6Pgdh polymorphism in wild bulb mite populations - prevalence, environmental correlates and life history trade-offs

doi:10.21203/rs.3.rs-3792019/v1

Genetic polymorphism in key metabolic genes plays a pivotal role in shaping phenotypes and adapting to varying environments. Polymorphism in the metabolic gene 6-phosphogluconate dehydrogenase (6Pgdh) in bulb mites, Rhizoglyphus robini is characterized by two alleles, S and F, that differ by a single amino acid substitution and correlate with male reproductive fitness. The S-bearing males demonstrate a reproductive advantage. Although the S allele rapidly fixes in laboratory settings, the persistence of polymorphic populations in the wild is noteworthy. This study examines the prevalence and stability of 6Pgdh polymorphism in natural populations across Poland, investigating potential environmental influences and seasonal variations. We found widespread 6Pgdh polymorphism in natural populations, with allele frequencies varying across locations and sampling dates but without clear geographical clines. This widespread polymorphism and spatio-temporal variability may be attributed to population demography and gene flow between local populations. We found some correlation between soil properties, particularly cation content (Na, K, Ca, and Mg) and 6Pgdh allele frequencies, showcasing the connection between mite physiology and soil characteristics and highlighting the presence of environment-dependent balancing selection. We conducted experimental fitness assays to determine whether the allele providing the advantage in male-male competition has antagonistic effects on life-history traits and if these effects are temperature-dependent. We found that temperature does not differentially influence development time or juvenile survival in different 6Pgdh genotypes. This study reveals the relationship between genetic variation, environmental factors, and reproductive fitness in natural bulb mite populations, shedding light on the dynamic mechanisms governing 6Pgdh polymorphism.

Rhizoglyphus robini

6-Phosphogluconate dehydrogenase

genetic polymorphism

balancing selection

metabolic gene

genotype-environment interaction for fitness

Balancing selection is the process through which polymorphism within a population is actively maintained over generations. Such maintenance of genetic variation is particularly important in genes that govern key metabolic traits, as variation in these genes has the potential to significantly impact the organism’s fitness and chances of survival (Koshiba et al. 2020, Whitt et al. 2002). Despite the general conservation of metabolic genes (Mukherjee et al. 2018, Kapahi et al. 2010), the presence of functional differences and selective pressures acting on them suggest, in some cases, the action of balancing selection. Metabolic genes can affect multiple aspects of an organism’s physiology and many traits. Therefore, balancing selection on these genes is likely to take the form of antagonistic pleiotropy, where one of the alleles is beneficial for one trait while the other for another trait, thus creating a tradeoff between different fitness components (Meyer & Zanger 1997, Kozyra et al. 2017, Di Bartolomeo et al. 2020). Antagonistic pleiotropy is already a recognized mechanism for the maintenance of polymorphism (Hedrick 1999).

This mechanism becomes even more intriguing when we consider the complex interplay of environmental factors, (Brown and Kelly 2018, Mérot et al. 2020) where antagonistic pleiotropy might be present in some environments, but not in others. Different enzyme variants can be favored at different environments, resulting in selection on such enzyme variants and influencing the geographic patterns of genetic diversity. For example, in Drosophila serrata (Rusuwa et al. 2022), the polymorphism in a single gene influencing circular hydrocarbon profile (CHC) is maintained in warm and humid climates but not in hot and dry ones. This is because one of the alleles is associated with male reproductive advantage, but lowers female desiccation resistance, resulting a trade-off between male reproduction and female stress response (termed also sexually antagonistic pleiotropy) and driving balancing selection in specific climate conditions (Rusuwa et al. 2022). The influence of environmental heterogeneity on functional polymorphism in metabolic genes was shown by Kerwin et al (2015) on the gene involved in glucosinolate production in Arabidopsis thaliana, which showed that no allele consistently outperformed another in different environments, emphasizing the importance of environmental heterogenity as an evolutionary force. Understanding the relationship between environmental factors and the functioning of enzyme variants is essential for gaining insight on how organisms adapt to their environment, utilize resources (Vieille & Zeikus 2001), defend against diseases (Hollman et al. 2016), and evolve (Rix et al. 2020).

