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\begin{document}

\title{The Rapid, Mass Invasion of New Zealand by North American Daphnia
pulex/pulicaria}



\author[1]{Zhiqiang Ye}%
\author[1]{Emily Williams}%
\author[1]{Chaoxian Zhao}%
\author[2]{Carolyn Burns}%
\author[1]{Michael Lynch}%
\affil[1]{Arizona State University}%
\affil[2]{University of Otago}%


\vspace{-1em}



  
  \date{May 05, 2024}


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\selectlanguage{english}
\begin{abstract}
Non-indigenous Daphnia `pulex' have been found in many lakes in New
Zealand (NZ) in the past 20 years, suggesting a recent invasion.
However, very little is known about the origin of invasive D. `pulex',
whether they are D. pulex or D. pulicaria, and whether they are
obligately asexual clones or cyclical parthenogens. Furthermore, the
source and time of arrival of the invasive genotype(s) are unclear. We
address these questions by genomic sequencing Daphnia populations from
13 lakes on the South Island and one on the North Island, NZ. Based on
\textasciitilde{}24,000 monomorphic species-specific markers, the
invasive Daphnia on the South Island were found to be D. pulicaria,
while those on the North Island are D. pulex/ pulicaria hybrids. Both
the South and North Island Daphnia are phylogenetically clustered with
North American D. pulicaria/pulex, thereby suggesting their North
American origins. We further found that the South Island Daphnia
populations are fixed heterozygotes for nearly all bi-allelic sites in
the nuclear genome and contain identical mitochondrial genomes,
suggesting the origin and proliferation from a single founder clone,
which we experimentally verified to be an obligate asexual. Estimates
from molecular data imply a colonization time for the South Island
populations of \textasciitilde{} 60 years ago, with a likely invasion
route associated with the introduction of salmonids from North America.

Key words: Daphnia pulex; Daphnia pulicaria; invasion; obligately
asexual; hybridization%
\end{abstract}%



\sloppy




\textbf{Introduction}

The \emph{Daphnia pulex} complex contains at least eight genetically
distinct species, widely found throughout the northern hemisphere
(Colbourne et al. 1998). Two of the species, \emph{D. pulex} and
\emph{D. pulicaria} , can form viable hybrids, but are not easily
distinguished morphologically, and typically are identified by allozyme
electrophoresis of the lactate dehydrogenase locus, with homozygous
\emph{D. pulicaria} having two ``fast'' alleles and homozygous \emph{D.
pulex} having two ``slow'' alleles (Hebert et al. 1989; Crease et al.
2011). Hybrids with the heterozygous slow/fast genotype are generally
found in disturbed ponds or deforested areas (Hebert et al. 1989). Due
to the frequent gene flow between \emph{D. pulex} and \emph{D.
pulicaria} (Hebert et al. 1989; Cristescu et al. 2012), some hybrids are
also homozygous for the Ldh locus, presumably resulting from
backcrossing with either \emph{D. pulex} or \emph{D. pulicaria} (Xu et
al. 2013), which further complicates the species identification.

Both \emph{D. pulex} and \emph{D. pulicaria} can reproduce by cyclical
parthenogenesis (CP), with extended periods of parthenogenesis
interspersed with sexual resting-egg production (Hebert 1978; Hebert et
al. 1993). However, some clones in each species have lost the ability to
engage in meiosis and have become obligate parthenogens (OP) (Hebert and
Crease 1980; Hebert et al. 1993; Paland et al. 2005). Interestingly, the
loss of meiosis is often limited to females, and some OP \emph{D.
pulex}can still produce functional males bearing haploid sperm. Such
males from OP \emph{D. pulex} can mate with females from CP \emph{D.
pulex}and form viable hybrids, \textasciitilde{}40\% of which have the
OP phenotype (Innes and Hebert 1988), indicating that partially dominant
meiosis-suppressing elements are carried by OP males. Further analysis
has shown that all OP \emph{D. pulex} share common haplotypes on
chromosomes VIII and IX that arose by introgression from \emph{D.
pulicaria} (Xu et al. 2015) and are transmitted through OP males without
recombination (Tucker et al. 2013). These observations suggest that all
OP \emph{D. pulex} may be descendants of a single hybridization event
between \emph{D. pulex} and \emph{D. pulicaria.} Initially, it was
thought that all F\textsubscript{1} hybrids between \emph{D. pulex}
and\emph{D. pulicaria} are obligate parthenogens (Crease and Lynch
1991), but Heier and Dudycha (2009) found that F\textsubscript{1}
hybrids between some CP \emph{D. pulex} females and CP \emph{D.
pulicaria} males have CP phenotypes and can backcross with both parental
species and themselves (Heier and Dudycha 2009). Thus, Xu et al. (2013)
argued that OP hybrids originated from a unique historical hybridization
and introgression event between female \emph{D. pulex} and male \emph{D.
pulicaria} .

