Genetic Analysis
We calculated pairwise FST values for all populations using HIERFSTAT v0.04-22 function pairwise.neifst (Goudet, 2005). Site-specific statistics were calculated by running the ‘populations’ program of Stacks on the filtered VCF file, using the –fstats argument to compare select populations from each quadrant. Genetic differentiation between states was assessed with AMOVA from R package Popper (Kamvar et al., 2014). Basic diversity statistics for each population were calculated using HIERFSTAT v0.04-22, and ADEGENET v2.1.3 (Jombart & Ahmed, 2011), including number of alleles (Nalleles), allelic richness (AR), observed heterozygosity (Ho), mean gene diversity (Hs), and inbreeding coefficient (FIS). Following this, we described clustering and membership probabilities for all samples utilizing their assigned populations by running discriminant analysis of principal components (DAPC) from ADEGENET. A STRUCTURE-like analysis was then completed using R package LEA (Frichot & François, 2015) to identify clusters and calculate membership probabilities without inputting population information (Jenkins et al., 2021).
Due to noticeable uniqueness of samples from Osceola County, Florida, the species identifications of these samples were confirmed by sequencing fragments of the 16S rRNA gene (Lv et al., 2014). The phylogenetic tree was created using the 16S rRNA sequences obtained from Osceola County, Florida samples, and sequences from Genbank that were previously assigned to the American or Southern Clade for I. scapularis , or positively identified as Ixodes affinis . Sequences were aligned in Geneious Prime 2022 (Auckland, New Zealand) using the Geneious alignment algorithm with the following parameters: Global alignment with free end gaps, 65% similarity (5.0/-4.0) cost matrix, 12 for gap open penalty, 3 for gap extension penalty, and doing 2 refinement iterations. The tree was generated using the maximum-likelihood algorithm with 500 iterations in MEGA 11 withRhipicephalus sanguineus (L34302) used as the outgroup (Stecher et al., 2020). The final tree was produced using the R package ggtree (Yu et al., 2017).
Isolation by distance was also tested for all populations, using Mantel tests (Wright, 1943) following the tutorial for the ADEGENET R package (Jombart & Ahmed, 2011). The coordinates used for the geographic distances are provided in Table 1. Comparisons were then visually grouped to show Northern-Northern, Northern-Southern, and Southern-Southern comparisons, as determined by the LEA analysis where populations in clusters 1 and 5 are labelled as Northern, and those in clusters 2, 3, and 4 are Southern.