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PLANT GENETIC RESOURCES

AFLP Fingerprinting of Phaseolus lunatus L. and Related Wild Species from South America

^a Biotechnology Research Unit, CIAT, Cali, Colombia
^b Genetic Resources Unit, CIAT, Cali, Colombia

j.tohme{at}cgiar.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
The taxonomic classification of the wild Lima bean complex needs to be assessed to select species for use in breeding programs and to identify genetic resources for conservation. The objectives of this study were to determine the genetic relationships among, and the phylogeny of, wild Lima bean (Phaseolus lunatus L.) and related species (P. augusti Harms, P. bolivianus Piper, P. pachyrrhizoides Harms, and P. rosei Piper) from South America and to identify specific genetic reserves for conservation. These relationships were investigated by means of amplified restriction fragment length polymorphism (AFLPs) on total genomic DNA. The 122 accessions formed a cluster that was distant from common bean (P. vulgaris L.), confirming earlier morphology and hybridology data. Two gene pools of wild Lima beans were confirmed. One was widely distributed in neotropical lowlands, while the other was restricted to the western Andes, in Ecuador and northern Peru. The study also revealed the existence of a third group of wild Lima bean distributed in the Departments of Boyacá and Cundinamarca, Colombia. The three species P. augusti, P. bolivianus, and P. pachyrrhizoides differed very little, certainly not sufficiently to merit a separate taxonomic ranking at the species level. The accessions could be grouped instead according to four geographic origins: Ecuador and northern Peru; Department of Junín, Peru; Departments of Cuzco and Apurímac, Peru; and Bolivia and northwestern Argentina. Results from this study should result in a better selection of parental materials in breeding programs and point to areas where germplasm collections and conservation are needed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
THE GENUS Phaseolus includes five grain legumes of worldwide or regional economic importance, and approximately 50 species, all of neotropical origin (Delgado Salinas, 1985; Lackey, 1983; Maréchal et al., 1978). Although floristic surveys of the tropical Western Hemisphere are largely incomplete, evidence suggests that more biological species exist in North America than in South America (Delgado Salinas, 1985). Approximately 45 species of wild beans are distributed from Panama to southern Canada (Debouck, 1991; Delgado Salinas, 1985), whereas only four to six wild species are native to South America.

Most South American Phaseolus sensu stricto species have their range of distribution apparently limited to the Andean orographic system from the Venezuelan Andes to the Sierra de Córdoba in Argentina. Exceptions are P. mollis Hooker, which is restricted to the Galapagos archipelago (Wiggins and Porter, 1971), and P. lunatus, which is present in the eastern tropics of the South American lowlands (Lewis, 1987). No wild Phaseolus species have been reported in Chile (Lackey, 1983).

The range of wild P. vulgaris, the ancestor of common bean, in South America, extends in the Andes from western Venezuela to San Luis in Argentina (Toro Ch. et al., 1990). Phaseolus polyanthus Greenman (synonyms: P. flavescens Piper, P. harmsianus Diels) is cultivated in the northern Andes (Berglund-Brücher and Brücher, 1974) and exists as a feral species in secondary forests in the Andes from western Venezuela, south to the Department of Apurímac, Peru (Schmit and Debouck, 1991). Wild P. lunatus, the Lima bean, exists as two major groups of morphologically distinct populations. One is distributed in the lowlands of eastern South America, stretching from the Caribbean coast, through Brazil and eastern Peru, to Salta, Argentina. The other group is distributed in the western Andes, in Ecuador and northern Peru (Debouck et al., 1987; Gutiérrez Salgado et al., 1995). Phaseolus vulgaris and the two wild forms of P. lunatus appear to be genuine floristic components of natural dry and subhumid South American forests. Molecular markers studies have shown intrinsic differences within the wild forms of common bean (Khairallah et al., 1992; Tohme et al., 1996) and Lima bean present in Mesoamerica (Gutiérrez Salgado et al., 1995; Maquet et al., 1994). These studies have also shown that wild forms are more polymorphic than the cultivated ones.

