John R. Gold

dDocent: a RADseq, variant-calling pipeline designed for population genomics of non-model organisms

Restriction-site associated DNA sequencing (RADseq) has become a powerful and useful approach for population genomics. Currently, no software exists that utilizes both paired-end reads from RADseq data to efficiently produce population-informative variant calls, especially for non-model organisms with large effective population sizes and high levels of genetic polymorphism. dDocent is an analysis pipeline with a user-friendly, command-line interface designed to process individually barcoded RADseq data (with double cut sites) into informative SNPs/Indels for population-level analyses. The pipeline, written in BASH, uses data reduction techniques and other stand-alone software packages to perform quality trimming and adapter removal, de novo assembly of RAD loci, read mapping, SNP and Indel calling, and baseline data filtering. Double-digest RAD data from population pairings of three different marine fishes were used to compare dDocent with Stacks, the first generally available, widely used pipeline for analysis of RADseq data. dDocent consistently identified more SNPs shared across greater numbers of individuals and with higher levels of coverage. This is due to the fact that dDocent quality trims instead of filtering, incorporates both forward and reverse reads (including reads with INDEL polymorphisms) in assembly, mapping, and SNP calling. The pipeline and a comprehensive user guide can be found at http://dDocent.wordpress.com.

Phylogenetic Relationships in the North American Cyprinid Genus Cyprinella (Actinopterygii: Cyprinidae) Based on Sequences of the Mitochondrial ND2 and ND4L Genes

Abstract Shiners of the cyprinid genus Cyprinella are abundant and broadly distributed in eastern and central North America. Thirty species are currently placed in the genus: these include six species restricted to Mexico and three barbeled forms formerly placed in different cyprinid genera (primarily Hybopsis). We conducted a molecular phylogenetic analysis of all species of Cyprinella found in the United States, using complete nucleotide sequences of the mitochondrial, protein-coding genes ND2 and ND4L. Maximum-parsimony analysis recovered a single most-parsimonious tree for Cyprinella. Among historically recognized, nonbarbeled Cyprinella, the mitochondrial (mt) DNA tree indicated that basal lineages in Cyprinella are comprised largely of species with linear breeding tubercles and that are endemic to Atlantic and/or Gulf slope drainages, whereas derived lineages are comprised of species broadly distributed in the Mississippi basin and the American Southwest. The Alabama Shiner, C. callistia, was basal in the mtDNA tree, although a monophyletic Cyprinella that included C. callistia was not supported in more than 50% of bootstrap replicates. There was strong bootstrap support (89%) for a clade that included all species of nonbarbeled Cyprinella (except C. callistia) and two barbeled species, C. labrosa and C. zanema. The third barbeled species, C. monacha, fell outside of Cyprinella sister to a species of Hybopsis. Within Cyprinella were a series of well-supported species groups, although in some cases bootstrap support for relationships among groups was below 50%. A derived clade consisting of C. spiloptera, C. whipplei, C. venusta, and the southwestern C. lutrensis group was strongly supported. The species C. lutrensis and C. lepida were not monophyletic, suggesting further study and revision within this group are warranted. In general, the most-parsimonious mtDNA tree was similar in terms of relationships among species to those proposed more than 40 years ago by R. H. Gibbs.

Microsatellite DNA Variation in Sandbar Sharks (Carcharhinus plumbeus) from the Gulf of Mexico and Mid-Atlantic Bight

Three polymorphic DNA microsatellite loci were developed in the sandbar shark (Carcharhinus plumbeus) and used to test the hypothesis that sharks from the midAtlantic Bight and western Gulf of Mexico comprise a single genetic stock. No significant differences in microsatellite allele frequencies were detected, a finding consistent with the null hypothesis. Polymorphic microsatellite loci appear to be scarce in sandbar sharks relative to other fish species. The three loci examined in this study had relatively low levels of heterozygosity, most likely attributable to the small number of dinucleotide repeat units per locus. T HE sandbar shark (Carcharhinus plumbeus) is

Selection and sex-biased dispersal in a coastal shark: the influence of philopatry on adaptive variation

