How well do rotifers invade populations? The interplay of sex propensity, migration rates and local genetic diversity

How well do rotifers invade populations? The interplay of sex propensity, migration rates and local genetic diversity | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 11 March 2025 V1 Latest version Share on How well do rotifers invade populations? The interplay of sex propensity, migration rates and local genetic diversity Authors : Cristina Arenas-Sánchez , Raquel Ortells 0000-0003-1389-603X [email protected] , María José Carmona , Eduardo M. García-Roger , and Javier Monterio-Pau 0000-0002-0864-8157 Authors Info & Affiliations https://doi.org/10.22541/au.174170730.00901673/v1 286 views 145 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Dispersal is a complex process that affects all living organisms, with the settlement phase being particularly critical. This phase depends on the interaction between the environmental conditions of the new habitat and the biological traits of both resident and immigrant populations. While these factors have been studied individually, their combined effects remain poorly understood. To address this gap, we conducted an invasibility experiment using the facultatively sexual rotifer Brachionus plicatilis as a model organism. We examined how the combination of migration rates, genetic diversity in the resident population, and the propensity for sexual reproduction in immigrants influence settlement success. Using whole-genome sequencing, we tracked immigrant private alleles to assess settlement. Our results indicate that all three factors –both individually and interactively–, significantly affect settlement success. Specifically, higher migration rates, lower genetic diversity in the resident population, and earlier sexual reproduction in immigrants were all associated with greater settlement success. Notably, high migration rates had the strongest impact when combined with early sexual reproduction in immigrants, while low genetic diversity in the resident population facilitated the settlement of immigrants with delayed sexual reproduction. These findings highlight the importance of considering multiple interacting factors to gain a more comprehensive understanding of dispersal dynamics. They also provide valuable insights into the ecological and evolutionary mechanisms governing the settlement in new habitats with pre-existing populations. Introduction Dispersal is a fundamental ecological and evolutionary process affecting all living organisms (Bonte & Dahirel 2017; Dieckmann et al. 1999; Havel & Shurin 2004). As an evolutionarily stable strategy, dispersal has been consistently favored throughout evolutionary history because it maximizes the likelihood of survival and reproductive success in contexts of high environmental variability (Bonte & Dahirel 2017; Comins et al. 1980; Dieckmann et al. 1999) and increases the chances of population persistence over time (Comins et al. 1980). This complex process involves three major phases ─departure, transfer and settlement─ influenced by individual and species traits as well as environmental factors (Bonte et al. 2012), that collectively interact to alter the cost and benefits of dispersal (Kolar & Lodge 2001; Montero-Pau et al. 2018; Tagg et al. 2005). While extensive research has examined the first two phases, the settlement phase remains less understood, particularly regarding how multiple factors interact to determine settlement success in new habitats (Kolar & Lodge 2001; Montero-Pau et al. 2018). Key factors such as propagule pressure (Richardson 2004), genetic diversity and reproductive strategies may be affecting settlement success. The number of migrants and the frequency of dispersal events increase the probability of successfully reaching a new habitat (Blackburn et al. 2016; Cassey et al. 2018; Vedder et al. 2021), thereby mitigating the effects of environmental filtering, demographic stochasticity and low genetic diversity in small founding populations (Simberloff 2009). In occupied habitats, local adaptation and the level of genetic diversity of the resident population pose additional challenges, with hybrid vigour or outbreeding depression playing pivotal roles (Ebert et al. 2002). While many studies have focused on the genetic diversity of migrants (Aguirre et al. 2013; Crawford & Whitney 2010; Gamfeldt et al. 2005; Hughes et al. 2019; Ottewell et al. 2014), fewer consider the genetic structure of resident populations. Finally, reproductive-related strategies such as high fecundity or early maturation strategies enhance colonization (Stevens et al. 2014). Also relevant to the success of dispersal is the mode of reproduction. In plants, for example, asexual (vegetative) reproduction serves as the primary mechanism for expanding established populations of perennials (Yang & Kim 2016). In variable environments, adaptive strategies should optimize the relationship between dispersal and sexual reproduction (e.g., sex-biased dispersal in vertebrates or amphicarpy in plants), since both strategies help to increase biological fitness (Auld & Rubio de Casas 2013). In this sense, facultative sexual organisms provide an exceptional framework for studying the relationship between dispersal and the investment in sexual or asexual reproduction (Kokko 2020), yet its role in dispersal remains understudied. Although some of these factors affecting success of the settlement phase of dispersal have been studied previously (e.g., Blackburn et al. 2016), they have not been examined in combination. To address this gap, a multifactorial approach is needed. We propose the facultatively sexual monogonont rotifer Brachionus plicatilis , as a model organism to study these factors in concert during settlement. Monogonont rotifers are common