Cryptic asexual reproduction in Caenorhabditis nematodes revealed by interspecies hybridization

Most animal species reproduce by sex. Theory predicts there are advantages to being able to switch reproduction between sexual and asexual modes. However, facultative sex is rarely observed in animals, implying that there are strong selective pressures that prevent asexuality arising from an obligately sexual ancestor. One of the critical steps in the evolution of asexuality from a sexual ancestor is the transition from haploid to diploid maternal inheritance. Here we report that interspecific hybridization between two sexual Caenorhabditis nematode species (C. nouraguensis females and C. becei males) results in two classes of viable offspring. The first class consists of fertile offspring, which are produced asexually by sperm-dependent parthenogenesis (also called gynogenesis or pseudogamy); these progeny inherit a diploid maternal genome but fail to inherit a paternal genome. The second class consists of sterile hybrid offspring, which inherit both a diploid maternal genome and a haploid paternal genome. Using whole-genome sequencing of individual viable worms, we show that diploid maternal inheritance in both asexually produced and hybrid offspring results from the inheritance of two randomly selected homologous chromatids from C. nouraguensis oocytes. This genetic mechanism of diploid maternal inheritance is indistinguishable from that of many obligately asexual species. Furthermore, we show that intraspecies C. nouraguensis crosses can also result in a low frequency of asexual reproduction through diploid maternal inheritance. Thus, C. nouraguensis provides a genetically tractable model to study the evolutionary origins of asexuality from obligately sexual species.


INTRODUCTION
Theory predicts that facultative sex, the ability to undergo both asexual and sexual reproduction, is the optimal reproductive strategy [1][2][3][4]. Advantages of asexuality include an immediate two-fold enhancement of fitness and an enhanced ability to disperse geographically by obviating the requirement of a mate for reproduction [5,6]. By contrast, the advantages of sexual reproduction include the production of genotypic diversity that could be used to adapt to a changing environment and the ability to purge deleterious alleles through recombination [7]. Most unicellular eukaryotes undergo facultative sex, taking advantage of their ability to switch between these two reproductive strategies as conditions dictate [8,9]. Despite the predicted benefits of facultative sex, most animal species are obligately sexual, suggesting that there must be strong selective pressures to prevent the origin or persistence of asexuality from an obligately sexual ancestor [4]. A better understanding of these selective pressures requires understanding how asexuality evolves from a sexual ancestor. However, very few such transitions are known, and even fewer occur in genetically tractable organisms.
Here we focus on one key aspect of how asexuality evolves from sexually reproducing organisms: diploid maternal inheritance. Sexual reproduction requires that diploid females and males generate haploid eggs and sperm, which then fuse to produce the next generation of diploid offspring.
By contrast, asexual females produce diploid eggs that either develop independently of sperm fertilization, known as parthenogenesis [10], or require fertilization but do not inherit the paternal genome, known as gynogenesis or pseudogamy [11]. Thus, understanding how egg production can be modified to result in diploid maternal inheritance provides insight into understanding the origins of asexuality.
There are several known mechanisms of generating diploid eggs in asexual species. In apomixis, eggs produced by mitotic rather than meiotic divisions result in offspring that are clones of univalent. Therefore, triploid individuals have six bivalents and six univalents in mature oocytes, which appear as twelve DAPI-staining bodies ( Figure 2B) [31].
We crossed C. nouraguensis (JU1825) females to C. becei (QG711) males, collected fertile F1 females and dissected and stained their germlines with DAPI. As controls, we examined the germlines of JU1825 and QG711 females after mating with conspecific males. Both parental species mostly have six DAPI-staining bodies, consistent with six diploid chromosomes (like C. elegans).
Most fertile F1 females also have six DAPI-staining bodies, indicating they are diploid ( Figure 2C).

