# Rare sexual reproduction by females of “obligate parthenogenetic” lineages of Daphnia cf. pulex

Cécile Molinier, Cassandra Clément, Héloïse Calzan, Thomas Lenormand, Christoph R. Haag

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Cécile Molinier, Cassandra Clément, Héloïse Calzan, Thomas Lenormand, Christoph R. Haag. Rare sexual reproduction by females of “obligate parthenogenetic” lineages of Daphnia cf. pulex. Journal of Evolutionary Biology, 2025, 38 (7), pp.930-938. 10.1093/jeb/voaf044. hal-05357615

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# Rare sexual reproduction by females of "obligate parthenogenetic" lineages of *Daphnia* cf. *pulex*

Cécile Molinier<fnref n="1" />,<fnref n="2" />,*, ID, Cassandra Clément<fnref n="1" />,<fnref n="3" />,*, Héloïse Calzan<fnref n="1" />, Thomas Lenormand<fnref n="1" />, ID,
Christoph R. Haag<fnref n="1" />, ID

## Abstract

Rare sexual reproduction in otherwise asexual lineages is predicted to strongly alleviate the long-term costs of asexuality while avoiding most of the cost of sex. Here, we test for rare sexual reproduction in females of obligate parthenogenetic (OP) lineages of *Daphnia* cf. *pulex*. We find that a small proportion of ~0.5% of offspring contain paternal alleles when they are produced in the presence of males from a so-called cyclical parthenogenetic lineage of the species, thus demonstrating sexual reproduction. Rare sexual reproduction occurred in two independent tests, using females from two different OP lineages, suggesting that it may be a regular feature in OP lineages of the species. Even though it could not be established whether the sexual events involved fertilization of haploid or diploid eggs, these results disclose a reproductive trait entirely new to *Daphnia* biology, aligning with recent results in other organisms. Indeed, the occurrence of rare or cryptic events of sexual reproduction may be a common feature in lineages previously characterized as obligately parthenogenetic, with significant implications for the evolution of obligate asexuality, its long-term costs, and its genomic consequences, including the evolution of polyploidy.

**Keywords:** *Daphnia*, asexual reproduction, cryptic sex, parthenogenesis, contagious asexuality

## Introduction

Asexual reproduction is predicted to have long-term costs compared to sexual reproduction, including lower efficacy of selection leading to an increased genetic load and a decreased rate of adaptation (Charlesworth et al., 1993; Hartfield, 2016; Hollister et al., 2015; Kondrashov, 1993; Muller, 1964; Normark & Moran, 2000). These costs, may explain the short evolutionary lifetime of asexual lineages (Keightley & Eyre-Walker, 2000; Lodé, 2013; Schurko & Logsdon, 2008; Simon et al., 2002), are expected to be most pronounced in obligate asexuals (or "obligate parthenogens" if asexuality occurs via unreduced female gametes), that is, species that exclusively reproduce asexually. Indeed, already a small amount of sexual reproduction may strongly reduce the long-term costs of asexuality, while maintaining most of its advantages (De Visser & Elena, 2007; Engelstäder, 2017; Hojsgaard & Hörandl, 2015; Neiman & Schwander, 2011; Peck & Waxman, 2000).

Obligate parthenogens are rare but they do occur in several branches of the tree of life (Bell, 1982). Yet, recent findings in several species considered to be obligate parthenogens, have revealed evidence for rare sexual reproduction, including in insects such as ants (Kuhn et al., 2021), stick insects (Freitas et al., 2023; Larose et al., 2023) and psyllids (Nokkala et al., 2015), as well as in brine shrimp (Boyer et al., 2021) and bdelloid rotifers (Laine et al., 2022; Vakhrusheva et al., 2020). The question of whether obligate parthenogens are

actually facultative parthenogens with a low frequency of sexual reproduction is important for understanding the long-term existence and evolution of these lineages (Bengtsson, 2009; Burke & Bonduriansky, 2017; D'Souza & Michiels, 2010; Green & Noakes, 1995). This has also significant implications for explaining the maintenance of sexual characters, including the formation of rare males and rare gametogenesis via standard meiosis (Molinier et al. (n. d); Defendini et al., 2023; Jalinsky et al., 2020; van der Kooi & Schwander, 2014).

