Bayesian analysis of population structure and the characterization of nine novel
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1 Page 1 of Bayesian analysis of population structure and the characterization of nine novel microsatellite markers for the study of a Caribbean coral reef Gobiid (Coryphopterus personatus) and related taxa. 4 5 J. Derek Hogan*, Juan A. Galarza, Ryan P. Walter* and Daniel D. Heath* *Great Lakes Institute for Environmental Research, University of Windsor, 401 Sunset Ave., Windsor, Ontario, Canada, N9B 3P4. Estacion Biologica Doñana (CSIC), Av. Americo Vespucio S/N, 41092, Seville, Spain Keywords: microsatellite, Coryphopterus, population structure, cross-amplification, coral reef fish Corresponding Author: J. Derek Hogan, Great Lakes Institute for Environmental Research, University of Windsor, 401 Sunset Ave., Windsor, Ontario, Canada, N9B 3P4. hoganh@uwindsor.ca, fax: Running Title: Microsatellite markers for Coryphopterus. 1
2 Page 2 of Abstract Nine polymorphic microsatellite loci were isolated for the Masked Goby (Coryphopterus personatus), a widespread and abundant coral reef fish from the Caribbean. These loci show a high degree of allelic diversity and observed levels of heterozygosity ranged from We found evidence of some deviations from Hardy-Weinberg equilibrium (HWE) in some loci and linkage disequilibrium (LD) among loci. However, a Bayesian analysis of population structure found evidence of four distinct genetic clusters within our samples, each showed significantly reduced levels of LD and improved adherence to HWE. Between two and four of these loci successfully amplified in other related goby species. These microsatellite markers will be useful for population genetic studies of this species and closely related Caribbean Gobiids. 2
3 Page 3 of The masked goby (Coryphopterus personatus) is a small marine goby, common on Caribbean coral reefs, found at depths greater than 5m. (Cole & Robertson 1988). Coryphopterus personatus spawn by depositing eggs on a substrate (Cole & Robertson 1988), when the larvae hatch they undergo a developmental stage in the pelagic environment away from the reef structure before settling to a reef as juveniles. Caribbean gobies are repeat spawners with extended spawning seasons (Thresher 1984). This lifehistory, allows for the potential for long distance dispersal from the natal reef, as well as for the mixing of cohorts and populations through fluctuations in ocean currents. For these reasons, C. personatus provides an excellent model for studying chaotic genetic patchiness in marine systems. The population genetics of this species is unstudied to date, however, studies of other Caribbean gobiid species found significant spatial genetic structure (Taylor and Hellberg 2003), suggesting that there may also be population or subpopulation structuring in C. personatus. Here we report nine novel polymorphic microsatellite DNA loci for studies of the population genetics of C. personatus. This paper adds to five previously published markers for this species (Hepburn et al. 2005) and is complemented by markers from other goby species that cross-amplify in C. personatus (Dufour et al. 2007). A microsatellite enriched genomic library was developed using a modified version of Hamilton et al. (1999). Genomic DNA was extracted from fin tissue of a single C. personatus using a phenol-chloroform (1:1) method (Sambrook et al. 1989). Approximately180 ng of genomic DNA was digested using RsaI restriction enzyme yielding a majority of fragments ranging in size from bp. Double stranded SNX linkers were ligated onto the DNA fragments using T4 DNA ligase. These restricted 3
4 Page 4 of and ligated DNA fragments were amplified then hybridized with biotinylated probes including (GACA) 7 and (AC) 7. Enriched DNA fragments were ligated into a p-gem cloning vector and transformed into competent E. coli following the manufacturers protocol (Promega, Madison WI). Positive clones were selected, PCR amplified using m13/puc primers, sequenced using ABI Prism BigDye Terminator Cycle kit (Applied Biosystems) and resolved on an ABI 3100 Genetic Analyser (Applied Biosystems). Polymerase chain reaction (PCR) primers were designed from sequences containing microsatellite inserts using Primer 3 software (Rozen & Skaletsky 2000). PCRs were performed in 25 µl reaction volumes. Each reaction contained 10 ng of template DNA, 1 X PCR buffer, 