MIPs: multi-locus intron polymorphisms in species identification and population genomics.

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).

Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers.Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.

Contrasting life-history traits of two sympatric smooth-hound species: implication for vulnerability.

In this study, life-history traits (maximum and average size, size at maturity and fecundity) of two congeneric smooth-hounds, Mustelus mustelus and Mustelus punctulatus, which share a geographical distribution and experience a similar fishing exploitation, were estimated and compared between species. The results indicated a lower maximum and average size, a lower size at maturity and a higher fecundity in M. punctulatus compared with those in M. mustelus. Considering that these two species co-occur in the same areas and are caught by the same fishing gears, the results indicate a higher vulnerability to exploitation of M. mustelus compared with that of M. punctulatus.

Connectivity Among Populations of the Top Shell Gibbula divaricata in the Adriatic Sea.

Genetic connectivity studies are essential to understand species diversity and genetic structure and to assess the role of potential factors affecting connectivity, thus enabling sound management and conservation strategies. Here, we analyzed the patterns of genetic variability in the marine snail Gibbula divaricata from five coastal locations in the central-south Adriatic Sea (central Mediterranean) and one in the adjacent northern Ionian Sea, using 21 described polymorphic microsatellite loci. Observed and expected heterozygosity varied from 0.582 to 0.635 and 0.684 to 0.780, respectively. AMOVA analyses showed that 97% of genetic variation was observed within populations. Nevertheless, significant, although small, genetic differentiation was found among nearly all of the pairwise F ST comparisons. Over a general pattern of panmixia, three groups of populations were identified: eastern Adriatic populations, western Adriatic populations, and a third group represented by the single northern Ionian Sea population. Nonetheless, migration and gene flow were significant between these groups. Gibbula divaricata is thought to have a limited dispersal capacity related to its lecithotrophic trochophore larval stage. Our results indicated high levels of self-recruitment and gene flow that is mainly driven through coastline dispersion, with populations separated by the lack of suitable habitats or deep waters. This stepping-stone mode of dispersion together with the high levels of self-recruitment could lead to higher levels of population structuring and differentiation along the Adriatic Sea. Large effective population sizes and episodic events of long-distance dispersal might be responsible for the weak differentiation observed in the analyzed populations. In summary, the circulation system operating in this region creates natural barriers for dispersion that, together with life-history traits and habitat requirements, certainly affect connectivity in G. divaricata. However, this scenario of potential differentiation seems to be overridden by sporadic events of long-distance dispersal across barriers and large effective population sizes.

Population genomics meet Lagrangian simulations: Oceanographic patterns and long larval duration ensure connectivity among Paracentrotus lividus populations in the Adriatic and Ionian seas.

Connectivity between populations influences both their dynamics and the genetic structuring of species. In this study, we explored connectivity patterns of a marine species with long-distance dispersal, the edible common sea urchin Paracentrotus lividus, focusing mainly on the Adriatic-Ionian basins (Central Mediterranean). We applied a multidisciplinary approach integrating population genomics, based on 1,122 single nucleotide polymorphisms (SNPs) obtained from 2b-RAD in 275 samples, with Lagrangian simulations performed with a biophysical model of larval dispersal. We detected genetic homogeneity among eight population samples collected in the focal Adriatic-Ionian area, whereas weak but significant differentiation was found with respect to two samples from the Western Mediterranean (France and Tunisia). This result was not affected by the few putative outlier loci identified in our dataset. Lagrangian simulations found a significant potential for larval exchange among the eight Adriatic-Ionian locations, supporting the hypothesis of connectivity of P. lividus populations in this area. A peculiar pattern emerged from the comparison of our results with those obtained from published P. lividus cytochrome b (cytb) sequences, the latter revealing genetic differentiation in the same geographic area despite a smaller sample size and a lower power to detect differences. The comparison with studies conducted using nuclear markers on other species with similar pelagic larval durations in the same Adriatic-Ionian locations indicates species-specific differences in genetic connectivity patterns and warns against generalizing single-species results to the entire community of rocky shore habitats.

