Women in tunicate research: Pioneers of the past and their present legacy.

The search for female scientists who pioneered the research on tunicates is hindered by the tradition of reporting only the first initials of authors' names on scientific publications using only the initials of their first names. While this practice has the theoretical merit of broadening the readership by preventing the possible bias that could be caused by the gender of the author(s) in some of the readers, it rendered the identification of female researchers active in, or before, the first half of the 20th century quite challenging. Sifting through several dozen electronic records, and with the help of references and/or quotes found online, we have stitched together the information that we were able to retrieve on the life of female scientists who authored some of the earliest publications on tunicates, and we have organized them in (approximate) chronological order. We have also compiled brief synopses of the findings of scientists active in the field of tunicate biology in more recent times, and organized them by subdiscipline.

Phylogenomic and morphological relationships among the botryllid ascidians (Subphylum Tunicata, Class Ascidiacea, Family Styelidae).

Ascidians (Phylum Chordata, Class Ascidiacea) are a large group of invertebrates which occupy a central role in the ecology of marine benthic communities. Many ascidian species have become successfully introduced around the world via anthropogenic vectors. The botryllid ascidians (Order Stolidobranchia, Family Styelidae) are a group of 53 colonial species, several of which are widespread throughout temperate or tropical and subtropical waters. However, the systematics and biology of this group of ascidians is not well-understood. To provide a systematic framework for this group, we have constructed a well-resolved phylogenomic tree using 200 novel loci and 55 specimens. A Principal Components Analysis of all species described in the literature using 31 taxonomic characteristics revealed that some species occupy a unique morphological space and can be easily identified using characteristics of adult colonies. For other species, additional information such as larval or life history characteristics may be required for taxonomic discrimination. Molecular barcodes are critical for guiding the delineation of morphologically similar species in this group.

Whole genome sequencing identifies an allele responsible for clear vs. turbid plaque morphology in a Mycobacteriophage.

BACKGROUND: Whole genome sequencing promises to revolutionize our ability to link genotypic and phenotypic variation in a wide range of model and non-model species. RESULTS: Here we describe the isolation and characterization of a novel mycobacteriophage named BGlluviae that grows on Mycobacterium smegmatis mc2155. BGlluviae normally produces turbid plaques but a spontaneous clear plaque was also recovered. The genomic DNA from pure populations of the BGlluviae phage and the clear plaque mutant were sequenced. A single substitution, at amino acid 54 (I to T), in the immunity repressor protein resulted in a clear plaque phenotype. CONCLUSIONS: This substitution is predicted to be located at the subunit interaction interface of the repressor protein, and thus prevents the establishment of lysogeny.

Evolution of Allorecognition in the Tunicata.

Allorecognition, the ability to distinguish self or kin from unrelated conspecifics, plays several important biological roles in invertebrate animals. Two of these roles include negotiating limited benthic space for colonial invertebrates, and inbreeding avoidance through self-incompatibility systems. Subphylum Tunicata (Phylum Chordata), the sister group to the vertebrates, is a promising group in which to study allorecognition. Coloniality has evolved many times independently in the tunicates, and the best known invertebrate self-incompatibility systems are in tunicates. Recent phylogenomic studies have coalesced around a phylogeny of the Tunicata as well as the Order Stolidobranchia within the Tunicata, providing a path forward for the study of allorecognition in this group.

Origin and Dispersal History of Two Colonial Ascidian Clades in the Botryllus schlosseri Species Complex.

Human-induced global warming and species introductions are rapidly altering the composition and functioning of Earth's marine ecosystems. Ascidians (Phylum Chordata, Subphylum Tunicata, Class Ascidiacea) are likely to play an increasingly greater role in marine communities. The colonial ascidian B. schlosseri is a cryptic species complex comprising five genetically divergent clades (A-E). Clade A is a global species, and Clade E has so far been identified in European waters only. Using the largest mitochondrial cytochrome oxidase I datasets yet assembled, we determine the origin and dispersal history of these species. Nucleotide diversity and Approximate Bayesian Computation analyses support a Pacific origin for Clade A, with two likely dispersal scenarios that both show the northwestern Atlantic populations establishing early in the history of the species. Both Discrete Phylogeographic Analysis and Approximate Bayesian Computation support an origin of Clade E on the French side of the English Channel. An unsampled lineage evolved from the French lineage, which reflects the conclusion from the median joining network that not all Clade E lineages have been sampled. This unsampled lineage gave rise to the haplotypes on the English side of the English Channel, which were the ancestors to the Mediterranean and Bay of Biscay populations. Clade E has a wider geographic range than previously thought, and shows evidence of recent range expansion. Both Clade A and Clade E should be considered widespread species: Clade A globally and Clade E within Europe.

