Intragenomic Variation Research Articles

Concerted evolution rapidly eliminates sequence variation in rDNA coding regions but not in intergenic spacers in Nicotiana tabacum allotetraploid

Nicotiana tabacum (tobacco) is a natural allotetraploid that formed from two diploid progenitors (N. sylvestris—S-genome, N. tomentosiformis—T-genome) within past 0.2 million years. Previous classical studies have shown that its 35S rDNA has been largely homogenised towards T-genome-like homeologs. However, the degree of conversion at single nucleotide resolution remains unknown. Here, we analysed intragenomic variation of rDNA at high resolution in natural tobacco, synthetic tobacco and the progenitors employing genomic, molecular and cytogenetic methods. In synthetic tobacco, we identified 13 highly (≥10% units) polymorphic sites in the 18S-5.8S-26S coding region. In contrast, only a single polymorphic site was detected in natural tobacco, indicating that gene conversion has removed most of the polymorphisms over shallow evolutionary times. However, the non-coding 26S-18S intergenic spacer (IGS) was highly polymorphic in both natural (57 polymorphic sites) and synthetic tobacco (128 polymorphic sites). In natural tobacco, most (64%) IGS polymorphisms were inherited from the N. tomentosiformis progenitor, while 36% appeared de novo indicating rapid rates of sequence divergence of IGS. FISH revealed that the T-genome-like units (harbouring N. tomentosiformis-type IGS) occurred on all four loci in tobacco variety 095-55, including those loci derived from N. sylvestris progenitor, while the variety SR-1 retained 1–2 S-genome loci unconverted and transcriptionally silenced. We discuss potential caveats associated with experimental and in silico approaches used for determination of rDNA polymorphisms. We also hypothesise that polyploidy-associated gene conversion may eliminate mutated and non-functional genes that have accumulated in progenitor genomes, thereby contributing to success of polyploidy species.

Using high-throughput sequencing of ITS2 to describe Symbiodinium metacommunities in St. John, US Virgin Islands.

Symbiotic microalgae (Symbiodinium spp.) strongly influence the performance and stress-tolerance of their coral hosts, making the analysis of Symbiodinium communities in corals (and metacommunities on reefs) advantageous for many aspects of coral reef research. High-throughput sequencing of ITS2 nrDNA offers unprecedented scale in describing these communities, yet high intragenomic variability at this locus complicates the resolution of biologically meaningful diversity. Here, we demonstrate that generating operational taxonomic units by clustering ITS2 sequences at 97% similarity within, but not across, samples collapses sequence diversity that is more likely to be intragenomic, while preserving diversity that is more likely interspecific. We utilize this ‘within-sample clustering’ to analyze Symbiodinium from ten host taxa on shallow reefs on the north and south shores of St. John, US Virgin Islands. While Symbiodinium communities did not differ between shores, metacommunity network analysis of host-symbiont associations revealed Symbiodinium lineages occupying ‘dominant’ and ‘background’ niches, and coral hosts that are more ‘flexible’ or ‘specific’ in their associations with Symbiodinium. These methods shed new light on important questions in coral symbiosis ecology, and demonstrate how application-specific bioinformatic pipelines can improve the analysis of metabarcoding data in microbial metacommunity studies.

Nucleotide diversity in the two co-resident genomes of allopolyploid cotton

Genetic diversity within and among populations lies at the heart of evolution. Unraveling the extent to which each intrinsic or extrinsic factor determines levels of diversity among genes, populations, and species is challenging, given the difficulty of isolating any single potentially important variable from all others. Allopolyploid species provide an opportunity to disentangle external and intrinsic factors, as the two (or more) homoeologous genomes co-occur in the same nucleus, often exhibiting high collinearity along homoeologous chromosomes. Here we evaluate the pace of molecular evolution and intraspecific, intragenomic diversity in two species of allopolyploid Gossypium, G. hirsutum and G. barbadense, using several hundred genes sequenced from multiple accessions of each species. Genic diversity in both species is low, having been influenced both by the polyploid bottleneck and a domestication bottleneck (for cultivated accessions), but with a directional bias in homoeolog diversity favoring the same genome in both allopolyploids. Total diversity is remarkably similar for the two homoeologous genomes overall, but the two copies of many gene pairs have accumulated statistically different diversity levels, and in a biased fashion with respect to genome. Domesticated accessions show reduced diversity in both genomes, as expected, but with a much greater reduction in one of the two homoeologous genomes. Furthermore, this biased reduction affects opposite homoeologous genomes in the two species. Interspecific introgression has played a role in shaping diversity within each species. Introgression was only detected for certain accessions, and only from G. barbadense into G. hirsutum in one of the two co-resident genomes.

