Polymorphisms of rDNA genes in Cyberlindnera yeast suggest birth-and-death evolution events.

In eukaryotes, the ribosome machinery is encoded by repeats of the ribosomal RNA genes: 26/28S, 18S, 5.8S, and 5S, structured in tandem arrays and frequently homogenized within a genome. This homogenization is thought to be driven by concerted evolution, evolving as a unit, which contributes to its target as the species barcode in modern taxonomy. However, high heterogeneity of rDNA genes has been reported, including in Saccharomycotina yeasts. Here, we describe the polymorphisms and heterogeneity of D1/D2 domains (26S rRNA) and the intergenic transcribed spacer of a new yeast species with affinities to the genus Cyberlindnera and their evolution. Both regions are not homogenized, failing the prediction of concerted evolution. Phylogenetic network analysis of cloned sequences revealed that Cyberlindnera sp. rDNAs are diverse and evolved by reticulation rather than by bifurcating tree evolution model. Predicted rRNA secondary structures also confirmed structural differences, except for some conserved hairpin loops. We hypothesize that some rDNA is inactive within this species and evolves by birth-and-death rather than concerted evolution. Our findings propel further investigation into the evolution of rDNA genes in yeasts.

Genomic Differences Between the Sexes in a Fish Species Seen Through Satellite DNAs.

Neotropical fishes have highly diversified karyotypic and genomic characteristics and present many diverse sex chromosome systems, with various degrees of sex chromosome differentiation. Knowledge on their sex-specific composition and evolution, however, is still limited. Satellite DNAs (satDNAs) are tandemly repeated sequences with pervasive genomic distribution and distinctive evolutionary pathways, and investigating satDNA content might shed light into how genome architecture is organized in fishes and in their sex chromosomes. The present study investigated the satellitome of Megaleporinus elongatus, a freshwater fish with a proposed Z1Z1Z2Z2/Z1W1Z2W2 multiple sex chromosome system that encompasses a highly heterochromatic and differentiated W1 chromosome. The species satellitome comprises of 140 different satDNA families, including previously isolated sequences and new families found in this study. This diversity is remarkable considering the relatively low proportion that satDNAs generally account for the M. elongatus genome (around only 5%). Differences between the sexes in regards of satDNA content were also evidenced, as these sequences are 14% more abundant in the female genome. The occurrence of sex-biased signatures of satDNA evolution in the species is tightly linked to satellite enrichment associated with W1 in females. Although both sexes share practically all satDNAs, the overall massive amplification of only a few of them accompanied the W1 differentiation. We also investigated the expansion and diversification of the two most abundant satDNAs of M. elongatus, MelSat01-36 and MelSat02-26, both highly amplified sequences in W1 and, in MelSat02-26's case, also harbored by Z2 and W2 chromosomes. We compared their occurrences in M. elongatus and the sister species M. macrocephalus (with a standard ZW sex chromosome system) and concluded that both satDNAs have led to the formation of highly amplified arrays in both species; however, they formed species-specific organization on female-restricted sex chromosomes. Our results show how satDNA composition is highly diversified in M. elongatus, in which their accumulation is significantly contributing to W1 differentiation and not satDNA diversity per se. Also, the evolutionary behavior of these repeats may be associated with genome plasticity and satDNA variability between the sexes and between closely related species, influencing how seemingly homeologous heteromorphic sex chromosomes undergo independent satDNA evolution.

The B Chromosomes of Prochilodus lineatus (Teleostei, Characiformes) Are Highly Enriched in Satellite DNAs.

B or supernumerary chromosomes are dispensable elements that are widely present in numerous eukaryotes. Due to their non-recombining nature, there is an evident tendency for repetitive DNA accumulation in these elements. Thus, satellite DNA plays an important role in the evolution and diversification of B chromosomes and can provide clues regarding their origin. The characiform Prochilodus lineatus was one of the first discovered fish species bearing B chromosomes, with all populations analyzed so far showing one to nine micro-B chromosomes and exhibiting at least three morphological variants (Ba, Bsm, and Bm). To date, a single satellite DNA is known to be located on the B chromosomes of this species, but no information regarding the differentiation of the proposed B-types is available. Here, we characterized the satellitome of P. lineatus and mapped 35 satellite DNAs against the chromosomes of P. lineatus, of which six were equally located on all B-types and this indicates a similar genomic content. In addition, we describe, for the first time, an entire population without B chromosomes.

Cytogenomic analysis unveils mixed molecular evolution and recurrent chromosomal rearrangements shaping the multigene families on Schistocerca grasshopper genomes.

