De Novo Genome Assembly of Auanema Melissensis, a Trioecious Free-Living Nematode

Abstract Nematodes of the genus Auanema are interesting models for studying sex determination mechanisms because their populations consist of three sexual morphs (males, females, and hermaphrodites) and produce skewed sex ratios. Here, we introduce a new undescribed species of this genus, Auanema melissensis n. sp., together with its draft nuclear genome. This species is also trioecious and does not cross with the other described species A. rhodensis or A. freiburgensis. Similar to A. freiburgensis, A. melissensis’ maternal environment influences the hermaphrodite versus female sex determination of the offspring. The genome of A. melissensis is ~60 Mb, containing 11,040 protein-coding genes and 8.07% of repeat sequences. Using the estimated ancestral chromosomal gene content (Nigon elements), it was possible to identify putative X chromosome scaffolds.

Auanema species display atypical segregation and inheritance patterns of the X chromosome, which is gametogenesis-and sexual morphdependent (Shen and Ellis, 2018;Tandonnet et al., 2018). These singularities lead to skewed sex ratios according to the reproduction mode (crossings and selfing) (Tandonnet et al., 2018). In A. rhodensis, the three sexual morphs are produced by selfing hermaphrodites and crossing females, independently of the environmental conditions (Chaudhuri et al., 2015). In A. freiburgensis, however, mothers exposed to crowding cues derived from high population densities produce mostly hermaphrodite offspring. In the absence of those crowding cues, mothers produce predominantly females and a few males (Zuco et al., 2018;Robles et al., 2021).
The genomes of A. rhodensis and A. freiburgensis are relatively small (~60 Mb) and organized into seven chromosomes (Tandonnet et al., 2019;Al-Yazeedi, pers. comm.). This number of chromosomes differs from the usual six, which is the karyotype of most other nematodes in the same clade (Clade V) (Gonzalez de la Rosa et al., 2021). By tracking the ancestral linkage groups in nematode evolution, the so-called Nigon elements, it is known that Auanema chromosomes have undergone more fission and fusion events than other clade V nematodes (Tandonnet et al., 2019;Gonzalez de la Rosa et al., 2021). The reasons for this evolutionary pattern are unknown.
In this study, we report a new species of the genus Auanema, which we named Auanema melissensis, along with its draft genome assembly and associated annotation. We also describe the morphological and biological characteristics of this nematode and compare them to those of A. rhodensis and A. freiburgensis.

Nematode culture
Auanema melissensis (aka, Auanema sp. JU1783) was maintained under the standard culture conditions of Caenorhabditis elegans (Chaudhuri et al., 2011b) at 20°C. Plates were seeded with the streptomycinresistant Escherichia coli strain OP50-1. Microbial contamination was prevented by adding 50 mg/ml of streptomycin and 10 mg/ml of nystatin to the nematode growth medium (NGM).

Determination of Trioecy
To determine if A. melissensis was trioecious, we randomly isolated eggs from the culture plates. The eggs were individually placed into 48-well plates seeded with Escherichia coli OP50-1 and allowed to develop to adulthood in isolation. The sexual morph was determined by morphological characters and the ability or not to reproduce on their own. Hermaphrodites laid eggs in the absence of a mating partner, whereas females could only reproduce if paired with a male. Males were distinguished by their blunt tails.

The sexual fate of dauer larvae
Dauers of other Auanema species invariably develop into self-reproducing hermaphrodites (Félix, 2004;Chaudhuri et al., 2011a;Zuco et al., 2018;Robles et al., 2021). To determine if this was also the case in A. melissensis, we isolated dauers onto individual plates seeded with E. coli OP50-1 and determined their sexual morph in adulthood, as described above.
Effect of crowding cue on the female/ hermaphrodite ratio To test if the crowding conditions have an effect on the proportion of each sexual morph produced, we counted the number of male, female and hermaphrodite progeny produced by hermaphrodite mothers placed in the presence and absence of crowding cues. The crowding cues were prepared by first washing a Petri dish (ϕ = 60 mm) containing a crowded culture of nematodes with 1 ml of M9 buffer. The resuspended nematode culture was placed in a 50 ml tube and left on a thermomixer for 16-24 hr at 20°C. The liquid culture was then transferred to a 1.5 ml microcentrifuge tube and centrifuged for 45 min, at 15,000 rpm. Once clear from nematodes, the supernatant with the crowding cues was placed in a clean 1.5 ml tube and used immediately.
For the treatment conditions, we used Petri dishes (ϕ = 60 mm) with NGM that were seeded with 50 ml of OP50-1 and supplemented with either 100 ml of supernatant with crowding cues or with M9 buffer (control). The crowding cue or M9 buffer was added directly to the OP50-1 lawn in two installments of 50 ml, letting the liquid dry after each installment.
Dauers (fated to become hermaphrodites) were isolated separately on either the "crowding conditions" or the "control conditions" plates and allowed to develop into adulthood at 20°C. Hermaphrodite mothers were moved to a new plate (under the same conditions) each day and egg collection was carried out >3 d ( Fig. S1 in Supplementary Materials). Eggs were placed individually on standard (non-treated) plates and the sexual morph was identified as described above.
We built a generalized linear model (GLM) in R using the "glm" function to test if the proportion of females and hermaphrodites depends on the experimental conditions. The data were fitted to a binomial distribution.

