BACKGROUND
an integrated understanding of the function and evolution of complex biological traits – such as eyes – is a major goal for biologists.
ideally, we will learn how specific genes evolved to influence particular phenotypes at multiple levels of organization.
eyes are an excellent system for establishing causative links between genotype and phenotype because their genetic components tend to be well-characterized and deeply conserved  <cit> .
however, we lack genomic or transcriptomic resources for many species that are amenable to the physiological, developmental, or evolutionary study of eyes and vision.
new tools for high throughput sequencing  and de novo assembly provide a solution to this problem, as they make the development of transcriptomic resources feasible for almost any organism, even invertebrate animals where few full genomes are available relative to species diversity  <cit> .
a remaining challenge is that it can be difficult to assign identities to the sequences that comprise transcriptomes from non-model organisms.
existing methods for annotating transcriptomes – e.g.
blast2go  <cit> , gotcha  <cit> , gofigure  <cit> , ontoblast  <cit> , and autofact  <cit>  – tend to rely upon similarities between new sequences and previously characterized genes, an approach which can give misleading results because there is no consistent method for predicting how similar an uncharacterized gene must be to a characterized one to share a common function.
phylogenetic analyses provide a more objective way to annotate transcriptomes: if a sequence falls in a clade of genes whose functions are characterized and similar to each other, we can use parsimony to infer that the sequence has a similar function.
a draw-back to phylogenetic analyses is that they tend to be time-consuming because of the need to re-calculate trees each time that new data are collected .
in response, we used existing tools for phylogenetic analysis in the osiris package  <cit>  of galaxy  <cit>  – an open-source workflow management system – to produce a computationally efficient, tree-based approach for annotating transcriptomes that we term phylogenetically-informed annotation .
first, we used tools in galaxy and protein sequences predicted from  <dig> fully-sequenced genomes to produce trees for  <dig> gene families from a metazoan light-interaction toolkit , i.e.
a set of genetic components that metazoans use to build eyes and other light-interacting structures.
lit  <dig>  includes genes that animals use to detect light , absorb light , and refract light , as well as transcription factors associated with the development of eyes and other light-interacting structures .
second, we designed a workflow in galaxy that uses e-values from blast  <cit>  to identify potential homologs of lit genes.
the workflow then employs a choice of multiple sequence alignment programs  and a repurposed evolutionary placement algorithm  to place these sequences using maximum likelihood on to the trees that we calculated earlier for genes from lit  <dig> .
finally, we tested our approach by using pia to search for lit genes in  <dig> new transcriptomes that we generated using the roche  <dig> platform.
these transcriptomes – generated from rna expressed in light-interacting tissues from a range of cephalopod mollusks, arthropods, and cubozoan cnidarians – are all from animals that are not traditional model organisms, but are well-suited for answering particular questions about the function and evolution of eyes and other light-interacting structures.
implementation
here, we describe the implementation of phylogenetically-informed annotation , a new approach for using phylogenetic methods to rapidly annotate transcriptomes from non-model organisms.
we focus on a set of  <dig> genes that we selected to form a metazoan light interaction toolkit .
we make lit-pia available through an instance of galaxy on a publicly accessible web server .
users can find a written tutorial and a screencast demonstration linked on that site.
in galaxy, we implemented a tool called pia, which executes a perl script that calls a series of bioinformatics tools, including blast  <cit> , a choice of multiple sequence alignment programs  and raxml  <cit> .
implementing pia in galaxy allows us to integrate the pia tool with other useful tools, such as tab2trees of the osiris package  <cit> , which visualizes multiple phylogenetic trees in a single pdf file.
furthermore, the galaxy instance allows for user-friendly annotation of lit genes using pia.
our pre-calculated trees for lit genes can be selected from a menu on our galaxy pia tool, and all the gene trees are also available on the bitbucket public repository and documentation for using the public website is available there  in the docs subdirectory.
