Phylogeography of a freshwater sculpin, Cottus nozawae, from the northeastern part of Honshu Island, Japan

 The fluvial sculpin, Cottus nozawae, is a coldwater-adapted fish distributed in Hokkaido Island and the northeastern part of Honshu Island (Tohoku District), Japan. Mitochondrial DNA (mtDNA) control region sequencing was used to investigate the geographic distribution of genetic variation and phylogeography of C. nozawae. Most populations possessed unique haplotypes, few being shared across river systems. Phylogenetic analysis of the sequences of the mtDNA control region and adjacent regions of C. nozawae revealed three distinct phylogenetic groups that differed by 3.05% to 3.11%, corresponding to distinct geographic regions, Hokkaido Island, northern Tohoku District, and Yamagata Prefecture (southwestern Tohoku District), respectively. The divergence times of three groups were estimated to be about 1.5 million years ago by applying a general rate for mtDNA, suggesting that the divergence among them might have occurred in the early Pleistocene. Divergence among the haplotypes within the group from the northern Tohoku District was also high (1.84%), no haplotypes being shared by local populations in different river systems in this region. Local populations from a single river system in this region comprise a distinct lineage that differed from other river systems. Such genetically divergent population structures among the different regions and river systems are considered to have resulted mainly from long-term isolation and restricted gene flow among river systems, probably promoted by the fluvial benthic life history and low dispersal ability of this species. Received: April 12, 2001 / Revised: December 1, 2001 / Accepted: December 19, 2001     

Patchy distribution of a fluvial sculpin,cottus nozawae, in the gakko river system at the southern margin of its native range

The distribution pattern of the freshwater sculpin,Cottus nozawae, was investigated in the Gakko River system, Tohoku District, northern Japan, that region representing the southern limit of the species distribution.C: nozawae was not found in the lower sections of the river, being instead restricted mainly to the upstream segments of some tributaries. The relationships between habitat variables and density ofC. nozawae analysed at reach scale, revealed that fish density increased with cover area, decreasing stream width and lower maximum water, temperature. Additionally, maximum water temperature was the best predictor of fish density according to stepwise multiple regression analysis. These data indicated that tributary populations ofC. nozawae were isolated from thermally unsuitable environments. Accordingly,C. nozawae was distributed patchily in headwater streams of this river system, in contrast to those on Hokkaido Island, where the species is distributed continuously throughout middle to upper stream courses.     

Genetic divergence between the sibling species of river-sculpin,Cottus amblystomopsis andC. nozawae,with special reference to speciation

Synopsis Polypatric speciation from an amphidromous ancestor has been suggested for a fluvial gobyRhinogobius flumineus; a similar history may explain life-history and genetic variation inCottus species. We used starch-gel electrophoresis to examine intra- and interspecific genetic differentiation of two siblingCottus species, amphidromousC. amblystomopsis and fluvialC. nozawae, which are presumed to be in an ancestor-descendant phyletic relationship. Genetic differentiation was markedly less withinC. amblystomopsis, than inC. nozawae. One group ofC. nozawae populations was closer toC. amblystomopsis (by Nei's genetic distance) than it was to other populations ofC. nozawae. This suggests that the differentiation of presently identifiable groups ofC. nozawae populations might have occurred polypatrically from an amphidromous ancestral population at different times. The two groups are presumed to be different species in spite of their quite similar morphological and ecological characteristics: we propose to call this type of speciation Polypatric and parallel speciation. This model of Polypatric and parallel speciation might be generalized to explain evolution of amphidromous or anadromous species to fluvial or lacustrine land-locked species.     

Genetic variations ofCottus nozawae populations from five tributaries of the Shiribetsu River of southern Hokkaido,Japan

To estimate genetic differentiation and heterogeneity in the landlocked river sculpin,Cottus nozawae, between tributary populations in the same river-system, 107 specimens were captured from 5 tributaries of the Shiribetsu river (course length 128 km), Hokkaido Island and surveyed for allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA (mtDNA). Three and two alleles were seen at theIdh-2 andPgm loci, respectively, but only one locus,Idh-2, out of twenty loci examined was regarded as polymorphic, since the frequency of the most common allele did not exceed 0.95. Three different mtDNA haplotypes were detected, there being replacement of them between the tributary populations. Heterogeneities of allele and haplotype frequencies were significant between some tributary populations, suggesting that genetic differentiation has occurred between them.     

