Sabino-Pinto, J. | bioRxiv Science

bioRxiv2024

Between a Warm Winter and a Cold Spell: Physiological Responses to Changing Winter Climate in Amphibians

Climate change is swiftly altering environmental winter conditions, leading to significant ecological impacts such as phenological shifts in many species. As a result, animals might face physiological mismatches due to longer or earlier activity periods and are at risk of being exposed to late spring freezes. Our study points for the first time to the complex physiological challenges that amphibians face as a result of changing thermal conditions due to winter climate change. We investigated the physiological responses to a period of warmer winter days and sudden spring freeze in the common toad (Bufo bufo) by acclimating them to 4{degrees}C or 8{degrees}C for 48 h or exposing them to 4{degrees}C or -2{degrees}C for 6 h, respectively. We assessed the daily energy demands, determined body condition and cold tolerance, explored the molecular responses to freezing through hepatic tissue transcriptome analysis, and measured blood glucose levels. Toads acclimated to higher temperatures showed a higher daily energy expenditure and a reduced cold tolerance suggesting faster depletion of energy stores and the loss of winter acclimation during warmer winters. Blood sugar levels were higher in frozen toads indicating the mobilization of cryoprotective glucose with freezing which was further supported by changed patterns in proteins related to glucose metabolism. Overall, our results emphasize that increased thermal variability incurs physiological costs that may reduce energy reserves and thus affect amphibian health and survival. This might pose a serious threat to breeding adults and may have subsequent effects at the population level.

ecology↗

bioRxiv2023

Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians.

Restriction site-Associated DNA sequencing (RADseq) has great potential for genome-wide systematics studies of non-model organisms. However, accurately assembling RADseq reads into orthologous loci remains a major challenge in the absence of a reference genome. Traditional assembly pipelines cluster putative orthologous sequences based on a user-defined clustering threshold. Because improper clustering of orthologs is expected to affect results in downstream analyses, it is crucial to design pipelines for empirically optimizing the clustering threshold. While this issue has been largely discussed from a population genomics perspective, it remains understudied in the context of phylogenomics and coalescent species delimitation. To address this issue, we generated RADseq assemblies of representatives of the amphibian genera Discoglossus, Rana, Lissotriton and Triturus using a wide range of clustering thresholds. Particularly, we studied the effects of the intra-sample Clustering Threshold (iCT) and between-sample Clustering Threshold (bCT) separately, as both are expected to differ in multi-species data sets. The obtained assemblies were used for downstream inference of concatenation-based phylogenies, and multi-species coalescent species trees and species delimitation. The results were evaluated in the light of a reference genome-wide phylogeny calculated from newly generated Hybrid-Enrichment markers, as well as extensive background knowledge on the species systematics. Overall, our analyses show that the inferred topologies and their resolution are resilient to changes of the iCT and bCT, regardless of the analytical method employed. Except for some extreme clustering thresholds, all assemblies yielded identical, well-supported inter-species relationships that were mostly congruent with those inferred from the reference Hybrid-Enrichment data set. Similarly, coalescent species delimitation was consistent among similarity threshold values. However, we identified a strong effect of the bCT on the branch lengths of concatenation and species trees, with higher bCTs yielding trees with shorter branches, which might be a pitfall for downstream inferences of evolutionary rates. Our results suggest that the choice of assembly parameters for RADseq data in the context of shallow phylogenomics might be less challenging than previously thought. Finally, we propose a pipeline for empirical optimization of the iCT and bCT, implemented in optiRADCT, a series of scripts readily usable for future RADseq studies.

evolutionary biology↗