RESULTS
new trees for  <dig> lit genes
we generated maximum likelihood trees for  <dig> genes from a metazoan light interaction toolkit .
from our efforts, we noted that many lit genes do not have orthologous relationships across metazoa.
for example, we find that lit genes with similar functions in distantly related taxa  are often paralogs, not orthologs, due to lineage-specific gene duplications.
although evidence suggests that orthologs tend to be more similar functionally than paralogs, this does not hold true in the case of all gene families  <cit> .
thus, we conclude that tree-building is a useful approach for inferring the function of certain lit genes, yet we still urge researchers to use caution when assigning functions to uncharacterized genes in the absence of functional tests and based on phylogenetic relationships alone.
further, how one selects the sequences used to build a tree may have a significant influence on the inferences drawn about the ancestral function and patterns of diversification of gene families.
thus, we advocate for an objective, repeatable approach to choosing sequences for gene trees , especially when using phylogenetic relationships between these sequences to make inferences about the functions of newly sequenced genes.
new transcriptomes for integrative and comparative vision research
we generated  <dig> transcriptomes for light-interacting tissues from a range of cephalopod mollusks, arthropods, and cubozoan cnidarians .
we sequenced transcriptomes from these taxa because they lacked genomic or transcriptomic resources, but are well-suited for answering certain questions about the function, development, and evolution of eyes and other light-interacting structures.
the sizes of our transcriptomes varied .
for example, the number of total bases in our transcriptomes averaged  <dig> ,000 ±  <dig> , <dig>  with a low of  <dig>  bp  and a high of  <dig> ,000 bp .
the number of isotigs per transcriptome ranged from  <dig>  to  <dig>   and the mean sizes of isotigs ranged from 530 bp  to  <dig>  bp .table  <dig>
collection data for the samples from which we generated  <dig> new transcriptomes
species
description
tissue
collection
location
lat.
long.
depth
chiroteuthis calyx
euprymna scolopes
galiteuthis armata
octopus bimaculoides
uroteuthis edulis
vampyroteuthis infernalis
asellus aquaticus
asellus aquaticus
asellus aquaticus
asellus aquaticus
benthesicymus bartletti
caecidotea bicrenata
caecidotea bicrenata
caecidotea forbesi
caecidotea forbesi
euphilomedes carcharodonta
hemisquilla californiensis
ischnura ramburii
limulus polyphemus
limulus polyphemus
limulus polyphemus
procambarus alleni
procambarus franzi
pseudosquilla ciliata
systellaspis debilis
telebasis salva
tripedalia cystophora
tripedalia cystophora
phylogenetically-informed annotation
to rapidly identify potential lit  <dig>  genes in our genetic datasets, we implemented pia in galaxy.
unlike past efforts at using phylogenetic methods to annotate transcriptomes, pia does not require the re-calculation of gene trees every time a new sequence is to be analyzed.
the output from pia is a gene tree or a set of gene trees in newick format that can be viewed using existing tools in galaxy, such as tab2trees from the osiris package  <cit> .
these trees include sequences identified from predicted protein databases associated with  <dig> fully-sequenced genomes .
the trees also include sequences marked as either landmarks or queries .
landmarks are genes  whose functions and/or patterns of expression have been characterized relatively well.
sequences marked "landmark1" – which are highlighted with red squares when the trees are viewed using our tab2trees tool – are well-characterized lit genes.
sequences marked "landmark2" are also well-characterized genes, but are those that have functions different than the lit genes that we are seeking.
for example, we included certain non-opsin gpcrs in our trees for opsins, but we labeled them as landmark2s because we have evidence that they are not involved in the detection of light.
queries marked “query” – which are noted by yellow circles in the output from the tab2trees tool – represent potential lit genes that pia has identified from a particular genetic dataset.
promising queries from a transcriptome  will tend to fall on short branches in phylogenetic positions that are sensible given established relationships between species .
the output from pia may also include query sequences that are close relatives, but not orthologs, of lit genes .
