Phylogenetic relationships among southern South American species of Camptosema, Galactia and Collaea (Diocleinae: Papilionoideae: Leguminosae) on the basis of molecular and morphological data Silvana M. Sede A,F , Daniela Tosto B , Paola Talia B , Melissa Luckow C , Lidia Poggio D and Renée Fortunato E A Instituto de Botánica Darwinion, Labardén 200, Casilla de Correo 22, B1642HYD, San Isidro, Buenos Aires, Argentina. B Unidad Integrada Facultad de Ciencias Exactas y Naturales, UBA y Centro de Investigaciones en Ciencias Veterinarias y Agronómicas (CICVyA), INTA, Argentina. C L. H. Bailey Hortorium, 462 Mann Library, Cornell University, Ithaca, New York 14853, USA. D Laboratorio de Citogenética y Evolución, Departamento de Ecología, Genética y Evolución, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina. E CONICET, Instituto de Recursos Biológicos, Centro de Investigación en Recursos Naturales, INTA, Argentina. F Corresponding author. Email: [email protected]Abstract. The neotropical genus Camptosema in its southern distribution is represented by the following four species: Ca. paraguariense, Ca. praeandinum, Ca. rubicundum and Ca. scarlatinum. Ca. rubicundum is the only species with all the diagnostic characters of the genus, i.e. tubular calyx. The other three taxa are related morphologically and cytologically to Galactia and Collaea, two closely allied genera in the subtribe Diocleinae. Individual and combined phylogenetic analyses of morphology, ITS and trnL-F were conducted to examine the position of these species as well as to explore their relationship with Galactia and Collaea species in southern South America. In none of the analyses does Camptosema species form a monophyletic group. Instead, they are scattered in different groups. These results together with cytological, morphological and AFLP data provide good support for re-evaluating the taxonomic position of these species within Camptosema. Introduction Camptosema Hook. & Arn. has ~20 species distributed from central to north-eastern Brazil, Argentina, Paraguay and Uruguay (Izaguirre de Artuccio and Beyhaut 1998; de Queiroz 1999). In southern South America the species of this genus are closely related to Galactia P.Browne and Collaea DC. (Fortunato and de Queiroz 1998; de Queiroz et al. 2003). Uncertainty about taxonomic circumscriptions has been caused by dissimilar criteria employed by the authors working within the group (Bentham 1859; Grisebach 1874; Taubert 1894; Burkart 1952, 1970). Bentham (1859) recognised Collaea, Camptosema and Galactia; however, he included in Collaea most of Galactia species. Burkart (1952) defined Camptosema as a genus of vines, with 3-foliolate leaves, long axillary inflorescences, tubular calyx, long unguiculate petals, pseudomonadelphous androecium, stipitate ovary, filiform style and straight stipitate fruit. Later on, Burkart (1970) adopted a broadened concept of Camptosema, transferring species described in other genera (Collaea and Dioclea Kunth). He also described a new species, Ca. praeandinum Burkart, which has a pubescent corolla and lacks a gynophore, and proposed the combination, Ca. paraguariense (Chodat & Hassl.) Hassl., a species which had been described under Galactia, justifying its position in Camptosema only by the presence of long gynophore, although it is a species with small flowers and campanulate calyx. Karyological studies in the tribe Phaseoleae indicate that x = 11 is the prevailing basic chromosome number (Goldblatt 1981). Species of Camptosema are characterised by n = 11 whereas Galactia and Collaea species show an aneuploid reduction, being characterised by n = 10 (Goldblatt 1981). This reduction in the chromosome number was corroborated for South American species (Sede et al. 2003, 2006; Sede 2005). Although Ca. rubicundun Hook. et Arn., the type species of the genus, is characterised by n = 11 (Goldblatt 1981; Sede et al. 2006), the chromosome number of Ca. praeandinum, Ca. paraguariense and Ca. scarlatinum (Mart. ex Benth.) Burkart reported was n = 10 (Sede et al. 2003, 2006) and did not agree with the proposed number for the genus. CSIRO PUBLISHING www.publish.csiro.au/journals/ajb Australian Journal of Botany, 2009, 57, 76–86 Ó CSIRO 2009 10.1071/BT08091 0067-1924/09/010076
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Phylogenetic relationships among southern South Americanspecies of Camptosema, Galactia and Collaea (Diocleinae:Papilionoideae: Leguminosae) on the basis of molecularand morphological data
AInstituto de Botánica Darwinion, Labardén 200, Casilla de Correo 22, B1642HYD, San Isidro,Buenos Aires, Argentina.
