A key evolutionary step determining osmoregulatory ability for freshwater colonisation in early life stages of fish.

Colonisation of freshwater habitats by marine animals is a remarkable evolutionary event that has enriched biodiversity in freshwater ecosystems. The acquisition of tolerance to hypotonic stress during early life stages is presumed to be essential for their successful freshwater colonisation, but very little empirical evidence has been obtained to support this idea. This study aimed to comprehend the evolutionary changes in osmoregulatory mechanisms that enhance larval freshwater tolerance in amphidromous fishes, which typically spend their larval period in marine (ancestral) habitats and the rest of their life history stages in freshwater (derived) habitats. We compared the life history patterns and changes in larval survivorship and gene expression depending on salinity among three congeneric marine-originated amphidromous goby species (Gymnogobius), which had been suggested to differ in their larval dependence on freshwater habitats. An otolith microchemical analysis and laboratory-rearing experiment confirmed the presence of freshwater residents only in G. urotaenia and higher larval survivorship of this species in the freshwater condition than in the obligate amphidromous G. petschiliensis and G. opperiens. Larval whole-body transcriptome analysis revealed that G. urotaenia from both amphidromous and freshwater-resident populations exhibited the greatest differences in expression levels of several osmoregulatory genes, including aqp3, which is critical for water discharge from their body during early fish development. The present results consistently support the importance of enhanced freshwater tolerance and osmoregulatory plasticity in larval fish to establish freshwater forms, and further identified key candidate genes for larval freshwater adaptation and colonisation in the goby group.

Environmental DNA phylogeography: Successful reconstruction of phylogeographic patterns of multiple fish species from cups of water.

Phylogeography is an integrative field of science linking micro- and macro-evolutionary processes, contributing to the inference of vicariance, dispersal, speciation, and other population-level processes. Phylogeographic surveys usually require considerable effort and time to obtain numerous samples from many geographical sites covering the distribution range of target species; this associated high cost limits their application. Recently, environmental DNA (eDNA) analysis has been useful not only for detecting species but also for assessing genetic diversity; hence, there has been growing interest in its application to phylogeography. As the first step of eDNA-based phylogeography, we examined (1) data screening procedures suitable for phylogeography and (2) whether the results obtained from eDNA analysis accurately reflect known phylogeographic patterns. For these purposes, we performed quantitative eDNA metabarcoding using group-specific primer sets in five freshwater fish species belonging to two taxonomic groups from a total of 94 water samples collected from western Japan. As a result, three-step data screening based on the DNA copy number of each haplotype detected successfully eliminated suspected false positive haplotypes. Furthermore, eDNA analysis could almost perfectly reconstruct the phylogenetic and phylogeographic patterns obtained for all target species with the conventional method. Despite existing limitations and future challenges, eDNA-based phylogeography can significantly reduce survey time and effort and is applicable for simultaneous analysis of multiple species in single water samples. eDNA-based phylogeography has the potential to revolutionize phylogeography.

Origin of scarlet gynogenetic triploid Carassius fish: Implications for conservation of the sexual-gynogenetic complex.

Conservation of sperm-dependent asexual (gynogenetic) species is challenging due to their complicated ecological dynamics, which requires the stable coexistence with their sperm-providing sexual relatives, who often share similar niches. A symbolic but vulnerable gynogenetic animal is the scarlet Carassius fish, or Hibuna, which is mainly found in Lake Harutori on Hokkaido, Japan. Although Hibuna in Lake Harutori has been protected as a symbol of the Natural Monument of Japan, it has recently suffered population decline. To establish an effective conservation strategy for Hibuna, we investigated its origin, reproductive mode, and genetic diversity, with reference to the surrounding wild populations, using nuclear microsatellites and mitochondrial gene sequences. Our genetic analyses revealed that the main ploidy of Hibuna was triploid or tetraploid, and it reproduces gynogenetically. However, no co-existing sexual diploid Carassius was detected among our samples, suggesting that the sexual diploids and the gynogenetic population including Hibuna would be at risk of co-extirpation. In addition, Hibuna showed high genetic/clonal diversity and most Hibuna had nonindigenous mitochondrial haplotypes that are mostly identical to those reported from goldfish. These results indicate that Hibuna most probably originated from hybridization between indigenous gynogenetic triploids and goldfish introduced about 100 years ago, involving rare sexual reproduction. This spontaneous long-term field experiment exemplifies the recently documented diversification process of gynogenetic Carassius via complex interploidy gene flow. Although the priority to be placed on the conservation of Hibuna is controversial, the maintenance of gynogenetic Carassius, including Hibuna, requires strategic conservation of sexual populations.