Sex‐biased dispersal is expected to homogenize nuclear genetic variation relative to variation in genetic material inherited through the philopatric sex. When site fidelity occurs across a heterogeneous environment, local selective regimes may alter this pattern. We assessed spatial patterns of variation in nuclear‐encoded, single nucleotide polymorphisms (SNPs) and sequences of the mitochondrial control region in bonnethead sharks (Sphyrna tiburo), a species thought to exhibit female philopatry, collected from summer habitats used for gestation. Geographic patterns of mtDNA haplotypes and putatively neutral SNPs confirmed female philopatry and male‐mediated gene flow along the northeastern coast of the Gulf of Mexico. A total of 30 outlier SNP loci were identified; alleles at over half of these loci exhibited signatures of latitude‐associated selection. Our results indicate that in species with sex‐biased dispersal, philopatry can facilitate sorting of locally adaptive variation, with the dispersing sex facilitating movement of potentially adaptive variation among locations and environments.

Contemporary population structure and post-glacial genetic demography in a migratory marine species, the blacknose shark, Carcharhinus acronotus

Patterns of population structure and historical genetic demography of blacknose sharks in the western North Atlantic Ocean were assessed using variation in nuclear‐encoded microsatellites and sequences of mitochondrial (mt)DNA. Significant heterogeneity and/or inferred barriers to gene flow, based on microsatellites and/or mtDNA, revealed the occurrence of five genetic populations localized to five geographic regions: the southeastern U.S Atlantic coast, the eastern Gulf of Mexico, the western Gulf of Mexico, Bay of Campeche in the southern Gulf of Mexico and the Bahamas. Pairwise estimates of genetic divergence between sharks in the Bahamas and those in all other localities were more than an order of magnitude higher than between pairwise comparisons involving the other localities. Demographic modelling indicated that sharks in all five regions diverged after the last glacial maximum and, except for the Bahamas, experienced post‐glacial, population expansion. The patterns of genetic variation also suggest that the southern Gulf of Mexico may have served as a glacial refuge and source for the expansion. Results of the study demonstrate that barriers to gene flow and historical genetic demography contributed to contemporary patterns of population structure in a coastal migratory species living in an otherwise continuous marine habitat. The results also indicate that for many marine species, failure to properly characterize barriers in terms of levels of contemporary gene flow could in part be due to inferences based solely on equilibrium assumptions. This could lead to erroneous conclusions regarding levels of connectivity in species of conservation concern.

Chromosomal NOR Phenotypes of Seven Species of North American Cyprinidae, with Comments on Cytosystematic Relationships of the Notropis volucellus Species-group, Opsopoeodus emiliae, and the Genus Pteronotropis

in the fifth (P. hubbsi), the single NOR was located terminally on the short arm of a large-sized acro-/subtelocentric chromosome. Pteronotropis signipinnis and P. welaka each possessed two pair of NOR-bearing chromosomes: one pair had a NOR located terminally on the short arm of a medium-sized submetacentric chromosome; whereas the other pair had a NOR located terminally on the short arm of a large-sized acro-/subtelocentric chromosome. Consideration of these and previous NOR data in North American cyprinids suggest that: 1) the N. volucellus species-group as presently constituted may not be monophyletic; 2) the species Opsopoeodus emiliae may belong in an assemblage which includes, among others, at least four species of the genus Cyprinella; 3) 0. emiliae may not be closely related to N. maculatus; and 4) P. hubbsi may belong in an assemblage which includes P. signipinnis and P. welaka, and hence may be a valid member of the genus Pteronotropis.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2011 - 31 January 2012: PERMANENT GENETIC RESOURCES NOTE

This article documents the addition of 473 microsatellite marker loci and 71 pairs of single‐nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Barteria fistulosa, Bombus morio, Galaxias platei, Hematodinium perezi, Macrocentrus cingulum Brischke (a.k.a. M. abdominalis Fab., M. grandii Goidanich or M. gifuensis Ashmead), Micropogonias furnieri, Nerita melanotragus, Nilaparvata lugens Stål, Sciaenops ocellatus, Scomber scombrus, Spodoptera frugiperda and Turdus lherminieri. These loci were cross‐tested on the following species: Barteria dewevrei, Barteria nigritana, Barteria solida, Cynoscion acoupa, Cynoscion jamaicensis, Cynoscion leiarchus, Cynoscion nebulosus, Cynoscion striatus, Cynoscion virescens, Macrodon ancylodon, Menticirrhus americanus, Nilaparvata muiri and Umbrina canosai. This article also documents the addition of 116 sequencing primer pairs for Dicentrarchus labrax.

