molecular phylogeny and diversiﬁcation history of

Molecular phylogeny and diversification history ofProsopis (Fabaceae: Mimosoideae)

SANTIAGO ANDRÉS CATALANO1*, JUAN CÉSAR VILARDI1, DANIELA TOSTO1,2 andBEATRIZ OFELIA SAIDMAN1

1Departamento de Ecología Genética y Evolución, Facultad de Ciencias Exactas y Naturales,Universidad Nacional de Buenos Aires. Intendente Güiraldes 2160, Ciudad Universitaria,C1428EGA - Capital Federal, Argentina.2Instituto de Biotecnología, CICVyA INTA Castelar, CC 25 Castelar 1712, Argentina

Received 29 December 2006; accepted for publication 31 May 2007

The genus Prosopis is an important member of arid and semiarid environments around the world. To studyProsopis diversification and evolution, a combined approach including molecular phylogeny, molecular dating, andcharacter optimization analysis was applied. Phylogenetic relationships were inferred from five different molecularmarkers (matK-trnK, trnL-trnF, trnS-psbC, G3pdh, NIA). Taxon sampling involved a total of 30 Prosopis speciesthat represented all Sections and Series and the complete geographical range of the genus. The results suggest thatProsopis is not a natural group. Molecular dating analysis indicates that the divergence between SectionStrombocarpa and Section Algarobia plus Section Monilicarpa occurred in the Oligocene, contrasting with a muchrecent diversification (Late Miocene) within each of these groups. The diversification of the group formed by speciesof Series Chilenses, Pallidae, and Ruscifoliae is inferred to have started in the Pliocene, showing a highdiversification rate. The moment of diversification within the major lineages of American species of Prosopis iscoincident with the spreading of arid areas in the Americas, suggesting a climatic control for diversification of thegroup. Optimization of habitat parameters suggests an ancient occupation of arid environments by Prosopisspecies. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 621–640.

ADDITIONAL KEYWORDS: ancestral environments – aridification – character optimization – moleculardating – penalized likelihood.

INTRODUCTION

The genus Prosopis L. (Fabaceae) has approximately45 species, which are important members of arid andsemiarid environments (Fig. 1). Some of these speciesare characteristic of the driest regions in the world.For example, Prosopis tamarugo F. Philippi, is one ofthe few tree species capable of surviving in theextremely arid climate of the Atacama desert inNorthern Chile. Other species are distinctive of thelarge deserts of North America (Tamaulipas, Sonora,Chiuahua) and of arid and semiarid regions of SouthAmerica (Monte, Patagonia, Puna, and Chaco).However, only a few representatives of the genus,

such as Prosopis africana (Guill., Perr., & Rich.)Taubert, are partially distributed in subhumid tropi-cal or subtropical regions. From a taxonomic point ofview, the genus is divided into five Sections, basedmainly on the presence and type of armature andshoot structure (Burkart, 1976). The two main Sec-tions (Algarobia and Strombocarpa) are native toNorth and South America and include approximately90% of all the species of the genus. Section Monili-carpa comprises only one species endemic to centralArgentina. Sections Anonychium and Prosopis haveexclusively Old World representatives. Prosopis afri-cana, the only species of Section Anonychium, isnative to the Soudano-Guinean zone and neighbour-ing areas of Africa, from Senegal in the west to Sudanand Kenya in the east (Pasiecznik et al., 2001).*Corresponding author. E-mail: [email protected]

Biological Journal of the Linnean Society, 2008, 93, 621–640. With 6 figures

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 621–640 621

mailto:[email protected]

Section Prosopis comprises three species that arenative to North Africa and Asia, stretching east toIndia, north to Georgia and Turkmenistan, and westto Algeria along the North African coast (Pasieczniket al., 2001).

Different studies have evaluated the relationshipsamong Prosopis species (Saidman & Vilardi, 1987,1993; Saidman et al., 1996, 1998; Ramírez et al.,1999; Bessega, Saidman & Vilardi, 2005; Bessega,Vilardi & Saidman, 2006). However, these generallyinvolved phenetic analyses and included species ofonly some of the Series and Sections of the genus. Inaddition, none of these studies have evaluated thenature of the Prosopis generic limits as either out-groups were not included (Ramírez et al., 1999;Bessega et al., 2005), or these were distantly relatedto Prosopis species (Bessega et al., 2006). The onlycladistic analysis performed (Bessega et al., 2006)suggested that most of the groups taxonomically rec-ognized are not monophyletic.

Several ideas were proposed about the timing ofProsopis diversification (Pasiecznik et al., 2001).Burkart (1976), based on the presence of the genus inboth New and Old World, suggested a late Cretaceousor Early Tertiary origin for the genus, previous to theformation of the Atlantic Ocean. Following this idea,Ramírez et al. (1999) suggested that the genus wouldhave originated 130 Mya. However, this time frameis strongly contradicted by both palaeonthologicalevidence (Herendeen, Crepet & Dilcher, 1992)and molecular dating (Lavin, Herendeen &

Wojciechowski, 2005) because both support the notionthat the subfamily Mimosoideae originated between42–50 Mya. Burkart & Simpson (1977) also consid-ered that Prosopis is an old genus that diverged earlyinto several principal lineages but that, within someof these lineages, more recent episodes of expansionand isolation produced further speciation. To date, noattempt has been made to evaluate the divergencetimes within Prosopis using molecular dating.

The observation of hybrid formation between somespecies of Section Algarobia, together with their highmorphological similarity and partially overlappinggeographical distributions, led to the considerationthat this group is a syngameon (Palacios & Bravo,1981). However, recent molecular studies have sup-ported the biological meaning of the specific limitswithin this group (Saidman & Vilardi, 1987;Burghardt, 1995; Bessega et al., 2000) and indicatethe possible existence of isolating mechanisms thatrestrict the introgression between its species. Previ-ous cladistic analysis (Bessega et al., 2006) suggestedthat these species do not form a monophyletic group.Nonetheless, in that analysis, taxon sampling of non-hybridizing species within Algarobia was relativelyscarce. A more complete taxon sampling within Alga-robia would give the opportunity to study the timingand evolution of isolating barriers within this group.

During recent years, different studies have sup-ported the hypothesis that dry-adapted taxa in differ-ent regions of the world diverged concomitantly withthe expansion of arid environments. That is the case

Figure 1. Natural distribution of Prosopis after Pasiecznik et al. (2001). Number of native species are indicated for eachregion.

622 S. A. CATALANO ET AL.

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 621–640

for Phylica (Richardson et al., 2001), Ruchoideae(Klak, Reeves & Hedderson, 2003), and Ehrharta(Verboom, Linder & Stock, 2003) in South Africa,Tiquillia (Moore & Jansen, 2006) and Agave (Good-Avila et al., 2006) in North America, and Rheum(Wang, Yang & Liu, 2005) in East Asia. Evidencesupporting this hypothesis is in all cases a temporalrelationship between the increase of arid environ-ments and the diversification of these groups. Due tothe affinity of Prosopis with arid environments, this isa promising hypothesis to be tested in this genus.

OBJECTIVES AND HYPOTHESIS

The present study aimed to investigate the mainpatterns of Prosopis diversification. We are particu-larly interested in answering the following questions.(1) Which are the relationships among the main lin-eages within Prosopis? (2) When did Prosopis diver-sification occur? (3) Has the spreading of aridenvironments driven Prosopis evolution, as suggestedfor other arid-adapted taxa? To answer these ques-tions, a combined approach was applied that includedmolecular phylogeny, molecular dating, and characteroptimization analyses.

MATERIAL AND METHODSMOLECULAR TECHNIQUES

Five different gene regions were sequenced includ-ing two nuclear markers: coding and noncodingsequences of the glyceraldehyde-3-phosphate dehy-drogenase gene (G3pdh), one intron of the nitratereductase gene (NIA) and three chloroplast markers:partial sequences of trnS-psbC, trnK-matK and trnL-trnF regions.

Total DNA was extracted from five day old cotyle-dons or silica dried leaves and/or stems with theDNeasy Plant Mini Kit (QIAGEN). All polymerasechain reaction reactions were conducted in a totalvolume of 50 mL, containing 80 ng of DNA template,1 ¥ Taq-Buffer, 2.5 mM MgCl2, 0.25 U Taq polymerase,0.2 mM of each DNTP and 0.04 mM of each primer. Theintergen between the psbC and trnS genes was ampli-fied using the primers psbC and trnS (Demesure,Sodzi & Petit, 1995) together with a new internalprimer psbCint (5′-CGTTCTTGCCAAGGCTGTAT-3′).Since a preliminary survey indicated that most of thevariation among sequences was located in the first500 bp from the 3′ end region of trnS gene, only thisregion was subsequently sequenced for the rest of thespecies. G3pdh region was amplified with primersGPDX7F and GPDXR9 (Strand, Leebens-Mack &Milligan, 1997) and a newly designed primerG3pdhintF (5′-GACTGGAGGGGTGGAAGAG-3′).NIA intron was amplified with a degenerate primer

pair: NIA3F-NIA3R (Howarth & Baum, 2002) andsequenced with a nondegenerate pair: NIAproF (5′-GAACCAGCAGTTGTTCATCAT-3′) and NIAproR (5′-ACTGGTGCTGGTGTTTTTGG-3′). trnL intron wasamplified and sequenced using the primers c, d, e andf (Taberlet et al., 1991). Partial sequences of trnKintron were amplified with Ac283R and trnK 3914Fprimers (Miller & Bayer, 2001). Partial sequences ofmatK were amplified and sequenced with primers685F and 1265R (Lavin et al., 2000). To amplify theregion trnS-psbC, trnK-matK and trnL-trnF, the fol-lowing cycling profile was used: 35 cycles of 1 min at94 °C, 1 min at 56 °C, and 1.5 min at 72 °C. In thecase of G3pdh and specific NIA amplification, thecycling profile was the same, except that annealingtemperature for these regions was set to 52 °C. Forthe amplification of NIA region with degenerateprimers, a stepdown programme was used thatincluded ten cycles in which melting temperaturedecreased 1 °C per cycle starting from 55 °C. Twentyadditional cycles were performed with 30 s at 94 °C,30 s at 45 °C, and 40 s at 72 °C. In all cases, theprogramme started with one cycle at 94 °C for 4 minand finished with 7 min at 72 °C.

