Annals of Botany 95: 601–608, 2005 doi:10.1093/aob/mci062, available online at www.aob.oupjournals.org Inter- and Intra-specific Variation among Five Erythroxylum Taxa Assessed by AFLP EMANUEL L. JOHNSON*, DAPENG ZHANG and STEPHEN D. EMCHE 1USDA ARS PSI ACSL, 10300 Baltimore Avenue, BARC-W, Beltsville, MD 20705, USA Received: 5 March 2004 Returned for revision: 23 September 2004 Accepted: 15 November 2004 Published electronically: 13 January 2005 Background and Aims The four cultivated Erythroxylum taxa (E. coca var. coca, E. novogranatense var. novogranatense, E. coca var. ipadu and E. novogranatense var. truxillense) are indigenous to the Andean region of South America and have been cultivated for folk-medicine and, within the last century, for illicit cocaine pro- duction. The objective of this research was to assess the structure of genetic diversity within and among the four cultivated alkaloid-bearing taxa of Erythroxylum in the living collection at Beltsville Agricultural Research Center. Methods Amplified fragment length polymorphism (AFLP) fingerprinting was performed in 86 Erythroxylum accessions using a capillary genotyping system. Cluster analysis, multidimensional scaling (MDS) and analysis of molecular variance (AMOVA) were used to assess the pattern and level of genetic variation among and within the taxa. Key Results A clear distinction was revealed between E. coca and E. novogranatense. At the intra-specific level, significant differentiation was observed between E. c. var. coca and E. c. var. ipadu, but the differentiation between E. n. var. novogranatense and E. n. var. truxillense was negligible. Erythroxylum c. var. ipadu had a significantly lower amount of diversity than the E. c. var. coca and is genetically different from the E. c. var. ipadu currently under cultivation in Colombia, South America. Conclusions There is a heterogeneous genetic structure among the cultivated Erythroxylum taxa where E. coca and E. novogranatense are two independent species. Erythroxylum coca var. coca is most likely the ancestral taxon of E. c. var. ipadu and a founder effect may have occurred as E. c. var. ipadu moved from the eastern Andes in Peru and Bolivia into the lowland Amazonian basin. There is an indication of artificial hybridization in coca grown in Colombia. ª 2005 Annals of Botany Company Key words: Erythroxylum coca var. coca, Erythroxylum coca var. ipadu, Erythroxylum novogranatense var. novogranatense, Erythroxylum novogranatense var. truxillense, AFLP markers, genetic variation, cultivated coca, DNA fingerprinting, cocaine, tropical plants. INTRODUCTION Since the 1970s, morphology, breeding systems and chemotaxonomic data were the primary descriptors used to The extensive living collection of Erythroxylum at the detail the differences between the cultivated Erythroxylum Beltsville Agricultural Research Center, Beltsville, MD, taxa (Bohm et al., 1982; Johnson et al., 1997, 1998, 2002, USA, which has been maintained since the early 1970s, 2003a; Johnson and Schmidt, 1999). With the identification contains the four cultivated alkaloid-bearing varieties: of molecular markers and their associated specificity, Erythroxylum coca var. coca Lam (E. c. var. coca); further assessment of the genetic diversity among the cult- Erythroxylum coca var. ipadu Plowman (E. c. var. ivated Erythroxylum taxa is warranted in order to accrete ipadu); Erythroxylum novogranatense var. novogranatense and refine the existing morphological and chemotaxonomic- (Morris) Hieron (E. n. var. novogranatense); Erythroxylum based classification system. While a variety of molecular novogranatense var. truxillense [Rusby] Plowman (E. n. var. assays could be used to assess the genetic diversity, each truxillense). The geographical, ecological and morpho- method differs in principle, application, the amount of poly- logical differences of these taxa were detailed as early as morphism detected, cost and time required. In a previous the 16th century (Ganders, 1979; Plowman, 1979, 1982, study, amplified fragment length polymorphism (AFLP) 1984; Rury, 1981; Schultes, 1981). However, it was not (Vos et al., 1995) was used to analyse 132 accessions of until the 1970s that cultivated coca was determined to be Erythroxylum to characterize and positively identify the four derived from two species of the genus Erythroxylum; E. c. cultivated taxa, as well as a feral taxon (Johnson et al., var. coca Lam and E. n. var. novogranatense (Morris) Hieron 2003b). The first objective of the current study was to (Plowman, 1979, 1982, 1984; Bohm et al., 1982). This clas- examine further the taxonomic status, and elucidate the sification, according to Plowman and Rivier (1983), has evolutionary relationship, of the four cultivated alkaloid- been supported