DOI: 10.5061/dryad.5tb2rbp69
title: Asymmetric density-dependent competition does not contribute to the maintenance of sex in a mixed population of sexual and asexual Potamopyrgus antipodarum

description: Asexual reproduction is expected to have a two-fold reproductive advantage
 over sexual reproduction, owing to the cost of producing males in sexual
 subpopulations.  The persistence of sexual females thus requires
 an advantage to sexual reproduction, at least periodically. 
  Here we tested the hypothesis that asexual females are more
 sensitive to limited resources.  Under this idea, fluctuations in
 the availability of resources (per capita) could periodically favor sexual
 females when resources become limited.  We combined sexual and
 asexual freshwater snails (Potamopyrgus antipodarum) together in nylon
 mesh enclosures at three different densities in an outdoor
 mesocosm.  After one month, we counted the brood size of fertile
 female snails.  We found that fecundity declined significantly
 with increasing density.  However, sexual females did not produce
 more offspring than asexual females at any of the experimental
 densities.  Our results thus suggest that the cost of sexual
 reproduction in P. antipodarum is not ameliorated by periods of intense
 resource competition.
description: <strong>Natural History </strong>
 All <em>P. antipodarum</em> snails were collected
 from Lake Alexandrina, a mesotrophic high-country lake on the South Island
 of New Zealand.  This lake experiences rapid plant growth in the spring,
 which corresponds to rapid increase in <em>P. antipodarum</em>
 populations (Talbot & Ward, 1987).  This growth in snail
 population size was especially apparent after algal blooms (Talbot
 & Ward, 1987), suggesting increases in resources correlates with
 higher brooding.  This pattern has been seen in other species, including
 the freshwater snail Helisoma trivolvis (Hoverman et al., 2005), and
 several species of <em>Daphnia </em>(Walls et al., 1991; Wu
 & Culver, 1994).  Female snails in Lake
 Alexandrina reach sexual maturity at about 3mm shell length.  Females are
 ovoviviparous with the lower oviduct forming a brood pouch (Winterbourn,
 1970a).  Asexuals reproduce by apomictic parthenogenesis.  Mated sexual
 female snails will store sperm (Fretter & Graham, 1962; Wallace,
 1992), indicating that male limitation is not an issue for sexually mature
 females.  In both reproductive modes, offspring are retained within the
 brood pouch until they reach a ‘crawl-away’ stage (Winterbourn, 1970a;
 Jokela et al., 1997).  <strong>Collection and
 Density </strong> In January 2020, a large
 (<em>N</em> &gt; 10,000), random sample of <em>P.
 antipodarum</em> was collected near site JMS at Lake Alexandrina, NZ
 (GPS coordinates: -43°56’12.1” S, 170°27’36.7”E; or see JMS on Figure 3 in
 Million et al., 2021).  Snails were collected by pushing a kick net
 through a bed of the macrophyte, <em>Isoetes kirki</em> at a
 depth of 1.5-3.0m.  We focused on this site at this depth because it
 contains a large population, with mixtures of sexual and asexual snails
 (Jokela & Lively, 1995).  In addition to snails, 6 water samples
 (125 mL each) were collected at sites around Lake Alexandrina to condition
 the water in the experiment with local microflora and fauna.  This water
 would also seed the experimental tank with natural algal and diatom
 propagules, that would contribute to resources available to the snails. 
 Six days prior to the experimental start, these water samples, and 2.25 mL
 of dried <em>Spirulina</em> powder were combined and mixed
 into an 800 L tank of aged tap water in Kaikoura, NZ.  Experimental
 enclosures were also placed within the tank allow for algal growth on the
 mesh (Supp. Video 1).  Each enclosure consisted of a 2L plastic bottle
 with eight cut out holes (four circles approx. 5.5cm in diameter, and four
 rectangles approx. 12 x 5.5 cm) to ensure water exchange, and a tagged
 float to keep the enclosures upright (Supp. Fig. 1A).  The bottle and
 float were sewn into 500µm nylon mesh bags, a mesh size which allows
 newborn snails to escape.  Adult snails were separated
 with a 1.7mm sieve, which allows juvenile snails (&lt;3mm) through,
 but retains adults.  Adult snails were randomly selected (males, sexual
 females, and asexual females), counted, and assigned to eight replicates
 each of sample sizes of 100, 400, and 900 snails for a total of 11,200
 snails.  An additional sample of 500 snails was frozen to determine the
 brooding status before the experiment.  Each group was placed within an
 experimental enclosure and returned to the tank with pre-conditioned
 water.  A submersible pump was added to the tank to gently circulate the
 enclosures during the day, which also ensured that the food was well mixed
 in the water column.  Density was maintained at experimental levels
 throughout the experiment, because newborn snails in the crawl-away stage
 were small enough to pass through the enclosure mesh.  We did not measure
 the production of crawl-away offspring, as we were instead interested in
 the brooding following a period of resource deprivation.