microinvertebrates typically found in the plankton of continental water bodies, where they play a crucial role in aquatic food webs (Serra et al. 2019; Starkweather 1987; Wallace et al. 2006). Like all monogonont rotifers, B. plicatilis exhibits cyclical parthenogenesis alternating an asexual and a sexual phase in their life cycle (Wallace & Smith 2009; Wallace et al. 2015). At temperate latitudes, rotifer populations are typically temporary, with their growing season (i.e., the period of time in which a rotifer population is active in the water column) starting from the hatching of diapausing eggs present in the sediment. The hatchlings of these eggs are diploid asexual females that reproduce by ameiotic parthenogenesis for several generations. During this asexual phase, populations are composed exclusively of asexual females that reproduce rapidly via clonal proliferation, producing genetically identical daughters through ameiotic parthenogenesis (Wallace et al. 2006). Consequently, rotifer populations can be conceptualized as assemblages of clones (Gómez & Carvalho 2000). In the genus Brachionus, the transition to the sexual phase is triggered by population density when the concentration of an infochemical ─secreted by the rotifers themselves─ reaches a critical threshold. This chemical induces asexual females to produce a proportion of their offspring as sexual daughters (Carmona et al. 1995; Carmona et al. 2011; Stelzer & Snell 2003). The sexual females produce haploid eggs which hatch into dwarf males if unfertilized but if fertilized become diapausing eggs that can resist adverse conditions (e.g., desiccation) and accumulate in the sediment (García-Roger et al. 2006). When favourable conditions resume, a fraction of the viable diapausing eggs will hatch. The remaining unhatched eggs form egg banks and can survive for decades in the sediment (Brendonck & De Meester 2003; García-Roger et al. 2006). Diapausing eggs are essential for surviving among growing seasons in temporary environments (García-Roger et al. 2019). Since the survival of a genotype is contingent upon the production of diapausing eggs, the total number of diapausing eggs produced is considered as a measure of between-season fitness (Campillo et al. 2011; Serra & King 1999). Interestingly, in this species, variation exists among genotypes regarding the propensity to reproduce sexually, resulting in different ecological strategies (Aparici et al. 2001; Carmona et al. 2009; Franch-Gras et al. 2017). Some genotypes reproduce sexually at low population densities, ensuring early diapausing egg production, while others delay sexual reproduction to maximize asexual proliferation (Aparici et al., 2001; Carmona et al., 2009; Franch-Gras et al., 2017). Whether this timing of sex influences settlement success remains an open question. Genetic diversity within a resident population could play an important role in the successful settlement of immigrants in rotifers, particularly in habitats colonized by only a few individuals (Montero-Pau et al. 2018). In natural rotifer populations genetic diversity decreases throughout the annual cycle due to clonal erosion (Carmona et al. 2009; Ortells et al. 2006). Intraclonal reproduction can lead to inbreeding depression in rotifers, whereas outbreeding might confer fitness advantages (Tortajada et al. 2009; Tortajada et al. 2010). Consequently, inbreeding depression in resident populations can create opportunities for the settlement of later immigrants. The hybrid offspring resulting from crosses between immigrants and residents might exhibit higher fitness, thereby promoting gene flow between populations. Diapausing eggs, due to their small size and resistance to desiccation, serve as effective propagules for dispersal (Arenas-Sánchez et al. 2023; Rivas et al. 2018; Rivas et al. 2019). The spread of diapausing eggs mediated by water or wind flows, or facilitated by animal vectors, is crucial in maintaining a metapopulation structure (Vanschoenwinkel et al. 2013). While zooplankton migration rates are generally assumed to be high, particularly through wind dispersal (Brendonck & Riddoch 1999; Cáceres & Soluk 2002; Cohen & Shurin 2003), recent research on rotifers has found that migration rates are not as high as expected but can increase substantially during the dry phases in temporary ponds (Arenas-Sánchez et al. in prep.; Bilton et al. 2001; Moreno et al. 2016; Tuytens et al. 2014; Vanschoenwinkel et al. 2008). For all these reasons, the success of the rotifer diapausing eggs that leave a population to settle in another water body may be influenced by specific factors, including (i) the number of immigrants, (ii) the genetic diversity of the resident population, and (iii) the likelihood of reproductive settlement of immigrants in the new habitat, all of which we specifically address in this paper. Building on these insights, we designed an experimental study to test how these three factors interact to influence the invasion success. We employed an experimental invasibility approach using clones of the rotifer B. plicatilis from two different locations—one serving as the resident population and the other as the invader, and tracking their frequencies using whole-genome sequencing. Our hypothesis is that higher migration rates, lower genetic diversity in the resident population, a higher propensity for sexual reproduction in immigrants, will increase the chances of settlement success. Material and methods Source populations and foundation of experimental clones In the experiments, we used B. plicatilis sensu stricto (hereafter B. plicatilis ) clones derived from diapausing eggs collected from the field in order to capture natural levels of genetic variation. Populations from two temporary ponds in Eastern Spain were sampled: Atalaya de los