Sexual Signals Persist over Deep Time: Ancient Co-option of Bioluminescence for Courtship Displays in Cypridinid Ostracods.

Although the diversity, beauty, and intricacy of sexually selected courtship displays command the attention of evolutionists, the longevity of these traits in deep time is poorly understood. Population-based theory suggests sexual selection could either lower or raise extinction risk, resulting in high or low persistence of lineages with sexually selected traits. Furthermore, empirical studies that directly estimate the longevity of sexually selected traits are uncommon. Sexually selected signals-including bioluminescent courtship-originated multiple times during evolution, allowing the empirical study of their longevity after careful phylogenetic and divergence time analyses. Here, we estimate the first transcriptome-based molecular phylogeny and divergence times of Cypridinidae. We report extreme longevity of bioluminescent courtship, a trait important in mate choice and probably under sexual selection. Our relaxed-clock estimates of divergence times coupled with stochastic character mapping show luminous courtship evolved only once in Cypridinidae-in a Sub-Tribe, we name Luxorina-at least 151 millions of years ago from cypridinid ancestors that used bioluminescence only in antipredator displays, defining a Tribe we name Luminini. This time-calibrated molecular phylogeny of cypridinids will serve as a foundation for integrative and comparative studies on the biochemistry, molecular evolution, courtship, diversification, and ecology of cypridinid bioluminescence. The persistence of luminous courtship for hundreds of millions of years suggests that sexual selection did not cause a rapid loss of associated traits, and that rates of speciation within the group exceeded extinction risk, which may contribute to the persistence of a diverse clade of signaling species. [Ancestral state reconstruction; Biodiversity; co-option; divergence time estimates; macroevolution; Ostracoda; phylogenomics; sexual selection.].

Similar enzymatic functions in distinct bioluminescence systems: evolutionary recruitment of sulfotransferases in ostracod light organs.

Genes from ancient families are sometimes involved in the convergent evolutionary origins of similar traits, even across vast phylogenetic distances. Sulfotransferases are an ancient family of enzymes that transfer sulfate from a donor to a wide variety of substrates, including probable roles in some bioluminescence systems. Here, we demonstrate multiple sulfotransferases, highly expressed in light organs of the bioluminescent ostracod Vargula tsujii, transfer sulfate in vitro to the luciferin substrate, vargulin. We find luciferin sulfotransferases (LSTs) of ostracods are not orthologous to known LSTs of fireflies or sea pansies; animals with distinct and convergently evolved bioluminescence systems compared to ostracods. Therefore, distantly related sulfotransferases were independently recruited at least three times, leading to parallel evolution of luciferin metabolism in three highly diverged organisms. Reuse of homologous genes is surprising in these bioluminescence systems because the other components, including luciferins and luciferases, are completely distinct. Whether convergently evolved traits incorporate ancient genes with similar functions or instead use distinct, often newer, genes may be constrained by how many genetic solutions exist for a particular function. When fewer solutions exist, as in genetic sulfation of small molecules, evolution may be more constrained to use the same genes time and again.

Collective synchrony of mating signals modulated by ecological cues and social signals in bioluminescent sea fireflies.

Individuals often employ simple rules that can emergently synchronize behaviour. Some collective behaviours are intuitively beneficial, but others like mate signalling in leks occur across taxa despite theoretical individual costs. Whether disparate instances of synchronous signalling are similarly organized is unknown, largely due to challenges observing many individuals simultaneously. Recording field collectives and ex situ playback experiments, we describe principles of synchronous bioluminescent signals produced by marine ostracods (Crustacea; Luxorina) that seem behaviorally convergent with terrestrial fireflies, and with whom they last shared a common ancestor over 500 Mya. Like synchronous fireflies, groups of signalling males use visual cues (intensity and duration of light) to decide when to signal. Individual ostracods also modulate their signal based on the distance to nearest neighbours. During peak darkness, luminescent 'waves' of synchronous displays emerge and ripple across the sea floor approximately every 60 s, but such periodicity decays within and between nights after the full moon. Our data reveal these bioluminescent aggregations are sensitive to both ecological and social light sources. Because the function of collective signals is difficult to dissect, evolutionary convergence, like in the synchronous visual displays of diverse arthropods, provides natural replicates to understand the generalities that produce emergent group behaviour.

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.

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.

Selection, drift, and constraint in cypridinid luciferases and the diversification of bioluminescent signals in sea fireflies.

