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Abstract Background Transposable elements (TEs) can represent one of the major sources of genomic variation across eukaryotes, providing novel raw materials for species diversification and innovation. While considerable effort has been made to study their evolutionary dynamics across multiple animal clades, molluscs represent a substantially understudied phylum. Here, we take advantage of the recent increase in mollusc genomic resources and adopt an automated TE annotation pipeline combined with a phylogenetic tree-based classification, as well as extensive manual curation efforts, to characterize TE repertories across 27 bivalve genomes with a particular emphasis on DDE/D class II elements, long interspersed nuclear elements (LINEs), and their evolutionary dynamics. Results We found class I elements as highly dominant in bivalve genomes, with LINE elements, despite less represented in terms of copy number per genome, being the most common retroposon group covering up to 10% of their genome. We mined 86,488 reverse transcriptases (RVT) containing LINE coming from 12 clades distributed across all known superfamilies and 14,275 class II DDE/D-containing transposons coming from 16 distinct superfamilies. We uncovered a previously underestimated rich and diverse bivalve ancestral transposon complement that could be traced back to their most recent common ancestor that lived ~ 500 Mya. Moreover, we identified multiple instances of lineage-specific emergence and loss of different LINEs and DDE/D lineages with the interesting cases of CR1- Zenon, Proto2, RTE-X, and Academ elements that underwent a bivalve-specific amplification likely associated with their diversification. Finally, we found that this LINE diversity is maintained in extant species by an equally diverse set of long-living and potentially active elements, as suggested by their evolutionary history and transcription profiles in both male and female gonads. Conclusions We found that bivalves host an exceptional diversity of transposons compared to other molluscs. Their LINE complement could mainly follow a “stealth drivers” model of evolution where multiple and diversified families are able to survive and co-exist for a long period of time in the host genome, potentially shaping both recent and early phases of bivalve genome evolution and diversification. Overall, we provide not only the first comparative study of TE evolutionary dynamics in a large but understudied phylum such as Mollusca, but also a reference library for ORF-containing class II DDE/D and LINE elements, which represents an important genomic resource for their identification and characterization in novel genomes.

Abstract The evolution of automixis – i.e., meiotic parthenogenesis – requires several features, including ploidy restoration after meiosis and maintenance of fertility. Characterizing the relative contribution of novel versus pre-existing genes and the similarities in their expression and sequence evolution is fundamental to understand the evolution of reproductive novelties. Here we identify gonads-biased genes in two Bacillus automictic stick-insects and compare their expression profile and sequence evolution with a bisexual congeneric species. The two parthenogens restore ploidy through different cytological mechanisms: in Bacillus atticus, nuclei derived from the first meiotic division fuse to restore a diploid egg nucleus, while in Bacillus rossius, diploidization occurs in some cells of the haploid blastula through anaphase restitution. Parthenogens’ gonads transcriptional program is found to be largely assembled from genes that were already present before the establishment of automixis. The three species transcriptional profiles largely reflect their phyletic relationships, yet we identify a shared core of genes with gonad-biased patterns of expression in parthenogens which are either male gonads-biased in the sexual species or are not differentially expressed there. At the sequence level, just a handful of gonads-biased genes were inferred to have undergone instances of positive selection exclusively in the parthenogen species. This work is the first to explore the molecular underpinnings of automixis in a comparative framework: it delineates how reproductive novelties can be sustained by genes whose origin precedes the establishment of the novelty itself and shows that different meiotic mechanisms of reproduction can be associated with a shared molecular ground plan.