Cryptic genetic divergence of the red dwarf rasbora, Microrasbora rubescens, in and around Inle Lake: implications for the origin of endemicity in the ancient lake in Myanmar.

Inle Lake, an ancient lake located in the Shan Plateau of Myanmar, is a biogeographically attractive region with high fish endemism. Some endemic species inhabit the lake as well as the surrounding areas. The genetic and ecological relationships between populations in the lake and surrounding areas provide important insights into the process underlying ichthyofaunal formation in Inle Lake. In this study, the authors focused on red dwarf rasbora Microrasbora rubescens, an endemic genus and species in this region, and estimated its population structure and evolutionary scenario based on genome-wide polymorphism, mtDNA and geometric morphometric analyses using samples from Inle Lake and three areas surrounding the lake. The results showed that M. rubescens comprises at least three genetically divergent lineages (Inle, Heho and Hopong) with distinct geographic structures consistent with nuclear and mtDNA data. In contrast, there was no clear regional differentiation in morphology. The divergence time estimation based on mtDNA suggests that the Hopong lineage diverged at 2.7 Ma and the Inle and Heho lineages diverged at 1.9 Ma - consistent with the nuclear DNA results. The deep divergence observed in the endemic species supports the ancient history of ichthyofaunal development in this region. The distinct regional differentiation and morphological conservatism of this species might have been shaped by niche conservatism in stagnant water environments that limit dispersal and morphological diversification. Future comprehensive genetic and morphological analyses and comparisons for other native species should reveal the geographic and ecological processes that shaped the ichthyofauna in this region.

Fish diversity of a spring field in Hopong Town, Taunggyi District, Shan State, Myanmar (the Salween River Basin), with genetic comparisons to some "species endemic to Inle Lake".

Hopong, a small town in the Salween (Thanlwin) River Basin, Myanmar, is located 35 km northeast of Inle Lake, a famous ancient lake with numerous endemic fish species. We surveyed the fish fauna of a spring pond in Hopong in 2016, 2019 and 2020 and identified 25 species. Of these, seven, including Inlecyprisauropurpureus and Sawbwaresplendens, had been considered endemic to Inle Lake and at least three species were genetically unique. Eight were suspected or definite introduced species, including Oreochromisniloticus and Gambusiaaffinis. We were unable to identify a nemacheilid species of the genus Petruichthys, which would need a taxonomic examination. The Hopong area is being developed rapidly and, hence, it is crucial to conserve its native fish species and the freshwater ecosystems.

Molecular evolution and convergence of the rhodopsin gene in Gymnogobius, a goby group having diverged into coastal to freshwater habitats.

Adaptive evolution of vision-related genes has been frequently observed in the process of invasion of new environments in a wide range of animal taxa. The typical example is that of the molecular evolution of rhodopsin associated with habitat changes in aquatic animals. However, few studies have investigated rhodopsin evolution during adaptive radiation across various habitats. In the present study, we examined the link between molecular evolutionary patterns in the rhodopsin gene and macroscopic habitat changes in Gymnogobius species (Gobiidae), which have adaptively radiated to diverse aquatic habitats including the sea, brackish waters, rivers and lakes. Analysis of amino acid substitutions in rhodopsin in the phylogenetic framework revealed convergent substitutions in 4-5 amino acids in three groups (four species), including two spectral tuning amino acid sites known to change rhodopsin's absorption wavelength. Positive selection was detected in the basal branches of each of these three groups, suggesting adaptive molecular convergence of rhodopsin. However, no significant correlation was observed between amino acid substitutions and the species' habitat changes, suggesting molecular adaptation to some unidentified micro-ecological environments. Taken together, these results emphasize the importance of considering not only macroscopic habitats but also micro-ecological environments when elucidating the driving forces of adaptive evolution of the visual system.

