Genetic diversity and relationships in Solanum subg ... - Springer

Plant Syst Evol (2011) 291:35–47DOI 10.1007/s00606-010-0371-5ORIGINAL ARTICLEGenetic diversity and relationships in Solanum subg.Archaesolanum (Solanaceae) based on RAPD and chloroplastPCR-RFLP analysesPeter Poczai • András Cseh • János Taller •David E. SymonReceived: 13 October 2008 / Accepted: 4 October 2010 / Published online: 27 October 2010Ó Springer-Verlag 2010Abstract The subgenus Archaesolanum is a groupcomposed of eight species with a characteristic chromosomenumber based on n = x = 23 and an area restrictedto the South Pacific. This subgenus is an isolated group ofSolanum for which extensive information about phylogeneticrelationships based on molecular genetic methods islacking. This study represents an approach to analyzegenetic relationships within this group. In this context,seven species were examined using random amplifiedpolymorphic DNA (RAPD) markers. In further analysis,the amplification products of two chloroplast regions(trnS-trnG and rbcL) were studied with polymerase chainreaction (PCR) restriction fragment length polymorphism(RFLP) method. Screening for the presence of uniquemitochondrial rearrangements was also carried out usingP. Poczai (&) J. TallerDepartment of Plant Sciences and Biotechnology,Georgikon Faculty, University of Pannonia, Festetics 7,Keszthely 8360, Hungarye-mail: guanine@ex1.georgikon.huJ. Tallere-mail: taller@ex1.georgikon.huA. CsehDepartment of Plant Genetic Resources and Organic Breeding,Agricultural Research Institute of the Hungarian Academyof Sciences, Brunszvik u. 2, Martonvásár 2462, Hungarye-mail: andrascseh@gmail.comD. E. SymonDepartment of Environment and Heritage,Plant Biodiversity Centre, State Herbarium of South Australia,PO Box 2732, Kent Town, SA 5071, Australiae-mail: symon.david@sa.gov.auuniversal mitochondrial primers for the detection offragment length polymorphisms. We identified two majorgroups within the subgenus; one was composed of themembers of ser. Avicularia and Laciniata, while the otherwas formed by species belonging to ser. Similia. It issuggested that the taxonomic status of series within theArchaesolanum clade should be revised. The hybrid originof S. laciniatum was also tested, and two hypothesesregarding its phylogeny are assumed.Keywords Archaesolanum Phylogenetic relationships Solanum PCR-RFLP RAPD Kangaroo applesIntroductionThe diverse genus Solanum L., with approximately 1,400species, has worldwide distribution, with center of diversityin South America. The species belonging to subg.Archaesolanum Bitter ex Marzell, often called kangarooapples, are described as a distinctive group with no obviousclose relatives. The subgenus is represented by eight species,which occur only in the South West Pacific region(New Guinea, Australia, Tasmania, and New Zealand). Thespecies are short-lived soft-wooded shrubs, 1–3 m tall,becoming straggly with age, unarmed, glabrescent, withlarge (up to 30 cm) deeply lobed leaves in the juvenilephase, becoming smaller (up to 10 cm) and entire in theadult stage, with violet–purple flowers in cymes growingat the axils of stem-fork sites (Symon 1984). The fruitsare greenish, yellowish, or scarlet; the succulent berriesproduce numerous seeds (approx. 100–600). White oryellowish stone cell aggregates are present in the driedcontents of the fruits, mixed with seeds. The fruits areeaten by birds, which are probably responsible for their123

36 P. Poczai et al.distribution throughout Australia, New Zealand, Tasmania,and New Guinea (Symon 1979).Plants belonging to this group were first collected byForster in Australia during the second voyage of CaptainJames Cook. Forster (1786) was the first to publish thename Solanum aviculare in the ‘‘Dissertatio inauguralisbotanico-medica de plantis esculentis insularum oceaniaustralis.’’ Since then, the group has been recognized byseveral Solanum researchers, e.g., Bitter (1927), Danert(1970), D’Arcy (1972, 1990), Symon (1981, 1994), andHunziker (2001). The subgenus was divided into threeseries by Gerasimenko (1970): ser. Avicularia Geras.,consisting of S. aviculare Forst. (2n = 2x = 46) andS. multivenosum Symon (2n = 4x = 92); ser. LaciniataGeras. composed of S. laciniatum Ait. (2n = 4x = 92),S. vescum F. Muell (2n = 2x = 46), and S. linearifoliumGeras. ex Symon (2n = 2x = 46); and ser. Similia Geras.,with S. capsiciforme (Domin) Baylis (2n = 2x = 46),S. simile F. Muell. (2n = 2x = 46), and S. symonii Eichler(2n = 4x = 92). Symon (1994) mentioned S. cheesemaniiGeras. and S. baylisii Geras. as separate species, but theyare now considered to be synonyms and varieties ofS. aviculare, as suggested by Baylis (1963).The basic chromosome number in subg. Archaesolanumis n = x = 23, in contrast to the n = x = 12 typical ofother members of genus Solanum (Randell and Symon1976). Despite the name, suggesting an archetypal Solanum,the chromosome number indicates a derived conditionwhich has itself become polyploid (Symon 1979),probably reached by aneuploid loss from n = x = 24(Randell and Symon 1976). It is clear that all species basedon secondary gametic numbers are polyploid; in the case ofsecondary polyploidy (Hair 1966) each number