J Plant Res (2011) 124:561–576
DOI 10.1007/s10265-010-0395-5
REGULAR PAPER
Rate accelerations in nuclear 18S rDNA of mycoheterotrophic
and parasitic angiosperms
Benny Lemaire • Suzy Huysmans • Erik Smets
Vincent Merckx
•
Received: 4 June 2010 / Accepted: 25 October 2010 / Published online: 25 December 2010
Ó The Author(s) 2010. This article is published with open access at Springerlink.com
Abstract Rate variation in genes from all three genomes
has been observed frequently in plant lineages with a
parasitic and mycoheterotrophic mode of life. While the
loss of photosynthetic ability leads to a relaxation of evolutionary constraints in genes involved in the photosynthetic apparatus, it remains to be determined how prevalent
increased substitution rates are in nuclear DNA of nonphotosynthetic angiosperms. In this study we infer rates of
molecular evolution of 18S rDNA of all parasitic and
mycoheterotorphic plant families (except Lauraceae and
Polygalaceae) using relative rate tests. In several holoparasitic and mycoheterotrophic plant lineages extremely high
substitution rates are observed compared to other photosynthetic angiosperms. The position and frequency of these
substitutions have been identified to understand the mutation dynamics of 18S rRNA in achlorophyllous plants.
Despite the presence of significantly elevated substitution rates, very few mutations occur in major functional
and structural regions of the small ribosomal molecule,
B. Lemaire (&) S. Huysmans E. Smets V. Merckx
Laboratory of Plant Systematics, Institute of Botany and
Microbiology, K.U. Leuven, Kasteelpark Arenberg 31,
PO Box 2437, 3001 Leuven, Belgium
e-mail: benny.lemaire@bio.kuleuven.be
E. Smets
Netherlands Centre for Biodiversity Naturalis,
PO Box 9517, 2300 RA Leiden, The Netherlands
E. Smets
National Herbarium of the Netherlands, Leiden University,
PO Box 9514, 2300 RA Leiden, The Netherlands
V. Merckx
Department of Plant and Microbial Biology,
University of California Berkeley, Berkeley, CA 94720, USA
providing evidence that the efficiency of the translational
apparatus in non-photosynthetic plants has not been
affected.
Keywords 18S rDNA Mycoheterotrophy Parasitism
Substitution rates Relative rate test
Introduction
In flowering plants, a fully heterotrophic mode of life is an
exceptional trait. Little over 1% of all described species
derive all of their carbon from other organisms (Kuijt 1969;
Heide-JØrgensen 2008). Based on the partners involved in
the interaction, two groups of heterotrophic plants can be
distinguished: parasitic and mycoheterotrophic plants. Parasitic plants directly penetrate host plants via their haustoria
to obtain water and (in)organic solutes (Nickrent et al. 1998).
These plants include hemiparasites and holoparasites, a
division based on the presence or absence of chlorophyll
during at least one part of their life cycle, respectively.
Parasitic plants are mainly restricted to eudicots, with the
exception of three magnoliid genera: Hydnora, Prosopanche
(Hydnoraceae) and Cassytha (Lauraceae). A parasitic
lifestyle has evolved at least 11 times independently in
angiosperms, occurring in 20 families and close to 4,500
extant species (Nickrent et al. 1998; Barkman et al. 2007;
Heide-JØrgensen 2008). The phylogenetic positions of
many parasitic lineages are still unknown at low taxonomic
level (APG 2003).
In contrast to parasitic plants, mycoheterotrophic plants
derive carbon from fungi (Leake 2005). With a few
exceptions, most mycoheterotrophic plant species exploit
mycorrhizal fungi that are simultaneously mycorrhizal with
neighboring photosynthetic plants. Because all carbon in
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this tripartite relationship is ultimately derived from autotrophic plants, mycoheterotrophic plants are considered to be
epiparasitic on green plants (Bidartondo et al. 2002; Leake
2004). However, some mycoheterotrophic orchids are associated with litter- and wood-decay fungi (Ogura-Tsujita et al.
2009; Selosse et al. 2010). Over 400 fully mycoheterotrophic
plant species have been reported in eudicots (Ericaceae,
Gentianaceae and Polygonaceae) and monocots (Burmanniaceae, Corsiaceae, Iridaceae, Orchidaceae, Petrosaviaceae,
Thismiaceae and Triuridaceae) (Leake 1994; Heide-JØrgensen 2008; Merckx et al. 2009). In addition, over 20,000
flowering plant species are thought to be at least partially
mycoheterotrophic, mostly initial mycoheterotrophs in the
Orchidaceae (Leake 2005; Selosse and Roy 2009).
With the loss of photosynthetic ability, genes required for
the photosynthetic apparatus will undergo random mutations
under relaxed natural selection (Conopholis americana,
Wimpee et al. 1991; Cuscuta, Funk et al. 2007; McNeal et al.
2009; Revill et al. 2005; Epifagus virginiana, dePamphilis and
Palmer 1990; Wolfe et al. 1992; dePamphilis et al. 1997;
Lathraea clandistina, Delavaut et al. 1995; Orobanche
hederae, Thalouarn et al. 1994). Although some chloroplast
genes are retained and functional in several holoparasitic
plants (Bungard 2004), many holoparasites have a reduced
plastid genome due to excessive gene loss and increased
substitution rates in the remaining genes (Wolfe et al. 1992).
In chloroplast genome analysis of non-photosynthetic plants
different explanations have been proposed why a reduced set
of genes have been retained and need to be translated within
the chloroplast as opposed to replacement by a product from
cytosolic orthologues: (1) import of proteins with hydrophobic membranes from the cytosol back into the organelle would
be impossible; (2) rate of synthesis of specific proteins can be
regulated within a individual plastids preventing deadly sideeffects of oxidative stress or lethal effects of accumulating
toxic products; (3) synthesis and assembly of components of
the photosynthetic complexes are tightly regulated within
plastids (see review by Barbrook et al. 2006). The plastid
genome of fully mycoheterotrophic plants remains largely
unstudied, but rbcL data suggest that the genome is prone to a
similar relaxation of purifying selection (Caddick et al. 2002;
Barrett and Freudenstein 2008). These observations demonstrate that non-photosynthetic plants are under reduced
selective constraints, which affect the structure and function
of genes involved in photosynthetic reactions.
Somewhat surprisingly, extreme variation in rates of
evolution has also been observed in the nuclear and/or
mitochondrial genes of some fully mycoheterotrophic
(Merckx et al. 2006, 2009; Petersen et al. 2006) and parasitic plants (Nickrent and Starr 1994; Nickrent et al. 1998;
Davis et al. 2004; Chase 2004; Barkman et al. 2007). The
causes for increased substitution rates in nuclear and
mitochondrial DNA of achlorophyllous plants are still
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J Plant Res (2011) 124:561–576
poorly documented and understood. Several hypotheses
related to the loss of photosynthesis, effective population
size, generation time and host specificity have been proposed to explain this phenomenon in parasitic plants.
However, none of these hypotheses could unequivocally
explain the rate increases in all parasitic plants (dePamphilis
and Palmer 1990; Nickrent and Starr 1994; dePamphilis
et al. 1997; Young and dePamphilis 2005).
In this study, we infer the variation of substitution rates
of nuclear 18S rDNA of nearly all angiosperm families by
comparing branch length variation of 37 fully mycoheterotrophic species, 33 parasitic plant species, and related
autotropic lineages with relative rate tests (RobinsonRechavi and Huchon 2000; Wilcox et al. 2004). Estimating
rate variation across taxa, representing most groups of
angiosperms, gives us the opportunity to address the following questions: How frequent can increased substitution
rates be observed in nuclear loci of hemiparasitic, holoparasitic and mycoheterotrophic plants? Are individual
achlorophyllous taxa or whole heterotrophic plant families
prone to substitution rates? Is loss of chlorophyll in
angiosperms associated with longer branches? Can we
provide evidence for another hypothesis, which could
cause the increased substitution rates in 18S rDNA of
mycoheterotrophic and parasitic plants? What are the exact
nucleotide positions of the substitution in 18S rRNA and
are they interfering with functional sites?
