Haploid gynogens facilitate disomic marker development in paleotetraploid sturgeons.

Acipenseriformes (sturgeons and paddlefishes) are of substantial conservation concern, and development of genomic resources for these species is difficult due to past whole genome duplication. Development of disomic markers for polyploid organisms can be challenging due to difficulty in resolving alleles at a single locus from those among duplicated loci. In this study, we detail the development of disomic markers for the endangered pallid sturgeon (Scaphirhynchus albus) found in North America. One of the strategies for pallid sturgeon conservation is to stock U.S. rivers with offspring of pure pallid sturgeon, but introgression with the sympatric shovelnose sturgeon (S. platorynchus) threatens pallid sturgeon genetic integrity. Currently, 19 microsatellite loci are used to differentiate between both species and their hybrids, but the markers are insufficient to robustly identify backcrosses. We performed double digest restriction site-associated DNA sequencing (ddRADseq) on shovelnose sturgeon haploid gynogens to produce a reduced-representation genomic reference. Contiguous sequences that were heterozygous within a haploid individual were flagged as potentially encompassing multiple loci. Approximately 60 individuals of each species from two management units were sequenced, and reads were mapped to the haploid reference to identify single nucleotide polymorphisms (SNPs) at individual loci. The final data set contained 11,082 microhaplotyped loci which offer at least an order of magnitude greater resolution for species discrimination than the current panel of 19 microsatellites. These markers will be used to examine a larger sample of Scaphirhynchus individuals throughout their ranges to determine the extent and trajectory of hybridization.

Improved genetic identification of acipenseriform embryos with application to the endangered pallid sturgeon Scaphirhynchus albus.

We produced pallid sturgeon Scaphirhynchus albus embryos at five pre-hatch developmental stages and isolated and quantified genomic DNA from four of the stages using four commercial DNA isolation kits. Genomic DNA prepared using the kit that produced the largest yields and concentrations were used for microsatellite DNA analyses of 10-20 embryos at each of the five developmental stages. We attempted to genotype the hatchery-produced embryos at 19 microsatellite loci and confirmed reliable genotyping by comparing the microsatellite genotypes to those of known parents. Embryos at stages 5 and 8 did not produce reliable genotyping while those at stages 14, 24 and 33 did. We used the same DNA isolation method on 262 wild-caught acipenseriform embryos collected from the lower Yellowstone River. A total of 200 of the wild embryos were successfully identified to stages 8 to 34 and the rest could not be staged. Using a combination of single nucleotide polymorphism and microsatellite markers, 249 of the wild-caught embryos were genetically identified as paddlefish Polyodon spathula, five were identified as shovelnose sturgeon Scaphirhynchus platorynchus and eight failed to amplify. None were identified as pallid sturgeon. This study demonstrates that early-stage wild-spawned acipenseriform embryos can be genetically identified less than 24 h post-spawn. This methodology will be useful for recovery efforts for endangered pallid sturgeon and can be applied to other acipenseriform species.

Complete mitochondrial genome of the nurse shark Ginglymostoma cirratum.

We determined the complete mitochondrial genome sequence of nurse shark Ginglymostoma cirratum. The circular DNA of 16692 bp comprises 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, a non-coding control region and a non-coding origin of light strand replication with typical gene order of vertebrates. The nurse shark formed a well-supported clade that included whale shark Rhincodon typus and zebra shark Stegostoma fasciatum within the Orectolobiformes in a phylogenetic tree constructed with other published mitochondrial genomes of sharks.

Primordial linkage of ß2-microglobulin to the MHC.

ß2-Microglobulin (ß2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, ß2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing ß2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and ß2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that ß2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.

Genetic heterogeneity in a cyclical forest pest, the southern pine beetle, Dendroctonus frontalis, is differentiated into east and west groups in the southeastern United States.