Understanding the genetic causes of evolutionary diversification is challenging because differences across species are complex, often involving many genes. However, cases where single or few genetic loci affect a trait that varies dramatically across a radiation of species provide tractable opportunities to understand the genetics of diversification. Here, we begin to explore how diversification of bioluminescent signals across species of cypridinid ostracods ("sea fireflies") was influenced by evolution of a single gene, cypridinid-luciferase. In addition to emission spectra ("colour") of bioluminescence from 21 cypridinid species, we report 13 new c-luciferase genes from de novo transcriptomes, including in vitro assays to confirm function of four of those genes. Our comparative analyses suggest some amino acid sites in c-luciferase evolved under episodic diversifying selection and may be associated with changes in both enzyme kinetics and colour, two enzymatic functions that directly impact the phenotype of bioluminescent signals. The analyses also suggest multiple other amino acid positions in c-luciferase evolved neutrally or under purifying selection, and may have impacted the variation of colour of bioluminescent signals across genera. Previous mutagenesis studies at candidate sites show epistatic interactions, which could constrain the evolution of c-luciferase function. This work provides important steps toward understanding the genetic basis of diversification of behavioural signals across multiple species, suggesting different evolutionary processes act at different times during a radiation of species. These results set the stage for additional mutagenesis studies that could explicitly link selection, drift, and constraint to the evolution of phenotypic diversification.

Phenotypic evolution shaped by current enzyme function in the bioluminescent courtship signals of sea fireflies.

Mating behaviours are diverse and noteworthy, especially within species radiations where they may contribute to speciation. Studying how differences in mating behaviours arise between species can help us understand how diversity is generated at multiple biological levels. The bioluminescent courtship displays of cypridinid ostracods (or sea fireflies) are an excellent system for this because amazing variety evolves while using a conserved biochemical mechanism. We find that the evolution of one aspect in this behavioural phenotype-the duration of bioluminescent courtship pulses-is shaped by biochemical function. First, by measuring light production from induced bioluminescence in 38 species, we discovered differences between species in their biochemical reactions. Then, for 16 species for which biochemical, phylogenetic and behavioural data are all available, we used phylogenetic comparative models to show that differences in biochemical reaction are nonlinearly correlated with the duration of courtship pulses. This relationship indicates that changes to both enzyme (c-luciferase) function and usage have shaped the evolution of courtship displays, but that they differentially contribute to these phenotypic changes. This nonlinear dynamic may have consequences for the disparity of signalling phenotypes observed across species, and demonstrates how unappreciated diversity at the biochemical level can lead to inferences about behavioural evolution.

Furcation and fusion: The phylogenetics of evolutionary novelty.

Novelty and innovation are fundamental yet relatively understudied concepts in evolution. We may study the history and provenance of novelty using phylogenetics, where key questions include when evolution occurs by tree-like branching and when it occurs by movement of distantly related parts in processes akin to horizontal transfer. Perfectly vertical inheritance, often an assumption of evolutionary trees, requires simultaneous co-duplication of the parts of a duplicating or speciating (processes I collectively call 'furcating') biological feature. However, simultaneous co-duplication of many parts usually requires variational processes that are rare. Therefore, instead of always being perfectly tree-like, evolution often involves events that incorporate or fuse more distantly related parts into new units during evolution, which herein I call 'fusion'. Exon shuffling, horizontal gene transfer, introgression, and co-option are such fusion processes at different levels of organization. The ubiquity of processes that fuse distantly related parts has wide ranging implications for the study of macroevolution. For one, the central metaphor of a tree of life will often be incomplete, to the point where we may consider a different metaphor, such as economic public goods, or a 'web of life'. Secondly, we often may need to expand commonly used phylogenetic models and methods, highlighting a need for an expansive toolkit for studying evolutionary history. Even though furcation - the splitting and individuation of biological features - does happen, fusion of distant parts may often be just as critical for the evolution of novelties, and must formally be incorporated into the metaphors, models, and visualization of evolutionary history. This will allow us to understand the timing, order of appearance, and diversification rates of developmental systems, including cell types, organs, behavior, and language, which very commonly evolve through co-option.

Ecological Engineering Helps Maximize Function in Algal Oil Production.

