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 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.     

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.     

Diverse migratory histories of Japanese Trachidermus and Cottus species (Cottidae) as inferred from otolith microchemistry

The migratory histories of Japanese freshwater sculpins, one Trachidermus and four Cottus species, were studied by examining strontium (Sr) and calcium (Ca) concentrations in their otoliths using wavelength dispersive X‐ray spectrometry on an electron microprobe. The Sr : Ca ratios in the otoliths changed with salinity of the habitat. The otoliths of Cottus nozawae showed consistently low Sr : Ca ratios, with an average of 3·37 × 10^?3 from the core to the edge, suggesting a freshwater resident life cycle. In contrast, the otolith Sr : Ca ratios for Trachidermus fasciatus and Cottus kazika changed along the life history transects possibly in accordance with their migration patterns from sea to fresh water. The ratios of T. fasciatus and C. kazika averaged 5·4 × 10^?3 and 5·3 × 10^?3 respectively, in the otolith region from the core to the points 450–890 μm, and changed to the lower levels, averaging 2·0 × 10^?3 and 2·7 × 10^?3, in the outer otolith region. These data suggest that both the species have a catadromous life cycle. The otoliths of Cottus hangiongensis had low Sr : Ca ratios in the two regions from the core to the points 15–30 μm and the points 415–582 μm to the edge, averaging 2·0 × 10^?3 and 1·9 × 10^?3, with significantly higher ratios in the narrow area between these regions, averaging 4·6 × 10^?3. Similar ontogenetic changes in otolith Sr : Ca ratios were found in the otoliths of Cottus amblystomopsis, suggesting their amphidromous life cycle. These findings suggest that otolith Sr : Ca ratios reflect individual life histories and that Japanese Trachidermus and Cottus species have diverse migratory histories.     

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     

Size-specific spawing pattern in ayu,Plecoglossus altivelis

The spawning pattern and relationship to body size at maturation of amphidromous and land-locked forms of ayu,Plecoglossus altivelis, was investigated. Ovarian eggs of prespawning females showed a multimodal size distribution, regardles of body size, indicating that all the females were potentially a batch spawners. The spawning schedules of individual fish were described under simulated spawning conditions. Most large-sized females died soon after their first spawning, whereas approximately half of the medium and small-sized females successfully completed a second spawning some two weeks after the first. Such a size-specific spawning pattern was common to both forms, but development of a third batch of ovarian eggs after the second spawning occurred only in the land-locked form. Spawning tactics in ayu were related to offspring survival and adult mortality during spawning season.     

Male mating success and female mate choice in the river sculpin,Cottus nozawae (Cottidae)

Synopsis Mating success of males and its correlates were investigated in a natural population of the polygynous fluvial sculpinCottus nozawae. Furthermore, the female mate preference of this species was examined experimentally under alternative conditions for mating in a stream. The mating success of individual males (the number of females with which a male mated) ranged between 0 and 8 with a mean of 2.41 in 1983 and 2.52 in 1989, in a population of which the sex ratio was about 1 : 2 in both years, skewed toward females. Mainly due to the excess of nests without egg masses and the few nests with one egg mass, the distribution of male mating success did not fit a Poisson distribution, indicating its non-randomness. Male mating success was not correlated either with the size of the nest rocks or with the male size, suggesting that these two variables are not determinants of mating success. The mate choice experiments demonstrated that females of this species more frequently chose smaller males as mates whose nests already contained eggs than large males without eggs. Additionally, an analysis of stomach contents of guarding males suggested that the parental males ate their own eggs during egg guarding (filial-cannibalism). Based on these results and on a comparison of reproductive characteristics with congeneric species, it is suggested that one of the most important determinants for female mate choice inCottus species may be whether or not parental males are filial egg cannibals.     

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.     

Egg and clutch sizes of a goby Rhinogobius giurinus in the Aizu River, Kii Peninsula, Japan

Egg and clutch sizes, and gonad somatic index (GSI) at maturity of the amphidromous goby Rhinogobius giurinus were examined in the Aizu River, Wakayama Prefecture, Japan. The goby tended to have a larger clutch size, with smaller eggs, and matured at the lower GSI, compared with those in the three sympatric amphidromous species of R. brunneus complex. Higher water temperature and abundant food conditions may affect the optimal egg size in R. giurinus. Besides, higher larval survival rate during downstream migration may affect lower GSI at maturity in this goby that usually inhabits lower reaches of the river.     

