Deleterious mutation load in the admixed mice population

Deleterious mutation loads are known to correlate negatively with effective population size (Ne). Due to this reason, previous studies observed a higher proportion of harmful mutations in small populations than that in large populations. However, the mutational load in an admixed population that derived from introgression between individuals from two populations with vastly different Ne is not known. We investigated this using the whole genome data from two subspecies of the mouse (Mus musculus castaneus and Mus musculus musculus) with significantly different Ne. We used the ratio of diversities at nonsynonymous and synonymous sites (dN/dS) to measure the harmful mutation load. Our results showed that this ratio observed for the admixed population was intermediate between those of the parental populations. The dN/dS ratio of the hybrid population was significantly higher than that of M. m. castaneus but lower than that of M. m. musculus. Our analysis revealed a significant positive correlation between the proportion of M. m. musculus ancestry in admixed individuals and their dN/dS ratio. This suggests that the admixed individuals with high proportions of M. m. musculus ancestry have large dN/dS ratios. We also used the proportion of deleterious nonsynonymous SNVs as a proxy for deleterious mutation load, which also produced similar results. The observed results were in concordance with those expected by theory. We also show a shift in the distribution of fitness effects of nonsynonymous SNVs in the admixed genomes compared to the parental populations. These findings suggest that the deleterious mutation load of the admixed population is determined by the proportion of the ancestries of the subspecies. Therefore, it is important to consider the status and the level of genetic admixture of the populations whilst estimating the mutation loads.


Introduction
Population genetic theories predict a negative relationship between effective population size (N e ) and the fraction of deleterious mutations (Kimura and Ohta, 1978;Kimura, 1983). According to this, populations with small N e accumulate more deleterious mutations than those with large N e . This is because the effect of genetic drift is high amongst small populations, and hence the purifying selection is inefficient in purging deleterious mutations. Initially, accumulation of deleterious mutations was assumed to occur only in the asexual population, which was called as "Muller's ratchet" (Muller, 1964;Felsenstein, 1974). Later, Lynch et al. (1995) showed the possibility of accumulation of harmful mutations in sexual populations, particularly when the population is small. The proportion of deleterious mutations has been estimated in a number of species, including humans, chimpanzees, mice, cows, chickens, fruit flies, and plants (Eyre-Walker and Keightley, 1999;Schultz et al., 1999;Li and Saunders, 2005;Rubin et al., 2010;Bourneuf et al., 2017;Sohail, et al., 2017). These studies showed that amongst mammals, humans have the highest proportion of deleterious mutations. This is because the ancestral effective population size of humans is known to be the smallest of the mammals examined so far (Keightley and Eyre-Walker, 2000). Comparisons involving different populations within a species also showed similar patterns. For example, previous studies reported that European Americans have a significantly higher proportion of deleterious mutations than African Americans (Lohmueller et al., 2008;Fu et al., 2014;Subramanian, 2016). This observation could be explained based on the population bottleneck that occurred in Europeans when they migrated out of Africa. This was further confirmed by another large-scale study that showed a positive correlation between deleterious mutation load and the distance from Africa (Henn et al., 2016). Human populations experienced serial population bottlenecks as they migrated out of Africa, and populations that are furthest from Africa might have experienced more bottlenecks than those proximal to Africa. Therefore, the latter harbour more deleterious mutations than the former (Henn et al., 2016).
The effects of bottlenecks were also observed in populations that migrated much more recently. For instance, French Canadians have more deleterious mutations than those who live in France or Non-French Canadians (Peischl et al., 2018). Similarly, people living on Islands such as Greenland were also found to have more deleterious mutations than their mainland counterparts (Pedersen et al., 2017). In both cases, severe bottlenecks occurred due to the limited number of individuals who formed the founders of these populations. Hence the reduction in the population size caused the accumulation of higher proportions of deleterious mutations in French Canadians and Greenlanders than in their mainland counterparts.
During domestication, only a small subset of wild animals was selected, and hence the effective population size of domesticated animals is significantly reduced compared to their wild counterparts (Moyers et al., 2018). Due to this reason, the proportion of deleterious mutations is expected to be higher in domesticated animals than in their wild progenitors. A number of studies provided empirical evidence for this prediction. For example, an earlier study showed that domesticated dog breeds have a much higher deleterious mutation load than wild wolves (Marsden et al., 2016). Similar results were reported by comparing the exomes of domesticated and wild yaks (Xie et al., 2018). Using 432 animals belonging to 54 worldwide cattle breeds, a previous study found a negative correlation between heterozygosity and deleterious mutational load, which suggests that breeds that have low genetic variation have higher deleterious mutational load than those with high genetic variation (Subramanian, 2021). Since heterozygosity is the product of N e and mutation rate (μ), and the mutation rate is largely similar across breeds, the above relationship implies a negative correlation between N e and the mutation load.
Apart from domestication, the difference in the natural population sizes also cause a variation in the mutational loads. For instance, the island population of kakapo (a parrot species endemic to New Zealand) was found to carry a much higher proportion of deleterious variants than the mainland population (Foster et al., 2021). Similarly, the deleterious mutational load of mammoths that lived on Wrangel island was much higher than that of those who lived on the mainland (Rogers and Slatkin, 2017). Since the population size of island species are smaller than that of mainland ones, the accumulation of deleterious mutations was the result of high genetic drift modulating the mutations of the former. Similarly, killifish populations in drylands harboured more deleterious variants than those from the wetlands (Willemsen et al., 2020). This is because the former is known to have a smaller population size than the latter. Furthermore, a study on Alpine ibex found evidence for the accumulation of mildly deleterious SNVs but the purging of highly deleterious SNVs during population bottleneck (Grossen et al., 2020).
Almost all of the previous studies used the ratio of nonsynonymous-to-synonymous diversities (dN/dS) as the proxy for the deleterious mutation loads. Their findings clearly demonstrated low and high mutation loads in populations with large and small N e . However, genetic admixture occurs quite often between populations with distinctly different N e . For instance, a previous study showed the fitness of Neanderthals was significantly less than that of humans because the former has smaller N e than the latter (Harris and Nielsen, 2016). Furthermore, this study revealed that non-African populations that admixed with Neanderthals (non-Africans) have a 0.5% reduction in fitness (Harris and Nielsen, 2016). However, it is important to understand how the mutational load of the admixed progenies derived from two populations (or subspecies) with widely different N e (and, in turn, mutation loads) is shaped by evolution. To investigate this, we obtained the whole genome data of Mus musculus castaneus and Mus musculus musculus and their hybrid or admixed populations. We specifically selected these because the subspecies M. m. musculus and M. m. castaneus have population sizes of 60,000-120,000 and 200,000-400,000, respectively (Salcedo et al., 2007;Geraldes et al., 2008), and previous studies have found a much higher mutational load in the former compared to that observed in the latter (Phifer-Rixey et al., 2012;Subramanian, 2018). We estimated the dN/dS ratios and the proportion of deleterious nonsynonymous SNVs (nSNVs) for the admixed mouse population and compared them with those of their parental populations. We also examined the correlation between the level of admixture and deleterious mutational loads.