Although asexual clones are generally thought to suffer from long-term
selective disadvantages resulting from reduced efficiency of purging
deleterious mutations (Lynch et al. 1993), the OP clones with hybrid
backgrounds from \emph{D. pulex} and \emph{D. pulicaria} are notably
invasive. For example, an obligately asexual \emph{D. pulex}
\selectlanguage{ngerman}×\emph{pulicaria} hybrid clone invaded Africa after the 1920s (Mergeay
et al. 2006) and has spread throughout the range of native sexual
\emph{D. pulex} and displaced the sexual population\emph{.} This asexual
hybrid clone is thought to have been initially introduced from the USA
via the introduction of largemouth bass in 1927-29 and completely
displaced all other \emph{D. pulex} genotypes in Africa within 60 years
(Mergeay et al. 2006). In addition, an OP \emph{D. pulex} invasion in
Japan, involving four hybrid clones and thought to have happened between
680 and 3400 years ago, is unlikely due to human activity (So et al.
2015).

Recently, non-indigenous \emph{Daphnia} species morphologically
determined to be from the \emph{D. pulex} complex have been found in
several lakes on the South Island of New Zealand (NZ) (Duggan et al.
2012). Although Duggan et al. (2012) considered the invasive South
Island clones to be \emph{D. pulex,} because their mitochondrial
cytochrome c oxidase subunit 1 (CO1) gene sequences formed a
monophyletic group with North American \emph{D.} \emph{pulex} , their
analysis only used a 658-bp fragment of CO1 nucleotide sequences from 7
South Island clones. Due to the frequent hybridization between \emph{D.
pulex} and \emph{D. pulicaria} (Hebert et al. 1989; Heier and Dudycha
2009; Cristescu et al. 2012), the mitochondrial genomes may actually
have either a \emph{D. pulicaria} or \emph{D. pulex} origin (Cristescu
et al. 2012; Marková et al. 2013). Although members of the South Island
populations are Ldh homozygotes (Duggan et al. 2012), it is unclear
whether this locus is homozygous for \emph{D. pulex} or \emph{D.
pulicaria} alleles, and again the interpretation may be clouded when
hybrids exist. Moreover, due to the limited sequencing information,
Duggan et al. (2012) were unable to estimate when the invasion occurred.

To obtain a better understanding of the NZ \emph{Daphniapulex/pulicaria}
invasion, we performed genome-wide analyses of\emph{Daphnia}
\emph{pulex/pulicaria} populations from 13 lakes across the South Island
and one lake on the North Island. The species designations and likely
sources of origin were determined via a survey of \emph{D. pulicaria}
-specific markers and a whole-genomic phylogenetic tree that includes
\emph{D. pulicaria} and \emph{D. pulex} clones collected across North
America, Europe, and China.

\textbf{Methods and methods}

\emph{Sampling and sequencing}

\emph{Daphnia} populations were collected from 13 lakes in the South
Island and 1 lake in the North Island, NZ in March and October 2019
(Fig. 1). In each location, \emph{Daphnia} were sampled by tows of a
conical plankton net and preserved in DNA/RNA Shield. DNA from each
pooled population sample (50-200 individuals) was extracted with Zymo
DNA Clean and Concentrator-25 Kit and sequenced on an Illumina HiSeq
2500 platform.

