ORIGINAL ARTICLE

Molecular evolution of 35S rDNA and taxonomic statusof Lycopersicon within Solanum sect. Petota

Nataliya Y. Komarova Æ Guido W. Grimm ÆVera Hemleben Æ Roman A. Volkov

Received: 7 March 2008 / Accepted: 12 August 2008 / Published online: 18 September 2008

� Springer-Verlag 2008

Abstract To clarify the taxonomic status of tomatoes

(‘‘Lycopersicon’’) and their relationship to the members of

sect. Petota of genus Solanum L., organization of the

rDNA external transcribed spacer (50 ETS) was studied in

33 Solanum and ‘‘Lycopersicon’’ species. Phylogenetic

reconstruction revealed that three major groups can be

distinguished. Non-tuber-bearing species of ser. Etuberosa

as well as tuber-bearing Central American diploids

appeared as a paraphyletic group. The first of two well-

defined clades embraced all tuber-bearing South American

species and Central American polyploids. The other clade

(named ‘‘tomato clade’’) contains non-tuber-bearing spe-

cies of ser. Juglandifolia and tomato species of ser.

Neolycopersicon, which appears to be imbedded in sect.

Petota. The new 50 ETS variant D characterized by a

cluster of downstream subrepeats is characteristic for the

tomato clade. The variant D originated directly from

the most ancestral variant A found in ser. Etuberosa and

the Central American diploids, whereas variants B and C

specific for the tuber-bearing South American species and

Central American polyploids represent a parallel lineage of

molecular evolution. The sequence analysis demonstrates

the existence of an evolutionary trend of parallel multi-

plication of specific motifs in 50 ETS in different groups of

sect. Petota.

Keywords External transcribed spacer � Networks �Phylogeny � Potato � Repeated sequence elements �Tomato

Introduction

The genus Solanum L. is by far the largest of the genera of

the family Solanaceae and is distributed worldwide

(D’Arcy 1991; Nee 1999). Members of the genus are of

wide economic significance and important models for

studies on structural and functional genomics. Clear

understanding of the phylogenetic relationships and evo-

lutionary trends within the genus is necessary to improve

the efficiency of breeding programs involving wild genetic

material, and for appropriate interpretation of the genomics

studies.

The taxonomic status of tomato and its relationship to the

species of Solanum remains a subject of debates, in par-

ticular in regard to the members of the sect. Petota Dumort.

In 1753 Linnaeus treated tomato and potato as members of

the genus Solanum. On the contrary, in 1768 Philip Miller

separated tomato into the genus Lycopersicon Mill. Later

phylogenetic relationships between these two groups of

plants were a subject of numerous studies based on com-

parison of morphology (Correll 1958; Rick 1988; Child

1990; Hawkes 1990; Child and Lester 1991; D’Arcy 1991),

N. Y. Komarova

Institute of Plant Sciences, University of Bern, Altenbergrain 21,

3013 Bern, Switzerland

G. W. Grimm

Institute of Geosciences, University of Tubingen, Sigwartstr. 10,

72076 Tubingen, Germany

V. Hemleben (&)

Department of General Genetics, ZMBP,

University of Tubingen, Auf der Morgenstelle 28,

72076 Tubingen, Germany

e-mail: vera.hemleben@zmbp.uni-tuebingen.de;

vera.hemleben@uni-tuebingen.de

R. A. Volkov

Department of Molecular Genetics and Biotechnology,

University of Chernivtsi, Kotsubinsky str. 2, 58012 Chernivtsi,

Ukraine

123

Plant Syst Evol (2008) 276:59–71

DOI 10.1007/s00606-008-0091-2

interspecific crossability (Menzel 1962; Rick 1969;

DeVerna et al. 1990; Matsubayashi 1991; Haider Ali

et al. 2001), distribution of repeated genome elements

(Schweizer et al. 1993; Stadler et al. 1995) and application

of molecular markers (Palmer and Zamir 1982; McClean

and Hanson 1986; Spooner et al. 1993; Peralta and Spooner

2001; Weese and Bohs 2007).

The 35S rDNA (nuclear locus coding for the 5.8S, 18S

and 25S rRNA; for review see Volkov et al. 2004) represent

a class of repeated sequences under control of concerted

evolution which is responsible for a high homogenization

level between repeats (Coen et al. 1982; Dover and Flavell

1984; Dvorak et al. 1987; Hemleben et al. 2000) as well as

between repeated elements within the same unit (Barker

et al. 1989; Schiebel et al. 1989; Volkov et al. 1996). The

high level of homogenization and an existence of regions

evolving with different rates make rDNA a very attractive

tool for molecular taxonomy and phylogeny. Regions cod-

ing for 18S, 5.8S and 25S rRNAs with the lowest rate of

evolution were successfully used for phylogenetic studies of

distantly related taxa (Lipscomb et al. 1998), whereas

comparison of more rapidly evolving internal transcribed

spacer regions (ITS1 and ITS2) were widely applied for

taxonomic reconstructions among members of the same or

closely related genera (Baldwin 1992; Grebenstein et al.

1998; Jobst et al. 1998; Goel et al. 2002; Grimm et al. 2005).

As far as studied sequence comparisons of the 50

external transcribed spacer (50 ETS) of 35S rDNA repre-

sent an even more powerful instrument for the

phylogenetic analyses at low taxonomic levels (King et al.