This study focuses on the metabolic gene 6-Phosphogluconate dehydrogenase (6Pgdh), which is also a target of sexual selection in bulb mite, Rhizoglyphus robini. 6Pgdh is one of the key enzymes in the Pentose Phosphate Pathway (PPP), an alternative to glycolysis. It produces NADPH and ribose-5-phosphate used in the synthesis of fatty acids, sterols and nucleotides (Ge et al. 2020). PPP is also a source of amino acid and vitamin B6 precursors (Tambasco-Studart et al. 2005). The pathway is essential for energy metabolism of a cell and plays an important role in stress response (Fahrendorf et al. 1995, Krüger et al. 2011, Hou et al. 2007). The expression levels and activities of PPP enzymes are associated with environmental factors (Watts & Lawrence 1990). Environmental heterogeneity has been shown to drive patterns of polymorphism in the 6Pgdh and some other genes involved in PPP in organisms ranging from invertebrates (González-Ruiz et al. 2023) to plants (Landi et al. 2021). However, to really understand how selection of genes involved in PPP is driven by environment, we need to connect geographical and temporal patterns of polymorphism with phenotypic differences between genotypes and environmental background of these differences.

6Pgdh coding sequence in bulb mites consists of 486 amino acids and is divided by four introns. While four SNPs have been found in the coding sequence (Skwierzyńska and Plesnar-Bielak 2018), only one is associated with amino acid substitution (arginine to methionine). The two alleles of 6Pgdh, S and F correlate with reproductive fitness of males. Males bearing the “winning” S allele gain higher reproductive success compared to males with the “losing” F allele (Konior et al. 2006, Łukasik et al. 2010), due to higher sperm production and copulation frequency (Skwierzyńska and Plesnar-Bielak 2018). Female fitness seems to be independent of the 6Pgdh genotype, but the S-bearing males reduce the fitness of their female partners, even though the exact mechanism behind it is unknown (Konior et al. 2006). As expected from the advantage in male competition, the S allele rapidly fixes in laboratory conditions, but surprisingly, the polymorphism seems to be maintained in some natural populations (Konior et al. 2006, Łukasik et al. 2010, personal observations). There is very limited knowledge regarding the ecological conditions linked to this polymorphism and we hypothesize the active maintenance of polymorphism of this metabolic gene is environment dependent. Nevertheless, a systematic study on the patterns of 6Pgdh variation in natural bulb mite populations has not been conducted before so it is not known how common and stable over time the polymorphism is.

Here, we study several natural bulb mite populations to look into the amount of polymorphism, its geographical variation across Poland. To start with, we aim to correlate 6Pgdh polymorphism levels with latitude and longitude, which we use as proxies for macro-climatic factors, as well as with local soil properties. Moreover, we investigate seasonal variation in 6Pgdh frequencies, by investigating frequency shifts between spring and autumn. Finally, we explore fitness differences between 6Pgdh genotypes at different temperatures. We test if the allele providing advantage in male-male competition has antagonistic effects on life history traits and if these effects are temperature-dependent, as earlier studies has suggested the effects of temperature on PPP (Kauffman et al. 1969) and on 6Pgdh allele frequencies (Plesnar-Bielak et al. 2020). So, we further complement our field study with experimental fitness assays at different temperatures. The expression of the S allele, that conveys reproductive advantage to males, might be associated with energetic costs that could result in reduced juvenile survival and/or longer development time for individuals bearing this allele.

DNA extraction and 6Pgdh genotyping

DNA was extracted from individual mites. Each individual was placed in 1% chelex solution (40μl) and was crushed. Then 3μl of proteinase-K (EurX) was added, and the mixture was incubated in a thermocycler (10 min 94°C, 15 min 75°C).

The 6Pgdh genotyping was done using Real-Time PCR with fluorogenic TaqMan probes specific for the missense single nucleotide polymorphism determining the F and S alleles. The probes were obtained from Thermofisher Scientific. The Bio-Rad CFX96 Real-Time PCR detection system was used for the genotyping. A TaqMan Genotyping Master Mix (Thermofisher) and Custom Genotyping Assay that included allele-specific primers and fluorescent probes were mixed in 10:1 ratio. 5.5μl of such a mix and 4.5μl of DNA were put in a 96 well plate for genotyping. PCR was performed in 41 cycles (15 sec 95°C, 1 min 60°C).

