The Evolution and Characterization of the RNA Interference Pathways in Lophotrochozoa.

In animals, three main RNA interference mechanisms have been described so far, which respectively maturate three types of small noncoding RNAs (sncRNAs): miRNAs, piRNAs, and endo-siRNAs. The diversification of these mechanisms is deeply linked with the evolution of the Argonaute gene superfamily since each type of sncRNA is typically loaded by a specific Argonaute homolog. Moreover, other protein families play pivotal roles in the maturation of sncRNAs, like the DICER ribonuclease family, whose DICER1 and DICER2 paralogs maturate respectively miRNAs and endo-siRNAs. Within Metazoa, the distribution of these families has been only studied in major groups, and there are very few data for clades like Lophotrochozoa. Thus, we here inferred the evolutionary history of the animal Argonaute and DICER families including 43 lophotrochozoan species. Phylogenetic analyses along with newly sequenced sncRNA libraries suggested that in all Trochozoa, the proteins related to the endo-siRNA pathway have been lost, a part of them in some phyla (i.e. Nemertea, Bryozoa, Entoprocta), while all of them in all the others. On the contrary, early diverging phyla, Platyhelminthes and Syndermata, showed a complete endo-siRNA pathway. On the other hand, miRNAs were revealed the most conserved and ubiquitous mechanism of the metazoan RNA interference machinery, confirming their pivotal role in animal cell regulation.

Mitochondrially mediated RNA interference, a retrograde signaling system affecting nuclear gene expression.

Several functional classes of short noncoding RNAs are involved in manifold regulatory processes in eukaryotes, including, among the best characterized, miRNAs. One of the most intriguing regulatory networks in the eukaryotic cell is the mito-nuclear crosstalk: recently, miRNA-like elements of mitochondrial origin, called smithRNAs, were detected in a bivalve species, Ruditapes philippinarum. These RNA molecules originate in the organelle but were shown in vivo to regulate nuclear genes. Since miRNA genes evolve easily de novo with respect to protein-coding genes, in the present work we estimate the probability with which a newly arisen smithRNA finds a suitable target in the nuclear transcriptome. Simulations with transcriptomes of 12 bivalve species suggest that this probability is high and not species specific: one in a hundred million (1 × 10-8) if five mismatches between the smithRNA and the 3' mRNA are allowed, yet many more are allowed in animals. We propose that novel smithRNAs may easily evolve as exaptation of the pre-existing mitochondrial RNAs. In turn, the ability of evolving novel smithRNAs may have played a pivotal role in mito-nuclear interactions during animal evolution, including the intriguing possibility of acting as speciation trigger.

Metal and metalloid concentrations in wild mammals from SW Europe: European hedgehog (Erinaceus europaeus) and badger (Meles meles).

In recent years, there have been increasing ecological and global concerns associated to Potentially Toxic Elements (PTEs). Thus, the relevance of wild mammals as biomonitors has been globally recognised. In the present study, Cd, Pb, Hg, Zn and As concentrations were quantified in European hedgehog and badger inhabiting SW Europe, and cumulative trends in relation to age and sex were evaluated. Liver and kidney samples were collected, mineralised and PTE content was determined by ICP-MS. Zn was the most abundant element quantified in both organs (239 and 89.8 mg kg-1 for hedgehogs and 179 and 164 mg kg-1 dw for badgers). In hedgehogs, very high Hg concentration were quantified (4.35 and 15.5 mg kg-1 dw in liver and kidney), and Cd was the most abundant for badgers (4.70 and 7.61 mg kg-1 dw in liver and kidney). Positive correlations were observed for the concentrations of PTE in the organs of both species. Age-dependence increased only Cd concentration, with levels in adult kidneys being significantly higher. In this study, European hedgehog and badger were used as biomonitors for the determination of PTEs to provide current reference values in relatively non-polluted areas of SW Europe, and to enhance the use of these species for future ecotoxicological studies.

Biodiversity of extant snails (Gastropoda, Mollusca) in the Pliocene Mountain Spur Natural Reserve (Northern Apennine, Italy).