Balancing selection on allorecognition genes in the colonial ascidian Botryllus schlosseri.

Allorecognition is the capability of an organism to recognize its own or related tissues. The colonial ascidian Botryllus schlosseri, which comprises five genetically distinct and divergent species (Clades A-E), contains two adjacent genes that control allorecognition: fuhcsec and fuhctm. These genes have been characterized extensively in Clade A and are highly polymorphic. Using alleles from 10 populations across the range of Clade A, we investigated the type and strength of selection maintaining this variation. Both fuhc genes exhibit higher within-population variation and lower population differentiation measures (FST) than neutral loci. The fuhc genes contain a substantial number of codons with >95% posterior probability of dN/dS > 1. fuhcsec and fuhctm also have polymorphisms shared between Clade A and Clade E that were present prior to speciation (trans-species polymorphisms). These results provide robust evidence that the fuhc genes are evolving under balancing selection.

Molecular evolution and in vitro characterization of Botryllus histocompatibility factor.

Botryllus schlosseri is a colonial ascidian with a natural ability to anastomose with another colony to form a vascular and hematopoietic chimera. In order to fuse, two individuals must share at least one allele at the highly polymorphic fuhc locus. Otherwise, a blood-based inflammatory response will occur resulting in a melanin scar at the sites of interaction. The single-locus genetic control of allorecognition makes B. schlosseri an attractive model to study the underlying molecular mechanisms. Over the past decade, several candidate genes involved in allorecognition have been identified, but how they ultimately contribute to allorecognition outcome remains poorly understood. Here, we report our initial molecular characterization of a recently identified candidate allodeterminant called Botryllus histocompatibility factor (bhf). bhf, both on a DNA and protein level, is the least polymorphic protein in the fuhc locus studied so far and, unlike other known allorecognition determinants, does not appear to be under any form of balancing or directional selection. Additionally, we identified a second isoform through mRNA-Seq and an EST assembly library which is missing exon 3, resulting in a C-terminally truncated form. We report via whole-mount fluorescent in situ hybridization that a subset of cells co-express bhf and cfuhc(sec). Finally, we observed BHF's localization in HEK293T at the cytoplasmic side of the plasma membrane in addition to the nucleus via a nuclear localization signal. Given the localization data thus far, we hypothesize that BHF may function as a scaffolding protein in a complex with other Botryllus proteins, rather than functioning as an allorecognition determinant.

Defining loci in restriction-based reduced representation genomic data from nonmodel species: sources of bias and diagnostics for optimal clustering.

Next generation sequencing holds great promise for applications of phylogeography, landscape genetics, and population genomics in wild populations of nonmodel species, but the robustness of inferences hinges on careful experimental design and effective bioinformatic removal of predictable artifacts. Addressing this issue, we use published genomes from a tunicate, stickleback, and soybean to illustrate the potential for bioinformatic artifacts and introduce a protocol to minimize two sources of error expected from similarity-based de-novo clustering of stacked reads: the splitting of alleles into different clusters, which creates false homozygosity, and the grouping of paralogs into the same cluster, which creates false heterozygosity. We present an empirical application focused on Ciona savignyi, a tunicate with very high SNP heterozygosity (~0.05), because high diversity challenges the computational efficiency of most existing nonmodel pipelines while also potentially exacerbating paralog artifacts. The simulated and empirical data illustrate the advantages of using higher sequence difference clustering thresholds than is typical and demonstrate the utility of our protocol for efficiently identifying an optimum threshold from data without prior knowledge of heterozygosity. The empirical Ciona savignyi data also highlight null alleles as a potentially large source of false homozygosity in restriction-based reduced representation genomic data.

