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A Systematic Approach to Species–Level Identification of Chile Pepper (Capsicum spp.) Seeds: Establishing the Groundwork for Tracking the Domestication and Movement of Chile Peppers through the Americas and Beyond1 KATHERINE L. CHIOU* AND CHRISTINE A. HASTORF Department of Anthropology, University of California, Berkeley, CA, USA *Corresponding author; e-mail: [email protected]

A Systematic Approach to Species–Level Identification of Chile Pepper (Capsicum spp.) Seeds: Establishing the Groundwork for Tracking the Domestication and Movement of Chile Peppers through the Americas and Beyond The chile pepper (Capsicum spp.), a plant held in great esteem throughout history, was independently domesticated in a series of places including highland Bolivia, central Mexico, the Amazon, the Caribbean, and other locales with a particularly long history of cultivation and use in the central Andes of South America. Though identification of chile pepper species through fruit morphology is possible and has been utilized by botanists studying modern and archaeological specimens, species–level identification of Capsicum seeds has remained undetermined. Given the greater abundance of seed remains in the archaeological record due to the higher likelihood of preservation, the ability to identify specific Capsicum domesticates has profound implications for tracking the domestication and spread of chile peppers prehistorically through the Americas and historically through trade and exchange to the rest of the world. This article presents a systematic procedure to identify Capsicum seeds to the species level created by adopting a morphometric approach to compare attributes of modern Capsicum seeds to archaeological seeds. Un Procedimiento Sistemático para la Identificación de Diversas Especies Chiles/Ajíes (Capsicum spp.) por medio de Sus Semillas: Estableciendo una Base para Rastrear la Domesticación y Movimiento de los Chiles/Ajíes a través de las Américas y el Resto del Mundo El chile/ají (Capsicum spp. L.), una planta que goza de gran estima a lo largo de la historia de la humanidad, fue domesticado independientemente en una serie de diferentes lugares, incluyendo el altiplano boliviano, México central, la Amazonia y el Caribe. Aunque hoy en día es possible la identificación de diferentes especies de chile/ají a través de la morfología de la fruta, la identificación utilizando solamente la semilla permanece una tarea difícil. Dada la gran abundancia de semillas en el registro arqueológico, el desarollo de esta habilidad tiene profundas implicaciones para el estudio de la domesticación y difusión de chile/ají en las America precolombina y el resto del mundo. El presente artículo propone un procedimiento sistemático

1 Received 7 November 2013; accepted 14 August 2014; published online 16 September 2014.

Electronic supplementary material The online version of this article (doi:10.1007/s12231-014-9279-2) contains supplementary material, which is available to authorized users.

Economic Botany, 68(3), 2014 pp. 316–336 © 2014, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A.

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CHIOU & HASTORF: CAPSICUM SPP. SEED IDENTIFICATION

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para identificar especies de Capsicum adoptando un acercamiento morfométrico para comparer las semillas modernas con restos arqueológicos. Key Words: Archaeobotany, Capsicum, Chile peppers, Seed identification, Seed morphology, Ethnobotany.

An Introduction to the Genus Capsicum Chile peppers, a staple of many cuisines around the world today, have their origins in the Western Hemisphere. Prior to the 15th century, chile peppers were cultivated to varying extents from Chile to the American Southwest (Fig. 1). The chile pepper (Capsicum spp.) consists of about 25 species, five of which represent domesticated taxa (Andrews 1984; Basu and De 2003; Davenport 1970; Eshbaugh 1976, 1980; Eshbaugh et al. 1983; Heiser 1971; Heiser and Smith 1953; Naj 1992; Perry 2012). These taxa include C. annuum L., C. baccatum L. var. pendulum (Willd.) Eshbaugh, C. chinense Jacq., C. frutescens L., and C. pubescens Ruiz & Pavon. The general consensus among botanists is that the nuclear origin area for the Capsicum genus is in highland Bolivia on the eastern slopes, which is also the purported origin of the domesticated C. pubescens. From there, the wild Capsicum species radiated outward through the pre–Holocene Americas due to dispersal by birds and only much later by humans (Andrews 1984, 2006; Eshbaugh et al. 1983; Pickersgill 1977, 1988, 2009). The evidence suggests that there was a range of wild Capsicum species throughout Central and South America by the time people arrived in the area. C. baccatum is thought to have been domesticated in lowland Bolivia or coastal Peru, while C. chinense and C. frutescens may have more tropical roots in the northeastern Amazon (Aguilar–Meléndez 2006; Aguilar–Meléndez et al. 2009; Eshbaugh 2012; Hernández–Verdugo et al. 1999, 2001; Moses and Umaharan 2012; Perry and K. Flannery 2007; Pickersgill 1972). C. annuum, on the other hand, was domesticated in Mexico (Aguilar–Meléndez 2006; Kraft et al. 2014; Pickersgill 1972). While botanists have painted a picture of Capsicum domestication based on the modern distribution of wild Capsicum taxa in the Americas as well as the presence of preserved Capsicum fruits with calyx morphology intact from archaeological sites, the lack of certainty surrounding the identification of Capsicum seeds to species– level has hindered this effort of tracing Capsicum species domestication and their movements, with some proclaiming Capsicum seed identification to

