ORIGINAL ARTICLE

Genetic diversity and relationships in Solanum subg.Archaesolanum (Solanaceae) based on RAPD and chloroplastPCR-RFLP analyses

Peter Poczai • Andras Cseh • Janos Taller •

David E. Symon

Received: 13 October 2008 / Accepted: 4 October 2010

� Springer-Verlag 2010

Abstract The subgenus Archaesolanum is a group

composed of eight species with a characteristic chromo-

some number based on n = x = 23 and an area restricted

to the South Pacific. This subgenus is an isolated group of

Solanum for which extensive information about phyloge-

netic relationships based on molecular genetic methods is

lacking. This study represents an approach to analyze

genetic relationships within this group. In this context,

seven species were examined using random amplified

polymorphic DNA (RAPD) markers. In further analysis,

the amplification products of two chloroplast regions

(trnS-trnG and rbcL) were studied with polymerase chain

reaction (PCR) restriction fragment length polymorphism

(RFLP) method. Screening for the presence of unique

mitochondrial rearrangements was also carried out using

universal mitochondrial primers for the detection of

fragment length polymorphisms. We identified two major

groups within the subgenus; one was composed of the

members of ser. Avicularia and Laciniata, while the other

was formed by species belonging to ser. Similia. It is

suggested that the taxonomic status of series within the

Archaesolanum clade should be revised. The hybrid origin

of S. laciniatum was also tested, and two hypotheses

regarding its phylogeny are assumed.

Keywords Archaesolanum � Phylogenetic relationships �Solanum � PCR-RFLP � RAPD � Kangaroo apples

Introduction

The diverse genus Solanum L., with approximately 1,400

species, has worldwide distribution, with center of diversity

in South America. The species belonging to subg.

Archaesolanum Bitter ex Marzell, often called kangaroo

apples, are described as a distinctive group with no obvious

close relatives. The subgenus is represented by eight spe-

cies, which occur only in the South West Pacific region

(New Guinea, Australia, Tasmania, and New Zealand). The

species are short-lived soft-wooded shrubs, 1–3 m tall,

becoming straggly with age, unarmed, glabrescent, with

large (up to 30 cm) deeply lobed leaves in the juvenile

phase, becoming smaller (up to 10 cm) and entire in the

adult stage, with violet–purple flowers in cymes growing

at the axils of stem-fork sites (Symon 1984). The fruits

are greenish, yellowish, or scarlet; the succulent berries

produce numerous seeds (approx. 100–600). White or

yellowish stone cell aggregates are present in the dried

contents of the fruits, mixed with seeds. The fruits are

eaten by birds, which are probably responsible for their

P. Poczai (&) � J. Taller

Department of Plant Sciences and Biotechnology,

Georgikon Faculty, University of Pannonia, Festetics 7,

Keszthely 8360, Hungary

e-mail: guanine@ex1.georgikon.hu

J. Taller

e-mail: taller@ex1.georgikon.hu

A. Cseh

Department of Plant Genetic Resources and Organic Breeding,

Agricultural Research Institute of the Hungarian Academy

of Sciences, Brunszvik u. 2, Martonvasar 2462, Hungary

e-mail: andrascseh@gmail.com

D. E. Symon

Department of Environment and Heritage,

Plant Biodiversity Centre, State Herbarium of South Australia,

PO Box 2732, Kent Town, SA 5071, Australia

e-mail: symon.david@sa.gov.au

123

Plant Syst Evol

DOI 10.1007/s00606-010-0371-5

distribution throughout Australia, New Zealand, Tasmania,

and New Guinea (Symon 1979).

Plants belonging to this group were first collected by

Forster in Australia during the second voyage of Captain

James Cook. Forster (1786) was the first to publish the

name Solanum aviculare in the ‘‘Dissertatio inauguralis

botanico-medica de plantis esculentis insularum oceani

australis.’’ Since then, the group has been recognized by

several Solanum researchers, e.g., Bitter (1927), Danert

(1970), D’Arcy (1972, 1990), Symon (1981, 1994), and

Hunziker (2001). The subgenus was divided into three

series by Gerasimenko (1970): ser. Avicularia Geras.,

consisting of S. aviculare Forst. (2n = 2x = 46) and

S. multivenosum Symon (2n = 4x = 92); ser. Laciniata

Geras. composed of S. laciniatum Ait. (2n = 4x = 92),

S. vescum F. Muell (2n = 2x = 46), and S. linearifolium

Geras. 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. cheesemanii

Geras. and S. baylisii Geras. as separate species, but they

are now considered to be synonyms and varieties of

S. aviculare, as suggested by Baylis (1963).

The basic chromosome number in subg. Archaesolanum

is n = x = 23, in contrast to the n = x = 12 typical of

other members of genus Solanum (Randell and Symon

1976). Despite the name, suggesting an archetypal Sola-

num, the chromosome number indicates a derived condi-

tion which 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 based

on secondary gametic numbers are polyploid; in the case of

secondary polyploidy (Hair 1966) each number generates

its own polyploid sequence as follows: in the original

series, 2n = 48 (4x) and 72 (6x); in the derived series,

2n = 46 (2x2) (S. aviculare) and 2n = 92 (4x2) (S. lacin-

iatum) (data from Baylis 1954). Whilst these zygotic

numbers are ‘‘diploid’’ and ‘‘tetraploid’’ with respect to the

secondary basic number, x2 = 23, the respective species

are relatively tetraploid and octoploid in terms of the ori-

ginal basic number, x = 12 (Hair 1966).