Hox and ParaHox genes (HPHGs) are key developmental genes that pattern regional identity along the anterior–posterior body axis of most animals. Here, we identified HPHGs in tadpole shrimps (Pancrustacea, Branchiopoda, Notostraca), an iconic example of the so-called “living fossils” and performed a comparative genomics analysis of HPHGs and the Hox cluster among major branchiopod lineages. Notostraca possess the entire Hox complement, and the Hox cluster seems to be split into two different subclusters, although we were not able to support this finding with chromosome-level assemblies. However, the genomic structure of Hox genes in Notostraca appears more derived than that of Daphnia spp., which instead retains the plesiomorphic condition of a single compact cluster. Spinicaudata and Artemia franciscana show instead a Hox cluster subdivided across two or more genomic scaffolds with some orthologs either duplicated or missing. Yet, branchiopod HPHGs are similar among the various clades in terms of both intron length and number, as well as in their pattern of molecular evolution. Sequence substitution rates are in fact generally similar for most of the branchiopod Hox genes and the few differences we found cannot be traced back to natural selection, as they are not associated with any signals of diversifying selection or substantial switches in selective modes. Altogether, these findings do not support a significant stasis in the Notostraca Hox cluster and further confirm how morphological evolution is not tightly associated with genome dynamics.

Abstract The class Branchiopoda, whose origin dates back to Cambrian, includes ~ 1200 species which mainly occupy freshwater habitats. The phylogeny and systematics of the class have been debated for long time, until recent phylogenomic analyses allowed to better clarify the relationships among major clades. Based on these data, the clade Anostraca (fairy and brine shrimps) is sister to all other branchiopods, and the Notostraca (tadpole shrimps) results as sister group to Diplostraca, which includes Laevicaudata + Spinicaudata (clam shrimps) and Cladoceromorpha (water fleas + Cyclestherida). In the present analysis, thanks to an increased taxon sampling, a complex picture emerges. Most of the analyzed mitogenomes show the Pancrustacea gene order while in several other taxa they are found rearranged. These rearrangements, though, occur unevenly among taxa, most of them being found in Cladocera, and their taxonomic distribution does not agree with the phylogeny. Our data also seems to suggest the possibility of potentially homoplastic, alternative gene order within Daphniidae.

Abstract Inferring the selective forces that orthologous genes underwent across different lineages can help us understand the evolutionary processes that have shaped their extant diversity and the phenotypes they underlie. The most widespread metric to estimate the selection regimes of coding genes—across sites and phylogenies—is the ratio of nonsynonymous to synonymous substitutions (dN/dS, also known as ω). Nowadays, modern sequencing technologies and the large amount of already available sequence data allow the retrieval of thousands of orthologous genes across large numbers of species. Nonetheless, the tools available to explore selection regimes are not designed to automatically process all genes, and their practical usage is often restricted to the single‐copy ones which are found across all species considered (i.e., ubiquitous genes). This approach limits the scale of the analysis to a fraction of single‐copy genes, which can be as low as an order of magnitude in respect to those which are not consistently found in all species considered (i.e., nonubiquitous genes). Here, we present a workflow named BASE that—leveraging the CodeML framework—eases the inference and interpretation of gene selection regimes in the context of comparative genomics. Although a number of bioinformatics tools have already been developed to facilitate this kind of analyses, BASE is the first to be specifically designed to allow the integration of nonubiquitous genes in a straightforward and reproducible manner. The workflow—along with all relevant documentation—is available at github.com/for‐giobbe/BASE.

Abstract Background The crustacean class Branchiopoda includes fairy shrimps, clam shrimps, tadpole shrimps, and water fleas. Branchiopods, which are well known for their great variety of reproductive strategies, date back to the Cambrian and extant taxa can be mainly found in freshwater habitats, also including ephemeral ponds. Mitochondrial genomes of the notostracan taxa Lepidurus apus lubbocki (Italy), L. arcticus (Iceland) and Triops cancriformis (an Italian and a Spanish population) are here characterized for the first time and analyzed together with available branchiopod mitogenomes. Results Overall, branchiopod mitogenomes share the basic structure congruent with the ancestral Pancrustacea model. On the other hand, rearrangements involving tRNAs and the control region are observed among analyzed taxa. Remarkably, an unassigned region in the L. apus lubbocki mitogenome showed a chimeric structure, likely resulting from a non-homologous recombination event between the two flanking trnC and trnY genes. Notably, Anostraca and Onychocaudata mitogenomes showed increased GC content compared to both Notostraca and the common ancestor, and a significantly higher substitution rate, which does not correlate with selective pressures, as suggested by dN/dS values. Conclusions Branchiopod mitogenomes appear rather well-conserved, although gene rearrangements have occurred. For the first time, it is reported a putative non-homologous recombination event involving a mitogenome, which produced a pseudogenic tRNA sequence. In addition, in line with data in the literature, we explain the higher substitution rate of Anostraca and Onychocaudata with the inferred GC substitution bias that occurred during their evolution.