BUnidad Integrada Facultad de Ciencias Exactas y Naturales, UBA y Centro de Investigacionesen Ciencias Veterinarias y Agronómicas (CICVyA), INTA, Argentina.
CL. H. Bailey Hortorium, 462 Mann Library, Cornell University, Ithaca, New York 14853, USA.DLaboratorio de Citogenética y Evolución, Departamento de Ecología, Genética y Evolución,Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
ECONICET, Instituto de Recursos Biológicos, Centro de Investigación en Recursos Naturales,INTA, Argentina.FCorresponding author. Email: [email protected]
Abstract. The neotropical genus Camptosema in its southern distribution is represented by the following four species:Ca. paraguariense, Ca. praeandinum, Ca. rubicundum and Ca. scarlatinum. Ca. rubicundum is the only species with allthe diagnostic characters of the genus, i.e. tubular calyx. The other three taxa are related morphologically and cytologicallyto Galactia and Collaea, two closely allied genera in the subtribe Diocleinae. Individual and combined phylogeneticanalyses of morphology, ITS and trnL-Fwere conducted to examine the position of these species as well as to explore theirrelationshipwithGalactia andCollaea species in southern SouthAmerica. In none of the analyses doesCamptosema speciesform a monophyletic group. Instead, they are scattered in different groups. These results together with cytological,morphological and AFLP data provide good support for re-evaluating the taxonomic position of these species withinCamptosema.
Introduction
Camptosema Hook. & Arn. has ~20 species distributed fromcentral to north-eastern Brazil, Argentina, Paraguay andUruguay(Izaguirre de Artuccio and Beyhaut 1998; de Queiroz 1999). Insouthern South America the species of this genus are closelyrelated toGalactia P.Browne andCollaeaDC. (Fortunato and deQueiroz 1998; de Queiroz et al. 2003).
Uncertainty about taxonomic circumscriptions has beencaused by dissimilar criteria employed by the authors workingwithin the group (Bentham 1859; Grisebach 1874; Taubert 1894;Burkart 1952, 1970). Bentham (1859) recognised Collaea,Camptosema and Galactia; however, he included in Collaeamost of Galactia species.
Burkart (1952) defined Camptosema as a genus of vines,with 3-foliolate leaves, long axillary inflorescences, tubularcalyx, long unguiculate petals, pseudomonadelphousandroecium, stipitate ovary, filiform style and straightstipitate fruit. Later on, Burkart (1970) adopted a broadenedconcept of Camptosema, transferring species described inother genera (Collaea and Dioclea Kunth). He also described
a new species, Ca. praeandinum Burkart, which has a pubescentcorolla and lacks a gynophore, and proposed the combination,Ca. paraguariense (Chodat & Hassl.) Hassl., a specieswhich had been described under Galactia, justifying itsposition in Camptosema only by the presence of longgynophore, although it is a species with small flowers andcampanulate calyx.
Karyological studies in the tribe Phaseoleae indicate thatx= 11 is the prevailing basic chromosome number (Goldblatt1981). Species of Camptosema are characterised by n= 11whereas Galactia and Collaea species show an aneuploidreduction, being characterised by n= 10 (Goldblatt 1981). Thisreduction in the chromosome number was corroborated forSouth American species (Sede et al. 2003, 2006; Sede 2005).Although Ca. rubicundun Hook. et Arn., the type species ofthe genus, is characterised by n= 11 (Goldblatt 1981; Sede et al.2006), the chromosome number of Ca. praeandinum, Ca.paraguariense and Ca. scarlatinum (Mart. ex Benth.) Burkartreported was n = 10 (Sede et al. 2003, 2006) and did not agreewith the proposed number for the genus.
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� CSIRO 2009 10.1071/BT08091 0067-1924/09/010076
Previous studies on the species of Camptosema, Collaea andGalactia include AFLP analyses by Sede et al. (2008). Theirstudy confirmed the present morphological circumscriptionof Collaea; however, the results were incongruent with thecurrent circumscription of Camptosema and Galactia.
Larger-scale phylogenetic analyses that included members ofthe subtribe Diocleinae have been done by using molecularcharacters. Chloroplast-DNA restriction sites (Bruneau et al.1995), rbcL (Kajita et al. 2001) and the internal transcribedspacer of nrDNA (Varela et al. 2004) all demonstrated thelack of monophyly of the subtribe, although species ofCamptosema were underrepresented or not included at all inthese studies.
