: While olfaction is one of the most important senses in most terrestrial mammals, it is absent in modern toothed whales (Odontoceti, Cetacea). Furthermore, behavioral evidence suggests that gustation is very limited. In contrast, their aquatic sistergroup, baleen whales (Mysticeti) retain small but functional olfactory organs, and nothing is known about their gustation. It is difficult to investigate mysticete chemosensory abilities because experiments in a controlled setting are impossible. RESULTS: Here, we use the functional regionalization of the olfactory bulb (OB) to identify the loss of specific olfactory functions in mysticetes. We provide the whole-genome sequence of a mysticete and show that mysticetes lack the dorsal domain of the OB, an area known to induce innate avoidance behavior against odors of predators and spoiled foods. Genomic and fossil data suggest that mysticetes lost the dorsal domain of the OB before the Odontoceti-Mysticeti split. Furthermore, we found that all modern cetaceans are revealed to have lost the functional taste receptors. CONCLUSION: These results strongly indicate that profound changes in the chemosensory capabilities had occurred in the cetacean lineage during the period when ancestral whales migrated from land to water.

Organization and distribution of glomeruli in the bowhead whale olfactory bulb.

Although modern baleen whales (Mysticeti) retain a functional olfactory system that includes olfactory bulbs, cranial nerve I and olfactory receptor genes, their olfactory capabilities have been reduced to a great degree. This reduction likely occurred as a selective response to their fully aquatic lifestyle. The glomeruli that occur in the olfactory bulb can be divided into two non-overlapping domains, a dorsal domain and a ventral domain. Recent molecular studies revealed that all modern whales have lost olfactory receptor genes and marker genes that are specific to the dorsal domain. Here we show that olfactory bulbs of bowhead whales (Balaena mysticetus) lack glomeruli on the dorsal side, consistent with the molecular data. In addition, we estimate that there are more than 4,000 glomeruli elsewhere in the bowhead whale olfactory bulb, which is surprising given that bowhead whales possess only 80 intact olfactory receptor genes. Olfactory sensory neurons that express the same olfactory receptors in rodents generally project to two specific glomeruli in an olfactory bulb, implying an approximate 1:2 ratio of the number of olfactory receptors to the number of glomeruli. Here we show that this ratio does not apply to bowhead whales, reiterating the conceptual limits of using rodents as model organisms for understanding the initial coding of odor information among mammals.

Pairwise comparison of orthologous olfactory receptor genes between two sympatric sibling sea kraits of the genus Laticauda in Vanuatu.

Olfaction-based reproductive isolation is widely observed in animals, but little is known about the genetic basis of such isolation mechanisms. Two species of sibling amphibious sea snakes, Laticauda colubrina and L. frontalis live in Vanuatu sympatrically and syntopically, but no natural hybrids have been reported. Adult females of both taxa possess distinctive lipids in the skin, and male L. frontalis distinguishes conspecific females based on olfactory cues. To shed light on the molecular basis of the evolution of olfaction-based isolation mechanisms, olfactory receptor (OR) gene repertoires of both taxa were identified using pyrosequencing-based technology, and orthologous OR gene sets were identified. Few species-specific gene duplications or species-specific gene losses were found. However, the nonsynonymous-to-synonymous substitution rate ratio was relatively higher between orthologous OR genes of L. frontalis and L. colubrina, indicating that L. frontalis and L. colubrina have evolved to possess different olfactory senses. We suggest that L. frontalis and L. colubrina have evolved allopatrically, and this may be a byproduct of the allopatric evolution, and that this dissimilarity may function as a premating isolation barrier, since L. frontalis has returned to the ancestral range (Vanuatu).