The evolution of annelids reveals two adaptive routes to the interstitial realm.

Many animals permanently inhabit the marine interstitium, the space between sand grains [1, 2]. Different evolutionary scenarios may explain the existence of interstitial animals [3, 4]. These scenarios include (1) that the interstitial realm is the ancestral habitat of bilaterians [5, 6], (2) that interstitial taxa evolved from larger ancestors by miniaturization, or (3) progenesis [3]. The first view mirrors the former hypothesis that interstitial annelids, called archiannelids, were at the base of the annelid radiation [7]. Based on morphological data, however, progenesis is generally favored for interstitial annelids today [3, 4, 8]. Herein, our phylogenomic approach revealed that interstitial archiannelids are robustly placed into two groups nested within the annelid tree. Evolution of the first group comprising among others Dinophilidae is best explained by progenesis. In contrast, the second group comprising Protodrilida and Polygordiidae appears to have evolved by stepwise miniaturization adapting from coarser to finer sediments. Thus, in addition to progenesis [3, 4], miniaturization, thought to be too slow for an adaptation to the interstitium [3], is an important second route allowing adaptation to interstitial environments. Both progenesis and miniaturization should be considered when investigating evolution of interstitial taxa [1, 3].

Expression study of the hunchback ortholog in embryos of the onychophoran Euperipatoides rowelli.

Zinc finger transcription factors encoded by hunchback homologs play different roles in arthropods, including maternally mediated control, segmentation, and mesoderm and neural development. Knockdown experiments in spider and insect embryos have also revealed homeotic effects and gap phenotypes, the latter indicating a function of hunchback as a "gap gene". Although the expression pattern of hunchback has been analysed in representatives of all four major arthropod groups (chelicerates, myriapods, crustaceans and insects), nothing is known about its expression in one of the closest arthropod relatives, the Onychophora (velvet worms). We therefore examined the expression pattern of hunchback in embryos of the onychophoran Euperipatoides rowelli. Our transcriptomic and phylogenetic analyses revealed only one hunchback ortholog in this species. The putative Hunchback protein contains all nine zinc finger domains known from other protostomes. We found no indication of maternally contributed transcripts of hunchback in early embryos of E. rowelli. Its initial expression occurs in the ectodermal tissue of the antennal segment, followed by the jaw, slime papilla and trunk segments in an anterior-to-posterior progression. Later, hunchback expression is seen in the mesoderm of the developing limbs. A second "wave" of expression commences later in development in the antennal segment and continues posteriorly along each developing nerve cord. This expression is restricted to the neural tissues and does not show any segmental pattern. These findings are in line with the ancestral roles of hunchback in mesoderm and neural development, whereas we find no evidence for a putative function of hunchback as a "gap gene" in Onychophora.

Phylogenetic analysis and expression patterns of Pax genes in the onychophoran Euperipatoides rowelli reveal a novel bilaterian Pax subfamily.

Pax family genes encode a class of transcription factors that regulate various developmental processes. To shed light on the evolutionary history of these genes in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we analyzed the Pax repertoire in the embryonic and adult transcriptomes of the onychophoran Euperipatoides rowelli. Our data revealed homologs of all five major bilaterian Pax subfamilies in this species, including Pax2/5/8, Pax4/6, Pox-neuro, Pax1/9/Pox-meso, and Pax3/7. In addition, we identified a new Pax member, pax-α, which does not fall into any other known Pax subfamily but instead clusters in the heterogenic Pax-α/ß clade containing deuterostome, ecdysozoan, and lophotrochozoan gene sequences. These findings suggest that the last common bilaterian ancestor possessed six rather than five Pax genes, which have been retained in the panarthropod lineage. The expression data of Pax orthologs in the onychophoran embryo revealed distinctive patterns, some of which might be related to their ancestral roles in the last common panarthropod ancestor, whereas others might be specific to the onychophoran lineage. The derived roles include, for example, an involvement of pax2/5/8, pox-neuro, and pax3/7 in onychophoran nephridiogenesis, and an additional function of pax2/5/8 in the formation of the ventral and preventral organs. Furthermore, our transcriptomic analyses suggest that at least some Pax genes, including pax6 and pax-α, are expressed in the adult onychophoran head, although the corresponding functions remain to be clarified. The remarkable diversity of the Pax expression patterns highlights the functional and evolutionary plasticity of these genes in panarthropods.