Microscopy and whole-body measurements
Pictures of ten males, hermaphrodites, and females were taken on day 1 of adulthood with a Zeiss Axio Zoom V16 microscope, using a microscopy camera Axiocam and processed with the Zeiss ZEN2 software. Pictures were stored and edited in TIFF file formats. All pictures were taken at 100´ magnification.
Measurements of whole-body adults of each sexual morph were taken using the ImageJ "Measure" function. Nematode length was measured, head to tail, and the scale was defined using image scale bars and the ImageJ "Set Scale" feature.

Time course
A time course following ten females and ten hermaphrodites over the first 3 d of adulthood was also conducted using the same microscope settings stated above. Measurements of whole-body adults were taken in two parts: from the tip of the head to the anus and from the anus to the tip of the tail. This avoided measurement errors in pictures where the worm had an extremely curled tail. We analyzed the data by performing a two-way repeated measures ANOVA to evaluate simultaneously the effect of the sexual morph and the age on the total body length variable. A repeated measures ANOVA was necessary as the same individuals were measured for three consecutive days. The normality of the data was assessed by performing Shapiro-Wilk tests. We performed pairwise comparisons (t-tests) to further analyze the differences between age and sex.

Male tail imaging
Typological characters of the male tail, which are considered to represent species-specific characters, were observed and micrographed using a light microscope Eclipse Ni (Nikon) facilitated with DIC optics and a digital camera MC170 HD (Leica) attached to the microscope. Dauer larvae were individually cultured on small (ϕ = 40 mm) Petri dishes and allowed to develop into self-reproducing hermaphrodites. Newly emerged F1 males were picked up using a stainless steel insect pin (Insect pin #00, Shiga Kontyu), and observed and micrographed using the silicon grease method (Kanzaki, 2013). Micrographs were edited with PhotoShop Elements 2021 (Adobe) to construct the figure plate.

Preparation of fixed museum specimens
The type material of each sexual morph was prepared according to the Natural History Museum of London specifications. Ten individuals of each sexual morph were placed in a microcentrifuge tube with 200 ml of 80% ethanol.

DNA and RNA extraction
A large mixed nematode population of A. melissensis cultured on NGM agarose plates was used to extract the genomic DNA. The nematode handling and DNA extraction were based on the genomic DNA preparation protocol from Dudley and Goldstein (2005) except for pelleting the DNA by centrifugation at 14,000 rpm for 15-30 min instead of winding out the DNA precipitate. After removing the supernatant, an ethanol precipitation step was performed (i.e., the DNA pellet was washed using cold 70% ethanol and centrifuged before discarding the supernatant).
For RNA extraction, we also used a mixed population of nematodes reared on NGM agar plates. Plates were washed using M9 and worms were pipetted into a 15 ml conical tube. The tube was centrifuged for 15 min to gather the nematodes in a "pellet" at the bottom of the tube. The "supernatant" was discarded, and the nematodes were transferred into 1.5 ml tubes. The tubes were placed on dry ice until RNA extraction. For RNA extraction, the nematodes were resuspended in 1 ml of lysis buffer and shredded by sonication until the solution was bubbly and homogeneous (five cycles). Subsequently, we used the RNAeasy Mini kit, following the manufacturer's protocol.