Facultative amphidromy involving estuaries in an annual amphidromous fish from a subtropical marginal range

Variation in the life cycle of diadromous fishes can be explained by differential food availability between marine and freshwater habitats, since migration is often interpreted as a mechanism for exploiting food resources. Theoretically, a migration pattern of mainly remaining in freshwater occurs in tropical and subtropical habitats where fluvial productivity possibly exceeds marine productivity. However, in Yakugachi River, Amami-Oshima Island, southern Japan, low nutrient concentrations in the river suggest that food availability is limited for the subtropical Ryukyu-ayu Plecoglossus altivelis ryukyuensis. Since Ryukyu-ayu is an amphidromous fish that mainly grows in rivers after spending 2 months in the sea, limited food availability in rivers would force this species to migrate to other habitats with better food availability. Otolith increment and Sr:Ca analyses of 48 adult Ryukyu-ayu collected from the Yakugachi River revealed that all individuals visited estuaries more than three times after moving upstream. Although the specific growth rates of this species in the river had no correlation with the salinity profile in the fluvial period, this movement may be an adaptive choice because the salinity profile significantly affected the body size at maturity. Our results highlighted individual-based variations in amphidromous migration for utilizing estuaries, which could be explained by relatively higher productivity in estuarine than in freshwater and marine habitats.     

Coloration and ontogenetic features of fluviatile species of <Emphasis Type="Italic">Rhinogobius</Emphasis> (Gobioidei: Gobiidae) in Amami-oshima Island,Ryukyu Islands,Japan

 The fluviatile Rhinogobius sp. 1 (hereinafter R. sp. 1) (Japanese name kibara-yoshinobori or English name yellow belly medium-egg type) is broadly distributed in the Ryukyu Islands ranging from Amami-oshima Island to Iriomote Island. However, based on the molecular evidence, this species has probably had multiple origins from amphidromous species. We compared the coloration of specimens at Amami-oshima I. with that of the other specimens (islands) in the Ryukyu Is. Amami-oshima I. specimens of R. sp. 1 were different from the specimens of other populations in having five to six cross lines on the caudal fin. We examined the early life history of fluviatile Rhinogobius at Amami-oshima I., based on rearing experiments, and estimated the age of wild specimens by counting otolith increments. Spawned egg capsules of “Rhinogobius sp. 1” at Amami-oshima I. were larger than those of amphidromous species. Their developmental stage in newly hatched larvae was similar to that of Okinawan R. sp. 1 in having a straight notochord; however, it had a flexed notochord earlier than that of Rhinogobius sp. 2 (hereinafter R. sp. 2) (aobara-yoshinobori or blue belly medium-egg type). The ontogenetic trajectory of “Rhinogobius sp. 1” in Amami-oshima I. was similar to Okinawan R. sp. 1 (30 days in larval duration) but different from R. sp. 2 (15 days). In amphidromous relatives of R. sp. 2, developmental timing during larval period was earlier than in the amphidromous relatives of R. sp. 1. It has been suggested that the phenomenon of fluvial landlocking has been accompanied somewhat by egg size increase (on the basis of a few examples). However, the ontogenetic trajectories of fluviatile Rhinogobius species may be a reflection of those of ancestral amphidromous species. Received: March 18, 2002 / Revised: November 7, 2002 / Accepted: December 10, 2002     

Polymorphic spawning-nest types in a local population of the fluvial sculpin, Cottus nozawae (Teleostei: Cottidae)

The nesting behavior of a fluvial sculpin Cottus nozawae population was investigated in a stream–reservoir system on the Sainai River, northern Honshu Island, Japan in 2003, 2004 and 2006. Except for the usual rock nest-spawning type (RSN), two new types of spawning nest were found: hole nest spawning (HNS) and crevice nest spawning (CNS). The HNS type was characterized by males digging one or more nest holes in the wall of the banks of the stream and reservoir, or on the sandy silt bottom of the reservoir. The CNS type involved the use of a crevice between large boulders or crevice of crag for their nests. The HNS type was dominant in the reservoir section, whereas the RNS type was dominant in the rapid riffles of stream sections. The HNS and CNS types appeared to be rarely or first observed in the Cottus species. These new nest-spawning types might have occurred in relation to the artificial environment changes in the river by dam construction.     

