Genetic diversity and ecogeographical niche overlap among hybridizing ox-eye daisies (Leucanthemum, Asteraceae) in the Carpathian Mountains: the impact of anthropogenic disturbances

This study examines the ecogeographical niche overlap and genetic diversity among three Leucanthemum species in the Carpathian Mountains: the lowland L. ircutianum (4x), the montane L. rotundifolium (2x), and the alpine L. gaudinii (2x). Previous research noted hybridization between L. rotundifolium and L. gaudinii, but our analysis reveals more extensive hybridization across all three species, with additional potential hybridization events among the hybrids. Over 600 individuals were genotyped using SNP analysis, followed by Principal Coordinate Analysis (PCoA), Neighbor-Net Network, and Structure clustering. These analyses uncovered distinct genetic groups corresponding to particular taxa and supported the formation of hybrids. Within species-specific clusters, L. rotundifolium is divided into western and south-eastern populations, while L. gaudinii and L. ircutianum demonstrate close genetic relationships. Our analyses suggest hybridization between all species pairs and the potential for triple hybrids. Genetic admixture is further supported by environmental background. Niche overlap analyses reveal substantial overlap among species, particularly in line with their vertical distribution. Climate envelope plots indicate a likely increase in competition due to climate change, leading to a reduction in available habitat for mountainous species and an intensification of hybridization. Anthropogenic influences are intensifying these hybridization trends. Among the studied species, L. gaudinii is most at risk of overwhelming hybridization, whereas L. ircutianum may experience habitat expansion. This study provides insights into the intricate ecological and genetic interrelations of the Carpathian ox-eye daisies, emphasizing the role of environmental changes and genetic diversity in understanding and responding to these dynamics.

Methods
Data Collection

The dataset was gathered through extensive fieldwork across the Carpathian Mountains, focusing on three species of ox-eye daisies (Leucanthemum ircutianum, L. rotundifolium, L. gaudinii and their hybrids). Over 600 individual plants were sampled from various elevations and habitats to capture the genetic diversity and distribution patterns of these species.

Genetic Analysis

DNA was extracted from dried leaf samples using a DNA extraction kit. The ploidy levels of the samples were determined using flow cytometry. For genetic analysis, DArTSeq technology was employed to sequence the DNA, providing a comprehensive overview of the genetic makeup of the collected samples. The raw genetic data were filtered to remove low-quality and monomorphic loci, resulting in a dataset with high-confidence genetic markers.

Environmental Data

Environmental data were collected for each sampling site, including bioclimatic variables and geographical coordinates. These data were used to model the ecological niches of the species and assess the overlap between them. Future climate scenarios were also incorporated to predict potential changes in species distributions.

Human Disturbance

Data on human infrastructure were sourced from detailed maps (openstreetmap.org), and a Human Disturbance Index (HDI) was created. This index quantifies the impact of human activities on the habitats of the sampled species, considering factors such as proximity to roads, buildings, and other man-made structures.

Data Processing

The genetic data underwent processing to ensure quality and accuracy. Loci with low call rates and reproducibility were excluded, and individuals with high levels of missing data were removed from the analysis. Clustering algorithms were used to identify genetic groups, and simulations were conducted to validate the presence of hybrids. Environmental niche models were constructed using both current and future climate data to understand the ecological dynamics of the species.