the s pecies level impossible a nd others misidentifying seeds as the incorrect species (Andrews 1984; Pickersgill 1969; Towle 1961). Based on the research presented here, we argue the opposite. We believe that the analysis of both quantitative and qualitative traits of archaeological Capsicum seeds can indeed lead to species–level identification. Analyses conducted on modern and early Capsicum starches, for example, have suggested that Capsicum (in particular, C. baccatum, C. frutescens, and C. pubescens) starch grains have species–diagnostic morphotypes (Perry and K. Flannery 2007). In this article, we present the results of our morphometric attribute analysis of seeds of domesticated members of the genus Capsicum, which we believe will prove useful to scholars engaged in Capsicum research. We (1) recorded data for 27 qualitative and quantitative attributes of modern C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens, (2) determined which eight qualitative and quantitative attributes provided the greatest utility in Capsicum seed identification, and (3) developed a seed identification guide for chile peppers. Our research will aid in the study of the unique histories of various Capsicum domesticates that are reflected in seed morphology (Chiou and Hastorf 2012; Chiou et al. 2014).

BACKGROUND ON CAPSICUM RESEARCH Capsicum spp. was first encountered by the European explorers in the late 15th century during a quest for expanding the spice trade by Columbus and his men on the West Indian island of Hispaniola. There, they encountered a plant that the native Arawak Indians called axí/ají. Like many of the explorers of the era, they were in search of a route to the spices of Asia, and in particular, black pepper (Piper nigrum L.). Upon encountering ají, the Spanish named it pimiento (pepper) as its flavor and spiciness reminded them of the Asian pepper. The chile portion of the common name derives from Nahuatl (chīlli). Across the Americas, there are many names for the chile pepper such as uchu in Quechua or huayca in Aymara. As with many of our continental plants, the Nahuatl or Caribbean words have dominated (Andrews 1984).

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Fig. 1. Distribution of Capsicum species at European contact ca. AD 1530 (redrawn from Andrews 1984; Heiser 1976; McLeod et al. 1982).

Chile peppers have long held a position of great esteem in the New World. Indeed, chile peppers were used in pre–Columbian times as an essential ingredient in the preparation of dishes (such as the Aztec chocolatl, a concoction made from ground cacao beans and flavored with vanilla and chile), serving as what one might call a “signature food” (Gasser and Kwiatkowski 1991; McNeil 2006; Perry 2012). Much like rice among the Japanese,

the chile pepper had close ties to notions of identity and self in the Americas (Ohnuki–Tierney 1993). In the Andes of South America, we know that certain cultivars of chile pepper are restricted to specific regions (i.e., highland, coast, and jungle), suggesting the possibility that different groups of people identified with these distinct species and used the different species for different dishes. Among the Nasca, for example, chile peppers are

