Molecular diversity of the copepod, Nannocalunus minor: Genetic evidence of species and population structure in the North Atlantic Ocean

The abundant calanoid copepod, Nannocalanus minor, is widespread from the Florida Straits (FS), throughout the Gulf Stream and the Sargasso Sea, to the eastern North Atlantic Ocean. Does the species represent a single, randomly-mating population across this extensive region, or does it comprise a number of genetically distinct populations or taxonomically distinct forms? What are patterns and pathways of dispersal of the copepod across the North Atlantic? These questions were addressed using population genetic analysis of DNA sequence variation of a 440 base-pair region of the mitochondrial 16S rRNA gene. This analysis separated N. minor into two genetically distinct types (distinguished by 10% sequence difference) that may represent the previously described N. m. forma major and N.m. forma minor. The two genetic types differed in size range and in geographic distribution: Type I individuals were larger and were most abundant in the western regions of the Gulf Stream; Type II individuals were smaller and became more abundant toward the eastern regions of the Gulf Stream. Significant differences in the size-frequency distributions of N. minor from different regions of the North Atlantic may result from mixtures of the two genetic types and environmental differences in food availability. Within N. minor Type I, mtDNA sequence variation defined 68 haplotypes among 155 individuals. The haplotype frequency distribution was skewed: there were 40 individuals of one haplotype, 31 individuals of a second, and 60 unique individuals. Haplotype diversity, h, was very similar across the sampled range: h = 0.886 in samples from the FS and 0.874 for samples from the Gulf Stream Meander Region (GSMR). Nucleotide diversity, pi, was significantly greater in the FS (pi = 0.00490) than in the GSMR (0.00414), largely due to a number of genetically divergent individuals. Haplotype abundances did not differ significantly either within the regions (among FS samples, P = 0.756; among GSMR samples, P = 0.336) or between the regions (P = 0.636). Molecular genetic analysis can reveal cryptic species among marine taxa, and is particularly useful for taxa characterized by morphological similarity

Investigating Coral Bleaching in a Changing Climate: Our State of Understanding and Opportunities to Push the Field Forward

[First Paragraph] Coral reefs throughout the world are facing the consequences of large-scale changes in Earth’s climate. In particular, ocean warming is leading to frequent coral bleaching, which is threatening the long-term stability of coral reefs. Coral bleaching is a stress response that results in the disassociation of the mutualistic symbioses (i.e., dysbiosis) between corals and their endosymbiotic algae (Symbiodinium spp.). In the past two decades, there have been four substantial bleaching events, which have affected large geographic areas across the globe, including the worst recorded bleaching event on the Great Barrier Reef in 2016 (Berkelmans et al. 2004; Eakin et al. 2010; Stella et al. 2016). These large-scale bleaching events, in combination with many local-scale stressors, have contributed substantially to global declines in coral populations. In addition, bleaching may lead to compromised coral immunity, possibly resulting in additional mortality by a range of post-bleaching diseases (Maynard et al. 2015, Randall et al. 2014). Given their link to patterns of global-climate change and projections of increased warming in the coming decades, mass coral bleaching events are a key concern. In addition, current climate projections estimate that global bleaching is expected to occur annually by late this century, with more than 90% of reefs facing long-term degradation (Frieler et al. 2012). Furthermore, in locations such as the Caribbean, frequent thermal anomalies and consecutive annual bleaching events are expected to be common in less than 25 years (van Hooidonk et al. 2015). In fact, large-scale bleaching two years in a row was documented for the first time in 2014-2015 in Hawaii and in the Florida Keys. However, not all corals (and other symbiotic cnidarians) are equally susceptible to thermal stress, and some corals have been shown to recover from bleaching more quickly than others. Likewise, not all reefs are equally susceptible, and depending on local conditions, susceptibility can vary from one event to the next. Such variability in resilience could be a cornerstone to reef persistence over the coming century. However, the research needed to test this hypothesis remains to be performed

Transcriptional Response of Two Core Photosystem Genes in Symbiodinium spp. Exposed to Thermal Stress

