The phylogeographic puzzle of Pseudoacanthocephalus toshimai,an amphibian acanthocephalan in northern Japan

The amphibian acanthocephalan, Pseudoacanthocephalus toshimai, was considered to be an island-endemic species in Hokkaido, Japan. However, the parasite was found from Rana ornativentris, Rana tagoi, Zhangixalus arboreus, and Bufo japonicus formosus in northern Honshu (Aomori and Iwate Prefectures), which is separated from Hokkaido by the Tsugaru Strait. The mitochondrial DNA-based phylogenetic and population genetic analyses of P. toshimai showed that the northern Honshu isolates are far distantly related to the Hokkaido isolates, and that a demographic population expansion occurred in Hokkaido during the recent geological past. The rich genetic diversity of P. toshimai in northern Honshu suggests a scenario that anuran hosts invaded Hokkaido together with P. toshimai via the land bridge of the Tsugaru Strait. However, the evolutionary history of Rana pirica, a main definitive host for P. toshimai in Hokkaido, is contradictory to the introduction scenario inferred from the parasite. The finding of several geographically mismatched isolates of P. toshimai from both northern Honshu and Hokkaido suggests a possibility that the migration of the parasite infrequently occurred between the two areas even after the land bridge disappeared. More detailed information on the evolutionary history of anurans is needed to resolve the biogeographical enigma of P. toshimai.     

Morphological and molecular differentiation of two new species of Pseudoacanthocephalus Petrochenko, 1958 (Acanthocephala: Echinorhynchidae) from amphibians and reptiles in the Philippines, with identification key for the genus

The genus Pseudoacanthocephalus Petrochenko, 1958 currently includes 14 species of acanthocephalans parasitic in amphibians and reptiles worldwide. This work describes two new species of Pseudoacanthocephalus from amphibians and reptiles collected in several localities on Luzon Island, Philippines. Pseudoacanthocephalus nickoli n. sp. was found in two species of frogs, Rana luzonensis Boulenger and Rana similis (Günther), and Pseudoacanthocephalus smalesi n. sp. was found in a scincid lizard, Sphenomorphus abdictus Brown & Alcala. Differential diagnoses of the two new species of Pseudoacanthocephalus from their congeners are provided. Comparative analysis of nuclear ribosomal rRNA sequences encompassing the 3′ end of 18S nuclear rDNA gene, internal transcribed spacer region (ITS1+5.8S+ITS2), and 5′ end of the 28S gene strongly corroborated the morphological evidence and demonstrated significant differences between the two new species as well as between these species and closely related species from continental China and Vietnam. No intraspecific sequence variability was detected among different individuals representing each of the examined species. This is the first report of Pseudoacanthocephalus in the Philippines. A key to known species of Pseudoacanthocephalus is provided.     

Frequent infections of mountain stream fish with the amphibian acanthocephalan,Pseudoacanthocephalus toshimai (Acanthocephala: Echinorhynchidae)

Pseudoacanthocephalus toshimai is an intestinal acanthocephalan parasite of amphibians in Hokkaido, the northernmost island of Japan. In this study, common freshwater fish of the families Salmonidae and Cottidae in mountain streams around the Kamikawa basin of Hokkaido were examined for acanthocephalan infections with P. toshimai. A total of 160 salmonids and 14 cottids were caught in 4 streams by bait fishing during summer and autumn seasons of 2019. Adult acanthocephalans were found only from the salmonids, namely, Salvelinus leucomaenis leucomaenis, Salvelinus malma krascheninnikovi, Oncorhynchus masou, and Oncorhynchus mykiss. The maximum prevalence reached 58.1% in S. leucomaenis, but the mean worm burden was at low levels (e.g., 3.1 in S. leucomaenis and 2.2 in S. malma). All of the acanthocephalans were identified to P. toshimai by morphological observation and DNA barcoding. Although the male acanthocephalans became sexually mature, the females never reached the gravid adult stage, suggesting that salmonids are unsuitable or aberrant hosts for P. toshimai. The infected fish were found exclusively from a small stream with bush, in which a large habitat of amphibians is included. Ligidium japonicum, a terrestrial isopod, collected from the habitat was highly infected with cystacanth larvae of P. toshimai. The observation of fish stomach contents directly demonstrated that small salmonids eat L. japonicum. The terrestrial isopods, which are washed away by rain into a stream, seem to be a source of salmonid infections with P. toshimai. The habitat of intermediate hosts should be emphasized in the taxonomy of the closely related genera Acanthocephalus and Pseudoacanthocephalus.     

