Advances in Genetic Diversity Analysis of Phaseolus in Mexico

genic and genomic microsatellites to analyse allele diversity and heterozygosity in P. vulgaris and P. acutifolius . Genic sequence SSRs were more polymorphic than genomic SSR. SSR distinguished between Mesoamerican and Andean gene pools, and separated genetic races into each gene pool and into wild from cultivated germplasm. Andean germplasm was more polymorphic at both inter- and intra-populational levels. Contrasting results were found by Benchimol et al. [67], who analysed Mesoamerican and Andean P. vulgaris accessions and reported low values of polymorphisms, likely due to the domestication process [68]. genetic structure, genetic relations, genetic patterns of dispersion,


Introduction
Mexico is a centre of origin, domestication and diversity of major crops worldwide, such as maize (Zea mays L.) and beans (Phaseolus spp). It is known that five species of Phaseolus have been domesticated: P. vulgaris L. (common bean), P. coccineus L. ('ayocote' bean), P. lunatus L. ('lima' bean), P. acutifolius Gray ('tepary' bean) and P. polyanthus Greenm. (= P. polyanthus McFad.) ('acalete' bean) [1]. The five species are well distributed through Mexico as wild, semidomesticated, and domesticated forms [2]. Beans are economically, socially, biologically and culturally important [3]. Owing to the importance of beans for Mexico and the world [4] extensive programmes focusing on the conservation, management and characterization of genetic resources of Phaseolus have been implemented in some countries [5] and international institutions such as Centro Internacional de Agricultura Tropical (CIAT) in Cali, Colombia; the Grupo de Mejoramiento de Leguminosas de la Misión Biológica de Galicia and Consejo Español de Investigación Científica (MBG-CSIC) in Spain; the USDA/ARS Western Regional Plant Introduction Station at USA, and the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) of Mexico who include bean collections in their germplasm banks, including all forms [6,7].
Collections at germplasm banks are usually classified as base, active, for the work, and core [5]. Core collections include the highest levels of genetic diversity of one species (from 70 to 80%) as Phaseolus spp. into the lower number of accessions and due its size offers an easy and cheaper management and improves and efficient the germplasm use [8]. In this sense researchers need to characterize and to evaluate the core collections. Traditional strategies were based on the use of high-heritability morpho-agronomic traits such as growth habit, flower colour; the use of biochemical markers as seed reserve proteins as phaseolins, etc. Recent advances in molecular biology have improved the capability of plant genetic resource characterization using methodologies based on DNA analysis such as dominant (random amplified polymorphic DNA, or RAPDs, and amplified fragment length polymorphisms, or AFLPs) and co-dominant (restriction fragment length polymorphisms, or RFLPs, and simple sequence repeats, or SSRs) molecular markers [9]. The sequencing of the common bean genome has been concluded and this information broadens our perspective about some facts and challenges related to origin, domestication, diversity patterns and breeding of common beans [10][11][12][13].

Beans in Mexico
The common bean is the second legume crop worldwide, behind soybeans [Glycine max (L.) Merr] [4]. Despite the major anthropocentric use of beans being their green pods and dry grains, in some Latin American and African countries people consume young leaves and flowers as fresh vegetables [14]. By 2013, more than 1.8 million hectares were cultured with beans, and grain yields were 0.74 t ha -1 [15]. Common beans are well adapted, and grow through the different agroecosystems of Mexico and different seasons [7], depending upon the genetic diversity of the native germplasm and breed cultivars developed for each region [3].
Beans are a basic food due to being a major source of proteins, minerals, fibre, carbohydrates and vitamins in the Mexican daily diet for most people, but mainly for those with low economic resources [3]. Beans are the perfect complement to the Mexican diet based on maize as 'tortillas' in order to substitute animal protein for proper nutrition [4,6]. Clinical studies showed that bean consumption prevents or improves both cholesterol and glucose levels in the blood [16]. However, the consumption of common beans has some problems, since they contain antinutritional compounds such as polyphenols (condensed tannins and anthocyanins), and inhibitors of proteases as trypsin, lectins and phytic acid [17,18]. These compounds limit bean consumption and prevent them need breeding programmes and/or industry treatments such as cooking.
Economic conditions and customer preferences cause a variation in bean consumption per cápita between countries, and between regions within countries. In Mexico, black beans are preferred in the southern regions, while 'Flor de Mayo' and 'Flor de Junio' beans are preferred in the central western regions. Finally 'pinto', 'bayo' and 'azufrado' beans are mostly consumed in the northern regions. While Mexican bean per capita consumption is 11 kg per year, some African countries report more than 40 kg [18].

