Integrating Sequence Capture and Restriction Site-Associated DNA Sequencing to resolve Recent Radiations of Pelagic Seabirds

The diversification of modern birds has been shaped by a number of radiations. Rapid diversification events make reconstructing the evolutionary relationships among taxa challenging due to the convoluted effects of incomplete lineage sorting (ILS) and introgression. Phylogenomic datasets have the potential to detect patterns of phylogenetic incongruence, and to address their causes. However, the footprints of ILS and introgression on sequence data can vary between different phylogenomic markers at different phylogenetic scales depending on factors such as their evolutionary rates or their selection pressures. We show that combining phylogenomic markers that evolve at different rates, such as paired-end double-digest restriction site-associated DNA (PE-ddRAD) and ultraconserved elements (UCEs), allows a comprehensive exploration of the causes of phylogenetic discordance associated with short internodes at different timescales. We used thousands of UCE and PE-ddRAD markers to produce the first well-resolved phylogeny of shearwaters, a group of medium-sized pelagic seabirds that are among the most phylogenetically controversial and endangered bird groups. We found that phylogenomic conflict was mainly derived from high levels of ILS due to rapid speciation events. We also documented a case of introgression, despite the high philopatry of shearwaters to their breeding sites, which typically limits gene flow. We integrated state-of-the-art concatenated and coalescent-based approaches to expand on previous comparisons of UCE and RAD-Seq datasets for phylogenetics, divergence time estimation and inference of introgression, and we propose a strategy to optimise RAD-Seq data for phylogenetic analyses. Our results highlight the usefulness of combining phylogenomic markers evolving at different rates to understand the causes of phylogenetic discordance at different timescales

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species

The first genome of the Balearic shearwater (Puffinus mauretanicus) provides a valuable resource for conservation genomics and sheds light on adaptation to a pelagic lifestyle

The Balearic shearwater (Puffinus mauretanicus) is the most threatened seabird in Europe and a member of the most speciose group of pelagic seabirds, the order Procellariiformes, which exhibit extreme adaptations to a pelagic lifestyle. The fossil record suggests that human colonisation of the Balearic Islands resulted in a sharp decrease of the Balearic shearwater population size. Currently, populations of the species continue to be decimated mainly due to predation by introduced mammals and bycatch in longline fisheries, with some studies predicting its extinction by 2070. Here, using a combination of short and long reads, we generate the first high-quality reference genome for the Balearic shearwater, with a completeness amongst the highest across available avian species. We used this reference genome to study critical aspects relevant to the conservation status of the species and to gain insights into the adaptation to a pelagic lifestyle of the order Procellariiformes. We detected relatively high levels of genome-wide heterozygosity in the Balearic shearwater despite its reduced population size. However, the reconstruction of its historical demography uncovered an abrupt population decline potentially linked to a reduction of the neritic zone during the Penultimate Glacial Period (∼194-135 ka). Comparative genomics analyses uncover a set of candidate genes that may have played an important role into the adaptation to a pelagic lifestyle of Procellariiformes, including those for the enhancement of fishing capabilities, night vision, and the development of natriuresis. The reference genome obtained will be the crucial in the future development of genetic tools in conservation efforts for this Critically Endangered species

Integrating Sequence Capture and Restriction Site-Associated DNA Sequencing to Resolve Recent Radiations of Pelagic Seabirds

The diversification of modern birds has been shaped by a number of radiations. Rapid diversification events make reconstructing the evolutionary relationships among taxa challenging due to the convoluted effects of incomplete lineage sorting (ILS) and introgression. Phylogenomic data sets have the potential to detect patterns of phylogenetic incongruence, and to address their causes. However, the footprints of ILS and introgression on sequence data can vary between different phylogenomic markers at different phylogenetic scales depending on factors such as their evolutionary rates or their selection pressures. We show that combining phylogenomic markers that evolve at different rates, such as paired-end double-digest restriction site-associated DNA (PE-ddRAD) and ultraconserved elements (UCEs), allows a comprehensive exploration of the causes of phylogenetic discordance associated with short internodes at different timescales. We used thousands of UCE and PE-ddRAD markers to produce the first well-resolved phylogeny of shearwaters, a group of medium-sized pelagic seabirds that are among the most phylogenetically controversial and endangered bird groups. We found that phylogenomic conflict was mainly derived from high levels of ILS due to rapid speciation events. We also documented a case of introgression, despite the high philopatry of shearwaters to their breeding sites, which typically limits gene flow. We integrated state- of-the-art concatenated and coalescent-based approaches to expand on previous comparisons of UCE and RAD-Seq data sets for phylogenetics, divergence time estimation, and inference of introgression, and we propose a strategy to optimize RAD-Seq data for phylogenetic analyses. Our results highlight the usefulness of combining phylogenomic markers evolving at different rates to understand the causes of phylogenetic discordance at different timescales.