Notes on the genus <Emphasis Type="Italic">Inocybe</Emphasis> of Japan. IV. Species having metuloids collected from Hokkaido,Honshu, and Kyushu

This fourth paper in the series considers five species of Inocybe occurring in Hokkaido, Honshu, and Kyushu. (1) Inocybe furfurea (section Tardae) is recorded from Hokkaido as new to Japan. (2) Inocybe luteola sp. nov. (section Tardae) is described from eastern Honshu (Chiba). It has smooth basidiospores and thick-walled caulocystidia descending to the middle of the stipe. (3) Inocybe napiformis sp. nov. [section Inocybe (= Cortinatae)], known from Hokkaido, appears close to Inocybe napipes, but characters of the metuloids distinguish the two species. (4) Inocybe grammata (section Marginatae) is recorded from Hokkaido and Nagano as new to Japan. (5) Inocybe pyriformis sp. nov. (section Marginatae) is described from Kyushu (Miyazaki). It has nodulose basidiospores and caulocystidia wholly covering the stipe surface. However, I. pyriformis is not typical in the section, lacking a marginate bulbous base in its stipe.     

Brachylaima lignieuhadrae n. sp. (Trematoda: Brachylaimidae) from land snails of the genus Euhadra in Japan

Land snails of the genus Euhadra (Gastropoda: Bradybaenidae) are indigenous to the Japanese Archipelago. The larvae of an unknown species, tentatively named as Brachylaima sp. B (Trematoda: Brachylaimidae), have been found from Euhadra brandtii sapporo in Hokkaido, the northernmost island of Japan. In this study, a large-scale snail survey covering a wide area of Japan was conducted to confirm the larval parasite from members of Euhadra and related genera. Sporocysts with cercariae were found only from Eu. brandtii sapporo in Hokkaido and Euhadra callizona in central Honshu at low prevalence (1.0–9.6%). The metacercariae were detected widely from 6 species of Euhadra and the related genera at high prevalence (7.1–100%). A molecular identification by DNA barcoding demonstrated almost all of the larvae to be Brachylaima sp. B. Adult worms experimentally raised from the metacercariae were morphologically most similar to Brachylaima ezohelicis in Hokkaido, but could be differentiated by the microstructure of the tegumental surface. We propose Brachylaima lignieuhadrae n. sp. for the unknown species, based on the morphology, DNA profile, host specificity, and geographic distribution. Phylogeography of the new species suggests a possibility that migratory birds serve as the definitive hosts.     

Alectrias markevichi sp. nov.—A new species of cockscombs (Perciformes: Stichaeidae: Alectriinae) from the sublittoral of the Sea of Japan and adjacent waters

A new species of cockscombs, Alectrias markevichi sp. nov., is described from 46 individuals from the sublittoral of Peter the Great Bay and Moneron Island (the Sea of Japan), South Kuril Strait, southern Hokkaido, and northeastern Honshu (the northwestern Pacific). A. markevichi sp. nov. differs from all the known species of the genus in the presence of only three pores in the occipital commissure (no pores near crest base and no medial tubule), only four pores in the preopercular canal, only one shortened spine at the end of dorsal fin, and 11?C18 preural vertebrae with wide neural processes.     

Male death resulting from hybridization between subspecies of the gypsy moth,Lymantria dispar