1993; Volkov et al. 1996; Baldwin and Marcos 1998; Bena

et al. 1998; Linder et al. 2000). Our recent studies of the

molecular evolution of the 5S and 35S rDNA and phy-

logeny of Solanum sect. Petota (Volkov et al. 2001, 2003)

revealed that the 50 ETS has a broad range of resolving

power allowing separation of major taxonomic groups

within the section as well as discrimination of closely

related species. In this study we analyzed the 50 ETS region

of eight wild and cultivated tomato species in order to

clarify their relationship to sect. Petota. Obtained results

also allowed us to find out common trends in the molecular

evolution of subrepeats in the 50 ETS of Solanum.

Materials and methods

Plant material and growth conditions

Seeds of the studied species were obtained from several

collections (Table 1). The seeds were germinated in the

dark at 21�C on MS-B5 (Duchefa, The Netherlands)

medium complemented with 10 g/l sucrose, 0.5 g/l MES,

1 g/l gibberellic acid, and 8.5 g/l plant agar, pH 5.7, with

the exception of S. nigrum (NIG) from which leaves were

collected in the field around Frankfurt-Main, Germany.

Before the germination seeds of S. cheesmanii (CHE),

S. sitiens (SIT) and S. lycopersicoides (LPD) were soaked

in 2.7% sodium hypochlorite for 30 min, thoroughly rinsed

Table 1 List of Solanum (including former Lycopersicon) species newly analyzed and accession numbers of IGS sequences obtained

Speciesa Abbreviation Plant accession nos. Sourceb Sequence accession nos.

S. lycopersicoides Dunal lpd LA2951 TGRC DQ118130

DQ118131

S. sitiens Johnston sit LA1974 TGRC DQ118128

DQ118129

S. cheesmaniae (Riley) Fosb.

[syn. Lycopersiconcheesmanii Riley]

che I

che II

LA0166

T675/88

TGRC

IPK

DQ118133

DQ118134

DQ118132

S. chmielewskii (Rick et al.)

Spooner et al. [syn. L.chmielewskii Rick et al.]

chm T1254/92 IPK DQ093388

DQ093389

S. hirsutum (Dunal) Macbride

[syn. L. hirsutum Dunal]

hir LYC 4/81 IPK DQ093390

DQ093391

S. pennellii Correll [syn. L.pennellii (Correll) D’Arcy]

pen SOL 165/85 IPK DQ093392

DQ093393

S. peruvianum L. [syn. L.peruvianum (L.) Mill.]

per LYC 3/88 IPK DQ093395

DQ093394

S. nigrum L. nig — TUB DQ248893

a Species names are shown according to Nee (1999)b IPK, the Institut fur Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany; TGRC, Tomato Genetics Resource Centre, Uni-

versity of California, Davis, USA; TUB, Herbarium Tubingense, University of Tubingen, Germany

60 N. Y. Komarova et al.

123

with sterile distillated water and dried during 6 days. The

treatment was repeated four times (C. M. Rick and F. H.

Borgnino, Department of Vegetable Crops, University of

California, Davis, personal communication). After germi-

nation, the plants were transferred to soil and grown under

light/dark conditions of 16/8 h at 24�C.

DNA isolation and restriction mapping

Genomic DNA was isolated from leaves using DNeasy

Plant kit (Qiagen, Valencia, CA). Restriction mapping of

rDNA was performed using BamHI, EcoRI and SspI

(Fermentas, Vilnus, Lithuania) according to Sambrook

et al. (1989). Two probes, Pro-18S (50 fragment of 18S

coding region of Nicotiana tomentosiformis) and Pro-25S

(30 fragment of 25S coding region of N. tomentosiformis),

were used for blot hybridization. Pro-25S was amplified by

PCR from rDNA clone pNtm-1 (Y08427; Volkov et al.

1999) using Hot-start Taq-polymerase (Qiagen) and a

sequence-specific primer pair. PCR products were purified

with Qiaquick PCR Purification kit (Qiagen). To prepare

Pro-18S, DNA of pNtm-1 was digested by XbaI and

BamHI. After electrophoretic separation the required

rDNA fragment was purified using Gel Band Purification

Kit (APBiotech, Piscataway, NJ). The fragments obtained

were used for generation of 32P-CTP labeled DNA probes

using Rediprime II Random Prime Labeling System

(Amersham Biosciences, Buckinghamshire, England).

Isolation, cloning and sequencing of rDNA spacer

region

Primers Pr1 and Pr2 (Volkov et al. 2003) deduced from the

sequences of the 25S and 18S rRNA coding regions of S.

tuberosum (TUB; Borisjuk and Hemleben 1993) were used

for PCR amplification of the complete intergenic spacer

(IGS) of Solanum species. PCR was performed with Tfl

DNA-Polymerase (Epicentre Technologies, Madison, WI)

applying the following program: initial DNA denaturation

at 94�C, 3 min; 10 cycles at 94�C, 1 min; 70�C, 4 min; 20

cycles at 94�C, 1 min; 70�C, 4 min ? 15 s/cycle; final

extension 72�C, 10 min. PCR products were either purified

with Qiaquick PCR Purification kit (Qiagen) or separated

in agarose gel, and bands of the appropriate size were

purified with Gel Band Purification Kit (APBiotech). To

check the identity of PCR products, restriction endonu-

cleases BamHI, EcoRI and SspI were used. Localization of

restriction enzyme recognition sites in the PCR products

was in agreement with those predicted from preliminary

Southern-blotting experiments, confirming that these PCR

products do really represent complete IGS.