Alternative life-history styles of Japanese freshwater sculpins revisited

Synopsis Japanese freshwater sculpins consist of 6Cottus and 1Trachidermus species, with various types of life-cycles, namely, catadromous, amphidromous, lacustrine land-locked and fluvial land-locked. Among them,C. amblystomopsis andC. nozawae have been regarded as sibling species. Because of very similar morphological and ecological adult characteristics they were formerly classified as a single species.C. amblystomopsis mainly inhabits the lower courses of rivers and produces many small eggs from which pelagic larvae are formed. In contrast,C. nozawae lives in the middle or upper courses of rivers and deposits few, large eggs, from which well-developed benthic young emerge, well on the way to the definitive phenotype as fully formed juveniles. These two species have distinctly different life-cycles: amphidromous forC. amblystomopsis and fluvial forC. nozawae. A comparison of the early ontogeny shows that, amongCottus species, the small-egg, amphidromousC. amblystomopsis is altricial, while the large-egg, fluvialC. nozawae is precocial. Assuming thatC. nozawae has been derived phylogenetically fromC. amblystomopsis or its ancestral relatives, it is reasonable to consider that the fluvial land-locked life-history style ofC. nozawae has evolved from the ancestral arnphidromous one through adapting to the upstream habitat by an increase in egg size and consequent truncation of the larval period.     

Biochemical evidence for reproductive isolation between the sympatric populations ofCottus amhlystomopsis andC. nozawae

An electrophoretic study on the biochemical genetics of two sibling river-sculpinsCottus amblystomopsis andC. nozawae was undertaken with the primary objective of clarifying the reproductive isolation between the sympatric populations in three rivers around Cape Erimo of Hokkaido, where their distributions overlap widely along the river courses. At the 3 lociAcp,Ldh and6Pgd, out of 20 examined loci, evident displacement of alleles were observed between the two species. In addition, no genetical evidences for hybridization between the two species were detected in the three rivers examined. These results strongly suggest that the two species are reproductively isolated from each other even when they are distributed sympatrically and their distributions overlap widely along the course of a river.     

Spatial variation in density of stream benthic fishes in northern Hokkaido, Japan: does riparian vegetation affect fish density via food availability?

The densities of two benthic fishes, the Siberian stone loach (Noemacheilus barbatulus) and the wrinklehead sculpin (Cottus nozawae), and the biomass of their food resources (i.e., periphyton and benthic invertebrates) were compared between forest and grassland streams in northern Hokkaido, Japan, to examine whether riparian deforestation had positive effects on the benthic fishes via enhancement of food availability. The comparisons indicated that riparian vegetation had little influence on periphyton, invertebrates, or fishes. Regression analysis indicated that spatial variations in loach and sculpin densities were explained more by substrate heterogeneity, competitor abundance, or both, rather than by food abundance. However, when the two species were combined as benthic insectivores, a strong correlation was found between total benthic fish density and invertebrate biomass. Our results suggest that, although total benthic fish abundance was food limited, riparian vegetation had no positive effects via food availability on the benthic fishes in our streams.     

Taxonomic review of the genus <Emphasis Type="Italic">Cottiusculus</Emphasis> (Cottoidei: Cottidae) with description of a new species from the Sea of Japan

A taxonomic review of the cottid genus Cottiusculus Jordan and Starks 1904 established three species, C. nihonkaiensis sp. nov., C. schmidti, and C. gonez. Cottiusculus nihonkaiensis sp. nov., which had been previously confused with C. schmidti, is described on the basis of 32 specimens (44.8–77.9 mm, SL) collected from the Sea of Japan. The new species is very similar to C. schmidti in having a curved barbless uppermost preopercular spine and the lateral line extending past the caudal fin base, but is distinguishable as follows: nasal spines simple or sometimes weakly bicuspid (vs. deeply bicuspid in C. schmidti); first dorsal fin not elongated in either males or females (vs. elongated in males); ventral lateral and lateral line cirri present (vs. absent). The former is known from the Sea of Japan coasts of Hokkaido, Honshu, Kyushu and the Korean Peninsula, and Volcano Bay, Hokkaido, and the latter from the Pacific coast of Tohoku District, Japan. Cottiusculus gonez, known from the Sea of Japan, Sea of Okhotsk and Pacific coast of Hokkaido, is characterized by having a simple nasal spine, the uppermost preopercular spine almost straight with two small cusps dorsally, posterior cusp barbed, and the lateral line almost reaching to the caudal fin base. A lectotype of C. gonez is designated here. Sequence differences in the cytochrome b gene among the above three species of Cottiusculus are also presented.     