thus, we urge users of pia to inspect carefully where queries fall on their respective gene trees and to make inferences about function accordingly.figure  <dig>
an example of results from phylogenetically-informed annotation .
here, we search a transcriptome generated for an eye from the squid chiroteuthis calyx for relatives of the gene g protein-coupled receptor kinase  <dig> , a component of the rhabdomeric phototransduction pathway.
sequences marked with red squares and labeled "landmark1" are homologs of gprk <dig> that have been well-characterized functionally and are thought to share similar functions.
in contrast, sequences labeled "landmark2" are well-characterized genes that are more distantly related to gprk <dig>  sequences marked with yellow circles and labeled "query" are protein sequences predicted from our transcriptome for c. calyx.
based on their phylogenetic positions and branch lengths, it is likely that one hit  represents an ortholog of gprk <dig> and that two hits  represent genes that are distant paralogs of gprk <dig>  we conclude that the eyes of c. calyx express an ortholog of gprk <dig>  a component of the rhabdomeric phototransduction pathway.
building additional gene trees will allow researchers to use pia to search for lit genes that are not included in our initial list or to search for new sets of genes.
for example, there has been much recent interest in the sets of genes that underlie the process of biomineralization in animals  <cit> .
extensive databases of these genes have been published for invertebrate taxa  that are not traditional model systems  <cit> .
by building trees for these sets of genes and applying our methods for pia, researchers will be able to survey new transcriptomes rapidly for genes that may be involved in biomineralization.
the distribution of lit genes across  <dig> new vision-related transcriptomes
across our  <dig> newly-sequenced transcriptomes, pia identified potential orthologs of  <dig> of the  <dig> genes included in lit  <dig>  .
we recovered certain genes from our transcriptomes far more often than others.
genes from lit  <dig>  that were expressed in ten or more of our transcriptomes included several components of the rhabdomeric phototransduction pathway, which is employed by the photoreceptors found in the eyes of many invertebrates  <cit> .
these components include: arrestin , gq alpha , gq beta , protein kinase c , r-opsin , phospholipase c , and transient receptor potential protein .
ten or more of our transcriptomes also contained the enzyme aminolevulinate synthase , a component of the heme synthesis pathway  <cit> , as well as an aldehyde dehydrogenase  that is related to the Ω-crystallins expressed in the lenses of the camera eyes of cephalopods  <cit>  and the mirror-based eyes of scallops  <cit> .
amino acid and nucleotide sequences for the potential lit genes that we identified from our  <dig> transcriptomes, along with the corresponding gene trees, are available on our publicly-accessible web server  under the shared data tab.figure  <dig>
the results of using phylogenetically-informed annotation  to search  <dig> new transcriptomes for light-interacting genes.
here, cells shaded in black represent orthologs of lit genes that are present in our transcriptomes.
we have named genes based on conventions for drosophila melanogaster whenever possible.
otherwise, gene names are given for mus musculus.
cells marked "c" are hits from our transcriptomes that may represent contamination or assembly errors.
the columns represent the following transcriptomes : 1 = chiroteuthis calyx eye; 2 = euprymna scolopes eye; 3 = galiteuthis armata eye; 4 = octopus bimaculoides skin; 5 = uroteuthis edulis eye; 6 = vampyroteuthis infernalis eye; 7 = asellus aquaticus cave head; 8 = asellus aquaticus embryos and hatchlings; 9 = asellus aquaticus hybrid head; 10 = asellus aquaticus surface head; 11 = benthesicymus bartletti eye; 12 = caecidotea bicrenata adult head; 13 = caecidotea bicrenata embryos; 14 = caecidotea forbesi adult head; 15 = caecidotea forbesi embryos; 16 = euphilomedes carcharodonta embryos; 17 = hemisquilla californiensis eyes; 18 = ischnura ramburii head; 19 = limulus polyphemus lateral eye; 20 = limulus polyphemus median eye; 21 = limulus polyphemus ventral eye; 22 = procambarus alleni eye; 23 = procambarus franzi eye; 24 = pseudosquilla ciliata eye; 25 = systellaspis debilis eye; 26 = telebasis salva head; 27 = tripedalia cystophora eyes; 28 = tripedalia cystophora planula larvae.
