Discussion
In this study, we show a new pattern of population structure that can arise at the secondary contact zone due to different courses of evolution in hosts and parasites. The main signature of this pattern is a conflicting arrangement of mitochondrial markers in the host and the parasite at the secondary contact zone (Figure 2). The host’s mitochondrial lineages, coming from different refugia, mix across the area of the secondary contact and re-establish a panmictic population. In contrast, the parasite’s mitochondrial lineages stop their dispersal at the secondary contact zone. In our model, the re-established panmixia of A. flavicollis across the secondary contac zone is strongly suggested by a previous study on mitochondrial and microsatellite markers (Martinů et al., 2018) and further corroborated by our recent RAD-seq analysis which did not find in A. flavicollis any genetic disruption analogous to the SW/SE split (ms in prep). For the louseP. serrata , we detected a sharp geographic division between the SE and SW lineages using short (379 bp) cytochrome oxidase I (COI) haplotypes sampled across Europe (Martinů et al., 2018). To obtain a more informative comparison of genetic distance within and between the SE/SW clusters, in the present study we demonstrate this split on near-complete mitochondrial genomes from 26 samples collected across the secondary contact zone (Figures. 3, 4). From a strictly theoretical point of view, the pattern produced by the mitochondrial data can be explained by several scenarios. The first explanation is based on the strong presumption that the louse population structure will be determined entirely by the hosts’ migrations, given that the lice are highly host-specific and intimate parasites. Consequently, the discrepancy shown in Figure 2 would be a sampling or methodological artifact. However, considering the geographic extent and the number of samples in our previous study (Martinů et al., 2018), we believe that a methodological artifact is a highly implausible explanation. This view is further supported by the present study of the complete mitochondrial sequences and the same genealogical pattern obtained for 23 complete genomes of the maternally inherited symbiont L. polyplacis(Figure 4).
A second theoretical possibility assumes that the lice speciated during their separation in refugia before secondary contact of their hosts, due to their shorter generation time. A similar case was reported by Hafner et al. (2019) for a recent secondary contact of two subspecies of pocket gophers and their lice. While the gophers established a hybrid zone, their lice had already speciated and their contact resulted in “competitive parapatry”, with one louse species replacing the other. The authors also pointed out that the distribution data on the pocket gophers and their chewing lice indicate many instances of range overlap, potentially representing zones of competitive parapatry or species replacements. There are two strong arguments against applying similar scenarios to our system. A theoretical objection is that since the twoA. flavicollis mtDNA lineages do not create a secondary contact zone or hybrid zone, but intermix across Europe, it is difficult to envisage a mechanism that would prevent dispersion of the two new louse species across the secondary contact zone. Since both louse mtDNA lineages share the same host species and live in identical ecological environments (as evidenced by sampling both lineages even from the same host individuals), their mutually exclusive distribution is obviously not due to different adaptations (i.e. different host/environment specificities). Also, competitive exclusion is a very unlikely cause as demonstrated by the frequent coexistence of the S-lineage and N-lineage (Martinů et al. 2018). An empirical argument rests on the comparison between the mtDNA and SNP data. If the two mitochondrial lineages were fully isolated non-interbreeding species, we would expect to see the same pattern (i.e. two clearly separated and distant clusters) for both the mtDNA and the SNP sets. However, the comparison in Figure 4 shows that the two sets of data provide very different pictures. In contrast to the two distant mtDNA clusters, the SNPs sets create three distinct clusters corresponding to the two pure SW/SE lineages and an interspersed hybrid cluster containing samples from both mtDNA lineages (Figures. 4, S4, S5).
