Figure 2. Many MLGs were identified at multiple time points throughout the epidemics in A) Little Appleton and B) Crooked, but, overall, distance between P. ramosa populations increased with time. C) For Little Appleton, this pattern is driven mostly by the difference between the genotypes at the beginning of the outbreak and those from later in the outbreak, whereas for Crooked (D), genetic distance between populations increased steadily with time between sampling dates. Genetic distance (C,D) is calculated between populations and is the average difference in allele frequencies between the populations summed over all loci (Prevosti et al., 1975).
DISCUSSION
We tracked P. ramosa genotypes through epidemics in two lakes, one with a large outbreak and one with a small outbreak. Parasite genetic diversity remained relatively constant over time, but, surprisingly, genetic diversity was much higher in the lake with the smaller outbreak (Crooked). We found signatures of evolution in both lakes: parasite genotypes structured by sample date in Little Appleton and the genetic distance between parasite populations increased with time between sampling dates in both outbreaks. In Little Appleton, there was a large change in genetic distance between the first sampling date and the second and relatively little change thereafter, whereas in Crooked, the increase in genetic distance occurred more gradually over time. Overall, our data show that parasite evolutionary trajectories may differ across outbreaks and that ecological drivers and feedbacks associated with epidemic size should be more extensively explored.
A priori , we would have expected greater diversity of P. ramosa in the larger outbreak, since the much larger number of infected hosts would presumably allow more opportunities for P. ramosa to infect. Why, then, did we see lower diversity in the much larger Little Appleton population? One possibility is that selection might have been more efficient during this larger outbreak; in general, selection is more efficient in larger populations (Weber, 1990). The observed structuring of parasite genotypes by sampling date in Little Appleton might indicate that different parasite genotypes were selected over time perhaps due to changes in the host genotypes present or other ecological factors impacting parasite fitness. Selection on parasites by hosts or abiotic conditions can lead to local adaptation (Lively et al., 2004; Koskella, 2014) and negative frequency dependent selection (Ebert, 2008). Importantly, both phenomena have been observed in the Daphnia -P. ramosasystem. P. ramosa assemblages are locally adapted to abiotic conditions (namely: light penetration into lakes; (Rogalski & Duffy, 2020)) and negative frequency dependent selection has been observed in theDaphnia-P. ramosa system over decadal time scales (Decaestecker et al., 2007) and has been implicated in other Daphnia -parasite systems from observations of Daphnia genotype turnover within epidemics (Wolinska & Spaak, 2009; Turko et al., 2018). Additional ecological factors such as predation could also influence parasite evolution in this system. It is noteworthy that (Gowleret al., 2022) documented the evolution of reduced spore production during this same epidemic in Little Appleton, potentially due to the selective pressure to shift from vegetative growth to the production of transmission spores earlier (thus generating fewer spores) in a likely high predation environment.
Notably, the study lakes differed in host species (i.e., cladoceran) diversity with Crooked home to more Daphnia species than Little Appleton (unpublished data). Species diversity is often correlated with genotypic diversity within species (Vellend & Geber, 2005). This could be important because diverse host populations often experience smaller epidemics (King & Lively, 2012; Ekroth et al., 2019; Gibson, 2021). Even if the diversity within our focal host did not differ between the two populations, the higher host species diversity in Crooked may have helped to minimize the parasite outbreak via a dilution effect (Keesinget al., 2006; Hall et al., 2009; Strauss et al., 2016). While different Daphnia species tend to become infected by distinct genotypes of P. ramosa(Duneau et al.,2011; Shaw, 2019), it is possible that these species can consume and kill parasite spores that infect D. dentifera . Future studies that track parasite evolution across several populations that vary in host diversity would help uncover the links between interspecific host diversity and genotypic diversity within parasite populations.
Migration is another important determinant of parasite diversity and evolution. In this system, most migration is likely through time as parasites from epidemics in previous years get resuspended from sediments (Decaestecker et al., 2004, 2007). The relative contribution to infections from spores from the spore bank vs. those produced in the ongoing epidemic is unknown, and this contribution likely changes through time and may depend on lake basin structure (Cácereset al., 2006; Hall et al., 2010; Penczykowski et al., 2014). The pattern from Little Appleton suggests that transmission from the sediments occurs at the beginning of an epidemic and after that, successful genotypes from the ongoing epidemic are amplified. The pattern from Crooked – where more different genotypes were found throughout the epidemic – may indicate instead that infection from the spore bank might continue throughout the season either due to feeding in the sediments or due to sediment resuspension into the water column. Future work that tracks the genotypes of free-living spores in the water column, as well as the genotypes in infected hosts would help determine the relative contributions of spores produced during an ongoing epidemic vs. those resuspended from sediment; ideally, this would be done in multiple populations that varied in the likelihood of spore resuspension (e.g., lakes that are weakly stratified vs. those with very strong stratification).
Our data also indicate that parasite genotypes might migrate between lakes as three genotypes were shared between Little Appleton and Crooked which are 9 miles (14.5 km) apart. Such distances are commonly traversed by waterfowl, which can move parasite spores and infected hosts (Green & Figuerola, 2005). It is also possible that hosts in these lakes could be related to each other due to long distance dispersal of ephippia by birds (Green & Figuerola, 2005) and that related hosts could become infected by related parasites in different lakes; future studies tracking the genetic diversity of both hosts and parasites in multiple lakes would help uncover whether this is the case. However, it is also possible that, if we had more loci available, we would discover that these were, in fact, not the same genotype. While the number of loci that we used in this study was sufficient to detect substantial diversity within and between lakes, using newly discovered hypervariable regions of the P. ramosagenome (Andras et al., 2020) would likely uncover additional variation that was not captured by our VNTR analysis.
We quantified the genetic structure of populations of the parasiteP. ramosa in infected D. dentifera hosts and found evidence of evolution within outbreaks, potentially acting on parasite diversity introduced from the spore bank. We hypothesize that intra- and interspecific host diversity, host population densities, and epidemic size all influence the evolution of P. ramosa within epidemics. Future studies that include more epidemics and measure host genotypic diversity as well as genotypes of spores in the water column could help disentangle the mechanisms underlying this evolution.
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