Research Article |
Corresponding author: Run-Zhi Zhang ( zhangrz@ioz.ac.cn ) Corresponding author: Si-Qin Ge ( gesq@ioz.ac.cn ) Academic editor: Michael Schmitt
© 2017 Jing Ren, Ming Bai, Xing-Ke Yang, Run-Zhi Zhang, Si-Qin Ge.
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:
Ren J, Bai M, Yang X-K, Zhang R-Z, Ge S-Q (2017) Geometric morphometrics analysis of the hind wing of leaf beetles: proximal and distal parts are separate modules. ZooKeys 685: 131-149. https://doi.org/10.3897/zookeys.685.13084
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The success of beetles is mainly attributed to the possibility to hide the hindwings under the sclerotised elytra. The acquisition of the transverse folding function of the hind wing is an important event in the evolutionary history of beetles. In this study, the morphological and functional variances in the hind wings of 94 leaf beetle species (Coleoptera: Chrysomelinae) is explored using geometric morphometrics based on 36 landmarks. Principal component analysis and Canonical variate analysis indicate that changes of apical area, anal area, and middle area are three useful phylogenetic features at a subtribe level of leaf beetles. Variances of the apical area are the most obvious, which strongly influence the entire venation variance. Partial least squares analysis indicates that the proximal and distal parts of hind wings are weakly associated. Modularity tests confirm that the proximal and distal compartments of hind wings are separate modules. It is deduced that for leaf beetles, or even other beetles, the hind wing possibly exhibits significant functional divergences that occurred during the evolution of transverse folding that resulted in the proximal and distal compartments of hind wings evolving into separate functional modules.
Chrysomelinae , evolution, variance, venation, wing folding
Coleoptera (known as beetles) are the largest insect order, containing 380,000 named living species classified into more than 160 families (
Given that large and thin hind wings are vulnerable to damage, they must be folded not only longitudinally but also transversely to be stored below the elytra for protection during ground locomotion and especially when entering narrow spaces. The hind wings unfold only when needed, such as before flying (
The apical area of beetle’s hind wings is folded under elytra when not flying. Transverse folding leads to some morphological changes of hind wings, for example, the wing size, but what additional changes are concomitant? In this study, the morphological variances of beetle hind wings were investigated using geometric morphometrics analysis based on Chrysomelinae beetles. Chrysomelinae beetles could be divided into two tribes, one is Timarchini which are not able to fly, hind wings are reduced or disappeared; the other is Chrysomelini which have functional hind wings (
Geometric morphometrics analysis approaches have been used successfully in evolutionary biology and systematics (
This study was based on hind wing images (see Suppl. material
The tpsUtil (Version 1.44, Copyright 2009, F. James Rohlf, Ecology & Evolution, SUNY at Stony Brook) and tpsDig (Version 2.12, Copyright 2008 F. James Rohlf, Ecology & Evolution, SUNY at Stony Brook) software programs were used for digitising landmarks. Figure
Leaf beetle hind wing (Chrysomela populi Linnaeus), with landmark locations (the dot with number), vein nomenclature and regional division. The nomenclature of the wing venation follows that of Kukalová-Peck & Lawrence (1993, 2004). Radial area: green, central area: blue, medial area: purple, anal area: yellow, apical (folding) area: red. Proximal part landmarks 1–6, 23, 24, and 26–36 mainly include radial, medial, and anal areas; distal part landmarks 7–22 and 25 include the central area, radial cell, and apical area. Abbreviations: Costa (C), Subcosta (Sc), Subcosta Anterior (ScA), Subcosta Posterior (ScP), Radius Anterior (RA), Radius Posterior (RP), Radial cross veins (r3, r4), Media Posterior (MP), Radio-medial cross veins (rp-mp1, rp-mp2), medial cross vein (cv), Cubitus Anterior (CuA), Medio-cubital Cross-vein or Arculus (mp-cua), Anal Anterior (AA), Anal posterior (AP). “+” indicates fused veins. The sub-number of veins reflects vein branches.