PHYLOGENETIC ANALYSIS

Thirty species of Prosopis were included in this analy-sis. Taxon sampling within Prosopis was planned toinclude representatives from the entire geographicalrange of the genus (Appendix 1). Moreover, taxonomicdiversity of the genus was totally represented asspecies of all Sections and Series were included.Three different analyses that involved different com-binations of taxa and molecular markers were per-formed. A first analysis was performed comprisingtrnL-trnF and trnK-matK (two-marker analysis)sequences of 80 legume species downloaded fromGenBank (see Supplementary material) plus fiveProsopis species that represented each Section of thegenus. In the second analysis (three-marker analy-sis), G3pdh, NIA, and trnS-psbC sequences from allthe sampled species of Prosopis plus Prosopidastrumangusticarpum, Acacia caven, Xerocladia viridiramis,and Lotus japonicus were analysed in a total evidencecontext (Kluge, 1989). As more than one sequence ofeach species was sampled in Algarobia clade, the totalnumber of terminals was 41. This was due to the highlevel of shared polymorphism found in previousanalyses within this group (Saidman & Vilardi, 1987;Bessega et al., 2000). A third analysis (five-markeranalysis) involved a combination of the first twodatasets.

Sequences were edited with Bioedit software (Hall,1999) and aligned with Clustal X (Thompson et al.,1997) with a posteriori minor manual changes. Par-

PROSOPIS PHYLOGENY AND EVOLUTION 623


simony based searches were performed using the TNTprogram (Goloboff, Farris & Nixon, 2003). Charactersand state transformations were considered as equallyweighted. Gaps were alternatively treated as a fifthstate, missing entries or as separate charactersSimmons & Ochoterena (2000). Since the results werevery similar, only those based on fifth state coding areshown and discussed. Within trnL-trnF region, a dele-tion of approximately 300 bp was found in someMimosoid species. Individual gaps representing thisdeletion were treated as missing data. As a model-based analysis alternative to the parsimony analysis,we also conducted a Bayesian analysis. Search strat-egies in parsimony and Bayesian analysis are indi-cated in Appendix 2.

ESTIMATING DIVERGENCE TIMES

To estimate divergence times, G3pdh and trnSsequences were concatenated and analysed together.Simultaneous analysis of gene sequences from mul-tiple loci was performed because the penalized likeli-hood method used for age estimation (see below) isprone to errors when dealing with zero lengthbranches or very short branches (Sanderson, 2003).The congruence between both datasets in branchlengths was evaluated by the likelihood ratio testdescribed by Lewis (1998) and Xiang et al. (2005). Thestatistics of this test is -2 [ln L - (ln L1 + ln L2)], whereL1 is the likelihood of the tree obtained from the firstgene, L2 is the likelihood of the tree obtained from thesecond gene and L is the likelihood obtained from thecombined analysis of both genes. The value of thisstatistic was compared to a c2 distribution withdegrees of freedom equal to the sum of 2N + 3 - P,where N is the number of terminals and P is thenumber of free parameters of the model used in thelikelihood calculation. NIA sequences were notincluded because it was not possible to obtain thesequence of this region from P. angusticarpum, one ofthe taxa that defines the unique fixed node in thecalibration step. Furthermore, X. viridiramis was notincluded because only a part of G3pdh was possible tobe sequenced. Branch lengths were estimated byMaximum Likelihood using PAUP* (Swofford, 2002).The evolutionary model was chosen with the hierar-chical likelihood ratio test as implemented inModeltest, version 3.06 (Posada & Crandall, 1998).Penalized likelihood implemented in the R8s software(Sanderson, 2003) was used for age estimation usingthe Powell algorithm and defining smoothing param-eter values with a cross validation procedure (Sander-son, 2002).

Divergence times were derived from molecular datacombining two calibration points. The time obtainedin Lavin et al. (2005) for the divergence between one

species of Section Algarobia (Prosopis pallida) andone species of genus Prosopidastrum (Prosopidastrummexicanum) was used to fix the node subtendingProsopidastrum and the American species of Prosopis(ASP). To take into account the error associated withthe estimation of Lavin et al. (2005), we repeated theanalysis considering the mean ± SD. A second calibra-tion point was used to give minimum age to the ASPdivergence. In this case, this fossil corresponds topollen grains obtained from Early Oligocene sedi-ments of British Columbia, Canada (Piel, 1971). Asthis Epoch extends from 28.4 and 33.7 Mya, theformer age was used to define the minimum age.Since the sister group of ASP + Xerocladia clade wasnot clearly defined in our phylogenetic analysis, twodifferent topologies were considered, one of them cor-responding to the results of the three-marker analysisand the other corresponding to the two-marker analy-sis. Error in age estimates was evaluated by boot-strapping following Sanderson (2002).

DIVERSIFICATION ANALYSIS

To study the possible changes in diversification rateover time a lineages through time (LTT) analysis (Neeet al., 1995) was performed. LTT analysis was repeat-edly performed with extreme values of divergencetimes obtained in the penalized likelihood analysis.The lack of resolution in the mesquite clade (seeResults) was considered in two ways for this analysis:(1) as a hard polytomy with all the lineages divergingsimultaneously and (2) as if the diversificationoccurred at a constant rate. Consequently, PHYLO-GEN program, version 1.1 (Rambaut, 2002), was usedto simulate trees under a Yule Model of diversification(Yule, 1924) with the final number of species equal tothe present in the mesquite group. Subsequently, thissubtree was grafted in the original tree, replacing thepolytomy in the mesquite clade. This was repeated for100 different simulated subtrees. As the generalpattern obtained was the same, LTT of one randomlychosen tree is shown. To examine the effects ofmissing species in LTT analysis, these were placed inthe most likely position on the tree on the grounds oftaxonomic treatment (Burkart, 1976) in accordnacewith Barraclough & Vogler (2002). To compare diver-gence rates within Prosopis with those obtained inother plant genera, per lineage net diversificationrate (NDR) sensu Coyne & Orr (2004) was calculated.As in LTT analysis, missing species were included intheir most likely place on the tree.

ESTIMATION OF ANCESTRAL HABITAT

To study the historical relationship of Prosopis witharid environments, maximum and minimum value ofhumidity index (HI) were gathered for the natural



geographical range of the species of Prosopis includedin the phylogenetic analysis plus X. viridiramis. HI isdefined as the ratio of precipitation to potentialevapotranspiration (UNEP, 1992). Species distribu-tions were defined from the bibliography (Ross, 1975;Burkart, 1976; Rzedowski, 1988; Roig, 1993) andfrom herbarium specimens (BAFC, SI, LIL, CTES).Climate data for each species (see Supplementarymaterial) were extracted from FAO World ClimateDatabase using the New LocClim software (FAO,2005). Regions were classified as a function of HIvalues following UNEP (1992): 0.05 < Hyper-Arid;0.05–0.20 Arid; 0.20–0.50 Semi-Arid; 0.50–0.65Dry-Subhumid; > 0.65 Humid. The phrase ‘aridenvironments’ is used here to collectively representhyperarid, arid and semiarid classes. Maximum andMinimum of HI (MaHI and MiHI, respectively) wereindependently optimized on the tree as in theMinMax coding (Hardy & Linder, 2005). Once thesevalues were optimized, they were used to delimit thetotal range of HI for each ancestral node. We consid-ered that MinMax coding is the most suitable for thischaracter because maximum drought tolerance mightbe partial or totally decoupled from maximum humid-ity tolerance. MaHI and MiHI were optimized usingthe implementation in TNT (Goloboff et al., 2003) ofFarris (1970) optimization to deal with continuouscharacters (Goloboff, Mattoni & Quinteros, 2006). Inthis case, linear changes are minimized along eachbranch. To evaluate how uncertainty in the state ofthe root can affect the results, the analysis wasrepeated with two extreme values: hyperarid andhumid. In addition, the effect of topological uncer-tainty within the mesquite clade (see Results) in theresults was evaluated by considering 10 000 differentresolutions of this clade. A strict association of aparticular node with arid environments was deter-mined when the value of MaHI was lower thanthe limit between humid and arid environments(HI < 0.50). A nonstrict association with arid environ-ments corresponded to a value of MiHI lower andMaHI higher than this limit. No association with arid

environment occurred when MiHI value was higherthan this limit.

RESULTSPHYLOGENETIC RESULTS

Two-marker analysisStatistics of the different phylogenetic analyses basedon parsimony are shown in Table 1. Strict consensussuggests that Prosopis is not monophyletic (Fig. 2).Forcing the monophyly of the genus gave a tree ninesteps longer than the optimum (Table 2). ASP formeda highly supported clade (BS = 4; J = 95) with X. viri-diramis (subsequently named ASP + Xerocladia). ASPdid not appear as monophyletic because X. viridira-mis formed a monophyletic group with P. tamarugo.However, this clade is not well supported and (BS = 1;J = 66) and forcing the monophyly of ASP gave a treeonly one step longer than the optimum. Prosopiscineraria appeared as sister group of this cladethough with low support (BS = 1; J < 50). Prosopisnigra of Section Algarobia formed a strongly sup-ported group (BS = 5; J = 98) with Prosopis argentinaof Section Monilicarpa. Bayesian analysis gave iden-tical results concerning the lack of monophyly ofProsopis and the relationships among APS. By con-trast, P. cineraria does not appear forming a mono-phyletic clade with ASP + Xerocladia. Posteriorprobabilities for the groups found in the Bayesiananalysis are given in Figure 2.

Three-marker and five-marker analysesStrict consensus obtained in this analysis agreed withtwo-marker analysis in rejecting Prosopis monophyly(Fig. 3). Forcing the monophyly of the genus gave atree six steps longer than the optimum (Table 2). Asin two-marker analysis, ASP formed a monophyleticgroup with X. viridiramis (APS + Xerocladia clade),though with lower support (BS = 1; J = 64). Inaddition, forcing the monophyly of the ASP gave atree only one steps longer than the optimum. Two

Table 1. Statistics of the different phylogenetic analyses based on parsimony

Two-marker analysis Three-marker analysis Five-marker analysis

Number of MPT 224 9 4680Length 5079 1552 6710Number of characters 4031 (1444–2587)† 1764 (648–701–415)* 5795Number of informative characters 1357 (609–748)† 391 (193–183–15)* 1458CI/RI 0.496/0.655 0.695/0.846 0.535/0.700

*Statistics for G3pdh, NIA and trnS-psbC characters, respectively.†Statistics for trnL-trnF and trnK-matK characters, respectively.CI, consistency index (excluding uninformative characters); MPT, most parsimonious trees; RI, retention index.