\emph{Reads mapping and SNP calling}

First, we trimmed adapter sequences from the sequence reads by applying
Trimmomatic (Bolger et al. 2014) to the FASTQ files. Then, we mapped the
adapter-trimmed sequence reads to the \emph{D. pulex} reference genome
(PA42 version 4.1) using BWA (Li and Durbin 2009). Coverage for each
site was calculated by counting the total mapped reads, and coverage for
each sample is calculated as the total mapped bases divided by the
mapped genomic regions. We examined the genomic distribution of the
sites coverage and set minimum and maximum sample-coverage cutoffs to
avoid analyzing problematic sites (Table 1; Fig. S1). SNPs were
identified by Samtools (Li et al. 2009) with ``samtools mpileup -uf
ref.fasta sorted.bam \textbar{} bcftools call -mv \textgreater{}
raw.vcf'' and ``bcftools filter -s LowQual -e '\%QUAL\textless{}20'
raw.vcf \textgreater{} flt.vcf''.

If each NZ population represents a single clone, all of its bi-allelic
sites should have equal numbers of reads mapped to the two parental
alleles. To check this, we searched for sites with reads for the two
parental alleles deviating from a 1:1 ratio. To minimize mapping bias,
1) we remapped the reads using bowtie with ``-q -m 1 -v 3 --best'' to
obtain unique mapped reads (Stevenson et al. 2013); 2) we removed
regions with \textgreater{}3 SNPs within 100 bp because additional
differentiating sites will interfere with read alignment (Stevenson et
al. 2013); 3) To reduce the mapping bias toward the reference alleles
(Degner et al. 2009), we generated an assembly by masking all of the
bi-allelic sites with ``N'' in the PA42 4.1 reference. Only bi-allelic
sites with reads for the two alleles deviating from a 1:1 ratio from
both the original and masked assemblies are considered true deviating
sites.

\emph{Characterizing} \emph{D. pulicaria-specific markers}

To identify \emph{D. pulicaria} -specific markers, we first searched for
homozygous SNPs shared by all 14 \emph{D. pulicaria} clones. Then, we
checked these SNPs in 42 \emph{D. pulex} clones collected across North
American, Europe, and China and eliminated those that appeared in any of
the \emph{D. pulex} clones. For the remaining SNPs, we checked the
corresponding loci in the genome in each of the \emph{D. pulex} clones
and only kept those that are homozygous for the same two bases across
all \emph{D. pulex} clones.

\emph{Phylogenetic analysis}

To generate clean mitochondrial and nuclear genomes for the phylogenetic
analysis, we removed regions that are subjected to transposition of
cytoplasmic mitochondrial DNA into the nuclear genome in the historical
past (numts). We discarded read pairs for which two reads in the pair
mapped to both mitochondrial genome and nuclear genome. First, trimmed
reads were mapped to the mitochondrial reference genome using BWA (Li
and Durbin 2009) with default parameters. We extracted paired-end reads
that aligned to the mitochondrial genome from each BAM file. GSNAP (Wu
and Watanabe 2005) was used to realign these reads to the mitochondrial
reference genome and nuclear genome separately following the pipeline
from MToolBox (Calabrese et al. 2014). Read pairs that mapped to both
the mitochondrial and nuclear genomes were removed from downstream
analysis.

We generated phylogenetic trees using nuclear and mitochondrial genomes,
separately. For the nuclear genome, a phylogenetic tree was constructed
using the consensus genomic sequences for each NZ population and\emph{D.
pulex} /\emph{D. pulicaria}clones. Consensus sequences for each
population/clone were generated using Samtools (Li et al. 2009) with
command: samtools mpileup -uf ref.fa aln.bam \textbar{} bcftools call -c
\textbar{} vcfutils.pl vcf2fq \textgreater{} cns.fq. We randomly
selected 1\% of the consensus sequences and repeated this 1000 times,
constructing maximum-likelihood trees for each subset of data using
iq-tree with GTR + I model and ultrafast bootstrap (Nguyen et al. 2014;
Hoang et al. 2017). The consensus tree was generated using the Consense
program, and branch lengths estimated by the Dnaml program in the PHYLIP
package (Felsenstein 1993). A separate phylogenetic tree based on
mitochondrial data was constructed with the maximum likelihood method
using MEGA 7 with 100 Bootstrap (Kumar et al. 2016).

\emph{Phenotypic tests}

The reproductive modes of NZ \emph{Daphnia} clones were determined using
a sexuality test. Because sexual \emph{Daphnia} need sperm fertilization
to produce diapausing embryos, the sexuality test is to check whether,
in the absence of males, diapausing embryos are deposited into ephippia
by females. The consistent presence of embryos in ephippia in the
absence of males implies an obligately asexual clone, whereas consistent
results of no ephippial embryos from at least three consecutive rounds
of tests was used to infer a male-requiring cyclical parthenogen. We
decapsulated a total of 32 ephippial embryos produced by clones from
Lake Alexandrina in the absence of males. All 30 contained embryos and
some of these hatched, consistent with the clones from this lake being
obligate asexuals.