Purified PCR products were directly sequenced using

several IGS specific primers (the list of primers is available

from the authors upon request) in combination with the Big

Dye Terminator Cycle Sequencing Kit and ABI Prism 310

sequencer (PE Applied Biosystems, Palo Alto, CA). For

cloning, PCR products were digested with NotI, ligated

into the corresponding site of pBlueScript KS(?) and

transformed into Escherichia coli strain XL-blue. Blue/

white colony selection was used for identification of

recombinant plasmids, and inserts of selected clones were

sequenced as mentioned above. The sequences obtained

appeared in the NCBI GenBank/EMBL nucleotide data-

base under the accession nos. listed in Table 1.

Sequence data analysis and phylogenetic reconstruction

Sequence alignment was obtained by CLUSTAL V method

(Higgins and Sharp 1989) applying MEGALIGN software

(DNASTAR 1998), and improved manually.

Maximum parsimony (MP), maximum likelihood (ML)

and neighbor-joining (NJ) phylograms were generated with

PAUP, version 4.0b10 (Swofford 2002). Under ML heu-

ristic searches were performed with stepwise addition, tree

bisection-reconnection (TBR) branch swapping and

‘‘MulTrees’’ option activated to find an optimal tree. Par-

simony analysis relied on ratchet analysis using a PRAP

generated command block (Muller 2004). A NJ phylogram

was computed with BioNJ algorithm and ML distances.

Gaps were treated as ‘‘missing’’. Node support was estab-

lished by non-parametric bootstrapping (BS; Felsenstein

1985) under MP and NJ with PAUP and posterior proba-

bilities (PP) using Bayesian analysis under ML with

MRBAYES 3.1 (Huelsenbeck and Ronquist 2001;

Ronquist and Huelsenbeck 2003). Bootstrap values were

obtained from 100,000 replicates (under MP ‘‘MulTrees’’

deactivated, single random addition tree as start; Muller

2005). Bayesian analysis used three parallel runs,

1,000,000 generations, one cold and three heated Monte–

Carlo Markov chains, and the tree of each 100th generation

was saved. Optimum was reached in two of the three

runs after 82,400 generations as identified by converging

log-likelihoods and relative stability of substitution

parameters. One run was stuck in a suboptimum and dis-

carded for computation of PP as well as the pre-optimum

saved trees.

The substitution model for maximum likelihood was

chosen using a hierarchal likelihood ratio test (hLRT) and

Akaike information criterion (AIC) of the models imple-

mented in MRMODELTEST 2.2 (Nylander 2004). AIC and

hLRT opted for a general time-reversible substitution model

allowing for gamma-distributed site variation (GTR ? C).

For ML tree reconstruction parameters were specified using

the AIC selected values by MRMODELTEST.

Neighbor-net (NN) splits graph analysis (Bryant and

Moulton 2002, 2004) implemented in SPLITSTREE

Molecular evolution of 35S rDNA and taxonomic status of Lycopersicon 61

123

version 4.6 (Huson and Bryant 2006; available at

http://www.splitstree.org) was used to infer the distribution

of incompatible splits based on uncorrected p-distances. In

addition, alternative splits supported by bootstrap replicates

and Bayesian inferred saved trees were visualized as split

networks (‘‘bipartition networks’’) with SPLITSTREE by

coding the partition table generated by PAUP and

MRBAYES as a split matrix. The frequency of each split

becomes an edge length in the bipartition network, and

contradictory splits can thereby be visualized (Grimm et al.

2006). Annotated NEXUS files used for analyses (PAUP,

MRBAYES, SPLITSTREE) can be supplied by the authors

upon request.

Results

Characterization and general organization of the IGS

region

In order to isolate the IGS region of 35S rDNA of Solanum

species of ser. Juglandifolia and Neolycopersicon (Table 1)

we have used primers located in the coding regions of the

25S and 18S rRNA. Regarding the very high level (more

than 99%) of intragenomic sequence similarity of indivi-

dual rDNA copies demonstrated recently for tomato

(S. lycopersicon, LYC) and several potato species (Volkov

et al. 2003; Komarova et al. 2004) we firstly applied direct

sequencing of the PCR products. For the species of ser.

Neolycopersicon a clearly readable sequence without any

visible internal heterogeneity was obtained for the complete

IGS except for the fragment between the 25S coding region

and upstream subrepeats in the IGS of S. peruvianum

(PER). However, for LPD and SIT of ser. Juglandifolia a

clearly readable sequence was obtained only for the 50 ETS

region. According, PCR products of these two species were

cloned and then sequenced. For each of these two species,

one recombinant plasmid was completely and several ones

partially sequenced for the IGS region. In the 50 ETS the

clones demonstrate more than 99% of similarity with the

corresponding sequences obtained by direct sequencing

indicating the high intensity of concerted evolution for the

rDNA region.

