# Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini)

Mengzhen Chen, Wanru Guo, Sunbin Huang, Xiaozhu Luo, Mingyi Tian, Weixin Liu

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Mengzhen Chen, Wanru Guo, Sunbin Huang, Xiaozhu Luo, Mingyi Tian, et al.. Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini). Insects, 2021, 12 (11), pp.1002. 10.3390/insects12111002. hal-03966013

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# Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini)

Mengzhen Chen, Wanru Guo, Sunbin Huang, Xiaozhu Luo, Mingyi Tian, Weixin Liu

► To cite this version:

Mengzhen Chen, Wanru Guo, Sunbin Huang, Xiaozhu Luo, Mingyi Tian, et al.. Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini). Insects, 2021, 12 (11), pp.1002. 10.3390/insects12111002. hal-03966013

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https://hal.science/hal-03966013v1

Submitted on 28 Jun 2024

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entific research documents, whether they are pub-
lished or not. The documents may come from
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Article

# Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini)

Mengzhen Chen <fnref n="1" />,†, Wanru Guo <fnref n="1" />,†, Sunbin Huang <fnref n="1" />,<fnref n="2" /> ID, Xiaozhu Luo <fnref n="1" />,<fnref n="3" />, Mingyi Tian <fnref n="1" />,* and Weixin Liu <fnref n="1" />,* ID

<figure><img src="image_3.png" /></figure>

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**Citation:** Chen, M.; Guo, W.; Huang, S.; Luo, X.; Tian, M.; Liu, W. Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini). *Insects* **2021**, *12*, 1002. https://doi.org/10.3390/insects12111002

Academic Editor: Markus Friedrich

Received: 28 September 2021

Accepted: 30 October 2021

Published: 8 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Simple Summary:** Cavernicolous ground beetles dwelling in China are one of the most diverse and underground-adapted coleopteran group in the world. The tribe Trechini is, among them, the most representative group constituting over 170 known species with a narrow and elongated body and long appendages or a stout body and short appendages. However, very little information about their morphology has been explored. The aim of this study was to analyze the morphological adaptations of this group using geometric morphological methods. The beetles were divided into four different morphological types, including aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling, and the analysis is based on the morphology of their head, pronotum, and elytra. Our findings indicate that the overall morphological variation of cave trechine beetles has gradually specialized from an anophthalmic to semi-aphaenopsian to aphaenopsian type. Different types have different directions of variation in the head, pronotum, and elytra, but the pronotum is more differentiated and morphologically diverse than the head and elytra.

**Abstract:** Cave-dwelling ground beetles in China represent the most impressive specific diversity and morphological adaptations of the cavernicolous ground beetles in the world, but they have not been systematically examined in quantitative terms. The present study focuses on the application of geometric morphological methods to address the morphological adaptations of the tribe Trechini, the most representative group in China. We have employed a geometric morphometry analysis of the head, pronotum, and elytra of 53 genera of Trechini, including 132 hypogean and 8 epigean species. Our results showed that the overall morphological variation of cave carabids has gradually specialized from an anophthalmic to semi-aphaenopsian to aphaenopsian type. There were extremely significant differences (*p* < 0.01) among four different adaptive types including aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling Trechini when their adaptability to a cave environment was used as the basis for grouping. Furthermore, there were differences in the phenotypic tree of the head, pronotum, and elytra, and an integrated morphology. To the best of our knowledge, this is the first report on the analysis of the head, pronotum, and elytra of four different adaptive types of ground beetles in order to clarify the morphological adaptations of cavernicolous carabids to the cave environment.

**Keywords:** adaptive type; cave environment; morphological variation; phenotypic development

<footnote n="1">¹ Department of Entomology, College of Plant Protection, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China; 18819427325@163.com (M.C.); g1597667714@163.com (W.G.); huangsunbin@163.com (S.H.); xiaozhu.luo@uni-jena.de (X.L.)</footnote>

<footnote n="2">² Mécanismes Adaptatifs et Évolution (MECADEV), Muséum National d'Histoire Naturelle, CP50, 57 Rue Cuvier, 75005 Paris, France</footnote>

<footnote n="3">³ Institute of Zoology and Evolutionary Research, Friedrich Schiller University Jena, Erbertstr. 1, 07743 Jena, Germany</footnote>

<footnote>* Correspondence: mytian@scau.edu.cn (M.T.); da2000wei@163.com (W.L.)</footnote>

<footnote>† These authors have contributed equally to this work.</footnote>
<figure><img src="image_2.png" /></figure>

