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.
Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds ...of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.
The cosmopolitan ascidian Ciona intestinalis is the most common model species of Tunicata, the sister-group of Vertebrata, and widely used in developmental biology, genomics and evolutionary studies. ...Recently, molecular studies suggested the presence of cryptic species hidden within the C. intestinalis species, namely C. intestinalis type A and type B. So far, no substantial morphological differences have been identified between individuals belonging to the two types. Here we present morphometric, immunohistochemical, and histological analyses, as well as 3-D reconstructions, of late larvae obtained by cross-fertilization experiments of molecularly determined type A and type B adults, sampled in different seasons and in four different localities. Our data point to quantitative and qualitative differences in the trunk shape of larvae belonging to the two types. In particular, type B larvae exhibit a longer pre-oral lobe, longer and relatively narrower total body length, and a shorter ocellus-tail distance than type A larvae. All these differences were found to be statistically significant in a Discriminant Analysis. Depending on the number of analyzed parameters, the obtained discriminant function was able to correctly classify > 93% of the larvae, with the remaining misclassified larvae attributable to the existence of intra-type seasonal variability. No larval differences were observed at the level of histology and immunohistochemical localization of peripheral sensory neurons. We conclude that type A and type B are two distinct species that can be distinguished on the basis of larval morphology and molecular data. Since the identified larval differences appear to be valid diagnostic characters, we suggest to raise both types to the rank of species and to assign them distinct names.
Complete mitochondrial (mt) genomes have been sequenced for thousands of animals and represent a molecule of choice for many evolutionary studies. Nevertheless, some animal groups have remained ...under-sampled. Ctenophora (comb jellies) is one such example, with only two complete mt sequences determined hitherto for this phylum, which encompasses ca. 150-200 described species. This lack of data derives from the extremely fast mt evolutionary rate in this lineage, complicating primer design and DNA amplification. Indeed, in the two ctenophore mt genomes sequenced to date, i.e. those of Mnemiopsis leidyi (order Lobata) and Pleurobrachia bachei (order Cydippida), both rRNA and protein coding genes exhibit an extraordinary size reduction and have highly derived sequences. Additionally, all tRNAs, and the atp6 and atp8 genes are absent. In order to determine whether these characteristics are shared by other ctenophores, we obtained the complete mt genomes of three benthic ctenophores belonging to the so far unsampled order of Platyctenida: Coeloplana loyai, Coeloplana yulianicorum and Vallicula multiformis.
The mt genomes of benthic ctenophores reveal the same peculiarities found in Mnemiopsis and Pleurobrachia, demonstrating that the fast evolutionary rate is a general trait of the ctenophore mt genomes. Our results also indicate that this high evolutionary rate not only affects the nucleotide substitution but also gene rearrangements. Indeed, gene order was highly rearranged among representatives of the different taxonomic orders in which it was close to random, but also quite variable within Platyctenida, in which the genera Coeloplana and Vallicula share only four conserved synteny blocks. However, the two congeneric Coeloplana species display exactly the same gene order. Because of the extreme evolutionary rate, our phylogenetic analyses were unable to resolve the phylogenetic position of ctenophores within metazoans or the relationships among the different Ctenophora orders. Comparative sequence-analyses allowed us to correct the annotation of the Pleurobrachia mt genome, confirming the absence of tRNAs, the presence of both rRNA genes, and the existence of a reassignment of codon TGA from tryptophan to serine for this species.
Since Platyctenida is an early diverging lineage among Ctenophora, our findings suggest that the mt traits described above are ancestral characteristics of this phylum.
Botryllus schlosseri is a widespread colonial ascidian commonly considered cosmopolitan and amply used as model for researches ranging from developmental biology to immunobiology. Recently, molecular ...data lead to hypothesize that the species named B. schlosseri may consist of more than a single taxon. Indeed, five highly divergent clades, named A-E, have been genetically identified and are referred as cryptic species. In this context, and lacking both a type and a detailed morphological description, we believe that it is necessary, as a taxonomic reference point, to designate a neotype and re-describe the species. Therefore, a sample from the Lagoon of Venice (Adriatic Sea, Italy) was deposited as neotype in the Natural History Museum of Venice (Italy), preserved both in formalin and in 90% ethanol. Here we provide a morphological description of the suggested neotype of B. schlosseri that takes into account several developmental stages (oozooid, zooid of first blastogenic generations, and mature zooid) and is carefully compared with the previous descriptions of samples coming from other European and non-European localities. Finally, we associate our morphological description to a "DNA barcode", consisting in a long fragment of the mitochondrial COI gene. Our description is associated to clade A, although at now we cannot guarantee that this association is univocal.
The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) ...technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.