Symon (1994) provided a preliminary phylogeny for

the subgenus based on morphology, but these concepts

have not been tested using molecular methods. To the

best of our knowledge, no study including all eight spe-

cies and investigating phylogenetic relationships within

the group using molecular tools has yet been published.

Many molecular studies on phylogenetic relationships

within the genus Solanum have included species repre-

senting the subgenus in their analysis. Olmstead and

Palmer (1997) included S. aviculare in an analysis

based on chloroplast restriction site data. Bohs and

Olmstead (2001) and Bohs (2005) gave information about

S. aviculare and S. laciniatum based on data from nuclear

internal transcribed spacers (ITS) and ndhF gene, which

indicated that the members of subg. Archaesolanum

formed a well-supported basal clade in Solanum. Using

RAPD, Poczai et al. (2008) also found that the group was

distinct from the other members of the genus. It thus

seems safe to say that the Archaesolanum clade represents

an isolated group whose closest relatives have not yet

been identified (Bohs 2005). Putative ancestors have

certainly not been recognized in Australia, nor are extra-

Australian relatives apparent (Symon 1979). Although

red-fruited species, such as S. dunalianum Gaudich.,

S. viride Spreng., and S. incanoalabastrum Symon, occur

in New Guinea, these all have stellate hairs and no stone

cells; 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 to

the separation of South America and Australia (Olmstead

and Palmer 1997). The ndhF data of Bohs and Olmstead

(1997) suggest that the ancestor of the Archaesolanum

clade arrived in Australia early in the evolutionary radi-

ation of Solanum.

Random amplified polymorphic DNA (RAPD) tech-

nique (Williams et al. 1990, 1993; Welsh and McClelland

1990) is a useful tool in population and evolutionary

genetics, since no prior knowledge of the genome structure

or sequence data is required. RAPD has been used in

several studies in the case of Solanum (e.g., Stedje and

Bukenya-Ziraba 2003; Van den Berg et al. 2002; Spooner

et al. 1996; Miller and Spooner 1999) to clarify phyloge-

netic relationships at various levels (Sheng et al. 2006;

Baeza et al. 2007; Liebst 2008).

Organelle DNA sequences have been used extensively

in phylogenetic studies on plants. Chloroplast DNA

(cpDNA) variation has proven to be immensely valuable in

reconstructing phylogenies at the species and higher taxo-

nomic level. PCR amplification with specific or universal

primers, followed by restriction digestion and electropho-

retic separation of the fragments (PCR-RFLP), is fre-

quently applied in plant phylogenetic studies (e.g., Cseh

and Taller 2008; Friesen et al. 1997; Wachowiak et al.

2006; Prentice et al. 2008). The combination of this tech-

nique with other molecular methods such as inter-simple

sequence repeat (ISSR) (Huang et al. 2002), RAPD

(Potokina et al. 1999), and amplified fragment length

polymorphism (AFLP) (Fu et al. 2004) is also widespread.

Compared with standard RFLP analysis using entire

cpDNA or probe cpDNAs, RFLP analysis of PCR-ampli-

fied cpDNA regions has several advantages: simple

procedure, small amounts of tissue required, and reduced

time and expense. Therefore, it is considered to be an easy

and advantageous tool for detecting cpDNA variations.

P. Poczai et al.

123

Another approach for extracting phylogenetic informa-

tion from both cpDNA and mitochondrial DNA (mtDNA)

is to analyze the distribution of major structural rear-

rangements. The mitochondrial genome evolves consider-

ably more slowly at the nucleotide sequence level than the

nuclear or the chloroplast genomes (Palmer 1990; Wolfe

et al. 1987), but the rate of rearrangements is extraordi-

narily faster in plant mtDNA than in cpDNA (Palmer and

Herborn 1988), possibly making it useful in investigations

on distant phylogenetic relationships. The presence or

absence of rearrangements in a particular gene or intron

can be assayed by hybridization using probes specific to the

gene/intron or with the use of the PCR technique. Primers

are synthesized for conserved sequences flanking the

region of interest, and the intervening sequence is amplified

by PCR (Downie and Palmer 1992). Comparing the size of

the resultant PCR product with a sequence of known length

on an agarose or polyacrylamide gel can indicate the

presence or absence of specific gene or intron rearrange-

ments (Bruzdzinski and Gelehrter 1989).

