Morphometric Analysis of Sunower (Helianthus annuus L.

) Achenes from Mexico and Eastern North America1

SOMAYEH S. TARIGHAT2, DAVID L. LENTZ*,2, STEPHEN F. MATTER2, ROBERT BYE3
2 3

AND

Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221 USA Jardn Botnico, Instituto de Biologa, Universidad Nacional Autnoma de Mxico, 04511 Mxico, DF, Mxico *Corresponding author; e-mail: david.lentz@uc.edu
Morphometric Analysis of Sunower (Helianthus annuus L.) Achenes from Mexico and Eastern North America. Sunower (Helianthus annuus L.) has played a major role in the evolution of agricultural systems in the Americas. The discovery of ancient domesticated remains from archaeological deposits in pre-Columbian Mexico offers new dimensions to widely accepted viewpoints on the domestication pattern of H. annuus. Although American sunower populations north of Mexico have been examined extensively, Mexican indigenous domesticated landraces have not been studied in any detail. In this study, we morphologically assessed wild and domesticated sunower achenes from Mexico and compared them to similar datasets from eastern North America. Additionally, we evaluated the utility of four computer-assisted shape measurements in discriminating between wild and domesticated sunower achenes (fruits) and compared variation in achene size among modern wild and cultivated populations from both Mexico and the U.S. We found that, of the shape parameters tested, none were informative in distinguishing wild achenes from domesticated varieties. Subsequent size analysis, using conventional parameters of length, width, and thickness, showed that modern wild populations from Mexico had smaller achenes compared to modern populations from eastern North America. Domesticated achenes unearthed from Mexican archaeological sites, however, were signicantly larger than the early domesticated specimens recovered from eastern North America. Our methodological results indicate that variation in archaeological sunower achenes is better described by conventional size parameters rather than computerized shape analysis. Anlisis Morfomtrico de Aquenios de Girasol (Helianthus annuus L.) de Mxico y Este de los Estados Unidos de Amrica. El girasol (Helianthus annuus L.) ha jugado un parte importante en la evolucin de los sistemas agrcolas en las Amricas. El descubrimiento de restos domsticos antiguos de yacimientos arqueolgicos en el Mxico precolombino ofrece nuevas dimensiones a los puntos de vista ampliamente aceptados en el patrn de domesticacin de H. annuus. Aunque las poblaciones de girasol de Mxico y Estados Unidos de Amrica (EUA) han sido examinadas ampliamente, los restos arqueolgicos domsticos de las razas indgenas de Mxico no han sido estudiados en detalle. En este estudio, se evaluaron morfolgicamente aquenios de girasol silvestre y domesticado de Mxico y se compararon con conjuntos de datos similares del este de EUA. Adems, se evalu la utilidad de cuatro mediciones de forma asistidas por computador en la discriminacin entre los aquenios (frutos) de girasol silvestre y domesticado adems la variacin en comparacin de tamao entre los aquenios modernos de poblaciones silvestres y cultivadas de Mxico y los EUA. Se encontr que de los parmetros probados ninguno fue determinante para distinguir entre los aquenios silvestres y las variedades domesticadas. El anlisis de tamao, utilizando por los parmetros convencionales de la longitud, anchura y espesor, mostr que las poblaciones modernas de Mxico presentan aquenios ms pequeos, en comparacin con las poblaciones modernas del Este de EUA. Sin embargo, los aquenios domesticados desenterrados de los sitios arqueol-

1 Received 19 April 2010; accepted 21 July 2011; published online 13 August 2011.

Economic Botany, 65(3), 2011, pp. 260270 2011, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

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gicos de Mxico fueron signicativamente ms grandes que los ejemplares domesticados y recuperados del Este de EUA. Nuestros resultados indican que la variacin metodolgica para aquenios de girasol arqueolgicos est mejor descrita por los parmetros de tamao convencional en lugar del anlisis de la forma informtica. Key Words: San Andres, Cueva del Gallo, shape factors, sunower, Helianthus annus.