The patterns of 6Pgdh polymorphism in the wild

Population sampling was carried out in Poland, which has a clear gradient of climatic and other environmental conditions from south-west to north-east that might affect 6Pgdh frequencies. Moreover, there is some record of variation in the level of 6Pgdh polymorphism in Poland and substantial genetic diversity within populations with little structuring between populations in this region (Kolasa et al. unpublished, Boroń et al. unpublished, Przesmyska and Rarwan 2023). Sampling was done between October 2021 and December 2022. The main sampling was done in late Spring/Summer (May, June) with some locations sampled also in Autumn (October, November) to see how stable 6Pgdh frequencies are across seasons.

Samples of bulbs of different plant species were collected from private gardens and botanical gardens across different regions in Poland (Table. 1) and checked for the presence of mites. Between 2 and 6 plant bulbs together with soil samples (taken only during Spring sampling and for a subset of samples) were collected per location, depending on availability.

Table. 1 :- Locations (with latitude and longitude) from which the samples were collected across Poland and the respective seasons during sample collection. The F-allele frequency, heterozygosity measures and results from the Hardy Weinberg test for each location are also shown.

Location	Latitude	Longitude	Season	F frequency	Heterozygosity	H-W test, P-value
Stanislaw Gorny	49.91062	19.62935	Autumn	0.145	0.184	2.005 0.157
Krakow OB.	50.06368	19.95549	Autumn	0.051	0.103	0.216 0.642
Mikolow	50.18060	18.82853	Autumn	0.596	0.538	0.734 0.392
Rudawa	50.12200	19.71217	Autumn	0.200	0.229	2.501 0.114
Lublin	51.29331	22.53616	Autumn	0.145	0.178	0.782 0.377
Marszyce	50.18099	19.85584	Autumn	0.130	0.148	4.733 0.029
Warszawa	52.25531	21.02248	Autumn	0.071	0.048	8.494 0.004
Brzeg	50.87413	17.46600	Spring	0.333	0.444	3.55e-15 1
Glucholazy.1	50.33271	17.37789	Spring	0.562	0.375	1.824 0.177
Brody	49.88037	19.72432	Spring	0.250	0.286	1.479 0.224
Rudawa	50.12200	19.71217	Spring	0.242	0.314	0.701 0.402
Piekary Slaskie	50.34695	18.98586	Spring	0.320	0.400	0.161 0.688
Stanislaw Gorny	49.91062	19.62935	Spring	0.576	0.424	0.571 0.449
Krakow	50.06368	19.95549	Spring	0.194	0.056	21.026 4.53e-06
Lublin.1	51.29331	22.53616	Spring	0.033	0.067	0.069 0.793
Lublin.2	51.29331	22.53616	Spring	0.416	0.500	0.005 0.944
Bory Tucholskie	53.61859	18.16236	Spring	0.000	0.000	n.a 1
Poznan	52.41385	16.92981	Spring	0.054	0.107	0.170 0.680
Warszawa	52.25531	21.02248	Spring	0.232	0.179	6.226 0.013
Glucholazy.2	50.33271	17.37789	Spring	0.500	0.000	41.589 1.12e-10
Przysieki	49.74042	21.38654	Spring	0.000	0.000	n.a.
Mikolow	50.18060	18.82853	Spring	0.026	0.000	9.248 0.002
Kepa Slupska	54.41783	17.05663	Spring	0.138	0.138	3.785 0.052
Lublin.3	51.29331	22.53616	Spring	0.250	0.250	2.454 0.117

In the lab, bulb mites, if present, were transferred to plastic containers (diameter ≈ 2.5cm) with plaster of Paris soaked with water (which are standard containers to keep large groups of mites). They were kept at 12°C and fed powdered yeast ad libitum. Ca. 40 individuals from each sample (location, see Table. 1) were genotyped within 2 months after collection to ensure that the individuals collected as juveniles reached adulthood.

