Barcoding and mitochondrial phylogenetics of Porites corals.

Coral reefs are the most diverse ecosystem on the planet based on the abundance and diversity of phyla and higher taxa. However, it is still difficult to assess the diversity of lower taxa, especially at the species level. One tool for improving the identification of lower taxa are genetic markers that can distinguish cryptic species and assess species boundaries. Here, we present one such approach for an important and challenging group of reef-building corals. Porites corals are the main reef-builders of many coral reefs in the Indo-Pacific, owing to the massive growth forms of some species. The current number of valid Porites species is controversial, inflated with many synonymies, and often based on gross colony morphology although several morphospecies believed to be widespread and common can only be distinguished based on detailed microstructure analyses by taxonomic experts. Here, we test the suitability of multiple regions of mtDNA as genetic barcodes to identify suitable markers for species differentiation and unambiguous identification. Resulting sequencing data was further used for the first phylogenetic analysis of Guam's Porites species. We tested eight different mitochondrial markers and analyzed four in detail for 135 Porites specimens: mtDNA markers were amplified for 67 Porites specimens from Guam, representing 12 nominal Porites species, and combined with 69 mitochondrial genomes, mostly from Hawaii. The combination of all 4 markers distinguished 10 common and 7 uncommon Central-West Pacific Porites species. Most clades separate species along taxonomic boundaries, which is uncommon for Porites corals and testifies to the suitability of our multi-marker approach, and a combination of the two most promising barcodes distinguished 8/10 common species. These barcodes are thus suitable to distinguish virtually cryptic species in one of the most important and challenging coral genera. They offer a cheap, fast and reliable way to identify Porites species for species-level research, monitoring and conservation.

Three new species of Nautilus Linnaeus, 1758 (Mollusca, Cephalopoda) from the Coral Sea and South Pacific.

Nautiloids are a charismatic group of marine molluscs best known for their rich fossil record, but today they are restricted to a handful of species in the family Nautilidae from around the Coral Triangle. Recent genetic work has shown a disconnect between traditional species, originally defined on shell characters, but now with new findings from genetic structure of various Nautilus populations. Here, three new species of Nautilus from the Coral Sea and South Pacific region are formally named using observations of shell and soft anatomical data augmented by genetic information: N.samoaensissp. nov. (from American Samoa), N.vitiensissp. nov. (from Fiji), and N.vanuatuensissp. nov. (from Vanuatu). The formal naming of these three species is timely considering the new and recently published information on genetic structure, geographic occurrence, and new morphological characters, including color patterns of shell and soft part morphology of hood, and will aid in managing these possibly endangered animals. As recently proposed from genetic analyses, there is a strong geographic component affecting taxonomy, with the new species coming from larger island groups that are separated by at least 200 km of deep water (greater than 800 m) from other Nautilus populations and potential habitats. Nautilid shells implode at depths greater than 800 m and depth therefore acts as a biogeographical barrier separating these species. This isolation, coupled with the unique, endemic species in each locale, are important considerations for the conservation management of the extant Nautilus species and populations.

Unified methods in collecting, preserving, and archiving coral bleaching and restoration specimens to increase sample utility and interdisciplinary collaboration.