Materials and methods
Taxon sampling
In total, 18S rDNA sequences of 178 angiosperm species
were used for this study, representing the 45 orders of the
APGII classification (APGII 2003). All families with parasitic and fully mycoheterotrophic species are represented
in our study by one or more taxa, with the exception of
Polygalaceae (Epirixanthes—fully mycoheterotrophic) and
Lauraceae (Cassytha—hemiparasitic). To evaluate the
substitution rates of mycoheterotrophic and parasitic taxa,
18S rDNA sequences of related autotrophic plants were
obtained from Genbank and included as reference points to
measure substitution rates. The outgroups used for the
heterotrophic lineages, according to recent phylogenetic
studies are showed in Table 1. All species sampled with
voucher information and Genbank accession numbers are
listed in the ‘‘Appendix’’.
DNA extraction, amplification and sequencing
Ten and six 18S rDNA sequences of mycoheterotrophic
and autotrophic plants, respectively, were newly obtained
J Plant Res (2011) 124:561–576
563
Table 1 List of mycoheterotrophic/parasitic taxa and outgroups investigated in this study
Mycoheterotroph/parasite
Outgroup
BRT (constrained)
BRT (unconstrained)
References
McNeal et al. (2007)
Hemiparasites
Convolvulaceae
Cuscuta gronovii
0.332/0.390/0.471
0.334/0.401/0.466
Convolvulus arvensis
0.164/0.202/0.252
0.167/0.216/0.260
Krameriaceae
Krameria ixine
0.117/0.155/0.208
0.137/0.173/0.221
Guiacum sanctum
0.096/0.137/0.176
Orobanchaceae
0.118/0.152/0.201
Pedicularis racemosa
0.128/0.164/0.206
0.120/0.157/0.192
Orthocarpus erianthus
0.143/0.184/0.231
0.142/0.179/0.219
Paulownia tomentosa
0.123/0.154/0.194
0.115/0.151/0.189
Lamium amplexicaule
0.139/0.171/0.214
0.128/0.165/0.202
Simpson et al. (2004)
Olmstead et al. (2001)
Santalales (Loranthaceae)
Nuytsia floribunda
0.114/0.152/0.195
0.106/0.139/0.181
Der and Nickrent (2008)
Tupeia antarctica
0.102/0.139/0.184
0.094/0.130/0.174
Malécot and Nickrent (2008)
Santalales (Misodendraceae)
Misodendrum linearifolium
0.193/0.242/0.308
0.194/0.245/0.298
Santalales (Olacaceae)
Erythropalum scandens
0.092/0.124/0.161
0.081/0.116/0.150
Olax aphylla
0.156/0.207/0.261
0.130/0.172/0.216
Santalales (Opiliaceae)
Opilia amentacea
Lepionurus sylvestris
0.179/0.227/0.293
0.170/0.215/0.269
0.161/0.205/0.267
0.149/0.196/0.250
Santalales (Santalaceae)
Lepidoceras chilense
0.170/0.208/0.270
0.166/0.210/0.262
Santalum album
0.126/0.166/0.231
0.138/0.175/0.221
Santalales (Schoepfiaceae)
Schoepfia arenaria
0.158/0.197/0.242
0.133/0.184/0.229
Santalales (Viscaceae)
Arceuthobium verticilliflorum
0.365/0.438/0.512
0.363/0.445/0.521
Dendrophtora domingensis
0.233/0.288/0.351
0.244/0.300/0.368
Gunnera manicata
0.131/0.167/0.212
0.112/0.150/0.189
Hamamelis virginiana
0.127/0.166/0.206
0.132/0.169/0.222
Plumbago auriculata
0.142/0.182/0.219
0.144/0.186/0.238
Holoparasites
Apodanthaceae
Pilostyles thurberi
0.189/0.245/0.296
0.189/0.235/0.282
Celastrus scandens
0.162/0.213/0.272
Balanaphoraceae
0.158/0.202/0.246
Ombrophytum subterraneum
0.386/0.457/0.547
0.383/0.456/0.555
Gunnera manicata
0.131/0.167/0.212
0.112/0.150/0.189
Hamamelis virginiana
0.127/0.166/0.206
0.132/0.169/0.222
Plumbago auriculata
0.142/0.182/0.219
0.144/0.186/0.238
Nickrent et al. (2004)
Stevens (2001)
Cynomoriaceae
Cynomorium coccineum
0.189/0.236/0.285
0.182/0.226/0.285
Gunnera manicata
0.131/0.167/0.212
0.112/0.150/0.189
Hamamelis virginiana
0.127/0.166/0.206
0.132/0.169/0.222
Plumbago auriculata
0.142/0.182/0.219
0.144/0.186/0.238
Nickrent et al. (2005)
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Table 1 continued
Mycoheterotroph/parasite
Outgroup
BRT (constrained)
BRT (unconstrained)
References
Bdallophyton americanum
0.256/0.303/0.346
0.248/0.308/0.374
Nickrent (2007)
Cytinus hypocistis
0.322/0.406/0.467
0.343/0.410/0.479
Muntingia calabura
0.136/0.172/0.217
0.147/0.187/0.233
Cytinaceae
Euphorbiaceae
Rafflesia keithii
0.930/1.091/1.274
0.942/1.085/1.267
Rhizanthes infanticida
0.982/1.136/1.310
0.977/1.132/1.317
Euphorbia pulcherrima
0.114/0.156/0.203
0.107/0.145/0.182
Hydnora africana
Hydnoraceae
0.293/0.359/0.410
0.364/0.432/0.497
Prosopanche americana
0.351/0.425/0.495
0.397/0.487/0.566
Aristolochia macrophylla
0.074/0.099/0.130
0.103/0.164/0.215
Davis et al. (2007)
Nickrent et al. (2002)
Lennoaceae
Pholisma arenarium
0.242/0.290/0.346
0.236/0.276/0.320
Hydrophyllum fendleri
0.173/0.215/0.270
0.162/0.204/0.243
Olmstead and Ferguson (2001)
Mitrastemonaceae
Mitrastemon yamamotoi
0.351/0.420/0.508
0.363/0.439/0.542
Vaccinium macrocarpon
0.143/0.183/0.225
0.133/0.170/0.207
Davis et al. (2007)
Orobanchaceae
Boschniakia rossica
0.126/0.169/0.214
0.127/0.165/0.199
Conopholis americana
0.140/0.178/0.221
0.132/0.173/0.210
Epifagus virginiana
0.135/0.173/0.214
0.130/0.168/0.204
Harveya speciosa
0.155/0.195/0.241
0.158/0.195/0.238
Lathraea clandestina
Orobanche fasciculata
0.154/0.194/0.242
0.178/0.220/0.276
0.146/0.190/0.229
0.171/0.217/0.256
Paulownia tomentosa
0.123/0.154/0.194
0.115/0.151/0.189
Lamium amplexicaule
0.139/0.171/0.214
0.128/0.165/0.202
Mycoheterotrophs
Family
Olmstead et al. (2001)
Burmanniaceae
Apteria aphylla
0.221/0.279/0.349
0.155/0.212/0.304
Burmannia oblonga
0.263/0.311/0.358
0.188/0.238/0.288
Burmannia sphagnoides
0.198/0.254/0.303
0.136/0.187/0.249
Campylosiphon purpurascens
0.147/0.188/0.246
0.083/0.122/0.160
Cymbocarpa refracta
0.201/0.254/0.326
0.138/0.192/0.256
Dictyostega orobanchoides
0.186/0.227/0.298
0.105/0.157/0.210
Gymnosiphon aphyllus
0.178/0.232/0.286
0.115/0.159/0.215
Gymnosiphon divaricatus
0.163/0.215/0.281
0.099/0.144/0.207
Hexapterella gentianoides
0.141/0.187/0.243
0.075/0.118/0.166
Burmannia bicolor
Dioscorea rockii
0.329/0.395/0.479
0.139/0.189/0.239
0.269/0.325/0.377
0.083/0.123/0.169
Merckx et al. (2006)
Corsiaceae
Arachnitis uniflora*
0.599/0.705/0.805
0.584/0.698/0.849
Luzuriaga latifolia
0.102/0.140/0.183
0.037/0.075/0.107
Fay et al. (2006)
Ericaceae
Hemitomes congestum*
0.149/0.199/0.256
0.147/0.187/0.238
Monotropa uniflora
0.191/0.242/0.293
0.151/0.198/0.248
Pityopus californicus*
0.165/0.210/0.252
0.184/0.233/0.283
Pterospora andromedea
0.157/0.200/0.241
0.156/0.191/0.232
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Cullings (2000)
J Plant Res (2011) 124:561–576
565
Table 1 continued
Mycoheterotroph/parasite
Outgroup
BRT (constrained)
BRT (unconstrained)
Sarcodes sanguinea
0.151/0.196/0.237
0.139/0.185/0.231
Arctostaphylos uva-ursi
0.104/0.142/0.182
0.097/0.135/0.174
Pyrola picta
0.120/0.159/0.197
0.114/0.150/0.191
References
Gentianaceae
Exacum paucisquamum*
0.251/0.294/0.352
0.229/0.269/0.315
Sebaea oligantha*
0.195/0.241/0.303
0.177/0.217/0.255
Voyria caerulia*
0.241/0.294/0.361
0.218/0.268/0.320
Voyria corymbosa*
0.275/0.327/0.387
0.257/0.303/0.352
Voyria aurantiaca*
Voyriella parviflora*
0.255/0.299/0.366
0.170/0.221/0.284
0.227/0.273/0.319
0.152/0.198/0.238
Sebaea grandis
0.187/0.244/0.308
0.174/0.219/0.263
Yuan et al. (2003)
Iridaceae
Geosiris sp.
