Kimberly R. Andrews

Harnessing the power of RADseq for ecological and evolutionary genomics

High-throughput techniques based on restriction site-associated DNA sequencing (RADseq) are enabling the low-cost discovery and genotyping of thousands of genetic markers for any species, including non-model organisms, which is revolutionizing ecological, evolutionary and conservation genetics. Technical differences among these methods lead to important considerations for all steps of genomics studies, from the specific scientific questions that can be addressed, and the costs of library preparation and sequencing, to the types of bias and error inherent in the resulting data. In this Review, we provide a comprehensive discussion of RADseq methods to aid researchers in choosing among the many different approaches and avoiding erroneous scientific conclusions from RADseq data, a problem that has plagued other genetic marker types in the past.

Defining Boundaries for Ecosystem-Based Management: A Multispecies Case Study of Marine Connectivity across the Hawaiian Archipelago.

Determining the geographic scale at which to apply ecosystem-based management (EBM) has proven to be an obstacle for many marine conservation programs. Generalizations based on geographic proximity, taxonomy, or life history characteristics provide little predictive power in determining overall patterns of connectivity, and therefore offer little in terms of delineating boundaries for marine spatial management areas. Here, we provide a case study of 27 taxonomically and ecologically diverse species (including reef fishes, marine mammals, gastropods, echinoderms, cnidarians, crustaceans, and an elasmobranch) that reveal four concordant barriers to dispersal within the Hawaiian Archipelago which are not detected in single-species exemplar studies. We contend that this multispecies approach to determine concordant patterns of connectivity is an objective and logical way in which to define the minimum number of management units and that EBM in the Hawaiian Archipelago requires at least five spatially managed regions.

ezRAD: a simplified method for genomic genotyping in non-model organisms

Here, we introduce ezRAD, a novel strategy for restriction site–associated DNA (RAD) that requires little technical expertise or investment in laboratory equipment, and demonstrate its utility for ten non-model organisms across a wide taxonomic range. ezRAD differs from other RAD methods primarily through its use of standard Illumina TruSeq library preparation kits, which makes it possible for any laboratory to send out to a commercial genomic core facility for library preparation and next-generation sequencing with virtually no additional investment beyond the cost of the service itself. This simplification opens RADseq to any lab with the ability to extract DNA and perform a restriction digest. ezRAD also differs from others in its flexibility to use any restriction enzyme (or combination of enzymes) that cuts frequently enough to generate fragments of the desired size range, without requiring the purchase of separate adapters for each enzyme or a sonication step, which can further decrease the cost involved in choosing optimal enzymes for particular species and research questions. We apply this method across a wide taxonomic diversity of non-model organisms to demonstrate the utility and flexibility of our approach. The simplicity of ezRAD makes it particularly useful for the discovery of single nucleotide polymorphisms and targeted amplicon sequencing in natural populations of non-model organisms that have been historically understudied because of lack of genomic information.

Rolling stones and stable homes: social structure, habitat diversity and population genetics of the Hawaiian spinner dolphin (Stenella longirostris)

Spinner dolphins (Stenella longirostris) exhibit different social behaviours at two regions in the Hawaiian Archipelago: off the high volcanic islands in the SE archipelago they form dynamic groups with ever‐changing membership, but in the low carbonate atolls in the NW archipelago they form long‐term stable groups. To determine whether these environmental and social differences influence population genetic structure, we surveyed spinner dolphins throughout the Hawaiian Archipelago with mtDNA control region sequences and 10 microsatellite loci (n = 505). F‐statistics, Bayesian cluster analyses, and assignment tests revealed population genetic separations between most islands, with less genetic structuring among the NW atolls than among the SE high islands. The populations with the most stable social structure (Midway and Kure Atolls) have the highest gene flow between populations (mtDNA ΦST < 0.001, P = 0.357; microsatellite FST = −0.001; P = 0.597), and a population with dynamic groups and fluid social structure (the Kona Coast of the island of Hawai’i) has the lowest gene flow (mtDNA 0.042 < ΦST < 0.236, P < 0.05; microsatellite 0.016 < FST < 0.040, P < 0.001). We suggest that gene flow, dispersal, and social structure are influenced by the availability of habitat and resources at each island. Genetic comparisons to a South Pacific location (n = 16) indicate that Hawaiian populations are genetically depauperate and isolated from other Pacific locations (mtDNA 0.216 < FST < 0.643, P < 0.001; microsatellite 0.058 < FST < 0.090, P < 0.001); this isolation may also influence social and genetic structure within Hawai’i. Our results illustrate that genetic and social structure are flexible traits that can vary between even closely‐related populations.

Recent novel approaches for population genomics data analysis

Next‐generation sequencing (NGS) technology is revolutionizing the fields of population genetics, molecular ecology and conservation biology. But it can be challenging for researchers to learn the new and rapidly evolving techniques required to use NGS data. A recent workshop entitled ‘Population Genomic Data Analysis’ was held to provide training in conceptual and practical aspects of data production and analysis for population genomics, with an emphasis on NGS data analysis. This workshop brought together 16 instructors who were experts in the field of population genomics and 31 student participants. Instructors provided helpful and often entertaining advice regarding how to choose and use a NGS method for a given research question, and regarding critical aspects of NGS data production and analysis such as library preparation, filtering to remove sequencing errors and outlier loci, and genotype calling. In addition, instructors provided general advice about how to approach population genomics data analysis and how to build a career in science. The overarching messages of the workshop were that NGS data analysis should be approached with a keen understanding of the theoretical models underlying the analyses, and with analyses tailored to each research question and project. When analysed carefully, NGS data provide extremely powerful tools for answering crucial questions in disciplines ranging from evolution and ecology to conservation and agriculture, including questions that could not be answered prior to the development of NGS technology.

