Orthologs at the Base of the Olfactores Clade.

Tunicate orthologs in the human genome comprise just 84 genes of the 19,872 protein-coding genes and 23 of the 16,528 non-coding genes, yet they stand at the base of the Olfactores clade, which radiated to generate thousands of tunicate and vertebrate species. What were the powerful drivers among these genes that enabled this process? Many of these orthologs are present in gene families. We discuss the biological role of each family and the orthologs' quantitative contribution to the family. Most important was the evolution of a second type of cadherin. This, a Type II cadherin, had the property of detaching the cell containing that cadherin from cells that expressed the Type I class. The set of such Type II cadherins could now detach and move away from their Type I neighbours, a process which would eventually evolve into the formation of the neural crest, "the fourth germ layer", providing a wide range of possibilities for further evolutionary invention. A second important contribution were key additions to the broad development of the muscle and nerve protein and visual perception toolkits. These developments in mobility and vision provided the basis for the development of the efficient predatory capabilities of the Vertebrata.

Extreme genome scrambling in marine planktonic Oikopleura dioica cryptic species.

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicate Oikopleura dioica around the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes, Drosophila, or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation in O. dioica suggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

HES and Mox genes are expressed during early mesoderm formation in a mollusk with putative ancestral features.

The mesoderm is considered the youngest of the three germ layers. Although its morphogenesis has been studied in some metazoans, the molecular components underlying this process remain obscure for numerous phyla including the highly diverse Mollusca. Here, expression of Hairy and enhancer of split (HES), Mox, and myosin heavy chain (MHC) was investigated in Acanthochitona fascicularis, a representative of Polyplacophora with putative ancestral molluscan features. While AfaMHC is expressed throughout myogenesis, AfaMox1 is only expressed during early stages of mesodermal band formation and in the ventrolateral muscle, an autapomorphy of the polyplacophoran trochophore. Comparing our findings to previously published data across Metazoa reveals Mox expression in the mesoderm in numerous bilaterians including gastropods, polychaetes, and brachiopods. It is also involved in myogenesis in molluscs, annelids, tunicates, and craniates, suggesting a dual role of Mox in mesoderm and muscle formation in the last common bilaterian ancestor. AfaHESC2 is expressed in the ectoderm of the polyplacophoran gastrula and later in the mesodermal bands and in putative neural tissue, whereas AfaHESC7 is expressed in the trochoblasts of the gastrula and during foregut formation. This confirms the high developmental variability of HES gene expression and demonstrates that Mox and HES genes are pleiotropic.

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.

Ascidian diversity of Costa Rica, including new records for the North Pacific / Diversidad de ascidias de Costa Rica, incluyendo nuevos reportes para el área del Pacífico Norte

Introduction: The class Ascidiacea has about 3 000 species, which fulfill various roles in the ecosystem, for example, they filter high amounts of particles, and are shelter and food for other animals. Furthermore, the group has a high number of invasive species reported. In Costa Rica, ascidians have been barely studied. Objective: In this study, we aim to describe the diversity of ascidians in Costa Rica from new samplings in Área de Conservación Guanacaste, North Pacific, and by compiling previous reports for the entire country in order to improve the group's state of knowledge. Methods: Samples were collected during two field trips between 2018 and 2019, at six sites within the Gulf of Santa Elena and three sites near the Murcielago Islands area. The specimens were dissected and analyzed in detail to determine the species. All the identifications were compared with the available information from literature and from museum collections. Literature, collections databases of the National Museum of Natural History, Smithsonian Institution and the new material collected were used to create an updated taxonomic list. Results: A total of eight species were obtained from 70 specimens. Of these, five are new reports for the country, which increase to 22 species the total number reported for Costa Rica. The most common species was Rhopalaea birkelandi, whose presence was higher in Santa Elena Bay. Conclusions: This study improved the knowledge of ascidian diversity in Costa Rica. Polyandrocarpa anguinea, reported for the first time, is considered invasive in other areas, which suggests the necessity of a continuous monitoring of its population. It is necessary to include more areas of the country since almost all the reported species come from the North Pacific; the diversity of ascidians from other parts of the country, especially the Caribbean, is still unknown.

