Armbruster, Georg F. J.

Korte, Anja

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RESEARCH NOTESGenomic nucleotide variation in the ITS1 rDNA spacerof land snailsGeorg F. J. Armbruster1 and Anja Korte21University of Basel, Department of Integrative Biology, Conservation Biology Unit, St. Johanns-Vorstadt 10, CH - 4056 Basel, Switzerland;2University of Leipzig, Institute of Zoology, Department of Molecular Evolution & Animal Systematics, Talstr. 33, 04103 Leipzig, GermanyRibosomal RNA is coded by the rDNA gene cluster. Hundredsof rDNA loci are consecutively arranged and form repeatedmotifs on chromosomes. The Internal Transcribed Spacer 1(ITS1) is part of the eukaryotic rDNA locus. This spacer is arelatively fast evolving, ‘non-coding’ element. It is often usedin phylogenetic reconstructions of closely related taxa. TheITS1 separates two functional regions: the 18S rDNA and the5.8S rDNA (Fig. 1).There are usually no or very few ITS1 nucleotide polymorph-isms within an animal species (0–3% intraspeciﬁc variation1–4).The phenomenon of very low intraspeciﬁc rDNA variation isexplained by the concept of concerted evolution in the genepool of a species under molecular drive.5–7 Yet, there areexceptions from the rule of very low intraspeciﬁc rDNA vari-ation, particularly in crustaceans8 and in some species-ﬂocks.For example, in the Cicindela dorsalis complex (tiger beetle), inthe Simulium damnosum group (West African black ﬂies), incorals with reticulate evolution and in a few plant species thegenomic variation in the ITS1 is extremely high, with somesequences showing as much as 20–35% intraspeciﬁc diver-gence.9–12 In the following contribution, we analyse the extentof ITS1 variation of four taxonomically robust species of landsnail of different family categories, i.e. within Acanthinula aculeata(Valloniidae sensu lato ), Arianta arbustorum (Helicoidea), Cochlo-dina ﬁmbriata (Clausiliidae), and Vallonia costata (Valloniidaesensu stricto ). We show that one species (A. aculeata ) exhibits ahigh level of ITS1 variation, whilst another species (V. costata )displays nearly no variation. The other two taxa displaymoderate variation. We also examine the ITS1 rDNA forhighly conserved sequence motifs and investigate the extent ofnucleotide variation within such motifs. Evolutionarily con-served regions in ribosomal spacers are suspected to be involvedin the maturation of the precursor rRNA.13–15 Finally, wediscuss the possible occurrence of rDNA pseudogenes in PCRamplicons and genome databases.DNA was extracted from 13 snail specimens. Polymerasechain reaction (PCR) of the ITS1 rDNA was performed withﬂanking 18S and 5.8S primers. PCR products were cloned inpCR blunt vectors (INVITROGEN), transformed in E. coli,and isolated and sequenced in both directions (AccessionNos AY267026-AY267030, AF124052-AF124053, AY546422-AY546468). Number of analysed sequences per species was asfollows: A. aculeata ¼ 23 cloned ITS1 molecules obtained fromthree individuals (collected in Germany and Switzerland), A.arbustorum ¼ 17 from four individuals (two unknown Dutchsites, Switzerland and Estonia), C. ﬁmbriata ¼ 9 from one indi-vidual (Switzerland), and V. costata ¼ 5 from ﬁve individuals(Germany, Great Britain and Austria). Details on samplingsites, DNA extraction and PCR are available in the ElectronicSupplementary Data.The consensus alignment of the 54 sequences is 662 bp long,and includes ITS1 sequences of 522 bp to 599 bp (see Sup-plementary Data and EMBL/EBI Alignment AccessionNo. ALIGN_000906). Genetic distances (D ) were calculatedamong all cloned sequences from the ITS1 regions usingKimura’s 2-parameter method implemented in MEGA,version 2.1.16 The D values give information about genomicvariability within the samples. The highest genomic D was0.061 (within A. aculeata ), whilst A. arbustorum, C. ﬁmbriata andV. costata showed lower D values (with maxima of 0.022, 0.018and 0.002, respectively). A neighbour-joining tree was recon-structed using MEGA (Fig. 2). Each sequence was correctlyassigned to its predicted species level, and the interspeciﬁcsignal was not confounded by genomic D values (Fig. 2).Approximately 190 nucleotide positions of the ITS1 are invar-iant in all cloned sequences (i.e. 32–36% of the alignmentlength). The invariant nucleotides are conserved amongspecies from the Helicoidea, Clausiliidae and Valloniidae. Thelast common ancestor of these three families lived in theUpper-Mesozoic, .70 million years ago (for origin of extantland snail family lineages, see Ref. 17). It is possible that theseinvariant ITS1 nucleotide positions are evolving under func-tional constraints, i.e. they could be involved in the processingpathway of the precursor rRNA.Subsequently, the ITS1 was analysed on variable nucleotidepositions: (1) in phylogenetically very ‘old’ and conservedregions; and (2) in phylogenetically unconserved segments.