Giorgio Bertorelle’s research while affiliated with University of Ferrara and other places

Environmental DNA (eDNA) analysis is a promising tool for monitoring wild animal populations and, more recently, their genetic variability. In this study, we used the mitochondrial Cytochrome B gene to develop and apply new eDNA metabarcoding assays targeting amphibian families and genera in order to estimate both inter‐ and intraspecific genetic diversity. We designed and tested seven new primer pairs (a) in silico against an amphibian reference database based on the target genera; (b) in vitro on tissue samples of the target species; and (c) in situ on water samples from 38 wetlands in the Province of Trento (Italy). Overall, most target species were amplified successfully, although some markers also amplified non‐target amphibian species. In addition, to complete the workflow, we compared the performance of three different bioinformatic pipelines (namely, MICCA with VSEARCH, and OBITools using ecotag or metabinkit), in retrieving reads and exact sequence variants from the metabarcoding datasets. Overall, the MICCA based pipeline retrieved more reads, but less putative haplotypes of amphibians. After comparing these sequences with previously known haplotypes from tissue‐based studies, when the aim is to both decrease the probability of detecting false haplotypes and retrieve the highest number of reads, we suggest using MICCA+VSEARCH, unless a direct comparison with tissue‐based genetic data is possible.

Islands are crucial evolutionary hotspots, providing unique opportunities for differentiation of novel biodiversity and long-term segregation of endemic species. Islands are also fragile ecosystems, where biodiversity is more exposed to environmental and anthropogenic pressures than on continents. The Ponza grayling, Hipparchia sbordonii, is an endemic butterfly species that is currently found only in two tiny islands of the Pontine archipelago, off the coast of Italy, occupying an area smaller than 10 km2. It has been classified as Endangered (IUCN) because of the extremely limited area of occurrence, population fragmentation, and the recent demographic decline. Thanks to a combination of different assemblers of long and short genomic reads, bulk transcriptome RNAseq, and synteny analysis with phylogenetically close butterflies, we produced a highly contiguous, chromosome-scale annotated reference genome for the Ponza grayling, including 28 autosomes and the Z sexual chromosomes. The final assembly spanned 388.61 Gb with a contig N50 of 14.5 Mb and a BUSCO completeness score of 98.5%. Synteny analysis using four other butterfly species revealed high collinearity with Hipparchia semele and highlighted 10 intrachromosomal inversions longer than 10 kb, of which two appeared on the lineage leading to H. sbordonii. Our results show that a chromosome-scale reference genome is attainable also when chromatin conformation data may be impractical or present specific technical challenges. The high-quality genomic resource for H. sbordonii opens up new opportunities for the accurate assessment of genetic diversity and genetic load and for the investigations of the genomic novelties characterizing the evolutionary path of this endemic island species.

The Australian lungfish is a primitive and endangered representative of the subclass Dipnoi. The distribution of this species is limited to south‐east Queensland, with some populations considered endemic and others possibly descending from translocations in the late nineteenth century shortly after European discovery. Attempts to resolve the historical distribution of this species have met with conflicting results based on descriptive genetic studies. Understanding if all populations are endemic or some are the result of, or influenced by, translocation events, has implications for conservation management. In this work, we analysed the genetic variation at three types of markers (mtDNA genomes, 11 STRs and 5196 nuclear SNPs) using the approximate Bayesian computation (ABC) algorithm to compare several demographic models. We postulated different contributions of Mary River and Burnett River gene pools into the Brisbane River and North Pine River populations, related to documented translocation events. We ran the analysis for each marker type separately, and we also estimated the posterior probabilities of the models combining the markers. Nuclear SNPs have the highest power to correctly identify the true model among the simulated datasets (where the model was known), but different marker types typically provided similar answers. The most supported demographic model able to explain the real dataset implies that an endemic gene pool is still present in the Brisbane and North Pine Rivers and coexists with the gene pools derived from past documented translocation events. These results support the view that ABC modelling can be useful to reconstruct complex historical translocation events with contemporary implications, and will inform ongoing conservation efforts for the endangered and iconic Australian lungfish.

