Intragenomic Variation Research Articles

Measuring rDNA diversity in eukaryotic microbial systems: how intragenomic variation, pseudogenes, and PCR artifacts confound biodiversity estimates

Molecular approaches have revolutionized our ability to study the ecology and evolution of micro-organisms. Among the most widely used genetic markers for these studies are genes and spacers of the rDNA operon. However, the presence of intragenomic rDNA variation, especially among eukaryotes, can potentially confound estimates of microbial diversity. To test this hypothesis, bacterially cloned PCR products of the internal transcribed spacer (ITS) region from clonal isolates of Symbiodinium, a large genus of dinoflagellates that live in symbiosis with many marine protists and invertebrate metazoa, were sequenced and analysed. We found widely differing levels of intragenomic sequence variation and divergence in representatives of Symbiodinium clades A to E, with only a small number of variants attributed to Taq polymerase/bacterial cloning error or PCR chimeras. Analyses of 5.8S-rDNA and ITS2 secondary structure revealed that some variants possessed base substitutions and/or indels that destabilized the folded form of these molecules; given the vital nature of secondary structure to the function of these molecules, these likely represent pseudogenes. When similar controls were applied to bacterially cloned ITS sequences from a recent survey of Symbiodinium diversity in Hawaiian Porites spp., most variants (approximately 87.5%) possessed unstable secondary structures, had unprecedented mutations, and/or were PCR chimeras. Thus, data obtained from sequencing of bacterially cloned rDNA genes can substantially exaggerate the level of eukaryotic microbial diversity inferred from natural samples if appropriate controls are not applied. These considerations must be taken into account when interpreting sequence data generated by bacterial cloning of multicopy genes such as rDNA.

Analysis of intragenomic variation of the rDNA internal transcribed spacers (ITS) in Halichondrida (Porifera: Demospongiae)

Three species of sponges, Axinella aruensis, Phakellia sp. and Acanthella cavernosa, were selected to investigate the extent of intragenomic variation (IGV) in the internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA within the order Halichondrida. The IGV was detected in both the spacers and in the three species. At least three non-identical sequences were found in any single individual with the number of polymorphic sites within species ranging from 6 to 77. Uncorrected distance values up to 29% were observed in the ITS-2 of Axinella aruensis. The levels of polymorphism found in this species are the highest reported for Porifera and comparable to those observed in hybrid species of scleractinean corals. Analyses of ITS sequences from multiple clones from individuals of Halichondrida are strongly recommended if these genetic markers are to be used to depict phylogenetic and/or phylogeographic relationships of closely related species and their populations. Data from other nuclear and mitochondrial markers should be used to complement conclusions derived from studies based on ITS sequences.

Empreinte moléculaire des communautés bactériennes et hétérogénéité intraspécifique de l'ADNr 16S: est-il raisonnable d'occulter le problème?

Molecular fingerprinting methods are currently used to study microbial communities by culture independent approaches. They are proposed as identification tool owing to the availability of rapid automated methods. The 16S rRNA gene (16S rDNA) is an efficient marker for bacterial identification and microbial communities analysis. However, the 16S rDNA polymorphism among strains of the same species is an underestimated pitfall of the fingerprinting approaches. We studied the 16S rDNA variability among strains of three bacterial species of medical interest. Total DNA was extracted from clinical isolates of Pseudomonas aeruginosa (N=20), Clostridium difficile (N=20) and Enterobacter cloacae (N=14). The Polymerase Chain Reaction (PCR) products obtained with consensus primers flanking the 16S rDNA variable regions V3 and V6-V7-V8 were separated by Temporal Temperature Gradient gel Electrophoresis (TTGE). DNA extracted from TTGE bands were sequenced and analysed. All the isolates of P. aeruginosa and of C. difficile displayed one single TTGE band with constant migration distances suggesting that there was no 16S rDNA polymorphism among strains in these two species. Oppositely, the isolates of E. cloacae gave complex TTGE patterns formed by multiple bands with variable migration distances. These patterns corresponded to 16S rRNA genes variable in a single genome as well as among strains of the species. Intra-species and/or intragenomic variability of 16S rDNA should be taken into account for pertinent interpretation of molecular fingerprint. For this purpose, a comprehensive description of the polymorphism of this marker is necessary.

