Morphological,distributional, and genetic characteristics of <Emphasis Type="Italic">Cottus pollux</Emphasis> in the Kyushu Island,Japan: indication of fluvial and amphidromous life histories within a single lineage

We evaluated the morphological, distributional, and genetic characteristics of the freshwater sculpin, Cottus pollux, in Kyushu Island, Japan. Based on pectoral fin ray number, the sculpins inhabiting 30 rivers were divided into two types [modes 13 (M13) and 15 (M15)]. We evaluated four environmental parameters: length of main river (L-MR), average gradient of main river (G-MR), distance from sampling site to river mouth (D-SM), and gradient around sampling site (G-S), and compared the two types. L-MR, D-SM, and G-S were significantly larger for the rivers that contained M13 fish than for those containing M15 fish. M13 individuals were distributed in the upstream areas of large river systems, suggesting a fluvial life history, whereas M15 individuals were distributed in the downstream areas of small river systems, indicating an amphidromous life history. We conducted phylogenetic analysis based on mitochondrial 12S rRNA [788 base pair (bp)] and control regions (386 bp). Mitochondrial DNA analysis showed that both M13 and M15 groups were genetically of C. pollux middle-egg type (ME). Our findings proposed the hypothesis that C. pollux ME in Kyushu Island exhibits dimorphism in both morphological and distributional traits. Additionally, haplotype distribution indicated that the fluvial M13 populations had higher genetic specificity in each river in contrast to the existence of one genetic group of amphidromous M15 individuals in Kyushu Island.     

Non-indigenous amphidromous sculpin Cottus pollux small-egg type (Teleostei: Cottidae) detected in rivers entering the Sea of Japan off Honshu Island, Japan

The amphidromous sculpin Cottus pollux small-egg type (SE) is an endemic species in rivers that flow into the Pacific Ocean on the east side of Honshu Island, Japan. However, our molecular genetic and morphological analyses using recently collected specimens showed that C. pollux SE inhabits some rivers flowing into the Sea of Japan, where this species is originally non-indigenous. Thus, these results suggest that C. pollux SE was anthropogenically introduced into these rivers. In addition, our data on the size distribution of juvenile fishes suggest that non-indigenous C. pollux SE reproduce at least in rivers of Ishikawa Prefecture.     

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.     

A fluvial population of Cottus pollux (middle-egg type) from the Honmyo River, Kyushu Island, Japan

 Specimens of the Cottus pollux species' group collected from the upper part of the Honmyo River, Nagasaki Prefecture, Japan, were subjected to morphological and allozyme analyses to place them into one of the recognized valid taxa, viz. small-egg type (SE type), middle-egg type (ME type), or large-egg type (LE type). They were identified as ME type on the basis of specific morphological characteristics, such as laterally depressed cross-sectional shape at posterior half of the body and deep caudal peduncle, and by having a diagnostic allozyme allele (MEP ^* 54). This ME-type population is the first recorded from Kyushu Island, Japan. An ecological survey of the population revealed that females spawned larger eggs (2.8–3.2 mm in diameter) than those of other amphidromous populations, from which well-developed yolk-sac larvae of about 8.0 mm TL were hatched out. In addition, ME-type specimens collected on 25 and 26 May 2001 included 15 sex-unknown juveniles ranging from 18.8 to 30.2 mm SL, suggesting that they represented larvae hatched out on a nearby spawning ground, with no experience of downstream migration into Isahaya Bay. This observation strongly suggests that the ME-type population in the Honmyo River has a fluvial lifestyle, being different from other amphidromous populations. The former population may have arisen from an amphidromous ancestor through changes in egg size and early ontogenetic development. Received: February 25, 2002 / Revised: May 21, 2002 / Accepted: June 17, 2002 Acknowledgments We thank Professor M. Azuma, Nagasaki University, for his kind guidance of our field survey in the Honmyo River and Dr. G.S. Hardy, Thames, New Zealand, for correcting the English. Thanks are also offered to Mrs. Y. Miyajima and Y. Masaoka, Kyushu Branch of Construction and Technology Institute Co., for their helpful cooperation in the field survey. Drs. H. Sakai, National Fisheries University and Y. Yamazaki, Toyama University, and Mrs. N. Okabe and Y. Suzuki of Yamagata Prefecture are thanked for their help in sample collection. This work was partly supported by a Grant-in-Aid (No. 13660171) from the Japan Ministry of Education, Culture, Sports, Science and Technology to A. Goto. Correspondence to:Akira Goto     

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.     