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Introduction
Recent reports of pre-Columbian sunower (Helianthus annuus L.) in Mexico (Bye et al. 2009; Lentz et al. 2001, 2008a, b; Pope et al. 2000) have focused attention on sunower populations from that country. Unfortunately, very little is understood about sunower in Mexico, either wild or domesticated. This study is an effort to provide comparable morphometric data for assessment of modern and archaeological sunower populations from Mexico and eastern North America. Aside from its importance as one of the worlds major oilseed crops (Steffansson 2007), the study of sunower bears an evolutionary signicance to our understanding of ancient cultures of the pre-Columbian Americas. Moreover, the study of germplasm from warmer climes takes on an additional signicance as global warming and other aspects of climate change become manifest. Archaeological records of H. annuus consist mainly of achenes because these are the parts of the plant most likely to be preserved. Conventionally, achene size has been the key indicator of domestication in H. annuus and, through the study of modern wild and domesticated populations, achenes longer than 7 mm have been categorized as structures derived from domesticated plants (Heiser 1985). Determining whether an unearthed achene is domesticated or wild and dating these remains play a key role in reconstructing the domestication trajectory of H. annuus. Seed identication and classication coupled with the application of novel methods, which are faster and more reliable, have signicant economic and technical importance in studies of seed populations (Granitto et al. 2000). In this context, modern computerized shape analysis methods have added to the knowledge base developed through conventional size analysis. Image processing methods applied to plant morphology have been useful in distinguishing wild from domesticated varieties as well as crop seeds from contaminants (Zayas et al. 1989). Russ and Rovner (1989) utilized computerassisted image analysis of plant phytoliths to

discriminate between wild Zea varieties and domesticated maize. The main measured shape factors, including formfactor, roundness, solidity, curl, and aspect ratio, proved to be informative in distinguishing wild and domesticated maize populations. Despite the growing application of computers in morphometric approaches, there are not many examples of computer-assisted studies involving seed populations (Rovner and Gyulai 2006). Travis and Draper (1985) used an image processing system to distinguish crop seeds from contaminant weed species by measuring the size of the seeds and one shape parameter (formfactor). Their results indicated that ve of seven crop species could be separated from weed species based on these characteristics and that most of the weed seeds could be distinguished from each other using this system of measurements. Granitto and colleagues (2000, 2002, 2005) also evaluated computerassisted image processing methods examining morphometric (size and shape) characteristics of seeds and these techniques have been shown to be highly effective in automated weed seed identication. Rovner and Gyulai (2006) developed and applied new seed taxonomic approaches using computer-assisted image processing. These fresh approaches include measuring shape parameters such as formfactor, curl, convexity, and roundness, which cannot be obtained through manual methods. Heretofore, standard classication of seeds in sunower has been based on size parameters usually represented by a composite value called a size index (length x width). Computer-analyzed shape features of seeds, which may be informative in distinguishing wild seeds from domesticated varieties, have not been utilized in examining H. annuus. Developing new means of seed differentiation would enhance classication and identication endeavors in archaeological contexts as well as in modern seed taxonomy. Fast and reliable computer-facilitated approaches could be of great benet in this eld of scientic research. We sought to test the utility of modern image processing approaches by measuring and assessing

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four shape parameters (i.e., formfactor, curl, convexity, and roundness) to discriminate between wild and domesticated sunower achenes. We used a dataset that included numerous samples of Mexican indigenous cultivars and wild populations (Figs. 1 and 2) collected in Mexico during the course of a previous study (Lentz et al. 2008a, b). Furthermore, we compared size information from modern data to records of archaeological evidence from Mexico and eastern North America (ENA) in search of further insights into domestication patterns of this important crop plant.

Materials and Methods

Groups of seeds collected from Mexico and ENA, both wild and domesticated, were used for this study. ENA indigenous domesticated landraces were obtained from the United States Department of Agriculture (USDA). Mexican wild and indigenous domesticated landraces and ENA wild populations were collected by Lentz

and Bye during eld studies conducted from 2001 through 2005. To minimize the potential effect of introgression between cultivated sunowers with conspecic wild H. annuus populations on achene size (Rieseberg et al. 1999), diligent sampling was conducted by collectors, who selected only plants exhibiting wild phenotype characteristics (i.e., multiple slender pedicles and small ower heads; Doebley et al. 2006). Overall, seeds from 27 wild and domesticated populations including 14 wild (10 Mexican and 4 eastern North American) and 13 domesticated lines (8 Mexican and 5 eastern North American) were examined in this study (Fig. 1). A complete description of the Mexican sunower domesticates can be found in Tarighat (2010). For most of the populations, 100 seeds were measured, but for ve populations, only 40 seeds were available for measurement. Three principal size dimensions, namely length, width, and thickness, were measured using a Vernier caliper (accurate to 0.01 mm)

