DOI: 10.5061/dryad.95x69p8rw
title: Data from: Trophic niche drives the evolution of craniofacial shape in Trinidadian guppies

description: Diverse clades of fishes adapted to feeding on the benthos repeatedly
 converge on steep craniofacial profiles and shorter, wider heads. But in
 an incipient radiation, to what extent is this morphological evolution
 measurable and can we distinguish the relative genetic vs. plastic
 effects? We use the Trinidadian guppy (Poecilia reticulata) to test the
 repeatability of adaptation and the alignment of genetic and environmental
 effects shaping poecilid craniofacial morphology. We compare wild-caught
 and common garden lab-reared fish to quantify the genetic and plastic
 components of craniofacial morphology across four populations from two
 river drainage systems (n=56 total). We first use microCT to capture 3D
 morphology, then place both landmarks and semilandmarks to perform
 size-corrected 3D morphometrics and quantify shape space. We find a
 measurable, significant, and repeatable divergence in craniofacial shape
 between high predation invertivore and low predation detritivore
 populations. As predicted from previous examples of piscine adaptive
 trophic divergence, we find increases in head slope and craniofacial
 compression among the benthic detritivore foragers. Furthermore, the
 effects of environmental plasticity among benthic detritivores produces
 exaggerated craniofacial morphological change along a parallel axis to
 genetic morphological adaptation from invertivore ancestors. Overall, many
 of the major patterns of benthic-limnetic craniofacial evolution appear
 convergent among disparate groups of teleost fishes.
description: <em>Collection and animal husbandry</em>
 We collected female and male guppies, <em>Poecilia
 reticulata</em>, from four distinct populations in the Northern
 Range mountains on the island of Trinidad in spring 2013 and spring 2014.
 Guppies were collected from two independent drainages: Aripo river
 (southern slope) and Yarra river (northern slope) (Figure 1A); these
 watersheds are completely isolated from one another, with no admixture
 occurring over the mountain divide (but some can occur within each
 drainage). The specific populations collected were from
 benthic detritivore, or low predation, and invertivore, or high predation,
 sites within the Aripo and Yarra drainages (four populations total).
 Twenty females and five males were collected at each of the four sites (n
 = 100 total); the fish were transported in bottles filled halfway with
 fresh stream water and treated with 2mg Tetracycline (1mg/L) and
 StressCoat (according to manufacturer directions) to minimize infection
 during transportation. The fish were housed in an open-air field
 laboratory in Verdant Vale, Arima, Trinidad, within 2-liter tanks each
 equipped with air stone at (~18+3 degrees C)
 temperatures and exposed to ambient light, which maintained a light:dark
 schedule at approximately 12:12 hours. Fish were fed twice daily on algae
 flakes (TetraMin Tropical Flakes) in rations that amounted to two minutes
 of feeding time per meal (Arendt & Reznick, 2005; D. N. Reznick,
 1982). 30% water changes were performed weekly throughout the husbandry
 period. We randomly selected 10 female guppies from
 each population to represent the “wild-caught” phenotype and euthanized
 them as quickly as possible after capture to minimize plastic response to
 laboratory settings. We did not record the exact duration that we had the
 fish in captivity, but it was often less than a week and never more than
 30 days.  These fish were euthanized via overdose of buffered 1 g/L
 tricaine methanesulfonate (Tricaine-S, Western Chemical Inc., Ferndale,
 WA, USA), fixed for 24 hours in 4% paraformaldehyde and preserved in 70%
 ethanol at -20 degrees C. A subset of live female (n =
 10) and male (n = 5) guppies from each of the four populations (n=60
 total) were exported from Trinidad with a permit issued by the Fisheries
 Division of the Ministry of Agriculture, Land and Fisheries, Republic of
 Trinidad and Tobago, and imported into the United States with a valid U.S.
 Fish and Wildlife Service Declaration of Importation permit (USFWS Form
 3-177). These fish were bred to produce the “lab-reared” treatment of the
 study. Lab-reared fish were housed in community
 aquaria, which are 38-liter tanks with open stocks of fish. Population
 density was regulated to reduce overcrowding. Open stock temperature was
 maintained at 25 to 26C and fish were fed newly hatched Artemia, Repashy
 Community Crave (a gel that is prepared in small amounts every few days),
 and occasional Omega one flakes, Daro worms and Daphnia, which were
 cultured in the laboratory. This mixed diet was used to minimize strong
 directional plasticity. It should be noted that we did not conduct a
 reciprocal feeding experiment; fish from all four represented populations
 were fed the same food throughout their life in the lab, which provides a
 common garden environment from which to compare the genetic basis of
 craniofacial traits. Lab-reared fish were housed for a minimum of two
 generations to remove any epigenetic effects. We selected 5-10 individual
 lab-reared female guppies from each of the four populations. These
 lab-reared fish were euthanized, fixed and preserved following the above
 procedure. All research reported here followed strict
 ethical guidelines and complied with the US federal
 government’s regulations. Procedures were approved by the Institutional
 Animal Care and Use Committee at Brown University (protocol: 1211035 to E.