The purpose of this study is to investigate genetic

relationships in subg. Archaesolanum based on RAPD

markers and PCR-RFLP analysis of two chloroplast

regions (trnS-trnG and rbcL). Another objective of this

study is to reveal mitochondrial rearrangements using

universal primers, which could be useful for further anal-

ysis of the group.

Materials and methods

Plant material and DNA extraction

Taxon sampling included seven species belonging to subg.

Archaesolanum, with two accessions from each of the

species S. aviculare, S. laciniatum, and S. simile and one

from each of S. linearifolium, S. capsiciforme, S. symonii,

and S. vescum. For outgroups, seven Solanum species,

representing different subgenera, were included in the

analysis. An accession from outside the genus, Capsicum

annuum, was also added in the experiments, according to

the results reported by Olmstead et al. (1999) and Bohs and

Olmsted (2001). Although we used only one accession for

each species in this study, our ongoing continuous studies

within different lineages of Solanum show that intraspecific

variation does not adversely affect phylogenetic relation-

ships between sections of Solanum, as was also shown

previously in other groups of the genus by Spooner and

Systma (1992). Voucher specimens were deposited at the

herbarium of University of Pannonia, Keszthely, Hungary.

Information about the accessions can be found in Table 1.

Genomic DNA was extracted from approximately 50 mg

of young fresh leaves using the procedure of Walbot and

Warren (1988). RAPD fingerprints were obtained from DNA

bulks, according to Spooner et al. (1997), where five plants

from each accession were bulked for DNA extraction.

Although fragments present in \15% of individuals com-

posing the DNA bulk are often observed to be lost from the

banding patterns of bulked samples (e.g., Divaret et al.

1999), we designed our study to sample as many alleles as

possible within the accessions. Additionally, the aim was to

examine more populations, rather than more individuals,

within a population. Thus the bulking strategy described by

Michelmore et al. (1991) was considered to be useful to

generate a group (e.g., population or accession) fingerprint

by combining DNA from a number of individuals. This

strategy may reduce the noise in the dataset due to markers

segregating within the groups (Bussel et al. 2005) and has

been used successfully in the case of Solanum species (Miller

and Spooner 1999; Rodrıguez and Spooner 1997; Spooner

et al. 1991, 1993, 1995, 1997; Clausen and Spooner 1998).

RAPD amplification

In the RAPD analysis a total of 40 primer pairs were used.

Each reaction was performed twice to verify reproducibil-

ity. The primers were paired arbitrarily, but palindromes

and complementarities within and between primers were

avoided. The sequence of each primer was generated ran-

domly, comprising 12 base oligonucleotides and *50–70%

GC content. The sequences of the primers are available

from the corresponding author upon request. PCR was

carried 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, Lithua-

nia), 2 ll 109 PCR buffer (1 mM Tris–HCl, pH 8.8 at

25�C, 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton

X-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, and

2 min at 72�C. A final amplification for 5 min at 72�C was

applied. 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 gels

were documented using the GeneGenius Bio Imaging Sys-

tem (Syngene, UK). The banding patterns were evaluated

and annotated with the program GeneTools (Syngene, UK).

Chloroplast region amplification and restriction

digestion

trnS-trnG region

The chloroplast intergenic spacer between trnS and trnG

was amplified using the primers described by Hamilton

Genetic diversity and relationships in Solanum subg. Archaesolanum

123

(1999). All PCR reactions were performed in a Master-

Cycler ep384 (Eppendorf, Germany) with the same com-

position as in the RAPD analysis, except that the MgCl2concentration was adjusted to 2 mM. The thermal cycler

program 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 for

1 min; with a final extension at 72�C for 7 min, as

described by Levin et al. (2006).

rbcL1-rbcL2 region

The sequence of the large subunit of the ribulose-1,5-bis-

phosphate carboxylase gene (rbcL) was amplified using the

primers described by Demesure et al. (1995). The 20 ll

reaction solution was the same as that described above. The

thermal 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 35

cycles; and a final cycle of 4 min at 72�C. Further infor-

mation about the primers used in the study is given in

Table 2.

The trnS-trnG amplification products were digested

with the restriction endonuclease enzymes HinfI, DdeI,

MboI, MspI, RsaI, TaqaI, and AluI (New England Biolabs

Inc., USA). The reaction conditions recommended by the

supplier were used for all enzymes. The restriction frag-

ments were separated on 2.3% high-resolution MetaPhor

agarose gel (Cambrex Bio Science Rockland, Inc., USA),

after which the products were visualized by ethidium-

bromide staining.

The rbcL1-rbcL2 region was digested with the same

enzymes except that instead of TaqaI the AluI and HhaI

restriction endonucleases were used in the analysis. The

Table 1 Accessions of Solanum species used in the analysis

Taxonomic positionb Taxon Collector Accession number Collection

locality

Origin

Ingroup

Subg. Archaesolanum

Ser. Avicularia Geras. S. aviculare (1) Forst. Unknown 874 750 027 A I.