Background: To date, there is a substantial lack of information about gastropods from the Tuscan-Emilian Apennine in the north of Italy, notwithstanding the availability of detailed and comprehensive literature on this molluscan class. We present a gastropod fauna from the Natural Reserve of the Pliocene Mountain Spur: to our knowledge, this is the first investigation of the extant gastropod fauna in the Tuscan-Emilian Apennine and one of the few in the Apennine's mountain chain as a whole. New information: We describe a gastropod fauna comprised by 25 species, belonging to 18 genera and 10 families: the general figure which is emerging is an assemblage of European and Mediterranean-European species, with a single Asian contribution. Several shells were collected as embedded in sandy-clayey soils and showed fossilisation traces: therefore, we regard these shells as subfossil samples. Namely, subfossil shells are from the species Pomatiaselegans, Granariafrumentum, Retinellaolivetorum, Xerolentaobviaobvia, Oxychiluscf.draparnaudi, Monachacartusiana and Monachacantiana. The present checklist is the first report in the Tuscan-Emilian Apennine and Emilia-Romagna areas for six taxa: Morlinaglabraglabra, Oxychiliusalliarius, Xerosectacespitum, Fruticicolafruticum, Xerogyraspadae and Xerolentaobviaobvia.

Mito-nuclear coevolution and phylogenetic artifacts: the case of bivalve mollusks.

Mito-nuclear phylogenetic discordance in Bivalvia is well known. In particular, the monophyly of Amarsipobranchia (Heterodonta + Pteriomorphia), retrieved from mitochondrial markers, contrasts with the monophyly of Heteroconchia (Heterodonta + Palaeoheterodonta), retrieved from nuclear markers. However, since oxidative phosphorylation nuclear markers support the Amarsipobranchia hypothesis instead of the Heteroconchia one, interacting subunits of the mitochondrial complexes ought to share the same phylogenetic signal notwithstanding the genomic source, which is different from the signal obtained from other nuclear markers. This may be a clue of coevolution between nuclear and mitochondrial genes. In this work we inferred the phylogenetic signal from mitochondrial and nuclear oxidative phosphorylation markers exploiting different phylogenetic approaches and added two more datasets for comparison: genes of the glycolytic pathway and genes related to the biogenesis of regulative small noncoding RNAs. All trees inferred from mitochondrial and nuclear subunits of the mitochondrial complexes support the monophyly of Amarsipobranchia, regardless of the phylogenetic pipeline. However, not every single marker agrees with this topology: this is clearly visible in nuclear subunits that do not directly interact with the mitochondrial counterparts. Overall, our data support the hypothesis of a coevolution between nuclear and mitochondrial genes for the oxidative phosphorylation. Moreover, we suggest a relationship between mitochondrial topology and different nucleotide composition between clades, which could be associated to the highly variable gene arrangement in Bivalvia.

One in a Million: Genetic Diversity and Conservation of the Reference Crassostrea angulata Population in Europe from the Sado Estuary (Portugal).

The production of cupped oysters is an important component of European aquaculture. Most of the production relies on the cultivation of the Pacific oyster Crassostrea gigas, although the Portuguese oyster Crassostrea angulata represents a valuable product with both cultural and economic relevance, especially in Portugal. The authors of the present study investigated the genetic diversity of Portuguese oyster populations of the Sado estuary, both from natural oyster beds and aquaculture facilities, through cox1 gene fragment sequencing. Then, a comparison with a wide dataset of cupped oyster sequences obtained from GenBank (up to now the widest available dataset in literature for the Portuguese oyster) was performed. Genetic data obtained from this work confirmed that the Pacific oyster does not occur in the natural oyster beds of the Sado estuary but showed that the species occasionally occurs in the oyster hatcheries. Moreover, the results showed that despite the founder effect and the bottleneck events that the Sado populations have experienced, they still exhibit high haplotype diversity. Risks are arising for the conservation of the Portuguese oyster reference populations of the Sado estuary due to the occurrence of the Pacific oyster in the local hatcheries. Therefore, researchers, local authorities, and oyster producers should work together to avoid the loss of this valuable resource.

