Allopolyploidy, diversification, and the Miocene grassland expansion Matt C. Estepa,b,1, Michael R. McKaina,c,1, Dilys Vela Diaza,d,1, Jinshun Zhonga,e,1, John G. Hodgea,c, Trevor R. Hodkinsonf, Daniel J. Laytona,c, Simon T. Malcomberg, Rémy Pasqueth,i,j, and Elizabeth A. Kellogga,c,2 aDepartment of Biology, University of Missouri–St. Louis, St. Louis, MO 63121; bDepartment of Biology, Appalachian State University, Boone, NC 28608; cDonald Danforth Plant Science Center, St. Louis, MO 63132; dDepartment of Biology, Washington University, St. Louis, MO 63130; eDepartment of Plant Biology, University of Vermont, Burlington, VT 05405; fDepartment of Botany, Trinity College Dublin, Dublin 2, Ireland; gDivision of Environmental Biology, National Science Foundation, Arlington, VA 22230; hInternational Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya; iUnité de Recherche Institut de Recherche pour le Développement 072, Laboratoire Évolution, Génomes et Spéciation, 91198 Gif-sur-Yvette, France; and jUniversité Paris-Sud 11, 91400 Orsay, France Edited* by John F. Doebley, University of Wisconsin–Madison, Madison, WI, and approved July 29, 2014 (received for review March 4, 2014) The role of polyploidy, particularly allopolyploidy, in plant di- inference of polyploidization events from chromosome numbers. versification is a subject of debate. Whole-genome duplications Although unavoidable, inferences from chromosome numbers precede the origins of many major clades (e.g., angiosperms, require assumptions about which numbers represent polyploids Brassicaceae, Poaceae), suggesting that polyploidy drives diversi- and when the polyploids arose. fication. However, theoretical arguments and empirical studies Phylogenetic trees of nuclear genes can reliably identify allo- suggest that polyploid lineages may actually have lower specia- polyploidization events (17–21) because they produce charac- tion rates and higher extinction rates than diploid lineages. We teristic double-labeled tree topologies in which the polyploid focus here on the grass tribe Andropogoneae, an economically and species appears twice (Fig. S1). In such trees, allopolyploids are readily identified and can be discovered even when no chromo- ecologically important group of C4 species with a high frequency of polyploids. A phylogeny was constructed for ca. 10% of the some counts are available (e.g., ref. 22). This tree-based method species of the clade, based on sequences of four concatenated will recover genetic allopolyploids; following the terminology low-copy nuclear loci. Genetic allopolyploidy was documented us- of Doyle and Egan (23), these polyploids may be descendants ing the characteristic pattern of double-labeled gene trees. At least of two species (taxonomic allopolyploids) or one (taxonomic EVOLUTION 32% of the species sampled are the result of genetic allopolyploidy autopolyploids). The gene-tree approach thus provides un- and result from 28 distinct tetraploidy events plus an additional six ambiguous evidence of allopolyploidization and provides an es- hexaploidy events. This number is a minimum, and the actual fre- timate of relative timing that does not require calibration of a tree. The approach does require identification of all or most quency could be considerably higher. The parental genomes of paralogues, which can be accomplished by investigating genes in most Andropogoneae polyploids diverged in the Late Miocene categories that tend to be retained after whole-genome dupli- coincident with the expansion of the major C grasslands that 4 cation, such as transcription factors. dominate the earth today. The well-documented whole-genome The grass family is a powerful system in which to study allopoly- duplication in Zea mays ssp. mays occurred after the divergence of ploidization. About 80% of its species are polyploid (24), and Zea and Sorghum. We find no evidence that polyploidization is Hunziker and Stebbins (25) have noted that the grasses are the “only followed by an increase in net diversification rate; nonetheless, large family in which high frequency of polyploids prevails through- allopolyploidy itself is a major mode of speciation. out the family.” The polyploids that Stebbins refers to and that we olyploidy (whole-genome duplication) is often linked with Significance Pthe acquisition of new traits and subsequent species di- versification, particularly in plants (1, 2). Ancient polyploidy Duplication of genomes following hybridization (allopoly- correlates with major land-plant radiations (3) and the origins of – ploidy) is common among flowering plants, particularly in the orders, large families, and major clades (4 7) although, in many grasses that cover vast areas of the world and provide food