Fertile interspecific F1 inherit two random homologous chromatids from each maternal C. nouraguensis bivalent
There are three mechanisms by which the fertile F1 animals could have diploid maternal inheritance. They could inherit two chromatids from each maternal bivalent in a C. nouraguensis oocyte, inherit one chromatid from each bivalent and undergo genome-wide diploidization by endoreplication, or inherit the original two maternal chromatids by producing diploid eggs by mitosis (apomixis). To distinguish between these possibilities, we used whole-genome sequencing of individual worms to determine the genetic identity of the chromatids inherited by fertile F1 individuals.
All four of the chromatids within a bivalent are genetically distinct; of the six possible ways of combining two chromatids, four have distinct chromosomal genotypes and two are indistinguishable by Illumina whole-genome sequencing ( Figure 3A, 3B and Figure S5). Thus, the genotype of the fertile F1 can indicate which two chromatids were inherited. Because recombination in Caenorhabditis species occurs predominantly on chromosome arms [32,33], inheriting two sister chromatids would usually result in homozygosity in the centers of chromosomes and heterozygosity on one of the arms, whereas inheriting two homologous chromatids would result in heterozygosity in the center of each chromosome ( Figure 3B). By contrast, endoreplication of single chromatids would lead to homozygosity along all chromosomes, and apomixis would lead to heterozygosity along all chromosomes.
We first crossed two genetically distinct C. nouraguensis strains, NIC59 and JU1825, to generate heterozygous C. nouraguensis (NIC59/JU1825) females, which we then crossed to C. becei (QG711) males. We collected eleven fertile F1 and performed whole-genome amplification and sequencing of each one ( Figure 3A). We determined the genotype of each fertile individual's chromosomes by calculating average NIC59 SNP frequencies in 50-kb windows across their genome (see Materials and Methods for details). An approximately 0.50 NIC59 allele frequency was inferred as heterozygosity (NIC59/JU1825), whereas an approximately 1.0 or 0.0 NIC59 allele frequency was inferred as homozygous NIC59 or JU1825, respectively. This experiment also allowed us to determine whether fertile F1 inherit only maternal C. nouraguensis DNA or whether some paternal C.
becei DNA is also present.
We found that nearly 100% of each fertile F1's reads map to the C. nouraguensis genome assembly; only a small percentage (0.1-0.2%) map to the C. becei genome assembly (Table S1 and Figure S6). These results resemble our controls that contain only C. nouraguensis DNA (samples: F1_NIC59_JU1825 and NIC59plusJU1825, Table S1 and Figure S6). Therefore, our sequencing results confirm that fertile F1 inherit only maternal C. nouraguensis DNA. Strikingly, wholechromosome genotyping data shows that the fertile F1 always inherited two homologous chromatids from each maternal bivalent ( Figure 3C, Figure S5 and Figure S6). Combining chromosome genotypes from the fertile F1 indicates that two randomly selected homologous chromatids were inherited ( Figure 3D). Based on these findings, we can rule out endoreplication and apomixis.
Instead, we conclude that diploid, fertile F1 inherit two homologous chromatids from each maternal bivalent in C. nouraguensis oocytes.

Sterile F1 inherit a diploid C. nouraguensis genome and a haploid C. becei genome
Having established that fertile F1 adults inherit only a diploid maternal genome, we next examined the genome-wide inheritance pattern in sterile F1 adults. We performed whole-genome sequencing of ten sterile F1 adults derived from the same interspecies crosses mentioned in the previous section ( Figure 3A).
Surprisingly, five of the sterile F1 appear to be triploid hybrids that inherited a diploid C.
nouraguensis autosomes was approximately double that of the C. becei autosomes ( Figure 4A). Furthermore, genotyping of the C. nouraguensis chromosomes revealed that these individuals inherited two homologous chromatids from each maternal C. nouraguensis bivalent, just like the fertile F1 ( Figure 4B and Figure S6). Three other sterile F1 males are also hybrids that inherited two homologous chromatids from each maternal bivalent. However, they are unlikely to be true, full triploids because the normalized coverage of the C. becei autosomes is considerably less than half that of the C. nouraguensis autosomes (males: F1_10, F1_16 and F1_23, Figure 4A). Instead, we hypothesize that the reduced coverage of the C. becei genome results from mosaicism, in which only a subset of cells in these individuals contain the paternal C. becei DNA. The C. becei autosomes within each individual hybrid mosaic exhibit similar levels of coverage (except perhaps Chr. V of F1_16), suggesting that most hybrid cells within these individuals have a complete haploid copy of the C. becei autosomal genome (diploid-triploid mosaic hybrids) ( Figure 4A).
Combining maternal chromosome genotypes from the sterile F1 indicates that two randomly selected homologous chromatids were inherited ( Figure 4C and Figure S5C). Additionally, the combined data on maternal genotype frequencies from both sterile and fertile F1 also indicate random homologous chromatid inheritance ( Figure 4D and Figure S5C). These results show that both the fertile and sterile F1 share the same mechanism of diploid maternal inheritance and are distinguished by whether they inherited the haploid C. becei genome.

The C. becei X-chromosome is toxic to F1 hybrids
In Caenorhabditis, males are hemizygous for the X-chromosome (XO) and produce haploid sperm that carry either a single X or no X. Therefore, roughly half of the F1 are expected to inherit the paternal C. becei X-chromosome. However, despite inheriting C. becei autosomes, none of the sterile F1 we sequenced show inheritance of the C. becei X-chromosome ( Figure S6). We collected additional viable F1 hybrids from the interspecies cross and determined whether any inherited the C. becei X-chromosome by PCR genotyping of two indel polymorphisms, one X-linked and one autosomal, that distinguish between C. nouraguensis and C. becei sequences ( Figure 5A). We found that none of the F1 inherited the C. becei X-chromosome ( Figure 5B), regardless of whether they were autosomal hybrids (22 females and 12 males) or had a maternal genotype (10 females).
Our failure to find any viable F1 carrying a C. becei X-chromosome suggests that it is toxic to F1 hybrids. Alternatively, this pattern could reflect an unusual segregation pattern specifically involving the X chromosome in hybrids. To distinguish between these possibilities, we PCR genotyped dead F1 embryos from the same interspecies cross. Consistent with the expected inheritance of the X-chromosome from males, we found that 50% of dead F1 embryos inherited both the C. becei paternal X-chromosome and the C. nouraguensis maternal X-chromosome (17/34), whereas 47% inherited only the maternal C. nouraguensis X-chromosome (16/34). One dead F1 embryo possessed only a paternal C. becei genotype, suggesting that it lost the maternal C. nouraguensis X-chromosome. Because dead F1 embryos can inherit the C. becei X-chromosome but viable F1 animals do not, we conclude that the C. becei X-chromosome is toxic to F1 hybrids. Previous work has described the "large X-effect" on hybrid inviability and sterility [25]. We hypothesize that a dominant incompatibility involving one or more loci on the C. becei X chromosome underlies its toxic effect on hybrids with C. nouraguensis.