Coral reefs are declining worldwide primarily because of bleaching and subsequent mortality resulting from thermal stress. Currently, extensive efforts to engage in more holistic research and restoration endeavors have considerably expanded the techniques applied to examine coral samples. Despite such advances, coral bleaching and restoration studies are often conducted within a specific disciplinary focus, where specimens are collected, preserved, and archived in ways that are not always conducive to further downstream analyses by specialists in other disciplines. This approach may prevent the full utilization of unexpended specimens, leading to siloed research, duplicative efforts, unnecessary loss of additional corals to research endeavors, and overall increased costs. A recent US National Science Foundation-sponsored workshop set out to consolidate our collective knowledge across the disciplines of Omics, Physiology, and Microscopy and Imaging regarding the methods used for coral sample collection, preservation, and archiving. Here, we highlight knowledge gaps and propose some simple steps for collecting, preserving, and archiving coral-bleaching specimens that can increase the impact of individual coral bleaching and restoration studies, as well as foster additional analyses and future discoveries through collaboration. Rapid freezing of samples in liquid nitrogen or placing at -80 °C to -20 °C is optimal for most Omics and Physiology studies with a few exceptions; however, freezing samples removes the potential for many Microscopy and Imaging-based analyses due to the alteration of tissue integrity during freezing. For Microscopy and Imaging, samples are best stored in aldehydes. The use of sterile gloves and receptacles during collection supports the downstream analysis of host-associated bacterial and viral communities which are particularly germane to disease and restoration efforts. Across all disciplines, the use of aseptic techniques during collection, preservation, and archiving maximizes the research potential of coral specimens and allows for the greatest number of possible downstream analyses.

Differential gene expression during substrate probing in larvae of the Caribbean coral Porites astreoides.

The transition from larva to adult is a critical step in the life history strategy of most marine animals. However, the genetic basis of this life history change remains poorly understood in many taxa, including most coral species. Recent evidence suggests that coral planula larvae undergo significant changes at the physiological and molecular levels throughout the development. To investigate this, we characterized differential gene expression (DGE) during the transition from planula to adult polyp in the abundant Caribbean reef-building coral Porites astreoides, that is from nonprobing to actively substrate-probing larva, a stage required for colony initiation. This period is crucial for the coral, because it demonstrates preparedness to locate appropriate substrata for settlement based on vital environmental cues. Through RNA-Seq, we identified 860 differentially expressed holobiont genes between probing and nonprobing larvae (p ≤ .01), the majority of which were upregulated in probing larvae. Surprisingly, differentially expressed genes of endosymbiotic dinoflagellate origin greatly outnumbered coral genes, compared with a nearly 1:1 ratio of coral-to-dinoflagellate gene representation in the holobiont transcriptome. This unanticipated result suggests that dinoflagellate endosymbionts may play a significant role in the transition from nonprobing to probing behaviour in dinoflagellate-rich larvae. Putative holobiont genes were largely involved in protein and nucleotide binding, metabolism and transport. Genes were also linked to environmental sensing and response and integral signalling pathways. Our results thus provide detailed insight into molecular changes prior to larval settlement and highlight the complex physiological and biochemical changes that occur in early transition stages from pelagic to benthic stages in corals, and perhaps more importantly, in their endosymbionts.

Genomic signatures of evolution in Nautilus-An endangered living fossil.

Living fossils are survivors of previously more diverse lineages that originated millions of years ago and persisted with little morphological change. Therefore, living fossils are model organisms to study both long-term and ongoing adaptation and speciation processes. However, many aspects of living fossil evolution and their persistence in the modern world remain unclear. Here, we investigate three major aspects of the evolutionary history of living fossils: cryptic speciation, population genetics and effective population sizes, using members of the genera Nautilus and Allonautilus as classic examples of true living fossils. For this, we analysed genomewide ddRAD-Seq data for all six currently recognized nautiloid species throughout their distribution range. Our analyses identified three major allopatric Nautilus clades: a South Pacific clade, subdivided into three subclades with no signs of admixture between them; a Coral Sea clade, consisting of two genetically distinct populations with significant admixture; and a widespread Indo-Pacific clade, devoid of significant genetic substructure. Within these major clades, we detected five Nautilus groups, which likely correspond to five distinct species. With the exception of Nautilus macromphalus, all previously described species are at odds with genomewide data, testifying to the prevalence of cryptic species among living fossils. Detailed FST analyses further revealed significant genome-wide and locus-specific signatures of selection between species and differentiated populations, which is demonstrated here for the first time in a living fossil. Finally, approximate Bayesian computation (ABC) simulations suggest large effective population sizes, which may explain the low levels of population differentiation commonly observed in living fossils.

A Phylogenomic Solution to the Origin of Insects by Resolving Crustacean-Hexapod Relationships.

