Juan Carlos Illera

Gene conversion rapidly generates major histocompatibility complex diversity in recently founded bird populations

Population bottlenecks can restrict variation at functional genes, reducing the ability of populations to adapt to new and changing environments. Understanding how populations generate adaptive genetic variation following bottlenecks is therefore central to evolutionary biology. Genes of the major histocompatibility complex (MHC) are ideal models for studying adaptive genetic variation due to their central role in pathogen recognition. While de novo MHC sequence variation is generated by point mutation, gene conversion can generate new haplotypes by transferring sections of DNA within and across duplicated MHC loci. However, the extent to which gene conversion generates new MHC haplotypes in wild populations is poorly understood. We developed a 454 sequencing protocol to screen MHC class I exon 3 variation across all 13 island populations of Berthelot’s pipit (Anthus berthelotii). We reveal that just 11–15 MHC haplotypes were retained when the Berthelot’s pipit dispersed across its island range in the North Atlantic ca. 75 000 years ago. Since then, at least 26 new haplotypes have been generated in situ across populations. We show that most of these haplotypes were generated by gene conversion across divergent lineages, and that the rate of gene conversion exceeded that of point mutation by an order of magnitude. Gene conversion resulted in significantly more changes at nucleotide sites directly involved with pathogen recognition, indicating selection for functional variants. We suggest that the creation of new variants by gene conversion is the predominant mechanism generating MHC variation in genetically depauperate populations, thus allowing them to respond to pathogenic challenges.

Goats, birds, and emergent diseases: apparent and hidden effects of exotic species in an island environment

Exotic species can have devastating effects on recipient environments and even lead to the outbreak of emergent diseases. We present here several hidden effects that the introduction of goats has had on the Lesser Short-toed Lark, Calandrella rufescens, the commonest native bird inhabiting the island of Fuerteventura (Canary Islands). Vegetation structure varied with grazing pressure, and indeed, vegetation was all but eradicated from the locality with greatest goat densities, which was also where the lowest density of Lesser Short-toed Larks was recorded. The impact of habitat impoverishment, however, was partially compensated for by changes in the foraging behavior of birds, which benefited from the abundant food provided to goats on farms. Capture-resighting methods showed that birds visiting farms outnumbered the estimates for birds obtained in the surrounding natural habitat, suggesting that there was recruitment from a much larger area. Stable isotope analyses of feathers indicated that island birds feed largely on the maize supplied at goat farms, showing poorer body condition than birds from populations not associated with farms (peninsular Spain and Morocco). Moreover, larks from Fuerteventura had a very high prevalence of poxvirus lesions compared with other bird populations worldwide and may increase the risk of contracting the disease by feeding on farms, where they aggregate and coexist atypically with domestic birds. The island birds also had lower average productivity, which may be the consequence of the emergent disease and/or the poor nutritional state resulting from feeding on a low-protein diet. Diseased and non-diseased birds from Fuerteventura showed similar body condition and annual survival rates. However, the isotopic traces of delta 13C indicate that the diet of diseased birds was more uniform than that of non-diseased birds, being based on food from goat farms. Our results show how the combination of species frequently introduced onto islands (goats, poultry, and associated pathogens) can create ecological traps for native species that are not always easy to identify. Moreover, we stress that nutrition and infectious diseases are important determinants of the well-being and dynamics of animal populations, and thus health research must be included in the design of monitoring programs and conservation strategies.