0.095/0.134/0.175
0.067/0.095/0.129
Aristea glauca
0.122/0.157/0.199
0.096/0.123/0.155
Reeves et al. (2001)
Orchidaceae
Corallorhiza maculata
0.165/0.205/0.246
0.112/0.171/0.221
Freudenstein et al. (2004)
Aplectrum hyemale
0.161/0.200/0.246
0.108/0.167/0.217
Molvray et al. (2000)
Erythrorchis cassythoides
0.288/0.335/0.385
0.226/0.297/0.361
Cyrtosia septentrionalis
0.277/0.329/0.378
0.231/0.293/0.367
Lecanorchis multiflora
0.265/0.305/0.361
0.216/0.270/0.335
Vanilla aphylla
0.273/0.313/0.366
0.219/0.279/0.346
Neottia nidus-avis
0.201/0.245/0.293
0.167/0.217/0.272
Eburophyton austinae
0.196/0.236/0.285
0.154/0.205/0.262
Rhizanthella gardneri
Diuris sulphurea
0.436/0.490/0.555
0.223/0.265/0.310
0.378/0.443/0.529
0.175/0.225/0.286
Wullschlaegelia calcarata
0.175/0.222/0.265
0.137/0.189/0.239
Orchis quadripunctata
0.237/0.274/0.324
0.177/0.236/0.295
Petrosaviaceae
Petrosavia stellaris
0.083/0.131/0.204
0.052/0.112/0.162
Japonolirion osense
0.073/0.117/0.180
0.056/0.102/0.152
Afrothismia hydra
0.469/0.554/0.657
0.431/0.522/0.638
Afrothismia winkleri
0.472/0.557/0.658
0.428/0.518/0.632
Haplothismia exannulata
0.285/0.333/0.394
0.210/0.268/0.333
Thismia rodwayi
0.332/0.385/0.459
0.253/0.327/0.428
Thismia aseroe
0.475/0.551/0.630
0.379/0.467/0.569
Tacca chantieri
0.148/0.195/0.248
0.095/0.135/0.185
Dioscorea rockii
0.139/0.189/0.239
0.083/0.123/0.169
Kupea martinetugei
Triuridaceae
0.378/0.472/0.557
0.341/0.416/0.500
Sciaphila ledermannii*
0.133/0.185/0.237
0.077/0.128/0.177
Sciaphila densiflora
0.131/0.188/0.246
0.076/0.132/0.175
Stemona japonica
0.103/0.156/0.206
0.075/0.119/0.164
Cameron et al. (2003)
Thismiaceae
Merckx et al. (2006)
Stevens (2001)
95% confidence intervals and median values of constrained and non-constrained Bayesian relative rates test (BRT) are indicated between ‘‘/’’.
Mycoheterotrophic/parasitic taxa are indicated in bold. Lineages with significant elevated substitution rates are underlined. Choice of outgroups
were based on previous phylogenetic studies. Newly obtained 18S rDNA sequences for this study are indicated with an asterisk
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566
for this study (Table 1; Appendix). Total DNA was extracted
from silica-dried material with the Puregene DNA extraction
kit (Gentra Systems, Landgraaf, The Netherlands) following
the manufacturer’s instructions. The 18S rDNA region was
amplified using primers NS1, NS2, NS3, NS4, NS5 and NS8
(White et al. 1990). Each amplification reaction was performed in 25 ll reaction mix containing 5 ll DNA, 4 ll H2O,
2.5 ll 109 PCR buffer, 0.75 ll 25 mM MgCl2, 10 ll of
2.2 mM forward and reverse primers, 2.5 ll 2 mM dNTP and
0.2 ll Taq DNA polymerase. The polymerase chain reactions
were run on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA) for 30 cycles starting at 94°C
for 1 min, followed by denaturation (94°C for 30 s), annealing
(44°C for 30 s), extension (72°C for 1 min) and a final
extension (72°C for 7 min). PCR products were cleaned
using a Nucleospin Extraction II kit (Machery-Nagel, Düren,
Germany) according to the manufacturer’s instructions.
Sequencing reactions were done using the ABI PRISM Big
Dye Terminator Cycle sequencing kit (Applied Biosystems)
with the same primers as listed above. All the samples
were sequenced on an ABI 310 Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA).
Phylogenetic analyses
The sequences were assembled and edited using Staden et al.
(1998). A preliminary sequence alignment was created with
Clustal X (Thompson et al. 1997) followed by manual
adjustments with MacClade 4.04 (Maddison and Maddison
2001) resulting in an unequivocal alignment with a length of
1708 nucleotide positions. The best fitting model of DNA
substitution was chosen by performing hierarchical Likelihood-ratio tests in MrModeltest v3.06 (Posada and Crandall
1998). The Likelihood-ratio tests and Akaike Information
Criterion selected the GTR?I?G model of evolution. To
reduce calculation time a starting tree for the Bayesian analyses was generated under Maximum Likelihood using Garli
v0.951 (Zwickl 2006) with the GTR?I?G model of evolution. An initial unconstrained Bayesian analysis of the 18S
rDNA dataset retrieved a moderate resolved topology with
conflicting nodes as compared to the relationships of APGII
(results not shown). However, the conflicting nodes in our
unconstrained phylogeny did not receive significant Bayesian
posterior probabilities. In order to improve the topology of
this single gene analysis according to the classification of
APGII and to compare the branch lengths of the heterotrophic
lineages with the according autotrophic relatives, the analysis
was rerun with 19 constraints using the ‘topologypr’ command in MrBayes (Huelsenbeck and Ronquist 2001;
Ronquist and Huelsenbeck 2003) (Fig. 1a, b). The constraints
resolved some major informal groups (e.g. magnoliids,
monocots, commelinids, eudicots, core eudicots, rosids
including eurosids I and II and asterids, including euasterids I
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J Plant Res (2011) 124:561–576
and II) as recovered by multi-gene analyses (APG 2003) and
forced five families and one order containing mycoheterotrophic and parasitic lineages in monophyletic groups
(Euphorbiaceae, Gentianaceae, Orchidaceae, Petrosaviaceae
and Santalales). Amborella was chosen as outgroup for the
analysis. Bayesian analyses were run on the K.U.Leuven
UNIX cluster (‘VIC’), running four Markov chains sampling
every 100 generations for four million generations. The first
10,000 sampled trees (25%) were regarded as ‘‘burnin’’ and
discarded. Convergence of the Markov chains was checked
using Tracer v.1.4 (Rambaut and Drummond 2007).