Article

# Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini)

Mengzhen Chen <fnref n="1" />,†, Wanru Guo <fnref n="1" />,†, Sunbin Huang <fnref n="1" />,<fnref n="2" /> ID, Xiaozhu Luo <fnref n="1" />,<fnref n="3" />, Mingyi Tian <fnref n="1" />,* and Weixin Liu <fnref n="1" />,* ID

<figure><img src="image_3.png" /></figure>

check for
updates

**Citation:** Chen, M.; Guo, W.; Huang, S.; Luo, X.; Tian, M.; Liu, W. Morphological Adaptation of Cave-Dwelling Ground Beetles in China Revealed by Geometric Morphometry (Coleoptera, Carabidae, Trechini). *Insects* **2021**, *12*, 1002. https://doi.org/10.3390/insects12111002

Academic Editor: Markus Friedrich

Received: 28 September 2021

Accepted: 30 October 2021

Published: 8 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Simple Summary:** Cavernicolous ground beetles dwelling in China are one of the most diverse and underground-adapted coleopteran group in the world. The tribe Trechini is, among them, the most representative group constituting over 170 known species with a narrow and elongated body and long appendages or a stout body and short appendages. However, very little information about their morphology has been explored. The aim of this study was to analyze the morphological adaptations of this group using geometric morphological methods. The beetles were divided into four different morphological types, including aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling, and the analysis is based on the morphology of their head, pronotum, and elytra. Our findings indicate that the overall morphological variation of cave trechine beetles has gradually specialized from an anophthalmic to semi-aphaenopsian to aphaenopsian type. Different types have different directions of variation in the head, pronotum, and elytra, but the pronotum is more differentiated and morphologically diverse than the head and elytra.

**Abstract:** Cave-dwelling ground beetles in China represent the most impressive specific diversity and morphological adaptations of the cavernicolous ground beetles in the world, but they have not been systematically examined in quantitative terms. The present study focuses on the application of geometric morphological methods to address the morphological adaptations of the tribe Trechini, the most representative group in China. We have employed a geometric morphometry analysis of the head, pronotum, and elytra of 53 genera of Trechini, including 132 hypogean and 8 epigean species. Our results showed that the overall morphological variation of cave carabids has gradually specialized from an anophthalmic to semi-aphaenopsian to aphaenopsian type. There were extremely significant differences (*p* < 0.01) among four different adaptive types including aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling Trechini when their adaptability to a cave environment was used as the basis for grouping. Furthermore, there were differences in the phenotypic tree of the head, pronotum, and elytra, and an integrated morphology. To the best of our knowledge, this is the first report on the analysis of the head, pronotum, and elytra of four different adaptive types of ground beetles in order to clarify the morphological adaptations of cavernicolous carabids to the cave environment.

**Keywords:** adaptive type; cave environment; morphological variation; phenotypic development

<footnote n="1">¹ Department of Entomology, College of Plant Protection, South China Agricultural University, 483 Wushan Road, Guangzhou 510642, China; 18819427325@163.com (M.C.); g1597667714@163.com (W.G.); huangsunbin@163.com (S.H.); xiaozhu.luo@uni-jena.de (X.L.)</footnote>

<footnote n="2">² Mécanismes Adaptatifs et Évolution (MECADEV), Muséum National d'Histoire Naturelle, CP50, 57 Rue Cuvier, 75005 Paris, France</footnote>

<footnote n="3">³ Institute of Zoology and Evolutionary Research, Friedrich Schiller University Jena, Erbertstr. 1, 07743 Jena, Germany</footnote>

<footnote>* Correspondence: mytian@scau.edu.cn (M.T.); da2000wei@163.com (W.L.)</footnote>

<footnote>† These authors have contributed equally to this work.</footnote>
# 1. Introduction

