Evolutionary history of the chili pepper Capsicum baccatum L. (Solanaceae): domestication in South America and natural diversification in the Seasonally Dry Tropical Forests

Evolutionary history of the chili pepper Capsicum baccatum L. (Solanaceae): domestication in... Abstract Plant domestication genetics has been of interest not only for evolutionary biologists but also for anthropologists and breeders, because of its past and present role in human cultural evolution. Capsicum baccatum, which grows in the Seasonally Dry Tropical Forests of South America (SDTF), was domesticated by native peoples and used as a spice. The centre of domestication has been discussed, and vast territories of Peru and Bolivia have been proposed. The aims of this research were to elucidate the geographical origin of cultivated C. baccatum and to infer geological and climate events that have influenced the distribution of its genetic variability. Samples were collected from 25 localities across the entire range of wild C. baccatum and analysed using DNA sequences and phylogeographical approaches; they were then compared with 20 samples of cultivated forms obtained from different South American markets. We found a main centre of C. baccatum domestication spanning Bolivian Amazonia and the inter-Andean valleys. We also inferred an ancient cultivation site between Bolivia and Argentina. Finally, we found two lineages of wild populations distributed in nuclei of SDTFs which have been fragmented during glacial periods, when aridity increased and expansion of Chaco vegetation fragmented the region. Capsicum baccatum, chloroplast DNA sequences, crop origin, haplotype network, molecular dating, Solanaceae, South America INTRODUCTION The domestication of plants is one of the most significant cultural and evolutionary transitions in human history. Investigating when, where and how domestication took place is of interest to crop evolutionists and of practical importance for plant breeders and conservationists. Research into the domestication and spatio-temporal origins of this process has increased significantly over the past decade through archaeological research, advances in DNA/RNA sequencing techniques, and methods used to recover and formally identify those changes in interactions among plants and animals that lead to domestication (Larson et al., 2014). Genetic studies that examine the geographical origins of a crop typically rely on genome-wide neutral markers, which are used to assess allele frequencies in a crop compared with populations of its wild relatives. Due to the strong genetic bottlenecks that occur during the domestication process, allelic diversity in the crop is expected to be a subset of that found in the wild population(s) from which it was derived. Thus, if populations of the wild progenitor are extant in the geographical location(s) where domestication occurred, the geographical origin(s) of domestication can potentially be pinpointed to a particular population or region (Gross & Olsen, 2010). One of the most successful applications of phylogeographical analysis has been the investigation of sites of origin of domesticated crops (Londo et al., 2007). Phylogeographical studies draw inferences about the history of population divergence based on associations between the geographical distribution of alleles and their genealogical relationships (Avise, 2000). The number of domestication events and regions in cultivated rice and peanuts was determined using a phylogeographical approach based on DNA sequence variation (Londo et al., 2007; Grabiele et al., 2012). In addition, such approaches have made a significant contribution to the interpretation of species in response to climate and geological changes, such as the glaciation events of the Neogene (Turchetto-Zolet et al., 2013; Daneck, Fér & Marhold, 2016; Zhang, Zhang & Sanderson, 2016). Capsicum baccatum L. is one of the most economically important species of the genus, with three varieties, the wild C. baccatum var. baccatum, and two [var. pendulum (Willd.) Eshbaugh and var. umbilicatum (Vell.) Hunz. and Barboza] varieties, cultivated commercially mainly in South America (Pickersgill, 1997; Djian-Caporalino et al., 2007; Peralta et al., 2008). The wild form of the species typically bears small, erect, deciduous fruits (Fig. 1A; D’Arcy & Eshbaugh, 1974; Barboza, 2013), whereas the domesticated forms usually bear larger, pendant or bell-shaped, persistent fruits (Fig. 1B–D). The fruits are used as a spice in regional cuisine, although they are also of interest for a wide range of other applications, such as a natural colouring agent, an ingredient in pharmaceuticals (as a rubefacient and stomachic), and a highly pungent extract used in self-defence sprays, animal repellents and insecticides (Bosland, 1996). The wild form of the species is distributed in South America, from Colombia, Peru, Bolivia, Brazil and Paraguay to north-west and north-east Argentina (Moscone et al., 2007; Barboza, 2013), and is recognized under various common names: ‘cumbari’, ‘ají quitucho’, ‘quitucho’ and ‘ají del monte’. Figure 1. View largeDownload slide Fruits of Capsicum baccatum. A, C. baccatum var. baccatum SVL. B, C. baccatum var. pendulum 642. C, C. baccatum var. pendulum 215. D, C. baccatum var. umbilicatum 063. E, C. baccatum ‘became wild’ PRF. Scale bar = 2 cm. Figure 1. View largeDownload slide Fruits of Capsicum baccatum. A, C. baccatum var. baccatum SVL. B, C. baccatum var. pendulum 642. C, C. baccatum var. pendulum 215. D, C. baccatum var. umbilicatum 063. E, C. baccatum ‘became wild’ PRF. Scale bar = 2 cm. Starch microfossils of C. baccatum in Peru date from 4000 years BP to European contact, indicating an early use of domesticated peppers in ancient America (Perry et al., 2007). Based on combinations of archaeological evidence and genetic analyses, researchers suggested that the centre of domestication of C. baccatum was the lowlands of Bolivia (Pickersgill, 1984; Eshbaugh, 1993; Perry et al., 2007). However, based on a Bayesian spatial clustering methodology with AFLP markers, Albrecht et al. (2012) found that the cultivated C. baccatum was domesticated independently at two sites, one in the Andes highlands (Peru and Bolivia) and the other in the lowland of Paraguay. More recently, however, based on ethnobotanical studies and bibliographic revisions, Clement et al. (2016) located the centre of domestication in the area of the upper Madeira River basin, in the Amazonia of Bolivia and Brazil. Capsicum has been the focus of taxonomic, cytogenetic and phylogenetic studies (Scaldaferro et al., 2006; Scaldaferro, Grabiele & Moscone, 2013; Barboza et al., 2011; Carrizo García et al., 2016). However, few studies have analysed the intraspecific genetic diversity across the total geographical distribution in order to reconstruct the natural evolutionary history of their species, and even fewer have attempted to elucidate the domestication origin of their cultivated species. Most genetic diversity studies are focused on germplasm bank characterization or on individuals cultivated in home gardens, always with an emphasis on the characterization of domesticated forms (Ibiza et al., 2011; Albrecht et al., 2012). In C. baccatum, Albrecht et al. (2012) using AFLP markers reported two principal genetic groups (western and eastern) in accessions from a germplasm repository; most of the specimens studied were cultivated forms and only 13 accessions came from wild localities, although they did not include wild specimens of the extremes of the natural distribution range in Argentina. The distribution of wild C. baccatum var. baccatum in South America is coincident with four nuclei of the Seasonally Dry Tropical Forests (SDTF). The SDTF is a wide and fragmented biome, extending from Mexico to Argentina and throughout the Caribbean (Prado & Gibbs, 1993; Pennington, Prado & Pendry, 2000). For much of the 20th century, the SDTF of Central and South America remained conceptually included within other neighbouring vegetation units of the continent, such as the Chaco, the Yungas or the Amazonian Dominions (e.g. Cabrera, 1971; Morrone, 2014); however, it has also been proposed as a new phytogeographical dominion (Mogni et al., 2015). Prado & Gibbs (1993) and Pennington et al. (2000) proposed that the dry forest nuclei are remnants (i.e. refugia) of a much larger, single formation (the Pleistocene dry forest arc) that spanned these disjunct dry forest regions during drier periods of the Pleistocene, e.g. the Last Glacial Maximum (LGM, c. 20000 years BP), extending into regions now covered by Amazon rainforest and Cerrado savannas. In this study we used a phylogeographical approach based on DNA sequence variation from two chloroplast gene regions to elucidate the evolutionary history of C. baccatum and to determine the effect of biome past fragmentation on its genetic variation. Our objectives were to (1) examine the DNA sequence variation in cultivated and wild C. baccatum, (2) explore the geographical pattern of cultivated and wild haplotypes, (3) determine the number of potential sites of domestication within South America, and (4) infer geological events and climate changes that have influenced the distribution of genetic variability of the species. We hypothesized that cultivated forms of C. baccatum would have multiple sites of domestication, from where they expanded to the rest of South