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The explosive adaptive radiation of Haplochromine cichlids in Lake Victoria, East Africa, produced 500 endemic species within only 15,000 years. A paedophage or an egg-eater is considered a unique example of trophic adaptation. Many field studies reported that more than 200 cichlids have extinct due to the upsurge of Nile perch, a carnivorous species introduced to the lake in the 1950s. Especially, piscivorous cichlids like paedophages were critically damaged by Nile perch predation. Here, we performed a genome-wide evolutionary study of the paedophages in Lake Victoria to understand their past demographic events and phylogenetic relationships. We discovered evidence of a recent, short-period, and severe bottleneck in a paedophage “matumbi hunter”. Interestingly, the signature of a strong bottleneck, as observed in matumbi hunter, was not detected in other species including paedophagus species. In addition, it was revealed that the population size of matumbi hunter started to decline 30 years ago and recover from 20 to 10 years ago, corresponding to the time of both disappearance and resurgence of Lake Victoria Haplochromines were reported. Although population structure analyses showed that matumbi hunter is composed of a unique genetic component, phylogenetic analyses supported its strong monophyly with other paedophagus species. These results suggest that the paedophages originated only once in Lake Victoria followed by the decline of genetic diversity in matumbi hunter. This study succeeded to demonstrate the demographic events triggered by invasive species and associated genomic consequences of the unique trophic group, promoting a holistic understanding of adaptive radiation.

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.

Background Aggression is an evolutionarily conserved behavior critical for animal survival. In the fish Betta splendens, across different stages of fighting interactions, fighting opponents suffer from various stressors, especially from the great demand for oxygen. Using RNA sequencing, we profiled differential alternative splicing (DAS) events in the brains of fish collected before fighting, during fighting, and after fighting to study the involvement of alternative splicing (AS) in the response to stress during the fight. Results We found that fighting interactions induced the greatest increase in AS in the ‘during-fighting’ fish, followed by that of the ‘after-fighting’ fish. Intron retention (IR) was the most enriched type among all the basic AS events. DAS genes were mainly associated with synapse assembly, ion transport, and regulation of protein secretion. We further observed that IR events significantly differentiated between winners and losers for 19 genes, which were associated with messenger RNA biogenesis, DNA repair, and transcription machinery. These genes share many common features, including shorter intron length and higher GC content. Conclusions This study is the first comprehensive view of AS induced by fighting interactions in a fish species across different stages of those interactions, especially with respect to IR events in winners and losers. Together, these findings facilitate future investigations into transcriptome complexity and AS regulation in response to stress under the context of aggression in vertebrates.

Currently, the insertions of SINEs (and other retrotransposed elements) are regarded as one of the most reliable synapomorphies in molecular systematics. The methodological mainstream of molecular systematics is the calculation of nucleotide (or amino acid) sequence divergences under a suitable substitution model. In contrast, SINE insertion analysis does not require any complex model because SINE insertions are unidirectional and irreversible. This straightforward methodology was named the “SINE method,” which resolved various taxonomic issues that could not be settled by sequence comparison alone. The SINE method has challenged several traditional hypotheses proposed based on the fossil record and anatomy, prompting constructive discussions in the Evo/Devo era. Here, we review our pioneering SINE studies on salmon, cichlids, cetaceans, Afrotherian mammals, and birds. We emphasize the power of the SINE method in detecting incomplete lineage sorting by tracing the genealogy of specific genomic loci with minimal noise. Finally, in the context of the whole-genome era, we discuss how the SINE method can be applied to further our understanding of the tree of life.

The evolutionary transition of vertebrates from water to land during the Devonian period was accompanied by major changes in animal respiratory systems in terms of physiology and morphology. Indeed, the fossil record of the early tetrapods has revealed the existence of internal gills, which are vestigial fish-like traits used underwater. However, the fossil record provides only limited data on the process of the evolutionary transition of gills from fish to early tetrapods. This study investigated the gills ofPolypterus senegalus, a basal ray-finned/amphibious fish which shows many ancestral features of stem Osteichthyes. Based on scanning electron microscopy observations and transcriptome analysis, the existence of motile cilia in the gills was revealed which may create a flow on the gill surface leading to efficient ventilation or remove particles from the surface. Interestingly, these cilia were observed to disappear after rearing in terrestrial or high CO2environments, which mimics the environmental changes in the Devonian period. The cilia re-appeared after being returned to the original aquatic environment. The ability of plastic changes of gills inPolypterusrevealed in this study may allow them to survive in fluctuating environments, such as shallow swamps. The ancestor of Osteichthyes is expected to have possessed such plasticity in the gills, which may be one of the driving forces behind the transition of vertebrates from water to land.

