Identification of Prosopis juliflora and Prosopis - Universidad Pública ...

2species are sympatric in each centre. Studies using morphological characters(Solbrig et al. 1977), isoenzymes (Saidman 1985), seed protein electrophoresis(Burghardt and Palacios 1997) and molecular markers (Ramírez et al. 1999)have shown the occurrence of intra- as well as inter-series hybrids in populationsof both sections Algarobia and Strombocarpa. These studies, however,support the division of the genus into sections. Pasiecznik et al. (2001) havesuggested the grouping of species of section Algarobia into ‘complexes’ or‘species groups’ principally according to their genetic similarity and geographicdistribution, but also to their habit and resource characters.Prosopis juliflora Swartz DC. and Prosopis pallida (Humbolt & Bonpland exWilld.) H.B.K. were grouped into the ‘P. juliflora–pallida complex’ by Pasieczniket al. (2001), a suggestion also made earlier by Felker (1990). These twospecies occur over a broad geographical area approximately limited by thetropics of Cancer and Capricorn, including Mexico, Guatemala, El Salvador,Honduras, Nicaragua, Costa Rica, Panama, Colombia, Venezuela, Ecuador,Peru and the Caribbean islands. The species of section Algarobia to the northare P. glandulosa Torrey, P. laevigata (Humbolt & Bonpland ex Willd.) M.C.Johnston and P. velutina Wooton, which may be considered a North Americancomplex. To the south of the P. juliflora–pallida complex lie all other species ofsection Algarobia, including P. chilensis (Molina) Stuntz emend. Burkart.The P. juliflora–pallida complex was divided into three geographically distinctgroups, termed ‘races’ by Pasiecznik et al. (2001). Group 1 comprisespopulations in Ecuador and Peru. P. pallida has been identified throughout thisarea while three varieties of P. juliflora have been identified in northern Peruand southern Ecuador (Burkart 1976). Burkart (1976) also reported a possibleP. juliflora · P. pallida hybrid in Ecuador. Populations in the north of this areadisplay some phenotypic characteristics typical of P. juliflora while those in thecentre and south appear to be purely P. pallida. Group 2 comprises populationsof P. juliflora in Colombia, Venezuela and the Caribbean islands. Severalauthorities have included the Caribbean in the native range, and if not nativethey became naturalised in prehistory (Timyan 1996). Group 3 also comprisespopulations of P. juliflora, in the Pacific coastal areas of Central America fromMexico to Panama, with some inland extensions. In the north of this region,populations of P. juliflora are thought to overlap with P. glandulosa andP. laevigata (Pasiecznik et al. 2001). However, the status of P. juliflora inMexico is uncertain (Pasiecznik et al. 2001), although recent work usingmolecular markers is beginning to resolve some of the underlying geneticvariation found here (Jua´rez-Mun˜ oz et al. 2002).Many Prosopis species are valuable multipurpose biotic resources in theirnative range and where introduced, providing timber, firewood, livestock feed,human food, shade, shelter and soil improvement (Harris et al. 1998). Severalspecies, for example the sub-tropical P. chilensis, P. glandulosa and P. velutina,and more importantly, the tropical species, P. juliflora and P. pallida, have beenintroduced in the last 200 years and are now widespread in, for example,Brazil, Sahelian Africa, South Africa, Pakistan, India and Australia. However,

paper moistened with sterile distilled water and germinated for 8–10 days at20–18 ° C day-night, respectively, 80% relative humidity and 12 h photoperiod.Cotyledons were then harvested from seedlings and stored individually at80 ° C.5DNA manipulationGenomic DNA was obtained from cotyledons according to the protocol describedby Lassner et al. (1989) with the following modifications: 100 mg ofplant material (average weight of two cotyledons of a single plant) were groundin a microcentrifuge tube containing 300 ll of extraction buffer (100 mM Tris–HCl, 50 mM EDTA, 500 mM NaCl,10 mM b-mercaptoethanol, pH 8.0).After grinding, 400 ll of extraction buffer and 400 ll of chloroform/isoamylalcohol (24:1 v/v) were added to the sample, mixed gently and incubated for12 min at 60 ° C. After