DOI: 10.5061/dryad.rjdfn2zm7
title: Data from: Seasonal plasticity in sympatric <em>Bicyclus</em> butterflies in a tropical forest where temperature does not predict rainfall

description: While temperature variation appears to be the main environmental cue for
 plasticity in adult traits in many species of Mycalesina, relying on
 temperature would result in a mismatch between adult phenotype and
 environment in some regions. We measured phenotypes of six species of
 Bicyclus butterflies (Nymphalidae: Satyrinae: Mycalesina) in a humid
 tropical forest with two rainy seasons per year and modest unimodal
 seasonal temperature variation, such that temperature does not predict
 rainfall and butterflies can reproduce year-round. The butterflies showed
 subtle temporal variation in body size and relative eyespot size, while
 relative androconia length was robust to temporal environmental variation.
 After higher temperatures, body size tended to be smaller, and relative
 eyespot size was larger for some species-eyespot combinations. This
 indicates that these butterflies follow the “hotter is smaller” rule and
 show developmental plasticity in eyespot size that is typical in this
 clade. Eyespot sizes tended to be correlated with each other, except Cu1
 in B. auricruda and some eyespots that always remained very small.
 Androconia length was not related to eyespot size. This pattern of
 correlations suggests conserved cue-use and shared mechanisms for eyespot
 size using both temperature and rainfall-related cues, with some
 exceptions.
description: <strong>Data collection</strong>
 Our study site was a sub-montane tropical forest near the
 Makerere University Biological Field Station (0°13’ - 0°41’N and 30°19’ -
 30°32’E) in Kibale National Park, Western Uganda. In this region, there
 are two rainy seasons per year, while temperature has a unimodal
 distribution so that there is a warm and a cool dry season (Valtonen et
 al. 2013). Thus, unlike in study sites of previous studies
 <em>(<em>Oostra et al. 2014a, van Bergen et al.
 2017</em>),</em> there is probably selection against
 developing a dry-season phenotype when temperatures are lower (and vice
 versa), at least during part of the year. During the study period, a data
 logger (Lascar EL-USB-2-LCD) was placed inside the forest, suspended 1
 meter above the ground, and protected from direct rain and sunlight by a
 plate of roofing zinc. The logger recorded temperature and relative
 humidity in half-hour intervals. In addition, daily rainfall and
 temperature data were obtained from a weather station at the field station
 (within 1km of all butterfly sampling locations; Chapman et al. 2018). To
 minimize the impact of our study on the local butterfly populations, we
 focused our sampling efforts on male specimens. Up to ten individuals of
 the six most abundant <em>Bicyclus</em> species (<em>B.
 collinsi</em> (Hewitson, 1873); <em>B. mollitia</em>
 (Karsch, 1895); <em>B. smithi</em> (Aurivillius, 1899);
 <em>B. auricruda</em> (Butler, 1868); <em>B.
 golo</em> (Aurivillius, 1893), and <em>B. graueri</em>
 (Rebel, 1914)) were collected weekly from baited traps for 14 consecutive
 months (23<sup>rd</sup> July 2013 to the
 26<sup>th</sup> of September 2014). All six species have
 eyespots in the distal region of the ventral wings (Figure 1). Whilst some
 species have multiple androconial brushes, all the investigated species
 have a prominent brush with its base located in the dorsal wing cell of
 the hindwing. This shared brush was selected as the androconial trait that
 could be compared across species. All four wings of
 collected butterflies were placed on a Nikon grey card, which was placed
 on graph paper (Figure 1) and photographed using a Nikon D7000 camera in a
 custom-made studio with constant light conditions (luminance and
 intensity) and the same manual settings of 1/125 shutter speed and F14
 aperture for all photographs. We then used a macro in ImageJ to measure a
 proxy of the wing area of each wing, the area covered by four ventral
 eyespots, and the length of the basal hindwing androconial brush (Figure
 2). <strong>Data
 analysis</strong> To obtain the average
 temperature for each day, we averaged the maximum and minimum temperatures
 provided by Chapman et al. (2018) rather than our own measurements, as
 these also cover the months before butterflies were collected (necessary
 for cross-correlation analyses). We averaged rainfall and humidity by
 two-week period and month. As a proxy of body size, we averaged the area
 of triangles measured from forewings and hindwings (Figure 2). In general,
 wing area is a well-established proxy for body size in these butterflies
 (e.g. Bergen et al. 2024). We calculated relative eyespot size as eyespot
 area divided by the body size proxy. Relative androconia length was
 calculated as the length of androconia divided by wing length. To avoid
 periods with missing data due to low abundance of butterflies, species’
 averages of traits were calculated per two weeks or four weeks, depending
 on species abundance. The few remaining missing data points (5 out of 135)
 were replaced by the average of data points from two weeks before and two
 weeks after (for biweekly data) or four weeks before and four weeks after
 (for four-weekly data). To assess whether butterflies
 showed seasonal dimorphism (distinct wet season and dry season morphs), we
 generated density plots of body size, eyespot size, and androconia length
 for each species using the R function <em>geom_density</em>
 from the package <em>ggplot2</em> (Wickham 2016, R_Core_Team
 2024). We visualized temporal trends using the <em>loess</em>
 function in R (R_Core_Team 2024). To estimate whether traits were linked
 to habitat seasonality (seasonal changes in average phenotype), we
 performed autocorrelation analyses using the function
 <em>ggAcf</em> in the R package <em>ggplot2</em>
 (Hyndman & Khandakar 2008). Autocorrelation analysis tests
 whether, within a single time series, there are correlations between data
 points that are a particular time lag apart, as would be the case with
 seasonal patterns (e.g, correlation between data points that are six
 months apart). To focus on seasonality, for each autocorrelation, we
 visually identified the lag with the most negative correlation coefficient
 and determined whether it was statistically significant, thus ignoring
 lags next to zero that typically show positive correlation coefficients.
