Santiago Herrera

Going where traditional markers have not gone before: utility of and promise for RAD sequencing in marine invertebrate phylogeography and population genomics

Characterization of large numbers of single‐nucleotide polymorphisms (SNPs) throughout a genome has the power to refine the understanding of population demographic history and to identify genomic regions under selection in natural populations. To this end, population genomic approaches that harness the power of next‐generation sequencing to understand the ecology and evolution of marine invertebrates represent a boon to test long‐standing questions in marine biology and conservation. We employed restriction‐site‐associated DNA sequencing (RAD‐seq) to identify SNPs in natural populations of the sea anemone Nematostella vectensis, an emerging cnidarian model with a broad geographic range in estuarine habitats in North and South America, and portions of England. We identified hundreds of SNP‐containing tags in thousands of RAD loci from 30 barcoded individuals inhabiting four locations from Nova Scotia to South Carolina. Population genomic analyses using high‐confidence SNPs resulted in a highly‐resolved phylogeography, a result not achieved in previous studies using traditional markers. Plots of locus‐specific FST against heterozygosity suggest that a majority of polymorphic sites are neutral, with a smaller proportion suggesting evidence for balancing selection. Loci inferred to be under balancing selection were mapped to the genome, where 90% were located in gene bodies, indicating potential targets of selection. The results from analyses with and without a reference genome supported similar conclusions, further highlighting RAD‐seq as a method that can be efficiently applied to species lacking existing genomic resources. We discuss the utility of RAD‐seq approaches in burgeoning Nematostella research as well as in other cnidarian species, particularly corals and jellyfishes, to determine phylogeographic relationships of populations and identify regions of the genome undergoing selection.

Environmental Impacts of the Deep-Water Oil and Gas Industry: A Review to Guide Management Strategies

The industrialization of the deep sea is expanding worldwide. Expanding oil and gas exploration activities in the absence of sufficient baseline data in these ecosystems has made environmental management challenging. Here, we review the types of activities that are associated with global offshore oil and gas development in water depths over 200 m, the typical impacts of these activities, some of the more extreme impacts of accidental oil and gas releases, and the current state of management in the major regions of offshore industrial activity including 18 exclusive economic zones. Direct impacts of infrastructure installation, including sediment resuspension and burial by seafloor anchors and pipelines, are typically restricted to a radius of approximately 100 m on from the installation on the seafloor. Discharges of water-based and low-toxicity oil-based drilling muds and produced water can extend over 2 km, while the ecological impacts at the population and community levels on the seafloor are most commonly on the order of 200-300 m from their source. These impacts may persist in the deep sea for many years and likely longer for its more fragile ecosystems, such as cold-water corals. This synthesis of information provides the basis for a series of recommendations for the management of offshore oil and gas development. An effective management strategy, aimed at minimizing risk of significant environmental harm, will typically encompass regulations of the activity itself (e.g. discharge practices, materials used), combined with spatial (e.g. avoidance rules and marine protected areas) and temporal measures (e.g. restricted activities during peak reproductive periods). Spatial management measures that encompass representatives of all of the regional deep-sea community types is important in this context. Implementation of these management strategies should consider minimum buffer zones to displace industrial activity beyond the range of typical impacts: at least 2 km from any discharge points and surface infrastructure and 200 m from seafloor infrastructure with no expected discharges. Although managing natural resources is, arguably, more challenging in deep-water environments, inclusion of these proven conservation tools contributes to robust environmental management strategies for oil and gas extraction in the deep sea.

Molecular systematics of the bubblegum coral genera (Paragorgiidae, Octocorallia) and description of a new deep-sea species

Bubblegum octocorals (Paragorgia and Sibogagorgia) play an important ecological role in many deep-sea ecosystems. However, these organisms are currently threatened by destructive fishing methods such as bottom trawling. Taxonomic knowledge of conservation targets is necessary for the creation and implementation of efficient conservation strategies. However, for most deep-sea coral groups this knowledge remains incomplete. For instance, despite its similarities with Paragorgia, Sibogagorgia is particular in lacking polyp sclerites, which are present in groups like Paragorgia and the Coralliidae. Although two kinds of sclerites are very similar between Paragorgia and Sibogagorgia, other characters challenge the monophyly of these genera. Here we help to clarify the taxonomy and evolutionary relationships of the bubblegum octocorals and related taxa by examining molecular data. We employed nucleotide sequences of mitochondrial (ND6, ND6-ND3 intergenic spacer, ND3, ND2, COI, msh1 and 16S) and nuclear (28S and ITS2) genomic regions from several taxa to infer molecular phylogenetics and to examine the correspondence of morphological features with the underlying genetic information. Our data strongly supported the monophyly of the genus Paragorgia, the family Coralliidae (precious corals), and a group of undescribed specimens resembling Sibogagorgia. Further morphological observations were congruent regarding the uniqueness of the undescribed specimens, here defined as a new species, Sibogagorgia cauliflora sp. nov., which occurs in both sides of the North American landmass at depths below 1700 m. This new species resembles S. dennisgordoni with branching in one plane but has fairly different radiate sclerites and significantly divergent DNA sequences. The existence of several diagnostic characters of Sibogagorgia in S. cauliflora indicates that they indeed belong to this genus. It is however remarkable that a small number of medullar canals are also found in this species; medullar canals have been considered as the main diagnostic character of Paragorgia. Thus, the evidence generated here indicates that the presence or absence of these canals per se is not a conclusively diagnostic character for either genus. The lack of internal-node resolution in the inferred phylogenetic hypotheses of these genera does not allow us to propose a clear scenario regarding the evolution of these traits.

