1 American Journal of Botany 98(9): T HREE NEW TEOSINTES ( Z EA SPP., POACEAE) FROM M É XICO 1 J. J. Sánchez G. 2, L. De La Cruz L. 2, V. A. Vidal M. 4, J. Ron P. 2, S. Taba 3, F. Santacruz-Ruvalcaba 2, S. Sood 5, J. B. Holland 5, J. A. Ruíz C.4, S. Carvajal 2, F. Aragón C.4, V. H. Chávez T.3, M. M. Morales R. 2, and R. Barba-González6 2 Centro Universitario de Ciencias Biol ó gicas y Agropecuarias, Universidad de Guadalajara. Km Carretera Guadalajara- Nogales, C.P Las Agujas, Zapopan, Jalisco, M é xico; 3 Centro Internacional de Mejoramiento de Ma í z y Trigo, Unidad de Recursos Gen é ticos, Apartado Postal M é xico, D.F. M é xico; 4 Instituto Nacional de Investigaciones Forestales Agr í colas y Pecuarias, Parque Los Colomos S/N, Col. Providencia, Guadalajara Jalisco, M é xico; 5 USDA-ARS Plant Science Research Unit, Department of Crop Science, Box 7620, North Carolina State University, Raleigh, North Carolina USA; and 6 Centro de Investigaci ó n y Asistencia en Tecnolog í a y Dise ñ o del Estado de Jalisco A.C., Av. Normalistas No. 800, Col. Colinas de la Normal, CP Guadalajara, Jalisco, M é xico Premise of the study : Teosinte species are the closest relatives of maize and represent an important but increasingly rare genetic resource for maize improvement and the study of evolution by domestication. Three morphologically and ecologically distinct teosinte populations were recently discovered in M é xico. The taxonomic status of these rare and endangered populations was investigated by detailed comparisons to previously characterized wild Zea species. Methods : Three new teosinte populations were compared to known teosinte taxa on the basis of morphological, ecogeographic, cytological, and molecular characteristics. Phenetic and phylogenetic analyses were performed using morphological and molecular data, respectively. Key results : The newly discovered populations are distinct from each other and from other Zea species to represent three new entities based on their unique combinations of morphological, ecological, ploidy, and DNA markers. A perennial diploid population from Nayarit is distinguished by early maturing plants, and having male inflorescences with few tassel branches and long spikelets. A perennial tetraploid population from Michoac á n is characterized by tall and late maturing plants, and having male inflorescences with many branches. An annual diploid population from Oaxaca is characterized by having male inflorescences with fewer branches and longer spikelets than those found in the sister taxa Z. luxurians and Z. nicaraguensis, plants with high thermal requirements, and very long seed dormancy. Conclusions : Evidence from multiple independent sources suggests placement of the three new populations of teosinte as distinct entities within section Luxuriantes of the genus Zea. However, more extensive DNA marker or sequence data are required to resolve the taxonomy of this genus. Key words: maize; Poaceae; teosinte; Zea. Humankind depends on a handful of staple crops, including maize ( Zea mays L., subsp. mays ), for survival and social stability ( Cassman, 1999 ; Roberts and Schlenker, 2009 ). Maize domestication occurred in M é xico approximately yr ago from Zea mays subsp. parviglumis Iltis & Doebley ( Matsuoka, et al., 2002 ; Doebley, 2004 ). One effect of domestication was a population genetic bottleneck that reduced genetic diversity in maize compared to its wild Zea relatives, collectively referred to as teosinte ( Wright et al., 2005 ; Ross-Ibarra et al., 2009 ). Teosinte, therefore, represents an important genetic resource for maize improvement. Teosinte harbors alleles that are absent or rare in maize but nevertheless could be useful for maize 1 Manuscript received 26 April 2011; revision accepted 15 June The authors thank the Comisi ó n Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO) for their support of collection missions during 2007 and 2008, Dr. Filiberto Herrera Cedano for helping relocate the teosinte at Huajicori, and IBUG (Instituto de Bot á nica, Universidad de Guadalajara Herbarium) staff for help during the description process. Support for SSR analysis was from a United States National Science Foundation grant IOS (Principal Investigator Dr. Edward Buckler). 7 Author for correspondence ( phone: (52-33) ext ) doi: /ajb breeding. Teosinte genes can improve productivity ( Cohen and Galinat, 1984 ; Magoja and Pischedda, 1994 ; Wang et al., 2008b ), disease resistance ( Wang et al., 2008b ), flooding tolerance ( Mano and Omori, 2007 ), and nutritional quality of maize ( Flint-Garcia et al., 2009 ; Swarup et al., 1995 and Wang et al., 2008a ). Conservation and intelligent use of this valuable genetic resource depends on an understanding of the genetic diversity among and within teosinte populations and their phylogenetic relationships, as well as knowledge of geographic distribution of teosinte populations. Maize and teosinte ( Zea spp.), are members of the Tribe Andropogoneae, Subtribe Tripsacinae of the family Poaceae (Carvajal, in press; Sanchez-Ken and Clark, 2010 ; Kellogg, 2000 ). Poaceae contains approx genera with some species ( Takhtajan, 2009 ). In M é xico and Central America, the genus Zea is very important because maize originated in this region, where it remains a staple crop with great cultural significance (Hernández, 1998). The distribution of teosinte is restricted to the western escarpment of tropical and subtropical areas of M é xico, Guatemala, Honduras, and Nicaragua, with isolated populations limited to areas varying in size from one hectare to several square kilometers ( Wilkes, 1967 ; S á nchez et al., 1998; Iltis and Benz, 2000 ). The distribution of teosinte in M é xico extends from the southern part of the cultural region known as Arid America, in the Western American Journal of Botany 98(9): , 2011; Botanical Society of America 1537
2 1538 American Journal of Botany [Vol. 98 Sierra Madre mountains of the state of Chihuahua and the Guadiana Valley in Durango, to Oaxaca in southern M é xico, including nearly the entire western part of Mesoamerica. Throughout M é xico there are teosinte populations whose morphological and genetic characteristics permit easy differentiation among them. Importantly, teosinte populations do not have a uniform geographic distribution; rather their distribution is closely related to specific climate, soil, and human cultural factors. Friar Bernardino de Sahag ú n in his Historia General de las Cosas de la Nueva Espa ñ a (written about 1570), mentioned teosinte (referred to as Cocopi ): Cocopi is similar to maize; just like the maize stalk It grows everywhere in between the fields, no one sows it. After Sahag ú n, the next reports of the existence of teosinte are those of Liebmann, who collected an herbarium specimen in Oaxaca in 1842 (US, herbarium catalog ); Lumholtz (1902) who found teosinte in the Nabogame Valley in southern Chihuahua; and Lopez y Parra (1908), who reported the presence of teosinte at several Mexican locations, including the states of Chiapas, Jalisco, M é xico, Guanajuato, and Sonora. During the first