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the second most depicted plant in Nasca ceramic iconography, commonly shown attached to mythical beings (Proulx 2009). Early Evidence for Chile Pepper in the Americas In terms of early chile pepper evidence, Capsicum seeds have purportedly been found in deposits dating to as far back as 10,000 B.P. at Guitarrero Cave located in modern–day Peru, though this date is disputed since Phaseolus remains from the site have been directly dated to around 3000–4000 B.P. (Kaplan and Lynch 1999; Pearsall 2008). Starch grains of Capsicum have also been recovered dating back to 6000 B.P. from Real Alto and Loma Alta in Ecuador (Perry and K. Flannery 2007). Indirectly dated Capsicum seeds from early deposits at Puebla in the Tehuacán Valley and Tamaulipas (Ocampo caves) of Mexico suggested a date of 9000–7000 B.P., though subsequent AMS dating of maize remains from Tehuacán have yielded a more recent age at 5600 cal. B.P. (Kraft et al. 2014; Long et al. 1989; Mangelsdorf et al. 1965; McClung de Tapia 1992; Smith 1967, 1987). Similarly, AMS dating of bottle gourd and squash from Ocampo have been dated to 6400– 6000 cal. B.P. (Kraft et al. 2014; Smith 1997). Overall, the literature claims that the chile pepper was cultivated by at least 7000–9000 B.P. (Chiou and Hastorf 2012; Kraft et al. 2014; Perry 2012; Pickersgill 1969). Recent research at the sites of Huaca Prieta and Paredones have pushed this date back further, with a directly dated Capsicum seed from the pre–mound occupational levels at Paredones radiocarbon–dated to 9330 +/– 40 cal. B.P. (Beta–343109: 10,430–10,650 cal. B.P., Chiou et al. 2014). Local and Global Perspectives on Capsicum through Space and Time The domestication of the Capsicum genus and its various species has been the subject of a fair amount of botanical work that has greatly informed our own research. According to Barbara Pickersgill, domesticated species of Capsicum are somewhat distinct and difficult to cross, producing sterile hybrids even when fertilization is successful, though this applies less so to the “annuum” complex that includes C. annuum, C. chinense, and C. frutescens (Pickersgill 1972; Eshbaugh 2012). Thus, cultivated species of Capsicum had distinct wild ancestors

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and were characteristic of different areas in pre– Conquest times (Smith and Heiser 1957). Given these traits, the Capsicum genus serves as a potential proxy for studying human interactions in the Americas, as “[g] enera in which several species have been domesticated may thus be useful indicators of cultural contact if the place of domestication of the individual species is accurately known and if the archaeological material can be assigned with certainty to a particular species or group” (Pickersgill 1972:99). Aguilar–Meléndez and colleagues’ genetic work (2009) has revealed that there are four major subspecies of C. annuum in Mexico, with the one in the Yucatan Peninsula being spatially discrete today; in the west and north, the other three overlap. These sub–species variants aid us in visualizing earlier local engagement with the wild species as well as the fluidity of the three sub–species in exchange and sharing. From ethnographic, ethnohistoric, and archaeological evidence, we know that chile peppers have been used in specific recipes for at least 5,000 years. Nevertheless, most of these varieties are quite localized, suggesting quite a strong belief in the quality of one’s local variety (Aguilar– Meléndez 2006; Hastorf 1998; Hugh–Jones 2001). Given that the presence or absence of different Capsicum species can reveal valuable information about cultural contacts, the ability to identify Capsicum plant parts, especially seeds given their common presence in the archaeological record, is crucial. The identification of Capsicum seeds in the archaeological record up to this point has remained somewhat unsystematic. Margaret Towle, for example, reported that the majority of Capsicum remains reported from coastal, pre–Columbian Peru were C. annuum, which presents an unlikely scenario given that there are at least two species of Capsicum (C. baccatum and C. chinense) that are native to the central and western Andes (Towle 1961). Subsequent research has suggested that C. annuum was relatively restricted to Mexico and Central America in the pre–Colombian times. Even though wild progenitors to C. annuum exist from Mexico to Colombia, genetic studies have shown that the most parsimonious scenario for C. annuum domestication is in Mexico, since all C. annuum plants have two pairs of acromere chromosomes, while most wild taxa have one except for wild species in Mexico (Aguilar–Meléndez 2006; Pickersgill 1972). Furthermore, at Huaca Prieta, previous work on Capsicum has revealed the presence of C. baccatum or C. chinense by the Late Preceramic (Pickersgill 1969). Pickersgill identified

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Capsicum pod remains housed in Margaret Towle’s collection at the Harvard Botanical Museum that were recovered from Junius Bird’s excavations in 1947–1948 (Bird 1985). These identifications were made based on calyx morphology (Pickersgill 1969). While most of her discussion centered on chile pepper pods, Pickersgill reported the range of diameters for the seeds that were also recovered. While these data may be useful for arriving at a general sense of seed size, they do not lend very much insight to species–level identification. After our review of the literature, we determined that the best course of action would be to complete a targeted study on seeds of all the domesticated chile pepper species to learn if they have the potential to be uniquely identifiable.