the transcriptomes that we generated for cephalopods contained between two  and sixteen  lit genes .
the majority of these genes represent components of the rhabdomeric phototransduction pathway, which is known to confer light-sensitivity to photoreceptors from the retinas of cephalopods  <cit> .
these components include r-opsins, subunits of the hetero-trimeric gq protein with which r-opsins interact, phospholipase c, and the ion channel trp.
we also recovered a number of lens crystallins, including relatives of the s- and Ω-crystallins indentified previously from the lenses of cephalopods  <cit> .
lastly, we found orthologs of lit genes that are associated with two vision-related features that may be unique to the eyes of cephalopods.
first, we found orthologs of the retinoid-binding protein ralbp, which is involved in regenerating the chromophores employed by the visual pigments of cephalopods  <cit> .
second, we found sequences that may represent reflectins, which are proteins that contribute to the biological mirrors that cephalopods use to camouflage their eyes  <cit> .
our transcriptomes from arthropods contained between three  and  <dig>  potential orthologs of genes from lit  <dig>  .
as in cephalopods, many of the genes we identified in arthropods represent components of the rhabdomeric phototransduction pathway.
we also identified a number of genes that are associated with the synthesis of pterins and ommochromes, types of pigment found previously in the compound eyes of certain arthropods  <cit> .
additionally, several transcriptomes contained genes  related to those that help synthesize the chromophores employed by the visual pigments in the eyes of the fruit fly drosophila melanogaster  <cit> .
finally, our transcriptomes for the adult rhopalia and planula larvae of the cubozoan cnidarian tripedalia cystophora contained thirteen and seven light-interacting genes, respectively .
the majority of these genes are related to those associated with phototransduction in bilaterians.
for example, we found a previously characterized opsin in our transcriptome for rhopalia from adult t. cystophora  <cit> , as well as a gs alpha subunit that is associated with light-detection in other cnidarians  <cit> .
we also found evidence of j-crystallins, which are lens crystallins unique to the camera-type eyes of cubozoans  <cit> .
it is important to note that the absence of a particular gene from a transcriptome is not necessarily informative.
even if a transcriptome is “complete”, it is only complete for a particular piece of tissue, from a particular animal, at a particular time.
thus, we have tried to draw general conclusions and points of future interest from the genes that we identified from our transcriptomes.
we hope researchers will apply the approach that we have developed here to re-visit these light-interacting tissues and sequence more deeply and more broadly so that statistically meaningful comparisons of gene expression may be drawn between them.
CONCLUSIONS
in this study, collaborators from multiple institutions worked together to produce new data and new approaches for studying genes expressed by eyes and other light-interacting tissues.
we used high throughput sequencing to discover orthologs of light-interacting genes expressed in  <dig> vision-related tissues from a range of cephalopod mollusks, arthropods, and cnidarians.
we have made available to vision researchers these genetic data, as well as new resources for analyzing high throughput genetic data.
specifically, we calculated trees to understand the evolutionary histories of  <dig> separate genes known to be involved with the function or development of light-interacting structures such as eyes.
these trees can now be used to annotate transcriptomes by comparing the evolutionary similarities between newly identified sequences and genes that have been characterized previously through studies of their expression patterns and functions.
these tools and analyses can be implemented by anyone using a set of online, flexible, user-friendly workflows implemented in galaxy.
these new data and tools will accelerate the understanding of genotype-phenotype connections and evolution in a diversity of animal visual systems.