The southern pine beetle, Dendroctonus frontalis Zimmerman (Coleoptera: Curculionidae) is an economically important pest species throughout the southeastern United States, Arizona, Mexico, and Central America. Previous research identified population structure among widely distant locations, yet failed to detect population structure among national forests in the state of Mississippi. This study uses microsatellite variation throughout the southeastern United States to compare the southern pine beetle's pattern of population structure to phylogeographic patterns in the region, and to provide information about dispersal. Bayesian clustering identified east and west genetic groups spanning multiple states. The east group had lower heterozygosity, possibly indicating greater habitat fragmentation or a more recent colonization. Significant genetic differentiation (θ(ST) = 0.01, p < 0.0001) followed an isolation-by-distance pattern (r = 0.39, p < 0.001) among samples, and a hierarchical AMOVA indicated slightly more differentiation occurred between multi-state groups. The observed population structure matches a previously identified phylogeographic pattern, division of groups along the Appalachian Mountain/Apalachicola River axis. Our results indicate that the species likely occurs as a large, stable metapopulation with considerable gene flow among subpopulations. Also, the relatively low magnitude of genetic differentiation among samples suggests that southern pine beetles may respond similarly to management across their range.

World phylogeography and male-mediated gene flow in the sandbar shark, Carcharhinus plumbeus.

The sandbar shark, Carcharhinus plumbeus, is a large, cosmopolitan, coastal species. Females are thought to show philopatry to nursery grounds while males potentially migrate long distances, creating an opportunity for male-mediated gene flow that may lead to discordance in patterns revealed by mitochondrial DNA (mtDNA) and nuclear markers. While this dynamic has been investigated in elasmobranchs over small spatial scales, it has not been examined at a global level. We examined patterns of historical phylogeography and contemporary gene flow by genotyping 329 individuals from nine locations throughout the species' range at eight nuclear microsatellite markers and sequencing the complete mtDNA control region. Pairwise comparisons often resulted in fixation indices and divergence estimates of greater magnitude using mtDNA sequence data than microsatellite data. In addition, multiple methods of estimation suggested fewer populations based on microsatellite loci than on mtDNA sequence data. Coalescent analyses suggest divergence and restricted migration among Hawaii, Taiwan, eastern and western Australia using mtDNA sequence data and no divergence and high migration rates, between Taiwan and both Australian sites using microsatellite data. Evidence of secondary contact was detected between several localities and appears to be discreet in time rather than continuous. Collectively, these data suggest complex spatial/temporal relationships between shark populations that may feature pulses of female dispersal and more continuous male-mediated gene flow.

Fine scale gene flow and individual movements among subpopulations of Centrolene prosoblepon (Anura: Centrolenidae).

Dispersal capabilities determine and maintain local gene flow, and this has implications for population persistence and/or recolonization following environmental perturbations (natural or anthropogenic), disease outbreaks, or other demographic collapses. To predict recolonization and understand dispersal capacity in a stream-breeding frog, we examined individual movement patterns and gene flow among four subpopulations of the Neotropical glassfrog, Centrolene prosoblepon, at a mid-elevation cloud forest site at El Copé, Panama. We measured male movement directly during a two year mark-recapture study, and indirectly with gene flow estimates from mitochondrial DNA sequences (mtDNA). Individuals of this species showed strong site fidelity: over two years, male frogs in all four headwater streams moved very little (mean = 2.33 m; mode = 0 m). Nine individuals changed streams within one or two years, moving 675-1,108 m. For those males moving more than 10 m, movement was biased upstream (p < 0.001). Using mtDNA ND1 gene sequences, we quantified gene flow within and among headwater streams at two spatial scales: among headwater streams within two adjacent watersheds (2.5 km2) and among streams within a longitudinal gradient covering 5.0 km2. We found high gene flow among headwater streams (phi(ST) = 0.007, p = 0.325) but gene flow was more limited across greater distances (phi(CT) = 0.322, p = 0.065), even within the same drainage network. Lowland populations of C. prosoblepon potentially act as an important source of colonists for upland populations in this watershed.

Fine-scale genetic population structure of southern pine beetle (Coleoptera: Curculionidae) in Mississippi forests.