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicits social and reproductive behaviors. Although the VNO is highly conserved across mammals, it has been lost in some species that have evolved alternate sensing systems during diversification. In this study, we investigate a newly identified VNO-specific gene, ancV1R, in the extant 261 species of mammals to examine the correlation between genotype (ancV1R) and phenotype (VNO). As a result, we found signatures for the relaxation of purifying selection (inactivating mutations and the elevation of dN/dS) on ancV1Rs in VNO-lacking mammals, such as catarrhine primates, cetaceans, the manatees, and several bat lineages, showing the distinct correlation between genotype and phenotype. Interestingly, we further revealed signatures for the relaxation of purifying selection on ancV1R in true seals, otters, the fossa, the owl monkey, and alcelaphine antelopes in which the existence of a functional VNO is still under debate. Our additional analyses on TRPC2, another predictive marker gene for the functional VNO, showed a relaxation of purifying selection, supporting the possibility of VNO loss in these species. The results of our present study invite more in-depth neuroanatomical investigation in mammals for which VNO function remains equivocal.

The vomeronasal type 2 receptor (V2R, also called OlfC) multigene family is found in a broad range of jawed vertebrates from cartilaginous fish to tetrapods. V2Rs encode receptors for food-related amino acids in teleost fish, whereas for peptide pheromones in mammals. In addition, V2Rs of teleost fish are phylogenetically distinct from those of tetrapods, implying a drastic change in the V2Rrepertoire during terrestrial adaptation. To understand the process of diversification of V2Rs in vertebrates from “fish-type” to “tetrapod-type”, we conducted an exhaustive search for V2Rs in cartilaginous fish (chimeras, sharks, and skates) and basal ray-finned fish (reedfish, sterlet, and spotted gar), and compared them with those of teleost, coelacanth, and tetrapods. Phylogenetic and synteny analyses on 1897V2Rs revealed that basal ray-finned fish possess unexpectedly higher number of V2Rs compared with cartilaginous fish, implying that V2Rgene repertoires expanded in the common ancestor of Osteichthyes. Furthermore, reedfish and sterlet possessed various V2Rs that belonged to both “fish-type” and “tetrapod-type”, suggesting that the common ancestor of Osteichthyes possess “tetrapod-type” V2Rs although they inhabited underwater environments. Thus, the unexpected diversity of V2Rs in basal ray-finned fish illuminates the process of how the osteichthyan ancestors adapt from water to land.

The vomeronasal organ (VNO) plays a key role in sensing pheromonal cues, which elicit innate responses and induce social and sexual behaviors. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the VNO. Previous studies have revealed that the V1R family shows extraordinary variety among mammalian species owing to successive gene gains and losses. Because species-specific pheromonal interaction may facilitate species-specific reproductive behaviors, understanding the evolution of V1Rs in terms of their origin, repertoire and phylogeny should provide insight into the mechanisms of animal diversification. Here I summarize recent studies about the V1R family from its initial discovery in the rat genome to extensive comparative analyses among vertebrates. I further introduce our recent findings for V1Rs in a broad range of vertebrates, which reveal unexpected diversity as well as shared features common among lineages.

Τo clarify the fracture behavior of electroslag welding (ESW) joints, we used the connection of box-column flange and interior diaphragm made of high-performance steel SA440C. Owing to the large heat input, the ductility and notch toughness of ESW joints are prone to degradation. In particular, because the mechanical properties of high-strength steel are sensitive to the high heat input, the performance of ESW joints may significantly degrade when used with SA440C. In addition, considering the case wherein the beam-to-column connection is under strong seismic excitation, an ESW joint located at the crossing of a column flange, beam flange, and interior diaphragm is subject to biaxial tensile forces, causing premature brittle fracturing. To clarify the performance of ESW joints under such conditions, biaxial tensile loading tests were conducted. Four specimens were fabricated from the built-up box column, with interior diaphragms welded by ESW. Tensile load was statically applied between the interior diaphragm and the beam flange under constant axial load in the column flange. The maximum strength was demonstrated to be governed by the Charpy V-notch (CVN) toughness at the fractured locations. With regard to CVN toughness at the test temperature of the heat-affected zone or weld metal larger than 50 J, the maximum strength was comparable to that of the base metal of SA440C.

Fishes use olfaction to gain various information vital for survival and communication. To understand biodiversity in fishes, it is important to identify what receptors individual fish use to detect specific chemical compounds. However, studies of fish olfactory receptors and their ligands are still limited to a few model organisms represented primarily by zebrafish. Here, we tested the neural activation of olfactory sensory neurons (OSNs) in East African cichlids, the most diversified teleost lineage, by in situ hybridization with a c-fos riboprobe. We confirmed that microvillous neurons contributed the most to the detection of amino acids, as in other fishes. Conversely, we found that ciliated neurons contributed the most to detection of conjugated steroids, known as pheromone candidates. We also found that V2Rs, the major expressed receptor in microvillous neurons exhibited differential responsiveness to amino acids, and further suggested that the cichlid-specific duplication of V2R led to ligand differentiation in cichlids by demonstrating a differential response to arginine. Finally, we established a nonlethal method to collect cichlid urine and showed how various OSNs, including V1R+ neurons, respond to male urine. This study provides an experimental basis for understanding how cichlids encode natural odors and illuminates how olfaction has contributed to the diversification of cichlids by combining with phylogenetic studies of olfactory receptors gene evolutions.