Dead F1 embryos have unusual maternal and paternal inheritance
To determine whether the unusual patterns of maternal and paternal inheritance seen in viable F1 also occur in the dead F1 embryos in the interspecies cross, we PCR genotyped the same dead F1 embryos described above at an autosomal indel polymorphism that distinguishes the two species ( Figure 5C). We found that 27 of the 35 dead F1 embryos (77%) had a heterozygous C. nouraguensis/C. becei genotype while eight (22%) had an only maternal C. nouraguensis genotype ( Figure 5C). These data suggest that although most embryos inherit both maternal and paternal DNA, a significant fraction fail to inherit paternal C. becei DNA for this marker. Aneuploidy caused by incomplete loss of the paternal genome may contribute to death of embryos.
To determine whether dead F1 embryos also inherit two homologous C. nouraguensis chromatids from each maternal bivalent, we PCR genotyped them at an autosomal indel polymorphism in the center of chromosome I that distinguishes NIC59 and JU1825 alleles. We found that 34/36 dead F1 embryos had a heterozygous NIC59/JU1825 genotype, whereas one embryo had a JU1825 genotype and one embryo had a NIC59 genotype ( Figure 5D). These data suggest that very few embryos are derived from a canonical female meiosis in which a haploid maternal complement is inherited. Instead, most embryos appear to have inherited at least two homologous chromatids from an aberrant female meiosis.
To better understand how female meiosis is modified so that at least two homologous chromatids are inherited, we quantified the number of polar bodies found in F1 embryos, of which the vast majority are expected to die. If most C. nouraguensis oocytes fertilized by C. becei sperm have aberrant meiotic segregations that result in the inheritance of two homologous chromatids, we might expect abnormalities in polar body number or morphology. We DAPI stained F1 embryos derived from crossing C. nouraguensis (JU1825) females to C. becei (QG711) males. As a control, we stained embryos derived from intraspecies JU1825 crosses. As expected, JU1825 embryos always have two polar bodies whose morphology and size appear similar across individuals ( Figure 5E and 5I). By contrast, hybrid embryos often have fewer than two polar bodies, potentially indicating failed or abnormal meiotic segregations. The polar bodies in hybrid embryos also often appear abnormal in their morphology ( Figure 5F-I). Together with our genotyping data, these observations indicate that meiotic segregations are frequently perturbed in C. nouraguensis females mated to C. becei males.

Diploid maternal inheritance occurs occasionally in C. nouraguensis intraspecies crosses
To determine whether diploid inheritance from C. nouraguensis oocytes can occur independently of interspecies hybridization, we designed an experiment in which C. nouraguensis sperm can fertilize C. nouraguensis oocytes but cannot contribute a paternal genome. In this scenario, a canonical female meiosis would result in haploid maternal genome inheritance and the embryos would fail to develop into viable adults [26]. However, if C. nouraguensis oocytes inherit a diploid maternal genome, they could complete embryogenesis and develop into viable adults.
We blocked the inheritance of the haploid sperm genome using UV irradiation to damage and inactivate the sperm genome while still allowing for fertilization and development of the maternal haploid embryo [34,35]. We exposed NIC59 and JU1825 adult males to a high dose of shortwave UV radiation and then crossed them to unexposed JU1825 or NIC59 adult virgin females, respectively.
We then screened for and PCR genotyped viable progeny at several autosomal loci to determine whether they inherited both maternal and paternal DNA (heterozygous NIC59/JU1825), or just maternal DNA ( Figure 6A).
First, we crossed JU1825 females to NIC59 UV-irradiated males. Only seven out of approximately 17,800 embryos survived to adulthood. Upon PCR genotyping a chromosome I indel polymorphism, we found that four of these seven individuals had a heterozygous NIC59/JU1825 genotype and therefore inherited both maternal and paternal alleles at this locus. We presume these animals represent rare cases in which the sperm genome was not destroyed. However, three females inherited only the maternal JU1825 allele ( Figure 6B). We PCR genotyped these females at two other unlinked autosomal indel polymorphisms. All had a maternal JU1825 genotype at all three loci, suggesting that they inherited only the maternal genome ( Figure 6C). We performed similar experiments with NIC59 females and UV-irradiated JU1825 males. Only two out of roughly 7,120 progeny survived to adulthood. These two females had only maternal NIC59 genotypes at all three autosomal loci ( Figure 6B and 6C). These results indicate that diploid maternal inheritance in C.
nouraguensis oocytes does not require fertilization by C. becei sperm and can occur even in intraspecies matings.

