REPORTS 22. Y. Nonaka et al., Eur. J. Biochem. 229, 249 (1995). 28. N. Gompel, B. Prud’homme, P. J. Wittkopp, V. Kassner, Supporting Online Material 23. H. E. Bulow, R. Bernhardt, Eur. J. Biochem. 269, 3838 S. B. Carroll, Nature 433, 481 (2005). www.sciencemag.org/cgi/content/full/312/5770/97/DC1 (2002). 29. J. Piatigorsky, Ann. N.Y. Acad. Sci. 842, 7 (1998). Materials and Methods 24. M. Weisbart, J. H. Youson, J. Steroid Biochem. 8, 1249 30. M. J. Ryan, Science 281, 1999 (1998). Figs. S1 and S7 (1977). 31. We thank S. Sower and S. Kavanaugh for agnathan Tables S1 to S4 25. Y. Li, K. Suino, J. Daugherty, H. E. Xu, Mol. Cell 19, 367 plasma and explant cultures, D. Anderson and References and Notes (2005). B. Kolaczkowski for technical expertise, and P. Phillips for 26. J. M. Smith, Nature 225, 563 (1970). manuscript comments. Supported by NSF-IOB-0546906, 27. J. W. Thornton, E. Need, D. Crews, Science 301, 1714 NIH-F32-GM074398, NSF IGERT DGE-0504627, and a 2 December 2005; accepted 13 February 2006 (2003). Sloan Research Fellowship to J.W.T. 10.1126/science.1123348 eroponerinae, Paraponerinae, Ponerinae, and Phylogeny of the Ants: Diversification Proceratiinae; our results exclude Ectatomminae and Heteroponerinae but add Agroecomyrmecinae. in the Age of Angiosperms The latter is represented by a single extant species, Tatuidris tatusia, and two fossil genera, and its Corrie S. Moreau,1* Charles D. Bell,2 Roger Vila,1 S. Bruce Archibald,1 Naomi E. Pierce1 placement within the poneroid clade is entirely novel. Both Ectatomminae and Heteroponerinae We present a large-scale molecular phylogeny of the ants (Hymenoptera: Formicidae), based on nested within the formicoid clade. Although the 4.5 kilobases of sequence data from six gene regions extracted from 139 of the 288 described poneroid clade received less support in the extant genera, representing 19 of the 20 subfamilies. All but two subfamilies are recovered as maximum likelihood and maximum parsimony monophyletic. Divergence time estimates calibrated by minimum age constraints from 43 fossils analyses (Table 1), it was strongly supported in indicate that most of the subfamilies representing extant ants arose much earlier than previously the Bayesian analysis (100% bpp). It seems proposed but only began to diversify during the Late Cretaceous to Early Eocene. This period likely that the five included subfamilies form also witnessed the rise of angiosperms and most herbivorous insects. a monophyletic group or, alternatively, a basal polytomy, but in either case they remain outside nts are a ubiquitous and dominant fea- subfamily (Leptanillinae) and sister to all other both the leptanilloid and the formicoid clades. ture of the terrestrial landscape, playing ants; the poneroid clade, containing 5 sub- The inclusion of Heteroponerinae within the A key roles in symbiotic interactions, soil families (Agroecomyrmecinae, Amblyoponinae, formicoid clade is also unexpected. As sug- aeration, and nutrient cycling. They have a rich Paraponerinae, Ponerinae, and Proceratiinae); gested by their name, heteroponerines have fossil record (1), yet the evolutionary history of and the formicoid clade, containing the remain- historically been placed in the poneromorph the È11,800 described modern species remains ing 13 subfamilies sampled in this study. All clade. Moreover, Ectatomminae, until recently poorly resolved. three clades were supported by 100% Bayesian also considered poneromorphs, appear to be Bolton_s(1) recent revision of ants (Hymenop- posterior probability (bpp) support, but only the closely related to Heteroponerinae. These find- tera: Formicidae) recognized 288 genera in 21 formicoid and leptanilloid clades were well ings, combined with the lack of stability for the Esubsequently reduced to 20 (2, 3)^ subfamilies. supported in the maximum likelihood analyses Bponeromorphs[ observed in morphological Several phylogenies have been proposed based EQ94% maximum likelihood bootstrap (ml bs)^. analyses (4–7), underscore the extent to which primarily on morphological characters, but Of the 19 subfamilies investigated here, 14 our understanding of ancestral ant morphology these reflect disagreement about the positions were recovered as monophyletic with strong sup- and behavior must be revised. of major lineages (fig. S2) (4–7). Recent mo- port, and none of the three monotypic taxa, each The phylogenetic position of Aneuretus, today lecular analyses have included only a moderate represented by a single extant species (Agro- restricted to Sri Lanka, has been hypothesized number of taxa and recovered only weak sup- ecomyrmecinae, Aneuretinae, and Paraponerinae), to be basal either to the Dolichoderinae or to port for most clades (8, 9), although more com- nested within another