Analysis of the sequences showed that in the IGS of the

species studied a conservative motif TATATAAGGGGGG

is present (Fig. 1), which represents the transcription ini-

tiation site (TIS) in eudicots (Perry and Palukaitis 1990;

Volkov et al. 1996). Upstream of TIS a unique AT-rich

sequence and a subrepeat region were found. The IGS

region downstream of TIS (i.e., 50 ETS) contains (1) a

variable region (VR) composed of several subrepeats and

(2) a conservative region (CR) without obvious subre-

peated elements.

Six 141-bp downstream subrepeats were found in VR of

all species of ser. Neolycopersicon, whereas eight subre-

peats are present in LPD and SIT. The downstream

subrepeats belong to the structural type III, which was

already described for tomato (Perry and Palukaitis 1990;

Borisjuk and Hemleben 1993; Komarova et al. 2004). No

downstream subrepeats but only one copy of the homolo-

gous sequence were found in the 50 ETS of NIG. Sequence

comparison showed that each of the type III subrepeats

contains a 40-bp conservative element (CE), which is

highly similar to those of the distantly related Nicotiana

(Borisjuk et al. 1997). Regarding the organization of VR,

three variants of 50 ETS (A, B and C) were described for

the members of sect. Petota (Volkov et al. 2003).

According to this classification, NIG possess the 50 ETS

structural variant A, whereas VR composed of several

tandemly arranged type III subrepeats characteristic for ser.

Neolycopersicon and Juglandifolia are recognized as

structural variants D1 and D2, respectively (Fig. 1).

Sequence alignment and generation of dendrograms

reflecting similarity of individual downstream subrepeats

(details not shown) revealed that the first and last subrepeats

in the 50 ETS exhibited high sequence similarity and appear

to have a common origin in all species of ser. Juglandifolia

and Neolycopersicon. In contrast, the ‘‘internal’’ down-

stream subrepeats, i.e., subrepeats II–V in ser. Neoly-

copersicon (variant D1) and II–VII in ser. Juglandifolia

(variant D2) can only be homologized within the series

(Fig. 1). Remarkably, in the species of ser. Juglandifolia

increased sequence similarity was found between subre-

peats IV and VI as well as between subrepeats III and V.

The possible explanation of this observation could be (1)

relatively late duplication or (2) intensive conversion of the

two adjacent subrepeats.

The approximately 700 bp long CR evolved mainly by

base substitutions, i.e., insertions and deletions (indels) are

absent making this region useful for alignment-based

phylogenetic reconstructions.

Phylogenetic reconstruction

The novel sequences of CR of tomatoes as well as the

respective sequence data set obtained recently for Solanum

species of sect. Petota (Table 2; Volkov et al. 2003; Ko-

marova et al. 2004) were combined and used for

phylogenetic inferences (Figs. 2, 3, 4). Phylograms under

different optimality criteria (NJ, Fig. 2; MP and ML, not

shown) were largely congruent considering the terminal

relationships that received high support. Tomato species of

ser. Neolycopersicon (variant D1) form a distinct and

highly supported sister clade with non-tuber-bearing spe-

cies of Solanum ser. Juglandifolia (LPD, SIT; variant D2),

both sister clades are referred to as tomato clade in the

62 N. Y. Komarova et al.

123

following. Within ser. Neolycopersicon PEN and HIR are

placed as sister taxa to a clade comprising LYC, CHE, PER

and CHM (Figs. 2, 3). Accessions are grouped accordingly

in the neighbor-net (Fig. 4), which highlights the close

relationship (i.e., low genetic distances) between LYC,

CHE, CHM and the more pronounced position of PER

within this group.

Topological ambiguity was either due to the position of

closely related species that are genetically weakly differ-

entiated in the CR or the unresolved phylogenetic

backbone (Fig. 2; box-like structure in Fig. 3). The bipar-

tition networks and neighbor-net (Figs. 3, 4) were largely

congruent and illustrated that low node support considering

the position of the tomato clade as sister clade to the

variants B/C-bearing potatoes (Fig. 2) was due to several,

equally supported alternatives promoting partly incongru-

ent relationships.

In phylograms rooted with DUL and NIG variant

A-bearing taxa are organized as a grade (Fig. 2), which

embraces the non-tuber-bearing species of ser. Etuberosa

(BRD and ETB) from Chile and Argentina as well as the

tuber-bearing Central American diploid species (BLB,

EHR, PNT, JAM and PLD) of several series with stellata

morphology of flowers (Hawkes 1990). The taxa appear to

be closer related to the out-group species (DUL of sect.

Dulcamara and NIG of sect. Solanum) than variant B and

C-bearing potatoes and tomatoes (Figs. 2, 4). Within one

clade (Fig. 2) tuber-bearing South American species of

several series and the Central American polyploids of ser.

Demissa with rotata morphology of flowers exhibiting VR

variants B and C are grouped.

Signal from the CR of 50 ETS is ambiguous consid-

ering the exact position of the root of sect. Petota and

relationships among the variant A-bearing potatoes

(Figs. 2, 3) and is possibly affected by outgroup-induced

long-branching artifacts (Sanderson et al. 2000; cf. branch

lengths in Figs. 2, 4). Depending on the method, either

the tuber-bearing BLB and EHR (NJ and ML;

BSNJ = 43; PPML = 0.84) or, alternatively, non-tuber-

bearing BRD and ETB (MP, BS = 43) may represent

sister taxa to all other species of sect. Petota including

tomatoes (Fig. 3).