Ojicos (ATA), an inland endorheic pond in Albacete (38.462097 N, 1.254912 W), which was designated as the resident population, and Poza Sur (TOS), a coastal pond in the Prat de Cabanes-Torreblanca Marsh Nature Reserve, Castellón (40.089170 N, 0.101480 E), which served as the immigrant population. These populations were previously characterized in phylogeographic and population genetic studies and belong to different phylogeographic groups (Gomez et al. 2000). The upper sediment layer of the ponds was collected during a dry period and stored in the laboratory at 4 °C in the dark, until the start of the experiment. Then, diapausing eggs were extracted from the sediment using a sucrose flotation technique (Gómez & Carvalho 2000). Based on their morphology, putative diapausing eggs of B. plicatilis were isolated in polystyrene 96-well plates containing 200 μL saline water (Instant Ocean®, Aquarium Systems; 6 g L -1 salinity) and incubated under standard hatching conditions (i.e., constant illumination with PAR of 35 µmol quanta m -2 s -1 , and 25°C temperature (García-Roger et al. 2006) for 28 days. During this period, wells were checked daily for hatchlings and, when observed, 50 μL of rotifer culture medium was added. This consisted of 12 g L -1 salinity artificial seawater (Instant Ocean®, Aquarium Systems), enriched with f/2 medium (Guillard & Ryther 1962) and containing the microalga Tetraselmis suecica (Kylin) Butcher 1959 (Microalgae Culture Collection of IATS-CSIC) as food at a concentration of approx. 500,000 cells mL -1 . Since diapausing eggs are the result of sexual reproduction, each hatched female represents a unique genotype. Clone cultures were established after a few days of parthenogenetic reproduction of the asexual females. Rotifer clones were transferred to 13 mL culture medium and maintained as a stock culture at 20 °C under constant illumination (PAR: 35 μmol quanta m -2 s -1 ). These conditions (thereafter standard culture conditions) were used for algal cultures, the maintenance of rotifer stock clones, and experiments (see below). Since B. plicatilis belongs to a cryptic species complex (Gómez et al. 2002; Mills et al. 2017; Suatoni et al. 2006), and that some of these species coexist with B. plicatilis in the studied ponds (Montero-Pau et al. 2011), particular care was taken to ensure accurate species identification. Thus, morphological discrimination was used to differentiate B. plicatilis clones among coexisting morphotypes (Ciros-Pérez et al. 2001; Dimas-Flores et al. 2013), and RFLP analysis of a fragment of the mitochondrial COI gene to discriminate species within the same morphotype (Franch-Gras et al. 2017). A total of 30 clones from TOS and eight clones from ATA were founded and maintained in stock cultures under standard conditions until their use in the experiments. Propensity for sexual reproduction bioassays The propensity to reproduce sexually of all the clones used in the experiment was studied by conducting bioassays to estimate the density threshold for sexual reproduction initiation. A total of 114 bioassays (38 clones x 3 replicates) were performed following procedures adapted from Carmona et al. (2009) and Franch-Gras et al. (2017). Rotifer clones were replicated and pre-cultured under standard conditions at a low density and medium renewal for three generations to control for maternal effects and avoid the induction of sexual reproduction before the bioassays (Stelzer & Snell 2006). To achieve this, three independent sublines were established by isolating a single asexual female carrying two eggs from each clonal stock culture and transferring her to an individual Petri dish containing 40 mL of culture medium (initial microalgae concentration of 250,000 cells mL -1 ). After 24 hours, when newborn daughters had hatched, all but one neonate female (F1) were removed. This individual was allowed to proliferate asexually for 48 hours, after which a second-generation (F2) neonate female was isolated and transferred to fresh culture medium to produce the third generation (F3). This method ensured independence across the three within-clone replicates from the onset of the pre-experimental culture. In the bioassays, each F3 neonate female was placed in a Petri dish containing 15 mL of fresh culture medium with an initial microalgae concentration of 500,000 cells mL -1 . F3 females were allowed to grow and reproduce, being monitored every 24 hours until the first male was observed, marking the onset of sexual reproduction. At this point, the culture was fixed with Lugol’s solution (final concentration 4%), and the density of females in the culture was recorded as an inverse measure of sex propensity (Aparici et al. 2001). Clones initiating sexual reproduction at lower densities were classified as having high sex propensity, indicative of an early sexual reproduction during the planktonic growing season. Conversely, clones requiring higher densities to initiate sexual reproduction were considered to have low sex propensity, reflecting a late sexual reproduction in the growing season. Among the TOS clones, five clones with the earliest initiation of sexual reproduction and five with the latest initiation were selected for use in the invasion experiment. Invasion experiment To test the three factors that may influence the settlement success of B. plicatilis, we emulated the invasion of immigrant clones into a locality already occupied by a resident population. A multifactorial design was implemented: (1) the propensity for sexual reproduction of the invaders (early vs. late, based on prior sex propensity bioassays), (2) the migration rate (9.4% vs. 2.5%), and (3) the genetic diversity of the resident population (monoclonal vs. multiclonal) (Figure 1). For the first factor, we selected the five clones from