Interploidy gene flow involving the sexual-asexual cycle facilitates the diversification of gynogenetic triploid Carassius fish.

Asexual vertebrates are rare and at risk of extinction due to their restricted adaptability through the loss of genetic recombination. We explore the mechanisms behind the generation and maintenance of genetic diversity in triploid asexual (gynogenetic) Carassius auratus fish, which is widespread in East Asian fresh waters and exhibits one of the most extensive distribution among asexual vertebrates despite its dependence on host sperm. Our analyses of genetic composition using dozens of genetic markers and genome-wide transcriptome sequencing uncover admixed genetic composition of Japanese asexual triploid Carassius consisting of both the diverged Japanese and Eurasian alleles, suggesting the involvement of Eurasian lineages in its origin. However, coexisting sexual diploid relatives and asexual triploids in Japan show regional genetic similarity in both mitochondrial and nuclear markers. These results are attributed to a unique unidirectional gene flow from diploids to sympatric triploids, with the involvement of occasional sexual reproduction. Additionally, the asexual triploid shows a weaker population structure than the sexual diploid, and multiple triploid lineages coexist in most Japanese rivers. The generated diversity via repeated interploidy gene flow as well as an increased establishment of immigrants is assumed to offset the cost of asexual reproduction and might contribute to the successful broad distribution of this asexual vertebrate.

Gudgeon fish with and without genetically determined countershading coexist in heterogeneous littoral environments of an ancient lake.

Countershading, characterized by a darker dorsal surface and lighter ventral surface, is common among many animals. This dorsoventral pigment polarity is often thought to be adaptive coloration for camouflage. By contrast, noncountershaded (melanistic) morphs often occur within a species due to genetic color polymorphism in terrestrial animals. However, the polymorphism with either countershaded or melanistic morphs is poorly known in wild aquatic animals. This study explored the genetic nature of diverged color morphs of a lineage of gudgeon fish (genus Sarcocheilichthys) in the ancient Lake Biwa and propose this system as a novel model for testing hypotheses of functional aspects of countershading and its loss in aquatic environments. This system harbors two color morphs that have been treated taxonomically as separate species; Sarcocheilichthys variegatus microoculus which occurs throughout the littoral zone and Sarcocheilichthys biwaensis which occurs in and around rocky areas. First, we confirmed that the divergence of dorsoventral color patterns between the two morphs is under strict genetic control at the levels of chromatophore distribution and melanin-related gene expression under common garden rearing. The former morph displayed sharp countershading coloration, whereas the latter morph exhibited a strong tendency toward its loss. The crossing results indicated that this divergence was likely controlled by a single locus in a two-allele Mendelian inheritance pattern. Furthermore, our population genomic and genome-wide association study analyses detected no genome-wide divergence between the two morphs, except for one region near a locus that may be associated with the color divergence. Thus, these morphs are either in a state of intraspecific color polymorphism or two incipient species. Evolutionary forces underlying this polymorphism appear to be associated with heterogeneous littoral environments in this lake. Future ecological genomic research will provide insight into adaptive functions of this widespread coloration, including the eco-evolutionary drivers of its loss, in the aquatic world.

Mesozoic origin and 'out-of-India' radiation of ricefishes (Adrianichthyidae).

The Indian subcontinent has an origin geologically different from Eurasia, but many terrestrial animal and plant species on it have congeneric or sister species in other parts of Asia, especially in the Southeast. This faunal and floral similarity between India and Southeast Asia is explained by either of the two biogeographic scenarios, 'into-India' or 'out-of-India'. Phylogenies based on complete mitochondrial genomes and five nuclear genes were undertaken for ricefishes (Adrianichthyidae) to examine which of these two biogeographic scenarios fits better. We found that Oryzias setnai, the only adrianichthyid distributed in and endemic to the Western Ghats, a mountain range running parallel to the western coast of the Indian subcontinent, is sister to all other adrianichthyids from eastern India and Southeast-East Asia. Divergence time estimates and ancestral area reconstructions reveal that this western Indian species diverged in the late Mesozoic during the northward drift of the Indian subcontinent. These findings indicate that adrianichthyids dispersed eastward 'out-of-India' after the collision of the Indian subcontinent with Eurasia, and subsequently diversified in Southeast-East Asia. A review of geographic distributions of 'out-of-India' taxa reveals that they may have largely fuelled or modified the biodiversity of Eurasia.