Population history of Berthelot's pipit: colonization, gene flow and morphological divergence in Macaronesia

The fauna of oceanic islands provide exceptional models with which to examine patterns of dispersal, isolation and diversification, from incipient speciation to species level radiations. Here, we investigate recent differentiation and microevolutionary change in Berthelots pipit (Anthus berthelotii), an endemic bird species inhabiting three Atlantic archipelagos. Mitochondrial DNA sequence data and microsatellite markers were used to deduce probable colonization pathway, genetic differentiation, and gene flow among the 12 island populations. Phenotypic differentiation was investigated based on eight biologically important morphological traits. We found little mitochondrial DNA variability, with only one and four haplotypes for the control region and cytochrome b, respectively. However, microsatellite data indicated moderate population differentiation (FST = 0.069) between the three archipelagos that were identified as genetically distinct units with limited gene flow. Both results, combined with the estimated time of divergence (2.5 millions years ago) from the Anthus campestris (the sister species), suggest that this species has only recently dispersed throughout these islands. The genetic relationships, patterns of allelic richness and exclusive alleles among populations suggest the species originally colonized the Canary Islands and only later spread from there to the Madeiran archipelago and Selvagen Islands. Differentiation has also occurred within archipelagos, although to a lesser degree. Gene flow was observed more among the eastern and central islands of the Canaries than between these and the western islands or the Madeiran Islands. Morphological differences were also more important between than within archipelagos. Concordance between morphological and genetic differentiation provided ambiguous results suggesting that genetic drift alone was not sufficient to explain phenotypic differentiation. The observed genetic and morphological differences may therefore be the result of differing patterns of selection pressures between populations, with Berthelots pipit undergoing a process of incipient differentiation.

Phylogenetic relationships, biogeography and speciation in the avian genus Saxicola

The avian genus Saxicola is distributed throughout Africa, Asia, Europe and various islands across Oceania. Despite the fact that the group has great potential as a model to test evolutionary hypotheses due to the extensive variability in life history patterns recorded between and within species, the phylogenetic relationships among species and subspecies of this genus are poorly understood. We undertook a systematic investigation of the relationships within this genus with three main objectives in mind, (1) to test the monophyly of the genus; (2) to ascertain geographical origin and dispersal sequence; and (3) to test for monophyly within the most morphologically diverse species, S. torquata and S. caprata. We studied sequence data from the mitochondrial cytochrome b gene from 11 of the 12 recognized species and 15 of the 45 described subspecies. Four clades, two exclusively Asian, one Eurasian, and the fourth encompassing Eurasia and Africa, were identified. Based on our analyses, monophyly of the genus Saxicola is not supported and an Asian origin for the genus can be inferred. Results from DIVA analyses, tree topology and nodal age estimates suggest independent colonisation events from Asia to Africa and from Asia to the Western Palearctic, with the Sahara desert acting as a natural barrier for S. torquata. Subspecies and populations of S. torquata are not monophyletic due to S. tectes, S. dacotiae and S. leucura grouping within this complex. Subspecies and populations of S. caprata are monophyletic. Importantly, within S. torquata and S. caprata, slight morphological traits and plumage colour pattern differences used to recognize subspecies are indicative of the greater cryptic diversification that has occurred within this genus.

Genetic and phenotypic divergence in an island bird : Isolation by distance, by colonization or by adaptation?

Discerning the relative roles of adaptive and nonadaptive processes in generating differences among populations and species, as well as how these processes interact, is a fundamental aim in biology. Both genetic and phenotypic divergence across populations can be the product of limited dispersal and gradual genetic drift across populations (isolation by distance), of colonization history and founder effects (isolation by colonization) or of adaptation to different environments preventing migration between populations (isolation by adaptation). Here, we attempt to differentiate between these processes using island populations of Berthelots pipit (Anthus berthelotii), a passerine bird endemic to three Atlantic archipelagos. Using microsatellite markers and approximate Bayesian computation, we reveal that the northward colonization of this species ca. 8500 years ago resulted in genetic bottlenecks in the colonized archipelagos. We then show that high levels of genetic structure exist across archipelagos and that these are consistent with a pattern of isolation by colonization, but not with isolation by distance or adaptation. Finally, we show that substantial morphological divergence also exists and that this is strongly concordant with patterns of genetic structure and bottleneck history, but not with environmental differences or geographic distance. Overall, our data suggest that founder effects are responsible for both genetic and phenotypic changes across archipelagos. Our findings provide a rare example of how founder effects can persist over evolutionary timescales and suggest that they may play an important role in the early stages of speciation.

