Holobiont dysbiosis or acclimatation? Shift in the microbial taxonomic diversity and functional composition of a cosmopolitan sponge subjected to chronic pollution in a Patagonian bay.

Dysbiosis and acclimatization are two starkly opposing outcomes of altered holobiont associations in response to environmental pollution. This study assesses whether shifts in microbial taxonomic composition and functional profiles of the cosmopolitan sponge Hymeniacidon perlevis indicate dysbiotic or acclimatized responses to water pollution. To do so, sponge and water samples were collected in a semi-enclosed environment (San Antonio Bay, Patagonia, Argentina) from variably polluted sites (i.e., eutrophication, heavy metal contamination). We found significant differences in the microbiome of H. perlevis with respect to the pollution history of the sites. Several indicators suggested that acclimatization, rather than dysbiosis, explained the microbiome response to higher pollution: 1) the distinction of the sponge microbiome from the water microbiome; 2) low similarity between the sponge and water microbiomes at the most polluted site; 3) the change in microbiome composition between sponges from the different sites; 4) a high similarity in the microbiome among sponge individuals within sites; 5) a similar ratio of common sponge microbes to opportunistic microbes between sponges at the most and least polluted sites; and 6) a distinctive functional profile of the sponge microbiome at the most polluted site. This profile indicated a more expansive metabolic repertoire, including the degradation of pollutants and the biosynthesis of secondary metabolites, suggesting a relevant role of these microbial communities in the adaptation of the holobiont to organic pollution. Our results shed light on the rearrangement of the H. perlevis microbiome that could allow it to successfully colonize sites with high anthropogenic impact while resisting dysbiosis.

The order Axinellida (Porifera: Demospongiae) in California.

Sponges are common and diverse in California, but they have received little study in the region, and the identities of many common species remain unclear. Here we combine fresh collections and museum vouchers to revise the order Axinellida for California. Seven new species are described: Endectyon (Endectyon) hispitumulus, Eurypon curvoclavus, Aulospongus viridans, Aulospongus lajollaensis, Halicnemia litorea, Halicnemia montereyensis, and Halicnemia weltoni. One new combination is also described, and two existing species are reduced to junior synonyms, resulting in a total of 13 species; a dichotomous key to differentiate them is provided. DNA data from 9 of the 13 species is combined with publicly available data to produce updated global phylogenies for the order.

Epidermal threads reveal the origin of hagfish slime.

When attacked, hagfishes produce a soft, fibrous defensive slime within a fraction of a second by ejecting mucus and threads into seawater. The rapid setup and remarkable expansion of the slime make it a highly effective and unique form of defense. How this biomaterial evolved is unknown, although circumstantial evidence points to the epidermis as the origin of the thread- and mucus-producing cells in the slime glands. Here, we describe large intracellular threads within a putatively homologous cell type from hagfish epidermis. These epidermal threads averaged ~2 mm in length and ~0.5 µm in diameter. The entire hagfish body is covered by a dense layer of epidermal thread cells, with each square millimeter of skin storing a total of ~96 cm threads. Experimentally induced damage to a hagfish's skin caused the release of threads, which together with mucus, formed an adhesive epidermal slime that is more fibrous and less dilute than the defensive slime. Transcriptome analysis further suggests that epidermal threads are ancestral to the slime threads, with duplication and diversification of thread genes occurring in parallel with the evolution of slime glands. Our results support an epidermal origin of hagfish slime, which may have been driven by selection for stronger and more voluminous slime.

Hagfishes are deep-sea animals, and they represent one of the oldest living relatives of animals with backbones. To defend themselves against predators, they produce a remarkable slime that is reinforced with fibers and can clog a predator's gills, thwarting the attack. The slime deploys in less than half a second, exuding from specialized glands on the hagfish's body and expanding up to 10,000 times its ejected volume. The defensive slime is highly dilute, consisting mostly of sea water, with low concentrations of mucus and strong, silk-like threads that are approximately 20 centimeters long. Where and how hagfish slime evolved remains a mystery. Zeng et al. set out to answer where on the hagfish's body the slime glands originated, and how they may have evolved. First, Zeng et al. examined hagfishes and found that cells in the surface layer of their skin (the epidermis) produce threads roughly two millimeters in length that are released when the hagfish's skin is damaged. These threads mix with the mucus that is produced by ruptured skin cells to form a slime that likely adheres to predators' mouths. This slime could be a precursor of the slime produced by the specialized glands. To test this hypothesis, Zeng et al. analyzed which genes are turned on and off both in the hagfishes' skin and in their slime glands. The patterns they found are consistent with the slime glands originating from the epidermis. Based on these results, Zeng et al. propose that ancient hagfishes first evolved the ability to produce slime with anti-predator effects when their skin was damaged in attacks. Over time, hagfishes that could produce and store more slime and eject it actively into a predator's mouth likely had a better chance of surviving. This advantage may have led to the appearance of increasingly specialized glands that could carry out these functions. The findings of Zeng et al. will be of interest to evolutionary biologists, marine biologists, and those studying the ecology of predator-prey interactions. Because of its unique material properties, hagfish slime is also of interest to biophysicists, bioengineers and those engaged in biomimetic research. The origin of hagfish slime glands is an interesting example of how a new trait evolved, and may provide insight into the evolution of other adaptive traits.