America. In addition, we expected that haplotype divergences would be concomitant with fragmentation of the STDF. MATERIALS AND METHODS Sampling of natural and cultivated specimens Leaf material was collected from 38 individuals belonging to C. baccatum var. baccatum at 25 locations distributed throughout its entire geographical range in southern South America (Table 1). Some individuals presented transitional forms with moderately sized semipendulous fruits (eight); these were considered ‘became wild’, i.e. originated in the crop but growing in the wild (represented by squares in the map and by symbols with dots in the haplotype network; Figs 1E, 2). In addition, fruits from different localities of South America were purchased from the main markets of the cities (13 locations), and then cultivated in the glasshouse for further species identification. Thus, a total of 20 individuals belonging to C. baccatum var. pendulum and var. umbilicatum were selected and introduced in our study (Table 1). Voucher specimens were deposited in the herbarium of the Botanical Museum of Cordoba, Argentina (CORD). Note that the low number of individuals analysed per population is because the wild form of the species is very rare and only one or two individuals were found at most of the sites. In addition, many of the collected sites registered in herbarium data have already been degraded by anthropogenic activity. Thus, only DNA extracted from herbarium specimens in several localities was available to us. Here, a total of 25 wild locations were sampled throughout its entire range of distribution, for the first time, covering the whole range of the species distribution. Table 1. Collection sites, geographical coordinates, altitude, code and voucher number, sample origin and haplotypes of the studied Capsicum baccatum specimens Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  * Sample sizes are given in parentheses. Collector’s names: GEB, G. Barboza; CC, C. Carrizo; MMC, M. Cerana; AAC, A. Cocucci; GAD, G. Damasceno Junior; LG, L. Guzmán; EK, E. Kowaljow; LAM, L. A. Mentz; EAM, E. Moscone; RN, R. Neumann; MVR, V. Romero; MAS, M. Scaldaferro; JGS, G. Seijo; ANS, A. Sérsic; PTS, P. Souza, RSW, R. S. Williams. View Large Table 1. Collection sites, geographical coordinates, altitude, code and voucher number, sample origin and haplotypes of the studied Capsicum baccatum specimens Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  * Sample sizes are given in parentheses. Collector’s names: GEB, G. Barboza; CC, C. Carrizo; MMC, M. Cerana; AAC, A. Cocucci; GAD, G. Damasceno Junior; LG, L. Guzmán; EK, E. Kowaljow; LAM, L. A. Mentz; EAM, E. Moscone; RN, R. Neumann; MVR, V. Romero; MAS, M. Scaldaferro; JGS, G. Seijo; ANS, A. Sérsic; PTS, P. Souza, RSW, R. S. Williams. View Large Figure 2. View largeDownload slide Geographical distribution and genealogical relationships of the chloroplast DNA (cpDNA) haplotypes found in Capsicum baccatum. A, partial map of South America showing the distribution of cpDNA haplotypes within the studied C. baccatum populations. Groups of populations and symbols representing the different varieties of the species are specified in the figure. A complete map of South America is shown in the left box. B, median-joining network showing relationships among the cpDNA haplotypes found. The size of the symbols is proportional to the haplotype frequencies. Capsicum baccatum var. pendulum is represented by striped symbols. Capsicum baccatum ‘became wild’ is represented by dotted symbols. Figure 2. View largeDownload slide Geographical distribution and genealogical relationships of the chloroplast DNA (cpDNA) haplotypes found in Capsicum baccatum. A, partial map of South America showing the distribution of cpDNA haplotypes within the studied C. baccatum populations. Groups of populations and symbols representing the different varieties of the species are specified in the figure. A complete map of South America is shown in the left box. B, median-joining network showing relationships among the cpDNA haplotypes found. The size of the symbols is proportional to the haplotype frequencies. Capsicum baccatum var. pendulum is represented by striped symbols. Capsicum baccatum ‘became wild’ is represented by dotted symbols. DNA Extraction, Amplification and Sequencing Fresh tissue was dehydrated in silica gel. Total DNA was extracted with a DNeasy plant mini kit (Qiagen, Hilden, Germany). Two non-coding regions of the chloroplast genome were amplified using primer pairs rpl32-trnL and trnQ-5’rps16 (Shaw et al., 2007). We analysed only chloroplast genomes to avoid the detection of signals of hybridization or introgression of cultivated individuals into wild C. baccatum nuclear genomes. In many traditional communities throughout Latin America, wild Capsicum species are often found growing close to cultivated peppers (Van Zonneveld et al., 2015). The PCR mix contained 2 µL of template DNA (10 ng), 0.625 U GoTaq DNA polymerase (Promega, Madison, WI, USA), 5 µL of Green GoTaq reaction buffer (Promega), 0.25 mM of each dNTP and 0.3 µM of each primer in a total volume of 25 µL. The PCR cycling conditions were DNA denaturation at 80 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, primer annealing at 50 °C for 1 min, followed by a ramp of 0.3 °C/s to 65 °C, and primer extension at 65 °C for 4 min; and a final extension step of 5 min at 65 °C. All reactions were carried out on an Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany). Amplification products were separated by electrophoresis on a 1% agarose gel, stained with Syber Safe (Invitrogen, Eugene, OR, USA), and visualized with a UV transilluminator. The PCR-amplified products were sequenced by Macrogen Inc. (Seoul, Korea). Sequences were deposited in GenBank (accession numbers: rpl32-trnL: MH160187–MH160204; trnQ-5’rps16: MH172185–MH172202). For all subsequent analyses, the two chloroplast regions were concatenated for each individual. Sequences were aligned using MEGA 6.0 (Tamura et al., 2013), with manual adjustments made as needed. Gaps were coded following the ‘simple indel coding’ method (Simmons & Ochoterena, 2000). Data analyses To examine genetic relationships among the haplotypes defined by DNAsp v. 6.11.01 (Rozas et al., 2003), a median-joining network was constructed using NETWORK v. 5.0.1.0 (Bandelt, Forster & Röhl, 1999). Ambiguous connections (loops) in the network were resolved using predictions from coalescent theory (Crandall & Templeton, 1993). Molecular diversity indices, i.e. number of haplotypes, percentage CG content, number of polymorphic sites, transitions, transversions and indels, and haplotype and nucleotide diversity (Table 2) for the species and groups of populations were calculated in ARLEQUIN v. 3.5 (Excoffier & Lischer, 2010). Table 2. Molecular diversity indices calculated for Capsicum baccatum var. baccatum and each group of populations identified (‘became wild’ accessions were excluded) Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Number of populations (P) and individuals (N), alignment size and range, number of haplotypes (H), percentage of CG content, number of polymorphic sites (Ps), transitions (ti), transversions (tv), indels (I), and haplotype (h) and nucleotide (π) diversity are shown. Alignment size without outgroups and range are indicated in base pairs (bp). Standard errors are indicated in parentheses. View Large Table 2. Molecular diversity indices calculated for Capsicum baccatum var. baccatum and each group of populations identified (‘became wild’ accessions were excluded) Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Number of populations (P) and individuals (N), alignment size and range, number of haplotypes (H), percentage of CG content, number of polymorphic sites (Ps), transitions (ti), transversions (tv), indels (I), and haplotype (h) and nucleotide (π) diversity are shown. Alignment size without outgroups and range are indicated in base pairs (bp). Standard errors are indicated in parentheses. View Large The phylogenetic tree was constructed and divergence times were estimated using BEAST version 1.6.2 (Drummond & Rambaut, 2007). The input file was prepared in BEAUti version 1.6.2 (provided in the BEAST package). To estimate haplotype divergences we obtained sequences of the rpl region in C. chacoense (GEB et al. 1793 and MAS 44) using the same methodology as in C. baccatum. In addition, sequences of Lycium L. and Solanum L. species were taken from GenBank. The split of Lycium from the genera Solanum and Capsicum was considered the root of the tree and was set to 21 Mya. Additional time constraints were set for the split between Solanum and Capsicum at 19 Mya, between S. tuberosum and S. lycopersicum at 8 Mya, and between C. baccatum and C. chacoense at 2.68 Mya; all dates were taken from Särkinen et al. (2013). The substitution model was GTR with a Gamma site heterogeneity model with four categories, following the results of MrModeltest version 2.2 (Nylander, 2004); the clock was set as an uncorrelated lognormal relaxed model, and the Yule process was selected as a prior for the distribution of divergence dates. The Monte Carlo Markov chain was set to run for 1.5 × 107 generations, sampling every 1000 cycles. RESULTS Sequence analysis of 38 individuals sampled from 25 natural locations yielded 17 distinct chloroplast DNA (cpDNA) haplotypes (Table 1, Fig. 2, Supporting Information