Introduction:
The evolutionary success of sex has been one of the greatest puzzles
evolutionary biologists have pondered for over a century
(Weismann,
1889). Although, theoretically, sexually-reproducing organisms should be
outcompeted by the asexual organisms due to inherent costs of sex, they
remain predominant in the world of eukaryotes. Numerous hypotheses have
been proposed to explain this paradox (see
Shcherbakov,
2010 for one recent hypothesis, reviewed in e.g., Hartfield &
Keightley, 2012). Some are linked to inherent ecological properties of
reproductive modes, such as the penalization of asexuals for their
inability to rapidly generate novel variants, thus increasing their
intraspecific competition (Doncaster, Pound, & Cox, 2000;
Peck,
Yearsley, & Waxman, 1998) or vulnerability
to
pathogens (Hamilton, 1980). Another popular class of theories emphasizes
an increased extinction risk of the clones (ultimately decreasing their
diversification), or the gradual deterioration of their genomes
(hereafter referred to as mechanisms of ‘clonal decay’). These are based
on the hypothesis that every asexual lineage will acquire harmful
mutations, and selection will not be efficient enough to maintain the
fittest genome (Kondrashov, 1982) because individual mutations may not
escape their genomic background due to a whole-genome linkage, leading
to accumulation of mutations in a stochastic (Muller’s ratchet) or a
deterministic (Kondrashov’s hatchet)
manner
(Kondrashov, 1993). Consistent with this hypothesis, most asexual
lineages appear to be evolutionarily short-lived, and occupy terminal
positions on the tree of life (reviewed in Janko, Drozd, Flegr, &
Pannell, 2008;
Schwander
& Crespi, 2009). Several genomic studies indeed have found evidence of
higher rates of accumulation of nonsynonymous mutations in asexuals
(e.g. Henry, Schwander, & Crespi, 2012; Hollister et al., 2015; Howe &
Denver, 2008; S. G. Johnson & Howard, 2007; Neiman, Hehman, Miller,
Logsdon, & Taylor,
2010;
Paland & Lynch, 2006).
Despite broad consensus that accumulation of mutation should ultimately
lead to the demise of asexual lineages, it remains unclear whether the
process proceeds fast enough to exterminate the asexuals before they
become competitive threats to the sexual populations. Some argue that
higher rates of mutation accumulation can considerably harm the
clonal
populations even over a short period of time (e.g. Neiman et al., 2010),
especially in synergy with other mechanisms
(Pound,
Cox, & Doncaster, 2004; West, Lively, & Read, 1999). However, a
non-negligible number of studies have failed to confirm higher loads of
mutations in asexual organisms (e.g. Allen, Light, Perotti, Braig, &
Reed, 2009; Brandt et al., 2017; Naito &
Pawlowska,
2016; Pellino et al., 2013; Warren et al., 2018), and several studies
have also demonstrated comparable or higher net diversification rates in
asexual
clades than in the sexual ones, suggesting these lineages may not
inherently suffer from a decreased diversification, an increased
extinction, or both (Fontaneto, Tang, Obertegger, Leasi, & Barraclough,
2012; M. T. J. Johnson, FitzJohn, Smith, Rausher, &
Otto,
2011; Liu et al., 2012).
Such contradictions do not necessarily invalidate detrimental effects of
mutation accumulation as a factor, but they indicate that the full
explanation is more complex. For example, asexuals may have some
mechanisms that temporarily delay the process of clonal decay, such as
‘minimal sex’, ameiotic recombination, gene conversions, increasing
ploidy with consequent genome refreshment, and masking mutations with
heterozygous states (e.g. Flot et al., 2013;
Halligan &
Keightley,
2003; Loewe & Lamatsch, 2008; Sémon & Wolfe, 2007). It is also
possible that relaxation of selection stemming from asexuality is not
drastic enough to universally cause the expected trends in mutation
accumulation patterns (Allen et
al.,
2009). It thus remains a major challenge in evolutionary biology to
identify the short-term-acting mechanisms that protect the sexuals from
competitive displacement by the asexuals.
So called sexual-asexual complexes, i.e., taxa in which related sexual
and asexual forms coexist, play prominent roles in studies of the
persistence of sex since both types of organisms enter the same
evolutionary arena for mutation, selection, and drift (Birky &
Barraclough,
2009;
Janko, Drozd, & Eisner, 2011). In some complexes, asexual lineages may
emerge from the sexual populations very
dynamically
(Janko et al., 2012), which poses an additional question regarding the
role of mutation accumulation process in saving sex: even if individual
clonal lineages become debilitated in a relatively short term, whole
asexual populations, composed of multiple frequently emerging clones,
may escape extinction and pose a serious short-term or long-term threat
to the sexual counterparts
(Paland,
Colbourne, & Lynch, 2005).
Interestingly,
Janko et al. (2008) showed that continuous influx of new clones into an
asexual population causes inevitable replacement of the old clones by
drift without invoking an age-dependent decay of the clonal lineages.
This implies that ages attained by the individual clonal lineages may
not correspond to their theoretical maxima determined by an
age-dependent decay, but rather by the rate of influx of the new clones
into an asexual community of a finite size. This drift-like clonal
turnover process proved sufficient to explain some
phylogenetic
data (Janko, 2014), suggesting that in some cases clones may vanish from
a population before mutation accumulation or other such processes
compromise their fitness.
Even less is known about the direct impact of mutation accumulation on
clone fitness. Indeed, if increased mutation accumulation in asexuals is
relevant to the maintenance of sex, it should have phenotypic
consequences when relatively old asexual lineages ‘… suffer in
comparison to younger asexual lineages (and sexuals) with regard to
important traits such as mitochondrial function and the rate of
offspring production and population
growth’
(Neiman et al. 2010). Unfortunately, studies investigating
fitness-related traits are scarce, and their results are contradictory;
age-dependent fitness deterioration has been demonstrated in
some
lineages of viruses, bacteria, and yeasts (Andersson & Hughes, 1996;
Chao, 1990; Goddard, Godfray, &
Burt,
2005), but not in water frogs
with
genomes transmitted clonally for over 25 ky (Guex, Hotz, &
Semlitsch,
2002).
In this study, we focused on the Cobitis taenia hybrid complex of
the European bottom dwelling loaches, and examined whether the genomes
of sexual, young asexual, and old asexual lineages differed in
accumulation of nonsynonymous mutations, and whether the old and the
young clones differed in traits that impacted their fitness.