Two species, P. augusti Harms and P. pachyrrhizoides Harms, from Huancavelica and Junín, Peru, respectively, were described by Harms (1921), the latter having larger peduncles and bracts. Macbride (1943), however, observed that few differences exist between these two taxa, although P. pachyrrhizoides sometimes displays asymmetrical, lobed, lateral leaflets. Phaseolus bolivianus Piper was described as a new species from Cochabamba, Bolivia (Piper, 1926). This species and P. pachyrrhizoides were mentioned in this review, but not P. augusti, and were not cross-referenced to either of them (Piper, 1926). Phaseolus bolivianus was not sufficiently distinct from P. augusti and later was considered synonymous (Macbride, 1943). In a numerical taxonomy analysis, Maréchal et al. (1978) placed P. augusti close to P. coccineus and endorsed the synonymous classification. Lackey (1983) recognized P. pachyrrhizoides and P. bolivianus, but not P. augusti. A species from Ecuador, P. rosei Piper, was considered as probably annual (Piper, 1926), whereas Toro Ch. et al. (1993) considered this species to be not different from the Andean wild form of Lima bean. In a catalogue of Peruvian flowering plants, Brako and Zarucchi (1993) recognized only P. augusti, P. lunatus, P. pachyrrhizoides, P. polyanthus, and P. vulgaris. Phaseolus augusti extends from Ecuador (Debouck et al., 1989), through Peru (Brako and Zarucchi, 1993) and Bolivia (Foster, 1958), to Argentina (Palacios and Vilela, 1993). In contrast, the distribution of P. pachyrrhizoides is restricted to the Peruvian Andes (Brako and Zarucchi, 1993; Macbride, 1943).

Several questions need to be answered: (i) are P. augusti and P. pachyrrhizoides related to P. lunatus, (ii) are they distinct species (iii) can P. bolivianus be merged taxonomically with P. augusti, and (iv) is P. rosei different from the Andean wild Lima bean. The AFLP fingerprinting technique (Vos et al., 1995) has been successfully applied to the study of genetic diversity of a core collection of wild common bean held at CIAT (Tohme et al., 1996). The objectives of this study were (i) to determine the genetic relationships among species from South America thought to be related to the Lima bean by means of AFLP fingerprinting and (ii) to identify specific genetic resources for conservation.

	Materials and methods

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
A diverse collection of 71 populations of South American species (with one or two individuals per population and totaling 122 genotypes) was obtained from the world collection of Phaseolus maintained at CIAT (Fig. 1 and Table 1) . Populations collected at the type locality for the species P. bolivianus (BBLCO123), P. pachyrrhizoides (PPEJU102), and P. rosei (LECCH112/113) were included. Five wild accessions and one cultivated line of P. vulgaris (ICA-Pijao) were also included to determine genetic distances.

View larger version (33K): [in this window] [in a new window]   Fig. 1 Distribution map of genotypes of Phaseolus species used in this study

Primers complementary to adapter sequences, having one additional nucleotide on their 3' end, were used to carry out a selective pre-amplification of the DNA template. The primer sequence complementary to the EcoRI end was: 5'-GACTGCGTACCAATTCA-3'(E + A). The MseI primer sequence was: 5'-GATGAGTCCTGAGTAAG-3' (M + G). Underlined letters correspond to the first selective nucleotide. Five microliters of digested and ligated DNA were separated and mixed with 25 µL of primer cocktail (75 ng E + A, 75 ng M + G, 200 µM dNTPs) and 20 µL of Taq polymerase cocktail (1 unit Taq polymerase, 1x PCR buffer). Samples were amplified with a PTC-100 programmable thermal controller (MJ Research, Inc, Watertown, MA) with the following PCR profile: 35 cycles of 30 s at 94°C, 30 s at 55°C, and 60 s at 72°C. To verify adequate amplification, 20 µL of the amplification reaction were observed as smears on an ethidium bromide stained agarose gel. The rest of the amplification product was diluted 20 fold in TE buffer. Two primer pairs were used to carry out the final selective amplification of the DNA restriction fragments. The primer complementary to the EcoRI end of the DNA template, E + AAC (5'-GACTGCGTACCA ATTCAAC-3'), was used in combination with the MseI primer M + GTA (5'-GATGAGTCCTGAGTAAGTA-3'). Another EcoRI primer, E + AGT (5'-GACTGCGTACCAATTCAGT-3') was used in combination with the MseI primer, M + GAC (5'-GATGAGTCCTGAGTAAGAC-3'). The primer combinations will be referred to as PE1A/PM1A, and PE1C/PM1C respectively. EcoRI primers (5 ng) were labeled radioactively with -³²P (0.2 µL [-³²P]ATP 3000 Ci/mmol, 1x One-Phor-All buffer, 0.1 U T4PNK, adjusted to a volume of 0.5 µL with water) in a water bath at 37°C for 30 min. The phosphonucleokinase was then inactivated by placing in a water bath at 70°C for 10 min.

