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. 2023 Jul 1;15(7):evad122. doi: 10.1093/gbe/evad122

The Transcriptomic Signature of Cyclical Parthenogenesis

Trung Viet Huynh 1,1, Alexander S Hall 2,2, Sen Xu 3,1,✉
Editor: Sarah Schaack
PMCID: PMC10340444  PMID: 37392457

Abstract

Cyclical parthenogenesis, where females can engage in sexual or asexual reproduction depending on environmental conditions, represents a novel reproductive phenotype that emerged during eukaryotic evolution. The fact that environmental conditions can trigger cyclical parthenogens to engage in distinct reproductive modes strongly suggests that gene expression plays a key role in the origin of cyclical parthenogenesis. However, the genetic basis underlying cyclical parthenogenesis remains understudied. In this study, we characterize the female transcriptomic signature of sexual versus asexual reproduction in the cyclically parthenogenetic microcrustacean Daphnia pulex and Daphnia pulicaria. Our analyses of differentially expressed genes (DEGs), pathway enrichment, and gene ontology (GO) term enrichment clearly show that compared with sexual reproduction, the asexual reproductive stage is characterized by both the underregulation of meiosis and cell cycle genes and the upregulation of metabolic genes. The consensus set of DEGs that this study identifies within the meiotic, cell cycle, and metabolic pathways serves as candidate genes for future studies investigating how the two reproductive cycles in cyclical parthenogenesis are mediated at a molecular level. Furthermore, our analyses identify some cases of divergent expression among gene family members (e.g., doublesex and NOTCH2) associated with asexual or sexual reproductive stage, suggesting potential functional divergence among gene family members.

Keywords: meiosis, Daphnia, cell cycle, asexuality, ameiosis

Significance.

In some eukaryotic species, individuals can alternate sexual or asexual reproduction depending on the environmental conditions (i.e., engaging in cyclically parthenogenetic reproduction). However, the genetic mechanisms underlying cyclical parthenogenesis remain understudied. This study reveals the gene expression changes associated with sexual and asexual reproduction in a cyclical parthenogen, the microcrustacean Daphnia pulex.

Introduction

The establishment of sexual reproduction is a defining event of early eukaryotic evolution (Cavalier-Smith 2002). It is characterized by the (1) evolution of meiosis, (2) uniparental transmission of the organelle genome, (3) selective cell–cell fusion of gametes, and (4) the regulation of diploid resting spore formation by such coupling (reviewed in Goodenough and Heitman 2014). In the ∼2.5-billion-year evolution of eukaryotes, most eukaryotic lineages remain sexual in spite of the evolutionary costs of sex (Otto 2009). However, the transition from sexual, meiotic reproduction to parthenogenetic reproduction has occurred independently in many phylogenetic groups except birds and mammals (Bell 1982; Simon et al. 2003; Neiman et al. 2014). With ∼1 in every 1,000 eukaryotic species reproducing parthenogenetically (Vrijenhoek 1998), chromosomally unreduced gametes can occur through various, distinct modified forms of meiosis (Maynard Smith 1978; Suomalainen et al. 1987; Neiman et al. 2014).

Interestingly, in contrast to obligately asexual reproduction where sex is completely abandoned, some eukaryotic lineages (e.g., rotifers, aphids, and cladoceran crustaceans) have evolved cyclical parthenogenesis that can confer the evolutionary benefits of both sexual and asexual reproduction (Bell 1982). Cyclically parthenogenetic females can alternate between sexual and asexual reproduction depending on the environmental conditions. The fact that the same genome can engage in meiotic or parthenogenetic production of gametes under different environmental conditions strongly suggests that environment-mediated transcriptomic changes play a critical role in the origin of cyclical parthenogenesis. However, to date, the transcriptomic signatures associated with the distinct phases of cyclical parthenogenesis remain unclear, resulting in a lack of understanding of the possible underlying genetic mechanisms.

