Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.104.031393 Genetic Diversity and Population Structure of Teosinte Kenji Fukunaga,*,1 Jason Hill,† Yves Vigouroux,*,2 Yoshihiro Matsuoka,*,3 Jesus Sanchez G.,‡ Kejun Liu,§,4 Edward S. Buckler** and John Doebley*,5 *Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706, †Department of Plant Biology, University of Minnesota, St. Paul, Minnesota 55108, ‡Centro Universitario de Ciencias Biologicas y Agropecuarias, Universidad de Guadalajara, Zapopan, Jalisco, Mexico CP45110, §Statistics Department, North Carolina State University, Raleigh, North Carolina 27695 and **USDA-ARS, Institute for Genomic Diversity, Cornell University, Ithaca, New York 14853 Manuscript received May 19, 2004 Accepted for publication January 3, 2005 ABSTRACT The teosintes, the closest wild relatives of maize, are important resources for the study of maize genetics and evolution and for plant breeding. We genotyped 237 individual teosinte plants for 93 microsatellites. Phylogenetic relationships among species and subspecific taxa were largely consistent with prior analyses for other types of molecular markers. Plants of all species formed monophyletic clades, although relationships among species were not fully resolved. Phylogenetic analysis indicated that the Mexican annual teosintes divide into two clusters that largely correspond to the previously defined subspecies, Z. mays ssp. parviglumis and ssp. mexicana, although there are a few samples that represent either evolutionary intermediates or hybrids between these two subspecies. The Mexican annual teosintes show genetic substructuring along geographic lines. Hybridization or introgression between some teosintes and maize occurs at a low level and appears most common with Z. mays ssp. mexicana. Phylogeographic and phylogenetic analyses of the Mexican annual teosintes indicated that ssp. parviglumis diversified in the eastern part of its distribution and spread from east to west and that ssp. mexicana diversified in the Central Plateau of Mexico and spread along multiple paths to the north and east. We defined core sets of collections of Z. mays ssp. mexicana and ssp. parviglumis that attempt to capture the maximum number of microsatellite alleles for given sample sizes. EOSINTE is a wild grass native to Mexico and Cen- analyses, a refined understanding of its phylogenetics Ttral America (Figure 1) and the closest wild relative and population structure can help guide further re- of cultivated maize (Zea mays ssp. mays L.). Teosinte search in all of these areas. represents an important resource for the study of maize Together, teosinte and maize compose the genus Zea, genetics (Evans and Kermicle 2001), quantitative ge- which has four species (Figure 1): (1) Z. luxurians netics (Lukens and Doebley 1999), molecular popula- (Durieu and Ascherson) Bird, an annual teosinte from tion genetics (Gaut et al. 2000), genome evolution Central America; (2) Z. diploperennis Iltis, Doebley and (Sanz-Alferez et al. 2003), and crop evolution (Doe- Guzman, a diploid perennial teosinte from Jalisco, Mex- bley 1990a). Notably, teosinte has become one of the ico; (3) Z. perennis (Hitchc.) Reeves and Mangels- best-characterized systems for plant molecular popula- dorf, a tetraploid perennial teosinte from Jalisco, Mex- tion genetics, including studies utilizing DNA samples ico; and (4) Z. mays, a polytypic annual species that recovered from archeological specimens (Jaenicke- includes four subspecies. The four subspecies are (1) Despre´s et al. 2003). The teosintes also represent an ssp. mays (maize); (2) ssp. mexicana (Schrader) Iltis, important potential resource for maize breeding, al- a large-spikeleted teosinte adapted to the drier high m) of northern and central 2700–1600ف) though they have not yet been extensively used in this elevations capacity. Given the breadth of use of teosinte in genetic Mexico; (3) ssp. parviglumis Iltis and Doebley, a small- spikeleted teosinte adapted to the moister middle eleva- (m) of southwestern Mexico; and (4 1800–400ف) tion ssp. huehuetenangensis (Iltis and Doebley) Doebley, an 1Present address: The International Research Center for Japanese Studies, 3-2 Oeyama-cho, Goryo, Nishikyo-ku, Kyoto 610-1192, Japan. annual teosinte found only in the province of 2Present address: Institut de Recherche pour le Developpement, Mont- Huehuetenango in western Guatemala (Doebley pellier 34730, France. 