Illumina sequencing and preprocessing
An Illumina whole genome sequencing (WGS) pair-end library of insert-size 200 bp and three mate-pair Illumina WGS libraries of insert-sizes 3 kb, 5 kb, and 8 kb were sequenced using a HiSeq2000 sequencer (Table S1 in Supplementary Materials). Raw reads were preprocessed using Skewer, version 0.2.2 (Jiang et al., 2014) to remove poor-quality sequence regions (<20) and reads smaller than 51 bases (Table S2 in Supplementary Materials). A preliminary genome was assembled to estimate the libraries' insert sizes and assess contamination. We used Blobtools, version 1.1.1 (Laetsch and Blaxter, 2017) to visualize and assess contamination levels ( Fig. S2 in Supplementary Materials). Our libraries contained little contaminants (<2% of the trimmed reads), which predominantly corresponded to E. coli. We assumed it was the strain OP50-1 as this strain was used as the food source of the nematode culture. The reads mapping to the genome of OP50-1 (GCA_000176815.1, ASM17681v1) were removed and not used for the final genome assembly (Table S2 in Supplementary Materials). Reads both runs were merged using TSEBRA (Gabriel et al., 2021).
were error-corrected using Fiona, version 0.2.10 (Schulz et al., 2014) using 60 Mb as an estimated genome size.
An Illumina paired-end RNA-seq library from a mixed population was sequenced using an Illumina NovaSeq 6000 sequencer (Table S1 in Supplementary Materials). Mature RNA was selected using an oligo-dT method. Raw reads were preprocessed using Skewer, version 0.2.2 (Jiang et al., 2014) to remove poor-quality sequences.

Genome assembly
The final de novo genome assembly was performed with MEGAHIT, version 1.2.6 (Li et al., 2015(Li et al., , 2016 followed by SOAPdenovo, version 2.04-r241 (Luo et al., 2012) with a k-mer length of 35, using the paired-end library for contig assembly and the mate-pair libraries for scaffolding, as this resulted in the best assembly strategy tested. Gaps were closed using SOAPdenovo's GapCloser. Genome completeness was assessed using BUSCO, version 5.2.2 using the nematoda_obd10 database (Simão et al., 2015).
The masked genome was then annotated using BRAKER2 version 2.1.6 (Brůna et al., 2021), which uses evidence-based data in conjunction with the ab initio predictors GeneMark (Brůna et al., 2020) and Augustus version 3.4.0 (Stanke et al., 2006). The first run of BRAKER2 was performed using transcriptomic data (RNA-seq reads and de-novo assembled transcriptome). The RNA-seq data generated in this study were pre-processed using Skewer (version 0.2.2) (Jiang et al., 2014) and the trimmed reads were used directly as gene hints by BRAKER2. We also assembled a de novo transcriptome from the trimmed RNA-seq reads using Trinity (version 2.10.0) (Grabherr et al., 2011). The second run of BRAKER2 used the curated protein database "metazoa_odb10" database from orthoDB (Grabherr et al., 2011;Kriventseva et al., 2019) as the evidence-based data. The results of We also observed a difference in pigmentation between females and hermaphrodites, which accentuated during the time course: hermaphrodites seemed to accumulate pigments in the gut whereas females may effectively lose them (Fig. 2B). The bursa is anteriorly open, supported by eight pairs of papillae (rays) arranged as follows <GP1, GP2, (GP3, CO), GP4, GP5d, GP6, (GP7d, GP8) phasmid>, where the distances between GP1-GP2, GP2-GP3 and GP6-GP7 are similar to each other, the distance between GP5d-GP6 is clearly larger and that between GP3-GP4 and GP4-GP5d is shorter; GP7d and GP8 are close to each other (Fig. 3).
In addition to its generic characters, A. melissensis n. sp. is characterized by its male tail characters described above. The new species described here is typologically identical to A. rhodensis, i.e., the male tail characters are shared by these two species (Kanzaki et al., 2017). These two species can be distinguished only by molecular phylogenetic status and mating experiments.
The species was originally isolated from a rotting starfruit (Averrhoa carambola) on September 21, 2009 on the Indian Ocean island of La Réunion in Saint Benoit (Melissa domain). The species epithet is after its type locality, Melissa domain.
Crosses between A. melissensis females and A. rhodensis or A. freiburgensis males (as well as the reciprocal crosses) did not result in progeny (Table 1), indicating that A. melissensis can be considered a distinct species from the two other known trioecious Auanema nematodes.
Similar to the other Auanema nematodes, all dauer larvae of A. melissensis developed into hermaphrodites (n = 48). To determine if the female versus hermaphrodite decision in A. melissensis is mediated by overcrowding cues as in A. freiburgensis (Zuco et al., 2018;Robles et al., 2021), we exposed hermaphrodite mothers to crowding cues. These cues were extracted from plates with high population densities of A. melissensis. Mothers exposed to the crowding cues produced a higher proportion of hermaphrodite progeny than under the control conditions (GLM P-value <0.01; Fig. 4, Table S5 in Supplementary Materials).