incubation, the two phases were separated by centrifugation(15 min, 14,000 rpm) and the supernatant was recovered and theDNA precipitated by adding 1.2 volumes of cold ( 20 ° C) 2-propanol andincubated for 45 min. The genomic DNA was collected by centrifugation at14000 rpm for 15 min. The pellet was rinsed with 70% ethanol-10 mM sodiumacetate, dried and resuspended in TE buffer (Tris HCl 10 mM, EDTA 1 mM,pH 7.8). The RNA was removed by treatment with 25 lg ml1 of RNAse. Thegenomic DNA was dissolved in sterile water to a final concentration of 20 ngll1 . General molecular biology protocols were used as described by Sambrooket al. (1989).In order to generate RAPD markers specific for the species studied, pools ofDNA containing equal amounts of DNA purified from ten samples of the samespecies were used as templates. Polymerase chain reactions (PCR) for thegeneration of RAPD markers were performed as described by Williams et al.(1990) in a reaction volume of 25 ll containing 120 ng of pooled DNA astemplate, 2 mM MgCl 2 , 67 mM Tris HCl (pH 8.8), 16 mM (NH 4 )SO 4 ,0.1 g l1 Tween-20, 200 lM each of the four nucleotide triphospates (ATP,CTP, GTP, and TTP), 0.2 lM of the primer oligonucleotide and one unit ofTaq polymerase (Eurobiotaq, Ecogen, Barcelona, Spain). The oligonucleotidesused as primers (20 primers) for the reaction were 10-mers belonging to the L,P, S and R Operon series (Operon Technologies Inc. Alameda, California,USA). Amplification reactions were performed in a PTC-200 (Peltier ThermalCycler, MJ Research, Watertown, Massachusetts, USA) using the followingprogramme: 4 min of initial denaturation at 94 ° C followed by 40 cycles ofPCR reaction (1 min denaturation at 94 ° C, 1 min annealing at 37 ° C, 1.3 minextension at 72 ° C). Amplification products were analysed by electrophoresis in1.5% (w/v) agarose gels in TAE buffer (400 mM Tris, 200 mM sodium acetate,20 mM EDTA, pH 8.3) and stained with ethidium bromide. U29 HindIII digestedDNA and k HindIII-EcoRI were used as molecular size markers. Inthose experiments in which the amplification products were detected by DNA

7presence or absence of each marker was scored in each accession and Nei’ssimilarity index (Nei and Li 1979) was applied to establish similarities. Thesimilarity values were used to cluster species using the unweighted pair groupmethod average (UPGMA) algorithm (Sneath and Sokal 1973).ResultsMolecular markers: section-specific, species-specific and complex-specificThe molecular markers obtained by PCR amplification that identified speciesbelonging to the section Algarobia, those that differentiated between P. julifloraand P. pallida and those that characterised the P. juliflora–pallida complexin this study are presented in Table 2. Twenty primers yielded profiles usefulfor classification of the accessions. Six primers: L02 (775) , L06 (800) , L09 (335) ,S03 (550) , S07 (1160) and R17 (1220) (Figure 1) produced molecular markers thatwere present in all the accessions analysed except P. cineraria. Thus, five newprimers were added to S07 (1160) , previously described by Ramı´rez et al. (1999)as a section-specific marker for section Algarobia. Assuming that theseamplification products are all derived from Prosopis species forming part of thesection Algarobia, it could be suggested that there are section-specific molecularmarkers.The markers L04 (325) ; P01 (550) ; P08 (730) ; P09 (570) ; P13 (1250) ; P14 (510) ;S07 (980) and S11 (460) were present in P. pallida seeds collected in Trujillo. Asthe amplification products in plant material collected in Piura, where P. julifloraand P. pallida are sympatric, are essentially identical to those present inmaterial from Trujillo, it may be concluded that the sample from Piura isP. pallida or a very closely related hybrid (Figure 2a). Seed from introducedpopulations presently identified as P. juliflora from Brazil, Cape Verde andSenegal, and seed collected in Brazil and classified as P. affinis Sprengel allshowed similar molecular profiles to‘ that amplified in DNA extracted fromseed collected in Trujillo and Piura. Thus, all of these accessions correspond toTable 2. Molecular markers found in the Prosopis samples.SectionP. juliflora–pallidaP. pallidaP. julifloraAlgarobiacomplex markersmarkersmarkersmarkersL02 (775) L01 (520) L04 (325) L04 (410)L06 (800) P01 (550) P08 (440)L09 (335) P08 (730) P12 (760)S03 (550) P09 (570) P12 (610)S07 (1160) P13 (1250) P14 (745)R17 (1220) P14 (510)S07 (980)S11 (460)