Tables S1, S2). In addition, almost all cultivated specimens (18 individuals from 12 locations) showed the same haplotype (H2), except for one accession (two individuals) from INTA Cerrillos, Salta province, Argentina (Fig. 1C), which exhibited haplotype H11. Individuals here considered ‘became wild’ also displayed haplotype H2, except for specimens from Santa Ana (SAN), in Misiones province, Argentina, which exhibited haplotype H3. Most haplotypes (15) were unique to only one accession. The length of the rpl32-trnL sequence varied from 1071 bp in haplotype H5 to 1080 bp in haplotypes H9–H11, whereas the intergenic spacer trnQ-5’rps16 was more variable, ranging between 1234 bp (H4) and 1351 bp (H6). Alignment of the final matrix included 2463 bp and required the introduction of 11 gaps, ranging from 1 to 148 bp in length. Haplotypes H9–H11 had one duplication of 8 bp in rpl32-trnL, and haplotypes H1–H7, H12–H14 and H16-H18 had one duplication of 20 bp in the trnQ-5’rps16 spacer. In addition, a hypervariable region with T and A nucleobases was found in the latter. The configuration of the haplotype network and the phylogenetic tree showed two main lineages that coincide with the geographical distribution (Figs 2B, 3). Group 1 consisted of the westernmost localities on the slopes of the Andes (hereafter West Group) and Group 2 included the eastern populations (hereafter East Group) (Fig. 2A). Median vectors (i.e. non-sampled or extinct haplotypes in the network) connect the haplotypes in the East Group (H12–H18) and with the haplotypes from the West Group (H1–H11). Within the West Group, H2 was the most frequent and widespread haplotype (N = 33), and H1 was the haplotype with the highest number of mutational connections, from which other related haplotypes were derived (H2, H4–H8). Furthermore, the East Group did not include any widespread haplotype. Thus, H1 could be considered the ancestral haplotype of the West Group while the East Group would have originated from a non-sampled or extinct haplotype ancestor (represented here by a median vector). The highest haplotype and nucleotide diversity were found in the East Group (Table 2). Secondary contact zones between divergent lineages were found in SRA and VEL. Finally, the phylogenetic tree (Fig. 3) from cpDNA sequences of C. baccatum showed that the West Group and the East Group diverged at approximately 2.3 Mya (95% confidence and highest posterior density intervals = 28–62 Mya). Other more recent divergences occurred at c. 210 and 140 kya. Figure 3. View largeDownload slide Chronogram showing relations among chloroplast haplotypes (H) and lineages found within Capsicum baccatum. Numbers above branches indicate the ages of nodes (Mya) from the molecular dating analysis. Numbers below branches indicate posterior probabilities from Bayesian inference analysis (BPP). Figure 3. View largeDownload slide Chronogram showing relations among chloroplast haplotypes (H) and lineages found within Capsicum baccatum. Numbers above branches indicate the ages of nodes (Mya) from the molecular dating analysis. Numbers below branches indicate posterior probabilities from Bayesian inference analysis (BPP). DISCUSSION Our results show two principal genetic groups in C. baccatum according to the phylogenetic tree (West and East Groups), agreeing with the genetic groups found by Albrecht et al. (2012) using AFLP markers. Only one of these lineages (West Group) gave origin to most of the cultivated varieties. As indicated by Albrecht et al. (2012), genetic diversity in the wild C. baccatum was greater than in the domesticated form of the species. In the most commercially cultivated C. annuum, studies using RFLPs, isozyme loci, RAPDs, AFLPs and even cytogenetic methods showed the lack of variability in the domesticated types (Lefebvre, Palloix & Rives, 1993; Paran, Aftergoot & Shifriss, 1998; McLeod et al., 1983; Scaldaferro et al., 2013). Thus, in C. baccatum, the domestication process involved a genetic bottleneck associated with a founder effect event due to the selection processes. Origin of C.Baccatum cultivated forms in South America We found two different haplotypes (H2 and H11) in the cultivated forms, with the red haplotype (H2) the most common, present in 12 cultivated accessions of the 13 studied. The geographical distribution of this haplotype in the wild suggests that lowlands of Bolivia and inter-Andean valleys of Peru have been the most important centres of C. baccatum domestication (Fig. 4A). Thus, phylogeographical methods support the early hypothesis of these regions as sites of domestication of this species, occurring by 4000 years BP at the latest, probably by Arawak people inhabiting the Llanos de Mojos (i.e. Moxeños), Guarani populations, both in Bolivia, or pre-Incas in Peru (see Fig. 4A) (Perry et al., 2007; Piperno, 2011; Brown et al., 2013; Clement et al., 2016). The upper Madeira River basin inhabited by Moxeño people was a probable region of origin of other crops, such as other chili peppers (C. pubescens), three legume seeds (Arachis hypogaea L., Canavalia plagiosperma Piper, Phaseolus vulgaris L.) and cocoyam [Xanthosoma sagittifolium (L.) Schott], important drugs, such as coca (Erythroxylum coca Lam.), and tobacco (Nicotiana tabacum L.), and the major root crop of Amazonia, manioc (Manihot esculenta Crantz) and peach palm (Bactris gasipaes Kunth) (Grabiele et al., 2012; Clement et al., 2016, and references herein). Capsicum baccatum var. umbilicatum originated from the west clade, as did var. pendulum, although this result is in disagreement with those reported by Albrecht et al. (2012). The only C. baccatum var. umbilicatum accession included in their analysis was collected from Brazil (São Paulo); it was deeply nested within the ‘eastern’ Group and closely related to other cultivars from Brazil, primarily those from Minas Gerais. Figure 4. View largeDownload slide Maps of South America showing the geographical distribution of haplotypes along with different addressed aspects. A, distribution of aboriginal populations in South America before the Spanish conquest and geographical distribution of wild and cultivated samples of Capsicum baccatum. B, schematic distributions of the Seasonally Dry Tropical Forests (SDTF; adapted from Prado, 2000; Caetano et al., 2008) and their relations with haplotype structure in the studied C. baccatum var. baccatum populations. Figure 4. View largeDownload slide Maps of South America showing the geographical distribution of haplotypes along with different addressed aspects. A, distribution of aboriginal populations in South America before the Spanish conquest and geographical distribution of wild and cultivated samples of Capsicum baccatum. B, schematic distributions of the Seasonally Dry Tropical Forests (SDTF; adapted from Prado, 2000; Caetano et al., 2008) and their relations with haplotype structure in the studied C. baccatum var. baccatum populations. Our results indicate that various accessions grow in nature, whose haplotypes or genetic variants do not correspond to the wild-type expected lineage according to phylogenetic and phylogeographical analyses. All of these wild specimens had the red haplotype (H2; PRF, PRN, SMA, TEY, SPO), and one derived from it (H3; SAN) in only one case. Fruit size is generally intermediate between that of cultivated and wild plants (Fig. 1E). One possible explanation could be hybridization with wild parental species. In many traditional communities throughout Latin America, wild and semi-wild Capsicum species are often found growing close to cultivated peppers, in home gardens or at the edges of cultivated fields, where introgressive hybridization takes place between the cultigens and closely related wild species or sub-species, spontaneously giving rise to novel or intermediate forms (Van Zonneveld et al., 2015). Notably, accessions here named ‘became wild’ have been found only in the east of the species distribution. The region comprising Paraguay, Brazil and Misiones province in Argentina was inhabited by Guarani peoples before arrival of Europeans (Fig. 4A). They were traditionally farmers and cultivated maize (acquired by interethnic contact), cassava (probably domesticated by them), peanut and sweet potato. Occupation of their lands dedicated to agriculture together with cultural erosion, among other factors, would have caused the abandonment of this intensive practice and many of the crops may have been neglected (Keller, 2012). Hence, we propose that these agricultural peoples once cultivated chili peppers (domesticated in the west), probably obtained through commercial practices. After European conquest, this activity declined, which might have caused the de-selection in attributes of the cultivated fruits (e.g. from large to small size fruit) (F. Zamudio IMBIV, pers. comm.); since then, plants with smaller fruits may have continued to grow in the wild and ‘become wild’. The fact that these specimens always carried the cultivated haplotype type (H2) reinforces the idea of their origin as abandoned crops. Chiou et al. (2014) noted the reduction of seed size after the Spanish conquest (after 1532 CE), suggesting a loss of selective pressure for seed size in this particular Capsicum species. The cultivated accession EAM & RN 215 from INTA Cerrillos has a different haplotype (H11), which could indicate a secondary origin of domestication for C. baccatum