Alternative splicing is highly variable among Daphnia pulex lineages in response to acute copper exposure.

BACKGROUND: Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. RESULTS: By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. CONCLUSIONS: Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies.

Speciation with gene flow and the genetics of habitat transitions.

Whether speciation can advance to completion in the face of initially high levels of gene flow is a very controversial topic in evolutionary biology. Extensive gene exchange is generally considered to homogenize populations and counteract divergence. Moreover, the role of introgressive hybridization in evolution remains largely unexplored in animals, particularly in freshwater zooplankton in which allopatric speciation is considered to be the norm. Our work investigates the genetic structure of two young ecological species: the pond species, Daphnia pulex and the lake species, Daphnia pulicaria. Phylogenetic and population genetics analyses were conducted on mitochondrial NADH dehydrogenase 5 (ND5) gene, the nuclear Lactate dehydrogenase (Ldh) gene and 21 nuclear microsatellite markers in 416 individuals from habitats with various degrees of permanence. The strong and consistent phylogenetic discordance between nuclear and mitochondrial markers suggests a complex evolutionary history of multiple independent habitat transition events that involved hybridization and introgression between lake and pond Daphnia. On the other hand, the low level of contemporary gene flow between adjacent populations indicates the presence of effective habitat isolating barriers. The Daphnia system provides strong evidence for a divergence-with-gene flow speciation model that involves multiple habitat transition events.

Evolutionary factors affecting Lactate dehydrogenase A and B variation in the Daphnia pulex species complex.

BACKGROUND: Evidence for historical, demographic and selective factors affecting enzyme evolution can be obtained by examining nucleotide sequence variation in candidate genes such as Lactate dehydrogenase (Ldh). Two closely related Daphnia species can be distinguished by their electrophoretic Ldh genotype and habitat. Daphnia pulex populations are fixed for the S allele and inhabit temporary ponds, while D. pulicaria populations are fixed for the F allele and inhabit large stratified lakes. One locus is detected in most allozyme surveys, but genome sequencing has revealed two genes, LdhA and LdhB. RESULTS: We sequenced both Ldh genes from 70 isolates of these two species from North America to determine if the association between Ldh genotype and habitat shows evidence for selection, and to elucidate the evolutionary history of the two genes. We found that alleles in the pond-dwelling D. pulex and in the lake-dwelling D. pulicaria form distinct groups at both loci, and the substitution of Glutamine (S) for Glutamic acid (F) at amino acid 229 likely causes the electrophoretic mobility shift in the LDHA protein. Nucleotide diversity in both Ldh genes is much lower in D. pulicaria than in D. pulex. Moreover, the lack of spatial structuring of the variation in both genes over a wide geographic area is consistent with a recent demographic expansion of lake populations. Neutrality tests indicate that both genes are under purifying selection, but the intensity is much stronger on LdhA. CONCLUSIONS: Although lake-dwelling D. pulicaria hybridizes with the other lineages in the pulex species complex, it remains distinct ecologically and genetically. This ecological divergence, coupled with the intensity of purifying selection on LdhA and the strong association between its genotype and habitat, suggests that experimental studies would be useful to determine if variation in molecular function provides evidence that LDHA variants are adaptive.

Evolution of the nuclear ribosomal DNA intergenic spacer in four species of the Daphnia pulex complex.

