Highly rapid and diverse sex chromosome evolution in the Odorrana frog species complex.

Sex chromosomes in poikilothermal vertebrates are characterized by rapid and diverse evolution at the species or population level. Our previous study revealed that the Taiwanese frog Odorrana swinhoana (2n = 26) has a unique system of multiple sex chromosomes created by three sequential translocations among chromosomes 1, 3, and 7. To reveal the evolutionary history of sex chromosomes in the Odorrana species complex, we first identified the original, homomorphic sex chromosomes, prior to the occurrence of translocations, in the ancestral-type population of O. swinhoana. Then, we extended the investigation to a closely related Japanese species, Odorrana utsunomiyaorum, which is distributed on two small islands. We used a high-throughput nuclear genomic approach to analyze single-nucleotide polymorphisms and identify the sex-linked markers. Those isolated from the O. swinhoana ancestral-type population were found to be aligned to chromosome 1 and showed male heterogamety. In contrast, almost all the sex-linked markers isolated from O. utsunomiyaorum were heterozygous in females and homozygous in males and were aligned to chromosome 9. Morphologically, we confirmed chromosome 9 to be heteromorphic in females, showing a ZZ-ZW sex determination system, in which the W chromosomes were heterochromatinized in a stripe pattern along the chromosome axis. These results indicated that after divergence of the two species, the ancestral homomorphic sex chromosome 1 underwent highly rapid and diverse evolution, i.e., sequential translocations with two autosomes in O. swinhoana, and turnover to chromosome 9 in O. utsunomiyaorum, with a transition from XY to ZW heterogamety and change to heteromorphy.

Verification of Natural Marking for Individual Identification Using a Duplex Marking Approach in Ijima's Sea Snakes, Emydocephalus ijimae (Reptilia: Elapidae).

The mark and recapture method for free-ranging animals provides valuable information in ecological studies. Recently, natural marking has become more frequently used for individual identification, but it almost inevitably induces problems associated with the corroboration of individual specificity and the persistence of the given markings. We employed a duplex natural marking approach to resolve this problem over a four-year field study of a banded hydrophine sea snake and tested the effectiveness of this approach in corroborating the accuracy of individual identification. We conducted monthly field surveys in southwestern Japanese waters and photographed the patterns of the last five bands on each captured sea snake. We converted the band patterns into profile codes with five sections (one section corresponding to each band), according to the scale configurations involved in the bands. We considered the bilateral band patterns as a duplex set of natural markings for individual identification and checked their accuracy mutually. We looked at 593 photos of recorded snakes and recognized 179 unique profile codes on both the left and right sides, 96 of which were recorded more than once on both side. A particular code for the left side was always accompanied by a particular code on the right side in the same combination. It is certain that the 593 recorded snakes consisted of 179 snakes and their recaptures. The perfect correspondence between the left and right side profile codes throughout the four years showed the high individual uniqueness and persistence of each pattern. This study also showed that the duplex natural marking approach is effective in verifying the accurate individual identification. The duplex natural marking approach can be applied to various animals to justify the usage of a given natural marker for individual identification, without the aid of combined artificial markings. The duplex method itself can be a combination of the first five bands and the next five bands on the same side in a single photo, or a combination of some patterns on the head and those on the body.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for quick identification of the Japanese salamander Hynobius tokyoensis.

Species identification using molecular techniques has recently become common for various taxa. Loop-mediated isothermal amplification (LAMP) is one of the easiest and least expensive molecular identification methods. Although few studies have developed LAMP assays for amphibians, we believe that LAMP is also useful for identifying endangered amphibians. Hynobius tokyoensis and H. lichenatus occur in Honshu, Japan, and have parapatric distributions. They are similar morphologically, especially at early developmental stages, including eggs and larvae. Hynobius tokyoensis has been listed as a national endangered species in Japan since 2020, and unambiguous identification of these species is therefore important for their conservation and management. In this study, we developed a LAMP primer set for the mitochondrial cytochrome b region to detect H. tokyoensis, and we evaluated the LAMP assay using total genomic DNA from four H. tokyoensis and three H. lichenatus individuals from across most of their ranges. Our LAMP primer set could distinguish these two species. This study should help to establish LAMP assays for other endangered species and morphologically similar species.

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

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

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

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

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

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

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.

Correction to: Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

Unfortunately, one of the co-author's family name has been incorrectly published in the original online publication. The correct family name should be Tsai.