Zapoteca tetragona

Xylia africana

Xerocladia viridiramis

Tetrapleura tetraptera

Prosopis tamarugo

Schleinitzia insularum

Samanea saman

Pseudosamanea guachapele

Pseudoprosopis gilletii

Prosopis cineraria

Prosopis africana

Prosopidastrum mexicanumProsopidastrum angusticarpum

Plathymenia reticulata

Piptadeniopsis lomentifera

Piptadenia viridiflora

Piptadeniastrum africanum

Pentaclethra eetveldtiana

Parkia biglandulosa

Paraserianthes lophantha

Pararchidendron pruinosum

Parapiptadenia rigida

Prosopis nigra

Newtonia buchananii

Neptunia gracilis

Mimozyganthus carinatus

Mimosa aculeaticarpaMicrolobius foetidus

Lysiloma divaricatum

Leucaena greggiiKanaloa kahoolawensis

Inga edulis

Havardia albicans

Gagnebina bernieriana

Fillaeopsis discophora

Faidherbia albida

Enterolobium contortisiliquum

Entada abyssinicaElephantorrhiza elephantina

Ebenopsis ebano

Dinizia excelsa

Dichrostachys paucifoliolata

Desmanthus acuminatus

Cylicodiscus gabunensis

Chloroleucon mangens

Cercidium andicola

Cedrelinga cateniformis

Cathormion umbellatum

Calpocalyx dinklagei

Calliandropsis nervosus

Calliandra carbonaria

Prosopis argentina

Anadenanthera colubrina

Amblygonocarpus andongensis

Albizia kalkora

Alantsilodendron alluaudianum

Adenanthera pavonina

Acacia visco

Acacia tortilis

Acacia spinescens

Acacia senegal

Acacia schweinfurthii

Acacia schottii

Acacia roemeriana

Acacia pulchella

Acacia platycarpa

Acacia pennatula

Acacia neovernicosa

Acacia modesta

Acacia melanoxylon

Acacia macracantha

Acacia karroo

Acacia hindsii

Acacia glomerosa

Acacia elata

Acacia drummondii

Acacia dolichostachyaAcacia coulteri

Acacia constricta

Acacia collinsiiAcacia cochlicantha

Acacia chiapensis

Acacia caven

Acacia boliviana

Acacia berlandieri

Acacia ampliceps

Lotus japonicus

2/66

>12/99

3/-

1/52

12/100

12/1006/98

12/100

>12/1001/-

2/-

9/98

9/99

1/-

1/55

3/76

1/-

1/-4/98

4/95

1/-

1/66

5/98

1/-

8/100

2/55

4/87

1/-

1/74

1/90

>12/100

11/1003/73

2/78

2/78

8/1001/72

3/93

1/58

2/796/99

>12/100

6/74

1/-4/92

1/-3/75

8/-6/97

1/-

2/-

10/100

9/1001/-

1/-5/75

1/-1/-

7/921/68

1/-3/-

4/-

4/91

5/1005/95

1/-

>>

1.000.55

0.981.00

0.68

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.001.00

0.53

1.00

1.00

1.00

1.000.99

1.00

0.991.00

1.00

1.00

1.00

1.001.00

1.00

0.93

1.00

1.00

1.00

0.79

1.00

1.00

0.99

1.00

1.00

0.98

0.78

0.96

1.00

1.00

0.68

1.00



subclades were recognized in ASP + Xerocladia clade.One of them is formed by species of Section Stromb-ocarpa plus X. viridiramis whereas the other (subse-quently named Algarobia s.l.) is formed by species ofSection Algarobia plus P. argentina of Section Monili-carpa (BS = 6; J = 100). Section Algarobia appeared asparaphyletic because P. argentina formed a well sup-ported clade (BS = 5; J = 100) with species of SeriesHumiles, Sericanthae, and Denudantes of SectionAlgarobia (Prosopis kuntzei–P. argentina clade).Forcing the monophyly of Section Algarobia gave atree ten steps longer. Series Denudantes (representedhere by Prosopis denudans, Prosopis ruizleali andProsopis castellanosii) appeared as monophyletic butSeries Sericanthae (P. kuntzei and Prosopis serican-tha) would be paraphyletic. The sister group of theP. kuntzei–P. argentina clade was a very well sup-ported clade (BS = 6, J = 100) formed by species ofseries Chilenses, Pallidae, and Ruscifoliae (mesquiteclade). However, the relationships among the speciesof this clade are unclear because the strict consensusis highly unresolved and individuals of the samespecies in most of the cases did not form monophyleticgroups. Section Strombocarpa was monophyletic withX. viridiramis as its sister clade. Within this section,two groups were well supported. One of them corre-sponded to Series Cavenicarpae (Prosopis ferox andP. tamarugo) whereas the other was formed by twoNorth American species of Series Strombocarpae:Prosopis pubescens and Prosopis palmeri. Bayesiananalysis showed very similar results (Fig. 3). The

main difference was related with the sister group ofAPS + Xerocladia. In this analysis, the sister groupwas formed by A. caven and P. angusticarpum.

The results obtained in the five-marker analysiswere very concordant with those obtained in the othertwo analyses (see Supplementary material) andthe support increased in several relevant nodes:ASP + Xerocladia clade (BS = 9; J = 96); Stromb-ocarpa clade (BS = 2; J = 83); Strombocarpa +X. viridiramis clade (BS = 2; J = 77). However, differ-ing from the two-marker analysis, P. cineraria did notappear as sister group of APS + Xerocladia. Strictconsensus indicated identical relationships withinAPS + Xerocladia clade than those given by the three-marker analysis, except that the groupings within themesquite clade are less resolved.

MOLECULAR DATING

The optimal model chosen by the hierarchical likeli-hood ratio test was Hasegawa–Kishino–Yano plusGamma (HKY + G). A likelihood ratio test signifi-cantly rejected the hypothesis of evolutionary rateconstancy for the molecular regions analysed (Clockconstrained –lnL = 4394.64; unconstrained –lnL =4363.50; d.f. = 39; P < 0.05). Therefore, divergencetimes were estimated in a penalized likelihoodapproach that does not assume rate constancy. Tostudy the possible effect of short branches withinmesquite clade in the age estimated for the othernodes outside this group, an analysis leaving only one

Figure 2. Strict consensus of the 224 optimal trees based two-marker analysis (trnL-trnF, trnK-matK) with parsimonyas optimality criteria. Absolute Bremer support (left) and jackknifing values over 50% (right) are indicated abovebranches. Posterior probabilities estimated in Bayesian analysis are indicated below branches. Prosopis species areunderlined.�

Table 2. Extra steps obtained in searches conducted with positive constrains for some groupings not appearing in mostparsimonious trees

Two-marker analysis(5079)

Three-marker analysis(1552)

Five-marker analysis(6710)

Section Strombocarpa +Prosopis cineraria

6 63 68

Prosopis + Prosopidastrum 14 3 14Prosopis + Xerocladia 4 5 5Genus Prosopis 9 6 11Section Algarobia NA 10 10OSP + Xerocladia 8 6 11OSP 4 5 11ASP 1 1 3

In parentheses: optimal lengths in unconstrained searches. ASP, Americans species of Prosopis; NA, not applicable; OSP,Old World species of Prosopis.



terminal was performed. The results differ onlyslightly (data not shown).

The common and most relevant pattern observed inthe different analyses was (Fig. 4; Table 3): (1) anancient divergence between the two main lineages ofASP within the Oligocene; (2) a more recent diver-gence, starting in the late Miocene, within Stromb-ocarpa and Algarobia s.l.; and (3) a divergence amongmesquite species starting in the Pliocene.

DIVERSIFICATION ANALYSIS

Phylogenetic analyses indicated that Old Worldspecies of Prosopis are not closely related with theASP. This was true for P. africana in all the analysesand for P. cineraria in two out of three analyses.

Taking this into account, the study of diversificationand climate affinity evolution was restricted to theASP + Xerocladia clade.

Lineage through time analysis clearly suggests anincrease in the diversification rate of the ASP groupfrom the Late Miocene (Fig. 5A, B) until the present.Within this period, a more detailed examination sug-gests two different moments of rate acceleration: onein the Late Miocene and the other in the Pliocene.This general pattern is neither affected by the inclu-sion of missing species in the analysis (Fig. 5B), norby the different topologies used to derive the chrono-grams (data not shown). The NDR estimated for dif-ferent clades within Prosopis and for other taxa isshown in Table 4.

P.vinalillo 2

P.vinalillo 1

P. torquata

P. tamaulipana

P. tamarugo

P. strombulifera

P. sericantha

P. ruscifolia

P. ruizleali

P. reptans

P.pubescens

P.africana

Prosopidastrum angusticarpum

P.palmeri

P.nigra 2

P.nigra 1

Lotus japonicus

P.kuntzei 2P.kuntzei 1

P. juliflora 2

P. juliflora 1

P.humilis

P.glandulosa

P. flexuosa 2

P. flexuosa 1

P. ferox

P.denudans

P.chilensis 2P.chilensis 1

P.castellanosii

P.campestris 2P.campestris 1

P.caldenia 2

P.caldenia 1

P.articulata

P.alpataco

P.albaP.affinis

Acacia caven

-

100

100

100

80

87

-

61

62

65

72100

99

64

100

5476

57

1

1

1

1

1

1

2

2

2

3

2/ -

3

3

5

4

4

6

1

6

5

P.k

untz

eiP

. arg

entin

a- clad

e

P.cineraria

2

13

3/ -

60

Alg

arob

ia s

.l cl

ade

Str

ombo

carp

acl

ade

Am

eric

ansp

ecie

sof

clad

eP

roso

pis

+X

eroc

ladi

a

3

4

P.argentina

Mes

quit

ecl

ade

//

/

/

/

/57

/

/

/

/

/

/

/

/

/

/

//

702 /965/

/

6

/

1.000.58

0.96

1.001.00

0.690.63

0.83

0.99

0.910.99

0.64

0.96

0.99

1.00

0.61

0.85

0.90

0.55

Xerocladia viridiramis

7620.58

/

Str

ombo

carp

a+

clad

eX

eroc

ladi

a

Figure 3. Strict consensus of the nine optimal trees obtained in the three-marker analysis (trnS-psbC, G3pdh, NIA) withparsimony as optimality criteria. Absolute Bremer support (left) and jackknifing values over 50% (right) are indicatedabove branches. Posterior probabilities estimated in Bayesian analysis are indicated below branches. Rectangles delimiteach Section: 1, Prosopis; 2, Anonychium; 3, Strombocarpa; 4, Algarobia; 5, Monilicarpa. Black bars indicate the cladeswhose names are used throughout the manuscript.