8P. pallida and do not correspond to any other species likely to occur in thatarea (Pasiecznik et al. 2001). Seeds of a putative P. chilensis · P. pallida hybridfrom Cape Verde also displayed an amplification pattern similar to that presentedby P. pallida (Figure 2a). The amplification profile corresponding to theputative P. chilensis · P. pallida hybrid showed bands present in P. pallida, andsome bands present in P. chilensis could also be seen (data not shown). Whilethis confirms P. pallida as one of the parents of the putative hybrid, P. chilensiswas not specifically analysed in this study and thus can only be tentativelyconfirmed as the other parent. When DNA derived from seeds of P. julifloracollected in Honduras was analysed it could be seen that different loci wereamplified. Five species-specific molecular markers were found: L04 (410) (Figure3), P08 (440) , P012 (760) , P12 (610) and P14 (745) . A similar profile was obtainedwhen DNA derived from seeds collected in Burkina Faso, presentlyidentified as P. juliflora, was amplified. Thus, the plant material collected inBurkina Faso is very likely to be P. juliflora and differs from P. pallida.When the oligonucleotide L01 was used as primer in amplification reactionsusing as template DNAs derived from plant material collected in Honduras(P. juliflora), Piura and Trujillo (P. pallida), Senegal, Cape Verde, Brazil,Burkina Faso, in the putative hybrid P. chilensis · P. pallida and the materialFigure 2. (a) Amplification of the marker S11 460 , specific to P. pallida, using oligonucleotide S11as primer and the DNAs purified from all accessions analysed in this paper as templates. (b)Southern hybridisation of the cloned marker S11 460 used as probe on membranes containing theamplification products using S11 as oligonucleotide primer and the genomic DNA from allaccessions analysed in this paper as templates. Lane names as Figure 1.

9Figure 3. Amplification of the marker L04 410 using oligonucleotide L04 as primer and the DNAspurified from all accessions analysed in this paper as templates. Note that this oligonucleotideamplifies a low molecular weight marker present in P. pallida accessions. Lane names as Figure 1.classified as P. affinis, it could be seen that a DNA fragment of approximately520 bp was present in all the samples (Figure 4a). This result suggests that theL01 (520) marker could be a P. juliflora–pallida complex-specific marker. DNAhybridisation experiments (see below) confirm this result (Figure 4b).RAPD markers identityThe identification of species on the basis of their RAPD profiles relies on theassumption that bands of the same size appearing in different individuals,populations and/or species correspond to identical sequences and not only to acircumstantial coincidences in size. In order to test this assumption, hybridisationexperiments were performed with cloned RAPD markers.It could be seen that the cloned RAPD marker S11 (460) highlighted a bandthat was present in plant material collected in Trujillo and Piura as well as inthe accessions from Brazil, Cape Verde and Senegal (Figure 2b). This markeralso appeared in samples collected in Brazil and classified as P. affinis as well asin the putative P. chilensis · P. pallida hybrid collected in Cape Verde. Theseresults strongly suggest that the marker S11 (460) identifies populations ofP. pallida. The cloned RAPD marker S11 (460) was absent in sample seeds fromHonduras and Burkina Faso which confirms that neither sample is P. pallida.DNA hybridisation experiments using the cloned marker L01 (520) as a probeshowed that accessions collected in Peru (Piura and Trujillo) and the populationcollected in Honduras belong to the P. juliflora–pallida complex (Figure4b). The presence of this cloned marker in DNA templates derived fromaccessions collected in Brazil and Africa indicate that they also belong to theP. juliflora–pallida complex. Figure 4b also clearly shows the presence ofthe complex-specific marker L01 (520) in the putative P. chilensis · P. pallidahybrid.