Controversies surrounding segments and parasegments in onychophora: insights from the expression patterns of four "segment polarity genes" in the peripatopsid Euperipatoides rowelli.

Arthropods typically show two types of segmentation: the embryonic parasegments and the adult segments that lie out of register with each other. Such a dual nature of body segmentation has not been described from Onychophora, one of the closest arthropod relatives. Hence, it is unclear whether onychophorans have segments, parasegments, or both, and which of these features was present in the last common ancestor of Onychophora and Arthropoda. To address this issue, we analysed the expression patterns of the "segment polarity genes" engrailed, cubitus interruptus, wingless and hedgehog in embryos of the onychophoran Euperipatoides rowelli. Our data revealed that these genes are expressed in repeated sets with a specific anterior-to-posterior order along the body in embryos of E. rowelli. In contrast to arthropods, the expression occurs after the segmental boundaries have formed. Moreover, the initial segmental furrow retains its position within the engrailed domain throughout development, whereas no new furrow is formed posterior to this domain. This suggests that no re-segmentation of the embryo occurs in E. rowelli. Irrespective of whether or not there is a morphological or genetic manifestation of parasegments in Onychophora, our data clearly show that parasegments, even if present, cannot be regarded as the initial metameric units of the onychophoran embryo, because the expression of key genes that define the parasegmental boundaries in arthropods occurs after the segmental boundaries have formed. This is in contrast to arthropods, in which parasegments rather than segments are the initial metameric units of the embryo. Our data further revealed that the expression patterns of "segment polarity genes" correspond to organogenesis rather than segment formation. This is in line with the concept of segmentation as a result of concerted evolution of individual periodic structures rather than with the interpretation of 'segments' as holistic units.

Unexplored character diversity in onychophora (velvet worms): A comparative study of three peripatid species.

Low character variation among onychophoran species has been an obstacle for taxonomic and phylogenetic studies in the past, however we have identified a number of new and informative characters using morphological, molecular, and chromosomal techniques. Our analyses involved a detailed examination of Epiperipatus biolleyi from Costa Rica, Eoperipatus sp. from Thailand, and a new onychophoran species and genus from Costa Rica, Principapillatus hitoyensisgen. et sp. nov.. Scanning electron microscopy on embryos and specimens of varying age revealed novel morphological characters and character states, including the distribution of different receptor types along the antennae, the arrangement and form of papillae on the head, body and legs, the presence and shape of interpedal structures and fields of modified scales on the ventral body surface, the arrangement of lips around the mouth, the number, position and structure of crural tubercles and anal gland openings, and the presence and shape of embryonic foot projections. Karyotypic analyses revealed differences in the number and size of chromosomes among the species studied. The results of our phylogenetic analyses using mitochondrial COI and 12S rRNA gene sequences are in line with morphological and karyotype data. However, our data show a large number of unexplored, albeit informative, characters in the Peripatidae. We suggest that analysing these characters in additional species would help unravel species diversity and phylogeny in the Onychophora, and that inconsistencies among most diagnostic features used for the peripatid genera in the literature could be addressed by identifying a suite of characters common to all peripatids.

Mesozoic retroposons reveal parrots as the closest living relatives of passerine birds.

The relationships of passerines (such as the well-studied zebra finch) with non-passerine birds is one of the great enigmas of avian phylogenetic research, because decades of extensive morphological and molecular studies yielded highly inconsistent results between and within data sets. Here we show the first application of the virtually homoplasy-free retroposon insertions to this controversy. Our study examined ~200,000 retroposon-containing loci from various avian genomes and retrieved 51 markers resolving early bird phylogeny. Among these, we obtained statistically significant evidence that parrots are the closest and falcons the second-closest relatives of passerines, together constituting the Psittacopasserae and the Eufalconimorphae, respectively. Our new and robust phylogenetic framework has substantial implications for the interpretation of various conclusions drawn from passerines as model organisms. This includes insights of relevance to human neuroscience, as vocal learning (that is, birdsong) probably evolved in the psittacopasseran ancestor, >30 million years earlier than previously assumed.