Introduction
Determining the appropriate delineation of populations and species is critical to understanding many biological systems. Several different species concepts are being used depending on the characteristics of the organisms of interest and the lens of researchers studying them (de Queiroz, 2005). Accurately identifying the differences across closely related species, clades, and populations is beneficial to assessing the existing diversity of the group in question (Sokal & Crovello, 1970). Vectors offer opportunities to study complex ecological interactions among biological groups with varying levels of differentiation and co-varying levels of association. In vector biology, these differences can significantly change the potential risk for vector-borne pathogen transmission within different environments (Arsnoe et al., 2019; Dolan et al., 1998; L. Eisen et al., 2017), and the organismal classification can influence how public health officials track disease risk in a region (Lane et al., 1991; Oliver et al., 1993).
The blacklegged tick, Ixodes scapularis , is a vector in the eastern and midwestern U.S. for at least seven human pathogens includingAnaplasma phagocytophilum, Borrelia mayonii, Borrelia miyamotoi, Ehrlichia muris eauclarensis, Babesia microti , Powassan virus, and most notably, Borrelia burgdorferi sensu stricto (s.s.) , a causative agent of Lyme disease (R. J. Eisen & Eisen, 2018). Lyme disease is a nationally notifiable condition in the U.S. (CDC, 1990) and cases have been continuously increasing for years (Bacon et al., 2008; Division of Vector-Borne Diseases, 2019; Rosenberg et al., 2018). More than 95% of human Lyme disease cases occur in the northeast and the upper Midwest regions of the U.S. (Division of Vector-Borne Diseases, 2019; Kuehn, 2013), with a low incidence of cases occurring throughout the rest of the U.S. (Figure 1). While the majority of human Lyme disease cases in the U.S. are due to transmission of B. burgdorferi s.s. byI. scapularis, other Ixodes species have been shown to be competent vectors (Couper et al., 2020; Fleshman et al., 2021).
The taxonomy of I. scapularis has been debated for many years. Currently, I. scapularis is subclassified to the Ixodes ricinus species complex, notably containing the main vectors of Lyme-causing Borrelia spirochetes. From 1979 (Krinsky, 1979) until 1996 (Oliver et al, 1993), there were three tick species from theI. ricinus -species complex located within the U.S., that were associated with transmitting B. burgdorferi s.s. : Ixodes pacificus in the West, I. scapularis in the Southeast, andI. dammini in the Northeast (Lane et al., 1991). However,I. scapularis were publicly believed to not be a large threat as they were thought to rarely bite humans (Oliver, 1996). Due to the geographical closeness and the intermediate characteristics found in nature of I. dammini and I. scapularis, the validity of the delination between the two was questioned (Spielman et al., 1979; Oliver et al., 1993). Laboratory mating experiments determined them to be conspecific species and I. dammini was synonymized withI. scapularis (Oliver et al., 1993). Once I. dammini , which was previously considered the main threat, was no longer considered a valid species, the concern for and surveillance of Lyme disease increased because the main vector, I. scapularis , is spread throughout the eastern U.S. (Oliver et al., 1993; Sanders, 1998). This new perception of I. scapularis led to questions on how populations of this species differ, especially when it came to Lyme disease prevalence in a region. Subsequent genetic studies redefinedI. scapularis into two distinct clades, delineating Northern populations (American clade) from Southern populations (Southern clade) (Norris et al., 1996).
Researchers have been using the American and Southern clades as a general way to classify Ixodes scapularis populations while the geographic range of the species has been expanding to the north and west. Populations have been documented in almost every state east of the Rocky Mountains while newly established populations are filling in previously uninhabited counties throughout the known range and can now even be found as far north as Québec (R. J. Eisen et al., 2016; Fleshman et al., 2021; Khatchikian et al., 2015; Ripoche et al., 2022). This continual range expansion is increasing the potential risk for Lyme disease throughout North America (Kelly et al., 2014; Kugeler et al., 2015; Leighton et al., 2012; Ogden et al., 2009). The Lyme endemic region is also expanding outward from the historical locations in the Northeast and upper Midwest, with some counties south of Virginia reporting increases in I. scapularis abundance (R. J. Eisen et al., 2016; Hickling et al., 2018) concomitant with increases in Lyme disease cases in humans over the past two decades (Division of Vector-Borne Diseases, 2019). States within the Ohio River Valley region are experiencing some of the highest rates of Lyme disease case spread in the U.S. For example, Lyme disease cases in Ohio have increased 10-fold over the past decade, likely due to the range convergence of the Northeastern and Upper Midwestern I. scapularis populations (Bisanzio et al., 2020).