DISCUSSION
In this study we investigated the hybridization of two sexual Caenorhabditis species, C. nouraguensis and C. becei. We found that although most offspring of C. nouraguensis females crossed to C. becei males die during embryogenesis, rare viable offspring are produced. About onethird of the viable offspring are fertile and result from a combination of diploid maternal inheritance and paternal genome loss, two traits that define gynogenetic reproduction. The remaining two-thirds of the viable offspring are sterile and possess a diploid maternal C. nouraguensis genome together with a haploid paternal C. becei genome. However, none of the sterile hybrids inherit the C. becei Xchromosome, although it is inherited at the expected frequency in dead embryos. Finally, we found that diploid maternal inheritance also occurs at a low frequency in intraspecies C. nouraguensis crosses. We hypothesize that the same mechanism of diploid maternal inheritance occurs in both intraspecies and interspecies crosses, but that fertilization of C. nouraguensis oocytes by C. becei sperm dramatically increases its frequency, suggesting that a signal from C. becei sperm alters C. nouraguensis female meiosis.

Mechanism of diploid maternal inheritance
We found that diploid maternal inheritance in fertile and sterile offspring almost always results from inheriting two randomly selected homologous chromatids from each maternal bivalent in the oocyte. We also found that dead F1 embryos almost always inherit at least two homologous chromatids from C. nouraguensis oocytes. Thus, diploid maternal inheritance is a general feature of C. nouraguensis oocytes when fertilized by C. becei sperm. We hypothesize that this diploid maternal inheritance reflects a stereotypical segregation mechanism that is a slight modification of canonical meiotic divisions ( Figure 7A).
In our model ( Figure 7B), meiotic prophase occurs normally in C. nouraguensis oocytes, generating six recombinant bivalents. Upon fertilization by C. becei sperm, the bivalents randomly bi-orient their homologs on the meiotic spindle, sister chromatid cohesion is lost between homologous chromosomes, and homologs segregate at anaphase I. However, instead of one set of homologs getting segregated into the first polar body, both sets are retained in the oocyte. Both half-bivalents within the oocyte then randomly bi-orient on the meiotic spindle, sister chromatid cohesion is lost, and sister chromatids segregate at anaphase II. One chromatid from each half-bivalent segregates into a polar body while the other is retained in the oocyte. Thus, the oocyte inherits two randomly selected homologous chromatids from each bivalent. This model suggests that diploid maternal inheritance could result from a modification of female meiosis such as failure of cytokinesis after anaphase I.
There is evidence that incomplete anaphase I and a failure of cytokinesis generates diploid eggs in Daphnia pulex, which undergoes obligate pathenogenesis [36].
Some loss-of-function mutations and gene knockdowns in C. elegans can possibly lead to diploid maternal inheritance [37,38], suggesting plausible cellular mechanisms underlying this phenomenon. During normal meiosis I in C. elegans, the meiotic spindle migrates and orients perpendicularly to the oocyte cortex. In anaphase I, one set of homologs segregates towards the cortex into the first polar body while the other is retained in the oocyte. However, weak knockdown of dynein heavy chain (dhc-1) causes the meiotic spindle to be oriented parallel to the cortex rather than perpendicular during meiosis I; as a result, homologous chromosomes segregate parallel to the cortex in anaphase I and no polar body is formed [38]. The half-bivalents that remain in the oocyte then segregate relatively normally during anaphase II and form a polar body. The oocyte presumably inherits two randomly selected homologous chromatids from each bivalent. A similar misalignment of the meiotic spindle at metaphase I is also thought to be the mechanism underlying the generation of diploid eggs in the obligately parthenogenetic species Drosophila mangabeirai [39]. We hypothesize that a misalignment of the meiotic spindle or a failure of cytokinesis may similarly result in diploid maternal inheritance in C. nouraguensis.

Paternal genome loss
In addition to diploid maternal inheritance, gynogenesis also requires paternal genome loss. In viable F1 offspring from the C. nouraguensis x C. becei cross, we found that the entire haploid C.

