DISCUSSION
We confirmed that the two populations of Serpula lacrymans var.lacrymans from the built environment in Japan and Europe are
highly divergent, as previously revealed by Kauserud et al.(2007), and that the divergence between the two populations predated
human influence. The results from the demographic modelling analyses
strongly support this hypothesis, indicating that the ancestral
population split more than 3,000 generations ago and led to the current
divergent populations in Japan and Europe. Based on genomic similarity
of the wild Serpula lacrymans var. shastensis and the
building occupant Serpula lacrymans var. lacrymans , we
have previously suggested that this species is one of few wood-decay
fungi that was highly pre-adapted to the built environment (Balasundaramet al. 2018). As a result, it established extensive populations
in temperate regions worldwide vectored by human translocation (Kauserudet al . 2007).
The split between the two invasive populations predates human
influence. The natural habitat of S. lacrymans is high alpine
regions with scattered large conifer logs (Kauserud et al. 2012).
From there, Serpula lacrymans has established independently at
least twice in the indoor habitat (Kauserud et al . 2007).
However, our dating of this split is influenced by the implemented
mutation rate, illustrated by an increase in number of generations from
3,067 to 18,770 when using 10-8 as alternative
mutation rate (to 10-7). Assuming a split
approximately 3,000 generations ago and a generation time of one year
under optimal fungal growth conditions, human-made habitats will have
existed, i.e. 3,000 years before present. Indeed, there are dated wooden
constructions circa 7,000 years ago from Europe (Tegel et al.2012). However, a longer generation time than one year (for
establishment, vegetative growth and fruiting) is plausible and the
mutation rate is uncertain, which suggest that the split of the
ancestral population probably predated the availability of the built
environment. Central and eastern Asia, where S. lacrymans can be
found in nature, were not covered by ice sheets during the last
glaciation 115 – 11 K BP (Tian & Jiang 2016). The dry and cold areas
may have driven S. lacrymans into refugia, thus splittingS. lacrymans into several sub-populations. Although speculative,
such a scenario also fits well with the estimated reduction in
population size at almost the same time as the split. Consequently, the
Himalayas, where the fungus has it westernmost natural distribution
(Kauserud et al. 2012), and another north Eastern Asian
population, might have functioned as separate source populations of the
current indoor populations in Europe and Japan. From previous population
studies of S . lacrymans , it is known that the European
population shares genetic material with specimens from the Himalayas,
and that the Japanese population is geographically and genetically
closer to the natural population in East Asia (Kauserud et al .
2012, 2007).
The genome-wide SNP data confirmed that the Japanese population is
genetically more variable than the European population and that more
genetic variation was retained during its founder event(s). This is
mirrored by the higher number of vegetative incompatibility (vic )
alleles and diversity of mating types present in the Japanese population
(Engh et al. 2010a). The two genetic regions in scaffold 1 and 3
with lower genetic diversity, cover two large genomic regions containing
many genes with various functions. One possible interpretation is that
these two genomic regions harbor inversions and therefore show lower
rates of recombination in the Japanese population (and in one New
Zealand isolate). However, better genome assemblies, which can be
achieved with long read sequence data is required to test this.
Currently, the function and effect of these possible genome
rearrangements are unknown.
The European population invaded the built environment about 250
generations ago. As noted above, genetic similarity between Himalayan
specimens collected in forests and the European indoor population has
previously been observed (Singh et al. 1993; Kauserud et
al. 2007). Our demographic modelling suggests that the colonization of
Europe happened between 200 and 400 generations ago. Assuming a
generation time of one year, this estimate of 200-400 generations fits
well with the species description of S. lacrymans in 1781 (Wulfen
1781), based on a collection from Europe (Austria). During this period,
and also somewhat earlier, there were considerable trade activities
between Europe and Asia which could easily have vectored the
colonization of S. lacrymans to Europe, e.g. through the trade
and transport of timber. Human activity might have further spreadS. lacrymans , from Europe to North America and Australia
(Kauserud et al. 2007) and is also considered the main agent for
dispersal of several plant pathogens, such as Microbotryum
lychnidis-dioicae and Ophiostoma ulmi (Brasier 1991; Fontaineet al. 2013). To better understand the colonization event ofS . lacrymans in Europe, the relatedness of European
isolates to isolates from the natural range in the Himalayas should be
further investigated.