Different phylogenomic methods support monophyly of enigmatic 'Mesozoa' (Dicyemida + Orthonectida, Lophotrochozoa).

Dicyemids and orthonectids were traditionally classified in a group called Mesozoa, but their placement in a single clade has been contested and their position(s) within Metazoa is uncertain. Here, we assembled a comprehensive matrix of Lophotrochozoa (Metazoa) and investigated the position of Dicyemida (= Rhombozoa) and Orthonectida, employing multiple phylogenomic approaches. We sequenced seven new transcriptomes and one draft genome from dicyemids (Dicyema, Dicyemennea) and two transcriptomes from orthonectids (Rhopalura). Using these and published data, we assembled and analysed contamination-filtered datasets with up to 987 genes. Our results recover Mesozoa monophyletic and as a close relative of Platyhelminthes or Gnathifera. Because of the tendency of the long-branch mesozoans to group with other long-branch taxa in our analyses, we explored the impact of approaches purported to help alleviate long-branch attraction (e.g. taxon removal, coalescent inference, gene targeting). None of these were able to break the association of Orthonectida with Dicyemida in the maximum-likelihood trees. Contrastingly, the Bayesian analysis and site-specific frequency model in maximum-likelihood did not recover a monophyletic Mesozoa (but only when using a specific 50 gene matrix). The classic hypothesis on monophyletic Mesozoa is possibly reborn and should be further tested.

Community structure of coral microbiomes is dependent on host morphology.

BACKGROUND: The importance of symbiosis has long been recognized on coral reefs, where the photosynthetic dinoflagellates of corals (Symbiodiniaceae) are the primary symbiont. Numerous studies have now shown that a diverse assemblage of prokaryotes also make-up part of the microbiome of corals. A subset of these prokaryotes is capable of fixing nitrogen, known as diazotrophs, and is also present in the microbiome of scleractinian corals where they have been shown to supplement the holobiont nitrogen budget. Here, an analysis of the microbiomes of 16 coral species collected from Australia, Curaçao, and Hawai'i using three different marker genes (16S rRNA, nifH, and ITS2) is presented. These data were used to examine the effects of biogeography, coral traits, and ecological life history characteristics on the composition and diversity of the microbiome in corals and their diazotrophic communities. RESULTS: The prokaryotic microbiome community composition (i.e., beta diversity) based on the 16S rRNA gene varied between sites and ecological life history characteristics, but coral morphology was the most significant factor affecting the microbiome of the corals studied. For 15 of the corals studied, only two species Pocillopora acuta and Seriotopora hystrix, both brooders, showed a weak relationship between the 16S rRNA gene community structure and the diazotrophic members of the microbiome using the nifH marker gene, suggesting that many corals support a microbiome with diazotrophic capabilities. The order Rhizobiales, a taxon that contains primarily diazotrophs, are common members of the coral microbiome and were eight times greater in relative abundances in Hawai'i compared to corals from either Curacao or Australia. However, for the diazotrophic component of the coral microbiome, only host species significantly influenced the composition and diversity of the community. CONCLUSIONS: The roles and interactions between members of the coral holobiont are still not well understood, especially critical functions provided by the coral microbiome (e.g., nitrogen fixation), and the variation of these functions across species. The findings presented here show the significant effect of morphology, a coral "super trait," on the overall community structure of the microbiome in corals and that there is a strong association of the diazotrophic community within the microbiome of corals. However, the underlying coral traits linking the effects of host species on diazotrophic communities remain unknown. Video Abstract.

The Genome of the Softshell Clam Mya arenaria and the Evolution of Apoptosis.