Mitochondrial Genomic Landscape: A Portrait of the Mitochondrial Genome 40 Years after the First Complete Sequence.

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.

HERMES: An improved method to test mitochondrial genome molecular synapomorphies among clades.

Mitochondrial chromosomes have diversified among eukaryotes and many different architectures and features are now acknowledged for this genome. Here we present the improved HERMES index, which can measure and quantify the amount of molecular change experienced by mitochondrial genomes. We test the improved approach with ten molecular phylogenetic studies based on complete mitochondrial genomes, representing six bilaterian Phyla. In most cases, HERMES analysis spotted out clades or single species with peculiar molecular synapomorphies, allowing to identify phylogenetic and ecological patterns. The software presented herein handles linear, circular, and multi-chromosome genomes, thus widening the HERMES scope to the complete eukaryotic domain.

Mitonuclear Coevolution, but not Nuclear Compensation, Drives Evolution of OXPHOS Complexes in Bivalves.

In Metazoa, four out of five complexes involved in oxidative phosphorylation (OXPHOS) are formed by subunits encoded by both the mitochondrial (mtDNA) and nuclear (nuDNA) genomes, leading to the expectation of mitonuclear coevolution. Previous studies have supported coadaptation of mitochondria-encoded (mtOXPHOS) and nuclear-encoded OXPHOS (nuOXPHOS) subunits, often specifically interpreted with regard to the "nuclear compensation hypothesis," a specific form of mitonuclear coevolution where nuclear genes compensate for deleterious mitochondrial mutations due to less efficient mitochondrial selection. In this study, we analyzed patterns of sequence evolution of 79 OXPHOS subunits in 31 bivalve species, a taxon showing extraordinary mtDNA variability and including species with "doubly uniparental" mtDNA inheritance. Our data showed strong and clear signals of mitonuclear coevolution. NuOXPHOS subunits had concordant topologies with mtOXPHOS subunits, contrary to previous phylogenies based on nuclear genes lacking mt interactions. Evolutionary rates between mt and nuOXPHOS subunits were also highly correlated compared with non-OXPHO-interacting nuclear genes. Nuclear subunits of chimeric OXPHOS complexes (I, III, IV, and V) also had higher dN/dS ratios than Complex II, which is formed exclusively by nuDNA-encoded subunits. However, we did not find evidence of nuclear compensation: mitochondria-encoded subunits showed similar dN/dS ratios compared with nuclear-encoded subunits, contrary to most previously studied bilaterian animals. Moreover, no site-specific signals of compensatory positive selection were detected in nuOXPHOS genes. Our analyses extend the evidence for mitonuclear coevolution to a new taxonomic group, but we propose a reconsideration of the nuclear compensation hypothesis.

Molecular Phylogenetics and Mitochondrial Evolution.

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

Mitochondrial phylogeny and taxonomic revision of Italian and Slovenian fluvio-lacustrine barbels, Barbus sp. (Cypriniformes, Cyprinidae).

BACKGROUND: Barbels are ray finned cyprinid fishes of the Old-World with partially unresolved, intricate taxonomy. Within the Barbus sensu lato paraphyletic assemblage, Barbus sensu stricto is a monophyletic tetraploid lineage of Europe, northern Africa and Middle East, including two monophyletic sibling genera: Barbus and Luciobarbus. Italy, Slovenia and northern Croatia are natively inhabited by several entities of the genus Barbus, whose relationships and taxonomic ranks are still unclear. Aim of the present work is to focus on phylogeography of Italian and Slovenian barbels, with an appraisal of their current taxonomy. RESULTS: One hundred fifty specimens were collected in 78 sampling sites from 33 main watersheds, widely distributed along Italian and Slovenian ichthyogeographic districts. We amplified two mitochondrial markers, cytochrome b (cytb) and control region (D-loop), to infer a robust phylogeny for our sample and investigate on species delimitation. Our results strongly indicate all Italian and Adriatic Slovenian fluvio-lacustrine barbels to be comprised into at least three distinct species. We provide a proposal of taxonomic revision and a list of synonymies for two of them and a new description under the International Code of Zoological Nomenclature rules for the third one. CONCLUSIONS: If nuclear data will confirm our findings, at least three specific entities should be acknowledged across our sampling area. Namely, the three species are (i) Barbus plebejus, in the Padano-Venetian district; (ii) Barbus tyberinus, in the Tuscany-Latium district; (iii) Barbus oscensis Rossi & Plazzi sp. nov., in the Tyrrhenian and southernmost-Adriatic parts of Apulia-Campania district. Finally, we briefly discuss the implications of such a taxonomic scenario on conservation policies.