Morphological and molecular characterization of four Sarcocystis spp., including Sarcocystis linearis n. sp., from roe deer (Capreolus capreolus) in Italy.

Fresh (frozen/thawed) muscle samples from four 2-12-year-old roe deer (Capreolus capreolus) from the Sondrio province in north-eastern Italy were examined under a dissecting microscope, and about 180 sarcocysts were isolated and identified to morphological type in wet mounts by light microscopy (LM). Seventy-seven of these sarcocysts were subsequently examined by molecular methods, comprising polymerase chain reaction (PCR) amplification and sequencing of the partial cytochrome c oxidase subunit I gene (cox1) of all isolates, as well as PCR amplification, cloning and sequencing of the complete18S ribosomal RNA (rRNA) gene of two isolates of each species found. By LM, three major sarcocyst types were recognised: spindle-shaped sarcocysts, 0.5-3mm long, either with no clearly recognisable protrusions (thin-walled) or with finger-like protrusions (thick-walled); and slender, thread-like sarcocysts, 2-3mm long, with hair-like protrusions. Sequencing of cox1 revealed that the sarcocysts belonged to four different species. Those with no visible protrusions either belonged to Sarcocystis gracilis (n=24) or to a Sarcocystis taeniata-like species (n=19), whereas those with finger- and hair-like protrusions belonged to Sarcocystis silva (n=27) and Sarcocystis capreolicanis (n=7), respectively. The 19 cox1 sequences of the S. taeniata-like species, comprising five haplotypes, differed from each other at 0-16 of 1038 nucleotide positions (98.5-100% identity). They differed from 25 previous cox1 sequences of S. taeniata from moose and sika deer (with 98.0-100% intraspecific identity), at 33-43 nucleotide positions (95.9-96.8% interspecific identity), and there were 20 fixed nucleotide differences between the two populations. In the phylogenetic analysis based on cox1 sequences, the two populations formed two separate monophyletic clusters. The S. taeniata-like species in roe deer was therefore considered to represent a separate species, which was named Sarcocystis linearis n. sp. At the 18S rRNA gene, however, the two species could not be clearly separated from each other. Thus, there was considerable intraspecific sequence variation in the 18S rRNA gene of S. linearis (98.1-99.9% identity between 24 sequences), which was similar both in magnitude and nature to the variation previously found in this gene of S. taeniata. The new 18S rRNA gene sequences of S. linearis shared an identity of 97.9-99.6% with those of S. taeniata (overlap between intra- and interspecific identity), and in the phylogenetic tree, sequences of the two species were interspersed. By scanning electron microscopy (SEM), the sarcocysts of S. linearis were found to possess regularly spaced, thin and narrow ribbon-like cyst wall protrusions (about 2.8-3.2μm long, 0.3-0.4μm wide and about 0.02-0.03μm thick), terminating in a plate-like structure of the same thickness but with an elliptic outline (about 0.3-0.4μm wide and 0.7-0.9μm long). The terminal plates were connected in the middle with the band-like portion of the protrusions like the board of a seesaw (tilting board). The terminal plates of adjacent protrusions were neatly arranged in a hexagonal pattern resembling tiles on a roof. Together, they formed an outer roof-like layer facing the surrounding cytoplasm of the host cell and completely covering the band-like proximal portion of the protrusions, which overlapped and were stacked in three to four layers close to the cyst surface. The sarcocyst morphology of S. linearis was consistent with that of an unnamed Sarcocystis sp. in roe deer previously found by transmission electron microscopy in several countries, including Italy. A few sarcocysts of S. gracilis and S. silva were also examined by SEM, confirming the presence of regularly distributed, short knob-like protrusions in S. gracilis (as seen in previous SEM studies) and revealing tightly packed, erect 6-7-μm-long villus-like protrusions having regularly distributed round depressions on their surface in S. silva. The sequencing of cox1 of 7, 24 and 27 new isolates of S. capreolicanis, S. gracilis and S. silva, respectively, recovered 7, 11 and 10 new haplotypes from each of the three species and expanded our knowledge on the intraspecific sequence variation at this marker. Similarly, the study revealed a more extensive intragenomic sequence variation at the 18S rRNA gene of S. capreolicanis and S. silva than known from previous studies and confirmed a near absence of such variation in the 18S rRNA gene of S. gracilis.