The PCR reaction mixture consisted of 5 µL of the diluted PCR +1/+1 product, along with 5 µL of primer mix (5 ng labeled EcoRI primer, 30 ng MseI primer, 200 µM dNTPs), and 10 µL of the amplification mixture (0.4 U Taq polymerase, 1x PCR buffer). The PCR reaction was carried out in a PTC-100 with the following profile: 12 cycles of 30 s at 94°C, 30 s at 65°C (-0.7°C/cycle), and 30 s at 72°C, 25 cycles of 30 s at 94°C, 30 s at 56°C, and 60 s at 72°C. Amplification products were mixed with an equal volume of stop solution (Amersham), denatured at 95°C, and 4 µL were loaded into a 6% (w/v) polyacrylamide gel in 1x TBE electrophoresis buffer. Gels were dried out on Whatman 3MM paper (Whatman Lab, Hillsboro, OR) and exposed at least fourteen hours to Kodak, X-OMAT LS 35.3 x 43.2 cm films (Sigma-Aldrich Co., Saint Louis, MO) at room temperature. In each gel, a common bean accession, ICA-Pijao, was included three times to allow comparisons of bands among gels. Data were computed as 1/0, corresponding to presence or absence, respectively, of heavy bands. Minor or slightly marked bands were ignored.

Genetic similarities among all accessions were calculated with the Nei-Li coefficient (Nei and Li, 1979), that is, S = 2a/(2a + b + c), where a = bands shared by both individuals, b = bands presented by individual (1) but not by (2), and c = bands presented by individual (2) but not by (1). Dendrograms were constructed with SAHN clustering of NTSYS-PC, version 1.80 (Rohlf, 1993), by the UPGMA method (unweighted paired grouped mean arithmetic average). Confidence intervals at 95% of the Nei-Li similarity indexes for selected nodes of the dendrogram were calculated by bootstrap analysis, by SAS (SAS Institute, 1989). Multiple correspondence analysis was performed with "CORRESP" of SAS; three dimensions were sufficient to explain most of the observed variation.

Heterogeneity or gene diversity indices for each observed gene pool were calculated according to formulas proposed by Nei (1987). Because alleles belonging to a single locus could not be identified in the AFLP fingerprinting, each band was considered as a character with two possible states (alleles): presence and absence. Heterogeneity was calculated for each band and then averaged out for the total measure. Heterogeneity values measured in this manner result in the overestimation of the number of real loci, whereas the number of alleles per locus is underestimated, and heterozygotes are not detected. Thus, heterogeneity measures have only relative value, and cannot be compared with values obtained by other molecular markers or even in other AFLP studies.

	Results and discussion

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
The whole set of accessions was evaluated with two combinations of primers, PE1A/PM1A and PE1C/PM1C, that were selected for the high number of bands and polymorphism found in previous studies of P. vulgaris (Tohme et al., 1996). Indeed, a sufficient level of polymorphism was found (Table 2) . For each primer, a subset of accessions was re-run through the whole process (DNA extraction, digestion-ligation, amplification, and band scoring). In all cases, the original band pattern was obtained, indicating high reproducibility of data.

