Development and characterization of 11 novel microsatellite loci for the roundscale spearfish Tetrapturus georgii and their cross‐species amplification among other istiophorid species

Eleven novel polymorphic microsatellite loci were developed and characterized for the recently validated roundscale spearfish Tetrapturus georgii. Characterization of these markers, based on 35 roundscale spearfish from the western North Atlantic, revealed two to 21 alleles per locus with an average expected heterozygosity (H_E) of 0·09–0·94, and all loci conformed to Hardy–Weinberg expectations. Cross‐amplification of these 11 loci against all other eight known istiophorid species indicates promising prospects for the utility of these markers for istiophorids in general.     

Development and characterization of 14 microsatellite loci in the Neotropical fish Geophagus brasiliensis (Perciformes,Cichlidae)

Fourteen polymorphic microsatellite loci were isolated and characterized for the Neotropical cichlid Geophagus brasiliensis and tested on 30 individuals belonging to a single population. Among the 14 loci described, four showed potential presence of null alleles, inferred from the excess of homozygous genotypes, and three of these loci showed significant deviations from Hardy–Weinberg equilibrium. Fifty‐nine different alleles were detected (ranging from two to eight alleles per locus), with estimates of observed and expected heterozygosity ranging from 0·167 to 0·700 and from 0·269 to 0·825. Cross‐amplification of primers was successful in five other cichlid species.     

Isolation and characterization of 16 polymorphic microsatellite loci for Euchiloglanis kishinouyei

A total of 16 novel polymorphic microsatellite marker loci were isolated from a genomic library of Euchiloglanis kishinouyei and further characterized using a sample from a wild population consisting of 40 individuals. The number of alleles among loci ranged from three to 17 and the observed and expected heterozygosities ranged from 0·030 to 0·950 and 0·163 to 0·980, respectively. The average polymorphic information content (PIC) of all loci was 0·450.     

Isolation and characterization of 20 polymorphic microsatellite loci in the migratory freshwater fish Leporinus obtusidens (Characiformes: Anostomidae) using 454 shotgun pyrosequencing

Twenty polymorphic microsatellite markers were developed for the Neotropical fish Leporinus obtusidens using a next generation sequencing approach and tested in two other characifomes species, Schizodon platae and Prochilodus lineatus. Microsatellite loci alleles in L. obtusidens ranged between 2 and 20 alleles per locus (mean = 5·7), with expected heterozygosity values ranging from 0·097 to 0·956 (mean = 0·578) and observed heterozygosity values ranging from 0·000 to 0·800 (mean = 0·400) in a sample of 20 specimens from the lower Paraná River (Argentina). Most of these markers will be a valuable tool for captive breeding and stocking programmes, as well as for analyses of population connectivity and genetic structure in this broadly distributed Neotropical migratory fish.     

Isolation and characterization of eight microsatellite loci in the brook lamprey Lampetra planeri (Petromyzontiformes) using 454 sequence data

Eight polymorphic microsatellite loci were developed for the brook lamprey Lampetra planeri through 454 sequencing and their usefulness was tested in 45 individuals of both L. planeri and the river lamprey Lampetra fluviatilis. The number of alleles per loci ranged between two and five; the Italian and Irish populations had a mean expected heterozygosity of 0·388 and 0·424 and a mean observed heterozygosity of 0·418 and 0·411, respectively.     

Isolation of novel microsatellite loci in the black goby Gobius niger and cross‐amplification in other gobiid species (Perciformes,Gobiidae)

Twelve microsatellite loci were isolated from and characterized for the black goby Gobius niger. These loci were tested on a total of 48 individuals from two geographically distant locations (Orbetello and Cattolica) and the number of alleles ranged from two to 18, with expected (H_e) and observed (H_o) heterozygosities ranging from 0·042 to 0·941 and from 0·042 to 0·917, respectively. The loci described were used to cross‐amplify other gobiid species belonging to Gobius, Zosterisessor, Lesueurigobius and Aphia.     

Isolation and characterization of 13 polymorphic microsatellite loci in Hypostomus ancistroides (Teleostei,Loricariidae) and cross‐amplification in related species

This study details 13 novel polymorphic microsatellite loci in the armoured catfish Hypostomus ancistroides, and assesses their utility for population genetic studies. The analysis of 30 individuals revealed a total of 99 different alleles (ranging from two to 15 alleles per locus), with an average of 7·62 alleles per locus, with observed and expected heterozygosities ranging from 0·103 to 0·931 and from 0·102 to 0·906, respectively. One of the 13 loci showed significant deviations from Hardy–Weinberg equilibrium, probably due to the presence of null alleles, inferred from the excess of homozygotes.     