Cultivated species of Phaseolus
As has been listed before, only five Phaseolus species have been domesticated, and wild and cultivated forms are well known: P. vulgaris L., P. coccineus, P. lunatus, P. acutifolius, and P. dumosus from over 70 Phaseolus species, subspecies and varieties reported for Mexico (Tables  1 and 2). Most Phaseolus species are well distributed through Mexico with the exception of cold humid temperate and extremely hot arid tropic climates. The species P. coccineus and P. leptostachyus are distributed in the largest numbers of climatic types [19].
The cultivated form of P. vulgaris is grown across Mexico, but the highest producers are the states of Zacatecas, Durango, Sinaloa, San Luis Potosí, Guanajuato and Chiapas. Common beans represent 95% of national bean consumption. Common beans show an annual cycle and exhibit broad variability in growth habits, biological cycle duration, adaptation to different altitudes and soil conditions, as well as a large variation in seed colour and shapes. The last decades the common bean crop has migrated from central and southern regions to the north. Bean crop growth has also changed from common cultivation association with maize or pumpkin to monoculture using cultivars with determinate growth habits. Wild populations of P. vulgaris are mainly distributed through major mountain systems: Sierra Madre Occidental, Sierra Madre del Sur, Eje Neovolcánico, and Sierra Madre Oriental from 760 to 2,250 m above sea level. This species has a climbing growth habit, thin pods and small seeds where greyish-spotted seed coat colours are common, although there are other colours including black, 'bayo', yellow, brown, etc. Pods are dehiscent when mature, and seeds can be latent due to seed coat hardness. In this species it is common to find segregant populations derived from the spontaneous crossing between wild plants and cultivated forms, due to both types cohabiting or co-existing by variable lengths of time or seasons [7,[20][21][22].
Ayocote beans, P. coccineus, are known as 'patol' or 'patola'. They are a perennial species that develop tuber roots, as well as being a vigorous species, with climbing plants. Ayocote beans are commonly grown at the highlands of the central states of Puebla, Tlaxcala, Hidalgo and Mexico. P. coccineus is alogamous due to incompatibility problems, and landraces and wild plants show a great level of genetic variability in seed colours, sizes and shapes. The species shows short-width pods with a low number of seeds per pod; the seeds are large and have thick coats. The cultivated form has an impermeable seed coat. The wild form is distributed in temperate regions from the highlands and mountains: Sierra Madre Occidental, Eje Neovolcánico and Sierra Madre Oriental, between 1,800 and 3,000 m above sea level [23][24][25][26].
Lima beans, P. lunatus, are named as 'comba' at the Balsas River depression (western Mexico) or 'ibes' at the Península of Yucatán (southeastern Mexico). As ayocotes, lima beans are perennial and climbing species, vigorous and with a late biological cycle. Seeds of cultivated populations are variable in colour, shape and size. The species tolerates high temperatures, drought stress and some insect pests. Lima beans are frequently inter-cropped with maize, but most of their vegetative development and reproductive phase is completed after the maturity of maize when high levels of sun radiation are available. The wild form is perennial and climbing, and is distributed in the lower-coastal lands of Mexico near the Gulf of Mexico, Pacific Ocean and the Peninsula of Yucatán. Some wild populations have been found at the Balsas River depression (states of Michoacán, Jalisco, Colima and Nayarit) at altitudes of up to 1,600 m above sea level (masl) [27,28].
The poor surface cropped with P. acutifolius is located mainly in northern and northeastern Mexico at altitudes of up to 1,800 masl. Tepary bean is an annual species with some landraces with an undetermined growth habit for monoculture. Despite the cultivated form having larger seeds than the wild population, some varieties conserve grain traits similar to their ancestors. Wild populations are distributed at Sierra Madre Occidental from the northern states of Sonora and Chihuahua to the central-eastern state of Michoacán [27].
Finally, the less cultivated Phaseolus species, P. dumosus (= P. polyanthus), named 'acalete' shows vigorous climbing growth habits, and is commonly cultured in the state of Oaxaca, associated with maize. Acalete seeds are medium to large and are highly variable in colour, shape and size. The seed coat is thick, as in P. coccineus, but is permeable to water. The wild form is inferred to be dispersed through the state of Oaxaca [19,27].