Sequence diversity and evolution of multigene families in Trypanosoma cruzi

Several copies of genes belonging to three multigene families present in the genome of Trypanosoma cruzi were sequenced and comparatively analyzed across six different strains of the parasite belonging to the T. cruzi I lineage (Colombiana, Silvio X10 and Dm28c), the T. cruzi II lineage (Esmeraldo and JG) and a hybrid strain (CL Brener). For all three gene families analyzed, our results support the division in T. cruzi I and II lineages. Furthermore, in agreement with its hybrid nature, sequences derived from the CL Brener clone clustered together with T. cruzi II sequences as well as with a third group of sequences. Paralogous sequences encoding Amastin, an amastigote surface glycoprotein and TcAG48, an antigenic RNA binding protein, which are clustered in the parasite genome, present higher intragenomic variability in T. cruzi II and CL Brener strains, when compared to T. cruzi I strains. Paralogous sequences derived from the TcADC gene family, which encode various isoforms of adenylyl cyclases and are dispersed throughout the T. cruzi genome, exhibit similar degree of variability in all strains, except in the CL Brener strain, in which the sequences were more divergent. Several factors including mutation rates and gene conversion mechanisms, acting differently within the T. cruzi population, may contribute to create such distinct levels of sequence diversity in multigene families that are clustered in the T. cruzi genome.

Morphological and molecular characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp. Nov.

K. Řeháková, J.R. Johansen, D.A. Casamatta, L. Xuesong and J. Vincent. 2007. Morphological and molecular characterization of selected desert soil cyanobacteria: three species new to science including Mojavia pulchra gen. et sp.nov. Phycologia 46: 481–502. DOI: 10.2216/06-92.1Four Nostocacean species from desert soils of the western United States, including the phycobiont of the lichen Collema tenax, were studied. Our strains could be forced into morphospecies previously described from Europe, but phylogenetic analysis indicated that they belonged in separate, distinct, and previously undescribed taxa. Partial 16S rRNA sequences of the strains CM1-VF10, CM1-VF14, CNP-AK1 and JT2-VF2 were determined and aligned with published Nostoc sequences from GenBank and our lab, as well as other Nostocales. All aligned sequences were analysed using parsimony, distance, and maximum likelihood methods, and trees based on three separate data sets were generated. Full 16S-23S internal transcribed spacer (ITS) regions were also characterized for our strains, and secondary structures of the ITS region were compared among these and N. commune and N. punctiforme. Intragenomic variability was documented among ITS regions in different operons for these taxa. One of the four strains (JT2-VF2) is distinct from Nostoc by both morphological and molecular criteria and is described as Mojavia pulchra gen. et sp. nov. Two other strains (CM1-VF10 and CM1-VF14) are described as Nostoc indistinguendum sp. nov. and Nostoc desertorum sp. nov., respectively. According to both morphological and molecular characteristics, the phycobiont of C. tenax is not N. commune, N. sphaericum or N. punctiforme as variously suggested in the lichenological literature, and the older name for this taxon, Nostoc lichenoides, is consequently validated in this paper.

Extensive variation in synonymous substitution rates in mitochondrial genes of seed plants.

