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.