half of the 20 th century, the work by Collins was the most important reference on the distribution of teosinte ( Collins, 1921 ). Wilkes later traveled throughout M é xico and found teosinte at most sites where it had previously been recorded or collected. In addition to his collections, he prepared maps of teosinte sites from southeastern Honduras to northern México (Wilkes, 1967, 1977, 1985, 2004 ). Teosinte species are represented by annual and perennial diploid species ( 2n = 20) along with perennial tetraploid species ( 2n = 40). The annual form of teosinte received the scientific name of Euchlaena mexicana Schrad., in 1832; Hitchcock discovered in 1910 a perennial tetraploid form which he called Euchlaena perennis ( Doebley, 1990 ). During the early years of the 20 th century, all annual forms of teosinte were included within Zea mexicana (Schrad.) Kuntze (i.e., Euchlaena mexicana Schrad.). Wilkes (1967) described six races of teosinte from M é xico and Guatemala based on ethnobotanical information, geography, cytology, and several morphological aspects. In 1978, a new species of perennial teosinte ( Zea diploperennis Iltis, Doebley & Guzman) was discovered ( Guzman 1978 ; Iltis et al., 1979). After this discovery, Iltis and Doebley (1980) proposed a hierarchical system of classification for Zea, based on the morphological and ecological features of the taxa. Iltis and Benz (2000) estimated that sufficient differences existed between the teosinte from the coastal plain of Nicaragua and Z. luxurians (Durieu & Asch.) Bird from southeastern Guatemala to consider the former a distinct species ( Z. nicaraguensis Iltis & Benz). Based on the studies of Doebley and Iltis (1980), Iltis and Doebley (1980), Doebley (1990), and Iltis and Benz (2000), the genus Zea contains eight taxa classified into two sections and five species. Section Luxuriantes Doebley & Iltis includes the perennial species Z. diploperennis Iltis, Doebley & Guzman, Z. perennis (Hitchc.) Reeves & Mangelsd., and the annuals Z. luxurians, and Z. nicaraguensis Iltis & Benz. Section Zea includes the annual Z. mays L., which has been divided into subspecies: Z. mays subsp. mexicana (Schrad.) Iltis (races Chalco, Central Plateau, Durango, and Nobogame); Z. mays subsp. parviglumis (race Balsas), Z. mays subsp. huehuetenangensis (Iltis & Doebley) Doebley (race Huehuetenango), and Z. mays subsp. mays for cultivated maize. The systematic collection of teosinte began during the 1960s and 1970s by Wilkes and Kato ( Wilkes, 1967 ; Kato, 1976 ), and continued during the last 25 years by S á nchez and coworkers; they have explored and collected teosinte in most of the geographical regions in M é xico (S á nchez and Ordaz, 1987; S á nchez et al., 1998, 2008; Ruiz et al., 2001). As a result of many collection missions, the most complete seed collections of teosinte are held at Universidad de Guadalajara (Jalisco, M é xico), Instituto Nacional de Investigaciones Forestales Agr í colas y Pecuarias (INIFAP, Texcoco, M é xico), and at Centro Internacional de Mejoramiento de Ma í z y Trigo (CIMMYT, Texcoco, M é xico). The discovery of the new teosinte populations in Oaxaca, Nayarit, and Michoac á n prompted their detailed ecogeographic, morphologic, cytogenetic and molecular characterization, and comparison to previously studied teosinte taxa. Here we report the results of analyses of two data sets: morpho-physiological and molecular descriptors. We concluded that the unusual combinations of climatic, morpho-physiological, and molecular characters associated with these populations are sufficiently distinct to clearly distinguish them from the remaining species in the genus Zea, indicating that they represent entities to be newly described. MATERIALS AND METHODS Plant material and study site The genetic material included in this study consists of accessions from each previously named teosinte species and races, in addition to the three new populations. Fifty-one accessions were grown during 2008 under greenhouse conditions at Centro Universitario de Ciencias Biol ó gicas y Agropecuarias (CUCBA) of the University of Guadalajara, located in Nextipac, Jalisco, M é xico. CUCBA is situated N, W at an elevation of 1650 m. Samples of seeds of the new teosinte populations were collected from 2006 to 2010 and stored at the germplasm banks at the Universidad de Guadalajara, INIFAP, and CIMMYT. In addition, rhizomes of two perennial populations were collected from their natural habitats, and plants of these collections are maintained in the greenhouses at CUCBA. Cytogenetic analysis The cytogenetic analyses were carried out at Centro de Investigaci ó n y Asistencia en Tecnolog í a y Dise ñ o del Estado de Jalisco, Asociaci ó n Civil (CIATEJ). Seeds of the new Zea populations and their presumed relatives were placed in petri dishes on moist filter paper in the dark. Germination took place in two to three days and radicles of about 1 cm long were collected and placed in a α -bromonaphtalene saturated solution overnight in ice-water. The radicles were rinsed in tap-water and fixed in a 3:1 ethanol:acetic acid solution for at least 12 h and stored at 20 C until use. The root tips were rinsed 3 times in mq water (water that has been purified using an ion exchange cartridge); subsequently the roots were rinsed in 10 mm citrate buffer (ph 4.5) for 15 m. The root tips were then placed in an embryo dish and incubated at 37 C for 2 h in a drop (50 µ l) of enzyme mixture containing 0.2% (w/v) pectolyase Y23, 0.2% (w/v) cellulase RS and 0.2% (w/v) cytohelicase in 10 mm citrate buffer (ph 4.5). The root tips were then placed on a slide and squashed in a drop of 1% aceto-orceine. Each sample was observed with a Leica DMR-A2 microscope; the selected chromosome images were captured with an Evolution QEi camera (Media Cybernetics Inc., Bethesda, Maryland, USA) with the Image Pro-Plus software. The images were sharpened with a High-Gauss filter. Ecological Descriptors The National Environmental Information System (NEIS) of INIFAP (Ruiz et al., 2008) was used to characterize the environmental conditions of the collecting sites by using the GIS Idrisi Andes (Clark Labs, Clark University, Worcester, Massachusetts, USA). Climatic information in this system is based on normal statistics calculated from data series. Ecological descriptors were determined for each taxa in terms of climatic ranges. Climatic ranges were established once the values for each variable were specified in every accession site. These values were searched with the GIS Idrisi Andes, using the climatic raster images and the geographical coordinates for each accession site. The extreme values for each variable were used to establish the climatic ranges. Ecological descriptors included altitude, annual mean precipitation (ap), annual moisture availability index (amai), May-October moisture availability index (m-omai), annual mean temperature (at), annual mean maximum temperature (axt), annual mean minimum temperature (ait), hottest month mean maximum temperature (hmxt), coldest month mean minimum temperature (cmit), and climate type (CT).
