Research Article |
Corresponding author: Hao Yu Liu ( liuhy@hbu.edu.cn ) Corresponding author: Yu Xia Yang ( yuxia0305@126.com ) Academic editor: André Nel
© 2023 Wei Zhao, Hao Yu Liu, Xue Ying Ge, Yu Xia 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:
Zhao W, Liu HY, Ge XY, Yang YX (2023) Evaluating the significance of wing shapes in inferring phylogenetic proximity among the generic taxa: an example of Cantharinae (Coleoptera, Cantharidae). Arthropod Systematics & Phylogeny 81: 303-316. https://doi.org/10.3897/asp.81.e101411
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The resolution of phylogenetic relationship among animals is still one of the most challenging problems in systematic zoology. Insect wing is a highly valued morphological character in the systematics, but few studies have been conducted to quantify wing shape variations for phylogenetic reconstruction. In this study, with Cantharinae as the subject, we conducted the GM analyses from hindwings of 16 representative genera. Further, we conducted the UPGMA based on Procrustes distance and Euclidean similarity measure of Mahalanobis distance, respectively, and NJ analysis of the Mahalanobis distance, as well as MP analysis using merged landmark dataset. In the meantime, we constructed the phylogenetic relationships among these genera based on the mitochondrial genomes, with a total of 41 sequences novel to Cantharinae, by BI and ML analyses. As a result, the CVA analysis demonstrated that the hindwing shapes of the cantharid genera are significantly different from one another. All the topologies produced by the GM data partially correspond with that of mitogenomic data. The close relationships of some genera are frequently recovered, including Cyrebion + Themus, Cantharis + Taiwanocantharis + Taocantharis, Stenothemus + Falsopodabrus + Habronychus. These results prove the importance and potential application of the hindwing shapes in recovering the relationships among the sibling genera.
Geometric morphometric, genera, hindwing, mitochondrial genome, phylogeny, Soldier beetles
Knowledge of how living (and extinct) taxa are related to one another underpins much of evolutionary biology (
Although many advantages of molecular over morphological phylogenetic have been recognized, morphological analysis still cannot be replaced or neglected in the construction of phylogenetic relationships. For many groups of poorly known organisms, the only known specimens of many species are represented merely by the holotype or type series. Collecting additional material can be prohibitive because of rarity of the species, inaccessibility of the habitat, destruction of known collection localities, legal protection of the habitat or species, or high costs of procurement. A high percentage of recently extant species have been exterminated in this century by human activities, especially through the destruction of tropical rainforests (
It is well-known that wing shape of insects exhibits a high heritability in nature (
The beetle family Cantharidae, commonly known solider beetles, is a large group with approximately 6,000 species in the world (
In the present study, taking the cantharid beetles as the subject, we are going to apply GM to analyze the hindwing shapes of 16 representative genera of Cantharinae, based on which to explore their relationships by the PM analysis. Meanwhile, we shall reconstruct the phylogeny among these genera based on the (nearly) complete mitochondrial genomes by both Maximum-likelihood (ML) and Bayesian inference (BI) analyses. Finally, we will examine the accuracy of PM of hind wing shapes, by comparison with the produced topology of mitogenomes. Based on the results, we are able to evaluate the reliability of the hindwing shapes in inferring phylogenetic relationships among the generic taxa, also shed new lights on reconstruction of phylogenetic relationships, especially for those taxa rare, inaccessible or extinct organism relying on the morphology.
Hind wings of the following Cantharinae species (Table
The species of subfamily Cantharinae used in the GM analysis and information for the representative species’ mitogenomes used for phylogenetic analysis.