Our model taxon consists of several parapatrically distributed sexual
species that inhabit Europe and coexist over much of the continent with
their hybrids that regularly establish successful, persistent clonal
lineages with a gynogenetic reproductive mode (i.e. females lay
unreduced eggs that require activation by sperm from sexual males to
trigger development). Production of clonal eggs is achieved via
“pre-meiotic endomitosis” when oogonial chromosomes are duplicated,
and subsequent meiotic divisions do not yield any variability since
crossovers occur among identical copies of chromosomes (Juchno, Arai,
Boroń, & Kujawa, 2016, Dedukh et al. 2019). Phylogenetic and crossing
experiments (Choleva et al., 2012;
Janko, Culling, Rab,
& Kotlik, 2005; Janko et al., 2012; Janko, Vasil’ev, et al., 2005;
Janko, Bohlen,
et
al. 2007) showed that range shifts related to the Pleistocene climatic
oscillations have repeatedly placed the Cobitis parental species
into a secondary reproductive contact, provoking the dynamic formation
of hybrid clones. Their diversity is created in a two-step process (Fig.
1 insert). First, diploid clonal F1 females form through crosses of the
parental species C. elongatoides with either C. taenia orC. tanaitica (Choleva et al. 2012; Janko et al. 2018); for
simplicity, the haploid genomes of those species have been hereafter
denoted as E, T, and N, respectively, and hybrid forms with particular
combinations of the parental genome (so-called ‘genomotypes’) have been
denoted by these letters, e.g. ETT indicates a triploid hybrid with one
elongatoides and two taenia genomes. In the second step, mating with
sexual males may occasionally result in the incorporation of sperm
genomes leading to the formation of secondary triploid clones.
Tetraploids are occasionally produced in an analogous way, but generally
die before maturity (Janko, Bohlen, et
al., 2007; Juchno et al., 2014).
Contemporary Cobitis populations are composed of a mixture of
clones of quite different evolutionary ages. Some clones are very
recent. Other clones originated during the early-Holocene with
establishment of the secondary contact zones between the parental
species. Molecular dating using mtDNA data indicates that the origin of
the oldest known lineage dates to ~0.3
Mya
(Janko, Culling, et al., 2005, Majtánová et al., 2016). This lineage,
referred to as the ‘hybrid clade I’, originated in the Ponto-Caspian
region as a diploid C. elongatoides-tanaitica (EN) hybrid,
and during its subsequent expansion it has incorporated additional
genomes on multiple occasions, resulting in a monophyletic clonal
assemblage of the EN, the EEN, the ENN, and the ETN clonal genomotypes.
Owing to a lack of evidence on the observation of asex → sex
transitions, the emergence of asexuality in the Cobitis appears
to be a unidirectional process that has been more or less continuously
occurring for at least hundreds of thousands of years, which should be
enough to observe the putative effects of clonal decay (Loewe &
Lamatsch, 2008). Mechanisms ensuring such long-term coexistence with
sexual forms have not been properly
understood,
although Kotusz et al. (2014) and Bartoš et al. (2019) documented niche
segregation between the Cobitis forms, suggesting some role of
ecological factors.
Dynamic emergence of asexuality, the presence of diploid and polyploid
clones, and a relatively well-resolved evolutionary history and ecology
all make the Cobitis an attractive model for investigation of
genomic and phenotypic consequences of asexual reproduction, allowing,
among other objectives, the disentanglement of polyploidy effects from
asexuality and/or hybridization. Here, we simultaneously analyzed the
genetic variability in nuclear and mitochondrial genes, together with
fitness data, to test predictions of mutation accumulation hypotheses.
Specifically, we investigated whether asexual genomes carried traces of
relaxed purifying selection which may be manifested byd N/d S ratios closer to 1
along the ‘asexual’ branches of the phylogenetic trees (Wertheim et al.,
2014), by generally higher rates of accumulation of the nonsynonymous
mutations, or by more radical amino acid substitutions in clonal genomes
(Pellino et al., 2013; Sharbrough, Luse, Boore, Logsdon, & Neiman
2018). We further compared mutation frequency spectra to test whether
the spread of novel nonsynonymous variants differed between the sexual
and the asexual lineages
(Hollister
et al., 2015). Finally, we extended our analyses to the phenotypic data
from additional individuals and tested whether traits related to the
body-condition and fecundity tended to decay in the aging clones.
Material & Methods
Origins and numbers of analyzed specimens are listed in Figure 1 and
Tables 1 & 2. The taxonomical identities of all fish used in this study
were determined with a set of previously published and validated species
diagnostic markers including allozyme and microsatellite loci and
flow-cytometric determination of ploidy (Janko, Flajšhans, et al., 2007;
Janko et al.,
2012).
Genomic data
Exome data were acquired using the exome capture method described in
Janko et al. (2018) and briefly below. Using probes for targeted
enrichment of gDNA loci we collected sequence data from nuclear exomic
and mtDNA segments of 22 females belonging to three hybridizing sexual
species, 26 hybrids of different ploidy, and the genome composition and
one specimen of C. paludica as an outgroup (Tab. 1, Fig. 1).
Samples of parental species represent all major phylogroups,sensuJanko,
Culling, et al. (2005), to capture maximum coverage of intraspecific
variability in our data. Hybrid specimens include two or more
individuals from several independent clonal lineages characterized
previously by
Janko
et al. (2012), to compare genetic variability within and between clonal
lineages.
Sequencing and SNP calling
Isolated gDNA was sheared with Bioruptor, tagged by indices, pooled, and
hybridized to custom-designed sequence-capture probes. Captured
fragments were sequenced on an Illumina NextSeq in 75 bp paired-end (PE)
mode. Fastq files were trimmed by
the
fqtrim tool (Pertea, 2015) with minimum read lengths of 20 bp, and 3’
end trimming when average base quality within a sliding window drops
below 15, which is a commonly recommended threshold (e.g. Suren et al.,
2016).
To further process the reads, we used a published Cobitisreference transcriptome (GGJF00000000;
Janko
et al., 2018), from which Janko et al. (2018) discarded potentially
paralogous contigs, as described in Gayral et al.
(2013).
In short, we considered as potentially paralogous contigs those that
possessed spurious heterozygosity patterns when the same heterozygotic
positions occurred across distantly related species (including the
outgroup). Such patterns are unlikely to originate from meaningful
biological processes, and probably result from mapping of paralogous
reads onto one contig present in the reference. Loci with such
properties were removed from the reference. For the purposes of this
study, we annotated the reference
transcriptome
with Blast2GO software (Conesa et al., 2005). Contigs were aligned
against the nr database (18.11.2015) by Blastx 2.2.31. Hits with
e-values < 0.0001 were accepted as significant. Otherwise,
default settings were used. Subsequently, we identified the longest open
reading frame (ORF) within each annotated
contig using the getorf tool from
Embassy package (v6.6.0.0).
