Research Article |
Corresponding author: Martina Šemeláková ( martina.semelakova@upjs.sk ) Academic editor: Martin Wiemers
© 2022 Barbora Mikitová, Martina Šemeláková, Ľubomir Panigaj.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Mikitová B, Šemeláková M, Panigaj Ľ (2022) Wing morphology and eyespot pattern of Erebia medusa (Lepidoptera, Nymphalidae) vary along an elevation gradient in the Carpathian Mountains. Nota Lepidopterologica 45: 233-250. https://doi.org/10.3897/nl.45.68624
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Butterfly wings play a crucial role during flight, but also in thermoregulation, intraspecific signalling and interaction with predators, all of which vary across different habitat types and may be reflected in wing morphology or colour pattern. We focused on the morphological variability of Erebia medusa in order to examine patterns and variations in the colouration and morphology of wings from areas representing different habitat types with different environmental characteristics. The barrier (larger fragments of forest) between populations of Erebia medusa along the elevation gradient of Kojšovská hoľa might be the aspect that hinders the movement of the population. The wing characteristics (shape, size, spotting) of males representing populations of Carpathian mountain habitats (Volovské vrchy, Ondavská vrchovina) located at different elevations were measured. The forewing shape analysis, using geometric morphometry based on 16 landmarks, showed significant differences between populations from different elevation levels. The pattern of the forewings also varied between populations. Morphological changes among individuals of Erebia medusa populations along the elevation gradient in the Carpathian Mountains showed that in the cold, highland habitats we observed smaller, narrower and elongated forewings with a reduced number of spots, while males from warmer habitats at low elevations had rounder, larger and more spotted forewings.
Introduction
The ecological role of individual butterfly species is largely reflected in the wings, whose shape, size and colour pattern often have adaptive value and provide information about important differences, even at the population level (
The wing eyespot pattern, which may serve different functions, can also play an irreplaceable role. While the pattern on the dorsal side is usually used for intraspecific communication (
Previous studies (Nice et al. 2005;
For the sedentary butterfly Erebia medusa, high intraspecific variability (numerous subspecies) and mosaic distribution throughout most of its Euro-Siberian region is characteristic (
We predicted that the morphological diversity between E. medusa populations would show changes that correlate with the average annual temperature, which varies within the elevation gradient. Our study is based on the hypothesis that i) morphological traits of males (size, shape and pattern of forewings) vary in response to various environmental conditions within an elevation gradient. We also focused on examining whether ii) the forewing size of individuals from higher elevations is smaller than the forewing size of individuals from lower and warmer regions, which induce longer feeding periods during larval development (
The studied species, the Woodland Ringlet – Erebia medusa (Denis & Schiffermüller, 1775) (Lepidoptera, Nymphalidae: Satyrinae) – inhabits areas from lowlands to sub-alpine zones (Sonderegger 2005) within a wide elevation range (
One hundred males of E. medusa from localities in the Volovské vrchy Mts. (Kojšovská hoľa – KH 1246 m a. s. l, Zlatá Idka – ZL 660 m a. s. l) and from the Ondavská vrchovina Mts. (Dobroslava – DB 335 m a. s. l) were analysed (Fig.
The front wings of each individual were photographed under standardised light conditions from the dorsal side using an Olympus digital camera MODEL NO. C-5060 white zoom DC GV connected to an Olympus SZ2-ILST stereomicroscope at 6.4× magnification using the program QuickPhoto MICRO 2.1 (Fig.
The left forewing of an Erebia medusa male used for (A) traditional and (B) geometric morphometrics. A. Studied traits. W – width, l – length, B – black pattern, W – white pattern. B. Distribution of landmarks (LM 1–16). Landmarks: 1 – intersection of veins at the base of the discal cell, 2 – Discal cell and Cu2 intersection, 3 – Discal cell and Cu1 intersection, 4 – Discal cell and M3 intersection, 5 –Discal cell and M2 intersection, 6 – Discal cell and M1 intersection, 7 – Discal cell and R4 Intersection, 8 – R4 and R5 intersection, 9 – R4 terminally, 10 – R5 terminally, 11 – M1 terminally, 12 – M2 terminally, 13 – M3 terminally, 14 – Cu1 terminally, 15 – Cu2 terminally, 16 – A terminally.