Fig. 1 Independent amplification of sequence elements occurring

downstream of TIS in 35S rDNA in Solanum. Ancestral variant A is

present in outgroup taxa (DUL, NIG) and in a polymorphic grade

containing species of tuber-bearing Central American series Bulbo-castana, Pinnatisecta, Polyadenia and the non-tuber-bearing South

American ser. Etuberosa. All tuber-bearing South American potato

species and Central American polyploids possess variants B and C

(Volkov et al. 2003), and species of ser. Neolycopersicon and

Juglandifolia (tomato clade) independently evolved variants D1 and

D2, respectively. IGS intergenic spacer region; ETS external

transcribed spacer region; NTS non-transcribed spacer region; TTRtranscription termination region; TIS transcription initiation site; CEconservative sequence element; VR and CR variable and conservative

regions in the 50 ETS

Molecular evolution of 35S rDNA and taxonomic status of Lycopersicon 63

123

Table 2 List of Solanum species and 50 ETS sequences used for phylogenetic reconstruction

Classification Species Sequence accession nos.

and abbreviations

Clone

name

Sequence

source

Subgenus Solanum

Section Petota Dumort

Subsect. Estolonifera Hawkes S. brevidens Phil. AF447409 (ETB) pBRD1 1

Ser. Etuberosa Buk. and Kameraz S. etuberosum Lindl. AF464144 (BRD) pETB8 1

Ser. Juglandifolia (Rydb.) Hawkes S. lycopersicoides Dunal DQ118130 (lpd 1) DS 2

DQ118131 (lpd 2) pSlpd14-3 2

S. sitiens Johnston DQ118128 (sit 1) DS 2

DQ118129 (sit 2) pSsit9-4 2

Ser. Neolycopersicon (Correll) Child. S. cheesmaniae (Riley) Fosb. DQ118134 (che I) DS 2

DQ118132 (CHE II) DS 2

S. chmielewskii (Rick et al.) Spooner et al. DQ093389 (chm) DS 2

S. lycopersicum L. X52215 (LYC I) – 3

AY366528 (LYCII-1) pTI-9 4

AY366529 (LYCII-2) pTII-4 4

S. hirsutum (Dunal) Macbride DQ093391 (hir) DS 2

S. pennellii Correll DQ093393 (pen) DS 2

S. peruvianum L. DQ093394 (per) DS 2

Subsect. Potatoe G. Don

Superseries Stellata Hawkes

Ser. Bulbocastana (Rydb.) Hawkes S. bulbocastanum Dunal AF464858 (BLB 2) pBLB2 1

AF464860 (BLB 4) pBLB4 1

Ser. Pinnatisecta (Rydb.) Hawkes S. cardiophyllum Lindl. (ssp. eherenbergii) AF447420 (Ehr) pEHR2 1

S. pinnatisectum AF447441 (PNT) pPNT5 1

S. jamesii AF464142 (JAM) pJAM 1

Ser. Polyadenia Correll S. polyadenium Greenm AF447439 (PLD) pLPD1 1

Ser. Circaeifolia Hawkes S. circaeifolium Bitter AF447412 (CRC) pCRC4 1

Ser. Commersoniana Bukasov S. commersonii Dunal AF459641 (CMM) DS 1

Ser. Yungasensa Correll S. chacoense Bitter AF447410 (CHC) pCHC1 1

Superseries Rotata Hawkes

Ser. Megistacroloba Card and Hawkes S. raphanifolium Card & Hawkes AF502961 (RAP) pRAP1 1

Ser. Conicibaccata Bitter S. laxissimum Bitter AF447426 (LXS) pLXS1 1

Ser. Tuberosa (Rydb.) Hawkes S. berthaultii Hawkes AF447407 (BER 1) pBER1 1

AF447408 (BER 2) pBER2 1

S. gourlayi Hawkes AF447422 (GRL) pGRL1 1

S. kurtzianum Bitter and Wittm. AF464871 (KTZ) pKTZ4 1

S. microdontum Bitter AF447430 (MCD) pMCD1 1

S. neorossii Hawkes and Hjerting AF447432 (NRS) pNRS1 1

S. okadae Hawkes and Hjerting AF447434 (OKA) pOKA1 1

S. phureja Juzepczuk and Bukasov AF447436 (PHU) pPHU1 1

S. tuberosum L. AY366530 (TBR 1) pKI-1-IGS 2

AY366531 (TBR 2) pKII-2-IGS 2

S. tuberosum B15 (dihaploid line) AF464864 (B15-2) pB15-2 1

AF464866 (B15-3) pB15-3 1

Ser. Acaulia Juzepczuk S. acaule Bitter AF447404 (ACL) pACL1 1

Ser. Demissa Bukasov S. demissum Lindl. AF464868 (DMS) pDMS2 1

Section Dulcamara (Moench) Dumort S. dulcamara L. AF447418 (DUL) pDUL32 1

Section Solanum S. nigrum L. DQ248893 (nig) DS 2

DS direct sequencing of PCR product without cloning was applied. Key to sequence source: 1 Volkov et al. 2003; 2 this study; 3 Perry and Palukaitis

1990; 4 Komarova et al. 2004

64 N. Y. Komarova et al.

123

Discussion

Organization of the IGS and taxonomic distribution of

the 50 ETS structural variants

The complete IGS of 35S rDNA of eight wild and culti-

vated tomato species was amplified and sequenced in order

to clarify taxonomic position of the species and to shed

light on the molecular evolution of 35S rDNA. Sequence

analysis revealed that the newly studied species of ser.