Landmark # | Position Description |
---|---|
1 | Proximal anterior point of humeral plate (HP) |
2 | The crossing point of BSc and Sc |
3 | The point of Sc getting to bifurcate into ScA and ScP |
4 | The crossing point of ScP and RA |
5 | The crossing point of ScA and RA |
6 | The crossing point of rp-m1 and RA |
7 | Proximal anterior point of radial cell |
8 | Distal anterior point of radial cell |
9 | Distal posterior point of radial cell |
10 | Anterior point of r4 (or the crossing point of r4 and radial cell) |
11 | Proximal posterior point of radial cell |
12 | Proximal point of r3 |
13 | Apical hinge |
14 | The anterior point of triangular area of radial cell’s distal side |
15 | The posterior point of triangular area of radial cell’s distal side |
16 | The proximal point of triangular area of radial cell’s distal side |
17 | The distal point of RA4 |
18 | The distal point of RA1 |
19 | The distal point of RP2 |
20 | The point of MP1+2 getting to bifurcate |
21 | The posterior point of r4, or the crossing point of r4 and rp-mp2 |
22 | The proximal point of RP |
23 | Anterior point of mp-cua |
24 | The crossing point of rm-mp1and MP |
25 | The posterior of medial spur |
26 | Posterior point of mp-cua |
27 | The point of AA getting to bifurcate |
28 | The point of AA1+2 getting to fuse with CuA3+4 |
29 | The posterior or distal point of AA3+4 |
30 | The proximal point of cv |
31 | The posterior or distal point of AA1+2+CuA3+4 |
32 | Anterior point of CuA1+2+MP4 |
33 | The distal point of cv |
34 | Posterior point of CuA1+2+MP4 |
35 | The base point of AP3+4 |
36 | The posterior point of AP3+4 |
MorphoJ (Version 1.06d) was used for landmark data analyses. MorphoJ is a software package enabling geometric morphometric analysis for two- and three-dimensional landmark data and designed for the analysis of actual biological data (
Principal component analysis (PCA): PCA is one of the most widely used methods for exploratory multivariate analysis (
Canonical variate analysis (CVA): CVA is a method used to find the shape features that best distinguish among multiple groups of specimens (
Partial least squares (PLS) analysis: MorphoJ offers an implementation of PLS analysis between blocks of landmarks within the same configuration. This analysis identifies the features of shape variation that most strongly co-vary between the blocks and indicates their relative contribution to the total covariation between blocks (
Modularity test: MorphoJ implements a method to evaluate hypotheses of modularity (
In geometric morphometrics, allometry is widely characterised by multivariate regression of shape on size (usually centroid size or log-transformed centroid size); such regressions often fit the data well and the allometric shape changes tend to affect the entire structures (
Based on the Procrustes fit data, PCA was carried out based on 96 specimens. The accumulative contribution ratio of the first three components was 68.04%, potentially indicating that the first three components represented the main shape variation of wing venation. Figure
Based on the PCA wing variance results, CVA was used to test whether the three features were useful on the distinguishing of the subtribe level. There were eleven subtribes (89 specimens) of Chrysomelinae involved in this study, except subtribe Hispostomina and Monarditina have one specimen for each (sample size is too small, there were no statistical significance); the left have 4–26 specimens for each subtribes. Iin CVA, there were nine subtribes with enough samples to do analysis. As the Figure
PCA and CVA results. A Centroid size graph of hind wing landmarks (Procrustes fit) BPCA results, the shape changes associated with the first three PCs: the relative size of the apical area which could be considered the main feature (variance contribution ratio was 45.01%) to influence of the overall variance of the hind wing, the changes of cross vein cv in the middle area (variance contribution ratio was 12.39%), and relative size of the anal area size (variance contribution ratio was 10.56%) CCVA results, the axis of CV1 and CV2 presented the first two large shape variance of all variance; points with different colours indicated different subtribes’ specimens; the ellipse is presented as an equal-frequency ellipse with a given probability level of 90%, which contains approximately 90% of the data points.