Male forewing length and width across localities were compared using traditional morphometric methods (
The obtained data (untransformed data) was evaluated using the statistical parameters of mean (M), standard deviation (SD) and coefficient of variation (CV). The normal distribution of forewing length, width and shape data, separated in terms of side (L, R), was confirmed by normality tests (the Shapiro-Wilk W test, the Anderson-Darling A test, the Jarque-Bera JB test) using PAST version 3.11 (
Differences in the morphological traits (wing width, wing length) among the sites were assessed using ANOVA and Tukey’s pairwise comparisons implemented in PAST version 3.19 (
The shape of the forewings was compared among the populations following the standard procedure consisting of the location of landmarks (at the costal margin and the nodes of veins), principal component analysis (PCA) based on the covariance matrix, canonical variate analysis (CVA) and multivariate analysis (ANOVA, Kruskal-Wallis test). ANOVA and CVA were used to compute morphological variabilities between the left and right forewing side.
The forewing shape variation among populations in the dataset were analysed using PCA. The results of CVA, which assessed the inter-location differences, were reported as respective P values for the Procrustes distances, after permutation tests (10,000 runs). The shape variation among localities was additionally analysed using ANOVA and post hoc tests. The mentioned statistical and geometric morphometric analyses were performed in MorphoJ (
We further analysed pattern elements from digital images and examined the presence and number of white and black spots on the dorsal side of the forewing margin. We examined the difference in the number of spots between populations in the program PAST version 3.1. The non-parametric Kruskal-Wallis test was performed to test the statistical significance of differences, and post hoc tests were performed to compare the samples. The mean and standard deviation (SD) values were calculated. Possible correlation between variables was examined using Spearman’s rank correlation coefficient (ρ). The program MorphoJ version 1.01 (
The intraspecific variability of wing morphology among the 100 samples (males) was analysed. All individuals were checked for measurement and digitisation errors, which were minimised (
Results of multiple linear regression on variables wing width and length, with temperature and elevation as fixed factors.
Dependent variable | Fixed effect | Coefficient | SE | t | df | p |
---|---|---|---|---|---|---|
FW length | (intercept) | 21.194 | 1.611 | 13.154 | 1 | 1.22 × 10-78 |
temperature | 1.310 | 0.333 | 2.172 | 1 | 0.032 | |
elevation | -0.262 | 0.603 | -0.786 | 1 | 0.434 | |
FW width | (intercept) | 11.225 | 0.956 | 11.745 | 1 | 2.59 × 10-79 |
temperature | 1.308 | 0.358 | 3.655 | 1 | 4.1 × 10-4 | |
elevation | -0.090 | 0.197 | -0.458 | 1 | 0.648 |
Comparison of Erebia medusa forewing length (A), width (B), length/width ratio (C) among the studied populations: KH – Kojšovská hoľa (1246 m a. s. l.); ZL – Zlatá Idka (660 m a. s. l.), DB – Dobroslava (335 m a. s. l). Data are shown as boxplots with minimum, median and maximum values and 25% and 75% percentiles.
The variation of forewing shape between populations was supported by ANOVA (F = 6.71, df = 56, p ˂ 0.0001) and further identified by principal component analysis (PCA) applied on the forewing landmarks. Changes in symmetry between the left and right forewings were not significant (ANOVA: F = 3.64, df = 28, p = 0.075). PCA revealed shape differences in the male forewings between the high elevation locality (Kojšovská hoľa, 1246 m a. s. l) and the low elevation sites. PC1 accounted for 29.3% and PC2 for 18.2% of the detected variation between populations. Canonical variate analysis (CVA) carried out with three groups along the first two canonical variate axes (CV1 and CV2) (Fig.
CVA results: A. the variation in the forewing shape of studied populations along the first two canonical variate (CV1 and CV2) axes with 95% confidence ellipses of means for each group. Kojšovská hoľa (KH), Zlatá Idka (ZL), Dobroslava (DB); B. wireframe graph of the morphometric overlap in forewing shape according to CV1 show deformation (dark blue line) from consensus configuration (sky blue line) associated to each canonical axis.