Unravelling population genetic structure with mitochondrial DNA in a notional panmictic coastal crab species: sample size makes the difference.

BACKGROUND: The extent of genetic structure of a species is determined by the amount of current gene flow and the impact of historical and demographic factors. Most marine invertebrates have planktonic larvae and consequently wide potential dispersal, so that genetic uniformity should be common. However, phylogeographic investigations reveal that panmixia is rare in the marine realm. Phylogeographic patterns commonly coincide with geographic transitions acting as barriers to gene flow. In the Mediterranean Sea and adjoining areas, the best known barriers are the Atlantic-Mediterranean transition, the Siculo-Tunisian Strait and the boundary between Aegean and Black seas. Here, we perform the so far broadest phylogeographic analysis of the crab Pachygrapsus marmoratus, common across the north-eastern Atlantic Ocean, Mediterranean and Black seas. Previous studies revealed no or weak genetic structuring at meso-geographic scale based on mtDNA, while genetic heterogeneity at local scale was recorded with microsatellites, even if without clear geographic patterns. Continuing the search for phylogeographic signal, we here enlarge the mtDNA dataset including 51 populations and covering most of the species' distribution range. RESULTS: This enlarged dataset provides new evidence of three genetically separable groups, corresponding to the Portuguese Atlantic Ocean, Mediterranean Sea plus Canary Islands, and Black Sea. Surprisingly, hierarchical AMOVA and Principal Coordinates Analysis agree that our Canary Islands population is closer to western Mediterranean populations than to mainland Portugal and Azores populations. Within the Mediterranean Sea, we record genetic homogeneity, suggesting that population connectivity is unaffected by the transition between the western and eastern Mediterranean. The Mediterranean metapopulation seems to have experienced a relatively recent expansion around 100,000 years ago. CONCLUSIONS: Our results suggest that the phylogeographic pattern of P. marmoratus is shaped by the geological history of Mediterranean and adjacent seas, restricted current gene flow among different marginal seas, and incomplete lineage sorting. However, they also caution from exclusively testing well-known biogeographic barriers, thereby neglecting other possible phylogeographic patterns. Mostly, this study provides evidence that a geographically exhaustive dataset is necessary to detect shallow phylogeographic structure within widespread marine species with larval dispersal, questioning all studies where species have been categorized as panmictic based on numerically and geographically limited datasets.

Multiple paternity and hybridization in two smooth-hound sharks.

Multiple paternity appears to be a common trait of elasmobranch mating systems, with its occurrence likely driven by convenience, due to females seeking to minimize the stress of male harassment. Here we use molecular markers to analyse the frequency of multiple paternity in two related viviparous sharks, Mustelus mustelus and Mustelus punctulatus. We first applied molecular methods to assign pregnant females, embryos and additional reference adults (N = 792) to one of the two species. Paternity analysis was performed using a total of 9 polymorphic microsatellites on 19 females and 204 embryos of M. mustelus, and on 13 females and 303 embryos of M. punctulatus. Multiple paternity occurs in both species, with 47% of M. mustelus and 54% of M. punctulatus litters sired by at least two fathers. Female fecundity is not influenced by multiple mating and in 56% of polyandrous litters paternity is skewed, with one male siring most of the pups. Genetic analyses also revealed hybridization between the two species, with a M. punctulatus female bearing pups sired by a M. mustelus male. The frequency of polyandrous litters in these species is consistent with aspects of their reproductive biology, such as synchronous ovulation and possible occurrence of breeding aggregations.

Transcriptomic profiling of male European eel (Anguilla anguilla) livers at sexual maturity.

