Island hitchhikers: pathogen agents of Madeira and Azores ticks.

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.

No evidence of inbreeding depression despite a historical severe bottleneck in the endangered Bermuda petrel (Pterodroma cahow).

The Bermuda petrel Pterodroma cahow is an island endemic seabird that belongs to the Procellariiformes, one of the most endangered orders of birds. Historical records suggest a significant population size decline following human settlement in Bermuda, bringing the species to near extinction. Since the 1950s, the population has been recovering aided by the implementation of an ongoing conservation plan. However, it still faces several threats, and negative genetic effects resulting from that drastic decline are to be expected, including inbreeding and genetic drift. We studied genetic diversity and levels of inbreeding, and their effects on individual fitness and mating choice. We also tested for a genetic signature of the recent demographic bottleneck. For this, we analyzed variation in thousands of nuclear single-nucleotide polymorphisms derived from double digest restriction site-associated DNA sequencing and 1 mitochondrial gene (cytochrome oxidase I). The results revealed that the Bermuda petrel suffered a recent genetic bottleneck and shows low mitochondrial diversity compared with other petrel species. Conversely, nuclear diversity was similar to that of other endangered petrels. Inbreeding levels were not high overall, although some individuals were highly inbred. However, we found no evidence that individual inbreeding or relatedness between mates affected hatching success, or that mate choice is influenced by kinship in this very small population.

A first look at sea-lavenders genomics - can genome wide SNP information tip the scales of controversy in the Limonium vulgare species complex?

BACKGROUND: Sea-lavenders (Limonium Mill., Plumbaginaceae) are a cosmopolitan group of diploid and polyploid plants often adapted to extreme saline environments, with a mostly Tethyan distribution, occurring in the Mediterranean, Irano-Turanian, Euro-Siberian and in the New World. The halophylic Limonium vulgare polyploid complex in particular, presents a large distribution throughout extreme salt-marsh habitats and shows little morphological but high taximetric variation, frequently blurring species delimitation. In this work we pursue three main goals: assert whether SNP data from polyploid individuals has the resolution to distinguish the seven sampled species, to better understand how genetically structured Limonium vulgare is, and attempt to identify specific molecular mechanisms for the differentiation between L. maritimum and L. vulgare. For this purpose, 95 individuals were genotyped using Genotyping by Sequencing (GBS), which were assembled as two independent datasets using IPYRAD. All analyses performed downstream of assembly were fully automated. Phylogenetic inference, PCA, and admixture plots were used to infer answers to the study's main goals. RESULTS: Close to 10,000 SNPs were obtained for each dataset. Phylogenetic analyses reveal that polyploid data can be used to infer species relationships. Population structure analyses suggest a genetically structured L. vulgare. A set of 34 SNPs were found to be fully segregated between L. vulgare and L. maritimum, two of which are potentially linked to proteins that might be involved in the speciation process. CONCLUSION: Despite polyploid data analyses shortcomings, GBS generated SNPs have the resolution to discern all seven included species. Limonium vulgare revealed pronounced genetic structure along a geographical north-south cline. L. maritimum always appears as a distinct genetic entity. Segregated SNPs between L. vulgare and L. maritimum indicate salinity response and morphological trait control genes as potentially interesting to follow up for studying these species' divergence process.

Repeated evolution of blanched coloration in a lizard across independent white-sand habitats.

The White Sands lizards of New Mexico are a rare and classic example of convergent evolution where three species have evolved blanched coloration on the white gypsum dunes. Until now, no geological replicate of the pattern had been described. However, one of the White Sands species, the lesser earless lizard (Holbrookia maculata), has been discovered to also inhabit the Salt Basin Dunes of Texas, where it has also evolved a blanched morph. We here present a first phenotypic and genetic description of the Salt Basin Dunes population of H. maculata. Phylogenetic inference based on a housekeeping gene (ND4) and a classic candidate gene in the melanin-synthesis pathway (Melanocortin 1 Receptor; Mc1r) shows the newly discovered population as an independent lineage, with no evidence of genetic parallelism in the coding region of Mc1r. Initial morphological data suggest that while this population displays convergent evolution in blanched coloration, there are divergent patterns in limb length and habitat use behavior between the gypsum environments. Our findings present the White Sands/Salt Basin Dunes as an exceptionally promising comparative model for studies of adaptation and convergent evolution.