ANCESTRAL CLIMATE ESTIMATION

Optimization analysis unambiguously indicated thatthe range of the most recent common ancestor(MRCA) of the clade formed by the ASP plus X. viri-diramis extended to semiarid or more xeric conditions

(Fig. 6). A strict association of this node with aridenvironments was obtained for all the reconstructionswhen the state of the root was defined as hyperaridand, in some of the reconstructions, when the state ofthe root was defined as humid. In the case of Strom-bocarpa clade, the different assignments of states to

Early Middle LatePLIOCENE

OLIGOCENEEarly Late

MIOCENEPLE

1.85.311.216.423.828.433.7

angusticarpum

P.ruizlealiP. strombulifera

P. reptansP. torquataP.palmeriP.pubescens

P. tamarugo

P.argentinaP. sericanthaP.humilisP.kuntzei 1P.kuntzei 2P.denudansP.castellanosii

P. ferox

P. flexuosa 1P.articulataP.caldenia 1P. juliflora 1P. juliflora 2P.glandulosaP. tamaulipanaP.vinalillo 2P.campestris 2P.nigra 2P.caldenia 2P. ruscifoliaP.alpatacoP.albaP.affinisP.chilensis 2P.campestris 1P.chilensis 1P.nigra 1P.vinalillo 1

P. flexuosa 2

Prosopidastrum

Figure 4. Chronogram resulting from penalized likelihood analysis. The topology corresponds with one of the mostparsimonious trees obtained in three-marker analysis (Fig. 3) and calibrated according to divergence times derived fromLavin et al. (2005). The approximate temporal spreading of arid zones in America is shown over the timescale with darkerzones representing the moment of its maximum historical extension. Grey bars over the nodes represent 95% confidenceinterval for node ages obtained by bootstrap techniques. PLE, Pleistocene.

Table 3. Divergence times (Mya) obtained in the penalized likelihood analysis

MRCA

Outgroup placement

Three-marker analysis Two-marker analysis

ASP + Xerocladia 28.96 (26.25–31.68) 29.37 (27.39–33.05)Algarobia s.l. 7.89 (7.15–8.62) 7.96 (7.58–9.14)Mesquites 3.65 (3.31–3.99) 3.69 (3.38–4.08)Strombocarpa 9.21 (8.35–10.07) 9.31 (9.18–11.07)

Ages estimated fixing the node subtending ASP + Xerocladia clade and Prosopidastrum to 33.2 Mya following the meanvalue obtained in Lavin et al. (2005). Ages estimated considering the mean ± one standard deviation are given inparentheses. When the topology corresponded to the two-marker analysis the node subtending ASP + Xerocladia node wasconstrained to a minimum age of 28.4 Mya following the fossil evidence.ASP, American species of Prosopis; MRCA, most recent common ancestor.



the root did not affect the results as, in all cases, itsMRCA appear to be restricted to arid environments.The same result was obtained for the MRCA ofX. viridiramis + Strombocarpa clade. In the case ofthe MRCA of Algarobia s.l., P. kuntzei–P. argentinaand mesquite clades, optimal reconstructions in-cluded in some cases ranges that fell completelywithin arid conditions whereas, in others, the rangesextended from arid to humid conditions.

DISCUSSIONSYSTEMATICS

The phylogenetic analysis performed in the presentstudy is the first to include species of all the Sectionsand Series of the genus and intends to test Prosopismonophyly by incorporating several species ofrelated genera. The results of this analysis suggestthat Prosopis would not be monophyletic. ASPappear to be more related with X. viridiramis, theonly species of this southern Africa genus, than withOld World representatives of Prosopis. A close rela-

tionship among Prosopis species and X. viridiramishas been previously indicated by Luckow et al.(2004). However, considering that only species ofSection Algarobia were included in that analysis, acloser relationship of ASP with X. viridiramis thanwith Old World Prosopis species is a novel and inter-esting result. Prosopis monophyly was also rejecteddue to the position of P. africana and, in some of theanalyses, the position of P. cineraria. It is suggestivethat many of the characters used to define the genusby Burkart (1976) would be, as stated by him, primi-tive within the whole subfamily (symplesiomorphic).If this is confirmed, the idea of Prosopis as naturalgroup might also be questioned at morphologicalgrounds.

A closer relationship among species of the Ameri-can sections of Prosopis (Strombocarpa, Algarobia,and Monilicarpa) than with species of the Old Worldsections agrees with the subgeneric classification pro-posed by (Guinet & Bessedik, 1984) based mainly onpalynological evidence. Particularly, they included allthe ASP in the subgenus Neoprosopis, whereas theother two subgenera corresponded to Section Proso-pis and Anonychium, the two Old World sectionsconsidered in Burkart (1976). However, the corre-spondence between the groups obtained in our analy-ses and the Subgenus Neoprosopis is not strictbecause ASP formed a monophyletic group withX. viridiramis. This close relationship among Ameri-can Sections of Prosopis contrasts with the ideastated by Burkart (1976) that Section Strombocarpais closely related to Section Prosopis because speciesof both Sections present comparatively smallerlegumes and share the ability to spread by means ofrootsuckers.

Species of Section Algarobia and P. argentinaformed a highly supported clade (Algarobia s.l.) thatis subsequently divided into two highly supportedclades. One of them corresponded to species of SeriesPallidae, Chilenses, and Ruscifoliae (mesquite clade),whereas the other corresponded to species of seriesSericanthae, Humiles, and Denudantes plus P. argen-tina of Section Monilicarpa (P. kuntzei–P. argentinaclade). This latter clade, endemic to southern SouthAmerica, includes all the subaphyllous and aphyllousspecies sampled for the phylogenetic analysis. Thisresult suggests that this condition might have origi-nated only once in Prosopis history.

The results obtained in the present study are con-sistent with the only previous cladistic analysisinvolving Prosopis species (Bessega et al., 2006) inshowing that the Section Algarobia is not monophyl-etic, and that P. argentina (Section Monilicarpa) isclose to species of Section Algarobia. However, severaldisagreements between these two studies can beobserved. Within Section Algarobia, the clade formed

Ln

(num

ber

ofli

neag

es)

Ln

(num

ber

ofli

neag

es)A

B

MIOCENEOLIGOCENE

23.828.433.7 16.4 11.2 5.3 1.8

0.5

1.0

1.5

2.0

2.5

3.0

3.5

1.0

1.5

2.5

3.0

3.5

2.0

E L E M LPLIOC PLE

Figure 5. Lineages-through-time plot (LTT) derived fromthe chronogram of Fig. 4. A, LTT including missingspecies. B, LTT with species included in the three-markeranalysis. Dotted lines represent LTT when the polytomy inthe mesquite clade is considered as hard. Black arrow-heads indicate the beginning of the diversification withinAlgarobia s.l. and Strombocarpa clades. Empty arrow-heads indicate the start of mesquite diversification. E,Early; M, Medium; L, Late; PLIOC, Pliocene; PLE, Pleis-tocene. The approximate temporal spreading of arid zonesin America is shown over the timescale with darker zonesrepresenting the moment of its maximum historicalextension.



Table 4. Comparison of net diversification rates (NDR) estimated within Prosopis with those obtained for other planttaxa

NDR Life forms

ProsopisMesquite clade 0.58–0.72* Trees†Prosopis kuntzei–Prosopis argentina clade 0.16–0.21* Trees and shrubsStrombocarpa clade 0.27–0.37* Trees and shrubsASP + Xerocladia clade 0.12–0.14* Trees and shrubs

Others taxaInga1 0.50 TreesLupinus2 1.92–3.84 Postrate herbs to treeletRuschioideae3 0.77–1.75 Stone plants to tree-like shrubsClarkia, section Peripetasma4 0.62 HerbsGentianella5 1.7–3.2 Herbs and small shrubsValeriana6 0.8–1.3 HerbsYucca7 0.21–0.27 Succulent rossete plants to arborescentEhrharta8 0.12–0.57 HerbsGaertnera9 0.71–0.83 Shrubs and small treesHawaiian silverwoods10 0.55 Vines and shrubs to treesAgave7 0.51 Succulent rossete plants to arborescent

*Range of rates values estimated with times derived from the different calibration strategies.†95% of the species are trees or present arboreal forms.1Richardson et al. (2001); 2Hughes & Eastwood (2006); 3Klak et al. (2003); 4Hey (1992); 5Von Hagen & Kadereit (2001);6Bell & Donoghue (2005); 7Good-Avila et al. (2006); 8Verboom et al. (2003); 9Malcomber (2002); 10Baldwin & Sanderson(1998).ASP, Americans species of Prosopis.

AAR-SA

SA-SH-HU

AR-SASA-SH-HU

AR-SASA-SH-HU

AR-SASA-SH-HU

B

Mes

quite

sS

trom

boca

rpa

AR-SA

AR-SA

AR-SA

HA-AR

HA-AR

P.ku

ntze

i-P.

arge

ntin

a

Xerocladia

ARAR-SA

Xerocladia

ARSA

HA-ARSA

SA-SH-HU

SA

SA-SH-HU

SA-SH-HU

SA

Mes

quite

sS

trom

boca

rpa

P.ku

ntze

i-P.

arge

ntin

a

Figure 6. Reconstruction of ancestral states of relevant nodes for maximum (above) and minimum (below) humidityindex (MaHI and MiHI, respectively). Instead of HI values, classes that represent each value are shown. A, optimizationresult when the state of the root was defined as hyperarid. B, optimization result when the state of the root was definedas humid. The presence of ambiguities in the optimization step and/or the different possible resolutions within themesquite clade produced, in some nodes, more than one optimal value for MiHI and MaHI.



by species of Series Ruscifoliae, Chilenses, and Pal-lidae (mesquite clade) is not monophyletic in theanalysis of Bessega et al. (2006). A similar situation isobserved in Section Strombocarpa, which is mono-phyltic in our analysis (Fig. 3) but not in that ofBessega et al. (2006). In addition, in the presentanalysis, we found that American Prosopis species aremore closely related among themselves than with OldWorld Prosopis species, whereas in the analysis ofBessega et al. (2006), P. cineraria, an Old Worldspecies, appears intermingled with New Worldspecies. The groupings obtained in our analysis aregenerally more in agreement with previous analyses(Ramírez et al., 1999; Bessega et al., 2005), and withtraditional taxonomy (Fig. 3). Although several ad hocexplanations could be given to account for the dis-agreement between these two analyses, it is possiblethat the wider sampling of species and molecular-markers performed in the present study might be thebest explanation for these differences.