Siamese fighting fish Betta splendens are notorious for their aggressiveness and males of this fish have been widely used to study aggression. However, an understanding of brain transcriptome signature associated with aggression in the context of male-male interaction in this fish remains to be understood. Herein, RNA-Seq transcriptome data from 37 brains samples collected at different fighting stages are described. These brain samples were collected before fighting (B), during fighting (D20 and D60), and after fighting (A0 and A30). The raw data were analyzed for differential gene expression using edgeR package in R. A criterion of FDR cut-off ≤ 0.05 and an absolute fold change (FC) of 0 or greater were used to identify top upregulated and downregulated genes in fighting groups (D20, D60, A0, and A30) relative to non-fighting group (B). The data presented hereafter enable fundamental studies on genes and molecular events mediating aggressive behavior in this fish and will lay a valuable foundation for future research on the aggression of vertebrates.

African cichlid fishes harbor an extraordinary diversity of sex-chromosome systems. Within just one lineage, the tribe Haplochromini, at least 6 unique sex-chromosome systems have been identified. Here we focus on characterizing sex chromosomes in cichlids from the Lake Victoria basin. In Haplochromis chilotes, we identified a new ZW system associated with the white blotch color pattern, which shows substantial sequence differentiation over most of LG16, and is likely to be present in related species. In Haplochromis sauvagei, we found a coding polymorphism in amh that may be responsible for an XY system on LG23. In Pundamilia nyererei, we identified a feminizing effect of B chromosomes together with XY- and ZW-patterned differentiation on LG23. In Haplochromis latifasciatus, we identified a duplication of amh that may be present in other species of the Lake Victoria superflock. We further characterized the LG5-14 XY system in Astatotilapia burtoni and identified the oldest stratum on LG14. This species also showed ZW differentiation on LG2. Finally, we characterized an XY system on LG7 in Astatoreochromis alluaudi. This report brings the number of distinct sex-chromosome systems in haplochromine cichlids to at least 13, and highlights the dynamic evolution of sex determination and sex chromosomes in this young lineage.

Territorial defense involves frequent aggressive confrontations with competitors, but little is known about how brain-transcriptomic profiles change between individuals competing for territory establishment. Our previous study elucidated that brain-transcriptomic synchronization occurs in a pair-specific manner between two males of the fishBetta splendensduring fighting, reflecting a mutual assessment process between them at the level of gene expression. Here we evaluated how the brain-transcriptomic profiles of opponents change immediately after shifting their social status (i.e., the winner/loser has emerged) and 30 min after this shift. We showed that unique and carryover hypotheses can be adapted to this system, in which changes in the expression of certain genes are unique to different fighting stages and in which the expression patterns of certain genes are transiently or persistently changed across all fighting stages. Interestingly, the specificity of the brain-transcriptomic synchronization of a pair during fighting was gradually lost after fighting ceased, because of the decrease in the variance in gene expression across all individuals, leading to the emergence of a basal neurogenomic state. Strikingly, this unique state was more basal than the state that existed in the before-fighting group and resulted in the reduced and consistent expression of genes across all individuals. In spite of the consistent and basal overall gene expression in each individual in this state, expression changes for genes related to metabolism, learning and memory, and autism still differentiated losers from winners. The fighting system using maleB. splendensthus provides a promising platform for investigating neurogenomic states of aggression in vertebrates.Author summaryCompetitive interactions involve complex decision-making tasks that are shaped by mutual feedback between participants. When two animals interact, transcriptomes across their brains synchronize in a way that reflects how they assess and predict the other’s fighting ability and react to each other’s decisions. Here, we elucidated the gradual loss of brain-transcriptomic synchrony between interacting opponents after their interaction ceased, leading to the emergence of a basal neurogenomic state, in which the variations in gene expression were reduced to a minimum among all individuals. This basal neurogenomic state shares common characteristics with the hibernation state, which animals adopt to minimize their metabolic rates to cope with harsh environmental conditions. We demonstrated that this unique neurogenomic state, which is newly characterized in the present study, is composed of the expression of a unique set of genes, each of which was presumably minimally required for survival, providing a hypothesis that this state represents the smallest unit of neurogenomic activity for sustaining an active life.

The revision of the sub-order Microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. The unexpected sister–taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. In order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, Leschenault’s rousette (Rousettus leschenaultia) and the Egyptian fruit bat (R. aegyptiacus). The sequences were compared with those of 22 other mammals, including nine bats, available in the database. We identified that megabat genomes are distinct in that they have extremely low activity of SINE retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (TAAR) and olfactory receptor (OR), and elevation of the dN/dS ratio in genes for immunity and protein catabolism. The adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding.