We explored the origin of all-female broods resulting from male death in a Hokkaido population of Lymantria dispar through genetic crosses based on the earlier experiments done by Goldschmidt and by testing for the presence of endosymbionts that are known to cause male killing in some insect species. The mitochondrial DNA haplotypes of the all-female broods in Hokkaido were different from those of normal Hokkaido females and were the same as those widely distributed in Asia, including Tokyo (TK). Goldschmidt obtained all-female broods through backcrossing, that is, F1 females obtained by a cross between TK females (L. dispar japonica) and Hokkaido males (L. dispar praeterea) mated with Hokkaido males. He also obtained all-male broods by mating Hokkaido females with TK males. Goldschmidt inferred that female- and male-determining factors were weakest in the Hokkaido subspecies and stronger in the Honshu (TK) subspecies. According to his theory, the females of all-female broods mated with Honshu males should produce normal sex-ratio broods, whereas weaker Hokkaido sexes would be expected to disappear in F1 or F2 generations after crossing with the Honshu subspecies. We confirmed both of Goldschmidt''s results: in the case of all-female broods mated with Honshu males, normal sex-ratio broods were produced, but we obtained only all-female broods in the Goldschmidt backcross and obtained an all-male brood in the F1 generation of a Hokkaido female crossed with a TK male. We found no endosymbionts in all-female broods by 4,′6-diamidino-2-phenylindole (DAPI) staining. Therefore, the all-female broods observed in L. dispar are caused by some incompatibilities between Honshu and Hokkaido subspecies.     

Integrative Taxonomic Approach for Describing a New Cryptic Species of Bush Frog (Raorchestes: Anura: Rhacophoridae) from the Western Ghats,India

A new cryptic species of bush frog Raorchestes honnametti sp. nov. is described from the south-eastern part of the Western Ghats, India. This newly described species belongs to the Charius clade and is morphologically similar to other clade members—R. charius and R. griet. Therefore, an integrative taxonomic approach based on molecular and bioacoustic analysis along with morphology was used to delimit the new species. Raorchestes honnametti sp. nov., is currently known only from Biligiri Rangaswamy Temple Tiger Reserve, a part of Biligiri Rangaswamy horst mountain range (a mountain formed due movement of two faults) formed during the Late Quaternary period (1.8–2.58 Ma). Discovery of cryptic species from a highly speciose and well-studied genus Raorchestes hints at the possible existence of several more cryptic species in this genus. We discuss the possible reasons for crypsis and emphasize the need for continued systematic surveys of amphibians across the Western Ghats.     

New and already known acanthocephalans from amphibians and reptiles in Vietnam, with keys to species of Pseudoacanthocephalus Petrochenko, 1956 (Echinorhynchidae) and Sphaerechinorhynchus Johnston and Deland, 1929 (Plagiorhynchidae)

Adults of 2 new species in 2 orders of acanthocephalans obtained from the intestines of terrestrial amphibians and reptiles collected between 1998 and 2004 in Vietnam are described here. Pseudoacanthocephalus nguyenthileae n. sp. (Palaeacnthocephala: Echinorhynchidae) was collected from 5 species of terrestrial amphibians: (1) the common Sunda toad Bufo melanostictus Schneider (Bufonidae); (2) Paa verucospinosa (Bourret); (3) Gunther's Amoy frog Rana guentheri Boulenger; (4) Taipei frog R. taipehensis Denburgh (Ranidae), and (5) the Burmese whipping frog Polypedates mutus (Smith) (Racophoridae); as well as from the Chinese cobra Naja atra Cantor (Reptilia: Elapidae) and house gecko Hemidactylus frenatus Dumeril and Bibron (Reptilia: Gekkonidae). Sphaerechinorhynchus maximesospinus n. sp. (Plagiorhynchidae: Sphaerechinorhynchinae) was isolated from a king cobra Ophiophagus hannah (cantor) (Reptilia: Elapidae). Cystacanths of Porrorchis houdemeri (Joyeux and Baer, 1935) Schmidt and Kuntz, 1967 (Plagiorhynchidae: Porrorchinae) obtained from the mesenteries of banded krait Bungarus fasciatus (Schneider) (Reptilia: Elapidae), a paratenic host, are reported for the first time. Keys to the species of Pseudoacanthocephalus and Sphaerechinorhynchus are included. Characteristic features distinguishing the new species from related taxa include: P. nguyenthileae has 15-19 (usually 16-18) proboscis hook rows, each with 5-6 hooks that progressively increase in length and size posteriorly. The largest, intermediate, and smallest proboscis hooks of S. maximesospinus are the middle, anterior, and posterior hooks, respectively; the proboscis and neck are enclosed in a membrane. Morphometric characteristics of P. nguyenthileae show host-related variability.     

Phylogenetic analyses of Japanese golden chanterelles and a new species description,Cantharellus anzutake sp. nov.

