Testing the single-clone origin hypothesis for the South Island
Daphnia populations
Because both North American Daphnia invasions in Africa and Japan
involved only one or just a few clones (Mergeay et al. 2006; So et al.
2015), we wanted to test if the NZ Daphnia populations are also
derived from just a few clones. If each NZ Daphnia population
represents
a single clone, assuming no mutation or gene conversion, all of its
bi-allelic sites should have similar numbers of reads mapped to the two
parental alleles as expected for a fixed heterozygote. To test this, we
performed binomial tests on all bi-allelic sites to check for deviations
from a 1:1 ratio. Over all 14 samples, with an average of 1.5 million
significant bi-allelic sites per sample, only 14 to 667 sites in each
population have unequal numbers of reads mapped to the two parental
alleles after correcting for multiple comparisons (Table 1), while a
typical sexually reproducing D. pulex population from North
America has >98% of heterozygous sites with minor-allele
frequencies significantly deviating from 0.5, and a strong skew of the
site-frequency spectrum towards low frequency alleles (Lynch et al.
2017). In principle, the tiny fraction of sites deviating from a 1:1
ratio might have arisen from an inherent bias toward more effective
mapping of one allele (Degner et al. 2009), although this seems very
unlikely, as it would have to occur in population-specific samples. A
more likely explanation is the low level of mutation and gene conversion
operating in all populations, including those that are obligately
asexual.
In further support of the hypothesis that all 13 South Island
populations originated from the same clone, we found that all of them
have identical
mitochondrial
genomes. Moreover, 6 of the 7 clones from four different South Island
lakes collected >7 years ago (Duggan et al. 2012) share
identical sequence for the CO1 gene as our South Island samples. The
only exception is a clone collected from Lake Wanaka by Duggan et al.
(2012) with one unique SNP, which is likely a de novo mutation.
To understand if the South and North Island Daphnia have the same
maternal origin, we compared their 15-kb mtDNA genome sequences, and
found 54 nucleotide differences, thereby ruling out origination from a
single clone.
To check if the South Island populations are obligate parthenogens, we
experimentally examined their ability to produce diapausing embryos
without fertilization. Cyclically parthenogenetic Daphnia produce
haploid diapausing eggs that require fertilization by sperm, while
obligately asexual Daphnia are capable of producing diploid
diapausing eggs in the absence of males. We found that in the absence of
males, the South Island Daphnia (from Lake Alexandrina) can
generate ephippia containing embryos, indicating that the life cycle can
be completed without sex. We also tested the male-producing ability for
the Daphnia from Lake Alexandrina and found consistent male
production. Our results suggest that the Daphnia invasion of
South Island involved a single obligately asexual clone capable of (but
not requiring) male production.
Due to the lack of live animals, we were not able to experimentally
examine the reproductive mode and male-producing ability in the North
Island Daphnia . Instead, we used informative markers to predict
their traits. Chromosomes VIII and IX in Daphnia pulex/pulicariacontain >30,000 markers that are informative with respect
to the origin of asexuality (Xu et al. 2015, Tucker et al. 2013). All of
these markers are heterozygous in obligate asexual hybrids but are
homozygous in CP D. pulex or D. pulicaria . The North
Island Daphnia carry the heterozygous markers at
>87% of these reference sites, suggesting obligate
asexuality. We also examined the male-producing ability in the North
Island Daphnia by reference to markers identified by Ye et al.
(2019), finding a complete absence of markers associated with the loss
of male-producing ability, thereby suggesting a capacity for male
production. Thus, our results indicate that the Daphnia invasion
of North Island also involved an obligately parthenogenetic clone,
capable of producing males.