City comfort: weaker metabolic response to changes in ambient temperature in urban red squirrels.

The ecophysiological responses of species to urbanisation reveal important information regarding the processes of successful urban colonization and biodiversity patterns in urban landscapes. Investigating these responses will also help uncover whether synurban species are indeed urban 'winners'. Yet we still lack basic knowledge about the physiological costs and overall energy budgets of most species living in urban habitats, especially for mammals. Within this context, we compared the energetic demands of Eurasian red squirrels (Sciurus vulgaris) from the core of an urban environment with those from a nearby forest. We measured oxygen consumption as a proxy for resting metabolic rate (RMR) of 20 wild individuals (13 urban, 7 forest), at naturally varying ambient temperature (Ta) in an outdoor-enclosure experiment. We found that the variation in RMR was best explained by the interaction between Ta and habitat, with a significant difference between populations. Urban squirrels showed a shallower response of metabolic rate to decreasing Ta than woodland squirrels. We suggest that this is likely a consequence of urban heat island effects, as well as widespread supplemental food abundance. Our results indicate energy savings for urban squirrels at cooler temperatures, yet with possible increased costs at higher temperatures compared to their woodland conspecifics. Thus, the changed patterns of metabolic regulation in urban individuals might not necessarily represent an overall advantage for urban squirrels, especially in view of increasing temperatures globally.

Disentangling direct from indirect effects of habitat disturbance on multiple components of biodiversity.

Human habitat disturbance affects both species diversity and intraspecific genetic diversity, leading to correlations between these two components of biodiversity (termed species-genetic diversity correlation, SGDC). However, whether SGDC predictions extend to host-associated communities, such as the intestinal parasite and gut microbial diversity, remains largely unexplored. Additionally, the role of dominant generalist species is often neglected despite their importance in shaping the environment experienced by other members of the ecological community, and their role as source, reservoir and vector of zoonotic diseases. New analytical approaches (e.g. structural equation modelling, SEM) can be used to assess SGDC relationships and distinguish among direct and indirect effects of habitat characteristics and disturbance on the various components of biodiversity. With six concrete and biologically sound models in mind, we collected habitat characteristics of 22 study sites from four distinct landscapes located in central Panama. Each landscape differed in the degree of human disturbance and fragmentation measured by several quantitative variables, such as canopy cover, canopy height and understorey density. In terms of biodiversity, we estimated on the one hand, (a) small mammal species diversity, and, on the other hand, (b) genome-wide diversity, (c) intestinal parasite diversity and (d) gut microbial heterogeneity of the most dominant generalist species (Tome's spiny rat, Proechimys semispinosus). We used SEMs to assess the links between habitat characteristics and biological diversity measures. The best supported SEM suggested that habitat characteristics directly and positively affect the richness of small mammals, the genetic diversity of P. semispinosus and its gut microbial heterogeneity. Habitat characteristics did not, however, directly impact intestinal parasite diversity. We also detected indirect, positive effects of habitat characteristics on both host-associated assemblages via small mammal richness. For microbes, this is likely linked to cross species transmission, particularly in shared and/or anthropogenically altered habitats, whereas host diversity mitigates parasite infections. The SEM revealed an additional indirect but negative effect on intestinal parasite diversity via host genetic diversity. Our study showcases that habitat alterations not only affect species diversity and host genetic diversity in parallel, but also species diversity of host-associated assemblages. The impacts from human disturbance are therefore expected to ripple through entire ecosystems with far reaching effects felt even by generalist species.