Introducción: La clase Ascidiacea tiene alrededor de 3 000 especies, las cuales llevan a cabo varias funciones en el ecosistema, por ejemplo: filtrar altas cantidades de partículas y servir de refugio y alimento para otros animales. Asimismo, para el grupo se han reportado un gran número de especies invasoras. En Costa Rica las ascidias han sido poco estudiadas. Objetivo: En el presente estudio buscamos describir la diversidad de ascidias en Costa Rica a partir de nuevos muestreos en el Área de Conservación Guanacaste, Pacífico Norte y mediante la recolección de reportes previos para el país para así aumentar el conocimiento del grupo. Métodos: Las muestras fueron colectadas en dos giras entre el 2018 y 2019, en seis sitios en el Golfo de Santa Elena y tres sitios cerca de las Islas Murciélago. Los especímenes fueron disectados y analizados en detalle para su identificación. Todas las identificaciones fueron comparadas con la información encontrada en la literatura y en colecciones de museos. Literatura, bases de datos de la colección del Museo Nacional de Historia Natural de la Institución Smithsonian y el nuevo material colectado, fueron utilizados para crear un listado taxonómico actualizado. Resultados: Se obtuvo un total de ocho especies del muestreo. De estas, cinco son nuevos reportes, incrementando el total de especies reportadas en Costa Rica a 22. La especie más común fue Rhopalaea birkelandi, cuya presencia fue mayor en Bahía Santa Elena. Conclusiones: Este estudio mejoró el conocimiento sobre la diversidad de ascidias en Costa Rica. Polyandrocarpa anguinea, reportada por primera vez, es considerada una especie invasora en otras áreas, lo que sugiere la necesidad de monitorear su población de forma continua. Es necesario incluir más áreas del país en futuros estudios puesto que casi todas las especies reportadas provienen del Pacífico Norte; la diversidad de otras partes del país, especialmente el Caribe permanece desconocida.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode.

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

The eventful history of nonembryonic development in tunicates.

Tunicates encompass a large group of marine filter-feeding animals and more than half of them are able to reproduce asexually by a particular form of nonembryonic development (NED) generally called budding. The phylogeny of tunicates suggests that asexual reproduction is an evolutionarily plastic trait, a view that is further reinforced by the fact that budding mechanisms differ from one species to another, involving nonhomologous tissues and cells. In this review, we explore more than 150 years of literature to provide an overview of NED diversity and we present a comparative picture of budding tissues across tunicates. Based on the phylogenetic relationships between budding and nonbudding species, we hypothesize that NED diversity is the result of seven independent acquisitions and subsequent diversifications in the course of tunicate evolution. While this scenario represents the state-of-the-art of our current knowledge, we point out gray areas that need to be further explored to refine our understanding of tunicate phylogeny and NED. Tunicates, with their plastic evolution and diversity of budding, represent an ideal playground for evolutionary developmental biologists to unravel the genetic and molecular mechanisms regulating nonembryonic development, as well as to better understand how such a profound innovation in life-history has evolved in numerous metazoans.

Phylogenetic Analyses of Glycosyl Hydrolase Family 6 Genes in Tunicates: Possible Horizontal Transfer.

