Linking phylogeography and recent dispersal in high mountains : Insights from two Iberian amphibians

Unveiling how genetic diversity and structure of high mountain populations evolved and what factors are currently shaping it is crucial to predict how species will respond to threats such as climate change, and this will ultimately help to design species-specific conservation measures. Therefore, an integrative approach that combines the study of phylogeographic processes and contemporary dispersal dynamics is required to shed light on the mechanisms underlying spatial patterns of present-day genetic diversity and population structure, which can help to predict species responses to ongoing or future environmental changes. The general aim of this thesis was to uncover major historical and contemporary factors explaining the current genetic diversity and structure of selected Iberian amphibians living in high mountains, namely the Pyrenean brook newt, Calotriton asper, and the midwife toads of the Alytes obstetricans complex. I integrated various techniques, including genetic and mark-recapture analyses, to provide new insights into the phylogeographic history of the study species, describe the genetic consequences of chytridiomycosis outbreaks in midwife toads, inform on current connectivity in C. asper, and characterize its process of lake recolonization following invasive fish removal. Specifically, I showed that C. asper likely endured the last glaciation in five distinct glacial refugia across the Pyrenees, and I detected evidence of extensive mito-nuclear discordances and admixture between taxa of the A. obstetricans complex across three Iberian mountain ranges. A. obstetricans populations hit by a relatively recent chytridiomycosis outbreak had a reduced effective size and were depleted of genetic diversity. Finally, although I found that the majority of C. asper dispersal events involved geographically close populations, I also found potential for occasional long-distance dispersal and revealed the importance of neighbouring fishless areas as source of individuals for restored lakes. Overall, the results of this thesis contribute to improve our understanding of the factors responsible for species structuring in high mountains and may be useful to guide future management decisions for C. asper and the A. obstetricans complex.Societas Europaea Herpetologica (SEH

New insights on patterns of genetic admixture and phylogeographic history in Iberian high mountain populations of midwife toads

Data Availability: Newly generated sequence data are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) under the following accession numbers: OP779379-OP779400 for ND4, OP820514-OP820517 for cyt-b, OP820518-OP820519 for β-fibint7, OP846560-OP846568 for 12S, and OP851584-OP851590 for 16S. Original sequence alignments and microsatellite genotypes are available in the Zenodo repository (https://doi.org/10.5281/zenodo.7270824).Funding: This work was supported by the European Commission projects LIFE+ LIMNOPIRINEUS (LIFE13 NAT/ES/001210, PI: M.V.), LIFE RESQUE ALPYR (LIFE20 NAT/ES/00347, PI: M.V.) and Biodiversa FISHME (BiodivRestor-280, PI: M.V.) and by the Spanish Government projects FUNBIO (RTI2018-096217-B-I00, PI: M.V.), BIOOCULT (2413/2017, PI: M.V.), (Organismo Autónomo Parques Nacionales 2399/2017; PI: J.B) and Picos de Europa National Park grants for 2015 and 2016 (PI: J.B.). F.L. had a doctoral grant funded by Fundação para a Ciência e a Tecnologia (FCT, grant number PD/BD/52598/2014). M.J.J. had an International collaboration grant (National Institute of Ecology) and a postdoctoral grant funded by FCT (grant number SFRH/BPD/109148/2015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Multiple Quaternary glacial refugia in the Iberian Peninsula, commonly known as “refugia within refugia”, allowed diverging populations to come into contact and admix, potentially boosting substantial mito-nuclear discordances. In this study, we employ a comprehensive set of mitochondrial and nuclear markers to shed light onto the drivers of geographical differentiation in Iberian high mountain populations of the midwife toads Alytes obstetricans and A. almogavarii from the Pyrenees, Picos de Europa and Guadarrama Mountains. In the three analysed mountain regions, we detected evidence of extensive mito-nuclear discordances and/or admixture between taxa. Clustering analyses identified three major divergent lineages in the Pyrenees (corresponding to the eastern, central and central-western Pyrenees), which possibly recurrently expanded and admixed during the succession of glacialinterglacial periods that characterised the Late Pleistocene, and that currently follow a ringshaped diversification pattern. On the other hand, populations from the Picos de Europa mountains (NW Iberian Peninsula) showed a mitochondrial affinity to central-western Pyrenean populations and a nuclear affinity to populations from the central Iberian Peninsula, suggesting a likely admixed origin for Picos de Europa populations. Finally, populations from the Guadarrama Mountain Range (central Iberian Peninsula) were depleted of genetic diversity, possibly as a consequence of a recent epidemic of chytridiomycosis. This work highlights the complex evolutionary history that shaped the current genetic composition of high mountain populations, and underscores the importance of using a multilocus approach to better infer the dynamics of population divergence.Spanish Government projects FUNBIO 2413/2017, RTI2018-096217-B-I00National Institute of Ecology SFRH/BPD/109148/ 2015European Commission BiodivRestor-280, LIFE13 NAT/ES/001210, LIFE20 NAT/ES/00347Fundação para a Ciência e a Tecnologia PD/BD/52598/201