De Queiroz et al. (2003) and Maxwell and Taylor (2003)analysed the phylogenetic relationships in the subtribeDiocleinaeby using morphological data. The study of de Queiroz et al.(2003) in particular included a broad sampling of the species ofGalactia and Camptosema. Their results indicated the artificialnature of the majority of the genera analysed in the subtribeDiocleinae, including Galactia and Camptosema, whichappeared as non-monophyletic.
On the basis of previous results, Camptosema praeandinum,Ca. paraguariense and Ca. scarlatinum, all distributed insouthern South America, were considered as taxonomicallyanomalous species. The objectives of the present work were touse DNA sequence data and morphological characters to analysethe position of these species of Camptosema as well as toexplore their relationship with Galactia and Collaea specieswith a similar geographic distribution.
Materials and methodsTaxon samplingIn the present study, 6 of the ~20 species of Camptosema, five ofthe seven species ofCollaea and 12 of the ~55 species ofGalactiawere sampled. Species of Galactia were chosen to represent thethree recognised sections, including one from section Galactia,one from section Collaearia and 10 from section Odonia.See Appendices 1 and 2 for a complete list of taxa sampled.
Outgroup selection was guided by previous molecularphylogenetic analyses in the tribes Millettieae and Phaseoleae(ITS/5.8S, Hu et al. 2002; rbcL, Kajita et al. 2001; matK,Wojciechowski et al. 2004) and morphological phylogeneticanalyses in the subtribe Diocleinae (Maxwell and Taylor 2003;de Queiroz et al. 2003).
MorphologyMorphological data were scored from herbarium specimensfrom BAB, G, MBM, and SI (Appendix 2) as well as datafrom the literature (Maxwell and Taylor 2003; de Queirozet al. 2003). In total, 46 morphological characters were coded(Appendix 3).
DNA isolation, amplification and sequencingTotal genomic DNA was extracted with the DNeasy Plant MiniKit (Qiagen, Valencia, California) and used as a template for thePCR amplification of the trnL-F intergenic spacer from thechloroplast genome (trnL-F hereafter) and the intergenic
spacer region from the nuclear rDNA (ITS hereafter),including ITS-1, the 5.8S subunit and ITS-2. Amplificationand sequencing were carried out with the primers pairsspecified as follows: ITS4 and ITS5 (White et al. 1990) forITS and primers e and f (Taberlet et al. 1991) for trnL-F. PCRreactions were performed in 25-uL final volume with 50–100 ngof template DNA, 0.2mM of each primer, 25mM of DNTPs,4mM MgCl2, 1� buffer and 1.5 units of Taq polymeraseprovided by Invitrogen Life Technologies (Carlsbad, CA, US).The reaction conditions were as follows: a first period ofdenaturation at 94�C for 5min, followed by 36 cycles ofdenaturation at 94�C for 30 s, annealing at 52�C for 1min andextension at 72�C for 1min 30 s. Final extension at 72�C for 7minterminated the reactions. PCR products were run out on a 1%TAE agarose gel stained with ethidium bromide. Purificationof PCR products and automated sequencing were performedby Cornell Biotechnology (Ithaca, NY, US) sequencingfacility and Macrogen Inc. (Seoul, Korea). Voucher informationand GenBank accession numbers are listed in Appendix 1.
Sequence alignmentAssembly and editing of sequences was performed with BioEditversion 5.0.9 (Hall 1999). Alignments were performed withDIALIGN 1 (Morgenstern et al. 1996), with a threshold T= 5.
Characterisation of ITS sequencesEvidence for ITS paralogues includes length variation, decreasedGC content, low stability of secondary structures andabsence of conserved motifs (Mayol and Rosselló 2001;Bailey et al. 2003).
Length variation and GC content were determined withBioEdit version 5.0.9 (Hall 1999). Free energy of RNAtranscripts and predicted secondary structures was determinedat the M. Zucker web server (http://mfold.bioinfo.rpi.edu/) withMFOLD program ver. 3.1. The presence of the conserved motif(Liu and Schardl 1994) GGCRY-(4 to 7n)-GYGYCAAGGAAwas searched at the ITS1 region.
Data matricesFive matrices were constructed with Winclada ver. 1.00.08(Nixon 2002), one for each individual data set (morphology,trnL-F and ITS) and two combined matrices (ITS + trnL-F,and ITS + trnL-F +morphology). Data matrices were submittedto TreeBASE (Study number 52264). The number ofterminals and informative characters for each matrix areshown in Table 1.