Horizontal gene transfer (HGT) is the movement of genetic material between different species. Although HGT is less frequent in eukaryotes than in bacteria, several instances of HGT have apparently shaped animal evolution. One well-known example is the tunicate cellulose synthase gene, CesA, in which a gene, probably transferred from bacteria, greatly impacted tunicate evolution. A Glycosyl Hydrolase Family 6 (GH6) hydrolase-like domain exists at the C-terminus of tunicate CesA, but not in cellulose synthases of other organisms. The recent discovery of another GH6 hydrolase-like gene (GH6-1) in tunicate genomes further raises the question of how tunicates acquired GH6. To examine the probable origin of these genes, we analyzed the phylogenetic relationship of GH6 proteins in tunicates and other organisms. Our analyses show that tunicate GH6s, the GH6-1 gene, and the GH6 part of the CesA gene, form two independent, monophyletic gene groups. We also compared their sequence signatures and exon splice sites. All tunicate species examined have shared splice sites in GH6-containing genes, implying ancient intron acquisitions. It is likely that the tunicate CesA and GH6-1 genes existed in the common ancestor of all extant tunicates.

DNA bar coding of Aplousobranchiata and Phlebobranchiata Ascidians (Phylum:Chordata) inferred from mitochondrial cytochrome oxidase subunit I (COI) gene sequence approach in Andaman and Nicobar Islands, India: a first report.

Ascidians (Phylum: Chordata) are sessile and filter-feeding marine animal, species identification of ascidians is possible by observing various morphological and anatomical features in various stages of life span. However, this method is labor intensive, time-consuming and very difficult for non-specialists particularly when dealing with field collections. Suborder Aplousobranchiata and Phlebobranchiata is the largest group of tunicates within, morphological and molecular data suggest that Didemnidae and Ascidiidae are monophyletic, but the monophyly of each genus and their phylogenetic relationships are still poorly understood. Therefore, this study was aimed to develop DNA barcodes of ascidians belonging to the orders of Aplousobranchiata and Phlebobranchiata species namely Diplosoma listerianum, Lissoclinum fragile, Didemnum psammatode, Phallusia fumigata and Phallusia ingeria collected from Andaman and Nicobar Islands were sequenced and submitted in Gen Bank. Colony structure, Scanning Electron Microscope (SEM) for spicules of colonial ascidians, larval type and zooids formation were found to be the most useful morphological characters for discriminating the species. Our BLAST results proved D. Listerianum KP842724 (98%) L. fragile KP842726 (100%) D. psammatode KP779902 (99%), P. fumigata KP779904 (99%) and P. ingeria KP842727 (100%) similarity and this is the first report of mitochondrial COI gene of these ascidians from Andaman and Nicobar Islands. We explored the usefulness of CO1 gene sequences for molecular level identification and mtDNA data in assessing a phylogenetic relationship of ascidian species.

Biological identification of ascidians from Vizhinjam Bay, southwest Coast of India using CO1 gene sequences.

Ascidians are ecologically important components of marine ecosystems, yet the taxonomy and diversity of ascidians remain largely unexplored. Only <60% of reported ascidian species in India have been taxonomically described and identified and the rest of the species remain unidentified due to uncertainty in the morphology-based identification. We explored the usefulness of CO1 gene sequences for molecular level identification and mtDNA data in assessing phylogenetic relationships of 15 ascidian species. The mean sequence divergences within and among the species fell into the mean divergence ranges found in ascidian group. Species that are most similar grouped together formed a cluster. Clusters of species in a clade indicate that the species are closely related. Species that are highly divergent formed a separate branch. This study has concluded that the CO1 gene sequence is an effective tool to ascertain the molecular taxonomical studies on ascidians.

Into the bloom: Molecular response of pelagic tunicates to fluctuating food availability.

The planktonic tunicates appendicularians and thaliaceans are highly efficient filter feeders on a wide range of prey size including bacteria and have shorter generation times than any other marine grazers. These traits allow some tunicate species to reach high population densities and ensure their success in a favourable environment. However, there are still few studies focusing on which genes and gene pathways are associated with responses of pelagic tunicates to environmental variability. Herein, we present the effect of food availability increase on tunicate community and gene expression at the Marquesas Islands (South-East Pacific Ocean). By using data from the Tara Oceans expedition, we show that changes in phytoplankton density and composition trigger the success of a dominant larvacean species (an undescribed appendicularian). Transcriptional signature to the autotroph bloom suggests key functions in specific physiological processes, i.e., energy metabolism, muscle contraction, membrane trafficking, and proteostasis. The relative abundance of reverse transcription-related Pfams was lower at bloom conditions, suggesting a link with adaptive genetic diversity in tunicates in natural ecosystems. Downstream of the bloom, pelagic tunicates were outcompeted by copepods. Our work represents the first metaomics study of the biological effects of phytoplankton bloom on a key zooplankton taxon.