Abstract Background Transposable elements (TEs) are common and often present with high copy numbers in cellular genomes. Unlike in cellular organisms, TEs were previously thought to be either rare or absent in viruses. Almost all reported TEs display only one or two copies per viral genome. In addition, the discovery of pandoraviruses with genomes up to 2.5-Mb emphasizes the need for biologists to rethink the fundamental nature of the relationship between viruses and cellular life. Results Herein, we performed the first comprehensive analysis of miniature inverted-repeat transposable elements (MITEs) in the 5170 viral genomes for which sequences are currently available. Four hundred and fifty one copies of ten miniature inverted-repeat transposable elements (MITEs) were found and each MITE had reached relatively large copy numbers (some up to 90) in viruses. Eight MITEs belonging to two DNA superfamilies (hobo/Activator/Tam3 and Chapaev–Mirage–CACTA) were for the first time identified in viruses, further expanding the organismal range of these two superfamilies. TEs may play important roles in shaping the evolution of pandoravirus genomes, which were here found to be very rich in MITEs. We also show that putative autonomous partners of seven MITEs are present in the genomes of viral hosts, suggesting that viruses may borrow the transpositional machinery of their cellular hosts’ autonomous elements to spread MITEs and colonize their own genomes. The presence of seven similar MITEs in viral hosts, suggesting horizontal transfers (HTs) as the major mechanism for MITEs propagation. Conclusions Our discovery highlights that TEs contribute to shape genome evolution of pandoraviruses. We concluded that as for cellular organisms, TEs are part of the pandoraviruses’ diverse mobilome.

The myth of a “typical” mitochondrial genome (mtDNA) is a rock-hard belief in the field of genetics, at least for the animal kingdom [...]

Notwithstanding the initial claims of general conservation, mitochondrial genomes are a largely heterogeneous set of organellar chromosomes which displays a bewildering diversity in terms of structure, architecture, gene content, and functionality. The mitochondrial genome is typically described as a single chromosome, yet many examples of multipartite genomes have been found (for example, among sponges and diplonemeans); the mitochondrial genome is typically depicted as circular, yet many linear genomes are known (for example, among jellyfish, alveolates, and apicomplexans); the chromosome is normally said to be “small”, yet there is a huge variation between the smallest and the largest known genomes (found, for example, in ctenophores and vascular plants, respectively); even the gene content is highly unconserved, ranging from the 13 oxidative phosphorylation-related enzymatic subunits encoded by animal mitochondria to the wider set of mitochondrial genes found in jakobids. In the present paper, we compile and describe a large database of 27,873 mitochondrial genomes currently available in GenBank, encompassing the whole eukaryotic domain. We discuss the major features of mitochondrial molecular diversity, with special reference to nucleotide composition and compositional biases; moreover, the database is made publicly available for future analyses on the MoZoo Lab GitHub page.