Characteristics of incipient gynogenesis
Asexuality is rare in animals, suggesting that there are strong selective pressures that prevent its evolution from a sexual ancestor. However, it is largely unknown how the transition from sexuality to asexuality might occur, nor how the costs and benefits of such a transition might influence the longevity of a nascent asexual lineage. Some obligately gynogenetic species are composed entirely of females, and require that their eggs be fertilized and activated by males of a closely related species [11]. We hypothesize that the rare gynogenetic reproduction seen in C. nouraguensis x C. becei hybridizations could serve as a transitional state between sexual and obligate or facultative interspecies gynogenetic reproduction. Furthermore, we hypothesize that the diploid maternal inheritance observed in C. nouraguensis intraspecies crosses could be used as a stepping-stone towards either gynogenetic or parthenogenetic reproduction. Both interspecies gynogenesis and intraspecies diploid maternal inheritance could be features of a nascent asexual lineage.
We have shown that diploid maternal inheritance in C. nouraguensis oocytes is the result of inheriting two homologous chromatids that have undergone meiotic recombination, and therefore fits a model of automixis in which two homologous chromatids are combined ("central fusion"). Because there is only a single recombination event that is biased towards the chromosome ends, inheriting two homologous chromatids either maintains heterozygosity across the entire chromosome or most of it ( Figure 3B). However, recombination does occur in the middle of chromosomes at a lower frequency [32], which will eventually result in genome-wide homozygosity and inbreeding depression after many generations of automixis. Most Caenorhabditis species appear to be dioecious (females and males), with some exhibiting genetic hyperdiversity [51,52]. Therefore, inbreeding depression could be a sizable hurdle to overcome in the evolution of this form of gynogenesis from an obligately sexual Caenorhabditis ancestor.
Given that two copies of each maternal autosome are almost always inherited in the interspecies C. nouraguensis x C. becei cross, we were surprised to observe that some diploid fertile F1 are males. Indeed, sequencing data show that the three fertile males have only a single X chromosome ( Figure S5 and Figure S6). Thus, missegregation of the X-chromosome during the modified female meiosis can result in only one X chromatid being inherited by the oocyte.
Interestingly, two of the three fertile males were also aneuploid (triploid) for a single C. nouraguensis autosome, indicating that widespread chromosome missegregation occurred while producing these individuals. Generating males at a low frequency can theoretically aid the spread and maintenance of gynogenesis by transmitting alleles required for diploid maternal inheritance into neighboring sexual populations [53]. The production of males even at a low frequency can also help propagate an intraspecific gynogenetic lineage by enabling the activation of the egg and inheritance of centrioles without relying on males from a related species, as seen in Mesorhabditis belari [54]. Thus, it is notable that gynogenetic reproduction in C. nouraguensis x C. becei hybridizations already exhibits the ability to produce males.
In conclusion, our study establishes C. nouraguensis as a new genetic model system to study rare asexuality in animals. Further study of this system may provide insights into the mechanisms of diploid maternal inheritance and paternal genome loss, as well as the obstacles associated with a transition from sexual to asexual reproduction.

Strain isolation and maintenance
All strains of Caenorhabditis used in this study were derived from single gravid females isolated from rotten fruit or flowers [55,56]. Strains of C. nouraguensis were kindly provided by Marie-Anne Félix ("JU" prefix) and Christian Braendle ("NIC" prefix). Most strains are outbred, except JU2079, QG2082 and QG2083, which are inbred lines derived from JU1825, QG704 and QG711, respectively. These inbred lines were used to generate the C. nouraguensis and C. becei genome assemblies. Strain stocks were stored at -80°. Strains were maintained at 25° on standard NGM plates spread with a thin lawn of E. coli OP50 bacteria [57].

Phylogenetic analysis
To construct a phylogeny, we used a subset of the coding sequences from Kiontke et al.

Quantifying strain viability
Strain viability was quantified as in [60]. Briefly, we crossed 10 virgin L4 females and males of the same strain on a single plate coated with palmitic acid. The plates were placed at 25° overnight, allowing the worms to mature to adulthood and begin mating. The adult worms were then moved to a new plate rimmed with palmitic acid, allowed to mate and lay eggs for 1.5 hours at 25°, and then removed. The eggs laid within that time were immediately counted. Two days later, we placed the plates at 4° for 1 hr and counted the number of healthy L4 larvae and young adults per plate. We defined the percentage of viable progeny as the total number of L4 larvae and young adults divided by the total number of eggs laid.

DIC imaging of embryogenesis
Twenty-four hours after the L4 stage, we picked 20-40 gravid females into a 30 µl pool of 1x Egg Buffer (25 mM HEPES pH 7.4, 118 mM NaCl, 48 mM KCl, 2 mM EDTA, 0.5 mM EGTA) on a glass coverslip and dissected out embryos by cutting the adults in half using two needles like scissors. The embryos were then transferred with a glass capillary tube into a fresh pool of Egg Buffer to dilute any contaminating bacteria and then placed onto a 2% agarose pad on a glass slide. The mounted embryos were put into a humid chamber for 20 minutes before adding a coverslip. The edges of the coverslips were sealed with petroleum jelly to prevent evaporation of the agarose pad.
DIC z-stack images were captured every 3 minutes for 10 hours using a Nikon Eclipse 80i compound microscope (60x oil lens, 1.40 NA). Images were processed using Image J [61].

Calculating the frequency of rare interspecific F1
For each interspecies cross we set up 20-25 plates, each with one virgin L4 female crossed to a single virgin L4 male. The plates were placed at 25° overnight, allowing the worms to mature to adulthood and begin mating and laying eggs. To prevent overcrowding, the adult couples were moved to a new plate every day for three days. The number of eggs laid on each plate was counted the day the parents were removed. Each plate was monitored for two days to check for the presence of rare viable F1, which were counted and moved onto a new plate.