Pheromones are crucial for eliciting innate responses and inducing social and sexual behaviors in mammals. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the vomeronasal organ (VNO). The V1R family shows extraordinary variety in gene number among vertebrates owing to successive gene gains and losses during evolution. Such diversity is believed to reflect a degree of dependence on the VNO. We investigated V1R evolution in primate lineages closely related to humans because these VNOs show a trend toward degeneration. We performed extensive phylogenetic analyses for V1Rs from a broad range of primate species. Although the decline of intact genes was evident in anthropoids (hominoids, Old World monkeys and New World monkeys), we found that a certain number of intact genes persist in New World monkeys. In one New World monkey species, the common marmoset (Callithrix jacchus), we examined seven putatively functional V1Rs using in situ hybridization and reverse transcription-PCR. Based on their mRNA expression patterns in the VNO and other organs, two types of V1Rs emerged: the canonical class with VNO-specific expression, and a second group having more ubiquitous expression in various organs as well as VNO. Furthermore, phylogenetic analysis revealed that the class with the more widespread expression had been retained longer in evolution than the VNO-specific type. We propose that the acquisition of a novel non-VNO-related function(s) may have led to the survival of a small but persistent number of V1Rs in anthropoid primates.

Terrestrial animals must have frequent contact with water to survive, implying that environmental DNA (eDNA) originating from those animals should be detectable from places containing water in terrestrial ecosystems. Aiming to detect the presence of terrestrial mammals using forest water samples, we applied a set of universal PCR primers (MiMammal, a modified version of fish universal primers) for metabarcoding mammalian eDNA. The versatility of MiMammal primers was tested in silico and by amplifying DNAs extracted from tissues. The results suggested that MiMammal primers are capable of amplifying and distinguishing a diverse group of mammalian species. In addition, analyses of water samples from zoo cages of mammals with known species composition suggested that MiMammal primers could successfully detect mammalian species from water samples in the field. Then, we performed an experiment to detect mammals from natural ecosystems by collecting five 500-ml water samples from ponds in two cool-temperate forests in Hokkaido, northern Japan. MiMammal amplicon libraries were constructed using eDNA extracted from water samples, and sequences generated by Illumina MiSeq were subjected to data processing and taxonomic assignment. We thereby detected multiple species of mammals common to the sampling areas, including deer (Cervus nippon), mouse (Mus musculus), vole (Myodes rufocanus), raccoon (Procyon lotor), rat (Rattus norvegicus) and shrew (Sorex unguiculatus). Many previous applications of the eDNA metabarcoding approach have been limited to aquatic/semiaquatic systems, but the results presented here show that the approach is also promising even for forest mammal biodiversity surveys.

Background The molecular basis of the incipient stage of speciation is still poorly understood. Cichlid fish species in Lake Victoria are a prime example of recent speciation events and a suitable system to study the adaptation and reproductive isolation of species. Results Here, we report the pattern of genomic differentiation between two Lake Victoria cichlid species collected in sympatry, Haplochromis pyrrhocephalus and H. sp. ‘macula,’ based on the pooled genome sequences of 20 individuals of each species. Despite their ecological differences, population genomics analyses demonstrate that the two species are very close to a single panmictic population due to extensive gene flow. However, we identified 21 highly differentiated short genomic regions with fixed nucleotide differences. At least 15 of these regions contained genes with predicted roles in adaptation and reproductive isolation, such as visual adaptation, circadian clock, developmental processes, adaptation to hypoxia, and sexual selection. The nonsynonymous fixed differences in one of these genes, LWS, were reported as substitutions causing shift in absorption spectra of LWS pigments. Fixed differences were found in the promoter regions of four other differentially expressed genes, indicating that these substitutions may alter gene expression levels. Conclusions These diverged short genomic regions may have contributed to the differentiation of two ecologically different species. Moreover, the origins of adaptive variants within the differentiated regions predate the geological formation of Lake Victoria; thus Lake Victoria cichlid species diversified via selection on standing genetic variation.

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 for400 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.

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.

Cichlid fishes in East Africa have undergone extensive adaptive radiation, which has led to spectacular diversity in their morphology and ecology. To date, genetic linkage maps have been constructed for several tilapias (riverine), Astatotilapia burtoni (Lake Tanganyika), and hybrid lines of Lake Malawi cichlids to facilitate genome-wide comparative analyses. In the present study, we constructed a genetic linkage map of the hybrid line of Lake Victoria cichlids, so that maps of cichlids from all the major areas of East Africa will be available. The genetic linkage map shown here is derived from the F2 progeny of an interspecific cross between Haplochromis chilotes and Haplochromis sauvagei and is based on 184 microsatellite and two single-nucleotide polymorphism (SNP) markers. Most of the microsatellite markers used in the present study were originally designed for other genetic linkage maps, allowing us to directly compare each linkage group (LG) among different cichlid groups. We found 25 LGs, the total length of which was 1133.2 cM with an average marker spacing of about 6.09 cM. Our subsequent linkage mapping analysis identified two putative sex-determining loci in cichlids. Interestingly, one of these two loci is located on cichlid LG5, on which the female heterogametic ZW locus and several quantitative trait loci (QTLs) related to adaptive evolution have been reported in Lake Malawi cichlids. We also found that V1R1 and V1R2, candidate genes for the fish pheromone receptor, are located very close to the recently detected sex-determining locus on cichlid LG5. The genetic linkage map study presented here may provide a valuable foundation for studying the chromosomal evolution of East African cichlids and the possible role of sex chromosomes in generating their genomic diversity.