Phylogenetic Relationships of Cyprinid Fishes in Subgenus Notropis Inferred from Nucleotide Sequences of the Mitochondrially Encoded Cytochrome b Gene

Abstract Cytochrome b sequences (1140 bp) from 16 species considered to be members of subgenus Notropis and several outgroup taxa were analyzed phylogenetically. Phylogenetic analysis of cytochrome b was sensitive to sampling of outgroup taxa, probably resulting from substitutional rate heterogeneity among outgroups. Maximum-likelihood analysis was more robust to these effects as compared to maximum parsimony. Both maximum parsimony and maximum-likelihood analysis supported removal of Notropis candidus and Notropis shumardi from subgenus Notropis. Monophyly of the 14 remaining species was supported by the maximum likelihood analysis. Regardless of the method of analysis or use of outgroup taxa, phylogenetic analysis of cytochrome b indicated strong support for the following hypothesis of relationships: ((Notropis rubellus, Notropis suttkusi), (Notropis amoenus, Notropis stilbius, ((Notropis atherinoides, Notropis oxyrhynchus), (Notropis amabilis, Notropis jemezanus)))). This phylogenetic hypothesis implies that morphological characters associated with increased olfaction were derived independently among members of subgenus Notropis, perhaps in response to life in more turbid habitats.

Systematics of the Blacktail Shiner (Cyprinella venusta) Inferred from Analysis of Mitochondrial DNA

RAGE,J.-C. 1987. Fossil history, p. 51-76. In: Snakes: ecology and evolutionary biology. R. A. Seigel, J. T. Collins, and S. S. Novek (eds.). MacMillan Publ. Co., New York. SAGHAI-MAROOF, M. A., K. M. SOLIMAN, R. A. JORGENSEN, AND R. W. ALLARD. 1984. Ribosomal DNA spacer length in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Nat. Acad. Sci., USA 81:8014-8019. SANGER, F., S. NICKLEN, AND A. R. COULSEN. 1977. DNA sequencing with chain-terminating inhibitors. Ibid. 74:5463-5467. WERMAN, S. D. 1992. Phylogenetic relationships of Central and South American pitvipers of the genus Bothrops (sensu lato): cladistic analyses of biochemical and anatomical characters, p. 21-40. In: Biology of the pitvipers. J. A. Campbell and E. D. Brodie Jr. (eds.). Selva Publ., Tyler, TX.

Haplotyping RAD loci: an efficient method to filter paralogs and account for physical linkage.

Next‐generation sequencing of reduced‐representation genomic libraries provides a powerful methodology for genotyping thousands of single‐nucleotide polymorphisms (SNPs) among individuals of nonmodel species. Utilizing genotype data in the absence of a reference genome, however, presents a number of challenges. One major challenge is the trade‐off between splitting alleles at a single locus into separate clusters (loci), creating inflated homozygosity, and lumping multiple loci into a single contig (locus), creating artefacts and inflated heterozygosity. This issue has been addressed primarily through the use of similarity cut‐offs in sequence clustering. Here, two commonly employed, postclustering filtering methods (read depth and excess heterozygosity) used to identify incorrectly assembled loci are compared with haplotyping, another postclustering filtering approach. Simulated and empirical data sets were used to demonstrate that each of the three methods separately identified incorrectly assembled loci; more optimal results were achieved when the three methods were applied in combination. The results confirmed that including incorrectly assembled loci in population‐genetic data sets inflates estimates of heterozygosity and deflates estimates of population divergence. Additionally, at low levels of population divergence, physical linkage between SNPs within a locus created artificial clustering in analyses that assume markers are independent. Haplotyping SNPs within a locus effectively neutralized the physical linkage issue without having to thin data to a single SNP per locus. We introduce a Perl script that haplotypes polymorphisms, using data from single or paired‐end reads, and identifies potentially problematic loci.