Fig. 1. Map of North America showing modern distribution of wild sunower (Lentz et al. 2008a; Heiser et al. 1969). Closed circles show the collection areas of modern wild H.annuus in Mexico and ENA. Stars indicate archaeological sites where domesticated sunower achenes were discovered. Three well-preserved achenes (330 250 B.C.E.) were unearthed at the Cuevo del Gallo site and a seed and achene (28752482 B.C.E.) were discovered at the San Andrs site. Two of the oldest ENA sites with clearly domesticated sunower remains were the Higgs site (1259829 B.C.E., Brewer 1973), and Marble Bluff (1264912 B.C.E., Fritz 1997).

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Fig. 2. Sample digital images of domesticated and wild Mexican sunowers set for computer-assisted image analysis. Images of multiple achenes were uploaded to the MATLAB image analysis program to obtain shape measurements.

for each individual seed. Due to the irregular shape of sunower seeds, the widest points for both length and width were recorded. A single value, size index = length (mm) x width (mm), was calculated for each seed. Shape factors were measured using an imageprocessing program (MATLAB version 7.4). Modern seeds were placed in groups of 50 seeds for domesticated and 100 seeds for wild populaTABLE 1.
Variable
Formfactor Convexity Curl Roundness

tions per image and randomly numbered by computer. Images of seeds of all populations, taken using a digital camera, were analyzed for four shape factors. Denitions of the shape features follow Russ (2007) and are provided in Table 1. Computer-assisted shape measurements were obtained using digital images of more than one object in an image. Color digital images were converted to binary (black and white) images, while an edge detection algorithm traced the exterior boundaries of each object using a threshold value. We optimized the threshold value for each image to get the best boundary detection. Parameters were then simultaneously measured in a two-dimensional binary image for each individual object. The MATLAB script developed for image analysis is available upon request from the corresponding author. We initiated the computer-assisted measurement process using four selected shape parameters: formfactor, roundness, convexity, and curl. Formfactor is the conguration of the perimeter relative to the area of an object. A perfect circle has the smallest ratio of perimeter to area and a formfactor value of 1. Departure from a true circle will result in a smaller formfactor value (Rovner and Gyulai 2006). Roundness is similar to formfactor but it is measured using the length (longest chord) of the objects rather than their perimeters. This property of roundness makes it more sensitive to how elongated the object is, whereas formfactor is more responsive to irregularity of the outline of the object. There are many examples where formfactor and roundness of a single object vary signicantly (Russ and Rovner 1989), suggesting that both parameters are important for a full evaluation of an object. Convexity is used to measure the degree of irregularity of an object, quantied by the ratio
2007).

DESCRIPTION AND FORMULA OF SHAPE FACTORS ASSESSED IN THIS STUDY (RUSS

Description
Departure of an objects perimeter from a smooth circle The ratio of a convex perimeter to perimeter Degree of departure of an object from a straight line Departure of an object from a circle

Formula
4 area/perimeter ; it is 1.0 for a perfect circle and diminishes for irregular shapes. Convex perimeter/perimeter. It diminishes if there are surface indentions. Length/*ber length 4 area/ x length2
2

*Fiber or skeleton of a shape is obtained by erosion (sequential removing) of edge pixels of that shape.

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of the true perimeter of the object to its tted convex hull. Curl is used to quantify the departure of an object from a straight line. Due to the signicantly high systematic error of measurement in sunower achenes when using the curl parameter and condent in the fact that curl provides the least discriminating power, we completed the analysis using only the remaining three feasible shape parameters: formfactor, convexity, and roundness. Assessing the utility of these three factors in discriminating between wild and domesticated seeds was one of our principal objectives in this study. Statistical analyses of the data were performed using JMP analysis software (version 7). Data points were inspected for normality and homogeneity of variance. For non-normal data we used a log10 transformation. To determine which continuous variables (shape or size features) could discriminate between the main sunower categories, our data sets were subdivided into four groups: ENA wild, Mexican wild, ENA indigenous domesticated, and Mexican indigenous domesticated. To distinguish among the achene categories we conducted a discriminant function
TABLE 2.