the immigrant population (TOS) with the earliest and the five with the latest propensities for sexual reproduction, as determined in the prior bioassays explained above. Five amictic females, each carrying two eggs, were introduced as immigrants into the resident populations. Each population was invaded by individuals of a single clone, with the five clones of each type acting as replicates. For the second factor, two different migration rates were achieved while maintaining a constant density (0.2 ind. mL -1 ) by adjusting volumes to 250 mL for the 9.4% migration rate and 1 L for the 2.5% migration rate. Consequently, the high migration treatments contained 5 immigrants with 48 residents (asexual females from ATA), whereas the low migration treatment contained 5 immigrants with 192 residents. For the third factor, the low genetic diversity treatment consisted of a single resident clone (monoclonal population) ─with intermediate propensity for sex─, while the high-density treatment mixed eight clones with an equal number of females from each. To obtain sufficient females for the experiment, all experimental clones were cultured in large volumes of medium to enable for high population sizes while preventing the early occurrence of sexual reproduction. In total, 40 experimental populations were established (2 migration rates x 2 levels of genetic diversity x 2 levels of sex propensity x 5 replicates). These experimental populations were initiated with a microalgae concentration of 250,000 cells mL⁻¹ and maintained under standard conditions with gentle agitation in an orbital shaker for 20 days. On day 10, the cultures were supplemented with concentrated algae to restore the initial cell concentration while ensuring a constant culture volume. At the end of the experiment, cultures were filtered to isolate the diapausing eggs produced, which were subsequently counted and collected for DNA extraction. For this purpose, the eggs were manually decapsulated using a small glass mortar in an Eppendorf tube, and DNA was purified using the JETFLEX Genomic DNA Purification Kit (Invitrogen) following the manufacturer’s protocol for tissue DNA extraction. To assess the settlement success, amplicon sequencing was performed to track the frequency of immigrant-specific alleles in the experimental populations. Private biallelic SNPs were identified for each population by whole-genome sequencing at low depth (10x) for each individual clone of TOS and for a mixture of the AYA clones. DNA extraction of the samples followed the protocol described above. A total of 150 paired-end genomic libraries were built and sequenced using Illumina technology. Reads were filtered by length and quality, with adapters removed using Trimmomatic. The clean reads then were mapped against the rotifer reference genome (NCBI: ASM1027981v1) and SNP calling was performed using FreeBayes. Custom Python scripts were used to filter low quality SNPs and genotypes, retaining SNPs that presented a homozygous allele common to all TOS samples but distinct to ATA. Finally, three SNPs were selected (two nuclear, QEOQ01000180.1_720451 and QEOQ01000348.1_68624, hereafter nSNP1 and nSNP2; and one mitochondrial, AP009407.1_2719, hereafter mSNP), all with flanking conserved regions, allowing for the design of primers for their amplification and genotyping. We genotyped these three SNPs in the experimental populations using amplicon sequencing on the Illumina platform, incorporating Unique Molecular Identifiers (UMIs) for increased accuracy. UMIs were added to the oligonucleotides used for amplifying specific regions, to enable accurate identification and counting of the original DNA molecules despite the potential biases introduced during PCR amplification. This approach enhances the reliability of genotyping by distinguishing true genetic variants from PCR artifacts. Data analysis To investigate the influence of (1) propensity for sexual reproduction, (2) migration rates and (3) genetic diversity of the resident population on settlement success, we first focused on nuclear SNPs as they reflect biparental inheritance patterns in gene flow. A Generalized Linear Model (GLM) was constructed only for the nSNP1, which had the highest number of reads. Preliminary tests of proportions (Wilson 1927) revealed no significant differences in the frequency of the two nuclear SNPs in any but one of the experimental populations, regardless of the combinations of factors tested. The only exception corresponded to a trial in which the number of reads for nSNP2 was abnormally low (only 2 reads, while the average number of reads in nuclear SNPs was 767). The GLM on read count data for nSNP1 included as a response variable a combination of the number of reads from immigrants (successes) and from residents (failures), assuming a binomial distribution of errors. The GLM incorporated the three previously mentioned factors as explanatory variables, along with all their interactions. The statistical significance of effects was assessed via chi-squared tests. The comparison of immigrant allele frequency patterns between nSNP1 and mSNP allowed us to assess the differential role of males and females of the invading clones in their settlement. To this end, we constructed a Generalized Linear Mixed-effects Model (GLMM) for the number of immigrant versus resident reads, assuming a binomial error distribution. As explanatory variables, the GLMM included SNP type (either nuclear or mitochondrial), and an eight-level treatment factor resulting from the combination of the levels of the propensity for sexual reproduction (early vs late), migration rate (low vs high) and genetic diversity of the resident population (monoclonal vs multiclonal), as well as the interaction between treatment and SNP type. Because read number data of the