Host specificity of Symbiodinium variants revealed by an ITS2 metahaplotype approach.

Analysis of the widely used ITS region is confounded by the presence of intragenomic variants (IGVs). In Symbiodinium, the algal symbionts of reef building corals, deep-sequencing analyses are used to characterise communities within corals, yet these analyses largely overlook IGVs. Here we consider that distinct ITS2 sequences could represent IGVs rather than distinct symbiont types and argue that symbionts can be distinguished by their proportional composition of IGVs, described as their ITS2 metahaplotype. Using our metahaplotype approach on Minimum Entropy Decomposition (MED) analysis of ITS2 sequences from the corals Acropora downingi, Cyphastrea microphthalma and Playgyra daedalea, we show the dominance of a single species-specific Symbiodinium C3 variant within each coral species. We confirm the presence of these species-specific symbionts using the psbA non-coding region. Our findings highlight the importance of accounting for IGVs in ITS2 analyses and demonstrate their capacity to resolve biological patterns that would otherwise be overlooked.

Characterization of the Two-Speed Subgenomes of Fusarium graminearum Reveals the Fast-Speed Subgenome Specialized for Adaption and Infection.

Fusarium head blight, caused by Fusarium graminearum, is one of the most severe diseases on wheat and barley worldwide. Although the genomic data of several strains were published, the intragenomic variation of F. graminearum was not well characterized. Here, we sequenced three Chinese strains and conducted genome-wide comparisons. Our data revealed that all the sequenced strains were distinct from each other and over 350 genes were functionally lost in each of them. Variants of each strain were unevenly distributed in a highly conserved pattern along the chromosomes, resulting in a conserved two-speed genome. The fast subgenome has a lower GC content, shorter gene length, and higher variation of exon numbers than the slow subgenome. Genes related to interaction and pathogenicity, under positive selection, and up-regulated in planta were all significantly enriched in the fast subgenome. Furthermore, we found that the fast subgenome coincided with facultative heterochromatin regions that were repressed in vegetative stage but activated during infection as measured by RNA-seq and ChIP-seq data, suggesting that the fast subgenome is epigenetically regulated. Taken together, our data demonstrated that F. graminearum has a highly conserved two-speed genome and the fast subgenome responsible for adaption and infection is under the control of heterochromatin.

Amphidoma languida (Amphidomatacea, Dinophyceae) with a novel azaspiracid toxin profile identified as the cause of molluscan contamination at the Atlantic coast of southern Spain

Azaspiracids (AZA) are a group of food poisoning phycotoxins that are known to accumulate in shellfish. They are produced by some species of the planktonic dinophycean taxon Amphidomataceae. Azaspiracids have been first discovered in Ireland but are now reported in shellfish from numerous global sites thus showing a wide distribution. In shellfish samples collected in 2009 near Huelva (Spain), AZA was also found along the Andalusian Atlantic coast for the first time. Analysis using LC–MS/MS revealed the presence of two different AZA analogues in different bivalve shellfish species (Chamelea gallina, Cerastoderma edule, Donax trunculus, and Solen vagina). In a number of samples, AZA levels exceeded the EU regulatory level of 160μg AZA-1 eq. kg−1 (reaching maximum levels of >500μg AZA-1 eq. kg−1 in Chamelea gallina and >250μg AZA-1 eq. kg−1 in Donax trunculus) causing closures of some local shellfish production areas. One dinophyte strain established from the local plankton during the AZA contamination period and determined as Amphidoma languida was in fact toxigenic, and its AZA profile disclosed it as the causative species: it contained AZA-2 as the main compound and the new compound AZA-43 initially detected in the shellfish. AZA-43 had the same mass as AZA-3, but produced different collision induced dissociation (CID) spectra. High resolution mass spectrometric measurements indicated that there is an unsaturation in the H, I ring system of AZA-43 distinguishing it from the classical AZA such as AZA-1, -2, and -3. Furthermore, the Spanish strain was different from the previously reported AZA profile of the species that consist of AZA-38 and AZ-39. In molecular phylogenetics, the Andalusian strain formed a monophyletic group together with other strains of Am. languida, but ITS sequences data revealed surprisingly high intragenomic variability. The first Andalusian case of AZA contamination of shellfish above the EU regulatory limit reported here clearly revealed the risk of azaspiracid poisoning (AZP) for this area and also for the Atlantic coast of Iberia and North Africa. The present study underlines the need for continuous monitoring of AZA and the organisms producing such toxins.