Apoptosis is a fundamental feature of multicellular animals and is best understood in mammals, flies, and nematodes, with the invertebrate models being thought to represent a condition of ancestral simplicity. However, the existence of a leukemia-like cancer in the softshell clam Mya arenaria provides an opportunity to re-evaluate the evolution of the genetic machinery of apoptosis. Here, we report the whole-genome sequence for M. arenaria which we leverage with existing data to test evolutionary hypotheses on the origins of apoptosis in animals. We show that the ancestral bilaterian p53 locus, a master regulator of apoptosis, possessed a complex domain structure, in contrast to that of extant ecdysozoan p53s. Further, ecdysozoan taxa, but not chordates or lophotrochozoans like M. arenaria, show a widespread reduction in apoptosis gene copy number. Finally, phylogenetic exploration of apoptosis gene copy number reveals a striking linkage with p53 domain complexity across species. Our results challenge the current understanding of the evolution of apoptosis and highlight the ancestral complexity of the bilaterian apoptotic tool kit and its subsequent dismantlement during the ecdysozoan radiation.

Annual Thermal Stress Increases a Soft Coral's Susceptibility to Bleaching.

Like scleractinian corals, soft corals contain photosymbionts (Family Symbiodiniaceae) that provide energy for the host. Recent thermal events have resulted in soft coral bleaching in four of five years on Guam, where they dominated back-reef communities. Soft coral bleaching was examined in Sinularia maxima, S. polydactyla, and their hybrid S. maxima x polydactyla. Results from annual field surveys indicated that S. maxima and the hybrid were more susceptible to bleaching than S. polydactyla, and this was related to differences in their Symbiodiniaceae communities in 2016 and 2017. The photosymbionts of S. polydactyla were apparently more stress tolerant and maintained higher photosynthetic potential through three years of bleaching, in contrast to the other species that exhibited a decline in photosynthetic potential after the first year of bleaching. Nonetheless, by the 2017 bleaching event all soft coral populations exhibited significant bleaching-mediated declines and loss of photosynthetic efficiency suggesting a declining resiliency to annual thermal stress events. While S. polydactyla initially looked to succeed the other species as the dominant space occupying soft coral on Guam back-reefs, cumulative bleaching events ultimately turned this "winner" into a "loser", suggesting the trajectory for coral reefs is towards continued loss of structure and function.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

Sequences of Circadian Clock Proteins in the Nudibranch Molluscs Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea.

While much is known about the genes and proteins that make up the circadian clocks in vertebrates and several arthropod species, much less is known about the clock genes in many other invertebrates, including nudibranchs. The goal of this project was to identify the RNA and protein products of putative clock genes in the central nervous system of three nudibranchs, Hermissenda crassicornis, Melibe leonina, and Tritonia diomedea. Using previously published transcriptomes (Hermissenda and Tritonia) and a new transcriptome (Melibe), we identified nudibranch orthologs for the products of five canonical clock genes: brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1, circadian locomotor output cycles kaput, non-photoreceptive cryptochrome, period, and timeless. Additionally, orthologous sequences for the products of five related genes-aryl hydrocarbon receptor nuclear translocator like, photoreceptive cryptochrome, cryptochrome DASH, 6-4 photolyase, and timeout-were determined. Phylogenetic analyses confirmed that the nudibranch proteins were most closely related to known orthologs in related invertebrates, such as oysters and annelids. In general, the nudibranch clock proteins shared greater sequence similarity with Mus musculus orthologs than Drosophila melanogaster orthologs, which is consistent with the closer phylogenetic relationships recovered between lophotrochozoan and vertebrate orthologs. The suite of clock-related genes in nudibranchs includes both photoreceptive and non-photoreceptive cryptochromes, as well as timeout and possibly timeless. Therefore, the nudibranch clock may resemble the one exhibited in mammals, or possibly even in non-drosopholid insects and oysters. The latter would be evidence supporting this as the ancestral clock for bilaterians.

A member of the Roseobacter clade, Octadecabacter sp., is the dominant symbiont in the brittle star Amphipholis squamata.