 The water in the tank was replaced halfway through the experiment
 with aged tap water held in a separate tank.  After 30 days in these
 conditions, all enclosures were photographed (Supp. Fig. 1B-D;
 representative photographs for each density), and the snails were removed
 to 20 l containers.  The experiment was constrained to 30 days due to the
 length of the field season, however the visual differences between
 enclosures indicated that this time was sufficient to induce resource
 differences, and the later dissection results indicated that the resource
 difference were sufficient to reduce brooding.
 <strong>Resource quantification</strong>
 Each of the enclosure photographs were analyzed in ImageJ
 (Schneider et al., 2012).  The background of the image was excluded from
 analysis using the polygon selection tool.  For each image, the mean grey
 value of the selection was measured and recorded.  Higher mean values for
 the grey scale were indicative of more resources per snail. 
 <strong>Tissue preservation, transport, and
 dissection</strong> Immediately following the
 removal of snails from the experimental enclosures, 10 snails per
 replicate per density were sexed and dissected.  The pilot samples from
 the 100-snail treatment were preserved by freezing for further analysis. 
 An additional sample of <em>N</em>=40 for each 100-snail
 replicate and <em>N</em>=100 for each 400- and 900-snail
 replicate were then frozen for transport to Indiana University in
 Bloomington, IN, USA.  The frozen snails were thawed, sexed and dissected
 in Bloomington, IN, USA to determine trematode infection status.  Because
 trematode infection is sterilizing, infected snails were excluded from
 further analysis.  Brooding status was defined as the presence or absence
 of eggs in any stage in the brooding pouch of the snail.  Brood size was
 measured as the number of eggs present in the brooding pouch – these eggs
 are identifiable even after freezing.  For all snail samples, head tissue
 was preserved by freezing at -80C, and reserved for DNA extraction and
 further analysis.  Body tissue was preserved by freezing at -80C and
 reserved for flow cytometric-based determination of DNA content, to
 estimate ploidy as a proxy for reproductive mode.
 <strong>Determination of DNA content through flow
 cytometry</strong> We used flow cytometry to
 quantify DNA content (following Osnas & Lively, 2006).  Each
 sample was homogenized in 200 µl of DMSO using a microtube homogenizer
 (Benchmark Scientific) with 1.5mm zirconium homogenizer beads.  After
 homogenizing, each sample was incubated at 4C for 30 minutes in a staining
 solution of 200ug/mL Propidium Iodide, 3.4 mM Trisodium citrate dihydrate,
 0.1% Nonidet P-40, 0.5 mM Tris, 466.7 ug/mL spermine, 693.3 ug/mL spermine
 tetrachloride, and 200 ug/mL Rnase A.  Each sample was filtered through
 Celltrix 50um mesh into a flat-bottomed Falcon plate to remove cell
 aggregates, homogenizer beads, and debris.  These samples were kept at 4C
 until they were analyzed, at which point they were transferred to a
 u-bottomed Falcon plate at a final volume of 200 µL per sample. 
 We recorded the fluorescence intensity of a maximum of 5000
 single cell nuclei per sample, using the Y1-A channel on the MACSQuant VYB
 cytometer running MACSQuantify software (Milteniyi Biotec).  The mean
 fluorescence intensity was recorded for the snail tissue in each sample. 
 We visually inspected the sample peak and used gating to create
 subpopulations of cells that corresponded to cell nuclei in the G1 phase. 
 We excluded from the data set any samples that yielded ambiguous or
 low-quality fluorescence peaks because we were unable to estimate ploidy
 for these individuals.  Each sample was initially analyzed using
 MACSQuantify software, then later processed using the R flowCore package
 (Ellis et al., 2019).  To estimate ploidy and thus reproductive mode for
 each sample, we used males (assumed to be diploid), which were collected
 from the same enclosures as the females, as an internal reference, and we
 used lab-reared triploid snails as an external reference.  Because
 triploids have three chromosomes for every two chromosomes in diploids,
 asexual snails were assumed to have 1.5 times the DNA of diploid sexual
 females.  However, recent work has shown that asexual triploid snails vary
 in DNA content (Neiman et al., 2011; Million et al., 2021).  Despite this
 variation, asexuals still tend to have higher DNA content than sexuals. 