Genus | Species for GM analysis | Number | Species for phylogenetic analysis | GenBank Accession | Voucher number | Locality of molecular material |
---|---|---|---|---|---|---|
Asiopodabrus | Asiopodabrus cheni | 3 | Asiopodabrus cheni | OQ221889 | 2CA132 | China: Zhejiang, Fengyang Mts |
Asiopodabrus satoi | OQ221851 | 2CA39 | China: Guangxi, Mao’er Mts | |||
Cantharis | Cantharis rufa | 3 | Cantharis jindrai | OQ221852 | 2CA70 | China: Beijing, Xiaolongmen |
Cantharis brunneipennis | 3 | Cantharis brunneipennis | OQ221875 | CAN197 | China: Shaanxi, Foping | |
Cantharis (Cyrtomoptila) plagiata | 3 | Cantharis (Cyrtomoptila) plagiata | MT364421 | CAN74 | China: Shaanxi, Yangxian | |
Cyrebion | Cyrebion subrufolineatus | 3 | Cyrebion subrufolineatus | OQ221853 | 2CA65 | China: Xizang, Mangkang |
Cyrebion gracilicornis | 3 | Cyrebion gracilicornis | OQ221870 | CAN24 | China: Hubei, Shennongjia | |
Falsopodabrus | Falsopodabrus tridentatus | 3 | Falsopodabrus tridentatus | OQ221854 | 2CA161 | China: Xizang, Cona |
Falsopodabrus rolciki | 3 | Falsopodabrus rolciki | OQ221876 | 2CA81 | China: Xizang, Bomê | |
Cephalomalthinus | Cephalomalthinus sp.1 | 3 | Cephalomalthinus sp.1 | OQ221871 | CAN182 | China: Hainan, Jianfengling |
Cephalomalthinus sp.2 | 3 | Cephalomalthinus sp.2 | OQ221877 | 2CA24 | China: Guangxi, Daming Mts | |
Habronychus | Habronychus (s. str.) sp.1 | 3 | Habronychus (s. str.) sp.1 | OQ221855 | CAN27 | China: Hubei, Shennongjia |
Habronychus (s. str.) sp.2 | 3 | Habronychus (s. str.) sp.2 | OQ221878 | CAN210 | China: Hubei, Huangbaoping | |
Habronychus (Monohabronychus) sp.1 | 3 | Habronychus (Macrohabronychus) sp. | OQ221884 | 2CA3 | China: Xizang, Medog | |
Habronychus (Monohabronychus) sp.2 | 3 | Habronychus (Macrohabronychus) chaoi | OQ221859 | 2CA162 | China: Xizang, Cona | |
Habronychus (Macrohabronychus) chaoi | 3 | Habronychus (Monohabronychus) sp. | OQ221873 | CAN98 | China: Hubei, Yi’en | |
Habronychus (Monohabronychus) sp. 3 | OQ221880 | CAN83 | China: Shaanxi, Yangxian | |||
Lycocerus | Lycocerus bilineatus | 3 | Lycocerus inopaciceps | OQ221874 | CAN198 | China: Shaanxi, Foping |
Lycocerus inopaciceps | 3 | Lycocerus curvatus | OQ221857 | CAN36 | China: Hubei, Shennongjia | |
Lycocerus orientalis | 3 | Lycocerus hubeiensis | OQ221858 | CAN123 | China: Hubei, Yichang | |
Lycocerus limbatus | 3 | Lycocerus orientalis | OQ221882 | 2CA44 | China: Jiangxi, Jinggang Mts | |
Lycocerus limbatus | OQ221883 | CAN16 | China: Hubei, Shennongjia | |||
Micropodabrus | Micropodabrus coleatus | 3 | Micropodabrus oudai | OQ221860 | CAN201 | China: Shaanxi, Fouping |
Podabrus | Podabrus annulatus | 3 | Podabrus annulatus | OQ221861 | 2CA47 | China: Beijing, Yanqing |
Pseudopodabrus | Pseudopodabrus atripes | 3 | Pseudopodabrus atripes | OQ221885 | 2CA27 | China: Guangxi, Daming Mts |
Prothemus | Prothemus kiukianganus | 3 | Prothemus semimetallicus | OQ221862 | CAN102 | China: Hunan, Wulingyuan |
Prothemus sanguineus | 3 | Prothemus sanguineus | OQ221872 | CAN96 | China: Hubei, Yi’en | |
Rhagonycha | Rhagonycha nigroimpressa | 3 | Rhagonycha nigroimpressa | OQ221863 | CAN100 | China: Hunan, Yongshun |
Rhagonycha prewalskii | 3 | Rhagonycha prewalskii | OQ221886 | CAN108 | China: Hebei, Xiaowutai Mts | |
Stenothemus | Stenothemus grahami | 3 | Stenothemus fukienensis | OQ221864 | 2CA137 | China: Zhejiang, Fengyang Mountain |
Stenothemus biimpressiceps | 3 | Stenothemus biimpressiceps | OQ221887 | 2CA99 | China: Zhejiang, Tianmu Mts | |
Taiwanocantharis | Taiwanocantharis parasatoi | 3 | Taiwanocantharis parasatoi | OQ221865 | 2CA28 | China: Guangxi, Daming Mts |
Taiwanocantharis chumbiensis | 3 | Taiwanocantharis sp. | OQ221881 | 2CA150 | China: Yunnan | |
Taocantharis | Taocantharis businskae | 3 | Taocantharis businskae | OQ221888 | CAN206 | China: Hubei, Huangbaoping |
Themus | Themus (Telephorops) coelestis | 3 | Themus (Telephorops) coelestis | OQ221866 | CAN1 | China: Hubei, Shennongjia |