Morphological variation associated with trophic niche expansion within a lake population of a benthic fish.

Ecological theory suggests that generalist species should have traits with multiple adaptive peaks. Consequently, in heterogeneous environments such adaptive landscapes may lead to phenotypic divergence that becomes fixed in populations via reproductive isolation, thus driving speciation. However, contrary to this expectation, the process of ecological diversification in wild populations is not always associated with obvious trait divergence and reproductive isolation due to some ecological and geographic constrains. To examine the ecological conditions that promote (or inhibit) divergence is quite important to improve our understanding of the underlying mechanisms. Here we examine how the patterns of trait variation (divergence/non-divergence) are determined in relation to ecological niche expansion and gene flow using a benthic fish, Pseudogobio esocinus, in the Lake Biwa system, Japan. The fish exhibited various patterns of morphological variation in mouth parts among populations. Lake fish tended to have a smaller mouth compared with river fish and also showed remarkable individual variations within some local samples. Lake fish utilized chironomid larvae as the primary prey, as in riverine fish. But, fish with smaller and narrower mouths utilized significantly higher proportions of amphipods (a novel prey unique to the lake) as their secondary prey. Microsatellite analysis detected no genetic structuring in the Lake Biwa catchment, suggesting no reproductive separation among eco-morphologically divergent individuals. Our results exemplify population niche expansion associated with continuous eco-morphological variation without divergence, and provide insights into the role of non-discrete diversification for thriving in heterogeneous environments.

Ecosystem size predicts the probability of speciation in migratory freshwater fish.

Predicting speciation is a fundamental goal of research in evolutionary ecology. The probability of speciation is often positively correlated with ecosystem size. Although the mechanisms driving this correlation are generally difficult to identify, a shared geographical and ecological context provides a suitable condition to study the mechanisms that promote speciation in large ecosystems by reducing the number of factors to be considered. Here, we determined the correlation between speciation and ecosystem size, and discuss the underlying mechanisms of this relationship, using a probable parallel ecotype formation for freshwater fish. Our population genetic analysis revealed that speciation of the landlocked goby, Rhinogobius sp. YB, of the Ryukyu Archipelago, Japan, from its migratory ancestor, R. brunneus, occurred in parallel across five islands. Logistic regression analysis showed that speciation probability could be predicted using island size. The results suggest that ecosystem size predicts the occurrence of adaptation and reproductive isolation, probably through its association with three possible factors: divergent selection strength, population persistence, and occurrence probability of habitat separation.

[Investigation of Factors Related to Stockpiling of Oral Care Hygiene Materials in Normal Times: Internet Survey Analysis].

OBJECTIVES: The aim of this study was to clarify the current status of stockpiling of oral care hygiene materials in case of a disaster and to determine methods to support stockpiling during disaster preparation. METHODS: We conducted an Internet questionnaire survey on stockpiling and disaster prevention measures among 300 adults. For statistical analysis, the χ² test, Mann-Whitney U-test and Spearman rank test were employed. Logistic regression analysis was conducted to review factors related to stockpiling. RESULTS: Among the questions on stockpiling and disaster prevention (14 of 15 items), seven items on disaster prevention measures and two on disaster prevention experiences significantly differed. Analysis of disaster prevention knowledge revealed that disaster prevention measures and disaster experiences significantly differed in terms of the presence or absence of stockpiling, albeit a positive correlation was noted with stockpiling. Logistic regression analysis was conducted with stockpiling as a dependent variable. As a result, the items 'I am aware about the hazard map of the residential area', 'I am aware about the type of oral care hygiene materials prepared for emergency' and 'Devising the living environment such that large furnitures and cupboards do not collapse during disasters' were effective. CONCLUSIONS: Stockpiling at home is considered necessary for self-sufficiency during a disaster on the basis of the experiences in disaster management, disaster prevention knowledge and disaster prevention measures. Hence, stockpiling is a strong measure against disaster prevention along with provision of appropriate knowledge about the necessity of oral care in daily life. Disaster prevention education that empowers a population to prepare itself by implementing disaster prevention measures, such as improving the home environment, is considered effective. In addition, considering that relying on stockpiling at an individual level is ineffective, it is necessary to create mechanisms and systems that jointly manage disaster situations according to local circumstances.