Male production was induced by adding the hormone methyl farnesoate (MF)
to the medium. Adult females for each clone were isolated and placed
into 50-mL tubes containing methyl farnesoate at a concentration of 400
nM (changed daily). We examined the sex of offspring using a dissection
microscope. Males can be visually distinguished from females based on
the enlarged antennules and flattened ventral carapace margin.
Successful production of males by \selectlanguage{english}[?]5 individuals was taken to be
evidence for a male-producing clone.

\textbf{Results}

\emph{Species identification of the Daphnia from New Zealand lakes}

We sampled \emph{Daphnia} populations from 13 lakes on the South Island
and one on the North Island, New Zealand (NZ) (Fig. 1; Table
S1).\emph{Daphnia} populations from each lake were determined to be
\emph{D. pulex} /\emph{D. pulicaria} \emph{sensu lato} based on
morphological features (Benzie 2005; Ebert 2005), although genetic
information is required to distinguish the two species. To obtain
preliminary genetic information in a simple manner, we pooled all
individuals (50-200) from each lake for whole-genomic sequencing. Each
population was sequenced to an average depth of sequencing coverage of
86x (Table 1), involving more than 100 million 150-bp reads per
population. On average, 1.5 million bi-allelic sites were identified for
each population (Table 1).

To determine the species of \emph{Daphnia} populations in NZ, we first
relied on the lactate dehydrogenase locus (Ldh), a commonly invoked
indicator for distinguishing \emph{D. pulex} and \emph{D.
pulicaria}(Hebert et al. 1989; Crease et al. 2011). For this locus,
non-hybrid\emph{D. pulicaria} have two ``fast'' allozyme alleles and
non-hybrid\emph{D. pulex} have two ``slow'' allozyme alleles. The
mobility difference is caused by the substitution of glutamine in
\emph{D. pulex}to glutamic acid in \emph{D. pulicaria}at position 229 of
the amino-acid sequence (Crease et al. 2011). We found that all the
South Island \emph{Daphnia} populations are monomorphic for glutamic
acid at position 229, suggesting that the South Island populations are
\emph{D. pulicaria} . However, the North Island population is
heterozygous for position 229, suggesting that it has a hybrid origin.

In addition to the Ldh locus, we identified 24,185 informative sites by
comparing 14 \emph{D. pulicaria} and 42 \emph{D. pulex} genomes (Table
S2)\emph{,} in which all \emph{D. pulicaria} are monomorphic for one
nucleotide and all \emph{D. pulex} are monomorphic for another
nucleotide (File S2). We found that all the South Island
\emph{Daphnia}populations have \textgreater{}23,390 (97\%) sites
homozygous for\emph{D. pulicaria-} specific nucleotides, while only 807
(3.3\%) sites are homozygous in the North Island population. Our result
is consistent with a \emph{D. pulicaria} origin of the South Island
\emph{Daphnia}populations, and a hybrid origin of the North Island
population.

To further confirm the species of \emph{Daphnia} in NZ, we constructed a
consensus maximum-likelihood (ML) tree using whole-genomic coding
sequences from 14 NZ populations, 14 \emph{D. pulicaria,} and 42\emph{D.
pulex} clones collected across North America, Czech Republic, and China
(Table S2). Within this tree, the South Island populations formed a
monophyletic clade with all North American \emph{D. pulicaria}clones,
further supporting the view that the South Island populations are
\emph{D. pulicaria} (Fig. 2). The North Island population is in a clade
with North American \emph{D. pulicaria} and \emph{D. pulex} , but
clusters with neither North American \emph{D. pulicaria} nor \emph{D.
pulex} (Fig. 2), confirming its hybrid origin. Unlike the situation for
nuclear data, for the ML tree based on full-length mitochondrial
genomes, \emph{D. pulex} and \emph{D. pulicaria} clones interlaced
outside of the NZ populations (Fig. S1). This discordant phylogeny
between the mitochondrial and nuclear genomes may represent historical
hybridization between the two species (Markova et al. 2013).

