Independent divergence of 13- and 17-y life cycles among three periodical cicada lineages Teiji Sotaa,1, Satoshi Yamamotob, John R. Cooleyc, Kathy B. R. Hillc, Chris Simonc, and Jin Yoshimurad,e,f aDepartment of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8502, Japan; bGraduate School of Global Environmental Studies, Kyoto University, Sakyo, Kyoto 606-8501, Japan; cDepartment of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06268-3043; dDepartment of Systems Engineering, Shizuoka University, Hamamatsu 432-8561, Japan; eMarine Biosystems Research Center, Chiba University, Kamogawa 299-5502, Chiba, Japan; and fDepartment of Environmental and Forest Biology, State University of New York College of Environmental Science and Forestry, Syracuse, NY 13210 Edited by Mary Jane West-Eberhard, Smithsonian Tropical Research Institute, Ciudad Universitaria, Costa Rica, and approved February 22, 2013 (received for review November 16, 2012) The evolution of 13- and 17-y periodical cicadas (Magicicada)is group (6, 14–18), and only rough divergence times among species enigmatic because at any given location, up to three distinct spe- have been inferred (8, 10). Thus, a comprehensive molecular cies groups (Decim, Cassini, Decula) with synchronized life cycles phylogeny covering all of the extant broods, their phylogeography, are involved. Each species group is divided into one 13- and one and divergence time has been lacking until now. 17-y species with the exception of the Decim group, which con- We conducted molecular phylogenetic and population genetic tains two 13-y species—13-y species are Magicicada tredecim, analyses by using nuclear and mitochondrial DNA markers for Magicicada neotredecim, Magicicada tredecassini, and Magicicada samples collected over a 30-y period (1978–2008). These samples tredecula; and 17-y species are Magicicada septendecim, Magici- represent all 15 extant broods and all known species (Table S1). cada cassini, and Magicicada septendecula. Here we show that the divergence leading to the present 13- and 17-y populations differs Results and Discussion considerably among the species groups despite the fact that each Phylogenetic analyses and divergence time estimation based on group exhibits strikingly similar phylogeographic patterning. The four nuclear and three mitochondrial genes (Table S2) clearly ∼ earliest divergence of extant lineages occurred 4 Mya with one reveal the monophyly of each of the three species groups EVOLUTION branch forming the Decim species group and the other subsequently (Decim, Cassini, and Decula) and the sister relationship be- splitting 2.5 Mya to form the Cassini and Decula species groups. tween Cassini and Decula (Fig. 1). A Bayesian relaxed clock The earliest split of extant lineages into 13- and 17-y life cycles analysis shows that the three species groups diverged 3.9 Mya. occurred in the Decim lineage 0.5 Mya. All three species groups Initially the Decim group diverged from the ancestor of Cassini + experienced at least one episode of life cycle divergence since the Decula, then Cassini and Decula separated 2.5 Mya (Fig. 1). last glacial maximum. We hypothesize that despite independent The mitochondrial gene genealogy further shows divergence origins, the three species groups achieved their current overlap- associated with regions, and partly with life cycles (Fig. 2, Figs. ping distributions because life-cycle synchronization of invading S1 and S2, and Table S3). We distinguished four mitochondrial congeners to a dominant resident population enabled escape from haplotype groups in Decim and three each in Cassini and Decula predation and population persistence. The repeated life-cycle diver- (Fig. 2 A–C). The geographic distributions of mitochondrial gences supported by our data suggest the presence of a common haplotype groups within each species group show similar divi- genetic basis for the two life cycles in the three species groups. sions among eastern, middle, and western regions (Fig. 2 D–F). In Decim, there is also a major divergence between northern and life-cycle shift | nurse brood | parallel evolution | speciation southern groups corresponding to formally distinguished mito- chondrial lineages A and B, respectively (15). Group A is di- eriodical cicadas (Magicicada) in the eastern United States vided into three groups, Ae, Am, and Aw, which occur in the Prepresent one of the most spectacular life history and eastern, middle, and western parts of the United States east of – population phenomena in nature (1 10). These periodical ci- the Great Plains, respectively. Populations of Ae and Am exhibit cadas spend most of their lives (13 y in the south, 17 y in the a 17-y cycle (Magicicada septendecim); those of Aw both 17-y north) as underground juveniles except for a brief 2- to 4-wk (M. septendecim) and 13-y (Magicicada neotredecim) cycles; and period when adults emerge simultaneously in massive num- those of