Genome characteristics
The genome of A. melissensis was sequenced using a combination of pair-end and mate-pair Illumina short reads (see Section "Material and Methods"). The best assembly obtained was 59.7 Mb long and estimated to be 88.9% complete using the program BUSCO (version 5.2.2) with the Nematoda_odb10 database (Table 2). This BUSCO score was very close to the score obtained for A. freiburgensis (89.1%) and A. rhodensis (89.8%) ( Table S3 in Supplementary Materials), which indicates that A. melissensis' genome contains most genes. Statistics on the A. melissensis genome are summarized in Table 2. Repeat annotation estimated that 4.8 Mb (8%) of the genome was repetitive (Table S6 in Supplementary Materials).

Nigon analysis and putative X scaffolds
We used the BUSCO output from A. melissensis to look at the profile of Nigon elements across the scaffolds. Most scaffolds with BUSCO genes corresponded to only one Nigon element (190/224, or 84.8%). Of the scaffolds containing BUSCO genes of several Nigons, 26 (11.6%), 6 (2.6%), and 2 (0.8%) had a mix between 2, 3, and 4 Nigon elements, respectively. The data was provided as an Excel data file in the attachment.
Using the Nigon element concept, it was possible to identify putative scaffolds of the X chromosome. We considered putative X scaffolds containing at least three orthologs pertaining to the Nigon X element (Table 3).

Phylogenetic position
The phylogenetic position of A. melissensis was determined relative to two other Auanema Auanema melissensis is placed as a sister taxon to A. freiburgensis, with A. rhodensis as an outgroup (Fig. 5). This phylogenetic position is the same as that previously reported, which was  constructed based on rDNA and RNA polymerase II sequences (Kiontke et al., 2007;Kanzaki et al., 2017;Winter et al., 2017).

Discussion
In this study, we report the genome and some biological characteristics of a non-described freeliving nematode species, A. melissensis. This nematode shares many similarities with A. rhodensis and A. freiburgensis: it is trioecious, has a small genome (~60 Mb), and has fewer genes than C. elegans (11,040). Crosses between A. melissensis and A. rhodensis or A. freiburgensis did not result in viable progeny, indicating reproductive isolation. The phylogenetic position based on the BUSCO single-copy orthologs places A. melissensis within Auanema, with A. freiburgensis as its closest relative. Nevertheless, A. melissensis has the same male tail morphological characteristics as A. rhodensis. This result indicates that A. melissensis can be regarded as a cryptic species of A. rhodensis, and can be distinguished from it with mating experiments and molecular sequences. Previous studies have also demonstrated other subtle differences in male sperm size (smaller) and sperm content (the presence of retained tubulin) in A. melissensis (aka Rhabditis sp. Table 3. Putative X scaffolds. Scaffolds containing at least 3 Nigon X BUSCO genes were considered putative X scaffolds.