In this study, we experimentally investigate transcriptomics of cyclical parthenogenesis in cladoceran microcrustaceans with Daphnia as a representative. There are about 600 species within the order Cladocera, occupying all kinds of aquatic habitats: freshwater ponds, lakes, and brackish and marine waters (Korovchinsky 1996; Forró et al. 2008). Although in other major crustacean clades, the emergence of asexual lineages does occur, for example, asexual ostracods (Butlin et al. 1998), all cladoceran species reproduce through cyclical parthenogenesis with some lineages even having become obligately asexual. This suggests that cyclical parthenogenesis is a derived trait in the Cladocera and the underlying genetic mechanism is most likely conserved in all cladoceran lineages, although lineage-specific modifications are possible.

Daphnia is one of the best known cladocerans because of its worldwide distribution in freshwater habitats. Daphnia females reproduce asexually under favorable environmental conditions (e.g., excellent food availability and low population density), producing numerous subitaneous eggs that directly develop into neonates in the females’ brood chamber (fig. 1A). The production of subitaneous eggs occurs through an ameiotic cell division in the germline. In this ameiotic division, the meiotic cellular machinery is modified so that recombination is suppressed, meiosis I is finished prematurely before the onset of anaphase I without the segregation of homologous chromosomes into daughter cells, and no cytokinesis occurs at the end of meiosis I (Ojima 1958; Zaffagnini and Sabelli 1972; Hiruta et al. 2010). Therefore, the ameiotic division is mitosis-like, leading to the production of genetically identical daughters (barring spontaneous mutations).

Fig. 1.

Fig. 1.

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(A) Cyclically parthenogenetic life history of Daphnia. (B) Female Daphnia at the four reproductive stages where RNA was collected. Reproductive stages were identified based on the size, color, and texture of the ovaries (ovals were drawn around the region containing ovaries). (C) PCA of the transcriptomic data.

The sex determination of the asexually produced offspring is environmentally controlled. Stressful conditions (e.g., high population density) and associated release of juvenoid hormone methyl farnesoate can trigger the male developmental program in the asexual progeny (Olmstead and Leblanc 2002). Therefore, males only appear in the Daphnia population under environmental stress. Furthermore, environmental stress also stimulates female Daphnia to switch to sexual production, producing eggs through meiosis (fig. 1A). Upon fertilization by sperm, the meiotic eggs (usually two such eggs are produced) develop into dormant embryos that are encapsulated in a protective case (ephippium). These dormant embryos can sustain harsh environmental conditions (i.e., desiccation of habitats) and hatch under suitable conditions.

Environmental conditions are thus the primary driver of reproduction mode and sex determination (two related but distinct aspects of reproduction) in Daphnia. For environmental signals to direct the choice of reproductive mode, environment-mediated gene expression changes most likely play a pivotal role in initiating different reproductive modes. To identify the genetic mechanisms underlying cyclical parthenogenesis in Daphnia, we examine gene expression profiles in two species of the North American Daphnia pulex species complex, D. pulex and Daphnia pulicaria. Although they share the same taxonomic name as the European D. pulex and D. pulicaria, they are distinct lineages (Cornetti et al. 2019), and we refer only to the North American D. pulex and D. pulicaria in this study. These two species are considered an ecological species pair which started their divergence about 0.8–1.2 million years ago (Omilian and Lynch 2009). Despite their extremely similar morphology and overlapping distribution range (Brandlova et al. 1972; Benzie 2005), they show habitat segregation and distinct ecological attributes (Dudycha and Tessier 1999), with D. pulex exclusively living in ephemeral ponds and D. pulicaria inhabiting stratified permanent lakes.

Because environment can trigger Daphnia females to alternate between meiotic and ameiotic production of eggs, we hypothesize that meiotic and ameiotic division are characterized by distinct gene expression profiles of meiosis and cell cycle genes. Furthermore, as Daphnia genomes contain many gene duplicates involved in meiosis and the cell cycle (Schurko et al. 2009), we ask whether these duplicates have divergent expression patterns in the meiotic and ameiotic reproduction phase, which would indicate functional divergence. To answer these questions, we assessed the transcriptomes of early and late stages of meiotic versus ameiotic females in multiple natural isolates of D. pulex and D. pulicaria. Our transcriptome data across isolates and species reveal conserved transcriptomic signatures that clearly distinguish meiosis and ameiosis in Daphnia.

