Eric Pante

Limitations of Mitochondrial Gene Barcoding in Octocorallia

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub‐class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral‐specific mitochondrial protein‐coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra‐ and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character‐based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.

marmap: A Package for Importing, Plotting and Analyzing Bathymetric and Topographic Data in R

In this communication we introduce marmap, a package designed for downloading, plotting and manipulating bathymetric and topographic data in R. marmap can query the ETOPO1 bathymetry and topography database hosted by the NOAA, use simple latitude-longitude-depth data in ascii format, and take advantage of the advanced plotting tools available in R to build publication-quality bathymetric maps. Functions to query data (bathymetry, sampling information…) are available interactively by clicking on marmap maps. Bathymetric and topographic data can also be used to calculate projected surface areas within specified depth/altitude intervals, and constrain the calculation of realistic shortest path distances. Such information can be used in molecular ecology, for example, to evaluate genetic isolation by distance in a spatially-explicit framework.

From Integrative Taxonomy to Species Description: One Step Beyond

Integrative taxonomy was formally introduced in 2005 as a comprehensive framework to delimit and describe taxa by integrating information from different types of data and methodologies (Dayrat 2005; Will et al. 2005). Even if debate remains about the hierarchy of the types of characters and criteria to use for species delimitation (Schlick-Steiner et al., 2009; Padial et al., 2010; Yeates et al., 2011), most, if not all taxonomists agree that objectively evaluating several lines of evidence within a formalized framework is the most efficient and theoretically-grounded approach to defining robust species hypotheses (Samadi and Barberousse 2006; de Queiroz 2007).The last ten years have seen a renewal of taxonomy, illustrated by the increasing number of published articles related to species concepts, species delimitation methodology and its application. In the early 90s, many systematists began to suspect that the majority of species would remain undescribed (Costello et al. 2013a; Erwin 1982; Mora et al. 2011 – but see Costello et al. 2013b) and that some of them will probably go extinct before we have a chance to describe them (Barnosky et al., 2011; Leakey and Lewin, 1995; Pimm et al., 2006). The use of molecular data, and in particular molecular barcoding (Hebert et al., 2003), was presented as one answer to this “taxonomic impediment” (as defined in Rodman and Cody, 2003), and welcomed as such by taxonomists. It thus adds to the toolkit of taxonomy, which continues its development as a synergic discipline involving morphological taxonomists, field ecologists, naturalists, and statisticians (Knapp 2008). Integrative taxonomy, used for many decades by taxonomists but only recently formalized concomitantly with the molecular revolution, is organised following a three-step workflow (see also Evenhuis 2007): first, we need to accumulate data on numerous specimens (from various types of data: DNA, morphology, ecology…); second, we need to circumscribe groups of organisms using concepts that ensure that these groups correspond to species (this second step may be coupled with the first, as biological data are continuously accumulated and species hypotheses re-discussed); and third, we need to provide a species description, i.e. a diagnosis and a name for the species recognized as new. Naming new species is a fundamental step when describing biodiversity and is the only way to ensure that scientists are talking about the same entity, and that all the data linked to conspecific specimens but produced by different researchers (or amateurs) can be associated in a comparative analysis (Patterson et al., 2010; Satler et al., 2013; Schlick-Steiner et al., 2007). Not linking biological data (should they be molecular, morphological, or ecological) to a formal species name will result in these data losing tremendous value (Goldstein and DeSalle 2011). Indeed, when authors publish data on entities that are not defined within the framework of a referencing system (e.g. solely identified by an alphanumeric label), they make it very difficult for other authors to build on these data. The best example is the need for taxa to be named to have a chance to be listed in an endangered species list and to benefit from a conservation program: no name, no surviving (Mace 2004). Beyond the need for communication among scientists, names are also key to communicating with non-scientist audiences. While it is now widely recognized that integrating several lines of evidence is the most efficient and theoretically grounded way to delimit new species (e.g. de Queiroz, 2007; Schlick-Steiner et al., 2009; Yeates et al., 2011), the formal naming of new entities may have become decoupled from species delimitation. Indeed, we noted that in several cases new delimited species were not accompanied by formal species description (see also Goldstein and DeSalle 2011). The aim of this article is therefore to test the hypothesis that integrative taxonomy, as defined in 2005 (Dayrat 2005; Will et al. 2005), and in particular the use of molecular data, helped to alleviate the taxonomic impediment by delimiting and describing new species. We reviewed part of the “integrative taxonomy” literature of the last eight years (2006-2013) and tested if authors that delimit new species also name them. We also looked at how the number and type of characters used, across different taxa, varies across articles.