\emph{Testing the single-clone origin hypothesis for the South Island
Daphnia populations}

Because both North American \emph{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 \emph{Daphnia} populations are also
derived from just a few clones. If each NZ \emph{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 \emph{D. pulex} population from North
America has \textgreater{}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 \textgreater{}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
\emph{de novo} mutation. To understand if the South and North Island
\emph{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 \emph{Daphnia} produce
haploid diapausing eggs that require fertilization by sperm, while
obligately asexual \emph{Daphnia} are capable of producing diploid
diapausing eggs in the absence of males. We found that in the absence of
males, the South Island \emph{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 \emph{Daphnia} from Lake Alexandrina and found consistent male
production. Our results suggest that the \emph{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 \emph{Daphnia} . Instead, we used informative markers to predict
their traits. Chromosomes VIII and IX in \emph{Daphnia
pulex/pulicaria}contain \textgreater{}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 \emph{D. pulex} or \emph{D.
pulicaria} . The North Island \emph{Daphnia} carry the heterozygous
markers at \textgreater{}87\% of these reference sites, suggesting
obligate asexuality. We also examined the male-producing ability in the
North Island \emph{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 \emph{Daphnia} invasion
of North Island also involved an obligately parthenogenetic clone,
capable of producing males.

\emph{Origin and colonization time of the NZ populations}

To infer the geographic origin of the \emph{D. pulicaria} that invaded
South Island, genetic data are required from throughout the natural
range of this species. Although whole nuclear and mitochondrial genomes
are available for samples from North America and New Zealand, the only
data available for \emph{D. pulicaria} from other continents are
mitochondrial CO1 gene sequences. For the maximum-likelihood tree using
mitochondrial CO1 gene sequences collected from \emph{D.
pulicaria}across North America, South America, Europe, Asia, New
Zealand, and the north polar region, the South Island \emph{Daphnia}
clustered with a subset of North American clones and a few clones from
South America (Fig. 3). Because South American \emph{D. pulicaria} are
thought to have been introduced from North America (Crease et al. 2012),
the phylogeny suggests that the South Island \emph{Daphnia} have a North
American origin.

To estimate when the South Island genotype diverged from North
American\emph{D. pulicaria} , we calculated the nuclear genome-wide
divergence between synonymous sites for these two groups. The divergence
time between the South Island genotype and North American \emph{D.
pulicaria}(T\textsubscript{1,} Fig. 2) can be estimated by the average
pairwise distance of synonymous sites (\emph{d} \textsubscript{s})
between North American \emph{D. pulicaria} and the South Island, NZ
haplotypes (\emph{d} \textsubscript{s} = 0.00023). Letting
T\textsubscript{1} =\emph{d} \textsubscript{s} /2*\selectlanguage{greek}μ, \selectlanguage{english}with \selectlanguage{greek}μ \selectlanguage{english}= 5.85 \selectlanguage{ngerman}×
10\selectlanguage{english}\textsuperscript{-9}being the mutation rate per site per generation
(obtained from a mutation-accumulation experiment; Keith et al. 2016),
and assuming \textasciitilde{}5 generations per year, we estimate that
the South Island genotype diverged from other North American \emph{D.
pulicaria \textasciitilde{}} 3400 years ago. Because the North
Island\emph{Daphnia} have a hybrid origin, and we do not have genomic
sequences available for North American hybrids, the divergence time
between the North Island genotype and North American hybrids of \emph{D.
pulex} x \emph{D. pulicaria} cannot be estimated by this means.