Algal biofuels have the potential to curb the emissions of greenhouse gases from fossil fuels, but current growing methods fail to produce fuels that meet the multiple standards necessary for economical industrial use. For example, algae grown as monocultures for biofuel production have not simultaneously and economically achieved high yields of the high-quality lipid-rich biomass desired for the industrial-scale production of bio-oil. Decades of study in the field of ecology have demonstrated that simultaneous increases in multiple functions, such as the quantity and quality of biomass, can occur in natural ecosystems by increasing biological diversity. Here, we show that species consortia of algae can improve the production of bio-oil, which benefits from both a high biomass yield and a high quality of biomass rich in fatty acids. We explain the underlying causes of increased quantity and quality of algal biomass among species consortia by showing that, relative to monocultures, species consortia can differentially regulate lipid metabolism genes while growing to higher levels of biomass, in part due to a greater utilization of nutrient resources. We identify multiple genes involved in lipid biosynthesis that are frequently upregulated in bicultures and further show that these elevated levels of gene expression are highly predictive of the elevated levels in biculture relative to that in monoculture of multiple quality metrics of algal biomass. These results show that interactions between species can alter the expression of lipid metabolism genes and further demonstrate that our understanding of diversity-function relationships from natural ecosystems can be harnessed to improve the production of bio-oil.IMPORTANCE Algal biofuels are one of the more promising forms of renewable energy. In our study, we investigate whether ecological interactions between species of microalgae regulate two important factors in cultivation-the biomass of the crop produced and the quality of the biomass that is produced. We found that species interactions often improved production yields, especially the fatty acid content of the algal biomass, and that differentially expressed genes involved in fatty acid metabolism are predictive of improved quality metrics of bio-oil. Other studies have found that diversity often improves productivity and stability in agricultural and natural ecosystems. Our results provide further evidence that growing multispecies crops of microalgae may improve the production of high-quality biomass for bio-oil.

Multimodal sensorimotor system in unicellular zoospores of a fungus.

Complex sensory systems often underlie critical behaviors, including avoiding predators and locating prey, mates and shelter. Multisensory systems that control motor behavior even appear in unicellular eukaryotes, such as Chlamydomonas, which are important laboratory models for sensory biology. However, we know of no unicellular opisthokonts that control motor behavior using a multimodal sensory system. Therefore, existing single-celled models for multimodal sensorimotor integration are very distantly related to animals. Here, we describe a multisensory system that controls the motor function of unicellular fungal zoospores. We found that zoospores of Allomyces arbusculus exhibit both phototaxis and chemotaxis. Furthermore, we report that closely related Allomyces species respond to either the chemical or the light stimuli presented in this study, not both, and likely do not share this multisensory system. This diversity of sensory systems within Allomyces provides a rare example of a comparative framework that can be used to examine the evolution of sensory systems following the gain/loss of available sensory modalities. The tractability of Allomyces and related fungi as laboratory organisms will facilitate detailed mechanistic investigations into the genetic underpinnings of novel photosensory systems, and how multisensory systems may have functioned in early opisthokonts before multicellularity allowed for the evolution of specialized cell types.

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 Genome Sizes of Ostracod Crustaceans Correlate with Body Size and Evolutionary History, but not Environment.

Within animals, a positive correlation between genome size and body size has been detected in several taxa but not in others, such that it remains unknown how pervasive this pattern may be. Here, we provide another example of a positive relationship in a group of crustaceans whose genome sizes have not previously been investigated. We analyze genome size estimates for 46 species across the 2 most diverse orders of Class Ostracoda, commonly known as seed shrimps, including 29 new estimates made using Feulgen image analysis densitometry and flow cytometry. Genome sizes in this group range ~80-fold, a level of variability that is otherwise not seen in crustaceans with the exception of some malacostracan orders. We find a strong positive correlation between genome size and body size across all species, including after phylogenetic correction. We additionally detect evidence of XX/XO sex determination in 3 species of marine ostracods where male and female genome sizes were estimated. On average, genome sizes are larger but less variable in Order Myodocopida than in Order Podocopida, and marine ostracods have larger genomes than freshwater species, but this appears to be explained by phylogenetic inertia. The relationship between phylogeny, genome size, body size, and habitat is complex in this system and provides a baseline for future studies examining the interactions of these biological traits.

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.

The Amphimedon queenslandica genome and the evolution of animal complexity.

Sponges are an ancient group of animals that diverged from other metazoans over 600 million years ago. Here we present the draft genome sequence of Amphimedon queenslandica, a demosponge from the Great Barrier Reef, and show that it is remarkably similar to other animal genomes in content, structure and organization. Comparative analysis enabled by the sequencing of the sponge genome reveals genomic events linked to the origin and early evolution of animals, including the appearance, expansion and diversification of pan-metazoan transcription factor, signalling pathway and structural genes. This diverse 'toolkit' of genes correlates with critical aspects of all metazoan body plans, and comprises cell cycle control and growth, development, somatic- and germ-cell specification, cell adhesion, innate immunity and allorecognition. Notably, many of the genes associated with the emergence of animals are also implicated in cancer, which arises from defects in basic processes associated with metazoan multicellularity.

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.

Eye-independent, light-activated chromatophore expansion (LACE) and expression of phototransduction genes in the skin of Octopus bimaculoides.