Juglandifolia and Neolycopersicon possess structural fea-

tures of the IGS described previously in species of sect.

Petota (Borisjuk and Hemleben 1993; Volkov et al. 2003)

such as presence of AT-rich sequence and subrepeats of the

same structural type I and II upstream of TIS.

Detailed inspection of the 50 ETS demonstrates that the

region downstream of TIS can be subdivided into two

structural subregions, VR and CR, differing by the mode

(indels vs. base substitutions, respectively) and rate of

molecular evolution. The VR and CR of Juglandifolia and

Neolycopersicon species correspond to the respective

subregions in the 50 ETS of Solanum species of sect. Petota

studied recently (Volkov et al. 2003).

Our new data show that the European outgroup species

NIG possesses the 50 ETS structural variant A, which

contains only one CE downstream of TIS. The variant A

was originally found in the rDNA of another European

species, DUL, in the non-tuber-bearing species of ser.

Etuberosa and also in tuber-bearing Central American

species of ser. Bulbocastana, Pinnatisecta and Polyadenia.

A 29-bp long sequence element containing XbaI-recogni-

tion site characteristic for variant A is replaced by the

second copy of CE producing the 50 ETS variant B, which

is specific for tuber-bearing South American species of ser.

Fig. 2 NJ phylogram based on

the conservative region (CR:

approximately 700 bp) of the 50

ETS upstream of 18S rDNA of

Solanum species. The

phylogram is based on ML-

adapted distances (GTR ? Csubstitution model). Support

(BSMP/BSNJ/PP) is indicated

near nodes; black dots indicate

that these nodes are incongruent

under different optimality

criteria (ML, MP, NJ).

Distribution of the 50 ETS

structural variants A, B, C and

D1/D2 is shown. Abbreviations

of species names are listed in

Table 2

Molecular evolution of 35S rDNA and taxonomic status of Lycopersicon 65

123

Commersoniana and Circaeifolia. Finally, all species of

superser. Rotata and S. chacoense have the 50 ETS variant

C containing two CE and additionally tandem duplications

around CE1. In the sect. Petota, the variant A is the

ancestral, whereas C is the most derived variant (Volkov

et al. 2003; Fig. 1). Species of series Juglandifolia and

Neolycopersicon analyzed here possess the 50 ETS variant

D characterized by the presence of a cluster of amplified

141-bp subrepeats of type III (Perry and Palukaitis 1990).

The series differ by the number of subrepeats in the cluster:

Six copies in ser. Neolycopersicon (variant D1) and eight

copies in ser. Juglandifolia (variant D2) were detected

(Fig. 1). Among the three 50 ETS variants described for

Solanum species of sect. Petota, the variant A shows the

highest similarity to the monomer of variant D. Also, XbaI-

containing sequence element absent (lost) in variants B and

C is conserved in the species of ser. Juglandifolia and

Neolycopersicon demonstrating that the variant D origi-

nated directly from the variant A.

Molecular evolution of downstream subrepeats

Comparison of the sequences of individual downstream

subrepeats within the same cluster demonstrated that in ser.

Juglandifolia each of eight subrepeats of LPD are homol-

ogous to the respective subrepeats of SIT. Similar, six

subrepeats in the 50 ETS of the species of ser.

Neolycopersicon are homologous to the corresponding

subrepeats of other species of this series. The first and last

copies of downstream subrepeats can be considered to be

homologous among taxa of both series. These data agree

with the model (Fig. 1) that the first duplication of down-

stream subrepeats occurred before the divergence of ser.

Juglandifolia and Neolycopersicon, whereas internal cop-

ies of subrepeats appeared independently after separation

of the two series, but predate the origin of modern species.

Therefore, several rounds of amplification were necessary

to produce downstream subrepeats currently existing in the

50 ETS of Juglandifolia and Neolycopersicon species.

In conclusion, the available data allow the reconstruc-

tion of the molecular evolution of downstream subrepeats

in the 50 ETS of Solanum species (Fig. 1). The results

demonstrate an evolutionary trend of parallel multiplica-

tion of the same sequence motifs downstream of TIS in

different groups of sect. Petota (i.e., homoplasy at molec-

ular level). A possible explanation of this phenomenon

could be an existence of selection pressure or molecular

drive. Especially, CE appears independently amplified

(alone or within subrepeats) in different groups of Solanum

species. Recently, it was proposed that this sequence ele-

ment may participate in the modulation of transcriptional

activity of rDNA, because in the synthetic allopolyploids

between different Solanum species a correlation between

the number of downstream subrepeats and differential

Fig. 3 Ambiguous support from CR, visualized by a bipartition

network based on 100,000 MP bootstrap replicates. Only splits are

shown that were found in more than 25% of bootstrap replicates,

congruent bipartitions can be found in Bayesian inferred trees and NJ

bootstrap replicates. A sister relationship (I) between the tomato clade

(exhibiting variants D1/D2) and BRD ? ETB clade is supported with

higher BS than the competing split II, favored in the reconstruction of

phylograms under ML, MP (not shown), and NJ (Fig. 2). Equally

supported topological alternatives characterize also relationships

among the focus group (III) and the root of the section as defined

by outgroup taxa DUL and NIG (IV)

66 N. Y. Komarova et al.

123

transcription of rDNA was detected (Komarova et al.