BACKGROUND: Concerted evolution refers to the pattern in which copies of multigene families show high intraspecific sequence homogeneity but high interspecific sequence diversity. Sequence homogeneity of these copies depends on relative rates of mutation and recombination, including gene conversion and unequal crossing over, between misaligned copies. The internally repetitive intergenic spacer (IGS) is located between the genes for the 28S and 18S ribosomal RNAs. To identify patterns of recombination and/or homogenization within IGS repeat arrays, and to identify regions of the IGS that are under functional constraint, we analyzed 13 complete IGS sequences from 10 individuals representing four species in the Daphnia pulex complex. RESULTS: Gene conversion and unequal crossing over between misaligned IGS repeats generates variation in copy number between arrays, as has been observed in previous studies. Moreover, terminal repeats are rarely involved in these events. Despite the occurrence of recombination, orthologous repeats in different species are more similar to one another than are paralogous repeats within species that diverged less than 4 million years ago. Patterns consistent with concerted evolution of these repeats were observed between species that diverged 8-10 million years ago. Sequence homogeneity varies along the IGS; the most homogeneous regions are downstream of the 28S rRNA gene and in the region containing the core promoter. The inadvertent inclusion of interspecific hybrids in our analysis uncovered evidence of both inter- and intrachromosomal recombination in the nonrepetitive regions of the IGS. CONCLUSIONS: Our analysis of variation in ribosomal IGS from Daphnia shows that levels of homogeneity within and between species result from the interaction between rates of recombination and selective constraint. Consequently, different regions of the IGS are on substantially different evolutionary trajectories.

Evolution of repeated sequences in the ribosomal DNA intergenic spacer of 32 arthropod species.

The evolution of a multigene family (MGF) is affected by the structure and function of its regulatory elements, specifically by the link between recombination and DNA transcription and/or replication. The ribosomal DNA (rDNA) MGF is often hierarchically repetitive, combining function with repetition in a single genic system. Its tandemly repeated operons contain the transcription unit of the 45S ribosomal RNA precursor alternating with an intergenic spacer (IGS) that commonly includes repeated transcription regulatory elements. To study the evolution of repeated sequences and the influence of repeat characteristics on their sequence divergence, we sequenced and characterized a single complete IGS from 11 daphniid species and analyzed their repeat arrays along with those from an additional 21 species of arthropods. We tested the hypotheses that sequence similarity is higher among tandemly arrayed repeats than among interleaved or dispersed repeats, and that the homogeneity of repeat arrays is affected by the number and the length of repeats, as well as by the presence of putative regulatory elements. We found that both tandem repeat organization and the presence of a TATA motif are significantly correlated with increased sequence similarity among homologous IGS repeats. We also observed that some repeat types are only found in a single species, while others appear to have persisted for >100 MY, with evidence for homologous repeat types in sister species. Taken together, these data suggest that both drift and natural selection influence repeat evolution within the IGS.

Metal exposure causes rDNA copy number to fluctuate in mutation accumulation lines of Daphnia pulex.

Ribosomal (r)DNA is a highly dynamic, conserved, multigene family whose sequence homogeneity is thought to be maintained by intra- and interchromosomal recombination, which are capable of changing rDNA copy number. It is generally not known how environmental stress such as sublethal exposure to environmentally relevant concentrations of metals impacts rDNA copy number. To determine how chronic metal exposure affects rDNA, we measured copy number of the 18S rRNA gene in 355 copper and nickel-exposed samples and 132 metal-free samples derived from 325 mutation accumulation (MA) lines of two genetically distinct Daphnia pulex lineages. The MA lines were sampled at four time points over 100+ generations of clonal propagation. The copy number of rDNA was also measured in 15 individuals sampled from a metal-free non-MA control population established from the same progenitor as one of the MA lineages. We found that mean rDNA copy number fluctuated across lines exposed to metals with a tendency to decrease over time. In contrast, mean rDNA copy number in the metal-free control lines and the non-MA population remained stable over time. It is generally accepted that extreme rDNA loss results in the loss of organism fitness. Thus, fluctuations in rDNA copy number, including losses, could affect the long-term viability of natural populations of Daphnia in metal-contaminated habitats.

Length variation in 18S rRNA expansion segment 43/e4 of Daphnia obtusa: ancient or recurring polymorphism?

Expansion segments in ribosomal DNA (rDNA) can show length variation at the level of the individual, yet our understanding of the evolutionary forces shaping this variation is incomplete. Previous studies of expansion segment 43/e4 of the 18S rRNA gene in Daphnia obtusa have examined this variation in six individuals; however, it is not known if the variation documented at this locus is representative of variation across the species' geographic range. Furthermore, it is unclear whether length variants found in multiple individuals share common ancestry, or were generated de novo through recombination. We quantified expansion segment length variant frequencies in 134 individual D. obtusa from 33 populations at 15 sites across the species range in the US, and used a phylogeographic approach to determine whether recombination continues to add to the standing crop of variation at this locus. We identified seven length variants across the sampling range, which spans almost 3000 km. Based on the phylogeographic distribution of length variants in the expansion segment, we conclude that they are shared ancient polymorphisms that have persisted despite the operation of molecular mechanisms that cause the concerted evolution of multigene families such as rDNA.

