Phylogenomic Analysis of Spiders Reveals Nonmonophyly of Orb Weavers

Department of Biological Sciences, The George WashingtonUniversity, Washington, D.C. 20052, USASummarySpiders constitute one of the most successful clades ofterrestrial predators [1]. Their extraordinary diversity, paral-leled only by some insects and mites [2], is often attributedto the use of silk, and, in one of the largest lineages, to ste-reotypedbehaviorsforbuildingforagingwebsofremarkablebiomechanical properties [1]. However, our understandingof higher-level spider relationships is poor and is largelybased on morphology [2–4]. Prior molecular efforts havefocused on a handful of genes [5, 6] but have provided littleresolution to key questions such as the origin of the orbweavers [1]. We apply a next-generation sequencingapproach to resolve spider phylogeny, examining the rela-tionships among its major lineages. We further explorepossible pitfalls in phylogenomic reconstruction, includingmissing data, unequal rates of evolution, and others. Ana-lyses of multiple data sets all agree on the basic structureofthespidertreeandallrejectthelong-acceptedmonophylyof Orbiculariae, by placing the cribellate orb weavers (Dein-opoidea) with other groups and not with the ecribellate orbweavers (Araneoidea). These results imply independentorigins for the two types of orb webs (cribellate and ecribel-late)oramuchmoreancestraloriginoftheorbwebwithsub-sequent loss in the so-called RTA clade. Either alternativedemands a major reevaluation of our current understandingof the spider evolutionary chronicle.Results and DiscussionOur analyses of multiple data matrices (Figure 1) result in awell-resolved phylogeny of the represented spider taxa thatislargelycongruentwiththecurrentlyacceptedmajorlineages(Figure 2) (see also the accompanying article [7]). Mesothelaeappear as the sister group to a well-supported Opisthothelae,divided into Mygalomorphae (‘‘tarantulas’’ and trap-door spi-ders) and Araneomorphae (‘‘true spiders’’). Araneomorphaedivides into Haplogynae and Entelegynae. The entelegynesconsist of two main lineages: Araneoidea (the bulk of the orbweavers) and a clade that includes the four representativesof the RTA clade (named for the retrolateral tibial apophysis[RTA]onthemalecopulatoryorgans)plusEresoidea(Oecobii-dae) and Deinopoidea (Uloboridae, the representative of theother superfamilywhosemembersspinorbwebs).Allof thesenodes ﬁnd near-maximal support in virtually all the analysesconducted (Figure 2). The notion of nonmonophyly of Orbicu-lariae based on molecular data is not entirely novel [8], but it isatoddswiththepredominant paradigmofspiderevolution, asit contradicts most morphological and behavioral dataanalyses[1].Forthisreason,molecularstudieshaveoftendis-missed results that do not recover monophyly of orbiculariansas artifactual or even ‘‘false’’ (e.g., [9]). However, the mosttaxon-intensive molecular analysis of this problem to date(362taxa,sixmarkers)didnotrecoverorbicularianmonophyly[10]. The results presented here required further testing sinceseveral potential pitfalls have been pointed out for phyloge-nomic analyses, including artiﬁcial support inﬂation [11].Consequently, we deﬁned multiple data matrices designedspeciﬁcally to account for the effects of common factorsaffecting phylogenomic analyses, including missing data [12,13], unequal rates of evolution in different lineages, composi-tional heterogeneity [14], and heterotachy [15].Supermatrix ApproachA ﬁrst set of matrices was thus constructed to discern the po-tential effects of missing data (supermatrices I to IV; Figure 1).Their analyses showed no variation in the relationships of themajorspiderlineages,andallmatricesshowedamaindivisionof Haplogynae and Entelegynae. Although some of the entele-gyne relationships are matrix dependent, nearly all analysesﬁndacladethatincludesDeinopoidea(Uloboridae),Eresoidea(Oecobiidae), and the RTA clade, and no analysis recoveredmonophyly of Orbiculariae (i.e., a sister group relationship ofDeinopoidea and Araneoidea) (Figure 2).In order to evaluate the effect of using genes with differentevolutionary rates or phylogenetic constrains [16], we con-structed three additional supermatrices including 100 geneswith different evolutionary rates each (measured as percent-age of identical sites). The three sets of genes found thesame overall topology, only differing in the internal relation-ships within Araneoidea or the RTA clade and in the relativeposition of Oecobiidae. All analyses placed Uloboridae withthe RTA clade (including or not Oecobiidae) with bootstrapsupport >99%, thus again refuting monophyly of Orbiculariae.Topological differences among the three sets of trees are notsupported. This result also contrasts with others suggestingthat resolving ancient divergences requires genes with strongphylogenetic signal [11] and that many have interpreted as ifonly certain rates would have such strong signal. In our case,the three sets of genes with different evolutionary rates arelargely congruent with our results from virtually all other ana-lyses. Other studies, however, show radically different resultsfrom different groups of genes [16]. Here we show that nearlyidentical phylogenetic hypotheses were inferred from sets ofgenes with radically different evolutionary rates, with orwithout missing data, or from 94 to 2,637 genes. This, oncemore, shows that phylogenomic data, even in the presenceof missing data, contain enough information to provide strongphylogenetic resolution for ancient arthropod divergences[17–19].In order to account for heterotachy, all supermatrices wereanalyzed with methods that implement mixture models (seethe Experimental Procedures). Again, the phylogenetic hy-potheses for the different matrices were similar. As an extrameasure, supermatrix III was selected for further analyses toaccount for the potential effects of this confounding factorby using the IL approach as implemented in PhyML v.3.0.3