Transposable element landscapes illuminate past evolutionary events in the endangered fern Vandenboschia speciosa.

Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises.

Gene expression changes elicited by a parasitic B chromosome in the grasshopper Eyprepocnemis plorans are consistent with its phenotypic effects.

Parasitism evokes adaptive physiological changes in the host, many of which take place through gene expression changes. This response can be more or less local, depending on the organ or tissue affected by the parasite, or else systemic when the parasite affects the entire host body. The most extreme of the latter cases is intragenomic parasitism, where the parasite is present in all host nuclei as any other genomic element. Here, we show the molecular crosstalk between a parasitic chromosome (also named B chromosome) and the host genome, manifested through gene expression changes. The transcriptome analysis of 0B and 1B females of the grasshopper Eyprepocnemis plorans, validated by a microarray experiment performed on four B-lacking and five B-carrying females, revealed changes in gene expression for 188 unigenes being consistent in both experiments. Once discarded B-derived transcripts, there were 46 differentially expressed genes (30 up- and 16 downregulated) related with the adaptation of the host genome to the presence of the parasitic chromosome. Interestingly, the functions of these genes could explain some of the most important effects of B chromosomes, such as nucleotypic effects derived from the additional DNA they represent, chemical defense and detoxification, protein modification and response to stress, ovary function, and regulation of gene expression. Collectively, these changes uncover an intimate host-parasite interaction between A and B chromosomes during crucial steps of gene expression and protein function.

Chromosomal Rearrangements during Turtle Evolution Altered the Synteny of Genes Involved in Vertebrate Sex Determination.

Sex-determining mechanisms (SDMs) set an individual's sexual fate by its genotype (genotypic sex determination, GSD) or environmental factors like temperature (temperature- dependent sex determination, TSD), as in turtles where the GSD "trigger" remains unknown. SDMs co-evolve with turtle chromosome number, perhaps because fusions/fissions alter the relative position/regulation of sexual development genes. Here, we map 10 such genes via FISH onto metaphase chromosomes in 6 TSD and 6 GSD turtles for the first time. Results uncovered intrachromosomal rearrangements involving 3 genes across SDMs (Dax1, Fhl2, and Fgf9) and a chromosomal fusion linking 2 genes (Sf1 and Rspo1) in 1 chromosome in a TSD turtle (Pelomedusa subrufa) that locate to 2 chromosomes in all others. Notably, Sf1 and its repressor Foxl2 map to Apalone spinifera's ZW chromosomes but to a macro- (Foxl2) and a microautosome (Sf1) in other turtles potentially inducing SDM evolution. However, our phylogenetically informed analysis refutes Foxl2 (but not Sf1) as Apalone's master sex-determining gene. The absence of common TSD-specific or GSD-specific rearrangements underscores the independent evolutionary trajectories of turtle SDMs. Further comparative analyses using more genes from the sexual development network are warranted to inform genome evolution and its contribution to enigmatic turnovers of vertebrate sex determination.

B-chromosome effects on Hsp70 gene expression does not occur at transcriptional level in the grasshopper Eyprepocnemis plorans.

As intragenomic parasites, B chromosomes can elicit stress in the host genome, thus inducing a response for host adaptation to this kind of continuous parasitism. In the grasshopper Eyprepocnemis plorans, B-chromosome presence has been previously associated with a decrease in the amount of the heat-shock protein 70 (HSP70). To investigate whether this effect is already apparent at transcriptional level, we analyze the expression levels of the Hsp70 gene in gonads and somatic tissues of males and females with and without B chromosomes from two populations, where the predominant B chromosome variants (B2 and B24) exhibit different levels of parasitism, by means of quantitative real-time PCR (qPCR) on complementary DNA (cDNA). The results revealed the absence of significant differences for Hsp70 transcripts associated with B-chromosome presence in virtually all samples. This indicates that the decrease in HSP70 protein levels, formerly reported in this species, may not be a consequence of transcriptional down-regulation of Hsp70 genes, but the result of post-transcriptional regulation. These results will help to design future studies oriented to identifying factors modulating Hsp70 expression, and will also contribute to uncover the biological role of B chromosomes in eukaryotic genomes.

The Ku70 DNA-repair protein is involved in centromere function in a grasshopper species.