The Japanese golden chanterelle commonly identified as Cantharellus cibarius was sampled in a broad range of forest vegetation. A total of 90 fresh and 11 herbarium specimens were examined microscopically, subjected to sequencing analysis of their nuclear ribosomal RNA (rDNA) and tef-1 genes, and their characteristics were compared with those of European C. cibarius. Based on morphological and ecological characteristics, basidioma samples from Japan were divided into four species. While specimens of Cantharellus sp. 4 from Hokkaido Island were included in the European C. cibarius clade phylogenetically, the other three species formed three unique clades. Among these, Cantharellus anzutake sp. nov. is sister to the clade of C. cibarius and was widely sampled from the northern limit of Honshu Island to the southern limit of Kumejima Island in Ryukyu Islands. Although C. anzutake was morphologically similar to C. cibarius, the two species were phylogenetically distinct. Other morphologically similar but genetically distinct chanterelle species from India exhibited macroscopic and microscopic differences compared with C. anzutake.     

Contrasting patterns of nuclear microsatellite genetic structure of Fraxinus mandshurica var. japonica between northern and southern populations in Japan

Aim The aim of this study is to detect extant patterns of population genetic structure of Fraxinus mandshurica var. japonica in Japan, and to provide insights into the post‐glacial history of this species during the Holocene. Location Hokkaido and Honshu islands, Japan (including the Oshima and Shimokita peninsulas). Methods We examined nine polymorphic nuclear microsatellite loci to assess genetic variation within and among 15 populations across almost the entire range of the species in Japan. Extant patterns of geographical structure were analysed using Bayesian clustering, Monmonier’s algorithm, analysis of molecular variance, Mantel tests and principal coordinates analysis. Recent bottlenecks within populations and regional genetic variation were also assessed. Results Northern populations (Hokkaido Island and the Shimokita Peninsula) formed a single homogeneous deme, maintaining the highest level of allelic diversity on the Oshima Peninsula. By contrast, southern populations (Honshu Island) demonstrated strong substructure on both coasts. Specifically, populations on the Pacific side of Honshu exhibited significant bottlenecks and erosion of allelic diversity but preserved distinct subclusters diverging from widespread subclusters on the Japan Sea side of this island. Main conclusions Genetic evidence and life history traits suggest that F. mandshurica occupied cryptic northern refugia on the Oshima Peninsula during the Last Glacial Maximum, which is reflected in the species’ extant northern distribution. Strong geographical structure in southern populations, in agreement with fossil pollen records, suggests geographical isolation by mountain ranges running north–south along Honshu. Given that this tree species is cold‐adapted and found in riparian habitats, populations on the Pacific side of Honshu probably contracted into higher‐elevation swamps during warm post‐glacial periods, leading to a reduction of effective population sizes and rare allelic richness.     

Phylogeography and genetic structure of disjunct Salix arbutifolia populations in Japan

Disjunct geographic distributions of boreal plant species at the southern edges of their ranges are expected to result in low genetic diversity and high genetic differentiation in the disjunct populations. This prediction was tested in a riparian willow, Salix arbutifolia, distributed in the northeastern Eurasian continent and the Sakhalin, Hokkaido, and Honshu Islands, using chloroplast DNA haplotypes and nuclear microsatellite genotypes. Hokkaido and Honshu populations shared a chloroplast haplotype identical to a closely related species, S. cardiophylla. This haplotype was divergent from haplotypes in the Eurasian continent (Primorsky) and the Sakhalin Island. In the nuclear microsatellites, most Hokkaido populations were genetically closer to Primorsky populations than to Sakhalin populations in spite of the geographical vicinity between Sakhalin and Hokkaido. The unexpected genetic divergence between Sakhalin and Hokkaido implies a complicated history of migration and colonization. The most peripheral populations in Honshu had the lowest genetic diversity and were most differentiated from the others. Thus, low genetic diversity and high genetic differentiation at the range periphery suggest substantial effects of genetic drift on genetic structure in the disjunct populations of Salix arbutifolia at the southern edge of its range.     