 an integrated understanding of the function and evolution of complex biological traits – such as eyes – is a major goal for biologists. ideally, we will learn how specific genes evolved to influence particular phenotypes at multiple levels of organization. eyes are an excellent system for establishing causative links between genotype and phenotype because their genetic components tend to be well-characterized and deeply conserved. however, we lack genomic or transcriptomic resources for many species that are amenable to the physiological, developmental, or evolutionary study of eyes and vision. new tools for high throughput sequencing and de novo assembly provide a solution to this problem, as they make the development of transcriptomic resources feasible for almost any organism, even invertebrate animals where few full genomes are available relative to species diversity. a remaining challenge is that it can be difficult to assign identities to the sequences that comprise transcriptomes from non-model organisms. existing methods for annotating transcriptomes – e.g. blast2go, gotcha, gofigure, ontoblast, and autofact – tend to rely upon similarities between new sequences and previously characterized genes, an approach which can give misleading results because there is no consistent method for predicting how similar an uncharacterized gene must be to a characterized one to share a common function. phylogenetic analyses provide a more objective way to annotate transcriptomes: if a sequence falls in a clade of genes whose functions are characterized and similar to each other, we can use parsimony to infer that the sequence has a similar function. a draw-back to phylogenetic analyses is that they tend to be time-consuming because of the need to re-calculate trees each time that new data are collected. in response, we used existing tools for phylogenetic analysis in the osiris package of galaxy – an open-source workflow management system – to produce a computationally efficient, tree-based approach for annotating transcriptomes that we term phylogenetically-informed annotation. first, we used tools in galaxy and protein sequences predicted from fully-sequenced genomes to produce trees for gene families from a metazoan light-interaction toolkit, i.e. a set of genetic components that metazoans use to build eyes and other light-interacting structures. lit includes genes that animals use to detect light, absorb light, and refract light, as well as transcription factors associated with the development of eyes and other light-interacting structures. second, we designed a workflow in galaxy that uses e-values from blast to identify potential homologs of lit genes. the workflow then employs a choice of multiple sequence alignment programs and a repurposed evolutionary placement algorithm to place these sequences using maximum likelihood on to the trees that we calculated earlier for genes from lit. finally, we tested our approach by using pia to search for lit genes in new transcriptomes that we generated using the roche platform. these transcriptomes – generated from rna expressed in light-interacting tissues from a range of cephalopod mollusks, arthropods, and cubozoan cnidarians – are all from animals that are not traditional model organisms, but are well-suited for answering particular questions about the function and evolution of eyes and other light-interacting structures. implementation here, we describe the implementation of phylogenetically-informed annotation, a new approach for using phylogenetic methods to rapidly annotate transcriptomes from non-model organisms. we focus on a set of genes that we selected to form a metazoan light interaction toolkit. we make lit-pia available through an instance of galaxy on a publicly accessible web server. users can find a written tutorial and a screencast demonstration linked on that site. in galaxy, we implemented a tool called pia, which executes a perl script that calls a series of bioinformatics tools, including blast, a choice of multiple sequence alignment programs and raxml. implementing pia in galaxy allows us to integrate the pia tool with other useful tools, such as tab2trees of the osiris package, which visualizes multiple phylogenetic trees in a single pdf file. furthermore, the galaxy instance allows for user-friendly annotation of lit genes using pia. our pre-calculated trees for lit genes can be selected from a menu on our galaxy pia tool, and all the gene trees are also available on the bitbucket public repository and documentation for using the public website is available there in the docs subdirectory.  