S. aviculare (2) Forst. var.

latifolium Bayl.

GTS Baylis 844 750 003 NZ I.

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.

Outgroup

Subg. Leptostemonum

Sect. 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. Minon

Sect. Bravantherum S. abutiloides Bitter & Lillo Unknown UHBG211-1455 Unknown II.

Subg. Potatoe

Sect. Dulcamara S. dulcamara L. P Poczai HU1GEO20060017 H III.

Subg. Solanum

Sect. Solanum S. americanum Miller. BG Redwood 904 750 023 USA I.

S. physalifolium Rusby var.

nitidibaccatum (Bitter) Edm.

Unknown 894 750 076 G I.

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)

P. Poczai et al.

123

separation and visualization procedure was the same as that

described above. The enzymes for the analysis were

selected based on the virtual digestion of the sequence data

of the fragment trnS-trnG from S. aviculare, submitted to

the NCBI database by Levin et al. (2005) under accession

number AY555458. For this procedure the program

NEBcutter (Vincze et al. 2003) was used.

Mitochondrial region amplification

The universal primers described by Demesure et al. (1995)

for the amplification of different mitochondrial regions

were tested to detect fragment length polymorphism

between the accessions. The contents and concentrations

used in the reaction mixture were the same as described for

the 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 at

72�C for 35 cycles; and a final cycle of 4 min at 72�C.

The amplified regions and further information about the

primers are summarized in Table 2.

RAPD data analysis

Only distinct, well-resolved, clear bands were scored.

Reliable bands are thought to refer to amplicons found in

replicate reactions. It was assumed that fragments of

equal length had been amplified from corresponding loci,

and the band conventionally assumed to represent a sin-

gle, dominant, nuclear locus with two possible alleles.

The amplified fragments were scored as: 1, for the pres-

ence, or 0, for the absence of homologous bands. From

this binary matrix, a distance matrix was computed

according to Nei and Li (1979) based on Dice’s similarity

coefficient (Dice 1945). A dendrogram was constructed

using the neighbor-joining method described by Saitou

and Nei (1987); the original matrix was bootstrapped

Table 2 Details of the primers used in the study of chloroplast and mitochondrial regions

Genea Primer pairs Size

(bp)

G ? C

(%)

Annealing

temperature (�C)

PCR

product (bp)

Chloroplast

primers

trnS-trnG trnS 50-GCCGCTTTAGTCCACTCAGC-30 20 60 52 *700–735

trnG 30-CACCATTTTCACACTAAGCAAG-50 22 41

rbcL1-rbcL2 rbcL1 50-ATGTCACCACCACAAACAGAGACT-30 24 46 60 *1,371

rbcL2 30-CCTCAGGACTTGATCGACGACGAACACTTC-50 31 52

Mitochondrial

primers

atp6F-atp6R atp6Fb 50-GGAGG(A=I)GGAAA(C=I)TCAGT(A=I)CCAA-30 22 48 58 *589–610

atp6R 30-TAGCATCATTCAAGTAAATACA-50 22 27

cobF-cobR cobF 50-AGTTATTGGTGGGGGTTCGG-30 20 55 58 *290–313

cobR 30-CCCCAAAAGCTCATCTGACCCC-50 22 59

cox1F-cox1R cox1Fb 50-GGTGCCATTGC(T=I)GGAGTGATGG-30 22 59 58 *1,466

cox1R 30-TGGAAGTTCTTCAAAAGTATG-50 21 33

nad3F-nad3R nad3F 50-AATTGTCGGCCTACGAATGTG-30 21 48 58 *237

nad3R 30-TTCATAGAGAAATCCAATCGT-50 21 33

nad5aF-nad5aR nad5aF 50-GAAATGTTTGATGCTTCTTGGG-30 22 41 58 *1,000

nad5aR 30-ACCAACATTGGCATAAAAAAAGT-50 23 30

nad5dF-nad5dR nad5dF 50-ATAAGTCAACTTCAAAGTGGA-30 21 33 58 *1,095–1,136

nad5dR 30-CATTGCAAAGGCATAATGAT-50 20 35

rps14F-rps14R rps14F 50-ATACGAGATCACAAACGTAGA-30 21 38 58 *114

rps14Rb 30-CCAAGACGATTT(C=I)TTTATGCC-50 21 38

nad4exon1-nad4exon2a nad4exon1 50-CAGTGGGTTGGTCTGGTATG-30 20 55 58 *2,058

nad4exon2a 30-TCATATGGGCTACTGAGGAG-50 20 50

a trnS-trnG, intergenic spacer between Ser-tRNA and Gly-tRNA; rbcL1-rbcL2, subunit of the ribulose-1,5-bisphosphate carboxylase gene; atp6 (or

atpF), F0-ATPase subunit 6 gene; cob, apocytochrome b gene; cox1 (or coxI), cytochrome c oxidase subunit 1 gene; nad3 (or nadC, nadhC, nadh3, or

nd3), NADH-ubiquinone oxidoreductase subunit 3 gene; nad5a (or nadF, ndhF, ndh5, nd5), NADH-ubiquinone oxidoreductase subunit 5 gene (intron

