Integrative taxonomy in Syllis prolifera (Annelida, Syllidae): from a unique cosmopolitan species to a complex of pseudocryptic species.

Syllis prolifera (Syllidae, Syllinae) is an abundant species of marine annelids commonly found in warm to temperate waters worldwide. Although morphological variability occurs among populations, S. prolifera has long been considered a cosmopolitan species, widely distributed in coastal environments, including acidified and polluted areas. However, the increasing number of cases of cryptic and pseudocryptic speciation in several polychaete families in recent years has led us to question whether S. prolifera represents a single globally distributed taxon or is a species complex. To address this question, we conducted an integrative study, combining morphological, ecological and molecular data of 52 S. prolifera specimens collected in different localities across the western Mediterranean Sea and the Gulf of Cadiz. Our phylogenetic and species delimitation analyses that included two mitochondrial DNA markers (COI and 16S rRNA ) were congruent in not considering S. prolifera a unique entity. Five distinct lineages that can also be recognised by certain morphological and ecological traits were identified from these analyses instead. Overall, our study does not support the homogeneity of S. prolifera across the Mediterranean Sea, providing a new example of pseudocrypticism in marine invertebrates.

Annelid adult cell type diversity and their pluripotent cellular origins.

Many annelids can regenerate missing body parts or reproduce asexually, generating all cell types in adult stages. However, the putative adult stem cell populations involved in these processes, and the diversity of cell types generated by them, are still unknown. To address this, we recover 75,218 single cell transcriptomes of the highly regenerative and asexually-reproducing annelid Pristina leidyi. Our results uncover a rich cell type diversity including annelid specific types as well as novel types. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. We find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors, in piwi+ cells. Finally, lineage reconstruction analyses reveal computational differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids by single cell transcriptomics and suggest that a piwi+ cell population with a pluripotent stem cell signature is associated with adult cell type differentiation.

Morphological and molecular study of Syllinae (Annelida, Syllidae) from Bermuda, with the description of five new species.

Although polychaetes from the Bermuda Archipelago have been studied since the beginning of the twentieth century, syllids have been particularly neglected in this area, which is surprising considering this family is usually a dominant group in marine benthic ecosystems. To fill this knowledge gap, we have carried out an extensive analysis of Bermudan Syllidae, combining morphological and molecular data including four nuclear and mitochondrial markers (cytochrome c oxidase subunit I, 18S rRNA, 16S rRNA and 28S rRNA). We have identified and established the phylogenetic position of five new species, Haplosyllis anitae n. sp., Haplosyllis guillei n. sp., Haplosyllis larsi n. sp., Haplosyllis vassiae n. sp. and Syllis laiae n. sp., together with Haplosyllis cf. cephalata. Overall, our results extend the knowledge on the diversity of Syllidae in Bermuda, increasing the number of species present in the area to 25. Our results also recover Opisthosyllis and Syllis as non-monophyletic genera, for which traditional diagnostic morphological features do not accurately reflect their evolutionary histories, and thus we propose that these groups should be reorganized based on molecular characters.

On the hormonal control of posterior regeneration in the annelid Platynereis dumerilii.

Regeneration is the process by which many animals are able to restore lost or injured body parts. After amputation of the posterior part of its body, the annelid Platynereis dumerilii is able to regenerate the pygidium, the posteriormost part of its body that bears the anus, and a subterminal growth zone containing stem cells that allows the subsequent addition of new segments. The ability to regenerate their posterior part (posterior regeneration) is promoted, in juvenile worms, by a hormone produced by the brain and is lost when this hormonal activity becomes low at the time the worms undergo their sexual maturation. By characterizing posterior regeneration at the morphological and molecular levels in worms that have been decapitated, we show that the presence of the head is essential for multiple aspects of posterior regeneration, as well as for the subsequent production of new segments. We also show that methylfarnesoate, the molecule proposed to be the brain hormone, can partially rescue the posterior regeneration defects observed in decapitated worms. Our results are therefore consistent with a key role of brain hormonal activity in the control of regeneration and growth in P. dumerilii, and support the hypothesis of the involvement of methylfarnesoate in this control.