3 September 2011] S á nchez et al. New Teosintes from Mexico 1539 Morphological and physiological descriptors To assess the relationships among teosinte taxa, 20 morphological and physiological traits were measured. Most of the traits measured were also previously studied by Doebley (1983) and S á nchez et al. (1998). Ten plants from each teosinte population were planted on July 2, 2008 in greenhouse conditions and the characters measured on five competitive plants per population. The morphological and physiological characteristics used can be grouped into four categories: 1. Vegetative characters of the plant (plant height, total number of leaves per plant, leaf width, leaf length, days to pollen shed, days to silk, number of lateral branches, number of tillers, and plant surface area); 2. Characters of the tassel (total number of tassel branches, tassel length, central spike length, length of branching space, and peduncle length); 3. Characters of the spikelet (spikelet width, outer glume length, outer glume width, and total vein number of the outer glume); and 4. Kernel (cupulate caryopsis) characteristics (weight of 100 kernels, and number of kernels per ear). Spikelet traits were measured, using a Zeiss Stemi SV6 binocular dissecting microscope, an Axiocam MRc digital camera, and Zeiss Axiovision 4.1 software. SSR marker analysis Eighteen highly polymorphic SSR loci (phi015, phi029, phi033, phi034, phi062, phi064, phi073, phi076, phi085, phi109188, phi115, phi127, phi233376, phi335539, phi389203, phi402893, phi427913, and phi96100) evenly distributed on the ten chromosomes were used to genotype 20 accessions representing the diversity of teosinte (17 diploids and 3 tetraploids); from each accession, 15 plants were analyzed. The plants were genotyped at the Department of Crop Science, North Carolina State University (NCSU), Raleigh, North Carolina, USA. Genomic DNA was extracted using Charge Switch DNA extraction kit by Invitrogen (Carlsbad, California, USA). Primers were selected based on informativeness and genomic distribution from the list published by Matsuoka et al. (2002). The repeat motif characteristics and forward and reverse primer sequences for each locus can be found at Maize Genetics and Genomics Database (www.maizegdb.org). All the forward primers were modified to include the M13 sequence (TGTAAAACGACGGCCAGT) at the 5 end for fluorescent labeling purposes ( Schuelke, 2000 ) PCR amplifications were performed in 15.0 µ l reactions containing 1.2 µ l of 10 buffer with 1.5 mm magnesium chloride, 0.96 µ l of dntps (2.5 mm of each dntp), 0.96 µ l of modified forward primer (1 µ M/ µ l), 0.72 µ l of reverse primer (10 µ M/ µ l), 0.72 µ l of fluorescent labeled M13 primer (10 µ M/ µ l), 0.18 µ l of Taq DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA) and ng of DNA in the Eppendorf Master Cycler (Eppendorf North America, Hauppage, New York, USA) thermal cyclers. PCR amplifications consisted of three minutes at 95 C, followed by 30 cycles of 95 C for 30 s, 64 C for 45 s and 72 C for 45 s and 10 cycles of 95 C for 30 s, 53 C for 45 s and 72 C for 45 s and final extension at 72 C for 10 min. PCR products were size-separated on automated sequencer (Applied Biosystems (Life Technologies, Carlsbad, California, USA) 3730 DNA Analyzer). Fragment (allele) size was determined with Genemarker analysis software (v. 1.85, Soft Genetics, LLC, State College, Pennsylvania, USA). Background amplicons were distinguished as chromatogram peaks and amplifications were rerun if more than two peaks per diploid genotype were observed. Fig. 1. Distribution of teosinte in M é xico. Locations include information from seed collections, herbarium specimens, and archaeological data.
