PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite markers in the white-faced capuchin monkey (Cebus capucinus) and cross-species amplification in other New World monkeys

Seventeen polymorphic microsatellite loci were identified for white-faced capuchin monkeys from an enriched genomic library in addition to one locus found through cross-species comparisons. In a sample of 187 wild individuals, these loci exhibited an average of five alleles and an observed heterozygosity of 0.62. The combined probability of exclusion of a random individual from parentage was 0.99. These loci were screened in 23 other New World monkeys and an average of seven loci was variable per species, suggesting that these loci could be of use in studies of other Neotropical primates.     

Isozyme Variation in a Tropical Pioneer Tree Species (Cecropia obtusifolia,Moraceae) with High Contents of Secondary Compounds1

We define 48 allozyme loci for a tropical pioneer tree species, Cecropia obtusifolia Bertol, which has high contents of secondary compounds. Our goals were to find the effects of extraction procedures on artifacts and variation in resolution of enzyme banding patterns; to explore the relationship among the variation of the loci sampled and the enzymes' molecular structure, metabolic function and substrate; to obtain estimates of the genetic variation in this species at Los Tuxtlas rain forest (México) and to explore the variation of allelic frequencies in six successive life-history stages of the species. The resolution of the isozymes bands and the actual banding pattern varied with the type and age of tissue, the collection and storage procedures, the extraction buffer, and other loading and running procedural details. However, artifactual variation was eliminated with a new extraction buffer for species with high contents of secondary compounds. Of the 26 enzymes resolved for C. obtusifolia, we found that enzymes with a greater number of substrates and an oligomeric quaternary structure tended to be more variable than their counterparts, but the relationship was not statistically significant. The proportion of polymorphic systems varied significantly with the metabolic pathway and the function of the enzymes. Enzymes involved in starch synthesis are significantly more variable (p < 0.05) than all others, except those involved in amino acids metabolism and the proportion of polymorphic enzymes is also significantly associated with the hnction of the enzyme, the hydrolases and isomerases are significantly more variable than lyases and oxidoreductases enzymes. The percentage of polymorphic loci for C. obtusifolia was estimated at P = 27.1%. The effective number of alleles was estimated at ne = 1.3 and ne = 2.4 for all loci and only polymorphic ones respectively and the average heterozygosity (H) for all 48 loci was estimated at H = 0.05. Allele frequencies varied throughout the life-cycle of the species, with significant differences for some alleles and loci among some life-cycle stages. “Tree seeds” allele frequencies differ significantly (P < 0.05) from “rainy dispersed seeds” in 7 of 8 loci and from “soil seeds” in Six of eight loci. Allele frequencies of all three seed categories (“tree seeds”, “rainy dispersed seeds”, and “soil seeds”) differed strongly from established individuals (seedlings, juveniles and adults), while allele frequencies of established individuals are relatively similar to one another. Seedling allele frequencies at most loci were also significantly different from those found in seeds collected from trees, seed-rain, and soil. Two alleles (at GOT-2 and FE-2) were only found in soil seeds and one allele (at LAP-2) was only found in seedlings.     

Isolation and characterization of 11 microsatellite markers from Sagittaria latifolia (Alismataceae)

We developed 11 microsatellite loci for Sagittaria latifolia, an aquatic plant common to wetlands of North America. From an (AG)-enriched library, we identified 66 unique microsatellite sequences for which primers could be designed. Twenty-two loci reliably amplified a clear single band of expected size, and 11 loci were scoreable and polymorphic. For these 11 loci, we genotyped a monoecious and a dioecious population, yielding four to 14 alleles per locus. Three loci exhibited significant linkage disequilibrium leaving eight independent variable loci. Eight loci also amplified in four other Sagittaria species. These microsatellite loci will be useful to compare genetic structure among monoecious and dioecious populations of S. latifolia.     

Genetic distance between two sibling species of the Aedes mariae complex (Diptera, Culicidae)