Fish use olfaction to sense a variety of nonvolatile chemical signals in water. However, the evolutionary importance of olfaction in species-rich cichlids is controversial. Here, we determined an almost complete sequence of the vomeronasal type 2 receptor-like (OlfC: putative amino acids receptor in teleosts) gene cluster using the bacterial artificial chromosome library of the Lake Victoria cichlid, Haplochromis chilotes. In the cluster region, we found 61 intact OlfC genes, which is the largest number of OlfC genes identified among the seven teleost fish investigated to date. Data mining of the Oreochromis niloticus (Nile tilapia) draft genome sequence, and genomic Southern hybridization analysis revealed that the ancestor of all modern cichlids had already developed almost the same OlfC gene repertoire, which was accomplished by lineage-specific gene expansions. Furthermore, comparison of receptor sequences showed that recently duplicated paralogs are more variable than orthologs of different species at particular sites that were predicted to be involved in amino acid selectivity. Thus, the increase of paralogs through gene expansion may lead to functional diversification in detection of amino acids. This study implies that cichlids have developed a potent capacity to detect a variety of amino acids (and their derivatives) through OlfCs, which may have contributed to the extraordinary diversity of their feeding habitats.

Five tRNA-derived short interspersed repetitive elements (SINEs), named SepiaSINE, Sepioth-SINE1, Sepioth-SINE2A, Sepioth-SINE2B and OegopSINE, were isolated from the genomes of three decabrachian species [Sepia officinalis (order Sepiida), Sepiotheuthis lessoniana (suborder Myopsida), and Mastigoteuthis cordiformes (suborder Oegopsida)], by random sequencing and genome screening. In addition, two tRNA-derived SINEs, named IdioSINE1 and IdioSINE2, were further detected from EST (expressed sequence tag) data of Idiosepius paradoxus (order Idiosepiida), using a GenBank FASTA search with a conserved sequence of the SepiaSINE as the query. All the isolated SINEs had a common and unique highly conserved 149-bp sequence in their central structures (Sepioth-SINE2B and IdioSINEs, however, had a continuous 73-bp deletion in the conserved region.), and are therefore grouped as the fourth SINE superfamily "Ceph-SINEs", following the CORE-SINE, V-SINE, and DeuSINE superfamilies. Our analysis suggested that the central conserved region called the "Ceph-domain" might have originated before the diversification of cephalopods (505 myr ago). A sequence alignment of Sepioth-SINE1, Sepioth-SINE2A, and Sepioth-SINE2B demonstrated that Sepioth-SINE2A has a chimeric structure shared with two other SINEs. The above relationship suggests possible template switching in the central conserved domain during reverse transcription for the birth of Sepioth-SINE2A, providing the possibility that the presence of the conserved domain contributed to yield a variety of SINEs during evolution. Furthermore, the distributions of the isolated SINEs showed that order Sepiida, suborders Oegopsida and Myopsida, and order Idiosepiida have their own independent SINE(s), and suggest that order Sepiida can be largely separated into two groups, with clarification of the phylogenetic relatedness between subfamily Sepioteuthinae and the other loliginid squids.

Although recent mammalian genome projects have uncovered a large part of genomic component of various groups, several repetitive sequences still remain to be characterized and classified for particular groups. The short interspersed repetitive elements (SINEs) distributed among marsupial genomes are one example. We have identified and characterized two new SINEs from marsupial genomes that belong to the CORE-SINE family, characterized by a highly conserved "CORE" domain. PCR and genomic dot blot analyses revealed that the distribution of each SINE shows distinct patterns among the marsupial genomes, implying different timing of their retroposition during the evolution of marsupials. The members of Mar3 (Marsupialia 3) SINE are distributed throughout the genomes of all marsupials, whereas the Mac1 (Macropodoidea 1) SINE is distributed specifically in the genomes of kangaroos. Sequence alignment of the Mar3 SINEs revealed that they can be further divided into four subgroups, each of which has diagnostic nucleotides. The insertion patterns of each SINE at particular genomic loci, together with the distribution patterns of each SINE, suggest that the Mar3 SINEs have intensively amplified after the radiation of diprotodontians, whereas the Mac1 SINE has amplified only slightly after the divergence of hypsiprimnodons from other macropods. By compiling the information of CORE-SINEs characterized to date, we propose a comprehensive picture of how SINE evolution occurred in the genomes of marsupials.

The family Mustelidae, which consists of Mustelinae, Lutrinae, Melinae, and Taxidiinae, is the largest family among Carnivora and is a highly diverse group. Recent molecular phylogenetic studies have clarified the phylogenetic relations among Mustelidae, but there remain several unresolved problems, particularly concerning the deep branchings. Whereas many studies support the monophyly of Mustelidae+Procyonidae among Musteloidea, the relations between Mustelidae+Procyonidae, Ailuridae, and Miphitidae are still unclear. To address these problems, we inferred a tree on the basis of the sequences of mitochondrial genomes and of multiple nuclear genes using the maximum likelihood method. Our results strongly support the hypothesis that the Taxidiinae branched at first, followed by the branching of the Melinae. After that, Mustelinae diversified, and Lutrinae evolved within Mustelinae. With respect to the deep branchings in Musteloidea, the Ailuridae/Mephitidae monophyly tree and the Mephitidae-basal tree are indistinguishable in log-likelihood score, and this problem remains unresolved.