D- and L-lactate dehydrogenases during invertebrate evolution.

BACKGROUND: The L-lactate and D-lactate dehydrogenases, which are involved in the reduction of pyruvate to L(-)-lactate and D(+)-lactate, belong to evolutionarily unrelated enzyme families. The genes encoding L-LDH have been used as a model for gene duplication due to the multiple paralogs found in eubacteria, archaebacteria, and eukaryotes. Phylogenetic studies have suggested that several gene duplication events led to the main isozymes of this gene family in chordates, but little is known about the evolution of L-Ldh in invertebrates. While most invertebrates preferentially oxidize L-lactic acid, several species of mollusks, a few arthropods and polychaetes were found to have exclusively D-LDH enzymatic activity. Therefore, it has been suggested that L-LDH and D-LDH are mutually exclusive. However, recent characterization of putative mammalian D-LDH with significant similarity to yeast proteins showing D-LDH activity suggests that at least mammals have the two naturally occurring forms of LDH specific to L- and D-lactate. This study describes the phylogenetic relationships of invertebrate L-LDH and D-LDH with special emphasis on crustaceans, and discusses gene duplication events during the evolution of L-Ldh. RESULTS: Our phylogenetic analyses of L-LDH in vertebrates are consistent with the general view that the main isozymes (LDH-A, LDH-B and LDH-C) evolved through a series of gene duplications after the vertebrates diverged from tunicates. We report several gene duplication events in the crustacean, Daphnia pulex, and the leech, Helobdella robusta. Several amino acid sequences with strong similarity to putative mammalian D-LDH and to yeast DLD1 with D-LDH activity were found in both vertebrates and invertebrates. CONCLUSION: The presence of both L-Ldh and D-Ldh genes in several chordates and invertebrates suggests that the two enzymatic forms are not necessarily mutually exclusive. Although, the evolution of L-Ldh has been punctuated by multiple events of gene duplication in both vertebrates and invertebrates, a shared evolutionary history of this gene in the two groups is apparent. Moreover, the high degree of sequence similarity among D-LDH amino acid sequences suggests that they share a common evolutionary history.

Rates of recombination in the ribosomal DNA of apomictically propagated Daphnia obtusa lines.

Ribosomal (r)DNA undergoes concerted evolution, the mechanisms of which are unequal crossing over and gene conversion. Despite the fundamental importance of these mechanisms to the evolution of rDNA, their rates have been estimated only in a few model species. We estimated recombination rate in rDNA by quantifying the relative frequency of intraindividual length variants in an expansion segment of the 18S rRNA gene of the cladoceran crustacean, Daphnia obtusa, in four apomictically propagated lines. We also used quantitative PCR to estimate rDNA copy number. The apomictic lines were sampled every 5 generations for 90 generations, and we considered each significant change in the frequency distribution of length variants between time intervals to be the result of a recombination event. Using this method, we calculated the recombination rate for this region to be 0.02-0.06 events/generation on the basis of three different estimates of rDNA copy number. In addition, we observed substantial changes in rDNA copy number within and between lines. Estimates of haploid copy number varied from 53 to 233, with a mean of 150. We also measured the relative frequency of length variants in 30 lines at generations 5, 50, and 90. Although length variant frequencies changed significantly within and between lines, the overall average frequency of each length variant did not change significantly between the three generations sampled, suggesting that there is little or no bias in the direction of change due to recombination.

Distribution of the DNA transposon family, Pokey in the Daphnia pulex species complex.

