Differentiation among maize (Zea mays L.) landraces from the Tarasca ...

Springer 2006

Genetic Resources and Crop Evolution (2007) 54:309–325 DOI 10.1007/s10722-005-4775-y

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Differentiation among maize (Zea mays L.) landraces from the Tarasca Mountain Chain, Michoacan, Mexico and the Chalquen˜o complex Javier O. Mijangos-Corte´s1,2,*, T. Corona-Torres1, D. Espinosa-Victoria1, A. Mun˜oz-Orozco3, J. Romero-Pen˜aloza3 and A. Santacruz-Varela1 1

Campus Montecillo, Colegio de Postgraduados, km 36.5 Carr. Me´xico-Texcoco, CP 56230, Montecillo, Texcoco, Edo. de Me´xico, Me´xico; 2Unidad de Biotecnologı´a, Centro de Investigacio´n Cientı´fica de Yucata´n A.C., Calle 43 # 130 Colonia Chuburna´ de Hidalgo, C.P. 97200, Me´rida, Yucata´n, Me´xico; 3Centro Regional Universitario del Centro Occidente, Universidad Auto´noma Chapingo; *Author for correspondence (e-mail: [email protected]) Received 31 January 2005; accepted in revised form 28 October 2005

Key words: Isozymes, Maize race, Phenetic relations, Tarasca Mountain Chain, Zea mays L.

Abstract The classification of Mexican maize (Zea mays L.) begun since the early 20th century, it was consolidated during the middle of this century, but recent additions and rearrangements have been performed by several authors employing new methods of analysis and collections from diverse origin; nevertheless, maize from the State of Michoacan, Mexico has received little attention in regard to its systematic classification. Maize populations from the Tarasca Mountain Chain in Michoacan are commonly considered in literature, as belonging to the Chalquen˜o race; however, closer observations indicate that significant differences do exist, suggesting the necessity of performing an in-depth study on this respect. Thirty nine native maize populations from the Tarasca Mountain Chain region were evaluated along with 19 typical populations of the Chalquen˜o, Celaya and Conico races coming from the States of Mexico, Puebla, Hidalgo, Quere´taro and Oaxaca. Populations were evaluated in Aranza, Michoacan and Montecillo, Mexico State. Seventeen morphological characters were scored and analyzed by one-way analyses of variance and multivariate techniques. Populations were also genetically analyzed through 17 isozyme loci. Native populations had some alleles not found either in the Chalquen˜o, Celaya or Conico races, and possess larger genetic diversity. Local populations were congregated into a discrete group apart from the typical Chalquen˜o populations, suggesting that landraces from the Tarasca Mountain Chain region might not be considered as belonging to the Chalquen˜o race, but they integrate by themselves a different race.

Introduction Maize genetic diversity in Mexico is explained by some factors as: (1) the geography with lots of microregions or ‘ecological niches’ throughout the country, with irregular orography and characterized by particular climatic and edaphic conditions differing from each other; thus, specific maize variants

must be adapted to every ecological niche (Mun˜oz 2003), and (2) ethnic groups that have selected for different uses and preserved the adequate kind of maize for their particular environments according to their own customs, knowledge, management practices and traditions (Herna´ndez and Alanis 1970). Mun˜oz et al. (2003) mentioned that in Mexico the diversity of native maize populations is finely

310 structured into varietal patterns within each ecological niche; those systems were developed by ethnic population and farmers from each location. The number of components of those varietal patterns turns out to be at least three and might be identified by their biological cycle as late, intermediate and early, grain color (white, yellow and dark, primarily), and their resistance to environmental factors, mainly the drought. Each group of varieties are planted in specific sites of the niche (valley, hills, hill side, or hill top) at specific periods of the year, depending on the altitude, temperature and soil moisture. The complex of conical races such as Chalquen˜o, Palomero Toluquen˜o, Conico, Arrocillo Amarillo and Cacahuacintle are commonly found at the high central valleys of Mexico and they are cultivated at altitudes above 2000 m (Eagles and Lothrop 1994). The Chalquen˜o race can be divided into three subraces: Chalquen˜o Crema, Chalquen˜o Palomo and Elotes Chalquen˜os, but it also can be found as a complex of variants of intermediate forms with other races such as Chalquen˜o-Conico, Chalquen˜oArrocillo and Mushito-Chalquen˜o (Herrera-Cabrera et al. 2004). The Chalquen˜o race has its center of distribution in the high central valleys of Mexico where it is broadly cultivated, it is cultivated from 2180 to 2800 m of altitude in almost all of the localities of Chalco in the State of Mexico and surrounding regions (Herrera-Cabrera et al. 2004); it is also widespread all over the Neovolcanic axis toward central-western Michoacan State where the races Conico and Celaya are cultivated likewise (Sa´nchez and Goodman 1992), being the Chalquen˜o the best distributed and adapted race over the region because of its late life cycle (Wellhausen et al. 1951). The Tarasca Mountain Chain is located at the center of Michoacan State in the Neovolcanic axis, from latitude 1858¢ N to 1954¢ N, and longitude from 101 W to 10229¢ W (Figure 1), it has a rough orography altitude ranging from 1000 to 3869 m a.s.l., on the Neovolcanic province (Escobar et al. 1996). It has many ecological niches as small valleys and hillsides where farmers plant their maize which is well adapted to the environmental and hygrothermal conditions (Mun˜oz et al. 1994). Some racial studies based on cluster analysis but including only a few accessions from the region