Results

Overview of Transcriptomic Data

To discover the transcriptomic programs that associate with the different reproductive modes in Daphnia, we collected transcriptomic data from four distinct reproductive stages—early meiosis (EM), late meiosis (LM), early ameiosis (EA), and late ameiosis (LA) (fig. 1B). Furthermore, to identify the common genetic mechanisms underlying cyclical parthenogenesis in Daphnia, we sampled multiple isolates from two Daphnia species (three isolates in D. pulex and two in D. pulicaria), yielding a total of 60 RNA-seq samples (see supplementary fig. S1 and supplementary table S2, Supplementary Material online).

We visualized the transcriptomic variation in our dataset by conducting a principal component analysis (PCA) using normalized RNA-seq read counts from the top 500 most variable genes (fig. 1C). The first principal component, largely corresponding to interspecific variation in our dataset, separates the two species and explains 27% of the total variance. The second principal component explains 16% of the total variance. Although a global separation of the reproductive modes and timepoints is not obvious at first sight, when concentrating on each genotype one by one, we can see a separation between the two reproductive modes (meiosis vs. ameiosis). Within each genotype, we also see the presence of a clear clustering for each timepoints (EA vs. LA and EM vs. LM), showing the existence of transcriptomic differences among the different stages. Separation between reproductive modes is greater than the separation observed between different timepoints of the same reproductive mode. This is especially true of the samples at meiotic stages, in which EM and LM show strong clustering. These observations validate that our data capture the interspecific and between-isolate transcriptomic diversity as well as the transcriptomic signatures of distinct reproductive stages.

Transcriptomic Signature of EM versus EA

Because there is no clear “control” or “treatment” group in the analysis below, we referred to differentially expressed genes (DEGs) as having higher expression in meiosis (i.e., upregulated during meiosis) or higher expression in ameiosis (i.e., downregulated during meiosis). Differential expression analysis between the early stage of meiosis and ameiosis (EM vs. EA) in each genotype resulted in a consensus set of 455 genes with higher EA expression and 319 with higher EM expression (fig. 2A; see supplementary table S3, Supplementary Material online for gene list). After mapping these consensus DEGs to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (304 annotated genes out of 774 mapped to 326 pathways), we found a striking trend that nearly all DEGs in meiosis and cell cycle–related pathways have higher expression in EM when compared with EA (figs. 2B and 3), clearly deviating from a random distribution of down- and upregulated DEGs (one proportion t-test with Holm correction P = 3.31 × 10−6). For example, all 18 DEGs in the cell cycle pathway had higher expression during in EM (supplementary fig. S2, Supplementary Material online), and 12 genes in the progesterone-mediated oocyte maturation pathway had higher expression during EM, whereas only 1 gene (protein kinase A) had lower expression in EM (supplementary fig. S3, Supplementary Material online). An equally noticeable pattern is that DEGs mapped to metabolic pathways had overwhelmingly lower expressions in EM compared with EA (figs. 2B and 3), again deviating from the random distribution of up- and downregulated DEGs (one proportion t-test with Holm correction P = 1.46 × 10−3).

Fig. 2.

Fig. 2.

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(A) Venn diagrams showing the number of DEGs in each genotype for the comparison between EM versus EA and LM versus LA. The top panels represent the number of DEGs with higher expression in ameiosis, whereas the bottom panels show DEGs with higher expression in meiosis when the two reproductive modes were compared against each other at similar timepoints. (B) Distribution of consensus genes among the top enriched KEGG pathways. (C) Alluvium graph illustrating the direction of differential expression of consensus DEGs that are concordant among all genotypes between early and late stages.

Fig. 3.

Fig. 3.

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Expression heat map of KEGG pathways enriched with the consensus set of DEGs.