Relative rate tests
Rates of molecular evolution of 18S rDNA data were
estimated using a Bayesian relative rates test according to
the method described by Wilcox et al. (2004). From 40,000
sampled trees of the Bayesian analysis with the topology
constraints enforced, 25% burnin phase excluded, 500
phylograms were randomly selected. For these sampled
trees, the distance from the most recent common ancestor
(MRCA) of the ingroup to each of the terminal taxa was
calculated with Cadence v1.0 (Wilcox et al. 2004). The
MRCA of the ingroup is the ancestral node shared by all
ingroup taxa. The values of these distances for every taxon
were plotted in Excel (Excel 2004 for Mac version 11.4.1)
in order to calculate the 95% confidence intervals. Based
on the assumption that the confidence interval of a given
taxon does not overlap with the confidence interval of
another taxon, we can state that a significant difference in
rate of molecular evolution between these two individual
taxa has occurred (Wilcox et al. 2004). In order to determine the effect of the implementation of topology constraints on the estimation of substitution rates, the Bayesian
relative rates test was repeated using phylograms sampled
during the unconstrained Bayesian analysis.
To test whether grouped phylogenetic lineages accumulated nucleotide substitutions at significantly increased rates
compared to an outgroup, we performed the relative rate test
using the program package RRTree (Robinson-Rechavi and
Huchon 2000). RRTree computes differences in molecular
rates between non-coding DNA sequences using Kimura’s
two parameter (K2P) (Kimura 1980) and Jukes and Cantor’s
one parameter (JC) model. The K2P substitution model was
selected for this study because it is the most complex model
presently implemented in RRTree.
Patterns of nucleotide substitutions in 18S rDNA
of mycoheterotrophic, parasitic and autotrophic
angiosperms
Conserved and variable nucleotides in the 18S rDNA
dataset were identified using the chart option in MacClade
J Plant Res (2011) 124:561–576
567
a
*
*
*
c
Sebaea grandis
Sebaea oligantha
Exacum paucisquamum
Exacum affine
Voyria aurantiaca
Voyria corymbosa
Voyria caerulia
Gentianaceae
Orphium frutescens
Pycnosphaera buchananii
Chelanthus purpurascens
Gentiana ascepiadea
E
U
Gentianella amarella
*
A
Anthocleista grandiflora
S
Voyriella parviflora
T
Bourreria succulenta
E
R
Ehretia cymosa
I
Phacelia bicolor
D
Hydrophyllum fendleri
S
Lennoaceae
Pholisma arenarium
I
Convolvulus arvensis
Convolvulaceae
Ipomoea hederacea
Cuscuta gronovii
Lathraea clandestina
Orobanche fasciculata
Pedicularis racemosa
Orthocarpus erianthus
Orobanchaceae
Epifagus virginiana
Conopholis americana
Boschniakia rossica
*
Harveya speciosa
Paulownia tomentosa
Lamium amplexicaule
Callicarpa dichotoma
Garrya elliptica
E
Scaevola aemula
U
Apium graveolens
A
*
Sambucus ebulus
S
T
Ilex opaca
*
E
Alangium chinense
R
Cornus florida
I
Hemitomes congestum
D
S
Monotropa uniflora
Pityopus californicus
II
Pterospora andromedea
Ericaceae
Sarcodes sanguinea
Arctostaphylos uva-ursi
Pyrola picta
Vaccinium macrocarpon
*
Clethra alnifoliaene
Mitrastemonaceae
Mitrastemon yamamotoi
Opilia amentacea
Opiliaceae
Lepionurus sylvestris
Schoepfiaceae
Schoepfia arenaria
Nuytsia floribunda
Loranthaceae
Tupeia antarctica
Dendrophthora domingensis
Viscaceae
Arceuthobium verticilliflorum
Santalum album
Santalaceae
Lepidoceras chilense
Ombrophytum subterraneum Balanaphoraceae
Misodendraceae
Misodendrum
linearifolium
*
Olax aphylla
Olacaceae
Erythropalum scandens
Gunnera manicata
Cynomorium coccineum
Cynomoriaceae
Peridiscus lucidus
Saxifraga integifolia
Crassula marnieran
Hamamelis virginiana
Pereskia aculeata
Plumbago auriculata
Rafflesia keithii
9x
Rhizanthes infanticida
Euphorbiaceae
* Ricinus communis
Ostodes paniculata
E
Euphorbia pulcherrima
U
Celastrus scandens
Apodanthaceae R
Pilostyles thurberi
O
Trema micrantha
S
I
Fagus grandifolia
D
Cucurbita pepo
S
Begonia xyloba
*
Bauhinia variegata
I
Krameriaceae
Krameria ixine
Guaiacum sanctum
Davidsonia pruriens
E
Cytinus hypocistis
Cytinaceae
U
Bdallophyton americanum
R
Muntingia calabura
O
S
Carica papaya
I
Citrus aurantium
D
*
Crossosoma californicum
S
Pelargonium cotyledonis
II
Epilobium angustifolium
Ranunculus taisanensis
Platanus occidentalis
Ceratophyllum demersum
Monocots
Magnoliids
Basal angiosperms
E
U
A
S
T
E
R
I
D
S
C
O
R
E
E
U
D
I
C
O
T
S
E
U
D
I
C
O
T
S
E
U
R
O
S
I
D
S
0
0.5
1.5
2.0
Distance to the MRCA
Fig. 1 a, b The majority-rule consensus phylogram of the Bayesian
analysis of the 18S rDNA data under topology constraints. The
implemented constrains are indicated with asterisks. Branches in bold
with a circle, a square, a diamond and an ellipse are, respectively,
mycoheterotrophic, holoparasitic, hemiparasitic and facultative
hemiparasitic lineages. c, d The Bayesian relative rates test shows
the relative branch lengths of all ingroup taxa using Amborella as
most recent common ancestor (MRCA). The squares indicate the
mean branch lengths. The triangles delimit the 95% confidence
intervals
4.04 (Maddison and Maddison 2001). Each character with
the corresponding character states for each angiosperm
taxon was optimized on the consensus tree obtained from
the Bayesian analyses. This approach provides a minimum
estimate of change for each site. The values of the numbers
of character substitutions or steps for every nucleotide site
123
568
J Plant Res (2011) 124:561–576
b
d
Eudicots
Basal eudicots
Burmannia biflora
Burmannia bicolor
Burmannia oblonga
Burmannia sphagnoides
Burmannia longifolia
Cymbocarpa refracta
Gymnosiphon divaricatus
Gymnosiphon aphyllus
Hexapterella gentianoides
Campylosiphon purpurascens
Dictyostega orobanchoides
Apteria aphylla
2x
Thismiaceae
3x
*
*
Luzuriaga latifolia
Smilax glauca
Sciaphila densiflora
Sciaphila ledermannii
Pandanus tectorius
Stemona japonica
Cyclanthus bipartitus
Acanthochlamys bracteata
Japonolirion osense
Petrosavia stellaris
*
*
M
R
C
A
*
*
Thismia aseroe
Thismia rodwayi
Haplothismia exannulata
Tacca palmata
Tacca chantieri
Dioscorea rockii
Stenomeris dioscoreifolia
Areca triandra
Musa acuminata
Juncus effusus
Tradescantia ohiensis
Bomarea hirtella
Tricyrtis latifolia
Colchicum autumnale
Chamaelirium luteum
Burmanniaceae
5x
Afrothismia hydra
Afrothismia winkleri
Arachnitis uniflora
Kupea martinetugei
Corsiaceae
Triuridaceae
M
O
N
O
C
O
T
S
Petrosaviaceae
Rhizanthella gardneri
Diuris sulphurea
Orchis quadripunctata
Neottia nidus-avis
Eburophyton austinae
Wullschlaegelia calcarata
Corallorhiza maculata
Aplectrum hyemale
Dendrobium nobile
Cymbidium goeringii
Oncidium spacelatum
Erythrorchis cassythoides
Cyrtosia septentrionalis
Epistephium subrepens
Eriaxis rigida
Lecanorchis multiflora
Vanilla aphylla
Cleistes divaricata
Isotria verticillata
Cypripedium calceolus
Spiranthes cernua
Apostasia stylidoides
*
Gladiolus buckerveldii
Aristea glauca
Geosiris sp.