China is very rich in cave-dwelling ground beetles. At present, 202 species of cave carabids belonging to 8 tribes and 71 genera have been recorded, among which the Tre-chini is the most diverse group including 175 species from 63 genera [1,2]. All of them are troglobionts and completely lack eyes, except for four troglophiles with more or less degenerated eyes [3]. In addition to the disappearance of their eyes, cavernicolous carabids underwent morphological modifications during long-term adaptation to the subterranean environment. These are manifested in the loss of pigment and metathoracic wings, as well as their more slender body and thin legs [4,5]. According to the adaptability of charac-teristic appearances and biological information, cave trechine beetles were divided into three morphological types [6–8]: aphaenopsian, semi-aphaenopisan, and anophthalmic (Figure 1). The former means that these carabids have an extreme elongation of their body and appendages, while the latter refers to their stout body and shorter appendages. The semi-aphaenopsian is considered to be a transitional type, with morphological characteris-tics lying between the above two. The surface-dwelling trechine beetles have darker body color and compound eyes (Figure 1).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Morphological characteristics of four different adaptive types of ground beetles. (a) Surface-dwelling (*Sinotrechiama yunnanus*); (b) anophthalmic (*Sinotroglydes yanwangi*); (c) semi-aphaenopsian (*Aspidaphaenops dudou*); and (d) aphaenopsian (*Giraffaphaenops clarkei*).</figcaption></figure>

Geometric morphometry is an approach that relies on the quantitative analysis of the geometry of the target structure and the further performance of statistical analyses [9]. Different types of data, such as landmark coordinates, outline curves, and surfaces are used to define the shape [10]. The original morphological information is usually obtained through Cartesian coordinates, which are used to remove the interference of non-morphological variation in the analysis so that the punctuation overprint analysis of all samples can be visualized and displayed [11]. Geometric morphometry began to be used in the 1980s, and in the 21st century it has been widely used in entomology, medicine, archaeology, and other fields [12–14]. Recently, geometric morphometry has developed from two-dimensional to three-dimensional. Three-dimensional scanning, electron microscope scanning, micro-CT scanning, etc., provide advanced technical support for the development of geometric morphometry [15–17].
# 1. Introduction

China is very rich in cave-dwelling ground beetles. At present, 202 species of cave carabids belonging to 8 tribes and 71 genera have been recorded, among which the Tre-chini is the most diverse group including 175 species from 63 genera [1,2]. All of them are troglobionts and completely lack eyes, except for four troglophiles with more or less degenerated eyes [3]. In addition to the disappearance of their eyes, cavernicolous carabids underwent morphological modifications during long-term adaptation to the subterranean environment. These are manifested in the loss of pigment and metathoracic wings, as well as their more slender body and thin legs [4,5]. According to the adaptability of charac-teristic appearances and biological information, cave trechine beetles were divided into three morphological types [6–8]: aphaenopsian, semi-aphaenopisan, and anophthalmic (Figure 1). The former means that these carabids have an extreme elongation of their body and appendages, while the latter refers to their stout body and shorter appendages. The semi-aphaenopsian is considered to be a transitional type, with morphological characteris-tics lying between the above two. The surface-dwelling trechine beetles have darker body color and compound eyes (Figure 1).

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Morphological characteristics of four different adaptive types of ground beetles. (a) Surface-dwelling (*Sinotrechiama yunnanus*); (b) anophthalmic (*Sinotroglycytes yanwangi*); (c) semi-aphaenopsian (*Aspidaphaenops dudou*); and (d) aphaenopsian (*Giraffaphaenops clarkei*).</figcaption></figure>

Geometric morphometry is an approach that relies on the quantitative analysis of the geometry of the target structure and the further performance of statistical analyses [9]. Different types of data, such as landmark coordinates, outline curves, and surfaces are used to define the shape [10]. The original morphological information is usually obtained through Cartesian coordinates, which are used to remove the interference of non-morphological variation in the analysis so that the punctuation overprint analysis of all samples can be visualized and displayed [11]. Geometric morphometry began to be used in the 1980s, and in the 21st century it has been widely used in entomology, medicine, archaeology, and other fields [12–14]. Recently, geometric morphometry has developed from two-dimensional to three-dimensional. Three-dimensional scanning, electron microscope scanning, micro-CT scanning, etc., provide advanced technical support for the development of geometric morphometry [15–17].
The application of geometric morphometry to coleopteran insects is very extensive. It is often used to explore morphological differences between species with sexual dimorphism [18,19], intersubspecies, sister groups [20,21], and high-level categories [22,23]. It is also possible to infer the ancestral form of an existing taxa in order to study its origin and evolution [24]. Geometric morphometry has gradually been applied to different groups of Coleoptera, including Carabidae [25–27], Lucanidae, Chrysomelidae [28], Scarabaeidae [29–31], and Silphidae [32]. However, studies of the geometric morphometry of cave-dwelling ground beetles have rarely been reported [33].

Based on a geometric morphometric approach, the present paper provides, for the first time, an analysis of the head, pronotum, and elytra of four different adaptive types of ground beetles in order to clarify the morphological adaptations of cavernicolous carabids to the cave environment. In addition, the phenotypic relationship was obtained with a clustering analysis in the genetic category to explore the morphological evolution of cave-dwelling ground beetles.