Species are hypotheses: avoid connectivity assessments based on pillars of sand.

Connectivity among populations determines the dynamics and evolution of populations, and its assessment is essential in ecology in general and in conservation biology in particular. The robust basis of any ecological study is the accurate delimitation of evolutionary units, such as populations, metapopulations and species. Yet a disconnect still persists between the work of taxonomists describing species as working hypotheses and the use of species delimitation by molecular ecologists interested in describing patterns of gene flow. This problem is particularly acute in the marine environment where the inventory of biodiversity is relatively delayed, while for the past two decades, molecular studies have shown a high prevalence of cryptic species. In this study, we illustrate, based on marine case studies, how the failure to recognize boundaries of evolutionary‐relevant unit leads to heavily biased estimates of connectivity. We review the conceptual framework within which species delimitation can be formalized as falsifiable hypotheses and show how connectivity studies can feed integrative taxonomic work and vice versa. Finally, we suggest strategies for spatial, temporal and phylogenetic sampling to reduce the probability of inadequately delimiting evolutionary units when engaging in connectivity studies.

Use of RAD sequencing for delimiting species

RAD-tag sequencing is a promising method for conducting genome-wide evolutionary studies. However, to date, only a handful of studies empirically tested its applicability above the species level. In this communication, we use RAD tags to contribute to the delimitation of species within a diverse genus of deep-sea octocorals, Chrysogorgia, for which few classical genetic markers have proved informative. Previous studies have hypothesized that single mitochondrial haplotypes can be used to delimit Chrysogorgia species. On the basis of two lanes of Illumina sequencing, we inferred phylogenetic relationships among 12 putative species that were delimited using mitochondrial data, comparing two RAD analysis pipelines (Stacks and PyRAD). The number of homologous RAD loci decreased dramatically with increasing divergence, as >70% of loci are lost when comparing specimens separated by two mutations on the 700-nt long mitochondrial phylogeny. Species delimitation hypotheses based on the mitochondrial mtMutS gene are largely supported, as six out of nine putative species represented by more than one colony were recovered as discrete, well-supported clades. Significant genetic structure (correlating with geography) was detected within one putative species, suggesting that individuals characterized by the same mtMutS haplotype may belong to distinct species. Conversely, three mtMutS haplotypes formed one well-supported clade within which no population structure was detected, also suggesting that intraspecific variation exists at mtMutS in Chrysogorgia. Despite an impressive decrease in the number of homologous loci across clades, RAD data helped us to fine-tune our interpretations of classical mitochondrial markers used in octocoral species delimitation, and discover previously undetected diversity.

Deep-sea origin and in-situ diversification of chrysogorgiid octocorals.

The diversity, ubiquity and prevalence in deep waters of the octocoral family Chrysogorgiidae Verrill, 1883 make it noteworthy as a model system to study radiation and diversification in the deep sea. Here we provide the first comprehensive phylogenetic analysis of the Chrysogorgiidae, and compare phylogeny and depth distribution. Phylogenetic relationships among 10 of 14 currently-described Chrysogorgiidae genera were inferred based on mitochondrial (mtMutS, cox1) and nuclear (18S) markers. Bathymetric distribution was estimated from multiple sources, including museum records, a literature review, and our own sampling records (985 stations, 2345 specimens). Genetic analyses suggest that the Chrysogorgiidae as currently described is a polyphyletic family. Shallow-water genera, and two of eight deep-water genera, appear more closely related to other octocoral families than to the remainder of the monophyletic, deep-water chrysogorgiid genera. Monophyletic chrysogorgiids are composed of strictly (Iridogorgia Verrill, 1883, Metallogorgia Versluys, 1902, Radicipes Stearns, 1883, Pseudochrysogorgia Pante & France, 2010) and predominantly (Chrysogorgia Duchassaing & Michelotti, 1864) deep-sea genera that diversified in situ. This group is sister to gold corals (Primnoidae Milne Edwards, 1857) and deep-sea bamboo corals (Keratoisidinae Gray, 1870), whose diversity also peaks in the deep sea. Nine species of Chrysogorgia that were described from depths shallower than 200 m, and mtMutS haplotypes sequenced from specimens sampled as shallow as 101 m, suggest a shallow-water emergence of some Chrysogorgia species.