We aligned reads to this reference with BWA MEM
(Li
& Durbin, 2009), followed by Picard tools v1.140 to mark duplicates
(Broad Institute, http://broadinstitute.github.io/picard).
Individuals’ variants were called with the GATK v3.4 HaplotypeCaller
tool, and all individuals were jointly genotyped using the GenotypeGVCFs
tool
(DePristo
et al., 2011; McKenna et al., 2010; Van der Auwera et al., 2013).
Variant recalibration was based on a previously used database of
species-diagnostic positions
(Janko
et al., 2018) representing a learning set for the variant quality score
recalibration tool VariantRecalibrator, and all variants were then
filtered with the ApplyRecalibration tool. All resulting high confidence
SNPs with coverage ≥ 5 were transferred to a database using custom
SQL/python scripts.
Four samples represent an
experimental family including two C. elongatoides-taenia F1
hybrid individuals and both their parents, which allowed us to
experimentally verify the efficiency of sequencing and SNP calling by
comparing F1 hybrids to their parents. We also compared our NGS data
with Sanger sequences of several loci obtained by Choleva et al. (2014)
to assess the accuracy of our method.
Tests of Relaxed Selection in
Mitochondrial genomes
Because haploid mtDNA data are not biased by the problem of phasing, we
first analyzed mitochondrial genomes by mapping reads to publishedC. elongatoides mitochondrion (accession no. NC_023947.1) and
applying the RELAX software (Wertheim et al. 2014) to individual
haplotypes to test the hypothesis that selection is relaxed along
asexual branches, resulting ind N/d S ratios closer to 1
than along sexual branches. Since the software requires phased sequences
representing single ORF with no heterozygous sites or stop codons, we
discarded all noncoding sequences (D-loop and tRNAs), and the
reverse-oriented ND6 gene from our mtDNA reference. We also masked
several observed heterozygous positions and stop codons with ’N’ using
the HyPhy
package
(Pond, Frost, & Muse,
2005),
since these may represent sequencing errors or mutated premature
termination codons. In case of overlapping ORFs in several mitochondrial
genes, we split the sequence at the start codon of the second ORF, and
padded the 3’ end of the first ORF with ’N’ to keep the correct reading
frame of the whole alignment.
The final alignment of coding mtDNA was uploaded to the PhyML server
(v3.0 build 20120412;
Guindon
et al., 2010), and
phylogenetic trees were computed with default settings. A substitution
model was selected based on AIC using Smart Model Selection (SMS 1.8.1.;
Lefort,
Longueville, & Gascuel, 2017; Fig. 2, Table S1). We then used both the
alignment and Newick tree file in HyPhy 3.8 to perform relaxed selection
analysis in RELAX with the assumption of strict sex → asex transitions,
which is justified based on known
pathways of asexual
hybridization
in Cobitis (Choleva et al., 2012). The software requires two
types of branches to be specified in each tree. As test branches, we
selected those leading exclusively to asexual individuals (i.e. asexual
branches). As reference branches, we considered the intraspecific tree
branches within all three sexual species (i.e. sexual branches; Fig. 2).
We left the interspecific branches unclassified, since sequence
evolution at the interspecific level is likely to bear traces of
stronger negative selection, as many mildly deleterious mutations
segregating within species would not become
fixed
between
species. Choleva et al. (2014) showed that C. tanaitica is fixed
for introgressed elongatoides-derived mitochondrion and appears
paraphyletic to C. elongatoides , potentially indicating repeated
introgression events in the past. Therefore, we also left one internal
branch within C. tanaitica unclassified, as we could not
determine whether it represents an intra- or interspecific branch (see
Fig. 2). We also left unclassified the branches leading to laboratory
progeny used in F1 crosses, as they represent non-natural experimental
strains with unknown reproductive modes in the F1 generation.
We compared the fit to data of the two models allowing site-specificd N/d Sratios, i.e. the
null model assuming the same ratios for asexual (test) and sexual
(reference) branches of the ML phylogenetic tree (see Fig. 2), and the
alternative model assuming thatd N/d S ratios differ
between types of branches.
Tests
of Relaxed Selection in nuclear DNA
While mitochondrial data allowed the application of sophisticated
methods (i.e. RELAX), we were also interested in trends in nuclear DNA,
which is highly heterozygous in hybrids and thus had to be analyzed
differently. Specifically, we inspected whether ratios of synonymous and
nonsynonymous mutations differ between sexual and asexual populations by
comparing the frequency spectra in each population, and testing whether
radicality of nonsynonymous substitutions differs among genomotypes.
Finally, as a more formal test of selection, we compared per-gene
distributions of d N/d Sratios among datasets. Details are provided below.
Types
of SNPs, N/S ratios, site frequency spectra, and radicality of amino
acid substitutions
All nDNA analyses are based on ORFs that represent annotated
protein-coding sequences with assigned GO terms. For methodological
simplicity (see e.g. Burgarella et al., 2015), we constrained all
downstream analyses to bi-allelic SNP positions, i.e. those with a
maximum of two alternative alleles observed across all investigated
specimens (note that less than ~0.001 of positions
contained more than two variants, similar to observations by
Ament-Velásquez et al., 2016). We further masked with ’N’ all triplets
containing more than one polymorphic site since we could not be sure
about their amino acid translation without knowing their phase. Also, to
enter the SNP database, each site had to be successfully sequenced in at
least 80% of individuals. After filtering, each SNP was categorized
into one of the three following categories. First, we identified sites
segregating within any of the three parental species (i.e. intraspecific
sexual polymorphisms). Second, we scored SNP variants that were
monomorphic within each species but differed between some pairs of
species (i.e. fixed interspecific sexual polymorphisms). Finally, we
noted the so-called ‘private asexual SNPs’, where all parental sexual
species appeared invariant, but asexual hybrids carried apomorphic
alleles. We assumed that such substitutions generally accumulated after
the origin of hybrid clonal lineages (see below).
To determine synonymity of any mutation, we translated nucleotide
sequences into amino acids (including premature stop codons) using R
package seqinr and compared numbers of synonymous and nonsynonymous
mutations among all SNP types (intraspecific and interspecific sexual,
and private asexual). Although some tools exist for direct comparisons
of selection efficiency and distributions of mutation fitness effects
between populations
(Eyre-Walker
& Keightley, 2009) and have even been applied to sexual-asexual
comparisons (e.g
Hollister
et al., 2015), some of their assumptions (segregation and recombination)
make their usage for asexuals problematic, hence we did not apply them.
Instead, we also estimated the frequency of each SNP in sexual
populations and within each clonal lineage, and tested whether patterns
differ between synonymous and nonsynonymous substitutions. For this
analysis, we assumed that the ancestral state of each intraspecific or
private asexual SNP corresponded to the variant present in the sister
species, while the other allele was assumed to be mutated.