var. pendulum or could reflect an ancient site of cultivation of the species where hybridization between cultivated and local samples may have occurred. Multiple domestications of different populations of the same species are rare, but there are some records, such as the best known examples of common bean (P. vulgaris) and Lima bean (P. lunatus L.) (Chacón, Pickersgill & Debouck, 2005; Serrano-Serrano et al., 2010). According to the haplotype network, the haplotype found in this accession is derived from a wild haplotype found in Aguas Blancas, at the frontier between Argentina and Bolivia. Ethnobotanical studies in Baritu National Park (Salta, Argentina) indicate that this region was occupied in pre-Hispanic times by a group of ‘Chiriguanos’ belonging to the Guarani group, who originated from the north-east of the Bermejo River basin (Fig. 4A). These avid farmers had commercial relationships with people from Tarija and Padcaya in Bolivia, and Santa Victoria in Argentina (Hilgert 1999) and they probably cultivated and commercialized C. baccatum. Phylogeographical implications Two principal genetic groups were identified in the wild C. baccatum accessions in relation to their geographical distribution in South America. One group included accessions from the western territories of the species distribution (Peru, Bolivia and north-western Argentina) and corresponds to SDTF nuclei of the Andean Piedmont and Peruvian inter-Andean valleys. The second group included samples from the eastern regions (Brazil, Paraguay and eastern Argentina) and corresponds to SDTF nuclei from Chiquitano and Misiones. Capsicum baccatum is in fact not considered in the species database of these forests (: database http://www.dryflor.info/data), and consequently, it was not considered in a recent study of these flora (Banda et al., 2016). Nonetheless the species could effectively be integrated into this database, highlighting the importance of conservation of this biome as a reserve of wild relatives from cultivated species. Our results supported by molecular dating indicate that a large split between the western and eastern groups of wild C. baccatum occurred at 2.3 Mya. A Late Pliocene to Early Pleistocene cooling and drying pulse was detected worldwide at about 2.5 Mya, in times of the Great Patagonian Glaciation (Rabassa, 2008). Ice covered part of the Andean region in Patagonia and part of the extra-Andean region, where extensive areas of frozen soils developed. The result would have been a displacement of the Dry Chaco region (Fig. 4B) northward, where the Chiquitano STDF nucleus exists today. Phylogeographical studies developed in Astronium urundeuva (Allemão) Engl. and Anadenanthera colubrina (Vell.) Brennan showed west and east groupings of populations located at sites that are comparable to our study sites (see fig. 2 in Caetano et al., 2008 and fig. 2 in Barrandeguy et al., 2016). The Pleistocene Arc Theory (PAT) suggests that present-day disjunct fragments of dry forests in central tropical South America provide evidence of a previously more continuous distribution during the Pleistocene that has been disrupted by dry-cold vs. humid-warm climatic cycles. Prado & Gibbs (1993) hypothesized that the increasing temperatures and precipitation over the last glacial–Holocene transition caused contraction and fragmentation of the Pleistocene dry forest arc into the present-day disjunct distribution of dry forest nuclei. We propose that, in contrast to expectation, the SDTFs in their southernmost distribution have been fragmented during glaciation events due to advance of the Chaco region from the south. This agrees with Pennington et al. (2004), who postulate that Chiquitano nucleus in Bolivia would have moved north rather than expanded during periods of glaciation. Population genetics studies remain very scarce and cover a relatively small part of the total distribution range of SDTF. Ecological niche modelling developed by Werneck et al. (2011) and Turchetto-Zolet et al. (2016) provides evidence that the SDTFs were even more disjunct during the LGM. Finally, areas of presumed long-term stability are identified and confirmed (Caatinga, Misiones, Piedmont and Chiquitano region), and possibly acted as current and historical refugia (Werneck et al., 2011). By contrast, the demographic history of Tabebuia impetiginosa (Mart. ex DC.) Standl. showed that the pattern of diversity found for the species is in agreement with distribution expansion during the LGM (Collevatti et al., 2012). This species is distributed in the northern relicts of SDTF in South America and Mesoamerica. The results obtained here contribute to the discussion regarding the PAT. Thus, the response of the SDTF to glaciation events could have depended on the STDF nucleus: northern nuclei have expanded over the Amazon forest, whereas the southernmost nuclei have undergone fragmentation because of increased area of drier regions, and moved their range north. Further studies in representative species from this biome are needed to evaluate the response to climatic change and to elaborate upon conservation actions. These forests are priority areas for conservation because they are the home of wild ancestors of many important crop species. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Table S1. Variable sites of the aligned chloroplast DNA sequences from the rpl32F-trnL region for the 18 haplotypes in Capsicum baccatum. Table S2. Variable sites of the aligned chloroplast DNA sequences from the trnQ-rps16 region for the 18 haplotypes in Capsicum baccatum. Database S1. Sequence dataset used in the phylogeographical analysis. ACKNOWLEDGEMENTS We are grateful to L. Boero, C. Carrizo, J. Daviña, A. Honfi, E. Kowaljow, M. Renny, V. Romero, G. Seijo and A. Sérsic who collected some leaf material. A. Romanutti provided valuable help in the laboratory and J. Brasca collaborated in the final editing of the manuscript. We thank three anonymous reviewers for their helpful comments. Financial assistance was provided by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP 114-200801-00326), Agencia de Promoción Científica y Tecnológica (PICT 573), Ministerio de Ciencia y Tecnología de la Provincia de Córdoba, and Secretaría de Ciencia y Tecnología (SECYT-UNC). MAS, GB and MCA are members of Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina and Universidad Nacional de Córdoba, Argentina. Dedicated to the Memory of Eduardo A. Moscone REFERENCES Albrecht E, Zhang D, Saftner RA, Stommel JR. 2012. Genetic diversity and population structure of Capsicum baccatum genetic resources. Genetic Resources and Crop Evolution  59: 517– 538. Google Scholar CrossRef Search ADS   Avise JC. 2000. Phylogeography: the history and formation of species . Cambridge: Harvard University Press. 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Evolutionary history of the chili pepper Capsicum baccatum L. (Solanaceae): domestication in South America and natural diversification in the Seasonally Dry Tropical Forests

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Abstract

Abstract Plant domestication genetics has been of interest not only for evolutionary biologists but also for anthropologists and breeders, because of its past and present role in human cultural evolution. Capsicum baccatum, which grows in the Seasonally Dry Tropical Forests of South America (SDTF), was domesticated by native peoples and used as a spice. The centre of domestication has been discussed, and vast territories of Peru and Bolivia have been proposed. The aims of this research were to elucidate the geographical origin of cultivated C. baccatum and to infer geological and climate events that have influenced the distribution of its genetic variability. Samples were collected from 25 localities across the entire range of wild C. baccatum and analysed using DNA sequences and phylogeographical approaches; they were then compared with 20 samples of cultivated forms obtained from different South American markets. We found a main centre of C. baccatum domestication spanning Bolivian Amazonia and the inter-Andean valleys. We also inferred an ancient cultivation site between Bolivia and Argentina. Finally, we found two lineages of wild populations distributed in nuclei of SDTFs which have been fragmented during glacial periods, when aridity increased and expansion of Chaco vegetation fragmented the region. Capsicum baccatum, chloroplast DNA sequences, crop origin, haplotype network, molecular dating, Solanaceae, South America INTRODUCTION The domestication of plants is one of the most significant cultural and evolutionary transitions in human history. Investigating when, where and how domestication took place is of interest to crop evolutionists and of practical importance for plant breeders and conservationists. Research into the domestication and spatio-temporal origins of this process has increased significantly over the past decade through archaeological research, advances in DNA/RNA sequencing techniques, and methods used to recover and formally identify those changes in interactions among plants and animals that lead to domestication (Larson et al., 2014). Genetic studies that examine the geographical origins of a crop typically rely on genome-wide neutral markers, which are used to assess allele frequencies in a crop compared with populations of its wild relatives. Due to the strong genetic bottlenecks that occur