nuclear and mitochondrial SNPs were not independent ─ i.e., they were measured in the same immigrant clones─, clone identity was incorporated as a random-effect factor. On this model, we performed a priori contrasts to explore for differences in the frequency of nuclear and mitochondrial immigrant alleles at each of the eight levels of the treatment factor. Finally, an additional GLM was generated to examine the influence of the three study factors on the number of diapausing eggs produced during the invasion experiment. To account for volume differences across experimental populations, we used egg density instead of number of eggs as the response variable. A Poisson distribution of errors was assumed, and the significance of effects was assessed with chi-squared tests. All analyses were conducted using R 4.4.1 statistical software (R Core Development Team). GLMs and GLMM, along with chi-square associated tests were run using the glmer and anova functions from package “lme” (Bates et al. 2015). The a priori comparisons were performed using the contrast function from package “emmeans” (Lenth 2025). Preliminary proportion tests comparing the frequency of nSNP1 vs nSNP2 in each experimental population were carried out using the prop.test function from the “stats” package (R Core Team 2024). Results The bioassays revealed substantial variation in the propensity for sexual reproduction among clones from the ATA (resident) and TOS (immigrant) populations. The average density for the initiation of sexual reproduction was 16.9 females mL -1 in ATA clones and 5.9 females mL -1 in TOS clones, with most clones initiating sexual reproduction at densities below 5 females mL -1 (Figure 2). Among TOS clones, those classified as having high sex propensity (i.e., earliest sexual reproduction initiation) exhibited an average density threshold of 1.7 individuals mL -1 , whereas low sex propensity clones (i.e., latest sexual reproduction initiation) initiated sexual reproduction at 16.9 females mL -1 . Changes in the frequency of the immigrant allele in the invasibility experiment revealed that all three experimental factors significantly influenced settlement success (Figure 3). Across all experimental conditions, the highest invasion success was observed in populations with high migration rates and monoclonal resident populations, particularly when immigrants had an early sexual reproduction. Conversely, the lowest settlement success occurred in low migration treatments with genetically diverse resident populations, regardless of immigrant sex propensity. GLM-based analyses confirmed significant positive effects of both migration rate ( p < 0.001) and propensity for sexual reproduction ( p < 0.001), while the genetic diversity of the resident population had a significant negative effect on settlement success ( p < 0.001). Interactions between these variables were also significant (migration rate x genetic diversity, p = 0.007; migration rate x propensity for sexual reproduction, p < 0.001; propensity for sexual reproduction x genetic diversity, p < 0.001; migration rate x propensity for sexual reproduction x diversity, p = 0.007) on settlement success. Comparisons of allele frequencies between mitochondrial and nuclear SNPs revealed significant differences in seven of the eight treatment combinations (Figure 4). A higher frequency of the nSNP1 was observed in the two combinations characterized by multiclonal resident populations and early sexual reproduction, regardless of migration rate. In the remaining combinations with significant differences, a higher frequency of the mSNP was observed. The only combination without significant differences corresponded to low migration rate, monoclonal resident populations, and immigrants with early sexual reproduction. The production of diapausing eggs varied widely across the experimental populations, with a clear effect of resident genetic diversity. Monoclonal resident populations consistently exhibited the lowest diapausing egg densities, ranging from 0.08 to 0.82 eggs mL⁻¹, whereas multiclonal resident populations had higher egg densities, ranging from 2.40 to 7.77 eggs mL⁻¹, irrespective of the migration rate (Figure 5). The GLM analysis confirmed that the level of genetic diversity within the resident population was the only factor having a significant positive effect on diapausing egg density ( p < 0.001). Discussion This study demonstrates that settlement success in B. plicatilis is significantly influenced by the interplay between migration rate, genetic diversity in the resident population, and the propensity for sexual reproduction in immigrants. Our findings highlight the importance of considering multiple interacting ecological and genetic factors when assessing dispersal dynamics, as their effects are not independent but rather synergistic. A key finding of this study is that early sexual reproduction increases the likelihood of invasion success in B. plicatilis clones. This effect is particularly noteworthy in a likely unfavourable scenario for the immigrants, characterized by a low migration rate and high diversity of the resident population. Under such conditions, clones that initiate sexual reproduction earlier were able to increase in frequency and successfully settled. This result provides new insights into the critical role of reproductive phenology in dispersal processes and support the hypothesis that interactions between migration rate and resident diversity modulate settlement success. The observed advantage of early sexual reproduction in the colonization process may arise from temporal niche differentiation. By initiating sexual reproduction early, immigrant clones can exploit a temporal window of reduced competition for resources and mates, thereby maximizing the production of diapausing