generatesits own polyploid sequence as follows: in the originalseries, 2n = 48 (4x) and 72 (6x); in the derived series,2n = 46 (2x 2 )(S. aviculare) and 2n = 92 (4x 2 )(S. laciniatum)(data from Baylis 1954). Whilst these zygoticnumbers are ‘‘diploid’’ and ‘‘tetraploid’’ with respect to thesecondary basic number, x 2 = 23, the respective speciesare relatively tetraploid and octoploid in terms of the originalbasic number, x = 12 (Hair 1966).Symon (1994) provided a preliminary phylogeny forthe subgenus based on morphology, but these conceptshave not been tested using molecular methods. To thebest of our knowledge, no study including all eight speciesand investigating phylogenetic relationships withinthe group using molecular tools has yet been published.Many molecular studies on phylogenetic relationshipswithin the genus Solanum have included species representingthe subgenus in their analysis. Olmstead andPalmer (1997) included S. aviculare in an analysisbased on chloroplast restriction site data. Bohs andOlmstead (2001) and Bohs (2005) gave information aboutS. aviculare and S. laciniatum based on data from nuclearinternal transcribed spacers (ITS) and ndhF gene, whichindicated that the members of subg. Archaesolanumformed a well-supported basal clade in Solanum. UsingRAPD, Poczai et al. (2008) also found that the group wasdistinct from the other members of the genus. It thusseems safe to say that the Archaesolanum clade representsan isolated group whose closest relatives have not yetbeen identified (Bohs 2005). Putative ancestors havecertainly not been recognized in Australia, nor are extra-Australian relatives apparent (Symon 1979). Althoughred-fruited species, such as S. dunalianum Gaudich.,S. viride Spreng., and S. incanoalabastrum Symon, occurin New Guinea, these all have stellate hairs and no stonecells; they are not related to the Archaesolanum group.Subgenus Archaesolanum represents an ambiguous case,either representing an early dispersal event in the genus,or a plausible case of vicariance dating to a time prior tothe separation of South America and Australia (Olmsteadand Palmer 1997). The ndhF data of Bohs and Olmstead(1997) suggest that the ancestor of the Archaesolanumclade arrived in Australia early in the evolutionary radiationof Solanum.Random amplified polymorphic DNA (RAPD) technique(Williams et al. 1990, 1993; Welsh and McClelland1990) is a useful tool in population and evolutionarygenetics, since no prior knowledge of the genome structureor sequence data is required. RAPD has been used inseveral studies in the case of Solanum (e.g., Stedje andBukenya-Ziraba 2003; Van den Berg et al. 2002; Spooneret al. 1996; Miller and Spooner 1999) to clarify phylogeneticrelationships at various levels (Sheng et al. 2006;Baeza et al. 2007; Liebst 2008).Organelle DNA sequences have been used extensivelyin phylogenetic studies on plants. Chloroplast DNA(cpDNA) variation has proven to be immensely valuable inreconstructing phylogenies at the species and higher taxonomiclevel. PCR amplification with specific or universalprimers, followed by restriction digestion and electrophoreticseparation of the fragments (PCR-RFLP), is frequentlyapplied in plant phylogenetic studies (e.g., Csehand Taller 2008; Friesen et al. 1997; Wachowiak et al.2006; Prentice et al. 2008). The combination of this techniquewith other molecular methods such as inter-simplesequence repeat (ISSR) (Huang et al. 2002), RAPD(Potokina et al. 1999), and amplified fragment lengthpolymorphism (AFLP) (Fu et al. 2004) is also widespread.Compared with standard RFLP analysis using entirecpDNA or probe cpDNAs, RFLP analysis of PCR-amplifiedcpDNA regions has several advantages: simpleprocedure, small amounts of tissue required, and reducedtime and expense. Therefore, it is considered to be an easyand advantageous tool for detecting cpDNA variations.123

Genetic diversity and relationships in Solanum subg. Archaesolanum 37Another approach for extracting phylogenetic informationfrom both cpDNA and mitochondrial DNA (mtDNA)is to analyze the distribution of major structural rearrangements.The mitochondrial genome evolves considerablymore slowly at the nucleotide sequence level than thenuclear or the chloroplast genomes (Palmer 1990; Wolfeet al. 1987), but the rate of rearrangements is extraordinarilyfaster in plant mtDNA than in cpDNA (Palmer andHerborn 1988), possibly making it useful in investigationson distant phylogenetic relationships. The presence orabsence of rearrangements in a particular gene or introncan be assayed by hybridization using probes specific to thegene/intron or with the use of the PCR technique. Primersare synthesized for conserved sequences flanking theregion of interest, and the intervening sequence is amplifiedby PCR (Downie and Palmer 1992). Comparing the size ofthe resultant PCR product with a sequence of known lengthon an agarose or polyacrylamide gel can indicate thepresence or absence of specific gene or intron rearrangements(Bruzdzinski and Gelehrter 1989).The purpose of this study is to investigate geneticrelationships in subg. Archaesolanum based on RAPDmarkers and PCR-RFLP analysis of two chloroplastregions (trnS-trnG and rbcL). Another objective of