MOLECULAR DATING AND FOSSIL RECORD

Molecular dating analysis suggests that the diver-gence between the two main groups within ASP +Xerocladia clade is remarkably ancient (26.2–33 Mya)considering that the diversification of the extantmembers of the whole subfamily Mimosoideae wouldhave occurred during the last 40 Myr (Lavin et al.,2005). However, most of the process of diversificationwithin each of the major clades occurred morerecently, in the Late Miocene. This contrast betweenearly divergence of the main groups and late diver-gence within these groups had already been estab-lished on the grounds of morphological evidence(Burkart & Simpson, 1977; Pasiecznik et al., 2001).

The number of fossil remains assigned to Prosopisis particularly abundant (Catalano et al. unpubl.data). However, as none of the fossils have beenanalysed in a phylogenetic context and some of thedescriptions are poorly detailed, their assignation toProsopis and its subgroups is, in some cases, verytentative. The oldest fossil that could be attributed tothe ASP clade (Guinet & Bessedik, 1984), which wasused as one of the calibration points in our moleculardating analysis, belongs to pollen grains (Prosopisquesneli) of the early Oligocene from British Colum-bia, Canada (Piel, 1971). A similar age has beenestimated for fruit remains (Prosopis lazarii) found inthe palaeoflora of Puebla, Mexico (Magallon-Puebla &Ceballos-Ferriz, 1994). Nevertheless, the affinity ofthis fossil with extant groups within the genus isuncertain. Recently, Anzótegui & Herbst (2004) havedescribed, based on leaflets remains, a new fossilspecies of Prosopis from the Middle Miocene of Argen-tina that appears to be related with extant members

of Section Strombocarpa. The age of this fossil iscompatible with the times estimated in the moleculardating analysis as both indicate that the divergencebetween the to major groups of Prosopis occurredbefore the middle Miocene. A more detailed compari-son of divergence times will require the combinedinclusion of fossil and extant species in a phylogeneticanalysis.

MESQUITES DIVERSIFICATION

The results obtained in the present study stronglysupport a close relationship of species of Series Chil-enses, Pallidae, Ruscifoliae (mesquite clade). Interest-ingly, many of these species were supposed to form asyngameon (Palacios & Bravo, 1981) because of theirability to hybridize naturally, their high morphologi-cal similarity and partially overlapping geographicaldistributions. However, several recent analysessuggest that hybrid formation does not produce sig-nificant gene flow among these species. First, molecu-lar studies showed that, in spite of their high geneticsimilarities, populations formed groupings coincidentwith their specific origin (Palacios & Bravo, 1981;Saidman & Vilardi, 1987; Burghardt, 1995; Bessegaet al., 2000). This was true even for populations ofdifferent species that were sympatric. In addition,Bessega et al. (2000) showed that sympatric speciesare not genetically more similar than allopatric ones.Second, the only analysis of hybrid viability availableto date (Naranjo, Poggio & Enus Zeiger, 1984) sug-gests that, at least for some species combinations,hybrids are partially or totally sterile. In the light ofour results, the high genetic similarity among thesespecies might now be explained by their recent diver-gence (< 4 Mya) without the need to invoke introgres-sion among them.

The average per lineage diversification rate esti-mated for the mesquite group is comparable to otherknown rapid plant radiations (Table 4). In particular,the diversification rate estimated for this group isfound to be higher than in Inga, another genus fromthe same subfamily that also comprises tree species.It is possible that this rapid and recent radiation ofmesquites might be the cause of the lack of resolutionobtained in the mesquite clade in the present study. Asimilar explanation for the lack of resolution has beenproposed in diverse plant genera (Hodges & Arnold,1995; Baldwin & Sanderson, 1998; Richardson et al.,2001; Hughes & Eastwood, 2006).

EVOLUTION OF REPRODUCTIVE ISOLATING

MECHANISM WITHIN ALGAROBIA

Molecular dating and phylogenetic results obtained inthe current study provides an improved insight into



the evolution of isolating mechanism within thegenus. In the case of the mesquite clade, as previouslyindicated, gene flow among its species would behighly restricted. This suggests the existence of iso-lating mechanism(s) that prevent introgressionamong them. The existence of hybrid between thesespecies in nature indicates that prezygotic reproduc-tive isolation barriers are probably weak (Palacios &Bravo, 1981) and stresses the potential importance ofpostzygotic isolating mechanisms within this group.The observation that hybrids between mesquitespecies are generally found in disturbed environ-ments (Palacios & Bravo, 1981; Verga, 1995) supportsthe idea that hybrids are not intrinsically inviable,but that their development is limited by the lack ofsuitable environmental conditions (ecological inviabil-ity; Coyne & Orr, 2004). Evidence for a second postzy-gotic isolating barrier has been obtained by Naranjoet al. (1984) who observed diminished fertility in somehybrid combinations. Given the times derived frommolecular dating, these postzygotic isolating mecha-nisms would have developed in less than 4 Myr, thetotal span for the diversification of this group(Table 3).

The phylogenetic results suggest that the ability tohybridize is not a characteristic extended throughoutthe Algarobia s.l. group. Indeed, the species of the twomain clades found within this group (Fig. 3) presentstrikingly different behaviour. Whereas, as previouslyindicated, mesquites do frequently hybridize, hybridformation between species of the P. kuntzei–P. argentina clade has not been documented. Althoughspecies of the P. kuntzei–P. argentina clade mighthave developed isolating mechanisms that preventhybrid formation, we consider that the reduced levelof sympatry between most of these species is a suffi-cient explanation for the difference. Furthermore, nohybrids have been observed between mesquite speciesand species of P. kuntzei–P. argentina clade even if inthis case they frequently appear in sympatry. Thissuggests the development of another barrier eitherintrinsic postzygotic or prezygotic in less than 8 Myr(Table 3), the estimated divergence time betweenthese two clades.

ANCESTRAL CLIMATE RECONSTRUCTION

During recent years, optimization techniques havebeen increasingly used to infer ancestral ecologicalconditions (Verboom et al., 2003; Graham et al., 2004;Hardy & Linder, 2005; Schrire, Lavin & Lewis, 2005).In the present study, an optimization analysis wasperformed to evaluate the historical association ofProsopis with arid environments. In particular, wewere interested in defining since when Prosopisspecies have occupied this kind of environment. The

analysis performed suggested an ancient associationof Prosopis with arid conditions (Fig. 6), alreadyestablished before the splitting of the two maingroups within ASP + Xerocladia clade (26.7–33 Mya).This result disagrees with Roig (1993) who proposedthat extant species of Prosopis occupying arid regionsin America have originated from ancestral lineages ofthis genus which were restricted to wetter regions.Palaeoenvironmental data suggest that, although thedevelopment of large arid areas in America started inthe late Miocene (see below), there were local aridconditions in North America (Axelrod, 1979a) andSouth America (Volkheimer, 1971; Jordan, Schluneg-ger & Cardozo, 2001) during much of the Tertiary. Itis possible therefore that ancestral lineages of Proso-pis had occupied these sites until the later spreadingof arid conditions.

DIVERSIFICATION PATTERN AND CAUSES

One of the predictions derived from the hypothesis ofaridity-driven diversification of Prosopis was a tem-poral correlation between the spreading of arid areasin the Americas and the diversification of the genus.Molecular clock dating showed that, except for thesplitting between the two main groups, the diver-gence among extant lineages within ASP clade wouldhave occurred in the Late Miocene or more recently,coincident with the estimated spreading of arid envi-ronments in America. Although with distinctive char-acteristics in each case, the development of aridenvironments occur simultaneously in North andSouth America starting in the Late Miocene andcontinuing in the Pliocene and Pleistocene. In NorthAmerica, the development of regional arid areas isthought to have started in the late Miocene toPliocene (Graham, 1999; Riddle & Hafner, 2006) asso-ciated with the uplift of the plateaus in WesternNorth America. The Sonora desert, where somespecies of the genus are present today, would havedeveloped from 15 to 8 Mya (van Devender, 2000).This process of aridification continued until the Pleis-tocene, reaching a considerable distribution in theinterglacials (Axelrod, 1979a,b). In South America,the spreading of large areas of semiarid-arid condi-tions possibly started in the late Miocene concomi-tantly with the uplift of the Andes in the Quechuadistrophic phase when it reached half of its presentelevation (Gregory-Wodzicki, 2000) and started to acteffectively as a barrier to moisture laden winds.During the ‘Diaguita’ diastrophic Phase, the finaluplift of the Pampean Mountain Range and theCentral Andes produced a rain-shadow effect thatresulted in the extremely xeric conditions existing atpresent on the areas located between them (Pascual,Ortiz-Jaureguizar & Prado, 1996; Alberdi. Bonadonna



& Ortiz-Jaureguizar, 1997). These climatic changeswould have produced the establishment of the biomeswhere Prosopis is established today (Axelrod, KalinArroyo & Raven, 1991; Pascual et al., 1996).

The possible correlation of the ASP diversificationwith the expansion of arid environments is also sup-ported by the analysis of the temporal changes ofdiversification rates. LTT analysis indicates a clearincrease in diversification rates from the late Mioceneuntil the present compared to previous times. Whenthis pattern is analysed in detail, two different shiftstowards higher rates may be distinguished. The firstof those occurred in the late Miocene and is coincidentwith the start of the divergence within each of the twomajor lineages of the ASP. The second shift appears tobe associated with the start of mesquites divergencein the Pliocene and is in agreement with the highdiversification rate estimated for this group (Table 4).A wrong assignment of missing taxa could havepotentially affected the conclusion derived from thisanalysis. However, as most of the species not sampledbelong to Series Chilensis, Ruscifoliae, and Pallidae,which formed the highly supported mesquite clade, awrong assignment seems to be improbable.

The combined assessment of the results obtained inthe present study is in agreement with the hypothesisthat Prosopis evolved and diversified concomitantlywith the spreading of arid areas in the Americas. Thisresult is coincident with those obtained for otherdry-lover plant groups distributed almost exclusivelyin North America, which is suggested to have under-gone a parallel radiation in the last 10 Myr (Good-Avila et al., 2006). To our knowledge, no previousstudy found this pattern for a group with mainlySouth American distribution, as is the case of Proso-pis. Consequently, the results of the present studysuggest an extension of this parallel radiation to acontinental scale.