We also estimated the expansion time of the South Island populations
(T\textsubscript{2,} Fig. 2). Assuming these populations descended from
a single primary colonization event, then the nucleotide diversity among
South Island haplotypes would have been acquired after the colonization,
and the expansion time can be estimated from the average synonymous
nucleotide divergence between the most distant population (Lake
Coleridge) and the remaining populations. We found that
the\emph{Daphnia} population from Lake Coleridge has an average of 24
synonymous substitutions (over a total of 7.05 million synonymous sites)
compared to the remaining populations, implying a expansion time of
T\textsubscript{2} = \emph{d} \textsubscript{s} /(2*\selectlanguage{greek}μ\selectlanguage{english}) = 3.4 \selectlanguage{ngerman}×
10\selectlanguage{english}\textsuperscript{-6} / (2 x 5.85 x 10\selectlanguage{english}\textsuperscript{-9} / site /
generation x 5 generations / year) = \textasciitilde{}58 years. This
calculation is supported by the presence of characteristic ephippia
of\emph{D. pulex/pulicaria} in a sediment-dated core taken in Lake Hayes
that suggested appearance of this species in the lake in the late 1950s
or early 1960s (Samiulah Khan, pers. comm). Moreover, we found that the
North Island \emph{Daphnia} has an identical ND5 nucleotide sequence and
only a single nucleotide difference on the COI gene relative to one of
the clones that invaded Japan between 680 and 3400 years ago (So et al.
2015), suggesting that these two have the same origin.

\textbf{Discussion}

\emph{Invasive Daphnia pulex/pulicaria in NZ}

In this study, we identified two independent invasions of \emph{Daphnia
pulex/pulicaria} on the South and North Island of NZ. Phylogenetic data
from mitochondrial genomes revealed that both invasive \emph{Daphnia
pulex/pulicaria} originated from North America. We examined\emph{Daphnia
pulex/pulicaria} populations from 13 lakes on the South Island, only to
find an average of 24 synonymous substitutions (over a total of 7.05
million synonymous sites) among populations and all the populations
share a single mitochondrial haplotype, suggesting that all South Island
\emph{Daphnia pulex/pulicaria} are decendants of a single clone. In
addition, all of the South Island \emph{Daphnia pulex/pulicaria} are
homozygous at the Ldh locus and have \textgreater{}97\% sites homozygous
for \emph{D. pulicaria} -specific nucleotides, indicating they are
non-hybrid \emph{D. pulicaria} . On the contrary, the North Island
\emph{Daphnia pulex/pulicaria} are heterozygous at the Ldh locus and
have \textgreater{}96.6\% sites heterozygous for \emph{D. pulicaria} -
and \emph{D. pulex} -specific nucleotides, thereby suggesting a hybrid
origin of the North Island population.

Both the South and North Island invasions involved obligately
asexual\emph{Daphnia} clones. The origin of asexuality for the North
Island\emph{Daphnia pulex/pulicaria} is likely caused by hybridization
between\emph{D. pulex} and \emph{D. pulicria} prior to the arrival of NZ
(Innes and Hebert 1988), while that for the South Island \emph{D.
pulicaria} is still not clear. Obligately asexual \emph{D. pulex} x
\emph{pulicaria}hybrids are notoriously invasive, invading many African
lakes within just a few decades (Mergeay et al. 2006), and invading
Japan between 680 and 3400 years ago (So et al. 2015). In Africa, the
invading \emph{D. pulex} x \emph{pulicaria} hybrids spread and replaced
the native sexual\emph{D. pulex} (Mergeay et al. 2006). This might be
due to obligate asexuals avoiding the cost of producing males, although
obligately asexual \emph{Daphnia} are often capable of producing males
(Lynch et al. 2008), and there are additional ecological matters to
consider. For example, the subset of obligate asexuals that are
successful are often generalists capable of outcompeting sexuals when
environmental dispersal is limited (Baker 1965; Parker 1977; Lynch
1984).

Although the NZ lakes in our study have historically been inhabited by
native NZ species of \emph{Daphnia} (Burns et al. 2017), we could not
find any native \emph{Daphnia} species in our samples, which is likely
due to them being completely displaced by just two invading clones from
North America, neither of which shows significant evidence of adaptive
divergence based on genome-wide sequence analyses. The invasive
asexual\emph{Daphnia} clones could be bearers of a serious pathogen for
the native \emph{Daphnia} , because complete displacement requires a
fortuitous ecological advantage over presumably well-adapted,
genetically variable, long-term residents of these lakes.