View larger version (26K): [in this window] [in a new window]   Fig. 2 Dendrogram developed from the UPGMA method of Nei-Li similarity values for both two primer combinations. Confidence intervals, using bootstrap analysis, are reported for key nodes. Figures at right refer to clusters discussed in the text. Cluster 1 refers to P. vulgaris accessions; Cluster 2 refers to P. lunatus genotypes from Colombia; Cluster 3 to small seeded wild Lima beans; Cluster 4 to slightly larger seeded wild Lima beans from Ecuador, Peru and Colombia; Cluster 5 includes P. pachyrrhizoides genotypes from Junin, Peru; Cluster 6 the same; Cluster 7 genotypes from Cuzco, Peru; Cluster 8 includes genotypes of P. augusti and P. pachyrrhizoides from Ecuador and northern Peru; Cluster 9 genotypes of P. augusti, P. bolivianus and P. pachyrrhizoides from Argentina, Bolivia and southern Peru; and Cluster 10 genotypes of P. augusti and P. pachyrrhizoides from Apurimac, Cuzco and Junin in Peru

View larger version (31K): [in this window] [in a new window]   Fig. 3 Multiple correspondence analysis (excluding P. vulgaris) showing separation of accessions of P. lunatus, P. augusti, P. pachyrrhizoides, and P. bolivianus according to geographic origin

View larger version (37K): [in this window] [in a new window]   Fig. 4 Multiple correspondence analysis (excluding P. vulgaris and P. lunatus) showing separation of accessions of P. augusti, P. pachyrrhizoides, and P. bolivianus according to geographic origin

Second, our results confirmed, with Clusters 2, 3, and 4, the existence of two major gene pools in wild Lima bean—also evidenced by polymorphism in seed storage proteins (Gutiérrez Salgado et al., 1995; Lioi, 1996), allozymes (Maquet et al., 1994), RAPDs of genomic DNA (Nienhuis et al., 1995; Fofana et al., 1997), and RFLPs of rRNA genes (Jacob et al., 1995). The wild form (Cluster 4), with slightly larger seeds and present in the western Andean range of Ecuador and northern Peru, clearly separates (0.58–0.71, P > 0.05, Fig. 2) from the form distributed in Central America and the eastern lowland South American tropics. Interestingly, in Cluster 3, the small-seeded wild Lima beans from Central America separate from those of South America, but the sample is too small to make a definitive conclusion. However, some Colombian genotypes from the Andean Departments of Boyacá and Cundinamarca, although close to the lowland neotropical wild Lima bean (Cluster 3), form Cluster 2. Some genotypes found in this region of Colombia, although having an Andean morphotype, had seed storage protein profiles close to that of wild "Mesoamerican" Lima bean (Toro Ch. et al., 1993), and fell within Cluster 4.

This finding raised the question of the origin of Cluster 2. Either it had resulted from crosses between the two gene pools of Lima bean, which are sympatric, or, because it does not fall exactly in between as would a hybrid group, it is related to something else. A comparison can perhaps be made with wild P. vulgaris from Ecuador and northern Peru with type "I" phaseolin: initially thought to result from crosses between the two major gene pools (Koenig et al., 1990). These beans were eventually shown to be a separate group with unique diversity (Kami et al., 1995; Khairallah et al., 1992; Tohme et al., 1996). Additional wild material from southwestern Colombia, particularly in the transition zone to Ecuador, is needed to answer this question.

The population of P. rosei collected at the type locality (LECCH112/113 in Cluster 4 in Fig. 2) falls within the Andean group of wild Lima beans (Cluster 4) as just another population among them. Phaseolus rosei would thus be an Andean wild form of Lima bean (and the formal name for it), as claimed elsewhere on the basis of morphological and biochemical evidence (Toro Ch. et al., 1993). This raises the question of whether one should separate, by a formal taxonomic nomenclature, the Andean gene pool of Lima bean from the one distributed in the lowland neotropics, and if so, at what level. For the time being, we favor maintaining the Lima bean as a single biological entity, with the name P. lunatus, and with two major gene pools (with a possibility of a third, minor, pool in Colombia), and passing "Phaseolus rosei" into synonymy.