10Figure 4. (a) Amplification of the marker L01 520 , specific to the P. juliflora–pallida complex, usingoligonucleotide L01 as primer and the DNAs purified from all accessions analysed in this paper astemplates. Note the absence of this marker in Lv., Vlt., Gl., and Ch. (b) Southern hybridisation ofthe cloned marker L01 520 used as probe on membranes containing the amplification products usingL01 as oligonucleotide primer and the genomic DNA from all accessions analysed in this paper astemplates. Lane names as Figure 1.Analysis of the similarity and clustering of Prosopis speciesThe RAPD profiles of the different Prosopis accessions were evaluated todetermine the degree of relatedness among them using numerical taxonomytechniques. Molecular markers present in two accessions were considered asshared markers only if they are of similar size. The clustering was performedusing 18 primers on seven accessions each from a different species. The numberof fragments amplified by each oligonucleotide in each sample was speciesspecificbut the average number of RAPD markers per sample was 120 (data notshown). The total number of bands per sample as well as the number of bandsshared in each pair-wise comparison (data not shown) allowed the constructionof a similarity matrix (Table 3) using the Nei and Li similarity index. The lowestsimilarity (0.09) resulted from the pair-wise comparison between an Old WorldProsopis species (P. cineraria, section Prosopis) and the rest of the accessions(section Algarobia), while the highest value (0.73) resulted from both the pairwisecomparisons P. glandulosa–laevigata and P. juliflora–pallida.Data from the similarity matrix were used to group the species using theUPGMA method (Figure 5). The cluster analysis revealed two major clusters;

11Table 3.Matrix of similarity among the Prosopis species studied.P. pallida(Tr)P. juliflora(Hnd)P. laevigata P. velutina P. glandulosa P. chilensis P. cinerariaP. pallida(Trujillo)P. juliflora 0.73(Honduras)P. laevigata 0.48 0.51P. velutina 0.47 0.53 0.71P. glandulosa 0.47 0.55 0.73 0.71P. chilensis 0.42 0.39 0.40 0.41 0.42P. cineraria 0.08 0.09 0.09 0.09 0.09 0.09one formed by P. juliflora and P. pallida (similarity 0.73), and a second comprisingthe North America species, P. laevigata, P. glandulosa and P. velutina(0.71). When the P. juliflora–pallida cluster was compared with the NorthAmerica cluster, the similarity value was 0.50. The similarity value betweenP. chilensis and the large cluster formed by P. laevigata, P. glandulosa,P. velutina, P. juliflora and P. pallida was 0.40. The North American cluster(P. laevigata, P. glandulosa and P. velutina) showed similarity values of 0.53and 0.47, respectively, when compared with P. juliflora (Honduras) andP. pallida (Trujillo) (data not shown). When P. chilensis was compared withP. juliflora (Honduras) and P. pallida (Trujillo) the similarity values were 0.40and 0.42, respectively.Figure 5. Dendrogram showing the estimates of the percentage of similarity among the Prosopisspecies studied. The Nei similarity index (Nei and Li 1979) and the UPGMA clustering algorithm(Sneath and Sokal 1973) were used.