Phylogenetic analysis of phenotypic characters of Tunicata supports basal Appendicularia and monophyletic Ascidiacea.

With approximately 3000 marine species, Tunicata represents the most disparate subtaxon of Chordata. Molecular phylogenetic studies support Tunicata as sister taxon to Craniota, rendering it pivotal to understanding craniate evolution. Although successively more molecular data have become available to resolve internal tunicate phylogenetic relationships, phenotypic data have not been utilized consistently. Herein these shortcomings are addressed by cladistically analyzing 117 phenotypic characters for 49 tunicate species comprising all higher tunicate taxa, and five craniate and cephalochordate outgroup species. In addition, a combined analysis of the phenotypic characters with 18S rDNA-sequence data is performed in 32 OTUs. The analysis of the combined data is congruent with published molecular analyses. Successively up-weighting phenotypic characters indicates that phenotypic data contribute disproportionally more to the resulting phylogenetic hypothesis. The strict consensus tree from the analysis of the phenotypic characters as well as the single most parsimonious tree found in the analysis of the combined dataset recover monophyletic Appendicularia as sister taxon to the remaining tunicate taxa. Thus, both datasets support the hypothesis that the last common ancestor of Tunicata was free-living and that ascidian sessility is a derived trait within Tunicata. "Thaliacea" is found to be paraphyletic with Pyrosomatida as sister taxon to monophyletic Ascidiacea and the relationship between Doliolida and Salpida is unresolved in the analysis of morphological characters; however, the analysis of the combined data reconstructs Thaliacea as monophyletic nested within paraphyletic "Ascidiacea". Therefore, both datasets differ in the interpretation of the evolution of the complex holoplanktonic life history of thaliacean taxa. According to the phenotypic data, this evolution occurred in the plankton, whereas from the combined dataset a secondary transition into the plankton from a sessile ascidian is inferred. Besides these major differences, both analyses are in accord on many phylogenetic groupings, although both phylogenetic reconstructions invoke a high degree of homoplasy. In conclusion, this study represents the first serious attempt to utilize the potential phylogenetic information present in phenotypic characters to elucidate the inter-relationships of this diverse marine taxon in a consistent cladistic framework.

Exotic species dominate marinas between the two most populated regions in the southwestern Atlantic Ocean.

Human occupation of coastal areas promotes the establishment of non-native species but information on bioinvasions is usually biased toward the Northern Hemisphere. We assessed non-native species' importance in sessile communities at six marinas along the most urbanized area of the Southwestern Atlantic coastline. We found 67 species, of which 19 are exotic. The most frequent species was the exotic polychaete Branchiomma luctuosum, while the most abundant was the exotic bryozoan Schizoporella errata that monopolized the substrata in three marinas. Along with S. errata, the exotic polychaete Hydroides elegans and ascidian Styela plicata dominated space in the three remaining marinas, while native species were in general rare. We show that communities associated with artificial substrata along this Brazilian urbanized area are dominated by exotic species and that using abundance data along with species identity can improve our understanding of the importance of exotic species for the dynamics of biological communities.

Wnt evolution and function shuffling in liberal and conservative chordate genomes.