Two sibling species of the mariae complex of the Aedes genus have been studied: Ae. mariae and Ae. zammitii. Thee mosquitoes are allopatric and show similar adaptations to rocky mediterranean coasts. The isolating mechanisms between the two species are well studied. A partial sterility of hybrid F1, limited to males, has been found with laboratory cross experiments. Strong pre-mating isolating mechanisms were shown in nature by means of release experiments, the frequency of hybrids never exceeding 2% without evidence of introgression. We analyzed genetic variation at 26 enzyme loci in a population of each species: 35% of loci were polymorphic, with an observed mean heterozygosity of 0.07 in Ae. mariae and 0.06 in Ae. zammitii; 6 loci allow discrimination between the two species at a probability of at least .99. Nei's measures of genetic identity and genetic distance are respectively I = 0.6096 and D = 0.2828. The distribution of genetic identities relative to loci is strongly bimodal, reflecting a different contribution of highly variable and fast evolving loci on one side and conservative and slowly evolving loci on the other. We consider, on the base of various experimental data (see Powell, 1975), in the first class ("fast evolving" loci) both variable substrate and regulatory enzymes, in the second ("slow evolving" loci) non regulatory enzymes. Values of genetic distance calculated separately for the "fast" (54% of the loci studied) and "slow" (46%) evolving loci are respectively Df = 0.41 and Ds = 0.03, showing that the main contribution to genetic distance in the first period of divergence is due to fast evolving loci, whose rate of evolution is about ten times more rapid with respect to slow evolving loci.     

Study of allelic polymorphism of (GATA)n-containing loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae)

The genesis of mini- and microsatellite loci, which is under extensive study in humans and some other bisexual species, have been virtually overlooked in species with clonal mode of reproduction. Earlier, using multilocus DNA fingerprinting, we have examined variability of some mini- and microsatellite DNA markers in parthenogenetic lizards from the genus Darevskia. In particular, mutant (GATA)n-restrictive DNA fragments were found in Darevskia unisexualis. In the present study, we examined intraspecific polymorphism of three cloned loci of D. unisexualis--Du323, Du215, and Du281--containing (GATA)7GAT(GATA)2, GAT(GATA)9, and (GATA)10TA(GATA) microsatellite clusters, respectively. Different levels of intrapopulation and interpopulation variability of these loci were found. Locus Du281 showed the highest polymorphism--six allelic variants (in the sample of 68 DNA specimens). Three alleles were found for locus Du215. The Du325 locus was electrophoretically invariant. The primers chosen for loci Du323, Du215, and Du281 were also used for PCR analysis of homologous loci in two presumptive parental bisexual species, D. valentini and D. nairensis. The PCR products of the corresponding loci of the parental species had approximately the same size (approximately 200 bp) as their counterparts in D. unisexualis, but the polymorphism levels of the paternal, maternal, and hybrid species were shown to be somewhat different. These data on the structure of the D. unisexualis loci provide a possibility to study genetic diversity in the parthenogenetic species D. unisexualis and other related unisexual and bisexual species of this genus, which can provide new information on the origin of parthenogenetic species and on the phylogenetic relationships in the genus Darevskia. These data can also be used for resolving problems of marking the lizard genome, which is still poorly studied.     

Microsatellite markers for the heavily exploited canary (Sebastes pinniger) and other rockfish species

The isolation and characterization of nine polymorphic microsatellite loci (eight tetranucleotide and one dinucleotide) from the canary rockfish Sebastes pinniger are described. Polymorphism at these loci revealed from six to 28 alleles, with expected heterozygosities ranging between 0.42 and 0.88, enabling high‐resolution genetic population structure investigation for this overfished species in the northeastern Pacific. They also amplify in 13 other congeneric species, providing highly variable loci for research on other rockfishes.     

Ten polymorphic microsatellite markers developed in the masson pine moth Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae)

A set of 10 polymorphic di‐ and trinucleotide microsatellite loci were developed for the forestry pest insect, masson pine moth, Dendrolimus punctatus Walker. The expected heterozygosity at these loci ranges from 0.285 to 0.859, and the observed allele numbers from five to 19. Cross species amplification of these loci in four other congeneric pine moth species indicates variable levels of loci conservation and thus cross‐applicability. Therefore, the microsatellite loci reported here should be useful for population genetic and other related studies in the masson pine moth and other closely related species.     

Genetic evidence for population expansion in Hydrotaea irritans (Fallèn) (Diptera: Muscidae)

Allozyme variation in the sheep headfly Hydrotaea irritans was studied on two spatial scales. Geographic variation among seven Danish and one Dutch population revealed significant but rather low genetic differentiation with F _ST = 0.01 over all loci. The Dutch population was on average not more different from the Danish populations than the Danish populations from each other. Allele frequencies were very skewed with the most common allele always exceeding 0.85 and usually 0.9 in frequency, but with many rare alleles at some loci. Tests for neutrality of the variation at the nine polymorphic loci revealed highly significant deviations from expected homozygosity in this species, which was not found in a comparative analysis of allozyme variation at similar loci of seven other Hydrotaea species. To explain the peculiar observed pattern of allozyme variation in H. irritans , it is suggested that this species has successfully expanded its range and spread through northern and central Europe in the recent past. Alternatively, H. irritans may have recently invaded a new niche, resulting in increased abundance of the species and subsequent dispersal to former areas of the species distribution.     