0.4 µm of each primer, 50 µm of each dntp and 0.5 U Taq polymerase (Sigma). The PCR conditions varied from locus to locus, however two main conditions were used; (1) Initial denaturation for 2 min at 94ºC then 30 cycles of 94ºC for 15 s, a locus specific annealing temperature for 15 s (Table 1), and 72ºC for 30 s, followed by a final elongation step at 72ºC for 90 s; (2) a touchdown protocol including an initial denaturation for 2 min at 94ºC followed by 38 cycles of 94ºC for 30 s, annealing temperatures 30 s, 72ºC for 45 s, and a final elongation step at 72ºC for 2 min. The first eight annealing temperatures for the touchdown ranged from 65 57ºC, decreasing by one degree each cycle, then 30 cycles with annealing temperatures of 57ºC. Individuals were genotyped using labelled forward primers (IR700, IR800, Integrated DNA Technologies) and visualized on a LiCor 4300 DNA analyser and analysed with GENE IMAGIR4.05 software. The variability in nine loci was tested with 48 C. personatus collected from a single reef in Belize. Observed and expected 4
5 Page 5 of heterozygosities were calculated and exact tests for goodness of fit to Hardy-Weinberg equilibrium for each locus were preformed using the Markov Chain method (100,000 dememorization steps followed by 1,000,000 permutations) in ARLEQUIN v3.11 (Excoffier et al. 2005). Sequential Bonferroni correction was applied for multiple tests (Rice 1989). Tests for linkage disequilibrium were based on 10,000 iterations in ARLEQUIN v3.11. The number of alleles per locus and the allele size ranges were calculated in MSA 4.05 (Dieringer & Schlötterer 2003). All loci were polymorphic and observed heterozygosities ranged from (Table 1). Five of nine loci showed significant deviations from HWE, and significant linkage disequilibrium (LD) was found in all loci ranging from 13 88% of pairwise comparisons. Bayesian analysis of population structure BAPS 5.2 (Corander and Marttinen 2006) was used to test for population substructuring among our samples. BAPS acts to find population structuring within samples by creating clusters with better fit to genetic population models (e.g. maximizing HWE and minimizing LD). Initial mixture clustering of individuals was performed in triplicate for each possible number of clusters (K). We tested between one and six clusters. Using the results from the mixture analysis we performed an admixture analysis where the minimum population size prior to estimating admixture was 10, in 200 iterations, with admixture simulated from 200 reference individuals in 20 iterations. BAPS found evidence for K = 4 clusters within our samples, with no admixture between clusters. Four clusters was considered most parsimonious based on the rate of change in the likelihood function with respect to K (Evanno et al. 2005). The change in likelihood between K = 4 and K = 5 was almost double that of any other change in likelihood. These 4 clusters were re-tested for goodness of fit to HWE 5
6 Page 6 of and for LD as above. The four clusters identified in BAPS 5.2 showed significant improvements in HWE and reduced LD in comparison with the original pooled samples (Table 1). To assess the possible effect of null alleles and population substructuring within our data set we used the R package Geneland (Guillot et al. 2005). Spatial coordinates were assign to each individual as described in Galarza et al. (2009) allowing them to vary within 1 km radius (i.e. to account for uncertainty of fish movement). A maximum of 10 populations was set and the inference algorithm was launched with 100,000 MCMC iterations using spatial information under the null allele model. The mode of the posterior distribution of the MCMC chain suggested a number between 4 and 5 different panmictic groups within our data. These results suggest that observed deviations from HWE and linkage disequilibrium in the original 48 samples are most likely caused by the pooling of multiple populations creating a Wahlund effect. Finally, these markers were tested for cross-amplification in five related Gobiid species from the Caribbean including C. lipernes, C. glaucofrenum, C. eidolon, Gnatholepis thompsoni and Elacatinus evelynae (Table 2). Our results suggest these markers will be useful for the assessment of gene flow and population structure in C. personatus and related taxa across the Caribbean Acknowledgements We thank M. Oullette for laboratory assistance. Xim Cerda and Raphaël Boulay for supporting research visit for J.A.G. This work was supported by an NSERC Canada Research Chair grant to D.D.H, the Global Environment Facility s Coral Reef Targeted 6