Genetic characterization, distribution and prevalence of avian pox and avian malaria in the Berthelot’s pipit (Anthus berthelotii) in Macaronesia

Exotic pathogens have been implicated in the decline and extinction of various native-island-bird species. Despite the fact that there is increasing concern about the introduction of diseases in island ecosystems, little is known about parasites in the islands of Macaronesia. We focus on Berthelot’s pipit (Anthus berthelotii), an endemic and widespread Macaronesian bird species, using a combination of field studies and molecular techniques to determine: (1) the range and prevalence of avian pox and malaria in Berthelot’s pipits throughout the species’ distribution, (2) the genetic characterization of both parasites in order to ascertain the level of host specificity. We sampled 447 pipits across the 12 islands inhabited by this species. Overall, 8% of all individuals showed evidence of pox lesions and 16% were infected with avian malaria, respectively. We observed marked differences in the prevalence of parasites among islands both within and between archipelagos. Avian pox prevalence varied between 0–54% within and between archipelagos and avian malaria prevalence varied between 0–64% within and between archipelagos. The diversity of pathogens detected was low: only two genetic lineages of avian malaria and one lineage of avian pox were found to infect the pipit throughout its range. Interestingly, both avian malaria parasites found were Plasmodium spp. that had not been previously reported in the Macaronesian avifauna (but that had been observed in the lesser kestrel Falco naumannii), while the avian pox was a host specific lineage that had previously been reported on two of the Canary Islands.

Biogeographical patterns and co-occurrence of pathogenic infection across island populations of Berthelot's pipit (Anthus berthelotii)

Pathogens can exert strong selective forces upon host populations. However, before we can make any predictions about the consequences of pathogen-mediated selection, we first need to determine whether patterns of pathogen distribution are consistent over spatiotemporal scales. We used molecular techniques to screen for a variety of blood pathogens (avian malaria, pox and trypanosomes) over a three-year time period across 13 island populations of the Berthelot’s pipit (Anthus berthelotii). This species has only recently dispersed across its range in the North Atlantic, with little subsequent migration, providing an ideal opportunity to examine the causes and effects of pathogenic infection in populations in the early stages of differentiation. We screened 832 individuals, and identified two strains of Plasmodium, four strains of Leucocytozoon, and one pox strain. We found strong differences in pathogen prevalence across populations, ranging from 0 to 65%, and while some fluctuations in prevalence occurred, these differences were largely stable over the time period studied. Smaller, more isolated islands harboured fewer pathogen strains than larger, less isolated islands, indicating that at the population level, colonization and extinction play an important role in determining pathogen distribution. Individual-level analyses confirmed the island effect, and also revealed a positive association between Plasmodium and pox infection, which could have arisen due to dual transmission of the pathogens by the same vectors, or because one pathogen lowers resistance to the other. Our findings, combined with an effect of infection on host body condition, suggest that Berthelot’s pipits are subject to different levels of pathogen-mediated selection both across and within populations, and that these selective pressures are consistent over time.

A multi-gene approach reveals a complex evolutionary history in the Cyanistes species group