Insects, the most diverse group of organisms, are nested within crustaceans, arguably the most abundant group of marine animals. However, to date, no consensus has been reached as to which crustacean taxon is the closest relative of hexapods. A majority of studies have proposed that Branchiopoda (e.g., fairy shrimps) is the sister group of Hexapoda [1-7]. However, these investigations largely excluded two equally important taxa, Remipedia and Cephalocarida. Other studies suggested Remipedia [8-11] or Remipedia + Cephalocarida [12, 13] as potential sister groups of hexapods, but they either did not include Cephalocarida or used only Sanger sequence data and morphology [9, 12]. Here we present the first phylogenomic study specifically addressing the origins of hexapods, including transcriptomes for two species each of Cephalocarida and Remipedia. Phylogenetic analyses of selected matrices, ranging from 81 to 1,675 orthogroups and up to 510,982 amino acid positions, clearly reject a sister-group relationship between Hexapoda and Branchiopoda [1-7]. Nonetheless, support for a hexapod sister-group relationship to Remipedia or to Cephalocarida-Remipedia was highly dependent on the employed analytical methodology. Further analyses assessing the effects of gene evolutionary rate and targeted taxon exclusion support Remipedia as the sole sister taxon of Hexapoda and suggest that the prior grouping of Remipedia + Cephalocarida is an artifact, possibly due to long branch attraction and compositional heterogeneity. We further conclude that terrestrialization of Hexapoda probably occurred in the late Cambrian to early Ordovician, an estimate that is independent of their proposed sister group [4, 8, 12, 14].

A family-level Tree of Life for bivalves based on a Sanger-sequencing approach.

The systematics of the molluscan class Bivalvia are explored using a 5-gene Sanger-based approach including the largest taxon sampling to date, encompassing 219 ingroup species spanning 93 (or 82%) of the 113 currently accepted bivalve families. This study was designed to populate the bivalve Tree of Life at the family level and to place many genera into a clear phylogenetic context, but also pointing to several major clades where taxonomic work is sorely needed. Despite not recovering monophyly of Bivalvia or Protobranchia-as in most previous Sanger-based approaches to bivalve phylogeny-our study provides increased resolution in many higher-level clades, and supports the monophyly of Autobranchia, Pteriomorphia, Heteroconchia, Palaeoheterodonta, Heterodonta, Archiheterodonta, Euheterodonta, Anomalodesmata, Imparidentia, and Neoheterodontei, in addition to many other lower clades. However, deep nodes within some of these clades, especially Pteriomorphia and Imparidentia, could not be resolved with confidence. In addition, many families are not supported, and several are supported as non-monophyletic, including Malletiidae, Nuculanidae, Yoldiidae, Malleidae, Pteriidae, Arcidae, Propeamussiidae, Iridinidae, Carditidae, Myochamidae, Lyonsiidae, Pandoridae, Montacutidae, Galeommatidae, Tellinidae, Semelidae, Psammobiidae, Donacidae, Mactridae, and Cyrenidae; Veneridae is paraphyletic with respect to Chamidae, although this result appears to be an artifact. The denser sampling however allowed testing specific placement of species, showing, for example, that the unusual Australian Plebidonax deltoides is not a member of Donacidae and instead nests within Psammobiidae, suggesting that major revision of Tellinoidea may be required. We also showed that Cleidothaerus is sister group to the cementing member of Myochamidae, suggesting that Cleidothaeridae may not be a valid family and that cementation in Cleidothaerus and Myochama may have had a single origin. These results highlight the need for an integrative approach including as many genera as possible, and that the monophyly and relationships of many families require detailed reassessment. NGS approaches may be able to resolve the most recalcitrant nodes in the near future.

Clarifying phylogenetic relationships and the evolutionary history of the bivalve order Arcida (Mollusca: Bivalvia: Pteriomorphia).