A dataset of fishes in and around Inle Lake, an ancient lake of Myanmar, with DNA barcoding, photo images and CT/3D models.

BACKGROUND: Inle (Inlay) Lake, an ancient lake of Southeast Asia, is located at the eastern part of Myanmar, surrounded by the Shan Mountains. Detailed information on fish fauna in and around the lake has long been unknown, although its outstanding endemism was reported a century ago. NEW INFORMATION: Based on the fish specimens collected from markets, rivers, swamps, ponds and ditches around Inle Lake as well as from the lake itself from 2014 to 2016, we recorded a total of 948 occurrence data (2120 individuals), belonging to 10 orders, 19 families, 39 genera and 49 species. Amongst them, 13 species of 12 genera are endemic or nearly endemic to the lake system and 17 species of 16 genera are suggested as non-native. The data are all accessible from the document "A dataset of Inle Lake fish fauna and its distribution (http://ipt.pensoft.net/resource.do?r=inle_fish_2014-16)", as well as DNA barcoding data (mitochondrial COI) for all species being available from the DDBJ/EMBL/GenBank (Accession numbers: LC189568-LC190411). Live photographs of almost all the individuals and CT/3D model data of several specimens are also available at the graphical fish biodiversity database (http://ffish.asia/INLE2016; http://ffish.asia/INLE2016-3D). The information can benefit the clarification, public concern and conservation of the fish biodiversity in the region.

Impacts of Dams and Global Warming on Fish Biodiversity in the Indo-Burma Hotspot.

Both hydropower dams and global warming pose threats to freshwater fish diversity. While the extent of global warming may be reduced by a shift towards energy generation by large dams in order to reduce fossil-fuel use, such dams profoundly modify riverine habitats. Furthermore, the threats posed by dams and global warming will interact: for example, dams constrain range adjustments by fishes that might compensate for warming temperatures. Evaluation of their combined or synergistic effects is thus essential for adequate assessment of the consequences of planned water-resource developments. We made projections of the responses of 363 fish species within the Indo-Burma global biodiversity hotspot to the separate and joint impacts of dams and global warming. The hotspot encompasses the Lower Mekong Basin, which is the world's largest freshwater capture fishery. Projections for 81 dam-building scenarios revealed progressive impacts upon projected species richness, habitable area, and the proportion of threatened species as generating capacity increased. Projections from 126 global-warming scenarios included a rise in species richness, a reduction in habitable area, and an increase in the proportion of threatened species; however, there was substantial variation in the extent of these changes among warming projections. Projections from scenarios that combined the effects of dams and global warming were derived either by simply adding the two threats, or by combining them in a synergistic manner that took account of the likelihood that habitat shifts under global warming would be constrained by river fragmentation. Impacts on fish diversity under the synergistic projections were 10-20% higher than those attributable to additive scenarios, and were exacerbated as generating capacity increased-particularly if CO2 emissions remained high. The impacts of dams, especially those on river mainstreams, are likely to be greater, more predictable and more immediately pressing for fishes than the consequences of global warming. Limits upon dam construction should therefore be a priority action for conserving fish biodiversity in the Indo-Burma hotspot. This would minimize synergistic impacts attributable to dams plus global warming, and help ensure the continued provision of ecosystem services represented by the Lower Mekong fishery.

Phylogeny and historical demography of endemic fishes in Lake Biwa: the ancient lake as a promoter of evolution and diversification of freshwater fishes in western Japan.