thisstudy is to reveal mitochondrial rearrangements usinguniversal primers, which could be useful for further analysisof the group.Materials and methodsPlant material and DNA extractionTaxon sampling included seven species belonging to subg.Archaesolanum, with two accessions from each of thespecies S. aviculare, S. laciniatum, and S. simile and onefrom each of S. linearifolium, S. capsiciforme, S. symonii,and S. vescum. For outgroups, seven Solanum species,representing different subgenera, were included in theanalysis. An accession from outside the genus, Capsicumannuum, was also added in the experiments, according tothe results reported by Olmstead et al. (1999) and Bohs andOlmsted (2001). Although we used only one accession foreach species in this study, our ongoing continuous studieswithin different lineages of Solanum show that intraspecificvariation does not adversely affect phylogenetic relationshipsbetween sections of Solanum, as was also shownpreviously in other groups of the genus by Spooner andSystma (1992). Voucher specimens were deposited at theherbarium of University of Pannonia, Keszthely, Hungary.Information about the accessions can be found in Table 1.Genomic DNA was extracted from approximately 50 mgof young fresh leaves using the procedure of Walbot andWarren (1988). RAPD fingerprints were obtained from DNAbulks, according to Spooner et al. (1997), where five plantsfrom each accession were bulked for DNA extraction.Although fragments present in \15% of individuals composingthe DNA bulk are often observed to be lost from thebanding patterns of bulked samples (e.g., Divaret et al.1999), we designed our study to sample as many alleles aspossible within the accessions. Additionally, the aim was toexamine more populations, rather than more individuals,within a population. Thus the bulking strategy described byMichelmore et al. (1991) was considered to be useful togenerate a group (e.g., population or accession) fingerprintby combining DNA from a number of individuals. Thisstrategy may reduce the noise in the dataset due to markerssegregating within the groups (Bussel et al. 2005) and hasbeen used successfully in the case of Solanum species (Millerand Spooner 1999; Rodríguez and Spooner 1997; Spooneret al. 1991, 1993, 1995, 1997; Clausen and Spooner 1998).RAPD amplificationIn the RAPD analysis a total of 40 primer pairs were used.Each reaction was performed twice to verify reproducibility.The primers were paired arbitrarily, but palindromesand complementarities within and between primers wereavoided. The sequence of each primer was generated randomly,comprising 12 base oligonucleotides and *50–70%GC content. The sequences of the primers are availablefrom the corresponding author upon request. PCR wascarried out on a 96-well RoboCycler (Stratagene, USA)using a 20 ll reaction mix which contained the following:10 ll sterile ion-exchanged water, 5 ng template DNA,1 lM of each primer, 0.2 mM dNTP (Fermentas, Lithuania),2 ll 109 PCR buffer (1 mM Tris–HCl, pH 8.8 at25°C, 1.5 mM MgCl 2 , 50 mM KCl, and 0.1% TritonX-100), and 0.5 U DyNazyme II (Finnzymes, Finland)polymerase. Reaction conditions were 1 min at 94°C,followed by 35 cycles of 30 s at 94°C, 1 min at 37°C, and2 min at 72°C. A final amplification for 5 min at 72°C wasapplied. Amplification products were separated on 1.5%agarose gels (Promega, USA) in 0.59 TBE buffer (300 V,1.5 h) and post-stained with ethidium bromide. The gelswere documented using the GeneGenius Bio Imaging System(Syngene, UK). The banding patterns were evaluatedand annotated with the program GeneTools (Syngene, UK).Chloroplast region amplification and restrictiondigestiontrnS-trnG regionThe chloroplast intergenic spacer between trnS and trnGwas amplified using the primers described by Hamilton123

38 P. Poczai et al.Table 1 Accessions of Solanum species used in the analysisTaxonomic position b Taxon Collector Accession number CollectionlocalityOriginIngroupSubg. ArchaesolanumSer. Avicularia Geras. S. aviculare (1) Forst. Unknown 874 750 027 A I.S. aviculare (2) Forst. var. GTS Baylis 844 750 003 NZ I.latifolium Bayl.Ser. Laciniata Geras. S. laciniatum Ait. (1) a Unknown A24 750 011 Unknown I.S. laciniatum Ait. (2) a DE Symon A24 750 098 A I.S. linearifolium Geras. Unknown 814 750 056 Unknown I.S. vescum F. Muell. D Martin 904 750 174 A I.Ser. Similia Geras. S. capsiciforme (Domin) Bayl. Unknown 884 750 213 Unknown I.S. simile F. Muell. (1) a CR Alcock A24 750 094 A I.S. simile F. Muell. (2) a Unknown 894 750 053 A I.S. symonii Eichler N Lovett 844 750 004 A I.OutgroupSubg. LeptostemonumSect. Acanthophora S. atropurpureum Schrank. Unknown UHBG211-1471 Unknown II.Androceras S. rostratum Dunal Unknown HU1GEO20060029 Unknown III.Cryptocarpum S. sisymbriifolium Lam. Unknown HU1GEO20060053 Unknown III.Subg. MinonSect. Bravantherum S. abutiloides Bitter & Lillo Unknown UHBG211-1455 Unknown II.Subg. PotatoeSect. Dulcamara S. dulcamara L. P Poczai HU1GEO20060017 H III.Subg. SolanumSect. Solanum S. americanum Miller. BG Redwood 904 750 023 USA I.S. physalifolium Rusby var. Unknown 894 750 076 G I.nitidibaccatum (Bitter) Edm.Genus Capsicum Capsicum annuum L. Unknown 884 750 092 Unknown I.Collection locality