**2. Materials and Methods**

**2.1. Studied Materials**

For the materials used in this study, we implemented the following principles: (1) Sampling as many genera and species as possible, including type species. (2) We did not deal with subgenus separately; species taxonomic treatment was based on the latest publications. (3) We used bibliographic data to obtain the morphological adaptation types of known taxa, e.g., [34–38].

A total of 140 species in 53 genera of Trechini (49 genera and 132 species from caves and 4 genera and 8 species of surface-dwelling beings) were examined in this study (Table A1). Among them, 97 specimens were deposited at South China Agricultural University, e.g., [39–41].

**2.2. Geometric Morphometric Approach**

**2.2.1. Image Acquisition**

Photographs of existing samples were taken with a Keyence VHX-5000 digital microscope (Figure S1). Due to the lack of specimens for 44 species, images were obtained from related original literature, e.g., [42–44] (Figure S1). For *Minimaphaenops (Enshiaphaenops) senecali* Deuve, 2016, we gathered the data from the specimen we collected as well as the additional figure of the type specimen from the original publication. After editing using Adobe Photoshop CS6, we imported the data into tps-Util 1.78 [45].

**2.2.2. Landmark Data**

The shape of the head, pronotum, and elytra and the positions of stable pores on the elytra were chosen as morphological indicators. We selected the configurations of 50 semilandmarks of each object, except for the stable pores on the elytra, which were represented by 6 landmarks (Figure 2). The landmarks and semilandmarks in each image were digitized using the tps-Dig 2.31 software (Landmark Data S1). The Tps-Small 1.34 software [46] was used to detect the data correlation of the.tps files after the landmarks to verify whether the data correlation met the requirements.
The application of geometric morphometry to coleopteran insects is very extensive. It is often used to explore morphological differences between species with sexual dimorphism [18,19], intersubspecies, sister groups [20,21], and high-level categories [22,23]. It is also possible to infer the ancestral form of an existing taxa in order to study its origin and evolution [24]. Geometric morphometry has gradually been applied to different groups of Coleoptera, including Carabidae [25–27], Lucanidae, Chrysomelidae [28], Scarabaeidae [29–31], and Silphidae [32]. However, studies of the geometric morphometry of cave-dwelling ground beetles have rarely been reported [33].

Based on a geometric morphometric approach, the present paper provides, for the first time, an analysis of the head, pronotum, and elytra of four different adaptive types of ground beetles in order to clarify the morphological adaptations of cavernicolous carabids to the cave environment. In addition, the phenotypic relationship was obtained with a clustering analysis in the genetic category to explore the morphological evolution of cave-dwelling ground beetles.

**2. Materials and Methods**

**2.1. Studied Materials**

For the materials used in this study, we implemented the following principles: (1) Sampling as many genera and species as possible, including type species. (2) We did not deal with subgenus separately; species taxonomic treatment was based on the latest publications. (3) We used bibliographic data to obtain the morphological adaptation types of known taxa, e.g., [34–38].

A total of 140 species in 53 genera of Trechini (49 genera and 132 species from caves and 4 genera and 8 species of surface-dwelling beings) were examined in this study (Table A1). Among them, 97 specimens were deposited at South China Agricultural University, e.g., [39–41].

**2.2. Geometric Morphometric Approach**

**2.2.1. Image Acquisition**

Photographs of existing samples were taken with a Keyence VHX-5000 digital microscope (Figure S1). Due to the lack of specimens for 44 species, images were obtained from related original literature, e.g., [42–44] (Figure S1). For *Minimaphaenops (Enshiaphaenops) senecali* Deuve, 2016, we gathered the data from the specimen we collected as well as the additional figure of the type specimen from the original publication. After editing using Adobe Photoshop CS6, we imported the data into tps-Util 1.78 [45].