have suggested that maize populations grown in the Tarasca Mountain Chain belong to the Chalquen˜o race with some local adaptations acquired through the time (Sa´nchez and Goodman 1992; Sa´nchez et al. 2000). Ortega and Sa´nchez (1989) carried out a study of maize diversity from the high lands of Mexico, they included two accessions from the Tarasca Mountain Chain region and suggested the existence of a different race; they found one of those accessions consistently associated with Chalquen˜o, and suggested that the populations from that zone could have evolved from Chalquen˜o. In a study of the definition of varietal performance Gil et al. (1995) found that farmers from the Tarasca region classify native populations (NP) into four groups: early white, late white, yellow and blue; based on grain color, yield capacity and life span. Blue populations had shorter life cycle (from 122 to 130 days to silk), it was followed by yellow, early white and late white populations; the late white populations showed a range of days to silk from 131 to 139 days. The four groups of populations had statistical differences in ear diameter, ranging from 4.07 in the blue group to 4.36 cm in the late white group; these morphological characters suggested important differences in comparison to the typical Chalquen˜o race as reported by Wellhausen et al. (1951). Despite the aforementioned studies, in general, it can be said that maize of the Tarasca Mountain Chain has been systematically misrepresented or excluded from racial descriptions (Ortega and Sa´nchez 1989; Sa´nchez and Goodman 1992; Sa´nchez et al. 2000). This study was performed to solve the ambiguous racial situation of the maize landraces from the Tarasca Mountain Chain of Michoacan and to describe their characters and genetic variability in order to establish its variation and relationships among them and their relationship with the Chalquen˜o complex from the central region of Mexico through morphological and isozymatic characterization. The study was based upon the hypothesis that the rough orography of the region, along with the cultural influence of a consolidated, traditional community, has led farmers to grow and select populations of maize for centuries, promoting their genetic isolation, creating new populations genetically different from those of the central highlands Chalquen˜o race.

311

Figure 1. Area of study of native maize populations in the Tarasca Mountain Chain region, Michoaca´n, Mexico. (a) Charapan; (b) Paracho; (c) Nahuatzen; (d) Tingambato; (e) Erongarı´ cuaro; (f) Salvador Escalante.

312 Materials and methods Germplasm Thirty nine native maize populations were collected from local farmers in 11 different communities of six municipalities distributed throughout the Tarasca Mountain Chain region (Table 1); a sample of 30 ears of each native population was taken. Sixteen varieties from the Chalquen˜o, two from Celaya and one from Conico races were used as checks (Table 1). Checks were selected in a way to include recently described variants of the Chalquen˜o race (Romero et al. 2002; Herrera-Cabrera et al. 2004).

Geographic region of study The physiography of Michoacan State is divided into two big provinces: the Sierra Madre del Sur and the Neovolcanic axis, the last one in turn is classified into eight subprovinces, one of those is the so-called Neovolca´nica Tarasca (Escobar et al. 1996), where the Tarasca Mountain Chain is located and this study was conducted. The orography is pretty rough, forming a huge diversity of microclimatic sites, with altitudes from 1000 to 3869 m, and characterized by a temperate subhumid climate, with summer rains between 1500 and 2000 mm, and high relative humidity (Mun˜oz et al. 1994); it has no permanent water currents but only some temporary streams; the soil is dated from the early tertiary Cenozoic period and Eocene, and mainly corresponds to the podzolic type, with 6–13% of organic matter and acidic pH from 5.4 to 6.4 (Pen˜a 1984); other characteristics are described in detail in Trinidad and Miranda (1984).

Field evaluations The experiments were performed in Aranza, Michoacan and Montecillo, Edo. de Mexico. Aranza is located at 1939¢50¢¢ N, 10201¢25¢¢ W, with 2200 m a.s.l., 1100 mm of precipitation and temperatures from 7 to 22 C. Montecillo is located at 1929¢ N, 9853¢ W, with 2250 m a.s.l.,

691 mm of precipitation and temperatures from 12.6 to 18.7 C. A randomized complete blocks design with three replications was used at each location and year. The experimental unit had two 5-m long rows 0.8 m apart, 66 seeds were planted per experimental unit. Five plants by replication were taken at random and measured for vegetative characters, and at the harvest five representative ears of each experimental unit were selected to measure ear and kernel traits.

Morphological traits Twenty one tracits were measured (Table 2), seven of them during the vegetative cycle: days to silk (dfh) as an estimator of the life cycle, counted from the day of planting to the day when 50% of plants showed receptive stigmas; plant height (ap) from the ground level to the top of tassel; ear height (am) from the ground level to the insertion node of the ear; number of leaves below the principal ear (hab) and over the principal ear (har), counted at the time of anthesis; the leaf of the upper ear was measured in length (lh) and width (ah). After anthesis five characters from tassel were measured: peduncle length (lpe), measured from the node to the first branch; tassel branching length (ltr), measured from the first to the last branch; central spike length (lce), measured from the last branch to the tip; total tassel length (lte), taken as the segment from the first branch to the tip; and finally the primary tassel branches were counted (nre). Five characters were measured from the ears after harvest; five ears were visually selected representing the plot variation, they were dried and weighted (ps5mz), and measured in length (lmz) from tip to bottom, and its diameter was measured in the middle (dmm); the number of rows of kernels (nh) were counted; and finally, the cob diameter (do) was measured in the mid part. Ten kernels from each of the five selected ears were arranged in line using modeling clay and measured in length (lg), width (ag), and thickness (eg), and the measures were divided by ten; after that, 100 kernels were weighted (wg).