Genetic evidence for the existence of cryptic species in theAnopheles albitarsis complex in Brazil: Allozymes and mitochondrial DNA restriction fragment length polymorphisms

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations ofAnopheles albitarsis in Brazil. Two sympatric species,An. deaneorum andAn. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (forHad-1, Pgi-1, Pep-1, Mpi-1, andIdh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species ofAn. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes.An. marajoara form CM had a higher variability (mean heterozygosity,H=0.22, and percentage of polymorphic loci,P=66.7) than did form IG and form MA (H=0.08 in both, andP=25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates ofF statistics, and genetic similarities, we propose thatAn. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1)An. deaneorum (currently one taxon) and (2) theAn. marajoara species complex, which further consists of at least three cryptic forms,marajoara form MA,marajoara form IG, andmarajoara form CM. The views of the authors do not purport to reflect the position of the Department of the Army or the Department of Defense of the United States. This research was conducted when the senior author was on the staff of the USDA-ARS Laboratory in Gainesville, Florida.     

Genetic differentiation,polyploidization and hybridization in northern EuropeanDactylorhiza (Orchidaceae): Evidence from allozyme markers

Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.     

Intra- and interspecific variation in body size ofProtohermes (Megaloptera: Corydalidae)

The life history of three populations ofProtohermes grandis and two populations ofProtohermes immaculatus (Megaloptera: Corydalidae) was compared. In general, the larvae lived in stream riffles for 2 years and the adults appeared in summer. Adult body size differed between these closely related species and also between the populations ofP. grandis. Dwarfism occurred inP. immaculatus, a species that is endemic to the small, isolated island, Amami Island. The population ofP. grandis on Yaku Island, located between Amami Island and the mainland Kyushu, had an intermediate body size between that ofP. immaculatus and the mainland population ofP. grandis. Despite being an insular population,P. grandis on Tsushima Island had a similar body size to mainlandP. grandis. In these populations with large adults, some larvae lived in the streams for 3 years. The size distribution of benthic animals, which are the prey available toProtohermes larvae, differed between the streams studied. The density of large prey was lowest on Amami Island, intermediate on Yaku Island, and highest on the mainland and Tsushima Island. Different size distributions of available prey may be caused by the differences of benthic fauna; most of Ecdyonuridae and Ephemerellidae (large mayflies) and Perlidae (large stoneflies) were not found on Amami and Yaku Islands. Thus, there is a tendency to dwarfism in the populations ofProtobermes inhabiting streams where the density of large prey is low.     

Evolutionary history of the genus Capra (Mammalia, Artiodactyla): discordance between mitochondrial DNA and Y-chromosome phylogenies

The systematics of the genus Capra remain controversial in spite of studies conducted using morphology, mtDNA, and allozymes. Here, we assess the evolutionary history of Capra (i) using phylogenetic analysis of two nuclear genes located on the Y-chromosome and (ii) previously published and new cytochrome b sequences. For the Y-chromosome phylogeny, we sequenced segments from the amelogenin (AMELY) and zinc finger (ZFY) genes from all of the eight wild taxa and from domestic goats (Capra hircus). Phylogenetic analysis of the Y-chromosome data revealed two well-defined clades. The domestic goat (C. hircus), the bezoar (Capra aegagrus), and the markhor (C. falconeri) belong to one clade (ML bootstrap value [BP]: 98%), suggesting that domestic goats originated from one or both of these wild species. The second clade (ML BP: 92%) is comprised of all the other wild species. Horn morphology is generally concordant with the Y-chromosome phylogeny. The mtDNA data also revealed two well-defined clades. However, the species in each clade are different from those inferred from the Y-chromosome data. To explain the discordance between Y-chromosome and mtDNA phylogenies, several hypotheses are considered. We suggest that a plausible scenario involves mtDNA introgression between ancestral taxa before the relatively recent colonization of Western Europe, the Caucasus Mountains, and East Africa by Capra populations.     

Long-term occurrence of hybrids between Japan Sea and Pacific Ocean forms of threespine stickleback, <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis>, in Hokkaido Island,Japan

The distribution patterns of two genetically divergent forms (Japan Sea and Pacific Ocean forms) of Gasterosteus aculeatus and their hybrids in the Hokkaido Island, Japan were investigated. Hybrid frequencies and the mating pattern of natural F1-hybrids and backcrosses, were examined by using allozyme analysis. The distribution patterns matched those previously reported. All three sympatric localities in eastern Hokkaido Island included hybrids between the two forms in each year examined. A low frequency of hybrids of sticklebacks has been maintained in areas of sympatry of the two forms in Hokkaido Island for several years. The proportion of F1-hybrids was significantly lower than expected by random mating in six samples from four sympatric populations, suggesting form-assortative mating. Mating crosses of natural F1-hybrids existed in both patterns. Natural F1-hybrids backcrossed more frequently with Pacific Ocean form than Japan Sea form. However, the proportions of backcross individuals in two of three samples were not significantly different from the expected proportions by random mating between F1-hybrids and their parent forms. In other samples, despite the Pacific Ocean form predominant in a population, F1-hybrid predominantly backcrossed with the Japan Sea form. These results support the hypothesis of gene flow from Pacific Ocean to Japan Sea forms.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