Annelid adult cell type diversity and their pluripotent cellular origins.

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

Distinct patterns of gene expression during regeneration and asexual reproduction in the annelid Pristina leidyi.

Regeneration, the ability to replace lost body parts, is a widespread phenomenon in the animal kingdom often connected to asexual reproduction or fission, since the only difference between the two appears to be the stimulus that triggers them. Both developmental processes have largely been characterized; however, the molecular toolkit and genetic mechanisms underlying these events remain poorly unexplored. Annelids, in particular the oligochaete Pristina leidyi, provide a good model system to investigate these processes as they show diverse ways to regenerate, and can reproduce asexually through fission under laboratory conditions. Here, we used a comparative transcriptomics approach based on RNA-sequencing and differential gene expression analyses to understand the molecular mechanisms involved in anterior regeneration and asexual reproduction. We found 291 genes upregulated during anterior regeneration, including several regeneration-related genes previously reported in other annelids such as frizzled, paics, and vdra. On the other hand, during asexual reproduction, 130 genes were found upregulated, and unexpectedly, many of them were related to germline development during sexual reproduction. We also found important differences between anterior regeneration and asexual reproduction, with the latter showing a gene expression profile more similar to that of control individuals. Nevertheless, we identified 35 genes that were upregulated in both conditions, many of them related to cell pluripotency, stem cells, and cell proliferation. Overall, our results shed light on the molecular mechanisms that control anterior regeneration and asexual reproduction in annelids and reveal similarities with other animals, suggesting that the genetic machinery controlling these processes is conserved across metazoans.

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics.

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.

Delegating Sex: Differential Gene Expression in Stolonizing Syllids Uncovers the Hormonal Control of Reproduction.

Stolonization in syllid annelids is a unique mode of reproduction among animals. During the breeding season, a structure resembling the adult but containing only gametes, called stolon, is formed generally at the posterior end of the animal. When stolons mature, they detach from the adult and gametes are released into the water column. The process is synchronized within each species, and it has been reported to be under environmental and endogenous control, probably via endocrine regulation. To further understand reproduction in syllids and to elucidate the molecular toolkit underlying stolonization, we generated Illumina RNA-seq data from different tissues of reproductive and nonreproductive individuals of Syllis magdalena and characterized gene expression during the stolonization process. Several genes involved in gametogenesis (ovochymase, vitellogenin, testis-specific serine/threonine-kinase), immune response (complement receptor 2), neuronal development (tyrosine-protein kinase Src42A), cell proliferation (alpha-1D adrenergic receptor), and steroid metabolism (hydroxysteroid dehydrogenase 2) were found differentially expressed in the different tissues and conditions analyzed. In addition, our findings suggest that several neurohormones, such as methyl farnesoate, dopamine, and serotonin, might trigger stolon formation, the correct maturation of gametes and the detachment of stolons when gametogenesis ends. The process seems to be under circadian control, as indicated by the expression patterns of r-opsins. Overall, our results shed light into the genes that orchestrate the onset of gamete formation and improve our understanding of how some hormones, previously reported to be involved in reproduction and metamorphosis processes in other invertebrates, seem to also regulate reproduction via stolonization.

The genus Syllis Savigny in Lamarck, 1818 (Annelida: Syllidae: Syllinae) from Australia (second part): four new species and re-description of twelve previously described species.