The role of ecological factors in determining phylogeographic and population genetic structure of two sympatric island skinks (Plestiodon kishinouyei and P. stimpsonii).

We conducted comparative phylogeographic and population genetic analyses of Plestiodon kishinouyei and P. stimpsonii, two sympatric skinks endemic to islands in the southern Ryukyus, to explore different factors that have influenced population structure. Previous phylogenetic studies using partial mitochondrial DNA indicate similar divergence times from their respective closest relatives, suggesting that differences in population structure are driven by intrinsic attributes of either species rather than the common set of extrinsic factors that both presumably have been exposed to throughout their history. In this study, analysis of mtDNA sequences and microsatellite polymorphism demonstrate contrasting patterns of phylogeography and population structure: P. kishinouyei exhibits a lower genetic variability and lower genetic differentiation among islands than P. stimpsonii, consistent with recent population expansion. However, historical demographic analyses indicate that the relatively high genetic uniformity in P. kishinouyei is not attributable to recent expansion. We detected significant isolation-by-distance patterns among P. kishinouyei populations on the land bridge islands, but not among P. stimpsonii populations occurring on those same islands. Our results suggest that P. kishinouyei populations have maintained gene flows across islands until recently, probably via ephemeral Quaternary land bridges. The lower genetic variability in P. kishinouyei may also indicate smaller effective population sizes on average than that of P. stimpsonii. We interpret these differences as a consequence of ecological divergence between the two species, primarily in trophic level and habitat preference.

The function of body coloration of the hai coral snake Sinomicrurus japonicus boettgeri.

BACKGROUND: Prey animals often protect themselves from visual hunting predators via their body coloration, which encompasses various visual effects. When a prey animal displays a certain color pattern on its body surface, its protective function and effect are largely dependent on how a predator would encounter and perceive the prey animal.Asian coral snakes of the genus Sinomicrurus,which are venomous, display black bands and stripes on their orange body coloration. The banded pattern has been characterized as an aposematic signal in the New World coral snakes, but the stripes generally occur in cryptic snakes. We investigated the function of this complex color pattern, which might be interpreted as aposematic and cryptic, in Sinomicrurus japonicusboettgeri. RESULTS: First, plasticine replica experiments were conducted to assess whether natural avian predators avoid the colorpattern of S.japonicus boettgeri;the results showed that they attacked the coral snake replicas and the control replicas with coloration similar to another prey snake, suggesting that the body coloration of S. japonicus boettgeri did not function aposematically in the wild. Second, we evaluated the chromatic contrast of the snake coloration with backgrounds from their natural habitats based on the avian predator visual systems. The body coloration of S. japonicus boettgeri showed the same, or lower, contrast levels with natural backgrounds than those of sympatric cryptic snakes, suggesting that the coloration was ineffective as an aposematic signal. CONCLUSIONS: These results imply that the body coloration of S. japonicus boettgeri would work as crypsis through background matching or disruptive camouflage rather than aposematism.

An investigation for population maintenance mechanism in a miniature garden: genetic connectivity or independence of small islet populations of the Ryukyu five-lined skink.

The Ryukyu five-lined skink (Plestiodon marginatus) is an island lizard that is even found in tiny islets with less than half a hectare of habitat area. We hypothesized that the island populations are maintained under frequent gene flow among the islands or independent of each other. To test our hypotheses, we investigated genetic structure of 21 populations from 11 land-bridge islands that were connected during the latest glacial age, and 4 isolated islands. Analyses using mitochondrial cytochrome b gene sequence (n = 67) and 10 microsatellite loci (n = 235) revealed moderate to high levels of genetic differentiation, existence of many private alleles/haplotypes in most islands, little contemporary migration, a positive correlation between genetic variability and island area, and a negative correlation between relatedness and island area. These evidences suggest a strong effect of independent genetic drift as opposed to gene flow, favoring the isolation hypothesis even in tiny islet populations. Isolation-by-distance effect was demonstrated and it became more prominent when the 4 isolated islands were excluded, suggesting that the pattern is a remnant of the land-bridge age. In a few island populations, however, the possibility of occasional overwater dispersals was partially supported and therefore could not be ruled out.

Phylogenetic relationships, genetic divergence, historical biogeography and conservation of an endangered gecko, Goniurosaurus kuroiwae (Squamata: Eublepharidae), from the Central Ryukyus, Japan.