The presence of I. scapularis alone is not enough to predict how rapidly Lyme disease risk can increase in a region because Lyme disease ecology is complex and involves multiple intersecting factors within the tick life cycle, such as dominant tick blood meal hosts and reservoir vertebrate hosts of B. burgdorferi (Bisanzio et al., 2020; Burtis et al., 2016; Gardner et al., 2020; Ogden et al., 2013). These intersecting factors all contribute to the vectorial capacity ofI. scapularis for B. burgdorferi , defined as all “vector-related variables affecting stability of pathogen transmission” (Spielman et al., 1984). In addition to variation in host preference, a key determinant of pathogen transmission by I. scapularis is likely in the host-seeking behaviors themselves, such as time spent host-seeking, or the height to which ticks will climb for host-seeking. Population level variation of host-seeking could be due to changes in humidity, because ticks are extremely sensitive to desiccation, and host community composition (i.e., mice are the most common host of tick larvae in the north, whereas lizards are important hosts in the south (Apperson et al., 1993; Ostfeld & Keesing, 2000)). However, previous research has also shown that genetic factors are likely contributing to a tick’s ability to acquire and transmit pathogens and genetics could even play a role in host-seeking behavior differences (Arsnoe et al., 2015; Ginsberg et al., 2017; Ginsberg et al., 2014). Thus, the genetic composition of the expanding I. scapularis populations merits consideration.
While there has yet to be a study directly linking tick host-seeking behaviors to genetic variants, underlying genetic mechanisms driving population-level differences in I. scapularis survivability and host-seeking behaviors have been hypothesized. The consistency of these behaviors within Northern and Southern I. scapularis populations when exposed to varying environments suggests that host-seeking behavior is driven in part by genetics, and not solely from environmental pressures (Arsnoe et al., 2015; Tietjen et al., 2020). Identifying the populations that are leading the range expansion fronts, like those in the Ohio River Valley and the Virginia-North Carolina border, will provide public health officials with an informed view of tick-borne disease risk in currently non-Lyme endemic areas experiencing invasion (or possibly re-invasion) by blacklegged ticks. However, due to the difficult and time-consuming efforts needed to directly measure vectorial capacity of ticks, it is far more efficient to identify genetic markers that are associated with tick populations that have high or low human Lyme disease prevalence. If the markers are sufficiently dense and spread throughout the genome, then it may also be possible to identify loci associated with the traits of interest (Anandan et al., 2022; Aubry et al., 2020; Dupuis & Siegmund, 1999; Rahman et al., 2021).
Research to increase our understanding of the population genetics ofI. scapularis has occurred for many decades. The main limitation so far has been that the studies either do not cover a wide portion of the genome or the wider geographic range of the species. They also cannot be easily compiled to accomplish both aspects as the targets and methodologies are non-compatible. Nonetheless, they have given insight into how populations of I. scapularis vary genetically, concluding there is significant genetic differentiation across the landscape, especially when comparing northern and southern populations (Gulia-Nuss et al., 2016; Ludwig, 2015; Norris et al., 1996; Van Zee et al., 2015). A study covering a large portion of the genome and known range of I. scapularis populations is needed to characterize the extent of differentiation within this species, as well as advancing an understanding that links phenotypes to genotypes.
Here, we investigate genome-wide variation within and among populations of I. scapularis grouped into four geographic regions that approximate major divisions described or hypothesized in previous research: Southeastern Atlantic, Southern Gulf, Upper Midwest, and Northeastern U.S. We aim to provide a basis for future research in uncovering how genetic variation among I. scapularis populations is driving observed phenotypic differences, thus driving Lyme disease prevalence in the U.S. Understanding the genotypic variation within and among I. scapularis populations, especially those at the fronts of range expansion and phenotypic change, is essential to understanding the ecology of these ticks and pathogens, which can mitigate Lyme disease risks and serve as a model system for disease ecology.