Monsters reveal patterns: bifurcated annelids and their implications for the study of development and evolution.

During recent decades, the study of anatomical anomalies has been of great relevance for research on development and its evolution. Yet most animal groups have never been studied under this perspective. In annelids, one of the most common and remarkable anomalies is anteroposterior axis bifurcation, that is animals that have two or more heads and/or tails. Bifurcated annelids were first described in the 18th century and have been occasionally reported since then. However, these animals have rarely been considered other than curiosities, one-off anomalies, or monsters, and a condensed but comprehensive analysis of this phenomenon is lacking. Such an analysis of the existing knowledge is necessary for addressing the different patterns of annelid bifurcation, as well as to understand possible developmental mechanisms behind them and their evolution. In this review we summarize reports of annelid bifurcation published during the last 275 years and the wide variety of anatomies they present. Our survey reveals bifurcation as a widespread phenomenon found all over the annelid tree. Moreover, it also shows that bifurcations can be classified into different types according to anatomy (lateral versus dorsoventral) or developmental origin (embryonic versus postembryonic, the latter occurring in relation to regeneration, reproduction, or growth). Regarding embryos, three different types of bifurcation can be found: conjoined twins (in clitellates); Janus embryos (two posterior ends with a single head which shows duplicated structures); and duplicitas cruciata embryos (with anterior and posterior bifurcation with a 90° rotation). In adults, we show that while lateral bifurcation can result in well-integrated phenotypes, dorsoventral bifurcation cannot since it requires the discontinuity of at least some internal organs. The relevance of this distinction is highlighted in the case of the Ribbon Clade, a group of syllid annelids in which some species reproduce by collateral and successive gemmiparity (which involves dorsoventral bifurcation), while others grow by branching laterally. Although most known cases of bifurcation came from accidental findings in the wild or were unintentionally produced, experimental studies resulting in the induction of bifurcation of both embryos and adults are also reviewed. In embryos, these experimental studies show how mechanical or chemical disruption of the zygote can result in bifurcation. In adults, the ventral nervous system and the digestive tract seem to play a role in the induction of bifurcation. Based on the reviewed evidence, we argue that the long-forgotten study of annelid developmental anomalies should be incorporated into the growing field of annelid EvoDevo and examined with modern techniques and perspectives.

Cellular proliferation dynamics during regeneration in Syllis malaquini (Syllidae, Annelida).

BACKGROUND: In syllids (Annelida, Syllidae), the regenerative blastema was subject of many studies in the mid and late XXth century. This work on syllid regeneration showed that the blastema is developed by a process of dedifferentiation of cells near the wound, followed by their proliferation and redifferentiation (cells differentiate to the original cell type) or, in some specific cases, transdifferentiation (cells differentiate to a cell type different from the original). Up to date, participation of stem cells or pre-existing proliferative cells in the blastema development has never been observed in syllids. This study provides the first comprehensive description of Syllis malaquini's regenerative capacity, including data on the cellular proliferation dynamics by using an EdU/BrdU labelling approach, in order to trace proliferative cells (S-phase cells) present before and after operation. RESULTS: Syllis malaquini can restore the anterior and posterior body from different cutting levels under experimental conditions, even from midbody fragments. Our results on cellular proliferation showed that S-phase cells present in the body before bisection do not significantly contribute to blastema development. However, in some specimens cut at the level of the proventricle, cells in S-phase located in the digestive tube before bisection participated in regeneration. Also, our results showed that nucleus shape allows to distinguish different types of blastemal cells as forming specific tissues. Additionally, simultaneous and sequential addition of segments seem to occur in anterior regeneration, while only sequential addition was observed in posterior regeneration. Remarkably, in contrast with previous studies in syllids, sexual reproduction was not induced during anterior regeneration of amputees lacking the proventricle, a foregut organ widely known to be involved in the stolonization control. CONCLUSIONS: Our findings led us to consider that although dedifferentiation and redifferentiation might be more common, proliferative cells present before injury can be involved in regenerative processes in syllids, at least in some cases. Also, we provide data for comparative studies on resegmentation as a process that differs between anterior and posterior regeneration; and on the controversial role of the proventricle in the reproduction of different syllid lineages.

Integrative anatomical study of the branched annelid Ramisyllis multicaudata (Annelida, Syllidae).

The sponge-dwelling Syllidae Ramisyllis multicaudata and Syllis ramosa are the only annelid species for which a branched body with one head and multiple posterior ends is known. In these species, the head is located deep within the sponge, and the branches extend through the canal system of their host. The morphology of these creatures has captivated annelid biologists since they were first discovered in the late XIXth century, and their external characteristics have been well documented. However, how their branched bodies fit within their symbiotic host sponges and how branches translate into internal anatomy has not been documented before. These features are crucially relevant for understanding the body of these animals, and therefore, the aim of this study was to investigate these aspects. In order to assess these questions, live observation, as wells as histology, immunohistochemistry, micro-computed tomography, and transmission electron microscopy techniques were used on specimens of R. multicaudata. By using these techniques, we show that the complex body of R. multicaudata specimens extends greatly through the canal system of their host sponges. We demonstrate that iterative external bifurcation of the body is accompanied by the bifurcation of the longitudinal organ systems that are characteristic of annelids. Additionally, we also highlight that the bifurcation process leaves an unmistakable fingerprint in the form of newly-described "muscle bridges." These structures theoretically allow one to distinguish original and derived branches at each bifurcation. Last, we characterize some of the internal anatomical features of the stolons (reproductive units) of R. multicaudata, particularly their nervous system. Here, we provide the first study of the internal anatomy of a branched annelid. This information is not only crucial to deepen our understanding of these animals and their biology, but it will also be key to inform future studies that try to explain how this morphology evolved.