Alisma plantago-aquatica
Acorus calamus
Chloranthus multistachys
Hydnora africana
Prosopanche americana
Aristolochia macrophylla
Lactoris fernandeziana
Canella winterana
Laurus nobilis
Magnolia tripetala
Austrobaileya scandens
Nuphar variegata
Amborella trichopoda
0.1 substitutions/site
Orchidaceae
Iridaceae
Hydnoraceae
M
A
G
N
O
L
I
I
D
S
0
0.5
1.5
2.0
Distance to the MRCA
Fig. 1 continued
are shown on a histogram constructed with Excel 2004
(for Mac version 11.4.1). This pattern of conserved and
variable nucleotides was examined within heterotrophic
(strict mycoheterotrophic and holoparasitic taxa) and
autotrophic species separately, both groups including 54
taxa. The autotrophic species included within this analysis are all sister group lineages of the heterotrophic taxa.
Hemiparasitic and partly mycoheterotrophic taxa were
removed from the analysis. In addition, the difference
between both groups was calculated and values of steps
per nucleotide site were depicted on a histogram. Positive
and negative values represent character substitutions
contributed by heterotrophic lineages and autotrophic
123
species, respectively. Furthermore, nucleotide substitutions of each nucleotide site were associated with the
secondary structures and functional regions proposed by
Wuyts et al. (2000) and Caetano-Anollés (2002). Nucleotides within the 18S rDNA alignment were divided into
915 stem and 793 loop positions, according to secondary
structures obtained by the European Small Subunit
Ribosomal RNA database (Van de Peer et al. 2000;
Wuyts et al. 2004). Stem regions are defined as those
bases that typically participate in base pairing, with the
remainder occurring in non-base pairing loop structures.
In this study, base pairing was restricted to Watson–Crick
nucleotide pairs and G–U pairs.
J Plant Res (2011) 124:561–576
Results
Evolutionary rates
The constrained phylogram (Fig. 1a, b) reveals heterogeneity in branch lengths among the taxa investigated.
Several mycoheterotrophic and parasitic plant species
show extremely elevated substitution rates resulting in long
terminal branches (e.g. Rafflesia keithii, Rhizanthes infanticida and Arachnitis uniflora). The results of the Bayesian
relative rates test are shown in Fig. 1 (panels c, d) and
listed in Table 1. The values of the confidence intervals for
every mycoheterotrophic and parasitic lineage with the
associated outgroups are given in Table 1. In order to
analyze the effect of the implementation of constraints on
the substitution rates of mycoheterotrophic and parasitic
lineages, a second Bayesian relative rates test was conducted on the same dataset using trees from the unconstrained analysis. Overall, the confidence intervals of the
substitution rates in the constrained and non-constrained
phylogenetic analyses showed no pronounced differences,
which mean that the implementation of topology constraints did not affect branch lengths (Table 1).
According to the Bayesian relative rate test the substitution rates of several hemiparasitic (3 out of 16), holoparasitic (8 out of 17) and mycoheterotrophic (8 out of 37)
species are significantly higher compared to their autotrophic relatives (Table 1). In general, most holoparasitic
plant families share substantial elevated substitution rates,
which are on average 1.8 (Bdallophyton americanum—
Cytinaceae) to 7.3-fold (Rhizanthes infanticida—Euphorbiaceae) higher compared to their autotrophic relatives.
Only the holoparasitic families, Apodanthaceae, Cynomoriaceae, Lennoaceae and Orobanchaceae, do not have
significantly increased substitution rates. Hemiparasitic
plants are characterized by less pronounced substitution
rates where only three species are placed on significant
longer branches: average of 1.6 (Dendrophthora domingensis—Viscaceae) to 2.4-fold (Arceuthobium verticilliflorum—Viscaceae) higher compared to their autotrophic
relatives. The (facultative) hemiparasites of the families
Krameriaceae and Orobanchaceae and the remaining
Santalalean lineages are not prone to increased substitution
rates in 18S rDNA. In mycoheterotrophic plants only eight
out of 37 fully mycoheterotrophic plants have significantly
elevated substitution rates with an average increase
between 1.7 (Haplothismia exannulata—Thismiaceae) and
5.0-fold (Arachnitis uniflora—Corsiaceae). The most pronounced increase of substitution rates among mycoheterotrophic species included all five taxa of Thismiaceae
and the single taxon of Corsiaceae. Although the branch
lengths of the two mycoheterotrophic species Rhizanthella
569
gardneri (Orchidaceae) and Kupea martinetugea (Triuridaceae) are considerably shorter they are still significantly
longer compared to their autotrophic sister groups. The
Bayesian relative rates test reveals no significant variation
in rates of molecular evolution in species of the mycoheterotrophic families Burmanniaceae, Ericaceae, Gentianaceae, Iridaceae and Petrosaviaceae. However, depending
on whether Burmannia bicolor or Dioscorea rockii
was used as autotrophic relative, the mycoheterotroph
Burmannia oblonga, is either placed on a significant or
non-significant long branch than its green relatives.
The RRTree test revealed similar results compared to
the Bayesian relative rate test (Table 2). However, significant increased substitution rates were observed in five
additional mycoheterotrophic (Exacum paucisquamum and
the genus Voyria) and holoparasitic groups (Cynomorium
coccineum, Pholisma arenarium and Pilostyles thurberi).
Furthermore, mycoheterotrophic/parasitic clades (i.e.
Triuridaceae and the Santalales clade), which contain both
lineages with and without significantly increased substitution rates according to the Bayesian relative rates test, are
indicated by significant P-values for the whole clade.
Patterns of nucleotide substitution in 18S rDNA
of heterotrophic and autotrophic angiosperms
Alignment of 18S rDNA angiosperm sequences showed an
alternation of conserved and variable regions. Using the
chart option in MacClade, patterns of nucleotide substitutions were generated, describing a mosaic of conserved and
variable nucleotides across the angiosperms (Fig. 2). Due
to missing data the first and last nucleotide positions of the
alignment could not be compared among all sequences.
This resulted in a substitution pattern starting and ending in
helix structure number 8 and 49, respectively.
Variable nucleotides tend to occur both in loop and stem
structures in both autotrophs and holoparasites/mycoheterotrophs, but highly variable regions (C10 steps) are
more abundant in loop structures (e.g. 11, 17, E23_2,
E23_7, E23_12, E23_13 and 49), than in stem structures
(e.g. E10_1, 11, 43 and 49) (Fig. 2a, b). Conserved regions
with no or few mutations appear both in stem and loop
structures (8, 9, 13, 14, 15, 16, 4, 19, 20, 21, 3, 22, 23,
E23_8, E23_9, E23_10, E23_11, 26, 23, 27, 28, 30, 31, 2,
32, 33, 34, 35, 36, 38, 39, 40, 41, 42 and 34) (Fig. 2a, b).
The loop and stem structures comprised 1943 and 1745 of
total steps, respectively.