313 Table 1. Landraces collected from the Tarasca Mountain Chain, Michoaca´n, Mexico and checks used in the study. Accession

Provenance (Locality, State)

Race

Local name

ASV-4 ASV-5 ASV-7 ASV-9 ASV-10 ASV-15 ASV-17 ASV-21 ASV-23 ASV-29 ASV-33 ASV-37 ASV-38 ASV-39 ASV-40 ASV-42 ASV-44 ASV-46 ASV-48 ASV-50 ASV-51 ASV-54 ASV-57 ASV-59 ASV-62 ASV-68 ASV-75 ASV-81 ASV-84 ASV-87 ASV-90 ASV-92 ASV-95 ASV-97 ASV-101 ASV-102 ASV-111 ASV-112 ASV-113

Aranza, Paracho Aranza, Paracho Aranza, Paracho Aranza, Paracho Aranza, Paracho Aranza, Paracho Nahuatzen, Nahuatzen Nahuatzen, Nahuatzen Nahuatzen, Nahuatzen Nurio, Paracho San Felipe de los Herreros, Charapan San Felipe de los Herreros, Charapan Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho Quinceo, Paracho San Isidro, Nahuatzen. San Isidro, Nahuatzen San Isidro, Nahuatzen San Isidro, Nahuatzen Zevina, Nahuatzen Picha´taro, Tingambato Picha´taro, Tingambato Picha´taro, Tingambato Picha´taro, Tingambato Zinciro, Erongarı´ cuaro Zirahue´n, Salvador Escalante Zirahue´n, Salvador Escalante Opopeo, Salvador Escalante Opopeo, Salvador Escalante Opopeo, Salvador Escalante San Gregorio, Salvador Escalante San Gregorio, Salvador Escalante San Gregorio, Salvador Escalante

NPa NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP NP

Azul Blanco Negro Azul Morado Blanco Amarillo Negro Prieto Azul Uaruti Blanco Negro Azul Blanco Ranchero Toluca Blanco Amarillo Blanco Toluca Blanco Negro Amarillo Amarillo Amarillo Blanco Amarillo Pinto Blanco Rojo Azul Rojo Chalca Amarillo Amarillo Blanco Aperlado Pinto Amarillo Pinto Blanco Blanco

Checks HGO-10 Dgo-189 Gto-142 Gto-208 MEX-2 MEX-158 MEX-43 MEX-304 Juchi 7o CSM Tlapala 4o CSM Cocoti 6o CSM COL-6538 COL-6949 COL-6951 Compuesto de Familias

Provenance (Locality, state) Xanov, Hidalgo Mezquital, Durango San Miguel de Allende, Guanajuato Leo´n, Guanajuato Toluca, Me´xico Timilpan, Me´xico Cuautitla´n, Me´xico Texcoco, Me´xico Juchitepec, Me´xico Juchitepec, Me´xico Cocotitla´n, Me´xico Cocotitla´n, Me´xico Zacapu, Michoaca´n Villa Jime´nez, Michoaca´n Nochixtla´n, Oaxaca

Racial classification Chalquen˜o Chalquen˜o Celaya Celaya Chalquen˜o Chalquen˜o Chalquen˜o Chalquen˜o Chalquen˜o Crema Chalquen˜o Crema Chalquen˜o Palomo Chalquen˜o-Cacahuacintle Chalquen˜o Celaya Chalquen˜o

314 Table 1. Continued. Checks

Provenance (Locality, state)

Racial classification

QRO-21 QRO-46 Sintetico Serdan Zac-66

Huimilpan, Quere´taro Pen˜amiller, Quere´taro Chalchicomula, Puebla Jerez de Garcı´ a Salinas, Zacatecas

Chalquen˜o Celaya Chalquen˜o Conico Norten˜o

a

Native population.

Isozyme polymorphism evaluation

Statistical analysis

An array of a set of 17 isozyme loci (Table 3), distributed over most of the maize chromosomes was selected for evaluation using the inbred lines B73, Mo17, Tx303 and Mo24W as checks, for which most of the alleles are known. Enzymes from 10 plants of each accession were extracted from the coleoptiles of six-day-old seedlings, subjected to starch gel electrophoresis and stained according to the protocols described by Stuber et al. (1988). The allelic frequencies were calculated by population for each allele through POPGENE software (Population Genetic Analysis) (Yeh et al. 1999).

Analysis of variance Analyses of variance were performed for the 21 traits of the 58 genotypes in the three environments (except for dfh, which was measured in two environments). The analyses were based on the model of a complete randomized blocks design: Yijk = l + Pi + Lj + cij + B(L)k(j) + Eijk; where Yijk is the variable value of the ith population in the jth environment in the kth block, l is the general average, Pi is the effect of the ith population, Lj is the effect of the jth environment, cij is the effect of the interaction between the ith population and the jth environment, B(L)k(j) is the effect of the kth block nested into the jth environment, and Eijk is the experimental error (SAS Institute 1985).