The Kuroiwa's eyelid gecko Goniurosaurus kuroiwae is an endangered species in a state of relict endemism in the Central Ryukyus, Japan, and is divided into five subspecies. We analyzed variations in sequence data for approximately 1900 base positions of mitochondrial 12S and 16S rRNA, and cytochrome b genes from samples representing all recognized subspecies of G. kuroiwae together with those from congeneric species in order to test the relevant previous phylogenetic hypotheses and discuss biogeographical implications in the degree and pattern of genetic divergence within G. kuroiwae. Our results, while confirming a previous molecular phylogenetic hypothesis proposed on the basis of much smaller data set, negate the relationships hypothesized on morphological grounds by explicitly supporting: 1) the primary dichotomy, with substantial genetic divergence, between G. k. splendens from the Amami Island Group and the remaining subspecies all from the Okinawa Island Group; and 2) the presence of at least six independent lineages within the latter, indicating non-monophyly for two of the subspecies, G. k. kuroiwae and G. k. orientalis, in the current taxonomic definitions. The marked genetic divergence between populations of the two island groups seems to have initiated in the middle Miocene, i.e., prior to formation of straits that have consistently been separating these two island groups since the early Pleistocene. All populations of G. kuroiwae are regarded as endangered from the viewpoint of conservation genetics.

Spatial and temporal instability of local biotic community mediate a form of aposematic defense in newts, consisting of carotenoid-based coloration and tetrodotoxin.

Most animals advertise their unprofitability to potential predators via conspicuous signals. Whether the strength of this aposematic signal indicates the quality and quantity of chemical defenses in animals is controversial. Here, we investigated the relationship between the conspicuousness of an aposematic signal and toxicity, which likely depends, at least in part, on dietary sources, in the newt Cynops pyrrhogaster. Our results indicate that the magnitude of the aposematic signal was not correlated with the amount of tetrodotoxin (TTX) and 6-epi TTX of wild individuals among populations. Using atoxic newts, reared from eggs, we compared the ability to accumulate TTX from diets between mainland and island populations. Newts of a mainland population that exhibited a less conspicuous signal accumulated more TTX than did equivalent newts of an insular population that displayed a more conspicuous signal; this was unrelated to variation in the toxicity of wild individuals of these two populations. We also found toxicity of wild newts changed over approximately one generation (10 years) in both populations. These results indirectly suggest that environmental variance, such as fluctuations in TTX resources in nature, may obscure differences in the ability of wild newts to accumulate TTX, and that this variation may be responsible for a lack of correlation between the strength of a newt's signal and its toxicity in the wild. These results imply that toxicity of wild individuals likely is a phenotypic trait largely dependent on environmental conditions.

Geographic genetic structure in two laticaudine sea kraits, Laticauda laticaudata and Laticauda semifasciata (Serpentes: Elapidae), in the Ryukyu-Taiwan region as inferred from mitochondrial cytochrome b sequences.

The Ryukyu-Taiwan region is an island arch with intervening waters of varying distances and depths. This study examines the geographic genetic structure of two sympatric sea kraits, Laticauda laticaudata and L. semifasciata, in the region, to infer factors affecting the extent of dispersal and other biogeographical traits of these amphibious reptiles. Sequence analyses of the mitochondrial cytochrome b gene revealed four and 16 haplotypes for L. laticaudata (136 individuals) and L. semifasciata (177 individuals), respectively. For both species, population pairwise F ST analyses revealed significant genetic differentiations among islands and island groups, which are separated by deep straits, suggesting that deep waters serve as obstacles for dispersal in both species. Significant genetic differentiation was detected even among islands of the same basin in L. laticaudata, but not in L. semifasciata, and the isolation by distance analyses revealed no significant correlation between geographic and genetic distances in the former species. These results further suggest that L. laticaudata has stronger site fidelity or degree of philopatry than L. semifasciata. Based on the geographic genetic patterns, the historical biogeography of the two species in the Ryukyu-Taiwan region is also discussed.

Allozyme variation in the camaenid tree snails Amphidromus atricallosus (Gould, 1843) and A. inversus (Müller, 1774).