Data analysesThe three data sets were tested for congruence with theincongruence length difference test, ILD (Farris et al. 1994),implemented inWinclada ver. 1.00.08 (Nixon 2002). Parsimony-uninformative characters were deactivated before performingeach test. The test was done on the basis of 1000 iterations,each finding a Wagner tree submitted to TBR swapping andholding a maximum of 10 trees for each submatrix.
Phylogenetic analyses were performed under the parsimonycriterion with TNT ver. 1.1 (Goloboff et al. 2003).Molecular and
Phylogenetic relationships among Camptosema, Galactia and Collaea Australian Journal of Botany 77
morphological characters were considered unordered, unlessotherwise stated in Appendix 3. Uninformative characterswere excluded from the analyses and gaps were treated asmissing data. Search strategy for the three matrices consistedof heuristic searches performed with 1000 series of randomaddition sequences, followed by TBR branch rearrangements(mult*1000) and by retaining 10 trees per series (hold/10). Treesfound were saved in memory and additionally TBR swapped,retaining a maximum of 10000 total trees (hold/10 000).
Data sets were analysed separately and combined as describedabove. A total-evidence approach (Kluge 1989; Nixon andCarpenter 1996) was utilised to permit full interaction of allcharacters and to allow secondary signal hidden within thedata sets to be revealed (Nixon and Carpenter 1996).
A strict consensus tree (Nelson 1979; Rohlf 1982) wasgenerated from the most parsimonious trees. Branch supportwas calculated with jackknife values (Farris et al. 1996) asimplemented in TNT (Goloboff et al. 2003), with a characterremoval probability of 36%, performing 10 000 replicates and a
heuristic search strategy of five addition sequences swappedwithTBR with one tree saved per replicate.
Results
Morphology
Themorphological data set comprised 28 taxa and 46 informativecharacters. The analysis of the morphological matrix resulted intwo trees (L = 154, consistency index CI = 0.37 and retentionindex RI = 0.65) (Table 1). The strict consensus tree is shown inFig. 1.
Neither Camptosema nor Galactia is monophyletic in thisanalysis. Five of the six species of Camptosema fall out as oneclade, which is sister to the rest of the ingroup. The remainingspecies of Camptosema, Ca. preaeandinum, is embedded withinGalactia, being a sister species toG. lindenii Burkart. Collaea ismonophyletic on the tree and is nestedwithinGalactia, as sister toG. martii DC.
Table 1. Attributes of the five data setsMPT=most parsimonious trees; CI = consistency index; RI = retention index
Fig. 1. Strict consensus of two most parsimonious trees (L = 154, CI = 0.37, RI = 0.65) resulting from the morphological data-matrix analysis. Numbers abovebranches refer to jackknife values.
78 Australian Journal of Botany S. M. Sede et al.
Galactia sect. Odonia is also not monophyletic. Most of thespecies in this section form a clade which is sister to a small cladecomposed of G. boavista (Vell.) Burkart (sect.Collaearia) +G. lindenii (sect. Galactia) +Ca. praeandinum.The remaining two species of section Odonia (G. neesiiDC. and G. martii) form a paraphyletic grade to the Collaeaclade (Fig. 1).
TrnL-F
The trnL-F data set consisted of 25 taxa and 529 characters, ofwhich 21 were parsimony informative. The analysis of thisdata set yielded nine trees (L = 32, CI = 0.81, RI = 0.84)(Table 1), the consensus of which provided little informationto resolve the relationships among species. Ca. paraguarienseis sister to the rest of the ingroup. A small clade with twospecies of Camptosema (Ca. coriaceum (Nees & Mart.) Benth.and Ca. coccineum (Mart. ex Benth.) Benth.) is sister to alarger clade of all remaining taxa, with very little internalresolution (Fig. 2).
ITS
The length of the sequences obtained in the present study is inaccordance with the estimated range for angiosperms, namely187–298 for ITS1 and 187–252 for ITS2 (Baldwin et al. 1995).The GC content was near 50% for the ITS1 and higher for theITS2 in most of the sequences. The conserved motif was foundin all the species. The optimal free energy is within theexpected range and the RNA foldings of nearly all sequencesare similar to the consensus structure of vascular plants(Baldwin et al. 1995).
The ITS data matrix consisted of 37 taxa and 772 characters,of which 291 were parsimony informative. The analysis yielded
14 trees (L = 1149, CI = 0.50, RI = 0.66) (Table 1); the strictconsensus tree is shown in Fig. 3.