To elucidate the origins of the endemic fish of Lake Biwa, an ancient lake in Japan, and the role of the lake in the diversification of freshwater fish in western Japan, we established a molecular phylogenetic framework with an absolute time scale and inferred the historical demography of a large set of fish species in and around the lake. We used mtDNA sequences obtained from a total of 190 specimens, including 11 endemic species of Lake Biwa and their related species, for phylogenetic analyses with divergence time estimations and from a total of 2319 specimens of 42 species (including 14 endemics) occurring in the lake for population genetic analyses. Phylogenetic analysis suggested that some of the endemic species diverged from their closest relatives earlier (1.3-13.0 Ma) than the period in which the present environmental characteristics of the lake started to develop (ca. 0.4 Ma), whereas others diverged more recently (after 0.4 Ma). In contrast, historical demographic parameters suggested that almost all species, including endemic and nonendemic ones, expanded their populations after the development of the present lake environment. In phylogeographic analyses, common or very close haplotypes of some species were obtained from Lake Biwa and other regions of western Japan. The phylogenetic and historical demographic evidence suggests that there was a time lag between phylogenetic divergence and population establishment and that phenotypic adaptation of some endemic species to the limnetic environment occurred much later than the divergences of those endemic lineages. Population structure and phylogeographic patterns suggest that Lake Biwa has functioned not only as the center of adaptive evolution but also as a reservoir for fish diversity in western Japan.

Genomic architecture of habitat-related divergence and signature of directional selection in the body shapes of Gnathopogon fishes.

Evolution of ecomorphologically relevant traits such as body shapes is important to colonize and persist in a novel environment. Habitat-related adaptive divergence of these traits is therefore common among animals. We studied the genomic architecture of habitat-related divergence in the body shape of Gnathopogon fishes, a novel example of lake-stream ecomorphological divergence, and tested for the action of directional selection on body shape differentiation. Compared to stream-dwelling Gnathopogon elongatus, the sister species Gnathopogon caerulescens, exclusively inhabiting a large ancient lake, had an elongated body, increased proportion of the caudal region and small head, which would be advantageous in the limnetic environment. Using an F2 interspecific cross between the two Gnathopogon species (195 individuals), quantitative trait locus (QTL) analysis with geometric morphometric quantification of body shape and restriction-site associated DNA sequencing-derived markers (1622 loci) identified 26 significant QTLs associated with the interspecific differences of body shape-related traits. These QTLs had small to moderate effects, supporting polygenic inheritance of the body shape-related traits. Each QTL was mostly located on different genomic regions, while colocalized QTLs were detected for some ecomorphologically relevant traits that are proxy of body and caudal peduncle depths, suggesting different degree of modularity among traits. The directions of the body shape QTLs were mostly consistent with the interspecific difference, and QTL sign test suggested a genetic signature of directional selection in the body shape divergence. Thus, we successfully elucidated the genomic architecture underlying the adaptive changes of the quantitative and complex morphological trait in a novel system.

Phylogeny, hybridization, and life history evolution of Rhinogobius gobies in Japan, inferred from multiple nuclear gene sequences.

Rhinogobius fishes (Gobiidae) are distributed widely in East and Southeast Asia, and represent the most species-rich group of freshwater gobies with diversified life histories (i.e., amphidromous, fluvial, and lentic). To reveal their phylogenetic relationships and life history evolution patterns, we sequenced six nuclear and three mitochondrial DNA (mtDNA) loci from 18 species, mainly from the mainland of Japan and the Ryukyu Archipelago. Our phylogenetic tree based on nuclear genes resolved three major clades, including several distinct subclades. The mtDNA and nuclear DNA phylogenies showed large discordance, which strongly suggested mitochondrial introgression through large-scale interspecific hybridization in these regions. On the basis of the molecular dating using geological data as calibration points, the hybridization occurred in the early to middle Pleistocene. Reconstruction of the ancestral states of life history traits based on nuclear DNA phylogeny suggests that the evolutionary change from amphidromous to freshwater life, accompanied by egg size change, occurred independently in at least three lineages. One of these lineages showed two life history alterations, i.e., from amphidromous (small egg) to fluvial (large egg) to lentic (small egg). Although more inclusive analysis using species outside Japan should be further conducted, the present results suggest the importance of the life history evolution associated with high adaptability to freshwater environments in the remarkable species diversification in this group. Such life history divergences may have contributed to the development of reproductive isolation.

Do stage-specific functional responses of consumers dampen the effects of subsidies on trophic cascades in streams?