ACKNOWLEDGEMENTS

We thank P. Steibel, H. Troiani, A. Vilela, andG. Humphreys for helping in sampling; N. Iusem andL. Maskin for helping in laboratory issues; F. Prevostifor helpful comments on the manuscript; E. Ulibarrifor helping in SI herbarium; P. Harris for P. africanamaterial; O. Ruiz for L. japonicus material; J. JuarezMuñoz for P. palmeri, Prosopis tamaulipana andProsopis articulata material; C. Pometti for A. cavenmaterial; M. Luckow for lending P. nigra and X. viri-diramis sequences and X. viridiramis DNA; A. Gott-lieb for helping in different steps of this study. S.A.C.is fellow of Consejo Nacional de InvestigacionesCientíficas y Técnicas (CONICET, Argentina). B.O.S.and J.C.V. are members of Carrera del InvestigadorCientífico of CONICET. This study was supported by

grants to B.O.S. from University of Buenos Aires(UBACYT_X321) Agencia Nacional de PromociónCientífica y Tecnológica (PICT 32064/05), and a grantto J.C.V. from CONICET (PIP 5122/04) grants. Theauthors wish to dedicate this manuscript to thememory of our mentor and advisor Professor CarlosA. Naranjo. Santiago A. Catalano wishes also to dedi-cate this manuscript to the memory of his father.

REFERENCES

Alberdi MT, Bonadonna FP, Ortiz-Jaureguizar E. 1997.Chronological correlation, paleoecology, and paleobiogeogra-phy of the Late Cenozoic South American ‘Rionegran’ land-mammal fauna: a review. Revista Española de Paleontología12: 249–255.

Anzótegui LM, Herbst R. 2004. Megaflora (hojas y frutos)de la Formación San José (Mioceno Medio) en río Seco,departamento Santa María, provincia de Catamarca, Argen-tina. Ameghiniana 41: 423–436.

Axelrod DI. 1979a. Age and origin of Sonoran desert vegeta-tion. Occasional Papers of the California Academy of Sci-ences 132: 1–74.

Axelrod DI. 1979b. Desert vegetation, its age and origin. In:Goodin JR, Northington DK, ed. Arid land plant resources.Lubbock, TX: International Center for Arid and SemiaridLand Studies, 1–72.

Axelrod D, Kalin Arroyo M, Raven P. 1991. Historicaldevelopment of temperate vegetation in the Americas.Revista Chilena de Historia Natural 64: 413–446.

Baldwin BG, Sanderson MJ. 1998. Age and rate of diver-sification of the Hawaiian silversword alliance (Composi-tae). Proceedings of the National Academy of Sciences of theUnited States of America 95: 9402–9406.

Barraclough TG, Vogler AP. 2002. Recent diversificationrates in North American tiger beetles estimated from adated mtDNA phylogenetic tree. Molecular Biology andEvolution 19: 1706–1716.

Bell CD, Donoghue MJ. 2005. Phylogeny and biogeographyof Valerianaceae (Dipsacales) with special reference to theSouth American valerians. Organisms, Diversity and Evo-lution 5: 147–159.

Bessega C, Ferreyra LI, Vilardi JC, Saidman BO. 2000.Unexpected low genetic differenatiation among allopatricspecies of Section Algarobia of Prosopis (Leguminoseae).Genetica 109: 255–266.

Bessega C, Saidman BO, Vilardi JC. 2005. Genetic rela-tionships among American species of Prosopis (Legumino-sae) based on enzyme markers. Genetics and MolecularBiology 28: 277–286.

Bessega C, Vilardi JC, Saidman BO. 2006. Genetic rela-tionships among American species of the genus Prosopis(Mimosoideae, Leguminosae) inferred from ITS sequences:evidence for long-distance dispersal. Journal of Biogeogra-phy 33: 1905–1915.

Bremer K. 1994. Branch support and tree stability. Cladis-tics 10: 295–304.



Burghardt AD. 1995. La identidad de cuatro especies deProsopis L. expresada a travéz de sus patrones electroforeti-cos. Mendeliana 12: 38–50.

Burkart A. 1976. A monograph of the genus Prosopis(Leguminosae subFam Mimosoideae). Journal of the ArnoldArboretum 57: 219–249.

Burkart A, Simpson BB. 1977. The genus Prosopis andannoted key to the species of the world. In: Simpson B, ed.Mesquite: its biology in two desert ecosystems. Stroudsburg,PA: Dowden, Hutchinson & Ross, 201–215.

Coyne JA, Orr HA. 2004. Speciation. Sunderland, MA:Sinauer Associates.

Demesure B, Sodzi N, Petit RJ. 1995. A set of universalprimers for amplification of polymorphic non-coding regionsof mitochondrial and chloroplast DNA in plants. MolecularEcology 4: 129–131.

van Devender TR. 2000. The deep history of the SonoranDesert. In: Phillips SJ, Comus PW, eds. A natural history ofthe Sonoran desert. Tucson, AZ: Arizona-Sonora DesertMuseum Press; Berkeley, CA: University of CaliforniaPress, 61–69.

FAO. 2005. New LocClim 1.06 local climate estimator. Agrom-eteorology group. Rome: FAO/SDRN.

Farris S. 1970. Methods for computing Wagner trees. Sys-tematic Zoology 34: 21–24.

Goloboff PA. 1999. Analyzing large data sets in reasonabletimes: solutions for composite optima. Cladistics 15: 415–428.

Goloboff PA, Farris JS, Nixon K. 2003. TNT: tree analysisusing new technology. Software and documentation avail-able at http://www.zmuc.dk/public/phylogeny

Goloboff PA, Mattoni CI, Quinteros AS. 2006. Continuouscharacters analyzed as such. Cladistics 22: 589–601.

Good-Avila SV, Souza V, Gaut BS, Eguiarte LE. 2006.Timing and rate of speciation in Agave (Agavaceae). Pro-ceedings of the National Academy of Sciences of the UnitedStates of America 103: 9124–9129.

Graham A. 1999. Late cretaceous and cenozoic history ofNorth American vegetation. New York, NY: Oxford Univer-sity Press.

Graham CH, Ron SR, Santos JC, Schneider CJ, MoritzC. 2004. Integrating phylogenetics and environmental nichemodels to explore speciation mechanisms in Dendrobatidfrogs. Evolution 58: 1781–1793.

Gregory-Wodzicki KM. 2000. Uplift history of the centraland northern Andes: a review. Geological Society of AmericaBulletin 112: 1091–1105.

Guinet PH, Bessedik M. 1984. Presence de Genre Prosopis(Leguminosae-Mimosoideae) a l’Aquitanien Basal dansl’aude (Languedoc-France). Pollen et Spores 26: 101–108.

Hall TA. 1999. BioEdit: a user-friendly biological sequencealignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.

Hardy CR, Linder HP. 2005. Intraspecific variability andtiming in ancestral ecology reconstruction: a test case fromthe Cape flora. Systematic Biology 54: 299–316.

Herendeen PS, Crepet WL, Dilcher DL. 1992. The fossilhistory of the leguminosae: phylogenetic and biogeographic

implications. In: Hendereen PS, Dilcher DL, eds. Advance inlegume systematics. Kew: The Royal Botanical Garden, 313–316.

Hey J. 1992. Using phylogenetic trees to study speciation andextinction. Evolution 46: 627–640.

Hodges SA, Arnold ML. 1995. Spurring plant diversifica-tion: are floral nectar spurs a key innovation? Proceedings ofthe Royal Society of London Series B, Biological Sciences262: 343–348.

Howarth DG, Baum DA. 2002. Phylogenetic utility of anuclear intron from nitrate reductase for the study of closelyrelated plant species. Molecular Phylogenetics and Evolu-tion 23: 525–528.

Hughes CE, Eastwood R. 2006. Island radiation on a con-tinental scale: exceptional rates of plant diversification afteruplift of the Andes. Proceedings of the National Academy ofSciences of the United States of America 103: 10334–10339.

Jordan TE, Schlunegger F, Cardozo N. 2001. Unsteadyand spatially variable evolution of the Neogene AndeanBermejo foreland basin, Argentina. Journal of South Ameri-can Earth Sciences 14: 775–798.

Klak C, Reeves G, Hedderson T. 2003. Unmatched tempoof evolution in Southern African semi-desert ice plants.Nature 427: 63–65.

Kluge AG. 1989. A concern for evidence and a phylogenetichypothesis of relationships among Epicrates (Boidae, Ser-pentes). Systematic Zoology 38: 7–25.

Lavin M, Thulin M, Labat J, Pennington RT. 2000.Africa, the odd man out: molecular biogeography of Dalber-gioid legumes (Fabaceae) suggests otherwise. SystematicBotany 25: 449–467.

Lavin M, Herendeen PS, Wojciechowski MF. 2005. Evo-lutionary rates analysis of Leguminosae implicates a rapiddiversification of lineages during the Tertiary. SystematicBiology 54: 575–594.

Lewis PO. 1998. Maximum likelihood as an alternative toparsimony for inferring phylogeny using nucleotidesequence data. In: Sotis DE, Soltis PS, Doyle JJ, eds.Molecular systematics of plants II DNA sequencing lluwer.Boston, MA: Academic Publisher, 132–163.

Luckow M, Miller JT, Jobson R, Murphy DJ. 2004.Generic relationships in the Mimosoideae (Fabaceae).Botany 2004, Alpine Diversity: Adapted to the Peaks, SaltLake City, Utah, USA. Session 19 (Colloquium) RupertBarneby and his legume legacy. Abstract 114, 29.

Magallon-Puebla S, Ceballos-Ferriz SRS. 1994. Fossillegume fruits from tertiary strata of Puebla, Mexico. Cana-dian Journal of Botany 72: 1027–1038.

Malcomber S. 2002. Phylogeny of Gaertnera Lam. (Rubi-aceae) based on multiple DNA markers: evidence of a rapidradiation in a widespread, morphologically diverse genus.Evolution 56: 42–57.

Miller JT, Bayer RJ. 2001. Molecular phylgenetics of Acacia(Fabaceae: Mimosoideae) based on the chloroplast matKconding sequence and flanking trnK intron spacer regions.American Journal of Botany 88: 697–705.

Moore MJ, Jansen RK. 2006. Molecular evidence for theage, origin, and evolutionary history of the American desert



http://www.zmuc.dk/public/phylogeny

plant genus Tiquilia (Boraginaceae). Molecular Phylogenet-ics and Evolution 39: 668–687.