Themus (Telephorops) cavipennis | 3 | Themus (Telephorops) cavipennis | OQ221867 | 2CA73 | China: Xizang, Medog | |
Themus (Themus) stigmaticus | 3 | Themus (Themus) stigmaticus | OQ221868 | CAN104 | China: Hebei, Xiaowutai Mts | |
Themus (Themus) luteipes | 3 | Themus (Themus) luteipes | OQ221869 | CAN69 | China | |
Themus (Haplothemus) hedini | 3 | Themus (Haplothemus) hedini | OQ221879 | CAN148 | China: Qinghai, Menyuan | |
Themus (Haplothemus) bimaculicollis | 3 | Themus (Haplothemus) bimaculicollis | OQ221856 | 2CA110 | China: Sichuan, Liziping |
The structure analyzed was the shape of hind wings, which was directly photographed by a stereomicroscope Nikon SMZ1500 and attached video camera Canon 450D connected to a HP computer. For the hind wings, a total of 13 landmarks of type II (Fig.
The GM method based on landmark data in inferring phylogenetic relationships among the generic taxa considering UPGMA, Maximum Parsimony and Neighbor-Joining as the optimality criterion (
The tps files produced in tps-DIG was used to perform GM analysis. To examine the shape variation, the digitized landmark data is analyzed using MorphoJ software (
The tps files produced in tps-DIG was also used to perform MP analysis in TNT 1.5 (
Meanwhile, both Maximum-likelihood and Bayesian inference analyses of mitochondrial genomes to examine the accuracy of phylogenetic morphometrics of hind wing shapes, we newly sequenced 41 species mitochondrial genomes and the detailed information was provided in Table
The individual genes were aligned and concatenated using PhyloSuite version 1.2.2 (
The first three principal components of the shape of hind wings explain 76.847% of the micromesh variation, which were 56.097%, 13.314% and 7.436%, respectively (see Supplementary material: Table S1). They were plotted to indicate variation along the first two relative warp two axes, which were shown as deformations of the least squares reference using thin-plate splines (Fig.
Difference in the hindwings shapes among the groups. Mahalanobis distances p-values (above) from permutation tests (10000 permutation rounds); Procrustes distances p-values (below) from permutation tests (10000 permutation rounds).
Asiopodabrus | Cantharis | Cyrebion | Falsopodabrus | Cephalomalthinus | Habronychus | Lycocerus | Micropodabrus | Podabrus | Prothemus | Pseudopodabrus | Rhagonycha | Stenothemus | Taiwanocantharis | Taocantharis | Themus | |
Asiopodabrus | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Cantharis | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Cyrebion | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Falsopodabrus | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Cephalomalthinus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Habronychus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Lycocerus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Micropodabrus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Podabrus | 0.0001 | 0.0002 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Prothemus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0083 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Pseudopodabrus | <0.0001 | 0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Rhagonycha | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
Stenothemus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0001 | <0.0001 | 0.0005 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 | <0.0001 |
Taiwanocantharis | 0.0001 | 0.0015 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0036 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — | <0.0001 | <0.0001 |
Taocantharis | 0.0001 | 0.0044 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | 0.0002 | — | <0.0001 |
Themus | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | <0.0001 | — |
The phylogenies of Cantharinae based on the mitochondrial genome data by both ML and BI analyses produced highly congruent topologies (Fig.