Camptosema,Galactia and sectionOdoniaofGalactia are notmonophyletic in this analysis, although the monophyly ofCollaea is supported (JK = 55%). Two species of Camptosema(Ca. coccineum and Ca. coriaceum) form a small clade, which issister to a larger one with the rest of the ingroup that is not fullyresolved.Within the latter, the remaining species ofCamptosemaare scattered throughout the cladogram. Ca. rubicundum andG. marginalis Benth. are sister species, Ca. paraguariense isunresolved relative to this clade with the remainder of theingroup. Ca. praeandinum is sister to G. boavista (sect.Collaearia) and Ca. scarlatinum is embedded in a clade withG. martii, G. neesii and Collaea.
Galactia is a paraphyletic group. A monophyletic Collaea(JK = 55%) is nested within it, as are the three species ofCamptosema, namely Ca. rubicundum, Ca. praeandinum andCa. scarlatinum. Sixof the ten species ofGalactia sectionOdoniaform a clade, whereas the other four species are dispersedthroughout the cladogram.
TrnL-F and ITS
The ILD test indicated congruence between trnL-F and ITS datasets (P = 0.99). Combining these two data sets resulted in amatrix of 36 taxa and 1220 characters, of which 322 werepotentially parsimony informative. Analysis of the matrixresulted in 10 trees (L = 1181, CI = 0.53, RI = 0.67) (Table 1)(tree not shown). Neither Camptosema nor Galactia ismonophyletic, whereas Collaea is supported as amonophyletic group. In general, the clades from the ITSanalysis are recovered with a little added resolution; Ca.paraguariense is resolved as basal to the large clade in theingroup, with the Ca. rubicundum and G. marginalis cladesister to the remainder of the group.
TrnL-F, ITS and morphology
The ILD indicated congruence between TrnL-F and morphology(P= 0.68) and between ITS and morphology (P = 0.99).Combining all three data sets yielded a matrix of 28 taxa and1075 characters, of which 240 were parsimony informative. Theanalysis yielded three trees (L = 720, CI = 0.52, RI = 0.65)(Table 1). The strict consensus tree is shown in Fig. 4. Thecharacter consistency and retention indices in the strictconsensus are shown in Tables 2 and 3. As in all previousanalyses, neither Camptosema nor Galactia is monophyletic,whereas the speciesofCollaea formamonophyletic group.Majorclades from the molecular analyses (ITS and ITS + trnL-F) arerecovered, although the position of G. texana (Scheele)A.Gray.–G. lindenii clade in this tree (Fig. 4) differs fromthe strict consensus of the ITS analysis (Fig. 3). As shown inFig. 4, the two species form a well-supported clade thatappears as a sister group to a clade including most species ofGalactia section Odonia. This large clade is sister to Ca.praeandinum–G. boavista clade.
Discussion
The topology derived from the combined analysis (Fig. 4) issimilar to the one obtainedwhen bothmolecular data sets (trnL-F
Fig. 2. Strict consensus of the nine most parsimonious trees (L = 32,CI = 0.81, RI = 0.84) resulting from the TrnL-F data-matrix analysis.Numbers above branches refer to jackknife values.
Phylogenetic relationships among Camptosema, Galactia and Collaea Australian Journal of Botany 79
and ITS, treenot shown)were combinedand to the topologyof theconsensus tree when the ITS data set alone was analysed (Fig. 3).However, it differs from the topology obtained with themorphological data alone, principally because of the positionof the species of Camptosema (Figs 1, 4).
Camptosema is notmonophyletic in any of the analyses. In theanalysis on the basis of morphology, five of the six species ofCamptosema included in the study forma clade (Fig. 1); however,the clade lacks jackknife (JK) support and is not recovered in thecombined analysis (Fig. 4).Within this clade,Ca. coccineum andCa. coriaceum are sister species, supported by a high jackknifevalue (JK = 95%) and defined by five morphological characters(coriaceous leaves (5), glabrous calyx inner surface (21), standardnot reflexed (28), conic nectary disc (36) and straight fruit (41)).This clade is recovered in all analyses, with a JK= 100% inthe combined analysis where it is sister group to the rest of theingroup (Fig. 4, Clade 1). Although these species show allthe diagnostic characters of the genus Camptosema (sensuBentham 1859; Burkart 1952; Camptosema s.s. hereafter), themorphological analysis of de Queiroz et al. (2003) foundthat these species were more closely related to CymbosemaBenth. and Dioclea than to Ca. rubicundum (the type species
of Camptosema). Although there is still uncertainty about therelationships of Clade 1, the present analysis supports the re-evaluation of the position Ca. coccineum and Ca. coriaceumwithin the subtribe Diocleinae. In the morphological analysis,Ca. praeandinum is embedded within a clade with G. lindenii(sect. Galactia) and G. boavista (sect. Collaearia), renderingCamptosema polyphyletic (Fig. 1) and supporting previousresults of de Queiroz et al. (2003). In the combined analysis,Ca. praeandinum is the sister species of G. boavista (Fig. 4,Clade 2). This relationship is defined by four morphologicalcharacters (standard with indument only at the upper portion(25), presence of indument on the margins of the wings (31), andcurved and sericeous fruit (41, 42)) as well as five basesubstitutions; consequently, the clade is supported by a highjackknife value (JK = 97%) (Fig. 4, Clade 2).