Genome data
Whole genome data for 11 Mus musculus castaneus, 11 Mus musculus musculus, 18 hybrids (M. m. castaneus' M. m. musculus) and one Mus spretus were obtained from a previous study (Fujiwara et al., 2022). We only used the single nucleotide variations (excluding INDELS), and positions with more than two alleles were excluded. To determine derived alleles, the genomic positions of the outgroup Mus spretus were used to orient the mutations. We only included the positions where the outgroup was homozygous, and at least one Mus musculus genome had an allele that was different from the outgroup. We excluded the mitochondrial genome and sex chromosomes and used only autosomes. We used the genome annotations and extracted the protein-coding genes, and using the software PAML (Yang, 2007) we determined the number of nonsynonymous and synonymous sites. To identify nonsynonymous (nSNVs) and synonymous SNVs (sSNVs), the program SNPEffect was used (Cingolani et al., 2012).
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Data analysis
The software plink was used to cluster the genomes based on their genetic relationship (Purcell et al., 2007), and the multidimensional scaling method was employed for this purpose. The VCF file was first converted into a binary bed format. The SNVs with <5% MAF (Minor Allele Frequency) were excluded, and genomes that contained >10% gap or unknown genotypes were excluded. Finally, the first two principal components that explain the maximum variation were used to plot the values and observe the population clusters. To determine the proportion of ancestries in each admixed genome of the hybrids the likelihood-based method admixture was used (Alexander et al., 2009). Using the cross-validation analysis, we determined the K value that had the lowest error and used that value (2) to denote the number of ancestral populations.
Using the number of nSNVs and sSNVs and their respective sites, we estimated the diversities at these sites and used them to compute their ratio (ω obs ) as: To determine the deleteriousness and to identify the harmful mutations, we used the PhyloP conservation scores (Hubisz et al., 2011). We obtained the basewise PhyloP scores based on 59 vertebrate genomes. 1 The score was available for each position of the mouse chromosomes. The PhyloP scores were then mapped to the genomic positions, and any SNV present in a genomic position with a PhyloP score > 2.0 was designated as deleterious in nature. Based on this, the nSNVs with a PhyloP score > 2.0 were considered deleterious nSNVs. The number of deleterious nSNVs (DN obs ) was divided by the number of all nSNVs (N obs ) to obtain the proportion of observed deleterious nSNVs (δ obs ) as given below: To determine the distribution of fitness effects of the SNVs, the software DFE was used (Eyre-Walker et al., 2006). The site frequency spectrums (SFS) of M. m. castaneus, M. m. musculus, and the hybrid mice populations were obtained. To model the distribution, first the lookuptable program of this software was used. The output of this program was used along with the SFS of each population to obtain the fitness effects of the SNVs.