abbreviations: A Australia, G Germany, H Hungary, NZ New Zealand, USA United States of America. Origin abbreviations:I Botanical and Experimental Garden of the Radboud University, The Netherlands. II Botanical Garden of the University of Hohenheim,Stuttgart, Germany. III Georgikon Botanical Garden of the University of Pannonia, Keszthely, Hungarya Numbers are abbreviations used in the further text, tables, and figures to distinguish between accessionsb According to D’Arcy (1972, 1992)(1999). All PCR reactions were performed in a Master-Cycler ep384 (Eppendorf, Germany) with the same compositionas in the RAPD analysis, except that the MgCl 2concentration was adjusted to 2 mM. The thermal cyclerprogram included an initial denaturation at 94°C for 4 min;40 cycles of 94°C for 45 s, 52°C for 1 min, and 72°C for1 min; with a final extension at 72°C for 7 min, asdescribed by Levin et al. (2006).rbcL1-rbcL2 regionThe sequence of the large subunit of the ribulose-1,5-bisphosphatecarboxylase gene (rbcL) was amplified using theprimers described by Demesure et al. (1995). The 20 llreaction solution was the same as that described above. Thethermal cycler program was the following: 94°C for 1 min;30 s at 94°C, 1 min at 60°C, and 2 min at 72°C for 35cycles; and a final cycle of 4 min at 72°C. Further informationabout the primers used in the study is given inTable 2.The trnS-trnG amplification products were digestedwith the restriction endonuclease enzymes HinfI, DdeI,MboI, MspI, RsaI, Taq a I, and AluI (New England BiolabsInc., USA). The reaction conditions recommended by thesupplier were used for all enzymes. The restriction fragmentswere separated on 2.3% high-resolution MetaPhoragarose gel (Cambrex Bio Science Rockland, Inc., USA),after which the products were visualized by ethidiumbromidestaining.The rbcL1-rbcL2 region was digested with the sameenzymes except that instead of Taq a I the AluI and HhaIrestriction endonucleases were used in the analysis. The123

Genetic diversity and relationships in Solanum subg. Archaesolanum 39Table 2 Details of the primers used in the study of chloroplast and mitochondrial regionsGene a Primer pairs Size(bp)G ? C(%)Annealingtemperature (°C)PCRproduct (bp)ChloroplastprimerstrnS-trnG trnS 5 0 -GCCGCTTTAGTCCACTCAGC-3 0 20 60 52 *700–735trnG 3 0 -CACCATTTTCACACTAAGCAAG-5 0 22 41rbcL1-rbcL2 rbcL1 5 0 -ATGTCACCACCACAAACAGAGACT-3 0 24 46 60 *1,371rbcL2 3 0 -CCTCAGGACTTGATCGACGACGAACACTTC-5 0 31 52Mitochondrialprimersatp6F-atp6R atp6F b 5 0 -GGAGG(A=I)GGAAA(C=I)TCAGT(A=I)CCAA-3 0 22 48 58 *589–610atp6R 3 0 -TAGCATCATTCAAGTAAATACA-5 0 22 27cobF-cobR cobF 5 0 -AGTTATTGGTGGGGGTTCGG-3 0 20 55 58 *290–313cobR 3 0 -CCCCAAAAGCTCATCTGACCCC-5 0 22 59cox1F-cox1R cox1F b 5 0 -GGTGCCATTGC(T=I)GGAGTGATGG-3 0 22 59 58 *1,466cox1R 3 0 -TGGAAGTTCTTCAAAAGTATG-5 0 21 33nad3F-nad3R nad3F 5 0 -AATTGTCGGCCTACGAATGTG-3 0 21 48 58 *237nad3R 3 0 -TTCATAGAGAAATCCAATCGT-5 0 21 33nad5aF-nad5aR nad5aF 5 0 -GAAATGTTTGATGCTTCTTGGG-3 0 22 41 58 *1,000nad5aR 3 0 -ACCAACATTGGCATAAAAAAAGT-5 0 23 30nad5dF-nad5dR nad5dF 5 0 -ATAAGTCAACTTCAAAGTGGA-3 0 21 33 58 *1,095–1,136nad5dR 3 0 -CATTGCAAAGGCATAATGAT-5 0 20 35rps14F-rps14R rps14F 5 0 -ATACGAGATCACAAACGTAGA-3 0 21 38 58 *114rps14R b 3 0 -CCAAGACGATTT(C=I)TTTATGCC-5 0 21 38nad4exon1-nad4exon2a nad4exon1 5 0 -CAGTGGGTTGGTCTGGTATG-3 0 20 55 58 *2,058nad4exon2a 3 0 -TCATATGGGCTACTGAGGAG-5 0 20 50a trnS-trnG, intergenic spacer between Ser-tRNA and Gly-tRNA; rbcL1-rbcL2, subunit of the ribulose-1,5-bisphosphate carboxylase gene; atp6 (oratpF), F0-ATPase subunit 6 gene; cob, apocytochrome b gene; cox1 (or coxI), cytochrome c oxidase subunit 1 gene; nad3 (or nadC, nadhC, nadh3, ornd3), NADH-ubiquinone oxidoreductase subunit 3 gene; nad5a (or nadF, ndhF, ndh5, nd5), NADH-ubiquinone oxidoreductase subunit 5 gene (intron1); nad5dF (or nadF, ndhF, ndh5,nd5), NADH-ubiquinone oxidoreductase subunit 5 gene (intron 2); rps14, ribosomal protein subunit 14 gene;nad4exon1 (or nadD, ndhD, ndh4, nd4) NADH-oxidoreductase subunit 4 gene (intron 1)b Inosine was used in the synthesis instead of the corresponding nucleotide because of the nucleotide variation between plant sequencesseparation and visualization procedure was the same as thatdescribed above. The enzymes for the analysis wereselected based on the virtual digestion of the sequence dataof the fragment trnS-trnG from S. aviculare, submitted tothe NCBI database by Levin et al. (2005) under accessionnumber AY555458. For this procedure the programNEBcutter (Vincze et al. 2003) was used.Mitochondrial region amplificationThe universal primers described by Demesure et al. (1995)for the amplification of different mitochondrial regionswere tested to detect fragment length polymorphismbetween the accessions. The contents and concentrationsused in the reaction mixture were the same as described forthe RAPD analysis. The PCR program was the following:94°C for 1 min; 30 s at 94°C, 1 min at 58°C, and 2 min at72°C for 35 cycles; and a final cycle of 4 min at 72°C.The amplified regions and further information about theprimers are summarized in Table 2.RAPD data analysisOnly distinct, well-resolved, clear bands were scored.Reliable bands are thought to refer to amplicons found inreplicate reactions. It was assumed that fragments ofequal length had been amplified from corresponding loci,and the band conventionally assumed to represent a single,dominant, nuclear locus with two possible alleles.The amplified fragments were scored as: 1, for the presence,or 0, for the absence of homologous bands. Fromthis binary matrix, a distance matrix was computedaccording to Nei and Li (1979) based on Dice’s similaritycoefficient (Dice 1945). A dendrogram was constructedusing the neighbor-joining method described by Saitouand Nei (1987); the original matrix was bootstrapped123