Why must common bean diversity be conserved?
Mexico is broadly recognized as the centre of origin, domestication and diversification of major crops including avocado, amaranth, cocoa, pumpkin, maize, beans, and others. These species have been well dispersed and cultivated worldwide and constitute a major source of economic input for many countries. One major Mexican institution advocated to the collection, study, documentation, preservation and promotion of Mexican genetic resources is the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) which created the Genetic Resources Unit, and recently a new National Bank of Germplasm (CNRG, INIFAP) located in Tepatitlán, Jalisco, where Phaseolus represents one of the most important species to be preserved and studied. Special emphasis must be given in further works to those species not properly represented in Mexican Germplasm Banks, without seed samples preserved under proper conditions or species only represented as herbarium samples ( Table 3) , showed the evident genetic erosion to be abnormally high in some regions. At these regions genetic erosion is so fast that can not will compensate by new genetic variability 'normally created' inside each ecosystem. Genetic erosion is significantly increased by human perturbation of agroecosystems. In addition, economical financing of a new re-collection of Phaseolus genetic resources must be revalued and consistently supported, assuming this involves future investment for genetic resource preservation and utilization for bean improvement.  Table 3. Species of Phaseolus geo-positioned, with herbarium registers and seed samples available and species without seed samples [29,30].

Species
In this sense, traditional strategies for germplasm analysis can be improved and better understood by the use of DNA-based strategies as molecular markers. Breeding programmes underutilize the genetic diversity available because of the necessity of pre-breeding exotic germplasm. The hybridization between wild and domesticated types of Phaseolus from the same gene pools offer greater potential for enhancing crop variation due the partial reproductive isolation between Andean and Mesoamerican domesticated gene pools. Former evaluations of wild and semi-wild Phaseolus accessions have shown resistance to insects and diseases [21,22,[31][32][33] and higher N, Fe, and Ca content in seeds, which could contribute to the improvement of nutritional grain quality and grain yield [34]. Acosta-Gallegos et al. [34] suggested Phaseolus pre-breeding based on the use of information on gene pool origins, domestication syndrome traits, molecular diversity, and mapping data of the wild forms; the indirect screening for biotic and abiotic stresses; and marker-assisted selection [35,36].

Common beans
One of the pioneer works on the study of Phaseolus genetic diversity was conducted by Debouck et al. [37] who described the ecological adaptations and geographical locations of  [38] analysed 151 bean accessions from the CORPOICA germplasm bank of Mosquera, Colombia. The data suggested higher morphoagronomic variation in Mesoamerican germplasm than Andean accessions. Rodiño et al. [39] reported the analysis of 388 bean landraces from Spain and Portugal on the basis of the use of morpho-agronomic traits and phaseolin patterns. Germplasm was classified as Andean (74.7%), Mesoamerican (16.8%) and mixed (8.4%). The data indicated that only 52 accessions should constitute one representative core collection from Spain and Portugal.
In Mexico, the core collection of common beans structured by INIFAP includes 200 accessions, and this was characterized using morpho-agronomic traits and AFLP molecular markers. The data indicated a high level of genetic variability and no duplicity of accessions (non-shared haplotypes) into the core collection, becoming itself in a representative sample of P. vulgaris variation through Mexico [7,40]. Rossi et al. [41] detected higher genetic diversity in the wild germplasm of beans from the Andean and Mesoamerican gene pools, while domesticated populations showed the largest linkage disequilibrium. Recent works of the analysis of P. vulgaris' genetic diversity have been published, where germplasm from different origins are analysed and broad genetic diversity ascertained into the genus and intra-and inter-specific and inter-and intra-population levels [42][43][44][45][46][47][48].