The Ku70 protein is involved in numerous cell functions, the nonhomologous end joining (NHEJ) DNA repair pathway being the best known. Here, we report a novel function for this protein in the grasshopper Eyprepocnemis plorans. We observed the presence of large Ku70 foci on the centromeres of meiotic and mitotic chromosomes during the cell cycle stages showing the highest centromeric activity (i.e., metaphase and anaphase). The fact that colchicine treatment prevented centromeric location of Ku70, suggests a microtubule-dependent centromeric function for Ku70. Likewise, the absence of Ku70 at metaphase-anaphase centromeres from three males whose Ku70 gene had been knocked down using interference RNA, and the dramatic increase in the frequency of polyploid spermatids observed in these males, suggest that the centromeric presence of Ku70 is required for normal cytokinesis in this species. The centromeric function of Ku70 was not observed in 14 other grasshopper and locust species, or in the mouse, thus suggesting that it is an autapomorphy in E. plorans.

Tandem Repeat DNA Provides Many Cytological Markers for Hybrid Zone Analysis in Two Subspecies of the Grasshopper Chorthippus parallelus.

Recent advances in next generation sequencing (NGS) have greatly increased our understanding of non-coding tandem repeat (TR) DNA. Here we show how TR DNA can be useful for the study of hybrid zones (HZ), as it serves as a marker to identify introgression in areas where two biological entities come in contact. We used Illumina libraries to analyse two subspecies of the grasshopper Chorthippus parallelus, which currently form a HZ in the Pyrenees. We retrieved a total of 152 TR sequences, and used fluorescent in situ hybridization (FISH) to map 77 families in purebred individuals from both subspecies. Our analysis revealed 50 TR families that could serve as markers for analysis of this HZ, using FISH. Differential TR bands were unevenly distributed between chromosomes and subspecies. Some of these TR families yielded FISH bands in only one of the subspecies, suggesting the amplification of these TR families after the geographic separation of the subspecies in the Pleistocene. Our cytological analysis of two TR markers along a transect of the Pyrenean hybrid zone showed asymmetrical introgression of one subspecies into the other, consistent with previous findings using other markers. These results demonstrate the reliability of TR-band markers for hybrid zone studies.

Epistatic selection on a selfish Segregation Distorter supergene - drive, recombination, and genetic load.

Meiotic drive supergenes are complexes of alleles at linked loci that together subvert Mendelian segregation resulting in preferential transmission. In males, the most common mechanism of drive involves the disruption of sperm bearing one of a pair of alternative alleles. While at least two loci are important for male drive-the driver and the target-linked modifiers can enhance drive, creating selection pressure to suppress recombination. In this work, we investigate the evolution and genomic consequences of an autosomal, multilocus, male meiotic drive system, Segregation Distorter (SD) in the fruit fly, Drosophila melanogaster. In African populations, the predominant SD chromosome variant, SD-Mal, is characterized by two overlapping, paracentric inversions on chromosome arm 2R and nearly perfect (~100%) transmission. We study the SD-Mal system in detail, exploring its components, chromosomal structure, and evolutionary history. Our findings reveal a recent chromosome-scale selective sweep mediated by strong epistatic selection for haplotypes carrying Sd, the main driving allele, and one or more factors within the double inversion. While most SD-Mal chromosomes are homozygous lethal, SD-Mal haplotypes can recombine with other, complementing haplotypes via crossing over, and with wildtype chromosomes via gene conversion. SD-Mal chromosomes have nevertheless accumulated lethal mutations, excess non-synonymous mutations, and excess transposable element insertions. Therefore, SD-Mal haplotypes evolve as a small, semi-isolated subpopulation with a history of strong selection. These results may explain the evolutionary turnover of SD haplotypes in different populations around the world and have implications for supergene evolution broadly.

Chromosome-Scale Genome Assembly of the Hexaploid Taiwanese Goosefoot "Djulis" (Chenopodium formosanum).

Djulis (Chenopodium formosanum Koidz.) is a crop grown since antiquity in Taiwan. It is a BCD-genome hexaploid (2n = 6x = 54) domesticated form of lambsquarters (C. album L.) and a relative of the allotetraploid (AABB) C. quinoa. As with quinoa, djulis seed contains a complete protein profile and many nutritionally important vitamins and minerals. While still sold locally in Taiwanese markets, its traditional culinary uses are being lost as diets of younger generations change. Moreover, indigenous Taiwanese peoples who have long safeguarded djulis are losing their traditional farmlands. We used PacBio sequencing and Hi-C-based scaffolding to produce a chromosome-scale, reference-quality assembly of djulis. The final genome assembly spans 1.63 Gb in 798 scaffolds, with 97.8% of the sequence contained in 27 scaffolds representing the nine haploid chromosomes of each sub-genome of the species. Benchmarking of universal, single-copy orthologs indicated that 98.5% of the conserved orthologous genes for Viridiplantae are complete within the assembled genome, with 92.9% duplicated, as expected for a polyploid. A total of 67.8% of the assembly is repetitive, with the most common repeat being Gypsy long terminal repeat retrotransposons, which had significantly expanded in the B sub-genome. Gene annotation using Iso-Seq data from multiple tissues identified 75,056 putative gene models. Comparisons to quinoa showed strong patterns of synteny which allowed for the identification of homoeologous chromosomes, and sub-genome-specific sequences were used to assign homoeologs to each sub-genome. These results represent the first hexaploid genome assembly and the first assemblies of the C and D genomes of the Chenopodioideae subfamily.