Project Overview and Goals This project was conceived after chile pepper seeds identified as Capsicum spp. from the Preceramic archaeological sites of Huaca Prieta and Paredones in the Chicama Valley of the desert North Coast of Peru and recovered through flotation of sediment samples during excavations led by Tom Dillehay of Vanderbilt University and Duccio Bonavia of the Academia Nacional de la Historia were sent to the McCown Archaeobotany Laboratory at the University of California, Berkeley for analysis. In attempting to analyze this rich data set spanning 4,000 years, which included a vast number of Capsicum seeds that appeared to display some variation, we realized that a systematic analysis of Capsicum seeds was essential to understand what was occurring at this site, which has some of the earliest evidence of agriculture in South America. Given the sparse nature of the literature on Capsicum seed identification (with some notable exceptions, such as Gunn and Gaffney 1974, which includes descriptions concerning seed coat, general seed dimensions, and the embryo of C. frutescens and C. annuum, Martin (1946), which includes drawing and description of the curved, linear C. baccatum embryo in comparison with other members of the Solanaceae family, and Minnis and Whalen (2010), which includes description of the first cultivated C. annuum chile pepper seed found in northwest Mexico/the American Southwest), we decided to record both qualitative and quantitative attributes of modern seeds we obtained from various sources listed in Appendix 1 (Electronic Supplementary

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Material, ESM), drawing inspiration from Maria Bruno, Christine Hastorf, and BrieAnna Langlie’s previous work on Andean Chenopodium (Bruno 2006; Bruno and Whitehead 2003; Langlie et al. 2011). Our goal was to determine diagnostic traits of modern Capsicum seeds that could be applied to our archaeological analysis, the results of which are presented elsewhere (see Chiou et al. 2014).

Methods for Modern Capsicum Seed Identification Studying a comparative collection of modern Capsicum seeds from all five domesticated species was crucial to the goals of this project, allowing us to determine diagnostic attributes from well–identified seeds that could be used to identify archaeological seeds. To that end, we amassed 44 distinct seed collections representing several examples of each of the domestic species—C. annuum, C. baccatum, C. chinense, C. frutescens, and C. pubescens—from different sources including vendors specializing in chile pepper cultivation and the USDA National Plant Germplasm System/Germplasm Resources Information Network (Appendix 1, ESM). These seeds were photographed using an Olympus SZ–61 stereomicroscope (10x–30x) and an Olympus digital camera (model DP72) housed in the McCown Archaeobotany Laboratory. Close–up scanning electron microscopy (SEM) images of the testa were taken using a Hitachi TM–1000 located in the Robert D. Ogg Electron Microscopy Laboratory on the UC Berkeley campus. The Olympus MicroSuite program was used to take various measurements of the whole seed, the attachment scar, and the testa in the transverse cross–section. Qualitative assessments were also made of the seed shape and testa texture. Initially, we identified and recorded 27 attributes for each of the 44 Capsicum seed collections that are listed and defined in Fig. 2. These attributes were selected based on the limited Capsicum literature (e.g., length and width measurements in Pickersgill 1969; Gunn and Gaffney 1974), previous research experience with seed morphology, and observations that were made concerning the nature of Capsicum seeds themselves (i.e., variation in “beak” length relative to the seed body, varying testa thickness at seed margins, and differences regarding the attachment scar).

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Fig. 2.


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Definitions of seed attributes analyzed in this study. Starred attributes are diagnostic.

Analysis After we recorded measurements for 27 attributes for each modern Capsicum seed species and variety, we ran a series of exploratory data analyses to seek the most productive differentiating measurements for seed identification, such as plotting two attributes against each other (x by y) and generating scatter plot matrices with multiple variables. From these plots, we determined that six quantitative attributes combined to form diagnostic identifications of Capsicum seeds to species–level. Combined with our qualitative attributes, we now have eight diagnostic characteristics. The two qualitative/ nominal attributes are seed shape and testa texture. Our six quantitative attributes are (1) the ratio of maximum seed length to perpendicular width, (2) beak angle, (3) beak prominence, (4) whole seed sphericity, (5) the ratio of the thickest portion of the testa to the thinnest portion, and (6) attachment

scar sphericity. These eight diagnostic attributes are defined below.