Ayocote beans
The genetic diversity of ayocote beans has been previously studied using morphological, agronomical and molecular markers, mainly using 'European' germplasm in order to detect high-yielding parents [49], highly tolerant germplasm to low temperatures [50], as well to characterize genetic relationships [51]. Spataro et al. [52] found clear differentiation between ayocote accessions [(wild, landraces, and P. dumosus (= P. coccineus ssp. dumosus)] as well as reduced gene diversity due the introduction of ayocote beans to Europe. Vargas-Vázquez et al. [24][25][26] reported that 80% of 798 P. coccineus accessions from Mexico originated from neovolcanic Axis and Eastern Sierra Madre with humid or semi-arid temperate climates, from 1500-2000 m above sea level and at 500-1000 mm of annual precipitation. Ayocote bean germplasm can be separated into two groups: late accessions adapted to minimum temperatures (2-5°C), with large seeds and pods, and early accessions adapted to 0-2°C, with small seeds and pods. Analysis of European domesticated P. coccineus, including botanical varieties albiflorus, bicolour and coccineus and domesticated and wild accessions from Mesoamerica using cpSSRs, nuclear SSRs and phenotypic traits, suggested a moderate-to-strong cytoplasmic bottleneck that followed the expansion of species into Europe and multiple domestication events into the species. An adaptive population differentiation was also found, suggesting that selection led to the diversification of P. coccineus in Europe [53].

Lima beans
The genetic diversity of P. lunatus from the Yucatán Peninsula was assessed based on morphological and phenological characters, and then related to ethnobotanical information obtained about intraspecific diversity recognized by farmers, their selection criteria, agronomic management, production purpose and percentage of cultivated area. Ethnobotanical and morpho-phenological data indicated 30 putative distinct landraces, two wild, and two weedy variants from 149 seed samples of P. lunatus germplasm, suggesting gene flow among them. Richness and diversity estimates were greatest, and evenness lowest, where there was minimal agricultural intensification, wild and weedy populations, and greater persistence of traditional culture [28]. Afterwards, Martínez-Castillo et al. [54] determined genetic diversity, structure and gene flow of 11 wild populations of P. lunatus in four regions of traditional agriculture in the Yucatán Peninsula, Mexico, using SSR loci. Strong genetic differentiation was found among populations due to isolation among agricultural regions, as well as low longterm gene flow and low rates of recent migration among populations. Positive correlation between agricultural intensification and increased diversity was found because wild populations are favoured by the intensification of disturbance in situations involving at least three years of fallow.
Recent low gene flow at both intraregional and interregional levels into the wild-weedydomesticated complex of P. lunatus under traditional agricultural conditions was found in four regions on the Yucatán Peninsula, Mexico, while gene flow from domesticated to wild populations was three times higher than in the opposite direction. This asymmetry was explained by regional agricultural practices and seed selection criteria. Domesticated alleles were shown to be entering wild populations of different agricultural regions, suggesting exchange of domesticated seeds between farmers of different regions. Thus, P. lunatus on the Yucatán Peninsula has a predominantly domesticated to wild gene flow, leading to genetic assimilation of the wild lima bean by its domesticated counterpart [55]. Afterwards, P. lunatus accessions collected in 1979 were compared with accessions collected in 2007 using SSR markers. The germplasm from 1979 was more diverse than that from 2007, suggesting the presence of a 'bottleneck' effect since alleles detected at each year of collection were different, as well as demonstrating allele drift due to the introduction of breed cultivars or changes in the selection criteria of germplasm [56].
Two wild Mesoamerican (MI and MII) gene pools with contrasting geographical distributions have been found in relation to P. lunatus. While the MI gene pool occurs in central western Mexico, including the Pacific coastal range, the MII gene pool is widespread and occurs towards the Gulf of Mexico, the Yucatán Peninsula, and Central and South America. Mesoamerican landraces clustered together with wild accessions from the MI gene pool (L haplotype), suggesting a unique domestication event in central western Mexico. The most likely domestication region is an area of the states of Nayarit-Jalisco or Guerrero-Oaxaca, and not areas such as the Peninsula of Yucatán where the crop is currently widespread and diverse. A strong founder effect due to domestication has been detected, and several recently diversified haplotypes identified [57]. The analysis of 67 wild populations of P. lunatus from Mexico with ten microsatellite loci confirmed not only the presence of the two gene pools (MI and MII), but also the possible existence of two subgroups within MI (MIa and MIb). While MI and MII are mainly divergent geographically, MIa and MIb overlap in their distribution. Thus, the genetic structure of the wild lima bean in Mexico is more complex than previously thought, and the presence of three gene pools (MIa, MIb, and MII), each one possessing relatively high levels of genetic diversity, is proposed [58]. Other work, including P. lunatus populations from different areas of America and germplasm, was analysed using two intergenic spacers of chloroplast DNA: atpB-rbcL and trnL-trnF. Three groups (AI, MI, MII) of genotypes were found, confirming the existence of Mesoamerican and Andean gene pools and multiple origins of domestication for the MA gene pool. For MI, western central Mexico was proposed as the domestication area, and for MII this was between Guatemala and Costa Rica [59].