Conservation of the endemic Pyrenean newt (Calotriton asper) in the age of invasive species: interlake dispersal and colonisation dynamics

info:eu-repo/semantics/publishedVersio

Multiple glacial refugia and contemporary dispersal shape the genetic structure of an endemic amphibian from the Pyrenees

Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species’ present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.info:eu-repo/semantics/publishedVersio

Multiple invasions, Wolbachia and human‑aided transport drive the genetic variability of Aedes albopictus in the Iberian Peninsula

Este artículo contiene 15 páginas, 7 figuras, 1 tabla.The Asian tiger mosquito, Aedes albopictus, is one of the most invasive species in the world. Native to the tropical forests of Southeast Asia, over the past 30 years it has rapidly spread throughout tropical and temperate regions of the world. Its dramatic expansion has resulted in public health concerns as a consequence of its vector competence for at least 16 viruses. Previous studies showed that Ae. albopictus spread has been facilitated by human-mediated transportation, but much remains unknown about how this has affected its genetic attributes. Here we examined the factors that contributed to shaping the current genetic constitution of Ae. albopictus in the Iberian Peninsula, where the species was first found in 2004, by combining population genetics and Bayesian modelling. We found that both mitochondrial and nuclear DNA markers showed a lack of genetic structure and the presence of worldwide dominant haplotypes, suggesting regular introductions from abroad. Mitochondrial DNA showed little genetic diversity compared to nuclear DNA, likely explained by infection with maternally transmitted bacteria of the genus Wolbachia. Multilevel models revealed that greater mosquito fluxes (estimated from commuting patterns and tiger mosquito population distribution) and spatial proximity between sampling sites were associated with lower nuclear genetic distance, suggesting that rapid short- and medium-distance dispersal is facilitated by humans through vehicular traffic. This study highlights the significant role of human transportation in shaping the genetic attributes of Ae. albopictus and promoting regional gene flow, and underscores the need for a territorially integrated surveillance across scales of this disease-carrying mosquito.The research leading to these results has received funding from the Spanish Ministry of Economy and Competitiveness (MINECO, Plan Estatal I+D+I CGL2013-43139-R), “la Caixa” Foundation (ID 100010434) under agreement HR18-00336, and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 853271).Peer reviewe

A fast and inexpensive genotyping system for the simultaneous analysis of human and Aedes albopictus short tandem repeats

Abstract Background: Determination of the interactions between hematophagous mosquitoes and their human hosts is of great importance for better understanding the transmission dynamics of mosquito-borne arboviruses and developing effective strategies to mitigate risk. Genetic analysis of human and mosquito DNA can play a key role in this but commercial kits for human short tandem repeat (STR) genotyping are expensive and do not allow for the simultaneous STR analysis of host and vector DNA. Here, we present an inexpensive and straightforward STR-loci multiplex system capable of simultaneously amplifying Ae. albopictus and human STRs from blood-fed mosquitoes. Additionally, we examine the effect of storage methods and post-feeding time on the integrity of host DNA. Methods: Thirty-five STRs (16 human and 19 Ae. albopictus STRs) subdivided in three multiplexes were tested for amplification and scoring reliability. Under laboratory conditions we compared the efficacy of two preservation methods (absolute ethanol vs. lysis buffer) on the integrity of host DNA in Ae. albopictus blood meals. Furthermore, we evaluated the effect of post-feeding time by sacrificing blood-fed mosquitoes at different time intervals after feeding, and we assessed our ability to detect multiple feedings. To determine if the system can be employed successfully under field conditions, we carried out a preliminary study using field-collected Ae. albopictus. Results: All 35 STRs amplified consistently in the laboratory. Lysis buffer performed better than absolute ethanol in terms of allele peak height and clarity of electropherograms. Complete human DNA profiles could be obtained up to 48h following the blood meal. Analysis of multiple feedings confirmed that peak heights can be used as a proxy to determine post-feeding time and thus derive the number of different people bitten by a mosquito. In the field trial, amplification was successful for 32 STRs. We found human DNA signal in 38 of the 61 field-collected mosquitoes (62%), of which 34 (89%) had ingested a single blood meal, while 4 (11%) contained double meals. Conclusions: Our new genotyping system allows fast and reliable screening of both host and vector species, and can be further adapted to other mosquito species living in close contact with humans