Hybridization between Cottus gobio and Cottus poecilopus in the Odra River drainage basin (Czech Republic)

Comparisons of morphology, nuclear gene sequencing and microsatellites were used to identify 19 hybrids between the related species Cottus poecilopus and Cottus gobio in three rivers of the Odra River drainage basin. All hybrids were the results of backcrossing and no F1 generation hybrid was found. The resulting progeny are fertile and continue in backcrossing with parental conspecifics. A high representation of backcrosses appears to indicate a lack of reproduction barriers within the hybridization process. It was impossible to differentiate individual back-cross categories on the sole basis of six loci. Mitochondrial haplotypes indicate that this is not a one-way process. In our study, hybridization was asymmetrical in favour of Cottus poecilopus.     

Estimates of nocturnal home-range size of the adult Japanese fluvial sculpin, <Emphasis Type="Italic">Cottus pollux</Emphasis> (Pisces: Cottidae) in relation to bottom topography and sampling intervals

I have estimated nocturnal home-range size for 24 individuals of the adult Japanese fluvial sculpin, Cottus pollux (large-egg type) by direct observation on a single night. On average, sculpin used 10.5 focal points (where they executed ambush predation) at night and stayed for 93.1 min at each point. Home-range size (mean 9.8 m², range 0.3–79.9 m²), which was calculated by use of the minimum convex polygon method, was positively correlated with the number of focal points. The swimming paths and focal points used by each sculpin often depended on the configuration of rocks on the streambed, suggesting the importance of bottom topography to home-range use by the sculpin. More than one-third of the sculpins returned to within 1 m² of the point of original capture and release; this provided evidence of their homing ability. Comparison of nocturnal home-range size and Schoener’s ratio (the amount of temporal autocorrelation) with the length of sampling intervals suggested that sampling intervals of 2 h through the dark period, which resulted in a 70% match with real home-range measurements and approximately half of the data sets became independent, provide the most accurate information for predicting the nocturnal home-range size of the sculpin.     

Migratory histories of three types of Cottus pollux (small-egg, middle-egg, and large-egg types) as revealed by otolith microchemistry

 Migratory histories of three types of Cottus pollux, the small-egg type (SE type), middle-egg type (ME type), and large-egg type (LE type), were studied by examining strontium (Sr) and calcium (Ca) in their otoliths with wavelength dispersive X-ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed both with ontogenetic development and with salinity of the habitat. Otolith Sr : Ca ratios of LE-type samples and the ME-type samples from the Honmyo River, Kyushu Island, showed consistently low ratios, averaging 1.8 × 10⁻³ and 2.4 × 10⁻³ from the core to the edge, respectively. In contrast, otolith Sr : Ca ratios of SE-type samples and the other four ME-type samples from Hokkaido and Honshu Islands fluctuated strongly along the life history transects in accordance with migration patterns from freshwater to the sea and vice versa. The otolith Sr : Ca ratios of SE-type samples showed low ratios from the core to a point around 15 μm, averaging 1.5 × 10⁻³, and subsequently increased sharply with a high Sr : Ca ratio phase to a point around 400 μm, averaging 5.5 × 10⁻³, and followed again a low ratio phase to the edge with averages of 3.1 × 10⁻³. Similar fluctuation patterns in otolith Sr : Ca ratios were found for the four ME-type samples. These findings clearly demonstrated that otolith Sr : Ca ratios reflected the sculpin's life histories, as being fluvial for the LE type and the Honmyo River ME type and amphidromous for the SE type and the other four populations of ME type. Received: August 1, 2002 / Revised: October 15, 2002 / Accepted: October 28, 2002 Acknowledgments We thank Dr. N. Miyazaki, University of Tokyo, for his kind guidance of our joint research. Thanks are also offered to Drs. H. Sakai, National Fisheries University, Y. Yamazaki, Toyama University, and R. Yokoyama, Hokkaido University, and Mrs. N. Okabe and Y. Suzuki of Yamagata Prefecture for their help in sample collection. This work was partly supported by a Grant-in-Aid (No. 13660171) from the Japan Ministry of Education, Science, Sports and Culture to A. Goto. Correspondence to:Akira Goto     

Geographic differentiation of <Emphasis Type="Italic">Cobitis shikokuensis</Emphasis> inferred from mtDNA RFLP analysis