4 1540 American Journal of Botany [Vol. 98 Table 1. Comparison of ecological descriptors for new teosinte populations and the most closely related previously described taxa (standards) from Zea section Luxuriantes (climate data values were characterized based on series). Ecological descriptor* Perennial diploids Perennial tetraploids Zea luxurians type Manantlán P. AnchaZea luxurians Zea nicaraguensis Z. diploperennis (Standard) Nayarit (New) Z. perennis (Standard) Michoacán (New) Guatemala (Standard) Nicaragua (standard) Oaxaca (New) Altitude (m) ap amai m-omai at axt ait hmxt cmit CT (A)Ca(w 2 )(w)(i) (A)Ca(w2 )(i ) (A)Ca(w 1 (w)(i ) (A)Ca(w2 )(w)(i ) (A)Cm(i) Am(i) A(fm)(i ) *ap = annual mean precipitation, amai = annual moisture availability index, m-omai = May-October moisture availability index, at = annual mean temperature, axt = annual maximum mean temperature, ait = annual minimum mean temp, hmxt = hottest month maximum mean temperature, cmit = coldest month minimum mean temperature, CT = climate type (K ö ppen-garc í a Classification, García, 1988 ). Phenetic data analysis For the morphological and physiological data of the 51 accessions, 19 variables were included in the analysis, which were standardized to zero mean and variance 1. A pair-wise distance matrix was generated using the Squared Euclidian Distance between accessions and a cluster analysis was conducted using the Increase in Sum of Squares Method of Clustan Graphics 8. The optimal number of clusters was estimated using the Tree Validation Procedure by random permutation of the original data; it compares a tree obtained for a given dataset with the family of trees generated by random permutation of the same data ( Wishart, 2006 ). Phylogenetic analysis For the data set of SSRs, allele frequencies for diploid populations were calculated by allele counting using Statistical Analysis System (SAS Institute, 2004). Estimating allele frequencies in the tetraploid teosintes was complicated because some marker genotypes were phenotypically indistinguishable. In this study we used Polysat ( Clark and Jasieniuk, 2011 ); this R program assumes allele copy number ambiguity in partial heterozygotes and estimates allele frequencies in polyploids using an iterative algorithm. Allele frequencies were used to compute Nei s genetic distance ( Nei et al., 1983 ) among individual accessions of teosinte. A midpoint-rooted phylogenetic tree was built using the neighbor-joining (NJ) method ( Saitou and Nei, 1987 ) with the Power Marker program package ( Liu and Muse, 2005 ) and visualized with FigTree 1.31 ( Rambaut, 2009 ). In addition, a midpoint-rooted tree was built using program ContML of Phylip 3.69 ( Felsenstein, 2010 ), which uses restricted maximum likelihood estimation (REML). Ten-thousand bootstrap repetitions of the ContML and with neighbor-joining (NJ) trees estimated the strength of support for the various branches ( Nei and Kumar, 2000 ). In the bootstraps, loci were randomly sampled with replacement. RESULTS AND DISCUSSION Discovery and collection of the three new teosinte populations After several successful expeditions in M é xico during the last decade, teosinte populations were newly discovered in several regions; the most important are three populations that clearly belong to Zea section Luxuriantes : Population 1. San Felipe Usila, Oaxaca (annual) In September 1992 Juan Ismael Calzada collected an herbarium specimen of a teosinte ( Calzada 18006, MO) at Paso Escalera close to San Felipe Usila, Oaxaca. The teosinte was identified by Patricia D á vila Aranda in 1992 as Zea mays subsp. mexicana ( Soreng et al., 2011 ). In 2004 Flavio Arag ó n Cuevas collected seeds from the same population at San Felipe Usila, Oaxaca ( Arag ó n, 2006 ), which was preliminarily determined as Z. luxurians. The natural distribution of Z. luxurians is currently restricted to southeastern Guatemala, however, an herbarium specimen from San Agustín, Oaxaca collected by Liebmann in 1842 had been identified as Z. luxurians ( Doebley and Iltis, 1980 ; Wilkes, 1986 ). Population 2. Huajicori, Nayarit (perennial) In 1991 and 1992, Ing. Filiberto Herrera Cedano working at INIFAP-Nayarit visited San Andres Milpillas, northern Huajicori county, and found a grass producing some small ears, which was identified as Euchlaena. This location was explored in 2007 and 2008 by some of the authors of this study (S á nchez, Vidal, De La Cruz, and Ron). After preliminary analysis of its root system, seed size and shape, and chromosome counts, it showed similarities to Zea diploperennis. Population 3. Uruapan-Ziracuaretiro, Michoac án (perennial) In 1982 Galinat and Pasupuleti wrote: The diploid perennial at Cerro de San Miguel is about 70 km from the tetraploid perennial near Ciudad Guzm á n, Jalisco, M é xico. It was originally introduced ca as a forage plant in both areas by two families who brought the seeds from southern Michoac á n (Dr. Elmer C. Johnson, personal letter Mar. 2, 1982).The introductions may include the original Hitchcock 1910 collection site. The question whence came the perennial teosintes? still seems open. During 2009 and early 2010 a perennial teosinte was discovered close to Uruapan, Michoac á n by Suketoshi Taba, head of the Maize Genetic Resources Unit at CIMMYT (Taba et al., 2011); in February 2010 the authors of this work determined that the teosinte was tetraploid. Chromosome counts Zea perennis from Piedra Ancha, Jalisco and the perennial from Michoac á n are both tetraploid species (2 n = 40), while Z. diploperennis, Z. luxurians, the perennial population from Huajicori, Nayarit and the annual population from San Felipe Usila, Oaxaca are diploid (2 n = 20, Appendix S1; see Supplemental Data with the online version of this article). The chromosome numbers for the new diploid Zea taxa have not been reported previously; cytological observations of the teosinte from Michoac á n were simultaneously carried out at CIATEJ (this work) and CIMMYT (Taba et al., 2011).
5 September 2011] S á nchez et al. New Teosintes from Mexico 1541 Fig. 2. Dendrogram of 51 accessions of teosinte, based on morphological variables (abbreviations are included in Appendix 1). Distribution and ecological descriptors The distributions of teosinte and climate types in M é xico are presented in Fig. 1 ; accessions included in this study were highlighted using special target symbols. The perennial diploid population is distributed exclusively in a very small valley of about five km 2 in the mountains of the Sierra Madre Occidental, in the northern part of the county of Huajicori, Nayarit at an average altitude of 1400 m. The San Andr é s Milpillas valley is surrounded by slopes covered with pine forests, and teosinte grows along stone walls or wire fences that border some maize fields. Furthermore, teosinte is found on the banks of the La Mesa and Charco Verde streams that cross the valley. The people living in the area are Southern Tepehuan, Mexicaneros, and Cora Indians, who know teosinte by the name Camalote. The climate is subhumid-semihot with summer rainfalls occurring mainly during June-October period (Ruiz et al., 2009). The growing season in the valley extends from June through December, although frosts may occur from December until March. Ecological descriptors regarding precipitation and moisture availability ( Table 1 ) differ from those teosintes belonging to Zea section Luxuriantes. The Nayarit
6 1542 American Journal of Botany [Vol. 98 Table 2. Principal morphological features of the new taxa of teosinte and their presumed closest relatives. Traits Penennial diploids Perennial tetraploids Zea luxurians type Las Joyas ( Z. diploperennis ) Nayarit (New) P. Ancha ( Z. perennis ) Michoacán (New) Zea luxurians (Guatemala) Zea nicaraguensis (Nicaragua) Oaxaca (New) Days to silk Days to pollen shed No. of leaves per plant Leaf length (cm) Leaf width (cm) Leaf area (cm 2 ) Plant height (cm) No. of lateral branches Number of tillers Kernels per ear Weight of 100 ker (g) No. of tassel branches Tassel length (cm) Branching space (cm) Central spike length (cm) Peduncle length (cm) Spikelet length (mm) Spikelet width (mm) Glume width (mm) No. veins of the spikelet teosinte seems to be adapted to more humid environments than Z. diploperennis from Manantl á n. According to the amai value ( Table 1 ) and the classification of hydric regions from UNEP ( Williams and Balling, 1996 ), teosinte from Nayarit grows in a humid to subhumid region, while teosinte from Manantl á n grows in a dry to subhumid region. The perennial tetraploid population was found near El Fresno, ca. 10 km E of Uruapan, Michoac á n at an average altitude of 1380 m. To date, the population is divided into several fragments each not larger than one hectare, located among avocado orchards. Part of the population was eliminated when the Morelia- Uruapan highway was built. The climate is subhumid-semihot with summer rainfalls occurring mainly during the months of June-October (Ruiz et al., 2009). The growing season extends from June through October and frosts may occur during December-January. Ecological descriptors for the perennial tetraploid population from Michoac á n ( Table 1 ) are similar to the population of the perennial tetraploid from Piedra Ancha, Jalisco, however, its climatic ranges are narrow and it seems to prefer environments with more rainfall. The Zea luxurians type was found in the county of San Felipe Usila, Oaxaca. San Felipe Usila is a Chinanteca Indian town along the Usila River; located in the area known as the valley of the Papaloapan River. The annual diploid teosinte is known by Fig. 3. Neighbor-joining tree of microsatellite diversity based on Nei et al., 1983 genetic distance. (abbreviations are included in Appendix 1. Zea section Luxuriantes colored in green; section Zea colored in orange. Numbers are bootstrap values).