Syllis Savigny in Lamarck, 1818, the type genus of Syllidae, is the largest and most diverse within the family. This genus presents many taxonomic and phylogenetic problems due mainly to the lack of molecular data and morphological synapomorphies for many of the species, but also to poor or inadequate descriptions. In order to improve the knowledge of the genus, we have undertaken a morphological revision of Australian species, based on type material and material from the Australian Museum. Sixteen species are herein described, of which four are new and twelve are redescriptions of alreadyknown species, with six new combinations: Syllis boggemanni, n. sp.; S. joaoi, n. sp.; S. karlae, n. sp.; S. marceloi, n. sp.; S. albanyensis (Hartmann-Schröder, 1984) n. comb.; S. erikae (Hartmann-Schröder, 1981) n. comb.; S. krohnii Ehlers 1864; S. lunaris (Imajima, 1966) n. comb.; S. lutea (Hartmann-Schröder, 1960); S. macrodentata (Hartmann-Schröder, 1982) n. comb.; S. monilaris Savigny in Lamarck, 1818; S. nigropunctata Haswell, 1886; S. pectinans Haswell, 1920; S. rosea (Langerhans, 1879); S. warrnamboolensis (Hartmann-Schröder, 1987) n. comb.; and S. yallingupensis (Hartmann-Schröder, 1982), n. comb.

The Syllis gracilis species complex: A molecular approach to a difficult taxonomic problem (Annelida, Syllidae).

Syllis gracilis is an emblematic member of the subfamily Syllinae (Syllidae, Annelida), which inhabits shallow, temperate coastal waters and can be found on algae, coral rubble, and sponges. Their distinctive ypsiloid chaetae, usually found in specimens from populations all around the world, led to the consideration of the species as cosmopolitan, even though four other species have similar chaetae: Syllis magellanica, S. picta, S. mayeri and S. ypsiloides. The discovery of deeply divergent lineages in the Mediterranean Sea, that were morphologically similar, questioned the cosmopolitanism of S. gracilis and suggested the possibility of it being a species complex. In order to assess the speciation patterns within the putative S. gracilis complex, we undertook species delimitation and phylogenetic analyses on 61 specimens morphologically ascribed to Syllis gracilis and closely related species using a multilocus molecular dataset (two mitochondrial and two nuclear markers). Our results suggest high levels of genetic differentiation between the S. gracilis populations analyzed, some of which have morphologically distinctive features. Five to eight distinct lineages (depending on the analysis) were identified, all with geographically restricted distributions. Although the presence of ypsiloid chaetae has been traditionally considered the main character to identify S. gracilis, we conclude that this feature is homoplastic. Instead, we propose that characters such as the degree of fusion of blades and shafts in chaetae, the morphology of the posterior chaetae or the animal color pattern should be considered to differentiate lineages within the S. gracilis species complex. Our study does not support the cosmopolitanism of S. gracilis, and instead provides morphological and molecular evidence of the existence of a complex of pseudo-cryptic species.

Two new species and new records of the genus Paraopisthosyllis Hartmann-Schröder, 1991 (Annelida: Syllidae) from northeastern Brazil and Philippine Islands.

Two new species of Paraopisthosyllis are described herein, P. correiae sp. nov. (State of Pernambuco, NE Brazil) and P. kuluguhin sp. nov. (Philippines). Paraopisthosyllis correiae sp. nov. represents the first record for the genus in Brazilian waters and it is also the second species known to occur outside the Pacific Ocean. This species is characterized by having a marked color pattern, with a transverse red stripe between dorsal cirri on each segment and an inverted V anteriorly and a V posteriorly on some segments, small papillae on body, large, distinct papillae on dorsal cirri, compound chaetae with elongated, bidentate blades, shafts with moderate subdistal serration on margin, pharyngeal tooth located on anterior third of pharynx, near the pharyngeal opening, and reproduction by schizogamy. Paraopisthosyllis kuluguhin sp. nov. represents the first report of the genus for the Philippines, together with P. pardus, originally known from Australia and also found in the Philippines in the present study. This new species is characterized by its color pattern, with dark patches irregularly distributed on anterior segments, forming one anterior pigmented short band and two lateral areas on each chaetiger after the proventricle, club-shaped dorsal cirri, and short, bidentate compound chaetae. Our findings allow us to synonymize the genus Basidiosyllis to Paraopisthosyllis and represent the first report of sexual stolons (scissiparity) in this genus, which supports its allocation into the subfamily Syllinae.