(1) Six highly conserved regions of the stylommatophoran-basommatophoran split 150 million years ago17 have beenfound previously.18 These six regions were concatenated andare referred to as ‘StyloBaso’ block (black boxes in Fig. 1).These six regions can be aligned across stylommatophoranland snails and basommatophoran freshwater snails,18 and arerelatively constant in length. The concatenated StyloBasobox ranged in length from 110 bp (in A. aculeata ) to 115 bp (inC. ﬁmbriata ). In A. aculeata, 18 variable positions (nucleotidesubstitutions and indels) occurred in the StyloBaso region(Table 1). Thus, even in sequence motifs of 150 million years,intraspeciﬁc ITS1 variation was found. (2) In the remainingpart of the ITS1 of A. aculeata (length between 450 and480 bp), 84 variable sites were detected. This means that atotal of 102 polymorphic nucleotide positions occurred in theITS1 of this species, i.e. 17–19% of the nucleotide positionswere variable (see Table 1). The other land snail speciesshowed lower percentage values of variable sites, with 0.2%(V. costata ), 3% (C. ﬁmbriata ), and 7% (A. arbustorum ). Thesepercentage values are only estimates because the probability ofdetection of variable nucleotide positions depends on thenumber of cloned sequences, and on sequence length. We there-fore performed a partial rarefaction analysis (with the MEGAsequence editor). As target groups we focused on A. aculeata(23 sequences; ITS1 alignment length ¼ 603 bp) andCorrespondence: G. F. J. Armbruster; e-mail: g.armbruster@unibas.chJournal of Molluscan Studies (2006) 72: 211–219# The Author 2006. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.A. arbustorum (17 sequences; ITS1 alignment length ¼ 589 bp).In each species, we analysed two randomly chosen sequencesand divided the number of observed polymorphic nucleotidesby alignment length. This was repeated with twenty replicatesfor each species. Finally, we calculated mean and standard devi-ation for both species. The mean value of the twenty replicates inA. aculeata was 0.029 (SD ¼ 0.012), whilst A. arbustorum had amean value of 0.016 (SD ¼ 0.007). The rarefaction analysis indi-cated that there were signiﬁcant differences in nucleotide vari-ation between A. aculeata and A. arbustorum (t-test with unequalvariance, P , 0.001).Focusing on the concept of concerted evolution, the questionarises of how effective is genomic homogenization of the ITS1rDNA? In experimental studies, a relaxation of concerted evol-ution was found in arbuscular mycorrhizal fungi.19 Also in theland snail A. aculeata, concerted evolution seems both relaxedand incomplete (with high nucleotide variation in PCR ampli-cons of three individuals from Switzerland and Germany).Arianta arbustorum and C. ﬁmbriata also showed a tendencytowards incomplete homogenization. In V. costata, however, con-certed evolution and genomic homogenization is nearly com-plete, indicated by just a single polymorphic nucleotide in ﬁveFigure 1. The ITS1 region with ﬂanking 18S and 5.8S rDNA. The 30-end of the 18S rDNA exhibits the animal-speciﬁc motif GATCATTA. The 50-terminus of the 5.8S rDNA starts in land snails with the signal AACTTTGA. Relative position and length of the six 150-million-year-old StyloBasoblocks are shown with black boxes (length of each region in bp indicated above each box; see text and Table 1). The StyloBaso consensus regionsare inherited from the last common ancestor of terrestrial pulmonate gastropods (Stylommatophora) and pulmonate freshwater snails(Basommatophora).18Figure 2.Neighbour-joining tree of 54 cloned ITS1 sequences of four land snail species. Bootstrap values higher than 95% are indicated (based on 1000replicates). Asterisks display clones with a complete lack of the ﬂanking 18S termination signal GATCATTA, i.e. putative genomic pseudogenes.Names refer to cloned sequences; see Supplementary Data for detailed information.RESEARCH NOTES212sequences from individuals from Great Britain, Austria andGermany (see Supplementary Data).Beside relaxed concerted evolution, an additional explanationfor the observed intragenomic