multifactorially through interdisciplinary bearing Erythroxylum taxa. The second objective was to research (Johnson et al., 2003b). Furthermore, breeding detect and quantify the inter- and intra-specific genetic vari- evidence and eco-geographical data suggests that the most ation in these taxa. Eighty-five Erythroxylum accessions, likely phylogeny for the four cultivated taxa is a linear evolu- which are representative of the four cultivated taxa in the tionary sequence, wherein E. c. var. coca is the ancestral living collection, were analysed using AFLP genotyping taxon that gave rise to E. n. var. truxillense which gave rise, in combination with cluster and ordination analysis. The in turn, to E. n. var. novogranatense (Bohm et al., 1982). resulting information provided insights into the structure * For correspondence. E-mail [email protected] and pattern of genetic diversity of Erythroxylum in the Annals of Botany 95/4 ª Annals of Botany Company 2005; all rights reserved 602 Johnson et al. — Variation among Cultivated Coca as Assessed by AFLP living collection at Beltsville Agricultural Research Center ethnobotany, morphological characterization, alkaloid and the Andes region of South America. content, breeding system, chemotaxonomic data and geo- graphical distribution have been summarized previously (Ganders, 1979; Plowman, 1979, 1981, 1982, 1983, 1984; MATERIALS AND METHODS Rury, 1981; Schultes, 1981; Bohm et al., 1982; Plowman Plant material and Rivier, 1983; Johnson et al., 1997, 1998, 1999, 2002, 2003b; Johnson and Schmidt, 1999). Due to the confusion Young expanding leaf tissue was harvested from 86 samples, by investigators for the four cultivated alkaloid-bearing taken from the living collection at Beltsville Agricultural taxa it was considered necessary to show how the taxa Research Center, of E. coca var. coca Lam, E. coca var. differed using AFLP DNA analysis. The living collec- ipadu Plowman, E. novogranatense var. novogranatense tion of Erythroxylum at Beltsville Agricultural Research (Morris) Hieron, E. novogranatense var. truxillense Center was authenticated by T. Plowman in 1988 and (Rusby) Plowman and E. ulei O.E. Schulz, as well as some re-authenticated by P. M. Rury in 1993. Plants derived F1 Erythroxylum coca var. ipadu propagules (Table 1). The from the living collection and authenticated by Rury were transferred to a Hawaiian field site. The Hawaiian T ABLE 1. Sample table of 86 Erythroxylum c. var. coca, field site was located on the Island of Kauai and was Erythroxylum c. var. ipadu, Erythroxylum n. var. novograna- selected by the US Department of Agriculture, Agricultural tense, E. n. var. truxillense and Erythroxylum ulei samples Research Service and the State of Hawaii because of the from the living collection at the Beltsville Agricultural similarity of soils to those found in the coca-growing Research Center regions of Bolivia and Peru. The pH of the soil at the Hawaiian field sites ranged from 4Á0to5Á7, which was Species Accession tag Origin Species Accession tag Origin ideal for coca growth. The harvested leaf tissues were sepa- rately placed in labelled Zip-Loc bags, immediately stored coca B102LS Bolivia novo B292LS Bolivia at 0 C and transported to the laboratory for DNA extraction coca B85LS Bolivia novo B253LS Bolivia coca B88LS Bolivia novo B205LS Bolivia and analysis. Erythroxylum ulei in the current study was coca B14LS Bolivia novo B300LS Bolivia only used to verify the consistency of the AFLP analysis and coca B104LS Bolivia novo B201-1LS Bolivia was not part of the quantitative analysis. coca B110LS Bolivia ipadu F-1 B508 Beltsville coca B56LS Bolivia ipadu F-1 B508 Beltsville coca B96LS Bolivia ipadu F-1 B501SS Beltsville Isolation of DNA from leaf tissue coca B180LS Bolivia ipadu F-1 B503SS Beltsville coca B105LS Bolivia ipadu F-1 B508 Beltsville Genomic DNA (i.e. total DNA) was extracted from leaf coca B63LS Bolivia ipadu F-1 B501SS Beltsville tissue of the five taxa using a modification of the Qiagen coca B127LS Bolivia ipadu F-1 B504 Beltsville DNA Stool Mini KitTM protocol (Qiagen Inc., Valencia, CA, coca B31LS Bolivia ipadu F-1 B503SS Beltsville coca B60LS Bolivia ipadu B501SS Bolivia USA). One hundred milligrams f. wt (i.e. 20 mg d. wt.) of coca B150LS Bolivia ipadu B503SS Bolivia leaf tissue were weighed and placed into a 2-mL lysing coca B94LS Bolivia ipadu B507 Bolivia matrix cylinder containing two 0Á53-cm ceramic spheres coca B98LS Bolivia ipadu B504 Bolivia (QBiogene, Inc., Carlsbad, CA, USA) with 35 mg of poly- coca B9LS Bolivia ipadu B505 Bolivia vinylpolypyrrolidone (Sigma Chem., Co., St Louis, MO, coca B80LS Bolivia ipadu B508 Bolivia coca B147RLS Bolivia ipadu F-1 B504 Beltsville USA). Then 1Á4 mL of the buffer ASL was added and coca B50LS Bolivia ipadu F-1