1990b). The four species of Z. mays have been placed 3Present address: Fukui Prefectural University, Matsuoka-cho, Yos- into two sections: section Zea, which contains only Z. hida-gun, Fukui 910-1195, Japan. mays, and section Luxuriantes, which is composed of the 4Present address: National Institute of Statistical Sciences, 19 T. W. Alexander Dr., Research Triangle Park, NC 27709-4006. other three species. Most of these teosinte species and 5Corresponding author: Laboratory of Genetics, University of Wiscon- subspecies have narrow geographic distributions con- sin, 445 Henry Mall, Madison, WI 53706. E-mail: [email protected] sisting of only a few local populations; however, ssp. Genetics 169: 2241–2254 (April 2005) 2242 K. Fukunaga et al. Figure 1.—Geographical distribution of the teosinte populations used in this study. Since many accessions come from geographi- cally very close locations, their symbols overlap on the map. mexicana and ssp. parviglumis are exceptions, being 1). Wilkes (1967) divided Z. mays ssp. mexicana into three widely distributed in Mexico (Figure 1). Recently, Iltis races: Central Plateau, Chalco, and Nobogame. Because many new populations have been discovered since Wilkes’ seminal and Benz (2000) classified Z. luxurians from Nicaragua work (Sanchez et al. 1998), we divided Z. mays ssp. mexicana as a new species, Z. nicaraguensis Iltis and Benz. Here, into five geographical groups: Central Plateau, Chalco, Du- we treat it as one geographical group of Z. luxurians. rango, Nobogame, and Puebla. Further extending Wilkes’ To clarify the phylogeny and population structure of analysis, we divided Z. mays ssp. parviglumis into two races, the teosintes, we used a set of 93 microsatellite or simple Balsas and Jalisco, and into five geographical groups, eastern Balsas, Central Balsas, Jalisco, Oaxaca, and southern Guerrero sequence repeat (SSR) loci and a sample of 237 individ- (Table 1, Figure 1). Two individual plants of the genus Tripsa- ual teosinte plants that cover the entire geographical cum (one individual each of T. zopilotense and T. peruvianum) distribution of the teosintes. We addressed four ques- were used as the outgroup in phylogenetic analyses. See sup- tions: (1) How are Zea taxa related to each other?, (2) plementary materials at http://genetics.org/supplemental/ How did the annual teosintes diversify in Mexico?, (3) for the complete passport data for the plants, including germ- How is genetic diversity structured in the Mexican an- plasm bank accession numbers and geographical coordinates. SSR genotypes: Ninety-three SSRs that are evenly distributed nual teosintes?, and (4) Has introgression among spe- throughout the genome were used to genotype all 237 Zea cies or subspecies played a role in teosinte evolution? plants and the two Tripsacum individuals. These SSRs were We also define core sets of teosinte accessions that best used in the previous analysis of maize and its wild progenitor capture the diversity of the teosintes. (Matsuoka et al. 2002b). The plants were genotyped at Celera AgGen (Davis, CA) following procedures published elsewhere (Matsuoka et al. 2002b). See supplementary materials at MATERIALS AND METHODS http://www.genetics.org/supplemental/ for a list of the SSRs, their repeat type, and their genomic locations. Plant materials: We sampled 237 teosinte plants from 172 Diversity analyses: Basic statistics, including the number of accessions representing the entire geographical distribution alleles, observed heterozygosity, gene diversity (or expected of teosinte from northern Mexico to western Nicaragua. For each heterozygosity), and the number of taxon-specific (private) accession, 1–5 individuals were assayed. The sample includes 93 alleles, were calculated for species, subspecies, and races using Z. mays ssp. mexicana individuals (69 accessions), 114 Z. mays ssp. PowerMarker (Liu 2002). In these analyses, individual plants parviglumis (82 accessions), 7 Z. mays ssp. huehuetenangensis (3 of possible hybrid origin as determined by population struc- accessions), 13 Z. luxurians (10 accessions), 6 Z. diploperennis ture analysis (see below) were excluded. (5 accessions), and 4 Z. perennis (3 accessions) (Figure 1, Table Phylogenetic trees: We used the FITCH program in the Genetic Diversity in Teosinte 2243 PHYLIP package (Felsenstein 1993) with the log-trans- vidual from each OTU as a representative. Individual plants of formed proportion-of-shared-alleles distance as implemented possible hybrid origin as determined by population structure in the computer program Microsat (http://hpgl.stanford.edu/ analysis (see below) were excluded. We calculated the log-trans- projects/microsat/). In FITCH, the J option was used to ran- formed proportion-of-shared-alleles distance among these in- domize the input order of samples. We constructed three dividuals and used this distance matrix in all tests. We then types of trees: (1) a tree with all individual plants, (2) a tree calculated correlation coefficients between the genetic dis- with individual plants pooled into operational taxonomic