The genus Syllis Savigny in Lamarck, 1818 (Annelida, Syllidae) from Australia. Molecular analysis and re-description of some poorly-known species.

The taxonomy and phylogenetic relationships within Syllis Savigny in Lamarck, 1818, the type genus of the family Syllidae, are still a matter of debate because the group does not show clear synapomorphies and because of the lack of molecular data for many of the species. In order to help understand some of the phylogenetic relationships within the genus Syllis, we have performed a morphological revision of part of the material collected during decades by the Australian Museum staff, and provide molecular data for species not sequenced before. In particular, seven poorly known Australian species of the genus Syllis Savigny in Lamarck, 1818 have been re-described in detail and sequenced to analyze their phylogenetic position: Syllis broomensis n. comb., S. crassicirrata (Treadwell, 1925) n. comb., Syllis cruzi Núñez & San Martín, 1991, S. edensis (Hartmann-Schröder, 1989), Syllis gracilis Grube, 1840, Syllis picta (Kinberg, 1866) n. comb., and S. setoensis (Imajima, 1966). The results obtained indicate the paraphyly of Typosyllis and a possible new organization of Syllis, which contains at least four well-supported clades.

Unveiling the Rosetta Stone of syllids: redescription and neotype designation of Syllis monilaris Savigny in Lamarck, 1818, type species of type genus of family Syllidae Grube, 1850 (Annelida).

Syllis monilaris Savigny in Lamarck, 1818, the type species of the genus Syllis Savigny in Lamarck, 1818, is redescribed based on two specimens deposited in the Museum für Naturkunde Berlin (Germany). One specimen is designated as neotype, since the original type material is considered to be lost, and there is a necessity to stabilize the nomenclature of the group. The species is large sized, with long dorsal cirri on anterior segments, becoming short and fusiform from midbody, it has thick compound chaetae with short, unidentate blades, not fused to shafts. The lack of chaetae with fused shafts and blades contradicts the division of the genus Syllis into subgenera as proposed by Langerhans (1879), who considered the subgenus Syllis as having thick fused chaetae on midbody, in addition to compound chaetae, and the subgenus Typosyllis with only compound chaetae. A discussion about the systematics of the genus is included, and according to this new data, Typosyllis is a junior synonymy of Syllis.

A new species and new record of the commensal genus Alcyonosyllis Glasby & Watson, 2001 and a new species of Parahaplosyllis Hartmann-Schröder, 1990, (Annelida: Syllidae: Syllinae) from Philippines Islands.

A new species of Alcyonosyllis (Annelida: Syllidae), A. aidae n.sp. is described from Luzón island, Philippines, associated with the alcyonacean Dendronephthya sp. (Nephthydae). This is the sixth known species of this genus living in the Indo-Pacific region. The new species differs from other Alcyonosyllis in having long and slender cirri with the first pair of dorsal cirri slightly thicker than remaining, bidentate chaetae with distal tooth larger than proximal one, and a distinct colour pattern, with a median longitudinal, slender reddish line, and two wider lateral bands, giving a tri-lineate appearance. A new report of the recently described species, A. hinterkircheri, previously known only from an area close to Bohol, in Philippines, is also included, being the first report of this species in Luzón Island. A new species of the genus Parahaplosyllis Hartmann-Schröder, 1990, is also described. Up to now, only the type species of the genus was known, from New South Wales, Australia; this is the second known species of this genus. It differs from P. brevicirra Hartmann-Schröder, 1990 by having unidentate dorsal simple chaetae (instead of bidentate ones as in P. brevicirra), ventral simple chaeta with shorter and less curved basal spur, more distinctly articulated dorsal cirri, with a long distal article, and a shorter proventricle. Finally, new different types of stolons are described for both genera.