\emph{Invasive history of NZ Daphnia populations}

To infer the source of invasive \emph{Daphnia pulex/pulicaria} in NZ, we
checked the possible routes of \emph{Daphnia pulex/pulicaria} invasion.
It is thought that \emph{D. thomsoni} (formerly \emph{D. carinata,}Burns
et al. 2017) and \emph{D. tewaipounamu} are the only
native\emph{Daphnia} species in NZ, although there are several
invasive\emph{Daphnia} . One recent invader, \emph{D. galeata,} now
common in lakes throughout the North Island of NZ, may have been
introduced to the South Island during translocation of farmed Chinese
grass carp (\emph{Ctenopharyngodon idella} ) for the purpose of weed
control in northern South Island waterways (Duggan and Pullan 2017). The
presence of \emph{D. pulex} in some shallow constructed ponds in
suburban Auckland, North Island, has also been attributed to
introductions of grass carp (Branford and Duggan 2017). However, Chinese
grass carp have only been used for the weed control on the North Island
and the northern tip of the South Island, as water temperatures in
southern NZ are generally too cold for these fish. Therefore, it is
highly unlikely that grass carp were a major vector for the introduction
of \emph{D. pulicaria} to the South Island.

Duggan et al. (2012) suggested introduced salmonids (trout, salmon) and
associated recreational fishing equipment as potential vectors
for\emph{D. pulicaria} on the South Island. The most common salmonid in
NZ, Chinook salmon (\emph{Oncorhynchus tshawytschca} ), was initially
introduced from the Sacramento River, California, USA to South Island,
NZ, between 1901 and 1907. Salmon fishing in NZ has become popular since
the 1970s and has attracted many international tourists. The \emph{D.
pulicaria} clone on the South Island has been known to produce
diapausing ephippia, which float on the water surface for long periods,
adhering to any surfaces they encounter, such as fishing equipment,
skin, fur, feathers, and clothing (Burns 2013). The movement of these
ephippia attached to recreational equipment, people, and wildlife may
facilitate the spread of \emph{D. pulicaria} in lakes in the South
Island, NZ. In support of this, the invasion time estimated from the
molecular data is roughly consistent with transport during this period.
As noted above, the North Island \emph{Daphnia} has only one nucleotide
difference on the COI gene compared to the JPN 1 clone that invaded
Japan (So et al. 2015), which suggests the possibility of a recent
transfer from Japan.

\emph{Distinguishing North American D. pulex and D. pulicaria}

North American \emph{D. pulex} and \emph{D. pulicaria} are thought to
have recently diverged from a common ancestor and adapted to distinct
environments while still experiencing introgression (Omilian and Lynch
2009; Cristescu et al. 2012). Such introgression may cause the \emph{D.
pulex} /\emph{pulicaria} hybrids to have mtDNA from either \emph{D.
pulex} or \emph{D. pulicaria} , thus biasing inferences based on trees
when derived from mtDNA data (Markova et al. 2013). Although the
nuclear-encoded lactate dehydrogenase locus (Ldh) has been used to
discriminate \emph{D. pulex} and \emph{D. pulicaria} , and was useful in
determining the species of NZ \emph{Daphnia} samples, inferring species
solely based on the Ldh locus can still be misinformative due to
hybridization and backcrossing (Xu et al. 2013). For example, an Ldh
SS\emph{Daphnia} that derived from crossing between SF \emph{D.
pulex/pulicaria} and SS \emph{D. pulex} may be erroneously inferred as a
non-hybrid clone. To eliminate the limitations of a single nuclear
marker to derive inferences, we generated a set of 24,185 \emph{D.
pulicaria} -specific nuclear markers that are monomorphic in every
non-hybrid \emph{D. pulicaria,} and unique with respect to North
American \emph{D. pulex} . These markers will be useful for diagnostic
purposes in other future studies involving these two species, including
the identification of hybrids. They may also serve as a resource for
studying genes involved in potential adaptive divergence between\emph{D.
pulex} and \emph{D. pulicaria.}

\textbf{Acknowledgments}

We thank Wei-chin Ho for helpful discussions. This work is supported by
NIH grant R35-GM122566-01 to Michael Lynch, financial support from Alan
Wilson @otago and the University of Otago to Carolyn Burns.

\textbf{Author Contributions}

Z.Y., C.W.B., and M.L. designed research; Z.Y., E.W., C.W.B., and M.L.
performed research; Z.Y., C.Z., and M.L. analyzed data; and Z.Y.,
C.W.B., and M.L. wrote the paper.

\textbf{Disclosure Declaration}

The authors confirm that there are no known conflicts of interest
associated with this publication.

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\emph{Data accession}

NZ population data for this study were deposited to NCBI under
BioProject ID PRJNA573527.