Population structure in Quercus suber L. revealed by nuclear microsatellite markers.

Quercus suber L. is a sclerophyllous tree species native to the western Mediterranean, a region that is considered highly vulnerable to increased temperatures and severe dry conditions due to environmental changes. Understanding the population structure and demographics of Q. suber is essential in order to anticipate whether populations at greater risk and the species as a whole have the genetic background and reproductive dynamics to enable rapid adaptation. The genetic diversity of Q. suber has been subject to different studies using both chloroplast and nuclear data, but population structure patterns remain unclear. Here, we perform genetic analyses on Q. suber using 13 nuclear microsatellite markers, and analysed 17 distinct locations across the entire range of the species. Structure analyses revealed that Q. suber may contain three major genetic clusters that likely result from isolation in refugia combined with posterior admixture and putative introgression from other Quercus species. Our results show a more complex structure scenario than previously inferred for Q. suber using nuclear markers and suggest that different southern populations contain high levels of genetic variation that may contribute to the resilience of Q. suber in a context of environmental change and adaptive pressure.

Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data.

Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Auchenorrhyncha, Aphrophoridae), presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.

evALLution: making basic evolution concepts accessible to people with visual impairment through a multisensory tree of life.

BACKGROUND: People with visual impairment have benefitted from recent developments of assistive technology that aim to decrease socio-economic inequality. However, access to post-secondary education is still extremelly challenging, especially for scientific areas. The under representation of people with visual impairment in the evolution research community is connected with the vision-based communication of evolutionary biology knowledge and the accompanying lack of multisensory alternatives for learning. RESULTS: Here, we describe the development of an inclusive outreach activity based on a multisensory phylogeny representing 20 taxonomic groups. We provide a tool kit of materials and ideas that allow both the replication of this activity and the adaptation of others, to include people with visual impairment. Furthermore, we provide activity evaluation data, a discussion of the lessons learned and an inclusive description of all figures and visual data presented.The presented baseline data show that people with visual impairment indeed have lack of access to education but are interested in and apt to understand evolutionary biology concepts and predict evolutionary change when education is inclusive. CONCLUSIONS: We show that, with creative investment, basic evolutionary knowledge is perfectly possible to be transmitted through multisensory activities, which everyone can benefit from. Ultimately, we hope this case study will provide a baseline for future initiatives and a more inclusive outreach community.

Using gradient Forest to predict climate response and adaptation in Cork oak.

Climate change is impacting locally adapted species such as the keystone tree species cork oak (Quercus suber L.). Quantifying the importance of environmental variables in explaining the species distribution can help build resilient populations in restoration projects and design forest management strategies. Using landscape genomics, we investigated the population structure and ecological adaptation of this tree species across the Mediterranean Basin. We applied genotyping by sequencing and derived 2,583 single nucleotide polymorphism markers genotyped from 81 individuals across 17 sites in the studied region. We implemented an approach based on the nearest neighbour haplotype 'coancestry' and uncovered a weak population structure along an east-west climatic gradient across the Mediterranean region. We identified genomic regions potentially involved in local adaptation and predicted differences in the genetic composition across the landscape under current and future climates. Variants associated with temperature and precipitation variables were detected, and we applied a nonlinear multivariate association method, gradient forest, to project these gene-environment relationships across space. The model allowed the identification of geographic areas within the western Mediterranean region most sensitive to climate change: south-western Iberia and northern Morocco. Our findings provide a preliminary assessment towards a potential management strategy for the conservation of cork oak in the Mediterranean Basin.