Attributes ofCalamagrostis langsdorffii,C. sachalinensis and their intermediates in Shikoku

The internal structure of theCalamagrostis langsdorffii-sachalinensis complex in Shikoku was examined on the basis of a number of new collections. Chromosome counts disclosed the occurrence of the following entities: 6X and 8X, as well as some aneuploids, ofC. longsdorffii; 6X ofC. sachalinensis; 6X and 8X of the intermediates between these two species. The examination of pollen of these collections and the herbarium specimens showed that all of them may be apomictic in seed formation and that the sexual tetraploids of the pertinent complex may not be distributed in Shikoku. Details of morphological features of the collections were analyzed, and the intermediacy in morphology of the group “intermediate” was confirmed by the distance diagram method. The collections ofC. langsdorffii from Shikoku generally showed an awn longer than that of the collections from Honshu and Hokkaido. On the basis of these and some other observations, relationships of the strains extant in Shikoku were discussed. For the derivation of the present-day composition of the pertinent complex, an hypothesis which is founded on the presumption of a past occurrence of sexual tetraploids of bothC. langsdorffii andC. sachalinensis in Shikoku has been advanced.     

The historical differentiation process inHemerocallis middendorfii (Liliaceae) of Japan based on restriction site variations of chlorplast DNA

Chloroplast DNA restriction site variation inHemerocallis middendorfii Trautv. & Meyer was investigated to deduce the historical differentiation process that had taken place in populations among three districts Hokkaido, Honshu and the Island of Tobishima in Japan as distinct from the morphological data. The populations selected were eight in total, consisting of two populations from Honshu, one each from the Islands of Sado and Tobishima and four from Hokkaido. Twelve endonuclease restriction enzymes were used. Six restriction site mutations were found among the populations investigated. A single, most parsimonious phylogenetic tree is generated, and revealed that the four populations of Hokkaido and two populations of the Islands of Sado and Tobishima are monophyly, respectively.     

Worldwide migration of parasitic mites as a result of bumblebee commercialization

We investigated the status of infestation by a tracheal mite, Locustacarus buchneri, in natural populations of a Japanese native bumblebee species, Bombus hypocrita, collected on Hokkaido Island and in the Aomori prefecture between 1997 and 2001. We also investigated mite infestation in commercial colonies of the European bumblebee, Bombus terrestris, imported from the Netherlands and Belgium, and the Japanese native species, B. ignitus, imported from the Netherlands, between 1997 and 2001. We detected mites in natural populations of the two B. hypocrita subspecies and in the commercial colonies. Analysis of variations in 535 bp sequences of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene showed that the mite haplotypes in the native populations and in the imported colonies did not overlap in 1997–1999, but in 2000–2001 some mites possessing European CO1 haplotypes were detected in the natural populations of Japanese native bumblebees. In addition, many mites possessing Japanese haplotypes were detected in the imported commercial colonies from Europe. Considering the fact that the Japanese native bumblebees, B. hypocrita, were once exported to Europe for commercialization, these results suggest that bumblebee commercialization has caused overseas migration and cross-infestation of parasitic mites among natural and commercial colonies. However, because the Japanese and European CO1 haplotypes were closely related, there was a possibility that the European haplotypes found in the mites in the Hokkaido Island revealed native variation. To clarify the status of mite invasion, further detailed analysis of genetic variation of the mite, using other genetic markers on additional samples, need to be performed.     

Genetic structure of <Emphasis Type="Italic">Cerasus jamasakura</Emphasis>, a Japanese flowering cherry,revealed by nuclear SSRs: implications for conservation

The genetic resources of a particular species of flowering cherry, Cerasus jamasakura, have high conservation priority because of its cultural, ecological and economic value in Japan. Therefore, the genetic structures of 12 natural populations of C. jamasakura were assessed using ten nuclear SSR loci. The population differentiation was relatively low (F _ST, 0.043), reflecting long-distance dispersal of seeds by animals and historical human activities. However, a neighbor-joining tree derived from the acquired data, spatial analysis of molecular variance and STRUCTURE analysis revealed that the populations could be divided into two groups: one located on Kyusyu Island and one on Honshu Island. Genetic diversity parameters such as allelic richness and gene diversity were significantly lower in the Kyushu group than the Honshu group. Furthermore, STRUCTURE analysis revealed that the two lineages were admixed in the western part of Honshu Island. Thus, although the phylogeographical structure of the species and hybridization dynamics among related species need to be evaluated in detail using several marker systems, the Kyusyu Island and Honshu Island populations should be considered as different conservation units, and the islands should be regarded as distinct seed transfer zones for C. jamasakura, especially when rapid assessments are required.