Rare production of male offspring occurs indeed in many putative obligate parthenogens, including in earthworms (Jaenike & Selander, 1979), water fleas (Innes & Hebert, 1988; Innes & Singleton, 2000), rotifers (Stelzer et al., 2010), parasitoid wasps (Sandrock & Vorburger, 2011), brine shrimp (Maccari et al., 2013), and aphids (Jaquiéry et al., 2014). Yet their existence alone does not prove rare sexual reproduction. Indeed, males may be non-functional (Schön et al., 2009; van der Kooi & Schwander, 2014) either because they are infertile or because the females do not engage in sexual reproduction. In the latter case, males may still be able to fertilize females of related sexual lineages, potentially leading to new asexual lineages, a phenomenon called "contagious asexuality," which has been reported from earthworms (Jaenike & Selander, 1979), water fleas (Innes & Hebert, 1988), rotifers (Stelzer & Lehtonen, 2016), parasitoid wasps (Sandrock &

<footnote n="1">¹CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France</footnote>

<footnote n="2">²Department Algal Development and Evolution, Max Planck Institute for Biology, Tübingen, Germany</footnote>

<footnote n="3">³Department of Biology, University of the Balearic Islands, Palma, Spain</footnote>

<footnote>*These authors have contributed equally to this work.</footnote>

<footnote>Handling editor: Richard Meisel, Guest editor: Karine Van Doninck</footnote>

<footnote>Corresponding author: Cécile Molinier, Max Planck Institute for Biology, Max-Planck-Ring 5 72076 Tübingen, Germany, Email: cecilemolinier94@gmail.com</footnote>
Vorburger, 2011), brine shrimp (Maccari et al., 2013), and aphids (Jaquiéry et al., 2014).

Here, we investigate whether rare sexual reproduction occurs in females of obligate parthenogenetic (OP) lineages of *Daphnia* cf. *pulex* (“D. pulex” hereafter, see Materials and Methods). The species has both OP and cyclical parthenogenetic (CP) lineages. Both CP and OP lineages produce live-born offspring asexually, but diapause offspring are produced sexually in CP lineages and asexually in OP lineages (Decaestecker et al., 2009; Innes, 1997). The OP lineages of this species are one of the most emblematic and best documented cases of contagious asexuality (Innes & Hebert, 1988; Lynch et al., 2008; Simon et al., 2003): They produce males at a low frequency due to residual environmental sex determination (Hebert et al., 1989), and, while some males may be sterile due to diploid or aneuploid sperm, others are fully functional, undergoing spermatogenesis via standard meiosis, and able to fertilize CP females if CP and OP females co-occur (Molinier et al., 2023; Xu et al., 2015). These reproduction events lead, at least sometimes, to novel asexual lineages, presumably due to asexuality genes being transmitted by the males of the OP lineages (Paland et al., 2005; Xu et al., 2013, 2015). Genomic evidence indeed shows that major asexuality-determining regions are heterozygous for an asexuality haplotype and a second haplotype “captured” from various CP backgrounds (Tucker et al., 2013).

So far, females of these OP lineages have been thought to be strictly asexual, for the production of live-born offspring, as well as for the production of diapause offspring. However, female offspring of experimental crosses between OP males and CP females have recently been found to produce diapause offspring both sexually and asexually, that is, they exhibit a mixed sexual and asexual reproduction mode during the production of diapause offspring (Molinier et al. n. d). This finding was surprising, but could be a specific feature of neo-asexuals produced by contagion in the laboratory. Alternatively, it might be possible that mixed sexual and asexual reproduction also occurs in OP females from nature but has not been uncovered so far because of the rarity of their sexual reproduction. In this paper, we test this possibility by investigating whether natural OP females can also produce diapause offspring sexually. To do so, we set up crossing tests designed to maximize the chance to observe events of sexual production of diapause offspring. As potential mothers, we used diploid OP lineages known to be unable to produce males (Ye et al., 2019; Molinier et al. n. d). Because sexually functional males from OP lineages are difficult to obtain in large numbers (Xu et al., 2015), we used males from CP lineages as potential fathers. We subsequently developed genetic markers specific to the CP lineages and tested amplification of these markers in diapause offspring (embryos obtained by dissection of diapause capsules). If OP females occasionally reproduce sexually, we would expect that these rare sexual events would have important implications for the genetics and long-term evolution of OP lineages and might necessitate revisiting previous results, which were based on the assumption that OP lineages never undergo sex.

