Isolation and characterization of microsatellite DNA markers in the rare species barfin flounder (Verasper moseri) and its closely related species spotted halibut (V. variegatus)

Barfin flounder and spotted halibut have been selected in Japan as target species for stock enhancement due to the fact that the number of individuals of these species in the wild has been decreasing in the last three decades. Barfin flounder is now considered to be a rare species as its situation is critical. The first microsatellite DNA markers for barfin flounder and spotted halibut are described in this study. Cross‐amplification of barfin flounder markers was examined in spotted halibut species and vice versa.     

Development of polymorphic microsatellite markers in barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

Isolation and characterization of 31 polymorphic microsatellite markers in barfin flounder (Verasper moseri) and the cross-species amplification in spotted halibut (Verasper variegatus)

The construction of a genetic linkage map and evaluation of population genetic diversity both require large numbers of polymorphic molecular markers. In the present study, we report 31 polymorphic microsatellite markers, of which 28 were isolated from two repeat-enriched libraries constructed from genomic DNA, and three were detected in two genes (MCH-R1 and MCH-R2) of barfin flounder (Verasper moseri). A total of 94 alleles were detected with an average of 3.0 alleles per locus. The number of alleles, observed and expected heterozygosity per locus ranged from two to six, from 0.30 to 1.00 and from 0.33 to 0.78, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0016) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated using the closely related species spotted halibut (Verasper variegatus). This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and studying the population genetic diversity of barfin flounder (Verasper moseri).     

Fine-scale population genetic structure in Alaskan Pacific halibut (<Emphasis Type="Italic">Hippoglossus stenolepis</Emphasis>)

Pacific halibut collected in the Aleutian Islands, Bering Sea and Gulf of Alaska were used to test the hypothesis of genetic panmixia for this species in Alaskan marine waters. Nine microsatellite loci and sequence data from the mitochondrial (mtDNA) control region were analyzed. Eighteen unique mtDNA haplotypes were found with no evidence of geographic population structure. Using nine microsatellite loci, significant heterogeneity was detected between Aleutian Island Pacific halibut and fish from the other two regions (F _ST range = 0.007–0.008). Significant F _ST values represent the first genetic evidence of divergent groups of halibut in the central and western Aleutian Archipelago. No significant genetic differences were found between Pacific halibut in the Gulf of Alaska and the Bering Sea leading to questions about factors contributing to separation of Aleutian halibut. Previous studies have reported Aleutian oceanographic conditions at deep inter-island passes leading to ecological discontinuity and unique community structure east and west of Aleutian passes. Aleutian Pacific halibut genetic structure may result from oceanographic transport mechanisms acting as partial barriers to gene flow with fish from other Alaskan waters.     

Development of 37 microsatellite loci for the great gray owl (<Emphasis Type="Italic">Strix nebulosa</Emphasis>) and other <Emphasis Type="Italic">Strix</Emphasis> spp. owls

We developed 37 great gray owl (Strix nebulosa) microsatellite primers from CA and TAGA enriched genomic libraries. Primers were tested in 15 great gray owls from California, USA and Alberta, Canada as well as two other Strix species, spotted owl (S. occidentalis) and barred owl (S. varia). These markers will have broad application in investigations of Strix population structure and genetic diversity.     

Genetic sex identification and the potential evolution of sex determination in Pacific halibut (Hippoglossus stenolepis)

The discovery of genetic markers linked to physiological traits in wild populations is increasingly desired for ecological and evolutionary studies, as well as to inform management decisions. However, identifying such markers often requires a large investment of both time and money. Serendipitously, in a recent microsatellite survey, we discovered three out of 16 microsatellite loci that were correlated to the female sex in Pacific halibut (Hippoglossus stenolepis). These three loci were screened in 550 Pacific halibut to determine their accuracy at identifying sex. Genetic assignment successfully identified sex in 92% of individuals from sample collections spanning 3,000 km and 9 years. All but two of 287 females had one copy of a characteristic allele for at least one of the three microsatellite loci, resulting in consistent heterozygote excess in females. This pattern is consistent with the hypothesis that females are the heterogametic sex in Pacific halibut, which thus may have a different sex-determination pattern than the closely related Atlantic halibut (Hippoglossus hippoglossus). A rapid divergence of sex-determining mechanisms could be either a cause or consequence of speciation between Pacific and Atlantic halibut. In either case, the ability to genetically identify sex in individual Pacific halibut provides a new tool for ecological studies, fisheries management, and insight into the evolution of sex determination in flatfish.     

