DOI: 10.5061/dryad.3ffbg79v5
title: Shell colouration is associated with sex, boldness, and innate immunity in wild adult eastern box turtles (Terrapene carolina carolina)

description: Animal colouration often plays a role in intraspecific communication. The
 badges of lizards and ornaments of birds are frequent examples of honest
 signals regarding physiological condition and behaviour. Turtles share
 evolved characteristics with both non-avian and avian reptiles and many
 express colour markings on the shell and head, yet the significance of
 colouration in turtles remains unclear. We assessed aspects of shell
 colouration (carapace spot hue, saturation, and brightness and percent
 melanism of the carapace and plastron) in adult eastern box turtles
 (Terrapene carolina carolina) from a wetland population and explored their
 association with age, sex, boldness, haemolysis capabilities, plasma
 triglycerides, plasma corticosterone, and body condition. We found
 evidence of varying bold temperaments based on high among- and low within-
 individual variation across several behaviours during and after handling.
 Findings further suggested that separate aspects of shell colouration were
 associated with sex, boldness temperament, and innate immunity.
 Specifically, males exhibited redder carapace spots than females, bolder
 turtles exhibited brighter carapace spots and less melanic (more spotted)
 plastrons, and turtles with higher in vitro haemolysis scores tended to
 have more saturated carapace spots. These associations suggest potential
 roles for shell colouration to signal aspects of behaviour and immune
 function, however, we discuss limitations arising from our methods such as
 the use of a single blood sample for physiological metrics. Further
 studies on these associations are needed to determine their stability,
 underlying mechanisms, and ecological implications.
description: # Shell colouration is associated with sex, boldness, and innate immunity
 in wild adult eastern box turtles (*Terrapene carolina carolina*) # by
 Warren & Klukowski ## Description of the Data and file structure The
 Excel data file '**Warren_Klukowski_BoxTurtle_Data.xlsx**' has
 three sheets containing some similar and some different data depending on
 their intended purpose / analysis. The **first** **sheet** contains the
 data from all behavioural assays (along with turtle characteristics and
 assay conditions) and is used for calculating the repeatability of these
 behaviours using GLMMs as well as testing the effects of turtle
 characteristics and assay conditions on the behaviours. The **second**
 **sheet** contains averages and one-time measures used to conduct the GLMs
 testing for the effects of (1) sex, age, and mean compiled boldness score
 on each of the colouration metrics (spot hue, spot saturation, spot
 brightness, plastron percent melanism, and carapace percent melanism), and
 (2) sex, mean compiled boldness score, and all colouration metrics on each
 of the physiological metrics (triglycerides, hemolysis, corticosterone,
 and vBCI). The **third** **sheet** contains information on pinch force and
 temperature differentials collected during this study from these same
 turtles but which is not discussed in the main manuscript published in
 AMRE and is only included for potential future use. **NOTE:** In the Excel
 data file, cells containing '**NA**' represent data points that
 are not available as they were failed to be collected during that specific
 assay for that particular individual turtle. The blank for cell G120 on
 sheet#1 (All_Assays_For_Repeatability) is also one of these missing data
 points but has been left blank to allow the R script to run appropriately
 when conducting the GLMM on this variable. **Sheet #1
 "All_Assays_For_Repeatability"** **headers:** * "id"
 and "subject": corresponds with individual turtles. The Rscript
 utilizes "id" but either is fine. Note that all 28 turtles were
 tested across 4 to 5 assays. * "EyeEmerge": The latency (in
 seconds) the turtle took to extend the head so that the eyes surpassed the
 margins of the shell during the 10-min observation period (note: turtle
 was assigned a minimum of 1 if immediately emerged and maximum of 600 if
 didn't at all). * "TwobodyLengths": The latency (sec) the
 turtle took to move 2-body lengths during the observation period (was
 assigned a minimum of 1 if immediately emerged and maximum of 600 if
 didn't at all). * "OneMeterMoveLatency": The latency (sec)
 the turtle took to move 1 meter during the observation period (was
 assigned a minimum of 1 if immediately emerged and maximum of 600 if
 didn't at all). * "OneMeterMoveScore": The yes/no scoring
 of one meter movement based on whether the turtle did or did not move 1 m
 during the observation period for that assay (0 = no, 0.5 = yes). *
 "TimeClosedUpDuringHandling_s": The amount of time (sec) the
 turtle spent closed up during the 3-min handling period (was assigned a
 minimum of 1 if closed up the whole time, and maximum of 180 if
 didn't close up at all). * "KicksScore": Represents whether
 the turtle did (0.5) or did not (0) kick with the limbs ("air
 walk") during the 3-min handling period. * "FinalScore":
 Represents the turtle's compiled boldness score for that assay,
 calculated using rankings from the behaviours above. See methods and table
 1 in AMRE publication for details on this score. *
 "FinalScoreAvg": Represent the mean compiled boldness score for
 turtles across all assays. * "testtime": The time of day that
 the assay took place. * "skycode": A ranking of cloud coverage
 and rainfall conditions during the assay (see methods in AMRE pub). *
 "testn": The number of assays for that turtle (1 - 5). *
 "irshelltemp" The infrared surface temperature (°C) of the
 turtle's carapace taken just prior to the start of the assay. *
 "sex": The sex of the turtle based on plastron indentation,
 where F = female and M = male. * "ageBC": The estimated age
 (years) of the turtle based on counts of annuli (scute rings). *
 "vBCI": The volumetric body condition of the turtle calculated
 using morphometric measures (see methods in AMRE pub).