## Materials and methods

We carried out two replicate crossing tests, using males of two natural CP lineages (TEX1 and KAP65) and females of two natural OP lineages (DIS47 and DIS85) of *D. pulex*. A lineage

is defined here as the descendants of a single female obtained from a natural population and maintained by parthenogenetic (clonal) reproduction of live-born offspring in the laboratory. All lineages used in the experiments are genetically distinct and originate from North America (Supplementary Table S1). They all belong to the “Panarctic” clade (sometimes called “American *D. pulex*” or “*Daphnia* cf. *pulex* sensu Hebert, 1995”), which is distinct from the European *D. pulex* s.s. (Colbourne et al., 1998; Dufresne, 2011; Hebert 1995; Mergeay et al., 2006; Taylor & Hebert, 1993). While females from many OP lineages are able to produce male offspring asexually due to residual environmental sex determination (Hebert et al., 1989), the two lineages used here are unable to do so due to an allele that inhibits male production (Molinier et al. n. d; Ye et al., 2019). This means that these lineages would be able to participate in sexual reproduction only as females (Galimov et al., 2011; Innes & Dunbrack, 1993; Tessier & Cáceres, 2004). The advantage of using OP lineages unable to produce males (rather than OP lineages able to produce males) was that potential rare sexual reproduction within the OP lineage, which would have been impossible to detect with the genetic markers used (see below), could be excluded. All stock lineages were maintained in the laboratory by parthenogenetic (clonal) reproduction under standard conditions, in glass beakers filled with culture medium (Klüttgten et al., 1994), 18 °C, 16:8 light–dark cycle, fed daily with freeze-dried microalgae *Tetraselmis chuii*. Maintenance routines designed to minimize the risk of contamination of stock cultures include splash-proof covers, complete replacement with individuals from a replicate culture at the slightest doubt about a splash while handling or any similar unforeseen event, and precautions during feeding (no contact between food dispenser and culture medium, as well as splash avoidance). Moreover, stock cultures regularly go through bottlenecks of 10–20 females, which reduces the risk of low-frequency contamination, and lineage identities are verified prior to experiments using microsatellites (Molinier et al. n. d).

The crossing tests were carried out in large volumes and at high densities to allow the production of many diapause offspring. Each of the two crossing tests (hereafter referred to as crossing test 1: DIS47 x TEX1 and crossing test 2: DIS85 x KAP65, Table 1) was initiated by transferring six adult females of the OP lineage from the stock culture to a new 400 ml glass beaker. Once these two new cultures had grown in number through the production of live-born offspring, we transferred each of them to an aquarium filled with 1 L of culture medium, gradually increasing the volume to 10 L as the population sizes increased. Males from the CP lineages were added on a weekly basis, once the cultures had reached a high density (judged by the presence of many adult females with empty brood pouches), which is conducive to the production of diapause offspring (Gerber et al., 2018; Kleiven et al., 1992).

Males from each CP lineage were obtained by transferring CP females from the stock culture into a separate beaker with a culture medium containing the juvenile hormone analogue methylfarnesoate at a concentration of 400 nM, which is known to induce male production (Toyota et al., 2015; Ye et al., 2019). The sex of the live-born offspring of these hormone-exposed females was morphologically examined under a stereo microscope, with males being identified based on the elongated first antennae protruding from the rostrum
<table><caption>**Table 1.** Crossing tests with female and male lineages used in each crossing test as well as the number of manually added males, the estimated number of females present at the end of the experiment, the estimated number of diapause capsules produced during the experiment, their fill rate (0.5* embryos/capsule), the number of embryos used for paternity tests, and the number of pools of 10 embryos showing cyclical parthenogenetic-specific bands as indication that at least one embryo of the pool was produced sexually.</caption><thead><tr><th>Crossing test</th><th>Female × male lineage</th><th>Nb of males added</th><th>Nb of females (± s.e.)</th><th>Diapause capsules (± s.e.)</th><th>Fill rate</th><th>Nb of embryos tested</th><th>Nb of pools with CP-specific bands</th></tr></thead><tbody><tr><td>Crossing test 1</td><td>DIS-47xTEX-1</td><td>1268</td><td>714.26 ± 222</td><td>933.33 ± 120</td><td>0.31</td><td>140</td><td>2</td></tr><tr><td>Crossing test 2</td><td>DIS-85xKAP-65</td><td>636</td><td>3 555.79 ± 762</td><td>4 283.33 ± 93</td><td>0.59</td><td>450</td><td>1</td></tr></tbody></table>

