DOI: 10.5061/dryad.d2547d868
title: Rapid evolution of sex role specialization in a hermaphrodite under sex-limited selection

description: The evolution of separate sexes from hermaphroditism is thought to have
 occurred independently many times, and may be linked to the evolution of
 sex chromosomes. Even though we have a good understanding of the
 theoretical steps in the evolution of sex chromosomes from a hermaphrodite
 ancestor, the initial stages are still hard to study in animals because
 many well-studied animal sex chromosome systems are old. We addressed this
 problem by experimentally selecting a hermaphrodite via sex-limited
 experimental evolution for several generations, simulating the early
 stages in the evolution of a sex chromosome. After 14 generations, a
 fitness assay revealed evidence of incipient sex role specialization in
 the female-selected lines, presumably reflecting the release from
 constraints usually imposed by selection on the other sex role.
 Importantly, however, this was not simply explained by morphology because
 testis and ovary sizes did not diverge among treatments. There was no
 evidence of a change in the male-selected lines. Our study shows that sex
 role specialization can occur rapidly as a result of sex-limited
 selection, which is consistent with genetic constraints between sex roles,
 and in line with the first predicted steps toward the evolution of a new
 sex chromosome system.
description: <strong>Experimental
 evolution protocol</strong> The experimental evolution lines consist of four
 replicate populations (denoted 1-4) within each selection regime
 (female-limited, male-limited, and control), resulting in 12 populations
 in total (denoted F1-4, M1-4, and C1-4). Populations with the same
 replicate number are not more closely genetically related to each other
 than to other replicates, but they are related in terms of handling (i.e.
 culturing was during the same time of the day, and worms with the same
 replicate number were placed in the same area of the incubator).
 A new
 generation starts with 48 mature, GFP(+) individuals from each replicate
 population being crossed with two worms each from the matched “source”
 population. Source populations are maintained in the same way as the other
 laboratory lines described above, and have the same genetic origin as the
 GFP(+) individuals from the same combination of treatment and replicate.
 They are used to provide mating partners for the GFP(+) individuals that
 neither carry the marker nor are exposed to the sex-limited selection. The
 trios of worms (1 GFP(+) and 2 GFP(-) individuals) are held in individual
 wells of 24-well tissue culture plates for one week, to provide
 opportunities for sperm competition and mate choice (figure 1). Therefore,
 the effective population size for each experimental line was approximately
 N<sub>e</sub> ≈ N + 0.5 = 144.5 (i.e. 48 GFP(+) individuals and 96 GFP(-)
 individuals; Caballero 1994, Falconer and Mackay
 1996), which is similar to
 several previous experimental evolution studies (e.g. Prasad et al. 2007, Michalczyk et al.
 2011, Innocenti et al. 2014, Buechel et al. 2016). Only 48 individuals per population experience
 sex-specific selection each generation, but we expected that this
 selection would be sufficient, given the success of other experimental
 evolution studies similar to our design (e.g. Morrow et al. 2008). After 7 days for mating, worms are isolated in
 new wells to lay eggs for one week. In the female-limited selection
 regime, the GFP(+) focal worms are isolated so that the GFP-marker is
 inherited via the female sex role, i.e. eggs. In the male-limited
 selection regime, the two GFP(-) mating partners are isolated for
 egg-laying, so that the GFP-marker is inherited via the male sex role,
 i.e. sperm. Finally, in the control lines, half of the selected worms are
 treated in the same way as the female-limited selection regime, and half
 are treated in the same way as the male-limited selection regime. Since
 the mating partners are not selected for one sex role or the other, the
 female-limited selection regime essentially mimics a gynodioecious system,
 while the male-limited selection regime mimics an androdioecious system.