Indumentum found in the upper portion of the petals is asynapomorphy of the Ca. praeandinum–G. boavista clade.Within the ingroup, this character state is found elsewhereonly in G. lindenii. In the remaining species of Camptosemaaswell as inmost species of the ingroup, with the exception of thegenus Collaea, where the indumentum covers the entire surface,the plesiomorphic state (glabrous petals) is found.
Fig. 3. Strict consensus of the 14most parsimonious trees (L = 1149, CI = 0.50, RI = 0.66) resulting from the ITS data-matrix analysis. Numbers above branchesrefer to jackknife values.
80 Australian Journal of Botany S. M. Sede et al.
Other morphological characters that could supporttransferring Ca. praeandinum from Camposema to sectionCollaearia of Galactia are the campanulate calyx, shortlystipitate gynoecium, cylindrical disc, incurved style andsessile legume. Moreover, the chromosome number andkaryotype of Ca. praeandinum (n = 10, Sede et al. 2006) agreewith those reported for G. boavista (n = 10, Bossi and Daviña2000; Sede et al. 2006). At the generic level, both Galactia andCollaea have n= 10 (e.g. Goldblatt 1981; Seijo and Vanni 1999;Sede et al. 2003, 2006; Sede 2005), whereas n= 11 ischaracteristic of the genus Camptosema (Goldblatt 1981),
including the type species for the genus (Ca. rubicundum,Sede et al. 2003).
In the topology derived from the combined analysis, Ca.paraguariense is a sister species to a large clade including allingroup species except the Ca. coccineum–Ca. coriaceum clade(Fig. 4). This relationship is strongly supported (JK = 98%) anddefined by three morphological characters (absence ofbrachyblasts (9), curved fruit (41) and compressed seed (44))as well as 11 base substitutions (Fig. 4). Ca. paraguariense wasoriginally described by Chodat andHassler (1904) as a species ofGalactia and later transferred by Hassler (1919) toCamptosema.
Fig. 4. Strict consensus of the three most parsimonious trees (L = 720, CI = 0.52, RI = 0.65) resulting from the combined (morphology, trnL-F and ITS) data-matrix analysis. Numbers above branches refer to jackknife values. Clades 1, 2, 3 and 4 refer to main clades discussed in the text.
Table 2. Character-consistency indices in the strict consensus tree resulting from the combined data-matrix analysis
Phylogenetic relationships among Camptosema, Galactia and Collaea Australian Journal of Botany 81
Although Ca. paraguariense shares some morphologicalcharacters with Camptosema s.s., such as reflexed standard,glabrous petals, long gynophore and pseudomonadelphousandroecium, the form and indument of the calyx and thestandard are more similar to those of the species of Galactiasect. Odonia. In the AFLP analysis performed by Sede et al.(2008), all individuals of Ca. paraguariense formed a well-supported cluster at the base of the tree separate from otherCamptosema species. Moreover, the chromosome number ofCa. paraguariense (n = 10, Sede 2005; Sede et al. 2006) doesnot agree with the proposed number of n= 11 for the genus(Goldblatt 1981).
In the combined analysis, Ca. rubicundum is sister toG. marginalis (Fig. 4, Clade 3). The clade is defined by 12base substitutions and it shows a high jackknife value(JK = 88%). No morphological characters support the group,although both species have a stipitate ovary and a reflexedstandard. The results from the AFLP analysis (Sede et al.2008) agree with these results as well.