2004). The presumptive functional importance of CE

seems to be rather consistent with the participation of

selection pressure than that of molecular drive in the evo-

lution but further studies are necessary to learn more about

the forces resulting in independent amplification of the

downstream repeated elements in sect. Petota.

Phylogeny of sect. Petota and taxonomic position

of tomato

According to the phylogenetic analyses of CR, either the

tuber-bearing BLB or, alternatively, the non-tuber-bearing

BRD and ETB may represent sister taxa to other species of

sect. Petota (Figs. 2, 3, 4). The first possibility agrees with

RAPD markers based systematics of sect. Petota (van den

Berg et al. 2001), whereas analysis of chloroplast DNA

(Spooner et al. 1993; Spooner and Castillo 1997) support

the second possibility. The latter is also in some agreement

with the waxy data presented by Weese and Bohs (2007)

who included six species of sect. Petota in their large three-

gene analysis of the genus Solanum. Ancient origin and

isolated taxonomic position of both series, Bulbocastana

and Etuberosa, were emphasized by Bukasov (1970).

Within sect. Petota, three distinct groups were identi-

fied. A grade comprising non-tuber-bearing and Central

American tuber-bearing diploid species possessing 50 ETS

structural variant A (Fig. 2) represents the remains of the

ancestral group from which two clades with derived

characters evolved, i.e., variants B/C-bearing potato clade

and the tomato clade. Earlier, applying restriction mapping

and sequencing of cpDNA (Spooner et al. 1993; Bohs and

Olmstead 1997, 1999; Olmstead and Palmer 1997; Olm-

stead et al. 1999) and nuclear GBSSI-gene (Peralta and

Spooner 2001) it was found that ser. Neolycopersicon (syn.

genus Lycopersicon) is deeply nested in genus Solanum

with sect. Petota as a sister group. Also in Weese and Bohs

(2007) L. esculentum (syn. S. lycopersicum) is consistently

recognized as sister taxon of S. juglandifolium by all three

genes. However, only few species of sect. Petota were

included in these studies. By significantly increasing the

taxon sampling including data from all main lineages

within sect. Petota (Volkov et al. 2003; this study), it is

obvious that ser. Neolycopersicon should be included in

sect. Petota, and combined with ser. Juglandifolia (herein

called ‘tomato clade’), the latter traditionally placed in

genus Solanum (Hawkes 1990; Nee 1999; Hunziker 2001).

The taxonomic position of ser. Neolycopersicon has

been a subject of debates between followers of Solanum

(von Wettstein 1891; Danert 1970; Spooner et al. 1993;

Child and Lester 2001) and Lycopersicon (Rick 1979;

D’Arcy 1982; Hawkes 1990; Hunziker 2001). Especially,

the discovery of PEN (Correll 1958), which occupies an

intermediate position between Solanum and Lycopersion

provoked new discussions about the boundary between the

genera. Addressing androecium peculiarities Hunziker

(2001) emphasized that PEN should remain in Solanum.

However, this proposal does not agree with the data of

Fig. 4 Distribution of

incompatible splits. Shown is a

neighbor-net (fit = 97.71, 759

characters) based on

uncorrected p-distances. The

overall topology is in agreement

with Figs. 2 and 3. Note that

within the variants B/C-bearing

potatoes, taxa exhibiting either

VR variant B or variant C are

separated and mirror the

amplification sequence inferred

for the VR subrepeats (Fig. 1)

Molecular evolution of 35S rDNA and taxonomic status of Lycopersicon 67

123

crossing experiments, because PEN can be easily crossed

with Lycopersicon, but not with Solanum species (Khush

and Rick 1963; Rick 1969; Haider Ali et al. 2001).

Moreover, all other wild species of ser. Neolycopersicon

can be crossed to cultivated tomato, and the hybrids

demonstrate normal chromosome pairing (Khush and Rick

1963; Rick 1969; Afify 1993). LYC can be also crossed to

LPD, but reduced pairing and recombination between

homologous chromosomes were reported (Menzel 1962;

Rick et al. 1986; Ji et al. 2001). No sexual hybrids were

obtained between TUB and LYC, and their chromosomes

do not conjugate if combined by protoplast fusion (Jac-

obsen et al. 1995; Garriga-Caldere et al. 1999). These

observations as well as reduced pairing of homologous

chromosomes in hybrids between LYC and LPD were used

as arguments against inclusion of LYC into Solanum (Ji

et al. 2001; Haider Ali et al. 2001; Hunziker 2001).

However, it should be kept in mind that the absence of

sexual hybrids between species of the same genus appears

to be normal, and reproductive isolation may take place

even between closely related species (Grant 1981). Our

molecular data demonstrate a correlation between genetic

distances, phylogenetic position (Figs. 2, 4) and cross-

ability between the species of Solanum sect. Petota

including tomato clade. Remarkably, in trigenomic hybrids

obtained by crossing of potato (?) tomato protoplast fusion

hybrids with PEN the chromosomes of PEN pairs with the

chromosomes of both LYC and TUB (Haider Ali et al.