Symbiotic associations with subcuticular bacteria (SCB) have been identified and studied in many echinoderms, including the SCB of the brooding brittle star, Amphipholis squamata. Previous studies on the SCB of A. squamata placed the isolated bacterium, designated as AS1, in the genus Vibrio (Gammaproteobacteria), but subsequent studies suggested that the SCB of echinoderms belong to the Alphaproteobacteria. This study examines the taxonomic composition of SCB associated with A. squamata from the Northwest Atlantic using the 16S rRNA gene and next generation sequencing. Results show the presence of a single dominant bacterial type, within the Roseobacter clade, family Rhodobacteraceae, which composes 70%-80% of the A. squamata microbiome. These Rhodobacteraceae sequences were identified as members of the genus Octadecabacter. Additionally, the original isolate, AS1, from the brittle star A. squamata also belongs in the genus Octadecabacter based on Sanger sequencing of cloned 16S rRNA gene sequences. By comparison, adjacent seawater and sediment porewater communities were significantly more diverse, hosting bacteria in the phyla Proteobacteria, Bacteroidetes, Cyanobacteria, Verrucomicrobia and Actinobacteria. Thus, a distinct SCB community is present in A. squamata that is dominated by a member of the genus Octadecabacter and is identical to the original isolate, AS1, from this brittle star.

Comparative Genomics of Color Morphs In the Coral Montastraea cavernosa.

Montastraea cavernosa is a common coral in the Caribbean basin found in several color morphs. To investigate the causes for brown and orange morphs we undertook a genomics approach on corals collected at the same time and depth in the Bahamas. The coral holobiont includes the host, symbiotic dinoflagellates (Symbiodinium spp.), and a diverse microbiome. While the coral host showed significant genetic differentiation between color morphs both the composition of the Symbiodinium spp. communities and the prokaryotic communities did not. Both targeted and global gene expression differences in the transcriptome of the host show no difference in fluorescent proteins while the metatranscriptome of the microbiome shows that pigments such as phycoerythrin and orange carotenoid protein of cyanobacterial origin are significantly greater in orange morphs, which is also consistent with the significantly greater number of cyanobacteria quantified by 16S rRNA reads and flow cytometry. The microbiome of orange color morphs expressed significantly more nitrogenase (nifH) transcripts consistent with their known ability to fix nitrogen. Both coral and Symbiodinium spp. transcriptomes from orange morphs had significantly increased expression of genes related to immune response and apoptosis, which may potentially be involved in maintaining and regulating the unique symbiont population in orange morphs.

Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria.

Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations.

Molecular clocks indicate turnover and diversification of modern coleoid cephalopods during the Mesozoic Marine Revolution.

Coleoid cephalopod molluscs comprise squid, cuttlefish and octopuses, and represent nearly the entire diversity of modern cephalopods. Sophisticated adaptations such as the use of colour for camouflage and communication, jet propulsion and the ink sac highlight the unique nature of the group. Despite these striking adaptations, there are clear parallels in ecology between coleoids and bony fishes. The coleoid fossil record is limited, however, hindering confident analysis of the tempo and pattern of their evolution. Here we use a molecular dataset (180 genes, approx. 36 000 amino acids) of 26 cephalopod species to explore the phylogeny and timing of cephalopod evolution. We show that crown cephalopods diverged in the Silurian-Devonian, while crown coleoids had origins in the latest Palaeozoic. While the deep-sea vampire squid and dumbo octopuses have ancient origins extending to the Early Mesozoic Era, 242 ± 38 Ma, incirrate octopuses and the decabrachian coleoids (10-armed squid) diversified in the Jurassic Period. These divergence estimates highlight the modern diversity of coleoid cephalopods emerging in the Mesozoic Marine Revolution, a period that also witnessed the radiation of most ray-finned fish groups in addition to several other marine vertebrates. This suggests that that the origin of modern cephalopod biodiversity was contingent on ecological competition with marine vertebrates.

The Last Common Ancestor of Most Bilaterian Animals Possessed at Least Nine Opsins.

The opsin gene family encodes key proteins animals use to sense light and has expanded dramatically as it originated early in animal evolution. Understanding the origins of opsin diversity can offer clues to how separate lineages of animals have repurposed different opsin paralogs for different light-detecting functions. However, the more we look for opsins outside of eyes and from additional animal phyla, the more opsins we uncover, suggesting we still do not know the true extent of opsin diversity, nor the ancestry of opsin diversity in animals. To estimate the number of opsin paralogs present in both the last common ancestor of the Nephrozoa (bilaterians excluding Xenoacoelomorpha), and the ancestor of Cnidaria + Bilateria, we reconstructed a reconciled opsin phylogeny using sequences from 14 animal phyla, especially the traditionally poorly-sampled echinoderms and molluscs. Our analysis strongly supports a repertoire of at least nine opsin paralogs in the bilaterian ancestor and at least four opsin paralogs in the last common ancestor of Cnidaria + Bilateria. Thus, the kernels of extant opsin diversity arose much earlier in animal history than previously known. Further, opsins likely duplicated and were lost many times, with different lineages of animals maintaining different repertoires of opsin paralogs. This phylogenetic information can inform hypotheses about the functions of different opsin paralogs and can be used to understand how and when opsins were incorporated into complex traits like eyes and extraocular sensors.