Generating rare interspecific F1 and testing their fertility
To collect enough rare viable F1 animals for fertility testing and genotyping, we set up six cross plates, each with 30 C. nouraguensis L4 females and 30 C. becei L4 males and let them develop into adults overnight at 25°. We monitored the plates for the presence of viable F1 larvae each day for 5-6 days. Each viable F1 was moved to its own new palmitic acid-rimmed plate, and its fertility was tested as either an L4 or young adult by backcrossing it to one or two C. nouraguensis individuals of the opposite sex. F1 individuals were classified as sterile if they mated to C. nouraguensis but produced no F2 embryos, while F1 individuals were classified as fertile if they produced F2 embryos. Mating was inferred by the presence of a male-deposited copulatory plug on the female vulva [62]. After fertility testing and PCR genotyping, we found that hybrid males produced no embryos when crossed to C. nouraguensis females and were classified as sterile. However, they often failed to produce a copulatory plug, so we cannot tell whether sterility is due to abnormal germline development or defective mating.
To track the fate of the two maternal homologous chromosomes in subsequent crosses, we crossed two genetically distinct strains of C. nouraguensis (JU1825 and NIC59) to generate heterozygous C. nouraguensis NIC59/JU1825 females. Specifically, we crossed virgin L4 JU1825 females to NIC59 males and vice versa to generate female offspring that are heterozygous NIC59/JU1825 at all nuclear loci and carry either JU1825 or NIC59 mitochondria, respectively. We denote the genotype of these heterozygous females by the following nomenclature: "(mitochondrial genotype); nuclear genotype". Therefore, the first cross produces C. nouraguensis (J); N/J females, while the second produces C. nouraguensis (N); N/J females (Table S1). We then crossed both types of C. nouraguensis N/J virgin females to C. becei QG711 males. We collected the rare viable F1 from these interspecies crosses and tested their fertility by backcrossing them to the C. nouraguensis strain matching their mitochondrial genotype in order to avoid known cytoplasmic-nuclear incompatibilities within this species [60].

Generating worm and embryo lysates for PCR
Single worm PCR: Each adult worm was placed in a PCR tube with 10 µl of lysis buffer (1x Phusion HF buffer + 1.0% Proteinase K) and frozen at -80° for at least 15 minutes. The worms were then lysed by incubating the tubes at 60° for 60 mins and 95° for 15 mins. 1 µl of worm lysate was used in each 10 µl PCR reaction. Control lysates were generated by mixing 10-15 worms in lysis buffer.
Single embryo PCR: Dead embryos were picked into a 30 µl pool of 2 mg/ml chitinase on an 18x18 mm coverslip. OP50 bacteria were washed off embryos by pipetting the chitinase solution up and down. Using a glass capillary tube, embryos were then transferred to a new pool of chitinase and washed again. Using a glass capillary tube, individual embryos were placed in a PCR tube with 5 µl of 2 mg/ml chitinase solution and incubated at room temperature for 2 hours. Afterwards, 5 µl of lysis buffer (2x Phusion HF Buffer + 2.0% Proteinase K) was added to each PCR tube and mixed by light vortexing. The samples were then frozen and lysed as in the single worm PCR protocol. 2.5 µl of embryo lysate was used in each 10 µl PCR reaction. Control lysates were generated by mixing 10-15 embryos in the same PCR tube.

PCR primers
The primers used in the PCR-RFLP polymorphism assays were chosen because they lie in conserved coding sequences that are easily amplified from a range of Caenorhabditis species [55].

Fixing and DAPI staining the female germline
Fixing and DAPI staining germlines was performed as in [64]. F1 L4 females were mated to C. Images were taken using a Nikon Eclipse 80i compound microscope and processed using Image J.

C. becei genome assembly and linkage map construction
The chromosomal reference assembly for C. becei is based on high-coverage paired-end and mate-pair short-read sequencing, low-coverage Pacific Biosciences long reads, and genetic linkage information from an experimental intercross family. We used two inbred lines, QG2082 and QG2083, derived from isofemale lines QG704 and QG711 respectively by sib-mating for 25 generations.
QG704 and QG711 were isolated from independent samples of rotten flowers (QG704) or fruit (QG711) collected in Barro Colorado Island, Panama, in 2012 [56]. We generated conservative de novo assemblies for the inbred lines, identified SNPs that distinguish them, and then used a sixgeneration breeding design to generate recombinant populations (G4BC2) from which we generated a SNP-based linkage map. With the map-scaffolded conservative assembly, we were able to evaluate more contiguous, less conservative genome assemblies for consistency, and we selected one of these as our final C. becei genome. Each of these steps is described in detail below.

Sequencing
We extracted genomic DNA from QG2083 by proteinase K digestion and isopropanol

Genome assembly for map construction
We assembled contigs and conservative scaffolds for QG2083 with the string graph assembler SGA [69]. Scaffolding required contigs to be linked by at least 5 unambiguously mapped mate-pair reads and full path consistency between contigs and all mapped mate-pair reads (option '--strict').  [73]). Fixed diallelic SNPs were supplemented with calls from reference mapping of the pooled G4BC2 data and, to mitigate potential mapping bias against highly divergent regions, SNP calls from whole genome alignment of a de novo assembly of G4BC2 data pooled with QG2083 data at 1:1 expected heterozygosity, using the heterozygous aware assembler Platanus [74] and Mummer [75]. This yielded a total of 1.63M diallelic SNPs across 6843 scaffolds spanning 89.2 Mb. These SNPs provide markers for genetic map construction.

