Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species.

Bitterlings are relatively small cypriniform species and extremely interesting evolutionarily due to their unusual reproductive behaviors and their coevolutionary relationships with freshwater mussels. As a group, they have attracted a great deal of attention in biological studies. Understanding the origin and evolution of their mating system demands a well-corroborated hypothesis of their evolutionary relationships. In this study, we provide the most comprehensive phylogenetic reconstruction of species relationships of the group based on partitioned maximum likelihood and Bayesian methods using DNA sequence variation of nuclear and mitochondrial genes on 41 species, several subspecies and three undescribed species. Our findings support the monophyly of the Acheilognathidae. Two of the three currently recognized genera are not monophyletic and the family can be subdivided into six clades. These clades are further regarded as genera based on both their phylogenetic relationships and a reappraisal of morphological characters. We present a revised classification for the Acheilognathidae with five genera/lineages: Rhodeus, Acheilognathus (new constitution), Tanakia (new constitution), Paratanakia gen. nov., and Pseudorhodeus gen. nov. and an unnamed clade containing five species currently referred to as "Acheilognathus". Gene trees of several bitterling species indicate that the taxa are not monophyletic. This result highlights a potentially dramatic underestimation of species diversity in this family. Using our new phylogenetic framework, we discuss the evolution of the Acheilognathidae relative to classification, taxonomy and biogeography.

Characteristics of Trees Infested by the Invasive Primary Wood-Borer Aromia bungii (Coleoptera: Cerambycidae).

The expanding distribution and tree damage of the invasive, primary wood-borer Aromia bungii (Coleoptera: Cerambycidae), which kills trees of the Rosaceae family, is a problem in intruded areas. However, the tree characteristics associated with infestation by A. bungii, which are useful for early detection or prioritizing preventive measures, are not well examined. We investigated the presence or absence of tree damage (response variable) in pre- and post- surveys along with tree characteristics (four explanatory variables; bark roughness, size, species, and vigor) on monitoring trees in uninvaded sites (survey for the first trees to be damaged) and already invaded sites (survey for the next trees to be damaged). We evaluated the variables using generalized linear mixed models for each site (i.e., a first trees model and a next trees model). Three tree characteristics (bark roughness, size, and vigor) were included as explanatory variables in both best models, indicating that trees with rough surface bark, large in size, and weakened conditions were more susceptible to A. bungii infestation. The reasons for the difference between the two models (species was only chosen in the next trees model) will be considered in our future work.

The life cycle and molecular phylogeny of a gorgoderid trematode recorded from the mussel Nodularia douglasiae in the Yodo River, Japan.

In 2009, a novel larval trematode of the family Gorgoderidae was found in the gonads of Nodularia douglasiae (Unionidae) from the lower reaches of the Yodo River, Osaka Prefecture, Japan. This is the first collection of trematodes in a unionid mussel in Japan. We investigated the morphology and life cycle of the trematode, and conducted a molecular phylogenetic analysis with other gorgoderid species, both those collected in the Yodo River water system and those reported in the literature. Immature adult worms were obtained from the ureters of the common carp Cyprinus carpio, the first known instance of a gorgoderid from these fish in Japan. Morphological characteristics and molecular data show that it belongs to the subfamily Gorgoderinae (genus Phyllodistomum sensu lato). Regarding the morphology, first intermediate host, and the infection site of adult worms, it resembles Phyllodistomum elongatum Nybelin, 1926 from Europe, but no comparable molecular data exist for Ph. elongatum. Three cytochrome c oxidase subunit I haplotypes were detected in the specimens analyzed, suggesting that the present species is indigenous to the Yodo River water system. The 28S ribosomal DNA data showed that this species is a member of the clade consisting of Ph. cf. symmetorchis, Ph. folium, Pseudophyllodistomum and Xystretrum. However, its phylogenetic position within the clade differs between the maximum likelihood and maximum parsimony trees, and the sister species of the present species remain unclear.

Isolation and characterization of 39 microsatellite loci in the endangered Japanese loach Leptobotia curta.

A microsatellite-enriched genomic library was obtained for the endangered Japanese loach Leptobotia curta, and 39 dinucleotide markers were successfully isolated and characterized. These markers had between one and nine alleles, with expected heterozygosity ranging from 0 to 0.839, in a population from the Lake Biwa-Yodo River system of Japan. Linkage equilibrium was observed in most loci, and only one locus showed significant deviation from Hardy-Weinberg equilibrium. These microsatellite markers will be useful for genetic diversity studies of wild and captive L. curta populations.

Low temperature requirement for embryonic development of Itasenpara bitterling Acheilognathus longipinnis.

The Itasenpara bitterling has an embryonic period up to 7 months, when the embryo experiences large seasonal temperature changes. We examined the temperature requisites for normal development during the embryonic stage. Fertilized eggs reared under any of the constant temperatures ranging from 5 degrees C to 30 degrees C did not achieve complete embryogenesis, and none reached the swim-up stage. The optimum temperature for normal embryonic development was found to be stage-dependent: 10-30 degrees C for fertilization, 15-25 degrees C for hatching, 5 degrees C for the requisite low temperature, 10-15 degrees C for eye pigmentation, and 20-30 degrees C for swim-up. These temperatures correlated well with the embryo's natural environmental conditions. Embryos raised at these temperatures sequentially grew normally, with 70% of the fertilized eggs achieving complete embryogenesis and, for the first time, developed to the swim-up stage. These results indicate that the low temperature, as required by the bitterling embryo, is an essential factor and correlates well with the embryo's natural ambient temperatures. Since the populations of Itasenpara bitterlings have been declining in Japan, this study is the first to provide additional information for successful artificial breeding of this endangered species.