Morphological data have indicated that toothed whales form a monophyletic group. However, research published in the last several years has made the issue of the monophyly or paraphyly of toothed whales a subject of debate. Our group previously characterized three independent loci in which SINE insertions were shared among dolphins and sperm whales, thus supporting the traditional, morphologically based hypothesis of toothed whale monophyly. Although in recent years a few additional molecular works proposed this topology, there is still skepticism over this monophyly from the view point of molecular systematics. When the phylogeny of rapidly radiated taxa is examined using the SINE method, it is important to consider the ascertainment bias that arises when choosing a particular taxon for SINE loci screening. To overcome this methodological problem specific to the SINE method, we examined all possible topologies among sperm whales, dolphins and baleen whales by extensively screening SINE loci from species of all three lineages. We characterized nine independent SINE loci from the genomes of sperm whales and dolphins, all of which cluster sperm whales and dolphins but exclude baleen whales. Furthermore, we characterized ten independent loci from baleen whales, all of which were amplified in a common ancestor of these whales. From these observations, we conclude that toothed whales form a monophyletic group and that no ancestral SINE polymorphisms hinder their phylogenetic assignment despite the short divergence times of the major lineages of extant whales during evolution. These results suggest that a small population of common ancestors of all toothed whales ultimately diverged into the lineages of sperm whales and dolphins.

Bov-A2 is a retroposon that is widely distributed among the genomes of ruminants (e.g., cow, deer, giraffe, pronghorn, musk deer, and chevrotain). This retroposon is composed of two monomers, called Bov-A units, which are joined by a linker sequence. The structure and origin of Bov-A2 has been well characterized but a genome-level exploration of this retroposon has not been implemented. In this study we performed an extensive search for Bov-A2 using all available genome sequence data on Bos taurus. We found unique Bov-A2-derived sequences that were longer than Bov-A2 due to amplification of three to six Bov-A units arranged in tandem. Detailed analysis of these elongated Bov-A2-derived sequences revealed that they originated through unequal crossing-over of Bov-A2. We found a large number of these elongated Bov-A2-derived sequences in cattle genomes, indicating that unequal crossing-over of Bov-A2 occurred very frequently. We found that this type of elongation is not observed in wild bovine and is therefore specific to the domesticated cattle genome. Furthermore, at specific loci, the number of Bov-A units was also polymorphic between alleles, implying that the elongation of Bov-A units might have occurred very recently. For these reasons, we speculate that genomic instability in bovine genomes can lead to extensive unequal crossing-over of Bov-A2 and levels of polymorphism might be generated in part by repeated outbreeding.

Recent comprehensive studies of DNA sequences support the monophyly of Afrotheria, comprising elephants, sirenians (dugongs and manatees), hyraxes, tenrecs, golden moles, aardvarks, and elephant shrews, as well as that of Paenungulata, comprising elephants, sirenians, and hyraxes. However, phylogenetic relationships among paenungulates, as well as among nonpaenungulates, have remained ambiguous. Here we applied an extensive retroposon analysis to these problems to support the monophyly of aardvarks, tenrecs, and golden moles, with elephant shrews as their sister group. Regarding phylogenetic relationships in Paenungulata, we could characterize only one informative locus, although we could isolate many insertions specific to each of three lineages, namely, Proboscidea, Sirenia, and Hyracoidea. These data prompted us to reexamine phylogenetic relationships among Paenungulata using 19 nuclear gene sequences resulting in three different analyses, namely, short interspersed element (SINE) insertions, nuclear sequence analyses, and morphological cladistics, supporting different respective phylogenies. We concluded that these three lineages diverged very rapidly in a very short evolutionary period, with the consequence that ancestral polymorphism present in the last common ancestor of Paenungulata results in such incongruence. Our results suggest the rapid fixation of many large-scale morphological synapomorphies for Tethytheria; implications of this in relation to the morphological evolution in Paenungulata are discussed.

Afrotheria is a newly recognized taxon comprising elephants, hyraxes, sea cows, aardvarks, golden moles, tenrecs, and elephant shrews, each of which originated in Africa. Although some members of this taxon were once classified into distantly related groups, recent molecular studies have demonstrated their close relationships. It was suggested that this group emerged as a result of physical isolation of the African continent during the successive breakup events of Gondowanaland. In this study, a novel family of SINEs, designated AfroSINEs, was isolated and characterized from the genomes of afrotherians. This SINE family is distributed exclusively among the afrotherian species, confirming their monophyletic relationships. Furthermore, a distinct subfamily, which shares a deletion in the middle region of the SINE, was identified. The distribution of this subfamily is apparently restricted to the genomes of hyraxes, elephants, and sea cows, suggesting monophyly of these three groups, which was previously proposed as Paenungulata. We characterized the structures of the AfroSINEs from all afrotherian representatives by PCR, and we discuss how they were generated as well as the phylogenetic relationships of their host species.

