Brain regions shared by onychophorans ((a, b) Euperipatoides rowelli) and chelicerates ((c, d) Eremobates pallipes). (a, c) The paired mushroom body (MB) calyces (Ca), lobes (lo) and heterolateral commissures (com) shown green. VNC, ventral nerve cord. Olfactory glomeruli (bracketed, glom) are coloured purple. In E. rowelli, glomeruli are supplied by the protocerebral antennae (ant). In E. pallipes glomeruli are supplied by axons from the pedipalps (1) and from the alveoli (2). (b, d) Visual pathways from the eyes are shown in carmine, with the first and second optic neuropils indicated (vis 1, 2). In onychophorans and chelicerates, vis 2 is connected to the midline arcuate body (arc bo). (e–g) Shared morphology of (e, f) onychophoran and (g and inset to g) opilionid synapses at the mushroom body pedunculus compared with synaptic structure at an equivalent locus in (inset to e) the honey bee. Apposition of extended asymmetric membrane densities (arrows) characterize synapses in E. rowelli and the opilionid Holonuncia sp. Open arrowheads indicate smaller asymmetric synaptic clefts present at the same level, sometimes within the same profile. (inset e) The equivalent synaptic level of the insect mushroom body comprises patches of small ribbon- or button-like presynaptic densities that converge onto post-synaptic sites. Scales a, b=200 μm; c, d=500 μm. Scales e–g and inset c=1 μm; inset a=0.5 μm. (See electronic supplementary material for larger version.)

Comparison of Arctonoe fragilis and examples of Mandibulata, with reference to midline neuropils. (a) Silver-stained brain of A. fragilis, showing the paired mushroom bodies (MB) and olfactory lobes (Olf Lo). Neuropils are bilaterally elaborated but there is no specialized midline region. Cell bodies of various sizes are clustered. (b) Allatostatin-stained midbrain of the millipede Orthoporus ornatus. Midline neuropil is absent (arrow). (c) Collaterals (bracketed) from heterolateral neurons extending across the brain define midline neuropil in the chilopod Scutigera coleoptrata. (d) Midline neuropil of the chilopod Scolopendra polymorpha. Neuropil is supplied by lateral fibres (arrowed), with perpendicular neurons (one indicated by arrowhead) extending through the neuropil. (e) Midline neuropil (bracketed) of Triops longicaudatis, showing the remote location of somata (arrows) supplying this neuropil. (f) Midline neuropil of the crab Hemigrapsus oregonensis. As in basal crustaceans, modular midline neuropil derives from heterolateral neurons (arrows) that originate from remote cell bodies. (g) The apterygote insect Machilis germanicus, showing similar arrangements with remote somata (arrows) providing midline neuropil. (h) An unidentified symphylan showing systems of heterolateral neurons, revealing (at arrow) midline modifications and some degree of lateralization (bracketed areas). (i) Fan-shaped body of a Tasmanian collembolan, Neanura sp, showing its associated heterolateral elements (arrows) supplying this midline neuropil. (j) Elaborate modular arrangements in the central complex of the cockroach Periplaneta americana. Heterolateral neurons supplying modular collaterals are arrowed. (k) Summary diagram showing the mandibulate (upper left) and chelicerate+onychophoran (upper right) midline neuropil ground patterns. Both are likely to have diverged from a simple rectilinear arrangement leading to the evolution of midline modules from remote heterolateral neurons (left) and from local midline columnar neurons (right). Scale bars=50 μm. (See electronic supplementary material.)

Arcuate midline neuropils shared by (a) onychophorans and (b–h) chelicerate taxa ((b) Limulus polyphemus (Xiphosura); (c) Centruroides sp. (Scorpionida); (d) Eremobates pallipes (Solfugida); (e) Heptathela kimurai (Aranaea); (f) Pardosa sp. (wolf spider, Aranaea); (g) Mastigoproctus giganteus (Uropygi); (h) Holonuncia sp. (Opilionidae)). Scales a–d, f, g=100 μm; e=50 μm; h=25 μm. (See electronic supplementary material for larger version.)

Homologous stratification in midline neuropils of (a, d, e) E. rowelli, the spiders (b, c) Delana cancerides and (f) Pardosa sp. (a) Anti-5HT staining of E. rowelli reveals a diffusely packed immunoreactive stratum (1) that is distinct from a palisade of immunoreactive columnar elements (2). (b, c) Anti-5HT staining of D. cancerides reveals homologous arrangements ((c) layers 1 and 2, and (b) purple in low resolution micrograph). (b) Anti-allatostatin staining (green) of D. cancerides reveals three levels: a graded concentration of profiles at level A, columnar processes at B (fibres detailed in inset, enlargement of bracketed area) and tangential processes at C. (d) Anti-allatostatin reveals homologous layers in E. rowelli (layer A–C). (e) Anti-5HT and (f) Bodian staining demonstrate identical layering (indicated by double arrows) through the midline neuropil of E. rowelli and arcuate body of Pardosa sp. Scales a–c, f=50 μm. e adjusted to depth of f. (See electronic supplementary material for larger version.)

(a) The results of a strict consensus of the three most parsimonious trees. All characters (see character matrix and character list, electronic supplementary material) are treated as unordered and unweighted. All trees support a sister relationship between Onychophora and Chelicerata. Hexapods and Crustacea show reciprocal monophyly and a sister relationship supporting the clade Tetraconata. (b) Bootstrap parsimony consensus tree showing those groups supported by a bootstrap value of 50% or more. Support for Onychophora+Chelicerata is strong at 90%.