Barcoding the Dendrobium (Orchidaceae) Species and Analysis of the Intragenomic Variation Based on the Internal Transcribed Spacer 2.

Many species belonging to the genus Dendrobium are of great commercial value. However, their difficult growth conditions and high demand have caused many of these species to become endangered. Indeed, counterfeit Dendrobium products are common, especially in medicinal markets. This study aims to assess the suitability of the internal transcribed spacer 2 (ITS2) region as a marker for identifying Dendrobium and to evaluate its intragenomic variation in Dendrobium species. In total, 29,624 ITS2 copies from 18 species were obtained using 454 pyrosequencing to evaluate intragenomic variation. In addition, 513 ITS2 sequences from 26 Dendrobium species were used to assess its identification suitability. The highest intragenomic genetic distance was observed in Dendrobium chrysotoxum (0.081). The average intraspecific genetic distances of each species ranged from 0 to 0.032. Phylogenetic trees based on ITS2 sequences showed that most Dendrobium species are monophyletic. The intragenomic and intraspecies divergence analysis showed that greater intragenomic divergence is mostly correlated with larger intraspecific variation. As a major ITS2 variant becomes more common in genome, there are fewer intraspecific variable sites in ITS2 sequences at the species level. The results demonstrated that the intragenomic multiple copies of ITS2 did not affect species identification.

Assessment of Genetic and Chemical Variability in Curcumae Longae Rhizoma (Curcuma longa) Based on DNA Barcoding Markers and HPLC Fingerprints.

Curcumae Longae Rhizoma (Curcuma longa L.) is an important traditional Chinese medicine with multiple beneficial effects. To elucidate the genetic and chemical differences among Curcumae Longae Rhizoma samples, three DNA barcoding markers (rbcL, matK, and ITS-LSU D1/D3) and HPLC fingerprinting were employed in this study. The discriminatory power of rbcL and matK was low, as they only detected one sequence type that showed 100% similarity with more than 20 congeneric species in the Barcode of Life Data Systems (BOLD) database. In contrast, ITS-LSU D1/D3 showed sufficient discriminatory power to precisely identify all of the market samples as C. longa L. in a BLAST search as well as differentiate each sample based on 2-10 ITS-LSU D1/D3 haplotypes with intragenomic variability (mean p-distance: 0.7%, range: 0-2.6%; mean number of differences: 9.6 sites, range: 0-38 sites). HPLC fingerprinting of 13 commercial samples showed a similarity that ranged from 0.769 to 0.996, indicating that the sample quality varied. A cluster analysis based on 5 common peak areas from the HPLC chromatogram resulted in two groups. Group I included 9 samples with a relatively high chemical content, and group II contained 4 samples with a low chemical content. A Mantel test revealed a low correlation (r=0.1721, p=0.047) between genetic and chemical differences. Our findings suggest that the integrated approach of ITS-LSU D1/D3 DNA barcoding and HPLC fingerprinting provides a comprehensive, precise, and convenient method to clarify the genetic and chemical differences in Curcumae Longae Rhizoma.

Identification and Analysis of Informative Single Nucleotide Polymorphisms in 16S rRNA Gene Sequences of the Bacillus cereus Group.

Analysis of 16S rRNA genes is important for phylogenetic classification of known and novel bacterial genera and species and for detection of uncultivable bacteria. PCR amplification of 16S rRNA genes with universal primers produces a mixture of amplicons from all rRNA operons in the genome, and the sequence data generally yield a consensus sequence. Here we describe valuable data that are missing from consensus sequences, variable effects on sequence data generated from nonidentical 16S rRNA amplicons, and the appearance of data displayed by different software programs. These effects are illustrated by analysis of 16S rRNA genes from 50 strains of the Bacillus cereus group, i.e., Bacillus anthracis, Bacillus cereus, Bacillus mycoides, and Bacillus thuringiensis These species have 11 to 14 rRNA operons, and sequence variability occurs among the multiple 16S rRNA genes. A single nucleotide polymorphism (SNP) previously reported to be specific to B. anthracis was detected in some B. cereus strains. However, a different SNP, at position 1139, was identified as being specific to B. anthracis, which is a biothreat agent with high mortality rates. Compared with visual analysis of the electropherograms, basecaller software frequently missed gene sequence variations or could not identify variant bases due to overlapping basecalls. Accurate detection of 16S rRNA gene sequences that include intragenomic variations can improve discrimination among closely related species, improve the utility of 16S rRNA databases, and facilitate rapid bacterial identification by targeted DNA sequence analysis or by whole-genome sequencing performed by clinical or reference laboratories.

