DOI: 10.5061/dryad.1vhhmgqx2
title: Body size as a magic trait in two plant-feeding insect species

description: When gene flow accompanies speciation, recombination can decouple
 divergently selected loci and loci conferring reproductive isolation. This
 barrier to sympatric divergence disappears when assortative mating and
 disruptive selection involve the same “magic” trait. Although magic traits
 could be widespread, the relative importance of different types of magic
 traits to speciation remains unclear. Because body size frequently
 contributes to host adaptation and assortative mating in plant-feeding
 insects, we evaluated several magic trait predictions for this trait in a
 pair of sympatric Neodiprion sawfly species adapted to different pine
 hosts. A large morphological dataset revealed that sawfly adults from
 populations and species that use thicker-needled pines are consistently
 larger than those that use thinner-needled pines. Fitness data from
 recombinant backcross females revealed that egg size is under divergent
 selection between the preferred pines. Lastly, mating assays revealed
 strong size-assortative mating within and between species in three
 different crosses, with the strongest prezygotic isolation between
 populations that have the greatest interspecific size differences.
 Together, our data support body size as a magic trait in pine sawflies and
 possibly many other plant-feeding insects. Our work also demonstrates how
 intraspecific variation in morphology and ecology can cause geographic
 variation in the strength of prezygotic isolation.
description: <strong>Geographic variation in host needle width and adult
 body size</strong>: Because <em>Neodiprion</em> eggs
 must be completely embedded in pockets that adult females carve within
 pine needles, egg size and female ovipositor size are likely constrained
 by needle width (Bendall et al. 2017). Given these constraints, we
 predicted that geographic variation in adult body size would correlate
 with geographic variation in host needle width. Although the ideal way to
 test this prediction would have been to collect host needles and sawfly
 adults from the same sites, we only had access to pine and sawfly samples
 that were collected at different times and without this hypothesis in
 mind. We therefore analyzed geographic variation in host needle width and
 adult body size separately, with the following expectations: <em>N.
 lecontei</em> adults and host needles will be bigger than those of
 <em>N. pinetum</em> and geographic trends in body size would
 mirror those observed for needle width (i.e., have slopes with the same
 sign and similar magnitude). To characterize geographic
 variation in pine needle width, we sampled needles from multiple trees and
 sites for each of 10 <em>Pinus</em> species used by
 <em>N. lecontei</em> (<em>P. taeda, P. palustris, P.
 echinata, P. elliottii, P. clausa, P. glabra, P. virginiana, P. rigida, P.
 resinosa,</em> and <em>P. banksiana</em>) and the single
 host species used by <em>N. pinetum</em> (<em>P.
 strobus</em>). For each pine species, we collected clippings from
 60-100 trees sampled from 6-10 sites (9-10 trees per site). Using digital
 calipers (Mitutoyo CD-6’PMX), we measured the width of 3 randomly sampled
 needles per tree and averaged these measurements to obtain a single needle
 width value per tree. In total, we sampled 878 individual pine trees from
 88 sites (Table S1). To determine how the needle width varies as a
 function of host species and latitude, we used linear regression to model
 needle width as a function of pine species, latitude, and their
 interaction. We used a Type III Analysis of Variance (ANOVA), implemented
 in the <em>car</em> v3.1-0 package (Fox and Weisberg 2019), to
 evaluate the significance of model terms and the
 <em>emmeans</em> v1.8.0 package (Lenth 2021) for post-hoc
 tests with false discovery rate (FDR) correction for multiple testing.
 These and all other statistical analyses were performed in R version 4.1.0
 (R Core Team, 2021). Because we were specifically interested in comparing
 body size clines between sawflies with needle width clines from their pine
 hosts, we also used linear regression to estimate the relationship between
 (1) needle width and latitude for all <em>N. lecontei</em>
 hosts (regardless of host species) and (2) needle width and latitude for
 <em>P. strobus</em>, the only <em>N. pinetum</em>
 host pine. To characterize geographic variation in body
 size for adult females and males of both species, we collected mid- to
 late-instar <em>N. lecontei</em> and <em>N.
 pinetum</em> larvae across the eastern United States between 2015
 and 2021 (Table S2 and Fig. S1) and reared the immature stages to adults
 in the lab using host plant clippings and standard lab protocols (Harper
 et al. 2016; Bendall et al. 2017). Upon emergence, live adults (which are
 non-feeding) were either preserved immediately or stored at 4°C to prolong
 life until needed for propagating lab lines or use in experimental assays.