BackgroundIt has long been known that rates of synonymous substitutions are unusually low in mitochondrial genes of flowering and other land plants. Although two dramatic exceptions to this pattern have recently been reported, it is unclear how often major increases in substitution rates occur during plant mitochondrial evolution and what the overall magnitude of substitution rate variation is across plants.ResultsA broad survey was undertaken to evaluate synonymous substitution rates in mitochondrial genes of angiosperms and gymnosperms. Although most taxa conform to the generality that plant mitochondrial sequences evolve slowly, additional cases of highly accelerated rates were found. We explore in detail one of these new cases, within the genus Silene. A roughly 100-fold increase in synonymous substitution rate is estimated to have taken place within the last 5 million years and involves only one of ten species of Silene sampled in this study. Examples of unusually slow sequence evolution were also identified. Comparison of the fastest and slowest lineages shows that synonymous substitution rates vary by four orders of magnitude across seed plants. In other words, some plant mitochondrial lineages accumulate more synonymous change in 10,000 years than do others in 100 million years. Several perplexing cases of gene-to-gene variation in sequence divergence within a plant were uncovered. Some of these probably reflect interesting biological phenomena, such as horizontal gene transfer, mitochondrial-to-nucleus transfer, and intragenomic variation in mitochondrial substitution rates, whereas others are likely the result of various kinds of errors.ConclusionThe extremes of synonymous substitution rates measured here constitute by far the largest known range of rate variation for any group of organisms. These results highlight the utility of examining absolute substitution rates in a phylogenetic context rather than by traditional pairwise methods. Why substitution rates are generally so low in plant mitochondrial genomes yet occasionally increase dramatically remains mysterious.

Patterns of Mutation and Selection at Synonymous Sites in Drosophila

That natural selection affects molecular evolution at synonymous sites in protein-coding sequences is well established and is thought to predominantly reflect selection for translational efficiency/accuracy mediated through codon bias. However, a recently developed maximum likelihood framework, when applied to 18 coding sequences in 3 species of Drosophila, confirmed an earlier report that the Notch gene in Drosophila melanogaster was evolving under selection in favor of those codons defined as unpreferred in this species. This finding opened the possibility that synonymous sites may be subject to a variety of selective pressures beyond weak selection for increased frequencies of the codons currently defined as "preferred" in D. melanogaster. To further explore patterns of synonymous site evolution in Drosophila in a lineage-specific manner, we expanded the application of the maximum likelihood framework to 8,452 protein coding sequences with well-defined orthology in D. melanogaster, Drosophila sechellia, and Drosophila yakuba. Our analyses reveal intragenomic and interspecific variation in mutational patterns as well as in patterns and intensity of selection on synonymous sites. In D. melanogaster, our results provide little statistical evidence for recent selection on synonymous sites, and Notch remains an outlier. In contrast, in D. sechellia our findings provide evidence in support of selection predominantly in favor of preferred codons. However, there is a small subset of genes in this species that appear to be evolving under selection in favor of unpreferred codons, which indicates that selection on synonymous sites is not limited to the preferential fixation of mutations that enhance the speed or accuracy of translation in this species.

An Assessment of Potential Diatom “Barcode” Genes (cox1, rbcL, 18S and ITS rDNA) and their Effectiveness in Determining Relationships in Sellaphora (Bacillariophyta)

Due to limited morphological differentiation, diatoms can be very difficult to identify and cryptic speciation is widespread. There is a need for a narrower species concept if contentious issues such as diatom biodiversities and biogeographies are to be resolved. We assessed the effectiveness of several genes (cox1, rbcL, 18S and ITS rDNA) to distinguish cryptic species within the model 'morphospecies', Sellaphora pupula agg. This is the first time that the suitability of cox1 as an identification tool for diatoms has been assessed. A range of cox1 primers was tested on Sellaphora and various outgroup taxa. Sequences were obtained for 34 isolates belonging to 22 Sellaphora taxa and three others (Pinnularia, Eunotia and Tabularia). Intraspecific divergences ranged from 0 to 5bp (=0.8%) and interspecific levels were at least 18bp (=c. 3%). Cox1 divergence was usually much greater than rbcL divergence and always much more variable than 18S rDNA. ITS rDNA sequences were more variable than cox1, but well-known problems concerning intragenomic variability caution against its use in identification. More information and less sequencing effort mean that cox1 can be a very useful aid in diatom identification. The usefulness of cox1 for determining phylogenetic relationships among Sellaphora species was also assessed and compared to rbcL. Tree topologies were very similar, although support values were generally lower for cox1.