The third hypothesis assumes that during their separation, the two parasite lineages reached a high degree of genetic differentiation resulting in a strong but not absolute postzygotic barrier, whilst lacking an efficient prezygotic barrier preventing them from mating. As a consequence, upon encountering each other they formed a narrow hybrid zone in which the majority of the inter-lineage matings fail or produce hybrids with lowered fitness. In this case, we would expect a sharp geographic division between the SW and SE populations with occasional hybrids identified by nuclear markers around the secondary contact zone. Based on the data presented in this study and the previous extensive analysis of mtDNA (Martinů et al., 2018), we consider this hypothesis to be the best explanation of the observed patterns. A decoupled genetic structure of a host and its parasite(s) is not exceptional. It has been reported in various host-parasite associations and caused by different biological and/or environmental circumstances (e.g du Toit, van Vuuren, Matthee, & Matthee, 2013; Hafner et al., 2019). However, to our knowledge, theApodemus -Polyplax association presented here is the first known example of genetic structuring caused by a parasite’s hybrid zone created in the absence of the reciprocal host’s hybrid zone. There are several possible factors behind the lack of evidence for similar patterns in nature. Firstly, only a few studies have dealt with hybrid zones in parasites, and they were usually approached in relation to their hosts’ hybrid zone (e.g. Theodosopoulos et al. 2019). This is understandable considering the prevailing view of parasites’ evolution being predominantly determined by their hosts. Secondly, it is likely that this pattern will emerge during secondary contact only at a specific ratio (or narrow range of ratios) of genetic diversification between host and parasite populations. If the diversification is too strong, it may either result in speciation of both counterparts (i.e. classical cospeciation Page, 2003), in speciation of the parasite and emergence of a hybrid zone in the host (Čížková et al. 2018; Hafner et al. 2019) or in hybrid zone for both counterparts (e.g. de Bellocq et al. 2018). On the contrary, if the diversification is too weak, both counterparts will re-establish panmictic populations. This only leaves a narrow window of time for hosts’ panmixia vs. parasite’s hybrid zone. Yet, such cases do not necessarily have to be rare in nature, they may just be understudied or unnoticed due to the a priori view that evolution in host-specific parasites is linked to their hosts. The case we present here shows that one possible indication of a decoupled pattern is a strong mtDNA structure in a highly panmictic host population.
Genetic incompatibility between two populations at the secondary contact zone can be caused by various mechanisms. Apart from the differences accumulated in the nuclear genetic information, interbreeding can also be prevented by the incompatibility of mitochondrial and nuclear genetic information (Wolff, Ladoukakis, Enríquez, & Dowling, 2014; Hill, 2019). In our system, the lice are known to have their mitochondrial DNA split into several circular minichromosomes (Cameron, Yoshizawa, Mizukoshi, Whiting, & Johnson, 2011; Song et al., 2019). The distribution of mitochondrial genes among the minichromosomes is not entirely conserved - there are several differences in the gene arrangements when comparing the species Polyplax spinulosa and P. asiatica (Dong et al., 2014). To address the theoretical possibility that the barrier between the SW and SE lineages is caused by the failure of nucleus-mitochondrion interaction due to different distributions of their mitochondrial genes on the minichromosomes, we reconstructed full minichromosomes (their coding part) from all sequenced samples. In all cases, we found the same gene arrangement. This observation does not rule out the nuclear-mitochondrial incompatibility as a cause of the barrier, but it shows that it would have to be due to point mutations rather than structural differences (Table S3). In a similar way, we were not able to detect any significant difference between the L. polyplacis genomes from the SE and SW lineages, indicating that neither differences in the symbionts’ metabolic capacities are causing the gene flow barrier.
It would be speculative to infer other genetic sources of incompatibility between SE and SW lineages without a detailed study of the louse nuclear genome and more extensive sampling in the secondary contact zone, which is beyond the scope of the current study. Nevertheless, based on evidence collected from three genetic resources, the two maternally inherited markers (Legionella and mtDNA) and nuclear SNP diversity, we were able to unambiguously distinguish between the three possible scenarios of host-parasite incongruence. We propose a new mechanism in host-parasite co-evolution, where a narrow hybrid zone is present in the parasite without a corresponding break in the genetic structure of its host. In this way, the panmictic population of the host is rid of the parasite lineage present on one side of the parasite’s hybrid zone, which is gradually replaced by a different parasite lineage on the other side. Given that this evolutionary scenario can easily pass unnoticed (due to the lack of structure in the host) we hypothesize that “parasite turnover zones” may be more common than is currently known, particularly in highly host-specific parasites.