Patterns of substitutions were separately analyzed in
autotrophic (Fig. 2a) and holoparasitic/mycoheterotrophic
lineages (Fig. 2b), showing a similar distribution pattern:
the positions of conserved and variable nucleotides are
almost identical in the autotrophic and achlorophyllous
123
570
J Plant Res (2011) 124:561–576
Table 2 Values of the relative rate test for comparing molecular evolutionary rates between parasitic/mycoheterotrophic lineages (Lineage 1)
and autotrophic relatives (Lineage 2)
K1/K2
P valuea
0.012
1.3
2.2 3 1022
0.068
-0.062
0.1
3.0 9 10-1
0.059
0.068
-0.009
0.9
4.5 9 10-2
B. biflora, B. bicolor
0.033
0.053
-0.020
0.6
8.0 9 10-5
Campylosiphon, Dictyostega
B. biflora, B. bicolor
0.063
0.093
-0.029
0.7
7.7 9 10-5
Cymbocarpa, Gymnosiphon clade,
Hexapterella
B. biflora, B. bicolor
0.044
0.068
-0.024
0.6
1.0 9 10-7
Convolvulaceae
Cuscuta
Convolvulus, Ipomoea
0.077
0.044
0.033
1.8
1.0 3 1027
Corsiaceae
Arachnitis
Luzuriaga, Smilax
0.105
0.020
0.084
5.2
1.0 3 1027
Cynomoriaceae
Cynomoriumb
Hamamelis, Peridiscus
0.053
0.039
0.014
1.4
3.9 3 1023
Cytinaceae
Bdallophyton, Cytinus
Muntingia
0.072
0.041
0.031
1.8
1.0 3 1027
Ericaceae
Ericaceae clade
Arctostaphylos, Pyrola, Vaccinium
0.054
0.053
0.001
1.0
7.4 9 10-1
Euphorbiaceae
Rafflesia, Rhizanthes
Family
Lineage 1
Lineage 2
K1
K2
Apodanthaceae
Pilostylesb
Celastrus
0.055
0.043
Burmanniaceae
Apteria
B. biflora, B. bicolor
0.006
Burmannia oblonga
B. biflora, B. bicolor
Burmannia sphagnoides
K1–K2
Euphorbia, Ostodes, Ricinus
0.150
0.035
0.115
4.3
1.0 3 1027
Gentianaceae
Exacum
b
Sebaea grandis
0.064
0.050
0.014
1.3
1.5 3 1023
Gentianaceae
Sebaea oligantha
Sebaea grandis
0.051
0.050
0.001
1.0
5.2 9 10-1
Gentianaceae
Voyria cladeb
Sebaea grandis, Orphium
0.063
0.052
0.011
1.2
1.4 3 1023
Gentianaceae
Voyriella
Sebaea grandis, Orphium
0.049
0.050
-0.001
1.0
8.3 9 10-1
Hydnoraceae
Hydnora, Prosopanche
Aristolochia, Lactoris
0.086
0.039
0.047
2.2
1.0 3 1027
Iridaceae
Krameriaceae
Geosiris
Krameria
Aristea, Gladiolus
Guaiacum
0.034
0.039
0.035
0.039
-0.001
0.000
1.0
1.0
7.6 9 10-1
9.9 9 10-1
Lennoaceae
Pholismab
Hydrophyllum, Phacelia, Ehretia
0.060
0.044
0.016
1.4
7.7 3 1024
Mitrastemonaceae
Mitrastemon
Arctostaphylos, Pyrola, Vaccinium
0.086
0.043
0.044
2.0
1.0 3 1027
Orchidaceae
Corallorhiza
Aplectrum, Dendrobium
0.048
0.045
0.003
1.1
1.2 9 10-1
Orchidaceae
Erytrorchis
Cyrtosia, Eriaxis, Epistephium
0.060
0.062
-0.002
1.0
6.0 9 10-1
Orchidaceae
Lecanorchis
Vanilla, Eriaxis, Epistephium
0.050
0.054
-0.004
0.9
2.7 9 10-1
Orchidaceae
Neottia
Eburophyton
0.060
0.055
0.006
1.1
1.2 9 10-1
Orchidaceae
Rhizanthella
Diuris, Orchis
0.088
0.050
0.038
1.8
2.0 3 1027
Orchidaceae
Wullschlaegelia
Eburophyton, Diuris
0.044
0.054
-0.010
0.1
7.4 9 10-3
Orobanchaceae
Orobanchaceae clade
Paulownia, Lamium, Callicarpa
0.044
0.425
0.002
0.1
5.6 9 10-1
Petrosaviaceae
Petrosavia
Japonolirion
0.035
0.031
0.004
1.1
2.5 9 10-1
Santalales
Santalales clade
Gunnera, Peridiscus, Plumbago
0.051
0.039
0.012
1.3
6.1 3 1026
Thismiaceae
Afrothismia clade
Dioscorea, Stenomeris, Tacca
0.113
0.041
0.072
2.8
1.0 3 1027
Thismiaceae
Haplothismia, Thismia clade
Dioscorea, Stenomeris, Tacca
0.082
0.042
0.040
1.9
1.0 3 1027
Triuridacaee
Kupea, Sciaphila clade
Pandanus, Stemona
0.056
0.037
0.019
1.5
5.7 3 1026
P values of lineages with significant increased substitution rates are indicated in bold
K1 mean divergence between lineage 1 and the most recent common ancestor of lineages 1 and 2; K2 mean divergence between lineage 2 and the
most recent common ancestor of lineages 1 and 2; K1/K2 rate ratio
a
Significance of the P values \0.05
b
Lineages with significant increased substitution rates which are not observed in the Bayesian relative rates test
histograms. However, more and highly variable nucleotides
are presented on the latter histogram. Figure 2c confirms this
result by calculating the difference of steps between mycoheterotrophic/holoparasitic taxa and autotrophic species. The
distribution pattern showed that most of the substitutions
occurred within achlorophyllous taxa, revealing that mainly
heterotrophic taxa contribute to the observed mutations.
123
Discussion
Rate heterogeneity
Both Bayesian relative rate test and the distance based
relative rate test showed similar results (Tables 1, 2),
providing evidence for their accuracy and robustness.
J Plant Res (2011) 124:561–576
Helix number
8
9
10 E10_1
11 8
571
12 13 14 15 16
17
18
19
20
21
22
E23_1
E23_2
23
20
Loops
E23_8 E23_13
E23_9 E23_14
E23_4 E23_10
E23_7 E23_11
24
25
26
23
27
28
29
30
38
39
40
41
42
43
44
31 32 34 36
33 35
37
15
36
45
46
47
48 32
49
34
10
0
16
00
00
12
Stems
0
80
0
40
-5
0
Steps
5
a
-10
-15
-20
Character number
20
Loops
15
10
0
16
00
00
12
Stems
0
0
80
40
-5
0
Steps
5
b
-10
-15
-20
Character number
10
c
0
00
00
16
12
-5
0
0
80
40
0
Steps
5
Character number
Tertiary
interaction
I-S bridges
A site I-S bridge
Tertiary
I-S bridges/E site
interaction
I-S bridges P/A site A site
P/E site
A site
P site
E site P site P/A site
I-S bridge
Fig. 2 Histograms showing the pattern of nucleotide substitutions in
18S rDNA across angiosperm taxa. The histogram describes for each
nucleotide site the amount of parsimony steps or character substitutions (y-axis; a, b loops above, stems below; c heterotrophic minus
autotrophic). Helix numbering according to Wuyts et al. (2000) is
shown at the top. The major functional and structural sites according
to Caetano-Anollés (2002) are indicated at the bottom. a Pattern of
nucleotide substitution of 54 autotrophic taxa. b Pattern of nucleotide
substitution of 37 mycoheterotrophic and 17 holoparasitic taxa.
c Difference between mycoheterotrophic/parasitic and autotrophic
values of character substitutions per nucleotide site. Light gray and
dark gray shading displays the different helix structures and
functional regions, respectively
The statistical tests showed remarkably long terminal
branches for the heterotrophic taxa Rafflesia keithii, Rhizanthes infanticida, Arachnitis uniflora, Afrothismia hydra,
A. winkleri and Thismia aseroe, compared to other angiosperms. Long terminal branches were not observed in
chlorophyllous relatives of these taxa. These significantly
increased substitution rates suggest that a non-photosynthetic mode of life could be a necessary condition for the
occurrence of extremely increased substitution rates in 18S
rDNA. However, our results show that the loss of photosynthesis in 29 mycoheterotrophic and nine holoparasitic
representatives does not necessarily imply significantly
increased substitution rates in 18S rDNA. Several nonphotosynthetic lineages in Apodanthaceae, Burmanniaceae,
Cynomoriaceae, Ericaceae, Gentianaceae, Iridaceae, Lennoaceae, Orobanchaceae, Orchidaceae, Petrosaviaceae and
Triuridaceae lack significantly accelerated substitution
rates, according to the Bayesian relative rates test. On the
other hand all sampled mycoheterotrophic and holoparasitic Balanaphoraceae, Corsiaceae, Cytinaceae, Euphorbiaceae, Hydnoraceae, Mitrastemonaceae and Thismiaceae
are characterized by elevated substitution rates (Fig. 1;
Tables 1, 2). Previous studies investigating the substitution
rates in mycoheterotrophic and parasitic plants confirm the
observation that there is no evolutionary trend between the
presence or absence of chlorophyll and elevated substitution rates (mycoheterotrophs: Cameron and Chase 2000;
Molvray et al. 2000; Merckx et al. 2006, 2009 and parasites: Nickrent and Starr 1994; Nickrent et al. 1998).