Mitochondrial DNA divergence among populations of the Japanese spinous loach Cobitis shikokuensis, endemic to Shikoku Island, was investigated by restricted fragment length polymorphism analysis. A total of 68 restriction sites on DNA fragments from the cytochrome b to D-loop regions and from the 12S rRNA to 16S rRNA regions, amplified by PCR, were analyzed. A total of 12 haplotypes (plus 6 in outgroups) were detected in 268 specimens collected from 19 localities in seven rivers (and 41 specimens from four localities in three rivers in outgroups). Three of the seven river populations of C. shikokuensis were shown to have unique haplotypes, and four of the seven river populations were monomorphic. The nested structure of the haplotype network for populations of C. shikokuensis exhibited two large clades corresponding to (1) populations from the Shimanto River and its neighbors and (2) two genetically divergent populations in the Shigenobu and Iwamatsu Rivers. The population from the Shimanto River, the largest river inhabited by C. shikokuensis, maintains great haplotype diversity as well as the allozyme diversity previously reported. On the other hand, populations from the Hiji River, the second largest river, which exhibited the highest allozyme diversity, were monomorphic in their mtDNA. The nested clade analysis (NCA) revealed that past fragmentation between the above two clades could occur in the initial distribution process of C. shikokuensis. The large genetic divergence of two river populations from the Shigenobu and Iwamatsu Rivers was inferred to be caused by a process of long distance colonization and fragmentation. MtDNA introgression into the Hiji River population from southern river populations was suggested. Taking genetic divergence into consideration, each river population of C. shikokuensis should be conserved separately as like a distinct species, and conservation programs for the small populations showing less genetic variability should be invoked as soon as possible.     

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.     

Sexual size dimorphism, growth and maturity of the Japanese fluvial sculpin,Cottus pollux (large egg type), in the Inabe River, Mie prefecture, central Japan

The population structure of the Japanese fluvial sculpin,Cottus pollux (large egg type), in the upper reaches of the Inabe River, Mie Prefecture, central Japan, was investigated by a mark-and-recapture method from July 1989 to January 1991. Breeding of the species occurred from mid February to early May, peaking from mid February to late March. The mean size of mature males observed in March 1990 was significantly larger than that of females, showing apparent sexual size dimorphism. Data analysis of the growth of 1658 marked individuals revealed that the species matured at 2 years of age in both sexes. Whereas 1 year old males reached ca. 50–70 mm SL, females were less than 50 mm SL at the same age, size dimorphism already being apparent. Immature males exhibited higher growth rates than females during their first and second years, some of the former outstripping mature males of the preceding year class in total length. After attaining sexual maturity, both males and females grew mainly from July to December, with no significant differences in mean growth rate between them. Sexual size dimorphism of the species seems to be attributable to different growth rates between the sexes during their immature stage.     

Year-round local movements of the japanese fluvial sculpin,Cottus pollux (large egg type), with special reference to the distribution of spawning nests

Year-round local movements of adult Japanese fluvial sculpin.Cottus pollux (large egg type), were investigated by a capture-mark-and-recapture method from July 1989 to July 1990 in the upper reaches of the Inabe River, central Japan. In the pre-breeding (July to January) and post-breeding seasons (June to July), the mean distance of movements in males and females was less than 20 m, and there was no apparent tendency to move into a particular channel-unit habitat, suggesting residential tendency in both sexes. In the breeding season (February to May), males tended to move into the raceways where most of the spawning nests were found, but females did not do likewise, indicating a sexual difference in movements in that season. Such sexual difference in movements was also confirmed by the records of individuals captured and recaptured more than three times: six (42.9%) of 14 males moved into the raceways in the breeding season, whereas only one (7.1%) of 14 females did so in that season. The reason behind this sexual difference in movements observed in the breeding season is discussed from the viewpoint of the patterns of spatial distribution between sexes during the pre-breeding and breeding seasons.     

Biotic and Abiotic Influences on Nest-hatching Outcome in the Japanese Fluvial Sculpin, Cottus pollux

Synopsis I examined the interrelationships among the timing of reproduction, physical characteristics of nests and hatching outcome of male Japanese fluvial sculpin, Cottus pollux, in a Japanese mountain stream with flood disturbance. Breeding took place from mid-February to early April, with bimodal peaks of breeding activity. Male body size had a significant effect on the timing of initial reproduction and mating success. Hatching outcome, by contrast, was not affected by male body size and male mating success. The timing of flood occurrence varied between years, and most nest-hatching failure was observed within 1 week after floods. Failed nests were exposed to faster current velocity than successful nests, and degree-days of failed nests negatively correlated with the magnitude of flood flow experienced. These data suggest that the timing and magnitude of floods substantially affected hatching outcome of the species. I also discuss the adaptive significance of bimodal breeding activity of males from life-history perspective in the face of variable stream flow conditions.     