40 P. Poczai et al.1,000 times to check the reliability of the branchingpatterns and the quality of the resulting phylogeneticgroups. These bootstrap values are shown at the nodes ofthe dendrogram as percentages. The FAMD program(Schlüter and Harris 2006) was used for all calculations.The tree obtained using FAMD was visualized and editedusing the TreeView program (Page 1996).Parsimony analysis of the restriction fragmentsAll calculations were carried out using the program packagePHYLIP (Phylogeny Inference Package) published byFelsenstein (1989). The two data sets (trnS-trnG andrbcL1-rbcL2) were analyzed separately and in combination.The discrete character data of the restriction fragmentswere coded into a series of (0 or 1) two-statecharacters. The further analysis was carried out using thebranch-and-bound algorithm of the DOLPENNY programto find all of the most parsimonious trees implied by thedata. The data analysis was performed with the use of theDollo parsimony method. The program was set to reportevery 100 trees and 1,000 groups. From the resulting outputtrees, a consensus tree was built with the CONSENSEprogram using the majority rule criterion.ResultsRAPD analysisThe 40 RAPD primer pairs generated 295 reliable fragmentsfrom all the accessions analyzed. RAPD analysisusing primer combinations clearly separated the accessionsused in the study. The dendrogram calculated from the datamatrix generated by the formula of Nei and Li (1979) ispresented in Fig. 1. We tried to root the resulting tree byadditionally adding/removing groups and taxa used asoutgroups. In all cases a consistently distinct Archaesolanumclade was present among the analyzed accessions,where no significant difference was found in the overalltopology of the groups within the Archaesolanum clade.The dendrograms in all rooted versions consistently producedtwo separate clades; one was composed of themembers of ser. Avicularia and Laciniata, namely twoaccessions of S. aviculare, two accessions of S. laciniatum,S. vescum, and S. linearifolium, which exhibit close affinitybased on the bootstrap values. S. vescum is sister to thisgroup composed of S. aviculare and S. laciniatum; S. linearifoliumoccupies a basal position in this clade. Thesecond major clade was formed by members of ser. Similia.S. capsiciforme is separated from the other members of thegroup. S. symonii and the two accessions of S. simile aregrouped together.Chloroplast and mitochondrial region analysisOne amplification product per sample was obtained for allthe accessions examined. The size of the fragmentsamplified with trnS-trnG primers was approximately710 bp among the species. The size of the rbcL1-rbcL2fragments was approximately 1,370 bp and showed novariation. Restriction enzyme assay with seven differentenzymes resulted in variable fragments in the case of thetrnS-trnG region. Approximately 10 bp differences wereobserved between the restriction fragments. The PCR-RFLP fragments of the region clearly separate two groupsin the subgenus. S. laciniatum, S. aviculare, and S. vescumcompose a group with larger fragments, while S. simile,S. symonii, and S. capsiciforme gave smaller restrictionproducts. S. linearifolium occupied an ‘‘intermediate’’position between the two groups according to the fragmentlengths of the PCR-RFLP analysis. An example is shown inFig. 2. The observed variability in the restriction productsindicates that there are differences between the Archaesolanumspecies in the trnS-trnG region (Table 3).The rbcL1-rbcL2 region was not informative in the caseof subg. Archaesolanum. After digestion with six restrictionendonuclease enzymes, no polymorphism wasdetectable. However, the selected enzymes had 4–9restriction sites in the 1,370-bp sequence. This similarity ofthe Archaesolanum species and their difference from theoutgroups indicates that, although this region is uninformativeat the subgeneric level, it could be useful in highertaxonomic analyses. As the rbcL region was not informativein the present case, it is not discussed in the following.Eight mitochondrial regions were amplified to detectdifferent fragment length patterns which could be attributedto the unique replication of the mitochondrial genome.For this reason the universal primers described by Demesureet al. (1995) were used, to reveal this type of variation.The size of the PCR products and the sequence of theprimers are given in Table 2. Polymorphic fragments weredetected with the use of nad5aF-nad5aR. These primers aredesigned for the amplification of an intron between exon 1(nad5aF) and exon 2 (nad5aR) of the nicotinamide adeninedinucleotide (NADH)-ubiquinone oxidoreductase subunit5 gene. While all the species had a 1,000-bp fragment,in the case of S. vescum, S. laciniatum, and S. linearifoliuman *690-bp size band was also detectable. Besidethese, S. linearifolium also had a unique *880-bp fragment(Fig. 3).Results of the parsimony analysisThe phylogenetic tree obtained from the analysis of theconsensus tree construction is given in Fig. 4. The topologyof the tree constructed from the chloroplast region123