BACKGROUND: The Pokey family of DNA transposons consists of two putatively autonomous groups, PokeyA and PokeyB, and two groups of Miniature Inverted-repeat Transposable Elements (MITEs), mPok1 and mPok2. This TE family is unusual as it inserts into a specific site in ribosomal (r)DNA, as well as other locations in Daphnia genomes. The goals of this study were to determine the distribution of the Pokey family in lineages of the Daphnia pulex species complex, and to test the hypothesis that unusally high PokeyA number in some isolates of Daphnia pulicaria is the result of recent transposition. To do this, we estimated the haploid number of Pokey, mPok, and rRNA genes in 45 isolates from five Daphnia lineages using quantitative PCR. We also cloned and sequenced partial copies of PokeyA from four isolates of D. pulicaria. RESULTS: Haploid PokeyA and PokeyB number is generally less than 20 and tends to be higher outside rDNA in four lineages. Conversely, the number of both groups is much higher outside rDNA (~120) in D. arenata, and PokeyB is also somewhat higher inside rDNA. mPok1 was only detected in D. arenata. mPok2 occurs both outside (~30) and inside rDNA (~6) in D. arenata, but was rare (≤2) outside rDNA in the other four lineages. There is no correlation between Pokey and rRNA gene number (mean = 240 across lineages) in any lineage. Variation among cloned partial PokeyA sequences is significantly higher in isolates with high number compared to isolates with an average number. CONCLUSIONS: The high Pokey number outside rDNA in D. arenata and inside rDNA in some D. pulicaria isolates is consistent with a recent increase in transposition rate. The D. pulicaria increase may have been triggered by insertion of PokeyA into a region of transcriptionally active rDNA. The expansion in D. arenata (thought to be of hybrid origin) may be a consequence of release from epigenetic repression following hybridization. Previous work found D. obtusa to be very different from the D. pulex complex; mean PokeyA is higher in rDNA (~75), rDNA array size is nearly twice as large (415), and the two are positively correlated. The predominance of Pokey in only one location could be explained by purifying selection against ectopic recombination between elements inside and outside rDNA.

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high-throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and "populations" of various species in our communities, we examine the impact of intra- and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59-84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31-63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group-specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.

Functional and ecological significance of rDNA intergenic spacer variation in a clonal organism under divergent selection for production rate.

It has recently been hypothesized that variation in the intergenic spacer (IGS) of rDNA has considerable developmental, evolutionary and ecological significance through effects on growth rate and body C : N : P stoichiometry resulting from the role of the IGS in production of rRNA. To test these ideas, we assessed changes in size and structure of the repetitive region of the IGS, juvenile growth rate (JGR), RNA and phosphorus (P) contents in clonal lineages of Daphnia pulex derived from a single female and subjected to divergent selection on weight-specific fecundity (WSF). As a result of selection, WSF diverged rapidly, with significant reductions within two generations. Other significant changes accompanying shifts in WSF were that juveniles produced by low-WSF females grew more rapidly and had higher RNA and P contents. An increased predominance of long IGS variants was observed in lineages with elevated JGRs and low WSF. The observed variations in IGS length were related to the number of subrepeat units carrying a promoter sequence in the repetitive region. These results strongly support the hypothesized relationships, indicate a genetic mechanism for the evolution of such associations and demonstrate that Daphnia (and perhaps other parthenogens) possess considerable potential for rapid adaptive change in major life-history traits.

Gene expression variation in duplicate lactate dehydrogenase genes: do ecological species show distinct responses?

Lactate dehydrogenase (LDH) has been shown to play an important role in adaptation of several aquatic species to different habitats. The genomes of Daphnia pulex, a pond species, and Daphnia pulicaria, a lake inhabitant, encode two L-LDH enzymes, LDHA and LDHB. We estimated relative levels of Ldh gene expression in these two closely related species and their hybrids in four environmental settings, each characterized by one of two temperatures (10°C or 20°C), and one of two concentrations of dissolved oxygen (DO; 6.5-7 mg/l or 2-3 mg/l). We found that levels of LdhA expression were 4 to 48 times higher than LdhB expression (p<0.005) in all three groups (the two parental species and hybrids). Moreover, levels of LdhB expression differed significantly (p<0.05) between D. pulex and D. pulicaria, but neither species differed from the hybrid. Consistently higher expression of LdhA relative to LdhB in both species and the hybrid suggests that the two isozymes could be performing different functions. No significant differences in levels of gene expression were observed among the four combinations of temperature and dissolved oxygen (p>0.1). Given that Daphnia dwell in environments characterized by fluctuating conditions with long periods of low dissolved oxygen concentration, we suggest that these species could employ regulated metabolic depression to survive in such environments.