Assessment of genetic variability and relatedness among atypical Aeromonas salmonicida from marine fishes,using AFLP-fingerprinting

Atypical strains of Aeromonas salmonicida are the causal agent of atypical furunculosis or ulcer disease in various fish species, including spotted wolffish Anarhichas minor, which is a promising species in the Norwegian fish-farming industry. Isolates of atypical A. salmonicida comprise a very heterogenous group showing large variety in biochemical, molecular and virulence characteristics. The genetic variability among atypical isolates from wolffish was characterised using amplified fragment length polymorphism analysis: AFLP-fingerprinting. Additional isolates from halibut Hippoglossus hippoglossus, turbot Scophthalmus maximus, cod Gadus morhua and several salmonid fishes were included for assessment of variability and relatedness among a total of 56 atypical isolates of A. salmonicida. They were compared to reference strains of A. salmonicida subspecies and to other Aeromonas species pathogenic in fishes. AFLP-fingerprints subjected to similarity analysis yielded a grouping of the isolates into several clusters, revealing genetic heterogeneity among the isolates. There seems to be a correlation between genetic similarity among isolates and the fish host. The Icelandic isolates, mainly from cod, formed a very homogeneous subcluster, which was closely related to the wolffish isolates. All atypical isolates from spotted and common wolffish grouped together in a large cluster and appear to be very homogeneous, even though they had been isolated over a period of 8 yr at different locations in Norway. On the other hand, most of the isolates from turbot and halibut grouped together into 2 different clusters, while the 9 atypical isolates from salmonids appeared in 4 different clusters. Thus, the atypical isolates of A. salmonicida from halibut, turbot and salmonid fishes seem to be more genetically diverse than those from wolffish and cod.     

Genetic structure in species with shallow evolutionary lineages: a case study of the rare flatfish Verasper variegatus

We examined the genetic population divergence of the spotted halibut Verasper variegatus. A previous report suggested two conservation units for this species along the Japanese Pacific coast. Extending the coverage of the genomes (29 microsatellites and three mitochondrial DNA segments) revealed hitherto-undetected genetic population boundaries. We screened population samples from the major habitats along the Japanese coast and the Yellow Sea coast (East Asian Continent). Significant genetic differentiation was found in every comparison between the habitats. In most cases, the nuclear and mitochondrial population divergences were incongruent, most likely caused by differences between the two genomes in the effects of genetic drift after recent population isolation and bottleneck events. We discuss the ecological and evolutionary mechanisms of the genetic structure as well as the units of conservation. The present study illustrates the merits of wider coverage of genomes in genetic population analysis especially for species with a shallow population history.     

Comparison of behavioural development between Japanese flounder (Paralichthys olivaceus) and spotted halibut (Verasper variegatus) during early life stages

Behavioural development was compared between two flatfish species (Japanese flounder and spotted halibut) from hatching to settlement (juvenile stage) in order to speculate on the ecology of their early life stages and to provide fundamental knowledge for improving seedling production techniques for stock enhancement. Fish were cultured under identical rearing conditions (500‐L tank maintained at 17.8 ± 0.4°C, 34 ppt, 10L : 14D light regime and an initial stocking density of 20 larvae L^?1). Behavioural observations were conducted at about 4‐day intervals from hatching to the juvenile stage. Fish were sampled randomly from the rearing tank, and one fish was transferred into a 250‐ml observation container. Behaviour was video‐recorded for 5 min without food and for an additional 5 min with live feed (rotifer or Artemia). All behavioural data were sorted according to eight developmental stages and compared among developmental stages and between species. The average standard length of the spotted halibut was significantly greater than that of the Japanese flounder in all developmental stages, while the development of Japanese flounder was faster than that of the spotted halibut. For Japanese flounder, feeding, swimming and Ohm‐posture (typical shivering behaviour observed during early life stages in flatfishes) frequency were highest before metamorphosis (mean ± SD; 1.0 ± 2.0 attacks min^?1, 24.0 ± 9.6 actions min^?1, 1.1 ± 1.1 counts min^?1, respectively). Spotted halibut expressed feeding behaviour frequently from the beginning of metamorphosis (3.6 ± 5.2 attacks min^?1), had relatively low swimming activity during all developmental stages, and showed a peak of Ohm‐posture frequency during the flexion stage (2.6 ± 1.0 counts min^?1).     

