Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that fewer than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation, and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database -- MirGeneDB (http://mirgenedb.org) -- to catalog this set of robustly supported miRNAs, which complements the efforts of miRBase, but differs from it by annotating the mature versus star products, and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

It is often assumed that similar behaviors in related species are produced by similar neural mechanisms. To test this, we examined the neuronal basis of a simple swimming behavior in two nudibranchs (Mollusca, Opisthobranchia), Melibe leonina and Dendronotus iris. The side-to-side swimming movements of Dendronotus [1] strongly resemble those of Melibe [2, 3]. In Melibe, it was previously shown that the central pattern generator (CPG) for swimming is composed of two bilaterally symmetric pairs of identified interneurons, swim interneuron 1 (Si1) and swim interneuron 2 (Si2), which are electrically coupled ipsilaterally and mutually inhibit both contralateral counterparts [2, 4]. We identified homologs of Si1 and Si2 in Dendronotus. (Henceforth, homologous neurons in each species will be distinguished by the subscripts (Den) and (Mel).) We found that Si2(Den) and Si2(Mel) play similar roles in generating the swim motor pattern. However, unlike Si1(Mel), Si1(Den) was not part of the swim CPG, was not strongly coupled to the ipsilateral Si2(Den), and did not inhibit the contralateral neurons. Thus, species differences exist in the neuronal organization of the swim CPGs despite the similarity of the behaviors. Therefore, similarity in species-typical behavior is not necessarily predictive of common neural mechanisms, even for homologous neurons in closely related species.

How neural circuit evolution relates to behavioral evolution is not well understood. Here the relationship between neural circuits and behavior is explored with respect to the swimming behaviors of the Nudipleura (Mollusca, Gastropoda, Opithobranchia). Nudipleura is a diverse monophyletic clade of sea slugs among which only a small percentage of species can swim. Swimming falls into a limited number of categories, the most prevalent of which are rhythmic left-right body flexions (LR) and rhythmic dorsal-ventral body flexions (DV). The phylogenetic distribution of these behaviors suggests a high degree of homoplasy. The central pattern generator (CPG) underlying DV swimming has been well characterized in Tritonia diomedea and in Pleurobranchaea californica. The CPG for LR swimming has been elucidated in Melibe leonina and Dendronotus iris, which are more closely related. The CPGs for the categorically distinct DV and LR swimming behaviors consist of nonoverlapping sets of homologous identified neurons, whereas the categorically similar behaviors share some homologous identified neurons, although the exact composition of neurons and synapses in the neural circuits differ. The roles played by homologous identified neurons in categorically distinct behaviors differ. However, homologous identified neurons also play different roles even in the swim CPGs of the two LR swimming species. Individual neurons can be multifunctional within a species. Some of those functions are shared across species, whereas others are not. The pattern of use and reuse of homologous neurons in various forms of swimming and other behaviors further demonstrates that the composition of neural circuits influences the evolution of behaviors.

The serotonergic systems in nudibranch molluscs were compared by mapping the locations of serotonin-immunoreactive (5-HT-ir) neurons in 11 species representing all four suborders of the nudibranch clade: Dendronotoidea (Tritonia diomedea, Tochuina tetraquetra, Dendronotus iris, Dendronotus frondosus, and Melibe leonina), Aeolidoidea (Hermissenda crassicornis and Flabellina trophina), Arminoidea (Dirona albolineata, Janolus fuscus, and Armina californica), and Doridoidea (Triopha catalinae). A nomenclature is proposed to standardize reports of cell location in species with differing brain morphologies. Certain patterns of 5-HT immunoreactivity were found to be consistent for all species, such as the presence of 5-HT-ir neurons in the pedal and cerebral ganglia. Also, particular clusters of 5-HT-ir neurons in the anterior and posterior regions of the dorsal surface of the cerebral ganglion were always present. However, there were interspecies differences in the number of 5-HT-ir neurons in each cluster, and some clusters even exhibited strong intraspecies variability that was only weakly correlated with brain size. Phylogenetic analysis suggests that the presence of particular classes of 5-HT-ir neurons exhibits a great deal of homoplasy. The conserved features of the nudibranch serotonergic system presumably represent the shared ancestral structure, whereas the derived characters suggest substantial independent evolutionary changes in the number and presence of serotonergic neurons. Although a number of studies have demonstrated phylogenetic variability of peptidergic systems, this study suggests that serotonergic systems may also exhibit a high degree of homoplasy in some groups of organisms.

Homologues of a neuron that contributes to a species-specific behavior were identified and characterized in species lacking that behavior. The nudibranch Tritonia diomedea swims by flexing its body dorsally and ventrally. The dorsal swim interneurons (DSIs) are components of the central pattern generator (CPG) underlying this rhythmic motor pattern and also activate crawling. Homologues of the DSIs were identified in six nudibranchs that do not exhibit dorsal-ventral swimming: Tochuina tetraquetra, Melibe leonina, Dendronotus iris, D. frondosus, Armina californica, and Triopha catalinae. Homology was based upon shared features that distinguish the DSIs from all other neurons: (1) serotonin immunoreactivity, (2) location in the Cerebral serotonergic posterior (CeSP) cluster, and (3) axon projection to the contralateral pedal ganglion. The DSI homologues, named CeSP-A neurons, share additional features with the DSIs: irregular basal firing, synchronous inputs, electrical coupling, and reciprocal inhibition. Unlike the DSIs, the CeSP-A neurons were not rhythmically active in response to nerve stimulation. The CeSP-A neurons in Tochuina and Triopha also excited homologues of the Tritonia Pd5 neuron, a crawling efferent. Thus, the CeSP-A neurons and the DSIs may be part of a conserved network related to crawling that may have been co-opted into a rhythmic swim CPG in Tritonia.