Eacock, A. | Search bioRxiv

bioRxiv2025

Historical Contingency Shapes Toxin Resistance in a Specialist Avian Predator

Adaptations to toxic diets can cascade through ecosystems, altering physiology, species interactions, and trophic dynamics. To uncover the molecular basis of toxin resistance in the black-headed grosbeak (Pheucticus melanocephalus), a specialist predator of cardiotonic steroid-defended monarch butterflies (Danaus plexippus), we investigated the evolution of target-site insensitivity in the toxins molecular target - the Na,K-ATPase. Functional assays of engineered Na,K-ATPases revealed that resistance in grosbeaks arises from a non-additive interaction between a substitution at position 111 (Q111E) and up to six nearby amino acid changes in the first extracellular loop of the protein. Using resurrected ancestral proteins, we show that the earliest of these six substitutions to evolve (V113L) altered the functional effects of Q111E, such that Q111E alone became maladaptive. Only after the accumulation of additional permissive substitutions could Q111E confer resistance, highlighting how intramolecular epistatic interactions and historical contingency constrained the evolutionary path to adaptation. Our phylogenetic analysis of Na,K-ATPase sequences from 360 birds further revealed that several of the specific grosbeak substitutions--particularly at sites 112, 114, and 116--show strong signatures of co-evolution with changes at site 111 across the avian tree, supporting the hypothesis that resistance evolves through repeated, interacting changes. Together, these results reveal the molecular mechanisms of convergent evolution of toxin resistance and demonstrate how genetic background can shape evolutionary outcomes across trophic levels.

evolutionary biology↗

bioRxiv2025

Ultra-sensitive detection of Phytophthora pluvialis by real-time PCR targeting a mitochondrial gene

Phytophthora pluvialis is a forest tree pathogen present in the USA, New Zealand, UK and Belgium. Reported hosts include Douglas-fir in the USA, New Zealand, UK and Belgium, as well as tanoak in the USA, radiata pine in New Zealand, Japanese larch and western hemlock in the UK. Disease symptoms range from needle lesions and casting on radiata pine through to twig and stem cankers, and crown dieback on western hemlock, Douglas-fir and Japanese larch. Current detection methods rely on isolation and culture, or PCR using a single-copy gene target with limited sensitivity at low pathogen titre. A qPCR assay targeting a multiple-copy mitochondrial gene was designed to increase sensitivity of P. pluvialis detection in forest samples, critical for informing biosecurity, long term disease management and ongoing research. The resulting assay has a detection limit of 12.8 fg mycelial DNA and can detect the pathogen on average 6.12 qPCR cycles before the single-copy target assay. In New Zealand forest samples, the assay was found to consistently detect P. pluvialis in all stages of radiata pine needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in radiata pine needle samples four weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has also enabled rapid and confident diagnostic support of the biosecurity response in the UK during recent detection of P. pluvialis. The assay has been used in applications requiring detection at low pathogen titre levels including asymptomatic infection, stream baiting, cast needles and during biosecurity responses, making it a useful tool for effective early detection and management of P. pluvialis in affected forests.

microbiology↗