Sex-specific gene expression differences in reproducing Syllis prolifera and Nudisyllis pulligera (Annelida, Syllidae).

Syllidae is an annelid family characterized by its complex life cycles involving some of the most outstanding annelid reproductive strategies. Syllid reproductive modes sometimes imply the modification of the posterior body to form independent reproductive units (schizogamy) or the development of swimming adults (epigamy). These modes of sexual reproduction have been studied for more than 150 years, and yet, little is known regarding their molecular background. Notably, while several studies during the last three decades have revealed details about molecular mechanisms involved in the reproduction of some few model annelids, studies focusing on syllids remain limited. Thus, we performed differential gene expression analyses of female, male, and non-reproducing individuals of Syllis prolifera (schizogamic) and Nudisyllis pulligera (epigamic), as representatives of two different reproductive strategies. For that, transcriptomes from specimens of three conditions (non-reproducing, male, female) were de novo assembled and annotated for S. prolifera and N. pulligera. We found rather similar gene expression profiles for female and non-reproducing individuals, while male gene expression is clearly different. Although previous studies have suggested that femininity in syllids might require additional signalling, our analyses support a scenario, where masculinity may also involve several specific genetic processes.

Comparative transcriptomics in Syllidae (Annelida) indicates that posterior regeneration and regular growth are comparable, while anterior regeneration is a distinct process.

BACKGROUND: Annelids exhibit remarkable postembryonic developmental abilities. Most annelids grow during their whole life by adding segments through the action of a segment addition zone (SAZ) located in front of the pygidium. In addition, they show an outstanding ability to regenerate their bodies. Experimental evidence and field observations show that many annelids are able to regenerate their posterior bodies, while anterior regeneration is often limited or absent. Syllidae, for instance, usually show high abilities of posterior regeneration, although anterior regeneration varies across species. Some syllids are able to partially restore the anterior end, while others regenerate all lost anterior body after bisection. Here, we used comparative transcriptomics to detect changes in the gene expression profiles during anterior regeneration, posterior regeneration and regular growth of two syllid species: Sphaerosyllis hystrix and Syllis gracilis; which exhibit limited and complete anterior regeneration, respectively. RESULTS: We detected a high number of genes with differential expression: 4771 genes in S. hystrix (limited anterior regeneration) and 1997 genes in S. gracilis (complete anterior regeneration). For both species, the comparative transcriptomic analysis showed that gene expression during posterior regeneration and regular growth was very similar, whereas anterior regeneration was characterized by up-regulation of several genes. Among the up-regulated genes, we identified putative homologs of regeneration-related genes associated to cellular proliferation, nervous system development, establishment of body axis, and stem-cellness; such as rup and JNK (in S. hystrix); and glutamine synthetase, elav, slit, Hox genes, ß-catenin and PL10 (in S. gracilis). CONCLUSIONS: Posterior regeneration and regular growth show no significant differences in gene expression in the herein investigated syllids. However, anterior regeneration is associated with a clear change in terms of gene expression in both species. Our comparative transcriptomic analysis was able to detect differential expression of some regeneration-related genes, suggesting that syllids share some features of the regenerative mechanisms already known for other annelids and invertebrates.

Expression of vasa, piwi, and nanos during gametogenesis in Typosyllis antoni (Annelida, Syllidae).

Although model species have proven to be crucial for developmental biology, the evo-devo approach requires a broader picture across phylogeny. Herein, we try to expand the range of studied annelids by presenting a transcriptome of Typosyllis antoni as a tool for the study of developmental and evolutionary processes in Syllidae. Moreover, we provide homologs of the stem-cell markers vasa, piwi, and nanos, and investigate their expression patterns in gamete-producing individuals for the first time in this group. We found no expression in females, while there is a distinct expression pattern in males. Based on this data, we argue that spermatogenesis starts in the gonads and finishes in the coelomic cavity, and it occurs simultaneously in a large number of segments. Surprisingly, no expression of the stem-cell markers was found in the segment addition zone of these reproducing animals (stolonizing). Preliminary explanations like a lack of growth during stolonization, or the absence of a common genetic program between germ and somatic stem cells, are discussed. Finally, no reservoir of primordial cells has been detected, suggesting a possible epigenic origin of the Primordial Germ Cells of this species, though this hypothesis needs to be further investigated.

The complete mitochondrial genome of the Endangered European brown frog Rana pyrenaica through RNAseq.

We sequenced the complete mitogenome of the Pyrenean frog Rana pyrenaica, which was determined from an Illumina Hi-seq RNAseq run (Illumina Inc., San Diego, CA). The genome is 17,213 bp in size, including 13 protein-coding genes, 21 transfer RNAs, two ribosomal RNAs and a control region. It shows the typical gene order of previously available frog mitogenomes, although it lacks the tRNAPhe . This is the first complete mitogenome described for a Western Palearctic brown frog species.