 \#----------------------------------------------- **Sheet #2
 "Avgs_single_measures_colour" headers:** * "TurtleID":
 ID of the turtle. * "AvgEyeEmergeLat": Represent the mean eye
 emergence latency (sec) for turtles across all assays. *
 "AvgCompiledBoldnessScore": Represent the mean compiled boldness
 score for turtles across all assays. * "Corticosterone_ng_ml":
 The turtle's plasma corticosterone levels (ng/ml). *
 "Triglycerides_mg_ml": The turtle's plasma triglycerides
 levels (mg/ml). * "HemolysisScore": The turtle's hemolysis
 score (see methods in AMRE pub). * "Sex": The sex of the turtle
 based on plastron indentation, where F = female and M = male. *
 "EstimateAge": The estimated age (years) of the turtle based on
 counts of annuli (scute rings). * "Weight_g": The turtle's
 weight in grams. * "CarapaceLength_mm": The length (mm) of the
 turtle's carapace. * "CarapaceWidth_mm": The width (mm) of
 the turtle's carapace. * "CarapaceHeight_mm": The height
 (mm) of the turtle's carapace. * "vBCI": The volumetric
 body condition of the turtle calculated using morphometric measures,
 including weight, shell length, width, and height (see methods in AMRE
 pub). * "CarapaceMelanismPercent": The percent of dark
 melanistic background (excluding spots) making up the carapace. *
 "PlastronMelanismPercent": The percent of dark melanistic
 background (excluding spots) making up the plastron. *
 "CarapaceHue": The calculated mean hue of carapace conspicuous
 spots. * "CarapaceSaturation": The calculated mean saturation of
 carapace conspicuous spots. * "CarapaceBrightness": The
 calculated mean brightness of carapace conspicuous spots. *
 "BleedTimeofDay": The time of day that a blood sample was
 collected from the turtle. * "TimetoBleed_s": The duration of
 time (in sec) that it took to bleed the turtle. * "testost":
 results on plasma testosterone levels (ng/ml) not evaluated in this study
 due to low sample size. * "Progest": results on plasma
 progestone levels (ng/ml) not evaluated in this study due to low sample
 size. \#--------------------------------- **Sheet #3
 "Avgs*_*For_PinchForce" headers:** * "ID": ID of the
 turtle. * "PinchForce_kg": The maximum pinch force (kg) measured
 from the turtle at the anterior gap of the shell following the final
 behavioral assay using a a pinch gauge dynamometer and poking and prodding
 the skin of the turtle's snout and forelimbs to elicit voluntary
 closure. * "AvgEyeEmergeLat": The mean eye emergence latency
 (sec), or time in seconds for the turtle to emerge the head so that the
 eyes surpassed the anterior margins of the carapace during the 10-minute
 observation period, for turtles across all assays. *
 "AvgFinalScore": The mean compiled boldness score for turtles
 across all assays. * "LMBoldCateg": Represents the placement of
 turtles into either "bolder" (AvgFinalScore ≥ 2) or
 "shier" categories (AvgFinalScore &lt; 2). *
 "Willing_Able_to_Close": Represents the placement of turtles
 into one of two categories concerning their perceived
 'willingness' to close the shell during the collection of pinch
 force measures: (1) "Quick/Able" for turtles that quickly and
 tightly closed the shell, or (2) "Reluctant/Unable" for turtles
 noted as highly reluctant (e.g., behaviorally pushed back and bit at
 gauge) or seemingly unable (e.g., lacked the ability to close completely)
 to close. * "FinalAssayEmergeLat": The observed eye emergence
 latency (sec) for turtles during their final behavioral assay test, just
 before pinch force measures were collected (note: turtle was assigned a
 minimum of 1 if immediately emerged and maximum of 600 if didn't at
 all). * "Sex": The sex of the turtle based on plastron
 indentation, where F = female and M = male. * "AgeClass":