cases, sharp changes in diversification rates are delayed for mil- and fuel. Here, we find that genome duplication has occurred lions of years after the polyploidization event (1). This phyloge- at a remarkable rate, accounting for at least a third of all netic pattern has led to the hypothesis that polyploidy causes or speciation events in a group of about 1,200 species. Much of promotes diversification. Good mechanistic reasons support such this genome duplication occurred during the expansion of the a hypothesis. Studies of naturally occurring and synthetic poly- C4 grasslands in the Late Miocene. We find no evidence that ploids find changes in gene expression, gene loss, release of allopolyploidy leads directly to a change in the net rate of di- transposons, and changes in morphology and physiology imme- versification or correlates with the origin of novel morpho- diately after polyploidy (4, 8–12). This pattern is particularly true logical characters. However, as a mode of speciation, the for allopolyploids, which originate from a cross between geneti- frequency of allopolyploidization is surprisingly high. cally distinct parents often representing different species; in such cases, the biological changes seen after polyploidization may not Author contributions: M.C.E., R.P., and E.A.K. designed research; M.C.E., M.R.M., D.V.D., reflect the effects of genome doubling per se, but rather the effect J.Z., J.G.H., D.J.L., and E.A.K. performed research; J.Z., J.G.H., T.R.H., D.J.L., S.T.M., R.P., of hybridization between distantly related progenitors (4). and E.A.K. contributed new reagents/analytic tools; M.R.M., D.V.D., J.Z., and E.A.K. analyzed data; and E.A.K. wrote the paper. Despite the appeal of the hypothesis that polyploidy causes diversification, there is evidence to the contrary. As noted by The authors declare no conflict of interest. Stebbins (13), “polyploidy has been important in the diver- *This Direct Submission article had a prearranged editor. sification of species and genera within families, but not in the Freely available online through the PNAS open access option. origin of the families and orders themselves,” implying that Data deposition: The sequences reported in this paper have been deposited in the Gen- polyploidy is only a minor force in diversification (see also ref. Bank database. For a list of accession numbers, see Table S1. 14). Surveys of angiosperms and ferns have found no evidence 1M.C.E., M.R.M., D.V.D., and J.Z. contributed equally to this paper. for increased speciation after polyploidization (15, 16), sup- 2To whom correspondence should be addressed. Email: [email protected]. ’ porting Stebbins s hypothesis. However, because of the large This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scale of the analyses, these studies necessarily had to rely on 1073/pnas.1404177111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1404177111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 discuss in this paper all formed after the well-documented whole- genome duplication at the origin of the family (26, 27); the grass 1 Chrysopogon WGD (rho) occurred about 80–90 million years ago (Mya) (28, 29). Here, we focus on the tribe Andropogoneae (subfamily 2 Zea/Tripsacum Panicoideae), a group of about 1,200 species in 90 genera. The tribe is morphologically diverse and contains some of our most eco- nomically important crop plants [maize (Zea mays), sorghum, and sugarcane (Saccharum officinarum)]. In addition, members of the 3 Apocopis tribe are ecologically dominant species of temperate and tropical 4 Miscanthus grasslands (e.g., Andropogon gerardii, Schizachyrium scoparium, 5 Miscanthus/Saccharum and Sorghastrum nutans of the North American tall grass prairie; Themeda triandra of East African and Australian grasslands), some 6 Polytrias of the most troublesome agricultural weeds (Imperata cylindrica, 7 Ischaemum Sorghum halepense, Ischaemum spp.), many important forage crops 8 Dimeria (Dichanthium spp., Bothriochloa spp., Andropogon spp.), and some burgeoning biofuels (Miscanthus and Saccharum). In short, the Andropogoneae feed, and increasingly fuel, the planet. 9 Hyparrhenia Of the species listed in the preceding paragraph, all but sorghum 10 Hyparrhenia are allopolyploid. We therefore sought to test whether each allo- 11 Hyparrhenia A polyploidy event leads only to one or a few species (the Stebbins A’ hypothesis), in which case, the allopolyploids should appear in 12 Hyparrhenia B the phylogeny as the result of numerous independent polyploidy 13 Schizachyrium 14 Schizachyrium events. Alternatively, allopolyploidy could correlate with di- C versification of a major clade or eventheentiretribe(thedi- 15 Andropogon “core versification hypothesis),