BACKGROUND: What impact gene loss has on the evolution of developmental processes, and how function shuffling has affected retained genes driving essential biological processes, remain open questions in the fields of genome evolution and EvoDevo. To investigate these problems, we have analyzed the evolution of the Wnt ligand repertoire in the chordate phylum as a case study. RESULTS: We conduct an exhaustive survey of Wnt genes in genomic databases, identifying 156 Wnt genes in 13 non-vertebrate chordates. This represents the most complete Wnt gene catalog of the chordate subphyla and has allowed us to resolve previous ambiguities about the orthology of many Wnt genes, including the identification of WntA for the first time in chordates. Moreover, we create the first complete expression atlas for the Wnt family during amphioxus development, providing a useful resource to investigate the evolution of Wnt expression throughout the radiation of chordates. CONCLUSIONS: Our data underscore extraordinary genomic stasis in cephalochordates, which contrasts with the liberal and dynamic evolutionary patterns of gene loss and duplication in urochordate genomes. Our analysis has allowed us to infer ancestral Wnt functions shared among all chordates, several cases of function shuffling among Wnt paralogs, as well as unique expression domains for Wnt genes that likely reflect functional innovations in each chordate lineage. Finally, we propose a potential relationship between the evolution of WntA and the evolution of the mouth in chordates.

A comparison of prokaryotic symbiont communities in nonnative and native ascidians from reef and harbor habitats.

Harbor systems represent passive gateways for the introduction of nonnative ascidians that compete with the surrounding benthos and may spread through localized dispersal, even populating adjacent natural reefs. To investigate the potential role of microbial symbionts in the success of ascidian introductions and spread, we evaluated the host-specificity of prokaryotic communities within two ascidian species commonly found off the North Carolina coast. Replicate samples of the native ascidian Eudistoma capsulatum, the nonnative ascidian Distaplia bermudensis and seawater were collected from artificial (harbor) and natural reef substrates. Prokaryotic communities in seawater samples and ascidian tunics were characterized via next-generation sequencing of partial 16S rRNA gene sequences. Ascidian microbiomes clustered strongly in response to host species, with significant differences in community structure between the two species and seawater. Further, symbiont community structure differed significantly between E. capsulatumindividuals collected from artificial and natural habitats, though this was not the case for D. bermudensis. These findings suggested that some ascidian species possess stable microbial symbiont communities that allow them to thrive in a wide range of habitats, while other species rely on the restructuring of their microbial communities with specific symbionts (e.g. Chelativorans) to survive under particular environmental conditions such as increased pollution.

A phylogenomic framework and timescale for comparative studies of tunicates.

BACKGROUND: Tunicates are the closest relatives of vertebrates and are widely used as models to study the evolutionary developmental biology of chordates. Their phylogeny, however, remains poorly understood, and to date, only the 18S rRNA nuclear gene and mitogenomes have been used to delineate the major groups of tunicates. To resolve their evolutionary relationships and provide a first estimate of their divergence times, we used a transcriptomic approach to build a phylogenomic dataset including all major tunicate lineages, consisting of 258 evolutionarily conserved orthologous genes from representative species. RESULTS: Phylogenetic analyses using site-heterogeneous CAT mixture models of amino acid sequence evolution resulted in a strongly supported tree topology resolving the relationships among four major tunicate clades: (1) Appendicularia, (2) Thaliacea + Phlebobranchia + Aplousobranchia, (3) Molgulidae, and (4) Styelidae + Pyuridae. Notably, the morphologically derived Thaliacea are confirmed as the sister group of the clade uniting Phlebobranchia + Aplousobranchia within which the precise position of the model ascidian genus Ciona remains uncertain. Relaxed molecular clock analyses accommodating the accelerated evolutionary rate of tunicates reveal ancient diversification (~ 450-350 million years ago) among the major groups and allow one to compare their evolutionary age with respect to the major vertebrate model lineages. CONCLUSIONS: Our study represents the most comprehensive phylogenomic dataset for the main tunicate lineages. It offers a reference phylogenetic framework and first tentative timescale for tunicates, allowing a direct comparison with vertebrate model species in comparative genomics and evolutionary developmental biology studies.