Palaeoceanographic changes in the late Pliocene promoted rapid diversification in pelagic seabirds

Aim: Palaeoceanographic changes can act as drivers of diversification and speciation, even in highly mobile marine organisms. Shearwaters are a group of globally distributed and highly mobile pelagic seabirds. Despite a recent well-resolved phylogeny, shearwaters have controversial species limits, and show periods of both slow and rapid diversification. Here, we explore the role of palaeoceanographic changes on shearwaters' diversification and speciation. We investigate shearwater biogeography and the evolution of a key phenotypic trait, body size, and we assess the validity of their current taxonomy

Birds of a feather flock together:a dataset for Clock and Adcyap1 genes from migration genetics studies

Birds in seasonal habitats rely on intricate strategies for optimal timing of migrations. This is governed by environmental cues, including photoperiod. Genetic factors affecting intrinsic timekeeping mechanisms, such as circadian clock genes, have been explored, yielding inconsistent findings with potential lineage-dependency. To clarify this evidence, a systematic review and phylogenetic reanalysis was done. This descriptor outlines the methodology for sourcing, screening, and processing relevant literature and data. PRISMA guidelines were followed, ultimately including 66 studies, with 34 focusing on candidate genes at the genotype-phenotype interface. Studies were clustered using bibliographic coupling and citation network analysis, alongside scientometric analyses by publication year and location. Data was retrieved for allele data from databases, article supplements, and direct author communications. The dataset, version 1.0.2, encompasses data from 52 species, with 46 species for the Clock gene and 43 for the Adcyap1 gene. This dataset, featuring data from over 8000 birds, constitutes the most extensive cross-species collection for these candidate genes, used in studies investigating gene polymorphisms and seasonal bird migration.</p

The evolutionary history of shearwaters: genomic analyses to resolve a radiation of pelagic seabrids

[eng] How populations differentiate and become new species is a foundational question to the field of evolutionary biology and has important implications for the generation of both local and global patterns of species-level biodiversity. Ernst Mayr emphasised the importance of geographical isolation as a driver of speciation: “populations in separate locations begin a process of differentiation, and once differentiation is sufficient the populations have become two species”. In marine environments, the lack of obvious physical barriers would suggest that panmixia, especially in highly mobile species, or isolation-by-distance, in other cases, will prevail. However, there is some counterintuitive evidence of fine-scale differentiation among populations and species in a number of mobile marine organisms, a phenomenon that has been described as the “marine species paradox”. Seabirds of the order Procellariiformes present some of the most extreme examples of this paradox. On the one hand, Procellariiformes are highly mobile pelagic seabirds with a high dispersal ability and perform some of the longest animal migrations on Earth. On the other hand, they show high philopatry to their breeding grounds, which is expected to limit gene flow and therefore reinforce genetic differentiation. This thesis aims to gain insights into the patterns and processes that contribute to genetic and phenotypic diversification, speciation and dispersal across multiple evolutionary timescales. To this end, I focus on shearwaters (Calonectris, Puffinus and Ardenna), a globally distributed and threatened group of Procellariiformes. Through an integrative approach combining two types of phylogenomic markers, which evolve at different nucleotide substitution rates, and state-of-the-art phylogenetic and introgression analyses, I inferred a robust phylogeny. This approach allowed to discover that the majority of the phylogenetic conflict in shearwaters is generated by high levels of incomplete lineage sorting (ILS) due to rapid speciation events. Divergence time estimation analyses highlighted a severe impact of the Pliocene marine megafauna extinction on shearwaters, probably caused by a sudden reduction in the availability of coastal habitat. Subsequently, the late Pliocene-early Pleistocene was inferred as a period of high and rapid speciation and dispersal, probably promoted by Pleistocene climatic shifts. Biogeographic analyses showed that surface ocean currents promote species dispersal and founder events are a main mode of speciation in shearwaters. Our analysis, combining genomic data with morphological and ecological evidence, did not support any of the current taxonomic classifications for the North Atlantic and Mediterranean Puffinus shearwaters, and so I propose a more accurate taxonomy for the group. Moreover, the detection of fine-scale genetic structure within Puffinus shearwater species, highlights the need for management of evolutionary significant units below the species level. Population genomics analyses identified genetic drift as the major process shaping the genomic landscapes of divergence. In conclusion, the marriage of these various investigations identifies a prevalence of ILS across different timescales, highlights the important role of paleoceanographic events in promoting diversification, and demonstrates the importance of neutral evolution at driving population differentiation in pelagic seabirds. Overall, this thesis showcases the use of multiple genomic approaches, leveraging phylogenetic and population genetic analyses across multiple timescales, to shed light on the evolutionary history of shearwaters.[cat] Una de les preguntes fundacionals del camp de la biologia evolutiva és com es diferencien les poblacions i esdevenen noves espècies, i té importants implicacions en l’establiment dels patrons locals i globals de biodiversitat específica. Ernst Mayr va subratllar la importància de l’aïllament geogràfic com a mecanisme promotor d’especiació: “les poblacions en localitats aïllades comencen un procés de diferenciació i, quan aquesta diferenciació és suficient, les poblacions esdevenen dues espècies”. En l’ambient marí, la manca de barreres físiques evidents suggereix que la panmixi (en espècies amb gran capacitat de moviment) i l’aïllament per distància (en espècies de mobilitat reduïda) són els patrons prevalents. En canvi, s’ha detectat diferenciació a petita escala entre diferents poblacions i diferents espècies en un elevat nombre d’organismes marins mòbils, un fenomen que es coneix com la “paradoxa de les espècies marines”. Els ocells marins de l’ordre dels Procellariiformes representen un dels exemples més extrems d’aquesta paradoxa. Per una banda, els Procellariiformes són ocells marins amb gran capacitat de moviment i alta capacitat dispersiva que duen a terme algunes de les migracions més llargues del planeta. Per altra banda, són espècies molt fidels a les seves zones de cria, fenomen que probablement limiti el flux gènic i reforci la diferenciació genètica. Aquesta tesi té com a objectiu caracteritzar els patrons i processos que contribueixen a la diversificació genètica i fenotípica, a l’especiació i a la dispersió a diferents escales evolutives. Per assolir aquest objectiu, m’he focalitzat en les baldrigues (Calonectris, Puffinus i Ardenna), un grup amenaçat de Procellariiformes distribuït per tot el planeta. Mitjançant una aproximació que combina dos tipus de marcadors filogenòmics que evolucionen amb diferents taxes de substitució nucleotídica, i anàlisis filogenètiques i de detecció d’introgressió infereixo una filogènia ben resolta. Aquesta aproximació ha permès descobrir que la majoria del conflicte filogenètic en les baldrigues és producte dels alts nivells de sorteig incomplet de llinatges (incomplete lineage sorting) degut a esdeveniments ràpids d’especiació. L’anàlisi dels temps de divergència ha demostrat un gran impacte de l’extinció de megafauna marina del Pliocè en les baldrigues, probablement a causa de la sobtada reducció en la disponibilitat d’hàbitats costaners. Posteriorment, el Pliocè tardà i el principi del Pleistocè van ser períodes de molta i ràpida especiació i dispersió, probablement a causa dels canvis climàtics del Pleistocè. Les anàlisis biogeogràfiques han demostrat que els corrents marins promouen la dispersió i que l’efecte fundador és un mecanisme important d’especiació en les baldrigues. Les nostres anàlisis, combinant dades genòmiques amb evidència morfològica i ecològica, no han donat suport a les classificacions taxonòmiques actuals del grup de les baldrigues del gènere Puffinus del nord de l’Atlàntic i del Mediterrani i, per tant, proposo una classificació taxonòmica més adequada pel grup. A més a més, la detecció d’estructura genètica a petita escala en les espècies d’aquest grup de baldrigues destaca la necessitat de gestió d’unitats evolutives significatives per sota del nivell d’espècie. Les anàlisis de genòmica de poblacions han identificat la deriva genètica com a principal promotor de divergència en el genoma. Per concloure, el conjunt de les investigacions d’aquesta tesi identifiquen la prevalença del sorteig incomplet de llinatges al llarg de diferents escales evolutives, destaquen l’important paper dels esdeveniments paleoceanogràfics com a promotors de diversificació i indiquen la importància de l’evolució neutra com a causa de diferenciació poblacional en ocells marins pelàgics. Globalment, aquesta tesi demostra la utilitat de diferents aproximacions genòmiques, mitjançant anàlisis filogenètiques i de genètica de poblacions en diferents escales evolutives per proporcionar nou coneixement sobre la història evolutiva de les baldrigues