A third implication of our results is that P. augusti, P. bolivianus, and P. pachyrrhizoides seem to form a continuum (Clusters 5–10) rather than three different clear-cut entities. Although we could not include the type for P. augusti, we found populations that matched the type description, particularly those from the southern range. These populations, the type locality population of P. bolivianus (BBLCO123 in Fig. 2), and P. pachyrrhizoides (PPEJU102 in Fig. 2) separate at a lower level than do the gene pools of Lima bean (Table 3). Their level of separation is comparable with the separation of gene pools in P. vulgaris (Cluster 1). Such a low level of separation makes us question the maintenance of these three taxa as separate species, instead of one polymorphic species. Germination habit is indeed polymorphic. While most populations of Clusters 5 to 10 had hypogeal germination and a tuberous root system, three populations (PPEAM104, PPECA107, and PPECU111) had epigeal germination and fibrous roots. Such varying modes of germination, while uncommon in the genus, has also been reported in P. leptostachyus Bentham (Delgado Salinas, 1985). Polymorphism also exists in leaflet shape. While all populations of Clusters 5 to 10 have ovate leaflets with variable pubescence, seven populations (APEPI41, AECLO43, AARJU99, PPEJU103, PPEAM104, PPECA107, and PPECU111) have lanceolate leaflets. One (APECU16) displays lobulate lateral leaflets (as noted previously by Macbride, 1943). In both cases, they did not separate clearly from the bulk of other populations. AFLPs also revealed polymorphism within populations. In five cases (ABLCH24-25, ABLCH26-27, APECU19-85, APECU38-39, and PPEAP9-121), individuals from the same population fell into other Clusters, indicating higher levels of polymorphism within populations than between them. Such levels of high polymorphism within populations, revealed by biochemical (Schmit et al., 1992) and molecular (Llaca et al., 1994; Schmit et al., 1993) markers, have also been found in wild P. coccineus, a polymorphic species from Mexico and Guatemala.

In contrast to results from isozyme analysis reported by Maquet and Baudoin (1996), we could not observe a clear-cut separation between the taxa P. augusti, P. pachyrrhizoides, and P. bolivianus. However we did see some groupings according to major geographic regions. These were (i) southern Peru (Apurímac, Cuzco) (Cluster 10), (ii) northwestern Argentina and central-southern Bolivia (Cluster 9), and (iii) northern Peru (Cajamarca, Piura) and southern Ecuador (Loja) (Cluster 8). The populations from Junín, Peru, however, were very different from each other, as was recognized earlier (Debouck, 1987), and did not link easily with other clusters. Clusters 5 and 6 display a relatively high number of polymorphisms over short geographic distances (PPEJU101 and PPEJU102, separated by roughly 15 km). Cluster 7 includes two populations with epigeal germination (BPECU45 and PPECU111). BPECU45 was initially collected as P. lunatus and temporarily classified as P. augusti. Cluster 8 includes populations from northern Peru and Ecuador. Cluster 9 includes populations from Bolivia and Argentina, and, although it has a long range, from Cochabamba (Bolivia) to Tucumán (Argentina), this cluster is somewhat less variable. Cluster 10 includes mostly populations from the Departments of Apurímac and Cuzco in southern Peru, and is relatively variable. Although we have not seen the type of P. augusti, we suspect it falls within the natural morphological variation of the taxa P. bolivianus and P. pachyrrhizoides. In our view, only one name should be kept and used for this somewhat polymorphic species, distributed from southern Ecuador to northern Argentina. Populations from Junín, and two others from Cuzco (BPECU45 and PPECU111) differ from each other and from the other groups, but not at a level that is high enough to justify a nomenclature treatment.