 Egg-laying worms are discarded after one week, and eggs are left to hatch
 in the wells. After one week of growth, offspring in each well are moved
 to petri dishes to mature. If an experimental line does not produce
 sufficient numbers of GFP(+) offspring, backup offspring from the previous
 generation are used, so that generations are mainly non-overlapping, but
 not completely so. During the first ten generations, each
 generation lasted for four weeks, but due to poor production of offspring
 within the female selection regime, we subsequently extended the
 maturation period to two weeks instead of one, which was successful in
 increasing offspring numbers. We based this decision on prior knowledge
 that in juveniles, testes mature slightly earlier than ovaries, so that
 female-limited selection could have resulted in longer ovary maturation
 times (Vizoso and Schärer
 2007). <br>
 <strong>Fitness
 assay</strong> Worms from the 14th experimental generation were
 collected and isolated in wells, directly after the completion of
 egg-laying to produce generation 15. More specifically, mating partners
 not used in the egg-laying were held in new wells instead of being
 discarded, and after egg-laying for generation 15 was complete, egg-laying
 worms were combined with the same partners again to provide 24 trios per
 selection regime and replicate. These trios of worms were again allowed to
 interact for 7 days. Next, all individuals were isolated in new wells to
 lay eggs for 7 days. Adult worms were then discarded, eggs were left to
 hatch, and juveniles to grow. The whole procedure (mating for 7 days
 followed by egg laying for 7 days, and growth of juveniles) was then
 repeated using the same trios of individuals, in order to increase total
 offspring production and thereby decrease the error in the individual
 fitness measurements. Fitness was measured as the number of GFP(+) and
 GFP(-) offspring per well (i.e. per GFP(+) focal individual). The total
 number of offspring produced via eggs was used as a measure of female
 fitness, and the proportion GFP(+) offspring produced by both GFP(-)
 mating partners was used as a measure of male fitness. This fitness assay
 builds on a standard fitness assay protocol commonly used in
 <em>Drosophila</em> (e.g. Abbott et al. 2013, Lund-Hansen et al.
 2020) and is essentially
 the same as the sex-limited selection protocol, except for the fact that
 all worms are given the opportunity to lay eggs.
 One possible source of error
 in our experimental set-up compared to the <em>Drosophila</em>
 studies mentioned above is the fact that the GFP(+) focal individuals used
 in the fitness assays are expected to be heterozygous for the marker,
 meaning that only half of all offspring produced by the GFP(+) focal
 individual are expected to inherit the GFP(+) phenotype. For female
 fitness, this is not an issue, since female fitness was measured as total
 number of offspring produced, regardless of GFP phenotype. However, it
 could result in an additional error in measures of male fitness, since in
 this case, the presence of the marker is essential for identifying the
 progeny of the GFP(+) focal individual. We have previously investigated
 rates of transmission of the GFP marker via eggs and sperm and found them
 to be highly correlated, though individually variable
 (Nordén and Abbott
 2017), presumably due to
 variation in copy number at the GFP locus (Wudarski et al. 2017). An analysis of gene expression in these lines
 suggests that we have not inadvertently selected on GFP(+) expression as a
 byproduct of the selection protocol (Cīrulis 2022), so we therefore believe that neither variable
 expression of the GFP gene nor biased transmission depending on gamete
 type is likely to have important confounding effects on our measurements
 of male fitness. We measured sex-specific fitness for 267 individuals in
 total (91 from the control regime, 91 from the male-limited regime, and 85
 from the female-limited regime).
 <strong>Phenotypic
 measurements</strong> Body size, relative testis size, and relative
 ovary size were estimated with a standard method used in this species
 (Schärer and Ladurner
 2003). Briefly, worms were
 first isolated in wells containing f/2 solution and starved overnight.
 Each worm was then anesthetized for ten minutes in a well containing a mix
 of 600 μl artificial seawater (ASW) and 1 ml MgCl<sub>2</sub>
 –solution (conc 7.14 mg/ml). It was then slightly squeezed between a
 microscope slide and a cover slip, with pieces of plastic film of a
 standard thickness used as spacers in between (Schärer and Ladurner 2003). A picture of the whole body was then taken at
 x40 magnification, and of the ovaries and testes at x200 magnification.
 Photos were processed and analyzed in the program ImageJ (version 1.51),
 where the area of the body and area of the ovaries (sum of left and right)
 and the testes (sum of left and right) were calculated, respectively.
