DOI: 10.5061/dryad.9kd51c5jm
title: Phenotypic correlates of pelvic spine coloration in the Threespine Stickleback (Gasterosteus aculeatus): Implications for function and evolution

description: Animal color patches may be static or plastic in expression and
 concealable or continuously visible, yet these aspects of coloration, and
 their consequences, have been little studied. We address them here using
 the threespine stickleback (Gasterosteus aculeatus). Despite a rich
 history of study of stickleback nuptial color pattern evolution,
 disagreement persists regarding selection pressures and function. However,
 little research has addressed the role of pelvic spine coloration, a
 potentially important, and substantially concealable, color pattern
 element. We investigated (i) whether male pelvic spine (along with throat
 and body) coloration is relatively static or plastic across the
 reproductive cycle, (ii) when pelvic spines are raised versus concealed
 across behavioral contexts, and (iii) associations between color patches
 and behavior in males. We found no significant variation in spine color
 across reproductive stages whereas body color was more plastic and
 intensely red during courtship and egg/fry care. Conspicuousness of pelvic
 spine coloration instead varied behaviorally, through increased erection
 frequency during social interactions and in response to a model predator.
 Spine erection frequency was positively associated with behaviors that
 enhance spine color visibility, i.e. flees and leads to nest. These
 findings suggest that stickleback use pelvic spines to display an
 intensely red color patch facultatively, either as a complement to similar
 body coloration or possibly as a substitute. In addition, elevated spine
 raising in the presence of a model predator, together with the presence of
 red spine coloration in females, raises the possibility that red spine
 coloration may also have an anti-predator function.
description: All data collected for this experiment was collected on male
 Threespine stickleback (<em>Gasterosteus aculeatus</em>)
 collected from Little Campbell River- Anadromous Site at the start of the
 reproductive season (May 2017 and May 2018) Please see publication for
 exact information on fish collection and husbandry. All
 data was uploaded into JMP versions 14.1.0 and 15.0.0 for statistical
 analyses. <strong>Comprehensive behavioral data as
 a complete data set can be found in the
 “BehavioralDATA_BORIS_WithPerMinCalculations_DRYAD” data sheet. Behavioral
 data was collected and analyzed in four steps: </strong>
 Step 1: fish were videotaped using an
 HD Sony camcorder positioned in front of the tanks during each of the
 unique behavioral interactions (including experimental contexts of a
 nesting male under various presentations, i.e. (i) presentation of a
 stimulus female within a UV transparent plexiglass container, (ii)
 presentation of a gravid female for mating, (iii) presentation of a rival
 male, and (iv) presentation of a model predator. Further, videos were
 recorded of control males within control tanks being presented with an
 empty UV transparent plexiglass container). Step 2: All videos were given random video
 identifiers by behavioral interaction type so as to prevent observer bias.
 No videos were analyzed until all videos were completed. Video analysis
 occurred during 2020 without direct knowledge of the color scores from
 each fish to again prevent observer bias. All videos were analyzed using
 BORIS behavioral software, which allowed for ethograms to be keyed in the
 system and behaviors quantified withing given videos. Not all behaviors
 could occur for each interaction type, so only videos that could be
 quantified were (see Supplemental Tables S1 and S2). After all videos were
 analyzed, the data from each fish across all interaction types was
 transferred to a comprehensive data sheet. Step 3: A master spreadsheet was created
 from all fish and videos, with data of behaviors scored imported from
 BORIS output and n/a denoting behaviors that could not occur (see
 Supplemental Tables S1 and S2). These behaviors were keyed in the data
 sheet as “_total”, which includes the total number of times that a
 behavior occurred within the video timeline scored. From there, “behavior
 per minute” calculations were employed to standardize values used in
 analysis. Each “total” value was divided by the total minutes in which the
 video was scored and recorded in the data sheet as “_perminute” to
 indicate the standardized values. Step 4: All statistical analysis of
 behaviors occurred within JMP versions 14.1.0 and 15.0.0. To analyze
 behavioral data by specific interaction type, the master spreadsheet was
 further broken down into the specific behavioral contexts. For example, if
 analyzing specific behavioral associations within the “female stimulus
 context”, only the behavioral values from the Stimulus Female interaction
 were included in analysis through “include/exclude and hide/unhide” row
 functions in JMP. Further, all Log1P values were generated within JMP from
 the “_permin” behavioral values, as well as all Principal Component
 Analysis (PCA) values for behavioral contexts noted within the
 publication. <strong>Color data was collected for
 three different areas of the body, including the ventral throat, lateral
 body, and pelvic spine. The comprehensive data with complete datasets can
 be found in the “ColorData_Stickleback_DRYAD” data sheet. It was collected
 as follows:</strong> 1. <strong>Ventral Throat
 Chroma</strong>: Following methods outlined within the publication;
 spectrophotometry was employed to collect reflectance values for two spots
 at 1nm intervals from 350-700nm along the ventral throat. The stimulation
 of the cones in the stickleback eye was then calculated for the 350-700nm
 range in order to provide quantam photon catches from each cone type used
 in matrix calculations of hue (h.theta, h.phi), and saturation/chroma
 (r.vec, i.e. the distance from the achromatic center). R.vec values were
 of particular interest here so were recorded for each fish at each stage,
 with the r.vec values from each fish averaged to create the final r.vec
 value used in analysis here (i.e.
 “MeanThroatColor_r.vec_ByStage”) 2.
 <strong>Lateral Body Coloration</strong>: Following methods
 outlined within the publication, photographs of the lateral body of each
 fish at each stage were taken. From there, photographs were uploaded into
 Adobe Photoshop, and RGB values were recorded for each of 9 spots across
 the body within 5 areas commonly known to express nuptial coloration,
 including the Lower jaw, operculum, pectoral plate, ventral area between
 the lower jaw and pelvic spines (i.e. “VPS”), and lateral plates. Color
 measurements were corrected relative to a gay card in order to standardize
 the RGB values by dividing the R, G, and B values from each point by the
 RGB values generated from four points along the grey card, creating
 R<sub>stand</sub>, G<sub>stand</sub>, and
 B<sub>stand</sub>. From here, Red Intensity (IR) was
 calculated by dividing the Rstand by the sum of
 R<sub>stand</sub> , G<sub>stand</sub>, and
 B<sub>stand</sub>. The final Red intensity (IR) values were
 recorded for each spot along the male at each stage and uploaded into the
 color spreadsheet Please see Fig. 2 of the main text for geographic
 location along the body for each spot analyzed here.
 Principal Component Analysis (PCAs) was used to create an overall
 value for lateral body coloration. All 9 spots were included and PCAs were
 generated for experimental males and control males separately, as outlined
 in the publication. 3.
 <strong>Pelvic Spine Coloration</strong>: Following methods
 outlined within the publication, photos of the erected pelvic spines were
 taken and uploaded into Adobe Photoshop. Using photoshop, R, G, and B
 values from each of 14 spots across 8 sections of the pelvic spine were
 recorded following the same steps outlined above. Final Red Intensity (IR)
 values were recorded for each spot and averaged to create the value
 analyzed here denoted as “SpineColor_Iaverage”.
description: Please refer to the README file.