The systematics of the bivalve order Arcida constitutes an unresolved conundrum in bivalve systematics. The current definition of Arcida encompasses two superfamilies: Limopsoidea, which includes the recent families Philobryidae and Limopsidae, and Arcoidea, which encompasses the families Arcidae, Cucullaeidae, Noetiidae, Glycymerididae and Parallelodontidae. This classification, however, is controversial particularly with respect to the position and taxonomic status of Glycymerididae. Previous molecular phylogenies were limited either by the use of only a single molecular marker or by including only a few limopsoid and glycymeridid taxa. The challenging nature of Arcida taxonomy and the controversial results of some of the previous studies, prompted us to use a broad range of taxa (55 species), three nuclear markers (18S rRNA, 28S rRNA and histone H3) and a wide range of algorithmic approaches. This broad but stringent approach led to a number of results that differ significantly from previous studies. We provide the first molecular evidence that supports the separation of Arcoidea from Limopsoidea, although the exact position of Glycymerididae remains unresolved, and the monophyly of Limopsoidea is algorithm-dependent. In addition, we present the first time-calibrated evolutionary tree of Arcida relationships, indicating a significant increase in the diversification of arcidan lineages at the beginning of the Cretaceous, around 140Ma. The monophyly of Arcida, which has been supported previously, was confirmed in all our analyses. Although relationships among families remain somehow unresolved we found support for the monophyly of most arcidan families, at least under some analytical conditions (i.e., Glycymerididae, Noetiidae, Philobryidae, and Limopsidae). However, Arcidae, and particularly Arcinae, remain a major source of inconsistency in the current system of Arcida classification and are in dire need of taxonomic revision.

Trans-Pacific RAD-Seq population genomics confirms introgressive hybridization in Eastern Pacific Pocillopora corals.

Discrepancies between morphology-based taxonomy and phylogenetic systematics are common in Scleractinian corals. In Pocillopora corals, nine recently identified genetic lineages disagree fundamentally with the 17 recognized Pocillopora species, including 5 major Indo-Pacific reef-builders. Pocillopora corals hybridize in the Tropical Eastern Pacific, so it is possible that some of the disagreement between the genetics and taxonomy may be due to introgressive hybridization. Here we used 6769 genome-wide SNPs from Restriction-site Associated DNA Sequencing (RAD-Seq) to conduct phylogenomic comparisons among three common, Indo-Pacific Pocillopora species - P. damicornis, P. eydouxi and P. elegans - within and between populations in the Tropical Eastern Pacific (TEP) and the Central Pacific. Genome-wide RAD-Seq comparisons of Central and TEP Pocillopora confirm that the morphospecies P. damicornis, P. eydouxi and P. elegans are not monophyletic, but instead fall into three distinct genetic groups. However, hybrid samples shared fixed alleles with their respective parental species and, even without strict monophyly, P. damicornis share a common set of 33 species-specific alleles across the Pacific. RAD-Seq data confirm the pattern of one-way introgressive hybridization among TEP Pocillopora, suggesting that introgression may play a role in generating shared, polyphyletic lineages among currently recognized Pocillopora species. Levels of population differentiation within genetic lineages indicate significantly higher levels of population differentiation in the Tropical Eastern Pacific than in the Central West Pacific.

Mixed asexual and sexual reproduction in the Indo-Pacific reef coral Pocillopora damicornis.

Pocillopora damicornis is one of the best studied reef-building corals, yet it's somewhat unique reproductive strategy remains poorly understood. Genetic studies indicate that P. damicornis larvae are produced almost exclusively parthenogenetically, and yet population genetic surveys suggest frequent sexual reproduction. Using microsatellite data from over 580 larvae from 13 colonies, we demonstrate that P. damicornis displays a mixed reproductive strategy where sexual and asexual larvae are produced simultaneously within the same colony. The majority of larvae were parthenogenetic (94%), but most colonies (10 of the 13) produced a subset of their larvae sexually. Logistic regression indicates that the proportion of sexual larvae varied significantly with colony size, cycle day, and calendar day. In particular, the decrease in sexual larvae with colony size suggests that the mixed reproductive strategy changes across the life of the coral. This unique shift in reproductive strategy leads to increasingly asexual replications of successful genotypes, which (in contrast to exclusive parthenogens) have already contributed to the recombinant gene pool.