These observations from consensus set of DEGs are supported by KEGG pathway and gene ontology (GO) term enrichment analyses using DEGs identified through our analyses with pooled samples. Pathways involved in meiosis and cell proliferation such as the oocyte meiosis pathway (P = 3.85 × 10−5) and cell cycle pathway (P = 5.04 × 10−11) were significantly enriched with DEGs with higher expression during EM compared with EA (fig. 4A). The higher expression of meiosis and cell cycle elements during EM was further supported by the enrichment of the DNA replication initiation (P = 0.011) and cell division (P = 0.097) GO terms during EM (fig. 4B). On the other hand, pathways and GO terms that were found to be enriched with DEGs with lower expression during EM were metabolism related. Among the metabolic pathways that were most enriched with DEGs with lower EM expression were pathways involved in sugar metabolism (P = 0.0013), especially amino sugars like uridine diphosphate (UDP) that are important in chitin metabolism (supplementary fig. S4, Supplementary Material online). In addition to metabolic pathways, we also observe the hormone signaling renin–angiotensin pathway (P = 0.071) containing substantially lowered expression during EM compared with EA (supplementary fig. S5, Supplementary Material online).

Fig. 4.

Fig. 4.

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Functional analysis of DEGs from the pooled sample analysis contrasting meiosis against ameiosis, including significantly enriched KEGG pathways at (A) early and (C) late stages (vertical dash line represents no expression change) and top 10 enriched GO terms in (B) early stage and (D) late stage analysis.

Taken together, these results strongly suggest that the onset of meiotic reproduction is associated with higher expression of meiosis and cell cycle genes and with lower expression of metabolic genes, whereas the transcriptomic signature of early ameiotic reproduction is lower expression of meiosis and cell cycle genes and higher expression of metabolic genes (fig. 3).

Transcriptomic Signature of LM versus LA

The consensus DEGs for the comparison between LM and LA stage consisted of 98 genes with higher LM expression and 71 genes with higher LA expression (fig. 2A; see supplementary table S4, Supplementary Material online). Although this represents a 4- to 5-fold decrease in the number of consensus genes compared with the analysis of early reproductive stages, we noted that a total of 51 and 20 genes maintained their differential expression pattern throughout both the early and late stages of ameiosis and meiosis (fig. 2C), respectively, including the persistent ameiotic-biased (higher expression found in ameiotic reproduction) expression of methyl farnesoate epoxidase and the persistent meiotic-biased (higher expression during meiotic reproduction) expression of never in mitosis gene A (NIMA)–related kinase 7. The former is an important enzyme in the methyl farnesoate signaling pathway, whereas the latter is an important regulator of mitosis (Fry et al. 2012).

Along with this decrease of consensus genes, KEGG enrichment analysis with pooled samples also showed fewer enriched pathways from 27 in the early stage analysis to only 12 pathways at late reproductive stages. This marked decrease is largely due to the loss of enriched pathways (from 12 during early stages to 1 during late stages) that are enriched for higher expression during meiosis (fig. 4A and C). Our analyses reveal that metabolic pathways are still enriched with genes exhibiting high expression during LA when compared with LM (fig. 4C).

The only pathway that was enriched of genes with higher expression during LM is the extracellular matrix (ECM)–receptor interaction pathway (fig. 4C). This is consistent with the loss of the cell division and DNA replication GO terms that were found during EM using pooled sample analysis, although oogenesis remains in the top ten enriched GO terms (not significant after correcting for multiple comparisons) (fig. 4B and D). Instead, we observed that many amino acid, lipid, and complex carbohydrate catabolism- and biosynthesis-related GO terms were enriched with DEGs possessing higher expression during LM (fig. 4D).

Thus, although transcription is strikingly less conserved among genotypes during the late stages of the meiotic and ameiotic cycles (i.e., lower number of consensus genes), many of the same metabolic and hormone (e.g., renin–angiotensin) pathways persistently had higher expression throughout the EA and LA stages.

Differential Expression within Gene Family

In total, 6,443 genes were placed into 1,691 gene families. Although more than half of these families consist of only two genes (i.e., paralogs), around 13% of families are more expansive and contain over five genes (supplementary fig. S6, Supplementary Material online). These gene families containing over five genes represent broad biochemical functional groups rather than paralogs and were therefore excluded from downstream analysis. The results presented below are solely based on our analyses using pooled samples.