Copy number of the transposon, Pokey, in rDNA is positively correlated with rDNA copy number in Daphnia obtuse [corrected].

Pokey is a class II DNA transposon that inserts into 28S ribosomal RNA (rRNA) genes and other genomic regions of species in the subgenus, Daphnia. Two divergent lineages, PokeyA and PokeyB have been identified. Recombination between misaligned rRNA genes changes their number and the number of Pokey elements. We used quantitative PCR (qPCR) to estimate rRNA gene and Pokey number in isolates from natural populations of Daphnia obtusa, and in clonally-propagated mutation accumulation lines (MAL) initiated from a single D. obtusa female. The change in direction and magnitude of Pokey and rRNA gene number did not show a consistent pattern across ∼ 87 generations in the MAL; however, Pokey and rRNA gene number changed in concert. PokeyA and 28S gene number were positively correlated in the isolates from both natural populations and the MAL. PokeyB number was much lower than PokeyA in both MAL and natural population isolates, and showed no correlation with 28S gene number. Preliminary analysis did not detect PokeyB outside rDNA in any isolates and detected only 0 to 4 copies of PokeyA outside rDNA indicating that Pokey may be primarily an rDNA element in D. obtusa. The recombination rate in this species is high and the average size of the rDNA locus is about twice as large as that in other Daphnia species such as D. pulicaria and D. pulex, which may have facilitated expansion of PokeyA to much higher numbers in D. obtusa rDNA than these other species.

Impact of ploidy level on the distribution of Pokey element insertions in the Daphnia pulex complex.

BACKGROUND: Transposable elements (TEs) play a major role in genome evolution. Their capacity to move and/or multiply in the genome of their host may have profound impacts on phenotypes and dramatic consequences on genome structure. The population dynamics and distribution of TEs are influenced by their mode of transposition, the availability of niches in host genomes, and host population dynamics. Theories predict an increase in the number of TE insertions following hybridization or polyploidization. Evolution of TEs in hybrids and polyploids has mostly been studied in plants; few studies have examined the impacts of hybridization and/or polyploidization on TEs in animals. Hybrids and polyploids have arisen multiple times in the Daphnia pulex complex and are thought to reproduce by obligate parthenogenesis. Our study examines the effects of ploidy level on polymorphism and number of Pokey element insertions in diploid and polyploid hybrid isolates from the Daphnia pulex complex. RESULTS: The polymorphism of Pokey insertion sites did not depend solely on either the ploidy level or the genetic background of their host; therefore, it may be the result of interactions between these parameters and other parameters such as Pokey activity, selection and/or drift. No significant effect of ploidy level was found on the number of Pokey insertions using TE display and qPCR. However, the load of Pokey insertion sites and the number of unique insertion sites were slightly (but not significantly) higher in polyploids than in diploids. CONCLUSIONS: These results suggest a lack of increase in the number of Pokey insertions following polyploidization but higher availability of Pokey insertion sites in polyploids than in diploids. Compared to previous TE display and qPCR results, the load of Pokey insertions in hybrid diploids was higher than in non-hybrid sexual and asexual diploids, which suggests an increase in the density of Pokey insertions following hybridization.

Evolution of a transposon in Daphnia hybrid genomes.