RAD sequencing enables unprecedented phylogenetic resolution and objective species delimitation in recalcitrant divergent taxa

Species delimitations is problematic in many cases due to the difficulty of evaluating predictions from species hypotheses. In many cases delimitations rely on subjective interpretations of morphological and/or DNA data. Species with inadequate genetic resources needed to answer questions regarding evolutionary relatedness and genetic uniqueness are particularly problematic. In this study, we demonstrate the utility of restriction site associated DNA sequencing (RAD-seq) to objectively resolve unambiguous phylogenetic relationships in a recalcitrant group of deep-sea corals with divergences >80 million years. We infer robust species boundaries in the genus Paragorgia by testing alternative delimitation hypotheses using a Bayes Factors delimitation method. We present substantial evidence rejecting the current morphological species delimitation model for the genus and infer the presence of cryptic species associated with environmental variables. We argue that the suitability limits of RAD-seq for phylogenetic inferences cannot be assessed in terms of absolute time, but are contingent on taxon-specific factors. We show that classical taxonomy can greatly benefit from integrative approaches that provide objective tests to species delimitation hypotheses. Our results lead the way for addressing further questions in marine biogeography, community ecology, population dynamics, conservation, and evolution.

Evolutionary and biogeographical patterns of barnacles from deep‐sea hydrothermal vents

The characterization of evolutionary and biogeographical patterns is of fundamental importance to identify factors driving biodiversity. Due to their widespread but discontinuous distribution, deep‐sea hydrothermal vent barnacles represent an excellent model for testing biogeographical hypotheses regarding the origin, dispersal and diversity of modern vent fauna. Here, we characterize the global genetic diversity of vent barnacles to infer their time of radiation, place of origin, mode of dispersal and diversification. Our approach was to target a suite of multiple loci in samples representing seven of the eight described genera. We also performed restriction‐site associated DNA sequencing on individuals from each species. Phylogenetic inferences and topology hypothesis tests indicate that vent barnacles have colonized deep‐sea hydrothermal vents at least twice in history. Consistent with preliminary estimates, we find a likely radiation of barnacles in vent ecosystems during the Cenozoic. Our analyses suggest that the western Pacific was the place of origin of the major vent barnacle lineage, followed by circumglobal colonization eastwards through the Southern Hemisphere during the Neogene. The inferred time of radiation rejects the classic hypotheses of antiquity of vent taxa. The timing and the mode of origin, radiation and dispersal are consistent with recent inferences made for other deep‐sea taxa, including nonvent species, and are correlated with the occurrence of major geological events and mass extinctions. Thus, we suggest that the geological processes and dispersal mechanisms discussed here can explain the current distribution patterns of many other marine taxa and have played an important role shaping deep‐sea faunal diversity. These results also constitute the critical baseline data with which to assess potential effects of anthropogenic disturbances on deep‐sea ecosystems.

Predicting RAD-seq Marker Numbers across the Eukaryotic Tree of Life

High-throughput sequencing of reduced representation libraries obtained through digestion with restriction enzymes—generically known as restriction site associated DNA sequencing (RAD-seq)—is a common strategy to generate genome-wide genotypic and sequence data from eukaryotes. A critical design element of any RAD-seq study is knowledge of the approximate number of genetic markers that can be obtained for a taxon using different restriction enzymes, as this number determines the scope of a project, and ultimately defines its success. This number can only be directly determined if a reference genome sequence is available, or it can be estimated if the genome size and restriction recognition sequence probabilities are known. However, both scenarios are uncommon for nonmodel species. Here, we performed systematic in silico surveys of recognition sequences, for diverse and commonly used type II restriction enzymes across the eukaryotic tree of life. Our observations reveal that recognition sequence frequencies for a given restriction enzyme are strikingly variable among broad eukaryotic taxonomic groups, being largely determined by phylogenetic relatedness. We demonstrate that genome sizes can be predicted from cleavage frequency data obtained with restriction enzymes targeting “neutral” elements. Models based on genomic compositions are also effective tools to accurately calculate probabilities of recognition sequences across taxa, and can be applied to species for which reduced representation data are available (including transcriptomes and neutral RAD-seq data sets). The analytical pipeline developed in this study, PredRAD (https://github.com/phrh/PredRAD), and the resulting databases constitute valuable resources that will help guide the design of any study using RAD-seq or related methods.