1); 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 sequences

Genetic diversity and relationships in Solanum subg. Archaesolanum

123

1,000 times to check the reliability of the branching

patterns and the quality of the resulting phylogenetic

groups. These bootstrap values are shown at the nodes of

the dendrogram as percentages. The FAMD program

(Schluter and Harris 2006) was used for all calculations.

The tree obtained using FAMD was visualized and edited

using the TreeView program (Page 1996).

Parsimony analysis of the restriction fragments

All calculations were carried out using the program pack-

age PHYLIP (Phylogeny Inference Package) published by

Felsenstein (1989). The two data sets (trnS-trnG and

rbcL1-rbcL2) were analyzed separately and in combina-

tion. The discrete character data of the restriction frag-

ments were coded into a series of (0 or 1) two-state

characters. The further analysis was carried out using the

branch-and-bound algorithm of the DOLPENNY program

to find all of the most parsimonious trees implied by the

data. The data analysis was performed with the use of the

Dollo parsimony method. The program was set to report

every 100 trees and 1,000 groups. From the resulting out-

put trees, a consensus tree was built with the CONSENSE

program using the majority rule criterion.

Results

RAPD analysis

The 40 RAPD primer pairs generated 295 reliable frag-

ments from all the accessions analyzed. RAPD analysis

using primer combinations clearly separated the accessions

used in the study. The dendrogram calculated from the data

matrix generated by the formula of Nei and Li (1979) is

presented in Fig. 1. We tried to root the resulting tree by

additionally adding/removing groups and taxa used as

outgroups. In all cases a consistently distinct Archaesola-

num clade was present among the analyzed accessions,

where no significant difference was found in the overall

topology of the groups within the Archaesolanum clade.

The dendrograms in all rooted versions consistently pro-

duced two separate clades; one was composed of the

members of ser. Avicularia and Laciniata, namely two

accessions of S. aviculare, two accessions of S. laciniatum,

S. vescum, and S. linearifolium, which exhibit close affin-

ity based on the bootstrap values. S. vescum is sister to this

group composed of S. aviculare and S. laciniatum; S. lin-

earifolium occupies a basal position in this clade. The

second major clade was formed by members of ser. Similia.

S. capsiciforme is separated from the other members of the

group. S. symonii and the two accessions of S. simile are

grouped together.

Chloroplast and mitochondrial region analysis

One amplification product per sample was obtained for all

the accessions examined. The size of the fragments

amplified with trnS-trnG primers was approximately

710 bp among the species. The size of the rbcL1-rbcL2

fragments was approximately 1,370 bp and showed no

variation. Restriction enzyme assay with seven different

enzymes resulted in variable fragments in the case of the

trnS-trnG region. Approximately 10 bp differences were

observed between the restriction fragments. The PCR-

RFLP fragments of the region clearly separate two groups

in the subgenus. S. laciniatum, S. aviculare, and S. vescum

compose a group with larger fragments, while S. simile,

S. symonii, and S. capsiciforme gave smaller restriction

products. S. linearifolium occupied an ‘‘intermediate’’

position between the two groups according to the fragment

lengths of the PCR-RFLP analysis. An example is shown in

Fig. 2. The observed variability in the restriction products

indicates that there are differences between the Archaeso-

lanum species in the trnS-trnG region (Table 3).

The rbcL1-rbcL2 region was not informative in the case

of subg. Archaesolanum. After digestion with six restric-

tion endonuclease enzymes, no polymorphism was

detectable. However, the selected enzymes had 4–9

restriction sites in the 1,370-bp sequence. This similarity of

the Archaesolanum species and their difference from the

outgroups indicates that, although this region is uninfor-

mative at the subgeneric level, it could be useful in higher

taxonomic analyses. As the rbcL region was not informa-

tive in the present case, it is not discussed in the following.

Eight mitochondrial regions were amplified to detect

different fragment length patterns which could be attrib-

uted to the unique replication of the mitochondrial genome.

For this reason the universal primers described by Deme-

sure et al. (1995) were used, to reveal this type of variation.

The size of the PCR products and the sequence of the

primers are given in Table 2. Polymorphic fragments were

detected with the use of nad5aF-nad5aR. These primers are

designed for the amplification of an intron between exon 1

(nad5aF) and exon 2 (nad5aR) of the nicotinamide adenine

dinucleotide (NADH)-ubiquinone oxidoreductase sub-

unit 5 gene. While all the species had a 1,000-bp fragment,

in the case of S. vescum, S. laciniatum, and S. linearifoli-

um an *690-bp size band was also detectable. Beside

these, S. linearifolium also had a unique *880-bp frag-

ment (Fig. 3).

