Theor Appl Genet (1999) 98: 202Ð212 ( Springer-Verlag 1999 B. Fofana á J. P. Baudoin á X. Vekemans D. G. Debouck á P. du Jardin Molecular evidence for an Andean origin and a secondary gene pool for the Lima bean (Phaseolus lunatus L.) using chloroplast DNA Received: 1 May 1998 / Accepted: 13 July 1998 Abstract Chloroplast DNA (cpDNA) diversity has Andean forms of the Lima bean are found to be more been examined using PCR-RFLP and RFLP strategies closely related to the 3 Andean wild species than its for phylogenetic studies in the genus Phaseolus. Mesoamerican forms. An Andean origin of the Lima Twenty-two species, including 4 of the 5 cultivated bean and a double derivative process during the evolu- species (P. lunatus ¸., the Lima bean; P. vulgaris L., the tion of P. lunatus are suggested. The 3 Andean species common bean; P. coccineus L., the runner bean and are proposed to constitute the secondary gene pool of P. polyanthus Greenman, the year-bean), represented P. lunatus, while its companion allies of Mesoamerican by 86 accessions were included in the study. Six PCR distribution can be considered as members of its primers designed from cpDNA and a total cpDNA tertiary gene pool. On the basis of these data, an over- probe were used for generating markers. Phylogenetic view on the evolution of the genus Phaseolus is also reconstruction using both Wagner parsimony and the discussed. neighbor-joining method was applied to the restriction fragment data obtained from each of the molecular Key words Intergenic regions á Molecular markers á approaches. P. vulgaris L. was shown to separate with Plant genetic resources á Phylogeny á Mesoamerica several species of largely Mesoamerican distribution, including P. coccineus L. and P. polyanthus Greenman, whereas P. lunatus L. forms a complex with 3 Andean Introduction species (P. pachyrrhizoides Harms, P. augusti Harms and P. bolivianus Piper) co-evolving with a set of com- In the context of conservation of plant genetic re- panion species with a Mesoamerican distribution. sources, a better knowledge of the phylogenetic rela- tionships within genera of crop species is of great importance to germplasm curators and plant breeders. Phylogenetic investigations are useful for identifying Communicated by P. M. A. Tigerstedt the wild progenitors of domesticated species (Doebley B. Fofana á J. P. Baudoin 1992) and suggesting putative members of their second- Unite« de Phytotechnie des Re«gions Intertropicales, ary and tertiary gene pools, which may help to define Faculte« des Sciences agronomiques de Gembloux, 2, priorities in sampling for ex situ collections as well as in Passages des De«porte«s, B-5030 Gembloux, Belgium the management of in situ conservation programs B. Fofana ( ) á P. du Jardin (Frankel et al. 1995). Unite« de Biologie ve«ge«tale, Faculte« des Sciences Phylogenetic studies on the origin of cultivated agronomiques de Gembloux, 2, Passages des De«porte«s, plants are classically based on evidence from morpho- B-5030 Gembloux, Belgium Fax:#32-81-600727 logy (Piper 1926; Mare«chal et al. 1978; Delgado 1985), E-mail: [email protected] seed-protein electrophoresis (Johnson 1972; Sullivan and Freytag 1986; Gepts et al. 1986) and allozyme X. Vekemans Laboratoire de Ge«ne«tique et d’Ecologie ve«ge«tales, variation (Doebley et al. 1984; Second 1982). From the Universite« Libre de Bruxelles, 1850, Chausse«e de Wavre, late 1980s, molecular markers involving chloroplast B-1160 Bruxelles, Belgium DNA (cpDNA) variation have been extensively used to D. G. Debouck resolve conflicting phylogenies in cultivated taxa Genetic Resources Unit, International Centre for Tropical (Doebley et al. 1987; Neale et al. 1988; Ogihara and Agriculture, Apartado Ae«reo 6713, Cali, Colombia Tsunewaki 1988; Wolf et al. 1997). The reason for 203 focusing on cpDNA variation lies in its conservative to Mare«chal et al. (1978), Baudoin (1988) and Debouck rate of evolution, both in terms of genome size and (1991), two groups of wild species are suspected to structure (Palmer 1987), its maternal inheritance, the belong to the clade of P. lunatus: (1) wild species of availability of cpDNA probes (Llaca et al. 1994; Jack Mesoamerican distribution such as P. ritensis Jones, P. et al. 1995) and, more recently, universal primers for maculatus Scheele, P. jaliscanus Piper, P. marechalii polymerase chain reaction (PCR) amplification of Delgado, P. salicifolius Piper and a specimen currently cpDNA sequences (Ogihara et al. 1991; Taberlet et al. not determined referred to here as P. sp.; and (2) wild 1991; Demesure et al. 1995; Fofana et al. 1997a). The species of Andean distribution such as P. augusti molecular techniques commonly applied to the study of Harms, P. bolivianus Piper and P. pachyrrhizoides cpDNA variation include (1) isolation of cpDNA fol- Harms. These two groups of species will be referred to lowed by digestion with restriction enzymes and elec- as the Mesoamerican and the Andean