 Worms were photographed in random order, and the observer was blind with
 respect to the selection regime. We determined the repeatability of the
 morphological measurements by measuring 20 pictures three times for testes
 size, ovary size, and body area. This assay was carried out at generation 25.
 After discarding poor-quality images, our final sample size was 121 for
 body area (40 from the control regime, 40 from the male-limited regime,
 and 41 from the female-limited regime), 116 for testes area (39 from the
 control regime, 37 from the male-limited regime, and 40 from the
 female-limited regime), and 115 for ovary area (40 from the control
 regime, 36 from the male-limited regime, and 39 from the female-limited
 regime). The repeatability of the morphological measurements was high
 (body: intraclass correlation coefficient, ICC = 0.998, p = &lt;
 0.001, testes: ICC = 0.940, p = &lt; 0.001 and ovary: ICC = 0.882, p=
 &lt; 0.001; Koo and Li, 2016, Vaz et al 2013). 
 <strong>Statistical
 analysis</strong> All statistical analysis was carried out in R
 Version 4.1.0 (R Core Team
 2021). For the fitness
 assay, we excluded data from worms that had no offspring in either sex
 role (which could for example occur if the worm was injured during
 handling) and individuals that had undefined male fitness (i.e. no
 offspring from either mating partner at all). The sex-specific fitness
 measures were standardized to make them directly comparable. Specifically,
 within each sex role we subtracted the mean within the control regime from
 all measurements, then divided by the pooled standard deviation. This
 standardization procedure results in the overall control mean being equal
 to zero, so that the difference in mean between the control and the
 female- or male-selected regimes reflects the magnitude of the change in
 sex-specific fitness measured in standard deviations. Changes in
 sex-specific fitness between selection regimes were analysed using a mixed
 model approach implemented in lme4 (Bates et al. 2015) with selection regime, sex role (male or
 female), and their interaction as fixed effects, and individual ID and
 replicate population nested within selection regime and sex role as random
 effects (Lund-Hansen et
 al. 2020, Manat et al. 2021). Since we have two types of sex-specific
 fitness measures per individual, the random effect of ID was to control
 for variance arising from overall fitness differences between individuals,
 and the nested effect of replicate population was included in order to
 avoid pseudoreplication (Arnqvist 2020). Posthoc comparisons were carried out using the
 pairs() function from emmeans (Lenth 2021). Because gonad size is correlated with overall
 body size, we used relative gonad areas as a measure of investment in
 testes and ovaries (i.e relative testes area was calculated as testes
 area/body area, and relative ovary area was calculated as ovary area/body
 area). Differences in body area, relative testes area, and relative ovary
 area were each analysed with mixed models where selection regime was a
 fixed effect, and replicate population nested within selection regime was
 a random effect. Although here we report results from analyses using
 relative gonad sizes, results were qualitatively similar if body area was
 instead included as a covariate (see Results).
 To examine whether there was a
 trade-off between investment in testes and ovaries, and if the magnitude
 of this trade-off differed between selection regimes, we first calculated
 the correlation between relative testes area and relative ovary area
 within each combination of selection regime and replicate population. We
 then used these 12 correlation coefficients as the dependent variable in a
 one-way anova analysis, with selection regime as the independent variable.
 The aim of this analysis was to test for consistent differences in the
 correlation coefficient between selection regimes. Since there was no
 evidence of any differences between treatments (see Results), we also
 tested if the mean correlation coefficient across all replicate
 populations was significantly different from zero using a one-sample
 t-test. Finally, we tested whether differences in
 morphology could explain differences in sex-specific fitness. Since
 morphological data and sex-specific fitness data were collected at
 different times, this analysis could not be carried out on the individual
 level, so instead, we used replicate population mean values. We used a
 regression approach with sex-specific fitness as the dependent variable,
 and morphological variable (body area, relative testes area, or relative
 ovary area) as the predictor variable. The effect of selection regime was
 not included since replication was so limited in this analysis. However,
 results were qualitatively similar when carrying out an ancova analysis
 including both the morphological variable and selection regime as
 predictors (data not shown).