Table 2. Morphological characters measured in maize populations from the Tarasca Mountain Chain, and checks. Michoaca´n, Mexico. Character classification

Character name

Abbreviation

Units

Vegetative

Days to silk Plant height Ear height Number of leaves below the ear Number of leaves above the ear Ear leaf length Ear leaf width Tassel peduncle length Tassel branching length Central spike length Total tassel length Tassel branches number Ear length Ear diameter Kernel row number Cob diameter Dry weight of five ears Kernel length Kernel width Kernel thickness 100 kernels dry weight

dfh ap am hab

days cm cm

Tassel

Ear

Kernel

har lh ah lpe ltr lce lte nre lmz dmm nh do ps5mz lg ag eg wg

cm cm cm cm cm cm cm cm cm g mm mm mm g

Variable selection The variables were screened by two statistical methods and finally a group of traits was selected for further analyses. The first method was the Pearson correlation coefficient using adjusted means; those traits with coefficients of 0.75 or higher and significant to a p < 0.001 were selected as candidate variables to be dropped from the data set to avoid multicolinearity problems. The second method was the Stepwise selection through the STEPDISC procedure of SAS, with a significant level to enter = 0.5 and significant level to stay = 0.5 (SAS Institute 1985). Variation and group discrimination The 39 populations native to the Tarasca Mountain Chain region were evaluated along with 19 checks, the adjusted means over the three environments of the selected traits were standardized to reduce the data to a common scale of mean 0 and variance 1. Principal component analysis (PCA), as computed with the SAS PRINCOMP procedure (SAS Institute 1985) was performed in a

315 17 · 58 matrix for reducing all variables to a number of uncorrelated indexes, with the maximum variance explained by a few of the first indexes and to know the traits primarily contributing to that explanation. The canonical discriminant analysis (CDA), as computed with SAS DISCRIM procedure (SAS Institute 1985) was performed for measuring how well it was possible to separate two or more groups, in this case race complexes or populations, on the basis of linear combinations of the predictor variables. Based upon the Hotelling’s T 2 statistic, the cubic clustering criterion (CCC), PCA and the phenogram, three groups were predetermined in this analysis. The best function for separating the groups is defined as the linear combination in which the F-ratio for a one-way ANOVA is maximized (Johnson 2000). Those analyses sort the data on a different basis and so can be used comparatively to verify the relationships (Johnson 2000). Relationships among genetic materials To determine the relationships among landraces and checks, a distance matrix was produced with the standardized data with the dissimilarity Euclidian coefficient and an UPGMA cluster analysis was performed, rendering a dendrogram through the NTSYS software (Rohlf 1993). The Table 3. Isozyme loci evaluated and chromosomal location. Locus

Symbol

Bina

Acid phosphatase-1 Acid phosphatase-4 b-Glucosidase-1 Malate dehydrogenase-1 Malate dehydrogenase-2 Malate dehydrogenase-3 Malate dehydrogenase-4 Malate dehydrogenase-5 Alcohol dehydrogenase-1 Catalase-3 Glutamate-oxaloacetate transaminase-1 Glutamate-oxaloacetate transaminase-2 Glutamate-oxaloacetate transaminase-3 Esterase-8 Phosphohexose isomerase-1 6-Phosphogluconate dehydrogenase-1 6-Phosphogluconate dehydrogenase-2

Acp1 Acp4 Glu1 Mdh1 Mdh2 Mdh3 Mdh4 Mdh5 Adh1 Cat3 Got1 Got2 Got3 E8 Phi1 Pgd1 Pgd2

9.03 1.11–1.12 10.03 8.03 6.07 3.08 1.07–1.08 5.03 1.10 4.19 3.08 5.08 5.03–5.04 3.01 1.11 6.01 3.05

a The Bin number is an interval between two fixed core maizegenomic marker loci, and includes the top marker on the map bins.

identification of the number of clusters was based upon two methodologies: the Hotelling’s T2 statistic and the CCC (SAS Institute 1985). The CCC procedure uses Ward’s minimum variance method, or other methods based on minimizing the withincluster sum of squares (Sarle 1983), the SAS procedure compute the number of clusters when they are less than 20% of the number of the original data (Johnson 2000), and is based on the assumption that a uniform distribution on a hyperrectangle will be divided into clusters shaped roughly like hypercubes; the performance of the CCC is evaluated by Monte Carlo methods (Sarle 1983). A second cluster analysis was performed with a combined data of the 17 morphological characters and the frequency of the 29 alleles detected from the isozyme loci; each allele was considered as an independent variable; the pooled dataset of 58 · 46 was standardized by subtracting the mean and dividing by the standard deviation; the data set was used to calculate Euclidian distances between populations and UPGMA cluster analysis was performed rendering a dendrogram with the NTSYS software (Rohlf 1993). Canonical discriminant analysis CDA was performed, detecting three genetic groups integrated by different number of native populations or checks; to characterize each genetic group the adjusted means of 17 variables previously selected of each genotype were calculated over the three environments. Analysis of variance was then recalculated using the groups generated by CDA as a variation factor using PROC GLM of SAS; the multiple average comparisons among groups were performed using the Tukey test (0.05) (SAS Institute 1985).

Results and discussion Analysis of variance A high degree of variation was found in the populations and checks, as revealed by the highly significant differences among populations throughout all of traits (Table 4), the total variation in most of the variables was due to the three main components: genetic, environmental and the interaction between them; but the greatest proportion of the

316 variation was attributed to the environmental effect that in most cases was higher than the other factors of variation. There were highly significant statistic differences among populations for most of the characters, except for ‘ah’, whose variation was mainly due to the environmental effect. It was clear, however that the genotypes showed lots of variation.