The European eel Anguilla anguilla has a complex life cycle that includes freshwater, seawater and morphologically distinct stages as well as two extreme long distance migrations. Eels do not feed as they migrate across the Atlantic to the Sargasso Sea but nevertheless reach sexual maturity before spawning. It is not yet clear how existing energy stores are used to reach the appropriate developmental state for reproduction. Since the liver is involved in energy metabolism, protein biosynthesis and endocrine regulation it is expected to play a key role in the regulation of reproductive development. We therefore used microarrays to identify genes that may be involved in this process. Using this approach, we identified 231 genes that were expressed at higher and 111 genes that were expressed at lower levels in sexually mature compared with immature males. The up-regulated set includes genes involved in lipid metabolism, fatty acid synthesis and transport, mitochondrial function, steroid transport and bile acid metabolism. Several genes with putative enzyme functions were also expressed at higher levels at sexual maturity while genes involved in immune system processes and protein biosynthesis tended to be down-regulated at this stage. By using a high-throughput approach, we have identified a subset of genes that may be linked with the mobilization of energy stores for sexual maturation and migration. These results contribute to an improved understanding of eel reproductive biology and provide insight into the role of the liver in other teleosts with a long distance spawning migrations.

New molecular tools for the identification of 2 endangered smooth-hound sharks, Mustelus mustelus and Mustelus punctulatus.

The smooth-hounds represent a significant proportion of the elasmobranch catch in the Adriatic basin of the Mediterranean Sea, where the common (Mustelus mustelus) and blackspotted (Mustelus punctulatus) smooth-hounds co-occur. The 2 species share several morphological and morphometric characters that lead to frequent misidentification. In order to provide information useful for their species identification, we performed a morphological identification of several Mustelus specimens to select individuals unambiguously attributed to 1 of the 2 species, and assayed these with 3 new molecular tests. First, we developed and validated a mitochondrial DNA assay based on species-specific amplification of the cytochrome c oxidase subunit 1 (COI). Second, a fragment analysis of 15 microsatellites cross-amplified from several triakid species was performed to identify diagnostic loci. Finally, a length difference was identified in the internal transcribed spacer 2 (ITS2) region and a diagnostic test based on its amplification was established. All the samples classified morphologically as M. mustelus and M. punctulatus showed a species-specific profile using all the 3 molecular tests. In addition, cross-amplification of microsatellites allowed identification of 9 highly polymorphic loci that will be useful for the study of the mating system and population differentiation of the 2 species.

Changes in the gene expression profiles of the brains of male European eels (Anguilla anguilla) during sexual maturation.

BACKGROUND: The vertebrate brain plays a critical role in the regulation of sexual maturation and reproduction by integrating environmental information with developmental and endocrine status. The European eel Anguilla anguilla is an important species in which to better understand the neuroendocrine factors that control reproduction because it is an endangered species, has a complex life cycle that includes two extreme long distance migrations with both freshwater and seawater stages and because it occupies a key position within the teleost phylogeny. At present, mature eels have never been caught in the wild and little is known about most aspects of reproduction in A. anguilla. The goal of this study was to identify genes that may be involved in sexual maturation in experimentally matured eels. For this, we used microarrays to compare the gene expression profiles of sexually mature to immature males. RESULTS: Using a false discovery rate of 0.05, a total of 1,497 differentially expressed genes were identified. Of this set, 991 were expressed at higher levels in brains (forebrain and midbrain) of mature males while 506 were expressed at lower levels relative to brains of immature males. The set of up-regulated genes includes genes involved in neuroendocrine processes, cell-cell signaling, neurogenesis and development. Interestingly, while genes involved in immune system function were down-regulated in the brains of mature males, changes in the expression levels of several receptors and channels were observed suggesting that some rewiring is occurring in the brain at sexual maturity. CONCLUSIONS: This study shows that the brains of eels undergo major changes at the molecular level at sexual maturity that may include re-organization at the cellular level. Here, we have defined a set of genes that help to understand the molecular mechanisms controlling reproduction in eels. Some of these genes have previously described functions while many others have roles that have yet to be characterized in a reproductive context. Since most of the genes examined here have orthologs in other vertebrates, the results of this study will contribute to the body of knowledge concerning reproduction in vertebrates as well as to an improved understanding of eel biology.

Permanent genetic resources added to molecular ecology resources database 1 April 2013-31 May 2013.