new trees for lit genes we generated maximum likelihood trees for genes from a metazoan light interaction toolkit. from our efforts, we noted that many lit genes do not have orthologous relationships across metazoa. for example, we find that lit genes with similar functions in distantly related taxa are often paralogs, not orthologs, due to lineage-specific gene duplications. although evidence suggests that orthologs tend to be more similar functionally than paralogs, this does not hold true in the case of all gene families. thus, we conclude that tree-building is a useful approach for inferring the function of certain lit genes, yet we still urge researchers to use caution when assigning functions to uncharacterized genes in the absence of functional tests and based on phylogenetic relationships alone. further, how one selects the sequences used to build a tree may have a significant influence on the inferences drawn about the ancestral function and patterns of diversification of gene families. thus, we advocate for an objective, repeatable approach to choosing sequences for gene trees, especially when using phylogenetic relationships between these sequences to make inferences about the functions of newly sequenced genes. new transcriptomes for integrative and comparative vision research we generated transcriptomes for light-interacting tissues from a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. we sequenced transcriptomes from these taxa because they lacked genomic or transcriptomic resources, but are well-suited for answering certain questions about the function, development, and evolution of eyes and other light-interacting structures. the sizes of our transcriptomes varied. for example, the number of total bases in our transcriptomes averaged,000 ± , with a low of  bp and a high of,000 bp. the number of isotigs per transcriptome ranged from to and the mean sizes of isotigs ranged from 530 bp to  bp.table collection data for the samples from which we generated new transcriptomes species description tissue collection location lat. long. depth chiroteuthis calyx euprymna scolopes galiteuthis armata octopus bimaculoides uroteuthis edulis vampyroteuthis infernalis asellus aquaticus asellus aquaticus asellus aquaticus asellus aquaticus benthesicymus bartletti caecidotea bicrenata caecidotea bicrenata caecidotea forbesi caecidotea forbesi euphilomedes carcharodonta hemisquilla californiensis ischnura ramburii limulus polyphemus limulus polyphemus limulus polyphemus procambarus alleni procambarus franzi pseudosquilla ciliata systellaspis debilis telebasis salva tripedalia cystophora tripedalia cystophora phylogenetically-informed annotation to rapidly identify potential lit genes in our genetic datasets, we implemented pia in galaxy. unlike past efforts at using phylogenetic methods to annotate transcriptomes, pia does not require the re-calculation of gene trees every time a new sequence is to be analyzed. the output from pia is a gene tree or a set of gene trees in newick format that can be viewed using existing tools in galaxy, such as tab2trees from the osiris package. these trees include sequences identified from predicted protein databases associated with fully-sequenced genomes. the trees also include sequences marked as either landmarks or queries. landmarks are genes whose functions and/or patterns of expression have been characterized relatively well. sequences marked "landmark1" – which are highlighted with red squares when the trees are viewed using our tab2trees tool – are well-characterized lit genes. sequences marked "landmark2" are also well-characterized genes, but are those that have functions different than the lit genes that we are seeking. for example, we included certain non-opsin gpcrs in our trees for opsins, but we labeled them as landmark2s because we have evidence that they are not involved in the detection of light. queries marked “query” – which are noted by yellow circles in the output from the tab2trees tool – represent potential lit genes that pia has identified from a particular genetic dataset. promising queries from a transcriptome will tend to fall on short branches in phylogenetic positions that are sensible given established relationships between species. the output from pia may also include query sequences that are close relatives, but not orthologs, of lit genes. thus, we urge users of pia to inspect carefully where queries fall on their respective gene trees and to make inferences about function accordingly.figure an example of results from phylogenetically-informed annotation. here, we search a transcriptome generated for an eye from the squid chiroteuthis calyx for relatives of the gene g protein-coupled receptor kinase, a component of the rhabdomeric phototransduction pathway. sequences marked with red squares and labeled "landmark1" are homologs of gprk that have been well-characterized functionally and are thought to share similar functions. in contrast, sequences labeled "landmark2" are well-characterized genes that are more distantly related to gprk sequences marked with yellow circles and labeled "query" are protein sequences predicted from our transcriptome for c. calyx. based on their phylogenetic positions and branch lengths, it is likely that one hit represents an ortholog of gprk and that two hits represent genes that