during the domestication process, allelic diversity in the crop is expected to be a subset of that found in the wild population(s) from which it was derived. Thus, if populations of the wild progenitor are extant in the geographical location(s) where domestication occurred, the geographical origin(s) of domestication can potentially be pinpointed to a particular population or region (Gross & Olsen, 2010). One of the most successful applications of phylogeographical analysis has been the investigation of sites of origin of domesticated crops (Londo et al., 2007). Phylogeographical studies draw inferences about the history of population divergence based on associations between the geographical distribution of alleles and their genealogical relationships (Avise, 2000). The number of domestication events and regions in cultivated rice and peanuts was determined using a phylogeographical approach based on DNA sequence variation (Londo et al., 2007; Grabiele et al., 2012). In addition, such approaches have made a significant contribution to the interpretation of species in response to climate and geological changes, such as the glaciation events of the Neogene (Turchetto-Zolet et al., 2013; Daneck, Fér & Marhold, 2016; Zhang, Zhang & Sanderson, 2016). Capsicum baccatum L. is one of the most economically important species of the genus, with three varieties, the wild C. baccatum var. baccatum, and two [var. pendulum (Willd.) Eshbaugh and var. umbilicatum (Vell.) Hunz. and Barboza] varieties, cultivated commercially mainly in South America (Pickersgill, 1997; Djian-Caporalino et al., 2007; Peralta et al., 2008). The wild form of the species typically bears small, erect, deciduous fruits (Fig. 1A; D’Arcy & Eshbaugh, 1974; Barboza, 2013), whereas the domesticated forms usually bear larger, pendant or bell-shaped, persistent fruits (Fig. 1B–D). The fruits are used as a spice in regional cuisine, although they are also of interest for a wide range of other applications, such as a natural colouring agent, an ingredient in pharmaceuticals (as a rubefacient and stomachic), and a highly pungent extract used in self-defence sprays, animal repellents and insecticides (Bosland, 1996). The wild form of the species is distributed in South America, from Colombia, Peru, Bolivia, Brazil and Paraguay to north-west and north-east Argentina (Moscone et al., 2007; Barboza, 2013), and is recognized under various common names: ‘cumbari’, ‘ají quitucho’, ‘quitucho’ and ‘ají del monte’. Figure 1. View largeDownload slide Fruits of Capsicum baccatum. A, C. baccatum var. baccatum SVL. B, C. baccatum var. pendulum 642. C, C. baccatum var. pendulum 215. D, C. baccatum var. umbilicatum 063. E, C. baccatum ‘became wild’ PRF. Scale bar = 2 cm. Figure 1. View largeDownload slide Fruits of Capsicum baccatum. A, C. baccatum var. baccatum SVL. B, C. baccatum var. pendulum 642. C, C. baccatum var. pendulum 215. D, C. baccatum var. umbilicatum 063. E, C. baccatum ‘became wild’ PRF. Scale bar = 2 cm. Starch microfossils of C. baccatum in Peru date from 4000 years BP to European contact, indicating an early use of domesticated peppers in ancient America (Perry et al., 2007). Based on combinations of archaeological evidence and genetic analyses, researchers suggested that the centre of domestication of C. baccatum was the lowlands of Bolivia (Pickersgill, 1984; Eshbaugh, 1993; Perry et al., 2007). However, based on a Bayesian spatial clustering methodology with AFLP markers, Albrecht et al. (2012) found that the cultivated C. baccatum was domesticated independently at two sites, one in the Andes highlands (Peru and Bolivia) and the other in the lowland of Paraguay. More recently, however, based on ethnobotanical studies and bibliographic revisions, Clement et al. (2016) located the centre of domestication in the area of the upper Madeira River basin, in the Amazonia of Bolivia and Brazil. Capsicum has been the focus of taxonomic, cytogenetic and phylogenetic studies (Scaldaferro et al., 2006; Scaldaferro, Grabiele & Moscone, 2013; Barboza et al., 2011; Carrizo García et al., 2016). However, few studies have analysed the intraspecific genetic diversity across the total geographical distribution in order to reconstruct the natural evolutionary history of their species, and even fewer have attempted to elucidate the domestication origin of their cultivated species. Most genetic diversity studies are focused on germplasm bank characterization or on individuals cultivated in home gardens, always with an emphasis on the characterization of domesticated forms (Ibiza et al., 2011; Albrecht et al., 2012). In C. baccatum, Albrecht et al. (2012) using AFLP markers reported two principal genetic groups (western and eastern) in accessions from a germplasm repository; most of the specimens studied were cultivated forms and only 13 accessions came from wild localities, although they did not include wild specimens of the extremes of the natural distribution range in Argentina. The distribution of wild C. baccatum var. baccatum in South America is coincident with four nuclei of the Seasonally Dry Tropical Forests (SDTF). The SDTF is a wide and fragmented biome, extending from Mexico to Argentina and throughout the Caribbean (Prado & Gibbs, 1993; Pennington, Prado & Pendry, 2000). For much of the 20th century, the SDTF of Central and South America remained conceptually included within other neighbouring vegetation units of the continent, such as the Chaco, the Yungas or the Amazonian Dominions (e.g. Cabrera, 1971; Morrone, 2014); however, it has also been proposed as a new phytogeographical dominion (Mogni et al., 2015). Prado & Gibbs (1993) and Pennington et al. (2000) proposed that the dry forest nuclei are remnants (i.e. refugia) of a much larger, single formation (the Pleistocene dry forest arc) that spanned these disjunct dry forest regions during drier periods of the Pleistocene, e.g. the Last Glacial Maximum (LGM, c. 20000 years BP), extending into regions now covered by Amazon rainforest and Cerrado savannas. In this study we used a phylogeographical approach based on DNA sequence variation from two chloroplast gene regions to elucidate the evolutionary history of C. baccatum and to determine the effect of biome past fragmentation on its genetic variation. Our objectives were to (1) examine the DNA sequence variation in cultivated and wild C. baccatum, (2) explore the geographical pattern of cultivated and wild haplotypes, (3) determine the number of potential sites of domestication within South America, and (4) infer geological events and climate changes that have influenced the distribution of genetic variability of the species. We hypothesized that cultivated forms of C. baccatum would have multiple sites of domestication, from where they expanded to the rest of South America. In addition, we expected that haplotype divergences would be concomitant with fragmentation of the STDF. MATERIALS AND METHODS Sampling of natural and cultivated specimens Leaf material was collected from 38 individuals belonging to C. baccatum var. baccatum at 25 locations distributed throughout its entire geographical range in southern South America (Table 1). Some individuals presented transitional forms with moderately sized semipendulous fruits (eight); these were considered ‘became wild’, i.e. originated in the crop but growing in the wild (represented by squares in the map and by symbols with dots in the haplotype network; Figs 1E, 2). In addition, fruits from different localities of South America were purchased from the main markets of the cities (13 locations), and then cultivated in the glasshouse for further species identification. Thus, a total of 20 individuals belonging to C. baccatum var. pendulum and var. umbilicatum were selected and introduced in our study (Table 1). Voucher specimens were deposited in the herbarium of the Botanical Museum of Cordoba, Argentina (CORD). Note that the low number of individuals analysed per population is because the wild form of the species is very rare and only one or two individuals were found at most of the sites. In addition, many of the collected sites registered in herbarium data have already been degraded by anthropogenic activity. Thus, only DNA extracted from herbarium specimens in several localities was available to us. Here, a total of 25 wild locations were sampled throughout its entire range of distribution, for the first time, covering the whole range of the species distribution. Table 1. Collection sites, geographical coordinates, altitude, code and voucher number, sample origin and haplotypes of the studied Capsicum baccatum specimens Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  * Sample sizes are given in parentheses. Collector’s names: GEB, G. Barboza; CC, C. Carrizo; MMC, M. Cerana; AAC, A. Cocucci; GAD, G. Damasceno Junior; LG, L. Guzmán; EK, E. Kowaljow; LAM, L. A. Mentz; EAM, E. Moscone; RN, R. Neumann; MVR, V. Romero; MAS, M. Scaldaferro; JGS, G. Seijo; ANS, A. Sérsic; PTS, P. Souza, RSW, R. S. Williams. View Large Table 1. Collection sites, geographical coordinates, altitude, code and voucher number, sample origin and haplotypes of the studied Capsicum baccatum specimens Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  Locality  Geographical coordinates (S, W)  Altitude (m)  Code and voucher number  Origin  Haplotype*  C. baccatum var. baccatum  Peru, Cusco District, Convención Province, Santa Teresa.  