analysis which generates a set of discriminant functions based on linear combinations of continuous predictor variables. These variables provide the most effective ways to distinguish among the seed categories and thereby render a measure of the overall similarity among them (Rashed et al. 2008). Six measured characters included achene length, width, thickness, achene formfactor, roundness, and convexity, all of which were used in the analysis as predictors for membership in one of the two wild or one of the domesticated groups. To determine if a signicant difference existed among sunower populations of Mexico and eastern North America, discriminant function analysis (DFA) and analysis of variance (ANOVA) were performed on size measurements. Separate one-way ANOVA tests comparing the size index variable (length x width) within wild populations and domesticated populations were carried out. The results were compared to size differences between archaeological specimens unearthed from Mexico and ENA (Tables 2 and 3). In addition, we analyzed climatic data (average annual temperature, average annual rainfall, and elevation) of wild

AVERAGE SIZE INDEX (LENGTH X WIDTH) OF ARCHAEOLOGICAL DOMESTICATED REMAINS RECOVERED FROM MEXICO AND EASTERN NORTH AMERICA. BOTH HIGGS (BREWER 1973) AND HAYES (CRITES 1993) ARE EARLY ENA SITES WITH SUNFLOWER EVIDENCE, BUT HAVE NO ACHENES.

Site
Patrick, TN Rose Island, TN Newt Kash Hollow, KY Marble Bluff, AR (34-23-345) Marble Bluff, AR (34-23-327) Edens Bluff, AR Salts Cave, KY Salts Cave, KY (feces) Mammoth Cave, KY Cueva del Gallo, Mex. San Andrs, Mex.
*

Time Period
Early, Middle Woodland (318 B.C.E.C.E. 287)* Early, Middle Woodland (318 B.C.E.C.E. 287)* Late Archaic, Early Woodland (1162369 B.C.E.)* Late Archaic (1264912 B.C.E.) Late Archaic (1032920 B.C.E.) Early, Middle Woodland (170 B.C.E.C.E. 50) Early Woodland (654416 B.C.E.)* 57 Early Woodland (970660 B.C.E.)* Early Woodland (539239 B.C.E.)* Formative (330250 B.C.E.) Late Archaic (28752575 B.C.E.)

No. of Achenes
3 4 14 19 14 4

Index (LxW)
21.2(30.8) 17.8(25.3) 29.2 27.2(39.2) 24.5(35.2) 25.9

References
1 1 2, 3 4 4 5

1,000 80 3 1

17.4(24.4) 16.8(23.7) 15.1(21.7) 57.5 36.9(51.9)

6,7 6,7 2, 6 5 8

Converted from conventional radiocarbon dates to calibrated dates using Fairbanks calibration curve (Fairbanks et al. 2005). Numbers in parentheses represent values that were enhanced by correction factors (see Yarnell 1978). These are often used to compensate for achene shrinkage during carbonization. References: 1 = Chapman and Shea 1981; 2 = Watson and Yarnell 1966; 3 = Yarnell 1978; 4 = Fritz 1997; 5 = Lentz et al. 2008b; 6 = Yarnell 1981; 7 = Gardner 1987; 8 = Lentz et al. 2001.

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TABLE 3.

ACHENE SIZE COMPARISON OF ANCIENT AND MODERN POPULATIONS FROM MEXICO AND EASTERN NORTH AMERICA. MODERN SAMPLES WERE DERIVED FROM BOTH WILD AND DOMESTICATED POPULATIONS COLLECTED FROM TWO REGIONS OF STUDY.

Categories
Archaeological ENA Domesticated Mexico Domesticated Modern ENA Domesticated Mexico Domesticated ENA Wild Mexico Wild

Mean Size Index

28.38 54.7 71.62 64.06 12.61 8.6

SD
5.79 3.96 0.24 0.16 0.018 0.018

sunower locations using three single linear regression tests to investigate the relationship between climatic factors and achene size index. Climatic data at each collection site, including temperature, rainfall, and elevation, for 14 wild populations in the specic year of seed collection, were derived from records of the Servicio Meteorological Nacinal de Mxico (SMN) and the National Climatic Data Center (NCDC) of the U.S. National Oceanic and Atmospheric Administration (NOAA).