Autosomal and sex-linked microsatellite loci in the green oak leaf roller Tortrix viridana L. (Lepidoptera, Tortricidae)

Eight microsatellite markers were developed for the lepidopteran species Tortrix viridana using an enrichment protocol. The loci were highly variable with number of alleles ranging from four to 38. Six of the eight loci were in Hardy-Weinberg equilibrium. The other two were linked to the Z-chromosome. Values of observed heterozygosity ranged for the autosomal loci from 0.510 to 0.957. All loci will be useful to study dispersal and the autosomal loci, as well for phylogeographical studies.     

New microsatellites primers for plaice,Pleuronectes platessa L. (Teleostei: Pleuronectidae)

Seven microsatellite loci were developed for Pleuronectes platessa, a commercially important flatfish found throughout northern European waters. The number of alleles per locus ranged from 11 to 33 and heterozygosities from 0.74 to 0.96. Cross‐reactivity was tested against six other flatfish species. Significant amplification was found in all species from one to all seven of the loci.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Microsatellite loci for the North American tortoises (genus Gopherus) and their applicability to other turtle species

We describe the development of nine microsatellite loci from the gopher tortoise (Gopherus polyphemus). Screening 270 individuals, we found these loci to be highly variable (from two to 15 alleles per locus) and thus likely to be applicable in parentage and population‐level analyses for G. polyphemus. All nine loci amplified readily in the other three species of Gopherus as well. This observation, together with successful cross amplification of several loci in two additional families (Cheloniidae, Kinosternidae), underscores their potential utility among turtles in general.     

Isolation of simple and compound polymorphic tetranucleotide microsatellites for the neotropical leaflitter frog Eleutherodactylus ockendeni (Leptodactylidae)

Few studies of population structure and genetic diversity exist for frogs in the Amazon of South America, an area renowned for exceptionally high species richness. We isolated seven highly variable tetranucleotide microsatellite loci for the neotropical leaflitter frog, Eleutherodactylus ockendeni using an enrichment method. Three of the repeats are simple, three are compound and one is imperfect. We screened all loci with 175 individuals from one geographical area in the upper Napo of Ecuador and found high polymorphism in all loci (> 14 alleles/locus). These markers are suitable for population genetics studies of E. ockendeni and perhaps other leaflitter frogs of the same genus.     

Identification of microsatellite loci in Collinsia verna (Veronicaceae)

We developed eight polymorphic microsatellite loci for Collinsia verna (Veronicaceae). In a sample of 18–35 individuals from a single population, we found two to 15 alleles per locus (mean 8.3). We also tested these loci for cross‐amplification in all 22 species in the tribe Collinseae. Overall, more than half the species in the tribe amplified one microsatellite while three species most closely related to C. verna (Collinsia violacea, Collinsia parviflora and Collinsia grandiflora) amplified multiple microsatellite loci. These microsatellite loci will be used in future studies of mating system in this tribe and other quantitative genetic and population genetic studies.     

Isolation and characterization of nuclear microsatellite loci from Ceratopetalum apetalum (Cunoniaceae)

We identified 16 polymorphic nuclear microsatellite loci from an AG-enriched library of Ceratopetalum apetalum. These loci were tested across two populations of C. apetalum, a single population of C. gummiferum and representatives of four other congeneric taxa. Fourteen loci were identified as polymorphic in at least one other species. These microsatellites will be used to look at population dynamics of species with varying distributions and presumed low dispersal capabilities within the genus Ceratopetalum.     

Distribution and biomass of microphytes measured by benthic chlorophyll a in a tropical lagoon (New Caledonia,South Pacific)

Starch-gel electrophoretic techniques were applied to the investigation of molecular genetic variation in populations of the rough periwinkle Littorina saxatilis. The investigation comprised two phases: a) technique development to resolve as many as possible of the allozyme loci reported in the literature as having been screened in the genus Littorina and in Melarhaphe neritoides; b) the use of these loci to assess levels of genetic variation in and patterns of genetic differentiation among populations of L. saxatilis from a relatively isolated group of populations from Galway Bay, Ireland. More than 43 allozyme loci (of which four were screened for the first time here in this species), coding for 37 enzymes, were investigated and thirteen of these loci (including two loci screened for the first time here) were found to be variable and reliably scorable. Samples from five pairs of transects were collected from Inismór, Aran Islands, from sites with known exposure levels; one transect within each pair was collected from an exposed site and the other from a nearby, but relatively sheltered site. UPGMA for eleven loci, (ARK and PGDH were excluded from cluster and FST analysis as they were unscorable in a few samples), showed that the samples cluster mostly by pair, reflecting their geographic origin and is indicative of little gene flow between populations. Levels of population differentiation were high among samples from the top of the shore, but unusually so at AAT-1 which showed nearly three times the mean FST value for the eleven loci. There was also a significant regression of frequency of AAT-1100 against level of exposure. In addition, among midshore samples, there was a consistently higher frequency of AAT-1100 in sheltered habitats. These results support the findings of others, indicating that this locus may be subject to natural selection.     