Las perturbaciones antropogénicas sobre los hábitats naturales pueden afectar tanto a la diversidad de las especies como a la diversidad genética intraespecífica, dando lugar a correlaciones entre estos dos elementos de la biodiversidad (denominados correlación de la diversidad genética de las especies, SGDC por sus siglas en inglés). Sin embargo, todavía queda sin explorar si las predicciones de la SGDC afectan a las comunidades de parásitos y microorganismos intestinales asociadas al hospedador. Adicionalmente, el rol que juegan las especies generalistas, especialmente aquéllas dominantes, suele ser descuidado, a pesar de la importancia de control que ejercen sobre la estructura de la comunidad, y su rol como fuente, reservorio y vector de enfermedades zoonóticas. Para poder evaluar las relaciones de SGDC y distinguir entre los efectos directos e indirectos que tienen las características del hábitat y las perturbaciones sobre los distintos componentes de la biodiversidad, se pueden utilizar nuevos enfoques analíticos como por ejemplo los modelos de ecuaciones estructurales (SEM, por sus siglas en inglés). Considerando seis modelos específicos y biológicamente sólidos, recopilamos las características del hábitat de 22 sitios ubicados en cuatro paisajes distintos situados en el centro de Panamá. Cada paisaje difería en el grado de perturbación antropogénica y fragmentación, medido por diferentes variables cuantitativas, como la cobertura del dosel, la altura del dosel y la densidad del sotobosque. En términos de biodiversidad, por un lado estimamos (1) la diversidad de especies de pequeños mamíferos y, por otro lado (2) la diversidad del genoma completo, (3) la diversidad de parásitos intestinales, y (4) la heterogeneidad de las comunidades microbianas del intestino de la especie generalista más dominante, la rata espinosa de Tomes Proechimys semispinosus. Para evaluar los vínculos entre las características del hábitat y las medidas de diversidad biológica se utilizó el modelado SEM. El SEM mejor apoyado sugirió que las características del hábitat afectan directa y positivamente a la abundancia de pequeños mamíferos, a la diversidad genética de P. semispinosus y a la heterogeneidad microbiana intestinal. Sin embargo, se observó que las características del hábitat no tienen un efecto directo en la diversidad de parásitos intestinales. Aparte de estos efectos directos, detectamos efectos indirectos y positivos de las características del hábitat en ambos conjuntos asociados al hospedador (diversidad de parásitos y microorganismos intestinales) a través de la abundancia de pequeños mamíferos. En el caso de las comunidades microbianas, esto está probablemente relacionado con la transmisión interespecífica, especialmente en hábitats compartidos y/o antropogénicamente alterados; mientras que la diversidad de hospedadores mitiga las infecciones de parásitos. El SEM reveló un efecto indirecto adicional pero negativo sobre la diversidad de parásitos intestinales a través de la diversidad genética de los hospedadores. Nuestro estudio muestra que los patrones de SGDC se filtran a través de las varias capas de diversidad biológica, añadiendo los ensamblajes asociados al hospedador como componentes biológicos afectados por las alteraciones del hábitat.

A framework for testing the impact of co-infections on host gut microbiomes.

Parasitic infections disturb gut microbial communities beyond their natural range of variation, possibly leading to dysbiosis. Yet it remains underappreciated that most infections are accompanied by one or more co-infections and their collective impact is largely unexplored. Here we developed a framework illustrating changes to the host gut microbiome following single infections, and build on it by describing the neutral, synergistic or antagonistic impacts on microbial α- and ß-diversity expected from co-infections. We tested the framework on microbiome data from a non-human primate population co-infected with helminths and Adenovirus, and matched patterns reported in published studies to the introduced framework. In this case study, α-diversity of co-infected Malagasy mouse lemurs (Microcebus griseorufus) did not differ in comparison with that of singly infected or uninfected individuals, even though community composition captured with ß-diversity metrices changed significantly. Explicitly, we record stochastic changes in dispersion, a sign of dysbiosis, following the Anna-Karenina principle rather than deterministic shifts in the microbial gut community. From the literature review and our case study, neutral and synergistic impacts emerged as common outcomes from co-infections, wherein both shifts and dispersion of microbial communities following co-infections were often more severe than after a single infection alone, but microbial α-diversity was not universally altered. Important functions of the microbiome may also suffer from such heavily altered, though no less species-rich microbial community. Lastly, we pose the hypothesis that the reshuffling of host-associated microbial communities due to the impact of various, often coinciding parasitic infections may become a source of novel or zoonotic diseases.

RADseq data reveal a lack of admixture in a mouse lemur contact zone contrary to previous microsatellite results.