lineage, validating their the Dolichoderinae þ Formicinae (12, 13). We prehensive studies are under way (10). status as separate subfamilies. However, the recover Aneuretus as basal to the Dolichoderinae, To evaluate competing phylogenetic hypothe- three sampled genera of Cerapachyinae were with both groups separated from Formicinae, ses, we analyzed 4.5 kb of sequence data (Fig. 1) paraphyletic in all analyses. The eight genera implying that the sting has been reduced in- from portions of five nuclear genes and one of Amblyoponinae grouped together in a clade dependently at least twice in the ants (Dolicho- mitochondrial gene from 139 ant genera and that lacked support, although the monophyly derinae and Formicinae). six Aculeatea Hymenoptera outgroups (n 0 149 of Amblyoponinae genera was well supported The ant fossil record is extensive, with more specimens) representing 19 of the 20 currently in an earlier molecular study (2). than 60 extant and 100 extinct genera. The oldest recognized extant subfamilies. The only ant The monophyly of the Leptanillinae was reliably dated fossils are È100 million years (My) subfamily not included was Aenictogitoninae, a strongly supported (100% bpp and ml bs), and old, from Early Cretaceous French and Bur- rare group known only from males collected at its basal position was recovered in all analyses. mese ambers (14, 15). These include both Ge- lights in equatorial Africa. The monophyly of Ward (6) noted that a basal position of Leptanil- rontoformica and Burmomyrma (Aneuretinae), the Formicidae itself was strongly supported in la within the poneroid group implies that tergo- with features typical of modern Bcrown group[ all analyses (Table 1). sternal fusion of abdominal segments III and IV ants, as well as Sphecomyrminae, with features Analyses with several methods (11)re- in the worker caste occurred early in ant evolu- typical of basal Bstem group[ ants. Although no sulted in a well-resolved phylogeny that tion and was lost secondarily in many lines. Our older sphecomyrmines are known, the presence of divided the family into three groups: the lep- results indicate that these characters are indeed stem and crown group ants in these roughly coeval tanilloid clade, a basal lineage containing 1 labile and homoplasious. Although the basal po- ambers implies an earlier history of Formicidae. sition of Leptanillinae was suggested in other The status of the Armaniinae/-idae as stem group 1 Museum of Comparative Zoology, Harvard University, 26 molecular studies (2, 10), previous phylogenetic ants is controversial (1, 3, 15), but if they are Oxford Street, Cambridge, MA 02138, USA. 2School of Computational Science, Florida State University, 150-R hypotheses based on morphology had failed to viewed as sister to Formicidae, this also implies Dirac Science Library, Tallahassee, FL 32306–4120, USA. place it in a basal position among extant ants. an extension of the minimum age of ants to the *To whom correspondence should be addressed. E-mail: Bolton (1) proposed a Bponeromorph[ clade, maximum age of Armaniinae/-idae, which has [email protected] including Amblyoponinae, Ectatomminae, Het- been estimated to be È125 My (16). www.sciencemag.org SCIENCE VOL 312 7 APRIL 2006 101 REPORTS Fig. 1. Phylogenetic chronogram of ants (A). Proportional lineages-through- (respective time scales are indicated above and below topology), and the time (LTT) plot for ants and distribution of clade ages for crown group ants overlap between these two regions corresponds to the greatest period of ant (B). Bayesian posterior probability clade support of 100% is indicated by diversification. The LTT plot for ants shows the number of lineages present (as thick lines on topology. Fossil calibration points are indicated on topology. a proportion of terminals) at sequential time points. The shaded area (green) Shaded areas (green) delineate the rise of angiosperm dominance depend- on the LTT plot and histogram likewise correspond to the overlapping region ing on whether minimum or maximum ages are used for fossil calibrations of the origin of angiosperm-dominated forests [(A) and (B)]. 102 7 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org REPORTS Table 1. Clade support for phylogenetic analyses and divergence time estimates. Support for Paleocene and the Late Cretaceous, 60 to 100 Ma clades recovered under Bayesian posterior probabilities, maximum likelihood bootstrap, and (21–24). The proliferation of angiosperms is maximum parsimony bootstrap (bpp/ml bs/mp bs). Divergence time estimations T1.96 SD of the thought to have driven the diversification of ma- bootstrapped samples. *, not applicable because only one taxon represents clade or outgroup; – , jor herbivorous groups such as beetles (25, 26) support