Microsatellite markers from Ceanothus roderickii (Rhamnaceae) using next-generation sequencing technology

? Premise of the study: Ceanothus roderickii is an endangered shrub endemic to California. To investigate the population genetics of this species, including the genetic consequences of population fragmentation and hybridization, 10 microsatellite markers were developed. ? Methods and Results: Using next-generation sequencing (454) data from a single C. roderickii individual, 10 microsatellite markers were developed. A group of 12 individuals representing all of the major C. roderickii populations were analyzed. All loci were found to be polymorphic, with a range from two to 12 alleles per locus. Observed heterozygosity ranged from 0.08 to 0.83 across loci. All 10 loci were also amplifiable in at least one other Ceanothus species. ? Conclusions: Results presented here indicate the utility of our new microsatellite primers in ongoing and future studies concerning population genetics and gene flow in C. roderickii, as well as the potential applicability of these primers in similar studies on other Ceanothus species.     

New studies on allozyme genetic distance and variability in akodontine rodents (Cricetidae) and their systematic implications

Allozyme genetic distance and variability were studied by horizontal starch gel electrophoresis in 11 species of rodents of the tribe Akodontini representing four different genera. One species of the phyllotine Calomys was used as an outgroup for comparisons. Thirty presumptive loci were assessed in a total of 172 specimens. Only three of 30 loci were found to be monomorphic in the whole sample. Levels of polymorphism ( P ) and mean heterozygosity ( H ) were dependent on esterase loci which increase H values by 6.26% on average. Nei's jackknife genetic distances among conspecific populations were found not to differ significantly with geographic distance. Phenetic and cladistic interspecific analysis coincided in indicating the monophyletic nature of the akodontine clade, and its splitting into two sharply differentiated subclades: one comprising species referred to as Abrothrix, Chelemys and Geoxus , another comprising species of Akodon (including Deltamys ) and Oxymycterus. Thus, the separateness of a southern Andean branch in the cladogenesis of the Akodontini was confirmed, as well as the early recognition of Abrothrix as a full genus, and the recent claim of Spotorno to include in this genus other species formerly classified as Akodon jelskii, Ak. andinus and Ak. olivaceus.     

A panel of microsatellite loci from two species of octopus, Pareledone turqueti (Joubin, 1905) and Pareledone charcoti (Joubin, 1905)

Eighteen dinucleotide microsatellite loci were isolated from two octopus species, Pareledone turqueti and Pareledone charcoti, which are endemic to the Southern Ocean. Genetic diversity was assessed in samples of P. charcoti and P. turqueti from Elephant Island and Shag Rocks respectively. All except one locus (which has proved to be polymorphic in other species) were variable in the focal species and amplified between six and 30 and between four and 28 alleles for P. charcoti and P. turqueti respectively; mean expected heterozygosities varied between 0.38 and 0.95 (P. charcoti) and between 0.34 and 0.97 (P. turqueti), with significant (P < 0.05) departures from Hardy–Weinberg equilibrium at seven loci; three of these loci provided significant (P < 0.05) evidence for null alleles. Two pairs of loci isolated from P. turqueti demonstrated significant (P < 0.05) linkage disequilibrium. We are presently using these genetic markers to quantify spatial genetic structure in the genus Pareledone.     

Isolation and characterization of 10 tetranucleotide microsatellite loci from the yellow-streaked greenbul (Phyllastrephus flavostriatus) and cross-species amplification in four closely related taxa

We describe the isolation of 10 tetranucleotide microsatellite loci from the yellow-streaked greenbul using an enrichment protocol. All loci were highly variable with the number of alleles ranging from 8 to 13, and observed heterozygosity ranging from 0.652 to 0.870. All loci were in Hardy-Weinberg equilibrium; however, loci Pfl12 and Pfl54 showed significant linkage disequilibrium. All 10 loci successfully amplified and were polymorphic in at least one of four related Phyllastrephus species. These loci should prove to be widely applicable to studies of phylogeography, hybridization and paternity in African greenbuls.