Correction to: Assessing genotype-phenotype associations in three dorsal colour morphs in the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) using genomic and transcriptomic resources.

Following publication of the original article [1], it has been brought to the authors' attention that in their paper (Rodrigues et al. 2016) they reported the genome size based on 2C values (diploid genome) when it is more common to present it as 1C value.

Genomic signatures of introgression between commercial and native bumblebees, Bombus terrestris, in western Iberian Peninsula-Implications for conservation and trade regulation.

Human-mediated introductions of species may have profound impacts on native ecosystems. One potential impact with largely unforeseen consequences is the potential admixture of introduced with autochthonous species through hybridization. Throughout the world, bumblebees have been deliberately introduced for crop pollination with known negative impacts on native pollinators. Given the likely allochthonous origin of commercial bumblebees used in Portugal (subspecies Bombus terrestris terrestris and B. t. dalmatinus), our aim was to assess their putative introgression with the native Iberian subspecies B. terrestris lusitanicus. We analysed one mitochondrial gene, cytochrome c oxidase subunit I (COX1) and genomic data involving thousands of genome-wide restriction-site-associated DNA markers (RAD-seq). In the mitochondrial COX1 analyses, we detected one relatively common haplotype in commercial bumblebees, also present in wild samples collected nearby the greenhouses where the commercial hives are used. In the RAD-seq analysis, we found a clear genetic differentiation between native and commercial lineages. Furthermore, we detected candidate hybrids in the wild, as well as putatively escaped commercial bumblebees, some of which being potentially fertile males. Although we cannot assess directly the fitness effects of introgressed alleles, there is a risk of maladaptive allele introgression to the local bumblebee subspecies, which can negatively impact autochthon populations. One immediate recommendation to farmers is for the proper disposal of hive boxes, after their use in greenhouses, so as to minimize the risk of escapees contaminating native populations. On the other hand, the feasibility of using local subspecies B. t. lusitanicus, preferably with local production, should be evaluated.

New insights into adaptation and population structure of cork oak using genotyping by sequencing.

Species respond to global climatic changes in a local context. Understanding this process, including its speed and intensity, is paramount due to the pace at which such changes are currently occurring. Tree species are particularly interesting to study in this regard due to their long generation times, sedentarism, and ecological and economic importance. Quercus suber L. is an evergreen forest tree species of the Fagaceae family with an essentially Western Mediterranean distribution. Despite frequent assessments of the species' evolutionary history, large-scale genetic studies have mostly relied on plastidial markers, whereas nuclear markers have been used on studies with locally focused sampling strategies. In this work, "Genotyping by sequencing" is used to derive 1,996 single nucleotide polymorphism markers to assess the species' evolutionary history from a nuclear DNA perspective, gain insights into how local adaptation is shaping the species' genetic background, and to forecast how Q. suber may respond to global climatic changes from a genetic perspective. Results reveal (a) an essentially unstructured species, where (b) a balance between gene flow and local adaptation keeps the species' gene pool somewhat homogeneous across its distribution, but still allowing (c) variation clines for the individuals to cope with local conditions. "Risk of Non-Adaptedness" (RONA) analyses suggest that for the considered variables and most sampled locations, (d) the cork oak should not require large shifts in allele frequencies to survive the predicted climatic changes. Future directions include integrating these results with ecological niche modeling perspectives, improving the RONA methodology, and expanding its use to other species. With the implementation presented in this work, the RONA can now also be easily assessed for other organisms.

Structure_threader: An improved method for automation and parallelization of programs structure, fastStructure and MavericK on multicore CPU systems.

Structure_threader is a program to parallelize multiple runs of genetic clustering software that does not make use of multithreading technology (structure, fastStructure and MavericK) on multicore computers. Our approach was benchmarked across multiple systems and displayed great speed improvements relative to the single-threaded implementation, scaling very close to linearly with the number of physical cores used. Structure_threader was compared to previous software written for the same task-ParallelStructure and StrAuto and was proven to be the faster (up to 25% faster) wrapper under all tested scenarios. Furthermore, Structure_threader can perform several automatic and convenient operations, assisting the user in assessing the most biologically likely value of 'K' via implementations such as the "Evanno," or "Thermodynamic Integration" tests and automatically draw the "meanQ" plots (static or interactive) for each value of K (or even combined plots). Structure_threader is written in python 3 and licensed under the GPLv3. It can be downloaded free of charge at https://github.com/StuntsPT/Structure_threader.