eggs without the constraints imposed by a dense resident population. This strategy would offset the trade-off between asexual proliferation, which fuels population growth, and sexual investment, which is essential for diapausing egg production ─a critical factor for clone survival under adverse conditions and for spatial dispersal (Innes & Singleton 2000; Montero-Pau et al. 2014; Serra & King 1999). Thus, early sexual reproduction during settlement likely ensures the production of at least some diapausing eggs before the advent of unfavourable conditions. This represents a key advantage in non-exploratory dispersal, where the duration of favourable conditions for growth and reproduction in the newly habitat reached cannot be anticipated. In the face of this uncertainty, a conservative strategy that facilitates the settlement of a minimal diapausing egg bank in the sediment of the new habitat would be to engage in sexual reproduction as soon as possible. Notably, this strategy has been recognized as adaptive in environments that fluctuate unpredictably (Franch-Gras et al. 2017; Tarazona et al. 2017). Natural variation in the timing of sexual reproduction among rotifer clones (Carmona et al. 2009; Gabaldon & Carmona 2015) suggests that this trait may underpin divergent evolutionary strategies, where early reproducers prioritize colonization, while late reproducers optimize local competition. This hypothesis could be tested by assessing whether disperser clones in the natural populations exhibit significantly earlier sex than their source population. Furthermore, early sexual reproduction may also contribute to the preservation of genetic diversity within diapausing egg banks, as clonal selection populations experience during the growing season progressively erodes genetic diversity (Carmona et al. 2009; De Meester et al. 2006; Gómez & Carvalho 2000; Ortells et al. 2006; Pfrender & Lynch 2000). High diversity within diapausing egg banks increases adaptive potential in novel environments (e.g., Hurst & Peck 1996; Tagg et al. 2005; West et al. 1999). Our analyses of nuclear and mitochondrial SNP frequencies revealed differential contributions of immigrant males and females to genetic turnover during the settlement process. In most treatments, mitochondrial SNP frequencies were higher than nuclear SNP frequencies, indicating that immigrant females contributed more to the gene pool than immigrant males. This suggests that immigrant females primarily mated with resident males, particularly in conditions where immigrants had low sex propensity, because resident females initiated sexual reproduction earlier. Therefore, by the time immigrant females began to reproduce, many resident males were already available to inseminate them. Conversely, in populations where resident genetic diversity was high and immigrants had early sexual reproduction, the frequencies of the nuclear SNP exceeded those of the mitochondrial SNP, indicating greater male-mediated gene flow. This pattern likely occurred because immigrant females initiated sexual reproduction earlier, allowing immigrant males to inseminate resident females. The presence of multiple resident clones with variability in the timing of sexual reproduction likely facilitates this process, as some resident clones may have initiated sexual reproduction early enough to coincide with the immigrants. Interestingly, no differences between mitochondrial and nuclear SNP frequencies were observed in the treatments with low migration rates, monoclonal resident population and early sexual reproduction of immigrant clones. This pattern suggests limited mating between immigrants and residents, with immigrant clones initiating sexual reproduction before resident clones became sexually active, effectively preventing genetic exchange. However, this pattern did not persist in the high migration treatments, likely because the increased number of migrants raised the likelihood of interactions with residents within a confined spatial volume. These findings underscore the complexity of reproductive timing in invasion success and highlight the differential contributions of males and females to the settlement process during dispersal. They suggest that reproductive timing is not only critical for the survival of dispersers but also influences patterns of hybridization and gene flow between resident and immigrant populations. The genetic diversity of the resident population appears to account for part of the colonization success. Our results showed that low genetic diversity in the resident population facilitates the invasion by immigrants, even under low migration rates. In monoclonal resident populations, the frequency of immigrant alleles consistently exceeded the initial migration rate, whereas in multiclonal populations, immigrant alleles remained closer to or even below their initial frequency. Interestingly, Ebert et al . (2002) experimentally demonstrated in Daphnia that crossovers between migrants and residents are advantageous when the resident population is inbred. Under such conditions, immigrants are expected to be more successful during the late phase of the growing season, when clonal selection has eroded genetic diversity in the resident population (De Meester et al. 2006; Louette et al. 2007; Ortells et al. 2006; Ortells et al. 2014; Pfrender & Lynch 2000). Alternatively, there may be a chance for successful settlement at the beginning of the growing season if initial hatching is low and so clonal diversity. The genetic structure of zooplanktonic populations is thought to be strongly influenced by founder effects (the so-called Monopolization Hypothesis; De Meester et al. 2002). According to this hypothesis, once a population of cyclic parthenogenetic organisms is established, invasion by immigrants is expected