The forewing eyespots of male specimens from high elevation populations on Kojšovská hoľa (1246 m a. s. l.) had on average fewer white (μ = 2.95) and black spots (μ = 4.16) than males in the low elevation populations of Zlatá Idka (660 m a. s. l.) and Dobroslava (335 m a. s. l) (white spots: μ = 3.72, black spots: μ = 4.61), with a median difference of 1 for both white and black spots. In summary, with increasing elevation and decreasing temperature males tend to have fewer eyespots on average. Moreover, the overall number of white spots is lower than the number of black spots on the forewings.
The number of black spots on the dorsal side of the male forewing significantly differs between populations (Kruskal-Wallis test: p = 0.046), with Dunn’s post hoc test showing the biggest difference between populations KH and ZL (p = 0.013). The white colour pattern in the centre of the eyespots was also significantly different between the populations (Kruskal-Wallis test p = 0.012). The exact differences in the number of white spots between the populations KH (1246 m a. s. l.) vs ZL (660 m a. s. l) (p = 0.015) and KH (1246 m a. s. l.) vs DB (335 m a. s. l) (p = 0.006) were detected using Dunn’s post hoc test. The frequencies also varied depending on the location (Figs
The morphology of Erebia medusa male forewings studied by methods of traditional and geometric morphometrics showed statistically significant differences between populations from low and high elevations differing in annual average temperatures. We found that larger butterflies were generally found in warmer habitats. Smaller and narrower shaped forewings with a fewer number of spots on the dorsal surface were detected in the cold habitat – Kojšovská hoľa (1246 m a. s. l.). The results of our study are consistent with previous findings of the impact of elevation (
According to the clinal variation detected as size differences between populations, our results are in line with the season-length effect described by the converse Bergmann’s rule, which states that depending on the season length, larger individuals can be found at lower elevations (
Results from geometric morphometrics demonstrate that the forewing shape of males inhabiting the cold, high elevation site (Kojšovská hoľa) was more angular, narrower and slightly elongated, while rounder wings were detected at low elevations. The obtained results are similar to the conclusions on wing shape variance of Speyeria diana (
Our findings of smaller male E. medusa in the high and cold environments of Kojšovská hoľa and larger males originating in the lowlands are in line with theory, which underlines the thermoregulatory function of such adjustments in the size and colour of the wings. However, our results on the number of eyespots are partly consistent with the findings of
In addition, the average number of eyespots on male forewings increased with average temperature and decreased with temperature and rising elevation. Our findings confirmed the observations from previous research on Erebia medusa in eastern Slovakia conducted by
Variability of shape and size, as well as pattern and colouration of wings, may be related to dispersal, migration, territoriality, courtship and interactions with predators that vary across habitats. Moreover, temporal isolation and a shift in flight time were observed among localities with respect to temperature, even within geographically close localities separated by elevation.
To summarise our observations, trends in size along an elevational gradient were in line with a converse Bergmann’s rule which states that size of body decreases with elevation. The wing shape variation showed that elongated wings, which allow for gliding flight to be maximised (
The specific flight behaviour of male butterflies and wing morphological traits may provide better insight into their morpho-functional role, but further research is needed for this purpose.
This study was partially supported by the Slovak Scientific Grant Agency (VEGA, grant 1/0346/18). The authors are grateful to David Lee McLean for proofreading the manuscript.
Supplementary File 1
Data type: JPG file.
Explanation note: Satellite view and elevation profile of the studied sites Kojšovská hoľa, (1246 m a. s. l) (A) and Zlatá Idka, (335 m a. s. l) (B).
Supplementary File 2
Data type: docx file.
Explanation note: Characterics of sampling sites: A) Kojšovská hoľa , B) Zlatá Idka, C) Dobroslava.
Supplementary File 3
Data type: docx file.
Explanation note: Average temperatures and occurrence of Erebia medusa according to sex during the flight period. KH – Kojšovská hoľa, ZL – Zlatá Idka, DB – Dobroslava.