Research Article 
Corresponding author: Yuxia Yang ( yuxia0305@126.com ) Academic editor: Lyubomir Penev
© 2015 Junyan Su, Kaile Guan, Jiaxu Wang, Yuxia Yang.
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:
Guan K, Su J, Wang J, Yang Y (2015) Significance of hind wing morphology in distinguishing genera and species of cantharid beetles with a geometric morphometric analysis. ZooKeys 502: 1125. doi: 10.3897/zookeys.502.9191

There remain some difficulties in delimitation of related genera or sibling species for cantharid beetles, because the traditionally taxonomic method and morphological characters have not been updated or introduced. In the present study, we firstly use the landmarkbased geometric morphometrics to analyze and compare the hind wings of nine species belonging to three genera of Cantharinae to ascertain whether this approach may be used as a reliable method in the study of the taxonomy of this group. The results show that the shape differences of the hind wings among genera seem more variable than that within each genus, and the variations for each species are different from one another, as shown in the principal component analyses. And the canonical variates analyses show that there are significant differences among the genera and the species of each genus, which demonstrates that the hind wing shape can be diagnostic for both generic and specific identification of the cantharid beetles. This study sheds new light into clarifying the taxonomic uncertainties of Cantharidae, and lays a foundation for further studies on the evolution of the cantharid hind wing shape.
Geometric morphometrics, hind wing morphology, Cantharidae, taxonomy
The Cantharinae represents a subfamily of beetles belonging to the family Cantharidae (
It is wellknown that wing shape of insects exhibits a high heritability in nature (
In Cantharidae, the venation of hind wings was suggested to be of diagnostic value in the subfamily level based on the comparative morphology by
Hind wings of the following Cantharinae species (Table
The number of specimens of each species used in the GM analysis.
Specific name  Number of specimens  

male  female  
Lycocerus asperipennis (Fairmaire, 1891)  9  11 
Lycocerus metallescens (Gorham, 1889)  12  15 
Lycocerus orientalis (Gorham, 1889)  13  13 
Prothemus kiukiangensis (Gorham, 1889)  10  11 
Prothemus limbolarius (Fairmaire, 1900)  10  10 
Prothemus purpuripennis (Gorham, 1889)  11  14 
Themus (Telephorops) coelestis (Gorham, 1889)  14  18 
Themus (Telephorops) impressipennis (Fairmaire, 1886)  10  12 
Themus (Haplothemus) licenti Pic, 1938  12  10 
The images of hind wings were captured using a stereomicroscope Nikon SMZ1500 and attached video camera Canon 450D connected to a HP computer. They were annotated using the TpsUtil software (
Landmarks of hind wing (according to veins nomenclature system by
No.  Junctions of veins  No.  Junctions of veins 

1  ScP (Subcosta Posterior) and RA  8  MP_{1+2} and MP_{3+4} 
2  RA (Radius Anterior) and RA_{3+4}  9  MP_{3+4} and CuA_{1} (Cubitus Anterior) 
3  RA_{1+2} and RA_{3+4}  10  MP_{4} and MP_{3} 
4  RA_{3+4} and r3 (radial crossvein)  11  CuA_{1} and CuA_{2} 
5  RA_{3+4} and r4  12  CuA and CuA_{1+2} 
6  r4 and RP (Radius Posterior)  13  AA (Anal Anterior) and CuA_{3+4} 
7  RP and MP_{1+2} (Media Posterior) 
To examine the wing shape variation, the digitized landmark data is analyzed using MorphoJ software (
The relative similarity and discrimination of the genera or species is analyzed using Canonical Variates Analysis (CVA). CVA finds shape values that maximize group means relative to variation within groups, by assuming that covariate matrices are identical (
To evaluate the role of wing size in discrimination among different genera or species, the centorid size (CS) was compared. In the absence of allometry, the CS is the only size measure uncorrelated with all the shape variables (
The shape variations of the hind wings in the genera Lycocerus, Prothemus and Themus is shown by the first two principal components of PCA (Fig.
Shape variables of the hind wings in the genera of Lycocerus, Prothemus and Themus. A principal component analysis (PCA) of hind wing configuration. Plot of PC1 (74.39% of total variation) and PC2 (8.52% variation) showing 90% confidence ellipses of population means B canonical variate analysis (CVA) of same matrix, also showing 90% confidence ellipses of population means. The averaged shape of each genus is depicted as deformations using thin plate splines.
Difference in the hind wing shapes among the genera Lycocerus, Pothemus and Themus. Mahalanobis distances (left) & Procrustes distances (right): pvalues (above); distances between populations (below).
Lycocerus  Pothemus  Themus  Lycocerus  Pothemus  Themus  