The candidate histocompatibility locus of a Basal chordate encodes two highly polymorphic proteins.

The basal chordate Botryllus schlosseri undergoes a natural transplantation reaction governed by a single, highly polymorphic locus called the fuhc. Our initial characterization of this locus suggested it encoded a single gene alternatively spliced into two transcripts: a 555 amino acid-secreted form containing the first half of the gene, and a full-length, 1008 amino acid transmembrane form, with polymorphisms throughout the ectodomain determining outcome. We have now found that the locus encodes two highly polymorphic genes which are separated by a 227 bp intergenic region: first, the secreted form as previously described, and a second gene encoding a 531 amino acid membrane-bound gene containing three extracellular immunoglobulin domains. While northern blotting revealed only these two mRNAs, both PCR and mRNA-seq detect a single capped and polyadenylated transcript that encodes processed forms of both genes linked by the intergenic region, as well as other transcripts in which exons of the two genes are spliced together. These results might suggest that the two genes are expressed as an operon, during which both genes are co-transcribed and then trans-spliced into two separate messages. This type of transcriptional regulation has been described in tunicates previously; however, the membrane-bound gene does not encode a typical Splice Leader (SL) sequence at the 5' terminus that usually accompanies trans-splicing. Thus, the presence of stable transcripts encoding both genes may suggest a novel mechanism of regulation, or conversely may be rare but stable transcripts in which the two mRNAs are linked due to a small amount of read-through by RNA polymerase. Both genes are highly polymorphic and co-expressed on tissues involved in histocompatibility. In addition, polymorphisms on both genes correlate with outcome, although we have found a case in which it appears that the secreted form may be major allorecognition determinant.

Molecular evolution of a polymorphic HSP40-like protein encoded in the histocompatibility locus of an invertebrate chordate.

Allorecognition, the ability to distinguish self from non-self, occurs in most organisms. Despite the ubiquity of the allorecognition process, the genetic basis for allorecognition remains unexplored in most taxa outside vertebrates and flowering plants. The allorecognition system in the colonial ascidian Botryllus schlosseri is a notable exception. We have recently identified a polymorphic gene within the fuhc locus that may play a role in allorecognition. The encoded protein, called Hsp40-L, is a Type II member of the J-protein family which usually functions as a co-chaperone with Hsp70. While many of the residues that interact with Hsp70 are conserved in Hsp40-L, it may not be a housekeeping protein because it is surprisingly polymorphic and expressed in the ampullae, the site of allorecognition. While the majority of the Hsp40-L protein appears to evolve under purifying selection, a section of the C-terminal region likely experiences balancing/directional selection, characteristic of other allorecognition proteins.

Evidence for selection on a chordate histocompatibility locus.

Allorecognition is the ability of an organism to differentiate self or close relatives from unrelated individuals. The best known applications of allorecognition are the prevention of inbreeding in hermaphroditic species (e.g., the self-incompatibility [SI] systems in plants), the vertebrate immune response to foreign antigens mediated by MHC loci, and somatic fusion, where two genetically independent individuals physically join to become a chimera. In the few model systems where the loci governing allorecognition outcomes have been identified, the corresponding proteins have exhibited exceptional polymorphism. But information about the evolution of this polymorphism outside MHC is limited. We address this subject in the ascidian Botryllus schlosseri, where allorecognition outcomes are determined by a single locus, called FuHC (Fusion/HistoCompatibility). Molecular variation in FuHC is distributed almost entirely within populations, with very little evidence for differentiation among different populations. Mutation plays a larger role than recombination in the creation of FuHC polymorphism. A selection statistic, neutrality tests, and distribution of variation within and among different populations all provide evidence for selection acting on FuHC, but are not in agreement as to whether the selection is balancing or directional.

The fester locus in Botryllus schlosseri experiences selection.