Genetic diversity and relationships in Solanum subg. Archaesolanum 41Fig. 1 Dendrogram constructedusing the neighbor-joining (NJ)method from the RAPD datamatrix calculated with theformula given by Nei and Li(1979). The numbers at the treenodes are bootstrap values as apercentageFig. 2 Restriction patterns ofthe trnS-trnG chloroplastregion, digestion with MboI.M Molecular weight size marker(100–1,000 bp). 1S. linearifolium; 2 S. aviculare(1), 3 S. aviculare (2); 4S.vescum; 5 S. laciniatum (1); 6S. laciniatum (2); 7S. capsiciforme; 8 S. symonii; 9S. simile (1); 10 S. simile (2)restriction data is almost identical to the tree topology ofthe RAPD data. The major difference is that S. symonii andS. capsiciforme formed a group together in the case of thechloroplast region. This arrangement was due to theabsence of a restriction site with RsaI in the trnS-trnGsequence unique for these species (Fig. 5).123

42 P. Poczai et al.Table 3 Restriction patterns of trnS-trnG; number of fragmentsobtained in the analysisAccessionDiscussionRestriction enzymeHinfI DdeI MboI MspI RsaI Taq a I AluIIngroupS. aviculare (1) 2 2 2 2 3 5 4S. aviculare (2) 2 2 2 2 3 5 4S. capsiciforme 2 2 2 2 2 4 3S. laciniatum (1) 2 2 2 2 3 5 3S. laciniatum (2) 2 2 2 2 3 5 3S. linearifolium 2 2 2 2 3 5 4S. simile (1) 2 2 2 2 3 4 4S. simile (2) 2 2 2 2 3 4 4S. symonii 2 2 2 2 2 4 3S. vescum 2 2 2 2 3 5 4OutgroupS. abutiloides 2 3 0 2 3 4 2S. americanum 2 3 2 2 2 3 2S. atropurpureum 2 2 0 0 2 5 0S. dulcamara 2 2 2 2 3 4 2S. physalifolium 2 3 2 2 3 4 2S. rostratum 2 2 0 2 3 6 3S. sisymbriifolium 2 2 0 2 3 4 3C. annuum 2 2 2 2 3 3 2This study represents an approach using genomic DNAfingerprint markers and other methods to study geneticrelationships in subg. Archaesolanum. The RAPD dataobtained in this study are a random sample, representing allthe polymorphic RAPDs in the germplasm examined.RAPDs have been shown to be useful as taxonomicmarkers for closely related species through concordantresults using other molecular marker systems for closelyrelated taxa elsewhere in sect. Petota (Cisneros and Quiros1995; Spooner et al. 1996). In addition, the results indicatethat S. aviculare and S. laciniatum are closely related,which is strongly supported by bootstrap values. S. aviculareis easily confused with S. laciniatum. PreviouslyS. laciniatum was treated as a variety of S. aviculare underthe name S. aviculare var. laciniatum (Aiton) Domin. Bothspecies were commonly cultivated in the former USSR,Australia, and New Zealand, and they were used in thealkaloid industry in the 1970s (Knapp 2006). Althoughthey are difficult to distinguish, there are some morphologicalparameters in which they differ. S. aviculare isdiploid (2n = 2x = 46) with bright-orange or red maturefruits containing approx. 600 seeds per fruit, whileS. laciniatum is tetraploid (2n = 4x = 92), with fruits thatare first green, later paling to yellow or orange-yellow, andcontaining approx. 200 seeds per fruit. Baylis (1954)clearly describes other characteristics: S. laciniatum haslarger pollen grains, flowers, seeds, and stone-cell masses(in the fruit pulp) than S. aviculare; its corolla is deeper incolor with relatively shallow lobes, the margins of whichflatten more completely, producing an emarginated apex.The analyses carried out provide results in re-examiningthe putative hybrid origin of S. laciniatum, indicating thatit is not of recent hybrid origin. There is a lack of additivityboth in the RAPD patterns and in the PCR-RFLP analysis.In the dendrogram constructed from RAPD data, S. laciniatumforms a group together with S. aviculare andS. vescum, and the same topology is present in the treeFig. 3 Amplification productsof nad5aF-nad5aR.M Molecular weight size marker(100 bp), the numbers indicateladder size in bp. 1S. linearifolium; 2 S. aviculare(1), 3 S. aviculare (2) 4S. vescum; 5 S. laciniatum (1);6 S. laciniatum (2); 7S. capsiciforme; 8 S. symonii; 9S. simile (1); 10 S. simile (2); 11Capsicum annuum123

Genetic diversity and relationships in Solanum subg. Archaesolanum 43Fig. 4 Majority rule consensustree based on the PCR-RFLPfragments obtained from thetwo chloroplast regions andmitochondrial fragment lengthpolymorphisms, showingphylogenetic relationships andclades in subg. Archaesolanum.The numbers at the nodes arebootstrap replicates as apercentageFig. 5 Restriction patterns ofthe trnS-trnG chloroplastregion, digestion with RsaI.M Molecular weight size marker(100–500 bp). 1S. linearifolium; 2. S. aviculare(1) 3 S. aviculare (2) 4S. vescum; 5 S. laciniatum (1);6 S. laciniatum (2); 7S. capsiciforme; 8 S. symonii; 9S. simile (1); 10 S. simile (2)from PCR-RFLP patterns. This topology is in agreementwith the crosses summarized by Symon (1994), who suggestedthat S. laciniatum may be a hybrid of S. aviculareand S. vescum. S. aviculare and S. vescum hybridizespontaneously when the species are grown next to eachother. The hybrids are only fertile when S. vescum isinvolved as the female parent (Baylis 1963).The presence of a specific fragment obtained from theamplification of the mtDNA region nad5aF-nad5aR alsoconfirms these results. This fragment is present in S. vescum,the hypothetical female parent, but it is absent fromS. aviculare, the putative male parent of S. laciniatum.According to the maternal inheritance of organelle genomes,this fragment in S. vescum and in S. laciniatum123