The European population of S. lacrymans holds low genetic
diversity, and the founding members were estimated to consist of only
four or five haplotypes (depending on mutation rate). Hence, it is
possible that only two or three heterokaryotic isolates colonized and
founded the entire European population. This assumption is strongly
supported by few mating (MAT) and vegetative incompatibility
(vic ) alleles detected in the European population where three
heterokaryotic isolates may account for the entire allelic diversity
(Kauserud et al. 2006; Engh et al. 2010b; Skrede et
al. 2013). Similar to S. lacrymans , the European population of
the invasive ash dieback fungus H. fraxineus is genetically
extremely homogenous and was established by only two haploid individuals
(McMullan et al. 2018). Two different subspecies of the Dutch Elm
disease parasite Ophiostoma novo-ulmi established as clonal
parasites in Europe. Later, sexual reproduction between these
populations produced a hybrid with high pathogenicity (Brasier & Kirk
2010). Hence, there are now multiple lines of evidence that invasive
fungi may establish on other continents through very tight founder
events followed by extensive population growth.
The small founder event establishing the European population is also
reflected in the slow LD decay and high levels of Fis observed
in the European population. A high degree of selfing or clonal dispersal
may cause such abnormal linkage decay curves (Nieuwenhuis & James
2016). Similar patterns of linkage disequilibrium were observed for
isolated and highly biparentally inbred wolf populations in Italy and
the Iberian peninsula (Pilot et al. 2014). The very low number of
isolates founding the European population of S. lacrymans has
probably also led to high levels of biparental inbreeding. The limited
number of mating alleles available will necessarily lead to a reduction
in mating opportunities between individuals, and the tetrapolar mating
system of S. lacrymans still allows 25% of the spores from the
same fruit body to mate. No putative clonal isolates were found in our
or previous studies (Kauserud et al. 2007; Engh et al.2010b; Maurice et al. 2014), supporting that selfing or clonal
dispersal is not the main explanation of the slow linkage decay.
Signatures of selection in the built environment. In both
populations, we observed signals indicative of balancing or purifying
selection acting on two loci. For these two loci we observed
significantly lower FST values than expected
(DXY was not different from the expectations). Genomic
regions of low differentiation could be caused by pleiotropic effects
(one locus having an effect on two or more phenotypes), where these
phenotypes have different selection pressure in the two populations. The
differences in Tajima’s D and π between the populations at these two
loci indicate that pleiotropic effects may be involved, i.e., the SNF1
complex at scaffold 8 show signals of purifying selection in the
European populations, but balancing selection in the Japanese
populations. The SNF1 complex is involved in gene expression regulation
as a response to starvation in yeast (Sanz 2003), but is also shown to
be involved in responses to several different environmental stressors
(summarized in Shashkova et al . 2015). The homolog of the main
SNF1 kinase was differentially expressed in response to varying
substrate composition during wood decay in S. lacrymans,supporting a role in substrate-dependent gene regulation (Hess et
al . 2021).
Another gene with an oxysterol-binding domain (as was found in the locus
at scaffold 27 in our analyses) evolved at a specifically rapid pace
(significantly higher dN/dS) in S. lacrymans compared to the
sister taxon S. himantioides (Balasundaram et al. 2018).
Oxysterol-binding proteins are involved in transportation of lipids in
eukaryotic cells (revised in Qui & Zeng 2019), and have shown to be
important to polarized hyphal formation and growth in Candida andAspergillus (Ghugtyal et al . 2015; Bühler et al .
2015). Maintaining diversity for both the oxysterol-binding and the SNF1
complex could therefore be important for S. lacrymans’ unique
ability for rapid growth and decay of large substrates.
In the European population, selective sweeps were found in genes related
to DNA replication (a DNA helicase with a Pif1 domain) and protein
modification (O-Fuct domain involved in glycosylation). In parallel to
this, associations between helicases and the environment have been found
in several landscape genomic studies, i.e. the wood decay fungusPhellopilus nigrolimitatus (Sønstebø et al . in prep.), the
ectomycorrhizal Suillus brevipes (Branco et al. 2017) and
the red bread mold Neurospora crassa (Ellison et al .
2011). Suggesting that modification of DNA replication may be the first
signals of local adaptation for recently founded populations. Thus, in
the European population we may be observing recent events, which
corresponds well with the demographic history of the European
population, with its small effective population size and the recent
bottleneck.