2001), demonstrating that the boundary between Solanum

and ‘‘Lycopersicon’’ appears elusive. According to our

molecular data (CR and VR) PEN is closely related to

‘‘Lycopersicon’’ (Figs. 1, 2, 3, 4), strongly confirming the

proposal of Nee (1999), who placed PEN together with

‘‘Lycopersicon’’ species in a ser. Neolycopersicon of sect.

Petota and emphasizing that the genus ‘‘Lycopersicon is an

offshoot of this line’’.

Differentiation within ser. Neolycopersicon

Interrelationship between species of ser. Neolycopersicon

was earlier studied using restriction mapping of chloroplast

(Palmer and Zamir 1982) or mitochondrial DNA (McClean

and Hanson 1986), RFLP (Miller and Tanksley 1990),

isoenzymes (Breto et al. 1993), sequencing of granule-

bound starch synthase gene (GBSSI; Peralta and Spooner

2001), internal transcribed spacer of 35S rDNA (ITS;

Marshal et al. 2001), satellite DNA (Schweizer et al. 1988),

microsatellite markers (Alvarez et al. 2001), AFLP

(Spooner et al. 2005; see also van den Berg and Groendijk-

Wilders 2007) and multilocus sequence analysis (Arunya-

wat et al. 2007). Results obtained by different authors

are partially controversial and the most recent accounts

(Arunyawat et al. 2007; Van den Berg and Groendijk-

Wilders 2007) demonstrate that speciation processes in this

group are complicated. Therefore, additional information is

required to finally clarify relationships within the former

genus Lycopersicon. Our data show that (1) LYC, CHE and

CHM represent a tightly related group with PER as a sister

taxon, whereas (2) HIR and PEN are more distantly related

species agreeing with the results of Peralta and Spooner

(2001), Marshal et al. (2001) and Alvarez et al. (2001). The

grouping of HIR and PEN as sister taxa reported by Miller

and Tanksley (1990) and Marshal et al. (2001) is supported

as one alternative (Fig. 3).

Utility of morphological and molecular traits

for phylogenetic systematics

Interpretation of phylograms in a strict cladistic manner

(Hennig 1966) requires one or two reversals depending on

the favored topology, if tubers evolved early in genus

Solanum sect. Petota (see Figs. 2, 3). Alternatively, the

ancestors of modern lineages could have the potential

(genetic prerequisites) to produce tubers or stolons as an

adaptation to similar environmental conditions as hypoth-

esized by Bukasov (1970), who suggested an independent

origin of tubers in different groups of potato. This is in

agreement to the phylogenetic reconstruction based on the

CR (Figs. 2, 3, 4) and the distribution of primitive (A) and

derived (B/C vs. D1/D2) VR variants. Among the variant

A-bearing grade, which falls into subclades of different and

ambiguous systematic affinity, non-tuber and tuber-bearing

lineages are found, whereas members of the clades with

derived VR variants are either all tuber-bearing (variants B/

C-showing potatoes) or all not tuber-bearing (tomato

clade). Thus, the occurrence of tubers in modern species

appears to have limited systematic relevance.

The taxonomic distribution of the 50 ETS structural

variants A–D (Fig. 1) perfectly correlates with the molec-

ular phylogeny and taxonomy of Solanum inferred from the

comparison of CR sequences (see Figs. 2, 3, 4). According

to the principles of cladistics (Hennig 1966), the aim of

phylogenetic systematics should be the identification of

monophyletic groups (clades), i.e., a group of taxa that has a

common ancestry. Clades are characterized by synapo-

morphies (shared derived characters). Respectively, the

derived 50 ETS structural variants B/C and D1/D2 represent

molecular synapomorphies in a strict sense and are char-

acteristic for the two main lineages in evolution of sect.

Petota. The ancestral variant A is retained in a polymorphic

grade and, hence, represents a symplesiomorphy (shared

primitive character). The recognition of such prominent

sequence patterns is prospective for taxonomy and (phylo-

genetic) systematics in addition to and independent of the

traditional approach in molecular phylogenetics, in which

only highly supported nodes are accepted as clades.

68 N. Y. Komarova et al.

123

Mode of VR evolution

The intensity of rearrangements of VR via indels and

amplifications differs obviously within the sect. Petota:

The ancestral 50 ETS variant A was conserved in a poly-

morphic grade of potatoes (Figs. 2, 3, 4), which members

exhibit mainly primitive morphologically characters

(Hawkes 1990). At least two structural changes occurred

during the evolution of potato clade embracing variants

B- and C-bearing species and several amplifications hap-

pened in course of the formation of the tomato clade (sers.

Neolycopersicon and Juglandifolia; see Fig. 1). Remark-

ably, in the tomato clade the increased frequency of

molecular changes in VR correlates with a high rate of base

substitutions in CR (branch lengths in Figs. 2, 3, 4).

Whether the intensive rearrangements of the 50 ETS reflect

the increased tempo of the whole genome alterations in the

tomato clade or simply represent an evolutionary favorable

local feature of the 50 ETS remains to be additionally

tested.

Acknowledgments This work was supported by the DAAD

(German Academic Exchange Service), the Alexander von Humboldt

Foundation, DFG (German Research Foundation) and DFFD

(Ukrainian Fundamental Researches State Fund). Authors are grateful

to C. M. Rick and F. H. Borgnino, University of California, Davis,

USA, and to the seed collection of the Institut fur Pflanzengenetik und

Kulturpflanzenforschung (IPK), Gatersleben, Germany, for providing

seed material.

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