Karyotypic Evolution of Sauropsid Vertebrates Illuminated by Optical and Physical Mapping of the Painted Turtle and Slider Turtle Genomes.

Recent sequencing and software enhancements have advanced our understanding of the evolution of genomic structure and function, especially addressing novel evolutionary biology questions. Yet fragmentary turtle genome assemblies remain a challenge to fully decipher the genetic architecture of adaptive evolution. Here, we use optical mapping to improve the contiguity of the painted turtle (Chrysemys picta) genome assembly and use de novo fluorescent in situ hybridization (FISH) of bacterial artificial chromosome (BAC) clones, BAC-FISH, to physically map the genomes of the painted and slider turtles (Trachemys scripta elegans). Optical mapping increased C. picta's N50 by ~242% compared to the previous assembly. Physical mapping permitted anchoring ~45% of the genome assembly, spanning 5544 genes (including 20 genes related to the sex determination network of turtles and vertebrates). BAC-FISH data revealed assembly errors in C. picta and T. s. elegans assemblies, highlighting the importance of molecular cytogenetic data to complement bioinformatic approaches. We also compared C. picta's anchored scaffolds to the genomes of other chelonians, chicken, lizards, and snake. Results revealed a mostly one-to-one correspondence between chromosomes of painted and slider turtles, and high homology among large syntenic blocks shared with other turtles and sauropsids. Yet, numerous chromosomal rearrangements were also evident across chelonians, between turtles and squamates, and between avian and non-avian reptiles.

Transcription of a B chromosome CAP-G pseudogene does not influence normal Condensin Complex genes in a grasshopper.

Parasitic B chromosomes invade and persist in natural populations through several mechanisms for transmission advantage (drive). They may contain gene-derived sequences which, in some cases, are actively transcribed. A further interesting question is whether B-derived transcripts become functional products. In the grasshopper Eyprepocnemis plorans, one of the gene-derived sequences located on the B chromosome shows homology with the gene coding for the CAP-G subunit of condensin I. We show here, by means of fluorescent in situ hybridization coupled with tyramide signal amplification (FISH-TSA), that this gene is located in the distal region of the B24 chromosome variant. The DNA sequence located in the B chromosome is a pseudogenic version of the CAP-G gene (B-CAP-G). In two Spanish populations, we found active transcription of B-CAP-G, but it did not influence the expression of CAP-D2 and CAP-D3 genes coding for corresponding condensin I and II subunits, respectively. Our results indicate that the transcriptional regulation of the B-CAP-G pseudogene is uncoupled from the standard regulation of the genes that constitute the condensin complex, and suggest that some of the B chromosome known effects may be related with its gene content and transcriptional activity, thus opening new exciting avenues for research.

Protein-coding genes in B chromosomes of the grasshopper Eyprepocnemis plorans.

For many years, parasitic B chromosomes have been considered genetically inert elements. Here we show the presence of ten protein-coding genes in the B chromosome of the grasshopper Eyprepocnemis plorans. Four of these genes (CIP2A, GTPB6, KIF20A, and MTG1) were complete in the B chromosome whereas the six remaining (CKAP2, CAP-G, HYI, MYCB2, SLIT and TOP2A) were truncated. Five of these genes (CIP2A, CKAP2, CAP-G, KIF20A, and MYCB2) were significantly up-regulated in B-carrying individuals, as expected if they were actively transcribed from the B chromosome. This conclusion is supported by three truncated genes (CKAP2, CAP-G and MYCB2) which showed up-regulation only in the regions being present in the B chromosome. Our results indicate that B chromosomes are not so silenced as was hitherto believed. Interestingly, the five active genes in the B chromosome code for functions related with cell division, which is the main arena where B chromosome destiny is played. This suggests that B chromosome evolutionary success can lie on its gene content.