Tepary beans
Since few genetic tools have been developed or tested for tepary bean, Blair et al. [60] validated one set of gene-derived and non-gene simple sequence repeat or microsatellite markers from the common bean in tepary bean cultivars and wild relative accessions. They then evaluated the genetic diversity and population structure of tepary bean accessions to determine if leaf morphology variants are valid as separate subgroups of wild tepary beans; if P. parvifolius was a separate variant or species; and if cultivated tepary beans originated from one domestication event or several events. The analysis of 140 tepary bean genotypes showed that a single domestication was likely as the cultivars were most closely related to accessions from Sinaloa and northern Mexico, and that diversity was much higher in the wild genotypes compared to the cultivated ones. P. parvifolius was classified as a separate species by population structure analysis while the variants P. acutifolius var. acutifolius and var. tenuifolius were admixed and inter-crossed. P. latifolius was not a valid species or variant of P. acutifolius, but represents a group of cultivars within the tepary bean. Other recent work was focused on the analysis of the agro-morphological variation of P. acutifolius germplasm in Botswana, where low genetic diversity was found [61].

Acalete beans
Total seed protein variability in a sample of 163 entries of year bean (P. polyanthus), including wild, feral and cultivated forms of the whole range of distribution in Latin America, was studied using I-dimensional SDS/PAGE and 2-dimensional IEF-SDS/PAGE. Ten different patterns were observed in this crop. Eight of these are found in the Mesoamerican materials, the other two of those in the northern Andes. The highest diversity is found in the wild ancestral forms present in central Guatemala with six patterns. The 'b' pattern predominant in all Mesoamerican cultivated materials and is also present at low frequency in Colombia. The 'k' pattern, predominant in the northern Andes, is present in Costa Rica. These results, together with information on indigenous names for the crop, suggest that there is a single gene pool domesticated from a wild ancestor still present in Guatemala, and distributed afterwards to the northern Andes, but with a clinal genetic drift from Mesoamerica to the Andean region [62].

Comparisons of genetic diversity among bean species
The diversity and relationships among species of Phaseolus complex were analysed using chloroplast DNA. Restriction patterns were used to identify polymorphisms and assess the type of mutations detected, and identify regions of high variability (Fig. 2). There is high cpDNA variability within P. coccineus but other species as P. vulgaris and P. coccineus subsp. glabeIIus show a very distinct cpDNA genotype compared to the former species. These evidences strongly suggests that P. coccineus subsp. glabellus belongs to a different but as yet undetermined section of the genus. In P. coccineus subsp. darwinianus (= P. polyanthus), the cpDNA lineage was in disagreement with data obtained from nuclear markers, and suggested a reticulated origin by hybridization between P. coccineus as the male parent and an ancestral P. polyanthus type, closely allied to P. vuIgaris. Molecular markers are an important strategy for elucidating phylogenetic relationships; in addition, accurate phylogenies will require analyses of both nuclear and cytoplasmic genomes [63]. Hamann et al. [64] identified 18 species from 90 genotypes using SSR markers, where the species P. vulgaris, P. lunatus, P. coccineus, P. acutifolius and P. polyanthus showed four specific (GATA) sequence patterns that help to clearly separate auto-pollinated (P. vulgaris and P. lunatus) from alogamous species as P. coccineus, but this species was found to have lower intraspecific variation. Gaitán-Solís et al. [65] later isolated, cloned and sequenced genomic DNA fragments into three gene libraries and then evaluated the polymorphisms of 68 SSRs. Markers were capable of separating germplasm on the basis of Phaseolus species: P. coccineus, P. polyanthus, P. acutifolius, and P. lunatus. Blair et al. [66] used genic and genomic microsatellites to analyse allele diversity and heterozygosity in P. vulgaris and P. acutifolius. Genic sequence SSRs were more polymorphic than genomic SSR. SSR distinguished between Mesoamerican and Andean gene pools, and separated genetic races into each gene pool and into wild from cultivated germplasm. Andean germplasm was more polymorphic at both inter-and intrapopulational levels. Contrasting results were found by Benchimol et al. [67], who analysed Mesoamerican and Andean P. vulgaris accessions and reported low values of polymorphisms, likely due to the domestication process [68].
In Italy, 66 genotypes representing 14 local varieties of P. vulgaris and nine of P. coccineus, collected through regions of Marche using ISSR markers, SSRs and cpSSRs, were analysed. Farmers' selection and adaptability to variable environments have provoked bean preservation and diversification. A total of 71% of local varieties of P. vulgaris come from Andean origins [69]. Chacón et al. [70] analysed 31 accessions of Phaseolus (27 from P. coccineus including both P. coccineus subspecies darwinianus and glabellus; three from P. vulgaris and one from P. lunatus) using restriction patterns of cpDNA. Molecular analysis clearly differentiated between Phaseolus species, and P. coccineus showed the highest molecular polymorphism values in both wild and cultivated accessions compared with all other species [71,72]. In Mexico, Ramírez et al. [73] characterized 107 common bean populations, 42 ayocote beans and one acalete bean on the basis of morpho-agronomic traits. Ayocote beans showed more diverse seed coat colours than common beans (54.8% purple, 26.2% black, 19.0% white and brown).