Lycocerus  —  <.0001  <.0001  —  <.0001  <.0001 
Pothemus  4.6396  —  <.0001  0.0456  —  <.0001 
Themus  10.8932  10.446  —  0.1323  0.1088  — 
In Lycocerus (Fig.
Shape variables of the hind wings in the Lycocerus species. A principal component analysis (PCA) of hind wing configuration. Plot of PC1 (49.02% of total variation) and PC2 (14.92% variation) showing 90% confidence ellipses of population means B canonical variate analysis (CVA) of same matrix, also showing 90% confidence ellipses of population means. The averaged shape of each species is depicted as deformations using thin plate splines.
Shape variables of the hind wings in the Prothemus species. A principal component analysis (PCA) of hind wing configuration. Plot of PC1 (38.40% of total variation) and PC2 (15.88% variation) showing 90% confidence ellipses of population means B canonical variate analysis (CVA) of same matrix, also showing 90% confidence ellipses of population means. The averaged shape of each species is depicted as deformations using thin plate splines.
Shape variables of the hind wings in the Themus species. A principal component analysis (PCA) of hind wing configuration. Plot of PC1 (32.87% of total variation) and PC2 (16.48% variation) showing 90% confidence ellipses of population means B canonical variate analysis (CVA) of same matrix, also showing 90% confidence ellipses of population means. The averaged shape of each species is depicted as deformations using thin plate splines.
Comparisons of centroid size variables among different groups: A Lycocerus, Prothemus and Themus B Lycocerus asperipennis, L. metallescens and L. orientalis; Prothemus chinensis, P. kiukiangensis and P. purpuripennis; Themus licenti, T. coelestis and T. impressipennis.
Difference in the hind wing shapes among the species of Lycocerus. Mahalanobis distances (left) & Procrustes distances (right): pvalues (above); distances between populations (below).
L. metallescens  L. asperipennis  L. orientalis  L. metallescens  L. asperipennis  L. orientalis  
L. metallescens  —  <.0001  <.0001  —  <.0001  0.0466 
L. asperipennis  5.6866  —  <.0001  0.0413  —  0.0003 
L. orientalis  4.2970  4.4457  —  0.0182  0.0321  — 
Difference in the hind wing shapes among the species of Prothemus. Mahalanobis distances (left) & Procrustes distances (right): pvalues (above); distances between populations (below).
P. chinensis  P. kiukiangensis  P. purpuripennis  P. chinensis  P. kiukiangensis  P. purpuripennis  

P. chinensis  —  <.0001  <.0001  —  <.0001  0.0002 
P. kiukiangensis  5.7352  —  <.0001  0.0376  —  <.0001 
P. purpuripennis  4.8174  5.5146  —  0.0247  0.0381  — 
Difference in the hind wing shapes among the species of Themus. Mahalanobis distances (left) & Procrustes distances (right): pvalues (above); distances between populations (below).
T. licenti  T. coelestis  T. impressipennis  T. licenti  T. coelestis  T. impressipennis  

T. licenti  —  <.0001  <.0001  —  <.0001  <.0001 
T. coelestis  6.7942  —  <.0001  0.0363  —  0.0001 
T. impressipennis  6.8548  3.9959  —  0.0311  0.016  — 
The result of PCA shows that the shape differences of the hind wings among the genera Lycocerus, Prothemus and Themus (Fig.
The CVA results (Figs
Tukey HSD for the CS among different groups: pvalues (above); mean differences (below). Asterisk (*) indicates the mean difference is significant at the 0.05 level.
CS among different genera  

Lycocerus  Prothemus  Themus  
Lycocerus  —  0.006  0 
Prothemus  218.52316401(*)  —  0.001 
Themus  483.54109456(*)  265.01793055(*)  — 
CS among the species of Lycocerus  
L. asperipennis  L. metallescens  L. orientails  
L. asperipennis  —  0.001  0.001 
L. metallescens  474.67493257(*)  —  1 
L. orientails  489.29359311(*)  14.61866054  — 
CS among the species of Prothemus  
P. chinensis  P. kiukiangensis  P. purpuripennis  
P. chinensis  —  0.005  0.002 
P. kiukiangensis  456.74308033(*)  —  1 
P. purpuripennis  460.37428735(*)  3.63E+00  — 
CS among the species of Themus  
T. coelestis  T. impressipennis  T. licenti  
T. coelestis  —  0.711  0.998 
T. impressipennis  183.8607895  —  0.992 
T. licenti  79.25669086  104.6040987  — 
Herein it can be concluded that the hind wing shape is useful for the discriminations of genera and species of Cantharinae. The geometric morphometrics represents a reliable tool not only in the taxonomic research but also in further study on the evolution of the hind wing shape of cantharid beetles.
We are grateful to Prof. Xingke Yang (IZAS) for his great support in providing the studied material. Thanks are due to the anonymous referee for their valuable comments on our manuscript and Mr. John MacDermott (USA) for correcting the English.
The present study was supported by the National Natural Science Foundation of China (Nos. 31172135, 41401064), the Knowledge Innovation of Chinese Academy of Sciences (Nos. KSCX2EWG4, KSCX2EWZ8), the Natural Science Foundation of Hebei Province (No. C2013201261) and the Foundation of the Key Laboratory of Zoological Systematics and Application of Hebei Province (No. 14967611D).