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•Phylogeny based on mt genomes places Diazonidae within Aplousobranchia.•The thorax regeneration of Rhopalea supports the aplousobranch nature of Diazonidae.•Our results support the ...loss of a colonial lifestyle in Rhopalea.•Mitochondrial phylogeny places Ciona as sister clade of Aplousobranchia.
The order Aplousobranchia (Chordata, Ascidiacea) contains approximately 1500 species distributed worldwide. Their phylogeny, however, remains unclear, with unresolved family relationships. While most Aplousobranchia are colonial, debates exist concerning the phylogenetic position of families such as the Diazonidae and Cionidae, which exhibit a solitary lifestyle and share morphological characteristics with both Aplousobranchia and Phlebobranchia orders. To clarify the phylogenetic position of the Diazonidae and Cionidae, we determined the complete mitochondrial sequence of the solitary diazonid Rhopalaea idoneta. The phylogenetic reconstruction based on the 13 mitochondrial protein coding genes strongly supports a positioning of Diazonidae well-nested within the Aplousobranchia rather than a positioning as a sister clade of the Aplousobranchia. In addition, we examined the regenerative ability of R. idoneta. Similar to colonial Aplousobranchia, R. idoneta was found to be able to completely regenerate its thorax. Ciona, also known to possess high regenerative abilities, is the Aplousobranchia sister clade rather than a member of the Phlebobranchia. Our results thus indicate that the colonial lifestyle was acquired in the Aplousobranchia, starting from a Ciona-like solitary ancestor and secondarily lost in Diazonidae representatives such as Rhopalaea. The solitary lifestyle of Rhopalaea is thus a derived characteristic rather than an ancestral trait.
According to the tRNA punctuation model, the mitochondrial genome (mtDNA) of mammals and arthropods is transcribed as large polycistronic precursors that are maturated by endonucleolytic cleavage at ...tRNA borders and RNA polyadenylation. Starting from the newly sequenced mtDNA of Ixodes ricinus and using a combination of mitogenomics and transcriptional analyses, we found that in all currently-sequenced tick lineages (Prostriata, Metastriata and Argasidae) the 3'-end of the polyadenylated nad1 and rrnL transcripts does not follow the tRNA punctuation model and is located upstream of a degenerate 17-bp DNA motif. A slightly different motif is also present downstream the 3'-end of nad1 transcripts in the primitive chelicerate Limulus polyphemus and in Drosophila species, indicating the ancient origin and the evolutionary conservation of this motif in arthropods. The transcriptional analyses suggest that this motif directs the 3'-end formation of the nad1/rrnL mature RNAs, likely working as a transcription termination signal or a processing signal of precursor transcripts. Moreover, as most regulatory elements, this motif is characterized by a taxon-specific evolution. Although this signal is not exclusive of ticks, making a play on words it has been named "Tick-Box", since it is a check mark that has to be verified for the 3'-end formation of some mt transcripts, and its consensus sequence has been here carefully characterized in ticks. Indeed, in the whole mtDNA of all ticks, the Tick-Box is always present downstream of nad1 and rrnL, mainly in non-coding regions (NCRs) and occasionally within trnL(CUN). However, some metastriates present a third Tick-Box at an intriguing site--inside the small NCR located at one end of a 3.4 kb translocated region, the other end of which exhibits the nad1 Tick-Box--hinting that this motif could have been involved in metastriate gene order rearrangements.
Accurate and timely monitoring of the evolution of SARS-CoV-2 is crucial for identifying and tracking potentially more transmissible/virulent viral variants, and implement mitigation strategies to ...limit their spread. Here we introduce HaploCoV, a novel software framework that enables the exploration of SARS-CoV-2 genomic diversity through space and time, to identify novel emerging viral variants and prioritize variants of potential epidemiological interest in a rapid and unsupervised manner. HaploCoV can integrate with any classification/nomenclature and incorporates an effective scoring system for the prioritization of SARS-CoV-2 variants. By performing retrospective analyses of more than 11.5 M genome sequences we show that HaploCoV demonstrates high levels of accuracy and reproducibility and identifies the large majority of epidemiologically relevant viral variants - as flagged by international health authorities - automatically and with rapid turn-around times.Our results highlight the importance of the application of strategies based on the systematic analysis and integration of regional data for rapid identification of novel, emerging variants of SARS-CoV-2. We believe that the approach outlined in this study will contribute to relevant advances to current and future genomic surveillance methods.
In vertebrates, the four transcription factors
,
,
and
are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic ...stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The
forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts.
is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified
orthologs of human
and
genes
as well as the closest homologs of the vertebrate-specific
gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of
using
hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover,
,
and
were expressed in candidate stem cells residing in their niches, while
was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that
,
and
may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.
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