Phylomitogenomics provides new perspectives on the Euphasmatodea radiation (Insecta: Phasmatodea).

Phasmatodea species diversity lies almost entirely within its suborder Euphasmatodea, which exhibits a pantropical distribution and is considered to derive from a recent and rapid evolutionary radiation. To shed light on Euphasmatodea origins and diversification, we assembled the mitogenomes of 17 species from transcriptomic sequencing data and analysed them along with 22 already available Phasmatodea mitogenomes and 33 mitogenomes representing most of the Polyneoptera lineages. Maximum Likelihood and Bayesian Inference approaches retrieved consistent topologies, both showing the widespread conflict between phylogenetic approaches and traditional systematics. We performed a divergence time analysis leveraging ten fossil specimens representative of most polyneopteran lineages: the time tree obtained supports an older radiation of the clade with respect to previous hypotheses. Euphasmatodea diversification is inferred to have started ~ 187 million years ago, suggesting that the Triassic-Jurassic mass extinction and the breakup of Pangea could have contributed to the process. We also investigated Euphasmatodea mitogenomes patterns of dN, dS and dN/dS ratio throughout our time-tree, trying to characterize the selective regime which may have shaped the clade evolution.

Clues of in vivo nuclear gene regulation by mitochondrial short non-coding RNAs.

Gene expression involves multiple processes, from transcription to translation to the mature, functional peptide, and it is regulated at multiple levels. Small RNA molecules are known to bind RNA messengers affecting their fate in the cytoplasm (a process generically termed 'RNA interference'). Such small regulatory RNAs are well-known to be originated from the nuclear genome, while the role of mitochondrial genome in RNA interference was largely overlooked. However, evidence is growing that mitochondrial DNA does provide the cell a source of interfering RNAs. Small mitochondrial highly transcribed RNAs (smithRNAs) have been proposed to be transcribed from the mitochondrion and predicted to regulate nuclear genes. Here, for the first time, we show in vivo clues of the activity of two smithRNAs in the Manila clam, Ruditapes philippinarum. Moreover, we show that smithRNAs are present and can be annotated in representatives of the three main bilaterian lineages; in some cases, they were already described and assigned to a small RNA category (e.g., piRNAs) given their biogenesis, while in other cases their biogenesis remains unclear. If mitochondria may affect nuclear gene expression through RNA interference, this opens a plethora of new possibilities for them to interact with the nucleus and makes metazoan mitochondrial DNA a much more complex genome than previously thought.

Comparative Transcriptomics in Two Bivalve Species Offers Different Perspectives on the Evolution of Sex-Biased Genes.

Comparative genomics has become a central tool for evolutionary biology, and a better knowledge of understudied taxa represents the foundation for future work. In this study, we characterized the transcriptome of male and female mature gonads in the European clam Ruditapes decussatus, compared with that in the Manila clam Ruditapes philippinarum providing, for the first time in bivalves, information about transcription dynamics and sequence evolution of sex-biased genes. In both the species, we found a relatively low number of sex-biased genes (1,284, corresponding to 41.3% of the orthologous genes between the two species), probably due to the absence of sexual dimorphism, and the transcriptional bias is maintained in only 33% of the orthologs. The dN/dS is generally low, indicating purifying selection, with genes where the female-biased transcription is maintained between the two species showing a significantly higher dN/dS. Genes involved in embryo development, cell proliferation, and maintenance of genome stability show a faster sequence evolution. Finally, we report a lack of clear correlation between transcription level and evolutionary rate in these species, in contrast with studies that reported a negative correlation. We discuss such discrepancy and call into question some methodological approaches and rationales generally used in this type of comparative studies.