Although our analysis of the expression patterns of genes in each family revealed no differential expression in 656 (44.8%) and 879 (60%) gene families in early and late stage analyses, respectively (fig. 5A and B), 2 notable divergent expression patterns within gene family were identified. In the remaining gene families with at least one gene showing differential expression, most consisted of both DEGs and non-DEGs members (represented in regions marked with an arrow followed by a dash in fig. 5A and B). For example, 349 (19.9%) and 292 (23.8%) gene families contained member genes that were differentially expressed and some nondifferentially expressed member genes in EA and EM, respectively; 330 (22.5%) and 164 (11.2%) gene families had at least one differentially expressed member gene and at least one nondifferentially expressed member gene in LA and LM, respectively.

Fig. 5.

Fig. 5.

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Venn diagrams showing gene families of different differential expression directions between member genes in early stage (A) and late stage (B) analysis. (C) Dot plots representing gene families with a bidirectional expression pattern in the early stage comparison. Expression patterns at the late stage are similar to the early stage shown here. Each gene is plotted by its log2 fold change, and significance is defined as adjusted P value < 0.05 and fold change > 1.5. DEG, differentially expressed genes.

Furthermore, some of these gene families exhibiting divergent expression contained some genes with significantly increased expression during ameiosis and other genes with significant differential expression in a different direction (i.e., increased expression during meiosis). We will refer to this divergent expression pattern as bidirectional expression (represented in regions containing two arrows in opposing directions in fig. 5A and B). In total, 96 unique gene families showed this bidirectional expression pattern in at least one stage of reproduction (supplementary table S5, Supplementary Material online). Fifty families were found exhibiting bidirectional expression exclusively at early stages and 24 at late stages, and 22 families maintained this pattern at both early and late stages. Especially interesting are the gene families involved in upstream transcriptional and signaling processes that have a paralog whose expression was specific to each reproductive cycle (fig. 5C).

Two such gene families represent transcription factors—DMRT4_5 (doublesex gene) and TFIIB (transcription factor II B). Daphnia possesses two copies of the doublesex gene with one copy (gene15158) having higher expression during the sexual cycle and the other (gene4231) with higher expression during the asexual cycle. A previous study has identified one copy of the doublesex gene to be essential to sex determination in Daphnia, whereas the function of other copy remains unknown (Kato et al. 2011). Furthermore, another family with this bidirectional expression pattern is the NOTCH2 receptor in the NOTCH signaling pathway (fig. 5C), previously found to modulate reproduction in another arthropod system (Duncan et al. 2016). We also identified three gene families related to the glutamate and GABA signaling, including two neurotransmitter receptors (i.e., GRIK1 and GABRB) and a modulator of neuroreceptors (i.e., DBI).

On the other hand, we found a small percentage of gene families whose member genes were all differentially expressed in the same direction between ameiosis and meiosis (represented by the region described as unidirectional and marked with a single arrow in fig. 5A and B). For gene families whose expressions were higher during early (60 gene families) and late ameiosis (37 families) stages, they contain 18 overlapping gene families. For gene families with higher expression in early (36 families) and late meiosis (9 families) stages, 6 gene families were in common. Consistent with the general trend identified for enriched pathways, most gene families with higher expression in ameiosis were associated with digestion and metabolism (with genes such as alpha-amylase, lactase-phlorizin hydrolase, and alpha-l-fucosidase), whereas gene families with higher expression in meiosis included several cyclins and other cell cycle regulators (supplementary table S6, Supplementary Material online and supplementary File 1, Supplementary Material online).

Discussion

Cyclical parthenogenesis represents a novel reproductive phenotype during the evolution of eukaryotes. Cladoceran crustaceans are the only clade in Crustacea that exclusively reproduce by cyclical parthenogenesis (McLaughlin 1980). To understand the genetic mechanisms underlying the evolution of cyclical parthenogenesis from sexual reproduction, we investigate the transcriptomic signature of sexual (meiotic) versus asexual (ameiotic) reproduction stages in two Daphnia species, D. pulex and D. pulicaria.