About the origin, domestication and classification of Phaseolus
Hernández-López et al. [74] published a review paper that analysed classic works focused on determining and locating the centres of origin and domestication of P. vulgaris, assuming that these areas are major sources of populations carrying useful genes for breeding, and because such populations can improve our understanding of the evolution, diversification and conservation of the species. Despite the broad and abundant information published over decades, new information is consistently published, new evidence found, and new strategies such as genomic and genetic techniques based on DNA analysis applied in these studies. The accumulated knowledge derived from varied sources including archaeology, agronomy, ethno-linguistic, ethnobotany, molecular biology, biochemistry, physics etc. is currently being applied in order to study and clarify origins, domestication and diversification patterns, phylogeographical relations, among others. Therefore, the use of tools based on molecular technologies and genomics should give definitive evidence on the origin, domestication and genetic diversity of Phaseolus [11,74,75].
As has been described by Muruaga-Martínez et al. (unpublished data), recent re-collection tours have been conducted in order to clarify the real and current state of genetic resources of Phaseolus across Mexico. One major problem for taxonomy, phylogeny or systematics studies in Phaseolus is that most specimens belong to herbariums and no 'fresh' plants are available. Thus, our work group assumed the necessity to re-collect Phaseolus specimens. We then subjected the germplasm to genetic analysis using molecular marker strategies and reproduced them under controlled conditions for future works, and to preserve endangered species and specimens. The first expeditions (2010-2012) yielded the collection of more than 100 samples (seeds), which comprised 19 species, including two subspecies of P. coccineus (P. coccineus griseus and P. coccineus striatus). Three species in this collection (P. albiviolaceus, P. maculatifolius and P. rotundatus) had not been studied before. Villarreal-Villagrán et al. [76] analysed them by using five trnT-trnL, trnL-trnF, rpl16, rpoC1-rpoC2, rps14-psaB non-coding regions of chloroplastic DNA amplified by PCR (polymerase chain reaction). Cluster analysis confirmed with strong bootstrap support that the genus Phaseolus is a monophyletic group that can subdivide itself into two major lineages: one includes P. pluriflorus, P. esperanzae, P. pedicellatus, P. microcarpus, P. glabellus, P. oligospermus, P. gladiolatus, P. zimapanensis and P. albiviolaceus; and the other includes P. filiformis, P. acutifolius, P. vulgaris, P. coccineus striatus, P. coccineus griseus, P. macvaughii, P. leptostachyus, P. lunatus, P. maculatus, P. maculatifolius and P. rotundatus. The topology of the dendrogram obtained agreed with the topology of Phaseolus recognized to this date, which was obtained using only the ribosomal ITS and chloroplast trnK locus [77]. The exception was P. albiviolaceus, a species not studied before, that according to traditional morphological criteria, belongs to the Pedicellatus group, but which in this study appeared with the Tuerckheimii group. The other two species that were characterized for the first time in a molecular phylogeny are P. maculatifolius and P. rotundatus, both of which were clustered within the polystachios group (Fig. 3).