Significant Asia‐Europe divergence in the middle spotted woodpecker (Aves,Picidae)

Population divergence could be strongly affected by species’ ecology and might not be a direct response to climate‐driven environmental change. We tested this in the middle spotted woodpecker (Dendrocoptes medius), a non‐migratory, low‐dispersal habitat specialist associated with old deciduous forests of the Western Palearctic. We present the first phylogeographic study of this species integrating genetic data (three mitochondrial loci, one autosomal and one Z‐linked intron) with species distribution modelling. Based on this species’ ecology, we predicted that the middle spotted woodpecker could have colonized its current range from multiple Last Glacial Maximum (LGM) refugia and that strongly structured populations could be expected. Indeed, we discovered a strong genetic divergence between Asian and European populations, with a split estimated at around one million of years ago. This was surprising given only slight intraspecific variation in plumage and morphology. Although there was no significant phylogeographic structure within the Asian and European groups, we cannot exclude the possibility of multiple refugia within either group during the LGM. This has to be further investigated with more extensive geographic sampling and larger number of variable independently evolving markers. Future studies should also investigate potential differences in vocalizations and ecology between the two groups. Lineages showing similar level of genetic differentiation including woodpeckers are often treated as species‐level taxa. Comparison of our results with the phylogeographic history of other woodpeckers, suggests that sympatric species with similar life‐histories might have idiosyncratic phylogeographic patterns probably resulting from different ecological requirements or historic stochasticity.     

Sequence and organization of the complete mitochondrial genomes of spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri).

In this work, the mitochondrial genomes for spotted halibut (Verasper variegatus) and barfin flounder (Verasper moseri) were completely sequenced. The entire mitochondrial genome sequences of the spotted halibut and barfin flounder were 17,273 and 17,588 bp in length, respectively. The organization of the two mitochondrial genomes was similar to those reported from other fish mitochondrial genomes containing 37 genes (2 rRNAs, 22 tRNAs and 13 protein-coding genes) and two non-coding regions (control region (CR) and WANCY region). In the CR, the termination associated sequence (ETAS), six central conserved block (CSB-A,B,C,D,E,F), three conserved sequence blocks (CSB1-3) and a region of 61-bp tandem repeat cluster at the end of CSB-3 were identified by similarity comparison with fishes and other vertebrates. The tandem repeat sequences show polymorphism among the different individuals of the two species. The complete mitochondrial genomes of spotted halibut and barfin flounder should be useful for evolutionary studies of flatfishes and other vertebrate species.     

The role of western Mediterranean islands in the evolutionary diversification of the spotted flycatcher Muscicapa striata,a long‐distance migratory passerine species

We investigated the evolutionary history of the spotted flycatcher Muscicapa striata, a long distance migratory passerine having a widespread range, using mitochondrial markers and nuclear introns. Our mitochondrial results reveal the existence of one insular lineage restricted to the western Mediterranean islands (Balearics, Corsica, Sardinia) and possibly to the Tyrrhenian coast of Italy that diverged from the mainland lineages around 1 Mya. Mitochondrial genetic distance between insular and mainland lineages is around 3.5%. Limited levels of shared nuclear alleles among insular and mainland populations further support the genetic distinctiveness of insular spotted flycatchers with respect to their mainland counterparts. Moreover, lack of mitochondrial haplotypes sharing between Balearic birds (M. s. balearica) and Corso‐Sardinian birds (M. s. tyrrhenica) suggest the absence of recent matrilineal gene flow between these two insular subspecies. Accordingly, we suggest that insular spotted flycatchers could be treated as one polytypic species (Muscicapa tyrrhenica) that differs from M. striata in morphology, migration, mitochondrial and nuclear DNA and comprises two subspecies (the nominate and M. t. balearica) that diverged recently phenotypically and in mitochondrial DNA and but still share the same nuclear alleles. This study provides an interesting case‐study illustrating the crucial role of western Mediterranean islands in the evolution of a passerine showing high dispersal capabilities. Our genetic results highlight the role of glacial refugia of these islands that allowed initial allopatric divergence of insular populations. We hypothesize that differences in migratory and breeding phenology may prevent any current gene flow between insular and mainland populations of the spotted flycatcher that temporarily share the same insular habitats during the spring migration.     