BACKGROUND: Allorecognition, the ability of an organism to distinguish self from non-self, occurs throughout the entire tree of life. Despite the prevalence and importance of allorecognition systems, the genetic basis of allorecognition has rarely been characterized outside the well-known MHC (Major Histocompatibility Complex) in vertebrates and SI (Self-Incompatibility) in plants. Where loci have been identified, their evolutionary history is an open question. We have previously identified the genes involved in self/non-self recognition in the colonial ascidian Botryllus schlosseri, and we can now begin to investigate their evolution. In B. schlosseri, colonies sharing 1 or more alleles of a gene called FuHC (Fusion Histocompatibility) will fuse. Protein products of a locus called fester, located ~300 kb from FuHC, have been shown to play multiple roles in the histocompatibility reaction, as activating and/or inhibitory receptors. We test whether the proteins encoded by this locus are evolving neutrally or are experiencing balancing, directional, or purifying selection. RESULTS: Nearly all of the variation in the fester locus resides within populations. The 13 housekeeping genes (12 nuclear genes and mitochondrial cytochrome oxidase I) have substantially more structure among populations within groups and among groups than fester. All polymorphism statistics (Tajima's D, Fu and Li's D* and F*) are significantly negative for the East Coast A-type alleles, and Fu and Li's F* statistic is significantly negative for the West Coast A-type alleles. These results are likely due to selection rather than demography, given that 10 of the housekeeping loci have no populations with significant values for any of the polymorphism statistics. The majority of codons in the fester proteins have ω values < 1, but 15-27 codons have > 95% posterior probability of ω values > 1. CONCLUSION: Fester proteins are evolving non-neutrally. The polymorphism statistics are consistent with either purifying selection or directional selection. The ω statistics show that the majority of the protein is experiencing purifying selection (ω < 1), but that 15-27 codons are undergoing either balancing or directional selection: ω > 1 is compatible with either scenario. The distribution of variation within and among populations points towards balancing selection and away from directional selection. While these data do not provide unambiguous support for a specific type of selection, they contribute to our evolutionary understanding of a critical biological process by determining the forces that affect loci involved in allorecognition.

Reproductive protein evolution in two cryptic species of marine chordate.

BACKGROUND: Reproductive character displacement (RCD) is a common and taxonomically widespread pattern. In marine broadcast spawning organisms, behavioral and mechanical isolation are absent and prezygotic barriers between species often operate only during the fertilization process. Such barriers are usually a consequence of differences in the way in which sperm and egg proteins interact, so RCD can be manifest as faster evolution of these proteins between species in sympatry than allopatry. Rapid evolution of these proteins often appears to be a consequence of positive (directional) selection. Here, we identify a set of candidate gamete recognition proteins (GRPs) in the ascidian Ciona intestinalis and showed that these GRPs evolve more rapidly than control proteins (those not involved in gamete recognition). Choosing a subset of these gamete recognition proteins that show evidence of positive selection (CIPRO37.40.1, CIPRO60.5.1, CIPRO100.7.1), we then directly test the RCD hypothesis by comparing divergence (omega) and polymorphism (McDonald-Kreitman, Tajima's D, Fu and Li's D and F, Fay and Wu's H) statistics in sympatric and allopatric populations of two distinct forms of C. intestinalis (Types A and B) between which there are strong post-zygotic barriers. RESULTS: Candidate gamete recognition proteins from two lineages of C. intestinalis (Type A and B) are evolving more rapidly than control proteins, consistent with patterns seen in insects and mammals. However, ω (dN/dS) is not significantly different between the sympatric and allopatric populations, and none of the polymorphism statistics show significant differences between sympatric and allopatric populations. CONCLUSIONS: Enhanced prezygotic isolation in sympatry has become a well-known feature of gamete recognition proteins in marine broadcast spawners. But in most cases the evolutionary process or processes responsible for this pattern have not been identified. Although gamete recognition proteins in C. intestinalis do appear to evolve more rapidly, on average, than proteins with other functions, rates of evolution are not different in allopatric and sympatric populations of the two reproductively isolated forms. That sympatry is probably human-mediated, and therefore recent, may explain the absence of RCD.

Creation and maintenance of variation in allorecognition Loci: molecular analysis in various model systems.

