Introduction
Speciation is a dynamic
evolutionary process through which populations segregate into
independently evolving lineages over time (De Queiroz, 2007). When gene
flow is restricted between populations, the accumulation of genetic
changes, through selection or local genetic drift, may lead to genetic
differentiation and potentially reproductive isolation (Coyne & Orr,
2004; Endler, 1973; Mayr, 1999; Richardson, Urban, Bolnick, & Skelly,
2014). This restriction of gene flow occurs through either geographic or
ecological isolation, though these are not mutually exclusive (Nosil,
2008). Thus, the level of gene flow between divergent populations is a
contributing factor to the rate of speciation, as well as to the spatial
level at which it occurs (Kisel & Barraclough, 2010).
Geographic isolation reduces migration between populations, and thus,
life-history traits influencing dispersal ability can drastically
influence the level of gene flow among populations (e.g. (Bolnick
& Otto, 2013; Slatkin, 1993). Species with low dispersal ability are
particularly likely to exhibit highly differentiated populations
resulting in the evolution of cryptic species over a limited spatial
scale (Pante, Puillandre, et al., 2015). In contrast, species with high
dispersal abilities are likely to maintain gene flow between
populations, therefore a process outside of geographic isolation is
needed to initiate the speciation process in these instances (Claramunt,
Derryberry, Remsen Jr, & Brumfield, 2012; Enbody et al., 2021; Palumbi,
1994). Two such mechanisms for
this are ecological and behavioral isolation whereby sympatric
populations occupy distinct ecological niches (Rundle & Nosil, 2005;
Wang & Bradburd, 2014). The difference between these populations, such
as habitat type, differences in mating times, or sexual selection reduce
the frequency of interbreeding and this can lead to genetic divergence
(Coyne & Orr, 2004; Dobzhansky, 1982). Ecological and behavioral
isolation can drive lineage divergence through selection, and subsequent
pre-zygotic isolation can further increase divergence through
reinforcement, accentuating the speciation process (Coyne & Orr, 2004).
However, with an increase in specialization, fragmented distributions of
either habitat or host may further reduce gene flow between populations
(Thompson, 1999; Thompson & Cunningham, 2002; West & Herre, 1994).
Each reproductive isolation mechanism can lead to similar morphological
adaptations and genomic signatures (Keller et al., 2013; Seehausen et
al., 2014), and this can make it challenging to interpret what factors
contributed to the speciation event. While the most accurate assumptions
about species delimitation are derived from a multifaceted approach
(Carstens, Pelletier, Reid, & Satler, 2013; Schlick-Steiner et al.,
2010), gene flow is directly tied to the fate of incipient species. By
using a population genetic approach to study microevolution and
incipient speciation, we can identify independent lineages and measure
the introgression between them to better understand the evolutionary
processes underlying species divergence. Species delimitation is
especially important when working with organisms responsible for
pathogen transmission, as misidentifications will lead to inaccurate
vector competency and surveillance data. Culicoides Latreille
(Diptera: Ceratopogonidae) biting midges are responsible for the
transmission of many disease-causing agents worldwide (Borkent, 2004;
Mellor, Boorman, & Baylis, 2000), including bluetongue virus (BTV) and
epizootic hemorrhagic disease virus (EHDV). These viruses can cause
severe symptoms and death in wild and domestic ungulates and are
responsible for substantial economic losses globally (Rushton & Lyons,
2015; Tabachnick, 1996).
In North America, one of the main
BTV and EHDV vectors is Culicoides sonorensis Wirth and Jones,
which belongs to the C. variipennis species complex. When
originally described, this group consisted of five subspecies (Wirth &
Jones, 1957), but it is currently composed of three distinct species
(C. occidentalis Wirth and Jones, C. sonorensis , andC. variipennis (Coquillet)) (Holbrook et al., 2000). Despite the
current taxonomic arrangement, species identification remains difficult
due to very subtle adult morphological differences and genetic
similarity. Additionally, cryptic species could make vector
incrimination and species distribution records potentially unreliable.
Measuring genetic divergence between species and populations can be
useful in vector biology as vectorial capacity and host association
become increasingly variable with increased genetic distance (Byrne &
Nichols, 1999; McCoy, Boulinier, Tirard, & Michalakis, 2001).
Population genetic studies of Culicoides species in Europe,
Africa, and Australia have consistently revealed frequent gene flow
between populations, even at continental scales (Jacquet et al., 2015;
Jacquet et al., 2016; Onyango et al., 2016; Onyango, Michuki, et al.,
2015). Their high dispersal ability, likely wind-mediated (Ducheyne et
al., 2007; Purse et al., 2005), decreases the likelihood of geographic
isolation between populations of Culicoides spp. Under laboratory
conditions, the species within the C. variipennis complex have
been shown to hybridize (Velten & Mullens, 1997), and while C.
occidentalis and C. variipennis are not known to be competent
vectors, both species occur sympatrically with C. sonorensis(Wirth & Jones, 1957). This lack of post-zygotic reproductive
isolation, coupled with a high dispersal ability and numerous sympatric
populations, makes this species complex an intriguing system in which to
study speciation and may also provide insights into the evolutionary
mechanisms responsible for vector competence in this group.
Here, we evaluated the geographic connectivity within and among the
species of the C. variipennis complex by assessing the level of
gene flow within and across populations. We used a high-throughput
ddRadSeq protocol to analyze 206 individuals collected from 17 sites
throughout the United States and Canada. We first estimated the overall
genetic similarity and population structure among these samples to
determine distinct lineages within the species complex. We then
estimated the level of gene flow between the inferred species, as well
as uncovered hybridization between sympatric species. As previous
attempts to separate these species using common barcoding genes have
been inconclusive, we sequenced a region of COI to compare to the
inferred SNP identifications. One species, was found to have two
distinct geographic haplogroups, while three other species shared a
single haplogroup. Additionally, we assessed the potential drivers of
divergence in this species complex by assessing loci under selection for
each species, as well as discuss the potential mechanisms controlling
reproductive isolation.