INTRASPECIFIC PHYLOGEOGRAPHY OF NORTH AMERICAN HIGHLAND FISHES: A TEST OF THE PLEISTOCENE VICARIANCE HYPOTHESIS

The highland fish fauna of eastern North America consists of Appalachian and Ozark centers of endemism separated by the intervening Glacial Till Plains. Clades within these areas are more closely related phylogenetically to each other than to clades occurring in the intervening formerly glaciated region, suggesting that the Pleistocene glaciations fragmented a widespread highland region and its associated fauna. Alternatively, it is possible that these faunal assemblages predate the glaciations or that recent dispersals may have been more important than vicariance in determining faunal compositions. We examined the relationships among mitochondrial DNA (mtDNA) haplotypes within five clades of highland fishes, each with a distribution suggestive of a Pleistocene vicariance event. Darters of the subgenera Litocara and Odontopholis have distributions and mtDNA relationships that are consistent with the Pleistocene integration and burial of the Teays-Mahomet valley, a major drainage of the early Pleistocene. The distribution and mtDNA relationships among subspecies of Erimystax dissimilis are not consistent with Pleistocene vicariance, but relationships among Appalachian haplotypes are consistent with the late Pleistocene integration of the modern Ohio River system. Both Cottus carolinae and the Fundulus catenatus species group have representatives in the Mobile basin consistent with pre-Pleistocene divergences. Three haplotype clusters were found in C. carolinae, corresponding to the Appalachian, Ozark, and upper Kanawha River populations. However, Appalachian and Ozark F. catenatus populations are paraphyletic with respect to each other. This, coupled with a relatively low degree of sequence divergence, suggests that no long-term barriers to gene flow exist for C. carolinae and F. catenatus. These three distinct phylogeographic patterns indicate that Pleistocene vicariance is not the only explanation for the Appalachian-Ozark distribution of highland fish communities.     

Growth differences in males of the river-sculpin Cottus hangiongensis along a river course,a correlate of life-history variation

Synopsis Growth patterns of the 1982 year-class, individual growth patterns, age at sexual maturity and longevity in the river-sculpin, Cottus hangiongensis (Cottidae), were investigated along the course of the Daitobetsu River of southern Hokkaido, Japan. Slow growth occurred in males from the lower reaches, while more rapid growth was found in males from upstream areas. Age at first sexual maturity in males generally increased from 2 or 3 years in downstream areas to 4–6 years in upstream sites. Longevity was estimated as 7 years throughout the river course except in the uppermost site where it was 6 years. The growth differences evident in male C. hangiongensis are likely a result of differences in sculpin population density and/or food abundance along the river course, and are apparently reflected in variation in their life-history as well.     

Genetic variation and differentiation of the two river sculpins,Cottus nozawae andC. amblystomopsis,deduced from allozyme and restriction enzyme-digested mtDNA fragment length polymorphism analyses

Genetic differentiation of the two sibling species,Cottus nozawae andC. amblystomopsis, from the northern part of Japan (Hokkaido Island and the Tohoku District) was investigated using allozyme variations and restriction fragment length polymorphisms of mitochondrial DNA. Although the two species are morphologically very similar, previously being thought to be a single species, they have different life-cycles;C. nozawae has a fluvial life-cycle with a small number of large-sized eggs, whereasC. amblystomopsis is an amphidromous species with a large number of small-sized eggs. Four populations ofC. amblystomopsis from Hokkaido Island and 24 populations ofC. nozawae (22 from Hokkaido Island and 2 from the Tohoku District) were sampled and examined Intrapopulational differentiation in the two species was measured by examining several indexes, including proportion of polymorphic loci (P), mean heterozygosity (H) and nucleotide diversity (π). All measurements were higher in theC. amblystomopsis populations, suggesting that intrapopulational variation inC. nozawae was less than inC. amblystomopsis and reflecting the difference in effective population sizes between them. Cluster analyses were performed using the UPGMA method, based on the data matrices of genetic distance (D) and the net nucleotide difference (δ) between populations. TheC. nozawae andC. amblystomopsis populations from Hokkaido Island composed a large cluster (Hokkaido group), while theC. nozawae populations from the Tohoku District composed a different cluster (Tohoku group). Bootstrap probabilities deduced from 1000 bootstrap replications for presence or absence of restriction sites showed that the mtDNA haplotypes detected within the Tohoku Group occurred in 99.9% of the bootstrap replicates outside the mtDNA haplotypes of the Hokkaido group, while those within the Hokkaido group occurred in 3.5–64.9% of bootstrap replicates. Consequently, the Hokkaido populations of the two species (Hokkaido group) were genetically close to each other, whileC. nozawae from the Tohoku District (Tohoku group) were distant from the Hokkaido group. These results suggest that the ancestral populations of the two species on Hokkaido Island shared the same gene pool, even after becoming geographically isolated from the ancestral population ofC. nozawae in the Tohoku District by the formation of the Tsugaru Straits.     