Population genetic structure of the round stingray Urobatis halleri (Elasmobranchii: Rajiformes) in southern California and the Gulf of California

The round stingray, Urobatis halleri, is a viviparous elasmobranch that inhabits inshore, benthic habitats ranging from the western U.S.A. to Panama. The population genetic structure of this species was inferred with seven polymorphic microsatellite loci in samples collected at three sites in coastal southern California, one near Santa Catalina Island, California and one in the eastern Gulf of California. Urobatis halleri is relatively common, but little is known of its movement patterns or population structure. Small F_ST values (?0·0017 to 0·0005) suggested little structure among coastal populations of southern and Baja California. The population sampled at Santa Catalina Island, which is separated by a deep‐water channel from the coastal sites, however, was significantly divergent (large F_ST, 0·0251) from the other populations, suggesting low connectivity with coastal populations. The Santa Catalina Island population also had the lowest allele richness and lowest average heterozygosity, suggesting recent population bottlenecks in size.     

MITOCHONDRIAL-DNA RESTRICTION-SITE POLYMORPHISMS IN THE TELEOST FUNDULUS HETEROCLITUS SUPPORT SECONDARY INTERGRADATION

Fundulus heteroclitus is a highly polymorphic fish distributed along the Atlantic coast of North America. Several loci show directional changes in gene frequency with latitude (i.e., clines). Such directional changes have classically been described by two general models: primary and secondary intergradation. Previously, it has not been possible to distinguish between these models for Fundulus heteroclitus on the basis of allelic isozymes or morphological data. However, recent analysis of mitochondrial-DNA (mtDNA) restriction electromorphs helps resolve this issue. Mitochondrial-DNA samples from 48 individuals representing four populations were digested with 17 restriction endonucleases. After electrophoresis, the sizes of the mtDNA fragments were used to analyze the phylogenetic relatedness of fish collected over most of the species range. The analysis clearly identified two major races within the species: a northern and a southern form. The distribution of the mtDNA electromorphs, combined with zoogeographical changes in allelic isozymes and in eggs and adult morphologies (published elsewhere), makes the secondary-intergradation hypothesis most compelling.     

Development of polymorphic microsatellite loci for conservation genetic studies of the coral reef fish Centropyge bicolor

A total of 23 novel polymorphic microsatellite marker loci were developed for the angelfish Centropyge bicolor through 454 sequencing, and further tested on two spatially separated populations (90 individuals each) from Kimbe Bay in Papua New Guinea. The mean ± s.e . number of alleles per locus was 14·65 ± 1·05, and mean ± s.e . observed (H_O) and expected (H_E) heterozygosity frequencies were 0·676 ± 0·021 and 0·749 ± 0·018, respectively. The markers reported here constitute the first specific set for this genus and will be useful for future conservation genetic studies in the Indo‐Pacific region.     

Development and characterization of microsatellite markers to study population genetic diversity of Przewalski's naked carp Gymnocypris przewalskii in China

Two populations of Przewalski's naked carp Gymnocypris przewalskii, 30 individuals per population, were screened for 10 microsatellite loci. Moderate allele variation was found in these loci with two to eight alleles per locus. The expected and observed heterozygosity ranged from 0·019 to 0·805 and from 0·160 to 0·575, respectively.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of seven-band grouper (Epinephelus septemfasciatus) and cross-species amplification

Seven-band grouper (Epinephelus septemfasciatus) is a commercially important fishery species. Sixty-six microsatellite loci were isolated from a dinucleotide-enriched genomic library of E. septemfasciatus. Twelve of these loci were polymorphic in a test population with alleles per locus ranging from two to five, and observed and expected heterozygosities per locus from 0.04 to 1.00 and from 0.28 to 0.76, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci after Bonferroni correction. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of E. septemfasciatus and other related species. Lili Zhao and Changwei Shao have contributed equally.     