This article documents the addition of 234 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser sinensis, Aleochara bilineata, Aleochara bipustulata, Barbus meridionalis, Colossoma macropomum, Delia radicum, Drosophila nigrosparsa, Fontainea picrosperma, Helianthemum cinereum, Liomys pictus, Megabalanus azoricus, Pelteobagrus vachelli, Pleuragramma antarcticum, Podarcis hispanica type 1A, Sardinella brasiliensis and Sclerotinia homoeocarpa. These loci were cross-tested on the following species: Acipenser dabryanus, Barbus balcanicus, Barbus barbus, Barbus cyclolepis, Drosophila hydei, Drosophila melanogaster, Drosophila obscura, Drosophila subobscura, Fontainea australis, Fontainea fugax, Fontainea oraria, Fontainea rostrata, Fontainea venosa, Podarcis bocagei, Podarcis carbonelli, Podarcis liolepis, Podarcis muralis and Podarcis vaucheri.

Genome evolution in the cold: Antarctic icefish muscle transcriptome reveals selective duplications increasing mitochondrial function.

Antarctic notothenioids radiated over millions of years in subzero waters, evolving peculiar features, such as antifreeze glycoproteins and absence of heat shock response. Icefish, family Channichthyidae, also lack oxygen-binding proteins and display extreme modifications, including high mitochondrial densities in aerobic tissues. A genomic expansion accompanying the evolution of these fish was reported, but paucity of genomic information limits the understanding of notothenioid cold adaptation. We reconstructed and annotated the first skeletal muscle transcriptome of the icefish Chionodraco hamatus providing a new resource for icefish genomics (http://compgen.bio.unipd.it/chamatusbase/, last accessed December 12, 2012). We exploited deep sequencing of this energy-dependent tissue to test the hypothesis of selective duplication of genes involved in mitochondrial function. We developed a bioinformatic approach to univocally assign C. hamatus transcripts to orthology groups extracted from phylogenetic trees of five model species. Chionodraco hamatus duplicates were recorded for each orthology group allowing the identification of duplicated genes specific to the icefish lineage. Significantly more duplicates were found in the icefish when transcriptome data were compared with whole-genome data of model species. Indeed, duplicated genes were significantly enriched in proteins with mitochondrial localization, involved in mitochondrial function and biogenesis. In cold conditions and without oxygen-carrying proteins, energy production is challenging. The combination of high mitochondrial densities and the maintenance of duplicated genes involved in mitochondrial biogenesis and aerobic respiration might confer a selective advantage by improving oxygen diffusion and energy supply to aerobic tissues. Our results provide new insights into the genomic basis of icefish cold adaptation.

Sequencing and characterization of striped venus transcriptome expand resources for clam fishery genetics.

BACKGROUND: The striped venus Chamelea gallina clam fishery is among the oldest and the largest in the Mediterranean Sea, particularly in the inshore waters of northern Adriatic Sea. The high fishing pressure has lead to a strong stock abundance decline, enhanced by several irregular mortality events. The nearly complete lack of molecular characterization limits the available genetic resources for C. gallina. We achieved the first transcriptome of this species with the aim of identifying an informative set of expressed genes, potential markers to assess genetic structure of natural populations and molecular resources for pathogenic contamination detection. METHODOLOGY/PRINCIPAL FINDINGS: The 454-pyrosequencing of a normalized cDNA library of a pool C. gallina adult individuals yielded 298,494 raw reads. Different steps of reads assembly and filtering produced 36,422 contigs of high quality, one half of which (18,196) were annotated by similarity. A total of 111 microsatellites and 20,377 putative SNPs were identified. A panel of 13 polymorphic transcript-linked microsatellites was developed and their variability assessed in 12 individuals. Remarkably, a scan to search for contamination sequences of infectious origin indicated the presence of several Vibrionales species reported to be among the most frequent clam pathogen's species. Results reported in this study were included in a dedicated database available at http://compgen.bio.unipd.it/chameleabase. CONCLUSIONS/SIGNIFICANCE: This study represents the first attempt to sequence and de novo annotate the transcriptome of the clam C. gallina. The availability of this transcriptome opens new perspectives in the study of biochemical and physiological role of gene products and their responses to large and small-scale environmental stress in C. gallina, with high throughput experiments such as custom microarray or targeted re-sequencing. Molecular markers, such as the already optimized EST-linked microsatellites and the discovered SNPs will be useful to estimate effects of demographic processes and to detect minute levels of population structuring.