(Wuerz et al., 2017), a diagnostic morphological character that is already visible in neonates. Male offspring were separated from female offspring and grown in hormone-free culture medium for several days. Their sex was morphologically re-examined a second time just before adding them to the test cultures. The number of males added weekly to the cultures varied between 54 and 226, according to the number of males obtained. In total, 1,268 and 636 males were added to the crossing tests 1 and 2, respectively, with the difference being explained by a lower male production in the CP lineage used for crossing test 2. Numbers of introduced males were relatively high in an attempt to avoid male limitation (Galimov et al., 2021), which may occur if the sex ratio is lower than about one male per 50 adult females. Given that the estimated number of adult females ranged from approximately 1,000 to 5,000 (based on the estimates at the end of the experiment), and considering that males typically survive for several weeks under our laboratory conditions, it is unlikely that male limitation was severe. Experimental procedures to limit the risk of contamination with CP females during the transfer of males, included the use of a fresh, high-temperature washed glass pipettes, as well as double-phenotyping of all males added to the cultures.

The two cultures were maintained for 13 (crossing test 1) and 9 (crossing test 2) weeks, with the difference being explained by the visibly lower number of diapausing capsules in crossing test 1 after 9 weeks. At the end of this period, the number of adult females in each aquarium was estimated by first reducing the total volume to 3 L for crossing test 1, or 1.88 L for crossing test 2. After thorough mixing, three independent samples of 22 to 29 ml each were taken (with replacement), and all adult females in these samples were counted (Supplementary Table S2). Subsequently, all diapause capsules that had accumulated at the bottom of each aquarium were collected. To estimate the total number of diapause capsules present at the end of the experiment, the capsules from each culture were resuspended in 50 ml of culture medium. After thorough mixing, three independent samples of 1 ml were taken with a pipette controller, and the number of diapause capsules in the samples were counted (Supplementary Table S3).

Per culture, 235 randomly sampled diapause capsules were opened using dissection needles, and the containing embryos were carefully removed (Supplementary Table S4). The dissection procedure was designed to minimize the risk of co-transferring paternal tissue, which may be present in trace amounts in the medium, along with the sampled offspring. Each diapause capsule was isolated individually from the initial sample, taking care to avoid transferring any debris such as tissue remains of recently deceased individuals or exuviae.

All remaining medium was then removed, and each diapause capsule was dissected in a drop of fresh water under a stereomicroscope. The isolated embryos were aspirated individually using a small amount of polymerase chain reaction (PCR)-grade water and placed directly into sampling tubes containing the PCR mix. Note that the 200 females used for the contamination control (see below) showed no evidence of cross-contamination with paternal tissue, despite being isolated using less stringent sampling procedures. The number of embryos per diapause capsule varies between zero and two, and we quantified the fill rate (i.e., the number of embryos per capsule divided by two; Barnard-Kubow et al., 2022). Additional diapause capsules were opened to obtain the 450 embryos for the genotyping tests in crossing test 2 (see below). Diapause embryos produced by the stock cultures of the different lineages were used to assemble the positive controls (see below). To prevent cross-contamination, embryos for the positive controls were isolated at a different time than those from the experimental cultures.

To develop genetic markers specific to each of the CP lineages, primers were designed based on insertion/deletion (“INDEL”) polymorphisms, which were identified using BBMap (https://jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/bbmap-guide). First, the genomic reads of each lineage (NCBI; accession number SRP155055, SRP176377) were mapped against the *D. pulex* reference genome version PA42_4.1 (NCBI; accession number GCA_900092285.2). Lineage-specific indel variants were identified by comparing the resulting VCF files between OP and CP lineages. In total, 22 loci with medium-sized (677–107,922 bp) INDEL variants were selected, five of them were common to both crossing tests, five specific to crossing test 1, and 12 specific to crossing test 2. All selected loci were (according to the VCF) homozygous within the lineages. Furthermore, we only selected loci at which the allele of the CP lineage was longer than the allele of the OP lineage (i.e., insertions specific to CP lineages or deletion specific to OP lineages). Hence it was possible to design PCR primers specific to the CP lineages (i.e., on the allele present in the CP lineages and absent in the OP lineages). A total of 20 pairs of primers were designed (both primers on the INDEL) using Primer3web v.4.0.0 (www.primer3plus.com), with the following characteristics: Primer size = 18-23, Tm = 58–63 °C, Max Tm Difference = 1 °C, Product Size = 150–900, GC% = 30–70, GC Clamp = 2. Initial PCR amplifications (30 cycles) were carried out for all pairs of primers using the Qiagen Multiplex PCR kit, with 1 µl of template DNA, 30 cycles (30 s of denaturation at 94 °C, 30 s of annealing at 58 °C, and 60 s of extension at 72 °C), and otherwise following the manufacturer’s recommendations. Initial amplification attempts were carried out using
genomic DNA extracted from adult individuals of each lineage using the HotShot DNA extraction protocol (Supplementary Material A). PCR products were assessed on a 1% agarose gel. Loci that consistently produced a clear band in the CP lineage while showing no band in the OP lineage used in the same crossing test were selected. Subsequently, these father-specific markers were evaluated on genomic DNA extracted (according to the protocol in Supplementary Material B) from pools of 10 diapause embryos from diapause capsules collected from the stock cultures (all 10 from CP or OP stock cultures for presence or absence of bands, respectively). Finally, the specificity of the amplifications was confirmed by ensuring consistent presence of bands, even when only one out of 10 embryos in a pool was obtained from the stock culture of the CP lineage, with the remaining embryos being obtained from the stock culture of the OP lineage. In the end, two loci were selected for the analyses of the diapause embryos produced by the crossing tests, one specific locus for each of the two crossing tests (Supplementary Table S5).