Allozyme Variation among Populations of Rana pirica (Amphibia: Anura)

Genetic variation of Rana pirica , an east Asian brown frog of the R. temporaria group, was elucidated by analysing 140 specimens from 11 populations from Hokkaido and Sakhalin, both locating near the eastern coasts of the Asian continent, and 12 specimens of R. ornativentris from Honshu, Japan mainland, as an outgroup, through horizontal starch gel electrophoresis. Rana pirica shows relatively low genetic differentiation, and high genetic affinities are found between Hokkaido and Sakhalin populations. Populations from these islands are morphologically somewhat differentiated but should be regarded as conspecific. Degree of genetic divergence among populations of amphibian species from Hokkaido, including R. pirica , is not so extensive as that reported for species from Japan mainland and relatively recent formation of amphibian fauna in Hokkaido is suggested.     

Phylogeography of Rhabdias spp. (Nematoda: Rhabdiasidae) collected from Bufo species in Honshu,Shikoku, and Kyushu,Japan including possible cryptic species

The genus Rhabdias Stiles & Hassall, 1905 comprises lung parasites of amphibians and reptiles worldwide. In Japan, 9 species have been recorded, including Rhabdias incerta Wilkie, 1930 which has been reported only in Bufo species. In this study, to assess the diversity of R. incerta, we performed molecular analyses of Rhabdias species sampled from three species/subspecies of Japanese toads namely Bufo japonicus, B. japonicus formosus, and B. torrenticola, collected in various regions of Honshu, Shikoku, and Kyushu, Japan. DNA sequence divergence was compared using mtDNA (COI) and nuclear DNA (28S) to identify possible cryptic species. Morphological analysis was performed through light microscopy and scanning electron microscopy (SEM). The results revealed that Bufo spp. serve as hosts for at least three Rhabdias species. Morphologically, most samples were identified as R. incerta but with a longer body and larger buccal cavity than originally described. Rhabdias incerta appears to be specific to the genus Bufo and is further subdivided into two or three phylogroups based on subspecies divisions and biogeography of their host. Some Rhabdias specimens collected in this study resemble R. tokyoensis Wilkie, 1930, parasitic in hosts from the order Caudata, which suggests host switching. Both molecular and morphological analyses suggested the presence of undescribed and cryptic Rhabdias species within toads collected in Japan. This study was the first to molecularly characterize Rhabdias species in Japan, including novel sequences of R. incerta and two undescribed species.     

Novel Genetic Variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a Novel Ehrlichia sp. in Wild Deer and Ticks on Two Major Islands in Japan

Wild deer are one of the important natural reservoir hosts of several species of Ehrlichia and Anaplasma that cause human ehrlichiosis or anaplasmosis in the United States and Europe. The primary aim of the present study was to determine whether and what species of Ehrlichia and Anaplasma naturally infect deer in Japan. Blood samples obtained from wild deer on two major Japanese islands, Hokkaido and Honshu, were tested for the presence of Ehrlichia and Anaplasma by PCR assays and sequencing of the 16S rRNA genes, major outer membrane protein p44 genes, and groESL. DNA representing four species and two genera of Ehrlichia and Anaplasma was identified in 33 of 126 wild deer (26%). DNA sequence analysis revealed novel strains of Anaplasma phagocytophilum, a novel Ehrlichia sp., Anaplasma centrale, and Anaplasma bovis in the blood samples from deer. None of these have been found previously in deer. The new Ehrlichia sp., A. bovis, and A. centrale were also detected in Hemaphysalis longicornis ticks from Honshu Island. These results suggest that enzootic cycles of Ehrlichia and Anaplasma species distinct from those found in the United States or Europe have been established in wild deer and ticks in Japan.     

Comparison of susceptibility to a toxic alien toad (Bufo japonicus formosus) between predators in its native and invaded ranges