The last species of Camptosema included in the presentstudy, Ca. scarlatinum, is sister to a group composed of twospecies ofGalactia (G.neesii andG.martii) and theCollaea clade(Fig. 4, Clade 4). Clade 4 is defined by one morphologicalcharacter (bracteole of the same size or longer than the calyxtube (14)) as well as three base substitutions and has a jackknifevalue of 82%. A relationship between Ca. scarlatinum,G. martii, G. neesii and Collaea has already been suggestedby de Queiroz et al. (2003), on the basis of inflorescenceswith a contracted rachis. Furthermore, in the analysis derivedfrom the AFLP data (Sede et al. 2008), Ca. scarlatinum alsogrouped with the Collaea clade.
Bentham (1859) originally placed Ca. scarlatinum withinCollaea. Taubert (1894) transferred it to Galactia, and later,Burkart (1970) moved it to Camptosema. The species has areflexed standard, presence of a gynophore, a straight styleand a pseudomonadelphous androecium, all characteristic ofthe genus Camptosema s.s. (Bentham 1859). However, thecampanulate calyx and sessile legume differ from thediagnostic characters of Camptosema s.s. (Bentham 1859) andthe chromosome number (n= 10, Sede et al. 2006) does not agreewith the n= 11 number proposed for the genus (Goldblatt 1981).
The genus Galactia is not supported as monophyleticwhereas the monophyly of Collaea is strongly corroborated inthe analyses derived from individual (morphology and ITS,Figs 1 and 3) and combined (Fig. 4) data sets. These resultsare in agreement with those of de Queiroz et al. (2003) andMaxwell and Taylor (2003).
In the combined analysis (Fig. 4), the Collaea cladeshows a jackknife value of 99% and is defined by eightmorphological characters (presence of petiole (4), caducousstipels (7), inflorescence shorter than the subtending leaf (8),strobiliform flower bracts (12), median pedicels (18), standardwith indument all over the outer surface (25), standardwith callous areas (26) and sericeous fruit (42)) and four basesubstitutions. The strobiliform flower bracts and thestandard with callous areas are diagnostic characters foundonly in species of Collaea, whereas other characters,such as digitate–trifoliolate leaves, umbelliform inflorescences,petals with indument and pseudomonadelphous to
monadelphous androecium are also found in Galactia andCamptosema.
The present combined analysis provides good support forsplitting upCamptosema, in agreementwith the hypothesis basedon morphological characters (de Queiroz et al. 2003). The Ca.coriaceum–Ca. coccineum clade is well supported and not mostclosely related to the type species of Camptosema. Therelationship of Ca. praeandinum with Galactia boavista (sect.Collaearia) is strongly supported, as is the relationship of Ca.scarlatinum with G. martii–G. neesii–Collaea clade. Both Ca.praeandinum and Ca. scarlatinum species have morphologicalcharacters as well as chromosome numbers that differ from thosetraditionally atributted toCamptosema s.s. Finally, the taxonomicposition of Ca. paraguariense is difficult to assess because of itsplacement as sister to most of the ingroup, as well as itsincongruent chromosome number and morphology comparedwith Camptosema s.s.
Our results constitute the framework for re-evaluating thetaxonomic position of southern South American Camptosemaspecies. However, a more extensive sampling within this genusand Galactia is needed before taxonomic rearrangements can becompleted.
Acknowledgements
This work was supported by UBA (Universidad de Buenos Aires) – GrantUBACyT EX317 ANPCyT (Agencia Nacional de Promoción Científica yTécnica, Argentina), Grant PICT 14119 CONICET (Consejo Nacional deInvestigaciones Científicas y Técnicas) and Grant PIP 02296 and IAPT(International Association of Plant Taxonomy). Collections weresupported by the Myndel Botanica Foundation. We thank INTA CerroAzul, Misiones, Cecilia Laporta, Fernando Fernandez, Silvina Soto andJulián Greppi for assistance and help during the field trips to Misiones andCorrientes, Argentina.
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Phylogenetic relationships among Camptosema, Galactia and Collaea Australian Journal of Botany 83
Appendix 1. List of specimens analysed in themolecular analyses, in alphabetical order by tribe and subtribe, andGenbankaccession numbers
Previously published sequences are indicated by Genbank number (*) and literature citation
Outgroup
Tribe AbreaeAbrus precatorius L. ITS AF467015* (Hu et al. 2002)
Tribe GenisteaeLupinus paraguariensis Chodat & Hassl. ITS AF007476* (Ainouche and Bayer 1999), trnL-F AF538709* (A Ainouche, MT Misset, RJ Bayer, unpubl.)
