Extensive Intraindividual Variation in Plastid Rdna Sequences from the Holoparasite Cynomorium Coccineum (Cynomoriaceae)

J Mol Evol (2004) 58:322–332 DOI: 10.1007/s00239-003-2554-y Extensive Intraindividual Variation in Plastid rDNA Sequences from the Holoparasite Cynomorium coccineum (Cynomoriaceae) Miguel A. Garcı´ a,1 Erica H. Nicholson,2 Daniel L. Nickrent2 1 Real Jardı´ n Bota´ nico, CSIC, Plaza de Murillo 2, 28014-Madrid, Spain 2 Department of Plant Biology & Center for Systematic Biology, Southern Illinois University, Carbondale, IL 62901-6509, USA Received: 30 June 2003 / Accepted: 6 October 2003 Abstract. Ribosomal genes are considered to have a indirect evidence that they have retained some degree high degree of sequence conservation between species of functionality. This intraindividual polymorphism and also at higher taxonomic levels. In this paper we is probably a case of plastid heteroplasmy but document a case where a single individual of Cy- translocation of ribosomal cistrons to the nucleus nomorium coccineum (Cynomoriaceae), a nonphoto- or mitochondria has not been tested and therefore synthetic holoparasitic plant, contains highly cannot be ruled out. divergent plastid ribosomal genes. PCR amplification a nearly complete ribosomal DNA cistron was per- Key words: 23S rRNA — Heteroplasmy — Ri- formed using genomic DNA, the products cloned, bosomal RNA — Chloroplast — Nonphotosynthetic and the 23S rDNA genes were sequenced from19 — RNA structural model colonies. Of these, five distinct types were identified. Fifteen of the sequences were nearly identical (11 or fewer differences) and these were designated Type I. Introduction The remaining types (II–V) were each represented by a single clone and differed fromType I by 93 to 255 Within eukaryotic species, subcellular organellar ge- changes. Compared with green vascular plants, we nomes are generally assumed to be homogeneous found that there are more substitutional differences in (homoplasmic). However, heteroplasmy, the presence the 23S rDNA sequences within a single individual of of different organellar genotypes within a single Cynomorium than among all sequenced photosyn- individual, is known for mitochondrial and plastid thetic vascular plants. Several trends of molecular genomes. Heteroplasmy can arise by biparental in- evolution observed in 16S rDNA fromother holo- heritance, mutation of organelle genomes after zygote parasitic angiosperms and heterotrophic green algae formation, or variation among uniparentally trans- have been also observed in Cynomorium 23S rDNA. mitted genes when vegetative segregation is incom- Higher-order structures were constructed for repre- plete (Chesser 1998). Most of the studies on sentatives of the five clone types, and in all cases these organellar heteroplasmy have been centered on possessed complete complements of the major struc- mitochondrial DNA (mtDNA), likely because most tural elements present in functional plastid 23S heteroplasmic mitochondrial mutations in animals rRNAs. These data indicate that such molecules may (including humans) are pathogenic (Chinnery et al. be subject to purifying selection, thus providing 2000). With regard to mitochondrial heteroplasmy, issues involving the transmission genetics, population genetics, and evolution of animal mtDNA were Correspondence to: Daniel L. Nickrent; email: nickrent@plant. reviewed by Rand (2001). In plants, evidence of siu.edu intragenic recombination and therefore the existence 323 of heteroplasmy in mtDNA has been described in between 7.3% in Cynomorium (Cynomoriaceae) to Silene acaulis by Sta¨ dler and Delph (2002). Hattori et 35.9% in Corynaea (Balanophoraceae) compared to al. (2002) documented seven types of mitochondrial Nicotiana. This increase was accompanied by what the nad3–orf156 sequences in nucleus–cytoplasmhybrids authors called an ‘‘A/T drift’’ phenomenon, whereby of wheat and Aegilops and the data suggested differ- the A + T content of the SSU rDNA sequences of ential amplification of the heteroplasmic copies be- several lineages of holoparasitic angiosperms was in- tween the hybrids and the parental lines. Plastid creased compared to photosynthetic relatives. To- heteroplasmy has been detected in somatic chimeras, gether with A/T drift, an increase in transversional vs. which arise by the sorting-out of normal and chlo- transitional mutations was observed, especially in the rophyll-deficient plastids, thus resulting in variegated most highly diverged sequences. Similar molecular plants with tissue sectors of homoplasmic cells (Bo¨ r- evolutionary phenomena have been observed in some ner and Sears 1986). Lee et al. (1988) and Johnson lineages of the nonphotosynthetic chlorophyte alga and Palmer (1989) demonstrated the existence of bi- Polytoma (Nedelcu 2001; Vernon et al. 2001). Despite parental inheritance and