A novelty of this study is that we distinguish and compare the transcriptomes of early and late stages of meiotic versus ameiotic female D. pulex and D. pulicaria, which has not been done in previous studies (e.g., Raborn et al. 2016; Zhang et al. 2016). This strategy allows us to investigate the transcriptomic signature close to the initiation of sexual/asexual reproduction and assess the persistence and divergence of transcriptomic signatures as Daphnia females advance in reproductive state. Furthermore, as transcriptomes are heavily affected by environment and genotype interaction, we use a consensus and pooled analytic approach across the five genotypes of D. pulex and D. pulicaria to remove transcriptomic noise and unveil the conserved transcriptomic program associated with the initiation of sexual/asexual reproduction. We focus on genes annotated with a KEGG ortholog (KO) number or GO term to understand how transcriptional changes in these genes may be associated with reproductive mode switching.

Taking advantage of these experimental and analytical procedures, this study provides novel insights into the transcriptomic signature associated with sexual versus asexual reproduction in Daphnia, which has not been observed in previous studies assessing transcriptomes of parthenogens (Hanson et al. 2013; Srinivasan et al. 2014; Warren et al. 2018; Parker et al. 2019). In comparison with ameiosis (i.e., asexual reproduction), the early stage of meiotic, sexual reproduction features genes with higher expression being enriched in meiosis and cell cycle pathways and genes with lower expression were enriched in metabolism pathways. In contrast, early asexual reproduction (e.g., ameiosis) is characterized by lower expression of genes in meiosis and cell cycle pathways and higher expression of genes in metabolism pathways.

Although our RNA-seq data are derived from female whole-body tissue, these patterns provide important clues as to genetic mechanisms underlying cyclical parthenogenesis in Daphnia. Daphnia females deposit all primary germ cells in the posterior end of the ovary (Kato et al. 2012), and several primary germ cells can embark on either meiotic or ameiotic division as the females enter sexual or asexual reproduction. It is likely that the up- and downregulation of meiosis and cell cycle genes are involved in determining the oogenesis pathway of primary germ cells. Among the underexpressed meiosis genes in asexual stage, we note an interesting candidate gene Cdc20, which is a member of the anaphase promoting complex and spindle checkpoint assembly. Cdc20 plays an important role in promoting homologous chromosome segregation and the onset of anaphase and in maintaining ploidy during meiosis (Yin et al. 2007; Jin et al. 2010; Cooper and Strich 2011). The underexpression of Cdc20 in the ameiosis of Daphnia could be primarily responsible for the observed cytological observations of no homologous chromosome segregation and no cytokinesis in ameiosis I.

Furthermore, the up- and downregulation of transcription related to metabolism in early asexual and sexual reproduction could be due to the food quality and availability in favorable and adverse environmental conditions. Because it is well known that nutrients and amino acids play an important role in regulating gene expression (Moir and Willis 2013; Haro et al. 2019), we suggest that these metabolism- and biosynthesis-related DEGs may contain the master regulator controlling the reproductive mode of Daphnia. Pinpointing the master regulators will be highly interesting but certainly remains a technically challenging task as it would involve intricate gene manipulation to modify the gene expression levels of genes of interests.

We note that the underexpression of meiosis genes and overexpression of metabolism genes in the EA for production of directly developing embryos in the cyclically parthenogenetic Daphnia is highly similar to the transcriptomic profile of early resting embryo production in obligately parthenogenetic Daphnia (Xu et al. 2021). Obligately parthenogenesis in D. pulex is distinct from cyclical parthenogenesis in that females parthenogenetically produce resting embryos, rather than through sexual reproduction. Notably, these obligately parthenogenetic D. pulex originated through complex hybridization and backcrossing between cyclically parthenogenetic D. pulex and D. pulicaria. By comparing the transcriptomes of early parthenogenetic resting embryo production in obligate parthenogens with that of the cyclically parthenogenetic parental species D. pulex and D. pulicaria (i.e., meiosis), Xu et al. identified that the early embryo production in obligate parthenogens is characterized with underexpression of meiosis and cell cycle genes and overexpression of metabolism genes compared with the parental species. This observation coincides well with the cytological evidence that parthenogenetic production of resting embryo and parthenogenetic production of directly developing embryos in obligately parthenogenetic Daphnia share the same ameiotic cell division (Zaffagnini and Sabelli 1972).