Variable selection The analysis of the Pearson correlations showed that ap/am (0.878), am/hab (0.773), ltr/lte, (0.794) and dmm/lg (0.759) were highly correlated, so one of each pair might be dropped from further analyses. The Stepwise method of variable selection excluded lce, nh, and eg characters with values of average squared canonical correlations over 0.14 (p < 0.001). Based on these two methods the traits

finally excluded from the subsequent analyses were hab, lce, nh and eg. Most of the characters measured have been selected in other maize racial or genetic diversity studies (Sa´nchez et al. 1992; Herrera et al. 2000; Sa´nchez et al. 2000). Herrera et al. (2000) found that some attributes are useful to measure or differentiate in a certain level of diversity (between races) but not among populations of the same race, although they share some variables in common, thus, the different groups of characters measure in a different way the amplitude of the genetic diversity. For racial classification Sa´nchez et al. (1992) found 24 variables through the function of the ratio of variance components (or repeatability), and a minimum of nine characters were proposed to be appropriate; some of the characters considered in the list were: number of leaves per plant, central spike internode length, kernel width, rachis segment length, ear diameter/length and kernel width/length. In a genetic diversity study of maize

Table 4. Analysis of variance of 21 traits of 39 native populations from the Tarasca Mountain Chain and 19 checks, mean squares and levels of significance. Variable

dfh ap am hab har lh ah lpe ltr lce lte nre lmz dmm nh do lg ag eg wg ps5 mz

Mean squares Populations

Environment

Pop · Env

Blo (Env)

Error

359.0143** 6587.4199** 3948.2024** 4.9034** 0.5400** 181.7134** 4.1572 ns 102.4441** 78.7963** 57.2446** 152.1288** 55.1144** 25.8830** 1.4878** 20.6363** 0.6310** 16.4579** 13.4200** 1.5191** 220.5224** 90506.4180**

15616.3605** 184983.3197** 120617.1040** 374.6102** 8.3463** 14077.2707** 41.5405** 5094.9662** 72302.8698** 532.0442** 67104.7304** 720.9747** 77.5838** 5.6216** 58.9090** 0.3237** 1.6724 ns 3.2369 ns 9.9316** 631.4967** 421253.2530**

78.0902** 1443.5075** 50.8386** 1.0280** 0.2353** 63.6741* 3.7674 ns 97.1867** 65.0154** 13.6920** 75.7591** 8.0890** 4.9263** 0.3275** 1.6619** 0.0769 ns 2.3304** 9.2007 ns 0.1176 ns 36.2148** 67892.2510**

79.1523** 570.1502 ns 517.0828** 3.0026** 0.2401 ns 71.1249* 3.8118 ns 29.3296 ns 6.4040 ns 5.2306 ns 16.6008 ns 3.5098 ns 10.9228** 0.4366* 1.6807* 0.0610 ns 3.1315* 8.0146 ns 0.0873 ns 18.5755 ns 48680.6810**

15.1055 750.4850 162.1613 0.6359 0.1562 32.0258 3.1798 21.2550 9.3748 7.4169 21.5240 4.8978 1.6915 0.1699 0.7866 0.0647 1.2932 8.3773 0.0994 12.4285 11829.6400

dfh, days to silk; ap, plant height; am, ear height; hab, leaves below the principal ear; har, leaves above the principal ear; lh, leaf length; ah, leaf width; lpe, tassel peduncle length; ltr, tassel branching length; lce, central spike length; lte, total tassel length; nre, number of primary tassel branches; lmz, ear length; dmm, mid-ear diameter; nh, number of row kernels; do, cob diameter; lg, kernel length; ag, kernel width; eg, kernel thickness; wg, 100 kernel weight; ps5 mz, dry weight of five ears. *Significant at 5%; **significant at 1%; ns: non-significant.

317 populations from a particular ecological region Herrera et al. (2000) identified 11 morphological characters, some of they were: days to silk, ear height, number of branches in the tassel, mid-ear diameter, kernel length, and kernel width. Those results suggest that each study must select its own set of characters, according to the environment of evaluation for genetic diversity and relationship analyses.

Principal component analysis The total variance of genetic material was explained by the first four principal components (PC) in a 73.3%; the first PC explained 31.5% of the total variance and the second one explained 20.1% with Eigenvalues of 5.4 and 3.4, respectively (Table 5). The main original traits contributing to the first Eigenvector were ap, am, lh, lte, and lmz; meanwhile, for the second Eigenvector the main variables were dmm, lg, and ps5mz (Table 5).

Table 5. Variable contribution to the Eigenvectors and Eigenvalues in the principal component analysis of native populations of maize from the Tarasca Mountain Chain and checks. Michoaca´n, Mexico. Variablesa

Days to silk Plant height Ear height Leaves below the principal ear Leaf length Leaf width Tassel peduncle length Tassel branching length Total tassel length Number of primary tassel branches Ear length Mid-ear diameter Cob diameter Kernel length Kernel width 100 kernel weight Dry weight of five ears Eigenvalues Proportion (%) Cumulative (%) b

PC, Principal component.