13°00′47″  72°37′10″  1208  CUZ  GEB 3419  wild  2 (1)  Bolivia, Santa Cruz Department, Andres Ibañez Province, Basilo.  18°05′26″  63°11′40″  499  BAS  AAC & ANS  wild  2 (2)  Bolivia, Santa Cruz Department, Ñuflo de Chavez Province, San Ramón.  16°35′33″  62°29′34″  866  SRA  JGS et al. 3448  wild  15 (1)  Bolivia, Santa Cruz Department, Velasco Province, Santa Rosa.  17°27′32″  63°24′29″  344  VEL  JGS et al. 3580  wild  8 (1)  Bolivia, Santa Cruz Department, Florida Province, Bermejo.  18°07′48″  63°38′01″  807  BER  GEB & CC 3646  wild  2 (2), 6 (1)  Bolivia, Chuquisaca Department, Luis Calvo Province, Ñancaroinza.  20°42′22″  63°17′42″  744  NAN  AAC & ANS 5488  wild  7 (1)  Bolivia, La Paz Department, Sud Yungas Province, Coroico.  15°28′00″  67°10′00″  720  COR  LG et al. 1998  wild  2 (3)  Brazil, São Paulo State, Mairinque.  23°32′45″  47°11′00″  859  SPO  MVR 8  became wild  2 (3)  Brazil, São Paulo State, Campinas.  22°53′59″  47°56′16″  683  JOA  PTS 10  wild  17 (1)  Brazil, Santa Catarina State, Rio do Sul.  27°28′01″  53°59′54″  280  RGS  LAM et al. 263  wild  14 (1)  Brazil, Mato Grosso do Sul State, Corumbá.  19°00′33″  57°39′11″  226  COU  GAD 19126  wild  18 (1)  Paraguay, Presidente Hayes Department, Nanawa.  25°16′32″  57°41′49″  63  PRN  MAS 40  became wild  2 (1)  Paraguay, Presidente Hayes Department, Puerto Falcón.  25°15′23″  57°42′55″  66  PRF  MAS 38  became wild  2 (1)  Paraguay, Paraguarí Department, Acahay.  25°52′56″  57°09′28″  280  ACA  RSW et al. 129  wild  16 (1)  Argentina, Jujuy Province, El Carmen Department, Dique Las Maderas.  24°26′56″  65°15′01″  1200  JUJ  AAC & ANS 5262  wild  1 (3)  Argentina, Jujuy Province, Ledesma Department, Agua de las Cañas.  23°45′09″  64°51′20″  718  CAN  GEB 1921  wild  5 (1)  Argentina, Salta Province, Oran Department, Aguas Blancas.  22°43′30″  64°21′54″  417  BLA  AAC & ANS 5469  wild  9 (2), 10 (1)  Argentina, Salta Province, La Caldera Department, La Caldera.  24°36′07″  65°22′51″  1380  SAL  AAC & ANS 5258  wild  1 (1)  Argentina, Salta Province, Capital Department, Sierra de Vélez.  24°45′33″  65°22′38″  1350  SVL  GEB 1805  wild  1 (2), 4 (1)  Argentina, Formosa Province, Pirané Department, Pirané.  25°37′08″  59°10′45″  87  PIR  MMC 823  wild  12 (1)  Argentina, Misiones Province, Santa Ana Department, Santa Ana.  27°20′10″  55°35′15″  102  ANA  MAS 47  wild  13 (1)  Argentina, Misiones Province, Gral. San Martín Department, Puerto Oro Verde.  26°49′40″  54°59′39″  171  SMA  GEB et al. 1038  became wild  2 (1)  Argentina, Misiones Province, Santa Ana, Department, Santa Ana.  27°20′40″  55°35′59″  124  SAN  MAS 48  became wild  3 (1)  Argentina, Misiones Province, San Ignacio Department, Teyucuaré.  27°16′12″  55°32′57″  181  TEY  MAS 49  became wild  2 (1)  Argentina, Corrientes Province, San Martín Department, Guaviraví.  29°23′05″  56°47′04″  62  GUA  MAS 50  wild  16 (1)  C. baccatum var. pendulum  Ecuador, Manabí Province, Puerto López, bought at market place.  1°33′33″  80°48′39″  12  061  MAS 61  cultivated  2 (2)  Peru, Piura Department, Piura, bought at market place.  5°11′13″  80°38′54″  43  019  MAS 19  cultivated  2 (2)  Peru, Cusco District, Cusco, bought at market place.  13°31′06″  71°58′41″  1208  573  MAS & EK 70  cultivated  2 (1)  Bolivia, Cochabamba Department, Pairumani Farmhouse, cultivated at Fundación Simón Patiño, Granja Modelo.  17°21′40″  66°19′28″  2632  320  MAS 43  cultivated  2 (2)  Bolivia, Santa Cruz Department, Santa Cruz de La Sierra, bought at market place.  17°47′39″  63°10′57″  423  642  GEB & CC 3642  cultivated  2 (1)  Bolivia, Santa Cruz Department, Valle Grande, bought at market place.  18°29′29″  64°06′26″  2050  062  MAS 62  cultivated  2 (2)  Bolivia, Tarija Department, Villa Montes, bought at market place.  21°15′57″  63°28′37″  392  037  MAS 37  cultivated  2 (1)  Brazil, Minas Gerais State, Belo Horizonte, bought at market place.  19°55′12″  43°56′40″  918  798  GEB et al. 798  cultivated  2 (1)  Brazil, São Paulo State, Santo Antonio de Posse, bought at market place.  23°34′09″  46°36′41″  673  056  MAS 56  cultivated  2 (2)  Argentina, Salta Province, Salta, bought at market place.  24°47′16″  65°24′38″  1196  247  EAM 247  cultivated  2 (1)  Argentina, Salta Province, cultivated at INTA Cerrillos.  24°53′03″  65°28′05″  1242  215  EAM & RN 215  cultivated  11 (2)  C. baccatum var. umbilicatum  Brazil, Rio de Janeiro State, Petrópolis, bought at market place.  22°30′49″  43°11′17″  732  057  MAS 57  cultivated  2 (1)  Argentina, Cordoba Province, Santa Rosa de Calamuchita, cultivated at a private home.  32°04′24″  64°32′13″  620  063  MAS 63  cultivated  2 (2)  * Sample sizes are given in parentheses. Collector’s names: GEB, G. Barboza; CC, C. Carrizo; MMC, M. Cerana; AAC, A. Cocucci; GAD, G. Damasceno Junior; LG, L. Guzmán; EK, E. Kowaljow; LAM, L. A. Mentz; EAM, E. Moscone; RN, R. Neumann; MVR, V. Romero; MAS, M. Scaldaferro; JGS, G. Seijo; ANS, A. Sérsic; PTS, P. Souza, RSW, R. S. Williams. View Large Figure 2. View largeDownload slide Geographical distribution and genealogical relationships of the chloroplast DNA (cpDNA) haplotypes found in Capsicum baccatum. A, partial map of South America showing the distribution of cpDNA haplotypes within the studied C. baccatum populations. Groups of populations and symbols representing the different varieties of the species are specified in the figure. A complete map of South America is shown in the left box. B, median-joining network showing relationships among the cpDNA haplotypes found. The size of the symbols is proportional to the haplotype frequencies. Capsicum baccatum var. pendulum is represented by striped symbols. Capsicum baccatum ‘became wild’ is represented by dotted symbols. Figure 2. View largeDownload slide Geographical distribution and genealogical relationships of the chloroplast DNA (cpDNA) haplotypes found in Capsicum baccatum. A, partial map of South America showing the distribution of cpDNA haplotypes within the studied C. baccatum populations. Groups of populations and symbols representing the different varieties of the species are specified in the figure. A complete map of South America is shown in the left box. B, median-joining network showing relationships among the cpDNA haplotypes found. The size of the symbols is proportional to the haplotype frequencies. Capsicum baccatum var. pendulum is represented by striped symbols. Capsicum baccatum ‘became wild’ is represented by dotted symbols. DNA Extraction, Amplification and Sequencing Fresh tissue was dehydrated in silica gel. Total DNA was extracted with a DNeasy plant mini kit (Qiagen, Hilden, Germany). Two non-coding regions of the chloroplast genome were amplified using primer pairs rpl32-trnL and trnQ-5’rps16 (Shaw et al., 2007). We analysed only chloroplast genomes to avoid the detection of signals of hybridization or introgression of cultivated individuals into wild C. baccatum nuclear genomes. In many traditional communities throughout Latin America, wild Capsicum species are often found growing close to cultivated peppers (Van Zonneveld et al., 2015). The PCR mix contained 2 µL of template DNA (10 ng), 0.625 U GoTaq DNA polymerase (Promega, Madison, WI, USA), 5 µL of Green GoTaq reaction buffer (Promega), 0.25 mM of each dNTP and 0.3 µM of each primer in a total volume of 25 µL. The PCR cycling conditions were DNA denaturation at 80 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, primer annealing at 50 °C for 1 min, followed by a ramp of 0.3 °C/s to 65 °C, and primer extension at 65 °C for 4 min; and a final extension step of 5 min at 65 °C. All reactions were carried out on an Eppendorf Mastercycler thermocycler (Eppendorf, Hamburg, Germany). Amplification products were separated by electrophoresis on a 1% agarose gel, stained with Syber Safe (Invitrogen, Eugene, OR, USA), and visualized with a UV transilluminator. The PCR-amplified products were sequenced by Macrogen Inc. (Seoul, Korea). Sequences were deposited in GenBank (accession numbers: rpl32-trnL: MH160187–MH160204; trnQ-5’rps16: MH172185–MH172202). For all subsequent analyses, the two chloroplast regions were concatenated for each individual. Sequences were aligned using MEGA 6.0 (Tamura et al., 2013), with manual adjustments made as needed. Gaps were coded following the ‘simple indel coding’ method (Simmons & Ochoterena, 2000). Data analyses To examine genetic relationships among the haplotypes defined by DNAsp v. 6.11.01 (Rozas et al., 2003), a median-joining network was constructed using NETWORK v. 5.0.1.0 (Bandelt, Forster & Röhl, 1999). Ambiguous connections (loops) in the network were resolved using predictions from coalescent theory (Crandall & Templeton, 1993). Molecular diversity indices, i.e. number of haplotypes, percentage CG content, number of polymorphic sites, transitions, transversions and indels, and haplotype and nucleotide diversity (Table 2) for the species and groups of populations were calculated in ARLEQUIN v. 3.5 (Excoffier & Lischer, 2010). Table 2. Molecular diversity indices calculated for Capsicum baccatum var. baccatum and each group of populations identified (‘became wild’ accessions were excluded) Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Number of populations (P) and individuals (N), alignment size and range, number of haplotypes (H), percentage of CG content, number of polymorphic sites (Ps), transitions (ti), transversions (tv), indels (I), and haplotype (h) and nucleotide (π) diversity are shown. Alignment size without outgroups and range are indicated in base pairs (bp). Standard errors are indicated in parentheses. View Large Table 2. Molecular diversity indices calculated for Capsicum baccatum var. baccatum and each group of populations identified (‘became wild’ accessions were excluded) Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Grouping  P  N  Size  Range  H  CG%  Ps  ti  tv  I  h  π  C. baccatum var. baccatum  19  30  2463  2306–2423  16  26.91  22  5  6  11  0.8966 (0.0398)  0.0140 (0.0070)  West Group  12  23  2432  2306–2423  10  26.91  13  3  2  8  0.8261 (0.0590)  0.0103 (0.0052)  East Group  7  7  2428  2338–2403  6  26.90  9  2  4  3  0.9524 (0.0955)  0.0161 (0.0092)  Number of populations (P) and individuals (N), alignment size and range, number of haplotypes (H), percentage of CG content, number of polymorphic sites (Ps), transitions (ti), transversions (tv), indels (I), and haplotype (h) and nucleotide (π) diversity are shown. Alignment size without outgroups and range are indicated in base pairs (bp). Standard errors are indicated in parentheses. View Large The phylogenetic tree was constructed and divergence times were estimated using BEAST version 1.6.2 (Drummond & Rambaut, 2007). The input file was prepared in BEAUti version 1.6.2 (provided in the BEAST package). To estimate haplotype divergences we obtained sequences of the rpl region in C. chacoense (GEB et al. 1793 and MAS 44) using the same methodology as in C. baccatum. In addition, sequences of Lycium L. and Solanum L. species were taken from GenBank. The split of Lycium from the genera Solanum and Capsicum was considered the root of the tree and was set to 21 Mya. Additional time constraints were set for the split between Solanum and Capsicum at 19 Mya, between S. tuberosum and S. lycopersicum at 8 Mya, and between C. baccatum and C. chacoense at 2.68 Mya; all dates were taken from Särkinen et al. (2013). The substitution model was GTR with a Gamma site heterogeneity model with four categories, following the results of MrModeltest version 2.2 (Nylander, 2004); the clock was set as an uncorrelated lognormal relaxed model, and the Yule process was selected as a prior for the distribution of divergence dates. The Monte Carlo Markov chain was set to run for 1.5 × 107 generations, sampling every 1000 cycles. RESULTS Sequence analysis of 38 individuals sampled from 25 natural locations yielded 17 distinct chloroplast DNA (cpDNA) haplotypes (Table 1, Fig. 2, Supporting Information Tables S1, S2). In addition, almost all cultivated specimens (18 individuals from 12 locations) showed the same haplotype (H2), except for one accession (two individuals) from INTA Cerrillos, Salta province, Argentina (Fig. 1C), which exhibited haplotype H11. Individuals here considered ‘became wild’ also displayed haplotype H2, except for specimens from Santa Ana (SAN), in Misiones province, Argentina, which exhibited haplotype H3. Most haplotypes (15) were unique to only one accession. The length of the rpl32-trnL sequence varied from 1071 bp in haplotype H5 to 1080 bp in haplotypes H9–H11, whereas the intergenic spacer trnQ-5’rps16 was more variable, ranging between 1234 bp (H4) and 1351 bp (H6). Alignment of the final matrix included 2463 bp and required the introduction of 11 gaps, ranging from 1 to 148 bp in length. Haplotypes H9–H11 had one duplication of 8 bp in rpl32-trnL, and haplotypes H1–H7, H12–H14 and H16-H18 had one duplication of 20 bp in the trnQ-5’rps16 spacer. In addition, a hypervariable region with T and A nucleobases was found in the latter. The configuration of the haplotype network and the phylogenetic tree showed two main lineages that coincide with the geographical distribution (Figs 2B, 3). Group 1 consisted of the westernmost localities on the slopes of the Andes (hereafter West Group) and Group 2 included the eastern populations (hereafter East Group) (Fig. 2A). Median vectors (i.e. non-sampled or extinct haplotypes in the network) connect the haplotypes in the East Group (H12–H18) and with the haplotypes from the West Group (H1–H11). Within the West Group, H2 was the most frequent and widespread haplotype (N = 33), and H1 was the haplotype with the highest number of mutational connections, from which other related haplotypes were derived (H2, H4–H8). Furthermore, the East Group did not include any widespread haplotype. Thus, H1 could be considered the ancestral haplotype of the West Group while the East Group would have originated from a non-sampled or extinct haplotype ancestor (represented here by a median vector). The highest haplotype and nucleotide diversity were found in the East Group (Table 2). Secondary contact zones between divergent lineages were found in SRA and VEL. Finally, the phylogenetic tree (Fig. 3) from cpDNA sequences of C. baccatum showed that the West Group and the East Group diverged at approximately 2.3 Mya (95% confidence and highest posterior density intervals = 28–62 Mya). Other more recent divergences occurred at c. 210 and 140 kya. Figure 3. View largeDownload slide Chronogram showing relations among chloroplast haplotypes (H) and lineages found within Capsicum baccatum. Numbers above branches indicate the ages of nodes (Mya) from the molecular dating analysis. Numbers below branches indicate posterior probabilities from Bayesian inference analysis (BPP). Figure 3. View largeDownload slide Chronogram showing relations among chloroplast haplotypes (H) and lineages found within Capsicum baccatum. Numbers above branches indicate the ages of nodes (Mya) from the molecular dating analysis. Numbers below branches indicate posterior probabilities from Bayesian inference analysis (BPP). DISCUSSION Our results show two principal genetic groups in C. baccatum according to the phylogenetic tree (West and East Groups), agreeing with the genetic groups found by Albrecht et al. (2012) using AFLP markers. Only one of these lineages (West Group) gave origin to most of the cultivated varieties. As indicated by Albrecht et al. (2012), genetic diversity in the wild C. baccatum was greater than in the domesticated form of the species. In the most commercially cultivated C. annuum, studies using RFLPs, isozyme loci, RAPDs, AFLPs and even cytogenetic methods showed the lack of variability in the domesticated types (Lefebvre, Palloix & Rives, 1993; Paran, Aftergoot & Shifriss, 1998; McLeod et al., 1983; Scaldaferro et al., 2013). Thus, in C. baccatum, the domestication process involved a genetic bottleneck associated with a founder effect event due to the selection processes. Origin of C.Baccatum cultivated forms in South America We found two different haplotypes (H2 and H11) in the cultivated forms, with the red haplotype (H2) the most common, present in 12 cultivated accessions of the 13 studied. The geographical distribution of this haplotype in the wild suggests that lowlands of Bolivia and inter-Andean valleys of Peru have been the most important centres of C. baccatum domestication (Fig. 4A). Thus, phylogeographical methods support the early hypothesis of these regions as sites of domestication of this species, occurring by 4000 years BP at the latest, probably by Arawak people inhabiting the Llanos de Mojos (i.e. Moxeños), Guarani populations, both in Bolivia, or pre-Incas in Peru (see Fig. 4A) (Perry et al., 2007; Piperno, 2011; Brown et al., 2013; Clement et al., 2016). The upper Madeira River basin inhabited by Moxeño people was a probable region of origin of other crops, such as other chili peppers (C. pubescens), three legume seeds (Arachis hypogaea L., Canavalia plagiosperma Piper, Phaseolus vulgaris L.) and cocoyam [Xanthosoma sagittifolium (L.) Schott], important drugs, such as coca (Erythroxylum coca Lam.), and tobacco (Nicotiana tabacum L.), and the major root crop of Amazonia, manioc (Manihot esculenta Crantz) and peach palm (Bactris gasipaes Kunth) (Grabiele et al., 2012; Clement et al., 2016, and references herein). Capsicum baccatum var. umbilicatum originated from the west clade, as did var. pendulum, although this result is in disagreement with those reported by Albrecht et al. (2012). The only C. baccatum var. umbilicatum accession included in their analysis was collected from Brazil (São Paulo); it was deeply nested within the ‘eastern’ Group and closely related to other cultivars from Brazil, primarily those from Minas Gerais. Figure 4. View largeDownload slide Maps of South America showing the geographical distribution of haplotypes along with different addressed aspects. A, distribution of aboriginal populations in South America before the Spanish conquest and geographical distribution of wild and cultivated samples of Capsicum baccatum. B, schematic distributions of the Seasonally Dry Tropical Forests (SDTF; adapted from Prado, 2000; Caetano et al., 2008) and their relations with haplotype structure in the studied C. baccatum var. baccatum populations. Figure 4. View largeDownload slide Maps of South America showing the geographical distribution of haplotypes along with different addressed aspects. A, distribution of aboriginal populations in South America before the Spanish conquest and geographical distribution of wild and cultivated samples of Capsicum baccatum. B, schematic distributions of the Seasonally Dry Tropical Forests (SDTF; adapted from Prado, 2000; Caetano et al., 