Contrasting Patterns of rDNA Homogenization within the Zygosaccharomyces rouxii Species Complex.

Arrays of repetitive ribosomal DNA (rDNA) sequences are generally expected to evolve as a coherent family, where repeats within such a family are more similar to each other than to orthologs in related species. The continuous homogenization of repeats within individual genomes is a recombination process termed concerted evolution. Here, we investigated the extent and the direction of concerted evolution in 43 yeast strains of the Zygosaccharomyces rouxii species complex (Z. rouxii, Z. sapae, Z. mellis), by analyzing two portions of the 35S rDNA cistron, namely the D1/D2 domains at the 5’ end of the 26S rRNA gene and the segment including the internal transcribed spacers (ITS) 1 and 2 (ITS regions). We demonstrate that intra-genomic rDNA sequence variation is unusually frequent in this clade and that rDNA arrays in single genomes consist of an intermixing of Z. rouxii, Z. sapae and Z. mellis-like sequences, putatively evolved by reticulate evolutionary events that involved repeated hybridization between lineages. The levels and distribution of sequence polymorphisms vary across rDNA repeats in different individuals, reflecting four patterns of rDNA evolution: I) rDNA repeats that are homogeneous within a genome but are chimeras derived from two parental lineages via recombination: Z. rouxii in the ITS region and Z. sapae in the D1/D2 region; II) intra-genomic rDNA repeats that retain polymorphisms only in ITS regions; III) rDNA repeats that vary only in their D1/D2 domains; IV) heterogeneous rDNA arrays that have both polymorphic ITS and D1/D2 regions. We argue that an ongoing process of homogenization following allodiplodization or incomplete lineage sorting gave rise to divergent evolutionary trajectories in different strains, depending upon temporal, structural and functional constraints. We discuss the consequences of these findings for Zygosaccharomyces species delineation and, more in general, for yeast barcoding.

Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia, Heterolobosea.

The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.

Intragenomic polymorphism and intergenomic recombination in the ribosomal RNA genes of strains belonging to a yeast species Pichia membranifaciens

ABSTRACTA concerted evolution model has been proposed to explain the observed lack of sequence variation among the multiple ribosomal RNA (rRNA) gene copies in many different eukaryotic species. Recent studies on the level of intragenomic variations in the rRNA gene repeats of fungi resulted in controversial conclusions. In this study, we clearly showed that significant polymorphisms of the internal transcribed spacers (ITS1 and ITS2) of ribosomal DNA (rDNA) exist within the genome of a strain of the yeast species Pichia membranifaciens. More interestingly, we showed that the intragenomic ITS sequence polymorphisms were formed by intergenomic rDNA recombination among different P. membranifaciens strains with significantly different ITS sequences. Intergenomic rDNA recombination was also responsible for the diversification of rDNA sequences in different strains of the species. After the events bring together different rDNA types in individual genomes of the P. membranifaciens strains compared, rDNA sequence heterogeneity has remained in the genome of one but eliminated by homogenisation in the genomes of other strains. Our findings show new clue for further investigation on the mechanism of concerted evolution of rRNA genes in eukaryotes.

Genome Analysis of Pseudoloma neurophilia: A Microsporidian Parasite of Zebrafish (Danio rerio).

Microsporidia are highly successful parasites that infect virtually all known animal lineages, including the model Danio rerio (zebrafish). The widespread use of this aquatic model for biomedical research has resulted in an unexpected increase in infections from the microsporidium Pseudoloma neurophilia, which can lead to significant physical, behavioral, and immunological modifications, resulting in nonprotocol variation during experimental procedures. Here, we seek to obtain insights into the biology of P.neurophilia by investigating its genome content, which was obtained from only 29 nanograms of DNA using the MiSeq technology and paired-end Illumina sequencing. We found that the genome of P.neurophilia is phylogenetically and genetically related to other fish-microsporidians, but features unique to this intracellular parasite are also found. The small 5.25-Mb genome assembly includes 1,139 unique open-reading frames and an unusually high number of transposable elements for such a small genome. Investigations of intragenomic diversity also provided strong indications that the mononucleate nucleus of this species is diploid. Overall, our study provides insights into the dynamics of microsporidian genomes and a solid sequence reference to be used in future studies of host-parasite interactions using the zebrafish D.rerio and P.neurophilia as a model.