Resource subsidies often weaken trophic cascades in recipient communities via consumers' functional response to the subsidies. Consumer populations are commonly stage-structured and may respond to the subsidies differently among the stages yet less is known about how this might impact the subsidy effects on the strength of trophic cascades in recipient systems. We show here, using a large-scale field experiment, that the stage structure of a recipient consumer would dampen the effects of terrestrial invertebrate subsidies on the strength of trophic cascade in streams. When a high input rate of the terrestrial invertebrates was available, both large and small fish stages switched their diet to the terrestrial subsidy, which weakened the trophic cascade in streams. However, when the input rate of the terrestrial invertebrates was at a moderate level, the terrestrial subsidy did not weaken the trophic cascade. This discrepancy was likely due to small fish stages being competitively excluded from feeding on the subsidy by larger stages of fish and primarily foraging on benthic invertebrates under the moderate input level. Although previous studies using single fish stages have clearly demonstrated that the terrestrial invertebrate input equivalent to our moderate input rate weakened the trophic cascade in streams, this subsidy effect might be overestimated given small fish stage may not switch their diet to the subsidy under competition with large fish stage. Given the ubiquity of consumer stage structure and interaction among consumer stages, the effects we saw might be widespread in nature, requiring future studies that explicitly involve consumer's stage structure into community ecology.

Loss of genetic diversity at an MHC locus in the endangered Tokyo bitterling Tanakia tanago (Teleostei: Cyprinidae).

Genetic diversity at a major histocompatibility complex (MHC) class II B gene was examined for two wild and three captive populations of the endangered Tokyo bitterling Tanakia tanago. A specific primer set was first developed to amplify the MHC II B exon 2 locus. Using single strand conformation polymorphism (SSCP) and sequencing analysis, 16 DAB3 alleles were detected with 56 nucleotide substitutions in the 276-bp region. In the putative antigen-binding sites of exon 2, the rate of nonsynonymous substitutions was significantly higher than that of synonymous substitutions (dN/dS = 2.79), indicating positive selection on the retention of polymorphism. The population from the Handa Natural Habitat Conservation Area and that from the Tone River system exhibited low variation (one and three alleles, respectively), whereas the captive population that originated from a mix of three distinct populations had the highest amounts of variation (14 alleles). The levels of heterozygosity at the MHC varied considerably among populations and showed significant correlations with those at putative neutral microsatellite markers, suggesting that genetic drift following a bottleneck has affected MHC variability in some populations. Comparisons between endangered and non-endangered fish species in previous reports and the present results indicate that the number of MHC alleles per population is on average 70% lower in endangered species than non-endangered species. Considering the functional consequence of this locus, attention should be paid to captive and wild endangered fish populations in terms of further loss of MHC alleles.

A RAD-based linkage map and comparative genomics in the gudgeons (genus Gnathopogon, Cyprinidae).

BACKGROUND: The construction of linkage maps is a first step in exploring the genetic basis for adaptive phenotypic divergence in closely related species by quantitative trait locus (QTL) analysis. Linkage maps are also useful for comparative genomics in non-model organisms. Advances in genomics technologies make it more feasible than ever to study the genetics of adaptation in natural populations. Restriction-site associated DNA (RAD) sequencing in next-generation sequencers facilitates the development of many genetic markers and genotyping. We aimed to construct a linkage map of the gudgeons of the genus Gnathopogon (Cyprinidae) for comparative genomics with the zebrafish Danio rerio (a member of the same family as gudgeons) and for the future QTL analysis of the genetic architecture underlying adaptive phenotypic evolution of Gnathopogon. RESULTS: We constructed the first genetic linkage map of Gnathopogon using a 198 F2 interspecific cross between two closely related species in Japan: river-dwelling Gnathopogon elongatus and lake-dwelling Gnathopogon caerulescens. Based on 1,622 RAD-tag markers, a linkage map spanning 1,390.9 cM with 25 linkage groups and an average marker interval of 0.87 cM was constructed. We also identified a region involving female-specific transmission ratio distortion (TRD). Synteny and collinearity were extensively conserved between Gnathopogon and zebrafish. CONCLUSIONS: The dense SNP-based linkage map presented here provides a basis for future QTL analysis. It will also be useful for transferring genomic information from a "traditional" model fish species, zebrafish, to screen candidate genes underlying ecologically important traits of the gudgeons.