We also tested whether radicality of acquired nonsynonymous mutations
could be higher in asexuals because lack of segregation in asexuals
should relax selection against mutations with strong effects, especially
when the clone possesses another functional copy of a given gene
(Hollister et al. 2015; Sharbrough et al. 2018). We performed two tests
of this hypothesis. We first calculated for all categories of SNPs the
proportions of polymorphic sites carrying premature stop codons, which
likely represent strong-effect mutations. Second, we scored the
”radicality” of nonsynonymous mutations with BLOSUM90 and PAM100
substitution matrices, and compared score distributions among sexual and
asexual datasets. We have chosen these matrices due to low numbers of
mutation per gene, which may leave some amino acid substitutions
unobserved.
d N/d Sratio
Thed N/d Sratio, i.e. the ratio of the number of nonsynonymous substitutions per
nonsynonymous site (d N) to the number of
synonymous substitutions per synonymous site
(d S), is a common technique used to detect
selection in pairs of protein-coding sequences. Comparisons ofd N/d S ratios between pairs
of sexual and asexual lineages is a suitable tool to address the
efficiency of selection with the prediction that asexuals should possess
higher d N/d S values due to
relaxed purifying selection (e.g.
Pellino et al., 2013). However, due
to the hybrid origin of asexuals, theird N/d S values are not
directly comparable to those from sexual species. Many of asexuals’
polymorphisms are inherited from substitutions fixed between parental
species brought together by hybridization, and we know theird N/d S are lower than those
of intraspecific polymorphisms segregating within parental species.
Thus, we also calculatedd N/d S ratios for all
possible elongatoides-taenia and elongatoides-tanaiticainterspecific pairs, which served as null distributions to be compared
with real asexual hybrids. This approach is based on the rationale thatd N/d S ratios of simulated
hybrids provide variability that would be expected in hybrids without
the effects of any process operating on asexual genomes.
For comparisons ofd N/d Sdistributions within and between species and genomotypes, we selected
ORFs with at least 50 intact codons fully resolved in all individuals,
to maintain representative sequence lengths. We calculatedd N/d S ratios for all pairs
of sequences using the Nei-Gojobori evolutionary pathway method and the
Jukes-Cantor model as implemented in the Bioperl module
Bio::Align::DNAStatistics
(http://www.bioperl.org; Stajich
et al., 2002). Since some sequence pairs differed in synonymous or
nonsynonymous mutations, but not both, which would introduce zeros into
numerators or denominators ofd N/d S ratios, we added a
small constant to each
estimate
(0.01; Pellino et al., 2013).
Estimating fitness effects in old vs. recent clones
Inspection of SNPs, albeit very instructive, may only provide an
indirect proxy for inferences about fitness decay in clones. The next
logical step, albeit rarely undertaken, is to test for age-dependent
fitness deterioration of clones. To make such a comparison, we examined
additional samples from 7 sites in the Central European hybrid zone,
where asexuals belonging to recent and ancient lineages coexist. All
specimens used for phenotypic analyses were genetically assessed by
standard markers to determine their genomotype (Janko et al. 2007).
Because diploid and triploid Cobitis hybrids differ in many
traits (Bobyrev,
Burmensky, Vasil’ev, Kriksunov, & Lebedeva, 2003; Juchno & Boron,
2010; Kotusz, 2008; Maciak et al.,
2011),
we restricted analyses to triploid females, and investigated traits
related to body condition, growth, and fecundity, predicting more
prominent fitness decay in older clones compared to recent ones (Neiman
et al., 2010). For this part of the study we captured and analyzed 48
EEN females belonging to the ancient (hybrid clade I) and 52 females
belonging to recent clonal lineages (EET and ETT genomotypes; Fig. 1,
Tab. 2). To address seasonal variation, we performed sampling before and
after the reproductive season (April and September, respectively). We
caught the fish by electrofishing, sacrificed them by overdose of
2-di-phenoxyethanol, and fixed them in 4% formaldehyde after removing a
piece of fin tissue for genotyping.
Each specimen was
measured (SL; 1 mm accuracy), weighed (Wt; 1g) and dissected for
internal organs: heart, gonad, liver, and spleen, which were weighed.
The bodies were cooked, then re-measured and re-weighed after dissection
of the vertebral column and entrails (We). Weight and length data were
used to estimate standard body-condition coefficients: relative SL/Wt
relationship (LWR) and Clark’s condition factor (CC =
We/SL3;
Ricker,
2010), followed by anatomical traits, such as the heart-somatic index
(HI), reflecting heart functional capacity, spleen-somatic index (SSI)
as a measure of immunocompetence, the hepato-somatic index (HSI) — a
measure of energy reserves (Šimková et al., 2015), and vertebrae index
(VI), which assesses skeleton ossification. Size of vertebrae was
measured along the horizontal axis using digital calipers (accurate to
0.1 mm) of the three sequential vertebrae starting at the plane of the
first dorsal pterygiophore. Age-growth dynamics were estimated according
to
Fedorčák,
Koščo, Halačka, and Manko (2017) on the basis of the total number of
annuli on the vertebral body (distances from the vertebra’s centre to
each annual ring, and to the vertebra edge were measured to the nearest
0.005 mm). Growth dynamics were interpreted as an indication of
inter-clonal competition ability for resources within the shared niche.
Fecundity, either absolute (i.e. total number of pre-matured oocytes in
a female) or relative (i.e. absolute fecundity per weight) was estimated
as the number of exogenous vitellogenic oocytes in
the
gonad according to Halačka, Lusková, and Lusk (2000), and Juchno and
Boron (2010). Differences in size-structure of oocytes among females
were estimated according to Halačka et al. (2000) by measuring the
diameters of 250 randomly chosen oocytes. We note that these latter
measures were performed on a subset of fish of similar size and age to
avoid biases due to age differences (Tab. 2).
To further test for fecundity differences, we allowed four and three
hybrid females belonging to ancient (EEN) and recent (ETT) clones,
respectively, to mate at will with C. elongatoides males under
seminatural conditions. We kept each triploid hybrid female in an
identical tank, together with a single male, with spawning substratum
following the design of Bohlen
(1999).
After each spawning event, we counted eggs from each clutch and let them
develop under standardized conditions until they hatched and reached the
third fry stage, when we evaluated their survival rate.