In comparison with the above mitophylogenetic topologies (Fig.
Comparing phylogenetic relationships of Cantharinae. A phylogenetic hypothesis based on Procrustes distances using UPGMA. B phylogenetic hypothesis based on Mahalanobis distances using Euclidean similarity measure. C phylogenetic hypothesis based on two landmark configurations using MP analysis. D Neighbor-Joining tree for genera of Cantharinae based on Mahalanobis distances with 1000 bootstrap replicates. The average shape of four groups were displayed near the clades in a, b, c and d.
Furthermore, the phylogeny of Cantharinae was reconstructed by MP analysis based on the two landmark configurations shown in Fig.
Moreover, the tree under NJ analysis based on Mahalanobis distance (Fig.
In the present study, the statistical test performed by TpsSmall suggested that our obtained data of hind wings (3 specimens were collected from each of 37 species amount to 111 samples, 13 landmarks for each sample) is acceptable for the geometric morphometric analysis. Further the CVA analysis suggested that all representative genera (a total of 16 genera) of Cantharinae can be distinguished from one another by the hind wing shapes, which is consistent with the previous study (
In insects, the wing shapes of geometric morphometric analyses are usually applied in distinguishing the sibling species or uncovering the cryptic species (
Prior to estimate the values of hindwing shape in inferring the phylogenetic relationships among the genera of Cantharinae, a phylogenetic analysis of mitochondrial genomes was constructed to make a standard reference. Mitochondrial genomes has been widely used in the phylogenetic studies in various insect groups, not only in higher grades (
Compared with the above mitophylogenetic tree, the topologies produced by geometric morphometric data of hindwing shapes recovered the close relationships of the aforementioned genera using different methods (Fig.
Conflict between morphological and molecular studies of phylogeny may be also resulted from differences in assumptions about the evolutionary process and differences in methods of analysis. The reasons for these differences may be allometric effects, homoplasy, accelerated evolution, genetic drift and, of course possible sampling or measurement errors (
In the present study, taking the Cantharinae as an example, we evaluated the taxonomic value of the hindwing shapes in inferring phylogenetic relationships among the generic taxa of subfamily Cantharinae. A total of 111 hindwing samples representing 37 species belonging to 16 genera of Cantharinae were analyzed by GM analysis. The statistical test performed by TpsSmall suggested that our obtained data is acceptable for the geometric morphometric analysis, and the CVA analysis demonstrated that all representative genera of Cantharinae can be well separated by the hind wing shape. With the constructed mitophylogeny as reference, the PM analyses of the hindwing shapes data using different methods (MP analysis of the two landmark configurations, NJ analysis of Mahalanobis distance, phonograms of both Procrustes distiance and Euclidean similartiy metrics of Mahalanobis distance) showed that the sister relationships of allied genera or morphologically defined genera complex are always recovered. However, some genera in distant relationships sometimes are grouped together under PM analysis, probably due to the convergent evolution in the hindwing shapes. No matter how, the landmark-based hindwings shape GM analyses prove to be feasible in phylogenetic reconstruction and be helpful in recovering the sister relationships of allied genera.
The present study was supported by the National Natural Science Foundation of China (Nos. 32270491, 31772507), the Natural Science Foundation of Hebei Province (No. C2022201005), the Interdisciplinary Research Program of Natural Science of Hebei University (No. DXK202103) and the Excellent Youth Scientific Research and Innovation Team of Hebei University (No. 605020521005).
We wish to express our deepest thanks to Prof. Xingke Yang (Institute of Zoology, Chinese Academy of Sciences, Beijing, China) for his guidance to the senior corresponding author in studying on the taxonomy of Cantharidae. We are very grateful to the editors and anonymous reviewers for careful scrutiny and useful comments for improving the manuscript.
Table S1
Data type: .pdf
Explanation note: Eigen values and contributions of the principal components analysis in hindwings shape.