to be highly challenging due to the high population densities achieved by the residents. However, this hypothesis does not account for how the genetic diversity of the resident population might influence the settlement process. Our results challenge the assumptions of the Monopolization Hypothesis by highlighting that these genetic interactions, rather than population density alone, may play a pivotal role in the success of immigrant invasion. As expected, migration rates exerted a strong positive effect on immigrant settlement success, supporting theoretical models predicting that high propagule pressure reduces genetic differentiation between populations (Montero-Pau et al. 2018). This effect was particularly pronounced when paired with early sexual reproduction, reinforcing the notion that both the number of immigrants and their reproductive timing are key determinants of invasion success. Our results also revealed that resident genetic diversity had a strong positive effect on diapausing egg production, with significant lower egg densities in monoclonal resident populations compared to multiclonal ones. One possible explanation is inbreeding depression in monoclonal populations, as evidence suggest that inbreeding in B. plicatilis reduces diapausing egg viability (Tortajada et al. 2009). Aditionally, intraclonal barriers to sexual reproduction may evolve to prevent mating between females and males from the same clone, further reducing diapausing egg production (Jezkova et al. 2024). Another contributing factor may be the duration of the sexual reproduction period. In multiclonal populations, clones exhibit variability in the timing of sex initiation, prolonging the period where both males and sexual females coexist, thereby increasing the window for diapausing egg production. In contrast, monoclonal populations initiate sexual reproduction synchronously, resulting in a shorter mating window and reduced diapausing egg yield. A useful exercise is to translate our results into a plausible ecological scenario. To do this, we consider a metapopulation system in a landscape of isolated water bodies that are not connected by watercourses. While generalizing our findings is not essential, the framework aligns more closely with a system of temporary water bodies that dry up at some point in an annual hydrological cycle. In fact, the habitats of the study populations share this characteristic, which is also common to many zooplanktonic species. In such a scenario, migration rates are expected to increase significantly during the dry phase of the ponds, when the sediments become more exposed to the wind (Arenas-Sánchez et al. in prep.; Moreno et al. 2016; Vanschoenwinkel et al. 2008). Asynchronous drying across ponds (which can be explained by their variability in size or depth) may create scenarios favoring successful colonization. This could be the case, for instance, if immigrants arrive at a pond late in the hydroperiod, when population’s genetic diversity has already been eroded. As the end of the growing season is approaching, early induction of sexual reproduction would allow immigrants to produce or contribute to diapausing egg production before the habitat becomes unsuitable. In addition, genetic (and phenotypic) diversity among immigrants is expected to rise as migration rates increase. This may enhance the likelihood that at least a subset of dispersers will successfully overcome the abiotic filters of the new environment. Consequently, it may also mitigate the severity of the genetic bottleneck associated with the colonization process. Although we have not included this factor in our experiment, as we always invaded a population with individuals of the same clone, a next step in our research would be to investigate the effect of genetic variability among dispersers in combination with the other factors. Overall, the interplay between migration rate, genetic diversity, and reproductive strategies reveals complex and interdependent dynamics governing immigrant settlement success. Our results emphasize that all three factors interact synergistically, affecting colonization success in ways that cannot be fully explained when considered in isolation. For example, high migration rates significantly increase settlement success when combined with early sexual reproduction in immigrants, whereas low genetic diversity in the resident population facilitates the success of immigrants with delayed sexual reproduction. These findings underscore the importance of considering these factors simultaneously, as analysing them independently may oversimplify the ecological dynamics and their impact on settlement. Therefore, it might be useful to consider that there could have been other factors relevant to this experiment. Over time, our laboratory populations may have experienced environmental deterioration due to the accumulation of metabolites. This may have favoured the early sexual reproduction clones over the late ones, as the production of diapausing eggs may be lower under these conditions. However, this could also occur in natural populations of modest volumes, such as temporary ponds. Another factor that could interact with those studied is the size of the resident population. We have already discussed the importance of the timing at which an immigrant arrives in a population because there is variation in genetic diversity. But another factor that changes over time is the number of individuals that make up the population, and we believe that this number should be low to increase the success of immigrants. Finally, another factor that could interact with the others in establishing in a new habitat is the relative investment in sexual versus asexual reproduction. As the production of diapausing eggs, which are essential for survival in adverse