Chrysogorgia from the New England and Corner Seamounts: Atlantic–Pacific connections

Recent exploration of the New England and Corner Seamounts revealed four new species of Chrysogorgia , described here using a combination of molecular and morphological data. These four species are characterized by a sinistral spiral, a character that, with one known exception, has only been reported for Pacific species. In addition, two species have a sclerite composition typical of the Pacific (‘squamosae typicae’). This faunal connection between the Atlantic and the Pacific is confirmed by analysis of the mitochondrial msh1 gene . The exceptional preservation of specimens collected with remotely operated vehicles allows us to discuss the effect of growth on some morphological characters.

Applicability of RAD‐tag genotyping for interfamilial comparisons: empirical data from two cetaceans

Restriction‐site‐associated DNA tag (RAD‐tag) sequencing has become a popular approach to generate thousands of SNPs used to address diverse questions in population genomics. Comparatively, the suitability of RAD‐tag genotyping to address evolutionary questions across divergent species has been the subject of only a few recent studies. Here, we evaluate the applicability of this approach to conduct genome‐wide scans for polymorphisms across two cetacean species belonging to distinct families: the short‐beaked common dolphin (Delphinus delphis; n = 5 individuals) and the harbour porpoise (Phocoena phocoena; n = 1 individual). Additionally, we explore the effects of varying two parameters in the Stacks analysis pipeline on the number of loci and level of divergence obtained. We observed a 34% drop in the total number of loci that were present in all individuals when analysing individuals from the distinct families compared with analyses restricted to intraspecific comparisons (i.e. within D. delphis). Despite relatively stringent quality filters, 3595 polymorphic loci were retrieved from our interfamilial comparison. Cetaceans have undergone rapid diversification, and the estimated divergence time between the two families is relatively recent (14–19 Ma). Thus, our results showed that, for this level of divergence, a large number of orthologous loci can still be genotyped using this approach, which is on par with two recent in silico studies. Our findings constitute one of the first empirical investigations using RAD‐tag sequencing at this level of divergence and highlights the great potential of this approach in comparative studies and to address evolutionary questions.

SNP detection from de novo transcriptome sequencing in the bivalve Macoma balthica: marker development for evolutionary studies.

Hybrid zones are noteworthy systems for the study of environmental adaptation to fast-changing environments, as they constitute reservoirs of polymorphism and are key to the maintenance of biodiversity. They can move in relation to climate fluctuations, as temperature can affect both selection and migration, or remain trapped by environmental and physical barriers. There is therefore a very strong incentive to study the dynamics of hybrid zones subjected to climate variations. The infaunal bivalve Macoma balthica emerges as a noteworthy model species, as divergent lineages hybridize, and its native NE Atlantic range is currently contracting to the North. To investigate the dynamics and functioning of hybrid zones in M. balthica, we developed new molecular markers by sequencing the collective transcriptome of 30 individuals. Ten individuals were pooled for each of the three populations sampled at the margins of two hybrid zones. A single 454 run generated 277 Mb from which 17K SNPs were detected. SNP density averaged 1 polymorphic site every 14 to 19 bases, for mitochondrial and nuclear loci, respectively. An scan detected high genetic divergence among several hundred SNPs, some of them involved in energetic metabolism, cellular respiration and physiological stress. The high population differentiation, recorded for nuclear-encoded ATP synthase and NADH dehydrogenase as well as most mitochondrial loci, suggests cytonuclear genetic incompatibilities. Results from this study will help pave the way to a high-resolution study of hybrid zone dynamics in M. balthica, and the relative importance of endogenous and exogenous barriers to gene flow in this system.

Getting to the Point: Accuracy of Point Count in Monitoring Ecosystem Change

Ecological monitoring programs depend on the robust estimation of descriptive parameters. Percent cover, gleaned from transects sampled with video imagery, is a popular benthic ecology descriptor often estimated using point counting, an image-based method for identifying substrate types beneath random points. We tested the hypothesis that the number of points needed to robustly estimate benthic cover in video imagery transects depends on cover itself, predicting that lower cover will require more points/frame to be accurately estimated. While this point may seem obvious to the statistically inclined, the justification of point density has been largely ignored in the literature. We examined the statistical behavior of point count estimates using computer-simulated 20 m-long transects patterned after data from a Bahamian reef. The minimum number of points necessary to insure accurate percent cover estimation, the Optimal Point Count (OPC), is a function of mean percent cover and spatial heterogeneity of the benthic community. More points are required to characterize reefs with lower cover and more homogeneously distributed coral colonies. These results show that careful consideration must be given to sampling design and data analysis prior to attempting to estimate benthic cover, especially in the context of long-term monitoring of degrading coral reef ecosystems.