Identifying spawning behavior in Pacific halibut, <Emphasis Type="BoldItalic">Hippoglossus stenolepis</Emphasis>, using electronic tags

Synopsis Identifying spawning behavior in Pacific halibut, Hippoglossus stenolepis, is particularly challenging because they occupy a deep, remote environment during the spawning season. To identify spawning events, a method is needed in which direct observation by humans is not employed. Spawning behavior of seven other flatfish, species has been directly observed in their natural environment by investigators using SCUBA. All of these flatfish species display almost identical spawning behavior that follows a routine. Therefore, it is reasonable to believe that this spawning behavior occurs in other flatfish species, including Pacific halibut. As part of a larger study, we recaptured two Pacific halibut on which Pop-up Archival Transmitting (PAT) tags had been attached during the winter spawning season. Because the tags were physically retrieved, we were able to collect minute-by-minute depth records for 135 and 155 days. We used these depth data to tentatively identify spawning events. On seven separate occasions between 20 January 2001 and 9 February 2001, one fish displayed a conspicuous routine only seen during the spawning season of Pacific halibut and the routine parallels the actions of other spawning flatfish directly observed by humans using SCUBA. Therefore, we propose this routine represents spawning behavior in Pacific halibut. The second tagged fish did not display the conspicuous routine, thus challenging the assumption that Pacific halibut are annual spawners. PAT tags may prove to be a useful tool for identifying spawning events of Pacific halibut, and that knowledge may be used for improved management in the future.     

Morphology of unfertilized mature and fertilized developing marine pelagic eggs in four types of multiple spawning flounders

Starry flounder Platichthys stellatus, spotted halibut Verasper variegates, turbot Scophthalmus maximus, and Japanese flounder Paralichthys olivaceus are four commercially cultivated multiple spawning flounders that spawn pelagic eggs. Through appropriate light and scanning electron microscope processing, the shape and surface structures (such as micropyle, pores, pore density, and paten) of unfertilized mature and fertilized developing eggs of the four species were observed and measured. First, individual or intraspecific comparisons of the surface structures of eggs at different developmental stages were made. Second, interspecific differences among the four species at the same developmental stage of unfertilized mature eggs were statistically computed and analyzed through one-way analysis of variance and hierarchical cluster analysis. Eggs of the same species collected at different stages of development tend to be different in morphology. Smoothing of the convoluted egg envelope surface and closure of the micropyle to serve as a final step of the polyspermy-preventing reaction are common after fertilization. Based on detailed morphology of micropyle of just-mature fertilizable eggs, turbot, starry flounder, and Japanese flounder each have a micropyle with a long canal but no distinct micropylar vestibule, type III of Riehl and Götting (Arch Hydrobiol 74:393–402, 1974). In contrast, spotted halibut has a micropyle with a distinct flat micropylar vestibule and a long canal, type II. Envelope surface microstructures, especially those in the micropyle region, are useful characters for egg identification among the four species. Cluster analysis using selected egg characters indicated the highest similarity between turbot and Japanese flounder and that starry flounder is obviously more similar to turbot and Japanese flounder than to spotted halibut.     