In order to test a large number of diapause offspring, pooled groups of ten offspring were used for each test. When two embryos from the same diapause capsule were used (Supplementary Table S4), both of them were placed in the same group. A total of 59 pools of 10 embryos were tested, 14 from crossing test 1 and 45 pools from crossing test 2 (Table 1). From each pool, genomic DNA was extracted using the Proteinase K DNA extraction protocol for diapause embryos (Supplementary Material B). Amplification of each locus separately (not multiplexed) was carried out using the Qiagen Multiplex PCR kit, with 6 µl of extract solution (see protocol) as a template and the same amplification conditions as described above for the initial PCR tests. The PCR products were assessed for the presence of bands on 1.5% agarose gels. All obtained bands were re-confirmed with a second independent PCR on the same DNA pool. All PCR runs also contained positive controls, that is, manually assembled pools containing one embryo obtained from the stock culture of the CP lineage and nine embryos obtained from the stock culture of the OP lineage of the corresponding crossing test.

To investigate the possibility of contamination of the crossing tests by CP females (e.g., due to accidental introduction of females during the introduction of CP males), 200 females from crossing test 1 (an estimated 28% of all females present) were genotyped at the end of the experiment. Only females that did not carry diapause capsules were selected (because diapause capsules may contain outcrossed diapause embryos). Pools of 10 females were used for DNA extraction (HotShot DNA extraction protocol, Supplementary Material A). PCR amplification and test for the presence of CP-specific bands were carried out using 1 µl of extracted DNA as a template and otherwise as described above for the pools of embryos.

## Results

Both crossing tests produced dense cultures with an estimated number of adult females of 714 (± 222 s.e.) and 3556 (± 762 s.e.) present at the end of the experiment in crossing tests 1 and 2, respectively (Table 1, Supplementary Table S2). Crossing test 1 produced an estimated number of 933 (± 120 s.e.) diapause capsule with a fill rate of 0.31 (i.e., 31 % of all available places, two per capsule, occupied by an embryo, N = 235 capsules), while crossing test 2 produced an estimated 4283 (± 93 s.e.) diapause capsules with a fill rate

of 0.54 (N = 235 capsules; Table 1, Supplementary Tables S3 and S4).

The 590 embryos, collected from a total of 343 diapause capsules (Supplementary Table S4) were grouped into 59 pools of 10 embryos (14 and 45 pools from crossing tests 1 and 2, respectively, Table 1) and tested for amplification of CP-specific alleles. Three pools tested positive for the CP-specific allele, two from crossing test 1 and one from crossing test 2 (Table 1), indicating that each of these three pools contained at least one sexually produced embryo. Two independent PCR reactions confirmed the presence of the CP-specific band in all three cases. The presence of the CP-specific bands was also confirmed in all positive controls (one embryo produced by a CP stock culture pooled with nine embryos from an OP stock culture, N = 7 control pools).