Soil analyses

Soil samples were collected from sampling points (10 sampling points) near the plant using teaspoons and ensuring that the points were within 1m² of the plant and was kept in 12°C for analysis. The dry weight (DW) of the soil samples was determined by measuring mass loss (water) after soil samples dried at 105±1°C for 24 h. Next, the organic matter content (OM) in soil dry weight was determined as the mass loss on ignition at 550±1°C for 24 h. The water holding capacity (WHC), which is the amount of water that a given soil can hold without leaking, was measured by a standard gravimetric method after soil soaking for 24 h in net-ended plastic pipes immersed in water. The organic carbon (C), total nitrogen (N), and total sulfur (S) were analyzed by dry combustion of ca 10 mg milled soil samples with an elemental analyser (Vario El III, Elementar Analysensysteme GmbH). The soil pH was measured in air-dried subsamples (2 g) shaken in deionised water (1:10 w:v) for 1 h at 200 rpm (pH-meter with glass electrode).

The total cations concentrations, that is phosphorus (P), calcium (Ca), potassium (K), magnesium (Mg), manganese (Mn), and sodium (Na) in each soil sample were determined after wet digestion of ca 0.5 g of DW in 10 ml of SupraPure-concentrated HNO3 and HClO4 (7:1 v/v) (Sigma-Aldrich). A flow injection analyser (FIA compact, MLE, Radebeul, Germany) was used to determine the P content. The total concentrations of the other elements were measured using atomic absorption spectrometry (AAS) with a flame nebulizer (Perkin-Elmer, AAnalyst200, Waltham, Massachusetts, USA). The accuracy of the mineralization process was determined using blank samples as well as standard certified material (CRM025-050, Sandy Loam 8, RT Corp.). Each analysis was performed in two subsamples from each soil sample, and the data were averaged and expressed based on the dry weight of the soil.

Laboratory population

For the life-history fitness experiments, we used a population enriched in the F allele that was established from a field population obtained in July 2020 from Łazany (49.9476, 20.1535) near Kraków. Several dozens of individuals collected from an onion were placed in a common container with powder yeast that served as food. Such obtained population was kept at 8°C, with the exception of a one-week period after we finished collecting individuals, when it was moved to 24°C to let juvenile individuals develop so that population would expand. The F-increased population was created in spring 2021, when the F allele frequency in the source population was about 0.23. To do it, we randomly paired virgin females and males from a source population. After the pairs mated and females laid eggs, both parents were genotyped. Eight offspring from pairs with parents having at least 2 copies of F allele (either both parents FS, or one FF and one SS, or one FF and one SF, or both parents FF) were transferred as larvae/protonymphs to a common container to establish a population with increased F allele frequency. We used two containers (with offspring from the same parental pairs moved to both of them) that established two subpopulations that were mixed and divided again after ca. 2 months. The population was let to expand freely for ca. 2 months at 24°C (which corresponds to 3-4 mite generations), before it was moved to 12°C to elongate generation time, slowing down population’s evolution and the loss of the F allele. At all these stages the population was kept at >90% humidity and constant darkness, with powdered yeast provided ad libitum as a food source.

Development time

Development time of the individuals with different genotypes was measured at three temperatures, 24°C (standard laboratory temperature), 12°C (average yearly ground temperature at 5cm depth in Poland) and 8°C (low temperature relevant to colder periods in Poland) with three replicates per temperature (see Fig. 1). Per each replicate, ten females from the F-increased population were kept in containers for 24 hours to lay eggs. After the females were removed, the containers with the eggs were placed to experimental temperatures. The eggs were allowed to develop. When they reached the stage of tritonymph (last juvenile stage), they were checked every 24 hours for emerging adults. Adults that emerged were taken out from the containers and date of emergence and sex were noted. Then, the individuals were genotyped for 6Pgdh. The checks continued till all the adults emerged.

Juvenile survival differences between genotypes

Juvenile survival differences were also tested at 24°C, 12°C and 8°C. For the assay, 50 females were put in a common container (five replicates per temperature) and allowed to lay eggs at 24°C for four days, after which they were removed (see Fig. 1). The containers were then transferred to their respective experimental temperatures (24°C, 12°C and 8°C). After the adults emerged, around 40 individuals from each replicate were genotyped for the 6Pgdh. We calculated the frequencies of the F allele at each temperature and used them as a proxy of juvenile survival differences between genotypes. If juvenile survival of individuals with different alleles is temperature-independent, we expect that allele frequencies in adults do not differ between temperatures. A higher frequency of a certain allele at a given temperature, indicates higher survival of the individuals bearing this allele.