Estat de conservació del cavilat (Cottus hispaniolensis) a la Val d’Aran

E. Carrillo, J. Ninot, T. Buchaca and M. Ventura. Blanes, LIFE+ LIMNOPIRINEUS Technical OfficeEl cavilat (Cottus hispaniolensis) és un dels peixos continentals més amenaçats de Catalunya i Espanya. La seva situació a la Val d’Aran va empitjorar dràsticament arran de les riuades del 2013, fins al punt d’arribar pràcticament a desaparèixer. Des d’aleshores, les poblacions s’han anat recuperant progressivament, amb un lent increment de la seva densitat mitjana i de les localitats ocupades. En part, aquesta recuperació s’ha produït gràcies a actuacions emmarcades en el projecte LIFE+ LimnoPirineus, que s’han dissenyat específicament per a la recuperació d’aquesta espècie dins l’espai de la xarxa Natura 2000 «Aigüestortes » (ES0000022).Peer reviewe

Non-native minnows cause much larger negative effects than trout on littoral macroinvertebrates of high mountain lakes

Este artículo contiene 10 páginas, 4 figuras, 1 tabla.Despite being naturally fishless, the widespread introduction of trout and minnows is threatening the conservation of high mountain lakes all over the world. Previous studies have reported that amphibians quickly disappear after trout introduction, followed by many conspicuous invertebrates. Here, we have studied the effects of minnows versus trout on the littoral macroinvertebrate community of 54 high mountain lakes from the Pyrenees, covering a gradient of environmental characteristics. The relative importance of fish compared to other variables in explaining macroinvertebrate communities was assessed using distance-based redundancy analysis (dbRDA) and multivariate regression tree (MRT) to find the main environmental thresholds. Both dbRDA and MRT approaches revealed that minnow density was the most important variable negatively determining community structure, followed by aquatic macrophytes, which increased taxa richness. The occurrence and abundance of relevant taxa was analysed in relation to fish densities and other environmental factors using binomial and gamma generalized linear models (GLM). GLM suggested that trout had an impact on the distribution of swimmer taxa and caused declines in the abundance of conspicuous clinger and burrower taxa. Minnows restricted the occurrence of more taxa than trout and negatively affected a wide variety of body sizes and functional groups. Indeed, we found that minnows were responsible for a dramatic biodiversity loss in the littoral macroinvertebrate community. The fast spread of minnows in high mountain areas is of great concern for the conservation of lake macroinvertebrates. Urgent measures to stop minnow introductions are strongly recommended.Economic support was provided by the European Commission LIFE + LIMNOPIRINEUS (LIFE13 NAT/ES/001210), LIFE RESQUE ALPYR (LIFE20 NAT/ES/00347), BiodivERsA FISHME (BiodivRestor-280) and FUNBIO (RTI2018-096217-B-I00) funded by MCIN/AEI/10.13039/ 501100011033 and BIOOCULT (2413/2017) funded by MTERD/OAPN and by “ERDF A way of making Europe”Peer reviewe

A fast and inexpensive genotyping system for the simultaneous analysis of human and Aedes albopictus short tandem repeats

Background. Determination of the interactions between hematophagous mosquitoes and their human hosts is of great importance for better understanding the transmission dynamics of mosquito-borne arboviruses and developing effective strategies to mitigate risk. Genetic analysis of human and mosquito DNA can play a key role in this, but commercial kits for human short tandem repeat (STR) genotyping are expensive and do not allow for the simultaneous STR analysis of host and vector DNA. Here, we present an inexpensive and straightforward STR-loci multiplex system capable of simultaneously amplifying Aedes albopictus and human STRs from blood-fed mosquitoes. Additionally, we examine the effect of storage methods and post-feeding time on the integrity of host DNA. Methods. Thirty-five STRs (16 human and 19 Ae. albopictus STRs) subdivided in three multiplexes were tested for amplification and scoring reliability. Under laboratory conditions we compared the efficacy of two preservation methods (absolute ethanol vs lysis buffer) on the integrity of host DNA in Ae. albopictus blood meals. We also evaluated the effect of post-feeding time by sacrificing blood-fed mosquitoes at different time intervals after feeding, and we assessed our ability to detect multiple feedings. To determine if the system can be employed successfully under field conditions, we carried out a preliminary study using field-collected Ae. albopictus. Results. All 35 STRs amplified consistently in the laboratory. Lysis buffer performed better than absolute ethanol in terms of allele peak height and clarity of electropherograms. Complete human DNA profiles could be obtained up to 48 h following the blood meal. Analysis of multiple feedings confirmed that peak heights can be used as a proxy to determine post-feeding time and thus derive the number of different people bitten by a mosquito. In the field trial, amplification was successful for 32 STRs. We found human DNA signal in 38 of the 61 field-collected mosquitoes (62%), of which 34 (89%) had ingested a single blood meal, while four (11%) contained double meals. Conclusions. Our new genotyping system allows fast and reliable screening of both host and vector species, and can be further adapted to other mosquito species living in close contact with humans.This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant agreement No. 853271)