 All adults were ultimately placed in 100% ethanol and stored at -20°C. We
 used a Neiko Tools Digital Caliper (model 01407A) to measure body length
 (i.e., from the tip of the head to the tip of the abdomen) to the nearest
 hundredth of a millimeter (mm) for 1,080 preserved adults (<em>N.
 lecontei</em> females: <em>N</em> = 328; <em>N.
 lecontei</em> males: <em>N</em> = 243; <em>N.
 pinetum</em> females: <em>N</em> = 279; <em>N.
 pinetum</em> males: <em>N</em> = 230; Table S2). To
 determine how body length differed as a function of latitude, species, and
 sex, we fit a linear model to the body size data, with latitude, species,
 sex, latitude x species, latitude x sex, and species x sex as fixed
 effects. We used a Type III ANOVA to assess the significance of model
 terms. Based on these results, we also fit individual geographic clines to
 each sex for each species. <strong>Divergent
 selection on egg size</strong>: We hypothesize that variation in
 needle width among pine populations and species generates divergent
 selection on body size (egg size) in <em>Neodiprion</em> that
 use different pine hosts via a combination of constraints imposed by thin
 needles (favors smaller eggs) and selection on early larval survival
 (favors larger eggs). To test this hypothesis, we first verified that
 <em>N. pinetum</em> (thin-needled specialist) and <em>N.
 lecontei</em> (uses hosts with thicker needles) differ in egg size.
 To do so, we used females reared from wild-caught larvae collected from
 different sites in Kentucky and Tennessee between 2013 and 2015 (Table
 S3). In <em>Neodiprion</em>, all egg maturation occurs within
 the cocoon, from which females emerge with a full complement of eggs ready
 for oviposition (i.e., these species are pro-ovigenic). To measure egg
 length and width, we dissected eggs out of the abdomens of recently
 eclosed adult females and photographed a random subset of eggs at 10X
 total magnification using a Zeiss Discovery V8 stereomicroscope with an
 Axiocam 105 color camera and ZEN lite 2012 software (Carl Zeiss
 Microscopy, LLC Thornwood, NY). We then used the ZEN lite software to
 measure the length and width of 5 eggs from each of 5 females from each
 species (<em>N</em> = 25 eggs per species). We then calculated
 egg area after García-Barros (2000) using the following
 equation:           egg size = (0.5236 x
 d<sup>2</sup> x h)<sup>1/3</sup>,
 where d = egg diameter (width) and h = egg length. To test for
 differences in egg size between <em>N. pinetum</em> and
 <em>N. lecontei</em>, we fit a linear mixed-effects model to
 the egg area data, with species as a fixed effect and female ID as a
 random effect (since multiple eggs were sampled from each female). To
 evaluate the significance of the species term, we used a Type II
 ANOVA. <em>N. lecontei</em> and
 <em>N. pinetum</em> differ in many traits that affect hatching
 success on different types of pines, including ovipositor size and number
 of eggs per needle (Bendall et al. 2017; Fig. 3A, 3B). To evaluate the
 effect of egg size independent of other traits, we used a backcross design
 to generate recombinant hybrid progeny between <em>N.
 lecontei</em> and <em>N. pinetum</em> after Bendall et
 al. (2017). All females were <em>lecontei</em>-backcross
 females (one set of <em>N. lecontei</em> chromosomes and one
 set of chromosomes with approximately 50% <em>N. lecontei</em>
 and 50% <em>N. pinetum</em> ancestry). We used a
 <em>lecontei</em>-backcross design because preference for the
 thin-needled <em>N. pinetum</em> host is dominant (Bendall et
 al. 2017), and we wanted to ensure a good sample size of females that laid
 on both hosts. Backcross females were released individually into mesh
 cages (60 x 40 x 40 cm) containing two seedlings of <em>Pinus
 banksiana</em> (<em>N. lecontei</em> host) and two
 seedlings of <em>P. strobus</em> (<em>N.