Currently it is still unclear why some heterotrophic
plants accumulate much more substitutions in their ribosomal DNA than autotrophic plants, but numerous potential
hypotheses explaining causes of accelerated substitution
rates have been proposed. The long-term effects of a small
effective population size resulting in a genetic bottleneck
effect (Wu and Li 1985), the influence of a short generation
123
572
time and the correlated higher number of mutation-generating reproductive events (Wu and Li 1985), an increased
tolerance of mutations due to a relaxation of selective
constraints, variations in mutation rate (Sniegowski et al.
2000), DNA repair efficiency (Modrich and Lahue 1996)
and speciation rates (Barraclough and Savolainen 2001)
are possible factors that trigger high substitution rates in
mycoheterotrophic and parasitic plants. None of these
hypotheses could unequivocally explain higher substitution
rates in parasitic plants (Nickrent and Starr 1994; dePamphilis et al. 1997; Young and dePamphilis 2005). However,
in mycoheterotrophic plants, the effective population
hypothesis may have played an important role in many
cases of elevated substitution rates. The genera Kupea
(Triuridaceae), Afrothismia (Thismiaceae) and Arachnitis
(Corsiaceae) have very limited distribution ranges: Kupea
martinetugei has only been reported from two localities in
Cameroon (Cheek et al. 2003), Afrothismia is restricted to
extremely scattered populations in the tropical rain forests
in Africa (Maas-van de Kamer 1998; Franke et al. 2004) and
Arachnitis is restricted in its distribution to disjunct areas in
the southern part of South America (Ibisch et al. 1996). A
very limited geographic distribution or reduced effective
population size may indeed explain the significantly
increased substitution rates of 18S rDNA in these mycoheterotrophic taxa. In parasitic lineages, however, Nickrent
and Starr (1994) postulated that the effective population
hypothesis could not be the only cause of higher substitution
rates. A very restricted distribution pattern has been observed
in the holoparasites Rafflesia (Euphorbiaceae), Rhizanthes
(Euphorbiaceae) and Prosopanche (Hydnoraceae), which
have accumulated significantly more substitutions compared
to their autotrophic relatives. In contrast, Arceuthobium
(Viscaceae) species have large and extremely widespread
populations and show also significantly increased evolutionary rates. However, it remains to be studied whether populations of non-photosynthetic plants with wide distribution
ranges are still mating (see Taylor et al. 2004).
An alternative hypothesis to explain the absence or
presence of accelerated substitution rates in mycoheterotrophic plants is the absolute time when photosynthesis
capacity was lost. Non-photosynthetic plants without significant increased substitution rates might have lost their
chlorophyll only recently. However dating analysis on
Burmanniaceae, which show no significant increase in
substitution rates, suggests a Late Cretaceaus/Eocene origin of mycoheterotrophy, rejecting this hypothesis in that
case (Merckx et al. 2008). Probably no single mentioned
hypothesis will be able to explain unequivocally the substitution patterns observed in mycoheterotrophic and parasitic plants, but most likely a combination of several
mechanisms is affecting divergent rDNA sequences.
123
J Plant Res (2011) 124:561–576
Nucleotide variability and functionality of 18S rDNA
The number of substitutions per site in 18S rDNA differs
greatly among sites. Alignment of 18S rDNA sequences
reveals the presence of conserved areas with few variable
substitutions interspersed (Nickrent and Soltis 1995; Van
de Peer et al. 1993, 1996). We observed similar results in
our dataset, showing a mosaic pattern with long stretches of
conserved nucleotides and short variable regions in 18S
rDNA (Fig. 2). Both stem and loop structures contain
variable and conservative nucleotides. A higher proportion
of highly variable nucleotides (C10 steps) and a higher
number of steps occurred in loops compared to stem
structures, suggesting differences in selective constrains
between both structures. This result confirms the pattern
found in eukaryotes, where loops evolve considerably
faster than stems (Smit et al. 2007).
More important is the similar distribution pattern
between heterotrophic and related autotrophic plants
(Fig. 2a, b). Despite a high number of variable nucleotides
in mycoheterotrophic and holoparasitic plants (Fig. 2c),
mutations seem to be strictly positioned to specific nucleotide sites, suggesting fixed selective constraints in the 18S
rRNA molecule. These variable nucleotide sites occur in
regions that are known to be highly variable within
eukaryotes (Wuyts et al. 2000, 2001). The variable regions
include mainly peripherally located eukaryotic-specific
structures attached to a conserved core structure. Consequently, most conserved nucleotide sites are near the
ribosome centre with increased nucleotide variability
towards the ribosome surface (Wuyts et al. 2001). Nucleotides at the periphery of the ribosome, which are prone to
substitutions, were located in structure number 6, 10,
E10_1, 11, 17, 18, E23, 29, 43, 45 and 46. These variable
nucleotides are under less functional constraints compared
to centrally located sites with a catalytic or binding activity, which have less freedom to mutate. In Fig. 2, functional structures involved in peptidyl transferase activity,
the translational cycle and interaction between rRNA
subunits delimited by Caetano-Anollés (2002) are presented. As expected, substitutions are rare in those functional sites even within the faster evolving heterotrophic
taxa. Based on these observation we can conclude that no
major interference with the functions of the ribosome
occurs and that the small ribosomal subunit retains its
functionality, despite extremely high substitution rates in
achlorophyllous taxa detected.
Acknowledgments The authors thank Martin Bidartondo, Jonathan
Kissling, Sainge Moses and Paul Maas for sending plant material and
DNA extractions. This work was supported by the Institute for the
Promotion of Innovation by Science and Technology in Flanders
(IWT Vlaanderen, no. 71488). VM is supported by the Fund for
J Plant Res (2011) 124:561–576
Scientific Research Flanders (FWO Vlaanderen). General financial
support was provided by the K.U.Leuven (OT/05/35).
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
Appendix
List of sampled species, Genbank accession numbers and
voucher information arranged into the major Angiosperm
groups. Newly obtained 18S rDNA sequences for this study
are indicated with an asterisk. Sequences obtained from
Genbank do not list voucher information.
Taxon-Genbank accession; Voucher.
OUTGROUP
Amborella trichopoda Baill.-U42497
BASAL ANGIOSPERMS
Austrobaileya scandens C.T. White-AF206858, Nuphar
variegata Engelm. ex Clinton-AF206972
MAGNOLIIDS
Aristolochia macrophylla Lam.-AF206855, Canella
winterana Gaertn.,-AF206879, Chloranthus multistachys
P’ei-AF206885, Hydnora africana Thunb.-L25681,
Lactoris fernandeziana Phil.-LFU42783, Laurus nobilis
Cav.-AF197580, Magnolia tripetala L.-AF206956, Prosopanche americana (R.Br.) Kuntze-L24047
MONOCOTS
Acanthochlamys bracteata P.C.Kao-AY952411, Acorus
calamus L.-L24078, Afrothismia hydra Sainge &
T.Franke-DQ786083, Afrothismia winkleri Sainge &
T.Franke-EU420992,
Alisma
plantago-aquatica
L.-AF197585, Aplectrum hyemale Torr.-U59937, Apostasia
stylidioides Rchb.f.-AF135207, Arachnitis uniflora* Phil.HQ448758; Cocucci 2122, Aristea glauca KlattAF206854, Areca triandra Roxb.-AY952409, Apteria
aphylla (Nutt.) Barnh. ex Small-DQ786035, Bomarea
hirtella Herb.-AF206871, Burmannia bicolor Mart.DQ786072, Burmannia biflora L.-DQ786070, Burmannia
longifolia Becc.-AF309398, Burmannia oblonga Ridl.DQ786064, Burmannia sphagnoides Becc.-AF309400,
Campylosiphon purpurascens Benth.-EU420996, Chamaelirium luteum Miq.-AF206884, Cleistes divaricata
573
Ames-AF135205, Colchicum autumnale L.-U42072,
Corallorhiza maculata Greene-U59940, Cyclanthus
bipartitus Poit.-AF168837, Cymbidium goeringii (Rchb.f.)