A fourth implication of our results is that we can make inferences about the phylogeny and evolution of this group of Phaseolus beans. According to Lackey (1983) and Polhill et al. (1981), woody stems, associated with tuberous roots and a perennial habit, may be regarded as a primitive character in the Phaseolinae subtribe. To a somewhat constant degree throughout the genus, these traits are also associated with hypogeal germination. Accordingly, the complex augusti–pachyrrhizoides with hypogeal germination probably constitute an ancestral stock. Two scenarios can be envisioned for the early formation of this stock. One is that it may have been formed in South America only. However, cpDNA evidence (Bruneau et al., 1995; Delgado Salinas et al., 1993) suggests that the genus does not have a polyphyletic origin, but instead forms a natural group, with a large number of species currently distributed in Central America. This leads us to a second scenario. Central America may have been where most speciation took (and is still taking) place. A group of species including the ancestral branch of Lima bean would have been formed there, giving millenia afterwards species such as P. maculatus Scheele and P. ritensis Jones (Debouck, 1991). Geographic isolation would explain why this group of Mesoamerican (Mexican) species is the tertiary gene pool, and why genetic distances from the group of species related to P. vulgaris are so noticeable. A few mutations in the ancestral stock would have led to the forms with epigeal germination, particularly P. lunatus. If this second scenario is correct, then the species P. lunatus would have an Andean origin. The tropical small-seeded pool of wild Lima bean would have separated from the northern Andes to diffuse towards Mexico (Sinaloa, Tamaulipas) and Argentina (Salta), where it is known today (Gutiérrez Salgado et al., 1995). The lower diversity observed in Argentina, compared with that in the northern and central Andes, would be compatible with migration from a nuclear area in northern South America. Some of the above statements need further support from additional analyses of cpDNA and mtDNA, and/or sequencing ITS (internal transcribed spacer) on larger and more geographically diverse samples. These analyses would help towards a better understanding of the Phaseolus group's evolution and the Lima bean's phylogenetic affinities within this group, thus aiding future plant breeding programs.Toro Tohme Debouck 1990

	ACKNOWLEDGMENTS

 
The authors thank Elizabeth de Páez for editorial comments, Jason Rauscher for reviewing the manuscript, Guillermo Valencia and Luis Eduardo Garzon for assistance in the greenhouse. We thank the Centro Internacional de Agricultura Tropical, the International Board for Plant Genetic Resources, and the United States Department of Agriculture for grants for field work. During field collection, DGD received the support of the following institutions, to whom we remain most grateful: Estación Experimental "Fabio Baudrit" of Costa Rica, Instituto Colombiano Agropecuario, Instituto Nacional de Investigaciones Agropecuarias of Ecuador, Instituto Nacional de Investigación Agraria of Peru, Centro Fitotécnico de Pairumani (Cochabamba) of Bolivia, Estación Experimental "Obispo Colombres" (Tucumán) of Argentina, and Instituto Nacional de Tecnología Agropecuaria of Argentina. This research was supported in part by a grant from the Belgian Administration for Development Cooperation, Government of the Kingdom of Belgium, with matching support from the Centro Internacional de Agricultura Tropical (CIAT).

Received for publication October 9, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 