Phylogeny of five Prosopium species with comparisons with other Coregonine fishes based on isozyme electrophoresis*

Phylogenetic relationships of Prosopium gemmifer, P. spilonotus, P. williamsoni, P. cylindraceum and P. coulteri species were determined based on variation at 37 allozyme loci, and compared with Stenodus and nine species of Coregonus. Of the three genera, Prosopium was the most distinct with Nei genetic distances to Coregonus of D=0·55—1·05 and to Stenodus of D=0·58—0·92. Stenodus and Coregonus were closely related but grouped as sister taxa with a mean genetic distance of 0·31 (range 0·22—0·40). These results agree with the degree of morphological differentiation among the genera as well as with observations concerning hybridization, e.g. many Coregonus species hybridize with Stenodus, but no natural hybrids are known between Prosopium and other coregonines. Within Prosopium, P. coulteri was the most divergent species branching out at D=0·51 from the others and followed by P. cylindraceum at D=0·28. The three remaining species are closely related. Among them P. williamsoni joins P. gemmifer and P. spilonotus at 0·04, and the distance between the two Bear Lake endemics is only 0·004. The distance between P. cylindraceum and the remaining Prosopium species is similar to distances between Stenodus and Coregonus. The small genetic distance between P. spilonotus and P. gemmifer suggests their recent divergence. Even though no fixed differences were found between them, diVerent alleles were present at two loci at large enough frequencies to indicate reproductive isolation. This confirms that P. gemmifer and P. spilonotus are good biological species as indicated by their distinct morphology and ecology.     

Isolation and characterization of polymorphic microsatellite loci in Acanthoscelides obtectus Say (Coleoptera: Bruchidae)

Six microsatellite loci were isolated from the bruchid Acanthoscelides obtectus Say (Coleoptera: Bruchidae). Each locus was polymorphic, with the number of alleles ranging from 3 to 18. We found high levels of within‐population variation at most loci, with heterozygosities ranging from 0 to 0.75. Cross‐species amplification of these loci was tested in two other species of the genus Acanthoscelides, A. obvelatus Bridwell and A. argillaceus Sharp.     

Isolation and characterization of polymorphic microsatellites in Parnassius apollo and Euphydryas aurinia (Lepidoptera)

Microsatellite loci were developed from Parnassius apollo and Euphydryas aurinia, two endangered Palaearctic butterfly species. Respectively, six and five polymorphic loci were characterized from an enriched partial genomic library. Genetic diversity range from three to 25 alleles for the first species and from seven to 21 for the second. Although the presence of null alleles is suspected, these polymorphic loci are likely to provide important information on the fine scale genetic structure among populations of these species.     

Isolation and characterization of polymorphic microsatellite loci in Wuchang bream (Megalobrama amblycephala)

Wuchang bream (Megalobrama amblycephala) is an economically important fish in China. From a (GT)₁₃‐enriched genomic library, 20 microsatellites were developed. Nine of these 20 loci were polymorphic in a test population with allele numbers ranging from two to four, and the observed and expected heterozygosities ranging from 0.2609 to 0.7826 and from 0.3739 to 0.7546, respectively. In the cross‐species amplifications, six of these nine loci were also polymorphic in white amur bream (Parabramis pekinensis). These polymorphic microsatellite loci are potentially useful for population genetics of Wuchang bream and its closely related species.     

Isolation and cross‐species amplification of microsatellite loci from the fucoid seaweeds Fucus vesiculosus,F. serratus and Ascophyllum nodosum (Heterokontophyta,Fucaceae)

The Fucaceae is a family of brown seaweeds that dominate and frequently co‐occur on North Atlantic rocky shores. We developed nine polymorphic microsatellite markers for the fucoid seaweeds Fucus vesiculosus, F. serratus and Ascophyllum nodosum using a combined, enriched library. Six of these loci were polymorphic in at least two species, showing from two to eight alleles with heterozygosities ranging from 0.41 to 0.85. Loci were also tested on F. spiralis, revealing five polymorphic microsatellite loci in this species.     

Microsatellite loci for the mayfly Baetis rhodani (Baetidae,Ephemeroptera)

The first microsatellite primers are described for the mayfly family Baetidae (order Ephemeroptera). Seven polymorphic loci were isolated and characterized for the species Baetis rhodani from two enriched genomic libraries. A total of 183 individuals were genotyped using these loci. Allelic diversity was high for all loci, and observed heterozygosities ranged between 0.382 and 0.772. A heterozygote deficiency was detected in some loci, suggesting the presence of ‘null’ alleles.