Intragenomic diversity and geographical adaptability of diploid cotton species revealed by cytogenetic studies

Cotton is one of the most important crops in Iran, and is cultivated in different regions of the country. Gossypium herbaceum is one of the A-genome cottons, which is a potentially important genetic resource for cotton breeding programs. Collecting native cultivars of this species growing in different regions is a vital step in broadening variability of the gene pool. The G. herbaceum is one of the two cultivated species under cultivation in Iran, which is specifically adapted to a given environment and includes more than 40 ecotypes, named as landrace cottons. The present paper reports the intragenomic characteristics analysis of 42 G. herbaceum cultivars in the cotton genebank using cytological methods. The karyological studies showed variations within the species in the size of chromosome, chromosome volume and karyotype formulae. All cultivars possessed 2n=26 chromosome, but varied with regard to number of SAT-chromosomes (ranging from 1 to 3) and the chromosomes carrying secondary constructions. Karyotypes were of symmetrical type, having small chromosomes. Analysis of variance revealed significant differences between the cultivars as well as the chromosomes. Cluster analysis could group the cultivars in four distinct clusters. The present study indicates genomic differences among diploid G. herbaceum cultivars, which can be used in cotton hybridization programs in Iran or other countries.

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2-15) and contain 2.4 unique ITS length variants (range: 1-12) and 2.8 unique sequence variants (range: 1-12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family.

Real-time PCR reveals a high incidence of Symbiodinium clade D at low levels in four scleractinian corals across the Great Barrier Reef: implications for symbiont shuffling

Reef corals form associations with an array of genetically and physiologically distinct endosymbionts from the genus Symbiodinium. Some corals harbor different clades of symbionts simultaneously, and over time the relative abundances of these clades may change through a process called symbiont shuffling. It is hypothesized that this process provides a mechanism for corals to respond to environmental threats such as global warming. However, only a minority of coral species have been found to harbor more than one symbiont clade simultaneously and the current view is that the potential for symbiont shuffling is limited. Using a newly developed real-time PCR assay, this paper demonstrates that previous studies have underestimated the presence of background symbionts because of the low sensitivity of the techniques used. The assay used here targets the multi-copy rDNA ITS1 region and is able to detect Symbiodinium clades C and D with >100-fold higher sensitivity compared to conventional techniques. Technical considerations relating to intragenomic variation, estimating copy number and non-symbiotic contamination are discussed. Eighty-two colonies from four common scleractinian species (Acropora millepora, Acropora tenuis, Stylophora pistillata and Turbinaria reniformis) and 11 locations on the Great Barrier Reef were tested for background Symbiodinium clades. Although these colonies had been previously identified as harboring only a single clade based on SSCP analyses, background clades were detected in 78% of the samples, indicating that the potential for symbiont shuffling may be much larger than currently thought.

Phylogeographic patterns of Armillariaostoyae in the western United States

SummaryNuclear ribosomal DNA regions (i.e. large subunit, internal transcribed spacer, 5.8S and intergenic spacer) were sequenced using a direct‐polymerase chain reaction method from Armillaria ostoyae genets collected from the western USA. Many of the A. ostoyae genets contained heterogeneity among rDNA repeats, indicating intragenomic variation and likely intraspecific hybridization. Intragenomic variation was verified by visually editing base‐sequence offsets in regions with insertions/deletions, and using sequence‐specific internal primers to resequence heterogeneous regions. Phylogenetic analyses with Bayesian Inference methods were used to define groups within A. ostoyae. Analysis of A. ostoyae from outside the western USA indicated the presence of a Circumboreal group of A. ostoyae that also occurs in Utah; two other phylogeographic groups were associated with the Rocky Mountain and Pacific Northwest regions of the USA. Mixed sequence types, an indication of intraspecific hybrids, were common in some geographic regions. Hybridization events may have influenced species evolution, contributing to variation in pathogenicity and virulence. The occurrence of these groups and intraspecific hybrids also indicates that paleogeography and paleoclimate may have influenced the phylogeography of A. ostoyae. In addition, other Armillaria species were examined for evolutionary relationships with the groups of A. ostoyae. These findings will provide a basis for future research relating ecological function to genetic diversity within A. ostoyae.