ITS1 variation may lie elsewhere.Polymorphisms within genomes may be attributed to solitary,incognito, pseudogene variants that ‘escape’ the homogenizationprocess of the rRNA gene cluster. Several studies focus on thistopic, for example in plants12,20 and humans.21–22 Threesequences in our data set could have been incognito variantssince they show a lack of the animal-speciﬁc termination motifGATCATTA of the 18S rDNA (see Fig. 1; Accession NosAY546427, AY546443, AY546444). These three sequences aremarked with an asterisk in Figure 2. A lack of this terminationmotif provides evidence that rDNA pseudogene sequencescould have been cloned from the PCR amplicons. Yet, allITS1 sequences do group conspeciﬁcally (Fig. 2), pointing tolittle background noise in phylogenetic reconstructions. Wetherefore suggest that signals of rDNA pseudogenes should befurther studied.In summary, the high number of invariant ITS1 nucleotidepositions could make land snails potential model organisms forfurther investigations of ITS1 rRNA processing. Nevertheless,efﬁciency of concerted evolution of ITS1 rDNA seems to varybetween land snail species. Extent of ITS1 variation shouldalways be elucidated, particularly if direct sequencing is usedand ‘unreadable’ sequencing proﬁles appear from PCR tem-plates. The occurrence of rDNA pseudogenes requires furtherstudy. For example, in the human genome database we founda full-length 18S copy on the Y-chromosome, which is anescaped variant of the rRNA gene cluster of chromosomenumber 13, 14, 15, 21 or 22 (data are available on request).The Y-chromosomal sequence shows 89% nucleotide identityto the original autosomal 18S rDNA (see Ref. 22: pp. 894-895). Hence, escaped gene copies can show a relatively highsimilarity to their orthologous counterparts. Such copies mightalso appear in PCR amplicons.ACKNOWLEDGEMENTWe thank B. Gautschi and B. Koller (Ecogenics GmbH,Schlieren, Switzerland) for excellent cooperation. Threereviewers gave valuable help to improve the manuscript. Thestudy was funded by the NLU budget of the ConservationBiology Unit of the University of Basel.REFERENCES1. SCHLO¨TTERER, C., HAUSER, M.T., VON HAESELER, A. &TAUTZ, D. 1994. Mol. Biol. Evol., 11: 513–522.2. ZHUO, L., SAJDAK, S.L. & PHILLIPS, R.B. 1994. Genome, 37:664–671.3. HOLDER, K., MONTGOMERIE, R. & FRIESEN, V.L. 1999.Evolution, 53: 1936–1950.4. GANDOLFI, A., BONILAURI, P., ROSSI, V. & MENOZZI, P.2001. Heredity, 87: 449–455.5. DOVER, G. 1982. Nature, 299: 111–117.6. ELDER, J.F. Jr, & TURNER, B.J. 1995. Quart. Rev. Biol., 70:297–320.7. DOVER, G. 2002. Trends Gen., 18: 587–589.8. HARRIS, D.J. & CRANDALL, K.A. 2000. Mol. Biol. Evol., 17:284–291.9. VOGLER, A.P. & DESALLE, R. 1994. Mol. Biol. Evol., 11:393–405.10. TANG, J., TOE, L., BACK, C. & UNNASCH, T.R. 1996. Mol.Biol. Evol., 13: 244–252.11. ODORICO, D.M. & MILLER, D.J. 1997. Mol. Biol. Evol., 14:465–473.12. BUCKLER IV, E.S., IPPOLITO, A. &HOLTSFORD, T.P. 1997.Genetics, 145: 821–832.13. MAI, J.C. & COLEMAN, A.W. 1997. J. Mol. Evol., 44: 258–271.14. JOSEPH, N., KRAUSKOPF, E., VERA, M.I. & MICHOT,B. 1999. Nucl. Acids Res., 27: 4533–4540.15. VENEMA, J. & TOLLERVEY, D. 1999. Ann. Rev. Genet., 33:261–311.16. KUMAR, S., TAMURA, K., JAKOBSEN, I.B. & NEI, M. 2001.Bioinformatics, 17: 1244–1245.17. TILLIER, S., MASSELOT, M. & TILLIER, A. 1996. In: Originand evolutionary radiation of the Mollusca. (J.D. Taylor ed.), 267–284.Oxford University Press, Oxford.18. ARMBRUSTER, G.F.J., VAN MOORSEL, C.H.M. &GITTENBERGER, E. 2000. J. Moll. Stud., 66: 570–573.19. PAWLOWSKA, T.E. & TAYLOR, J.W. 2004. Nature, 427:733–737.20. RUGGIERO, M.V. & PROCACCINI, G. 2004. J. Mol. Evol., 58:115–121.21. GONZALEZ, I.L. & SYLVESTER, J.E. 1997. Genome Res., 7:65–70.22. INTERNATIONAL HUMAN GENOME SEQUENCINGCONSORTIUM 2001. Nature, 409: 860–921.doi:10.1093/mollus/eyi056Advance Access Publication: 23 September 2005Table 1. Summary of variable nucleotide positions in the ITS1 rDNA ofthe four species (see text).Species(no. of individualsanalysed/no. ofcloned sequences)Consensus regions of landsnails and basommato-phoran freshwatersnails (six black boxes)Remaining partof the ITS1Concatenatedlength (bp)VariablepositionsConcatenatedlength (bp)VariablepositionsA. aculeata (3/23) 110–113 18 450–480 84A. arbustorum (4/17) 111–112 11 443–462 32C. fimbriata (1/9) 115 2 450–452 15V. costata (5/5) 114 1 407 0RESEARCH NOTES213

Genomic nucleotide variation in the ITS1 rDNA spacer of land snails

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