 In addition, the fluorescent peak of asexual nuclei tends to be wider than
 sexual nuclei (see Figure 2 in Neiman, et al., 2011; Figure 3 in Soper et
 al., 2013).  Therefore, we have adapted our analysis of flow cytometry
 files to conservatively identify diploids and triploids, using the
 intensity and standard deviation of the fluorescence peak for each snail. 
 This analysis identified two populations: one sexual and one asexual, with
 minor overlap (Supp. Fig. 2).  The overlapping snails were excluded from
 further analysis as a conservative estimate of ploidy.  We confirmed that
 including these snails did not affect the statistical results or overall
 conclusions of this experiment.
 <strong>Statistical analyses</strong>
 All statistical analyses were performed in R version 3.6.3 (R
 Team, 2020).  We used the dplyr package to organize the data, and the
 ggplot2 package for all graphs (Wickham et al., 2020; Wickham, 2016).  The
 mean grey values (generated by Image J) for all enclosure photographs were
 analyzed using a generalized linear model with Density as the independent
 variable.  We also analyzed a pairwise comparison of all densities with
 linear contrasts.  To analyze snail brooding, we created a generalized
 linear model, with brooding/non-brooding as a binomial response variable,
 and density treatment, reproductive mode, and the interaction between the
 two variables as independent factors.  Further, we created a generalized
 linear model with a Gaussian distribution of brood size (excluding
 non-brooders) as the dependent variable, and with density treatment,
 reproductive mode, and the interaction between the two variables as
 independent effects.  We tested the normality of residuals for this model
 using the Shapiro-Wilk normality test, and the assumption of equal
 variance using the Studentized Breusch-Pagan test.  We calculated
 estimated marginal means with 95% confidence intervals for each model,
 using the emmeans package (Lenth, 2021), and the fixed effect F statistics
 for each independent variable using the rstatix package (Kassambara,
 2020).  To generate a combined metric for fitness,
 including brooding and non-brooding snails, which creates a dataset that
 is not normally distributed, we also computed a non-parametric test.  We
 performed a Kruskal-Wallis test for brood size on group.  Group variable
 was a factor which combined the factor variables of density and
 reproductive mode.  Finally, we performed the Dunn test (FSA package; Ogle
 et al., 2021) with the Bonferroni correction for multiple comparisons as a
 post-hoc analysis to determine which groups significantly differed from
 each other group. Finally, a post-hoc power analysis was performed using
 the pwr package (Champely, 2020).
description: Licence and Restriction statement There are no
 licences or restrictions placed on access to the dataset or any associated
 files. The data file “JEB_Resource Availability
 Analysis.csv” contains 4 columns: A. Density: numeric
 description (100, 400, 900) of the density treatment for each
 bag. B. Replicate: numeric description (1-8) of the
 replicate of the bag. C. Area: numeric value
 (328263-417370) of the area (in pixels) of the photograph analyzed in
 ImageJ. D. Mean_Grey: numeric value (191.432-214.991)
 of mean grey value of the area analyzed in Image J. The
 data file “JEB_Density and Sex.csv” contains 10 columns:
 A. Density: numeric description (100, 400, 900) of the density
 treatment for each bag. B. Replicate: numeric
 description (1-8) of the replicate of the bag. C. Tube:
 Identification of the sample (snail) analyzed. D.
 Length: numerical value of the length of the snail shell (in
 mm). E. Sex: factor (M/F) - sex of the snail, male (M)
 or female (F). F. Brood: numerical value of the number
 of eggs carried by the snail. G. Binary_Brood: Binary
 Value for brooding - 0 = nonbrooding snail, 1 = brooding snail.
 H. Binary_Infection: Binary value for infection - 0 = uninfected
 snail, 1 = infected snail. I. Fluorescence_Peak: The
 fluorescence intensity at the peak of the histogram (see
 "Determination of DNA content through flow cytometry" section of
 methods) J. Fluorescence_SD: The standard deviation of
 the fluorescence intensity peak (see "Determination of DNA content
 through flow cytometry" section of methods) Code
 Description The statistical analyses were performed in
 R using the file “JEB_Density and Sex.Rmd” in R Studio.