DEFINITIONS Seed Shape The general shape of Capsicum seeds is relatively distinct from species to species with some overlap. The range of seed shapes are drawn in Fig. 3 with archaeological correlations from Huaca Prieta and Paredones included. Testa Texture Testa texture refers to the appearance of the seed coat (Fig. 4). Reticulation is defined by a 3–D netted pattern on the seed surface smooth to broad reticulation (A) refers to small curved undulations of troughs and ridges in the testa surface. Tight

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Fig. 3. Capsicum seed shape categories (including body and attachment morphology) based on modern and archaeological seed data.

reticulation (B) refers tightly concentrated ridges and dramatic (C) refers to very large and pronounced ridges, particularly at the outer margins. The texture of the seed coat is diagnostic especially in the case of C. pubescens, which displays an exaggerated reticulation pattern on the outer margins of the seed as well as does C. baccatum, which exhibits a tighter reticulation pattern (as opposed to the other species that tend to have a smooth surface). Maximum Length: Perpendicular Width The length and width measurements are illustrated in Fig. 5. The length measurement was taken from the beak and the width measurement was taken perpendicular to the length measurement. All seeds were measured in the same manner (see Fig. 5A). This measurement was more diagnostic than measuring without the beak (placing the seed

in an upright position and measuring on vertical and horizontal axes). Beak Angle The beak is defined as the protruding area of the seed that differentiates Capsicum seeds from other similar–looking seeds of the family Solanaceae (Gunn and Gaffney 1974; Minnis and Whalen 2010). Figure 5 illustrates the morphometrics taken with the whole seed and depicts how beak angle was measured. The beak angle (Fig. 5B) gives us a sense of how much the beak diverges from the rest of the body. A high beak angle, for example, is one of the diagnostic attributes for C. frutescens seeds. Beak Prominence Beak prominence is depicted in Fig. 6 and refers to the extent to which the beak protrudes from the

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Fig. 4. Testa texture categories of the five Capsicum domesticates: A) smooth to broad reticulation B) tight reticulation and C) dramatic reticulation.

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Fig. 5. Seed morphometrics including (A) maximum length (measured from the beak): perpendicular width and (B) beak angle (measured from vertical beak edge).

rest of the seed body and is an ordinal scale based on a rank: 1 refers to no protrusion and 5 is considered prominently protruding. Whole Seed Sphericity Sphericity refers to how spherical (or in the two– dimensional sense, circular) a shape is and was calculated using the Olympus MicroSuite program. Photos such as those in Fig. 7 show examples of five modern taxa with several morphometrics. Note the sphericity measurement and the general shape of the seed associated with each one.

directed selection pressure during domestication (Bruno 2006; Bruno and Whitehead 2003; Flannery 1973; Fritz and Smith 1988; Smith 2006). Noticing a great amount of variation in the measurements of the testa (especially at the outer margins), we decided to calculate a ratio of the thick testa to the thin testa and trace that measurement. Three measurements are taken for each of the thick and thin testa areas, averaged and compared in a ratio (Fig. 8). We found this to be a useful measurement, as there is a range of these ratio values. C. annuum has a small thick to thin testa ratio (3.44) whereas C. pubescens displays the greatest thick to thin testa ratio (5.92).

Ratio of Thick Testa to Thin Testa Attachment Scar Sphericity We made a transverse cross–section cut of each seed to measure testa thickness, often an important measurement in studying domestication, as testas tend to get thinner as a result of

Fig. 6.

The attachment scar or hilum refers to the area that is attached to the placental wall of the chile pepper fruit. We noticed that the shape of the

Beak prominence ranking scale (from no beak [1] to extremely prominent vertical beak [5]).

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Fig. 7.

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Comparative examples of morphometric data for the five domesticated Capsicum taxa.

attachment scar varied and decided to measure sphericity of the attachment scar shape (Fig. 9).