BACKGROUND: Transposable elements play a major role in genome evolution. Their capacity to move and/or multiply in the genome of their host may have profound impacts on phenotypes, and may have dramatic consequences on genome structure. Hybrid and polyploid clones have arisen multiple times in the Daphnia pulex complex and are thought to reproduce by obligate parthenogenesis. Our study examines the evolution of a DNA transposable element named Pokey in the D. pulex complex. RESULTS: Portions of Pokey elements inserted in the 28S rRNA genes from various Daphnia hybrids (diploids and polyploids) were sequenced and compared to sequences from a previous study to understand the evolutionary history of the elements. Pokey sequences show a complex phylogenetic pattern. We found evidence of recombination events in numerous Pokey alleles from diploid and polyploid hybrids and also from non-hybrid diploids. The recombination rate in Pokey elements is comparable to recombination rates previously estimated for 28S rRNA genes in the congener, Daphnia obtusa. Some recombinant Pokey alleles were encountered in Daphnia isolates from multiple locations and habitats. CONCLUSIONS: Phylogenetic and recombination analyses showed that recombination is a major force that shapes Pokey evolution. Based on Pokey phylogenies, reticulation has played and still plays an important role in shaping the diversity of the D. pulex complex. Horizontal transfer of Pokey seems to be rare and hybrids often possess Pokey elements derived from recombination among alleles encountered in the putative parental species. The insertion of Pokey in hotspots of recombination may have important impacts on the diversity and fitness of this transposable element.

In and out of the rRNA genes: characterization of Pokey elements in the sequenced Daphnia genome.

BACKGROUND: Only a few transposable elements are known to exhibit site-specific insertion patterns, including the well-studied R-element retrotransposons that insert into specific sites within the multigene rDNA. The only known rDNA-specific DNA transposon, Pokey (superfamily: piggyBac) is found in the freshwater microcrustacean, Daphnia pulex. Here, we present a genome-wide analysis of Pokey based on the recently completed whole genome sequencing project for D. pulex. RESULTS: Phylogenetic analysis of Pokey elements recovered from the genome sequence revealed the presence of four lineages corresponding to two divergent autonomous families and two related lineages of non-autonomous miniature inverted repeat transposable elements (MITEs). The MITEs are also found at the same 28S rRNA gene insertion site as the Pokey elements, and appear to have arisen as deletion derivatives of autonomous elements. Several copies of the full-length Pokey elements may be capable of producing an active transposase. Surprisingly, both families of Pokey possess a series of 200 bp repeats upstream of the transposase that is derived from the rDNA intergenic spacer (IGS). The IGS sequences within the Pokey elements appear to be evolving in concert with the rDNA units. Finally, analysis of the insertion sites of Pokey elements outside of rDNA showed a target preference for sites similar to the specific sequence that is targeted within rDNA. CONCLUSIONS: Based on the target site preference of Pokey elements and the concerted evolution of a segment of the element with the rDNA unit, we propose an evolutionary path by which the ancestors of Pokey elements have invaded the rDNA niche. We discuss how specificity for the rDNA unit may have evolved and how this specificity has played a role in the long-term survival of these elements in the subgenus Daphnia.

Copy number variation of ribosomal DNA and Pokey transposons in natural populations of Daphnia.