Results
Discriminant function analysis (DFA) indicated that size factors (length and width) contributed signicantly to the discriminant function and consistently maximized the difference between the two wild and domesticated groups (Wilks Lambda=0.06, F3, 2532 =11417.8, P<

0.0001) (Fig. 3). According to the DFA of shape factors, the morphological differences among populations were signicant (Wilks Lambda= 0.81, F3, 2532 =195.5, P=0.001); however, the difference between wild and domesticated achenes was much less than that seen using size factors (Fig. 4). Size measurements of modern wild and domesticated populations are presented in Table 4. Analysis of variance (ANOVA) results (Table 5) showed that the achenes of modern wild sunower from ENA (average size index=12.61) were signicantly larger than modern Mexican wild achenes (index size=8.6) (F1, 1338 =1258.8, P<0.0001). The average index size of indigenous domesticated seeds from Mexico (X 64:06) was smaller than the size of those from ENA (X 71:62) (F1, 1190 =21.4, P<0.0001). Additionally, our sunower populations showed high within-population variation in their achene size index (ANOVA; ENA wild F3, 396 =192.4, P< 0.0001; Mexican wild F9, 930 =138.7, P<0.0001). ANOVA results of archaeological sunower specimens, however, showed that Mexican domesticated achenes were signicantly larger than domesticated samples from ENA (F1, 9 =22.53, P=0.001) (Table 5). Linear regression analysis testing the association between climatic factors and achene size yielded nonsignicant results for rainfall (R2 = 0.071, P=0.35) and elevation (R2 =0.013, P= 0.69). Average annual temperature, on the other hand, was signicantly associated with size index (R2 =0.543, P=0.0026) in an inverse manner

Wild

Domesticated

Fig. 3. Discriminant function analysis (DFA) of size measurements. The left cluster represents the wild achenes grouped together, while domesticated achene populations formed a distinct group.

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Fig. 4. Discriminant function analysis (DFA) of shape parameters. No separation can be detected between the wild and domesticated achene types.

TABLE 4. SIZE MEASUREMENTS OF MODERN WILD AND DOMESTICATED SUNFLOWER ACHENE POPULATIONS FROM MEXICO AND EASTERN NORTH AMERICA.
Length (mm) Categories
Mexico Indigenous Domesticated

Width (mm) Max

5.0 7.6 6.1 8.0 5.5 7.7 8.6 7.2 10.3 9.0 5.2 9.1 11.3 2.8 2.5 2.3 2.5 2.9 2.5 2.4 2.5 2.1 2.1 2.7 2.8 3.1 3.0

Thickness (mm) Max

3.3 5.6 4.0 5.2 3.8 5.8 5.7 4.8 6.6 5.3 3.7 5.2 6.4 1.6 1.5 1.6 1.5 2.0 1.5 2.0 1.5 1.4 1.2 1.8 2.2 1.9 1.7

Name/ID No.
34110 34116 34111 34055 34109 33656 33655 34175 Mandan Seneca Hopi Dye Arikara Hidatsa San Luis Potosi Nuevo Leon Coahuila Tamaulipas Chihuahua Zacatecas Sonora Durango Guanajuato Veracruz Missouri Tennessee Arkansas Illinois

No.
100 100 100 100 100 40 52 100 100 100 100 100 100 100 100 100 56 100 100 84 100 100 100 100 100 100 100

Max
10.4 12.4 12.2 12.3 12.5 11.9 15.4 13.3 13.1 14.8 12.2 15.1 15.6 4.5 4.6 4.7 4.3 5.9 4.9 5.3 4.9 4.4 4.3 5.3 5.4 6.1 5.6

Mean
9.4 11.4 11.3 11.1 10.8 11.3 11.8 12.8 11.5 11.3 9.9 11.0 12.0 3.8 3.9 4.0 4.0 4.9 4.0 4.4 4.5 4.1 4.1 4.8 4.8 5.3 5.1