Multigene families are essential components of eukaryotic genomes and play key roles either structurally and functionally. Their modes of evolution remain elusive even in the era of genomics, because multiple multigene family sequences coexist in genomes, particularly in large repetitive genomes. Here, we investigate how the multigene families 18S rDNA, U2 snDNA, and H3 histone evolved in 10 species of Schistocerca grasshoppers with very large and repeat-enriched genomes. Using sequenced genomes and fluorescence in situ hybridization mapping, we find substantial differences between species, including the number of chromosomal clusters, changes in sequence abundance and nucleotide composition, pseudogenization, and association with transposable elements (TEs). The intragenomic analysis of Schistocerca gregaria using long-read sequencing and genome assembly unveils conservation for H3 histone and recurrent pseudogenization for 18S rDNA and U2 snDNA, likely promoted by association with TEs and sequence truncation. Remarkably, TEs were frequently associated with truncated copies, were also among the most abundant in the genome, and revealed signatures of recent activity. Our findings suggest a combined effect of concerted and birth-and-death models driving the evolution of multigene families in Schistocerca over the last 8 million years, and the occurrence of intra- and interchromosomal rearrangements shaping their chromosomal distribution. Despite the conserved karyotype in Schistocerca, our analysis highlights the extensive reorganization of repetitive DNAs in Schistocerca, contributing to the advance of comparative genomics for this important grasshopper genus.

Chromosomal and Genomic Dynamics of Satellite DNAs in Characidae (Characiformes, Teleostei) Species.

Satellite DNAs (satDNAs) are tandemly repeated DNA sequences with great abundance in eukaryotic genomes. A single species may carry up to hundreds of satDNA families, which is collectively called as "satellitome," each showing its own dynamics and evolution rates. In this context, all live species contain a satDNA library that may be partially or totally shared with other related species/populations. In the late few years, next-generation sequencing (NGS) and novel bioinformatic tools facilitated the massive characterization of these sequences at low costs, and consequently, comparing satDNAs between species. In this study, we characterized two novel satDNAs (MsaSat03-80 and MsaSat04-142) in three characid fish (Astyanax paranae and Astyanax fasciatus and two populations of Moenkhausia sanctaefilomenae) and mapped their chromosomal location to unveil the evolutionary dynamics of satDNA repeats in those species. Our results evidenced that MsaSat03 is present in the genomes of all analyzed species, but is clustered only in the chromosomes of M. sanctaefilomenae, exhibiting a conserved number and location of sites. Conversely, MsaSat04 sequences is restricted to M. sanctaefilomenae and shows a differential distribution between the two analyzed populations. Altogether, our analyses point to a complex history of satDNA families in characid fish and the utility of NGS data for comparative satDNA analysis.

Intragenomic nuclear RNA variation in a cryptic Amanita taxon.

Amanita cf. lavendula collections in eastern North America, Mexico, and Costa Rica were found to consist of four cryptic taxa, one of which exhibited consistently unreadable nuclear rDNA ITS1-5.8S-ITS2 (fungal barcode) sequences after ITS1 base 130. This taxon is designated here as Amanita cf. lavendula taxon 1. ITS sequences from dikaryotic basidiomata were cloned, but sequences recovered from cloning did not segregate into distinct haplotypes. Rather, there was a mix of haplotypes that varied among themselves predominantly at 28 ITS positions. Analysis of each of these 28 variable bases showed predominantly two alternate bases at each position. Based on these findings and additional sequence data from the nuclear rDNA 28S, RNA polymerase II subunit 2 (RPB2) and mitochondrial rDNA small subunit (SSU) and 23S genes, we speculate that taxon 1 represents an initial hybridization event between two divergent taxa followed by failure of the ribosomal repeat to homogenize. Homogenization failure may be a result of repeated hybridization between divergent internal transcribed spacer (ITS) types with inadequate time for concerted evolution of the ribosomal repeat or, alternately, a complete failure of the ribosomal homogenization process. To our knowledge, this finding represents the first report of a geographically widespread taxon (Canada, eastern USA, Costa Rica) with apparent homogenization failure across all collections. Findings such as these have implications for fungal barcoding efforts and the application of fungal barcodes in identifying environmental sequences.

Uncovering the molecular organization of unusual highly scattered 5S rDNA: The case of Chariesterus armatus (Heteroptera).