 L. Brainerd), Harvard University (protocol: 20–03-2 to G. V. Lauder) and
 University of California Riverside (#:20200003 to D. N.
 Reznick). <em>CT scanning and
 segmentation</em>             We scanned all
 specimens using a micro Computed Tomography scanner (Bruker SkyScan 1173,
 Kontich, Belgium). To scan a group of fish, we first wrapped 1-7
 individuals in Kimwipes and placed them within 5 mL transport tubes with a
 small amount of ethanol to keep the specimens moist and stabilized within
 the tube. We µ-CT scanned each tube using 0.5° increments over 180° with
 the x-ray source set at 38kV and 190µA. This resulted in image stacks with
 an isotropic resolution between 6.8µm and 9.0µm that were then
 reconstructed using NRecon (Bruker, Kontich, Belgium). We used Mimics (v.
 22.0; Materialise NV, Leuven, Belgium) to segment individuals from each
 scan and isolate the pre-pectoral skeleton, then exported scans as 3D
 models for morphometric analysis. We removed any scans where the heads
 showed obvious distortion such as a lateral skew of the craniofacial
 morphology. Finally, we used MeshLab (v. 2020.07; Cignoni et al., 2008) to
 simplify our meshes (Quadratic Edge Collapse Decimation filter) to reduce
 necessary computing power for visualization. We used Python to batch
 process the meshes and reduced each scan to 1x10<sup>6</sup>
 faces. <em>3D Geometric Morphometric
 Analysis</em> We placed 11 fixed landmarks and
 21 sliding semilandmarks across the craniofacial apparatus to capture its
 three-dimensional shape (Figure 2; supplemental figure 1). We use these
 landmarks to capture elongation and compression across all three spatial
 axes, as well as the craniofacial slope, orientation of the mouth
 (including the articulation of the lower jaw and the quadrate), and shape
 of the suspensorium. While many past studies have included landmarks on
 the oral jaws, we exclude these points due to confounding variation
 induced from inconsistent positioning during fixation. However, each of
 the traits that we measure has been used to infer functional differences
 between populations or species of fish that have diverged along a trophic
 axis (Albertson et al., 2003b, 2005; Cooper et al., 2010, 2011). We
 restricted landmarks to the left side of the head and the midline to avoid
 capturing asymmetries within individuals. In cases where the left side of
 the head was damaged we instead landmarked the right side and reflected
 the points about the x axis to correct the chirality. We collected sliding
 semilandmarks by first placing an arbitrary number of points along either
 the maxilla or preopercle to fully capture the curvature of the structure,
 then resampling these curves to a fixed number of equally spaced
 semilandmarks (8 on the maxilla, 13 on the preopercle) bounded by two
 fixed landmarks on each structure. All landmarking was done in 3D Slicer
 (v. 4.11; Fedorov et al., 2012).             We created
 three data sets, one with all the data points, one with just the
 lab-reared fish, and one with just the wild-caught fish. Using Geomorph
 (v. 4.0.0; D. Adams et al., 2021) we conducted a geometric morphometric
 analysis on each dataset. First, we subjected each coordinate data set to
 a generalized Procrustes analysis using bending energy. This method
 compares the shapes of each individual after accounting for size,
 rotation, and position. Next, we regressed the shape variable on
 log-transformed geometric centroid size for each of our data sets. In all
 cases there was a significant allometric effect, so we took the residuals
 of these regressions to attain an allometry-free measure of shape
 (Outomuro & Johansson, 2017). For each data set we then used the
 function procD.lm to run multivariate regressions of allometry-corrected
 shape on feeding niche, drainage, and the interaction effect between the
 two. For the data set containing both lab-reared and wild-caught fish we
 also included the rearing condition as an explanatory variable. To
 visualize the distribution of each data set in shape space the shape data
 were subjected to a principal component analysis (PCA) using the gm.prcomp
 function and plotted in the morphospace defined by the first two principal
 component axes. Finally, we subjected the lab-reared and wild-caught data
 sets to a trajectory analysis (RRPP, V1.3.1)(Collyer & Adams,
 2022) to determine whether the evolved path through morphospace from high
 predation to low predation populations differed between river drainage
 systems. We plotted population mean shapes and the linear trajectories
 between them to visualize their evolutionary paths. The
 regression and PCA analyses on the full dataset revealed fish differed
 based on feeding niche and drainage, but that the rearing condition
 primarily affected the magnitude of the divergence and not the direction
 of the divergence in shape space. Therefore, we only visualized the shape
 differences between benthic detritivore and invertivore individuals in the
 wild-caught individuals since these were further diverged than were the
 lab-reared fish. We did this by plotting the size-corrected and
 allometry-corrected mean position of each landmark for benthic detritivore
 and invertivore fish separately for each drainage. We placed lines between
 landmarks on the same bone and filled in the area between homologous lines
 to aid in visualization. All geometric morphometric analyses were run
 using Geomorph (v. 4.0.0)(D. Adams et al., 2021) in RStudio
 (v.1.3.1056). Although these landmarks inform
 craniofacial elongation among these populations, they do not allow us to
 measure head length since this is typically measured from the oral jaws to
 the caudal end of the neurocranium. Instead, we infer head length based on
 the distance along the anterior-posterior axis from the dorsal most point
 on the posterior end of the preopercle (Supp. Figure 2) to the dorsal tip
 of the maxilla. To compare our results using this nontraditional metric to
 past studies on head length we additionally measured the distance from the
 dorsal tip of the maxilla to the posteroventral margin of the
 basioccipital. We ran a linear regression between these two measurements
 to check if our metric of craniofacial elongation was positively
 correlated with head length. Similarly, we compared our measurement of
 head width, the distance from the widest point of the preopercle to the
 midline plane, to a more common measurement of width, the distance from
 the lateral most point on the operculum (Supp. Figure 2) to the midline
 plane of the fish.