7 Page 7 of Research for Capacity Building and Management project and GLIER post-doctoral fellowship from the University of Windsor to J.D.H References Cole KS, Robertson DR (1988) Protogyny in the Caribbean reef goby Coryphopterus personatus: gonad ontogeny and social influences on sex change. Bulletin of Marine Science, 42, Corander J, Marttinen P (2006) Bayesian identification of admixture events using multilocus molecular markers. Molecular Ecology, 15, Dieringer D, Schlotterer C (2003) MICROSATELLITE ANALYSER (MSA): a platform independent analysis tool for large microsatellite data sets. Molecular Ecology Resources, 3, Dufour BA, Hogan TM, Heath DD (2007) Ten polymorphic microsatellite markers in the invasive Round Goby (Neogobius melanostomus) and cross-species amplification. Molecular Ecology Notes, 7, Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Molecular Ecology, 14, Excoffier L, Laval G, Schneider S (2005) Arlequin ver. 3.0: An integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1,
8 Page 8 of Galarza JA, Carreras-Carbonell J, Macpherson E, et al. (2009) The influence of oceanographic fronts and early-life-history traits on connectivity among littoral fish species. Proceedings of the National Academy of Sciences 106, Guillot G, Estoup A, Mortier F, Cosson JF (2005) A Spatial Statistical Model for Landscape Genetics. Genetics 170, Hamilton MB, Pincus EL, Di Fiore A, Fleisher RC (1999) Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites. Biotechniques, 27, Hepburn RI, Mottillo EP, Bentzen P, Heath DD (2005) Polymorphic microsatellite loci for the Masked Goby, Coryphopterus personatus (Gobiidae). Conservation Genetics, 6, Rice WR (1989). Analyzing tables of statistical tests. Evolution, 43, Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology (eds. Krawetz S, Misener S), pp Humana Press, Totowa, NJ. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual, 2 nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Taylor MS, Hellberg ME (2003) Genetic evidence for local retention of pelagic larvae in a Caribbean reef fish. Science, 299, Thresher RE (1984) Reproduction in reef fishes. T.F.H. Publications, Neptune City, New Jersey, USA. 8
9 Page 9 of 11 Table 1 Microsatellite loci characterized in Coryphopterus personatus. Locus / GenBank Accession no. Primer Sequences (5-3 ) Repeat Motif T a (ºC) N N a Allele size range (bp) BAPS Cluster H O (H E ) Cper26 CACAAACAGACAAACACC (CA) GQ CATGGTCTGCACTGCTCCT (0.66) Cper30 TCAACGAAGGCAAATAACCA (TG) * GQ CGCAGTGACATGCTGTAAGG (0.43) % LD % LD BAPS Cper92 CTACTCGCACCTGCAATCAA (TG) * - - * GQ CAAACACAAGCCTCACCAAA (0.50) Cper99 CGCAGTGACATGTTGTAAGGA (CA) GQ TCAACGAAGGCAAATAACCA (0.34) Cper103 TGGGTTTAATCGAAGCCAGT (TG) GQ CGCACCTTTCGAGGAGTTT (0.55) Cper119 ATGATGTGCACAACCGGATA (TG) * - - * GQ TGAGACATCACCTATAAAATCCTGA (0.92) Cper163 CGTCCATTATTTTAACCCGTCA (TG) * * * - * GQ AACATTCCCATTCACCAACC (0.54) Cper184 ATTGTTTCACCCAGCACCTC (TC) GQ GGAGAGGACAAATATGAAAAGCA (0.87) Cper188 ACTCACGCATTTGCCTCTTC (CA) * * GQ ACTGACACACATGCCCTCCT (0.71) : indicates touchdown PCR reaction used; T a : annealing temperature; N: number of genotypes; N a : number of alleles observed; H O : observed heterozygosity; H E : expected heterozygosity; *: locus deviates significantly from Hardy-Weinberg equilibrium after sequential Bonferroni correction for multiple tests; % LD: the per-locus proportion of pair-wise comparisons that showed significant linkage disequilibrium; % LD BAPS : the per-locus proportion of pair-wise comparisons that
10 Page 10 of 11 showed significant linkage disequilibrium within the BAPS clusters. All possible loci pairs were used in both LD analyses, both pre- and post-baps clustering (n = 45).
11 Page 11 of 11 Table 2 Results of cross-species amplification of nine microsatellite primers developed for C. coryphopterus. Bold numbers indicate number of alleles found if amplification occurred. Dashes indicate no amplification. C l = Coryphopterus lipernes, C g = Coryphopterus glaucofrenum, C e = Coryphopterus eidolon, G t = Gnathopelis thompsoni, E e = Elacatinus evelynae. All N = 5 except C e N = 2. Locus C l C g C e G t E e Cper Cper Cper Cper Cper Cper Cper Cper Cper
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