2.3.2 Postcopulatory trait measurements of male specimens
Abdomens of male O. babirussa specimens were dissected
into 1x phosphate-buffered solution (PBS) to measure the following
postcopulatory traits: testes weight and sperm length (Figure 2). Testes
were isolated and transferred onto pre-weighed aluminium sheets
and dried in a Memmert Gravity Basic Digital Oven D overnight. Then,
total weight was measured on the Mettler Toledo ML104 Newclassic ml
Analytical Balance. Weight of the testes were calculated by subtracting
the weight of the aluminium sheet from the total weight.
To measure the sperm length, seminal vesicles containing the mature
sperm were first isolated and transferred onto a drop of PBS on a
frosted slide. Then, sperms were teased out from the vesicles using an
insect pin. Slides were dried in the oven and sperms were fixed onto the
slides with a solution of three parts methanol and one part acetic acid
for two minutes. Next, the slides were washed in 1x PBS for one minute
and the sperms were stained for five minutes in the dark with
4′,6-diamidino-2-phenylindolev (DAPI), which binds to DNA to form a
fluorescent complex to allow for visualisation of sperm heads under a
fluorescent microscope. Following that, the slides were washed in 1x PBS
and placed in the dark to dry. When the slides were dried completely,
one to two drops of glycerol were added on the stained regions,
coverslips were placed, and the edges were sealed with clear nail polish
and left to dry in the dark. The sperms were visualized using an Olympus
BX50 fluorescence microscope and measured using μManager and ImageJ V.
1.51 software. Based on previous studies, five to ten sperms were
measured per specimen (García-González & Simmons, 2007; Simmons &
Kotiaho, 2002; Werner & Simmons, 2011).
2.4. Statistical
analyses
Box plots of average pronotum width were constructed with confidence
intervals using the R packages ggplot2 (Wickham, 2016),dplyr (Wickham et al. , 2020) and plotrix (Lemon,
2006) and data for the four populations were tested for normal
distribution using the Shapiro-Wilk test. As the data were not normally
distributed, the non-parametric Kruskal-Wallis test was conducted
instead of ANOVA to determine if there were significant sexual size
dimorphism within and across the four study populations of O.
babirussa . Post-hoc analyses using Dunn test were also conducted to
determine which populations differ from the other for male and female
body size. In addition, the sexual dimorphism index (SDI) was
calculated following the formulation by Gibbons and Lovich (1990), where
the size of the larger sex is divided by the size of the smaller sex. A
negative sign is arbitrarily added to the SDI as the males are larger
(Gibbons and Lovich, 1990).
To determine whether populations differed with respect to
relative investments in precopulatory and postcopulatory traits, the
static allometries were calculated by first constructing log-log
scatterplots of trait size against pronotum width. We used a linear
regression to assess testes size and sperm length. As the log-log
scatter plot of horn length against pronotum width displayed a clear
non-linear relationship, we followed the recommendations by Knell (2009)
and fitted (1) linear model, (2) quadratic model, (3) cubic model and
(4) breakpoint model using the R package segmented (Muggeo, 2008)
to the pooled data with all four populations to characterise the trait
size-body size relationship (see Appendix3, Figure S5). Model selection
was then collected with the Akaike information criterion (AIC). The
breakpoint model had the lowest AIC score for horn length (see
Appendix3, Table S2), indicating that this model is the best model
for explaining the relationship between the variables (Knell, 2009).
Next, allometric coefficients (β1and β2 ) of the traits of
interest (y 1 and y 2) were
obtained for each population from the following equations generated by
the breakpoint model:
Equation 1: log(𝑦1 )= β1 log
(𝑥1 )+𝑐
Equation 2: log(𝑦2 )= β2 log
(𝑥2 )+𝑐