Cheaters must prosper: reconciling theoretical and empirical perspectives on cheating in mutualism.

Cheating is a focal concept in the study of mutualism, with the majority of researchers considering cheating to be both prevalent and highly damaging. However, current definitions of cheating do not reliably capture the evolutionary threat that has been a central motivation for the study of cheating. We describe the development of the cheating concept and distill a relative-fitness-based definition of cheating that encapsulates the evolutionary threat posed by cheating, i.e. that cheaters will spread and erode the benefits of mutualism. We then describe experiments required to conclude that cheating is occurring and to quantify fitness conflict more generally. Next, we discuss how our definition and methods can generate comparability and integration of theory and experiments, which are currently divided by their respective prioritisations of fitness consequences and traits. To evaluate the current empirical evidence for cheating, we review the literature on several of the best-studied mutualisms. We find that although there are numerous observations of low-quality partners, there is currently very little support from fitness data that any of these meet our criteria to be considered cheaters. Finally, we highlight future directions for research on conflict in mutualisms, including novel research avenues opened by a relative-fitness-based definition of cheating.

Evolutionary relatedness does not predict competition and co-occurrence in natural or experimental communities of green algae.

The competition-relatedness hypothesis (CRH) predicts that the strength of competition is the strongest among closely related species and decreases as species become less related. This hypothesis is based on the assumption that common ancestry causes close relatives to share biological traits that lead to greater ecological similarity. Although intuitively appealing, the extent to which phylogeny can predict competition and co-occurrence among species has only recently been rigorously tested, with mixed results. When studies have failed to support the CRH, critics have pointed out at least three limitations: (i) the use of data poor phylogenies that provide inaccurate estimates of species relatedness, (ii) the use of inappropriate statistical models that fail to detect relationships between relatedness and species interactions amidst nonlinearities and heteroskedastic variances, and (iii) overly simplified laboratory conditions that fail to allow eco-evolutionary relationships to emerge. Here, we address these limitations and find they do not explain why evolutionary relatedness fails to predict the strength of species interactions or probabilities of coexistence among freshwater green algae. First, we construct a new data-rich, transcriptome-based phylogeny of common freshwater green algae that are commonly cultured and used for laboratory experiments. Using this new phylogeny, we re-analyse ecological data from three previously published laboratory experiments. After accounting for the possibility of nonlinearities and heterogeneity of variances across levels of relatedness, we find no relationship between phylogenetic distance and ecological traits. In addition, we show that communities of North American green algae are randomly composed with respect to their evolutionary relationships in 99% of 1077 lakes spanning the continental United States. Together, these analyses result in one of the most comprehensive case studies of how evolutionary history influences species interactions and community assembly in both natural and experimental systems. Our results challenge the generality of the CRH and suggest it may be time to re-evaluate the validity and assumptions of this hypothesis.

Using phylogenetically-informed annotation (PIA) to search for light-interacting genes in transcriptomes from non-model organisms.

BACKGROUND: Tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes (i.e. the suite of genes expressed in a tissue) feasible for almost any organism. Yet a challenge for biologists is that it can be difficult to assign identities to gene sequences, especially from non-model organisms. Phylogenetic analyses are one useful method for assigning identities to these sequences, but such methods tend to be time-consuming because of the need to re-calculate trees for every gene of interest and each time a new data set is analyzed. In response, we employed existing tools for phylogenetic analysis to produce a computationally efficient, tree-based approach for annotating transcriptomes or new genomes that we term Phylogenetically-Informed Annotation (PIA), which places uncharacterized genes into pre-calculated phylogenies of gene families. RESULTS: We generated maximum likelihood trees for 109 genes from a Light Interaction Toolkit (LIT), a collection of genes that underlie the function or development of light-interacting structures in metazoans. To do so, we searched protein sequences predicted from 29 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the Osiris package of Galaxy (an open-source workflow management system). Next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based Evolutionary Placement Algorithm (implemented in RAxML) to place sequences of potential LIT genes on to our pre-calculated gene trees. Finally, we implemented PIA in Galaxy and used it to search for LIT genes in 28 newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. Our new trees for LIT genes are available on the Bitbucket public repository ( http://bitbucket.org/osiris_phylogenetics/pia/ ) and we demonstrate PIA on a publicly-accessible web server ( http://galaxy-dev.cnsi.ucsb.edu/pia/ ). CONCLUSIONS: Our new trees for LIT genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures. We also introduce PIA, a high throughput method for using phylogenetic relationships to identify LIT genes in transcriptomes from non-model organisms. With simple modifications, our methods may be used to search for different sets of genes or to annotate data sets from taxa outside of Metazoa.