are distant paralogs of gprk we conclude that the eyes of c. calyx express an ortholog of gprk a component of the rhabdomeric phototransduction pathway. building additional gene trees will allow researchers to use pia to search for lit genes that are not included in our initial list or to search for new sets of genes. for example, there has been much recent interest in the sets of genes that underlie the process of biomineralization in animals. extensive databases of these genes have been published for invertebrate taxa that are not traditional model systems. by building trees for these sets of genes and applying our methods for pia, researchers will be able to survey new transcriptomes rapidly for genes that may be involved in biomineralization. the distribution of lit genes across new vision-related transcriptomes across our newly-sequenced transcriptomes, pia identified potential orthologs of of the genes included in lit. we recovered certain genes from our transcriptomes far more often than others. genes from lit that were expressed in ten or more of our transcriptomes included several components of the rhabdomeric phototransduction pathway, which is employed by the photoreceptors found in the eyes of many invertebrates. these components include: arrestin, gq alpha, gq beta, protein kinase c, r-opsin, phospholipase c, and transient receptor potential protein. ten or more of our transcriptomes also contained the enzyme aminolevulinate synthase, a component of the heme synthesis pathway, as well as an aldehyde dehydrogenase that is related to the Ω-crystallins expressed in the lenses of the camera eyes of cephalopods and the mirror-based eyes of scallops. amino acid and nucleotide sequences for the potential lit genes that we identified from our transcriptomes, along with the corresponding gene trees, are available on our publicly-accessible web server under the shared data tab.figure the results of using phylogenetically-informed annotation to search new transcriptomes for light-interacting genes. here, cells shaded in black represent orthologs of lit genes that are present in our transcriptomes. we have named genes based on conventions for drosophila melanogaster whenever possible. otherwise, gene names are given for mus musculus. cells marked "c" are hits from our transcriptomes that may represent contamination or assembly errors. the columns represent the following transcriptomes: 1 = chiroteuthis calyx eye; 2 = euprymna scolopes eye; 3 = galiteuthis armata eye; 4 = octopus bimaculoides skin; 5 = uroteuthis edulis eye; 6 = vampyroteuthis infernalis eye; 7 = asellus aquaticus cave head; 8 = asellus aquaticus embryos and hatchlings; 9 = asellus aquaticus hybrid head; 10 = asellus aquaticus surface head; 11 = benthesicymus bartletti eye; 12 = caecidotea bicrenata adult head; 13 = caecidotea bicrenata embryos; 14 = caecidotea forbesi adult head; 15 = caecidotea forbesi embryos; 16 = euphilomedes carcharodonta embryos; 17 = hemisquilla californiensis eyes; 18 = ischnura ramburii head; 19 = limulus polyphemus lateral eye; 20 = limulus polyphemus median eye; 21 = limulus polyphemus ventral eye; 22 = procambarus alleni eye; 23 = procambarus franzi eye; 24 = pseudosquilla ciliata eye; 25 = systellaspis debilis eye; 26 = telebasis salva head; 27 = tripedalia cystophora eyes; 28 = tripedalia cystophora planula larvae. the transcriptomes that we generated for cephalopods contained between two and sixteen lit genes. the majority of these genes represent components of the rhabdomeric phototransduction pathway, which is known to confer light-sensitivity to photoreceptors from the retinas of cephalopods. these components include r-opsins, subunits of the hetero-trimeric gq protein with which r-opsins interact, phospholipase c, and the ion channel trp. we also recovered a number of lens crystallins, including relatives of the s- and Ω-crystallins indentified previously from the lenses of cephalopods. lastly, we found orthologs of lit genes that are associated with two vision-related features that may be unique to the eyes of cephalopods. first, we found orthologs of the retinoid-binding protein ralbp, which is involved in regenerating the chromophores employed by the visual pigments of cephalopods. second, we found sequences that may represent reflectins, which are proteins that contribute to the biological mirrors that cephalopods use to camouflage their eyes. our transcriptomes from arthropods contained between three and potential orthologs of genes from lit. as in cephalopods, many of the genes we identified in arthropods represent components of the rhabdomeric phototransduction pathway. we also identified a number of genes that are associated with the synthesis of pterins and ommochromes, types of pigment found previously in the compound eyes of certain arthropods. additionally, several transcriptomes contained genes related to those that help synthesize the chromophores employed by the visual pigments in the eyes of the