Species‐level phylogeographical history of the endemic species Calligonum roborovskii and its close relatives in Calligonum section Medusa (Polygonaceae) in arid north‐western China

Quaternary climatic oscillations appear to have influenced the genetic diversity and evolutionary history of arid‐adapted plants. To understand the processes involved and reveal evolutionary relationships, haplotypes were examined from Calligonum roborovskii, an endemic species occurring in the arid zones across the desert regions of north‐western China, and seven other species also from Calligonum section Medusa, including C. gobicum, C. mongolicum and the narrow endemic species C. ebi‐nuricum, C. pumilum, C. taklimakanense, C. trifarium and C. yengisaricum. Forty‐three haplotypes were identified in 422 individuals from 51 natural populations, from variation of two plastid DNA intergenic spacers (rpl32‐trnL and ycf6‐psbM). A high level of total genetic diversity was found across species for which more than two populations were examined, including C. gobicum, C. mongolicum, C. pumilum and C. roborovskii. A distinct isolation‐by‐distance pattern in each of these species was suggested by the Mantel test, indicating that restricted gene flow caused high genetic differentiation among populations. Three haplotypes were shared by two or three species each, but the other 40 haplotypes were species‐specific. The 43 haplotypes split into three major clades, but not species‐specific lineages; most of the Calligonum species were not reciprocally monophyletic, probably due to incomplete lineage sorting or introgression. The identified haplotypes were dated to 1.97 Mya (95% highest posterior density: 2.95–0.99 Mya) and diverged until the late Pleistocene, possibly linked to aridification and enlargement of deserts caused by climate changes. Variation of desert habitats during the Pleistocene might play a key role in causing the divergence.     

Effects of a Severe Flood on the Movements of Japanese Fluvial Sculpin

I studied the movements of adult Japanese fluvial sculpin, Cottus pollux, in a Japanese mountain stream. An exceptionally severe flood in late September had negative impacts on refuge abundance, condition and population density of the sculpin. The mean distance moved monthly correlated positively with water discharge, but not with water temperature or with population density. Overall, the mean distance sculpins moved after the flood was significantly greater than before the flood, and sculpins tended to move into riffle-raceways after the flood. Comparisons of refuge-site limitation for adults and water depths between habitats indicated that the flood affected riffle-raceways less than pools. Fish in poorer conditions were likely to move extensively, and the condition of fish captured initially in pools deteriorated more significantly than that of sculpins captured in riffle-raceways. Movement bias into riffle-raceways by the sculpins after the flood suggests they sought suitable habitat with available refuges. The results suggest vulnerability to flood disturbance of the sculpins inhabiting the interstitial spaces of the substrate.     

Size-mediated dominance and aggressive behavior of male Japanese fluvial sculpin Cottus pollux (Pisces: Cottidae) reduce nest-site abundance and mating success of conspecific rivals

Despite growing evidence for plasticity in the mating patterns of nest-holding animals in relation to the changes in nest abundance, the effects of aggressive interaction by dominant males on nest availability for conspecific rivals remains unclear. To quantify the effects of male–male competition on nest-site choice and mating success of the male Japanese fluvial sculpin Cottus pollux, we conducted experiments on 5 males from different 5 size classes under both sufficient and shortage nest-abundance conditions. Nest-choice experiments showed that both male size class and nest-abundance condition had significant effects on the nesting rates of males. Following the nest-choice experiments, 10 gravid females were added in the experimental tanks. Mating experiments revealed that male size, nesting rate before addition of females, and the number of courtship attempts on females were valid variables of male mating success, regardless of nest-abundance conditions. After achieving initial mating success, the largest nesting male exhibited more frequent aggressive interaction with other conspecific males than he did before obtaining eggs in his nest. Our results suggest that size-mediated dominance and aggressive behavior of males may disrupt nest acquisition of other conspecific males, and may consequently result in extreme variation in mating success among males even under sufficient nest-abundance conditions.