Statistical Analyses of Data
We used R packages nlme (Pinheiro
et al., 2016) and lme4 (Bates, Maechler, Bolker, & Walker, 2015) to
analyze differences among fish types in clutch sizes, survival rates,
SNP data, site frequency spectra, amino acid radicality, andd N/d S ratios. To address
specifics of each dataset, we used several linear models including
Linear Mixed Effects models (LME), Generalized Linear Models (GLM), and
Generalized Linear Mixed Effects models (GLME). We obtained p-values for
the effect in question by Likelihood ratio tests (LRT) against the null
model. Details about these statistical methods are provided in
Supplementary online materials.
Results
Molecular data
On average, we mapped 17,684,155 reads per sample (all samtools bitwise
flags except 0x4) onto a reference Cobitis transcriptome
(Janko
et al., 2018). Within each of the 20,601 contigs, we identified open
reading frames (ORF) altogether providing us with 3,945 nuclear ORFs
(3,179,571 bp total length) with annotation and sufficient read coverage
to identify SNP variants. Mapping
the reads onto a published C. elongatoides mitogenome
(NC_023947.1) sufficiently covered on average 82.1% of the reference,
with most reads falling into coding regions (10,902 sites).
Test of Relaxed Selection on mtDNA
Smart model selection (SMS), used by the PhyML server, selected GTR + G
as the best model of sequence evolution (Table S1); the resulting tree
is depicted in Fig. 2. Comparison of the two models in RELAX by
likelihood ratio test (LRT) did not indicate a preference for the
alternative model over the null model (∆AICc = 0.3; K = 2.98, p = 0.129,
LR = 2.30), which assumed no differences between sexual and asexual
branches, and suggested that 86.72% of sites are explained byd N/d S values of 0.02,
7.08% by values of 0.44, and the remaining 6.2% by values of 1.56.
Nuclear DNA
SNP categories, relative ages of clones, and reliability of SNP calling
Across all nuclear contigs, we discovered 161,139 bi-allelic SNPs, of
which 149,248 were variable within the ingroup. Focusing specifically on
annotated ORFs, we categorized 28,878 sites as intraspecific
polymorphisms segregating within any of the sexual species. These SNPs
were characterized by the prevalence of nonsynonymous substitutions over
synonymous ones. We further scored 10,532 fixed interspecific SNP
variants, which, in contrast to the intraspecific SNPs, showed the
opposite trend, with prevailing synonymous substitutions (Fig. 3A). This
difference between SNP types was highly significant (GLME with binomial
family of error distribution, LRT against null model, p -value =
7.874699e-05).
Moreover, the site-frequency spectra within each species showed that
mutations inducing synonymous changes were significantly more frequent
than mutations changing the amino-acid (LME, LRT p = 1.61937e-107; Fig.
S1).
We further noted 8,840 of the so-called ‘private asexual SNPs’. The
hypothesis that such sites most likely accumulated after the hybrid
origin of clonal lineages is corroborated by the fact that their
proportion in hybrids’ genomes was tightly correlated with the mtDNA
distance of any clone from its nearest sexual haplotype (Pearson’sr = 0.94, p -value = 7.135e-14). Specifically, the ‘hybrid
clade I’, i.e. the oldest asexual lineage, possessed the most divergent
mtDNA haplotypes, and the highest proportion of private SNPs
(~1.5 % of all SNPs). Hybrids from Central Europe with
a putative Holocene origin
(Janko
et al., 2012) possessed haplotypes identical or closely related to those
segregating in current sexual populations, and simultaneously had a
moderate proportion of private SNPs (~0.2-0.4 %), while
the two experimental F1 hybrids possessed the least divergent mtDNA
haplotypes, and had a very low proportion of private SNPs, which
probably represent false-positives due to sequencing errors. The rarity
of private mutations in F1s also corroborates the quality of performed
SNP calling, which is further strengthened by the fact that our SNP
calling recovered 100% matches to sequences of several mitochondrial
and nuclear genes previously published
by
Choleva
et al. (2014).
Patterns of heterozygosity
Studied specimens systematically differed in their level of
heterozygosity. C. elongatoides possessed the highest average
heterozygosity of all parental species, and C. taenia the lowest
(Fig. 3B), consistent with its small effective population size
(Janko et al.,
2018).
Asexual individuals had significantly higher genome-wide heterozygosity
than sexuals (Fig. 3B; t-test p = 8.963e-14) with 98.5 – 99.8% of
‘private asexual SNPs’ occurring in heterozygous states. The vast
majority of fixed interspecific SNPs were in heterozygous states in
hybrids’, consistent with expectations. However, every hybrid possessed
a small proportion of sites with only one parental allele; i.e. sites
with so-called loss of heterozygosity (LOH). The proportion of LOH sites
in each individual was rather low, and correlated significantly with
putative ages of clones, i.e. either with K2P mtDNA distances from the
nearest sexual individual (Pearson’s r = 0.974, 95% c.i. = 0.940-0.988,
p-value = 3.016e-16) or with proportion of private asexual mutations
(Pearson’s r = 0.955, 95% c.i. = 0.902-0.980, p-value = 3.286e-14).
Hence, the proportion of LOH sites was near zero in experimental F1
hybrids, and highest (~10%) inelongatoides-tanaitica (Fig. 4).
Mutation accumulation and radicality in clones and polyploids
Private asexual SNPs had a significantly higher probability of being
nonsynonymous than SNPs fixed among parental species (GLME with binomial
family of error distribution, LRT p = 0.0008). Both intraspecific sexual
polymorphisms and private asexual SNPs had similar proportions of
nonsynonymous SNPs (binomial GLME, LRT p = 0.369; Fig. 3A).
Nevertheless, we noticed that old clones had a significantly lower
proportion of nonsynonymous SNPs than young clones (binomial GLME, LRT p
= 0.00055) or than sexual species when measured on intraspecific sexual
polymorphisms (binomial GLME, LRT p = 0.12). Altogether, these data
indicate no tendency toward a higher nonsynonymous mutation rate in
clones, and even suggests the possibility of lower rates in the old
clonal lineage.
To evaluate the impact of polyploidization on mutation accumulation, we
compared diploid and triploid asexuals within both major types of
hybrids (i.e. ET vs EET and ETT in elongatoides-taenia and EN vs. EEN in
elongatoides-tanaitica hybrid types). We noticed that triploids
possessed significantly higher proportions of private asexual SNPs
compared to diploids (Fig. 3A; ET vs EET & ETT: t -test p =
0.006; EN vs EEN: t -test p = 0.001). We also noticed that
variance in proportions of private asexual SNPs was generally higher
among triploids than among diploids, but these differences were not
significant due to small sample sizes (ET vs EET & ETT: F test p =
0.06; EN vs EEN: F test p = 0.4). To compare rates of nonsynonymous
mutation accumulation between di- and triploids we applied the binomial
GLME separately to elongatoides-taenia and elongatoides-tanaitica
hybrids, treating individuals within groups as random factors. Private
asexual SNPs in diploid ET hybrids accumulated a significantly higher
proportion of nonsynonymous mutations compared to EET or ETT triploids
(binomial GLME LRT p = 0.047) but the differences between EN diploids
and EEN triploids were not significant (binomial GLME LRT p = 0.42).
