microRNAs (miRNAs) are approximately 22-nucleotide noncoding RNA regulatory genes that are key players in cellular differentiation and homeostasis. They might also play important roles in shaping metazoan macroevolution. Previous studies have shown that miRNAs are continuously being added to metazoan genomes through time, and, once integrated into gene regulatory networks, show only rare mutations within the primary sequence of the mature gene product and are only rarely secondarily lost. However, because the conclusions from these studies were largely based on phylogenetic conservation of miRNAs between model systems like Drosophila and the taxon of interest, it was unclear if these trends would describe most miRNAs in most metazoan taxa. Here, we describe the shared complement of miRNAs among 18 animal species using a combination of 454 sequencing of small RNA libraries with genomic searches. We show that the evolutionary trends elucidated from the model systems are generally true for all miRNA families and metazoan taxa explored: the continuous addition of miRNA families with only rare substitutions to the mature sequence, and only rare instances of secondary loss. Despite this conservation, we document evolutionary stable shifts to the determination of position 1 of the mature sequence, a phenomenon we call seed shifting, as well as the ability to post-transcriptionally edit the 5' end of the mature read, changing the identity of the seed sequence and possibly the repertoire of downstream targets. Finally, we describe a novel type of miRNA in demosponges that, although shows a different pre-miRNA structure, still shows remarkable conservation of the mature sequence in the two sponge species analyzed. We propose that miRNAs might be excellent phylogenetic markers, and suggest that the advent of morphological complexity might have its roots in miRNA innovation.

The causal basis of vertebrate complexity has been sought in genome duplication events (GDEs) that occurred during the emergence of vertebrates, but evidence beyond coincidence is wanting. MicroRNAs (miRNAs) have recently been identified as a viable causal factor in increasing organismal complexity through the action of these Ϸ22-nt noncoding RNAs in regulating gene expression. Because miRNAs are continuously being added to animalian genomes, and, once integrated into a gene regulatory network, are strongly conserved in primary sequence and rarely secondarily lost, their evolutionary history can be accurately reconstructed. Here, using a combination of Northern analyses and genomic searches, we show that 41 miRNA families evolved at the base of Vertebrata, as they are found and/or detected in lamprey, but not in either ascidians or amphioxus (or any other nonchordate taxon). When placed into temporal context, the rate of miRNA acquisition and the extent of phenotypic evolution are anomalously high early in vertebrate history, far outstripping any other episode in chordate evolution. The genomic position of miRNA paralogues in humans, together with gene trees incorporating lamprey orthologues, indicates that although GDEs can account for an increase in the diversity of miRNA family members, which occurred before the last common ancestor of all living vertebrates, GDEs cannot account for the origin of these novel families themselves. We hypothesize that lying behind the origin of vertebrate complexity is the dramatic expansion of the noncoding RNA inventory including miRNAs, rather than an increase in the protein-encoding inventory caused by GDEs.genome duplication ͉ lamprey ͉ macroevolution ͉ shark ͉ chordate

SUMMARY Molecular paleoecology is the application of molecular data to test hypotheses made by paleoecological scenarios. Here, we use gene regulatory analysis to test between two competing paleoecological scenarios put forth to explain the evolution of complex life cycles. The first posits that early bilaterians were holobenthic, and the evolution of macrophagous grazing drove the exploitation of the pelagos by metazoan eggs and embryos, and eventually larvae. The alternative hypothesis predicts that early bilaterians were holopelagic, and new adult stages were added on when these holopelagic forms began to feed on the benthos. The former hypothesis predicts that the larvae of protostomes and deuterostomes are not homologous, with the implication that larval-specific structures, including the apical organ, are the products of convergent evolution, whereas the latter hypothesis predicts homology of larvae, specifically homology of the apical organ. We show that in the sea urchin, Strongylocentrotus purpuratus, the transcription factors NK2.1 and HNF6 are necessary for the correct spatial expression profiles of five different cilia genes. All of these genes are expressed exclusively in the apical plate after the mesenchyme-blastula stage in cells that also express NK2.1 and HNF6. In addition, abrogation of SpNK2.1 results in embryos that lack the apical tuft. However, in the red abalone, Haliotis rufescens, NK2.1 and HNF6 are not expressed in any cells that also express these same five cilia genes. Nonetheless, like the sea urchin, the gastropod expresses both NK2.1 and FoxA around the stomodeum and foregut, and FoxA around the proctodeum. As we detected no similarity in the development of the apical tuft between the sea urchin and the abalone, these molecular data are consistent with the hypothesis that the evolution of mobile, macrophagous metazoans drove the evolution of complex life cycles multiple times independently in the late Precambrian.

Both the monophyly and inter-relationships of the major annelid groups have remained uncertain, despite intensive research on both morphology and molecular sequences. Morphological cladistic analyses indicate that Annelida is monophyletic and consists of two monophyletic groups, the clitellates and polychaetes, whereas molecular phylogenetic analyses suggest that polychaetes are paraphyletic and that sipunculans are crown-group annelids. Both the monophyly of polychaetes and the placement of sipunculans within annelids are in conflict with the annelid fossil record-the former because Cambrian stem taxa are similar to modern polychaetes in possessing biramous parapodia, suggesting that clitellates are derived from polychaetes; the latter because although fossil sipunculans are known from the Early Cambrian, crown-group annelids do not appear until the latest Cambrian. Here we apply a different data source, the presence versus absence of specific microRNAs-genes that encode approximately 22 nucleotide non-coding regulatory RNAs-to the problem of annelid phylogenetics. We show that annelids are monophyletic with respect to sipunculans, and polychaetes are paraphyletic with respect to the clitellate Lumbricus, conclusions that are consistent with the fossil record. Further, sipunculans resolve as the sister group of the annelids, rooting the annelid tree, and revealing the polarity of the morphological change within this diverse lineage of animals.

Recent phylogenetic investigations have confirmed that hemichordates and echinoderms are sister taxa. However, hemichordates share several cardinal characterstics with chordates and are thus an important taxon for testing hypotheses of homology between key chordate characters and their putative hemichordate antecedents. The chordate dorsal nervous system (DNS) and endostyle are intriguing characters because both hemichordate larval and adult structures have been hypothesized as homologues. This study attempts to test these purported homologies through examination of the expression pattem of a Ptychodera flava NK2 gene, PfNK2.1, because this gene is expressed both in the DNS and endostyle/thyroid in a wide range of chordate taxa. We found that PfNK2.1 is expressed in both neuronal and pharyngeal structures, but its expression pattem is broken up into distinct embryonic and juvenile phases. During embryogenesis, PfNK2.1 is expressed in the apical ectoderm, with transcripts later detected in presumable neuronal structures, including the apical organ and ciliated feeding band. In the developing juvenile we detected PfNK2.1 signal throughout the pharynx, including the stomochord, and later in the hindgut. We conclude that the similar utilization of NK2.1 in apical organ development and chordate DNS is probably due to a more general role for NK2.1 in neurogenesis and that hemichordates do not possess a homologue of the chordate DNS. In addition, we conclude that P. flava most likely does not possess a true endostyle; rather during the evolution of the endostyle NK2.1 was recruited from its more general role in pharynx development.