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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.

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.

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.

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.

Although mitochondrial inheritance in metazoans is typically strictly maternal, doubly uniparental inheritance (DUI) is probably the major exception to this widespread rule. DUI has been found in many species of bivalve molluscs, belonging to several different families. Based on current understanding, the detection of DUI generally relies on the detection of two distinct mitochondrial DNA lineages: a female-transmitted one, that dominates somatic tissues in males and females and eggs, and a male-transmitted one, that dominates the male germline and sperm. When a new species with DUI is identified, novel data are available to make a better inference on the evolution of this phenomenon within the Bivalvia. In this study, mitochondrial heteroplasmy in Pseudocardium sachalinense (Schrenck, 1862) is described. This species belongs to the family of Mactridae, in which DUI has not been previously demonstrated: this finding allowed to upgrade the present knowledge about the distribution of DUI.

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.

Bivalves and gastropods are the two largest classes of extant molluscs. Despite sharing a huge number of features, they do not share a key ecological one: gastropods are essentially epibenthic, although most bivalves are infaunal. However, this is not the ancestral bivalve condition; Cambrian forms were surface crawlers and only during the Ordovician a fundamental infaunalization process took place, leading to bivalves as we currently know them. This major ecological shift is linked to the exposure to a different redox environoments (hypoxic or anoxic) and with the Lower Devonian oxygenation event. We investigated selec- tive signatures on bivalve and gastropod mitochondrial genomes with respect to a time calibrated mitochondrial phylogeny by means of dN/dS ratios. We were able to detect 1) a major signal of directional selection between the Ordovician and the Lower Devonian for bivalve mitochondrial Complex I, and 2) an overall higher directional selective pressure on bivalve Complex V with respect to gastropods. These and other minor dN/dS patterns and timings are discussed, showing that the Ordovician infaunalization event left heavy traces in bivalve mitochondrial genomes.

The Order Phasmatodea (stick and leaf insects) includes many well-known species of cryptic phytophagous insects. In this work, we sequenced the almost complete mitochondrial genomes of two stick insect species of the genus Bacillus. Phasmatodea pertain to the Polyneoptera, and represent one of the major clades of heterometabolous insects. Orthopteroid insect lineages arose through rapid evolutionary radiation events, which likely blurred the phylogenetic reconstructions obtained so far; we therefore performed a phylogenetic analysis to resolve and date all major splits of orthopteroid phylogeny, including the relationships between Phasmatodea and other polyneopterans. We explored several molecular models, with special reference to data partitioning, to correctly detect any phylogenetic signal lying in rough data. Phylogenetic Informativeness analysis showed that the maximum resolving power on the orthopteroid mtDNA dataset is expected for the Upper Cretaceous, about 80 million years ago (Mya), but at least 70% of the maximum informativeness is also expected for the 150–200 Mya timespan, which makes mtDNA a suitable marker to study orthopteroid splits. A complete chronological calibration has also been computed following a Penalized Likelihood method. In summary, our analysis confirmed the monophyly of Phasmatodea, Dictyoptera and Orthoptera, and retrieved Mantophasmatodea as sister group of Phasmatodea. The origin of orthopteroid insects was also estimated to be in the Middle Triassic, while the order Phasmatodea seems to appear in the Upper Jurassic. The obtained results evidenced that mtDNA is a suitable marker to unravel the ancient splits leading to the orthopteroid orders, given a proper methodological approach

Bivalve mitochondrial genomes show many uncommon features, like additional genes, high rates of gene rearrangement, high A-T content. Moreover, Doubly Uniparental Inheritance (DUI) is a distinctive inheritance mechanism allowing some bivalves to maintain and trans- mit two separate sex-linked mitochondrial genomes. Many bivalve mitochondrial features, such as gene extensions or additional ORFs, have been proposed to be related to DUI but, up to now, this topic is far from being understood. Several species are known to show this unusual organelle inheritance but, being widespread only among Unionidae and Mytilidae, DUI distribution is unclear. We sequenced and characterized the complete female- (F) and male-transmitted (M) mitochondrial genomes of Meretrix lamarckii, which, in fact, is the sec- ond species of the family Veneridae where DUI has been demonstrated so far. The two mitochondrial genomes are comparable in length and show roughly the same gene content and order, except for three additional tRNAs found in the M one. The two sex-linked genomes show an average nucleotide divergence of 16%. A 100-aminoacid insertion in M. lamarckii M-cox2 gene was found; moreover, additional ORFs have been found in both F and M Long Unassigned Regions of M. lamarckii. Even if no direct involvement in DUI pro- cess has been demonstrated so far, the finding of cox2 insertions and supernumerary ORFs in M. lamarckii both strengthens this hypothesis and widens the taxonomical distribu- tion of such unusual features. Finally, the analysis of inter-sex genetic variability shows that DUI species form two separate clusters, namely Unionidae and Mytilidae+Veneridae; this dichotomy is probably due to different DUI regimes acting on separate taxa.

Taxon sampling is a key point in phylogenetic studies. Incomplete, biased, or improper taxon sampling can lead to misleading results in reconstructing phylogenies. Several methods are available to optimize taxon choice, involving genetic distance evaluations, interruption of long branches, etc. However, all are conceived as a posteriori tests, i. e. they can only be carried out after data collection, when most of the experimental work is already done. We think it would be very useful to have an a priori test, allowing to compute and predict goodness of taxon sampling before the phylogenetic work starts. The established taxonomy of the group under study (although not necessary the “true” one), which is usually available from literature, may help in this: given a master list of organisms, it is possible to measure to which degree different subsamples do represent the whole assemblage. For this reason, we proposed a new method to assess the representativeness of a given taxon sampling, by developing the Clarke and Warwick statistics on taxonomic distinctness. Our method aims to measure the phylogenetic representativeness of a given sample or set of samples, and it is based entirely on the pre-existing available taxonomy of the ingroup. Moreover, our method also accounts for instability and discordance in systematics: we conceived a randomization algorithm on master lists, mimicking taxonomic revisions, therefore addressing test reliability. A Python-based script suite, called PhyRe, was developed to implement all analyses, and it is available for free download at www.mozoolab.net. We showed that this method is sensitive and allows direct discrimination between representative and unrepresentative samples. It is also informative about the addition of taxa to improve taxonomic coverage of the ingroup. Provided that the investigators’ expertise is mandatory in this field, phylogenetic representativeness makes up an objective touchstone in planning phylogenetic studies.

Despite huge fossil, morphological and molecular data, bivalves' early evolutionary history is still a matter of debate: recently, established phylogeny has been mostly challenged by DNA studies, and little agreement has been reached in literature, because of a substantial lack of widely-accepted methodological approaches to retrieve and analyze bivalves' molecular data. Here we present a molecular phylogeny of the class based on four mitochondrial genes (12s, 16s, cox1, cytb) and a methodological pipeline that proved to be useful to obtain robust results. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the utility of Bayesian inference and the importance of molding and implementing non-trivial evolutionary models. Therefore, our analysis allowed to target many taxonomic questions of Bivalvia, and to obtain a complete time calibration of the tree depicting bivalves' earlier natural history main events, which mostly dated in the late Cambrian.