The apical area of the hind wings of beetles can be folded transversely under elytra (
PLS1 presented 87.22% of the total covariance, indicating that PLS1 represented the main covariance of two blocks. Figure
Based on the PLS analysis results, a modularity analysis was performed to evaluate whether the proximal and the distal parts of beetle hind wings are separate modules. In the same manner, the landmarks 7–22 and 25 involving transverse folding were extracted as the distal part and the remaining landmarks 1–6, 23–24 and 26–36 as the proximal part, which was our hypothesised partition (Figure
Modularity test results. A The hypothesized partition: proximal part landmarks 1-6, 23, 24, and 26–36 and distal part landmarks 7–22, 25; different colour presents different modules B The partition with minimal covariance in all evaluated 104 partitions by RV coefficient C The partition with minimal covariance in all evaluated 106 partitions by RV coefficient.
The powerful visualisation tools of geometric morphometrics and the typical large amount of shape variables give rise to a specific exploratory style of analysis, allowing the identification and quantification of previously unknown shape features (
In this study, it was mainly focused on the wing variance of different species based on subtribe level of leaf beetles. Three main features of leaf beetle’s hind wing variance based on PCA results (Figure
The PCA results presented three phylogenetic features of hind wings of leaf beetles, and the changes of apical area was the most obvious (variance contribution ratio was 45.01%, Figure
In our study, PLS analysis showed that the distal part of hind wing, which is involved in transverse folding and includes the apical area, central area, and radial cell, exhibited independent shape variance in the total variance of the hind wing (Figure
Various studies of Drosophila’s wings using morphometric approaches have addressed the question of whether anterior and posterior wing compartments are distinct modules reflecting phenotypic and genetic variation (
What is the nature of the modularity interactions of hind wings? Generally, it can be developmental, functional, or genetic (
The separate modules of proximal and distal parts of Chrysomelid hind wings have been tested. The main reason of the separate modules could be attribute to its special function: transverse folding in hind wings of beetles. Although there were only 96 chrysomelid beetles considered in our study, it could be believed that other beetles feature the same modularity of hind wings, given that the transverse folding of the hind wing is a common feature of beetles. However, more data are needed to confirm this hypothesis. Here, the apical part of the hind wings of leaf beetles has an important influence on hind wing shape variance by PCA (Figure
Taking the PCA, CVA, PLS analyses and the modularity test into account, it was concluded that areas of the beetle hind wing relevant to transverse folding importantly influence hind wing shape variances and are relatively independent. In addition, the proximal and distal parts of a beetle’s hind wing are separate modules. The changes of apical area, anal area and middle area of hind wings were useful features to distinguishing subtribe level of leaf beetles. For beetles, hind wing folding is a morphological and functional compromise between fore wing evolution to elytra and the maintenance of hind wing flying function. This separate function modules could allow the hind wings to be folded at rest and to unfold when flying. In addition, the separate function could explain why beetles are the most prosperous animals in evolution. Our discovery could provide the theoretical basis and a new perspective for studies of the morphological evolution of wings and wing folding mechanisms.
Experiments were planned by Jing Ren, Si-Qin Ge, and Run-Zhi Zhang. Species identification was completed by Si-Qin Ge. Experiments were conducted by Jing Ren. Analysis and interpretation of the results was performed by Jing Ren, Ming Bai, Run-Zhi Zhang, and Xing-Ke Yang. The paper was written by Jing Ren, Si-Qin Ge, and Run-Zhi Zhang.
All authors declare there are no potential competing interests.
We are very grateful to Professor Christian Peter Klingenberg (Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom) for providing technical guidance of MorphoJ software and are very grateful to Adam Ślipiński (CSIRO Ecosystem Sciences, Australian National Insect Collection, Canberra, Australia) and Mauro Daccordi (Museo Civico di Storia Naturale, Verona, Italy) for loaning a portion of the research specimens. The project is partly supported by grants from the National Science Foundation of China (Grant Nos. 31472028, 31672345, 31672347).
Images of hind wings.
Data type: Species data
Sample information.
Data type: Specimens data
Coordinates data of landmarks.
Data type: Distribution data