fruit fly drosophila melanogaster. finally, our transcriptomes for the adult rhopalia and planula larvae of the cubozoan cnidarian tripedalia cystophora contained thirteen and seven light-interacting genes, respectively. the majority of these genes are related to those associated with phototransduction in bilaterians. for example, we found a previously characterized opsin in our transcriptome for rhopalia from adult t. cystophora, as well as a gs alpha subunit that is associated with light-detection in other cnidarians. we also found evidence of j-crystallins, which are lens crystallins unique to the camera-type eyes of cubozoans. it is important to note that the absence of a particular gene from a transcriptome is not necessarily informative. even if a transcriptome is “complete”, it is only complete for a particular piece of tissue, from a particular animal, at a particular time. thus, we have tried to draw general conclusions and points of future interest from the genes that we identified from our transcriptomes. we hope researchers will apply the approach that we have developed here to re-visit these light-interacting tissues and sequence more deeply and more broadly so that statistically meaningful comparisons of gene expression may be drawn between them.  in this study, collaborators from multiple institutions worked together to produce new data and new approaches for studying genes expressed by eyes and other light-interacting tissues. we used high throughput sequencing to discover orthologs of light-interacting genes expressed in vision-related tissues from a range of cephalopod mollusks, arthropods, and cnidarians. we have made available to vision researchers these genetic data, as well as new resources for analyzing high throughput genetic data. specifically, we calculated trees to understand the evolutionary histories of separate genes known to be involved with the function or development of light-interacting structures such as eyes. these trees can now be used to annotate transcriptomes by comparing the evolutionary similarities between newly identified sequences and genes that have been characterized previously through studies of their expression patterns and functions. these tools and analyses can be implemented by anyone using a set of online, flexible, user-friendly workflows implemented in galaxy. these new data and tools will accelerate the understanding of genotype-phenotype connections and evolution in a diversity of animal visual systems. 
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, which places uncharacterized genes into pre-calculated phylogenies of gene families. next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based evolutionary placement algorithm to place sequences of potential lit genes on to our pre-calculated gene trees. we generated maximum likelihood trees for genes from a light interaction toolkit, a collection of genes that underlie the function or development of light-interacting structures in metazoans. 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. to do so, we searched protein sequences predicted from fully-sequenced genomes and built trees using tools for phylogenetic analysis in the osiris package of galaxy. our new trees for lit genes are available on the bitbucket public repository and we demonstrate pia on a publicly-accessible web server. finally, we implemented pia in galaxy and used it to search for lit genes in newly-sequenced transcriptomes from the light-interacting tissues of a range of cephalopod mollusks, arthropods, and cubozoan cnidarians. we also introduce pia, a high throughput method for using phylogenetic relationships to identify lit genes in transcriptomes from non-model organisms. tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes 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. our new trees for lit genes will be a valuable resource for researchers studying the evolution of eyes or other light-interacting structures.
 tools for high throughput sequencing and de novo assembly make the analysis of transcriptomes 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, which places uncharacterized genes into pre-calculated phylogenies of gene families.  we generated maximum likelihood trees for genes from a light interaction toolkit, 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 fully-sequenced genomes and built trees using tools for phylogenetic analysis in the osiris package of galaxy. next, to rapidly annotate transcriptomes from organisms that lack sequenced genomes, we repurposed a maximum likelihood-based evolutionary placement algorithm 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 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 and we demonstrate pia on a publicly-accessible web server.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords bioinformaticseyesevolutiongalaxynext-generation sequence analysisorthologyphototransductiontranscriptomesvisionissue-copyright-statement© the author 2014 