Supplemental Information for: The first genome of the Balearic shearwater (Puffinus mauretanicus) provides a valuable resource for conservation genomics and sheds light on adaptation to a pelagic lifestyle

Figures and tables. -- Table S1. Summary of the RepeatMasker analysis results. -- Table S2. Annotated predicted protein-coding genes, their lengths and their functional annotation based on matches against the curated databases. -- Table S3. Annotated predicted transcripts, their lengths and their functional annotation based on matches against the curated databases. -- Table S4. Mean coverage and standardized coverage (gene coverage/whole mitogenome coverage) for each annotated mitochondrial gene. -- Table S5. Genes with evidence of positive selection, and their functional annotation, in the Procellariiformes included in the comparative genomics study. -- Table S6. GOs terms enriched in Procellariiformes dataset (positive selection) with its description and p-values. Dispensable GOs are collapsed. -- Table S7. RELAX results: list of genes under selection (both relaxed or intensified) in Procellariiformes. -- Table S8. GO terms enriched in the Procellariiformes (from RELAX dataset; Table S7). Dispensable GOs are collapsed. -- Table S9. Outlier Families in the Procellariiformes branch (BadiRate analysis), with its pvalue, FDR_P-value, and its functional annotation. -- Figure S1. Annotated mitogenome of P. mauretanicus. -- Figure S2. Species tree made with CDS supermatrix 4D positions with IQ-TREE. -- Figure S3. Species tree made with amino acid supermatrix with IQ-TREE. -- Figure S4. Species tree made with ASTRAL using CDS gene tree quartets. -- Figure S5. Species tree made with ASTRAL using amino acid gene tree quartets.Peer reviewe