 pinetum</em> host). Cages were checked daily for oviposition and
 once this occurred, live females (which tend to remain at the bottom of
 the egg-bearing branch until death) were preserved in 100% ethanol for
 subsequent morphological and molecular work. After preserving females, the
 number of eggs laid on each seedling was counted and monitored daily for
 hatching. Because females almost always cluster their eggs in a single
 branch tip, host choice was scored as a binary trait (<em>P.
 strobus</em> or <em>P. banksiana</em>). Once a hatchling
 was observed, eggs were given an additional 48 hours to provide sufficient
 time for hatching. All hatchlings were then removed by washing them off
 the pine seedling in ethanol. Hatchlings were counted and the hatching
 success for each female was calculated as the proportion of eggs that
 hatched (number of hatchlings/number of eggs). After
 oviposition, females almost always have eggs left in the abdomen. We used
 these remaining eggs to quantify the average egg size for each female. To
 measure egg size, we first rehydrated preserved female abdomens by soaking
 them in five decreasing concentrations of ethanol (95%, 80%, 65%, 50%, and
 25%) for 10 minutes each. After the lowest ethanol concentration, we
 soaked females in deionized water for 24 hours at room temperature. We
 then dissected the eggs from each female’s abdomen and placed them in 300
 mL of a modified Ringer’s dissection solution (7.5 g/L NaCl, 0.35 g/L KCl,
 and 0.21 g/L MgCl2). We then imaged 1-10 eggs per female at 10X total
 magnification and measured eggs and calculated the area as described
 above. We then averaged these values to obtain a single average egg area
 per female. Although ethanol preservation and rehydration may cause
 preserved eggs to differ in size from fresh eggs, this approach should
 nevertheless reveal <em>relative</em> egg size differences
 among females (i.e., which females laid the largest or smallest eggs)
 because all eggs were treated in the same way. We are also assuming here
 that the size of leftover eggs correlates positively with the size of laid
 eggs, which will require future experiments to confirm.
 In total, we measured hatching success, egg size, and host
 preference for N = 38 <em>lecontei</em>-backcross females.
 After removing two females that laid eggs on both pine species, our final
 sample size was <em>N</em> = 36 backcross females. To evaluate
 the effect of egg size (egg area) and host (<em>P.
 banksiana</em> or <em>P. strobus</em>) on hatching
 success, we used the glm function (lmerTest v3.1-3; Kuznetsova et al.
 2017) to fit a logistic regression model to the hatching data (proportion
 hatched ~ egg area + host + area*host), followed by a Type III ANOVA to
 evaluate the significance of model terms. A significant egg area x host
 interaction would support our hypothesis that there is a trade-off between
 egg fit and egg provisioning.
 <strong>Size-assortative mating and reproductive
 isolation</strong>: To determine whether
 <em>Neodiprion</em> adults mate assortatively by size and to
 quantify reproductive isolation between <em>N. pinetum</em>
 and <em>N. lecontei</em>, we first sampled larval colonies of
 both species from several locations throughout the eastern United States
 from June to August 2019 (Table S4 and Fig. S2). In both species, females
 tend to mate once and then lay a single clutch of eggs in one pine branch
 terminus. Thus, distinct larval clusters typically represent full-sib
 families. To maintain broad-scale geographic differences in behavior or
 morphology, we grouped field-collected larvae by state (Michigan,
 Kentucky, Indiana, and North Carolina) and then reared larvae to adults
 for mating assays and line propagation. Methods for line propagation are
 described elsewhere (Harper et al. 2016; Bendall et al. 2017). To minimize
 the evolutionary change in the lab, we used adults that were reared either
 from wild-caught colonies or first-generation lab colonies.
 No-choice mating assays were performed from September to October
 2019. We used no-choice assays because they are consistent with mating
 behaviors in the wild (Benjamin 1955; Wilson et al. 1992). Because
 reproductive isolation can vary across geographic space (Jiang et al.