Results of the parsimony analysis

The phylogenetic tree obtained from the analysis of the

consensus tree construction is given in Fig. 4. The topology

of the tree constructed from the chloroplast region

P. Poczai et al.

123

restriction data is almost identical to the tree topology of

the RAPD data. The major difference is that S. symonii and

S. capsiciforme formed a group together in the case of the

chloroplast region. This arrangement was due to the

absence of a restriction site with RsaI in the trnS-trnG

sequence unique for these species (Fig. 5).

Fig. 1 Dendrogram constructed

using the neighbor-joining (NJ)

method from the RAPD data

matrix calculated with the

formula given by Nei and Li

(1979). The numbers at the tree

nodes are bootstrap values as a

percentage

Fig. 2 Restriction patterns of

the trnS-trnG chloroplast

region, digestion with MboI.

M Molecular weight size marker

(100–1,000 bp). 1S. linearifolium; 2 S. aviculare(1), 3 S. aviculare (2); 4 S.vescum; 5 S. laciniatum (1); 6S. laciniatum (2); 7S. capsiciforme; 8 S. symonii; 9S. simile (1); 10 S. simile (2)

Genetic diversity and relationships in Solanum subg. Archaesolanum

123

Discussion

This study represents an approach using genomic DNA

fingerprint markers and other methods to study genetic

relationships in subg. Archaesolanum. The RAPD data

obtained in this study are a random sample, representing all

the polymorphic RAPDs in the germplasm examined.

RAPDs have been shown to be useful as taxonomic

markers for closely related species through concordant

results using other molecular marker systems for closely

related taxa elsewhere in sect. Petota (Cisneros and Quiros

1995; Spooner et al. 1996). In addition, the results indicate

that S. aviculare and S. laciniatum are closely related,

which is strongly supported by bootstrap values. S. avicu-

lare is easily confused with S. laciniatum. Previously

S. laciniatum was treated as a variety of S. aviculare under

the name S. aviculare var. laciniatum (Aiton) Domin. Both

species were commonly cultivated in the former USSR,

Australia, and New Zealand, and they were used in the

alkaloid industry in the 1970s (Knapp 2006). Although

they are difficult to distinguish, there are some morpho-

logical parameters in which they differ. S. aviculare is

diploid (2n = 2x = 46) with bright-orange or red mature

fruits containing approx. 600 seeds per fruit, while

S. laciniatum is tetraploid (2n = 4x = 92), with fruits that

are first green, later paling to yellow or orange-yellow, and

containing approx. 200 seeds per fruit. Baylis (1954)

clearly describes other characteristics: S. laciniatum has

larger pollen grains, flowers, seeds, and stone-cell masses

(in the fruit pulp) than S. aviculare; its corolla is deeper in

color with relatively shallow lobes, the margins of which

flatten more completely, producing an emarginated apex.

The analyses carried out provide results in re-examining

the putative hybrid origin of S. laciniatum, indicating that

it is not of recent hybrid origin. There is a lack of additivity

both in the RAPD patterns and in the PCR-RFLP analysis.

In the dendrogram constructed from RAPD data, S. lacin-

iatum forms a group together with S. aviculare and

S. vescum, and the same topology is present in the tree

Table 3 Restriction patterns of trnS-trnG; number of fragments

obtained in the analysis

Accession Restriction enzyme

HinfI DdeI MboI MspI RsaI TaqaI AluI

Ingroup

S. aviculare (1) 2 2 2 2 3 5 4

S. aviculare (2) 2 2 2 2 3 5 4

S. capsiciforme 2 2 2 2 2 4 3

S. laciniatum (1) 2 2 2 2 3 5 3

S. laciniatum (2) 2 2 2 2 3 5 3

S. linearifolium 2 2 2 2 3 5 4

S. simile (1) 2 2 2 2 3 4 4

S. simile (2) 2 2 2 2 3 4 4

S. symonii 2 2 2 2 2 4 3

S. vescum 2 2 2 2 3 5 4

Outgroup

S. abutiloides 2 3 0 2 3 4 2

S. americanum 2 3 2 2 2 3 2

S. atropurpureum 2 2 0 0 2 5 0

S. dulcamara 2 2 2 2 3 4 2

S. physalifolium 2 3 2 2 3 4 2

S. rostratum 2 2 0 2 3 6 3

S. sisymbriifolium 2 2 0 2 3 4 3

C. annuum 2 2 2 2 3 3 2

Fig. 3 Amplification products

of nad5aF-nad5aR.