Theoretical prediction
To predict the genomic heterozygosity or diversity for admixed genomes (H adm ) by using the allele frequencies of their ancestral populations we used the equation developed by Boca et al. (2020), as given below: where p j is the frequency of allele j, N is the number of alleles, γ i is the proportion of admixture of ancestor i, and K is the number of ancestral populations contributed to the genetic makeup of the admixed populations. d adm calculated for nonsynonymous (dN admn ) and synonymous sites (dS adms ) and their ratio of the predicted diversities (ω pre ) was computed as: Similarly, to predict the proportion of deleterious nonsynonymous SNVs (δ pre ) based on the nonsynonymous (N i ) and deleterious nonsynonymous SNVs (DN i ) of the ancestral populations, we used the following formula:

Statistical analyses
The average dN/dS ratios and the mean proportion of deleterious nSNVs, along with the standard errors, were estimated for each pure and admixed genome. The significance between the mean estimates was determined using the Z test. A regression and correlation analysis was performed to study the relationship between the proportion of genetic admixture and dN/dS ratios or the proportion of deleterious nSNVs. The nonparametric Spearman's rank correlation was used to determine the strength of the correlation. 2 Furthermore, using the parametrical Pearson correlation also produced similar strength of the relationship. The statistical significance of the Pearson correlation was determined by converting the correlation coefficient r to the normal deviation Z, and this was accomplished using the online software r to Frontiers in Ecology and Evolution 04 frontiersin.org P. 3 In-house Perl scripts were used to map the PhyloP scores, calculate the point estimates and standard errors, and perform statistical tests.

Results
Population structure and admixture To understand the genetic relationship between parental and admixed populations, the Principal Component Analysis (PCA) was performed. The results showed distinct clusters for parental populations ( Figure 1A). The genomes of M. m. musculus are on the left and those of M. m. castaneus on the right ends of the plot. In contrast, the admixed individuals spread in between their parental populations. The hybrid genomes close to M. m. musculus suggest a high proportion of musculus ancestry and a low proportion of castaneus ancestry in their genome. Those close to M. m. castaneus suggest the opposite. To estimate the actual proportion of these ancestries, we used the maximum likelihood-based software Admixture. Figure 1B shows the unadmixed parental populations in single colours, which confirms the purity of these genomes. The admixed individuals are shown in two colours suggesting the levels of admixture, and the size of each colour on the columns indicates the proportions of the corresponding ancestry. The admixed mice genomes have 10%-95% of M. m. musculus ancestry.

The ratio of nonsynonymous and synonymous diversities
To estimate the deleterious mutation load in each genome, we first calculated the dN/dS ratio for each genome. The average estimate and standard error were computed for M. m. castaneus, M. m. musculus, and the admixed populations. Figure 2A shows the mean estimates were very distinct for the populations, and the differences between them were highly significant (p < 0.00001, using a Z test). The ratio of M. m. musculus was higher than that of M. m. castaneus, and importantly that observed for the admixed population was intermediate between the two. We then plotted the dN/dS ratios of each admixed individual against the proportion of M. m. musculus ancestry of these genomes ( Figure 2B). Our results showed a highly significant (rho = 0.89, p < 0.00001, Spearman's rank correlation) positive correlation between the two variables. The dN/dS ratio was small for admixed individuals with less proportion of M. m. musculus ancestry, and it was high for those with a high fraction of the musculus ancestry.