conditions, is associated with sexual reproduction, we might expect higher invasion success in clones with higher ratios of sexual to asexual offspring. It would be valuable to examine this factor alongside others to determine whether it truly plays a decisive role in the settlement process. In addition to its role in settlement, early sexual reproduction may also play a crucial role in dispersal dynamics, aligning with other known dispersal-facilitating traits in zooplankton. Our results suggest that its early occurrence during the settlement phase provides a significant advantage to dispersing organisms. Early sexual reproduction can thus be considered as another adaptive trait, similar to other traits of dispersing propagules in rotifers and other zooplankton organisms, such as buoyancy, adhesiveness, or size, which influence dispersal success (Arenas-Sánchez et al. 2023; Cáceres et al. 2007; Moreno et al. 2016; Vanschoenwinkel et al. 2009). In conclusion, our study provides a broad and detailed understanding of the factors influencing settlement success during invasion, focusing on the interplay between migration rates, genetic diversity, and reproductive strategies. This experimental approach represents a significant step forward in the study of rotifer dispersal and sets the stage for future research to bridge the gap between laboratory findings and real-world ecological complexity, ultimately providing insights relevant to future studies of dispersal and population dynamics in other organisms in both theoretical and applied contexts. 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Rest of data is available on request from the authors. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial relationship that could be interpreted as a potential conflict of interest. There are no disputes regarding the ownership of the data presented in the paper, and all contributions have been attributed appropriately via co-authorship or acknowledgement, as appropriate to the situation. Author Contributions Designed research: CA-S, RO, MJC, EMG-R, JM-P. Developing methods: CA-S, RO, MJC, EMG-R, JM-P. Performed research: CA-S, EMG-R, JM-P. Contributed new reagents or analytical tools: EMG-R, JM-P. Data analysis: CA-S, EMG-R, JM-P. Data interpretation: CA-S, RO, MJC, EMG-R, JM-P. Preparation of figures and tables: CA-S. Writing: CA-S, RO, MJC, EMG-R, JM-P. Supervision: RO, MJC, EMG-R, JM-P. Funding acquisition: RO, MJC, EMG-R, JM-P. Project administration: RO, JM-P. Figures Figure 1. Schematic representation of the experimental setup used to investigate the effect of migration rates, timing of sexual reproduction and genetic diversity of residents on settlement success. Timing of sexual reproduction is related with propensity of sexual reproduction. Figure 2. Density at which sexual reproduction is initiated for ATA and TOS clones as an inverse measure of sex propensity. Figure 3. Frequency of the immigrant allele for the nSNP1 for each factor combination. Migration rate (9.4 and 2.5 %, high and low respectively), genetic diversity of the resident population (monoclonal and multiclonal) and timing of sexual reproduction (early and late). The x-axis shows the density at which sexual reproduction is initiated, being an inverse measure of sex propensity. The dotted line shows the percentage of initial immigrants (equal to the migration rate). 95% confidence interval based on binomial distribution for the frequency is shown. Figure 4. Mean frequency of the immigrant allele for nSNP1 and mSNP for each factor combination. Migration rate (9.4 and 2.5 %, high and low respectively), genetic diversity of the resident population (monoclonal and multiclonal) and timing of sexual reproduction (early and late). The dotted line shows the percentage of initial immigrants (equal to the migration rate). Values are averaged on clones and standard errors around the mean are showed. Black asterisk indicates significant differences (p-value < 0.05) between mitochondrial and nuclear SNP frequency according to the GLMM analysis. Figure 5. Density of diapausing eggs produced at the end of the experiment for each factor combination. Migration rate (9.4 and 2.5 %, high and low respectively), genetic diversity of the resident population (monoclonal and multiclonal) and propensity for sexual reproduction (early and late). Information & Authors Information Version history V1 Version 1 11 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords dispersal invasibility experiment settlement success sexual reproduction snp genotyping zooplankton Authors Affiliations Cristina Arenas-Sánchez University of Valencia Cavanilles Institute of Biodiversity and Evolutionary Biology View all articles by this author Raquel Ortells 0000-0003-1389-603X [email protected] University of Valencia Cavanilles Institute of Biodiversity and Evolutionary Biology View all articles by this author María José Carmona University of Valencia Cavanilles Institute of Biodiversity and Evolutionary Biology View all articles by this author Eduardo M. García-Roger University of Valencia Cavanilles Institute of Biodiversity and Evolutionary Biology View all articles by this author Javier Monterio-Pau 0000-0002-0864-8157 University of Valencia Cavanilles Institute of Biodiversity and Evolutionary Biology View all articles by this author Metrics & Citations Metrics Article Usage 286 views 145 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Cristina Arenas-Sánchez, Raquel Ortells, María José Carmona, et al. How well do rotifers invade populations? The interplay of sex propensity, migration rates and local genetic diversity. Authorea . 11 March 2025. DOI: https://doi.org/10.22541/au.174170730.00901673/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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