\emph{D. pulex/D. pulicaria} genomic data can be accessed at BioProject
ID PRJNA573529 and accession numbers SAMN03964756-SAMN03964769 (Xu et
al. 2015), and the \emph{D. obtusa} genomic sequences used in this study
can be accessed at NCBI under accession number SAMN12816670. The\emph{D.
pulex} genome assembly PA42 v4.1 is available at GenBank under accession
GCA\_900092285.2 (Ye et al. unpublished) and the corresponding
annotation file is provided in File S1.

\textbf{Table 1} . Summary of sample information. Coverage denotes the
mean depth of genomic coverage and chosen coverage range (in square
brackets) of sites analyzed in each population. Mapped sites: sites
covered by at least one read. Deviating sites are bi-allelic sites
(minor allele frequency \textgreater{}0.05) with read numbers for the
two parental alleles deviating significantly from 1:1 after
Benjamini-Hochberg procedure (FDR=0.05). Sites with more than two
alleles are not used in our analysis.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Samples & Mapped sites & Mapped sites & Coverage (X) & Bi-allelic sites
& Deviating sites & & &\tabularnewline
\midrule
\endhead
Alexandrina & 168,224,903 & 168,224,903 & 98 {[}40-200{]} & 1,540,667 &
288 & & &\tabularnewline
Coleridge & 166,329,006 & 166,329,006 & 84 {[}20-150{]} & 1,491,775 &
252 & & &\tabularnewline
Hawea & 165,334,107 & 165,334,107 & 51 {[}20-120{]} & 1,416,048 & 35 & &
&\tabularnewline
Hayes & 168,826,070 & 168,826,070 & 139 {[}50-250{]} & 1,538,123 & 667 &
& &\tabularnewline
Johnson & 168,204,869 & 168,204,869 & 125 {[}50-250{]} & 1,523,441 & 572
& & &\tabularnewline
Ohau & 168,392,915 & 168,392,915 & 102 {[}50-200{]} & 1,539,681 & 340 &
& &\tabularnewline
Pukaki & 168,109,981 & 168,109,981 & 94 {[}50-200{]} & 1,536,485 & 238 &
& &\tabularnewline
Sullivan's Dam & 167,880,110 & 167,880,110 & 88 {[}50-200{]} & 1,525,683
& 224 & & &\tabularnewline
Moke & 164,531,082 & 164,531,082 & 43 {[}20-100{]} & 1,509,765 & 41 & &
&\tabularnewline
Tekapo & 166,918,220 & 166,918,220 & 78 {[}30-150{]} & 1,494,053 & 175 &
& &\tabularnewline
Von & 166,794,759 & 166,794,759 & 82 {[}30-150{]} & 1,488,926 & 196 & &
&\tabularnewline
Wakatipu & 167,805,336 & 167,805,336 & 107 {[}50-200{]} & 1,513,257 &
411 & & &\tabularnewline
Wanaka & 164,530,961 & 164,530,961 & 42 {[}20-100{]} & 1,370,378 & 14 &
& &\tabularnewline
Kapoai & 175,278,062 & 175,278,062 & 48{[}20-100{]} & 2,059,905 & 147 &
& &\tabularnewline
& & & & & & & &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure 1.} Location of lakes on the South Island, New Zealand,
from which the \emph{Daphnia} in this study were collected (South
Island, New Zealand. \emph{Google Maps} , 16 October 2019). Lake names
are highlighted in blue. Lake Kapoai, North Island, not shown.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2.} Maximum likelihood tree using nuclear genome
sequences. South Island, NZ \emph{Daphnia} samples (red circles); North
Island, NZ \emph{Daphnia} sample (red ring); \emph{D. pulex} samples
(black circles); \emph{D. pulicaria} samples are marked with blue
circles. \emph{Inset} on the right shows the phylogeny of the South
Island and North American \emph{D. pulicaria} in more detail. Bootstrap
\textgreater{}75 are shown. T\textsubscript{1} is the divergent time
between the North American and the South Island \emph{D.
pulicaria.}T\textsubscript{2} is the expansion time of South Island
\emph{D. pulicaria} .\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 3.} Maximum-likelihood tree using partial nucleotide
sequences from the COI gene. Clones from different continents are color
coded: South Island, New Zealand (red), Asia (dark green), South America
(blue), North America (black), Europe (pink), and polar region (light
green).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