 2013; Rougemont et al. 2015), we assayed mating outcomes in three
 different crosses, each containing a different combination of <em>N.
 lecontei</em> and <em>N. pinetum</em> adults from
 different U.S. states: Cross 1 = North Carolina <em>N.
 lecontei</em> x Indiana <em>N. pinetum</em>; Cross 2 =
 Kentucky <em>N. lecontei</em> x Kentucky <em>N.
 pinetum</em>; and Cross 3 = Kentucky <em>N.
 lecontei</em> x Michigan <em>N. pinetum</em>. Each assay
 consisted of two arenas (plain white 8 x 11 pieces of printer paper) that
 were divided into six equally sized sections (dividing lines drawn with a
 black Sharpie pen; Fig. S3). Each arena was recorded by either a Logitech
 Carl Ziess Tessar HD 1080p or Microsoft LifeCam Cinema Model 1393 camera.
 We placed a small petri dish (5 cm x 1.5 cm) in each section of each
 arena, within which we placed a single male and female. Each assay (pair
 of arenas) consisted of three replicates of each of the four possible
 female-male pair types for a particular cross: (1) <em>N.
 lecontei</em> female x <em>N. lecontei</em> male, (2)
 <em>N. lecontei</em> female x <em>N. pinetum</em>
 male, (3) <em>N. pinetum</em> female x <em>N.
 lecontei</em> male, and (4) <em>N. pinetum</em> female x
 <em>N. pinetum</em> male (i.e., each assay contained 12
 female-male pairs total). Based on the availability of males and females
 from each geographic region, we were able to complete 12 assays for Cross
 1 (<em>N</em> = 36 replicates per pair type; 144 female-male
 pairs total), 5 assays for Cross 2 (<em>N</em> = 15 replicates
 per pair type; 60 female-male pairs total), and 5 assays for Cross 3
 (<em>N</em> = 15 replicates per pair type; 60 female-male
 pairs total).  During each assay, which lasted 2 hours,
 all mating events were recorded for each male-female pair. Each pair was
 assigned an arbitrary identifier and observed blind with respect to pair
 type. For an interaction to be considered a mating, the pair in question
 had to be properly aligned and physically connected (Fig. 1C) for at least
 1 minute to ensure a sufficiently secure attachment for sperm transfer.
 Although we have not experimentally determined the minimum duration that a
 pair must be connected for sperm transfer to occur, previous observations
 have shown that sawfly pairs that mate for approximately 1 minute produce
 daughters (Author, personal observation), which indicates that
 fertilization has occurred as pine sawflies are haplodiploid (Knerer and
 Atwood 1973). At the end of each assay, the overall mating outcome of each
 pair was recorded as a “1” if the pair mated at least once or as a “0” if
 the pair never mated.  All males and all unmated females were immediately
 preserved in 100% ethanol. Mated females were allowed to lay eggs for
 further lab line propagation but were preserved live after egg laying
 (except for 17 females that we were unable to find). To confirm recorded
 mating outcomes, we reviewed all videos (again, blind with respect to each
 pairs’ identity) and scored each pair as described above. While reviewing
 videos, we also counted the number of mating attempts made by each male.
 For a behavior to be counted as a “mating attempt,” we required that the
 male curled his abdomen in a “U-shape” under the female’s abdomen in an
 attempt to initiate copulation. We then coded male behavior in each pair
 as a binary trait: “1” if the male made any attempt and “0” if the male
 made no attempts. We used our mating assays to quantify
 the strength of prezygotic isolation between <em>N.
 lecontei</em> and <em>N. pinetum</em> both within each
 of the three crosses and globally (all crosses combined), following Sobel
 and Chen (2014). This method requires observed and expected mating
 frequencies for conspecific and heterospecific pairs. To obtain the
 observed frequency of conspecific matings, we divided the total number of
 conspecific pairs that mated (either <em>N. lecontei</em>
 female x <em>N. lecontei</em> male or <em>N.