Min
8.1 10.4 10.0 9.1 8.6 10.0 8.9 11.9 9.1 8.0 8.4 7.1 10.0 2.7 3.1 3.3 3.6 4.2 2.9 3.3 3.9 3.7 3.8 4.1 4.3 4.5 4.3

Mean
4.3 6.5 5.3 5.8 4.8 6.7 6.8 6.4 8.3 5.7 4.2 5.9 7.5 2.0 1.9 1.8 2.3 2.4 2.2 2.0 2.2 1.9 1.9 2.3 2.4 2.8 2.6

Min
3.2 5.5 4.2 3.7 3.4 5.2 5.4 5.3 5.7 4.1 3.3 3.8 5.5 1.4 1.4 1.4 1.8 1.4 1.7 1.5 1.7 1.7 1.7 1.8 1.8 2.3 2.2

Mean
2.7 4.7 3.2 3.7 2.8 4.4 4.2 3.9 5.3 3.4 2.8 3.4 4.2 1.1 1.2 1.1 1.3 1.4 1.2 1.2 1.2 1.1 1.1 1.3 1.3 1.6 1.4

Min
1.7 3.5 2.0 2.2 2.0 2.8 2.3 3.0 3.6 1.9 1.9 2.4 3.2 0.5 0.8 0.8 0.8 0.9 0.7 0.7 0.8 0.9 0.9 0.9 0.9 1.3 1.1

ENA Indigenous Domesticated

Mexico Wild

ENA Wild

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TABLE 5.

ANOVA RESULTS OF ACHENE SIZES COMPARING MODERN WILD AND DOMESTICATED ACHENES COLLECTED IN MEXICO AND EASTERN NORTH AMERICA (ENA). THE LAST ROW SHOWS THE RESULTS COMPARING THE MEAN SIZE INDICES OF PRE-COLUMBIAN DOMESTICATED SPECIMENS DISCOVERED FROM MEXICO AND ARCHAEOLOGICAL DOMESTICATED SAMPLES COLLECTED FROM ENA.

Evaluations
Modern ENA Wild vs. Mexican Wild Modern ENA Dom. vs. Mexican Domesticated Ancient ENA Dom. vs. Mexican Domesticated

Error DF
1338 1194 9

MSE
3.31 392.6 47.3

F
1361.84 42.67 22.53

P-value
<0.0001 <0.0001 0.001

where increasing temperature was associated with smaller size index (Fig. 5). In the Mexican populations, higher values of average annual temperature were recorded for eastern and central Mexico where smaller achene size indices were observed (Table 6).

Discussion
Morphometric analysis of sunower achene populations provided a number of important insights into both modern populations and historical interpretations of this valuable crop plant. The computer-assisted evaluations of sunower achenes conducted using discriminant function analysis (DFA) revealed that shape analysis techniques were less effective than techniques commonly utilized in conventional methods to distinguish between wild and domesticated populations of sunower. The four shape parameters measured in this study (curl, roundness, convexity, and formfactor) did not bear sufcient recognition robustness to recommend
15 14 13

their use in establishing quantiable differences between wild and domesticated achenes. In sunower populations, the transition from wild to domesticated form is accompanied by dramatically altered achene size but, evidently, not the shape features evaluated in this study. Computer-assisted approaches may continue to offer cost-effective, rapid, and accurate methods for discriminating between seed populations if appropriate parameters are selected for the species or structures being evaluated, but the parameters tested in this study could not be successfully applied to sunower achenes. Technical enhancements in differentiation and classication of sunower achene populations should focus on comparative aspects that have been demonstrated to be effective at discriminating between wild and domesticated types (i.e., achene length or size index). Analysis of variance of achene size indicates that modern wild sunower populations from eastern North America (ENA) have signicantly

R2 = 0.54,P = 0.0026

Achene Size Index

12 11 10 9 8 7 10 12.5 15 17.5 20 22.5 25

Temperature
Fig. 5. Graph showing the inverse relationship between temperature and average size index in modern wild populations sampled from Mexico and eastern North America.

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TABLE 6.

RESULTS OF THREE SINGLE REGRESSION ANALYSES ASSESSING ASSOCIATION OF MEAN ACHENE SIZE INDEX WITH THREE CLIMATIC FACTORS IN MODERN WILD POPULATIONS.