7 September 2011] S á nchez et al. New Teosintes from Mexico 1543 Fig. 4. Spikelets and cupulate caryopses of the perennial diploid teosinte population from Huajicori, Nayarit, M é xico. Bar = 5 mm. Chinanteca Indians as Cu Jah, and is distributed on the banks of the Usila River and in maize fields near the towns Paso Escalera, Arroyo Tambor, Arroyo Iguana, Congregaci ó n Santa Flora, Arroyo Tigre, and Arroyo Aguacate. The valley area is about 8 to 10 km 2 at an average altitude of 80 m. The climate in the area is hot humid with year-round rainfall with a pattern similar to a monzonic, because of the presence of cyclonic air masses in the months of July through October; however at San Felipe Usila, most of the annual precipitation falls during the period May-December. The most distinctive characteristic here is the humid environment ( Fig. 1 ) with intermediate thermal conditions. In contrast, Guatemalan and Nicaraguan teosintes prefer predominantly semihot (at = C) and very hot (at > 26 C) climates, respectively. Morphology The results of numerical taxonomy for the morphological data set are presented for the 51 accessions in the dendrogram of Fig. 2. Three major groups can be identified including Zea mays subsp. parviglumis, Z. mays subsp. mexicana and all the elements of section Luxuriantes. The Tree Validation Procedure of Clustan Graphics ( Wishart, 2006 ), based on 1000 random trials and by estimating the greatest departure from randomness, found the optimal number of clusters and subdivided the teosinte accessions into ten subgroups. The perennial species ( Z. perennis, ZP, and Z. diploperennis ZD) are sister species, while Z. luxurians (ZL) and Z. nicaraguensis (ZN) are sisters to them. The new teosintes from Oaxaca and Michoac á n belong to the same group; however, they seem to have several important morphological differences in comparison to Z. luxurians from southeastern Guatemala and Z. nicaraguensis from coastal Nicaragua and to Z. perennis from southern Jalisco, respectively ( Table 2 ). Each new population is most closely related morphologically to a cluster that contains multiple entities, indicating that the new populations are not simply members of previously described taxa. Furthermore, the new teosinte from Oaxaca is morphologically more distant from Z. luxurians and Z. nicaraguensis than these two previously named species are from each other ( Fig. 2 ). The ten subgroups identified in Fig. 2 generally correspond to the groups described previously by S á nchez et al. (1998). Table 2 presents means of 20 morphological variables used to differentiate the new populations from their suspected most closely related species. Within Zea mays subsp. mexicana and subsp. parviglumis, there are clear geographic patterns; populations from northern Guerrero (B1), eastern Michoac á n, southwestern M é xico, and southern Guerrero (B2) cluster near other populations of the same region; tropical teosintes from Jalisco are dispersed in the three groups of subsp. parviglumis. A small group of very late populations from southern Guerrero, southern Jalisco and Z. mays subsp. huehuetenangensis (B3) are separated from the rest of subsp. parviglumis. Populations of Chalco (CH) and Nobogame (NA) races group with populations from the same regions, while Central Plateau (MC) accessions are more dispersed throughout the subsp. mexicana division. Phylogenetic analysis The NJ tree based on Nei s genetic distances estimated from SSR fingerprints ( Fig. 3 ) showed significant divergence between Zea sections Zea and Luxuriantes as well as species differentiations within sections. The close relationship between Z. luxurians (ZL) from Guatemala and Z. nicaraguensis (ZN) is well supported, and similar results were reported by Wang et al. (2011) based on RAPD and ITS data. The new populations from Nayarit, Michoac á n, and Oaxaca were well-separated from their presumed closest relatives. The tree based on ContML (online Appendix S2) showed similar divergence between Zea and Luxuriantes sections as well as species differentiations within sections. It should also be noted that the number of SSR loci used in this study is low, bootstrapping the 18 SSR loci with all individual accessions did not provide strong support for many nodes, and the highest support was found for populations of the same race. In both analyses (Nei s genetic distance and ContML), grouping of populations from Zea luxurians with Z. nicaraguensis,
8 1544 American Journal of Botany [Vol. 98 Z. perennis accessions with the perennial teosinte population from Nayarit and populations of the same race within Z. mays subsp. mexicana was very well supported. The new populations were well-separated from their presumed relatives; however, teosinte from San Felipe Usila, Oaxaca is closer to Z. luxurians and Z. nicaraguensis in the ContML (Figure S2, Supplemental Data with the online version of this article) than in the NJ tree ( Fig. 3 ). Zea mays subsp. huehuetenangensis from Huehuetenango, Guatemala (HU) is closer to Zea section Luxuriantes, when NJ was the method of analysis ( Fig. 3 ), corroborating the conclusion of S á nchez et al. (1998) based on chromosomal knob frequencies. However, the Huehuetenango teosinte is closer to section Zea when ContML was used (online Appendix S2). Doebley (1990) previously noted that the relationship between the Huehuetenango teosinte and the other members of the genus is uncertain, with relationships based on isozymes conflicting with those based on chloroplast restriction site data. The new diploid perennial population from Huajicori, Nayarit is most closely related to the tetraploid Zea perennis, while the new tetraploid teosinte from Uruapan, Michoac á n is most closely related to Z. diploperennis (ContML tree; online Appendix S2); that is, within the perennials, the new populations are more closely related to the previously named perennial species with a different ploidy level than those with the same ploidy level. Tiffin and Gaut (2001) investigated DNA sequence diversity at four nuclear loci from the tetraploid Z. perennis and the closely related diploid Z. diploperennis ; their results support an autopolyploid origin of Z. perennis from a Z. diploperennis -like ancestor. Based on the SSR results presented here, it seems that the diploids could have diverged first, then the two tetraploids may have formed by independent autopolyploidy events; they probably diversified by subsequent adaptation to different environmental conditions, giving rise to Z. perennis and the tetraploid taxon from Michoac á n. Although our study included representatives