A Phylogeographic Split in Buxus balearica (Buxaceae) as Evidenced by Nuclear Ribosomal Markers: When ITS Paralogues Are Welcome

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict.

Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data

Repeat families within genomes are often maintained with similar sequences. Traditionally, this has been explained by concerted evolution, where repeats in an array evolve "in concert" with the same sequence via continual turnover of repeats by recombination. Another form of evolution, birth-and-death evolution, can also explain this pattern, although in this case selection is the critical force maintaining the repeats. The level of intragenomic variation is the key difference between these two forms of evolution. The prohibitive size and repetitive nature of large repeat arrays have made determination of the absolute level of intragenomic repeat variability difficult, thus there is little evidence to support concerted evolution over birth-and-death evolution for many large repeat arrays. Here we use whole-genome shotgun sequence data from the genome projects of five fungal species to reveal absolute levels of sequence variation within the ribosomal RNA gene repeats (rDNA). The level of sequence variation is remarkably low. Furthermore, the polymorphisms that are detected are not functionally constrained and seem to exist beneath the level of selection. These results suggest the rDNA is evolving via concerted evolution. Comparisons with a repeat array undergoing birth-and-death evolution provide a clear contrast in the level of repeat array variation between these two forms of evolution, confirming that the rDNA indeed does evolve via concerted evolution. These low levels of intra-genomic variation are consistent with a model of concerted evolution in which homogenization is very rapid and efficiently maintains highly similar repeat arrays.

Intragenomic rDNA ITS2 Variation in the Neotropical Anopheles (Nyssorhynchus) albitarsis Complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.

Intragenomic diversity and phylogenetic systematics of wild rosemaries (Rosmarinus officinalis L. s.l., Lamiaceae) assessed by nuclear ribosomal DNA sequences (ITS)

Nuclear ribosomal sequences (ITS) were used to study species boundaries and to infer phylogenetic patterns in wild rosemaries (Rosmarinus officinalis, R. eriocalyx, R. tomentosus). Intragenomic polymorphisms (overlapping peaks and in some cases unreadable sequences) were found throughout the sequencing electrophoretograms of most Rosmarinus accessions. Sequencing the cloned ITS products from representative individuals resulted in 25 ribotypes differing at 59 variable sites. Average sequence divergence among clones was 1.75%, and the most divergent sequences differed by 3.48%. No single ribotype was shared between any two-paired species. The highest values of intragenomic divergence were similar in R. officinalis (1.63%) and R. eriocalyx (1.14%–2.12%), and contrast with those shown by R. tomentosus (0.97%). Sequence data suggest that most divergent rDNA sequences within individuals belong to paralogous loci that apparently are not pseudogenes. A detailed inspection of direct and cloned sequences does not show evidence that the intragenomic polymorphism found is due to interspecific hybridization. Phylogenetic analyses of cloned sequences suggested that both R. officinalis and R. tomentosus were monophyletic, whereas R. tomentosus clones were nested within a paraphyletic R. eriocalyx.

Fish-isolated Naegleria strains and their phylogeny inferred from ITS and SSU rDNA sequences

Effort was made to identify Naegleria strains isolated from organs of fish, using phylogenetic analyses of SSU rDNA and ITS sequences. Eighteen fish-isolated strains studied enlarged substantially the so far available set of Naegleria strains characterized by both molecular markers. The phylogenetic analyses of separate and concatenated SSU rDNA and ITS sequences revealed phylogenetic relationships of strains under study; however, they failed to solve classification of fish-isolated strains into species. The sequence similarity of strain-representatives of Naegleria species as well as data obtained on intragenomic variation of ITS sequences discouraged the authors from the definition of new species. The results of the present study provide evidence of a need to re-evaluate the current practice of setting boundaries between species of the genus Naegleria. Sequences obtained in this study have been deposited in GenBank with accession numbers DQ768714-DQ768743.