12DiscussionThe value of molecular markers in clarifying the taxonomy of the genusProsopis has been demonstrated in this study and in an earlier work byRamı´rez et al. (1999). Results from RAPDs analysis mostly confirm thoseobtained from flavonoid studies (Solbrig et al. 1977) and electrophoresis ofseed proteins (Burghardt and Palacios 1997), which divide Prosopis intoidentifiable sections and species. Morphological analysis has also generally, butnot always, agreed with these findings (Pasiecznik et al. 2001; Harris et al.2003) and most recently, ploidy determination has helped to distinguishP. juliflora from other Prosopis species. However, the results obtained fromRAPDs analysis need to be treated with caution. The presence of a speciesspecificmarker in this study distinguishes the species only from others testedand does not guarantee that a similar band may not occur in other, as yetuntested, species. The use of bulked DNA from 10 seedlings, in some casesfrom more than one mother tree, allows the possibility that some individualgenotypes of a species might lack the specific marker. For example, the speciesspecificmarker for P. pallida in this study occurred in all, and only in, samplesthat were also identified as P. pallida on the basis of leaf morphology andploidy. This suggests that the molecular marker was effective in identifyingP. pallida among the samples in this study even if there is some risk in applyingthe technique to identify a single P. pallida individual in future.Ramı´rez et al. (1999) analysed Prosopis species from Asia, Africa and SouthAmerica. The present study aimed to analyse species from North and CentralAmerica that were not included in the earlier work, thereby extending thecoverage of the native range of the genus. Two of the species studied,P. chilensis and P. cineraria were common to both studies. In the present study,molecular markers were also applied to resolve the long-standing taxonomicconfusion regarding the identity of P. juliflora and P. pallida, especially whereintroduced. Although molecular studies have been applied to resolve taxonomicissues in the Argentinean centre, there has, until now, been no molecularstudy in the region from Mexico to Peru, where P. juliflora and P. pallida arefound and which is the origin of most of the material now widespreadthroughout the semi-arid tropics.Ramı´rez et al. (1999) found a molecular marker (S11 (730) ) which could beused to identify New World Prosopis species. In addition, markers were foundthat were specific to each of the New World sections Monilicarpa, Strombocarpaand Algarobia (Ramírez et al. 1999). In the present study, six markerswere found in all species from section Algarobia, which were absent inP. cineraria (section Prosopis) and it could be suggested that these were specificto section Algarobia. One of these (S07 (1160) ) was also found by Ramı´rez et al.(1999). This strongly suggests this marker as genuinely specific to sectionAlgarobia, now observed in 12 species of the section (series Ruscifoliae, Pallidaeand Chilenses), but was absent in P. kuntzei Harms (series Sericanthae)(Ramı´rez et al. 1999).

Pasiecznik et al. (2001) noted similarities between P. juliflora and P. pallidasufficient to lead them to treat the two species together as the P. juliflora–pallida complex, based on their overlapping geographic range, being the onlytruly tropical American Prosopis species, and having similar morphology andresource characters. The marker L01 (520) was found in both P. juliflora andP. pallida but not in the other species tested, and along with the high similarityindex (0.73), this supports the hypothesis of Pasiecznik et al. (2001). However,recent work has suggested a clear division of these species using ploidy (Harriset al. 2003), although further detailed studies are still required in areas of thenative range where both species have been identified in sympatry to fullyconfirm this.Although this paper did not aim to look for molecular markers specific toNorth American species, the data obtained could differentiate them but alsoclearly show a equivalently high similarity value at 0.70 among P. glandulosa,P. laevigata and P. velutina. Jua´rez-Mun˜ oz et al. (2002) used RAPDs to studythe inter- and intra-genetic variation between four wild Mexican populations ofP. glandulosa, P. laevigata