Predictable transcriptome evolution in the convergent and complex bioluminescent organs of squid.

Despite contingency in life's history, the similarity of evolutionarily convergent traits may represent predictable solutions to common conditions. However, the extent to which overall gene expression levels (transcriptomes) underlying convergent traits are themselves convergent remains largely unexplored. Here, we show strong statistical support for convergent evolutionary origins and massively parallel evolution of the entire transcriptomes in symbiotic bioluminescent organs (bacterial photophores) from two divergent squid species. The gene expression similarities are so strong that regression models of one species' photophore can predict organ identity of a distantly related photophore from gene expression levels alone. Our results point to widespread parallel changes in gene expression evolution associated with convergent origins of complex organs. Therefore, predictable solutions may drive not only the evolution of novel, complex organs but also the evolution of overall gene expression levels that underlie them.

Osiris: accessible and reproducible phylogenetic and phylogenomic analyses within the Galaxy workflow management system.

BACKGROUND: Phylogenetic tools and 'tree-thinking' approaches increasingly permeate all biological research. At the same time, phylogenetic data sets are expanding at breakneck pace, facilitated by increasingly economical sequencing technologies. Therefore, there is an urgent need for accessible, modular, and sharable tools for phylogenetic analysis. RESULTS: We developed a suite of wrappers for new and existing phylogenetics tools for the Galaxy workflow management system that we call Osiris. Osiris and Galaxy provide a sharable, standardized, modular user interface, and the ability to easily create complex workflows using a graphical interface. Osiris enables all aspects of phylogenetic analysis within Galaxy, including de novo assembly of high throughput sequencing reads, ortholog identification, multiple sequence alignment, concatenation, phylogenetic tree estimation, and post-tree comparative analysis. The open source files are available on in the Bitbucket public repository and many of the tools are demonstrated on a public web server (http://galaxy-dev.cnsi.ucsb.edu/osiris/). CONCLUSIONS: Osiris can serve as a foundation for other phylogenomic and phylogenetic tool development within the Galaxy platform.

Eye-specification genes in the bacterial light organ of the bobtail squid Euprymna scolopes, and their expression in response to symbiont cues.

The squid Euprymna scolopes has evolved independent sets of tissues capable of light detection, including a complex eye and a photophore or 'light organ', which houses the luminous bacterial symbiont Vibrio fischeri. As the eye and light organ originate from different embryonic tissues, we examined whether the eye-specification genes, pax6, eya, six, and dac, are shared by these two organs, and if so, whether they are regulated in the light organ by symbiosis. We obtained sequences of the four genes with PCR, confirmed orthology with phylogenetic analysis, and determined that each was expressed in the eye and light organ. With in situ hybridization (ISH), we localized the gene transcripts in developing embryos, comparing the patterns of expression in the two organs. The four transcripts localized to similar tissues, including those associated with the visual system ∼1/4 into embryogenesis (Naef stage 18) and the light organ ∼3/4 into embryogenesis (Naef stage 26). We used ISH and quantitative real-time PCR to examine transcript expression and differential regulation in postembryonic light organs in response to the following colonization conditions: wild-type, luminescent V. fischeri; a mutant strain defective in light production; and as a control, no symbiont. In ISH experiments light organs showed down regulation of the pax6, eya, and six transcripts in response to wild-type V. fischeri. Mutant strains also induced down regulation of the pax6 and eya transcripts, but not of the six transcript. Thus, luminescence was required for down regulation of the six transcript. We discuss these results in the context of symbiont-induced light-organ development. Our study indicates that the eye-specification genes are expressed in light-interacting tissues independent of their embryonic origin and are capable of responding to bacterial cues. These results offer evidence for evolutionary tinkering or the recruitment of eye development genes for use in a light-sensing photophore.