The proportion of deleterious nSNVs
To further examine the harmful mutation loads, we used the proportion of deleterious nSNVs as the measure to quantify the load. We computed this using homozygous nSNVs, and heterozygous nSNVs and then combined these two to obtain the proportion for all 3 http://vassarstats.net/tabs_z.html deleterious nSNVs. As shown in Figure 3A, the mean estimate of the proportion of all deleterious nSNVs was the highest for M. m. musculus, lowest for M. m. castaneus, and intermediate for the admixed population. The differences between the mean estimates were also statistically significant (at least p < 0.00001). Similar results were observed for the proportion of deleterious homozygous and heterozygous nSNVs as well (Figures 3B,C). Although the differences between the mean estimates were statistically significant, the magnitude of the differences was high for the heterozygous and low for the homozygous SNVs.
To understand the relationship between the proportion of M. m. musculus admixture/ancestry and the deleterious mutation load, a correlation analysis was conducted. For this purpose, first, we plotted the proportion of M. m. musculus ancestry against the proportion of all deleterious nSNVs of each genome ( Figure 4A). This produced a highly significant positive relationship (rho = 0.91, p < 0.00001), which suggests that the admixed individuals with high proportions of M. m. musculus ancestry also have high proportions of deleterious nSNVs and those with low proportions have less proportions of these SNVs. Similarly, highly significant positive relationships were also observed for the homozygous (rho = 0.94, p < 0.00001) and heterozygous SNVs (rho = 0.81, p < 0.0001; Figures 4B,C).

Expected and observed mutational loads in admixed populations
Previous results revealed the patterns of mutation load observed in admixed populations. In order to understand the patterns expected solely based on the allele frequencies of the ancestral parent populations, we used the Equation (3) developed by a previous study to estimate the expected heterozygosity (see Methods). Using this formula, we first calculated the predicted nonsynonymous (dN adm ) and synonymous (dS adm ) diversities and obtained the predicted ratio of these diversities (ω pre ). We then compared this ratio with that observed for the admixed genomes (ω obs ). The observed nonsynonymous (dN) and synonymous (dN) diversities were 11% smaller than those corresponding predicted diversities (dN adm and dS adm , respectively). However, the observed ratio of these diversities (ω obs ) was not statistically different from that expected (ω pre ; p = 0.33; Figure 5). We also developed an Equation (5) to estimate the predicted proportion of deleterious SNVs (δ pre ) in admixed genomes based on the level of contributions of their ancestral populations (see Methods). The proportion of deleterious SNVs observed for the admixed populations was compared (δ obs ) with the predicted proportion of these SNVs ( Figure 5). Our results did not reveal any significant difference between the predicted and observed proportions (p = 0.32).

Distribution of fitness effects
To infer the fitness effects of nonsynonymous SNVs, we used the software DFE by providing the site frequencies of nonsynonymous and synonymous SNVS as input. This analysis produced the fraction of nSNVs belonging to different fitness effect categories. The results revealed that the M. m. musculus population has the highest fraction of mildly deleterious or nearly neutral (Nes < 10) nSNVs and lowest Frontiers in Ecology and Evolution 05 frontiersin.org fractions of moderately (Nes 10-100) and highly deleterious nSNVs (Nes > 100; Figure 6).  Frontiers in Ecology and Evolution 06 frontiersin.org