of all known Zea taxa, we consider that molecular data available to date were not sufficient to determine with certainty the taxonomic status of the new populations. A detailed comparative study (based on more extensive DNA marker or sequence data) of the relationships within and among sections is needed to resolve longstanding questions about the taxonomy of this group, to verify the taxonomic status of the new populations, and to present a new formal classification of the genus Zea. The question, whence came the tetraploid teosintes? ( Galinat and Pasupuleti, 1982 ) still remains unresolved. Description of the new teosinte populations According to the results of the current study, the following morpho-physiological characteristics are useful to distinguish the new populations from previous described taxa. Geographical information for all populations studied here, are presented in Appendix 1. Population 1: M é xico. Oaxaca: San Felipe Usila (Arroyo Tambor) This annual taxon is diploid ( 2n = 20). Tall maizelike plants in their native habitat (300 to 400 cm), twenty leaves per plant, each leaf ca. 4.4 cm wide and 34 cm long; few tillers produced in the wild and 17 tillers per plant under greenhouse conditions. Male inflorescences with 6 to 10 erect tassel branches, branching axis 3-5 cm long, central spike cm long, spikelets (Fig. 6 ) in sessile-pedicellate pairs ( mm long), glumes of the spikelet scaberulous, 3.7 mm wide and having many veins (38-54). Female inflorescences consist of slender distichous spikes, each with 6-8 brown trapezoidal cupulate caryopses, these mm on long side, mm on short side; weight g per 100 mature caryopses, the heaviest among the teosintes. A very distinctive trait of this teosinte is its very long seed dormancy; without seed scarification, that is, removal of the covering tissues, the seed will not germinate until after ca. 18 months of dormancy. Plants of this population are characterized by high thermal requirements and very late maturity outside of San Felipe Usila (more than 200 days to flowering in Nextipac, Jalisco under greenhouse conditions). Fig. 5. Spikelets and cupulate caryopses of the perennial tetraploid teosinte population from Ziracuaretiro, Michoac á n, M é xico. Bar = 2.5 mm.
9 September 2011] S á nchez et al. New Teosintes from Mexico 1545 Population 2: M é xico. Nayarit: Huajicori (San Andr és Milpillas) Perennial diploid (2n = 20) plants have both thick, short, tuberous rhizomes and long, slender, cord-like rhizomes. Ten leaves per plant, each ca. 2.9 cm wide and 39 cm long; few tillers found in the wild and 26 tillers per plant in the greenhouse. Primary culms usually unbranched or with one to six inconspicuous lateral branches. Male inflorescences with none to two erect tassel branches, branching axis cm long, central spike 8-14 cm long, spikelets ( Fig. 4 ) in sessile-pedicellate pairs ( mm long), glumes of the spikelet glabrous, often purple-tinged, ca. 3.9 mm wide and having veins. Female inflorescences consist of slender distichous spikes, each with 4-6 light brown trapezoidal-cylindric cupulate caryopses and speckled dark brown or nearly black, mm on long side, mm on short side; weight 9 g per 100 mature caryopses. Plants of population 2 are characterized by very early maturity outside its natural distribution (20 to 25 days less than Zea diploperennis under greenhouse conditions), and being short plants in their native habitat (160 to 200 cm tall). Population 3: M é xico. Michoac á n: Ziracuaretiro (El Fresno) Perennial tetraploid (2n = 4 = 40) plants with cordlike and short, tuberous rhizomes. Teosinte from Ziracuaretiro is morphologically more similar to Zea diploperennis than to Z. perennis. The plants in their native habitat are typically m tall, with 10 to 16 leaves per plant, each leaf ca. 4.2 cm wide and 67 cm long; 2 to 20 tillers occur in the wild; primary culms usually unbranched or with one to four inconspicuous lateral branches. Male inflorescences with 5 to 15 erect tassel branches, branching axis 3-11 cm long, central spike cm long, spikelets ( Fig. 5 ) in sessile-pedicellate pairs ( mm long), glumes of the spikelet glabrous, often purple-tinged, ca. 3.7 mm wide and having 8-24 veins. Female inflorescences consist of slender distichous spikes, each with 5-10 light brown and gray trapezoidal-cylindric cupulate caryopses, speckled brown or nearly black, these mm on long side, mm on short side; weight 10 g per 100 mature caryopses. This tetraploid population and Zea perennis are the only polyploids in the genus. CONCLUSIONS The potential for discovering new variants with still unknown genetic properties for use in crop improvement programs fully justifies teosinte preservation efforts. For example, Zea luxurians and Z. nicaraguensis growing in areas that receive frequent rainfall possess unique flooding resistance traits such as the capacity to form root aerenchyma under nonflooding conditions ( Mano and Omori, 2007 ; Mano et al., 2009 ); the new teosinte population from San Felipe Usila (Oaxaca), grows in an area that receives more than twice the amount of rain compared to southwestern Guatemala and coastal Nicaragua and thus could represent another important source of flooding tolerance alleles. Nault (1983) found that Z. perennis and Z. diploperennis have resistance to several important viruses to which all other Zea species are susceptible. Similarly, one of the few sources of resistance to Striga spp.(orobanchaceae), which are menacing root parasites of significant importance in much of Africa and parts of Asia, is Z. diploperennis (Rich and Ejeta, 2008). Thus, we expect that the new teosinte populations described may similarly carry unique useful genes that can be exploited for maize improvement. It is difficult to estimate the danger of extinction for the new teosinte populations with precision. However, based on field observations over the last five years, all populations are threatened; they are rare and endemic to very limited areas. For these populations, especially the perennials, cattle farming and the establishment of pastures and fruit orchards (e.g., avocado orchards in the natural distribution of the perennial tetraploid populations), the introduction of mechanical tilling, opening of new roads that have made several isolated regions accessible, urbanization, introduction of modern maize hybrids, and use of herbicides are among other human activities that are affecting the stability of the populations. Fig. 6. Spikelets and cupulate caryopses of the annual teosinte population from San Felipe Usila, Oaxaca, M é xico. Bar = 5 mm.