Landscape genetics reveals unique and shared effects of urbanization for two sympatric pool‐breeding amphibians

Metapopulation‐structured species can be negatively affected when landscape fragmentation impairs connectivity. We investigated the effects of urbanization on genetic diversity and gene flow for two sympatric amphibian species, spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus), across a large (>35,000 km²) landscape in Maine, USA, containing numerous natural and anthropogenic gradients. Isolation‐by‐distance (IBD) patterns differed between the species. Spotted salamanders showed a linear and relatively high variance relationship between genetic and geographic distances (r = .057, p < .001), whereas wood frogs exhibited a strongly nonlinear and lower variance relationship (r = 0.429, p < .001). Scale dependence analysis of IBD found gene flow has its most predictable influence (strongest IBD correlations) at distances up to 9 km for spotted salamanders and up to 6 km for wood frogs. Estimated effective migration surfaces revealed contrasting patterns of high and low genetic diversity and gene flow between the two species. Population isolation, quantified as the mean IBD residuals for each population, was associated with local urbanization and less genetic diversity in both species. The influence of geographic proximity and urbanization on population connectivity was further supported by distance‐based redundancy analysis and multiple matrix regression with randomization. Resistance surface modeling found interpopulation connectivity to be influenced by developed land cover, light roads, interstates, and topography for both species, plus secondary roads and rivers for wood frogs. Our results highlight the influence of anthropogenic landscape features within the context of natural features and broad spatial genetic patterns, in turn supporting the premise that while urbanization significantly restricts interpopulation connectivity for wood frogs and spotted salamanders, specific landscape elements have unique effects on these two sympatric species.     

COMPARATIVE POPULATION STRUCTURE AND GENE FLOW OF A BROOD PARASITE,THE GREAT SPOTTED CUCKOO (CLAMATOR GLANDARIUS), AND ITS PRIMARY HOST,THE MAGPIE (PICA PICA)

The amount of gene flow is an important determinant of population structure and therefore of central importance for understanding coevolutionary processes. We used microsatellite markers to estimate population structure and gene flow rates of the great spotted cuckoo (Clamator glandarius) and its main host in Europe, the magpie (Pica pica), in a number of populations (seven and 15, respectively) across their distribution range in Europe. The genetic analysis shows that there exists a pattern of isolation by distance in both species, although the cuckoo data are only indicative due to a small sample size. Gene flow seems to be extensive between nearby populations, higher for magpies than cuckoos, and especially high for magpie populations within the area of distribution of the great spotted cuckoo. There is no correlation between genetic distances between magpie populations and genetic distances between cuckoo populations. We discuss the implications of extensive gene flow between magpie populations in sympatry with cuckoos for the population dynamics of hosts, in particular for the occurrence of egg rejection behavior in host populations and how the different rates of migration for both species can affect the dynamics of coevolutionary processes.     

Inheritance and gene mapping of spotted to non-spotted trait gene <Emphasis Type="Italic">CmSp-1</Emphasis> in melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L<Emphasis Type="Italic">.</Emphasis> var. <Emphasis Type="Italic">chinensis</Emphasis> Pangalo)

Melon (Cucumis melo L.) is one of the most popular and highly nutritious vegetable species within Cucurbitaceae. Because appearance is used as an important indicator of quality, the spotted to non-spotted trait associated with this product somewhat influences the buying habits of consumers. We tested a six-generation family to determine the inheritance and genetic basis of this trait. Genetic groups F₁, F₂, BC₁P₁, and BC₁P₂ were from a cross between “IM16559” (non-spotted) and “IM16553” (spotted). Our genetic analysis showed that the spotted to non-spotted trait was controlled by a single dominant gene that we named CmSp-1. Whole-genome resequencing-bulked segregant analysis (WG-BSA) demonstrated that this gene was located on the end of chromosome 2, in the intersections of 22,160,000 to 22,180,000 bp and 22,260,000 to 26,180,000 bp, an interval distance of 3.94 Mb. Insertion-deletion (InDel) markers designed based on WG-BSA data were used to map this gene. Using 13 InDel markers, we produced a genetic map indicating that CmSp-1 was tightly linked to markers I734-2 and I757, with genetic distances of 1.8 and 0.4 cM and an interval distance of 280.872 kb. The closest marker was I757. Testing of 107 different melon genotypes presented an accuracy of 84.11% in predicting the phenotype. By being able to locate CmSp-1 in melon, we can now use the findings to identify potential targets for further marker-assisted breeding and cloning projects.