Eigenvectorsb PC1

PC2

0.2168 0.3791 0.3251 0.2056 0.3791 0.1634 0.0209 0.2509 0.3256 0.0414 0.3423 0.0312 0.1691 0.0759 0.2187 0.2486 0.2399 PC1 5.3504 31.47 31.47

0.0018 0.0570 0.0301 0.0734 0.0059 0.1102 0.0594 0.2256 0.1995 0.1442 0.2020 0.5127 0.2547 0.4722 0.0886 0.3220 0.4002 PC2 3.4155 20.09 51.56

The genetic material was assembled into three groups on a biplot with the first two principal components that explained 51.56% of the total variance. Group III was defined grouping most of the NP (33 out of 39), and it was characterized by larger ap, am, lh, lte, and lmz but from mid to lower dmm, lg and ps5mz; in contrast, groups I and II congregated checks predominantly (Figure 2). The populations’ arrangement suffered some modifications when the relationship (Figure 3) and discriminant studies were performed. Group I was built up by 11 populations, most of them checks, but one NP (ASV-90), an accession from Erongaricuaro commonly called ‘Chalca’, a name most likely applied in relation to the Chalco Valley in Central highlands Mexico, where it was likely acquired through seed exchange via the Zacapu Valley, rather than through gene flow. This accession keeps striking morphological similarity with the typical Chalquen˜o race. The two Celaya checks and some typical Chalquen˜os from Mexico State grouped here; this group was integrated by genotypes with slightly greater ear diameter, ear weight and longer kernels. Gto-142 and Gto-208 populations were situated in this group, which agrees with Herrera-Cabrera et al. (2004) who found those populations as closely related and classified as Chalquen˜o-Conico; also the Mex-43 and Mex-304 populations were situated into this cluster since such populations belong to the same regional genetic group (TexcocoCuautitlan), according to Romero et al. (2002). Group II gathered 13 genotypes with shorter plants (plant and ear height). It includes seven checks from Puebla, Queretaro and Mexico and six NP, all of those from Nahuatzen and Salvador Escalante, but the native populations clearly formed a subgroup characterized by small and slender ears with short kernels. The checks subgroup were differentiated by longer ears, and this assembling agree with Romero et al. (2002), who found that some accessions of the regional genetic group from Puebla (such as Sintetico Serdan) were closely related to Chalquen˜o populations. Group III that clustered most of the native populations was integrated by the tallest plants, with big leaves and tassels and longer ears, but with thin ears and short kernels. This group included two ancient populations of Palomero Toluquen˜o collected at the Tarasca Mountain Chain (ASV-42 and ASV-48). The typical Conico race collection

318 (Zac-66) included in this group agree with Romero et al. (2002), who found a close relationship of this race with some populations from the Tarasca Mountain Chain, as well as the population ‘Compuesto de Familias’ from Oaxaca that segregated as an independent entity in the relationship study (Figures 3 and 4). This analysis can be seen as a preliminary group formation as it integrated groups with partial information (51.56%), but it helped to find the important traits differentiating the populations involved in the study.

Relationship analysis with morphological characters An arrangement of four major groups was identified at the Euclidian distance of 0.25 according to the CCC and Hotelling’s T 2 statistic procedures (Figure 3). The ‘Compuesto de Familias’ population from Oaxaca showed an independent origin in the dendrogram, and formed one group by itself (Group IV); this population turned out to be a different subrace from Chalquen˜o and not related to the populations of the Tarasca Mountain Chain in the study of Romero et al. (2002).

Figure 2. Principal component analysis of 39 native populations from the Tarasca Mountain Chain, Michoaca´n, Mexico and 19 checks.

319

Figure 3. Dendrogram through the UPGMA method for 39 native populations and 19 checks based on a matrix of Euclidian distances derived from 17 morphological traits.

320 Most of the NP (61.5%) were assembled into the biggest cluster built up by 27 populations and 2 checks (Group III), maintaining the Conico population (Zac-66) and the ancient populations Toluca Blanco (ASV-42 and ASV-48) as in the PCA.

Group II clustered 35.9% of NP, being consistent in assembling the same checks as in PCA with the addition of one Celaya (Gto-208) and one Chalquen˜o (Juchi 7o CSM). The PCA and Cluster analysis suggested that genetic material studied

Figure 4. Dendrogram through the UPGMA method for 39 native populations and 19 checks based on a matrix of Euclidian distances derived from the combined dataset of 17 morphological traits and 29 isozyme alleles.

321 can be differentiated into three main groups, one formed by the NP clearly differentiated from the checks, the second group with checks and the third one with populations representing a transition between the first and second groups.

Canonical discriminant analysis Squared Mahalanobis distances among the means of the three groups (from group one) were 42.9 and 106.7 for groups two and three, respectively. The eigenvalue for PC1 was 13.81 with a canonical correlation of 0.97 that explained 96.5%; the PC2: showed an eigenvalue of 0.5 with a canonical correlation of 0.58. The first Eigenvalue was the unique significant Eigenvalue (p=0.001), and can be interpreted in such a way that the means of the three groups were over a line (one dimension); thus PC2 did not have practical importance. The discriminant function classified correctly all groups, and the relative location of group means over the axis were 7.22 for group I, 0.88 for group II and – 3.10 for group III. The function discriminated among groups mainly by means of characters dmm, do, lg, wg, and ps5mz; thus, the typical landraces of the Tarasca Mountain Chain (Group III) had thinner ears (4.0 cm), thinner cobs (2.1 cm), shorter kernels (11.9 mm) with less kernel weight (296 mg/ kernel), so the ear weight was lower than in the checks (Table 6). The Group of checks (Group I) was characterized by having bigger ears: ear and cob diameter of 4.9 and 2.4 cm, respectively, and kernels of 14.5 mm in length and 0.358 mg in weight. Additionally, in the collection six different kernel colors could be seen (data not shown) that traditional farmers plant for different purposes. Some studies have suggested that the maize populations found in the Tarasca Mountain Chain belong to the Chalquen˜o race with adaptation to specific environmental conditions of the high valleys of Michoacan (Sa´nchez and Goodman 1992; Sa´nchez et al. 2000); but the NP complex from this region showed here a clear morphological differentiation from the Chalquen˜o complex. Based upon the CDA analysis some NP and some populations of the Chalquen˜o race had some traits with similar phenotypic expression (Table 6), mainly vegetative, but they differed significantly in some important characters, specially the ear; this