• Berglund-Brücher O., Brücher H. Murutungo, eine semi-domestizierte wildbohne (Phaseolus flavescens Piper) aus den tropischen gebirgen Südamerikas. Angew. Bot. 1974;48:209-220.
• Brako L., Zarucchi J.L. Catalogue of the flowering plants and gymnosperms of Peru. St. Louis: Missouri Botanical Garden, 1993.
• Bruneau A., Doyle J.J., Doyle J.L. Phylogenetic relationships in Phaseoleae: evidence from chloroplast DNA restriction site characters. In: Crisp M.D., Doyle J.J., eds. Advances in legume systematics. Kew, England: Part 7 Phylogeny. Royal Botanic Gardens, 1995:309-330.
• Debouck D.G. Recolección de germoplasma de Phaseolus en el centro y centro-sur del Perú. Rome: International Board for Plant Genetic Resources, 1987.
• Debouck D.G. Systematics and morphology. In: van Schoonhoven A., Voysest O., eds. Common beans, research for crop imporvement. Wallingford, England: Commonwealth Agricultural Bureaux International, 1991:55-118.
• Debouck D.G., Castillo R., Tohme J. Observations on little-known Phaseolus germplasm of Ecuador. FAO/IBPGR (Int. Board Plant Genet. Resour.). Plant Genet. Resour. Newsl. 1989;80:15-21.
• Debouck D.G., Liñan Jara J.H., Campana Sierra A., De la Cruz Rojas J.H. Observations on the domestication of Phaseolus lunatus L. FAO/IBPGR (Int. Board Plant Genet. Resour.). Plant Genet. Resour. Newsl. 1987;70:26-32.
• Delgado Salinas A. Systematics of the genus Phaseolus (Leguminosae) in North and Central America. Austin, TX: Ph.D. dissertation. University of Texas, 1985.
• Delgado Salinas A., Bruneau A., Doyle J.J. Chloroplast DNA phylogenetic studies in New World Phaseolinae (Leguminosae: Papilionoideae: Phaseoleae). Syst. Bot. 1993;18:6-17.
• Fofana B., Vekemans X., du Jardin P., Baudoin J.P. Genetic diversity in Lima bean (Phaseolus lunatus L.) as revealed by RAPD markers. Euphytica 1997;95:157-165.[ISI]
• Foster R.C. A catalogue of the ferns and flowering plants of Bolivia. Contr. Gray Herb. Harv. Univ. 1958;184:1-223.
• González, D.O., N. Palacios, G. Gallego, and J. Tohme. 1995. Protocolos para marcadores moleculares. Centro Internacional de Agricultura Tropical (CIAT). Cali, Colombia.
• Gutiérrez Salgado A., Gepts P., Debouck D.G. Evidence for two gene pools of the Lima bean, Phaseolus lunatus, in the Americas. Genet. Resour. Crop Evol. 1995;42:15-28.
• Harms H. Einige neue Phaseolus-Arten. Notizbl. Bot. Gart. Mus. Berlin-Dahlem 1921;7:503-508.
• Jacob M., Zink D., Nagl W. RFLPs of the rRNA genes in the genus Phaseolus. Genet. Resour. Crop Evol. 1995;42:97-106.
• Kami J., Becerra Velásquez V., Debouck D.G., Gepts P. Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris. Proc. Natl. Acad. Sci. (USA) 1995;92:1101-1104.[Abstract/Free Full Text]
• Khairallah M.M., Sears B.B., Adams M.W. Mitochondrial restriction fragment length polymorphisms in wild Phaseolus vulgaris L.: insights on the domestication of the common bean. Theor. Appl. Genet. 1992;84:915-922.[ISI]
• Koenig R.L., Singh S.P., Gepts P. Novel phaseolin types in wild and cultivated common bean (Phaseolus vulgaris, Fabaceae). Econ. Bot. 1990;44:50-60.[ISI]
• Lackey J.A. A review of generic concepts in American Phaseolinae (Fabaceae, Faboideae). Iselya 1983;2:21-64.
• Leonard M.F., Stephens L.C., Summers W.L. Effect of maternal genotype on development of Phaseolus vulgaris L. x P. lunatus L. interspecific hybrid embryos. Euphytica 1987;36:327-332.
• Lewis G.P. Legumes of Bahia. Kew, England: Royal Botanic Gardens, 1987.
• Lioi L. Phaseolin diversity in wild Lima bean (Phaseolus lunatus L.) in American centres of origin. Genet. Resour. & Crop Evol. 1996;43:575-580.