When testing the accumulation of large-effect mutations, we found as
expected, that interspecific SNPs are significantly less likely to carry
premature stop codon mutations than both intraspecific and private
asexual SNPs (contingency table, p < 1e-15). Asexuals, when
considered as one group, had significantly higher incidence of premature
stop codons than intraspecific sexual datasets (contingency table, p =
0.016). In our analyses of old and recent clones, we found that the
incidence of stop codons did not differ between intraspecific sexual
SNPs and private asexual SNPs in old clones (contingency table, p =
0.99), but their incidence was significantly elevated in young clones
(contingency table, p = 2.4e-6).
We also scored the ”radicality” of nonsynonymous mutations using the
PAM100 substitution matrix, and compared score distributions among
sexual and asexual datasets (Fig. 5A). Again, fixed interspecific
nonsynonymous substitutions were significantly less radical than
intraspecific segregating mutations (LME, LRT p = 0.0009) or private
asexual SNPs (LME, LRT p = 0.004). However, the radicality of mutations
segregating within sexual species (intraspecific polymorphisms) did not
differ from that of nonsynonymous private asexual SNPs (LME, LRT p =
0.339). We observed no significant differences between old and young
clones (LME, LRT p = 0.084). Analogous results were obtained with the
BLOSUM90 matrix (data not shown).
No evidence for relaxed selection fromd N/d S ratios and mutation
frequency spectra
A more formal test of selection
was based on the per-gene distributions ofd N/d Sratios in all genomotypes (Fig. 5B). A subset of 1,993 ORFs passed a
threshold of 50 or more codons fully resolved in all investigated
individuals. To address the hybrid origin of asexuals’ variability, we
compared the d N/d S ratio
of asexuals with that estimated from elongatoides-taenia orelongatoides-tanaitica sequence pairs, as well as estimates from
laboratory F1 hybrid offspring. The usefulness of this approach was
corroborated by the fact that elongatoides-taenia pairwised N/d S values did not
differ from those in F1-generation ET hybrids (LME, LRT p = 0.43).
Most genes in all comparisons had values below 1, consistent with the
influence of negative selection. Within-species pairwise comparisons
yielded significantly higher values than interspecific comparisons, and
values observed within asexual genomotypes (LME, LRT p <
1e-8). However, thed N/d Sratios calculated from interspecific pairs of sequences were
significantly higher than values from old clones (LME, LRT p = 0.01),
young clones (LME, LRT p = 0.02) or asexuals in general (LME, LRT p =
0.004), which was at odds with predictions based on mutation
accumulation hypotheses. Moreover, we found no differences between old
and young clones (LME, LRT p = 0.35).
Looking at the distribution of private asexual SNPs we found that
nonsynonymous substitutions often appeared as singletons, while
synonymous substitutions were significantly more likely to be shared by
two or more members of the same clone, (Fig. 5C; binomial GLME, LRT p =
5.7e-6). Since the ancient clonal group (hybrid clade I) had the highest
sample size, we also looked at its mutation frequency spectra and
noticed that nonsynonymous substitutions are again significantly less
frequent than synonymous ones (Poisson GLM, LRT p = 1.1e-6).
No evidence for fitness deterioration
in old clones
We found a significant correlation between fish length and weight
(ANCOVA, F1, 56 = 115.13, p = 3.346e-15) and also a
significant effect of season, with spring samples having higher SSI
(t95 = 8.34, p << 0.000), HSI
(t97 = 3.75, p = 0.000), and GSI (t97 =
4.740, p << 0.000) values, and gonads with higher
counts of generally smaller oocytes compared to autumn samples (Fig.
6A). Some effect of location was also present as fishes from the
Złotnica River site had smaller relative SL/Wt ratios
(F1, 56 = 33.56, p = 3.291e-07) and slower growth rates
(F1, 251 = 103.3101, p < 2.2e-16) than those
from the remaining sites.
Taking the aforementioned effects into account, we found no differences
between old and young clones in condition-related measures: LWR (ANCOVA,
F1, 56 = 0.0018, p = 0.9667), CC (t98 =
-0.838, p = 0.404; Fig. 6B), HI (t90 = 0.265, p =
0.791), HSI (t98 = 1.08, p = 0.282), VI
(t58 = 1.713, p = 0.92), or in growth rates
(F1, 251 = 2.11, p = 0.147), except for a significantly
higher SSI in old clones (t95 = -2.05, p = 0.043).
Looking at traits related to fecundity, the old clonal lineage possessed
significantly higher GSI values than the younger clonal lineages (in
spring: t17 = 2.617, p = 0.018, in fall:
t78 = 5.968, p < 0.000). Egg counts followed
this pattern, showing statistical significance in Spring samples:
absolute fecundity: t17 = 3.069, p = 0.007; relative
fecundity: t17 = 2.723, p = 0.014 (Fig. 6C,D). We also
noted differences between lineages in egg size structure. The old clonal
lineage possessed larger counts of smaller eggs than the younger clones
in the Spring period, while the opposite trend was observed in the
autumn (permutation Kolmogorov-Smirnov test, p <<
0.0001 in both seasons; Fig. 6A).
A reproductive experiment corroborated the aforementioned differences,
since females of the oldest clone had significantly larger clutches
compared to those from the younger clones (average of 394 and 176 eggs
per clutch, respectively; GLME with female ID as a random factor, Z =
4.364 p = 1.28e-05). We noted generally low survival rates (on average
~16% of eggs per clutch hatched and survived till third
fry stage), consistent with previous experiments
(Janko,
Bohlen, et al., 2007; Juchno et al., 2014; Bohlen pers.comm.), but we
found no significant differences between old and recent clones in
survival (zero-inflated GLME, Z = -0.001, p = 0.999).
Discussion
Various mechanisms have been proposed to explain why sexual reproduction
evolved and became a dominant force in nature but no clear answer has
been obtained. We tested whether the mutational deterioration of clones,
one of the most cited candidate processes, may serve as an efficient
short-term mechanism explaining the stable coexistence of sexual species
with clonal lineages, some reaching ages of up to 300 ky.
No evidence for accumulation of deleterious mutations and fitness decay
in asexuals:
Negative selection prevailed among >3000 studied nuclear
and mitochondrial loci, as evident from higher proportions of
nonsynonymous and more radical mutations, from higherd N/d S values observed
within the parental species rather than between the parental species, as
well as from generally lower intrapopulation frequencies of
nonsynonymous mutations compared to the synonymous ones. This is in line
with the hypothesis that nonsynonymous or radical intraspecies
polymorphisms may be often mildly deleterious, and may be consequently
removed by negative selection before reaching fixation.