The southern pine beetle, Dendroctonus frontalis Zimmerman, is the most destructive insect pest of pine forests in the southeastern United States, Mexico, and Central America. Southern pine beetle aggressively attacks pine trees, and when in epidemic stages, they are capable of killing even the most healthy pine trees in a short period of time. Despite the amount of destruction caused by the southern pine beetle and the amount of monetary loss faced by the timber industry and recreation, the population genetics of this species has been limited to comparisons among distant geographic locations. This study investigates the fine-scale genetic population structure of the southern pine beetle in Mississippi. Very little genetic differentiation was observed among samples. Bayesian assignment testing failed to detect multiple groups within all samples; estimates of genetic differentiation and genetic distance were very low in magnitude; and a Mantel test did not reveal a significant relationship between genetic distance and geographic distance. These results suggest that management of the southern pine beetle needs to consider the potential movements of individuals within and among national forests and should be focused on a large scale, at least as big as continuously forested areas and possibly even multiple forests. These results further suggest that removal of beetle-infested trees is important.

Fine scale gene flow and individual movements among subpopulations of Centrolene prosoblepon (Anura: Centrolenidae)

Dispersal capabilities determine and maintain local gene flow, and this has implications for population persistence and/or recolonization following environmental perturbations (natural or anthropogenic), disease outbreaks, or other demographic collapses. To predict recolonization and understand dispersal capacity in a stream-breeding frog, we examined individual movement patterns and gene flow among four subpopulations of the Neotropical glassfrog, Centrolene prosoblepon, at a mid-elevation cloud forest site at El Copé, Panama. We measured male movement directly during a two year mark-recapture study, and indirectly with gene flow estimates from mitochondrial DNA sequences (mtDNA). Individuals of this species showed strong site fidelity: over two years, male frogs in all four headwater streams moved very little (mean = 2.33 m; mode = 0 m). Nine individuals changed streams within one or two years, moving 675-1,108 m. For those males moving more than 10 m, movement was biased upstream (p < 0.001). Using mtDNA ND1 gene sequences, we quantified gene flow within and among headwater streams at two spatial scales: among headwater streams within two adjacent watersheds (2.5 km2) and among streams within a longitudinal gradient covering 5.0 km2. We found high gene flow among headwater streams (phi(ST) = 0.007, p = 0.325) but gene flow was more limited across greater distances (phi(CT) = 0.322, p = 0.065), even within the same drainage network. Lowland populations of C. prosoblepon potentially act as an important source of colonists for upland populations in this watershed.

La capacidad de dispersión determina y mantiene el flujo genético local, y esto tiene implicaciones para la persistencia poblacional y/o la recolonización que sigue a perturbaciones ambientales. Examinamos patrones individuales de movimiento y flujo genético entre subpoblaciones de Centrolene prosoblepon (Anura: Centrolenidae) en un sitio de elevación media en El Copé, Panamá. Medimos directamente el movimiento de los machos durante un estudio de marcado-recaptura, e indirectamente con estimaciones de flujo genético a partir de secuencias de ADN mitocondrial (mtDNA). Los individuos mostraron fuerte fidelidad a su lugar: por más de dos años, las ranas macho de los cuatro arroyos al inicio del río se movieron muy poco (promedio = 2.33 m; moda = 0 m). Nueve individuos cambiaron de corriente de agua en uno o dos años, moviéndose 675-1 108 m. Usando la secuencia genética ND1 del ADN mitocondrial, medimos el flujo genético en dos escalas espaciales: entre arroyos que originan el río (2.5 km2) y entre arroyos con un gradiente longitudinal en 5.0 km2. Encontramos un flujo genético alto entre los arroyos al inicio del río (f = 0.007, p = 0.325 y otro más limitado en distancias mayores (f = 0.322, p = 0.065).

Development of DNA microsatellite markers in the multiband butterflyfish (Chaetodon multicinctus).

Twelve polymorphic microsatellite loci were developed in the multiband (pebbled) butterflyfish Chaetodon multicinctus. The loci were scored in 45 individuals from Hawaii. There were five to 21 alleles per locus with observed heterozygosity ranging from 0.419 to 0.883. Four of the primer sets also reliably amplified polymorphic loci in Chaetodon quadrimaculatus. We expect these markers to be useful for studies of genetic population structure and kinship, for example to determine whether new recruits settling onto reefs are related.