MicroRNAs and phylogenomics resolve the relationships of Tardigrada and suggest that velvet worms are the sister group of Arthropoda

Abstract

Morphological data traditionally group Tardigrada (water bears), Onychophora (velvet worms), and Arthropoda (e.g., spiders, insects, and their allies) into a monophyletic group of invertebrates with walking appendages known as the Panarthropoda. However, molecular data generally do not support the inclusion of tardigrades within the Panarthropoda, but instead place them closer to Nematoda (roundworms). Here we present results from the analyses of two independent genomic datasets, expressed sequence tags (ESTs) and microRNAs (miRNAs), which congruently resolve the phylogenetic relationships of Tardigrada. Our EST analyses, based on 49,023 amino acid sites from 255 proteins, significantly support a monophyletic Panarthropoda including Tardigrada and suggest a sister group relationship between Arthropoda and Onychophora. Using careful experimental manipulations--comparisons of model fit, signal dissection, and taxonomic pruning--we show that support for a Tardigrada + Nematoda group derives from the phylogenetic artifact of long-branch attraction. Our small RNA libraries fully support our EST results; no miRNAs were found to link Tardigrada and Nematoda, whereas all panarthropods were found to share one unique miRNA (miR-276). In addition, Onychophora and Arthropoda were found to share a second miRNA (miR-305). Our study confirms the monophyly of the legged ecdysozoans, shows that past support for a Tardigrada + Nematoda group was due to long-branch attraction, and suggests that the velvet worms are the sister group to the arthropods.

Fig. 1.

Phylogenomics and miRNAs suggest velvets worm are the sister group to the arthropods within a monophyletic Panarthropoda. (A) Phylogenetic tree derived using Bayesian analysis of the EST data under the best-fitting CAT-GTR+Γ model supports tardigrades as the sister group of Lobopodia (Onychophora + Arthropoda). Support values represent posterior probabilities. Asterisks indicate a PP value of 1.0. Note that for Nematoda alone, the branch lengths are not shown to scale. (B) MiRNA distribution is consistent with the results obtained from the phylogenomic analysis. Single gray/black rectangles represent a miRNA gain. Clades are color-coded to highlight congruence between ESTs and miRNAs (see text for more details).

Fig. 2.

Model selection, signal dissection and taxon pruning experiments show LBA explains previous support for a tardigrade/nematode clade. As in Fig. 1, these are trees from the EST data; node values represent posterior probabilities, and asterisks indicate a PP of 1.0. The node where the Tardigrada join the tree is identified by a circle. Clades have been collapsed for clarity. (A) Tardigrades are recovered as the sister group of Nematoida under the poorly fitting GTR+Γ model of sequence evolution (for Δ-likelihoods and SDs; Fig. S1 and Methods). (B) Tree recovered from the analysis of the slowest-evolving 90% of the sites in our dataset (Fig. S3A). The PP values are reported in italics, whereas support values obtained from the analysis of the complete dataset are in roman type (Fig. 1A). (C) Topology recovered from the 10% fastest evolving sites in our dataset, under CAT-GTR+Γ. The fast-evolving sites support Tardigrada as the sister group of Nematoda. (D) Phylogeny generated under a reduced-taxon set (one onychophoran, one tardigrade, and no nematomorph) designed to exacerbate LBA artifact.