Scaffolds
Number  (2071) were used to build a concatenated alignment, which was subsequently used to construct the phylogenetic tree. The supermatrix alignment was 1,018,758 amino acids in length.
JU1783) compared to those of A. rhodensis and A. freiburgensis (Winter et al., 2017). Similar to other Auanema species, the dauer larvae, a non-feeding developmental arrested stage usually linked to stress resistance and dispersal, obligatorily develops into a hermaphrodite adult (Félix, 2004;Chaudhuri et al., 2011a;Zuco et al., 2018;Robles et al., 2021). Similar to A. freiburgensis, crowding cues perceived by A. melissensis hermaphrodite mothers influence the sexual fate of their progeny (Zuco et al., 2018;Robles et al., 2021). In the absence of such cues, mothers produced primarily males and females. These outcrossing individuals can bring together new combinations of alleles that may increase fitness. The production of hermaphrodite-fated offspring, which obligatorily pass through the mobile and dispersive dauer stage, can colonize a new habitat without the need to find a mate. In A. freiburgensis, the molecular mechanisms controlling this maternal non-Mendelian inheritance involve the modulation of energy-sensing signaling activation of AMPK, downregulation of the insulin signaling (inhibition of daf-18), inhibition of the intracellular nutrient sensor mTORC1 complex and enhanced histone acetylation increase the proportion of hermaphrodite offspring (Robles et al., 2021). We hypothesize that similar mechanisms would be at play in A. melissensis.
In A. freiburgensis, it has been proposed that females and hermaphrodites may play different roles in the life cycle, with each sexual morph exhibiting adaptations specific to their part, stabilizing trioecy in the population (Adams et al., 2022). A. freiburgensis hermaphrodites invest resources in the expansion of the intestine, the major metabolic organ, which may enable them to meet the high energy cost of dispersal and reproduction (Adams et al., 2022). In contrast, the obligate outcrossing female diverts resources from intestinal development to invest in mate-finding behavior (Adams et al., 2022). Here we show that the sexual morphs of A. melissensis exhibit similar developmental differences. Unmated A. melissensis females are significantly shorter, with lesser developed intestines, than age-matched hermaphrodites, consistent with females limiting investment in growth and development. Further future analysis of sexual morph specialization in A. melissensis could help uncover how trioecy persists in the Auanema genus.
In this report, we also identified putative X chromosome scaffolds in the A. melissensis genome, using the concept of Nigon elements. Nigon elements are defined as groups of genes originally found on the seven ancestral chromosomes of Rhabditida (Tandonnet et al., 2019;Gonzalez de la Rosa et al., 2021). By finding A. melissensis orthologs to Nigon X genes it was possible to identify possible X chromosome scaffolds. This is particularly relevant in the Auanema species, as segregation and inheritance peculiarities concerning the X chromosome were observed in the related species A. rhodensis. In A. rhodensis, during the meiosis of XX hermaphrodites, the X homologs do not recombine either during oogenesis or spermatogenesis resulting in the production of nullo-X oocytes and 2X sperm (Tandonnet et al., 2018). This is in contrast with A. rhodensis XX females for which the X homologs pair and recombine during meiosis (Tandonnet et al., 2018). The X chromosome is also inherited from the father by the son in the event of a cross (female-male or hermaphrodite-male) due to the asymmetric partitioning of the cytoplasm during male spermatogenesis resulting in only X-bearing sperm being produced (Shakes et al., 2011;Tandonnet et al., 2018;Al-Yazeedi et al., 2022). To this date, the mechanisms controlling the unusual X chromosome segregation are unknown. Testing whether other trioecious members of the Auanema genus undergo similar processes and their possible consequences would be an interesting future avenue of research and the genomic X sequences are an important basis for such exploration.

Consent for Publication
Not Applicable.

Ethics Approval and Consent to Participate
Not Applicable.

Availability of Data and Materials
The raw data are available using the accession number PRJEB51845. The genome assembly and annotations are available using the GenBank accession number GCA_943334845.

Author's Contributions
ST designed the study, wrote the manuscript, assembled, and annotated the genome, and performed the Nigon, phylogenomics and statistical analyses. AT and SD performed the experiments showing the species is trioecious and different from A. rhodensis and A. freiburgensis. PP extracted the RNA and maintained the laboratory strain. MH carried out the experiments on the effect of the crowding cue on the sex ratios, took the male, female and hermaphrodite pictures and prepared the museum specimens. SA took the pictures, conducted the time course, and determined the differences in gut pigmentation between females and hermaphrodites. NK took pictures of the male tail and annotated its morphology. TG designed the study, extracted the DNA, and sequenced the genomic libraries. IN and MAF discovered and isolated the strain. APS designed the study and wrote the manuscript.

Literature Cited
Supplementary Materials Table S1. Genomic and transcriptomic raw data used in this study.      For each replicate, we calculated the percentage of each sexual morph produced. Figure S1: Diagram of the experimental design to test if the crowding cues experienced by hermaphrodite mothers change the sex ratios of the F1 generation. Dauer larvae were isolated onto five 6 cm plates under each condition (control and crowded) and left to mature into a hermaphrodite. F1 eggs were then collected and placed individually onto non-treated 3.5 cm plates. Egg collection occurred >3 d and hermaphrodite mothers were moved to new plates (of their original condition) each day. Eggs were allowed to develop until adulthood and sexed.