BackCLIP: a tool to identify common background presence in PAR-CLIP datasets.

MOTIVATION PAR-CLIP, a CLIP-seq protocol, derives a transcriptome wide set of binding sites for RNA-binding proteins. Even though the protocol uses stringent washing to remove experimental noise, some of it remains. A recent study measured three sets of non-specific RNA backgrounds which are present in several PAR-CLIP datasets. However, a tool to identify the presence of common background in PAR-CLIP datasets is not yet available. RESULTS We used the measured sets of non-specific RNA backgrounds to build a common background set. Each element from the common background set has a score that reflects its presence in several PAR-CLIP datasets. We present a tool that uses this score to identify the amount of common backgrounds present in a PAR-CLIP dataset, and we provide the user the option to use or remove it. We used the proposed strategy in 30 PAR-CLIP datasets from nine proteins. It is possible to identify the presence of common backgrounds in a dataset and identify differences in datasets for the same protein. This method is the first step in the process of completely removing such backgrounds. AVAILABILITY The tool was implemented in python. The common background set and the supplementary data are available at https://github.com/phrh/BackCLIP. CONTACT phreyes@gmail.com SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Predicting genome sizes and restriction enzyme recognition-sequence probabilities across the eukaryotic tree of life

High-throughput sequencing of reduced representation libraries obtained through digestion with restriction enzymes – generically known as restriction-site associated DNA sequencing (RAD-seq) – is a common strategy to generate genome-wide genotypic and sequence data from eukaryotes. A critical design element of any RAD-seq study is a knowledge of the approximate number of genetic markers that can be obtained for a taxon using different restriction enzymes, as this number determines the scope of a project, and ultimately defines its success. This number can only be directly determined if a reference genome sequence is available, or it can be estimated if the genome size and restriction recognition sequence probabilities are known. However, both scenarios are uncommon for non-model species. Here, we performed systematic in silico surveys of recognition sequences, for diverse and commonly used type II restriction enzymes across the eukaryotic tree of life. Our observations reveal that recognition-sequence frequencies for a given restriction enzyme are strikingly variable among broad eukaryotic taxonomic groups, being largely determined by phylogenetic relatedness. We demonstrate that genome sizes can be predicted from cleavage frequency data obtained with restriction enzymes targeting ‘neutral’ elements. Models based on genomic compositions are also effective tools to accurately calculate probabilities of recognition sequences across taxa, and can be applied to species for which reduced-representation data is available (including transcriptomes and ‘neutral’ RAD-seq datasets). The analytical pipeline developed in this study, PredRAD (https://github.com/phrh/PredRAD), and the resulting databases constitute valuable resources that will help guide the design of any study using RAD-seq or related methods.High-throughput sequencing of reduced representation libraries obtained through digestion with restriction enzymes–generally known as restriction-site associated DNA sequencing (RAD-seq)–is now one most commonly used strategies to generate single nucleotide polymorphism data in eukaryotes. The choice of restriction enzyme is critical for the design of any RAD-seq study as it determines the number of genetic markers that can be obtained for a given species, and ultimately the success of a project. In this study we tested the hypothesis that genome composition, in terms of GC content, mono-, di- and trinucleotide compositions, can be used to predict the number of restriction sites for a given combination of restriction enzyme and genome. We performed systematic in silico genome-wide surveys of restriction sites across the eukaryotic tree of live and compared them with expectations generated from stochastic models based on genome compositions using the newly developed software pipeline PredRAD (https://github.com/phrh/PredRAD). Our analyses reveal that in most cases the trinucleotide genome composition model is the best predictor, and the GC content and mononucleotide models are the worst predictors of the expected number of restriction sites in a eukaryotic genome. However, we argue that the predictability of restriction site frequencies in eukaryotic genomes needs to be treated in a case-specific basis, because the phylogenetic position of the taxon of interest and the specific recognition sequence of the selected restriction enzyme are the most determinant factors. The results from this study, and the software developed, will help guide the design of any study using RAD sequencing and related methods.