With over 1 million living species described and a rich 520 Myr fossil record, arthropods are the most species-rich clade of animals on Earth, accounting for nearly 80 per cent of animal biodiversity [1
]. Four main euarthropod sub-phyla are recognized: Hexapoda (including insects); Crustacea (lobsters, water fleas and others); Myriapoda (e.g. millipedes and centipedes); and Chelicerata (including arachnids, horseshoe crabs and possibly sea spiders). After many years of debate, a consensus has emerged that these four classes (or sub-phyla) form a monophyletic group called the Euarthropoda [2
]. The relationships between the four euarthropod groups remain disputed, however, as is the validity of their close relationship to tardigrades (water bears) and onychophorans (velvet worms) in a more inclusive clade called Arthropoda (named Panarthropoda by Nielsen [4
Within the Euarthropoda, the main point of disagreement concerns the position of the myriapods, which were long thought to be most closely related to the hexapods [5
]. Myriapods and hexapods notably share a distinctive head composed of five segments distinguished by their unique appendages—the antennal, intercalary (appendage-less), mandibular, and usually two pairs of maxillae (the second being the insect labium). Molecular data, however, have shown crustaceans, which differ in having a second antennal rather than an intercalary segment, to be the closest sister group of hexapods in a clade named Pancrustacea or Tetraconata [6
]. When compared with chelicerates, the detailed similarities of the arrangement of head segments and associated appendages in Pancrustacea and myriapods strongly support their sister group relationship within a wider clade that has been named the Mandibulata in recognition of the similarity of their biting mouthparts (see the electronic supplementary material). Considering the complex shared features of myriapod and pancrustacean head morphology, it is surprising that the majority of published molecular phylogenetic analyses do not support the Mandibulata, instead placing the myriapods as the sister group of the chelicerates in an assemblage that has been named the Myriochelata or Paradoxopoda [8
]. Molecular support for Myriochelata was initially obtained using large and small subunit rRNAs [10
] and later Hox genes [8
], mitochondrial protein-coding sequences [11
] and combined datasets of both nuclear and mitochondrial genes [9
]. Myriochelata was also supported by several phylogenomic analyses [12
]. However, recently, a dataset of 62 nuclear protein-coding genes found support for Mandibulata [16
]. Regier et al
] did not identify the factors underpinning the difference between their new results and those of previously published phylogenies that supported Myriochelata. Consequently, and in light of the varying results from these molecular samples, the Mandibulata versus Myriochelata controversy remains an open question.
Uncertainty in deep arthropod phylogeny has recently been reinforced as Mayer & Whitington [17
] proposed various putative synapomorphies of the Myriochelata, including a revised character polarity for the well-studied neuro-developmental pattern [18
], and the mechanism of dorsoventral patterning. Here, debate surrounds the ancestral conditions, specifically whether nervous tissue forms from immigration of single or clusters of cells, and whether or not the neuroectoderm invaginates in each developing segment.
In a similar conflict between molecules and morphology, arthropods share features including segmentation and appendages with tardigrades and onychophorans [1
], yet a close relationship between these three phyla has not been clearly supported by molecular analyses. A close relationship between onychophorans and euarthropods is widely accepted, but affinities of tardigrades are less clear, to the extent that they have been linked with nematodes in several phylogenomic studies [13
]. Recently, a mitogenomic study of the Ecdysozoa supported a monophyletic origin of these three groups, although support is model-dependent [19
There are two explanations for the discrepancies between different molecular datasets and between molecules and morphology. First, morphology may mislead—mandibles might have evolved independently in pancrustaceans and myriapods or been lost in chelicerates; similarly, segmentation and legs may have appeared separately in arthropods, onychophorans and tardigrades. The second explanation is that some molecular data may be affected by errors—either stochastic (unlikely with phylogenomic scale datasets) or systematic such as compositional bias or long-branch attraction (LBA) [20
]. The possibility of systematic error is suggested by some datasets being equivocal regarding myriapod [7
] or tardigrade affinities [12
To resolve the phylogenetic relationships of the arthropods and their ecdysozoan outgroups, we present analyses of three independent datasets. The first is a phylogenomic dataset of 198 protein-coding genes, which includes new data from the pivotal myriapods. The second is a novel set of arthropod microRNAs (miRNAs), small non-coding regulatory genes implicated in the control of cellular differentiation and homeostasis. The third is a comprehensive dataset of 393 morphological characters, including the recently proposed morphological homologies of Myriochelata [17
] and recent gene expression data [25
] alongside new and traditional characters supporting the Mandibulata.
In addition, we have explored the nature of the conflict between molecular datasets supporting alternative arthropod phylogenies by assaying the potential effects of systematic error on our phylogenomic dataset using an experimental approach coupling targeted taxon-sampling, the use of alternative models of molecular evolution, and the analyses of subsets of slowly evolving sites extracted from our full dataset.