wild allies of P. trophoretic separation; (2) restriction digestion of total lunatus, respectively. In a recent study, Maquet (1995) DNA followed by Southern analysis using cpDNA- concluded that on the basis of seed protein patterns specific probes [hereafter called the probed-restriction and allozyme variation P. lunatus was more closely fragment length polymorphism (RFLP) method]; and related to its Andean wild allies than to the Mesoameri- (3) direct sequencing of cpDNA regions. These methods can ones. This evidence was used by the author to are powerful, but they restrict most analyses to small suggest an Andean origin for the species P. lunatus. sample sizes due to their inherently high cost and time However, phylogenetic information on several wild investments (Weatherhead and Montgomerie 1991). species of Mesoamerican distribution such as Recently, some PCR-based methods like the amplifica- P. xolocotzii Delgado, P. filiformis Benth., P. angustis- tion of coding and/or non-coding sequences followed simus A. Gray, P. oligospermus Piper, P. hintonii De- by restriction digestion (hereafter called the PCR- lgado, P. grayanus Woot, Standley, P. microcarpus RFLP method) have increasingly been used in phylo- Mart., P. pedicellatus Benth., P. leptostachyus Benth. genetic studies (Pe«rez de la Rosa and Farjon 1995; and the eastern United States species P. polystachyus Tsumura et al. 1995). Furthermore, because non-cod- B.S.P. is still scarce. All of the above-mentioned species ing sequences of the chloroplast genome are expected belong to the section Phaseolus of the genus with the to evolve more rapidly than coding sequences (Wolfe exception of P. hintonii that belongs to section Xan- and Sharp 1988; Wolfe et al. 1987), primers have been thotricha (Delgado 1985). In addition, no phylogenetic designed for the amplification of intergenic regions of studies based on molecular data have been carried out cpDNA (Taberlet et al. 1991; Demesure et al. 1995; with both Mesoamerican and Andean wild species. Dumolin-Lape` gue et al. 1997; Fofana et al. 1997a). In this study, we investigate phylogenetic relation- Phaseolus is a large, diverse genus of at least 50 ships among 22 species belonging to the genus species that grow naturally in warm tropical and sub- Phaseolus, with special emphasis on the group of spe- tropical regions of the New World, from Sinaloa, cies currently described as wild allies of P. lunatus.We Mexico, to Salta, Argentina (Debouck et al. 1987; specifically address the issues whether the putative wild Delgado 1985). Phylogenetic relationships among allies of P. lunatus are phylogenetically closer to P. Phaseolus species have been investigated using mor- lunatus than to the P. vulgarisÐP. coccineus complex, phological (Mare«chal et al. 1978; Debouck 1991), bio- whether the Andean wild allies are more closely related chemical (Sullivan and Freytag 1986; Jaaska 1996; to P. lunatus than the Mesoamerican wild allies and Pueyo and Delgado 1997) as well as molecular markers whether the two gene pools of P. lunatus form together (Delgado et al. 1993; Hamann et al. 1995; Schmit et al. a monophyletic group with respect to other taxa. We 1993; Llaca et al. 1994; Vekemans et al. 1998). These use variation in cpDNA assessed by two distinct tech- studies identified a complex of species including the niques, i.e. PCR-RFLP of intergenic regions (IGRs) cultivated P. vulgaris L., P. coccineus L. and P. polyan- and total cpDNA probed-RFLP, and two phylogenetic thus Greenman (hereafter called the P. vulgarisÐP. reconstruction methods, i.e. neighbor-joining and coccineus complex) and showed that P. lunatus L., the Wagner parsimony. Lima bean, which is ranked second in economical im- portance among the cultivated species of the genus Phaseolus, was very distantly related to that complex. Materials and methods Furthermore, detailed studies in P. lunatus showed convincingly that the whole primary gene pool of the Plant material and DNA extraction Lima bean is divided into two main groups: a Me- soamerican group and an Andean group (Debouck et Young leaves were collected in the greenhouse from 52 accessions of al. 1989; Maquet et al. 1990; Nienhuis et al. 1995, P. lunatus including 40 wild accessions, 1 weedy, and 11 landraces as Fofana et al. 1997b). Each group comprises both wild well as from 34 accessions corresponding to 21 wild species includ- ing P. vulgaris, P. coccineus and P. polyanthus (Table 1). These plant and cultivated forms, but the evolutionary relation- materials were chosen either in the world seed bank of the Genetic ships between these two groups and companion species Resource Unit of the CIAT, Cali, Colombia (for codes G, DGD, or of the Lima bean remain poorly understood. According PL) or in the base collection of the Belgium National Botanic 204 Table 1 List, biological status and origin of di¤erent species Accession Species Status Origin! and ecotypes of the Lima Number bean 1 G25221 P.