Seahorses (Hippocampus spp.) are flagship animals inspiring numerous conservation programs. They are the first marine genus to be fully listed on the Convention on International Trade in Endangered Species (CITES) Appendix II due to their substantial vulnerability to overexploitation and habitat loss. The peculiar life history of these fishes has been widely addressed through evolutionary and ecological analyses. Yet, no study has integrated current knowledge to approach species-based conservation status, including trends in abundance, diversity, and threats, hindering seahorses' worldwide effective assessment and management. Here, we bridge these gaps by taking advantage of the available geographic, ecological and genomic (ultra-conserved elements and whole genomes) data in the Hippocampus species at a global scale, and present the most comprehensive study of the seahorses' conservation status to date. Specifically, we explore species distributions and perform ecological niches modelling, phylogenomic mapping, comparative genomic applications, demographic inferences, and conservation gap analyses. We consider their evolutionary history, demographic changes and genomic erosion including genetic load, which have been largely disregarded by current conservation policies. We investigate the degree and type of protective measures currently granted to seahorses, and the biological and ecological factors that are contributing to their existing and future extinction risk. Our results raise questions on whether the current conservation indicators and practices are effective in preserving the present diversity and function of these iconic fishes, as well as their future evolutionary potential and ecological resilience. These insights provide a broad, more complete picture on the status and trends of seahorses and inform successful conservation initiatives. Finally, as more diverse datasets become available in the light of the "omics" and "open-access" era, we discuss the unprecedented opportunities to address interdisciplinary studies required for proper research-based conservation, assessment, and management of biodiversity.

Reduction in population size, with its predicted effects on population fitness, is the most alarming anthropogenic impact on endangered species. By introducing compatible individuals, genetic rescue (GR) is a promising but debated approach for reducing the genetic load unmasked by inbreeding and for restoring the fitness of declining populations. Although GR can improve genetic diversity and fitness, it can also produce loss of ancestry, hampering local adaptation, or replace with introduced variants the unique genetic pools evolved in endemic groups. We used forward genetic simulations based on empirical genomic data to assess fitness benefits and loss of ancestry risks of GR in the Apennine brown bear (Ursus arctos marsicanus). There are approximately 50 individuals of this isolated subspecies, and they have lower genetic diversity and higher inbreeding than other European brown bears, and GR has been suggested to reduce extinction risks. We compared 10 GR scenarios in which the number and genetic characteristics of migrants varied with a non‐GR scenario of simple demographic increase due to nongenetic factors. The introduction of 5 individuals of higher fitness or lower levels of deleterious mutations than the target Apennine brown bear from a larger European brown bear population produced a rapid 10–20% increase in fitness in the subspecies and up to 22.4% loss of ancestry over 30 generations. Without a contemporary demographic increase, fitness started to decline again after a few generations. Doubling the population size without GR gradually increased fitness to a comparable level, but without losing ancestry, thus resulting in the best strategy for the Apennine brown bear conservation. Our results highlight the importance for management of endangered species of realistic forward simulations grounded in empirical whole‐genome data.

Nonadaptive hypotheses on the evolution of eukaryotic genome size predict an expansion when purifying selection is weaker. Accordingly, species with huge genomes, such as lungfish, are expected to show a genome-wide relaxation signature of selection compared with other organisms. However, few studies have empirically tested this prediction using genomic data in a comparative framework. Here we show i) that the newly assembled transcriptome of the Australian lungfish, Neoceratodus forsteri, is characterized by an excess of pervasive transcription, or transcriptional leakage, possibly due to suboptimal transcriptional control, and ii) a significant relaxation signature in coding genes in lungfish species compared with other vertebrates. Based on these observations, we propose that the largest known animal genomes evolved in a nearly neutral scenario whereby genome expansion is less efficiently constrained.

