Genetic diversification of the Kanehira bitterling Acheilognathus rhombeus inferred from mitochondrial DNA, with comments on the phylogenetic relationship with its sister species Acheilognathus barbatulus.

The Kanehira bitterling, Acheilognathus rhombeus, is a freshwater fish, discontinuously distributed in western Japan and the Korean Peninsula. Unusually among bitterling it is an autumn-spawning species and shows developmental diapause. Consequently, the characterization of its evolutionary history is significant not only in the context of the fish assemblage of East Asia, but also for understanding life-history evolution. This study aimed to investigate the phylogeography of A. rhombeus and its sister species Acheilognathus barbatulus, distributed in China, using a mitochondrial analysis of the ND1 gene from 311 samples collected from 50 localities in Japan and continental Asia. Phylogenetic analysis revealed that A. barbatulus is included in A. rhombeus and genetically closer to Japanese A. rhombeus than to Korean A. rhombeus. Divergence of Korean A. rhombeus and A. barbatulus from Japanese A. rhombeus was estimated to be from the late Pliocene (3.44 Mya) and the early Pleistocene (1.98 Mya), respectively. Each event closely coincided with the time of the Japan Sea opening. Japanese A. rhombeus comprised seven lineages: three in Honshu and four in Kyushu. One lineage in central Kyushu was genetically closer to the Honshu lineages than to other lineages in northern Kyushu. Divergence of Japanese lineages was estimated to be from the early to middle Pleistocene (0.55-0.93 Mya), during a period of geological and paleoclimatic change, including volcanic activity. Population expansion in the late Pleistocene (<0.10 Ma) was suggested in many of the lineages, which accords with other freshwater fishes. Biogeographically the ancestral A. rhombeus/A. barbatulus was likely to have repeatedly colonized Japan from the continent through land bridges in the late Pliocene and the early Pleistocene. However, the close genetic relationship between Japanese A. rhombeus and A. barbatulus suggests another possibility, with the second colonization occurring in reverse, from Japan to China. The small genetic distance between them indicates that the colonization occurred later than colonization events of other freshwater fishes, including other bitterling species.

Phylogenetic relationships of bitterling fishes (Teleostei: Cypriniformes: Acheilognathinae), inferred from mitochondrial cytochrome B sequences.

Bitterling (Teleostei: Acheilognathinae) are small cyprinid fishes with a discrete distribution in East Asia and Europe. We used a complete mitochondrial cytochrome b sequence (1141 bp) from 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus), sampled across the major part of their distribution, to elucidate their phylogeny and biogeography, focusing particularly on their origin and dispersal. Based on high support value, the monophyletic Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In the latter clade, the monophyly of Rhodeus was poorly supported, though it was topologically nested in Tanakia. On the basis of molecular-clock calibration, both clades diverged in the middle Miocene, with Tanakia-Rhodeus diverging slightly earlier than Acheilognathus. The Tanakia-Rhodeus clade expanded its distribution westward from the Far East, eventually reaching Europe, while Acheilognathus dispersed in the temperate regions of East Asia. A feature common to both clades is that most extant species, including Japanese endemics, appeared by the end of the Pliocene, corresponding with the present delineation of the Japanese archipelago. Autumn-spawning species with an embryonic diapause, unique to bitterling among cyprinid fishes, formed two distinct lineages (barbatulusrhombeus and longipinnis-typus) within Acheilognathus. The estimated time of divergence of the two lineages was approximately from the late Pliocene, a period characterized by glaciations. The timing of divergence suggests that the shift of spawning from spring to autumn, coupled with embryonic diapause, convergently emerged twice in the evolution of bitterling, possibly as an adaptation to the climate of the late Pliocene.

Classical and molecular cytogenetic characterization of allochthonous European bitterling Rhodeus amarus (Cyprinidae, Acheilognathinae) from Northern Italy.

A cytogenetical study was carried out on 34 specimens of the European bitterling Rhodeus amarus (Teleostei: Cyprinidae, Acheilognathinae) from four rivers of the Venice district (NE Italy). This allochthonous fish species was accidentally introduced in the North-East of Italy about 20 years ago and is now rapidly spreading all over the rivers of the Northern part of the country. All the studied specimens are characterised by the same karyotype (2n = 48: 8M + 20SM + 20ST), i.e., the typical one of the native populations of the species. However, a polymorphism in the number of NOR bearing chromosomes has been found. In fact, in addition to the main species-specific NORs, on the short arms of chromosome pair 7, two to five additional 18S rDNA sites have been revealed by FISH in different specimens. Sequential staining with silver nitrate, chromomycin A(3) and DAPI revealed that most of the additional sites are inactive and CMA(3)-positive. Data herein reported confirm that in spite of an overall morphological karyological conservativeness, significant differences for the finer cytogenetic features can be found within the Acheilognathinae with the 2n = 48 and NF = 76 karyotype.

Phylogeography, Historical Demography, and Genetic Structure of the Rose Bitterling, Rhodeus ocellatus (Kner, 1866) (Cypriniformes: Acheilognathidae), in East Asia.

Yao-Feng Tsao, Wen-Wen Lin, Chia-Hao Chang, Takayoshi Ueda, Nian-Hong Jang-Liaw, Ya-Hui Zhao, and Hsiao-Wei Kao (2016) Rose bitterling, Rhodeus ocellatus, is a small cyprinid fish distributed in East Asia. To infer its phylogeography and genetic structure, specimens from Taiwan, China, and Japan were collected, and complete mitochondrial cytochrome b (cyt b) DNA sequences were amplified and sequenced. Phylogenetic analyses identified seven mitochondrial lineages (A-G). Among them, three lineages (A, B, and C) distributed in mainland China. Lineages D, E, and F distributed in Japan, Korea, and Taiwan, respectively. Lineage G distributed in both China and Japan. The results of the Bayesian Binary MCMC analysis (BBM) suggested that the most recent common ancestor of R. ocellatus was from Lower Yangtze region. Divergence times among lineages inferred by molecular clock ranged from 7.55 to 1.44 million years ago. We propose that topography and climate changes by uplift of the Tibetan Plateau in the Late Miocene-Pliocene and the glacial-interglacial cycles in the Pleistocene might account for population expansion and genetic differentiation. Divergence times among lineages A, B, and C in Yangtze River basin ranged from 7.55 to 2.27 million years ago that might result from changes of flow directions of rivers from westward to eastward driven by the uplift of the Tibetan Plateau. The glacial-interglacial cycles in the Pleistocene might further cause population expansion to the northward of lineage G at about 0.19 million years ago. Lineage D in Japan was dispersed from the mainland China before the opening of the Sea of Japan, and lineage F in Taiwan was dispersed from the mainland China through the land bridge in the Pleistocene. Because of the genetic differentiation is statistically significant among populations, protection of genetic diversity and distinctness of R. ocellatus should be considered in the future conservation management.