Phylogenomics offers resolution of major tunicate relationships.

Tunicata, a diverse clade of approximately 3000 described species of marine, filter-feeding chordates, is of great interest to researchers because tunicates are the closest living relatives of vertebrates and they facilitate comparative studies of our own biology. The group also includes numerous invasive species that cause considerable economic damage and some species of tunicates are edible. Despite their diversity and importance, relationships among major lineages of Tunicata are not completely resolved. Here, we supplemented public data with transcriptomes from seven species spanning the diversity of Tunicata and conducted phylogenomic analyses on data sets of up to 798 genes. Sensitivity analyses were employed to examine the influences of reducing compositional heterogeneity and branch-length heterogeneity. All analyses maximally supported a monophyletic Tunicata within Olfactores (Vertebrata + Tunicata). Within Tunicata, all analyses recovered Appendicularia sister to the rest of Tunicata and confirmed (with maximal support) that Thaliacea is nested within Ascidiacea. Stolidobranchia is the sister taxon to all other tunicates except Appendicularia. In most analyses, phlebobranch tunicates were recovered paraphyletic with respect to Aplousobranchia. Support for this topology varied but was strong in some cases. However, when only the 50 best genes based on compositional heterogeneity were analysed, we recovered Phlebobranchia and Aplousobranchia reciprocally monophyletic with strong support, consistent with most traditional morphology-based hypotheses. Examination of internode certainty also cast doubt on results of phlebobranch paraphyly, which may be due to limited taxon sampling. Taken together, these results provide a higher-level phylogenetic framework for our closest living invertebrate relatives.

Ambiguities in the taxonomic assignment and species delineation of botryllid ascidians from the Israeli Mediterranean and other coastlines.

Based on mtCOI sequences comparisons, recent studies reassigned the 'dwarf Botrylloides leachii' from the Levant as Botrylloides nigrum. Here we conducted a survey of the literature and of deposited mtCOI sequences of botryllid ascidians, elucidating ambiguities in their taxonomy. We found that the species, dwarf morph of Botrylloides leachii, Botrylloides nigrum, Botryllus aster and Botryllus arenaceus are grouped together on a single molecular taxon. Then, results of three additional markers (18S, 28S, H3) contradicted literature suggestions, revealing minute distances between Botrylloides leachii and the 'dwarf Botrylloides leachii'. Moreover, only Botrylloides leachii and the 'dwarf Botrylloides leachii' develop giant ampullae as an allorecognition response. Our results raise the possibility that inadequate identification, together with faults in molecular assignment, including queries regarding the efficacy of the mtCOI as the exclusive barcoding tool in botryllid ascidians, is the major culprits responsible for the emerged inconsistencies between the mtCOI sequences and traditional taxonomy. Thus, we assign the Levantine dwarf form as Botrylloides aff. leachii.

DNA barcoding and phylogenetic analysis of five ascidians (Phlebobranchia) distributed in Gulf of Mannar, India.

DNA barcoding has played a significant role in biodiversity assessment as well as its conservation. This technique involves sequencing of mitochondrial marker gene including a short COI gene, known as barcode gene. It has proved its efficiency in identifying several species and resolving the limitations incurred during identification through conventional taxonomy. This study involves the use of DNA barcoding of ascidian species belonging to order Phlebobranchia. A total of 14 individuals, covering two families, three genera and five species, were barcoded. COI gene sequences of all the five species were deposited for the first time in NCBI as well as BOLD. The NJ tree revealed identical phylogenetic relationship among the individuals collected from three different stations. Mean Kimura 2-parameter (K2P) distances within-species, genus, family and order were 0.08%, 6.69%, 9.49% and 18.58%, respectively. This result concludes that COI gene sequencing is the efficient tool in identifying ascidians of the order Phlebobranchia. We report for the first time the COI gene sequences of four species of ascidians studied.