 To test if temperature and rainfall drive temporal variation in butterfly
 traits, we performed cross-correlation analyses using the function ggCcf
 in the R package ggplot2 (Wickham, 2016). In addition, we performed such
 analyses for relative humidity (related to both rainfall and temperature).
 Cross-correlation analysis tests whether two time series are correlated
 with each other with a certain lag. We expected the lag to range within
 the length of development time, i.e., time between egg hatching and adult
 eclosion (about 6 weeks; Molleman et al*.* 2016, van Bergen et al. 2017)
 and development time with an added month to account for adult life span
 (Molleman et al. 2007) and an extra month if the effect is mediated by
 host-plant growth (Valtonen et al., 2013; total 4 months lag). For each
 cross-correlation, we visually identified the lag with the highest
 correlation coefficient and noted the sign and whether it was
 statistically significant, focusing on lags of less than six months. Among
 butterfly species, we tested for correlation within traits (no lags),
 which may indicate an evolutionarily conserved mechanism across species.
 Within species, we tested for the degree of correlation among traits,
 which would indicate a linked developmental mechanism. Given the low
 number of species included in our sampling, we did not account for
 phylogenetic non-independence among species in our cross-correlation
 analyses.
description: # Seasonal plasticity in sympatric *Bicyclus* butterflies in a tropical
 forest where temperature does not predict rainfall The study aims to
 investigate the seasonality in climatic conditions (Temperature, Rainfall,
 and Humidity) and the traits (Body size, Androconia length, Eyespots VFW
 Cu1, VFW M1, WHW Cu1, VFW M1) of 6 species of *Bicyclus* butterflies (*B.
 collinsi*, *B. mollitia*, *B. smithi*, *B. auricruda*, *B. golo, and* B.
 graueri) and the relationship between the climatic conditions and the
 traits. The following analyses were done: 1.     Correlation analyses
 a.     Between traits with species b.     Within traits between species
 2.     Temporal trends a.     Seasonal climatic patterns b.     Seasonal
 trait dimorphism 3.     Autocorrelation analyses a.     Between traits
 with species b.     Within traits between species 4.     Cross-correlation
 analyses a.     Between traits and climatic variables ## Description of
 the data and file structure Data are given in 3 Excel files (.xlsx) and
 the R-codes are given in 2 R-files (R). Descriptions of each file are
 given below. The values in the data files were averaged per two or four
 weeks as described in the main text of the publication – “To obtain the
 average temperature for each day, we averaged the maximum and minimum
 temperatures provided by Chapman et al. (2018) rather than our own
 measurements as these also cover the months before butterflies were
 collected (necessary for cross-correlation analyses). We averaged rainfall
 and humidity by two-week period and month. As a proxy of body size, we
 averaged the area of triangles measured from forewings and hindwings
 (Figure 2). In general, wing area is a well-established proxy for body
 size in these butterflies (e.g. Bergen et al. 2024). We calculated
 relative eyespot size as eyespot area divided by the body size proxy.
 Relative androconia length was calculated as the length of androconia
 divided by wing length. To avoid periods with missing data due to low
 abundance of butterflies, species’ averages of traits were calculated per
 two weeks or four weeks, depending on species abundance. The few remaining
 missing data points (5 out of 135) were replaced by the average of data
 points from two weeks before and two weeks after (for biweekly data) or
 four weeks before and four weeks after (for four-weekly data).”