 Placement of turtles into age class 1 (10 - 14 years old), class 2 (15 -
 19 years old), or class 3 (20+ years old). * "AgeBestCount": The
 estimated age (years) of the turtle based on counts of annuli (scute
 rings). * "Weight_g": The turtle's weight in grams. *
 "CarapaceLength_mm": The straight-line length (mm) of the
 turtle's carapace measured dorsally down the midline (i.e., from the
 anterior-most marginal scute to the posterior-most marginal scute) of the
 carapace using manual calipers. * "CarapaceWidth_mm": The
 maximum width (mm) of the turtle's carapace measured ventrally using
 manual calipers. * "CarapaceHeight_mm": The maximum height (mm)
 of the turtle's carapace measured laterally using manual calipers. *
 "vBCI": The volumetric body condition of the turtle calculated
 using morphometric measures, including weight, shell length, width, and
 height (see methods in AMRE pub). * "AvgWristEm":  The mean
 wrist emergence latency (sec), or time in seconds for the wrist to emerge
 during the 10-minute observation period of the behavioral trial, for
 turtles across all their assays. * "Avg2BodyLengths":  The mean
 2-body length movement latency (sec), or time in seconds for the turtle to
 reach 2-body lengths in distance during the 10-minute observation period
 of the behavioral trial, for turtles across all their assays. *
 "Avg1mMoveLat":  The mean 1 m movement latency (sec), or time in
 seconds for the turtle to reach at least 1 m in distance during the
 10-minute observation period of the behavioral trial, for turtles across
 all their assays. * "AvgTotDef": The mean number of defensive
 behaviors recorded during the 3-minute handling period for turtles across
 all their assays. * "FinalDay_IR_ShellTemp": The observed shell
 temperature (°C) of the turtle measured just before the final behavioral
 assay using a handheld infrared temperature reader. *
 "FullStudyTempDifferential": The temperature (°C) differential
 of turtles calculated as the difference between the average shell
 temperature (captured every 30-minutes via an iButton affixed to the
 carapace) and the average soil surface temperature (captured every
 30-minutes via an iButton placed on the soil surface of a nearby shaded
 region of the wetland) across the total time of the turtle's
 involvement in the study. * "DayTimeTempDifferential": The
 temperature (°C) differential of turtles calculated as the difference
 between the average shell temperature (captured every 30-minutes via an
 iButton affixed to the carapace) and the average soil surface temperature
 (captured every 30-minutes via an iButton placed on the soil surface of a
 nearby shaded region of the wetland) during only the daytime portions
 (0900-1700 h) of the turtle's involvement in the study. *
 "NightTimeTempDifferential": The temperature (°C) differential
 of turtles calculated as the difference between the average shell
 temperature (captured every 30-minutes via an iButton affixed to the
 carapace) and the average soil surface temperature
 (captured every 30-minutes via an iButton placed on the soil surface of a
 nearby shaded region of the wetland) during only the nighttime portions
 (2300-0500 h) of the turtle's involvement in the study. The included
 R script file ("RScript_BoxTurtles_Warren_Klukowski") shows how
 we analyzed these data. Comments within the R script will inform you on
 which excel sheet to have active for certain analyses. We swapped between
 active excel sheets by opening the excel data document and manually moving
 the desired sheet to be the first in the order of sheets, then we would
 save that excel document and have R Studio read it again to import that
 particular dataset. ## Sharing/access Information N/A