• Llaca V., Delgado Salinas A., Gepts P. Chloroplast DNA as an evolutionary marker in the Phaseolus vulgaris complex. Theor. Appl. Genet. 1994;88:646-652.
• Macbride J.F. Flora of Peru—Family Leguminosae. Field Mus. Nat. Hist. Publ. Bot. Ser. 1943;13:1-507.
• Maquet A., Baudoin J.P. New lights on phyletic relations in the genus Phaseolus. Annu. Rep. Bean Improv. Coop. (USA) 1996;39:203-204.
• Maquet A., Wathelet B., Baudoin J. Etude du réservoir génétique de la légumineuse alimentaire Phaseolus lunatus L. par l'analyse électrophorétique d'isozymes. Bull. Rech. Agron. Gembloux 1994;29:369-381.
• Maréchal R., Mascherpa J.-M., Stainier F. Etude taxonomique d'un groupe complexe d'espèces des genres Phaseolus et Vigna (Papilionaceae) sur la base de données morphologiques et polliniques, traitées par l'analyse informatique. Boissiera 1978;28:1-273.
• Mok D.W.S., Mok M.C., Rabakoarihanta A. Interspecific hybridization of Phaseolus vulgaris with P. lunatus and P. acutifolius Theor. Appl. Genet. 1978;52:209-215.
• Nei M. Molecular evolutionary genetics. New York: Columbia University Press, 1987.
• Nei M., Li W. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl Acad. Sci. (USA) 1979;76:5269-5273.[Abstract/Free Full Text]
• Nienhuis J., Tivang J., Skroch P., dos Santos J.B. Genetic relationships among cultivars and landraces of lima bean (Phaseolus lunatus L.) as measured by RAPD markers. J . Am. Soc. Hortic. Sci. 1995;120:300-306.[Abstract/Free Full Text]
• Palacios R.A., Vilela A.E. Las especies argentinas de Phaseolus, un recurso genético que necesita protección. In: Clausen et al A.M., ed. Recursos genéticos hortícolas. Mar del Plata, Argentina: Instituto Nacional de Tecnología Agropecuaria, 1993:123-139.
• Piper C.V. Studies in American Phaseolinae. Contr. US Nat. Herb. 1926;22:663-701.
• Polhill R.M., Raven P.H., Stirton C.H. Evolution and systematics of the Leguminosae. In: Polhill R.M., Raven P.H., eds. Advances in legume systematics – Part 1. Kew, England: Royal Botanic Gardens, 1981:1-26.
• Rohlf F.J. NTSYS-PC numerical taxonomy and multivariate analysis system. Setauket, NY: Version 1.8. Exeter Publ, 1993.
• SAS Institute. SAS/STAT users guide, 6th ed Cary, NC: SAS Institute, 1989.
• Schmit V., Baudoin J.P., Wathelet B. Contribution à l'étude des relations phylétiques au sein du complexe Phaseolus vulgaris L.–Phaseolus coccineus L.–Phaseolus polyanthus Greenman. Bull. Rech. Agron. Gembloux 1992;27:199-207.
• Schmit V., Debouck D.G. Observations on the origin of Phaseolus polyanthus Greenman. Econ. Bot. 1991;45:345-364.
• Schmit V., du Jardin P., Baudoin J.P., Debouck D.G. Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa, including P. vulgaris and P. coccineus. Theor. Appl. Genet. 1993;87:506-516.
• Tohme J., González D.O., Beebe S., Duque M.C. AFLP analysis of gene pools of a wild bean core collection. Crop Sci. 1996;36:1375-1384.[Abstract/Free Full Text]
• Toro Ch O., Lareo L., Debouck D.G. Observations on a noteworthy wild lima bean, Phaseolus lunatus L., from Colombia. Annu. Rep. Bean Improv. Coop. (USA) 1993;36:53-54.
• Toro Ch., O., J. Tohme, and D.G. Debouck. 1990. Wild bean (Phaseolus vulgaris L.): description and distribution. Centro Internacional de Agricultura Tropical, Cali, Colombia.
• Vos P., Hogers R., Bleeker M., Reijans M., van de Lee T., Hornes M., Frijters A., Pot J., Peleman J., Kuiper M., Zabeau M. Aflp: a new technique for Dna fingerprinting. Nucleic Acids Res. 1995;23:4407-4414.[Abstract/Free Full Text]
• Wiggins I.L., Porter D.M. Flora of the Galapagos Islands. Stanford, CA: Stanford University Press, 1971.

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