About the genetic diversity analyses of Phaseolus
The analysis of Mexican common bean core collection using SSR and AFLP markers revealed that the highest genetic diversity is found in central Mexico and Chiapas, which seems to be an important diversity centre in the south. SSR analysis indicated a reduced number of shared haplotypes among accessions and core collection has no duplicated accessions [40]. Hernández-López et al. [21,22] evaluated the diversity and genetic relations of one collection of bean populations produced after the random crossing among wild and domesticated or cultivated bean genotypes throughout Mexico to assess its usefulness for Phaseolus breeding due the detection of six SCAR markers associated with common blight (Xanthomonas axonopodis pv. phaseoli) resistance, as well as four for anthracnose (Colletotrichum lindemuthianum). The results indicated significant morphological variability in the common bean germplasm. AFLP marker analysis revealed high genetic diversity in those germplasms from north-central and central Mexico. Germplasm from Morelos, Guanajuato, Querétaro, Durango, and Tamaulipas showed the highest genetic diversity indexes. Cluster analysis was not consistent with classification forms and their distribution based on geographical or agro-ecological origin. Germplasm from Guanajuato and Tlaxcala showed the highest SCAR frequencies for both diseases. Genetic diversity and SCAR detection for resistance to anthracnose and common blight was analysed in two P. coccineus collections, one originating from the 'Huasteco Karst' located mainly in the state of Puebla, Mexico. The other group of ayocote accessions comes from the state of Veracruz. Analysis of germoplasm from Huasteco Karst revealed great genetic variability among and within accessions as well as high genetic differentiation among germplasm. Resistance to anthracnose was more frequent into the germplasm [31,32]. Ayocote beans from  results indicated high genetic variability among and within accessions of ayocote beans from the states of Puebla and Veracruz; the data also suggest them to be an important source of allele useful for inclusion in breeding programmes of P. coccineus or P. vulgaris [33].

Concluding remarks
In order to understand genetic variation patterns and to reinforce the richness and genetic potential of Phaseolus, it is necessary to preserve, characterize and take advantage of Mexican germplasm. Knowledge of genetic diversity in the common bean could give us a better view for conservation as well as management and use of these plants' genetic resources. Despite significant effort and financial support by the Mexican government, this is not enough. Last year a new national centre for genetic resources was constructed and financed (Centro Nacional de Recursos Genéticos, CNRG, INIFAP) as well as initiatives to preserve not only beans but also other major Mexican crops such as maize, amaranth, cocoa, pumpkin, agave, avocado, etc. (SINAREFI-SAGARPA, Sistema Nacional de Recursos Fitogenéticos para la Alimentación y la Agricultura). We suggest that this financial support is maintained or improved, and research consistently supported through mid-and long-term projects.
Collection tours have demonstrated fast genetic erosion in most regions where wild types of Phaseolus were sought. Unfortunately, the social and economic situation in Mexico has provoked the migration of farmers and rural people, who have originally maintained and preserved genetic resources and their diversity in order to improve our lives. Thus, basic crops are leaving and are frequently changed by 'highly profitable' crops. The growth of urban populations has increased pollution and should have consequences through the alteration of natural reservoirs of natural populations inside 'Reservas de la Biósfera' or 'protected areas'. The Mexican government offers incipient economic support to those farmers that preserve genetic diversity in situ on their land. This strategy is appropriate for genetic resource conservation, but is currently not enough.
Genetic diversity observed in Phaseolus germplasm collections represent an important sample of total genetic variability contained in the genus. Molecular marker strategies and other recent and advanced techniques such as sequencing, genomics, proteomics and other '-omics' could help us to better understand genetic structure, genetic relations, genetic patterns of dispersion, and variation, promising germplasm to be used as parents or in breeding programmes based on their outstanding traits, etc. Biotechnology will not substitute traditional strategies of germplasm characterization or the taking advantage of Phaseolus germplasm. Biotechnology should be one allied or even more strengthen traditional breeding to accelerate and improve breeding methods and analysis strategies of genotypes.
Finally, we suggest that genetic diversity is a major challenge for botanists and taxonomists, biotechnologists and breeders, as well as to the governmental institutions of Mexico, towards maintaining natural populations both in situ and ex situ, to avoiding their loss, to increasing the strategies for their use, and to exploiting their benefits.