1. To manage biological invasions effectively, the impacts of alien species on the demography and traits of native species must be known, but determining those impacts can be challenging. We used a comparative approach to gain insight into the impacts that an alien toad (Bufo japonicus formosus) might have on native Japanese predatory amphibians. We compared the susceptibility of native predator species to alien toad toxins in the alien-invaded range and the susceptibility of closely related native predator species to the toxins in the alien toad's native range to investigate the impacts of an alien on a native species.
2. Bufo japonicus formosus is native to Honshu, but was recently introduced to Hokkaido and Sado. In laboratory experiments, we compared individual mortality of predators exposed to a toad hatchling between novel predators on the toad-invaded islands and ecologically similar congeneric or conspecific species on Honshu, where the toad is native. We also compared (1) the percentage of individuals that consumed a toad hatchling and (2) toxin resistance (i.e. survival and growth of individuals after toad consumption) between these two groups of predators, as mechanistic components behind the susceptibility of the predators to the toxic prey.
3. The mortality of Rana pirica from all populations after consumption of a toad hatchling was almost 100%, and that of Hynobius retardatus ranged from 14 to 90%, depending on the population. In contrast, the mortality of Rana ornativentris and Hynobius nigrescens was near 0% regardless of population. These differences between congeneric predators were mostly due to differences in their toxin resistance.
4. These results suggest that the alien toad is a potential threat to the novel amphibian predators on Hokkaido, although they also imply that the novel predators on Hokkaido have the potential to develop toxin resistance through adaptive evolution. However, this counteradaptation may have a higher chance of evolving in H. retardatus than in R. pirica because of differences in their genetic backgrounds.

     

Brachylaima asakawai sp. nov. (Trematoda: Brachylaimidae), a rodent intestinal fluke in Hokkaido,Japan, with a finding of the first and second intermediate hosts

In the 1970’s and 1980’s, an unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) had been recorded from the intestines of Rattus norvegicus and Apodemus speciosus in Hokkaido, Japan. The rodent fluke was characteristic in extending a bilateral vitellarium till the level of posterior margin of anterior testis and in keeping almost the same-sized spherical ovary and testes. In this study, the rodent fluke was rediscovered from A. speciosus, Apodemus argenteus, and Myodes rufocanus in Hokkaido. The resultant parasite collection enabled us to make a mitochondrial DNA (mtDNA) barcode for tracking its intermediate hosts. The metacercaria of the rodent fluke was detected frequently from the kidney of three species of land snails (Discus pauper, Succinea lauta, and Ainohelix editha). However, its sporocyst with cercariae was found only from the hepatopancreas of D. pauper, a fairly small snail. The wide-spectrum of the second intermediate host seems to increase the chance of transmitting the parasite to various mammals and birds. The use of indigenous land snails as the first and second intermediate hosts, the distinctiveness of the mtDNA sequence, and the characteristic morphology of all the developmental stages prompted us to propose Brachylaima asakawai sp. nov. for the rodent intestinal fluke in Hokkaido. The present field survey suggests that the life cycle of the new species is primarily dependent on a predator-prey relationship between rodents and D. pauper.     

Morphology,Molecular Genetics,and Bioacoustics Support Two New Sympatric Xenophrys Toads (Amphibia: Anura: Megophryidae) in Southeast China

Given their recent worldwide declines and extinctions, characterization of species-level diversity is of critical importance for large-scale biodiversity assessments and conservation of amphibians. This task is made difficult by the existence of cryptic species complexes, species groups comprising closely related and morphologically analogous species. The combination of morphology, genetic, and bioacoustic analyses permits robust and accurate species identification. Using these methods, we discovered two undescribed Xenophrys species, namely Xenophrys lini sp. nov. and Xenophrys cheni sp. nov. from the middle range of Luoxiao Mountains, southeast China. These two new species can be reliably distinguished from other known congeners by morphological and morphometric differences, distinctness in male advertisement calls, and substantial genetic distances (>3.6%) based on the mitochondrial 16s and 12s rRNA genes. The two new species, together with X. jinggangensis, are sympatric in the middle range of Luoxiao Mountains but may be isolated altitudinally and ecologically. Our study provides a first step to help resolve previously unrecognized cryptic biodiversity and provides insights into the understanding of Xenophrys diversification in the mountain complexes of southeast China.     

Notes on the genus <Emphasis Type="Italic">Inocybe</Emphasis> of Japan: II

Three species of the genus Inocybe are reported as new species or a new record from Japan. Inocybe ovoidea sp. nov. and Inocybe furcata sp. nov. are described from Hokkaido. The distribution of caulocystidia in the former is noted. Inocybe oblectabilis is redescribed based on specimens collected in Honshu. This is the first record of I. oblectabilis for Japan.