PLOTTING THE DISCRETE DIAGNOSTIC ATTRIBUTES Discriminant analysis is useful in investigating differences among multiple groups, indicating which variables contribute most to group separation and is often used in ecological, biological, paleontological, and archaeobotanical analyses involving morphometrics (Hammer and Harper 2006;

Pearsall et al. 1995; Strauss 2010; Zhao et al. 1998). It is appropriate for datasets with continuous outcome variables and a priori established groups that are based on extrinsic criteria and when applied, classifies objects into groups by analyzing the relationships between variables of the objects and the boundaries defined in terms of these variables. In our study, we conducted linear discriminant analysis (LDA) on the modern Capsicum seed data set using our six diagnostic, quantitative attributes in JMP 7 (JMP. Version 7 1989, Perry et al. 2007). In Fig. 10, it appears that all five species separate

Fig. 8. Transverse cross–section morphometrics of the testa. Three measurements were taken of the thin and thick portions of the seed margin and averaged. A ratio was generated consisting of thick testa average: thin testa average.

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Fig. 9. Attachment scar morphometrics: (A) attachment scar width: attachment scar length and (B) attachment scar area.

completely. The two canonical axes explain 96% of the variance, and the circles in the figure represent 95% confidence intervals on group centroid means. The lines radiating outwards are the correlation coefficients between the first two axes and each of the six variables. For instance, the line representing the variable Beak Angle shows that this variable is positively correlated with the first canonical axis. Overall, the model does an excellent job of

Fig. 10.

attributes.

achieving classification. It must be noted, however, that low sample counts in some of the groups affect the discriminant analysis, ultimately overemphasizing group differences. We expect to see increasing overlap among the groups as we collect more modern seed data. Nevertheless, the discriminant functions only misclassified one seed or 2.22% of the sample (Table 1, Table 2). It is interesting to note that, save for one seed (a

Discriminant analysis of seeds from the five domesticated Capsicum taxa with six quantitative diagnostic

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TABLE 1. EIGENVALUES OF CANONICAL AXES.

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Eigenvalue

Percent

Cumulative percent

Canonical correlation

9.92980329 3.01528713 0.48254802 0.04030466

73.7292 22.3886 3.5829 0.2993

73.7292 96.1178 99.7007 100.0000

0.95315636 0.86657476 0.57051345 0.19683275

C. annuum in the C. chinense group), C. chinense, C. annuum, and C. frutescens are somewhat separate. This trio is closely related geographically and genetically, forming the “annuum complex,” one of the three Capsicum groups with the other groups named the “baccatum complex” and the “pubescens complex” (Eshbaugh et al. 1983; Eshbaugh 2012; Jensen et al. 1979; McLeod et al. 1979; Stommel and Albrecht 2012). Sexual crosses are possible among species within a complex with varying degrees of difficulty. The taxonomy of the “annuum complex” is a subject of debate, with some opposition to the splitting of C. annuum, C. chinense, and C. frutescens into three distinct species (Eshbaugh 2012). It is worth noting that the “pubescens complex” is the most isolated of the three groups (Stommel and Albrecht 2012). Our data suggest that despite this close relationship, morphological distinctions still exist. From our study of the five modern domestic Capsicum taxa, we believe that measuring the six attributes we have chosen is sufficient to identify both modern and archaeological whole domestic Capsicum seeds.

Discussion Our morphometric attributes revolve around measurements of the whole seed, the attachment scar, and the testa in cross–section. Measurements of the whole seed include that of the traditional length and width measurements and beak angle, along with other measurements easily calculated by Microsuite and defined in Table 3 (see Fig. 5 for length, width, and beak angle measurements). Figure 9A and B

TABLE 2. COUNTS: ACTUAL ROWS BY PREDICTED COLUMNS. Annuum Baccatum Chinense Frutescens Pubescens

