Next-Generation Phylogeography: A Targeted Approach for Multilocus Sequencing of Non-Model Organisms

The field of phylogeography has long since realized the need and utility of incorporating nuclear DNA (nDNA) sequences into analyses. However, the use of nDNA sequence data, at the population level, has been hindered by technical laboratory difficulty, sequencing costs, and problematic analytical methods dealing with genotypic sequence data, especially in non-model organisms. Here, we present a method utilizing the 454 GS-FLX Titanium pyrosequencing platform with the capacity to simultaneously sequence two species of sea star (Meridiastra calcar and Parvulastra exigua) at five different nDNA loci across 16 different populations of 20 individuals each per species. We compare results from 3 populations with traditional Sanger sequencing based methods, and demonstrate that this next-generation sequencing platform is more time and cost effective and more sensitive to rare variants than Sanger based sequencing. A crucial advantage is that the high coverage of clonally amplified sequences simplifies haplotype determination, even in highly polymorphic species. This targeted next-generation approach can greatly increase the use of nDNA sequence loci in phylogeographic and population genetic studies by mitigating many of the time, cost, and analytical issues associated with highly polymorphic, diploid sequence markers

Shallow gene pools in the high intertidal: extreme loss of genetic diversity in viviparous sea stars (Parvulastra)

We document an extreme example of reproductive trait evolution that affects population genetic structure in sister species of Parvulastra cushion stars from Australia. Self-fertilization by hermaphroditic adults and brood protection of benthic larvae causes strong inbreeding and range-wide genetic poverty. Most samples were fixed for a single allele at nearly all nuclear loci; heterozygotes were extremely rare (0.18%); mitochondrial DNA sequences were more variable, but few populations shared haplotypes in common. Isolation-with-migration models suggest that these patterns are caused by population bottlenecks (relative to ancestral population size) and low gene flow. Loss of genetic diversity and low potential for dispersal between high-intertidal habitats may have dire consequences for extinction risk and potential for future adaptive evolution in response to climate and other selective agents

Discordant distribution of populations and genetic variation in a sea star with high dispersal potential

Patiria miniata, a broadcast-spawning sea star species with high dispersal potential, has a geographic range in the intertidal zone of the northeast Pacific Ocean from Alaska to California that is characterized by a large range gap in Washington and Oregon. We analyzed spatial genetic variation across the P. miniata range using multilocus sequence data (mtDNA, nuclear introns) and multilocus genotype data (microsatellites). We found a strong phylogeographic break at Queen Charlotte Sound in British Columbia that was not in the location predicted by the geographical distribution of the populations. However, this population genetic discontinuity does correspond to previously described phylogeographic breaks in other species. Northern populations from Alaska and Haida Gwaii were strongly differentiated from all southern populations from Vancouver Island and California. Populations from Vancouver Island and California were undifferentiated with evidence of high gene flow or very recent separation across the range disjunction between them. The surprising and discordant spatial distribution of populations and alleles suggests that historical vicariance (possibly caused by glaciations) and contemporary dispersal barriers (possibly caused by oceanographic conditions) both shape population genetic structure in this species

Shallow gene pools in the high intertidal: extreme loss of genetic diversity in viviparous sea stars ( Parvulastra

We document an extreme example of reproductive trait evolution that affects population genetic structure in sister species of Parvulastra cushion stars from Australia. Self-fertilization by hermaphroditic adults and brood protection of benthic larvae causes strong inbreeding and range-wide genetic poverty. Most samples were fixed for a single allele at nearly all nuclear loci; heterozygotes were extremely rare (0.18%); mitochondrial DNA sequences were more variable, but few populations shared haplotypes in common. Isolation-with-migration models suggest that these patterns are caused by population bottlenecks (relative to ancestral population size) and low gene flow. Loss of genetic diversity and low potential for dispersal between high-intertidal habitats may have dire consequences for extinction risk and potential for future adaptive evolution in response to climate and other selective agents

Life‐history predicts past and present population connectivity in two sympatric sea stars

Life-history traits, especially the mode and duration of larval development, are expected to strongly influence the population connectivity and phylogeography of marine species. Comparative analysis of sympatric, closely related species with differing life histories provides the opportunity to specifically investigate these mechanisms of evolution but have been equivocal in this regard. Here, we sample two sympatric sea stars across the same geographic range in temperate waters of Australia. Using a combination of mitochondrial DNA sequences, nuclear DNA sequences, and microsatellite genotypes, we show that the benthic-developing sea star, Parvulastra exigua, has lower levels of within- and among-population genetic diversity, more inferred genetic clusters, and higher levels of hierarchical and pairwise population structure than Meridiastra calcar, a species with planktonic development. While both species have populations that have diverged since the middle of the second glacial period of the Pleistocene, most P. exigua populations have origins after the last glacial maxima (LGM), whereas most M. calcar populations diverged long before the LGM. Our results indicate that phylogenetic patterns of these two species are consistent with predicted dispersal abilities; the benthic-developing P. exigua shows a pattern of extirpation during the LGM with subsequent recolonization, whereas the planktonic-developing M. calcar shows a pattern of persistence and isolation during the LGM with subsequent post-Pleistocene introgression

Next Generation Extractants for Cesium Separation from High-Level Waste: From Fundamental Concepts to Site Implementation

This project seeks a fundamental understanding and major improvement in cesium separation from high-level waste by cesium-selective calixcrown extractants. Systems of particular interest involve novel solvent-extraction systems containing specific members of the calix[4]arene-crown-6 family, alcohol solvating agents, and alkylamines. Questions being addressed bear upon cesium binding strength, extraction selectivity, cesium stripping, and extractant solubility. Enhanced properties in this regard will specifically benefit applied projects funded by the USDOE Office of Environmental Management to clean up sites such as the Savannah River Site (SRS), Hanford, and the Idaho National Environmental and Engineering Laboratory. The most direct beneficiary will be the SRS Salt Processing Project, which has recently identified the Caustic-Side Solvent Extraction (CSSX) process employing a calixcrown as its preferred technology for cesium removal from SRS high-level tank waste

Next Generation Extractants for Cesium Separation from High-Level Waste: From Fundamental Concepts to Site Implementation

This project seeks a fundamental understanding and major improvement in cesium separation from high-level waste by cesium-selective calixcrown extractants. Systems of particular interest involve novel solvent-extraction systems containing specific members of the calix[4]arene-crown-6 family, alcohol solvating agents, and alkylamines. Questions being addressed pertain to cesium binding strength, extraction selectivity, cesium stripping, and extractant solubility. Enhanced properties in this regard will specifically benefit cleanup projects funded by the USDOE Office of Environmental Management to treat and dispose of high-level radioactive wastes currently stored in underground tanks at the Savannah River Site (SRS), the Hanford site, and the Idaho National Environmental and Engineering Laboratory.1 The most direct beneficiary will be the SRS Salt Processing Project, which has recently identified the Caustic-Side Solvent Extraction (CSSX) process employing a calixcrown as its preferred technology for cesium removal from SRS high-level tank waste.2 This technology owes its development in part to fundamental results obtained in this program