Mutualistic symbioses between scleractinian corals and endosymbiotic dinoflagellates (Symbiodinium spp.) are the foundation of coral reef ecosystems. For many coral-algal symbioses, prolonged episodes of thermal stress damage the symbiont\u27s photosynthetic capability, resulting in its expulsion from the host. Despite the link between photosynthetic competency and symbiont expulsion, little is known about the effect of thermal stress on the expression of photosystem genes in Symbiodinium. This study used real-time PCR to monitor the transcript abundance of two important photosynthetic reaction center genes, psbA(encoding the D1 protein of photosystem II) and psaA (encoding the P700 protein of photosystem I), in four cultured isolates (representing ITS2-types A13, A20, B1, and F2) and two in hospite Symbiodinium spp. within the coral Pocillopora spp. (ITS2-types C1b-c and D1). Both cultured and in hospite Symbiodinium samples were exposed to elevated temperatures (32°C) over a 7-day period and examined for changes in photochemistry and transcript abundance. Symbiodinium A13 and C1b-c (both thermally sensitive) demonstrated significant declines in both psbA and psaA during the thermal stress treatment, whereas the transcript levels of the other Symbiodinium types remained stable. The downregulation of both core photosystem genes could be the result of several different physiological mechanisms, but may ultimately limit repair rates of photosynthetic proteins, rendering some Symbiodinium spp. especially susceptible to thermal stress

Assessing Symbiodinium diversity in scleractinian corals via next-generation sequencing-based genotyping of the ITS2 rDNA region.

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)-based pipeline for Symbiodinium ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut-off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field-collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU-based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field-based studies.We would like to thank the KAUST BioScience Core Lab and S. Neelamegam for 454 library generation and sequencing. We would also like to thank Y. Sawall and A. Al-Sofyani for provision and collection of coral samples, and three anonymous reviewers for helpful comments. This project was funded by a KAUST Academic Excellence Alliance (AEA) Award to CRV and CJH, baseline research funds to CRV and a National Science Foundation grant to TCL (OCE-09287664).This is the final published version. It first appeared at http://onlinelibrary.wiley.com/doi/10.1111/mec.12869/abstract

Improved Resolution of Reef-Coral Endosymbiont (Symbiodinium) Species Diversity, Ecology, and Evolution through psbA Non-Coding Region Genotyping

Ribosomal DNA sequence data abounds from numerous studies on the dinoflagellate endosymbionts of corals, and yet the multi-copy nature and intragenomic variability of rRNA genes and spacers confound interpretations of symbiont diversity and ecology. Making consistent sense of extensive sequence variation in a meaningful ecological and evolutionary context would benefit from the application of additional genetic markers. Sequences of the non-coding region of the plastid psbA minicircle (psbAncr) were used to independently examine symbiont genotypic and species diversity found within and between colonies of Hawaiian reef corals in the genus Montipora. A single psbAncr haplotype was recovered in most samples through direct sequencing (∼80–90%) and members of the same internal transcribed spacer region 2 (ITS2) type were phylogenetically differentiated from other ITS2 types by substantial psbAncr sequence divergence. The repeated sequencing of bacterially-cloned fragments of psbAncr from samples and clonal cultures often recovered a single numerically common haplotype accompanied by rare, highly-similar, sequence variants. When sequence artifacts of cloning and intragenomic variation are factored out, these data indicate that most colonies harbored one dominant Symbiodinium genotype. The cloning and sequencing of ITS2 DNA amplified from these same samples recovered numerically abundant variants (that are diagnostic of distinct Symbiodinium lineages), but also generated a large amount of sequences comprising PCR/cloning artifacts combined with ancestral and/or rare variants that, if incorporated into phylogenetic reconstructions, confound how small sequence differences are interpreted. Finally, psbAncr sequence data from a broad sampling of Symbiodinium diversity obtained from various corals throughout the Indo-Pacific were concordant with ITS lineage membership (defined by denaturing gradient gel electrophoresis screening), yet exhibited substantially greater sequence divergence and revealed strong phylogeographic structure corresponding to major biogeographic provinces. The detailed genetic resolution provided by psbAncr data brings further clarity to the ecology, evolution, and systematics of symbiotic dinoflagellates

Host–symbiont combinations dictate the photo-physiological response of reef-building corals to thermal stress

High sea surface temperatures often lead to coral bleaching wherein reef-building corals lose significant numbers of their endosymbiotic dinoflagellates (Symbiodiniaceae). These increasingly frequent bleaching events often result in large scale coral mortality, thereby devasting reef systems throughout the world. The reef habitats surrounding Palau are ideal for investigating coral responses to climate perturbation, where many inshore bays are subject to higher water temperature as compared with offshore barrier reefs. We examined fourteen physiological traits in response to high temperature across various symbiotic dinoflagellates in four common Pacific coral species, Acropora muricata, Coelastrea aspera, Cyphastrea chalcidicum and Pachyseris rugosa found in both offshore and inshore habitats. Inshore corals were dominated by a single homogenous population of the stress tolerant symbiont Durusdinium trenchii, yet symbiont thermal response and physiology differed significantly across coral species. In contrast, offshore corals harbored specific species of Cladocopium spp. (ITS2 rDNA type-C) yet all experienced similar patterns of photoinactivation and symbiont loss when heated. Additionally, cell volume and light absorption properties increased in heated Cladocopium spp., leading to a greater loss in photo-regulation. While inshore coral temperature response was consistently muted relative to their offshore counterparts, high physiological variability in D. trenchii across inshore corals suggests that bleaching resilience among even the most stress tolerant symbionts is still heavily influenced by their host environment