Discussion
Population genetic theories predict a higher proportion of deleterious mutations in a population with a small N e compared to that with a large N e (Crow and Kimura, 1970;Phifer-Rixey et al., 2012;Marsden et al., 2016;Subramanian, 2021). Many previous studies provided empirical evidence for this prediction using genome data from humans and other vertebrates (Lu et al., 2006;Mezmouk and Ross-Ibarra, 2014;Renaut and Rieseberg, 2015;Kono et al., 2016;Marsden et al., 2016;Pedersen et al., 2017;Ramu et al., 2017;Makino et al., 2018;Peischl et al., 2018;Xie et al., 2018;Bosse et al., 2019;Robinson et al., 2019;Dussex et al., 2021;Subramanian, 2021). Using the dN/dS ratio as the measure to quantify deleterious mutation load, these studies consistently showed that the populations with small N e had a higher ratio than those with large N e . However, the mutation load of the populations that derive from genetic admixture between two parental populations with distinctly different N e is not known. In the present study, using the dN/dS ratio and the proportion of deleterious nSNVs as proxies to quantify deleterious mutation load in mouse populations that result from the admixture between the subspecies M. m. castaneus and M. m. musculus. We report two main findings. First, the mean dN/dS ratio and the proportion of deleterious nSNVs estimated for the admixed mouse population was intermediate between those of observed for their parental populations. Second, these measures positively correlated with the fraction of M. m. musculus ancestry (or negatively correlated with the fraction of M. m. castaneus ancestry).
The results of this study can be explained by the fact that the N e of M. m. castaneus (200,000-400,000) is much higher than that of M. m. musculus (60,000-120,000; Salcedo et al., 2007;Geraldes et al., 2008). Since deleterious mutation load negatively correlates N e (Kimura and Ohta, 1978;Kimura, 1983), this load is expected to be higher in M. m. musculus compared to that in M. m. castaneus. Our results exactly showed this, as the dN/dS ratio (Figure 2A) and the proportion of deleterious nSNVs (Figure 3) were found to be higher for the former than those for the latter. On the other hand, the deleterious mutation load for the population that derived from the admixture of these two subspecies is expected to be less than that of M. m. musculus as they have a fraction of M. m. castaneus ancestry, which will tend to reduce the overall load of the admixed individuals. Similarly, the deleterious mutation load of the admixed population is expected to be higher than that of M. m. castaneus as they have a fraction of M. m. musculus ancestry, which will tend to increase the overall load of the hybrids. The proportion of deleterious nonsynonymous SNVs (nSNVs) (δ obs ) computed for M. m. castaneus, M. m. musculus, and admixed populations. (A) All deleterious nSNVs, (B) Homozygous deleterious nSNVs, and (C) Heterozygous deleterious nSNVs. The error bars show the standard error of the mean. The differences between the proportion of deleterious nSNVs of the hybrid and M. m. musculus were significant for all (p < 0.00001), homozygous (p < 0.0341), and heterozygous SNVs (p < 0.00001). Similarly, the differences between the hybrid and M. m. castaneus were also significant for All (p < 0.00001), homozygous (p < 0.00001), and heterozygous SNVs (p < 0.0001).
Frontiers in Ecology and Evolution 07 frontiersin.org Due to this reason, the dN/dS ratio ( Figure 2A) and the proportion of deleterious nSNVs (Figure 3) observed for admixed genomes were higher than those of M. m. castaneus and less than those of M. m. musculus population. The above prediction was further confirmed by the positive correlation between the proportion of M. m. musculus ancestry and the dN/dS ratio ( Figure 2B) or the proportion of deleterious nSNVs (Figure 4). Note that if we used the M. m. castaneus ancestry we found a negative correlation with the same strength and same level of statistical significance. Previous studies on dogs, cows, and yak used deleterious heterozygous and homozygous nSNVs counts to compare mutational loads between populations with different N e (Marsden et al., 2016;Xie et al., 2018;Subramanian, 2021). However, populations with small N e are expected to have a higher number of deleterious as well as neutral (or benign) homozygous nSNVs than those with large N e . Therefore, the above studies observed a high number of deleterious and neutral homozygous SNVs in the genomes of the former and low in the latter. Since the deleterious and neutral homozygous SNVs show similar patterns, the use of SNV counts to measure the mutational load is not accurate. Due to this reason, we used the proportion of deleterious homozygous nSNVs, which was calculated by dividing the number of deleterious homozygous nSNVs by the number of all (benign + deleterious) homozygous nSNVs (Figures 3B,4B) and this proportion removes the bias expected in using the nSNV counts. A similar measure was used for heterozygous deleterious nSNVs as well ( Figures 3C, 4C).
Although we observed the ratio of nonsynonymous and synonymous diversities in the admixed genomes it is important to examine the loads expected in these based on the allele frequencies of their parental populations. Although the observed nonsynonymous and synonymous diversities were significantly smaller than those predicted, their ratios were not. This is because the rate of reduction in the observed estimates of both nonsynonymous and synonymous sites was almost the same (~11%). This result and the similarity between the predicted and observed proportion of deleterious nSNVs Relationship between the proportion of M. m. musculus ancestry/ admixture and the proportion of deleterious nSNVs estimated for admixed individuals. (A) All deleterious nSNVs, (B) Homozygous deleterious nSNVs, and (C) Heterozygous deleterious nSNVs. The relations were highly significant for all (rho = 0.91, p < 0.00001), homozygous (rho = 0.94, p < 0.00001), and heterozygous SNVs (rho = 0.81, p < 0.0001). The best-fitting curves are shown. Column graph comparing the observed (ω obs ) and predicted (ω pre ) ratios of nonsynonymous and synonymous diversities and the proportion of deleterious SNVs observed (δ obs ) and predicted (δ pre ). The differences between the observed and predicted values of the ratios (p = 0.33) and the proportions (p = 0.32) were not statistically significant. Distribution of fitness effects of nonsynonymous SNVs (nSNVs). The X-axis shows the level of deleteriousness of nSNVs in terms of the product of effective population size (N e ) and selection coefficient (s). The nSNVs were grouped into mildly (N e s < 10), moderately (N e s 10-100) and highly (N e s > 100) deleterious in nature. The double asterisks indicate statistical significance at the 1% level (Z-test, two-tailed). The actual p-values for each comparison are given in the main text (see Results).
Frontiers in Ecology and Evolution 08 frontiersin.org suggest that the level of admixture more likely determines the mutational loads of the admixed progenies. The distribution of fitness effects of nonsynonymous nSNVs revealed a higher proportion of mildly deleterious SNVs and a lower proportion of moderately and highly deleterious nSNVs in M. m. musculus than M. m. castaneus. This suggests that some of the highly deleterious nSNVs that were removed from M. m. castaneus population were present or segregating in the M. m. musculus population. This is because these nSNVs were deleterious for the former but became or behaved as mildly deleterious in the latter population. The potential reason is that the presence or removal of the nSNVs is determined by the product of effective population size (N e ) and selection coefficient (s). Therefore, for a large population, the N e s will be larger than that of a small population for the same vale of s. Hence, the fitness consequences of nSNVs in small populations will be relatively less deleterious than that in large populations. Therefore, some of the nSNVs that were highly deleterious for M. m. castaneus were mildly deleterious for M. m. musculus. The genetic Admixture between M. m. castaneus and M. m. musculus results in the loss of some of the moderately deleterious nSNVs whilst gaining or accumulating some of the mildly deleterious ones.
Although many previous studies estimated the mutation load in pure or unadmixed populations, how these loads are modulated by the proportion of ancestry in admixed genomes was not clearly demonstrated before. Hence, the analyses performed here make this study novel and unique. Due to this new approach, the results of this study have opened up new dimensions in understanding the diversity and mutational load of admixed populations in comparison to their ancestral/parental populations. Eventually, this approach will help to recognise the role of gene flow between populations, particularly in exchanging harmful mutations.

