The diversity and evolution of chelicerate hemocyanins

Abstract

BackgroundOxygen transport in the hemolymph of many arthropod species is facilitated by large copper-proteins referred to as hemocyanins. Arthropod hemocyanins are hexamers or oligomers of hexamers, which are characterized by a high O2 transport capacity and a high cooperativity, thereby enhancing O2 supply. Hemocyanin subunit sequences had been available from horseshoe crabs (Xiphosura) and various spiders (Araneae), but not from any other chelicerate taxon. To trace the evolution of hemocyanins and the emergence of the large hemocyanin oligomers, hemocyanin cDNA sequences were obtained from representatives of selected chelicerate classes.ResultsHemocyanin subunits from a sea spider, a scorpion, a whip scorpion and a whip spider were sequenced. Hemocyanin has been lost in Opiliones, Pseudoscorpiones, Solifugae and Acari, which may be explained by the evolution of trachea (i.e., taxon Apulmonata). Bayesian phylogenetic analysis was used to reconstruct the evolution of hemocyanin subunits and a relaxed molecular clock approach was applied to date the major events. While the sea spider has a simple hexameric hemocyanin, four distinct subunit types evolved before Xiphosura and Arachnida diverged around 470 Ma ago, suggesting the existence of a 4 × 6mer at that time. Subsequently, independent gene duplication events gave rise to the other distinct subunits in each of the 8 × 6mer hemocyanin of Xiphosura and the 4 × 6mer of Arachnida. The hemocyanin sequences were used to infer the evolutionary history of chelicerates. The phylogenetic trees support a basal position of Pycnogonida, a sister group relationship of Xiphosura and Arachnida, and a sister group relationship of the whip scorpions and the whip spiders.ConclusionFormation of a complex hemocyanin oligomer commenced early in the evolution of euchelicerates. A 4 × 6mer hemocyanin consisting of seven subunit types is conserved in most arachnids since more than 400 Ma, although some entelegyne spiders display selective subunit loss and independent oligomerization. Hemocyanins also turned out to be a good marker to trace chelicerate evolution, which is, however, limited by the loss of hemocyanin in some taxa. The molecular clock calculations were in excellent agreement with the fossil record, also demonstrating the applicability of hemocyanins for such approach.

Cross-bracing uncalibrated nodes in molecular dating improves congruence of fossil and molecular age estimates

“…Relationships among the paralog groups were identical to those recovered in a previous study [31]. Within the chelicerate orders, relationships of the three xiphosuran genera could only be assessed by one paralog (Hc1/A is the only hemocyanin sequenced for Tachypleus tridentatus), but are consistent with a recent multilocus phylogeny of Xiphosura using a separate set of molecular loci [36].…”

“…In cross-calibration sensu [17], calibrations are thus repeatedly applied to nodes corresponding to the same divergences using the same prior distributions, but these priors are not formally linked. Results of this analysis, in accordance with previous efforts in molecular dating using concatenated hemocyanin sequences, retrieved large (≥75 Myr) 95% HPD intervals for several derived nodes corresponding to the divergences of, and within, chelicerate orders [31] (Figure 2A, Additional file 1: File S1). The largest HPD interval is that of basal scorpion divergence, with ages ranging from 50.0 Ma (paralog Hc3B) to 362.9 Ma (paralog Hc2).…”

“…To an existing dataset of chelicerate hemocyanin sequences [31], we added orthologs of hemocyanins from four spiders (Frontinella communis, Leucauge venusta, Liphistius malayanus, and Neoscona arabesca); an amblypygid (Damon variegatus); a scorpion (Centruroides sculpturatus); and two previously unknown orthologs of the Atlantic horseshoe crab (Limulus polyphemus); Hc1/A and HcVI. Putative hemocyanins were extracted from transcriptomic assemblies using reciprocal best hits and orthology determined by phylogenetic placement.…”

“…Some variation in the 4 × 6 hemocyanin macromolecule of arachnids occurs in entelegyne spiders, wherein certain lineages have lost multiple subunits [26,27,30]. Hemocyanins do not occur in various terrestrial chelicerate orders that lack book lungs (e.g., Acariformes [mites]) or in sea spiders (Pycnogonida), although a recent study reported the presence of a single hemocyanin ortholog in the EST library of the pycnogonid Endeis spinosa [31]. The function of this ortholog in sea spiders is not presently known.…”

“…First, up to eight paralogs of hemocyanins have been reported in terrestrial chelicerates, proffering multiple targets for cross-bracing [31]. Second, the poor fossil record of terrestrial chelicerates has greatly impeded inference of evolutionary history through molecular dating.…”