Evolution of Two Short Interspersed Elements in Callorhinchus milii (Chondrichthyes, Holocephali) and Related Elements in Sharks and the Coelacanth.

Short interspersed elements (SINEs) are non-autonomous retrotransposons. Although they usually show fast evolutionary rates, in some instances highly conserved domains (HCDs) have been observed in elements with otherwise divergent sequences and from distantly related species. Here, we document the life history of two HCD-SINE families in the elephant shark Callorhinchus milii, one specific to the holocephalan lineage (CmiSINEs) and another one (SacSINE1-CM) with homologous elements in sharks and the coelacanth (SacSINE1s, LmeSINE1s). The analyses of their relationships indicated that these elements share the same 3'-tail, which would have allowed both elements to rise to high copy number by exploiting the C. milii L2-2_CM long interspersed element (LINE) enzymes. Molecular clock analysis on SINE activity in C. milii genome evidenced two replication bursts occurring right after two major events in the holocephalan evolution: the end-Permian mass extinction and the radiation of modern Holocephali. Accordingly, the same analysis on the coelacanth homologous elements, LmeSINE1, identified a replication wave close to the split age of the two extant Latimeria species. The genomic distribution of the studied SINEs pointed out contrasting results: some elements were preferentially sorted out from gene regions, but accumulated in flanking regions, while others appear more conserved within genes. Moreover, data from the C. milii transcriptome suggest that these SINEs could be involved in miRNA biogenesis and may be targets for miRNA-based regulation.

Evolution of sex-dependent mtDNA transmission in freshwater mussels (Bivalvia: Unionida).

Doubly uniparental inheritance (DUI) describes a mode of mtDNA transmission widespread in gonochoric freshwater mussels (Bivalvia: Palaeoheterodonta: Unionida). In this system, both female- and male-transmitted mtDNAs, named F and M respectively, coexist in the same species. In unionids, DUI is strictly correlated to gonochorism and to the presence of the atypical open reading frames (ORFans) F-orf and M-orf, respectively inside F and M mtDNAs, which are hypothesized to participate in sex determination. However, DUI is not found in all three Unionida superfamilies (confirmed in Hyrioidea and Unionoidea but not in Etherioidea), raising the question of its origin in these bivalves. To reconstruct the co-evolution of DUI and of ORFans, we sequenced the mtDNAs of four unionids (two gonochoric with DUI, one gonochoric and one hermaphroditic without DUI) and of the related gonochoric species Neotrigonia margaritacea (Palaeoheterodonta: Trigoniida). Our analyses suggest that rearranged mtDNAs appeared early during unionid radiation, and that a duplicated and diverged atp8 gene evolved into the M-orf associated with the paternal transmission route in Hyrioidea and Unionoidea, but not in Etherioidea. We propose that novel mtDNA-encoded genes can deeply influence bivalve sex determining systems and the evolution of the mitogenomes in which they occur.

SmithRNAs: Could Mitochondria "Bend" Nuclear Regulation?

Typically, animal mitochondria have very compact genomes, with few short intergenic regions, and no introns. Hence, it may seem that there is little space for unknown functions in mitochondrial DNA (mtDNA). However, mtDNA can also operate through RNA interference, as small non coding RNAs (sncRNAs) produced by mtDNA have already been proposed for humans. We sequenced sncRNA libraries from isolated mitochondria of Ruditapes philippinarum (Mollusca Bivalvia) gonads, a species with doubly uniparental inheritance of mitochondria, and identified several putative sncRNAs of mitochondrial origin. Some sncRNAs are transcribed by intergenic regions that form stable stem-hairpin structures, which makes them good miRNA-like candidates. We decided to name them small mitochondrial highly-transcribed RNAs (smithRNAs). Many concurrent data support that we have recovered sncRNAs of mitochondrial origin that might be involved in gonad formation and able to affect nuclear gene expression. This possibility has been never suggested before. If mtDNA can affect nuclear gene expression through RNA interference, this opens a plethora of new possibilities for it to interact with the nucleus, and makes metazoan mtDNA a much more complex genome than previously thought.