structure was 9 mm thinner in NP than in Chalquen˜o as a result of a reduced cob diameter, shorter kernels and lower ear weight. Some accessions from the Tarasca Mountain Chain have been differentiated as a regional genetic group from other populations from low lands of Michoacan and the Chalquen˜o race, suggesting genetic divergence (Romero et al. 2002). Ortega and Sa´nchez (1989) found that, based on morphological traits, two accessions from the Tarasca Mountain Chain, Michoacan were divergent from Chalquen˜o although something related, but when Euclidian distance was applied one accession grouped with the Conico race and the other one with the Chalquen˜o group; they concluded that there were enough differences with the other races to be considered different, probably derived from Chalquen˜o.

Relationship analysis through morphological and isozyme combined data The morphological characterization showed significant differences between NP and checks as well

Table 6. Morphological data of groups of populations assembled in the cluster analysis with 39 native populations from the Tarasca Mountain Chain and 18 checksa. Trait

Groups 1

Days to silk Plant height (cm) Ear height (cm) Number of leaves above the ear Leaf length (cm) Leaf width (cm) Tassel peduncle length (cm) Tassel branching part length (cm) Total tassel length (cm) Tassel branches number Ear length (cm) Ear diameter (cm) Cob diameter (cm) Kernel length (mm) Kernel width (mm) 100 kernel weight (g) Weight of five Ears (g) a

106.0 329.1 155.4 4.4 92.7 10.4 33.1 29.5 64.1 8.8 15.0 4.9 2.4 14.5 8.0 35.8 826.9

3b

2 a a a a a a a b a a a a a a a a a

105.6 339.4 167.5 4.3 92.2 10.2 31.1 32.1 66.2 9.1 16.2 4.4 2.1 13.3 8.4 33.0 712.7

a a a a a a a ab a a a b b b a a b

106.6 337.9 163.8 4.2 90.6 10.2 31.7 32.2 66.5 8.2 16.1 4.0 2.1 11.9 8.4 29.6 603.3

a a a a a a a a a a a c b c a b c

Means with different letters within files are statistically different Tukey (0.05). b Group of native populations.

322 as genetic variation (Table 4), so the analysis was complemented with isozyme markers in order to get more robust results and conclusions. The combined dataset of morphological and isozyme characters showed that the populations studied clustered into four groups and some independent populations. Groups were defined mainly by the populations’ provenance, and can be interpreted as integrated by the similarities that the gene pool share among populations. There was a clear differentiation among native populations and checks at a distance of 8.3 (Figure 4). Twenty one native populations clustered into a single group where no checks were included (Group A). Seven native populations from Paracho and two from Salvador Escalante showed an independent performance, explained by the individual genetic constitution (presence and frequency of some alleles). These represented approximately 77% of the total native populations genetically related (Figure 4). Results agree with Romero et al. (2002) in such a way that the populations of the region gathered as a group. All the native populations from Salvador Escalante included in the Group A gathered into a single subgroup. Group B was formed by five populations, two Chalquen˜o and one of the Celaya; again, native population ‘Chalca’ (ASV 90) from Erongarı´ cuaro clustered along with checks in this group; when this population was collected farmers pointed out that this population was introduced some time ago from Zacapu, Michoacan (a low Valley lying at a transitional region) nearby the Tarasca Mountain Chain (Figure 4). Group C included three checks, one Chalquen˜o population from Durango State and two Celaya populations from Guanajuato State, all of them located in the region of the Conico race. Group D included most of the Chalquen˜o populations from the region of the Mexico Central Plateau that includes the States of Quere´taro, Hidalgo and Mexico. Nine native populations were distributed in two of the three groups of checks; seven NP belong to Group D, being four of them from Nahuatzen municipality. Finally the checks Chalquen˜o Juchi 7o CSM from Mexico State, Chalquen˜o Col 6949 from Michoaca´n State, Conico Zac-66 from Zacatecas State and Compuesto Familias from Oaxaca State showed an independent performance.