The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae)

Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera.

Contrasting evolutionary patterns of 28S and ITS rRNA genes reveal high intragenomic variation in Cephalenchus (Nematoda): Implications for species delimitation

Concerted evolution is often assumed to be the evolutionary force driving multi-family genes, including those from ribosomal DNA (rDNA) repeat, to complete homogenization within a species, although cases of non-concerted evolution have been also documented. In this study, sequence variation of 28S and ITS ribosomal RNA (rRNA) genes in the genus Cephalenchus is assessed at three different levels, intragenomic, intraspecific, and interspecific. The findings suggest that not all Cephalenchus species undergo concerted evolution. High levels of intraspecific polymorphism, mostly due to intragenomic variation, are found in Cephalenchus sp1 (BRA-01). Secondary structure analyses of both rRNA genes and across different species show a similar substitution pattern, including mostly compensatory (CBC) and semi-compensatory (SBC) base changes, thus suggesting the functionality of these rRNA copies despite the variation found in some species. This view is also supported by low sequence variation in the 5.8S gene in relation to the flanking ITS-1 and ITS-2 as well as by the existence of conserved motifs in the former gene. It is suggested that potential cross-fertilization in some Cephalenchus species, based on inspection of female reproductive system, might contribute to both intragenomic and intraspecific polymorphism of their rRNA genes. These results reinforce the potential implications of intragenomic and intraspecific genetic diversity on species delimitation, especially in biodiversity studies based solely on metagenetic approaches. Knowledge of sequence variation will be crucial for accurate species diversity estimation using molecular methods.

Haplotype Detection from Next-Generation Sequencing in High-Ploidy-Level Species: 45S rDNA Gene Copies in the Hexaploid Spartina maritima.

Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5′-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.

Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy?

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.

The distribution of intra-genomically variable dinoflagellate symbionts at Lord Howe Island, Australia

The symbiotic dinoflagellates of corals and other marine invertebrates (Symbiodinium) are essential to the development of shallow-water coral reefs. This genus contains considerable genetic diversity and a corresponding range of physiological and ecological traits. Most genetic variation arises through the accumulation of somatic mutations that arise during asexual reproduction. Yet growing evidence suggests that occasional sexual reproductive events also occur within, and perhaps between, Symbiodinium lineages, further contributing to the pool of genetic variation available for evolutionary adaptation. Intra-genomic variation can therefore arise from both sexual and asexual reproductive processes, making it difficult to discern its underlying causes and consequences. We used quantitative PCR targeting the ITS2 locus to estimate proportions of genetically homogeneous symbionts and intra-genomically variable Symbiodinium (IGV Symbiodinium) in the reef-building coral Pocillopora damicornis at Lord Howe Island, Australia. We then sampled colonies through time and at a variety of spatial scales to find out whether the distribution of these symbionts followed patterns consistent with niche partitioning. Estimated ratios of homogeneous to IGV Symbiodinium varied between colonies within sites (metres to tens of metres) and between sites separated by hundreds to thousands of metres, but remained stable within colonies through time. Symbiont ratios followed a temperature gradient, with the local thermal maximum emerging as a negative predictor for the estimated proportional abundance of IGV Symbiodinium. While this pattern may result from fine-scale spatial population structure, it is consistent with an increased susceptibility to thermal stress, suggesting that the evolutionary processes that generate IGV (such as inter-lineage recombination and the accumulation of somatic mutations at the ITS2 locus) may have important implications for the fitness of the symbiont and that of the coral host.

Intragenomic variations of multicopy ITS2 marker in Agrodiaetus blue butterflies (Lepidoptera, Lycaenidae).

The eukaryotic ribosomal DNA cluster consists of multiple copies of three genes, 18S, 5. 8S and 28S rRNAs, separated by multiple copies of two internal transcribed spacers, ITS1 and ITS2. It is an important, frequently used marker in both molecular cytogenetic and molecular phylogenetic studies. Despite this, little is known about intragenomic variations within the copies of eukaryotic ribosomal DNA genes and spacers. Here we present data on intraindividual variations of ITS2 spacer in three species of Agrodiaetus Hübner, 1822 blue butterflies revealed by cloning technique. We demonstrate that a distinctly different intragenomic ITS2 pattern exists for every individual analysed. ITS2 sequences of these species show significant intragenomic variation (up to 3.68% divergence), setting them apart from each other on inferred phylogenetic tree. This variation is enough to obscure phylogenetic relationships at the species level.