44 P. Poczai et al.represents a unique mitochondrial structure, which supportsthe hybridization theory. Although this type ofmitochondrial structure is present in S. linearifolium too, itseparates from them, forming another fragment ofapproximately 880 bp. This fragment could be a promisingitem for further analysis of the subgenus.Hybrid speciation could occur at least two ways in thiscase: It is possible that S. laciniatum is developed throughdiploid hybrid speciation involving S. aviculare andS. vescum as female parent, and than the entire genome isduplicated through autopolyploidy. This alternativehybridization can only be achieved if S. laciniatum is anancient hybrid. This could be supported by the uniquemitochondrial rearrangement identified. This diploidhybrid speciation could result from a normal sexual eventwhere each gamete has a haploid complement of thenuclear chromosomes from its parent, but gametes thatform the zygote come from different species (in this casefrom S. vescum and S. aviculare). From the crossesreported by Baylis (1963) it is known that partial fertilityexists between S. vescum as female parent and S. aviculare,and backcrossing is often possible, like in the typicalcase of diploid hybrid speciation. However, this hypothesiscould not explain the extensive genomic homology detectedbetween S. aviculare and S. laciniatum. In the data setgenerated by the selected RAPD primers the number ofpolymorphic bands was very low, which was also reportedin our previous analysis with a different set of primers(Poczai 2007; Poczai et al. 2008).Another alternative hypothesis might be that S. laciniatumwas developed through autopolyploid formation,where the normal genome of S. aviculare is duplicated inits entirety and produced tetraploid offspring which werepostzygotically isolated from their parent. Crosses madebetween S. aviculare and S. laciniatum led to very fewseeds, and no successful germination could be detected(Gerasimenko 1969). This hypothesis could be an explanationfor the lack of additive bands in S. laciniatum fromS. vescum and S. aviculare, in the RAPD and cpDNAregion PCR-RFLP profile, and it also would be a reason forthe presence of the high genetic similarity betweenS. aviculare and S. laciniatum. However, the latterhypothesis suggesting an autopolyploid speciation eventfor S. laciniatum seems considerably more reasonable thanhybrid introgression through S. vescum.To clarify the relationship of S. laciniatum to the othermembers of the group will require much intensive phylogeneticanalysis. If S. laciniatum resulted from an ancienthybridization, additional evidence on the hybrid origincould be provided with particular analysis of DNAsequences. To resolve these events, reticulate evolutionshould be taken into consideration for further investigationsand cytogenetic analysis should also be carried out toresolve the origin of S. laciniatum. In addition, improvedtaxonomic sampling and/or use of more sensitive markerswould be required for more comprehensive understandingof the evolutionary history of S. laciniatum/S. aviculare.Such study based on different DNA fingerprinting markersand sequences is underway and will be summarized in acompanion paper.Preliminary phylogeny constructed from the crossessummarized by Symon (1994) and based on morphologysuggests S. multivenosum as a possible parent of S. laciniatum.However, the role of S. multivenosum in this phylogeneticconcept remains uncertain, because the presentstudy did not include samples from S. multivenosum. Thistaxon must be included in further investigations on thephylogenetic relationships in subg. Archaesolanum inorder to clarify its position with respect to S. aviculare,S. laciniatum, and S. vescum. The species S. multivenosumis endemic in high-altitude ([2,000 m) sites in Papua NewGuinea, so access to good material is very difficult. Possiblyfor this reason, no plant material or herbarium specimenwas recorded in the Solanaceae Source, a globalproject for taxonomy, or by the Botanical and ExperimentalGarden of Radboud University Nijmegen, which iswhy it was not included in the present analysis.S. linearifolium formed a basal branch of the clustercomposed of S. aviculare, S. laciniatum, and S. vescum inall the trees. Based on this topology, its closest relative isS. vescum, but the two accessions did not form a separategroup. This relationship can be explained by morphologicalparameters. The well-developed sinus tissues are diagnosticin the case of S. linearifolium, and the strongly wingedstems (from the sessile, decurrent