Habitat selection in amphidromous Gobiidae of Reunion Island: Sicyopterus lagocephalus (Pallas, 1770) and Cotylopus acutipinnis (Guichenot, 1863)

Freshwater populations of the Indo-pacific region are characterized by a large proportion of amphidromous species. In this paper, we analyse habitat selection by two amphidromous sympatric Gobiidae: Sicyopterus lagocephalus and Cotylopus acutipinnis in Reunion Island. A sampling method using Point Abundance Sampling (PAS) was conducted in 12 rivers. We used mixed logistic models in order to examine the presence probability of species according to location, downstream-upstream gradient, microhabitat variables (depth, velocity and predominant substrata) and presence of conspecifics and sympatric species. Presence probabilities varied between the sampled rivers. We observed a positive attraction between identical developmental stages of both species, which suggests that social interactions or similar preferences for environmental cues influenced their distribution. The presence probabilities of both species’ juveniles decreased from downstream to upstream. We showed that traditional microhabitat variables weakly explained the spatial distribution of both S. lagocephalus and C. acutipinnis, in Reunion Island. We suggest that weak habitat selection for these species is consistent with the amphidromous life style because of the unpredictability of juvenile settlement and the extreme hydrological variations in tropical rivers.     

Occurrence and body size changes of drifting land-locked Ryukyu-ayu Plecoglossus altivelis ryukyuensis larvae in the San-numata River, Okinawa-jima Island, Japan

The occurrence and body size of drifting land-locked Ryukyu-ayu, Plecoglossus altivelis ryukyuensis, larvae were investigated in the San-numata River, Okinawa-jima Island from November 2000 to March 2001. The water temperature in the river fluctuated from 13.8 to 17.9°C during the sampling period. The drifting larvae occurred from mid-December to mid-March with an estimated spawning peak during late November and early December. The notochord length of the drifting larvae ranged from 4.9 to 6.7 mm (5.7 ± 0.3 mm, mean ± SD), and became longer as the spawning months progressed. These results imply that the land-locked population possesses the same maturation attributes as the original amphidromous population.     

Comparison of biological and demographic parameters forMononychellus progresivus andOligonychus gossypii on cassava: influence of temperature

The effect of five constant temperatures (16, 22, 26, 31 and 36°C) on biological (survival and duration of developmental stages, fecundity and longevity of females, sex-ratio) and demographic parameters (R _o , G, r _m and λ) of the two main phytophagous mites that attack cassava in Africa,Mononychellus progresivus Doreste andOligonychus gossypii (Zacher), was studied in the laboratory. Experiments were performed simultaneously on the two mite species reared on the same cassava variety (1M20) under controlled conditions: 70±10% r.h. and 12L∶12D. The lower thermal threshold was 13°C forM. progresivus and 11°C forO. gossypii. Both species developed in the range from 22 to 36°C. The shortest development time was obtained at 31°C for both species; it was 7.2 days forM. progresivus and 8.2 days forO. gossypii. Maximum fecundity of both species was recorded at 26°C with 42.1 eggs forM. progresivus and 36.3 eggs forO. gossypii The highest intrinsic rate of increase (r _m ) was obtained at 31°C for both species with 0.289 and 0.214 forM. progresivus andO. gossypii, respectively.     

Simultaneous maturation of two discrete oocyte batches in ayu

Ayu Plecoglossus altivelis including amphidromous and landlocked populations, are known for batch spawning. Unusual clutches at ovulation were observed from rearing stocks that were naturally induced to sexual maturation. Two peaks occurred simultaneously in the frequency distribution of egg size, compared to normal fish, which have clutches with unimodal size distribution. The eggs in the small size class could be fertilized, and small-sized larvae hatched from these eggs. To explain the alteration in egg size through divergence from amphidromous to landlocked populations, the role of batch allocation among oocytes is discussed.     

Tropical and temperate freshwater amphidromy: a comparison between life history characteristics of Sicydiinae,ayu, sculpins and galaxiids

Amphidromy is a distinctive form of diadromy, but differences in the life histories of tropical and temperate amphidromous fishes suggest that there are two types of freshwater amphidromy. The life histories of Sicydiinae gobies, ayu (Plecoglossus altivelis), Japanese sculpins (Cottus) and galaxiids (Galaxiidae), suggest that the Sicydiinae are representatives of tropical freshwater amphidromy, whereas ayu, sculpins and galaxiids are representatives of temperate freshwater amphidromy. The Sicydiine larval stage may be required to occur in the ocean for all species, but ayu, sculpins and galaxiids have landlocked or fluvial forms with larvae that do not need to enter the ocean for larval feeding and growth. This suggests that Sicydiine larvae have a high oceanic dependency whereas ayu, sculpins and galaxiid larvae have a low oceanic dependency. Freshwater amphidromous fish in tropical and temperate zones appear to have developed two different strategies in the evolution of their life histories. It is likely that the evolutionary direction of the larval stage of tropical amphidromy is to remain in the sea and that of temperate amphidromy is towards having the ability to remain in freshwater if needed. Tropical and temperate amphidromy appear to be biologically informative categories and evaluations of this hypothesis will facilitate better understanding of the various forms of amphidromy in the future.     