CC-BY-NC-SA; International audience; Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.

Fish use olfaction to detect a variety of nonvolatile chemical signals, and thus, this sense is key to survival and communication. However, the contribution of the olfactory sense to social—especially reproductive—interactions in cichlids is still controversial. To obtain insights into this issue, we investigated the genes encoding V1Rs—possible candidates for reproductive pheromone receptors—among East-African cichlids. Interestingly, we found an excess of nonsynonymous over synonymous substitutions in four of six V1R genes in multiple cichlid lineages. First, we found that highly dimorphic V1R2 allele groups were shared among the cichlids inhabiting all East-African Great Lakes emerged through the episodic accumulation of the nonsynonymous substitutions prior to the radiation of the Lake Tanganyika species flock. We further detected such episodic events in V1R1 of the tribe Tropheini, and in V1R3 and V1R6 of the tribe Trematocarini. The excess of nonsynonymous substitutions in these examples were indicated as dN/dS > 1, which were all statistically significant by Fisher’s exact test. Furthermore, we speculate that the amino acid changes in these episodic events are likely functional switch because they occurred in the putative ligand-binding pocket. Our finding of the occurrence of multiple episodic events and the unexpected gene diversity in one unique gene family is suggestive of the contribution of the V1R to the species diversification and the social interaction in cichlids.

Coelacanths are known as “living fossils,” as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ∼2.74 Gbp and contains a high proportion (∼60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.

Coelacanths are known as “living fossils” because their morphology has changed very little from that in the fossil record. To elucidate why coelacanths have evolved so slowly is thus of primary importance in evolutionary biology. In the present study, we determined the entire sequence of the HOX cluster of the Tanzanian coelacanth (Latimeria chalumnae) and compared it with that of the Indonesian coelacanth (L. menadoensis), which was available in the literature. The most intriguing result was the extremely small genetic divergence between the two coelacanths. The synonymous divergence of the HOX coding region between the two coelacanths was estimated to be 0.07%, which is ~ 11-fold smaller than that of human–chimp. When we applied the estimated divergence time of the two coelacanths of 6 million years ago (MYA) and 30 MYA, which were proposed in independent mitochondrial DNA analyses, the synonymous substitution rate of the coelacanth HOX cluster was estimated to be ~ 11-fold and 56-fold smaller than that of human–chimp, respectively. Thus, the present study implies that the reduction of the nucleotide substitution rate in coelacanth HOX genes may account for the conservation of coelacanth morphology during evolution.

We determined the complete mitochondrial (mt) genomes of the deep-sea squid (Bathyteuthis abyssicola; supperfamily Bathyteuthoidea), the bob-tail squid (Semirossia patagonica; order Sepiolida) and four giant cuttlefish (Sepia apama, S. latimanus, S. lycidas and S. pharaonis; order Sepiida). The unique structures of the mt genomes of Bathyteuthis and Semirossia provide new information about the evolution of decapodiform mt genomes. We show that the mt genome of B. abyssicola, like those of other oegopsids studied so far, has two long duplicated regions that include seven genes (COX1-3, ATP6 and ATP8, tRNA(Asn), and either ND2 or ND3) and that one of the duplicated COX3 genes has lost its function. The mt genome of S. patagonica is unlike any other decapodiforms and, like Nautilus, its ATP6 and ATP8 genes are not adjacent to each other. The four giant cuttlefish have identical mt gene order to other cuttlefish determined to date. Molecular phylogenetic analyses using maximum likelihood and Bayesian methods suggest that traditional order Sepioidea (Sepiolida+Sepiida) is paraphyletic and Sepia (cuttlefish) has the sister-relationship with all other decapodiforms. Taking both the phylogenetic analyses and the mt gene order analyses into account, it is likely that the octopus-type mt genome is an ancestral state and that it had maintained from at least the Cephalopoda ancestor to the common ancestor of Oegopsida, Myopsida and Sepiolida.

Recent rapid generation of genomic sequence data has allowed many researchers to perform comparative analyses in various mammalian species. However, characterization of transposable elements, such as short interspersed repetitive elements (SINEs), has not been reported for several mammalian groups. Because SINEs occupy a large portion of the mammalian genome, they are believed to have contributed to the constitution and diversification of the host genomes during evolution. In the present study, we characterized a novel SINE family in the anteater genomes and designated it the MyrSINE family. Typical SINEs consist of a tRNA-related, a tRNA-unrelated and an AT-rich (or poly-A) region. MyrSINEs have only tRNA-related and poly-A regions; they are included in a group called t-SINE. The tRNA-related regions of the MyrSINEs were found to be derived from tRNA Gly . We demonstrate that the MyrSINE family can be classified into three subfamilies. Two of the MyrSINE subfamilies are distributed in the genomes of both giant anteater and tamandua, while the other is present only in the giant anteater. We discuss the evolutionary history of MyrSINEs and their relationship to the evolution of anteaters. We also speculate that the simple structure of t-SINEs may be a potential evolutionary source for the generation of the typical SINE structure.