and P. juliflora and found a genetic variation of72.48% between them and 27.52% within them, and postulated that P. julifloragave rise to the other two species. It may be postulated that these NorthAmerican species could be grouped together into a species complex as has beensuggested by Pasiecznik et al. (2001), and South American species of sectionAlgarobia also show patterns of clustering (Ramírez et al. 1999).Several authors have observed that sympatric Prosopis species, where noreproductive isolation exists, are able to form stable fertile hybrids (Hunzikeret al. 1975; Simpson 1977a). Hybrids have been identified in North AmericaJohnston 1962; Solbrig et al. 1977) and South America (Hunziker et al. 1975,1986; Burkart 1976; Saidman 1985, 1990). Tentative evidence for a P. pallida· P. chilensis hybrid was provided by this study. Burkart (1976) described aputative hybrid between P. juliflora and P. pallida in northern Ecuador butnoted that it could be a form of P. juliflora var. horrida (Kunth) Burkart.Johnston (1962) noted that P. inermis H.B.K. (syn. P. juliflora var. inermis(H.B.K.) Burkart) may be a synonym of P. pallida. Harris et al. (2003) haveidentified a putative hybrid of P. juliflora and P. pallida in Cape Verde based onploidy, but suggest that while hybrids may form they could be sterile.Pasiecznik et al. (2001) also divided the P. juliflora–pallida complex intothree geographically distinct groups; (1) Peru and Ecuador, (2) Colombia,Venezuela and the Caribbean islands, and (3) Pacific coastal Central American.Accessions from groups 1 and 3 were analysed in this study and found to begenetically distinct. Range-wide analysis is, however, required in order tofurther elucidate the genetic relationships within the P. juliflora–pallida complex.Burkart (1976) divided section Algarobia into six series. According toBurkart (1976), P. chilensis, P. juliflora, P. glandulosa, P. laevigata andP. velutina are all of series Chilenses, while P. pallida is in series Pallidae. Thisstudy clearly differentiated P. pallida and P. juliflora from each other and from13

14other Prosopis species using their RAPDs profiles. However, similarity valuesclearly show that P. juliflora is more closely related to P. pallida than to any ofthe tested species of series Chilenses. Ramı´rez et al. (1999) found the similaritiesbetween species of series Ruscifoliae, Pallidae and Chilenses were inconsistentwith the classification advanced by Burkart (1976). While Ramı´rez et al.(1999) found that P. kunzei of series Sericanthae was distinct and no species ofseries Denudantes or Humiles have been analysed, these studies may suggest areconsideration of the series rank within section Algarobia.There is considerable confusion as to the identity of Prosopis species introducedinto Africa, Asia, Australia and Northeast Brazil. The distinct profilesobserved with P. pallida and P. juliflora allow for the accurate identification ofthese species. In Burkina Faso, the accession analysed showed strong similaritiesto P. juliflora form Honduras. Therefore, it can be confirmed thatP. juliflora is present in Burkina Faso. The accession collected in Cape Verdehad also been identified as P. juliflora but perfectly matches P. pallida fromPeru and is distinct from P. juliflora. As this accession is representative of thedominant Prosopis used in reforestation programmes, it may be suggested thatmost Prosopis in Cape Verde has been incorrectly identified and is actuallyP. pallida. Similarly, in Senegal, the accession that had been identified asP. juliflora matches P. pallida from Peru, thus confirming that P. pallida ispresent in Senegal. The first recorded introduction of Prosopis into Africa wasin the early 19th century to Richard Toll, Senegal, then a French colony, wherethe accession tested in this study was collected. At this time, P. pallida wasintroduced to Hawaii from Peru via a herbarium in Paris, France (Esbenshade1980), but no records exists as to the origin of the Senegalese material. However,while the accession from Richard Toll has been identified as P. pallida,P. juliflora has also been identified by Burkart (1976) indicating that bothspecies are present in Senegal.In the north east of Brazil, two accessions were analysed, one of which hadbeen identified as P. juliflora and the other as