B show only a 13-y cycle (Magicicada tredecim). At the bers. With few exceptions, at any given location, all of the pe- boundary of Ae and Am, a few populations possess both Ae and riodical cicadas share the same life cycle and emerge on the same Am haplotypes. The Cassini group consists of three haplotype “ ” schedule, forming a single-year class referred to as a brood. groups, Ce, Cm, and Cw, again occurring in the eastern, middle, Surprisingly, each brood consists of multiple species from three and western regions, respectively (Fig. 2). Populations with Ce species groups (Decim, Cassini, Decula). and Cm show 17-y cycles only (Magicicada cassini), whereas These three groups were considered to have diverged from thoseofCwshowboth17-and13-ycycles(M. cassini and each other allopatrically and to have later become sympatric and formed 13- and 17-y life cycles (2). The prolonged, prime- numbered life cycles were hypothesized to have evolved in re- Author contributions: T.S. and J.Y. designed research; T.S., S.Y., J.R.C., K.B.R.H., C.S., and sponse to Pleistocene climatic cooling (9, 11) to avoid the adverse J.Y. performed research; T.S. and S.Y. analyzed data; and T.S., J.R.C., and C.S. wrote effect of low population density on mating success (9, 12, 13). the paper. Another view hypothesized that the long synchronized life cycles The authors declare no conflict of interest. evolvedinassociationwiththepredator avoidance strategy This article is a PNAS Direct Submission. (2, 4, 8) and that this took place before both the glacial periods Freely available online through the PNAS open access option. and the split of the three species groups (10) based on approx- Data deposition: The sequences reported in this paper have been deposited in the DNA imate genetic distances among species groups (8). To test these Data Bank of Japan (accession nos. AB740543–AB740917). hypotheses, phylogenetic information about the relationships of 1To whom correspondence should be addressed. E-mail: [email protected]. species, broods, and populations is essential. However, phyloge- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. netic studies of Magicicada have been largely limited to the Decim 1073/pnas.1220060110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1220060110 PNAS Early Edition | 1of6 Downloaded by guest on September 23, 2021 Sample code, Haplotype Life Species species, brood group cycle group en490 septendecula V en531 septendecula XIV en464 septendecula I en591 tredecula XXIII Dm en525 septendecula X en566 tredecula XXII De + Dm MRCA en542 tredecula XIX 0.16 [0.06-0.31] en561 tredecula XIX en524 septendecula IX -/91 en735 septendecula XIV Decula De en470 septendecula II en506 septendecula V Decula MRCA 13- & 17-year en515 septendecula VI 0.23 [0.10-0.45] 1/99 en485 septendecula IV en036 septendecula VI en727 septendecula XIII Dw en535 tredecula XIX Dw MRCA .99/95 en596 tredecula XXIII 0.08 [0.02-0.19] Cassini + Decula en480 septendecula III MRCA en326 cassini VIII Cm MRCA -/81 2.51 [1.65-3.56] en331 cassini IX 1/98 0.08 [0.02-0.19] en271 cassini V 1/99 en262 cassini I Cm en336 cassini X .87/86 en711 cassini XIV Ce MRCA .89/- en701 cassini XIV 0.12 [0.03-0.24] 17-year en356 cassini X 1/100 en332 cassini IX Ce Cassini MRCA Cassini en266 cassini II 0.32 [0.15-0.56] 1/99 1/75 en317 cassini V en283 cassini III en438 tredecassini XXII Cw MRCA en402 tredecassini XXIII 0.16 [0.04-0.33] en391 tredecassini XIX Cw .72/- en291 cassini IV en353 cassini X .95/81 13- & 17-year Magicicada MRCA en685 cassini XIII 3.89 [3.08-4.69] 1/100 en121 septendecim X Ae MRCA en086 septendecim IX 0.11 [0.03-0.23] en001 septendecim I en061 septendecim VII Ae 1/- Ae+Am+Aw MRCA en051 septendecim VI 0.27 [0.12-0.46] en641 septendecim XIV Fig. 1. Phylogeny and divergence en010 septendecim II times of Magicicada resulting from Am MRCA en013 septendecim V 17-year a Bayesian relaxed clock analysis 0.09 [0.03-0.20] en671 septendecim XIV 1/78 with nuclear and mitochondrial en152 septendecim X Am data showing different histories of en011 septendecim V .99/84 en101 septendecim IX divergence into 13- and 17-y species en606 septendecim XIII Decim among the three species groups. en172 neotredecim XIX Outgroup taxa are not shown. Bars en257 neotredecim XXIII show 95% highest probability den- Decim MRCA en181 neotredecim XIX Aw 1/86 1/91 sity (HPD) intervals of estimated 0.53 [0.26-0.90] 1/100 en227 neotredecim XXIII en021 septendecim divergence times. For major nodes, IV 13- & 17-year Aw MRCA en032 septendecim III divergence times and 95% HPD 0.12 [0.04-0.25] en242 tredecim XXIII intervals in brackets are described. en224 tredecim XXII Node supports are posterior prob- en196 tredecim XIX 1/100 B abilities of the Bayesian inference B MRCA en251 tredecim XXIII 0.13 [0.03-0.29] en166 tredecim XIX 13-year and bootstrap percentages in a en201 tredecim XIX maximum-likelihood analysis (shown Pliocene Pleistocene when >0.70 or >70%).