exhibit the attachment scar measurements we recorded that included length, width, and area (as well as sphericity of the attachment scar shape). The measurements taken on the transverse cross section of each Capsicum seed is illustrated in Fig. 5, while Fig. 6 illustrates the ranking scale we used to rank beak protrusion. All 27 attributes were recorded for the 44 modern seeds in our study sample. Figure 7 exhibits a selection of five seeds, one from each modern taxon with their beak angle, maximum length: perpendicular width, beak prominence, and whole seed sphericity data noted for a sense of the variation among species in regards to the seed in its entirety. As is evident in Fig. 7, C. baccatum displays the most prominent beak, with C. pubescens generally lacking or having very little beak protrusion. Furthermore, C. chinense tends to be the most circular in shape, which explains its higher sphericity value. Furthermore, while the beak of C. baccatum tends to protrude straight up on the right side of the side at an angle of near 0°, C. chinense, C. frutescens, and C. annuum have much higher beak angles. Figure 11 shows the variation in attachment scar shape among the five different Capsicum seeds as well as their sphericity. On average, C. baccatum tends to have the most linear–shaped attachment scar with a sphericity of 67°). Its testa texture is generally smooth, much like C. chinense and C. pubescens.

Identifying Capsicum pubescens Seeds C. pubescens is easily distinguished in modern seeds by its black seed color while all the others are naturally yellow or tan. Its shape generally resembles that of an oval or the letter “D.” C. pubescens seeds are also characterized by very thick margins and generally have the greatest thick testa to thin testa ratio. The surface of C. pubescens seeds is also highly reticulated, with uniquely dramatic reticulation around the seed margin. Furthermore, C. pubescens displays little to no beak protrusion.

Conclusion From the data presented above, we determined that Capsicum seed identification is possible using the six quantitative and two qualitative attributes discussed above. As can be seen in Fig. 10, the species groups remain mostly separate, even among the members in the C. annuum–C. chinense–C. frutescens complex, which some have argued should be subsumed under a single species (Eshbaugh 2012). Though our research does not necessarily support the

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validity of these separate taxonomic designations, it is worth noting from an archaeobotanist’s perspective that seeds from these taxa are less distinctive looking than C. baccatum and C. chinense. It is also worth adding that when applying our identification guide developed from modern, dried chile pepper seeds to seeds recovered from archaeological contexts, there are other issues to contend with, such as preservation of diagnostic parts of the seed, taphonomic processes such as carbonization, and seed browning (Chiou and Hastorf 2012; Boonsiri et al. 2007; Lee et al. 1991; Minnis and Whalen 2010). Minnis and Whalen (2010), for example, experimented with the effects of charring (the state in which most archaeobotanical macroremains are preserved outside of areas with unique preservation conditions such as deserts) on Capsicum spp. seeds and found that the variation in size change was 10% to 69%, with cultivated seeds averaging a 27% decrease in seed size and wild seeds averaging a 22% decrease in seed size. Thus, for those considering application of our identification guide to archaeobotanical material, it would be worth keeping those factors in mind. This development of an identification system for Capsicum seeds will allow for archaeologists, archaeobotanists, and botanists to identify well– preserved archaeological seeds to species–level. Using this method, archaeologists can pursue such Capsicum–related topics as determining how selective pressures on chile peppers shifted through time, how specific cultivars of Capsicum moved from the origin of domestication to other regions, and determining the path of chile peppers around the world following the arrival of Europeans in the Americas. In terms of future work, we will continue adding data from seeds of modern domesticates to enrich our data base as well as incorporate seeds from wild Capsicum species such as C. baccatum var. baccatum, C. baccatum var. praetermissum, C. eximium, C. tovarii, C. galapagoense, C. chacoense, etc. Data from these wild taxa could help untangle the complex story of the chile pepper domestication. Acknowledgments We would like to thank Professor Tom D. Dillehay for initiating our work on Capsicum. Araceli Aguilar–Meléndez was the original inspiration for a systematic study of these plants. We acknowledge Dr. Guanwei Min’s training and advice concerning SEM imaging of the Capsicum seeds. Special thanks to the USDA Germplasm

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Resources Information Network (USDA–GRIN) and especially Dr. Mark Bohning, Dr. Bob Jarret, and Tiffany Fields for helping us find rarer specimens of Capsicum. Our chile pepper seed vendors, and in particular Beth Boyd from Bayou Traders, worked with us to obtain various Capsicum species. We also thank our peers in the McCown Archaeobotany Laboratory, especially Alan Farahani, Rob Cuthrell, and Theresa Molino for offering advice and critical insight into our project. Alan Farahani, in particular, assisted us with statistics and commented on our drafts. We would especially like to acknowledge the helpful comments from our anonymous reviewers contacted by Economic Botany who aided in improving our manuscript.

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