We have discovered a new SINE that appears to have acquired a novel role in signal transduction in the lineage of a group of artiodactyls. SINEs are a group of retroposons which have been amplified and integrated into genomes by retroposition (Okada and Ohshima 1995) The majority of SINEs are believed to be parasitic and to represent junk DNA in host organisms. However, in certain cases, SINEs appear to have gained novel functions and to have become involved in biological processes. More than 10 such examples have been recorded, in which sequences of SINEs have become promoters that modify the expression of preexisting functional genes or provide a new polyadenylation signal (Britten 1996; Makalowski 1995). In other cases, SINEs have been integrated into the coding region and are translated into peptides, with resulting generation of protein variability. In fact, more than 30 SINEs have been found in the coding regions of mRNAs (Makalowski 1995). However, no examples of such messages playing an explicit role in an important biological process have yet been reported (Makalowski 1995). We recently characterized the CHR-1 SINEs, which are distributed exclusively among whales, cows, and hippopotamuses (Shimamura et al. 1997). In a BLASTN search (Altschul et al. 1990), we found an example of a CHR-1 SINE that was inserted into the coding region of mRNA for the EP3 subtype of receptor for bovine prostaglandin (PG) E2. It appears that this message with an integrated SINE plays a key role in the signal transduction that is mediated by the bovine EP3 receptor for PGE2. Prostaglandin E2 has a broad range of biological effects in diverse tissues that are mediated through its binding to specific receptors on plasma membranes. Four subtypes of receptors (EP1, EP2, EP3, and EP4) for PGE2 have been recognized on the basis of their respective responses to agonists and antagonists (Coleman et al. 1989). It has been proposed that the EP3 receptor mediates various very different effects of PGE2, such as inhibition of neurotransmitter release (Ohia and Jumblatt 1990), contraction of the uterus (Krall et al. 1984), inhibition of the reabsorption of sodium ions and water in kidney tubules (Nakao et al. 1989), and stimulation of catecholamine release from adrenal chromaffin cells (Tanaka et al. 1990). The EP3 receptor is a protein with seven transmembrane domains. The bovine EP3 receptor exists as

Baleen whales (suborder Mysticeti) comprise 11 extant species that are classified into four families. Although several phylogenetic hypotheses about these taxa have been proposed, their phylogenetic relationships remain confused. We addressed this problem using short interspersed repetitive element (SINE) insertion data, which now are regarded as almost ideal shared, derived characters at the molecular level. We reconstructed the phylogenetic relationships of baleen whales by characterizing 36 informative SINE loci. One of the intriguing conclusions is that balaenopterids and eschrichtiids radiated very rapidly during a very short evolutionary period. During this period, speciation occurred in balaenopterids and eschrichtiids while newly inserted SINE loci remains polymorphic. Later on, these SINEs were sorted incompletely into each lineage. Thus, there are now inconsistencies among species regarding the presence or absence of a given SINE. This is in sharp contrast to the phylogeny of toothed whales, for which no SINE inconsistencies have been found. Furthermore, we found monophyletic groupings between humpback and fin whales as well as between (sei+Bryde's) whales and blue whales, both of which have not previously been recognized. The comprehensive SINE insertion data, together with the mitochondrial DNA phylogeny that was recently completed (Sasaki, T., M. Nikaido, H. Healy et al. 2005. Mitochondrial phylogenetics and evolution of mysticete whales. Syst. Biol. 56:77-90; Rychel, A. L., T. W. Reeder, and A. Berta. 2004. Phylogeny of mysticete whales based on mitochondrial and nuclear data. Mol. Phylogenet. Evol. 32:892-901), provide a nearly complete picture of the evolutionary history of baleen whales.

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.

Advances in genome sciences are demonstrating the dynamic nature of noncoding DNA regions, which are comprised largely of repetitive elements with no apparent function. Retroposons are one class of mobile genetic elements that amplify and move about the genome via a copy-and-paste mechanism that employs an RNA intermediate. Short and long interspersed elements (SINEs and LINEs, respectively) are types of retroposons of particular interest because of their active role in shaping the architecture of genomes and their diagnostic value as evolutionary markers for studies of phylogeny and population biology. Although the use of SINEs and LINEs for molecular systematic studies is proliferating, a comprehensive laboratory protocol that explicitly outlines how to isolate and characterize retroposons for systematic studies in a detailed, step-by-step fashion has been lacking. The present chapter addresses this gap in the literature by focusing on the strategy for isolating new SINEs from a genomic library, the screening process, the sequencing and characterization of clones into subfamilies, quantification of copy number in host taxa, and the critical diagnosis of phylogenetically informative SINE and LINE insertion patterns. Practical limits to the method are discussed in relation to sampling design, systematic character theory, and the empirical distribution of elements observed in eukaryotic lineages. Major steps in the experimental process are illustrated with case examples from a diversity of taxonomic groups and by published results in the molecular biology and systematics literature.