10 1546 American Journal of Botany [Vol. 98 Priority of conservation efforts may be based on levels of human disturbance, levels of genetic diversity and inbreeding depression, and their uniqueness in the phylogenetic trees. There is no doubt that all three new populations of teosinte require special protection by Mexican institutions (Secretar í a de Medio Ambiente y Recursos Naturales, Secretar í a de Agricultura, Ganader í a, Desarrollo Rural, Pesca y Alimentaci ó n); in addition, ecogeographic studies, monitoring, and management of teosinte populations must be intensified to reduce the danger of extinction. LITERATURE CITED Arag ó n, C. F Nueva poblaci ó n de teocintle en Oaxaca. XXI Congreso Nacional y Primero Internacional de Fitogen é tica, 3 al 8 de septiembre de 2006, Tuxtla Guti é rrez, Chiapas. No. 65. Carvajal, S. In press. Sistema de las plantas vasculares de la Flora de Jalisco y Á reas Colindantes. Fasc. Supl. I. Universidad de Guadalajara. Cassman, K. 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Information for the taxa used in this study (Species or population, race, abbreviation, bank accession code, country, state, county, collection site, altitude (m), latitude and longitude of the collection site). Accessions deposited in the germplasm bank of Universidad de Guadalajara at Zapopan, Jalisco, M é xico. Zea diploperennis Iltis, Doebley & Guzman : ZD_Las_Joyas, JSG-RMM- LCL-551, M é xico, Jalisco, C.de Garc í a Barrag á n, Las Joyas, 1870 m, N, W. México, Nayarit, Huajicori : Nayarit_NEW, JVLJ-690, México, Nayarit, Huajicori, San Andr é s Milpillas, 1394 m, N, W; Nayarit1_NEW, JVLJ-692, México, Nayarit, Huajicori, San Andrés Milpillas, 1400 m, N, W; Nayarit2_NEW, JVLJ- 693, M é xico, Nayarit, Huajicori, Arroyo La Mesa, 1392 m, N, W. Zea perennis (Hitchc.) Reeves & Mangelsd. : ZP_P_Pancha, JSG-LCL-694, M é xico, Jalisco, San Gabriel, Piedra Ancha, 2140 m, N, W; ZP_La_Mesa, JSG-LCL-695, México, Jalisco, Zapotlán el Grande, Loma de La Mesa, 2174 m, N, W; ZP_P_ Ancha, RMM-12, M é xico, Jalisco, San Gabriel, Piedra Ancha, 2050 m, N, W. México, Michoacán, Ziracuaretiro: Michoacan_NEW, JLRM-712, México, Michoac á n, Ziracuaretiro, El Fresno, 1385 m, N, W. Zea luxurians (Durieu & Asch.) Bird : ZL_Jutiapa, G1, Guatemala, Jutiapa, Jutiapa, Las Majadas, 878 m, N, W; ZL_Agua_ Blanca, G3, Guatemala, Jutiapa, Agua Blanca, Km 162 Agua Blanca- Ipala, 895 m, N, W. Zea nicaraguensis Iltis & Benz : Z_nicaraguensis, CIMMYT-11083, Nicaragua, Chinandega, Somotillo, Rancho Apacunca, 9 m, N, W. M é xico, Oaxaca, San Felipe Usila : Oaxaca_NEW, JSG-593, México, Oaxaca, San Felipe Usila, Arroyo Tambor, 83 m, N, W. Zea mays L.subsp. parviglumis Iltis & Doebley (Race Balsas): B1_ Oxtotitlan, JLHNM-669, México, Guerrero, Teloloapan, Oxtotitlán, 1098 m, N, W; BA_Olinala, JLNCM-646, México, Guerrero, Olinal á, Vista Hermosa, 1580 m, N, W; BA_Quechultenango, JLNCM-649, M é xico, Guerrero, Quechultenango, Vista Hermosa-Colotlipa, 1020 m, N, W; B3_ Tecoanapa, JSG-RMM-LCL-487, M é xico, Guerrero, Tecoanapa, Los Saucitos-Tecoanapa, 590 m, N, W; B3_El_Rincón, JSG-RMM-LCL-489, México, Guerrero, Mochitlán, El Rincón de la Vía, 744 m, N, W; B2_El_Salado, JSG-RMM-LCL-494, M é xico, Guerrero, Mochitl á n, El Salado-Mazatl á n, 1132 m, N, W; B1_Ixcateopan, JSG-RMM-LCL-495, México, Guerrero, Ixcateopan de Cuauht é moc, Ixcateopan, 1890 m, N, W; B1_Zacatlancillo, JSG-RMM-LCL-500, México, Guerrero, Teloloapan, Zacatlancillo, 1740 m, N, W; B1_SM_Palmas, JSG- RMM-LCL-504, M é xico, Guerrero, Huitzuco de los Figueroa, San Miguel de las Palmas, 1173 m, N, W; B2_Am_Grandes, JSG-RMM-LCL-511, M é xico, Guerrero, General Canuto A. Neri, Amates Grandes, 1110 m, N, W; B3_T_Colorada, JSG- RMM-LCL-568, M é xico, Guerrero, Juan R. Escudero, Tierra Colorada, 300 m, N, W; B1_Llano_Merced, JSG-RMM- LCL-573, M é xico, Guerrero, Pedro Ascencio Alquisiras, Km 2 Llano de la Merced, 1580 m, N, W; B2_R_Nuevos, MGB- CIMMYT-29, M é xico, Guerrero, Teloloapan, Ranchos Nuevos, 1640 m,