P. affinis. Molecular markerssuggest that both may in fact be P. pallida. It is accepted that Prosopis seed wasintroduced into the northeast of Brazil in 1947 from Peru by S.C. Harland(Silva 1990). This can now be assumed to have been P. pallida. However,Prosopis has been identified in the north east of Brazil as long ago as 1879(Bentham 1879 in Burkart 1976) and two distinct types of Prosopis have beennoted in north east Brazil (Silva 1990). Pasiecznik et al. (2001) state that it ispossible that trade between the Caribbean and Brazil may have led to theintroduction of P. juliflora to the north east of Brazil in the 1800s and this maybe the origin of the Prosopis noted by Bentham. Later introductions from Perubeginning in the 1940s, however, are clearly P. pallida. While P. pallida is usedfor afforestation and is the most common, it cannot be stated that all Prosopisin Northeast Brazil is P. pallida. The identification of P. affinis in the Northeastof Brazil by Lima and Silva (1991) was based on the revised taxonomy ofProsopis in northern Peru by Ferreyra (1987). This was the first description ofP. affinis in Peru, with Ferreyra (1987) distinguishing it primarily on the basis

of pod coloration. Burkart (1976) noted P. affinis as native from Uruguay toParaguay. As the P. affinis identified in Brazil is probably P. pallida, it may alsobe assumed that the P. affinis identified in Peru by Ferreyra is also P. pallida,confirming the exclusion of this species from the P. juliflora–pallida complex byPasiecznik et al. (2001).Molecular markers have proven to be very useful tools in this study indifferentiating populations of the genus Prosopis at different taxonomic ranks.This study has allowed the identification and differentiation of the Prosopisspecies tested and has resolved the identity of introduced populations. This,and parallel studies using ploidy and leaf morphology (Harris et al. 2003), havehelped to resolve some of the outstanding taxonomical problems and has aidedin the development of a practical field guide for the identification of tropicalProsopis species, and P. juliflora and P. pallida in particular (Pasiecznik et al.2004) for the benefit of foresters, farmers, extensionists, researchers anddevelopment workers worldwide.15AcknowledgementsThis work was supported by a grant from the Accio´n Integrada, H.B. 1999–0035 of the Spanish Ministerio de Educacio´n y Cultura.ReferencesAlban L., Matorel M., Romero J., Grados N., Cruz, G. and Felker P. 2002. Cloning of elite,multipurpose trees of the Prosopis juliflora/pallida complex in Piura, Peru. Agroforest. Syst. 54:173–182.Burghardt A.D. and Palacios R.A. 1997. Electrophoretic characterisation of the American sectionsof Prosopis L. (Leguminosae: Mimosoideae). Bull. Int. Group Study Mimosoideae 20: 71–83.Burkart A. 1976. A monograph of the genus Posopis (Leguminosae subfam. Mimosoideae). (Part 1and 2). Catalogue of the recognised species of Prosopis. J. Arnold Arboretum 57: 219–249, 450–525.Church G.M. and Gilbert W. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. 81: 1991–1995.Esbenshade H.W. 1980. Kiawe: a tree crop in Hawaii. Int. Tree Crops J. 1: 125–130.Felker P. 1990. Prosopis spp. In: Burns R.M. and Mosquera M. (eds), Useful Trees of TropicalNorth America. North American Forestry Commission Compendium Publication, No.3. USDAForest Service, Washington DC, USA pp. 85–96.Ferreyra R. 1987. Estudio sistémico de los algarrobos de la costa norte del Perú. Dirección deInvestigacio´n Forestal y de Fauna. Min. de Agricultura, Lima, Peru.Griffith A.L. 1961. Acacia and Prosopis in the Dry Forests of the Tropics. FAO, Rome, Italy.Harris P.J.C., Pasiecznik N.M., Bradbury M. and Ramírez L. 1998. Problems and potential ofProsopis. In: Prendergast H.D.V., Etkin N.L., Harris D.R. and Houghton P.J. (eds), Plants forFood and Medicine. Royal Botanic Gardens, Kew, UK pp. 277–293.Harris P.J.C., Pasiecznik N.M., Smith S.J., Billington J. and Ramírez L. 2003. Differentiation ofProsopis juliflora (Sw.) DC. and P. pallida (H. & B. ex. Willd.) H.B.K. using foliar characters andploidy. Forest Ecol. Manage. 180: 153–164.Hunziker J.H., Poggio L., Naranjo C.A. and Palacios R.A. 1975. Cytogenetics of some species andnatural hybrids in Prosopis (Leguminosae). Can. J. Genet. Cytol. 17: 253–262.

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