Climatic Factor
Temperature Rainfall Elevation

R2
0.543 0.071 0.013

P-value
0.0026* 0.35 0.69

Relationship
Inverse None None

larger size indices than modern wild populations from Mexico. Wild sunower populations examined in this study showed high variability in their size indices both within and between populations. Our expectation was that this variation would be highly inuenced by climatic factors, but our results suggest a more complicated relationship. Regression analysis revealed no signicant association between size index and two climatic factors, rainfall and elevation, but indicated an inverse relationship between size and temperature. The largest wild achenes in Mexico were recorded for populations collected in the northern state of Chihuahua, where we found the coolest temperatures in the corresponding year. Temperature seems to be the important climatic factor affecting sunower achene size in North America. This observation makes sense in light of the fact that sunower is essentially a temperate plant and is well adapted to the climate of the middle latitudes. Accordingly, our results reect existing variation of achenes that largely can be attributed to the wild sunower genome and environmental factors. The observed pattern of size variation between modern wild populations becomes more intriguing when we consider them in relationship to the achene sizes from archaeological contexts. Based on available archaeological records of ancient domesticated H. annuus from both regions, ancient cultivated sunowers from Mexico produced achenes larger than the oldest H. annuus discovered in ENA (Lentz et al. 2008b). These observations raise provocative questions about the trajectory of crop development in the two regions and the cultural exchanges between ENA and Mesoamerica during pre-Columbian times. Pre-Columbian Mexico has been identied as a cradle of domestication for other important crop plants such as squash (Cucurbita pepo), which was grown in the highlands of Mexico in 10,000 B.P. (Smith 2006; Sanjur et al. 2002), and maize (Zea mays), which is believed to have been domes-

ticated in southern Mexico more than 6,300 years ago (Burger et al. 2008; Matsuoka et al. 2002; Piperno and Flannery 2001). It is reasonable to hypothesize, given the backdrop and long history of previous domestication processes in Mexico, that the occupants of early Mexico had the intellectual understanding, adequate chronological space, and the necessary wild germplasm available to complete the domestication process by 4600 B.P., the time of the rst domesticated sunower evidence from Mexico at the San Andres site. This position, however, has not been without controversy. Two domesticated sunower disseminules were recovered from San Andres: an achene and a seed. These disseminules came from different test pits, but dated to exactly the same time period. Heiser veried the identication of the San Andres achene soon after its discovery in 2000, but recanted his identication eight years later, long after the specimen had been consumed by the radiocarbon dating process (Heiser 2008a). The San Andres seed has attracted far less discussion. Our publication of the Cueva del Gallo sunower nds from Morelos, Mexico (Lentz et al. 2008a, b), engendered a spirited response (Brown 2008; Heiser 2008b; Rieseberg and Burke 2008; Smith 2008), but none of these commentaries seriously questioned the basic premise of the article, that the nds from central Mexico represented fully domesticated H. annuus achenes. These new data, therefore, reinforced our earlier contention that sunower was indeed a crop of the pre-Columbian Mesoamericans. It is our hope and expectation that future archaeological, paleoethnobotanical, and molecular studies in Mexico and ENA will help to clarify the relationship of the ancient sunower populations in these two regions.

Conclusion
In conclusion, the four shape parameters we evaluated (formfactor, roundness, convexity, and curl) were less effective than conventional methods in separating wild and domesticated sunower achenes despite their utility in other seed populations. Our assessment of achene size variation in modern wild sunowers showed that populations from Mexico had signicantly smaller achenes than populations from eastern North America. Observed size diversity in modern populations probably is a result of both genetic and environmental variation. Conversely, domesticated archaeological achenes discovered in Mexico were signicantly larger than early specimens recovered from eastern North

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America. Collectively these data will provide useful guideposts as more archaeological specimens of H. annuus from ENA and Mexico are unearthed.

Acknowledgments
This work was supported by the National Science Foundation (Grant Number BCS0228049), the National Geographic Society (Grant Number 7030-01), and a Faculty Research Support Grant from the University of Cincinnati. We thank Abbas Shirinifard from the Physics Department, Indiana University, for developing the MATLAB image processing script, an invaluable contribution to this project. We also thank Dr. Theresa Culley for her instrumental guidance. Kim Thompson and Edelmira Linares kindly offered editorial help.

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