heteroplasmic cells with the high level of sequence divergence for such SSU both chlorophyll-deficient and wild-type plastids in rRNAs, indirect evidence that these molecules have Medicago. In other cases, such as Gossypium (Lax retained functionality exists. Specifically, they retain et al. 1987), where uniparental inheritance occurs, most or all of the higher-order structures compared to heteroplasmy can occur when segregation of wild- Noller–Woese–Gutell structural models. Moreover, type and mutant plastids is incomplete and hetero- most variability occurs in regions that also vary in plasmic gametes are produced. Persistent plastid other organisms with fully functional rRNAs. heteroplasmy can be transmitted by slow vegetative In this study, we analyzed the plastid large-subunit segregation as found by Gillhamet al. (1991). Rice (LSU or 23S) rDNA sequences, inferred rRNA sec- plants derived fromanther culture contain plastids ondary structures obtained froma single individual with complete genomes as well as ones with large- of Cynomorium coccineum, and compared these with scale deletions that result in albino phenotypes sequences and structures fromother plants. The ob- (Harada et al. 1991; Yamagishi 2002). Nuclear mu- jective was to determine whether the molecular evo- tations may induce chlorophyll-deficient phenotypes lutionary trends observed in the 16S rRNA molecules but also can be originated by exposure to inhibitors also exist in these 23S rRNA molecules. Moreover, of plastid protein synthesis (Zubko and Day 1998). we wished to explore further the phenomenon of These authors found that exposure to spectinomycin heteroplasmy in this parasitic plant. leads to transmittable variegation in Brassica napus, resulting in heteroplasmic cells with normal and Materials and Methods ribosome-deficient plastids. As pointed out by Frey (1999), plastid DNA has A single specimen of Cynomorium coccineum L. was collected April great potential for accumulating mutations but the 19, 1996, in Ca´ diz, Spain (Nickrent 4063; voucher at SIU). Total lack of phenotypic markers makes detection difficult, genomic DNA was obtained by grinding the fresh tissue to a especially for pseudogenes and noncoding regions. In powder on liquid nitrogen and extracting in 2· CTAB buffer as most cases, detection of intraindividual plastid DNA described by Nickrent (1994). Nearly the entire plastid rDNA ci- polymorphisms in coding genes involves point mu- stron was PCR amplified using a TaKaRa long PCR kit (Takara Bio Inc., Shiga, Japan) with the SSU rDNA plastid-specific primer tations, as is the case for a synonymous transition in 323 for (Nickrent et al. 1997a) and the 23S rDNA 2749rev primer the psbC gene in Actinidia (Chat et al. 2002) or the (Table 1). The reactions were conducted in a Perkin–Elmer Ther- psbA gene that confers triazine resistance in Senecio mal Cycler with one cycle at 94°C for 3 min and 35 cycles at 94°C vulgaris (Frey et al. 1999). Plastid 16S and 23S rDNA for 1 min, 55°C for 1 min, 72°C for 3 min, followed by a final gene sequences are highly conserved, thus are less extension at 72°C for 5 min. A 4.3-kb PCR product was obtained and gel purified using Gel Elute spin columns (Sigma Aldrich, Inc., likely to show intraindividual polymorphisms. In this St. Louis, MO). The PCR product was cloned using the PCRII paper, we document a case of intraindividual varia- vector froma TA cloning kit (Invitrogen Corporation, Carlsbad, tion in the highly conserved plastid rRNA genes of CA) and 19 colonies were selected. Only one of the clones (Cyno0) Cynomorium coccineum, a perennial holoparasitic was obtained froma different PCR reaction but fromthe same (nonphotosynthetic) angiosperm. genomic DNA extraction. The plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia, CA). The Molecular evolutionary studies of plastid-encoded presence of inserts was checked by restriction digestion (HindIII) ribosomal genes in nonphotosynthetic organisms followed by electrophoresis of the fragments in an agarose gel. have been limited exclusively to small-subunit (SSU, Manual sequencing was conducted using the SequiThermEXCEL 16S) rRNA. In several lineages of nonasterid holo- II DNA Sequencing kit (EpiCentre Technologies, Madison, WI) parasitic angiosperms, Nickrent et al. (1997a) showed with the internal primers listed in Table 1. Complete 23S rDNA sequences for 19 clones were obtained an increased rate of nucleotide substitution in 16S fromthe Cynomorium sample and deposited in the NCBI database rDNAs genes compared to photosynthetic angio- (Table 2). For comparative analyses, additional plastid 23S rDNA sperms. The divergence in these holoparasites ranged sequences were obtained fromGenBank. These were Pinus thun- 324 Table 1. Primers used to sequence plastid 23S rDNA Primer sequence

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