Lastly, our analyses of the transcriptional differences between members of the same gene families provide initial evidence of possible functional divergence of gene duplicates. For example, we identified that paralogs of the doublesex, TFIIB, and NOTCH2 receptor gene show meiosis- and ameiosis-specific upregulation, indicating possible neofunctionalization of the paralogs. Detailed phylogenetic and functional analyses of these paralogs in Daphnia, cladocerans, and other crustacean outgroup lineages would be necessary to determine whether these paralogs experienced neofunctionalization.

Materials and Methods

Daphnia Culture and Sampling

Three isolates of cyclically parthenogenetic D. pulex and two of D. pulicaria were used in this study to identify the transcriptomic changes associated with ameiotic (asexual) versus meiotic (sexual) reproduction in females. These isolates were originally collected from ephemeral ponds and lakes in North America (supplementary table S1, Supplementary Material online). Each isolate was maintained as a clonal culture in COMBO artificial lake water (Kilham et al. 1998) at 18 °C under a 12:12 h light/dark photoperiod and was fed with the algae Scenedesmus obliquus.

Collection of Females at Different Reproductive Stages

Under a dissection microscope, we collected females of each isolate at EA, LA, EM, and LM stages (fig. 1B) that show distinct size, color, and texture of the ovary (Rossi 1980). Females at EA stage possessed ovaries that are small, light, and clear. Ovaries at EM stage have a creamy color in contrast to the clear ovaries of EA. Ovaries of LA and LM stages are large and dark, but they can be distinguished from each other by their texture. Ovaries in LM are smooth because they typically only contain two embryos, whereas LA ovaries have a lumpy texture due to the presence of many embryos. Some Daphnia females were carrying offspring from a previous clutch in their brood pouch. In such cases, those offsprings were removed from the brood pouch after the whole female was suspended in RNAlater® (ThermoFisher), and the offsprings were excluded from the RNA extraction step. For each Daphnia isolate, three biological replicates of around 20 females each were collected for each reproductive stage, yielding a total of 60 whole-body samples for RNA-seq.

To eliminate the impact of environmental factors on gene expression, all the isolates were acclimatized to the same culture conditions for two generations before we started collecting females. All isolates were exposed to crowding at a concentration of around 30 animals per 25 mL to induce some of the females to enter the sexual cycle. Both sexual and asexual females were sampled from the same culture where they coexisted, minimizing the impact of environmental conditions on our gene expression data.

RNA Isolation, Library Preparation, and Sequencing

We extracted total RNA from each sample using the Quick-RNA tissue/Insect RNA extraction kit (Zymo Research). The RNA quality of each sample was assessed on an Agilent Bioanalyzer. We purified mRNA from each total RNA sample using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) and prepared sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit following the manufacturer's instruction. Sequencing of the libraries was performed on an Illumina HiSeq2500 platform with 150 bp paired-end reads. The raw data are deposited at NCBI SRA PRJNA764929.

Transcriptomic Analyses of Ameiosis versus Meiosis

The raw transcript abundance of genes in each RNA-seq sample was quantified using the quasi-mapping approach in the software package Salmon (Patro et al. 2017) on its default parameters, with D. pulex PA42-3.0 transcriptome (Ye et al. 2017) as the reference. To validate whether our transcriptomic data correctly captured the transcriptomic diversity of different reproductive stages, isolates, and species, we performed PCA using transcript count data normalized in the software DESeq2 (Love et al. 2014). The PCA was performed with the plotPCA function of the DESeq2 package, using the top 500 genes with the most variance.