Statistical analysis

6Pgdh genotype frequencies in field samples were tested for Hardy-Weinberg equilibrium with likelihood ratio test implemented in Hardyweinberg package in R (Graffelman and Weir 2016). The frequencies from the samples collected in spring (when most of the samples were collected) were checked for their relationship with latitude and longitude of the location to look for geographical cline 6Pgdh polymorphism. We applied a quasibinomial model accounting for overdispersion (using glm function in R v3.6.1) with a vector of S and F allele counts at each location as a response variable and latitude and longitude as independent variables. For plotting the data points on the map of Poland, QGIS (v3.34.0-Prizren) was used along with the map shape file obtained from GADM data (v4.1).

To test for a correlation between 6Pgdh frequencies and soil characteristics, we first summarized soil parameters with Principal Component Analysis. Then, we run a generalized linear model with a vector of S and F allele counts at each location as a response variable and PC1 and PC2 as independent variables. Again, quasibinomial distribution was used to account for overdispersion in our data.

To check how genotype affects development time at different temperatures, we used a linear mixed model fit with the number of days taken for development (transformed with square root) as the response variable and with genotype and temperature (factor) as the dependent variables and population ID as random factor. We also checked to see if the effect of sex of the individuals was important to the model using AIC scores, but the effect of sex did not improve the model and the conclusions remained unchanged and hence the effect of sex was removed from the main model. The function lmer was used for the analysis in R (the package lmertest, lme4, v1.1-26)

The allele frequencies of the individuals that survived till adulthood at each temperature were obtained from the juvenile survival experiment. To analyze the data, a binomial model was used with a vector of S and F allele counts in each replicate as the response variable and temperature as the dependent variable using the glm function in R (glm2 package, v1.2.1).

The patterns of 6Pgdh polymorphism in the wild

We found 6Pgdh polymorphism in a majority (15 out of 17) of populations (Fig. 2), with only two of them having just one allele (S). In polymorphic populations, F-allele frequencies varied from 0.026 to 0.60, with a mean frequency of 0.23 (SD = 0.19). Mean frequency was similar for the samples collected in Autumn (mean±SD 0.191±0.18) and Spring (0.243±0.19). 6Pgdh allele frequencies varied a lot between Autumn and Spring, but the changes were not consistent in their direction, with F frequency increasing in Spring in some locations, but decreasing in others (Fig. 3). Similarly, samples taken from the same botanical garden substantially differed in allele frequencies, even in the same season. Eighteen samples were in Hardy-Weinberg equilibrium. In 6 samples (Table 1), we found deviation from Hardy-Weinberg equilibrium and observed heterozygosity was lower than expected in all these cases. We did not find evidence for geographical clines in 6Pgdh frequencies in Poland (longitude: effect estimate -0.285, t = -1.78, p = 0.089, latitude: effect estimate-0.406, t = -1.88, p = 0.075).

The association of 6Pgdh polymorphism with soil properties

PC1 and PC2 explained 44.3 and 19.8 % of the total variance in soil parameters, respectively (Fig. 4a). PC1 was mainly influenced by organic matter, S, N and C content (Fig. 4b). Higher PC1 values were also associated with lower pH. PC2 values were negatively correlated with Na, K, Ca and Mg content. We found a significant negative relationship between F allele frequencies and PC2 (t₁₁=-2.99, p=0.017), but not PC1 (t₁₁=1.584, p=0.152). (Fig. 4c)

Laboratory fitness experiments

Development time increased with decreasing temperature (F_{2; 8} = 7521.85, p < 0.01). There was no difference in development time between genotypes (F_{2; 953} = 1.53, p = 0.22) or genotype by temperature interaction (F_{4; 954} = 1.33, p = 0.25) (Fig. 5). The effect of sex was also not significant (F_{1; 950} = 1.31, p = 0.22).