Genetic map construction
We generated F2 animals from reciprocal crosses between QG2083 and QG2082. We then performed 160 single-pair random-mating intercrosses among F2s to generate G3s, and again among G3s to generate G4s. These intercrosses allow additional meioses to expand the genetic map. We backcrossed G4 females to QG2082 males, generating progeny carrying one recombinant version of the genome and one intact QG2082 genome. To increase the quantity of DNA representing these genomes, we crossed females to QG2082 males again and allowed the resulting G4BC2 populations to grow for a single generation before DNA extraction and library preparation. Data for 87 G4BC2 populations with at least 0.5x expected coverage were mapped to the QG2083 scaffolds and genotyped by HMM using a modified MSG pipeline (assuming six crossovers per assembled genome, genotyping error rates of 0.001, and a scaling parameter on transition probabilities of 1x10 -

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, taking the dominant parental assignment as a single genotype for each scaffold) [76]. Markers were filtered in r/qtl based on missing data (<50%) and segregation distortion (c 2 test p > 1x10 -10 ), followed by removal of redundant markers [77]. Based on genotyping error rate estimates we further required a minimum of 70 SNP calls per scaffold and a minimum length of 1 kb, yielding 1337 nonredundant scaffold markers spanning 81.5 Mb. Six linkage groups were recovered over a wide range of minimum pairwise LOD scores, and within linkage group marker ordering was carried out by the minimum spanning tree method implemented in ASMap [78].

Assembly of the C. becei draft genome
Linkage group scaffolds were aligned against multiple Platanus de novo assemblies using the sequencing data from inbred lines and subsampling of the approximately 350x pooled data from G4BC2 lines, with the UCSC chain/net pipeline [79]. Genome evolution in the Caenorhabditis genus occurs largely through intrachromosomal rearrangement, and 84-94% of the net for each C. becei linkage group aligned to the homologous C. elegans chromosome. The assembly most concordant with genetic data was selected, based on the number and aligned length of (1) scaffolds mapping to multiple linkage groups and (2)

C. nouraguensis genome assembly
The C. nouraguensis reference genome was generated from JU2079, an inbred strain derived from 28 generations of JU1825 sibling matings (Marie-Anne Félix, personal communication).
Genomic DNA and RNA were purified from mixed-stage individuals and sent to Mark Blaxter at the University of Edinburgh for Illumina sequencing. 125-bp paired-end reads were generated from two libraries, one with inserts of ~400 bp (~55 million read-pairs), the other with inserts of ~550 bp (~54 million read-pairs) (SRA project accession PRJEB10884). After generating a preliminary assembly, we used BlobTools [80] for taxonomic classification. Some contigs matched E. coli (provided as food) and some matched an unsequenced bacterial species in the Firmicutes phylum. We extracted the Firmicutes contigs and used GSNAP [81] to remove matching read-pairs, and to remove any readpairs matching the E. coli REL606 genome (Genbank accession NC_012967), a strain closely related to OP50. We also removed reads failing Illumina's 'chastity filter' and used cutadapt to trim poor quality sequences (phred score < 10) and adapter sequences from the 3' ends of reads. We then performed error-correction using Musket [82] with a k-mer size of 28, and performed de novo genome assembly, scaffolding and gap closure using Platanus [74] with an initial k-mer size of 21. We then used REAPR [71] to break any misassembled scaffolds. The resulting assembly is approximately 73 Mb in size. We used BUSCO [83] to measure its completeness using a set of conserved genes, and find that our assembly contains 81.5% of conserved single-copy orthologs as a single copy, 9.8% as duplicates, and 3.6% as fragments, very similar to what is seen in the finished C. elegans genome assembly [83]. Using MUMMER [75], we ordered and oriented our C. nouraguensis scaffolds based on synteny to the C. becei genome assembly. Using this approach, some scaffolds remain unplaced and others may be misplaced, either because of true differences between the two species' genomes, or because MUMMER's alignments are noisy.
We also generated assemblies of the mitochondrial genomes of C. becei strain QG2083, and C. nouraguensis strain JU2079 using reads generated by Mark Blaxter's lab (EMBL ENA accession ERR1018617), filtered as above to remove E. coli reads, and down-sampled to 10 million read-pairs for C. nouraguensis. We then used the Assembly by Reduced Complexity (ARC) [84] pipeline to assemble mitochondrial genomes, using the C. elegans mitochondrial genome sequence as a starting point.

Whole-genome amplification and library preparation
After testing the fertility of rare viable offspring from crosses of C. nouraguensis JU1825/NIC59 females to C. becei males (see above), we prepared Illumina whole-genome sequencing libraries as follows. We first transferred each worm individually to a blank NGM plate with no OP50 lawn for 30 minutes to reduce the amount of contaminating bacterial DNA. We then performed whole-genome amplification using the Qiagen REPLI-g Single Cell kit [85]. We picked individual worms into 4 µl of PBS sc and froze them at -20° for at least an hour. We thawed the samples and added 3 µl of Buffer D2, mixed well, and incubated at 65° for 20 minutes rather than the recommended 10 minutes to aid in worm lysis.