Romero et al. (2002), using morphological analysis found close relationship among the Zac66 check of Conico race and a group of maize populations from the Tarasca Mountain Chain, that agree with our morphological analysis, but when isozyme data were involved, that check showed genetic unrelatedness at an 8.3 Euclidian distance and moved away from that group; those authors considered Compuesto Familias from Oaxaca State as a subrace of Chalquen˜o and did not found it related with populations from the Tarasca Mountain Chain. This situation was confirmed by our results. Personal communications from farmers provide evidence of occasional seed movement from the Chalco-Amecameca region, State of Mexico to the State of Michoacan via the Zacapu Valley region, where farmers introduce new seeds when their seed degenerates (Romero 2002). This seed movement could promote some gene flow between populations from the Central Mexico Plateau and populations from the transitional region, but surely to a lesser extent with the populations of the Tarasca region where a few non-local populations are tested by farmers. As it was observed, in the morphological analysis some landraces shared some phenotypic similarities with the Chalquen˜o race (Group II), but when isozyme data were involved the relationship somewhat diminished among groups (Group D and B). It is important to mention that the Chalquen˜o race is cultivated as the predominant race in Chalco, Amecameca, Juchitepec, Toluca-Atlacomulco region and Texcoco-Cuautitlan regio´n in the Mexican Central Plateau (Herrera 1999), there is an active seed movement among those regions through seed purchase and exchange associated to a relatively dynamic agricultural activity. There are remarkable morphological differences among native populations from the Tarasca Mountain Chain and the complex of the Chalquen˜o race, explained not only by differences of climatic and edaphic characteristic between regions (Table 7), but also by contrasting social and cultural factors prevailing in each region such as social organization, land property, agriculture labor organization and parallel economic activities that influence directly the prevalence of maize types; in addition, the rough orography has imposed natural barriers that contributes to genetic isolation.

323 The Tarasca Mountain Chain is a high-altitude land in the state of Michoacan, where the Tarasco indigenous community settled down; they are organized in little rural communities and have a communal land property, practicing agriculture based on family labor, a structure acquired before the Spanish colonization and maintained during the colonial period because of the geographic isolation (Escobar et al. 1996). Small valleys are used mainly for maize cultivation that is the most important economic and social activity, the scarcity of water in the region does not allow irrigation in most of the region, so the crop uses the water retained by the soil during the rainy season, little external inputs are used and the production is destined to the family subsistence, animal feed and the surplus is sold in the market. The seed is traditionally obtained from the last harvest and manually selected by the farmer (Maturana and Sa´nchez 1970). Many tests of non-local varieties introductions have failed so far in the region because they are not adapted to the environmental conditions. At the end of the cycle landraces are superior to the introduced ones and the non-local varieties are abandoned. One explanation to this phenomenon is the soil characteristics that significantly differ among the involved regions. The Tarasca Mountain Chain soil shows different important characteristics

like microcrystals that come from its volcanic origin. Physical and chemical characteristics of these soils are relevant in relation to the productivity of crops; for example, the acidic reaction ranges from strong to neutral (pH 4.9–6.9), the ample range of organic mater content (0.4–9.6) but irregular in depth, high capacity of cationic interchange (12– 75), with medium humidity retention. The phosphorus is highly retained reaching in some places up to 85% or higher, the phosphorous is less soluble as the soil pH is more acidic. The andisol soil with crystal characteristics shows low productivity as the result of low nutrient and water retention capacity (Alcala´ et al. 2001). In contrast, the soil of the eastern State of Mexico where the Chalquen˜o complex is widely cultivated is a broad, flat region that has a lakebottom origin, rich in clay, high content of siliceous amorphous material, and alkaline reaction; some are saline with sodium at the exchange sites. It has poor drainage and high pH values, ranging from 6.3 to 8.8 and low levels of organic mater from 0.07 to 2.17% with a high capacity of cationic interchange (21.8–50.1). The bad drainage of soil increases the salt concentration that move to the surface by capillarity (Segura et al. 2000). Gutie´rrez et al. (1998) found that the carbonate accumulation mainly has lacustrian origin and it is related with old beaches, where concentric nodules

Table 7. Geographic and climatic characteristics and soil properties of the Tarasca Mountain Chain and the Central Mexico Plateau Regions.

Altitude (m.a.s.l.) Latitude (N) Longitude (W) Rainfall (mm) Temperaturea (C) Soil characteristics Sand (%) Lime (%) Clay (%) Apparent Density (g cm 3) pH Organic Mater (%) Capacity of Cationic Interchange (cmol kg 1) a

Minimum and maximum value through the year. Escobar et al. (1996), INEGI-Me´xcio. c Herrera (1999), meteorological station of Tecamac, Mexico. d Alcala´ et al. (2001). e Segura et al. (2000). b

Tarasca Mountain Chainb

Central Mexico Plateauc

2100–2470 1924¢–1939¢ 10138¢–10215¢45¢¢ 862–1200 2–37

2240–2298 1916¢–1935¢ 9853¢–9855¢ 586–691 5–34

Tarasca Mountain Chaind 11–74 9–78 1–26 1–1.5 4.9–6.9 0.4–9.6 12–75

Central Mexico Plateaue 7–61 25–73 13–48 1.3–1.7 6.3–8.8 0.2–2.2 22–50

324 were formed. The carbonates are deposited as layers formed by accumulation of calcium during the process of dissolution and evaporation. This work reinforce the idea that the native populations of the Tarasca Mountain Chain are a different genetic group from the Chalquen˜o race as it can be seen in the divergence of populations in the relationship analysis (Figure 4). Taking into account that the maize domestication center is likely placed in Michoacan, Mexico (Miranda 2003), it could be hypothesized that Chalquen˜o comes from ancient native populations originally developed in western Mexico and then migrated to central Mexico where the Chalquen˜o race differentiation took place. This idea is supported by recent findings by Matsuoka et al. (2002) who concluded through a phylogenetic study using DNA simple sequence repeats that the basal origin of cultivated maize is the highland region of western Mexico.

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