M Molecular weight size marker

(100 bp), the numbers indicate

ladder 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 annuum

P. Poczai et al.

123

from PCR-RFLP patterns. This topology is in agreement

with the crosses summarized by Symon (1994), who sug-

gested that S. laciniatum may be a hybrid of S. aviculare

and S. vescum. S. aviculare and S. vescum hybridize

spontaneously when the species are grown next to each

other. The hybrids are only fertile when S. vescum is

involved as the female parent (Baylis 1963).

The presence of a specific fragment obtained from the

amplification of the mtDNA region nad5aF-nad5aR also

confirms these results. This fragment is present in S. ve-

scum, the hypothetical female parent, but it is absent from

S. aviculare, the putative male parent of S. laciniatum.

According to the maternal inheritance of organelle gen-

omes, this fragment in S. vescum and in S. laciniatum

Fig. 4 Majority rule consensus

tree based on the PCR-RFLP

fragments obtained from the

two chloroplast regions and

mitochondrial fragment length

polymorphisms, showing

phylogenetic relationships and

clades in subg. Archaesolanum.

The numbers at the nodes are

bootstrap replicates as a

percentage

Fig. 5 Restriction patterns of

the trnS-trnG chloroplast

region, 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)

Genetic diversity and relationships in Solanum subg. Archaesolanum

123

represents a unique mitochondrial structure, which sup-

ports the hybridization theory. Although this type of

mitochondrial structure is present in S. linearifolium too, it

separates from them, forming another fragment of

approximately 880 bp. This fragment could be a promising

item for further analysis of the subgenus.

Hybrid speciation could occur at least two ways in this

case: It is possible that S. laciniatum is developed through

diploid hybrid speciation involving S. aviculare and

S. vescum as female parent, and than the entire genome is

duplicated through autopolyploidy. This alternative

hybridization can only be achieved if S. laciniatum is an

ancient hybrid. This could be supported by the unique

mitochondrial rearrangement identified. This diploid

hybrid speciation could result from a normal sexual event

where each gamete has a haploid complement of the

nuclear chromosomes from its parent, but gametes that

form the zygote come from different species (in this case

from S. vescum and S. aviculare). From the crosses

reported by Baylis (1963) it is known that partial fertility

exists between S. vescum as female parent and S. avicu-

lare, and backcrossing is often possible, like in the typical

case of diploid hybrid speciation. However, this hypothesis

could not explain the extensive genomic homology detec-

ted between S. aviculare and S. laciniatum. In the data set

generated by the selected RAPD primers the number of

polymorphic bands was very low, which was also reported

in our previous analysis with a different set of primers

(Poczai 2007; Poczai et al. 2008).

Another alternative hypothesis might be that S. lacini-

atum was developed through autopolyploid formation,

where the normal genome of S. aviculare is duplicated in

its entirety and produced tetraploid offspring which were

postzygotically isolated from their parent. Crosses made

between S. aviculare and S. laciniatum led to very few

seeds, and no successful germination could be detected

(Gerasimenko 1969). This hypothesis could be an expla-

nation for the lack of additive bands in S. laciniatum from

S. vescum and S. aviculare, in the RAPD and cpDNA

region PCR-RFLP profile, and it also would be a reason for

the presence of the high genetic similarity between

S. aviculare and S. laciniatum. However, the latter

hypothesis suggesting an autopolyploid speciation event

for S. laciniatum seems considerably more reasonable than

hybrid introgression through S. vescum.

To clarify the relationship of S. laciniatum to the other

members of the group will require much intensive phylo-

genetic analysis. If S. laciniatum resulted from an ancient

hybridization, additional evidence on the hybrid origin

could be provided with particular analysis of DNA

sequences. To resolve these events, reticulate evolution

should be taken into consideration for further investiga-

tions and cytogenetic analysis should also be carried out to

resolve the origin of S. laciniatum. In addition, improved

taxonomic sampling and/or use of more sensitive markers

would be required for more comprehensive understanding

of the evolutionary history of S. laciniatum/S. aviculare.

Such study based on different DNA fingerprinting markers

and sequences is underway and will be summarized in a

companion paper.

Preliminary phylogeny constructed from the crosses

summarized by Symon (1994) and based on morphology

suggests S. multivenosum as a possible parent of S. lacin-

iatum. However, the role of S. multivenosum in this phy-

logenetic concept remains uncertain, because the present

study did not include samples from S. multivenosum. This

taxon must be included in further investigations on the

phylogenetic relationships in subg. Archaesolanum in

order to clarify its position with respect to S. aviculare,

S. laciniatum, and S. vescum. The species S. multivenosum

is endemic in high-altitude ([2,000 m) sites in Papua New

Guinea, so access to good material is very difficult. Pos-

sibly for this reason, no plant material or herbarium spec-

imen was recorded in the Solanaceae Source, a global

project for taxonomy, or by the Botanical and Experi-

mental Garden of Radboud University Nijmegen, which is

why it was not included in the present analysis.