Naranjo CA, Poggio L, Enus Zeiger S. 1984. Phenolchromatography, morphology and cytogenetics in threespecies and natural hybrids of Prosopis (Leguminosae-Mimosoideae). Plant Systematic and Evolution 144: 257–276.

Nee S, Holmes EC, Rambaut A, Harvey PH. 1995. Infer-ring population history from molecular phylogenies. Philo-sophical Transactions of the Royal Society of London, SeriesB 349: 25–31.

Palacios RA, Bravo LD. 1981. Hibridación natural en Proso-pis (Leguminosae) en la region chaqueña argentina. Eviden-cias morfologicas y cromatográficas. Darwiniana 23: 3–35.

Pascual R, Ortiz-Jaureguizar E, Prado JL. 1996. Landmammals: paradigm of Cenozoic South American geobioticevolution. In: Arratia G, ed. Contribution of Southern SouthAmerica to Vertebrate Paleontology. Munich: MünchnerGeowissenschaftliche Abhandlungen (A), 265–319.

Pasiecznik NM, Felker P, Harris PJC, Harsh LN, CruzG, Tewari JC, Cadoret K, Maldonado LJ. 2001. Theprosopis juliflora-prosopis pallida complex: a monograph.Coventry: HDRA.

Piel KM. 1971. Palynolgy of Oligocene sediments from BritishColumbia. Canadian Journal of Botany 49: 1885–1920.

Posada D, Crandall KA. 1998. Modeltest: testing the modelof DNA substitution. Bioinformatics 14: 817–818.

Rambaut AP. 2002. Phylogen, Version 1.0. Oxford: Depart-ment of Zoology, University of Oxford.

Ramírez L, De La Vega A, Razkin N, Luna V, HarrisPJC. 1999. Analysis of the relationships between species ofthe genus Prosopis revealed by the use of molecularmarkers. Agronomie 19: 31–43.

Richardson JE, Pennington RT, Pennington TD,Hollingsworth PM. 2001. Rapid diversification of aspecies-rich genus of Neotropical rain forest trees. Science293: 2242–2245.

Riddle BR, Hafner DJ. 2006. A step-wise approach to inte-grating phylogeographic and phylogenetic biogeographicperspectives on the history of a core North American warmdeserts biota. Journal of Arid Environments 66: 435–461.

Roig FA. 1993. Informe nacional para la selección de germo-plasma en especies de Prosopis en la República Argentina.Mendoza, Argentina: IADIZA-CRICYT, 1–37.

Ronquist FR, Huelsenbeck JP. 2005. Mr Bayes 3. Baye-sian phylogenetics inference under mixed models. Bioinfor-matics 19: 1572–1574.

Ross JH. 1975. Flora of Southern Africa. Vol. 16, Part I. Mimo-soideae, Fabaceae. Pretoria: Deptartment of Agriculture.

Rzedowski J. 1988. Analisis de la distribución geográficadel complejo Prosopis (Leguminosae, Mimosoideae) enNorteamerica. Acta Botanica Mexicana 3: 7–19.

Saidman B, Vilardi J. 1987. Analysis of the genetic simi-larities among seven species of Prosopis (Leguminoseae:Mimosoideae). Theoretical and Applied Genetics 75: 109–116.

Saidman BO, Vilardi JC. 1993. Genetic variability andgermplasm conservation in the genus Prosopis. In: Puri S,

ed. Nursery technology of forest tree species of arid andsemiarid regions. New Delhi: Winrock-Oxford & IBH Pub-lishing Co. PVT. Ltd., 187–198.

Saidman BO, Vilardi JC, Pocoví MI, Acreche N. 1996.Isozyme studies in Argentine species of the Section Strom-bocarpa, Genus Prosopis (Leguminosae). Journal of Genetics75: 139–149.

Saidman BO, Vilardi JC, Montoya S, Dieguez MJ, HoppHE. 1998. Molecular markers: a tool for the understandingof the relationships among species of Prosopis (Legumino-sae, Mimosoideae). In: Puri S, ed. Tree improvement: appliedresearch and technology transfer. Enfield, NH: Science Pub-lishers, Inc., 324.

Sanderson MJ. 2002. Estimating absolute rates of molecularevolution and divergence times: a penalized likelihoodapproach. Molecular Biology and Evolution 19: 101–109.

Sanderson MJ. 2003. r8s: inferring absolute rates of molecu-lar evolution and divergence times in the absence of amolecular clock. Bioinformatics 19: 301–302.

Schrire BD, Lavin M, Lewis GP. 2005. Global distributionpatterns of the Leguminosae: insights from recent phylog-enies. In: Friis I, Balslev H, eds. Plant diversity and com-plexity patterns: local, regional and global dimensions.Biologiske Skrifter 55: 375–422.

Simmons MP, Ochoterena H. 2000. Gaps as characters insequence-based phylogenetic analyses. Systematic Biology49: 369–381.

Strand AE, Leebens-Mack J, Milligan BG. 1997. NuclearDNA-based markers for plant evolutionary biology. Molecu-lar Ecology 6: 113–118.

Swofford DL. 2002. PAUP* phylogenetic analysis using par-simony (*and other methods), Version 4. Sunderland, MA:Sinauer Associates.

Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universalprimers for amplification of three non-coding regions ofchloroplast DNA. Plant Molecular Biology 17: 1105–1109.

Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F,Higgins DG. 1997. The CLUSTAL–windows interface: flex-ible strategies for multiple sequence alignment aided byquality analysis tools. Nucleic Acids Research 25: 4876–4882.

UNEP. 1992. World atlas of desertification. London: EdwardArnold.

Verboom GA, Linder HP, Stock WD. 2003. Phylogenetics ofthe grass genus Ehrharta: evidence for radiation in thesummer-arid zone of the South African cape. Evolution 57:1008–1021.

Verga AR. 1995. Genetische untersuchungen an Prosopis chil-ensis und Prosopis flexuosa (Mimosaceae) im trockenenChaco Argentiniens. Göttingen: Universität Göttingen.

Volkheimer W. 1971. Aspectos paleoclimáticos del Terciarioargentino. Revista del Museo Argentino de Ciencias Natu-rales Bernardino Rivadavia 1: 243–262.

Von Hagen KB, Kadereit JW. 2001. The phylogeny of Gen-tianella (Gentianaceae) and its colonization of the southernhemisphere as revealed by nuclear and chloroplast DNAsequence variation. Organisms Diversity and Evolution 1:61–79.



Wang A, Yang M, Liu J. 2005. Molecular phylogeny, recentradiation and evolution of gross morphology of the rhubarbgenus Rheum (Polygonaceae) inferred from chloroplast DNAtrnL-F sequences. Annals of Botany 96: 489–498.

Xiang Q, Manchester SR, Thomas DT, Zhang W, Fan C.2005. Phylogeny, biogeography, and molecular dating of

cornelian cherries (Cornus, Cornaceae): traking tertiaryplant migration. Evolution 59: 1658–1700.

Yule GU. 1924. A mathematical theory of evolution based onthe conclusions of Dr. J. C. Willis. Philosophical Transac-tions of the Royal Society of London, Series B 213: 21–87.

SUPPLEMENTARY MATERIAL

The following material is available for this article online:

Figure S1. Strict consensus of 4680 most parsimonious trees obtained in the five-marker analysis (trnS-psbC,G3pdh, NIA, trnL-trnF, trnK-matK).Table S1. Sequences downloaded from GenBank.Table S2. Maximum and minimum humidity index for species included in the ancestral climate estimationanalysis.

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1095-8312.2007.00907.x(This link will take you to the article abstract).

Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementarymaterials supplied by the authors. Any queries (other than missing material) should be directed to thecorresponding author for the article.



http://www.blackwell-synergy.com/doi/abs/10.1111/j.1095-8312.2007.00907.x

AP

PE

ND

IX1

Lis

tof

spec

ies

sequ

ence

din

the

pres

ent

stu

dy,

indi

cati

ng

the

Sec

tion

and

Ser

ies

tow

hic

hth

eybe

lon

g,vo

uch

ersp

ecim

en,

the

nat

ura

ldi

stri

buti

onan

dac

cess

ion

nu

mbe

rs

Spe

cies

Sec

tion

–S

erie

sV

ouch

ersp

ecim

en/s

eed

ban

kac

cesi

onN

atu

ral

dist

ribu

tion

Gen

Ban

kA

cces

ion

nu

mbe

rsG

3pdh

trn

S-p

sbC

NIA

trn

L-t

rnF

trn

K/m

atK

Pro

sopi

sci

ner

aria

(L.)

Dru

ceP

roso

pis

DA

NID

AN

°01

089/

82A

FE

F16

5221

EF

1653

09E

F16

5269

EF

1652

92E

F16

5287

/E

F16

5248

Pro

sopi

saf

rica

na

(Gu

ill.,

Per

r.,

&R

ich

.)Ta

ube

rt

An

onyc

hiu

mH

DR

AN

°63

AF

EF

1652

09E

F16

5297

EF

1652

53E

F16

5291

EF

1652

86/

EF

1652

51

Pro

sopi

sst

rom

buli

fera

(Lam

.)B

enth

amS

trom

boca

rpa–

Str

ombo

carp

aeH

un

zike

r&

Gam

erro

1135

9(S

I)S

AE

F16

5241

EF

1653

27E

F16

5281

––

Pro

sopi

sre

ptan

sB

enth

amS

trom

boca

rpa–

Str

ombo

carp

aeS

aidm

an36

(BA

FC

)S

A,

NA

EF

1652

37E

F16

5333

––

–

Pro

sopi

sto

rqu

ata

(Cav

anil

les

exL

agas

ca)

Str

ombo

carp

a–S

trom

boca

rpae

Hu

nzi

ker

9733

(SI)

SA

EF

1652

44E

F16

5330

––

–

Pro

sopi

spu

besc

ens

Ben

tham

Str

ombo

carp

a–S

trom

boca

rpae

Eva

ns

N°

15(U

SD

A-U

SA

)N

AE

F16

5236

EF

1653

23–

––

Pro

sopi

spa

lmer

iS

.W

atso

nS

trom

boca

rpa–

Str

ombo

carp

ae–

NA

EF

1652

34E

F16

5322

EF

1652

78–

–

Pro

sopi

sfe

rox

Gri

seba

chS

trom

boca

rpa–

Cav

enic

arpa

eH

un

zike

ret

al.