Tribe MillettieaeAustrosteenisia blackii (F.Muell.) Geesink. ITS AF467020* (Hu et al. 2002)Lonchocarpus rugosus Benth. ITS AF467062* (Hu et al. 2002)Lonchocarpus sp. ITS AF467064* (Hu et al. 2002)Millettia grandis Skeels. ITS AF467474* (Hu et al. 2002)Millettia sp. ITS AF467480* (Hu et al. 2002)
Tribe Phaseoleae – subtribe DiocleinaeCanavalia bonariensis Lindl. ITS AY293839* (Varela et al. 2004)Can. brasiliensis Mart. ex Benth. ITS AF467034* (Hu et al. 2002)Cleobulia multiflora Mart. ex Benth. ITS AY881100* (TB Grangeiro, DMM Jorge, ES Varela, JPMS Lima, WM Bezerra, EP Nunes, MAO Alves, unpubl.)Cymbosema roseum Benth. ITS AY293836* (Varela et al. 2004)Dioclea bicolor Benth. ITS AY293833* (Varela et al. 2004)Dioclea virgata (Rich.) Amshoff. ITS AY293835* (Varela et al. 2004)
Phylogenetic relationships among Camptosema, Galactia and Collaea Australian Journal of Botany 85
Appendix 3. List of morphological characters used in the phylogenetic analysis
Vegetative character
1. Habit: trees (0); lianas, vines or shrubs (1); subffrutex or herbs (2). 2. Leaf rachis: absent (0); present (1). 3. Number of leaflets: one (0); three (1). 4. Petiole:absent (0); present (0).5.Leaf texture:membranous, papyraceousor chartaceous (0); coriaceous (1).6.Leaf shape:narrowlyoblong (0); broadly elliptic-obovate(1). 7. Stipels: absent (0); semipersistent (caducous) (1); persistent (2).
Inflorescence
8.Lengthof inflorescence v. lengthof subtending leaf: same size or longer (0); shorter (1).9.Brachyblast: absent (0); present (1).10.Woodiness of the rachis:not woody (0); woody (1). 11. Inflorescence type: elongate raceme (0); umbelliform/corymbiform raceme (1). 12. Flower bracts: not strobiliform (0);strobiliform (1). 13. Bracteole persistence: caducous (0); persistent (1). 14. Length of the bracteole v. length of the calyx tube: shorter (0); of the same size orlonger (1). 15. Bracteole position: at the base of the calyx (0); below the base of the calyx (1). 16. Bracteole shape: obovate to orbicular (0); subulate to triangular(1). 17. Flower bud shape: ovate to cylindrical (0); orbicular (1). 18. Length of the pedicels: short (0 to <7mm) (0); median (7 to <15mm) (1); large (15 to<30mm) (2) (states were determined with ANOVA, followed by comparisons with Scheffée’s test (Sokal and Rohlf 1995)).
Flower
19. Calyx color: red (0); green (1). 20. Calyx shape: campanulate (0); tubular (1). 21. Calyx inner surface indument: glabrous (0); only at the lobes (1); all overthe surface (2) (ordered). 22. Upper lobe shape: triangular to lanceolate (0); elliptic to ovate (1); broadly rectangular (2). 23. Petal color: red (0); blue lilac (1).24. Standard shape: orbicular (0); obovate to elliptic (1); oblong (2). 25. Standard outer surface indument: glabrous (0); only at the upper portion (1); all overthe surface (2) (ordered). 26. Callous areas: absent (0); present (1). 27. Callous areas indument: absent (0); present (1). 28. Standard reflexion: absent (0);present (1). 29. Standard auricles: vestigial or absent (0); present (1). 30.Wing shape: elliptic to oblong (0); broadly obovate (1). 31. Indument on themarginsof the wings: absent (0); present (1). 32. Keel apex: straight (0); incurved (1). 33. Keel upper margin: entire (0); toothed (1). 34. Stamen connation:pseudomonadelphous (0); diadelphous (1). 35. Anther indument: absent (0); present (1); 36. Nectary disc shape: cylindrical (0); conic (1); truncate (2).37.Nectarydiscmargin: irregular (0); lobulate (1).38.Ovary: sessile to subsessile (0); short-stipitate (1); long-stipitate (1).39.Style:geniculate at thebaseor themiddle (0); straight (1).
Fruit
40. Fruit margin: straight (0); undulate (1). 41. Fruit shape: straight (0); curved (1). 42. Fruit indument: glabrous (0); pilose (1); sericeous (2). 43. Beakposition: central (0); lateral (1).