Assessing genotype-phenotype associations in three dorsal colour morphs in the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) using genomic and transcriptomic resources.

BACKGROUND: Colour polymorphisms are common among animal species. When combined with genetic and ecological data, these polymorphisms can be excellent systems in which to understand adaptation and the molecular changes underlying phenotypic evolution. The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera, Aphrophoridae), a widespread insect species in the Holarctic region, exhibits a striking dorsal colour/pattern balanced polymorphism. Although experimental crosses have revealed the Mendelian inheritance of this trait, its genetic basis remains unknown. In this study we aimed to identify candidate genomic regions associated with the colour balanced polymorphism in this species. RESULTS: By using restriction site-associated DNA (RAD) sequencing we were able to obtain a set of 1,837 markers across 33 individuals to test for associations with three dorsal colour phenotypes (typicus, marginellus, and trilineatus). Single and multi-association analyses identified a total of 60 SNPs associated with dorsal colour morphs. The genome size of P. spumarius was estimated by flow cytometry, revealing a 5.3 Gb genome, amongst the largest found in insects. A partial genome assembly, representing 24% of the total size, and an 81.4 Mb transcriptome, were also obtained. From the SNPs found to be associated with colour, 35% aligned to the genome and 10% to the transcriptome. Our data suggested that major loci, consisting of multi-genomic regions, may be involved in dorsal colour variation among the three dorsal colour morphs analysed. However, no homology was found between the associated loci and candidate genes known to be responsible for coloration pattern in other insect species. The associated markers showed stronger differentiation of the trilineatus colour phenotype, which has been shown previously to be more differentiated in several life-history and physiological characteristics as well. It is possible that colour variation and these traits are linked in a complex genetic architecture. CONCLUSIONS: The loci detected to have an association with colour and the genomic and transcriptomic resources developed here constitute a basis for further research on the genetic basis of colour pattern in the meadow spittlebug P. spumarius.

4Pipe4--A 454 data analysis pipeline for SNP detection in datasets with no reference sequence or strain information.

BACKGROUND: Next-generation sequencing datasets are becoming more frequent, and their use in population studies is becoming widespread. For non-model species, without a reference genome, it is possible from a panel of individuals to identify a set of SNPs that can be used for further population genotyping. However the lack of a reference genome to which the sequenced data could be compared makes the finding of SNPs more troublesome. Additionally when the data sources (strains) are not identified (e.g. in datasets of pooled individuals), the problem of finding reliable variation in these datasets can become much more difficult due to the lack of specialized software for this specific task. RESULTS: Here we describe 4Pipe4, a 454 data analysis pipeline particularly focused on SNP detection when no reference or strain information is available. It uses a command line interface to automatically call other programs, parse their outputs and summarize the results. The variation detection routine is built-in in the program itself. Despite being optimized for SNP mining in 454 EST data, it is flexible enough to automate the analysis of genomic data or even data from other NGS technologies. 4Pipe4 will output several HTML formatted reports with metrics on many of the most common assembly values, as well as on all the variation found. There is also a module available for finding putative SSRs in the analysed datasets. CONCLUSIONS: This program can be especially useful for researchers that have 454 datasets of a panel of pooled individuals and want to discover and characterize SNPs for subsequent individual genotyping with customized genotyping arrays. In comparison with other SNP detection approaches, 4Pipe4 showed the best validation ratio, retrieving a smaller number of SNPs but with a considerably lower false positive rate than other methods. 4Pipe4's source code is available at https://github.com/StuntsPT/4Pipe4.