12 1548 American Journal of Botany N, W; BJ_El_Tablillo, JSG-JRP-ERG-545, México, Jalisco, Guachinango, El Tablillo, 1123 m, N, W; BJ_El_Saucito, JSG-LOS-142, M é xico, Jalisco, Jilotl á n de los Dolores, El Saucito, 1460 m, N, W; B3_Talpitita, JSG-RMM- LCL-555, M é xico, Jalisco, Villa Purificaci ó n, 1 Km N Talpitita, 526 m, N, W; BJ_La_Lima, RMM-11, México, Jalisco, Tolim á n, La Lima, 1450 m, N, W; BJ_San_Lorenzo, RMM-3, M é xico, Jalisco, Ejutla, San Lorenzo, 984 m, N, W; B2_Tlatlaya, JSG-LCL-670, México, México, Tlatlaya, Colonia Moctezuma, 870 m, N, W; BA_Sto_Tomas, JSG-LCL-676, México, México, Santo Tomás, Santo Tomás de los Pl á tanos, 1345 m, N, W; B2_Huixtitla, JSG-RMM- LCL-528, M é xico, M é xico, Amatepec, Huixtitla, 1008 m, N, W; B2_Quenchendio, JSG-RMM-458, México, Michoacán, Huetamo, Quenchendio, 635 m, N, W; B2_Taretan, JSG-RMM-558, M é xico, Michoac á n, Taretan, Col. Emiliano Zapata, 1170 m, N, W; B2_Pto_DLa_Cruz, JSG-RMM- LCL-517, M é xico, Michoac á n, Car á cuaro, Puerto de la Cruz, 870 m, N, W; B2_K37_Temascal, JSG-RMM-LCL-537, M é xico, Michoac á n, Tzitzio, Km 37 Temascal-Huetamo, 1030 m, N, W; BA_Morelos, JSG-RMM-LCL-474, México, Morelos, Tepoztlán, Amatlán de Quetzalcóatl, 1654 m, N, W; BA_Oaxaca, JSG-RMM-LCL-483, México, Oaxaca, San Jer ó nimo Coatl á n, San Cristobal Honduras, 1272 m, N, W. Zea mays L. subsp. mexicana (Schrad.) Iltis (Race Nobogame): NA_ Tejamanil, JSG-SRV-604, M é xico, Chihuahua, Guadalupe y Calvo, Arroyo Tejamanil, 1920 m, N, W; NA_Tarahumares, JSG- SRV-606, M é xico, Chihuahua, Guadalupe y Calvo, Tarahumares, 1951 m, N, W. Zea mays L. subsp. mexicana (Schrad.) Iltis (Race Durango): DU_Gavilanes, JSG-RMM-428, M é xico, Durango, Durango, Puente Gavilanes, 1875 m, N, W; DU_Fco_Villa, JSG-RMM-429, M é xico, Durango, Durango, Francisco Villa Nuevo, 1875 m, N, W; DU_Tuitan, JSG-SRV-EAM-705, México, Durango, Nombre de Dios, San Jos é de Tuit á n, 1870 m, N, W. Zea mays L. subsp. mexicana (Schrad.) Iltis (Race Central Plateau): MC_ Uriangato, JSG-RMM-447, M é xico, Guanajuato, Uriangato, Uriangato, 1880 m, N, W; MC_San_Jerónimo, JSG-465, México, Jalisco, Ayotl á n, San Jer ó nimo, 1597 m, N, W; MC_Indaparapeo, JACV-T-067, México, Michoacán, Indaparapeo, 1 km Oeste de Indaparapeo, 1872 m, N, W; MC_ Churintzio, JSG-426, México, Michoacán, Churintzio, Cerro Churintzio, 1950 m, N, W; MC_El_Salitre, JSG-427, México, Michoac á n, Ixtl á n, El Salitre, 1569 m, N, W; MC_ Cojumatlán, JSG-LOS-75, México, Michoacán, Cojumatlán de Régules, 5-7 km SW Cojumatl á n, 1700 m, N, W; MC_SA_ del_maíz, JSG-RMM-450, México, Michoacán, Copándaro, San Agustín del Ma í z, 1860 m, N, W; MC_Cd_Hidalgo, JSG- RMM-LCL-536, M é xico, Michoac á n, Hidalgo, Km 152 Cd. Hidalgo- Morelia, 2102 m, N, W. Zea mays L. subsp. mexicana (Schrad.) Iltis (Race Chalco): CH_Boyeros, JSG-JMHC-639, México, México, Texcoco, San Martín Netzahualcoyotl, 2254 m, N, W; CH_Chapultepec, JSG-RMM-LCL- 471, M é xico, M é xico, Chapultepec, Chapultepec, 2595 m, N, W; CH_Amecameca, JSG-RMM-LCL-479, México, México, Amecameca, Amecameca, 2471 m, N, W; CH_L_ Cárdenas, JSG-LCL-559, México, Michoacán, Erongarícuaro, Lázaro C á rdenas, 2410 m, N, W; CH_Opopeo, JSG-RMM- 463, M é xico, Michoac á n, Salvador Escalante, Opopeo, 2225 m, N, W; CH_SJ_Atenco, JSG-432, M é xico, Puebla, San Juan Atenco, Ejido San Antonio, 2520 m, N, W; CH_SN_Ranchos, JSG-437, M é xico, Puebla, S.N. de los Ranchos, Ca ñ ada Grande, 2475 m, N, W; CH_Tlaxcala, JSG-JMHC-631, México, Tlaxcala, Tenancingo, Tenancingo, 2311 m, N, W. Zea mays L. subsp. huehuetenangensis (Iltis & Doebley) Doebley (Race Huehuetenango): HU_Monajil_Buxup, H2, Guatemala, Huehuetenango, Santa Ana Huista, Monajil-Buxup, 1025 m, N, W.