BACKGROUND: Despite their ubiquity and high diversity in eukaryotic genomes, DNA transposons are rarely encountered in ribosomal DNA (rDNA). In contrast, R-elements, a diverse group of non-LTR retrotransposons, specifically target rDNA. Pokey is a DNA transposon that targets a specific rDNA site, but also occurs in many other genomic locations, unlike R-elements. However, unlike most DNA transposons, Pokey has been a stable component of Daphnia genomes for over 100 million years. Here we use qPCR to estimate the number of 18S and 28S ribosomal RNA genes and Pokey elements in rDNA (rPokey), as well as other genomic locations (gPokey) in two species of Daphnia. Our goals are to estimate the correlation between (1) the number of 18S and 28S rRNA genes, (2) the number of 28S genes and rPokey, and (3) the number of rPokey and gPokey. In addition, we ask whether Pokey number and distribution in both genomic compartments are affected by differences in life history between D. pulex and D. pulicaria. RESULTS: We found differences in 18S and 28S gene number within isolates that are too large to be explained by experimental variation. In general, Pokey number within isolates is modest (< 20), and most are gPokey. There is no correlation between the number of rRNA genes and rPokey, or between rPokey and gPokey. However, we identified three isolates with unusually high numbers of both rPokey and gPokey, which we infer is a consequence of recent transposition. We also detected other rDNA insertions (rInserts) that could be degraded Pokey elements, R- elements or the divergent PokeyB lineage recently detected in the Daphnia genome sequence. Unlike rPokey, rInserts are positively correlated with rRNA genes, suggesting that they are amplified by the same mechanisms that amplify rDNA units even though rPokey is not. Overall, Pokey frequency and distribution are similar in D. pulex and D. pulicaria suggesting that differences in life history have no impact on Pokey. CONCLUSIONS: The possibility that many rDNA units do not contain a copy of both 18S and 28S genes suggests that rDNA is much more complicated than once thought, and warrants further study. In addition, the lack of correlation between rPokey, gPokey and rDNA unit numbers suggests that Pokey transposition rate is generally very low, and that recombination, in combination with natural selection, eliminates rPokey much faster than gPokey. Our results suggest that further research to determine the mechanisms by which Pokey has escaped complete inactivation by its host (the usual fate of DNA transposons), would provide important insights into transposon biology.

Transcontinental phylogeography of the Daphnia pulex species complex.

Daphnia pulex is quickly becoming an attractive model species in the field of ecological genomics due to the recent release of its complete genome sequence, a wide variety of new genomic resources, and a rich history of ecological data. Sequences of the mitochondrial NADH dehydrogenase subunit 5 and cytochrome c oxidase subunit 1 genes were used to assess the global phylogeography of this species, and to further elucidate its phylogenetic relationship to other members of the Daphnia pulex species complex. Using both newly acquired and previously published data, we analyzed 398 individuals from collections spanning five continents. Eleven strongly supported lineages were found within the D. pulex complex, and one lineage in particular, panarctic D. pulex, has very little phylogeographical structure and a near worldwide distribution. Mismatch distribution, haplotype network, and population genetic analyses are compatible with a North American origin for this lineage and subsequent spatial expansion in the Late Pleistocene. In addition, our analyses suggest that dispersal between North and South America of this and other species in the D. pulex complex has occurred multiple times, and is predominantly from north to south. Our results provide additional support for the evolutionary relationships of the eleven main mitochondrial lineages of the D. pulex complex. We found that the well-studied panarctic D. pulex is present on every continent except Australia and Antarctica. Despite being geographically very widespread, there is a lack of strong regionalism in the mitochondrial genomes of panarctic D. pulex--a pattern that differs from that of most studied cladocerans. Moreover, our analyses suggest recent expansion of the panarctic D. pulex lineage, with some continents sharing haplotypes. The hypothesis that hybrid asexuality has contributed to the recent and unusual geographic success of the panarctic D. pulex lineage warrants further study.

Development of primers for the mitochondrial cytochrome c oxidase I gene in digenetic trematodes (Platyhelminthes) illustrates the challenge of barcoding parasitic helminths.

The phylum Platyhelminthes is a diverse group of flatworms that includes parasites with serious impacts on human health, animal husbandry, aquaculture and wildlife management. Here we present degenerate primers for the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene in flatworms. Although amplicons were obtained from a wide taxonomic range in the Cestoda and Trematoda, COI fragments from many taxa in these classes did not amplify. Primers specific to trematodes in the family Diplostomidae were also developed. Amplification success was much higher with diplostomid-specific primers and sequences were obtained from 504 of 585 specimens of Diplostomum and Tylodelphys. Sequences from the barcode region resolved all specimens to the species level, with mean divergence between congeners of 19% (3.9-25%). Because many of our specimens were small, we initially amplified part of the nuclear small subunit ribosomal (r) RNA gene to evaluate the quality and quantity of DNA in our specimens. Short sequences (~380 nt) of this gene were recovered from most specimens and can be used to distinguish specimens at the family level and often the generic level. We suggest that rRNA genes could be used to screen samples of completely unknown taxonomy, after which specific COI primers could be used to obtain species-level identifications.