The selective sweep in the Japanese population was in a non-coding
region. Thus, we cannot conclude about the function of this sweep.
However, within a 10 kb window of this sweep there are three predicted
genes, of which one is a Cytochrome P450. Cytochrome P450s are
monooxygenase enzymes known to be involved in the detoxification of
polyphenols and other defense molecules secreted by other organisms or
encountered during wood decay (Cresnar and Petric 2011; Ichinose 2013;
Xu et al . 2015). For fungi, cytochrome P450s were recently
suggested to be involved in heavy metal tolerance in Suillus
luteus (Bazzicalupo et al. 2019) and during pathogenic
interactions between Heterobasidion annum and conifer trees
(Karlsson et al. 2008). Wood type dependent difference in gene
expression of cytochrome P450s have previously been found for two wood
decay species (Wymelenberg et al . 2011). Further, in our recent
study of S. lacrymans (Hess et al . 2021), a larger
repertoire of differentially expressed cytochrome P450s was associated
with significantly faster decay of recalcitrant pine wood in a Japanese
isolate compared to an isolate from the European population. However, in
the experiments in the current study we did not detect systematic
differences in the ability to decay different substrates between the
Japanese and the European populations, rather that there is high strain
to strain variation in this trait. More detailed functional studies are
therefore required to better understand the functions of individual
cytochrome P450s.
The New Zealand population is admixed between European and Asian
populations. Inclusion of two isolates from New Zealand enabled us to
test whether this population has an admixed ancestry, as hypothesized in
Kauserud et al . (2007). Indeed, isolate ICMP18202 possessed a
combination of European and Japanese alleles, in support of this
hypothesis. Similarly, in other invasive fungi, like B.
dendrobatidis , O. ulmi/novo-ulmi, H. fraxineus and the
oomycete pathogen Phytophthora alni , secondary admixture between
independently evolved lineages have been observed (Ioos et al.2006; Brasier & Kirk 2010; McMullan et al. 2018; O’Hanlonet al. 2018). The merging of genetic lineages in founder areas
may lead to novel and more aggressive genotypes, as shown for most of
these species (Ioos et al. 2006; Stukenbrock & McDonald 2008;
Brasier & Kirk 2010; McMullan et al. 2018; O’Hanlon et
al. 2018). In our wood decay experiment, the New Zealand isolate
ICMP18202 decayed pine wood rapidly (Figure 8), though more isolates are
needed to confirm this pattern. Since only one of the two isolates
showed signals of admixture, the admixture could be a recent event.
Further, the admixture was heterogeneously distributed in the genome
(mainly scaffolds 1 and 3), indicating limited cycles of recombination.
It could also be that PCAdmix assigned genomic windows more
readily to homogenous genomic regions with low diversity, and hence,
more readily detects admixture in scaffolds 1 and 3 with lower diversity
as compared to the rest of the genomes of the Japanese isolates. The
reasons for reduced diversity and admixture in large genomic regions in
the admixed isolate from New Zealand are currently unknown. This could
be a consequence of genomic areas of low recombination, resulting from
chromosomal rearrangements. In the future, more thorough analyses of
genomic synteny, based on assembled genomes, among these three
populations may shed light on their effect on population divergence.
Furthermore, the isolate(s) that contributed to the admixture were not
necessarily of Japanese origin, but could originate from other locations
in Asia. Thus, including more isolates from the full geographic
distribution of S. lacrymans may contribute to better understand
the admixture event in New Zealand.
Concluding remarks. By using full genome data in combination
with growth experiments, we revealed genomic differences among isolates
of S. lacrymans from Europe, Japan and New Zealand. We estimated
that the source population of the European population split from the
Japanese population 3,000 to 19,000 generations ago, and that the
European population established about 200 – 400 generations ago through
a tight bottleneck event. Two genes related to mycelial growth seemed
particularly important for survival of both indoor populations and
probably evolved before the species became invasive in the built
environment. However, population specific selective sweeps identified
more recent events, which show that decay of various substrates in Japan
and rapid adaptation of DNA replication and protein modification in
Europe influence population survival. We were able to confirm that one
isolate from New Zealand held genomic signatures of admixed ancestry.
However, more samples from New Zealand and the native Asian populations
are needed to infer, with better certainty the source populations ofS. lacrymans and for in-depth analyses of the evolutionary
history of the populations in the built environment.