2008) and their relations with haplotype structure in the studied C. baccatum var. baccatum populations. Our results indicate that various accessions grow in nature, whose haplotypes or genetic variants do not correspond to the wild-type expected lineage according to phylogenetic and phylogeographical analyses. All of these wild specimens had the red haplotype (H2; PRF, PRN, SMA, TEY, SPO), and one derived from it (H3; SAN) in only one case. Fruit size is generally intermediate between that of cultivated and wild plants (Fig. 1E). One possible explanation could be hybridization with wild parental species. In many traditional communities throughout Latin America, wild and semi-wild Capsicum species are often found growing close to cultivated peppers, in home gardens or at the edges of cultivated fields, where introgressive hybridization takes place between the cultigens and closely related wild species or sub-species, spontaneously giving rise to novel or intermediate forms (Van Zonneveld et al., 2015). Notably, accessions here named ‘became wild’ have been found only in the east of the species distribution. The region comprising Paraguay, Brazil and Misiones province in Argentina was inhabited by Guarani peoples before arrival of Europeans (Fig. 4A). They were traditionally farmers and cultivated maize (acquired by interethnic contact), cassava (probably domesticated by them), peanut and sweet potato. Occupation of their lands dedicated to agriculture together with cultural erosion, among other factors, would have caused the abandonment of this intensive practice and many of the crops may have been neglected (Keller, 2012). Hence, we propose that these agricultural peoples once cultivated chili peppers (domesticated in the west), probably obtained through commercial practices. After European conquest, this activity declined, which might have caused the de-selection in attributes of the cultivated fruits (e.g. from large to small size fruit) (F. Zamudio IMBIV, pers. comm.); since then, plants with smaller fruits may have continued to grow in the wild and ‘become wild’. The fact that these specimens always carried the cultivated haplotype type (H2) reinforces the idea of their origin as abandoned crops. Chiou et al. (2014) noted the reduction of seed size after the Spanish conquest (after 1532 CE), suggesting a loss of selective pressure for seed size in this particular Capsicum species. The cultivated accession EAM & RN 215 from INTA Cerrillos has a different haplotype (H11), which could indicate a secondary origin of domestication for C. baccatum var. pendulum or could reflect an ancient site of cultivation of the species where hybridization between cultivated and local samples may have occurred. Multiple domestications of different populations of the same species are rare, but there are some records, such as the best known examples of common bean (P. vulgaris) and Lima bean (P. lunatus L.) (Chacón, Pickersgill & Debouck, 2005; Serrano-Serrano et al., 2010). According to the haplotype network, the haplotype found in this accession is derived from a wild haplotype found in Aguas Blancas, at the frontier between Argentina and Bolivia. Ethnobotanical studies in Baritu National Park (Salta, Argentina) indicate that this region was occupied in pre-Hispanic times by a group of ‘Chiriguanos’ belonging to the Guarani group, who originated from the north-east of the Bermejo River basin (Fig. 4A). These avid farmers had commercial relationships with people from Tarija and Padcaya in Bolivia, and Santa Victoria in Argentina (Hilgert 1999) and they probably cultivated and commercialized C. baccatum. Phylogeographical implications Two principal genetic groups were identified in the wild C. baccatum accessions in relation to their geographical distribution in South America. One group included accessions from the western territories of the species distribution (Peru, Bolivia and north-western Argentina) and corresponds to SDTF nuclei of the Andean Piedmont and Peruvian inter-Andean valleys. The second group included samples from the eastern regions (Brazil, Paraguay and eastern Argentina) and corresponds to SDTF nuclei from Chiquitano and Misiones. Capsicum baccatum is in fact not considered in the species database of these forests (: database http://www.dryflor.info/data), and consequently, it was not considered in a recent study of these flora (Banda et al., 2016). Nonetheless the species could effectively be integrated into this database, highlighting the importance of conservation of this biome as a reserve of wild relatives from cultivated species. Our results supported by molecular dating indicate that a large split between the western and eastern groups of wild C. baccatum occurred at 2.3 Mya. A Late Pliocene to Early Pleistocene cooling and drying pulse was detected worldwide at about 2.5 Mya, in times of the Great Patagonian Glaciation (Rabassa, 2008). Ice covered part of the Andean region in Patagonia and part of the extra-Andean region, where extensive areas of frozen soils developed. The result would have been a displacement of the Dry Chaco region (Fig. 4B) northward, where the Chiquitano STDF nucleus exists today. Phylogeographical studies developed in Astronium urundeuva (Allemão) Engl. and Anadenanthera colubrina (Vell.) Brennan showed west and east groupings of populations located at sites that are comparable to our study sites (see fig. 2 in Caetano et al., 2008 and fig. 2 in Barrandeguy et al., 2016). The Pleistocene Arc Theory (PAT) suggests that present-day disjunct fragments of dry forests in central tropical South America provide evidence of a previously more continuous distribution during the Pleistocene that has been disrupted by dry-cold vs. humid-warm climatic cycles. Prado & Gibbs (1993) hypothesized that the increasing temperatures and precipitation over the last glacial–Holocene transition caused contraction and fragmentation of the Pleistocene dry forest arc into the present-day disjunct distribution of dry forest nuclei. We propose that, in contrast to expectation, the SDTFs in their southernmost distribution have been fragmented during glaciation events due to advance of the Chaco region from the south. This agrees with Pennington et al. (2004), who postulate that Chiquitano nucleus in Bolivia would have moved north rather than expanded during periods of glaciation. Population genetics studies remain very scarce and cover a relatively small part of the total distribution range of SDTF. Ecological niche modelling developed by Werneck et al. (2011) and Turchetto-Zolet et al. (2016) provides evidence that the SDTFs were even more disjunct during the LGM. Finally, areas of presumed long-term stability are identified and confirmed (Caatinga, Misiones, Piedmont and Chiquitano region), and possibly acted as current and historical refugia (Werneck et al., 2011). By contrast, the demographic history of Tabebuia impetiginosa (Mart. ex DC.) Standl. showed that the pattern of diversity found for the species is in agreement with distribution expansion during the LGM (Collevatti et al., 2012). This species is distributed in the northern relicts of SDTF in South America and Mesoamerica. The results obtained here contribute to the discussion regarding the PAT. Thus, the response of the SDTF to glaciation events could have depended on the STDF nucleus: northern nuclei have expanded over the Amazon forest, whereas the southernmost nuclei have undergone fragmentation because of increased area of drier regions, and moved their range north. Further studies in representative species from this biome are needed to evaluate the response to climatic change and to elaborate upon conservation actions. These forests are priority areas for conservation because they are the home of wild ancestors of many important crop species. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Table S1. Variable sites of the aligned chloroplast DNA sequences from the rpl32F-trnL region for the 18 haplotypes in Capsicum baccatum. Table S2. Variable sites of the aligned chloroplast DNA sequences from the trnQ-rps16 region for the 18 haplotypes in Capsicum baccatum. Database S1. Sequence dataset used in the phylogeographical analysis. ACKNOWLEDGEMENTS We are grateful to L. Boero, C. Carrizo, J. Daviña, A. Honfi, E. Kowaljow, M. Renny, V. Romero, G. Seijo and A. Sérsic who collected some leaf material. A. Romanutti provided valuable help in the laboratory and J. Brasca collaborated in the final editing of the manuscript. We thank three anonymous reviewers for their helpful comments. Financial assistance was provided by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP 114-200801-00326), Agencia de Promoción Científica y Tecnológica (PICT 573), Ministerio de Ciencia y Tecnología de la Provincia de Córdoba, and Secretaría de Ciencia y Tecnología (SECYT-UNC). MAS, GB and MCA are members of Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina and Universidad Nacional de Córdoba, Argentina. Dedicated to the Memory of Eduardo A. Moscone REFERENCES Albrecht E, Zhang D, Saftner RA, Stommel JR. 2012. Genetic diversity and population structure of Capsicum baccatum genetic resources. Genetic Resources and Crop Evolution  59: 517– 538. Google Scholar CrossRef Search ADS   Avise JC. 2000. Phylogeography: the history and formation of species . Cambridge: Harvard University Press. 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