F allele frequencies did not differ between groups developed at different temperatures (effect estimate 0.002, z = 0.17, p = 0.86), indicating there is no temperature-dependent difference in juvenile survival between genotypes (Fig. 5)

Genetic polymorphisms in key metabolic genes can potentially influence a wide range of phenotypic traits and may therefore be important for adaptive polymorphism. 6Pgdh is an example of such a gene with different allelic variants influencing fitness in vertebrates (Rivera et al. 2006, Chen et al. 2023), plants (Oostermeijer et al. 1995) and invertebrates (Begun & Aquardo 1994, Kilias and Alahiotis 1985), including bulb mites (Rhizoglyphus robini). The maintenance of such polymorphism under natural conditions is surprising, particularly in the case of bulb mites, given the strong reproductive advantage of the S allele leading to its rapid fixation in the laboratory. In some environments, however, reproductive advantage may be balanced by metabolic costs that could lead to a trade-off between sexual and non-sexual fitness, leading to the maintenance of stable polymorphism under these conditions (Robinson et al. 2006, Höglund et al. 1998). Our study investigates the abundance of 6Pgdh polymorphism in natural populations, explores potential environmental factors influencing these patterns in the wild, and examines potential trade-offs in laboratory settings.

We show that 6Pgdh polymorphism is indeed common in natural populations. The majority of screened populations were 6Pgdh-polymorphic, but the actual allele frequencies varied. The abundance of polymorphism aligns with certain observations reported in prior studies (Łukasik et al. 2010). For example, a natural population from Poland found by Konior et al. (2006) was polymorphic in respect to 6Pgdh with F-allele frequency of 0.11. Screening of ten populations in Poland by Skwierzyńska and Plesnar-Bielak (2018) found only one polymorphic population, suggesting 6Pgdh polymorphism to be rare. However, the search was aimed at finding a population of a relatively high polymorphism level to use in laboratory experiments. Hence, the sampling might have not been suitable for detecting moderate F frequencies, which were not uncommon in the current study.

6Pgdh frequencies varied between populations and sampling dates, but there was no seasonal or latitudinal pattern. The high variability of 6Pgdh allele frequencies is supported by previous observations in bulb mites. For example, the F frequency in a natural population was 0.34 in 2003, but it decreased below 0.05 at the same site a year later (Łukasik et al. 2010). This lack of geographic or seasonal patterns suggests that perhaps factors other than temperature may play a more significant role in shaping the frequencies of 6Pgdh alleles or that the effect of temperature may occur at a much finer scale. Our results from the laboratory experiments support the former. We found no evidence that temperature differentially affects two life-history traits: development time and juvenile survival, in individuals with different 6Pgdh genotypes. This suggests that temperature does not have an effect on the allele fitness or the distribution of alleles on neither microhabitat level (life-history assays) nor macrohabitat scale (field study). Similarly, a prior laboratory investigation demonstrated that there is no reversal in F-allele fitness across temperatures, supporting our conclusion (Plesnar-Bielak et al. 2020). On the other hand, our observations contrast with those in other species. For example, two studies in Drosophila (D.melanogaster and D.simulans) showed clear latitudinal clines of 6Pgdh allele frequencies in different regions across the world, likely associated with climatic conditions (Oakeshott et al. 1983, Begun and Aquadro 1994). Similarly, latitudinal clines in 6Pgdh frequencies have been associated with other selective factors such as water salinity in Atlantic killifish (Fundulus heteroclitus) (Powers et al. 1986). Stockwell and Mulvey (1998) also explicitly demonstrated that it was water salinity, and not temperature, that influenced the polymorphism levels in white sands pupfish, Cyprinodon tularosa (Stockwell & Mulvey 1998).

Our study found a significant effect of soil properties on the level of polymorphism, with higher amounts of cations (Na, K, Ca and Mg) in the soil corresponding to higher frequencies of the F allele. Bulb mites, being subterranean organisms reliant on soil, can experience direct or indirect effects of soil composition on their physiology. Soil properties can affect plant diversity and soil fauna indirectly (Kudureti et al. 2023). Indeed, evidence for soil properties differentially affecting fitness of multiple mite species have already been observed. Soil properties such as pH, nitrogen and carbon content, among other variables have been shown to affect community composition (Nielsen et al. 2010) and diversity (de Moraes et al. 2011) in oribatid mite group, suggesting they drive fitness differences at inter-species level. Our results suggest that soil properties can differentially affect mite fitness at the intra-species level too. The effects of soil could be mediated by factors like vegetation or soil microbial community composition (Li et al. 2023, Pineda et al. 2017). For example, host-microbiome interactions in bulb mites affect nutrition (Zindel et al. 2013), which in turn impacts fitness and traits like development rate and body size (Leigh & Smallegange 2014). Nutritional conditions can also influence fitness of different alleles of the same metabolic genes. It has been shown in Drosophila melanogaster, where diet quality affected fitness of the allelic variants of the "foraging" gene (Burns et al. 2012). In general, the patterns of 6Pgdh polymorphism could be associated with environmental quality, mediated by the relationship between soil properties and microbiome. However, resolving this issue would require more direct experimental verification.