Allorecognition is the ability of an organism to differentiate self or close relatives from unrelated conspecifics. Effective allorecognition systems are critical to the survival of organisms; they prevent inbreeding and facilitate fusions between close relatives. Where the loci governing allorecognition outcomes have been identified, the corresponding proteins often exhibit exceptional polymorphism. Two important questions about this polymorphism remain unresolved: how is it created, and how is it maintained. Because the genetic bases of several allorecognition systems have now been identified, including alr1 and alr2 in Hydractinia, fusion histocompatibility in Botryllus, the het (vic) loci in fungi, tgrB1 and tgrC1 in Dictyostelium, and self-incompatibility (SI) loci in several plant families, we are now poised to achieve a clearer understanding of how these loci evolve. In this review, we summarize what is currently known about the evolution of allorecognition loci, highlight open questions, and suggest future directions.

Introgression despite substantial divergence in a broadcast spawning marine invertebrate.

Understanding the relationship between reproductive isolation and time since divergence is critical to our understanding of speciation. One group for which we know little about the relationship between hybridization/introgression and time since divergence is the marine broadcast spawners. Here, we investigate the distribution of closely related cryptic species of marine broadcast spawners (Type A and B Ciona intestinalis) in areas of potential sympatry to determine whether these two types occur together and if so, whether they show evidence of hybridization and introgression. Then we combine our data with other studies to investigate general patterns of reproductive isolation versus divergence in marine broadcast spawners. We found that Type A and B C. intestinalis occurred sympatrically in 2007, and that 21 individuals show evidence of introgression in sympatry (out of approximately 500). Type A and B C. intestinalis are 12.4% divergent at mitochondrial COI (mtCOI), and in comparison with other marine broadcast spawning species at mtCOI, these two types may be near the upper limit of the range of divergence values in which introgression is still possible. However, introgression at divergence levels similar to those found in Ciona does exist, prompting questions about the strength of postmating prezygotic reproductive barriers in marine broadcast spawners.

Polymorphism and divergence within the ascidian genus Ciona.

The genus Ciona, a widely distributed group of solitary ascidians, has long been an important model in embryology and developmental biology. Ciona has also recently attracted the attention of evolutionary biologists because of the remarkably high levels of heterozygosity found within single individuals. Surprisingly, genealogical relationships in Ciona have received little attention. Here, we expand our knowledge of relationships among the members of the Ciona genus and estimate levels of polymorphism in natural populations. Previous studies have documented the outgroup status of Ciona savignyi among the shallow-water Ciona and revealed the existence of two distinct forms (Types A and B) of the widespread Ciona intestinalis. Here, using gene genealogies of six nuclear gene loci, we show Types A and B to be well-supported monophyletic groups. In spite of their morphological similarity, Type A vs. Type B divergences range from 0.035 to 0.124. In contrast, the morphologically distinct Ciona roulei is embedded within Type B in all genealogies, and a new species, Ciona sp., appears to be associated with Type B/C. roulei to the exclusion of Type A. Levels of polymorphism in natural populations are similar to levels reported in other organisms that are considered to be highly polymorphic.

Multimodal warning signals for a multiple predator world.

Aposematism is an anti-predator defence, dependent on a predator's ability to associate unprofitable prey with a prey-borne signal. Multimodal signals should vary in efficacy according to the sensory systems of different predators; however, until now, the impact of multiple predator classes on the evolution of these signals had not been investigated. Here, using a community-level molecular phylogeny to generate phylogenetically independent contrasts, we show that warning signals of tiger moths vary according to the seasonal and daily activity patterns of birds and bats-predators with divergent sensory capacities. Many tiger moths advertise chemical defence using conspicuous colouration and/or ultrasonic clicks. During spring, when birds are active and bats less so, we found that tiger moths did not produce ultrasonic clicks. Throughout both spring and summer, tiger moths most active during the day were visually conspicuous. Those species emerging later in the season produced ultrasonic clicks; those that were most nocturnal were visually cryptic. Our results indicate that selective pressures from multiple predator classes have distinct roles in the evolution of multimodal warning displays now effective against a single predator class. We also suggest that the evolution of acoustic warning signals may lack the theoretical difficulties associated with the origination of conspicuous colouration.