The aoudad (Ammotragus lervia) is a Vulnerable bovid endemic of North Africa. Although legally protected in almost every country of its native distribution, the aoudad continues to be hunted for meat and trophy in both North Africa and the countries where it has been introduced. The species was subject to past translocations planned irrespectively of the genetic diversity and local adaptations of source and receiving populations, and no management or conservation units have ever been designed. We aim to provide here important insights on the taxonomy of the aoudad subspecies and the genetic diversity of most of its wild and captive populations. We collected 127 invasive and non-invasive samples from five of the six subspecies of aoudad recognized to date. We could successfully retrieve genetic data for 74 samples. Of those, 36 provided both nuclear (11 microsatellites) and mitochondrial (Cyt-b) data, 31 just nuclear data, and six just mitochondrial data. We implement Bayesian approaches to infer the population structure and phylogenetic relationships between the different populations/subspecies and backtrack the maternal lineages of introduced individuals in European populations. Our results support the presence of four genetically different wild populations, corresponding to three distinct mitochondrial lineages plus a fourth group restricted to Egypt identified by the nuclear markers. We also provide genetic evidence on the affiliation of some introduced European populations with respect to the native ones. The genetic diversity instead of variation within all wild populations was low. This might be a consequence of small effective population size and/or high inbreeding degree, probably related to hunting, decline in habitat availability and quality (i.e. overgrazing, and frequent drought), and high inbreeding degree. Our results provide important information for the aoudad conservation, including reintroductions and reinforcement actions of wild populations, and the exchange of individuals among captive stocks.

Purifying selection is the most pervasive type of selection, as it constantly removes deleterious mutations arising in populations, directly scaling with population size. Highly expressed genes appear to accumulate fewer deleterious mutations between divergent species' lineages, pointing towards gene expression as an additional driver of purifying selection. However, estimates of the effect of gene expression on segregating deleterious variants in natural populations are lacking, as well as an understanding of the relative contribution of population size and gene expression to overall purifying selection pressure. Here, we analyse genomic and transcriptomic data from two natural populations of closely related sister species with different demographic histories, the Emperor (Aptenodytes forsteri) and the King penguins (A. patagonicus), and demonstrate that purifying selection at the population-level depends on the level of gene expression, with larger effects than population size. Deleterious segregating variants spread less in the population when they are in genes with higher expression rate. Leveraging realistic forward simulations, we estimate that the top 10% of the most highly expressed genes in a genome experience a selection pressure corresponding to an average selection coefficient of -0.1, which decreases to a selection coefficient of -0.01 for the top 50%. Gene expression appears to be a fundamental driver of purifying selection in natural populations, also effective at small population size. We suggest gene expression could be used as a proxy for gene selection coefficients (i.e., distribution of fitness effects), which are notoriously difficult to derive in non-model species under real-world conditions.

We present a genome assembly of Caretta caretta (the Loggerhead sea turtle; Chordata, Testudines, Cheloniidae), generated from genomic data from two unrelated females. The genome sequence is 2.13 gigabases in size. The assembly has a busco completion score of 96.1% and N50 of 130.95 Mb. The majority of the assembly is scaffolded into 28 chromosomal representations with a remaining 2% of the assembly being excluded from these.

Southeastern Canada is inhabited by an amalgam of hybridizing wolf-like canids, raising fundamental questions regarding their taxonomy, origins, and timing of hybridization events. Eastern wolves (Canis lycaon), specifically, have been the subject of significant controversy, being viewed as either a distinct taxonomic entity of conservation concern or a recent hybrid of coyotes (C. latrans) and grey wolves (C. lupus). Mitochondrial DNA analyses show some evidence of eastern wolves being North American evolved canids. In contrast, nuclear genome studies indicate eastern wolves are best described as a hybrid entity, but with unclear timing of hybridization events. To test hypotheses related to these competing findings we sequenced whole genomes of 25 individuals, representative of extant Canadian wolf-like canid types of known origin and levels of contemporary hybridization. Here we present data describing eastern wolves as a distinct taxonomic entity that evolved separately from grey wolves for the past ∼67,000 years with an admixture event with coyotes ∼37,000 years ago. We show that Great Lakes wolves originated as a product of admixture between grey wolves and eastern wolves after the last glaciation (∼8,000 years ago) while eastern coyotes originated as a product of admixture between "western" coyotes and eastern wolves during the last century. Eastern wolf nuclear genomes appear shaped by historical and contemporary gene flow with grey wolves and coyotes, yet evolutionary uniqueness remains among eastern wolves currently inhabiting a restricted range in southeastern Canada.