We further found that clonal genomes accumulated novel mutations in a
time-dependent manner, so that the older clonal lineages had higher
incidence of private asexual SNPs compared to the younger clones or the
experimental F1 hybrids, consistent with mutation accumulation models.
Nevertheless, the way in which new mutations accumulated contrasts with
expectations of a reduced efficacy of selection in asexuals. Namely,
despite hundreds of thousands of years of clonality in some lineages,
any potential relaxation of selection was too weak to provide detectable
traces in the asexuals’ mitochondria, and there was no evidence for
increased proportions of nonsynonymous or radical mutations among
private asexual SNPs in the ancient clone. We also comparedd N/d S ratios of real
asexuals to values of hybrids simulated from the sequences of the sexual
individuals. This approach, which accounts for the effect of fixed
interspecific polymorphisms, showed lower-than-expectedd N/d S values in natural
clones, with no difference between the old and the recent clones, again
contrasting with predictions of mutation accumulation hypotheses.
The fitness-related data are also at odds with the prediction of clonal
decay since the ancient ‘hybrid clade I’ outperformed the young clones
in some important traits, such as GSI and fecundity. Given that
proportions of asexuals in mixed sexual-asexual populations often
correlate with their competitive success (Hellriegel & Reyer, 2000;
Vrijenhoek & Parker, 2009), the evolutionary success of the oldestCobitis clonal lineage is further indicated by its occupation of
the largest distribution range of all Cobitis clones (Janko,
Culling, et al., 2005) and its
ability to achieve higher
densities than co-occurring younger clones at many sites (Janko et al.,
2012). We have to note that old and recent clones used in our study have
different genomic compositions (elongatoides-tanaitica versuselongatoides-taenia genome combinations, respectively),
suggesting their dissimilarity may also reflect inherited differences
between parental species rather than asexuality-specific processes.
Nevertheless, the absence of a detectable effect on fitness continues to
indicate that even the oldest Cobitis clones successfully coexist
and compete for resources with recent clones and sexual species.
How do asexuals escape mutational deterioration?
Altogether, the acquired data provide no evidence for reduced efficacy
of the selection in asexuals, suggesting that mutation accumulation may
not notably hamper the demographic and the competitive performances of
the Cobitis clones even after 300 ky of asexuality. What are the
reasons behind these observations?
First, the observed patterns were unlikely to be methodological
artifacts, since our study covered most of the Cobitisdistribution range and provided a relatively high number of SNPs that
were validated in the experimental F1 hybrids and previously published
sequences, and estimated values of the phenotypic traits did not deviate
from previous reports of the Cobitis genomotypes, including the
polyploids (Halačka et
al.,
2000; Juchno & Boron, 2010). The inherent biological nature of the
studied organisms may have affected the data since comparisons between
asexuals and their sexual counterparts (or simulated hybrids) conflate
the variability frozen by hybridization in the distant past with that
segregating within the contemporary sexual populations. Additionally,
comparisons between the old and the recent clones potentially suffer
owing to the presence of different species in their ancestries. Despite
these pitfalls, we clearly documented that mutations did accumulate in
time, as predicted by the clonal decay model, but ~300
ky of asexuality did not leave detectable traces of the deleterious
mutation accumulation or decreased clonal fitness. This study adds to
the growing list of publications that have failed to detect traces of
clonal decay (e.g. Pellino et al., 2013; Warren et al., 2018).
What does the growing amount of negative evidence tell us about the
validity of mutation-accumulation theories for evolution of sex and
clonality? Commonly, negative results have been attributed to special
mechanisms enabling investigated asexuals to avoid mutation
accumulation, such as deviations from a strict clonality including
minimal sex, ameiotic recombination, gene conversions, beneficial
effects of polyploidization, or an increased efficiency of DNA
repair (e.g.
Maciver, 2016; Roach & Heitman, 2014; Schön & Martens, 2003; Warren et
al., 2018). However, given the available data, it is unlikely that any
such mechanism may have efficiently slowed down the clicks of the
ratchet in the Cobitis .
Refuting cryptic sexuality in predominantly clonal organisms is
notoriously difficult
(e.g. Birky, 2010),
but available evidence argues against its role in the Cobitis .
Extensive crossing experiments have never documented allelic
segregation (Choleva
et al., 2012; Janko, Bohlen, et al., 2007; Janko et al.,
2018),
and Majtánová et al.
(2016)
reported remarkable stability of the hybrids’ karyotypes with no
large-scale chromosomal restructuring or recombination. The current
study also shows that the vast majority of the private hybrid SNPs occur
in heterozygous states (between 98.5%–99.8%),
which corroborates the
hypothesized clonal reproduction, with new mutations occurring on one
chromosome with little possibility of recombination between the
homologues or coalescence between the alleles. Nevertheless, the
existence of LOH at fixed interspecific positions indicates that asexual
organisms’ alleles may occasionally recombine, convert, or vanish due to
hemizygous deletions. However, three lines of evidence argue against a
major impact of LOH on the speed of ratchet clicks in theCobitis .
First, even the oldest Cobitis clones accumulated LOH events in
only ~10% of the studied genes over
~300 ky, which strikingly contrasts with other asexual
organisms, where such processes have been hypothesized to interfere with
the accumulation of deleterious mutations. For example, Tucker,
Ackerman, Eads, Xu, and Lynch (2013) and Sunnucks, England, Taylor, and
Hales (1996) showed orders of magnitude of higher rates of conversions
& deletions of individual genes, and even entire chromosomal arms.
Second, the efficiency of LOH events in erasing potentially deleterious
mutations is expected to increase with the clonal age. This is because,
rare LOH events may likely happen on different genes from new mutations
in the recent clones, while in the older clones any emerging LOH event
has a higher chance of occurring in the genes with existing mutations.
Therefore, if LOH events were to attenuate the ratchet by overwriting
deleterious mutations, we would expect an exponential correlation
between the proportions of LOH and the private asexual SNPs because LOH
would erase a disproportionately higher proportion of acquired mutations
in old clones compared to the younger ones. As evidenced in Fig. 4, our
data offer no support for such a deviation from linearity (note that we
focused on diploid clones here since the presence of three homologs in
triploids complicates the detection of LOH). Finally, only <
1.5 % of the private SNPs were in homozygous state, probably resulting
from LOH events. If we assume that LOH events are more or less
symmetrical with respect to preserving or losing the new or ancestral
allele, it suggests that only ~3% of the private
asexual mutations could have been converted. Of course, if new mutations
tend to be deleterious, the selection might have preferred one direction
of LOH but altogether, our data indicate that genome restructuring
likely has no major effect on the clicks of the ratchet.