1045

1(S

I)S

AE

F16

5223

EF

1653

11–

––

Pro

sopi

sta

mar

ugo

F.P

hil

ippi

Str

ombo

carp

a–C

aven

icar

pae

DA

NID

AN

°01

215/

83S

AE

F16

5242

EF

1653

28–

EF

1652

93E

F16

5289

/E

F16

5250

Pro

sopi

sar

gen

tin

aB

urk

art

Mon

ilic

arpa

BN

GP

roso

pis

N°

15-8

8.S

AE

F16

5212

EF

1653

00E

F16

5261

EF

1652

90E

F16

5288

/E

F16

5249

Pro

sopi

sse

rica

nth

aG

ille

sex

Hoo

ker

&A

rnot

t

Alg

arob

ia–

Ser

ican

thae

Sai

dman

&V

ilar

di96

1(B

AF

C)

SA

EF

1652

40E

F16

5326

EF

1652

80–

–

Pro

sopi

sku

ntz

eiH

arm

sA

lgar

obia

–S

eric

anth

aeS

aidm

an&

Vil

ardi

516

(BA

FC

);S

aidm

an&

Vil

ardi

521

(BA

FC

)

SA

EF

1652

30E

F16

5231

EF

1653

18E

F16

5319

EF

1652

76E

F16

5277

––

Pro

sopi

sru

scif

olia

Gri

seba

chA

lgar

obia

–R

usc

ifol

iae

Sai

dman

&V

ilar

di42

0(B

AF

C)

SA

EF

1652

39E

F16

5325

EF

1652

57–

–

Pro

sopi

svi

nal

illo

Stu

cker

tA

lgar

obia

–R

usc

ifol

iae

Sai

dman

&V

ilar

di50

5(B

AF

C);

Sai

dman

&V

ilar

di51

1(B

AF

C)

SA

EF

1652

45E

F16

5246

EF

1653

31E

F16

5332

EF

1652

83E

F16

5284

––

Pro

sopi

sd

enu

dan

sB

enth

amA

lgar

obia

–D

enu

dan

tes

Cat

alan

o33

(BA

FC

)S

AE

F16

5222

EF

1653

10E

F16

5270

––

Pro

sopi

sru

izle

ali

Bu

rkar

tA

lgar

obia

–D

enu

dan

tes

Cat

alan

o13

(BA

FC

)S

AE

F16

5238

EF

1653

24E

F16

5279

––



Pro

sopi

sca

stel

lan

osii

Bu

rkar

tA

lgar

obia

–D

enu

dan

tes

Cat

alan

o8

(BA

FC

)S

AE

F16

5218

EF

1653

06E

F16

5267

––

Pro

sopi

sh

um

ilis

Gil

les

exH

ooke

r&

Arn

ott

Alg

arob

ia–

Hu

mil

esC

atal

ano

1(B

AF

C)

SA

EF

1652

27E

F16

5315

EF

1652

75–

–

Pro

sopi

sca

mpe

stri

sG

rise

bach

Alg

arob

ia–

Pal

lida

eC

atal

ano

18(B

AF

C);

Cat

alan

o20

(BA

FC

)S

AE

F16

5216

EF

1652

17E

F16

5304

EF

1653

05E

F16

5265

EF

1652

66–

–

Pro

sopi

saf

fin

isS

pren

gel

Alg

arob

ia–

Pal

lida

eS

aidm

anet

al.

526

(BA

FC

)S

AE

F16

5208

EF

1652

96E

F16

5256

––

Pro

sopi

sar

ticu

lata

S.

Wat

son

Alg

arob

ia–

Pal

lida

e–

NA

EF

1652

13E

F16

5301

EF

1652

62–

–

Pro

sopi

sta

mau

lipa

na

Bu

rkar

tA

lgar

obia

–P

alli

dae

–N

AE

F16

5243

EF

1653

29E

F16

5282

––

Pro

sopi

sch

ilen

sis

(Mol

ina)

Str

un

tzem

end.

Bu

rkar

t

Alg

arob

ia–

Ch

ilen

ses

Sai

dman

&V

ilar

di91

1(B

AF

C);

Sai

dman

&V

ilar

di92

2(B

AF

C)

SA

EF

1652

19E

F16

5220

EF

1653

07E

F16

5308

EF

1652

68E

F16

5285

––

Pro

sopi

sju

lifl

ora

(Sw

artz

)D

CA

lgar

obia

–C

hil

ense

sH

un

zike

r10

039

(SI)

;H

un

zike

r10

040

(SI)

NA

,CA

,SA

EF

1652

28E

F16

5229

EF

1653

16E

F16

5317

EF

1652

58E

F16

5274

––

Pro

sopi

sn

igra

(Gri

seba

ch)

Hie

ron

ymu

s

Alg

arob

ia–

Ch

ilen

ses

Sai

dman

&V

ilar

di43

5(B

AF

C);

Sai

dman

315

(BA

FC

)

SA

EF

1652

32E

F16

5233

EF

1653

20E

F16

5321

EF

1652

59E

F16

5260

––

Pro

sopi

sca

lden

iaB

urk

art

Alg

arob

ia–

Ch

ilen

ses

Sai

dman

&V

ilar

di35

0(B

AF

C);

Sai

dman

&V

ilar

di13

8(B

AF

C)

SA

EF

1652

14E

F16

5215

EF

1653

02E

F16

5303

EF

1652

63E

F16

5264

––

Pro

sopi

sfl

exu

osa

DC

Alg

arob

ia–

Ch

ilen

ses

Sai

dman

&V

ilar

di30

2(B

AF

C);

Cat

alan

o10

(BA

FC

)

SA

EF

1652

24E

F16

5225

EF

1653

12E

F16

5313

EF

1652

71E

F16

5272

––

Pro

sopi

sgl

and

ulo

saTo

rrey

Alg

arob

ia–

Ch

ilen

ses

Eva

ns

N°

5(U

SD

A-U

SA

)N

AE

F16

5226

EF

1653

14E

F16

5273

––

Pro

sopi

sal

pata

coR

.A.

Ph

ilip

piA

lgar

obia

–C

hil

ense

sC

atal

ano

34(B

AF

C)

SA

EF

1652

11E

F16

5299

EF

1652

55–

–

Pro

sopi

sal

baG

rise

bach

Alg

arob

ia–

Ch

ilen

ses

Sai

dman

&V

ilar

di41

3(B

AF

C)

SA

EF

1652

10E

F16

5298

EF

1652

54–

–

Pro

sopi

das

tru

man

gust

icar

pum

Pal

acio

s&

Hoc

NA

Pet

er24

4(L

P)

SA

EF

1652

35E

F16

5295

––

–/E

F16

5252

Xer

ocla

dia

viri

dir

amis

Tau

b.N

AG

erm

ish

uiz

en82

44(P

RE

)A

FE

F58

5492

EF

5854

93–

Su

pp.

data

Su

pp.

data

Aca

cia

cave

n(M

ol.)

Mol

ina

NA

Sai

dman

etal

.78

9(B

AF

C)

SA

EF

1652

07E

F16

5294

EF

1652

06S

upp

.da

taS

upp

.da

ta

Lot

us

japo

nic

us

L.

NA

–S

AE

F16

5247

Su

pp.

data

–S

upp

.da

taS

upp

.da

ta

BN

GP

roso

pis,

Ban

coN

acio

nal

deG

erm

opla

sma

deP

roso

pis

(Cor

doba

,A

rgen

tin

a);

DA

NID

A,

Dan

ida

For

est

See

dC

entr

e,D

enm

ark;

GR

S-U

SD

A-U

SA

,U

nit

edS

tate

sD

epar

tmen

tof

Agr

icu

ltu

re;

HD

RA

,H

enry

Dou

bled

ayR

esea

rch

Ass

ocia

tion

,U

nit

edK

indo

m;

SI,

Dar

win

ion

Her

bari

um

,S

anIs

idro

,A

rgen

tin

a;B

AF

C,

Her

bari

um

ofF

acu

ltad

deC

ien

cias

Exa

ctas

yN

atu

rale

sU

BA

,Bu

enos

Air

es,A

rgen

tin

a;A

F,A

fric

a;A

S,A

sia;

CA

,Cen

tral

Am

eric

a;N

A,N

orth

Am

eric

a;S

A,S

outh

Am

eric

a;S

upp

.da

ta,

acce

ssio

nn

um

ber

incl

ude

din

Su

pple

men

tary

mat

eria

l(T

able

S1)

.



APPENDIX 2SEARCH STRATEGIES IN PARSIMONY AND

BAYESIAN ANALYSES

In three-marker analysis, parsimony searchesinvolved 100 Random Addition Sequences (RAS) fol-lowed by ten cycles of Tree Drifting. In the two-marker and five-marker analyses, the strategyinvolved 200 RAS followed by Tree Drifting andRandom Sectorial Searches (Goloboff, 1999). Thesetwo algorithms intend to overcome the problem oflocal optima during tree searches. In Tree Drifting,suboptimal solutions are accepted with a certainprobability in different moments of the swappingwhereas, in Sectorial Searches, reduced datasets arecreated for different parts of the tree which are analy-sed by tree-bisection-reconnection (TBR). Then, thebest tree for each sector is replaced in the originaltree. In all cases, trees found using these strategieswere used as starting trees for a final round of TBRkeeping a maximum of 10 000 trees. Constrainedsearches forcing groups not found in the most parsi-monious trees were run with the same settings.

Support was evaluated with jackknifing procedurewith an independent character removal probability of0.36. For each jackknifing replicate, search strategywas the same than previously described except thatthe number of RAS was decreased to 10. AbsoluteBremer Support (Bremer, 1994) was calculated savingup to 6000 suboptimal trees in successive steps up totrees 12 steps longer than the optimal length.

Bayesian phylogenetic analyses were performedwith the Metropolis-coupled Markov chain MonteCarlo algorithm implemented in MrBayes, version3.1.2 (Ronquist & Huelsenbeck, 2005). The optimalmodel was selected with Modeltest (Posada & Cran-dall, 1998) and the values of each parameter weredetermined during each run. The analysis involvedtwo independent runs of 2 ¥ 106 generations sampledevery 100 generations after a burnin period of0.50 ¥ 106 generations. Each run involved threeheated chains. Convergence was assessed evaluatinglikelihood plots and standard deviation of the splitfrequencies.



molecular phylogeny and diversiﬁcation history of

Documents

darker zones representing

high morphological similarity

postzygotic isolating mechanisms

penalized likelihood approach

recent common ancestor

posterior probabilities estimated

penalized likelihood analysis

absolute bremer support