Microsatellites have been a workhorse of evolutionary genetic studies for decades and are still commonly in use for estimating signatures of genetic diversity at the population and species level across a multitude of taxa. Yet, the very high mutation rate of these loci is a double-edged sword, conferring great sensitivity at shallow levels of analysis (e.g. paternity analysis) but yielding considerable uncertainty for deeper evolutionary comparisons. For the present study, we used reduced representation genome-wide data (restriction site-associated DNA sequencing (RADseq)) to test for patterns of interspecific hybridization previously characterized using microsatellite data in a contact zone between two closely related mouse lemur species in Madagascar (Microcebus murinus and Microcebus griseorufus). We revisit this system by examining populations in, near, and far from the contact zone, including many of the same individuals that had previously been identified as hybrids with microsatellite data. Surprisingly, we find no evidence for admixed nuclear ancestry. Instead, re-analyses of microsatellite data and simulations suggest that previously inferred hybrids were false positives and that the program NewHybrids can be particularly sensitive to erroneously inferring hybrid ancestry. Combined with results from coalescent-based analyses and evidence for local syntopic co-occurrence, we conclude that the two mouse lemur species are in fact completely reproductively isolated, thus providing a new understanding of the evolutionary rate whereby reproductive isolation can be achieved in a primate.

Disparate roost sites drive intraspecific physiological variation in a Malagasy bat.

Many species are widely distributed and individual populations can experience vastly different environmental conditions over seasonal and geographic scales. With such a broad ecological reality, datasets with limited spatial and temporal resolution may not accurately represent a species and could lead to poorly informed management decisions. Because physiological flexibility can help species tolerate environmental variation, we studied the physiological responses of two separate populations of Macronycteris commersoni, a bat widespread across Madagascar, in contrasting seasons. The populations roost under the following dissimilar conditions: either a hot, well-buffered cave or within open foliage, unprotected from the local weather. We found that flexible torpor patterns, used in response to prevailing ambient temperature and relative humidity, were central to keeping energy budgets balanced in both populations. While bats' metabolic rate during torpor and rest did not differ between roosts, adjusting torpor frequency, duration and timing helped bats maintain body condition. Interestingly, the exposed forest roost induced extensive use of torpor, which exceeded the torpor frequency of overwintering bats that stayed in the cave for months and consequently minimised daytime resting energy expenditure in the forest. Our current understanding of intraspecific physiological variation is limited and physiological traits are often considered to be fixed. The results of our study therefore highlight the need for examining species at broad environmental scales to avoid underestimating a species' full capacity for withstanding environmental variation, especially in the face of ongoing, disruptive human interference in natural habitats.

Evidence of MHC class I and II influencing viral and helminth infection via the microbiome in a non-human primate.

Until recently, the study of major histocompability complex (MHC) mediated immunity has focused on the direct link between MHC diversity and susceptibility to parasite infection. However, MHC genes can also influence host health indirectly through the sculpting of the bacterial community that in turn shape immune responses. We investigated the links between MHC class I and II gene diversity gut microbiome diversity and micro- (adenovirus, AdV) and macro- (helminth) parasite infection probabilities in a wild population of non-human primates, mouse lemurs of Madagascar. This setup encompasses a plethora of underlying interactions between parasites, microbes and adaptive immunity in natural populations. Both MHC classes explained shifts in microbiome composition and the effect was driven by a few select microbial taxa. Among them were three taxa (Odoribacter, Campylobacter and Prevotellaceae-UCG-001) which were in turn linked to AdV and helminth infection status, correlative evidence of the indirect effect of the MHC via the microbiome. Our study provides support for the coupled role of MHC diversity and microbial flora as contributing factors of parasite infection.

Bioaccumulation of trace elements affects chick body condition and gut microbiome in greater flamingos.

Elevated concentrations of trace elements represent a major concern to wetland ecosystems, since river estuaries are geochemical endpoints that accumulate pollution. Although the negative impact of environmental exposure of highly toxic elements such as Pb and Hg has received substantial attention, we still lack a comprehensive understanding of the effects that these and other common trace elements have on natural populations. We used greater flamingos as a study system within three sites that represent a gradient of pollution. Controlling for environmental sediment exposure, we assessed if signatures of bioaccumulation in feathers for ten trace elements (As, Cd, Cr, Cu, Hg, Ni, Pb, Se, Sn and Zn) are associated with two known proxies of health: body condition and the gut bacterial microbiome. We found evidence of an adverse effect of Se, Hg, and Pb bioaccumulation on body condition. Furthermore, bioaccumulation of the elements As, Cu, Se, Pb and Zn influenced different aspects of the gut microbiome. Bioaccumulation of Se led to a shift in the microbiome composition, largely driven by an enrichment of Bacteroides plebeius, which is linked to the breakdown of sulphated polysaccharides of algae. Bacteroides plebeius was negatively associated with chick body condition, suggesting an adverse effect of a microalgae diet rich in Se. Pb bioaccumulation was linked with a decrease in microbial diversity (adjusted-R2 = 10.4%) and an increase in heterogeneity of the microbial community (adjusted-R2 = 10.5%), an indication of impaired gut homeostasis. As, Cu and Zn had more nuanced effects on gut microbiome heterogeneity according to breeding site and bioaccumulation concentration. Our results therefore suggest that in addition to well-studied elements, bioaccumulation of poorly studied elements also adversely affect health of natural populations.