Rchb.f.-AJ271248,
Cymbocarpa
refracta
MiersDQ786038, Cypripedium calceolus L.-AF069208, Cyrtosia
septentrionalis (Rchb.f.) Garay-AF135198, Dendrobium
nobile Lindle.-AB027309, Dictyostega orobanchoides
(Hook.) Miers-DQ786056, Dioscorea rockii Prain & BurkillDQ786090, Diuris sulphurea R.Br.-AF135196, Eburophyton austinae A. Heller-U59949, Epistephium subrepens
Hoehne-AF135200, Eriaxis rigida Rchb.f.-AF135201,
Erythrorchis cassythoides (A. Cunn. ex Lindl.) GarayAF135199, Geosiris sp. Bail-EU816707, Gladiolus buckerveldii (L. Bolus) Goldblatt L54062, Gymnosiphon aphyllus
Blume-AF309402, Gymnosiphon divaricatum (Benth.)
Benth. & Hook.-DQ786043, Haplothismia exannulata
Airy Shaw-DQ786082, Hexapterella gentianoides Urb.DQ786057, Isotria verticillata Raf.-AF135204, Japonolirion
osense Nakai-AF206942, Juncus effusus L.-AF206944,
Kupea martinetugei Cheek & Williams-EU816706, Lecanorchis multiflora J.J.Sm.-AF135203, Luzuriaga latifolia
Poir.-AF233091, Musa acuminata Colla-AF069226, Neottia
nidus-avis (L.) Rich.-U59948, Oncidium sphacelatum
Lindl.-U59939, Orchis quadripunctata Cirillo ex
Ten.-AF135206, Pandanus tectorius Parkinson ex J.P.du
Roi-AY952391, Petrosavia stellaris Becc.-AF206987, Rhizanthella gardneri R.S.Rogers-AF135197, Sciaphila densiflora Schltr.-EU816704, Sciaphila ledermannii* Engl.HQ448766; Merckx 128, Smilax glauca Walter-AF207022,
Spiranthes cernua Rich.-AF135195, Stemona japonica
Franch. & Sav.-AF207028, Stenomeris dioscoreifolia
Planch.-DQ786087, Tacca chantrieri André-DQ786086,
Tacca palmata Blume-EU421000, Thismia aseroe
Becc.-AF309404,Thismia rodwayi F.Muell.-AF309403,
Tradescantia ohiensis Raf.-AF069213, Tricyrtis latifolia
Maxim.-AF207046, Vanilla aphylla Wight-AF135202,
Wullschlaegelia calcarata Benh.-EU816708
BASAL EUDICOTS
Ceratophyllum demersum L.-U42517, Gunnera manicata
Linden-U43787, Platanus occidentalis L.-U42794,
Ranunculus taisanensis Hayata-D29780
CORE EUDICOTS
Arceuthobium verticilliflorum Engelm.-L24042, Crassula
marnieriana Huber & Jacobsen-U42525, Cynomorium
coccineum L.-AF039069, Dendrophthora domingensis
Eichler-X16601,
Erythropalum
scandens
BlumeDQ790111, Hamamelis virginiana L.-AF094551, Lepidoceras chilense (Molina) Kuijt-EF464459, Lepionurus
sylvestris Blume-DQ790101, Misodendrum linearifolium
123
574
DC.-L24397, Nuytsia floribunda R.Br.-DQ790103, Olax
aphylla R.Br.-L24405, Ombrophytum subterraneum
(Asplund)
B.Hansen-L24406,
Opilia
amentacea
Wall.-L24407, Pereskia aculeata Mill.-AF206986, Peridiscus lucidus Benth.-AY372815, Plumbago auriculata
Lam.-U42795, Santalum album L.–L24416, Saxifraga
integrifolia Hook.-U42810, Schoepfia arenaria Britton–
X16606, Tupeia antarctica Cham. & Schltdl.-L24425
ROSIDS
Bauhinia variegata L.-AF525295, Begonia oxyloba Welw.
ex. Hook.f.-AY968392, Bdallophyton americanum (R. Br.)
Harms-AY739089, Carica papaya L.-U42514, Celastrus
scandens L.-AY674581, Citrus aurantium L.-U38312,
Crossosoma californicum Nutt.-U42529, Cucurbita pepo
L.-AF206895, Cytinus hypocistis L.-AY739092, Davidsonia
pruriens F.Muell.-AF206897, Epilobium angustifolium
L.-AF206907, Euphorbia pulcherrima Willd. ex KlotzschL37582, Fagus grandifolia Ehrh.-AF206910, Guaiacum
sanctum L.-U42824, Krameria ixine L.-AF206948, Muntingia calabura L.-U42539, Ostodes paniculata BlumeAB268104, Pelargonium cotyledonis L’Hér-AF206982,
Pilostyles thurberi A.Gray-AY739081, Rafflesia keithii
Meijer-L24041, Rhizanthes infanticida H.Bänziger & B.
Hansen-L24048, Ricinus communis L.-AB233559, Trema
micrantha Blume-AF207044
ASTERIDS
Alangium chinense (Lour.) Harms-AF206843, Anthocleista grandiflora Gilg-AJ236026, Apium graveolens
L.-AF206852, Arctostaphylos uva-ursi Spreng.-L49272,
Boschniakia rossica (Cham. & Schltdl.) Standl.-U59951,
Bourreria succulenta Jacq.-U38319, Callicarpa dichotoma
Raeusch.-AJ236048, Chelonanthus purpurascens* (Aubl.)
Struwe, S.Nilsson & V.A.Albert-HQ448759; BINCO-FG43,
Clethra alnifolia L.-AF419793, Convolvulus arvensis
L.-AJ236013, Conopholis americana (L.) Wallr.-U59954,
Cornus florida L.-X17370, Exacum paucisquamum*
(C.B.Clarke) Klack-HQ448760; Yuan CN2k1-31, Cuscuta
gronovii Wild. ex Roem. & Schult –L24747, Ehretia cymosa
Thonn.-U59938, Epifagus virginiana (L.) W.P.C.BartonU59955, Exacum affine Balf.f.-AJ236023, Garrya elliptica Dougl. ex Lindl.-U42540, Gentiana asclepiadea*
L.-HQ448773; 19801931, Gentianella amarella* (L.) H.
Sm.-HQ448761; Fay 14626, Harveya speciosa Bernh.U59950, Hemitomes congestum* A.Gray-HQ448762;
Bidartondo s.n., Hydrophyllum fendleri A. Heller-AJ236019,
Ilex opaca Soland.-AF206938, Ipomoea hederacea Jacq.U38310, Lamium amplexicaule L.-L49287, Lathraea clandestine L.-U59941, Mitrastemon yamamotoi Makino
-AY739090, Monotropa uniflora L.-L25680, Orobanche
123
J Plant Res (2011) 124:561–576
fasciculata Nutt.-U59961, Orthocarpus erianthus Benth.U38316, Orphium frutescens* E.Mey.-HQ448763; J. Kissling and L. Zeltner 44, Paulownia tomentosa Steud.AJ236039, Pedicularis racemosa Dougl. ex Hook.-U59959,
Phacelia bicolor Torr. Ex S.Watson-L49292, Pholisma arenarium Nutt.-U59935, Pityopus californicus* (Eastw.)
Copel.-HQ448764; Bidartondo s.n., Pterospora andromedea
Nutt.-U59943, Pycnosphaera buchananii* N.E.Br.HQ448765; Bingham MG9370, Pyrola picta Sm.-U59936,
Sambucus ebulus L.-AJ236005, Sarcodes sanguinea Torr.U59945, Scaevola aemula R.Br.-AJ236008, Sebaea grandis* Steud.-HQ448767; Dessein et al. 752, Sebaea oligantha*
Schinz-HQ448768; Merckx 103, Vaccinium macrocarpon
Ait.-AF419808, Voyria aurantiaca* Splitq.-HQ448769; Maas
et al. 9610, Voyria caerulea* Aubl.-HQ448770; Maas et al.
9636, Voyria corymbosa* Splitq.-HQ448771; Maas et al.
9611, Voyriella parviflora* Miq.-HQ448772; Maas et al. 9678.
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