Quaternary climatic oscillations have been considered decisive in shaping much of the phylogeographic structure around the Mediterranean Basin. Within this paradigm, peripheral islands are usually considered as the endpoints of the colonization processes. Here, we use nuclear and mitochondrial markers to investigate the phylogeography of the blue tit complex (blue tit Cyanistes caeruleus, Canary blue tit C. teneriffae and azure tit C. cyanus), and assess the role of the Canary Islands for the geographic structuring of genetic variation. The Canary blue tit exhibits strong genetic differentiation within the Canary Islands and, in combination with other related continental species, provides an ideal model in which to examine recent differentiation within a closely related group of continental and oceanic island avian species. We analysed DNA sequences from 51 breeding populations and more than 400 individuals in the blue tit complex. Discrepancies in the nuclear and mitochondrial gene trees provided evidence of a complex evolutionary process around the Mediterranean Basin. Coalescent analyses revealed gene flow between C. caeruleus and C. teneriffae suggesting a dynamic process with multiple phases of colonization and geographic overlapping ranges. Microsatellite data indicated strong genetic differentiation among the Canary Islands and between the Canary archipelago and the close continental areas, indicating limited contemporary gene flow. Diversification of the blue tit complex is estimated to have started during the early Pliocene (≈ 5 Ma), coincident with the end of Messinian salinity crisis. Phylogenetic analyses indicated that the North African blue tit is derived from the Canary blue tits, a pattern is avian ‘back colonization’ that contrasts with more traditionally held views of islands being sinks rather than sources.

Local climate determines intra- and interspecific variation in sexual size dimorphism in mountain grasshopper communities

The climate is often evoked to explain broad‐scale clines of body size, yet its involvement in the processes that generate size inequality in the two sexes (sexual size dimorphism) remains elusive. Here, we analyse climatic clines of sexual size dimorphism along a wide elevation gradient (i) among grasshopper species in a phylogenetically controlled scenario and (ii) within species differing in distribution and cold tolerance, to highlight patterns generated at different time scales, mainly evolutionary (among species or higher taxa) and ontogenetic or microevolutionary (within species). At the interspecific level, grasshoppers were slightly smaller and less dimorphic at high elevations. These clines were associated with gradients of precipitation and sun exposure, which are likely indicators of other factors that directly exert selective pressures, such as resource availability and conditions for effective thermoregulation. Within species, we found a positive effect of temperature and a negative effect of elevation on body size, especially on condition‐dependent measures of body size (total body length rather than hind femur length) and in species inhabiting the highest elevations. In spite of a certain degree of species‐specific variation, females tended to adjust their body size more often than males, suggesting that body size in females can evolve faster among species and can be more plastic or dependent on nutritional conditions within species living in adverse climates. Natural selection on female body size may therefore prevail over sexual selection on male body size in alpine environments, and abiotic factors may trigger consistent phenotypic patterns across taxonomic scales.

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits.

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n=10 per group): control (iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10–60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60–120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.ResumenSe estudian los efectos de 5 anestésicos sobre las concentraciones de corticosterona, cortisol y glucosa en el conejo NZW. Se utilizan 60 animales divididos en 6 grupos (n=10 por grupo): control (inyección de solución salina iv), fentanil/droperidol (1 mg/kg sc), ketamina (10 mg/kg iv) junto a xilazina (3 mg/kg iv) o diacepán (2 mg/kg iv), pentobarbital (30 mg/kg iv) y tiopental (20 mg/kg iv). Las concentraciones plasmáticas de glucocorticoides se miden mediante la técnica EIA de competición y la glucosa con un autoanalizador. Las muestras de sangre se recogen a los 0, 10, 30, 60, 120 min y 24 horas tras la administración de los anestésicos/solución salina. En los grupos tratados con pentobarbital o fentanil/ droperidol se observa una disminución significativa de los glucocorticoides plasmáticos a los 10–60 min, mientras que la administración de ketamina/diacepán o del tiopental estimula la liberación de glucocorticoides, principalmente durante la recuperación. Sin embargo, tras la administración de ketamina/ xilazina no se observan cambios en los niveles de glucocorticoides, excepto un aumento significativo de cortisol a los 60–120 min. Los niveles de glucosa aumentan significativamente tras la administración de ketamina/diacepán y principalmente, de ketamina/xilazina. Los datos presentes sugieren que la ketamina/xilazina apenas afecta a los niveles de glucocorticoides y alcanza un adecuado nivel de anestesia quirúrgica, por lo que sería el anestésico de elección, aunque el efecto hiperglicémico tras el tratamiento ha de ser considerado en cualquier experimento realizado en conejos.