One cluster of 5S rDNA per haploid genome is the most common pattern among Heteroptera. However, in Chariesterus armatus, highly scattered signals were noticed. We isolated and characterized the entire 5S rDNA unit of C. armatus aiming to a deeper knowledge of molecular organization of the 5S rDNA among Heteroptera and to understand possible causes and consequences of 5S rDNA chromosomal spreading. For a comparative analysis, we performed the same approach in Holymenia histrio with 5S rDNA restricted to one bivalent. Multiple 5S rDNA variants were observed in both species, though they were more variable in C. armatus, with some of variants corresponding to pseudogenes. These pseudogenes suggest birth-and-death mechanism, though homogenization was also observed (concerted evolution), indicating evolution through mixed model. Association between transposable elements and 5S rDNA was not observed, suggesting spreading of 5S rDNA through other mechanisms, like ectopic recombination. Scattered organization is a rare example for 5S rDNA, and such organization in C. armatus genome could have led to the high diversification of sequences favoring their pseudogenization.

A Glimpse into the Satellite DNA Library in Characidae Fish (Teleostei, Characiformes).

Satellite DNA (satDNA) is an abundant fraction of repetitive DNA in eukaryotic genomes and plays an important role in genome organization and evolution. In general, satDNA sequences follow a concerted evolutionary pattern through the intragenomic homogenization of different repeat units. In addition, the satDNA library hypothesis predicts that related species share a series of satDNA variants descended from a common ancestor species, with differential amplification of different satDNA variants. The finding of a same satDNA family in species belonging to different genera within Characidae fish provided the opportunity to test both concerted evolution and library hypotheses. For this purpose, we analyzed here sequence variation and abundance of this satDNA family in ten species, by a combination of next generation sequencing (NGS), PCR and Sanger sequencing, and fluorescence in situ hybridization (FISH). We found extensive between-species variation for the number and size of pericentromeric FISH signals. At genomic level, the analysis of 1000s of DNA sequences obtained by Illumina sequencing and PCR amplification allowed defining 150 haplotypes which were linked in a common minimum spanning tree, where different patterns of concerted evolution were apparent. This also provided a glimpse into the satDNA library of this group of species. In consistency with the library hypothesis, different variants for this satDNA showed high differences in abundance between species, from highly abundant to simply relictual variants.

Dissecting the Satellite DNA Landscape in Three Cactophilic Drosophila Sequenced Genomes.

Eukaryote genomes are replete with repetitive DNAs. This class includes tandemly repeated satellite DNAs (satDNA) which are among the most abundant, fast evolving (yet poorly studied) genomic components. Here, we used high-throughput sequencing data from three cactophilic Drosophila species, D. buzzatii, D. seriema, and D. mojavensis, to access and study their whole satDNA landscape. In total, the RepeatExplorer software identified five satDNAs, three previously described (pBuM, DBC-150 and CDSTR198) and two novel ones (CDSTR138 and CDSTR130). Only pBuM is shared among all three species. The satDNA repeat length falls within only two classes, between 130 and 200 bp or between 340 and 390 bp. FISH on metaphase and polytene chromosomes revealed the presence of satDNA arrays in at least one of the following genomic compartments: centromeric, telomeric, subtelomeric, or dispersed along euchromatin. The chromosomal distribution ranges from a single chromosome to almost all chromosomes of the complement. Fiber-FISH and sequence analysis of contigs revealed interspersion between pBuM and CDSTR130 in the microchromosomes of D. mojavensis Phylogenetic analyses showed that the pBuM satDNA underwent concerted evolution at both interspecific and intraspecific levels. Based on RNA-seq data, we found transcription activity for pBuM (in D. mojavensis) and CDSTR198 (in D. buzzatii) in all five analyzed developmental stages, most notably in pupae and adult males. Our data revealed that cactophilic Drosophila present the lowest amount of satDNAs (1.9-2.9%) within the Drosophila genus reported so far. We discuss how our findings on the satDNA location, abundance, organization, and transcription activity may be related to functional aspects.

Orthologs, turn-over, and remolding of tRNAs in primates and fruit flies.