A novel workflow to improve genotyping of multigene families in wildlife species: An experimental set-up with a known model system.

Genotyping complex multigene families in novel systems is particularly challenging. Target primers frequently amplify simultaneously multiple loci leading to high PCR and sequencing artefacts such as chimeras and allele amplification bias. Most genotyping pipelines have been validated in nonmodel systems whereby the real genotype is unknown and the generation of artefacts may be highly repeatable. Further hindering accurate genotyping, the relationship between artefacts and genotype complexity (i.e. number of alleles per genotype) within a PCR remains poorly described. Here, we investigated the latter by experimentally combining multiple known major histocompatibility complex (MHC) haplotypes of a model organism (chicken, Gallus gallus, 43 artificial genotypes with 2-13 alleles per amplicon). In addition to well-defined 'optimal' primers, we simulated a nonmodel species situation by designing 'cross-species' primers based on sequence data from closely related Galliform species. We applied a novel open-source genotyping pipeline (ACACIA; https://gitlab.com/psc_santos/ACACIA), and compared its performance with another, previously published pipeline (AmpliSAS). Allele calling accuracy was higher when using ACACIA (98.5% versus 97% and 77.8% versus 75% for the 'optimal' and 'cross-species' data sets, respectively). Systematic allele dropout of three alleles owing to primer mismatch in the 'cross-species' data set explained high allele calling repeatability (100% when using ACACIA) despite low accuracy, demonstrating that repeatability can be misleading when evaluating genotyping workflows. Genotype complexity was positively associated with nonchimeric artefacts, chimeric artefacts (nonlinearly by levelling when amplifying more than 4-6 alleles) and allele amplification bias. Our study exemplifies and demonstrates pitfalls researchers should avoid to reliably genotype complex multigene families.

Challenges of next-generation sequencing in conservation management: Insights from long-term monitoring of corridor effects on the genetic diversity of mouse lemurs in a fragmented landscape.

Long-term genetic monitoring of populations is essential for efforts aimed at preserving genetic diversity of endangered species. Here, we employ a framework of long-term genetic monitoring to evaluate the effects of fragmentation and the effectiveness of the establishment of corridors in restoring population connectivity and genetic diversity of mouse lemurs Microcebus ganzhorni. To this end, we supplement estimates of neutral genetic diversity with the assessment of adaptive genetic variability of the major histocompatibility complex (MHC). In addition, we address the challenges of long-term genetic monitoring of functional diversity by comparing the genotyping performance and estimates of MHC variability generated by single-stranded conformation polymorphism (SSCP)/Sanger sequencing with those obtained by high-throughput sequencing (next-generation sequencing [NGS], Illumina), an issue that is particularly relevant when previous work serves as a baseline for planning management strategies that aim to ensure the viability of a population. We report that SSCP greatly underestimates individual diversity and that discrepancies in estimates of MHC diversity attributable to the comparisons of traditional and NGS genotyping techniques can influence the conclusions drawn from conservation management scenarios. Evidence of migration among fragments in Mandena suggests that mouse lemurs are robust to the process of fragmentation and that the effect of corridors is masked by ongoing gene flow. Nonetheless, results based on a larger number of shared private alleles at neutral loci between fragment pairs found after the establishment of corridors in Mandena suggest that gene flow is augmented as a result of enhanced connectivity. Our data point out that despite low effective population size, M. ganzhorni maintains high individual heterozygosity at neutral loci and at MHC II DRB gene and that selection plays a predominant role in maintaining MHC diversity. These findings highlight the importance of long-term genetic monitoring in order to disentangle between the processes of drift and selection maintaining adaptive genetic diversity in small populations.