Transposable elements are widely distributed within genomes where they may significantly impact their evolution and cell functions. Short interspersed elements (SINEs) are non-autonomous, fast-evolving elements, but some of them carry a highly conserved domain (HCD), whose sequence remained substantially unchanged throughout the metazoan evolution. SINEs carrying the HCD called V are absent in amniote genomes, but V-like sequences were found within the miniature inverted-repeat transposable element (MITE) MER6 in Homo sapiens. In the present work, the genomic distribution and evolution of MER6 are investigated, in order to reconstruct the origin of human V domain and to envisage its possible functional role. The analysis of 85 tetrapod genomes revealed that MER6 and its variant MER6A are found in primates, while only the MER6A variant was found in bats and eulipotyphlans. These MITEs appeared no longer active, in line with literature data on mammalian DNA transposons. Moreover, they appeared to have originated from a Mariner element found in turtles and from a V-SINE from bony fishes. MER6 insertions were found within genes and conserved in mRNAs: in line with previous hypothesis on functional role of HCDs, the MER6 V domain may be important for cell function also in mammals.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR.

At least 12 genes (FH, HIF2A, MAX, NF1, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL) have been implicated in inherited predisposition to phaeochromocytoma (PCC), paraganglioma (PGL), or head and neck paraganglioma (HNPGL) and a germline mutation may be detected in more than 30% of cases. Knowledge of somatic mutations contributing to PCC/PGL/HNPGL pathogenesis has received less attention though mutations in HRAS, HIF2A, NF1, RET, and VHL have been reported. To further elucidate the role of somatic mutation in PCC/PGL/HNPGL tumourigenesis, we employed a next generation sequencing strategy to analyse “mutation hotspots” in 50 human cancer genes. Mutations were identified for HRAS (c.37G>C; p.G13R and c.182A>G; p.Q61R) in 7.1% (6/85); for BRAF (c.1799T>A; p.V600E) in 1.2% (1/85) of tumours; and for TP53 (c.1010G>A; p.R337H) in 2.35% (2/85) of cases. Twenty-one tumours harboured mutations in inherited PCC/PGL/HNPGL genes and no HRAS, BRAF, or TP53 mutations occurred in this group. Combining our data with previous reports of HRAS mutations in PCC/PGL we find that the mean frequency of HRAS/BRAF mutations in sporadic PCC/PGL is 8.9% (24/269) and in PCC/PGL with an inherited gene mutation 0% (0/148) suggesting that HRAS/BRAF mutations and inherited PCC/PGL genes mutations might be mutually exclusive. We report the first evidence for BRAF mutations in the pathogenesis of PCC/PGL/HNPGL.

R2 is an extensively investigated non-LTR retrotransposon that specifically inserts into the 28S rRNA gene sequences of a wide range of metazoans, disrupting its functionality. During R2 integration, first strand synthesis can be incomplete so that 5' end deleted copies are occasionally inserted. While active R2 copies repopulate the locus by retrotransposing, the non-functional truncated elements should frequently be eliminated by molecular drive processes leading to the concerted evolution of the rDNA array(s). Although, multiple R2 lineages have been discovered in the genome of many animals, the rDNA of the stick insect Bacillus rossius exhibits a peculiar situation: it harbors both a canonical, functional R2 element (R2Brfun) as well as a full-length but degenerate element (R2Brdeg). An intensive sequencing survey in the present study reveals that all truncated variants in stick insects are present in multiple copies suggesting they were duplicated by unequal recombination. Sequencing results also demonstrate that all R2Brdeg copies are full-length, i. e. they have no associated 5' end deletions, and functional assays indicate they have lost the active ribozyme necessary for R2 RNA maturation. Although it cannot be completely ruled out, it seems unlikely that the degenerate elements replicate via reverse transcription, exploiting the R2Brfun element enzymatic machinery, but rather via genomic amplification of inserted 28S by unequal recombination. That inactive copies (both R2Brdeg or 5'-truncated elements) are not eliminated in a short term in stick insects contrasts with findings for the Drosophila R2, suggesting a widely different management of rDNA loci and a lower efficiency of the molecular drive while achieving the concerted evolution.