Given the observation of 2/14 and 1/45 positive pools (i.e., proportions of positive pools a of 0.143 and 0.022, Figure 1) in the two crossing tests, respectively, the frequency of sexual events can be estimated under certain assumptions. First, assuming that each sexual event occurred independently, the proportion of sexually produced offspring, s, can be estimated from the proportion of positive pools a. More precisely, because of the slight possibility that positive pools contained more than one sexually produced offspring, s can be calculated from the proportion of negative pools $1-a = (1-s)^{10}$, that is, the expected proportion of negative pools is equal to the probability that none of 10 randomly selected embryos was produced sexually. This gives $s = 0.015$ and $s = 0.0023$ for the two crossing tests, respectively (average between the two cultures: $s = 0.0088$).

Within cultures, sexual events may not have been entirely independent. Most importantly, two embryos from the same diapause capsule may show a correlated probability to undergo a sexual event. As these two embryos were in all cases put into the same pool of ten embryos, the proportion of positive pools should not be affected. We can then obtain an alternative estimate of s by assuming diapause capsules as independent. From $1-a = (1-s)^N$, with N = 5.79 and 5.82 diapause capsules (crossing test 1 and 2, respectively, Supplementary Table S4), we obtain estimates that are higher by a factor of 1.71 (average between the two cultures: $s = 0.015$).

The difference in s between the two crossing tests may be affected by the OP lineage used but also by any other difference between the two cultures (e.g., different CP lineages, different numbers of males added). However, even judging by Fisher's exact test, which assumes independence of data points, the difference is non-significant (tested on the numbers of positive vs. negative pools, which was our unit of observation, P = 0.014). Non-independence of data points leads to overdispersion compared to binomial errors, which means that, with any source of non-independence, there is even less evidence for a difference in proportions of sexual events between the two crossing tests.

The contamination tests on the culture for crossing test 1 all came back negative. All 200 tested females belonged to the OP lineage. Yet, when compared to the frequency of sexually produced embryos in this culture, that is, two out of 140 tested offspring, the difference was not statistically significant (Fisher's exact test, P = 0.169). Again, independence of data points may not be strictly met but the conclusion of non-significance is robust against non-independence.

As a second test for contamination via accidentally transferred females of the CP lineage, we estimated the probability
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Proportions of pools with cyclical parthenogenetic-specific (CP) bands as indication that at least one embryo of the pool of 10 embryos was produced sexually (grey) for each crossing test and for the combination of both crossing tests, as well as the proportion of rare sexual event calculated from both crossing tests combined (black).</figcaption></figure>

of mis-identification of males from the fact that not a single female was detected during the second check of 1,268 and 636 males from crossing tests 1 and 2, respectively. Thus, in the total of 1,904 individuals re-examined, no potential errors from the initial morphological check were detected. The upper 95% binomial confidence limit (Agresti & Coull, 1998) of zero out of 1,904 gives a maximum rate of incorrectly identifying a female as a male of *e* = 0.0017 (a single *e* is used as there is no a priori reason why it should differ between the two cultures). For a CP female to be accidentally transferred to a culture, this error would have to occur twice in the same individual. For the results to be potentially explained by such accidental transfers, at least one female would have had to be transferred in both cultures. The joint probability of this to occur, under binomial assumptions and an *e* of 0.0017 is 6.7*10⁻⁶. The detailed calculations are given in Supplementary Material C. Note that this is for the maximum *e* compatible with the data. The true value of *e* may well be lower.

## Discussion

The findings of this study suggest that rare sexual reproduction (about 1 in 200 diapause offspring in our experiment) occurs in some so-called “OP” lineages of *D. pulex* in the presence of males. Several lines of evidence suggest that the

alternative explanation of contamination is unlikely. First, the experimental procedures including double-phenotyping of males were specifically chosen to minimize the risk of contamination. Second, the error rate of male phenotyping was low, leading to exceedingly low probability of contamination by transfer of females that were mis-identified as males. Third, rare sexual events were detected in both independent crossing tests, meaning that both cultures would have had to be contaminated. This contrasts with previous experiments with similar setups, which did not reveal evidence of contamination (see Supplementary Material D for details). Fourth, genotyping of a large number of females in the culture with the highest number of detected sexual events did not yield any evidence of contamination. Each culture was initiated with just six females. Any contaminations due to contaminated stock cultures would thus have had substantial initial frequencies (at least one out of six). Fifth, the CP-specific alleles detected in the positive pools differed between the two cultures, as males of two distinct CP lineages were used. Thus, contamination of the initial cultures could only explain the results if each culture had been contaminated by a CP female carrying exactly the CP-specific allele as the male donor lineage used for that culture. While we cannot entirely rule out errors such as contamination, all lines of evidence point to rare sexual production of diapause offspring occurring in both OP lineages, when exposed to males from CP lineages as
most likely explanation of the results. While these results are not generalizable to all OP lineages, the fact that we observed rare sex in two out of two tested lineages suggests that it is not a chance event restricted to a single lineage but may be more widespread among OP lineages.