To characterize the transcriptomic profiles of ameiosis versus meiosis, we examined the gene expression differences contrasting early/late stage of meiosis versus ameiosis (i.e., EM vs. EA and LM vs. LA) and contrasting early and late stage within meiosis and ameiosis by pooling all the Daphnia genotypes. Specifically, we identified the DEGs for each of the four reproductive stage comparisons, using the Wald negative binominal test with the design formulae ∼clone + stage in DESeq2 (Love et al. 2014). A gene was considered differentially expressed if it had an adjusted P value < 0.05 (Benjamini and Hochberg method) and a fold change > 1.5.

In addition to the analyses based on pooling all sampled genotypes, we also identified DEGs contrasting EM versus EA and LM versus LA in each individual genotype. We then generated a consensus set of the genes that are differentially expressed in the same comparison across all the examined Daphnia isolates and species.

Enrichment Analysis of KEGG Pathways and GO Terms

Mapping DEGs into functional pathways can provide stronger power for making biological discoveries compared with examining the functional significance of individual genes alone. We therefore produced KEGG pathway maps to visualize how DEGs may interact and to understand their collective functional significance.

First, we reconstructed the KEGG pathway maps for the D. pulex PA42-3.0 transcriptome (Ye et al. 2017). We queried all the protein sequences in the KEGG Automatic Annotation Server (KAAS) using the GHOSTX program (Moriya et al. 2007). A total of 10,135 out of the 18,440 genes in the transcriptome were assigned a KO number, and 6,282 genes annotated with a KO number were placed to a KEGG pathway map.

In order to increase the power to detect biologically relevant pathways, up- and downregulated DEGs were analyzed separately (Hong et al. 2014). We identified the KEGG pathways enriched for up- or downregulated genes using hypergeometric tests with Holm–Bonferroni corrected P values. Pathways significantly enriched for up- or downregulated genes (P < 0.05) were then visually inspected using the software Gene Annotation Easy Viewer (GAEV) (Huynh and Xu 2019), which displays DEGs on KEGG pathway maps with colors indicating the direction and magnitude of the differential expression.

Insight from KEGG pathways were supplemented with information from GO enrichment analysis to find differentially activated biological processes between reproductive stages. A total of 11,447 genes out of the 18,440 total genes were annotated with at least one GO term. All GO enrichment tests were performed using the R package topGO (Alexa and Rahnenfuhrer 2019) with the weight01 method, its default combination of elim and weight algorithms. P values were adjusted with the Holm method with a cutoff of 0.05.

Divergence of Expression within Gene Family

Divergence of expression within gene family associated with ameiosis and meiosis may indicate functional divergence between paralogous genes. We define a gene family as a group of at least two genes that have been annotated with the same KEGG ortholog number by the KAAS. We investigated whether genes within the same gene family are differentially expressed in the same direction between meiosis and ameiosis using the differential expression pattern of the consensus gene set.

Supplementary Material

evad122_Supplementary_Data
Click here for additional data file. (6.1MB, zip)

Acknowledgments

We thank the members of Xu Lab for help with the experiments and constructive discussion. We thank three anonymous reviewers for their constructive comments on an earlier version of this manuscript. This work is supported by NIH grant R35GM133730 to S.X.

Contributor Information

Trung Viet Huynh, Department of Biology, University of Texas at Arlington, Arlington, Texas, USA.

Alexander S Hall, Department of Biology, University of Texas at Arlington, Arlington, Texas, USA.

Sen Xu, Department of Biology, University of Texas at Arlington, Arlington, Texas, USA.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Author contributions

S.X. and A.S.H. designed this study. A.S.H. performed the tissue collection and molecular experiments. T.V.H. performed data analyses. T.V.H. and S.X. wrote the manuscript with input from A.S.H.

Data availability

The raw reads are deposited at NCBI SRA PRJNA764929. The scripts used in this study are available at https://github.com/UtaDaphniaLab/The-transcriptomic-signature-of-cyclical-parthenogenesis.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

evad122_Supplementary_Data
Click here for additional data file. (6.1MB, zip)

Data Availability Statement

The raw reads are deposited at NCBI SRA PRJNA764929. The scripts used in this study are available at https://github.com/UtaDaphniaLab/The-transcriptomic-signature-of-cyclical-parthenogenesis.

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