S. linearifolium formed a basal branch of the cluster

composed of S. aviculare, S. laciniatum, and S. vescum in

all the trees. Based on this topology, its closest relative is

S. vescum, but the two accessions did not form a separate

group. This relationship can be explained by morphological

parameters. The well-developed sinus tissues are diagnos-

tic in the case of S. linearifolium, and the strongly winged

stems (from the sessile, decurrent leaves) in the case of

S. vescum (Baylis 1954).

The other clade separated in the dendrogram is com-

posed of S. capsiciforme, S. symonii, and S. simile (two

accessions). These species belong to ser. Similia. The

separation of this group based on morphology was con-

firmed by data derived from the present investigation using

molecular genetic markers. S. symonii is often confused

with S. simile, as the habit, green fruits, and small flowers

of the two species are very similar. This morphological

similarity between the two species can be detected at DNA

level according to the RAPD data. In this dendrogram the

two species form a group together, which is supported by

strong bootstrap values of 100%. Despite the morphologi-

cal similarity, the chromosome numbers of the two species

are not the same; S. symonii is tetraploid (2n = 4x = 92),

whereas S. simile is diploid (2n = 2x = 46). Crosses

between S. simile and S. symonii and the other members of

the subgenus have been made, but no fertile hybrids could

be obtained (Baylis 1963).

S. capsiciforme is sister to all the other members of this

group in Symon’s (1994) preliminary phylogeny based on

P. Poczai et al.

123

morphology. In the present study it occupied a distinctive

place in the cluster composed of members of ser. Similia.

In the analysis of the restriction patterns of the two chlo-

roplast region trnS-trnG it formed a group with S. symonii.

However, the RAPD data separated S. capsiciforme

from the members of the ser. Similia, and S. symonii is

grouped together with the two accessions of S. simile. This

difference between the results can be explained by the

different nature of the two methods. RAPD amplifies

fragments from the whole genome, but mostly from the

nuclear genome, while the restriction analysis in this study

focused on specific regions of the chloroplast genome,

detecting site variations, in the light of which S. symonii is

more closely related to S. simile at the nuclear genomic

level than to S. capsiciforme. However, there is evidence

from the trnS-trnG restriction site variation that both

S. symonii and S. capsiciforme lack a cleavage site of the

RsaI endonuclease enzyme. This indicates that S. capsici-

forme and S. symonii could share a common maternal

ancestor, but additional data will be required to prove this

hypothesis.

Series Similia can be easily distinguished morphologi-

cally from the other series of subg. Archaesolanum, and the

results of the present study show that this is confirmed by

molecular genetic methods. Such a clear distinction cannot

be made between the members of ser. Avicularia and

Laciniata, which form a single clade in the RAPD data,

while the same topology could be observed in the tree

obtained from the chloroplast data. S. aviculare, the type

species for ser. Avicularia, and S. laciniatum, the type

species for ser. Laciniata, grouped together. From this

topology it is concluded that the existence of these two

taxonomic groups must be reconsidered. S. multivenosum

should also be included in further studies, and more

molecular data will be required to clarify the position of the

Avicularia/Laciniata clade. New formal taxonomic desig-

nations for the series in subg. Archaesolanum will be

needed. It would be reasonable to rise the series to sec-

tional level, since the Archaesolanum group is recognized

as a subgenus. As both the RAPD and cpDNA region

analyses separated two groups, it is suggested to form two

sections in subg. Archaesolanum. The first of these might

be sect. Similia, consisting of former members of ser.

Similia. As the Avicularia/Laciniata clade consists of

members from both ser. Avicularia and ser. Laciniata, a

new section should also be formed by uniting these two

groups, which could be sect. Avicularia, since S. aviculare

was the first name published by Forster (1786).

Subgenus Archaesolanum is a unique group in the

genus. The chromosome structure hypothesized to be

derived from secondary polyploidy and distinctive habit

puts them in the focus of phylogenetic interest. This study

aimed to summarize information about the phylogeny of

this exotic group and to use molecular genetic techniques

to provide more insight into the relationships between the

species of the subgenus. Although implications based on

genetic relationships within this group have been formu-

lated, some burning questions remain unanswered. Little is

known about within- and among-population genetic

diversity of each species, and the closest relatives of the

groups have even not been unambiguously identified.

Analysis of divergence through time and the combination

of different phylogenetic methods capable of resolving

complex evolutionary events would result in certain

information about the group’s radiation, dispersal, and

phylogeny.

Acknowledgments Thanks are due to Gerard M. van der Weerden

for rapid seed transfer, to the workers of the Botanical and Exper-

imental Garden of the Radboud University, Nijmegen, and to Linda

Magyar, Kinga Matyas, and Istvan Cernak for their excellent

assistance. This research represents a partial fulfillment of the

requirements for the degree of Doctor of Philosophy (PhD) in Plant

Genetics and Biotechnology at the University of Pannonia. This

work was carried out in 2007 and it was supported by the Hungarian

State PhD Grant provided for the first author at the University of

Pannonia.

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