The detection of CP-specific bands indicates that CP fathers have contributed genetic material to the investigated embryos, and thus demonstrate sexual reproduction in the strict sense of fertilization of eggs produced by OP females. What remains unknown is the ploidy of the offspring and thus the question of whether the fertilized eggs had been produced by standard ("canonical") meiosis or, as for parthenogenetic reproduction, by a modified meiosis that maintains ploidy, in which case fertilization by haploid sperm produced by a CP male likely would have led to a triploid offspring. Triploid lineages of *Daphnia* are known from nature (Beaton & Hebert, 1988; Dane et al., 2020; Lynch et al., 2008; Pecl et al., 2024; Vergilino et al., 2009), so the possibility certainly exists. If the sexually produced embryos in our experiment were indeed triploid, our observations could provide a model for how triploid lineages arise. The suggestion would be that the generation of triploid offspring may occur regularly in populations where CP and OP lineages coexist. Furthermore, it would support the hypothesis that triploidy is a secondary consequence (Vergilino et al., 2009) rather than a direct cause of asexuality (Otto & Whitton, 2000). Other, more remote possibilities include leakage of only a part of the paternal chromosomes into the zygote (leading to aneuploid offspring) or even a chimeric embryo with paternally-derived cell lineages. Such events (especially paternal leakage) are known to occur in some asexual animals and sometimes even lead to viable offspring thus contributing to diversity among asexual lineages and to gene flow from sexual into asexual backgrounds (Hedtke et al., 2008; Lamatsch & Stöck, 2009; Neiman & Sharbrough, 2023).

The alternative explanation is that OP females rarely undergo oogenesis through standard meiosis, resulting in a haploid egg that can be fertilized and give rise to diploid offspring. That some OP lineages retain fully functional meiotic pathways is demonstrated by the fact that standard meiosis resulting in viable haploid sperm occurs in asexually produced males of these lineages (Tucker et al. 2013; Xu et al. 2015; Molinier et al. n. d). Moreover, mixed sexual and asexual production of diapause offspring also occurs in females of laboratory-generated CP-OP hybrids (i.e., hybrids between females from CP lineages and rare males from OP lineages; Molinier et al. n. d). As natural OP lineages are themselves the result of natural hybridization between OP and CP lineages (contagious asexuality, Innes & Hebert, 1988; Lynch et al., 2008; Xu et al., 2013), it is possible that their capacity for rare sexual production of diapause offspring is a remnant of more frequent mixed reproduction due to their hybrid origin (~65% of diapause offspring produced sexually in laboratory-generated hybrid F1 lineages; evidence based on 30 F1 lineages from a single CP x OP cross, Molinier et al. n. d). Obviously, the proportions of sexually produced offspring may differ among natural OP lineages, as well as among F1 lineages from different crosses. In particular, given that we studied only two natural OP lineages, it is possible that other OP lineages are truly obligate parthenogens with zero sexual events in the presence of males. Moreover, whether meiosis occurs in females of laboratory generated CP-OP hybrids is still unknown, but given the high frequency at which they

reproduce sexually (Molinier et al. n. d), it would be surprising if they reproduced sexually only via unreduced eggs, systematically producing triploid offspring. Rare sexual reproduction via fertilization of haploid eggs in OP females would reinforce the idea that obligate (or nearly obligate) parthenogenesis in *Daphnia* has evolved via a mixed sexual--asexual intermediate (Hebert, 1981). Evolution of obligate parthenogenesis via facultative intermediates has also been suggested for other obligate parthenogens (*Drosophila*, Kramer & Templeton, 2001; aphids, Simon et al., 2002; stick insects, Larose et al., 2023), though empirical evidence is still scarce (Simon et al., 2003; van der Kooi & Schwander, 2014). Facultative asexual intermediates may be absent in other groups, perhaps particularly when asexuality evolves through hybridization (Janko et al., 2018; Stöck et al., 2021).