leaves) in the case ofS. vescum (Baylis 1954).The other clade separated in the dendrogram is composedof S. capsiciforme, S. symonii, and S. simile (twoaccessions). These species belong to ser. Similia. Theseparation of this group based on morphology was confirmedby data derived from the present investigation usingmolecular genetic markers. S. symonii is often confusedwith S. simile, as the habit, green fruits, and small flowersof the two species are very similar. This morphologicalsimilarity between the two species can be detected at DNAlevel according to the RAPD data. In this dendrogram thetwo species form a group together, which is supported bystrong bootstrap values of 100%. Despite the morphologicalsimilarity, the chromosome numbers of the two speciesare not the same; S. symonii is tetraploid (2n = 4x = 92),whereas S. simile is diploid (2n = 2x = 46). Crossesbetween S. simile and S. symonii and the other members ofthe subgenus have been made, but no fertile hybrids couldbe obtained (Baylis 1963).S. capsiciforme is sister to all the other members of thisgroup in Symon’s (1994) preliminary phylogeny based on123

Genetic diversity and relationships in Solanum subg. Archaesolanum 45morphology. In the present study it occupied a distinctiveplace in the cluster composed of members of ser. Similia.In the analysis of the restriction patterns of the two chloroplastregion trnS-trnG it formed a group with S. symonii.However, the RAPD data separated S. capsiciformefrom the members of the ser. Similia, and S. symonii isgrouped together with the two accessions of S. simile. Thisdifference between the results can be explained by thedifferent nature of the two methods. RAPD amplifiesfragments from the whole genome, but mostly from thenuclear genome, while the restriction analysis in this studyfocused on specific regions of the chloroplast genome,detecting site variations, in the light of which S. symonii ismore closely related to S. simile at the nuclear genomiclevel than to S. capsiciforme. However, there is evidencefrom the trnS-trnG restriction site variation that bothS. symonii and S. capsiciforme lack a cleavage site of theRsaI endonuclease enzyme. This indicates that S. capsiciformeand S. symonii could share a common maternalancestor, but additional data will be required to prove thishypothesis.Series Similia can be easily distinguished morphologicallyfrom the other series of subg. Archaesolanum, and theresults of the present study show that this is confirmed bymolecular genetic methods. Such a clear distinction cannotbe made between the members of ser. Avicularia andLaciniata, which form a single clade in the RAPD data,while the same topology could be observed in the treeobtained from the chloroplast data. S. aviculare, the typespecies for ser. Avicularia, and S. laciniatum, the typespecies for ser. Laciniata, grouped together. From thistopology it is concluded that the existence of these twotaxonomic groups must be reconsidered. S. multivenosumshould also be included in further studies, and moremolecular data will be required to clarify the position of theAvicularia/Laciniata clade. New formal taxonomic designationsfor the series in subg. Archaesolanum will beneeded. It would be reasonable to rise the series to sectionallevel, since the Archaesolanum group is recognizedas a subgenus. As both the RAPD and cpDNA regionanalyses separated two groups, it is suggested to form twosections in subg. Archaesolanum. The first of these mightbe sect. Similia, consisting of former members of ser.Similia. As the Avicularia/Laciniata clade consists ofmembers from both ser. Avicularia and ser. Laciniata, anew section should also be formed by uniting these twogroups, which could be sect. Avicularia, since S. avicularewas the first name published by Forster (1786).Subgenus Archaesolanum is a unique group in thegenus. The chromosome structure hypothesized to bederived from secondary polyploidy and distinctive habitputs them in the focus of phylogenetic interest. This studyaimed to summarize information about the phylogeny ofthis exotic group and to use molecular genetic techniquesto provide more insight into the relationships between thespecies of the subgenus. Although implications based ongenetic relationships within this group have been formulated,some burning questions remain unanswered. Little isknown about within- and among-population geneticdiversity of each species, and the closest relatives of thegroups have even not been unambiguously identified.Analysis of divergence through time and the combinationof different phylogenetic methods capable of resolvingcomplex evolutionary events would result in certaininformation about the group’s radiation, dispersal, andphylogeny.Acknowledgments Thanks are due to Gerard M. van der Weerdenfor rapid seed transfer, to the workers of the Botanical and ExperimentalGarden of the Radboud University, Nijmegen, and to LindaMagyar, Kinga Mátyás, and István Cernak for their excellentassistance. This research represents a partial fulfillment of therequirements for the degree of Doctor of Philosophy (PhD) in PlantGenetics and Biotechnology at the University of Pannonia. 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