Cephalopods are renowned for changing the color and pattern of their skin for both camouflage and communication. Yet, we do not fully understand how cephalopods control the pigmented chromatophore organs in their skin and change their body pattern. Although these changes primarily rely on eyesight, we found that light causes chromatophores to expand in excised pieces of Octopus bimaculoides skin. We call this behavior light-activated chromatophore expansion (or LACE). To uncover how octopus skin senses light, we used antibodies against r-opsin phototransduction proteins to identify sensory neurons that express r-opsin in the skin. We hypothesized that octopus LACE relies on the same r-opsin phototransduction cascade found in octopus eyes. By creating an action spectrum for the latency to LACE, we found that LACE occurred most quickly in response to blue light. We fit our action spectrum data to a standard opsin curve template and estimated the λmax of LACE to be 480 nm. Consistent with our hypothesis, the maximum sensitivity of the light sensors underlying LACE closely matches the known spectral sensitivity of opsin from octopus eyes. LACE in isolated preparations suggests that octopus skin is intrinsically light sensitive and that this dispersed light sense might contribute to their unique and novel patterning abilities. Finally, our data suggest that a common molecular mechanism for light detection in eyes may have been co-opted for light sensing in octopus skin and then used for LACE.

Opsins in Limulus eyes: characterization of three visible light-sensitive opsins unique to and co-expressed in median eye photoreceptors and a peropsin/RGR that is expressed in all eyes.

The eyes of the horseshoe crab Limulus polyphemus have long been used for studies of basic mechanisms of vision, and the structure and physiology of Limulus photoreceptors have been examined in detail. Less is known about the opsins Limulus photoreceptors express. We previously characterized a UV opsin (LpUVOps1) that is expressed in all three types of Limulus eyes (lateral compound eyes, median ocelli and larval eyes) and three visible light-sensitive rhabdomeric opsins (LpOps1, -2 and -5) that are expressed in Limulus lateral compound and larval eyes. Physiological studies showed that visible light-sensitive photoreceptors are also present in median ocelli, but the visible light-sensitive opsins they express were unknown. In the current study we characterize three newly identified, visible light-sensitive rhabdomeric opsins (LpOps6, -7 and -8) that are expressed in median ocelli. We show that they are ocellar specific and that all three are co-expressed in photoreceptors distinct from those expressing LpUVOps1. Our current findings show that the pattern of opsin expression in Limulus eyes is much more complex than previously thought and extend our previous observations of opsin co-expression in visible light-sensitive Limulus photoreceptors. We also characterize a Limulus peropsin/RGR (LpPerOps1). We examine the phylogenetic relationship of LpPerOps1 with other peropsins and RGRs, demonstrate that LpPerOps1 transcripts are expressed in each of the three types of Limulus eyes and show that the encoded protein is expressed in membranes of cells closely associated with photoreceptors in each eye type. These finding suggest that peropsin was in the opsin repertoire of euchelicerates.

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.

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.

Phylotranscriptomics to bring the understudied into the fold: monophyletic ostracoda, fossil placement, and pancrustacean phylogeny.

An ambitious, yet fundamental goal for comparative biology is to understand the evolutionary relationships for all of life. However, many important taxonomic groups have remained recalcitrant to inclusion into broader scale studies. Here, we focus on collection of 9 new 454 transcriptome data sets from Ostracoda, an ancient and diverse group with a dense fossil record, which is often undersampled in broader studies. We combine the new transcriptomes with a new morphological matrix (including fossils) and existing expressed sequence tag, mitochondrial genome, nuclear genome, and ribosomal DNA data. Our analyses lead to new insights into ostracod and pancrustacean phylogeny. We obtained support for three epic pancrustacean clades that likely originated in the Cambrian: Oligostraca (Ostracoda, Mystacocarida, Branchiura, and Pentastomida); Multicrustacea (Copepoda, Malacostraca, and Thecostraca); and a clade we refer to as Allotriocarida (Hexapoda, Remipedia, Cephalocarida, and Branchiopoda). Within the Oligostraca clade, our results support the unresolved question of ostracod monophyly. Within Multicrustacea, we find support for Thecostraca plus Copepoda, for which we suggest the name Hexanauplia. Within Allotriocarida, some analyses support the hypothesis that Remipedia is the sister taxon to Hexapoda, but others support Branchiopoda + Cephalocarida as the sister group of hexapods. In multiple different analyses, we see better support for equivocal nodes using slow-evolving genes or when excluding distant outgroups, highlighting the increased importance of conditional data combination in this age of abundant, often anonymous data. However, when we analyze the same set of species and ignore rate of gene evolution, we find higher support when including all data, more in line with a "total evidence" philosophy. By concatenating molecular and morphological data, we place pancrustacean fossils in the phylogeny, which can be used for studies of divergence times in Pancrustacea, Arthropoda, or Metazoa. Our results and new data will allow for attributes of Ostracoda, such as its amazing fossil record and diverse biology, to be leveraged in broader scale comparative studies. Further, we illustrate how adding extensive next-generation sequence data from understudied groups can yield important new phylogenetic insights into long-standing questions, especially when carefully analyzed in combination with other data.