Relationships of nine Italian Chrysotoxum species were analysed using morphological and molecular data. The morphology-derived cladogram revealed three well-defined groups: (i) C. cautum, (ii) the arcuatum group (C. arcuatum, C. fasciolatum) and (iii) the festivum group (C. festivum - C. vernale, C. bicinctum, C. elegans, C. octomaculatum and C. parmense). Trees inferred from COI-tRNALeu-COII sequences were largely in agreement, but they identified (i) C. parmense as an isolated branch, (ii) C. festivum and C. vernale as separate entities, (iii) C. elegans within a paraphyletic C. festivum clade. ITS2 trees were partially unresolved but C. parmense sequence emerged as a sister to the festivum group. The monophyly of the festivum group derived from morphological data was rejected by a phylogenetic test performed on combined molecular data set. The diagnostic value of some morphological characters commonly used to identify Chrysotoxum species is therefore questioned.

Retrotransposons of the R2 superclade specifically insert within the 28S ribosomal gene. They have been isolated from a variety of metazoan genomes and were found vertically inherited even if their phylogeny does not always agree with that of the host species. This was explained with the diversification/extinction of paralogous lineages, being proved the absence of horizontal transfer. We here analyze the widest available collection of R2 sequences, either newly isolated from recently sequenced genomes or drawn from public databases, in a phylogenetic framework. Results are congruent with previous analyses, but new important issues emerge. First, the N-terminal end of the R2-B clade protein, so far unknown, presents a new zinc fingers configuration. Second, the phylogenetic pattern is consistent with an ancient, rapid radiation of R2 lineages: being the estimated time of R2 origin (850-600 Million years ago) placed just before the metazoan Cambrian explosion, the wide element diversity and the incongruence with the host phylogeny could be attributable to the sudden expansion of available niches represented by host's 28S ribosomal genes. Finally, we detect instances of coexisting multiple R2 lineages showing a non-random phylogenetic pattern, strongly similar to that of the "library" model known for tandem repeats: a collection of R2s were present in the ancestral genome and then differentially activated/repressed in the derived species. Models for activation/repression as well as mechanisms for sequence maintenance are also discussed within this framework.

Homologous Replacement is used to modify specific gene sequences of chromosomal DNA in a process referred to as "Small Fragment Homologous Replacement", where DNA fragments replace genomic target resulting in specific sequence changes. To optimize the efficiency of this process, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF-SV-40). To obtain the highest correction frequency with this system, several parameters were considered: fragment synthesis and concentration, cell cycle phase and methylation status of both fragment and recipient genome. In addition, different drugs were employed to test their ability to improve technique efficiency. SFHR-mediated genomic modification resulted to be stably transmitted for several cell generations and confirmed at transcript and genomic levels. Modification efficiency was estimated in a range of 0.01-0.5%, further increasing when PARP-1 repair pathway was inhibited. In this study, for the first time SFHR efficiency issue was systematically approached and in part addressed, therefore opening new potential therapeutic ex-vivo applications.

Abstract??Tunga penetransis a widely distributed sand flea, infecting men and domestic animals. It originated in South America, but it is now also endemic of Sub-Saharan Africa due to a recent accidental introduction. Previous genetic analyses indicated a very limited variability in the Ecuadorian populations; on the other hand, samples from Madagascar resulted to be genetically isolated. To better examine the genetic variability ofT. penetrans, a wider sampling was analysed for mitochondrial (cytochrome oxidase II) and nuclear (ribosomal internal transcribed spacer [ITS] 2) DNA sequences. The mitochondrial marker confirms the low genetic variability, with few haplotypes characterizing the majority of individuals from different populations. Haplotype distribution is in agreement with a recent colonization of Africa and with a rapid spreading across this continent. Moreover, a consistent gene flow between the Pacific and Atlantic South American populations emerges, possibly due to human and/or animal cross-Andean dispersal. On the other hand, the ITS2 marker depicts a sharply diverging pattern with samples collected in Ecuador unequivocally distinguished from the Brazilian and African ones. On the whole, taking into account also the molecular features of the marker used, data here presented are better interpreted in the light of a high dispersal ability ofT. penetrans, probably reducing the phylogeographic signal. [ABSTRACT FROM AUTHOR]