 Furthermore, we note that in the data and code files we used the older
 notions of the eyespots, whereas in the manuscript we used the latest
 notion. The eyespot notions can be interpreted as below. | **Latest notion
 as in the manuscript** | **Older Notions as in the data and code file** |
 | :------------------------------------- |
 :--------------------------------------------- | | VFW Cu1 | VFW\_2 | |
 VFW M1 | VFW\_5 | | VHW Cu1 | VHW\_2 | | VHW M1 | VHW\_5 |
 **BicCorellationDataSM.xlsx** contains the data for the Correlation
 analyses and the Temporal trends. **2WeekSM.xlsx** contains the data for
 the Autocorrelation and Cross-correlation analyses for the species *B.
 collinsi*, *B. mollitia,* and *B. smithi*. **MonthSM.xlsx** contains the
 data for the Autocorrelation and Cross-correlation analyses for the
 species *B. auricruda*, *B. golo, and* *B. grauer*.
 **SM_Bicyclus_CorrelationTests.R** contains the R code for the Correlation
 analyses and the Temporal trends. **SM_Bicyclus_ACF_CCF.R** contains the R
 code for the Autocorrelation and Cross-correlation analyses for all the
 species. The meaning of the abbreviated column names of all three Excel
 files age given below. In any of the Excel files, "NA"
 means that for the case we do not have an observed value; a yellow
 coloured cell means that for this case we do not have an observed value,
 but the entered value is the average of the previous and next values from
 the same column. | Column name | Explanation | | |
 :----------------------------- |
 :-------------------------------------------------------------------------------------------- | :---------------------------------- | | Average AndroLength | Average length of androconia | | | Average Body Size | Average body size, i.e., wing area | | | Average Celsius. mean | Mean temperature in Celsiuss | | | Average de Humidity. mean | Mean humidity in percentage | | | Average DewPoint.mean | Mean dewpoint | | | Average Humidity. mean | Mean humidity in percentage | | | Average Rainfall | Mean rainfall in mm | | | B.auricruda | Butterfly species Bicuclus auricruda | | | B.auricruda (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus auricruda | | | B.auricruda (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus auricruda | | | B.auricruda (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus auricruda | | | B.auricruda (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus auricruda | | | B.golo | Butterfly species Bicuclus golo | | | B.golo (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus golo | | | B.golo (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus golo | | | B.golo (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus golo | | | B.golo (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus golo | | | B.graueri | Butterfly species Bicuclus graueri | | | B.graueri (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus graueri | | | B.graueri (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus graueri | | | B.graueri (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus graueri | | | B.graueri (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus graueri | | | B.mandanes | Butterfly species Bicuclus mandanes | | | B.mandanes (Log\_VFW\_2) | Log eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus mandanes | | | B.mandanes (Log\_VFW\_5) | Log eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus mandanes | | | B.mandanes (Log\_VHW\_2) | Log eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus mandanes | | | B.mandanes (Log\_VHW\_5) | Log eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus mandanes | | | B.mandanes (Relative\_VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus mandanes | | | B.mandanes (Relative\_VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus mandanes | | | B.mandanes (Relative\_VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus mandanes | | | B.mandanes (Relative\_VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus mandanes | | | B.mandanes (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus mandanes | | | B.mandanes (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus mandanes | | | B.mandanes (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus mandanes | | | B.mandanes (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus mandanes | | | B.mollitia | Butterfly species Bicuclus mollitia | | | B.mollitia (Log\_VFW\_2) | Log eyespot of ventral forewing segment 2 of butterfly species Bicuclus mollitia | | | B.mollitia (Log\_VFW\_5) | Log eyespot of ventral forewing segment 5 of butterfly species Bicuclus mollitia | | | B.mollitia (Log\_VHW\_2) | Log eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus mollitia | | | B.mollitia (Log\_VHW\_5) | Log eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus mollitia | | | B.mollitia (Relative\_VFW\_2) | Relative eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus mollitia | | | B.mollitia (Relative\_VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus mollitia | | | B.mollitia (Relative\_VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus mollitia | | | B.mollitia (Relative\_VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus mollitia | | | B.mollitia (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus mollitia | | | B.mollitia (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus mollitia | | | B.mollitia (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus mollitia | | | B.mollitia (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus mollitia | | | B.smithi | Butterfly species Bicuclus smithi | | | B.smithi (Log\_VFW\_2) | Log eyespot of ventral forewing segment 2 of butterfly species Bicuclus smithi | | | B.smithi (Log\_VFW\_5) | Log eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus smithi | | | B.smithi (Log\_VHW\_2) | Log eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus smithi | | | B.smithi (Log\_VHW\_5) | Log eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus smithi | | | B.smithi (Relative\_VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus smithi | | | B.smithi (Relative\_VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus smithi | | | B.smithi (Relative\_VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus smithi | | | B.smithi (Relative\_VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus smithi | | | B.smithi (VFW\_2) | Eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus smithi | | | B.smithi (VFW\_5) | Eyespot of the ventral forewing segment 5 of the butterfly species Bicuclus smithi | | | B.smithi (VHW\_2) | Eyespot of the ventral hindwing segment 2 of the butterfly species Bicuclus smithi | | | B.smithi (VHW\_5) | Eyespot of the ventral hindwing segment 5 of the butterfly species Bicuclus smithi | | | BiarARL | Androconia length of butterfly species Bicuclus auricruda | | | BiarBS | Body size, i.e., wing area of butterfly species Bicuclus auricruda | | | BiarRES(VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus auricruda | | | BiarRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus auricruda | | | BiarRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus auricruda | | | BiarRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus auricruda | | | BigoARL | Androconia length of butterfly species Bicuclus golo | | | BigoBS | Body size, i.e., wing area of butterfly species Bicuclus golo | | | BigoRES(VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus golo | | | BigoRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus golo | | | BigoRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus golo | | | BigoRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus golo | | | BigrARL | Androconia length of butterfly species Bicuclus graueri | | | BigrBS | Body size, i.e., wing area of butterfly species Bicuclus graueri | | | BigrRES(VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus graueri | | | BigrRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus graueri | | | BigrRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus graueri | | | BigrRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus graueri | | | BimaARL | Androconia length of butterfly species Bicuclus mandanes | | | BimaBS | Body size, i.e., wing area of butterfly species Bicuclus mandanes | | | BimaRES(VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus mandanes | | | BimaRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus mandanes | | | BimaRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus mandanes | | | BimaRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus mandanes | | | BimoARL | Androconia length of butterfly species Bicuclus mollitia | | | BimoBS | Body size, i.e., wing area of butterfly species Bicuclus mollitia | | | BimoRES(VFW\_2) | Relative eyespot of the ventral forewing segment 2 of the butterfly species Bicuclus mollitia | | | BimoRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus mollitia | | | BimoRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus mollitia | | | BimoRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus mollitia | | | BismARL | Androconia length of butterfly species Bicuclus smithi | | | BismBS | Body size, I.Wingwing area of butterfly species Bicuclus smithi | | | BismRES(VFW\_2) | Relative eyespot of ventral forewing segment 2 of butterfly species Bicuclus smithi | | | BismRES(VFW\_5) | Relative eyespot of ventral forewing segment 5 of butterfly species Bicuclus smithi | | | BismRES(VHW\_2) | Relative eyespot of ventral hindwing segment 2 of butterfly species Bicuclus smithi | | | BismRES(VHW\_5) | Relative eyespot of ventral hindwing segment 5 of butterfly species Bicuclus smithi | | | dew | Dewpoint | | | Eyespot size (VFW\_2) | Eyespot on the ventral forewing segment 2 of butterflies | | | Eyespot size (VFW\_5) | Eyespot of the ventral forewing segment 5 of butterflies | | | Eyespot size (VHW\_2) | Eyespot of the ventral hindwing segment 2 of butterflies | | | Eyespot size (VHW\_5) | Eyespot of the ventral hindwing segment 5 of butterflies | | | hum | Humidity in percentage | | | Log Average AndroLength | Average of log androconia lengths | | | Log Average Body Size | Average of log body sizes, i.e., wing area | | | Log Eyespot size (VFW\_2) | Log eyespot of the ventral forewing segment 2 of butterflies | | | Log Eyespot size (VFW\_5) | Log eyespot of the ventral forewing segment 5 of butterflies | | | Log Eyespot size (VHW\_2) | Log eyespot of the ventral hindwing segment 2 of butterflies | | | Log Eyespot size (VHW\_5) | Log eyespot of the ventral hindwing segment 5 of butterflies | | | Month | Month number from the start of sampling | | | rain | Rainfall in mm | | | Relative Androlenth | Relative androconia length | | | Relative Eyespot size (VFW\_2) | Relative eyespot of the ventral forewing segment 2 of butterflies | | | Relative Eyespot size (VFW\_5) | Relative eyespot of the ventral forewing segment 5 of butterflies | | | Relative Eyespot size (VHW\_2) | Relative eyespot of the ventral hindwing segment 2 of butterflies | | | Relative Eyespot size (VHW\_5) | Relative eyespot of the ventral hindwing segment 5 of butterflies | | | Species | Butterfly species | | | Tmean | Mean temperature in Celsius | | | Two-Week Period | Two-week | d number from the start of sampling | | twp | Two-week period from the start of sampling | | #### Code/Software SM_Bicyclus_ACF_CCF.R: This code imports Bicyclus butterfly body size and relative eyespot size data from Excel, converts selected columns into time series objects with two-week intervals, and then plots their autocorrelation functions (ACF) using ggplot2 with customized axis labels and themes. SM_Bicyclus_CorrelationTests.R: In *B. mandanes*, the relative traits (columns 22, 24, and 26) show Pearson correlations calculated and saved, highlighting significant relationships among relative eyespot sizes and androconia length, which were further visualized but only significant correlations were emphasized.