description: # Data from: Trophic niche drives the evolution of craniofacial shape in
 Trinidadian guppies This
 is the morphometric data set that was used to attain the results presented
 in "Trophic niche drives the evolution of craniofacial shape in
 Trinidadian guppies". There are 4 main components to the data set: 1)
 3D models of guppy skulls (.ply format), 2) landmark files that correspond
 to each 3D model (.fcsv output from 3D Slicer), 3) an R code that runs the
 analysis on this data, and 4) supporting files used by the code. Given
 these data and the R file, any user can output the statistical and
 graphical results presented in the manuscript. ## Description of the data
 and file structure 3D files' filename structure: Each 3D file is
 named to reflect the ecological context of that individual. Specifically,
 files have several elements that are separated by underscore characters.
 These are: * Aripo or Yarra- Reflects the river drainage that the fish was
 collected in. * HP or LP- Reflects the amount of predation present in the
 streams the fish were collected from, either high predation (HP) or low
 predation (LP). This is highly correlated with their trophic niche where
 HP fish are limnetic invertivores and LP fish are benthic detritivores. *
 F or Fwc- "F" reflects that the fish is female (there were no
 males used in this dataset). Fish labeled "F" were raised under
 common garden conditions in the lab. Fish labeled "Fwc" were
 wild caught individuals. * Individual ID- Each fish received an individual
 ID (e.g. A1 or B4). The letter in the name simply reflects which batch the
 fish was CT scanned in. * ML- Indicates that the 3D file was simplified in
 MeshLab (v. 2020.07) to lower the file size. Landmark filename structure:
 Each landmark file is named to reflect the ecological context of that
 individual. Specifically, files have several elements that are separated
 by underscore characters. These are: * LR or WC- LR means the fish was lab
 reared in a common garden environment. WC means that the fish was wild
 caught. * Aripo or Yarra- Reflects the river drainage that the fish was
 collected in. * HP or LP- Reflects the amount of predation present in the
 streams the fish were collected from, either high predation (HP) or low
 predation (LP). This is highly correlated with their trophic niche where
 HP fish are limnetic invertivores and LP fish are benthic detritivores. *
 F- Reflects that the fish is female (there were no males used in this
 dataset). * Individual ID- Each fish received an individual ID (e.g. A1 or
 B4). The letter in the name simply reflects which batch the fish was CT
 scanned in. CSV files: * curveslideAsymm- This is a list of landmarks that
 should be treated as sliding semilandmarks in the geometric morphometric
 analysis, and is read into R to be used with the Geomorph package. Each
 row indicates a different landmark that should be considered a sliding
 semilandmark. The first and third columns refer to other landmarks between
 which the semilandmark can slide. The second column indicates precisely
 which landmark is the semilandmark. * LolliLinks- This is a list of line
 segments used for visualization in figure 6. * SpecimenData- This file
 lists all of the landmark files and all the information included in the
 filenames to make it easier for the R program to parse. Additionally, this
 has a column to let the program know whether to use the left or ride side
 of the head for analysis. ## Code/Software In order to run the R code, a
 user just needs to change the directories listed at the beginning of the
 script to whatever location the appropriate files are in. Additionally,
 this code was originally run with the following packages: * Geomorph (v.
 4.0.0) * ggplot2 (v. 3.4.3) * ggthemes (v. 4.2.4) * Tcltk (v. 4.0.2) *
 Janitor (v. 2.1.0) * Morpho (v. 2.10) * MASS (v. 7.3-51.6) * Shapes (v.
 1.2.6) * Plotly (v. 4.9.4.1) * Dplyr (v. 1.1.3)