 pinetum</em> female x <em>N. pinetum</em> male) by the
 total number of attempted conspecific crosses. To obtain the observed
 frequency of heterospecific matings, we divided the total number of
 heterospecific pairs that mated (either <em>N. lecontei</em>
 female x <em>N. pinetum</em> male or <em>N.
 pinetum</em> female x <em>N. lecontei</em> male) by the
 total number of attempted heterospecific crosses. For the expected
 conspecific and heterospecific mating frequencies under random mating, we
 used 0.5. We then calculated reproductive isolation using the following
 equation:             RI = 1 – 2 x (H/H+C);
 where H = heterospecific pairs and C = conspecific pairs. This
 equation yields reproductive isolation values ranging from 0 (no
 reproductive isolation) to 1 (complete reproductive isolation). To further
 evaluate evidence of prezygotic isolation, we used one-tailed Fisher’s
 exact tests in each cross and globally (<em>fisher.test</em>
 function) to determine whether heterospecific pairs were significantly
 less likely to mate than conspecific pairs. The Sobel and Chen (2014)
 equation and our statistical analysis ignores possible asymmetries within
 pair types. Therefore, to determine whether mating outcomes differed
 between the four different pair types (<em>N. lecontei</em>
 female x <em>N. lecontei</em> male; <em>N.
 lecontei</em> female x <em>N. pinetum</em> male;
 <em>N. pinetum</em> female x <em>N. lecontei</em>
 male; <em>N. pinetum</em> female x <em>N.
 pinetum</em> male), we also used the
 f<em>isher.multcomp</em> function from the RVAideMemoire
 v0.9-81-2 package (Hervé 2022), with FDR correction for multiple testing.
 For plotting proportion data, we used the DescTools v0.99.45 package
 (Signorell et al. 2022) to calculate 95% Clopper-Pearson confidence
 intervals for each cross and pair type. To determine
 whether body size influenced mating outcomes, we measured body size for
 <em>N</em> = 511 preserved males and females from our mating
 assays. Because body length is likely to be especially important for
 proper alignment during mating (Fig. 1C and Video S1), we used body length
 as our measure of body size. Specifically, we used a Neiko Tools Digital
 Caliper (model 01407A) to measure each individual from the tip of the head
 to the tip of the abdomen to the nearest hundredth of a millimeter. To
 reduce measurement error, each sawfly was measured independently by two
 individuals, and the average body length (in mm) was used. To calculate a
 size differential for each pair, we subtracted the male body length from
 the female body length. We then used logistic
 regression to evaluate how species, pair type, and size differences affect
 binary mating outcomes in each of the three crosses and globally. For each
 cross, we modeled mating outcome as a function of female species, male
 species, female species x male species interaction, and size differential.
 For the global model, cross was included as a random effect. Then, we used
 Type III ANOVAs to evaluate the significance of model terms. If the two
 species differ in willingness/motivation to mate, we expected significant
 male species or female species terms. If the type of male-female pair
 (conspecific versus heterospecific) affects mating outcomes independent of
 body size, we expected significant female species x male species
 interaction terms. And if there is size-based assortative mating
 independent of male-female pair type, we expected significant effects of
 size differentials in our models, with pairs that mated having a smaller
 size differential than those that did not. We also used logistic
 regression to evaluate how species, pair type, and size differences affect
 male mating attempts (coded as a binary trait) in each cross and globally.
 Then, we used Type III ANOVAs to evaluate the significance of model
 terms. To further explore how variation in body size
 within and between species relates to variation in the strength of
 prezygotic isolation among the three crosses, we compared the size
 differences for all intraspecific pairs (<em>N. lecontei</em>
 female x <em>N. lecontei</em> male and <em>N.
 pinetum</em> female x <em>N. pinetum</em> male) to the
 size differences for all interspecific pairs (<em>N.
 lecontei</em> female x <em>N. pinetum</em> male and
 <em>N. pinetum</em> female x <em>N. lecontei</em>
 male) within each cross and globally. We then performed one-sided t-tests
 using the <em>t.test</em> function to evaluate whether the
 size differential for interspecific pairs was significantly greater than
 the size differential for intraspecific pairs.
description: All data files are .csv files and can be opened in R.