A Connection between Colony Biomass and Death in Caribbean Reef-Building Corals

Increased sea-surface temperatures linked to warming climate threaten coral reef ecosystems globally. To better understand how corals and their endosymbiotic dinoflagellates (Symbiodinium spp.) respond to environmental change, tissue biomass and Symbiodinium density of seven coral species were measured on various reefs approximately every four months for up to thirteen years in the Upper Florida Keys, United States (1994–2007), eleven years in the Exuma Cays, Bahamas (1995–2006), and four years in Puerto Morelos, Mexico (2003–2007). For six out of seven coral species, tissue biomass correlated with Symbiodinium density. Within a particular coral species, tissue biomasses and Symbiodinium densities varied regionally according to the following trends: Mexico≥Florida Keys≥Bahamas. Average tissue biomasses and symbiont cell densities were generally higher in shallow habitats (1–4 m) compared to deeper-dwelling conspecifics (12–15 m). Most colonies that were sampled displayed seasonal fluctuations in biomass and endosymbiont density related to annual temperature variations. During the bleaching episodes of 1998 and 2005, five out of seven species that were exposed to unusually high temperatures exhibited significant decreases in symbiotic algae that, in certain cases, preceded further decreases in tissue biomass. Following bleaching, Montastraea spp. colonies with low relative biomass levels died, whereas colonies with higher biomass levels survived. Bleaching- or disease-associated mortality was also observed in Acropora cervicornis colonies; compared to A. palmata, all A. cervicornis colonies experienced low biomass values. Such patterns suggest that Montastraea spp. and possibly other coral species with relatively low biomass experience increased susceptibility to death following bleaching or other stressors than do conspecifics with higher tissue biomass levels

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships.journal articl

Building consensus around the assessment and interpretation of Symbiodiniaceae diversity

Within microeukaryotes, genetic variation and functional variation sometimes accumulate more quickly than morphological differences. To understand the evolutionary history and ecology of such lineages, it is key to examine diversity at multiple levels of organization. In the dinoflagellate family Symbiodiniaceae, which can form endosymbioses with cnidarians (e.g., corals, octocorals, sea anemones, jellyfish), other marine invertebrates (e.g., sponges, molluscs, flatworms), and protists (e.g., foraminifera), molecular data have been used extensively over the past three decades to describe phenotypes and to make evolutionary and ecological inferences. Despite advances in Symbiodiniaceae genomics, a lack of consensus among researchers with respect to interpreting genetic data has slowed progress in the field and acted as a barrier to reconciling observations. Here, we identify key challenges regarding the assessment and interpretation of Symbiodiniaceae genetic diversity across three levels: species, populations, and communities. We summarize areas of agreement and highlight techniques and approaches that are broadly accepted. In areas where debate remains, we identify unresolved issues and discuss technologies and approaches that can help to fill knowledge gaps related to genetic and phenotypic diversity. We also discuss ways to stimulate progress, in particular by fostering a more inclusive and collaborative research community. We hope that this perspective will inspire and accelerate coral reef science by serving as a resource to those designing experiments, publishing research, and applying for funding related to Symbiodiniaceae and their symbiotic partnerships

Data from: Symbiodinium population genetics: testing for species boundaries and analyzing samples with mixed genotypes

Population genetic markers are increasingly being used to study the diversity, ecology, and evolution of Symbiodinium, a group of eukaryotic microbes that are often mutualistic with reef-building corals. Population genetic markers can resolve individual clones, or strains, from samples of host tissue, however samples may comprise different species that may confound interpretations of gene flow and genetic structure. Here we propose a method for resolving species from population genetic data using tests of genetic recombination. Assigning individuals to genetically recombining populations prior to further analyses avoids critical errors in the interpretation of gene flow and dispersal. To demonstrate the effectiveness of the approach we first apply this method to a simulated dataset. We then use the method to resolve two species of host-generalist Symbiodinium that commonly co-occur in reef-building corals collected from Indo-West Pacific reefs. We demonstrate that the method is robust even when some hosts contain genotypes of two distinct species. Finally, we examine population genetic datasets from two recently published papers in Molecular Ecology. We show that each strongly supports a two species interpretation, which significantly changes the original conclusions presented in these studies. When combined with available phylogenetic and ecological evidence, the use of population genetic data offers a robust method for unambiguously delimiting morphologically cryptic species