SNP calling, genotyping, and coverage calculations
Using GSNAP [81], we mapped the reads from each sample to a combined reference genome containing the C. nouraguensis and C. becei nuclear and mitochondrial genome assemblies, the E.
coli REL606 genome sequence and the Firmicutes contigs described above. We allowed GSNAP to report only a single map position for each read (--npaths=1). We further filtered read mappings, requiring mapq value of at least 20 to select uniquely mapping reads.
After preprocessing bam files by marking duplicates (using picard's MarkDuplicates, http://broadinstitute.github.io/picard/) and realigning indels (using GATK's RealignerTargetCreator and IndelRealigner tools [73]), we called SNPs in C. nouraguensis scaffolds using samtools mpileup [86] (ignoring indels, disabling the per-base alignment quality option and increasing the depth downsampling parameter to 6660). We counted reads matching each allele using GATK's VariantAnnotator tool [73], disabling the down-sampling option and using the "ALLOW_N_CIGAR_READS" option. We noticed that the distribution of allele frequencies in our 'heterozygous' control samples was skewed towards the reference allele, likely because those reads are easier to map to the reference assembly. We therefore used this first round of SNP calls to remap all reads to the combined reference genome using GSNAPs "SNP-tolerant" mode that allows reads to map to either haplotype. SNPs were called a second time, after one additional level of read mapping filtering, where we used the R/Bioconductor Rsamtools package [87] to select only reads where the full length of the read could be aligned to the reference genome.
We then used the R/Bioconductor VariantAnnotation package [87] to select high-quality fixed differences between the NIC59 and JU1825 strains as follows: we required a SNP quality score of at least 100; that opposite alleles be fixed (non-reference frequencies of <5% and >95%) in the two strains; that read depth in some C. nouraguensis control samples be within typical range for that sample (5-50 for JU1825_bulk and NIC59_bulk, and 100-450 for the combined JU2079 samples); and that read depth be 0 in our C. becei control samples (QG711_bulk and the two QG2083 libraries). 337,493 SNPs passed these filters (an average of one SNP every 217 bp). To estimate mean allele frequencies in 50-kb windows across the genome, we counted the total number of reads matching NIC59 and JU1825 alleles across the window and divided the NIC59 count by the total count. We then used a circular binary segmentation algorithm, implemented in the Bioconductor package DNAcopy [88], to estimate the locations of breaks between different haplotypes as well as the average allele frequency in each segment.
Using the same filtered bam files, we determined coverage at each base position using the samtools depth tool [86]. Examining coverage and aligned bases in our control samples We only analyzed embryos at early stages of embryogenesis (1-4 cell stage). In normal embryos at this stage, the DNA in the two polar bodies appears as two distinct clumps at the periphery of the embryo that are easily distinguishable from other embryonic nuclei. Polar bodies in hybrid embryos were considered to have abnormal morphologies if they were unlike those observed in the JU1825 control. This encompasses a range of abnormalities such as not being round, being larger, or having easily distinguishable chromatids.

Male UV irradiation
Virgin C. nouraguensis L4 males and L4 females were collected and separately placed onto two new NGM plates seeded with OP50 and rimmed with palmitic acid. We added a few females to the male plate to coax the males to stay on the surface of the plate. The next day, now young adult males were picked onto a blank NGM plate rimmed with palmitic acid. This plate was placed in a CL-seeded with OP50 and rimmed with palmitic acid. Each plate had thirty irradiated males and thirty young adult virgin females. The next day, rare viable larvae were picked from the cross plates onto a new NGM plate seeded with OP50 and rimmed with palmitic acid. When these progeny reached adulthood, they were individually frozen, lysed and PCR genotyped.
We tested the following UV exposures: 15,000, 40,000, 50,000, 60,000, 70,000, 80,000, 150,000 and 250,000 µJ/cm 2 . Males treated with lower exposures (15,000 and 40,000 µJ/cm 2 ) were able to produce many viable progeny, indicating that their sperm DNA was not damaged enough to induce paternal genome loss. Males treated with the highest exposures (150,000 and 250,000 µJ/cm 2 ) mostly stopped moving and never recovered. We found that males treated with 70,000 µJ/cm 2 were healthy enough to mate and produce many dead fertilized embryos, but relatively few viable progeny.

DECLARATION OF INTERESTS
The authors declare no competing interests.   . Both sterile and fertile F1 exhibited a more strongly female-biased sex ratio than that seen in intraspecies crosses ( Figure S1). See also Figures S1-S3 and Videos S1-S4.    Figures S5 and S6 and Table S1. (I) Hybrid embryos can have fewer than two and abnormal polar bodies. By contrast, embryos derived from intraspecies JU1825 crosses always have two polar bodies. "Two abnormal" refers to embryos that have two polar bodies, but one or both have an abnormal structure. "One abnormal" refers to embryos that have a single polar body with an abnormal structure.  random homologous chromatids from a bivalent. Upon fertilization, the bivalent randomly bi-orients its homologs on the meiotic spindle and cohesion is lost between homologous chromosomes as is normal. Homologs segregate but cytokinesis fails (Anaphase I) and both half-bivalents remain in the oocyte. Each half-bivalent then bi-orients on the meiotic spindle, sister chromatid cohesion is lost, and sister chromatids segregate. One chromatid from each half-bivalent segregates into the second polar body while the other is retained in the oocyte (Anaphase II and Cytokinesis). Thus, the oocyte inherits two random homologous chromatids from a bivalent.