Many OP lineages regularly produce males, at least some of which can produce viable offspring sexually by mating with CP females (Innes & Hebert, 1988; Lynch et al., 2008; Xu et al., 2015). Our results indicate that OP females are capable of sexual reproduction as well. It is therefore possible that cryptic sexual events occur in natural populations of OP lineages, even in the absence of coexistence with CP lineages. Whether such events lead to viable offspring is unknown. In our study, the offspring (resulting from crossing with CP males) were sampled at the diapause stage, indicating that they were viable and developed to an embryonal stage consisting of ~3,500 cells (Chen et al., 2018). However, given their low numbers and difficulties in hatching CP x OP hybrid diapause embryos in the laboratory (Molinier et al. n. d; Innes & Hebert, 1988; Xu et al., 2015), studying their hatchability, further survival, and fecundity was not feasible. The evolutionary significance of rare sexual reproduction in OP lineages will obviously depend on the fitness of these offspring. Yet the possibility that cryptic sexual events may occur within OP lineages (even if rare) offers an alternative explanation for genomic features of OP lineages, including observations of homozygosity and recombination (Omilian et al., 2006; Tucker et al., 2013; Xu et al., 2011).

Cryptic sexual events have recently been documented also in other taxa formerly believed to be obligate parthenogens such as ants (Kuhn et al., 2021), stick insects (Freitas et al., 2023) and psyllids (Nokkala et al., 2015), brine shrimp (Boyer et al., 2021), and bdelloid rotifers (Laine et al., 2022; Vakhrusheva et al., 2020). While the notion of obligate parthenogenesis currently still holds for other taxa oribatid mites (Brandt et al., 2021; Oztoprak et al., 2023; Tran Van et al., 2021) or the grasshopper *Warramaba virgo* (Kearney et al., 2022), these recent discoveries show that demonstrating strict asexuality is not straightforward, as absence of evidence is not evidence of absence. It is likely that similar discoveries will be made in the future in other systems. Future studies will be needed to explore whether the overrepresentation of invertebrates in these models arises from traits that favour rare sexual reproduction in OP lineages within this group or is instead a consequence of methodological factors. Moreover, the finding that both males and females can be vectors of contagious asexuality would suggest that new asexual lineages may arise more frequently than anticipated. It also opens the possibility that such contagion events could occur even when males are no longer produced or are unfunctional. Cryptic sex has profound implications for the long-term maintenance of asexual lineages. It strongly alters the evolutionary consequences of asexuality by providing many of the benefits of
sexual reproduction while minimizing its costs (Engelstädter, 2017; Gerber et al., 2018; Kokko, 2020; Otto, 2009; Otto & Lenormand, 2002; Peck & Waxman, 2000). Because largely asexual life cycles with rare or occasional sex appear optimal in terms of the costs and benefits of sex, it has been suggested that the “true paradox of sex” is why these life cycles are not more common (Burke & Bonduriansky, 2017). Our and other recent results suggest that these life cycles may well be detected in many “obligate parthenogens” if scrutinized in sufficient detail.

## Supplementary material

Supplementary material is available at *Journal of Evolutionary Biology* online.

## Data availability

The data underlying this article are available in the article and in the Dryad Digital Repository, at https://doi.org/10.5061/dryad.kd51c5bj8.

## Author contributions

Cécile Molinier (Conceptualization [equal], Formal analysis [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Cassandra Clément (Formal analysis [equal], Methodology [equal], Visualization [equal], Writing—original draft [equal], Writing—review & editing [equal]), Héloïse Calzan (Data curation [supporting], Methodology [supporting]), Thomas Lenormand (Conceptualization [equal], Investigation [equal], Supervision [equal], Writing—review & editing [equal]), and Christoph Haag (Conceptualization [equal], Formal analysis [equal], Funding acquisition [lead], Supervision [equal], Writing—original draft [equal], Writing—review & editing [equal])

## Acknowledgments

The authors are grateful to M. Lynch for kindly providing live *D. pulex* lineages. We thank S. Xu for sharing the DNA extraction protocol for the embryos. We thank R. Zahab, M.-P. Dubois and the Genomics, Molecular Ecology and Experimental Evolution platform (GEMEX) at CEFE, as well as the Génotypepage-Séquençage (GenSeq) platform (Labex Centre Méditerranéen de l'Environnement et de la Biodiversité) for the genotyping of microsatellite markers. We thank the editor and two reviewers for their constructive comments.

## Funding

This work was funded by grant ANR-17-CE02-0016-01, GENASEX, from the French National Research Agency.

## Conflicts of interest

None declared.

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