Flexible migration of Japanese freshwater gobies Rhinogobius spp. as revealed by otolith Sr:Ca ratios

The migratory histories of six Rhinogobius spp., the cross‐band type (R. sp. CB), the large‐dark type (R. sp. LD), the dark type (R. sp. DA), the cobalt type (R. sp. CO), the orange type (R. sp. OR) and R. flumineus, were studied by examining strontium (Sr) and calcium (Ca) concentrations in their otoliths using wavelength dispersive X‐ray spectrometry on an electron microprobe. The Sr:Ca ratios in the otoliths changed both with the ontogenetic development and with the salinity level of the habitat. Most fishes had high Sr:Ca ratios around the otolith core in spite of the fact that those fishes live most of their lives in a freshwater environment. The high ratios in the otoliths were thought to be a physiological effect in those fishes. Thereafter, the Sr:Ca ratios changed remarkably along the life‐history transect, showing intraspecies and interspecies variations. The otolith Sr:Ca ratios of Rhinogobius sp. CB, R. sp. LD and R. sp. CO collected from three rivers connected to the sea were low around the core, subsequently increased sharply to the points 180–345 μm from the core and then decreased again towards the edge. They were thought to reflect the typical amphidromous life history. The R. sp. CO, however, remain in a brackish‐water environment after migration from the sea, while the other species showed typical amphidromous lives with complete freshwater residence after migration from the sea. The five species (R. sp. CB, R. sp. LD, R. sp. CO, R. sp. DA and R. sp. OR) collected above dams had never migrated to the sea, spending their whole life in a freshwater environment, although Rhinogobius species, except for the fluvial type, were thought to have an amphidromous life history according to previous studies. These species are thought to have a landlocked life cycle. The otolith Sr:Ca ratios of R. flumineus showed consistently low ratios towards the edge except for only around the core, although they were collected from a river connected to the sea. These species could have a fluvial life history corresponding to a previous study. The present study clearly suggests that the migratory histories of Rhinogobius spp. are highly different both within and between species and that they have flexible migratory patterns allowing them to utilize the full range of salinity during their life history.     

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.     

陆封型和洄游型香鱼体肾组织的蛋白质组学分析

香鱼（Plecoglossusaltivelis）是典型的降海洄游鱼类,由于地理原因,一些香鱼在繁殖季无法回到海中产卵,形成陆封型香鱼。陆封型和洄游型香鱼相比,其体型明显偏小。鱼类的体肾组织可以调控渗透压,从而影响生长,因此我们采用蛋白质组学的方法研究陆封型和洄游型香鱼体肾组织蛋白质组的差异。双向电泳（2一DE）分析筛选到25个差异表达蛋白点,质谱分析成功鉴定出21个蛋白。与洄游型香鱼相比,陆封型香鱼体肾组织中NADH脱氢酶、异柠檬酸脱氢酶、Or．．微管蛋白、热适应相关蛋白65（Wap65）、热休克蛋白60（HSP60）、丙酮酸脱氢酶（E1）、碳酸酐酶、烯醇酶和乳酸脱氢酶表达量较高。同时陆封型香鱼的B一肌动蛋白、铁蛋白、氨基酰化酶、甲硫氨酸腺苷转移酶、过氧化物酶、丙氨酸乙醛酸转移酶和磷酸烯醇丙酮酸羧激酶则表达量较低。它们主要参与能量代谢、应激反应、氨基酸代谢等过程。由于wap65、HSP60和E13种蛋白在蛋白质组学结果中表达改变,且与应激和能量代谢有关,可能影响鱼类生长,因此采用荧光定量PCR（RT—PCR）验证陆封型和洄游型香鱼体肾wap65、HSP60和E1基因mR-NA表达差异,结果表明这3个基因mRNA表达均在陆封型香鱼体肾中较高,与蛋白质组学结果一致。综上,陆封型香鱼的几个糖酵解相关酶和应激蛋白表达量比洄游型香鱼高,而蛋白代谢相关酶表达量较低,揭示陆封型和洄游型香鱼在环境应激和能量代谢等层面有明显的差异。