ENSO drove 2500-year collapse of eastern Pacific coral reefs.

Cores of coral reef frameworks along an upwelling gradient in Panamá show that reef ecosystems in the tropical eastern Pacific collapsed for 2500 years, representing as much as 40% of their history, beginning about 4000 years ago. The principal cause of this millennial-scale hiatus in reef growth was increased variability of the El Niño-Southern Oscillation (ENSO) and its coupling with the Intertropical Convergence Zone. The hiatus was a Pacific-wide phenomenon with an underlying climatology similar to probable scenarios for the next century. Global climate change is probably driving eastern Pacific reefs toward another regional collapse.

Population genetics of an ecosystem-defining reef coral Pocillopora damicornis in the Tropical Eastern Pacific.

BACKGROUND: Coral reefs in the Tropical Eastern Pacific (TEP) are amongst the most peripheral and geographically isolated in the world. This isolation has shaped the biology of TEP organisms and lead to the formation of numerous endemic species. For example, the coral Pocillopora damicornis is a minor reef-builder elsewhere in the Indo-West Pacific, but is the dominant reef-building coral in the TEP, where it forms large, mono-specific stands, covering many hectares of reef. Moreover, TEP P. damicornis reproduces by broadcast spawning, while it broods mostly parthenogenetic larvae throughout the rest of the Indo-West Pacific. Population genetic surveys for P. damicornis from across its Indo-Pacific range indicate that gene flow (i.e. larval dispersal) is generally limited over hundreds of kilometers or less. Little is known about the population genetic structure and the dispersal potential of P. damicornis in the TEP. METHODOLOGY: Using multilocus microsatellite data, we analyzed the population structure of TEP P. damicornis among and within nine reefs and test for significant genetic structure across three geographically and ecologically distinct regions in Panama. PRINCIPAL FINDINGS/CONCLUSIONS: We detected significant levels of population genetic structure (global R(ST) = 0.162), indicating restricted gene flow (i.e. larvae dispersal), both among the three regions (R(RT) = 0.081) as well as within regions (R(SR) = 0.089). Limited gene flow across a distinct environmental cline, like the regional upwelling gradient in Panama, indicates a significant potential for differential adaptation and population differentiation. Individual reefs were characterized by unexpectedly high genet diversity (avg. 94%), relatively high inbreeding coefficients (global F(IS) = 0.183), and localized spatial genetic structure among individuals (i.e. unique genets) over 10 m intervals. These findings suggest that gene flow is limited in TEP P. damicornis populations, particularly among regions, but even over meter scales within populations.

Interspecific hybridization and restricted trans-Pacific gene flow in the Tropical Eastern Pacific Pocillopora.

Coral reefs in the Tropical Eastern Pacific (TEP) are among the most isolated in the world. This isolation has resulted in relatively low species diversity but comparatively high endemism. The dominant reef-building corals of the TEP are the Pocillopora corals, a ubiquitous Indo-Pacific genus commonly regarded as inferior reef-builder. In addition to being the dominant reef-builders in the TEP, the Pocilloporids have undergone a reproductive shift from internally brooding larvae through most of their Indo-Pacific range to free-spawning in the TEP. Using genetic data from the internally transcribed spacer (ITS) regions of the nuclear ribosomal DNA gene cluster, we show here that this apparent reproductive shift coincides with interspecific hybridization among TEP Pocillopora species. We document a pattern of one-way gene flow into the main TEP reef builder P. damicornis from one or both of its TEP congeners - P. eydouxi and P. elegans. Our data provide preliminary evidence that trans-Pacific gene flow in P. damicornis between the Central and Eastern Pacific is restricted as well (Phi(ST) = 0.419, P < 0.0001). In combination, these results suggest that Eastern Pacific corals exist in relative isolation from their Central Pacific counterparts and interact with each other differently via hybridization.