Surviving in a toxic world: transcriptomics and gene expression profiling in response to environmental pollution in the critically endangered European eel.

BACKGROUND: Genomic and transcriptomic approaches have the potential for unveiling the genome-wide response to environmental perturbations. The abundance of the catadromous European eel (Anguilla anguilla) stock has been declining since the 1980s probably due to a combination of anthropogenic and climatic factors. In this paper, we explore the transcriptomic dynamics between individuals from high (river Tiber, Italy) and low pollution (lake Bolsena, Italy) environments, which were measured for 36 PCBs, several organochlorine pesticides and brominated flame retardants and nine metals. RESULTS: To this end, we first (i) updated the European eel transcriptome using deep sequencing data with a total of 640,040 reads assembled into 44,896 contigs (Eeelbase release 2.0), and (ii) developed a transcriptomic platform for global gene expression profiling in the critically endangered European eel of about 15,000 annotated contigs, which was applied to detect differentially expressed genes between polluted sites. Several detoxification genes related to metabolism of pollutants were upregulated in the highly polluted site, including genes that take part in phase I of the xenobiotic metabolism (CYP3A), phase II (glutathione-S-transferase) and oxidative stress (glutathione peroxidase). In addition, key genes in the mitochondrial respiratory chain and oxidative phosphorylation were down-regulated at the Tiber site relative to the Bolsena site. CONCLUSIONS: Together with the induced high expression of detoxification genes, the suggested lowered expression of genes supposedly involved in metabolism suggests that pollution may also be associated with decreased respiratory and energy production.

Reconciling deep calibration and demographic history: bayesian inference of post glacial colonization patterns in Carcinus aestuarii (Nardo, 1847) and C. maenas (Linnaeus, 1758).

A precise inference of past demographic histories including dating of demographic events using bayesian methods can only be achieved with the use of appropriate molecular rates and evolutionary models. Using a set of 596 mitochondrial cytochrome c oxidase I (COI) sequences of two sister species of European green crabs of the genus Carcinus (C. maenas and C. aestuarii), our study shows how chronologies of past evolutionary events change significantly with the application of revised molecular rates that incorporate biogeographic events for calibration and appropriate demographic priors. A clear signal of demographic expansion was found for both species, dated between 10,000 and 20,000 years ago, which places the expansions events in a time frame following the Last Glacial Maximum (LGM). In the case of C. aestuarii, a population expansion was only inferred for the Adriatic-Ionian, suggestive of a colonization event following the flooding of the Adriatic Sea (18,000 years ago). For C. maenas, the demographic expansion inferred for the continental populations of West and North Europe might result from a northward recolonization from a southern refugium when the ice sheet retreated after the LGM. Collectively, our results highlight the importance of using adequate calibrations and demographic priors in order to avoid considerable overestimates of evolutionary time scales.

PERMANENT GENETIC RESOURCES: Isolation and characterization of microsatellite loci in the Mediterranean shore crab Carcinus aestuarii (Decapoda, Portunidae).

We characterized nine polymorphic microsatellites in the Mediterranean shore crab Carcinus aestuarii (Decapoda: Portunidae). Microsatellites were isolated from a partial genomic library enriched for multiple motifs. All loci were polymorphic, with number of alleles ranging from two to 16 and a mean observed heterozygosity of 0.75. Seven loci were in Hardy-Weinberg equilibrium, and two showed weak heterozygote deficiency. No linkage disequilibrium was found between loci. In addition, we tested Hardy-Weinberg and linkage equilibrium of three Carcinus maenas loci, already reported to cross-amplify in C. aestuarii. These molecular markers will be potentially useful to investigate genetic structure of this species.