Polyploidization is another mechanism that may refresh a clonal genome
by temporarily masking deleterious mutations, although it also frees
gene copies to mutate, ultimately increasing the per-genome mutation
rate (Otto & Whitton, 2000). Consistent with this expectation, we found
higher numbers of private asexual SNPs in triploid loaches than in
diploids but the differences were relatively small. This may suggest
that the contemporary triploids may have spent considerable parts of
their history as diploid clones before polyploidization, leaving
relatively little time for accumulation of extra mutations. Importantly,
we found no evidence for the increased nonsynonymous mutation rates in
triploids. Hence, although polyploidization likely plays a very
important role in clonal evolution, at least in the Cobitis , its
major benefits do not seem to stem from a deleterious mutation masking;
after all, the diploid clones have persisted at the Balkan together with
their triploid derivatives at least since the last interglacial period
(Janko, Culling, Rab, & Kotlik, 2005). Major benefits of triploidy may
instead relate to other effects, such as metabolic changes invoked by
the modified cell architecture, the altered dosage of parental genomes,
or the modified crosstalk between the allopolyploids’ genomes due to
different stoichiometric relations between transcription factors and
their binding sites (Bartoš et al., 2019; Beukeboom & Vrijenhoek, 1998;
Maciak et al, 2011).
The null model of clone persistence assumes delayed effects of
accumulated mutations
In summary, although we may not rule out the existence of some
mechanisms counteracting the effects of clonality, the aforementioned
candidate mechanisms do not appear strong enough to stop or considerably
slow the ratchet in the Cobitis . Instead, we propose that the
apparent absence of signs of clonal decay, as in the Cobitis and
other similar cases, may have a much simpler reason, which we propose as
a null hypothesis for any tests of clonal decay. Namely, the simplest
plausible explanation is that the relaxation of a purifying selection is
not strong enough to allow sufficient mutation accumulation that will
compromise the performance of clones over several hundreds of thousands
of generations.
Consistent with this explanation, we found similar frequency spectra of
non-/synonymous mutations in the sexual and the asexual populations,
indicating that selection efficiently restricts the spread of putatively
deleterious mutations even in the clones. The
counter-intuitive
observations of lower nonsynonymous mutation loads and the incidence of
premature stop codons in old clones may thus be explained by the
classical population genetic theory, since a selection-based removal of
the deleterious mutations requires varying amounts of time depending on
the selection coefficient, the population size, and the genetic
background. It follows that recent clones, despite having acquired lower
absolute numbers of mutations, will possess a higher fraction of
nonsynonymous private asexual mutations, or more radical SNPs.
Different mutation loads in the old and the young clones may thus
reflect a time-lag necessary to remove the slightly deleterious
mutations (S. G. Johnson & Howard 2007) rather than some special
mechanisms that alleviate the mutation accumulation. Paradoxically, the
delay in fitness decay may be caused directly by clonal reproduction
because the maintenance of heterozygosity may mask the negative effects
of acquired deleterious recessives (Halligan & Keightley, 2003) until
new mutations accumulate in high numbers (Otto, 2007) or become exposed
to selection in the homozygous states (Guex et al., 2002; Leslie &
Vrijenhoek, 1978; Leslie & Vrijenhoek, 1980). This was in line with
recent reanalysis of published sequences of asexual animals (Janko et
al., 2011), which showed that significant deviations from neutrality,
indicative of selection against aging clones, could only be detected in
asexual complexes when clones achieved substantial ages beyond 1 million
generations.
Hence, even if a clonal lineage were to accumulate deleterious mutations
since its birth, it may take considerable time before they start to
negatively affect the evolutionary life span of that clone. In younger
asexual complexes, the replacement of clonal lineages may be dominated
by a more neutral drift-like process of the clonal turnover, which does
not depend on the effects of accumulated mutations (Janko et al., 2008).
By analogy to a genetic drift, the clonal turnover predicts that a major
portion of clonal diversity may be formed by the young clonal lineages,
which did not have enough time to spread, while ancient lineages will be
less diverse but more geographically widespread. Interestingly, both
predictions have been met in the Cobitis and in some other taxa
(Janko et al., 2012; Janko, 2014; Quattro et al., 1992).
Conclusions
To date, the existence of successful clones in nature has usually been
explained by their special abilities to avoid accumulation of the
deleterious mutations. Our study shows that even if clones clearly
accumulate mutations as they age, the relaxation of selection due to
asexuality may not be sufficient to cause their fitness to deteriorate,
even after hundreds of thousands of generations of asexuality. Thus,
although mutation accumulation may ultimately drive any clone to
extinction, individual clones may be replaced by more recent clones in a
drift-like process of the clonal turnover if the influx rates of new
clones are high (Janko, 2008). Clones in such complexes may simply not
be given an opportunity to become old enough for accumulated mutations
to start to manifest their effects. Successful and widespread clones may
therefore often represent merely temporary winners in the ongoing
turnover race.
This may seem trivial, but it has potentially serious implications for
our understanding of the persistence of sex: Mutation accumulation may
be relevant to the survival of clones after they reach some substantial
age, but before they do so, the persistence of sexual species in mixed
sexual-asexual complexes must rely on different mechanisms that offer
short-term advantages to sex.
Acknowledgements
We would like to thank Jan Šipoš and Petr Pyszko for valuable advice on
linear mixed effect models, Jacek Stefaniak for preparing maps of
samples, and Jan Eisner for help with the fitting of second-order
polynomial models to our data. This research was supported by The Czech
Science Foundation project no. 13-12580S to KJ, University of Ostrava
project no. SGS29/PřF/2016 to JKoč and by the National Science Centre in
Poland project no.
DEC-2011/03/B/NZ8/02095
to JKot. Computational resources were provided by the ELIXIR-CZ project
(LM2015047), part of the international ELIXIR infrastructure.
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[dataset] Kočí J., Röslein J., Pačes J., Kotusz J., Halačka K.,
Koščo J., Fedorčák J., Iakovenko N., Janko K.; 2019; No evidence for
accumulation of deleterious mutations and fitness degradation in clonal
fish hybrids: Abandoning sex without regrets; Institutional server (IMG
CAS, link pending)
Data accessibility
Raw sequencing data will be available from a server at IMG CAS. Input
files are provided as supplementary files.
Author contributions
KJ designed the experiments. KH, JF, JKoš, NI, performed the
experiments. JKoč, JR, JP, JKot, KJ analysed data. JKoč, JKot, JR, and
KJ contributed to writing the MS.
Figures