Importantly, soil nutrients cannot solely explain the levels of 6Pgdh found in this study. The minerals or nutrients can explain the variation across space, but they cannot explain the variation across seasons. It’s because even though the primary nutrients (nitrogen, potassium, phosphorus) can vary between seasons (Hu et al. 2022), there is little evidence of such being the case for the other minor nutrients and minerals. The shifts in 6Pgdh levels might instead be a result of population demography and intense gene flow between local populations. Indeed, gene flow and dispersal are common in bulb mite Rhizoglyphus robini (Deere et al. 2021, Díaz et al. 2000). In addition, the biology of the species – likely founding populations by small number of individuals (founder events) – may contribute to large differences and presumably erratic patterns of allele frequency changes in natural populations. Indeed, some of the population we have sampled were quite small, with high numbers of juvenile individuals, suggesting they had been founded recently. A detailed study on the structure of genetic variation within and between bulb mite populations using genome-wide data would help to clarify this issue.

To conclude, the study shows that the 6Pgdh polymorphism is indeed common in bulb mites. We also found significant influence of soil properties on the polymorphism levels. Additionally, we found that the patterns of 6Pgdh polymorphism varied across locations and seasons, although there was no pattern to this change. Gene flow, driven by the migration of individuals between bulb mite populations, could be a crucial factor contributing to the observed variations in nature. In suspicion of a genotype-by-environment interaction for fitness, we looked at climatic variables such as temperature and its influence on the patterns of 6Pgdh, but we found no evidence of such. Similarly, the life-history assays performed in the lab did not provide any evidence of temperature influencing fitness of the allelic variants.

In summary, soil properties can potentially explain the distribution of 6Pgdh alleles of bulb mites in the wild, but not the spatio-temporal variation. Perhaps there are other environmental factors contributing to this variation or perhaps it’s the result of gene flow between populations. Complementing this study with additional experiments to test the effects of different factors on more traits (related to reproductive success) may help us understand more about how selection works in nature and about environment dependent balancing selection in general.

Acknowledgements

We thank the following people and institutions from providing us with plant material for sampling mites: Krystyna Pietrasik , Aleksandra Walczyńska, Marzena Marszałek, Aneta Grząbka („PZD Okręg Poznań” garden), Jochanna Łupkowska and Marian Ptak (“ROD Omex” garden), Ewa Majewska (Polish Association of Allotment Gardeners, Opole District), Sebastian Chmielewski, Marta Labocha-Derkowska, Michał Czarnecki (The Botanical Garden of Maria Currie-Skłodowska University in Lublin), University of Warsaw Botanic Garden, Krzysztof Kapała (Botanic Garden of the Jagiellonian University), Magdalena Pardon (Silesian Botanical Garden). We are also grateful to Genomics and Experimental Evolution Group members (Jagiellonian University) for their comments on the previous version of the manuscript. The study was funded by a grant from Polish National Science Centre grant no UMO-2019/33/B/NZ8/02442 awarded to Agata Plesnar-Bielak.

Funding

The study was funded by a grant from Polish National Science Centre grant no UMO-2019/33/B/NZ8/02442 awarded to Agata Plesnar-Bielak.

Competing interests

The authors have no financial or non-financial interests to disclose.

Author contributions

Agata Plesnar-Bielak and Pranav Unnikrishnan designed the study, Pranav Unnikrishnan, Szymon Grzesik, and Magdalena Trojańska performed the experiments, Agata Plesnar-Bielak collected material in the field, Beata Klimek performed soil analyses, Pranav Unnikrishnan and Agata Plesnar-Bielak analysed the data, Pranav Unnikrishnan and Agata Plesnar-Bielak wrote the manuscript. All authors read and approved the final manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

6Pgdh polymorphism in wild bulb mite populations - prevalence, environmental correlates and life history trade-offs

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