We examined allozyme variation in two camaenid tree snails, Amphidromus atricallosus and A. inversus, across two principal regions of Thailand and from Singapore, plus for A. inversus, one site in peninsular Malaysia. Using horizontal starch gel electrophoresis, 13 allozyme loci (11 polymorphic) were screened for A. atricallosus and 18 (5 polymorphic) for A. inversus. Heterozygosity was higher in A. atricallosus (Hexp=0.018-0.201, mean=0.085) than in A. inversus (Hexp=0-0.023, mean= 0.002). Genetic heterogeneity among samples was higher in A. inversus (Fst=0.965) than in A. atricallosus (Fst=0.781). Within A. atricallosus, populations were more differentiated in southern Thailand (Fst=0.551) than in eastern Thailand (Fst=0.144). The high Fst and low Hexp in populations of A. inversus suggest that this species is likely to have experienced a series of strong bottlenecks, perhaps occurring chiefly on offshore continental-shelf islands. The low Fst values of A. atricallosus in eastern Thailand suggest frequent gene flows among populations in this region. The southern and eastern samples of A. atricallosus exhibited fixed allele differences at four loci and great genetic distance (Nei's D=0.485-0.946), suggesting that these two samples may actually represent, or else be evolving into, separate species.

Parapatric distribution of the lizards Plestiodon (formerly Eumeces) latiscutatus and P. japonicus (Reptilia: Scincidae) around the Izu Peninsula, Central Japan, and its biogeographic implications.

The scincid lizard Plestiodon latiscutatus is found in the Izu Islands and Izu Peninsula of central Japan, whereas P. japonicus, a close relative, is found over the entire main island group of Japan, except the Izu Peninsula. The precise area of occupancy of these species was surveyed around the Izu Peninsula. Species identification was made through comparison of mitochondrial DNA partial sequences of specimens from the Izu Peninsula with those from the other regions, since morphological differences between these species have not yet been characterized. This study determined that these species are deeply diverged from each other in mitochondrial DNA sequence, and that the ranges of these species overlap only in a narrow zone. The results imply that gene flow between these species, if any, is restricted to a low level, without physical barriers. The boundary between the geographic ranges of these species was established as occurring along the lower Fuji River, Mt. Fuji, and the Sakawa River. This region is concordant with that of the old sea that is assumed to have separated the Izu Peninsula from other parts of the Japanese main island group until the middle Pleistocene. This pattern suggests that P. latiscutatus and P. japonicus were differentiated allopatrically before the connection of land areas of the Izu Peninsula and Honshu, the main island of Japan, and come into secondary contact through this connection. Thus, the species boundary is likely to have been maintained in situ, without physical barriers, since the secondary contact in the middle Pleistocene.

Extensive Natural Hybridization Between Two Geckos, Gekko tawaensis and Gekko japonicus (Reptilia: Squamata), Throughout Their Broad Sympatric Area.

The status of natural hybridization between the two Japanese geckos, Gekko tawaensis and Gekko japonicus, was surveyed by use of 15 diagnostic allozyme loci. Of 438 specimens examined, 9 were identified as F(l), 1 was a first backcross with G. japonicus, and 15 were identified as more successive generations. Hybridizations were detected at 7 of the 15 localities interspersed throughout a broad sympatric area of the two species, forming a mosaic hybrid zone. A comparison of species-hybrid composition between 2-year samples from a single locality and a 5-year interval showed no evidence for progressive introgression or establishment of a hybrid swarm, despite constant emergences of F(l) hybrids. Nonprevalence of the hybrid genotypes was also indicated by the analysis using hybrid index scores for all other localities examined. These results suggest that strong selection acts against hybrid genotypes. Morphological features of hybrid individuals were also provided.

Discovery of sympatric cryptic species within Gekko hokouensis (Gekkonidae: Squamata) from the Okinawa Islands, Japan, by use of allozyme data.

An electrophoretic survey of samples of the gekkonid lizard, Gekko hokouensis, from the East Asian islands demonstrated that two genetically divergent, but morphologically almost identical, entities occur on five islands of the Okinawa Group, Ryukyu Archipelago, Japan. These entities, while sharing all of the external character states diagnostic of G. hokouensis, exhibited fixed allele differences at six to eight out of 30 loci examined and great overall genetic distances [Nei's (1978) D = 0.489-0.654]. On Kumejima and Tonakijima Islands of the Okinawa Group, the two entities were collected together from identical microhabitats. These results indicate that the two entities represent separate biological species. Genetic comparisons of these two cryptic species from the Okinawa Group with 'G. hokouensis' from other island groups revealed that one occurs broadly in the insular region of East Asia, whereas the other is restricted to the Okinawa Group. Implications of the present findings for the morphological evolution of 'G. hokouensis' are also discussed.