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.