An equilibrium partitioning model connecting gene expression and cis-motif content

Thermodynamic favorability of transcription factor (TF) binding to DNA is a significant factor in the control of gene expression. Theoretical and in vitro measures link the relative equilibrium energy of a particular DNA binding protein to the sequence variation among binding sites in a genome. Extending this principle, we investigate whether biological variation in expression levels of active proteins leads to regulation of different sets of genes, based on inferred affinities of sites upstream of those genes. The TF-concentration-dependent variation in the repertoire of genes regulated by a particular TF is expected to follow patterns of chemical partitioning over DNA sites having differing affinity, and we develop a new modeling approach to test this hypothesis. Based on computational TF binding site discovery and genome-wide expression data available in Saccharomyces cerevisiae, we explore motif content for sets of genes and conditions having varying concentrations of different transcription factors which turn those genes on or off. We find cases of significant correlation between the level of intragenomic motif sequence variation and modeled TF protein levels that actuates regulation of corresponding sets of genes, and discuss the observed TF motif variants for several yeast transcription factors, as well as the potential biological functions of genes that are regulated by differential response to these high and low concentrations of particular TFs. These findings suggest that motif sequences of transcription factor binding sites may often be linked with the expression state of corresponding DNA-binding proteins.

FIRST ANALYSIS OF GENETIC VARIABILITY IN CARINTHIAN POPULATIONS OF THE WHITE-CLAWED CRAYFISH AUSTROPOTAMOBIUS PALLIPES

Several authors have speculated about the nativeness of the white-clawed crayfish (Austropotamobius pallipes) in the Austrian province of Carinthia, since this is the only occurrence of the species within the Danube river system. Phylogeographic analyses based on mitochondrial DNA sequences showed that Carinthian A. pallipes are closely related to specimens from Slovenia, Croatia and north-eastern Italy. These analyses additionally pointed to an absence of variability among populations in Carinthia. In the present study a larger number of individuals sampled from three different brooks in western Carinthia was investigated by analysing sequences of the mtDNA cytochrome c oxidase gene (COI), microsatellite DNA and microsatellite length variation in the nuclear ribosomal DNA internal transcribed spacer region (ITS1). Not only COI sequences revealed low degrees of genetic variability, but also microsatellite loci displayed low allelic diversity and low heterozygosity. Analysis of ITS1 microsatellite length variation in turn showed high levels of intragenomic variability, since single individuals manifested up to seven distinct fragments. Microsatellite data and ITS1 microsatellite length variation analyses also revealed significant differentiation among some population pairs. Since genetic divergence was mainly based on differences in allele frequencies and not on the occurrence of alternative alleles, we assume that Carinthian populations became isolated rather recently and that the occurrence of this species in this region is more likely to be a result of human introduction than of natural postglacial colonisation.

Periodic Extinctions of Transposable Elements in Bacterial Lineages: Evidence from Intragenomic Variation in Multiple Genomes

Most previous work on the evolution of mobile DNA was limited by incomplete sequence information. Whole genome sequences allow us to overcome this limitation. I study the nucleotide diversity of prominent members of five insertion sequence families whose transposition activity is encoded by a single transposase gene. Eighteen among 376 completely sequenced bacterial genomes and plasmids carry between 3 and 20 copies of a given insertion sequence. I show that these copies generally show very low DNA divergence. Specifically, more than 68% of the transposase genes are identical within a genome. The average number of amino acid replacement substitutions at amino acid replacement sites is Ka = 0.013, that at silent sites is Ks = 0.1. This low intragenomic diversity stands in stark contrast to a much higher divergence of the same insertion sequences among distantly related genomes. Gene conversion among protein-coding genes is unlikely to account for this lack of diversity. The relation between transposition frequencies and silent substitution rates suggests that most insertion sequences in a typical genome are evolutionarily young and have been recently acquired. They may undergo periodic extinction in bacterial lineages. By implication, they are detrimental to their host in the long run. This is also suggested by the highly skewed and patchy distribution of insertion sequences among genomes. In sum, one can think of insertion sequences as slow-acting infectious diseases of cell lineages.