BACKGROUND: Transfer RNAs (tRNAs) are ubiquitous in all living organism. They implement the genetic code so that most genomes contain distinct tRNAs for almost all 61 codons. They behave similar to mobile elements and proliferate in genomes spawning both local and non-local copies. Most tRNA families are therefore typically present as multicopy genes. The members of the individual tRNA families evolve under concerted or rapid birth-death evolution, so that paralogous copies maintain almost identical sequences over long evolutionary time-scales. To a good approximation these are functionally equivalent. Individual tRNA copies thus are evolutionary unstable and easily turn into pseudogenes and disappear. This leads to a rapid turnover of tRNAs and often large differences in the tRNA complements of closely related species. Since tRNA paralogs are not distinguished by sequence, common methods cannot not be used to establish orthology between tRNA genes. RESULTS: In this contribution we introduce a general framework to distinguish orthologs and paralogs in gene families that are subject to concerted evolution. It is based on the use of uniquely aligned adjacent sequence elements as anchors to establish syntenic conservation of sequence intervals. In practice, anchors and intervals can be extracted from genome-wide multiple sequence alignments. Syntenic clusters of concertedly evolving genes of different families can then be subdivided by list alignments, leading to usually small clusters of candidate co-orthologs. On the basis of recent advances in phylogenetic combinatorics, these candidate clusters can be further processed by cograph editing to recover their duplication histories. We developed a workflow that can be conceptualized as stepwise refinement of a graph of homologous genes. We apply this analysis strategy with different types of synteny anchors to investigate the evolution of tRNAs in primates and fruit flies. We identified a large number of tRNA remolding events concentrated at the tips of the phylogeny. With one notable exception all phylogenetically old tRNA remoldings do not change the isoacceptor class. CONCLUSIONS: Gene families evolving under concerted evolution are not amenable to classical phylogenetic analyses since paralogs maintain identical, species-specific sequences, precluding the estimation of correct gene trees from sequence differences. This leaves conservation of syntenic arrangements with respect to "anchor elements" that are not subject to concerted evolution as the only viable source of phylogenetic information. We have demonstrated here that a purely synteny-based analysis of tRNA gene histories is indeed feasible. Although the choice of synteny anchors influences the resolution in particular when tight gene clusters are present, and the quality of sequence alignments, genome assemblies, and genome rearrangements limits the scope of the analysis, largely coherent results can be obtained for tRNAs. In particular, we conclude that a large fraction of the tRNAs are recent copies. This proliferation is compensated by rapid pseudogenization as exemplified by many very recent alloacceptor remoldings.

New tools and insights to assist with the molecular identification of Simulium guianense s.l., main Onchocerca volvulus vector within the highland areas of the Amazonia onchocerciasis focus.

Following the success of the Onchocerciasis Elimination Programme for the Americas (OEPA), there is now just one Latin American onchocerciasis focus where onchocerciasis transmission is described as 'on-going:' the Amazonia Onchocerciasis focus. In the hyperendemic highland areas of the Amazonia focus, Simulium guianense s.l. Wise are the most important vectors of the disease. Populations of S. guianense s.l. are, however, known to vary in their cytogenetics and in a range of behaviours, including in their biting habits. In the hypoendemic lowland areas of the Amazonia focus, for example, S. guianense s.l. are generally regarded as zoophilic and consequently unimportant to disease transmission. Robust tools, to discriminate among various populations of S. guianense s.l. have, however, not yet been developed. In the work reported here, we have assessed the utility of a ribosomal DNA sequence fragment spanning the nuclear ribosomal ITS-1, ITS-2 and 5.8S sequence regions and a ∼850 nucleotide portion of the mitochondrial cytochrome oxidase gene (CO1) for species-level identification and for resolving the within species substructuring. We report here how we have generated 78 CO1 sequences from a rich set of both zoophilic and anthropophilic populations of S. guianense s.l. that were collected from eight sites that are broadly distributed across Brazil. Consistent with previous findings, our analysis supports the genetic isolation of Simulium litobranchium from S. guianense s.l. In contrast with previous findings, however, our results did not provide support for the divergence of the two species prior to the radiation of S. guianense s.l. In our analysis of the S. guianense s.l. ribosomal DNA sequence trace files we generated, we provide clear evidence of multiple within-specimen single nucleotide polymorphisms and indels suggesting that S. guianense s.l. ribosomal DNA is not a good target for conventional DNA barcoding. This is the first report of S. guianense s.l. within individual ribosomal DNA variation and thus the first evidence that the species is not subject to the normal effects of concerted evolution. Collectively, these data illustrate the need for diverse sampling in the development of robust molecular tools for vector identification and suggest that ribosomal DNA might be able to assist with resolving S. guianense s.l. species substructuring that C01 barcoding has hitherto failed to.

Intragenomic variation in the second internal transcribed spacer of the ribosomal DNA of species of the genera Culex and Lutzia (Diptera: Culicidae)

Culex is the largest genus of Culicini and includes vectors of several arboviruses and filarial worms. Many species of Culex are morphologically similar, which makes their identification difficult, particularly when using female specimens. To aid evolutionary studies and species distinction, molecular techniques are often used. Sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) from 16 species of the genus Culex and one of Lutzia were used to assess their genomic variability and to verify their applicability in the phylogenetic analysis of the group. The distance matrix (uncorrected p-distance) that was obtained revealed intragenomic and intraspecific variation. Because of the intragenomic variability, we selected ITS2 copies for use in distance analyses based on their secondary structures. Neighbour-joining topology was obtained with an uncorrected p-distance. Despite the heterogeneity observed, individuals of the same species were grouped together and correlated with the current, morphology-based classification, thereby showing that ITS2 is an appropriate marker to be used in the taxonomy of Culex.