Conclusion
Using the whole genome data, this study revealed the patterns of deleterious mutation load in a mouse population, which derived from genetic admixture between two distinct populations belonging to the subspecies M. m. castaneus and M. m. domesticus. The results showed the dN/dS ratio and the proportion of deleterious nSNVs of admixed populations were intermediate compared to their parents. This could be observed only if the parental populations had significant variation in their mutational load, which is driven by their effective population sizes. Our study also revealed a significant positive correlation between the proportion of admixture and the dN/dS ratio or the proportion of deleterious nSNVs in admixed individuals. This suggests the role of admixture in shaping the deleterious mutation loads. Whilst this was observed in mice, it is highly likely that similar patterns are expected in other vertebrates as well. Therefore, it is important to consider the status and the level of genetic admixture of the populations whilst estimating the mutation loads.

Data availability statement
Publicly available datasets were analysed in this study. This data can be found at: https://www.ddbj.nig.ac.jp/bioproject/ accession: PRJDB11027.

Author contributions
SS conceived the idea, designed and supervised the research, and wrote the manuscript with inputs from other authors. UR performed research and data analysis, and contributed to writing the manuscript. AE supervised the research and contributed to writing and editing the manuscript. All authors contributed to the article and approved the submitted version.

Funding
The research was supported by the University of the Sunshine Coast DVCR grant awarded to SS.

Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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