Comparative phylogeography of the ocean planet.

Understanding how geography, oceanography, and climate have ultimately shaped marine biodiversity requires aligning the distributions of genetic diversity across multiple taxa. Here, we examine phylogeographic partitions in the sea against a backdrop of biogeographic provinces defined by taxonomy, endemism, and species composition. The taxonomic identities used to define biogeographic provinces are routinely accompanied by diagnostic genetic differences between sister species, indicating interspecific concordance between biogeography and phylogeography. In cases where individual species are distributed across two or more biogeographic provinces, shifts in genotype frequencies often align with biogeographic boundaries, providing intraspecific concordance between biogeography and phylogeography. Here, we provide examples of comparative phylogeography from (i) tropical seas that host the highest marine biodiversity, (ii) temperate seas with high productivity but volatile coastlines, (iii) migratory marine fauna, and (iv) plankton that are the most abundant eukaryotes on earth. Tropical and temperate zones both show impacts of glacial cycles, the former primarily through changing sea levels, and the latter through coastal habitat disruption. The general concordance between biogeography and phylogeography indicates that the population-level genetic divergences observed between provinces are a starting point for macroevolutionary divergences between species. However, isolation between provinces does not account for all marine biodiversity; the remainder arises through alternative pathways, such as ecological speciation and parapatric (semiisolated) divergences within provinces and biodiversity hotspots.

Unbroken: RADseq remains a powerful tool for understanding the genetics of adaptation in natural populations

Recently, Lowry et al. addressed the ability of RADseq approaches to detect loci under selection in genome scans. While the authors raise important considerations, such as accounting for the extent of linkage disequilibrium in a study system, we strongly disagree with their overall view of the ability of RADseq to inform our understanding of the genetic basis of adaptation. The family of RADseq protocols has radically improved the field of population genomics, expanding by several orders of magnitude the number of markers available while substantially reducing the cost per marker. Researchers whose goal is to identify regions of the genome under selection must consider the LD of the experimental system; however, there is no magical LD cutoff below which researchers should refuse to use RADseq. Lowry et al. further made two major arguments: a theoretical argument that modeled the likelihood of detecting selective sweeps with RAD markers, and gross summaries based on an anecdotal collection of RAD studies. Unfortunately, their simulations were off by two orders of magnitude in the worst case, while their anecdotes merely showed that it is possible to get widely divergent densities of RAD tags for any particular experiment, either by design or due to experimental efficacy. We strongly argue that RADseq remains a powerful and efficient approach that provides sufficient marker density for studying selection in many natural populations. Given limited resources, we argue that researchers should consider a wide range of trade‐offs among genomic techniques, in light of their study question and the power of different techniques to answer it.

Trade‐offs and utility of alternative RADseq methods: Reply to Puritz et al.

Puritz et al. provide a review of several RADseq methodological approaches in response to our ‘Population Genomic Data Analysis’ workshop (Sept 2013) review (Andrews & Luikart 2014). We agree with Puritz et al. on the importance for researchers to thoroughly understand RADseq library preparation and data analysis when choosing an approach for answering their research questions. Some of us are currently using multiple RADseq protocols, and we agree that the different methods may offer advantages in different cases. Our workshop review did not intend to provide a thorough review of RADseq because the workshop covered a broad range of topics within the field of population genomics. Similarly, neither the response of Puritz et al. nor our comments here provide sufficient space to thoroughly review RADseq. Nonetheless, here we address some key points that we find unclear or potentially misleading in their evaluation of techniques.

Microsatellites for next-generation ecologists: a post-sequencing bioinformatics pipeline.

Microsatellites are the markers of choice for a variety of population genetic studies. The recent advent of next-generation pyrosequencing has drastically accelerated microsatellite locus discovery by providing a greater amount of DNA sequencing reads at lower costs compared to other techniques. However, laboratory testing of PCR primers targeting potential microsatellite markers remains time consuming and costly. Here we show how to reduce this workload by screening microsatellite loci via bioinformatic analyses prior to primer design. Our method emphasizes the importance of sequence quality, and we avoid loci associated with repetitive elements by screening with repetitive sequence databases available for a growing number of taxa. Testing with the Yellowstripe Goatfish Mulloidichthys flavolineatus and the marine planktonic copepod Pleuromamma xiphias we show higher success rate of primers selected by our pipeline in comparison to previous in silico microsatellite detection methodologies. Following the same pipeline, we discover and select microsatellite loci in nine additional species including fishes, sea stars, copepods and octopuses.

Genetic Analyses and Simulations of Larval Dispersal Reveal Distinct Populations and Directional Connectivity across the Range of the Hawaiian Grouper (Epinephelus quernus)

Integration of ecological and genetic data to study patterns of biological connectivity can aid in ecosystem-based management. Here we investigated connectivity of the Hawaiian grouper Epinephelus quernus, a species of management concern within the Main Hawaiian Islands (MHI), by comparing genetic analyses with simulated larval dispersal patterns across the species range in the Hawaiian Archipelago and Johnston Atoll. Larval simulations revealed higher dispersal from the MHI to the Northwestern Hawaiian Islands (NWHI) than in the opposite direction and evidence for a dispersal corridor between Johnston and the middle of the Hawaiian Archipelago. Genetic analyses using mitochondrial DNA (mtDNA) control region sequences and microsatellites revealed relatively high connectivity across the Hawaiian Archipelago, with the exception of genetically distinct populations and higher mtDNA diversity in the mid-Archipelago. These analyses support the preservation of the mid-archipelago as a source of genetic diversity and a region of connectivity with locations outside the Hawaiian Archipelago. Additionally, our evidence for directional dispersal away from the MHI lends caution to any management decisions that would rely on the NWHI replenishing depleted MHI stocks.