**2.2.2. Landmark Data**

The shape of the head, pronotum, and elytra and the positions of stable pores on the elytra were chosen as morphological indicators. We selected the configurations of 50 semilandmarks of each object, except for the stable pores on the elytra, which were represented by 6 landmarks (Figure 2). The landmarks and semilandmarks in each image were digitized using the tps-Dig 2.31 software (Landmark Data S1). The Tps-Small 1.34 software [46] was used to detect the data correlation of the.tps files after the landmarks to verify whether the data correlation met the requirements.
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Landmark and semilandmark configurations of ground beetle specimens. (a) The right side of the head (50 positions); (b) right side of the pronotum (50 positions); and (c) right side of the elytra (56 positions).</figcaption></figure>

### 2.2.3. Statistical Analysis

The morphological data obtained by the standardized processing of different cave environment-adapted types were imported into the MorphoJ 1.07A software. Generalized Procrustes Analysis (GPA) was used to perform Procrustes superimposition on the overall samples to extract shape variables [47,48]. We used calculational processing to ensure that the sum of the squares of the distances between landmarks of the same serial number was minimized. Additionally, we calculated the overall average shape to compare the degree of difference between the individual and overall average shape (measured by Procrustes distance). On this basis, we applied principal component analysis (PCA).

We selected the first two principal components (PC) to construct scatterplots to show the morphological differences of carabids in different cave environment adaptation types. An energy map of the extreme points of the coordinate origin arrangement was obtained from a thin-plate spline (TPS) analysis using the MorphoJ 1.07A software, where differences in the landmarks were displayed in a visual form.

On the basis of PCA, we set the different cave environment adaptation types as the basis for grouping and performed canonical variate analysis (CVA). The results were displayed through the Mahalanobis distance and Procrustes distance.

### 2.2.4. Clustering Analysis

The original TPS file was split into 53 subfiles according to genera using the tps-Util 1.78 software; then we used the tps-Super 2.05 software to calculate the average form of each genus. Procrustes distances between genera were preformed using the tps-Small 1.34 software; then we used the unweighted group averaging method (UPGMA) in the NTSYSpc 2.10e software [49] to analyze the Procrustes distance matrix.

## 3. Results

### 3.1. *Internal Correlation of the Original Data*

The original data were converted from camber Kendall space to Euclidean tangent space. For the head, pronotum, and elytra, the correlation coefficients of the data before
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Landmark and semilandmark configurations of ground beetle specimens. (a) The right side of the head (50 positions); (b) right side of the pronotum (50 positions); and (c) right side of the elytra (56 positions).</figcaption></figure>

### 2.2.3. Statistical Analysis

The morphological data obtained by the standardized processing of different cave environment-adapted types were imported into the MorphoJ 1.07A software. Generalized Procrustes Analysis (GPA) was used to perform Procrustes superimposition on the overall samples to extract shape variables [47,48]. We used calculational processing to ensure that the sum of the squares of the distances between landmarks of the same serial number was minimized. Additionally, we calculated the overall average shape to compare the degree of difference between the individual and overall average shape (measured by Procrustes distance). On this basis, we applied principal component analysis (PCA).

We selected the first two principal components (PC) to construct scatterplots to show the morphological differences of carabids in different cave environment adaptation types. An energy map of the extreme points of the coordinate origin arrangement was obtained from a thin-plate spline (TPS) analysis using the MorphoJ 1.07A software, where differences in the landmarks were displayed in a visual form.

On the basis of PCA, we set the different cave environment adaptation types as the basis for grouping and performed canonical variate analysis (CVA). The results were displayed through the Mahalanobis distance and Procrustes distance.

### 2.2.4. Clustering Analysis

The original TPS file was split into 53 subfiles according to genera using the tps-Util 1.78 software; then we used the tps-Super 2.05 software to calculate the average form of each genus. Procrustes distances between genera were preformed using the tps-Small 1.34 software; then we used the unweighted group averaging method (UPGMA) in the NTSYSpc 2.10e software [49] to analyze the Procrustes distance matrix.

## 3. Results

### 3.1. *Internal Correlation of the Original Data*

The original data were converted from camber Kendall space to Euclidean tangent space. For the head, pronotum, and elytra, the correlation coefficients of the data before
and after the conversion were 0.999995, 0.999992, and 1.000000, respectively, which met the requirements.

## 3.2. PCA of the Morphological Variation in Head, Pronotum and Elytra

Principal component analysis was performed on the morphological data of the head, pronotum, and elytra for 141 species of carabid beetles, with 96, 96, and 108 principal components being obtained, respectively. Among them, the first principal component (PC1) accounted for 89.86%, 84.95%, and 39.35% of the overall variance, while the second principal component (PC2) accounted for 3.94%, 7.75%, and 27.20%, respectively. Using PC1 and PC2, which affect the morphological variation, as the abscissa and ordinate, respectively, a scatterplot of the morphological variation was obtained, and a 90% equal frequency ellipse was constructed based on the cave adaptation type of these carabid beetles (Figure 3a,c,e).

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Principal component analysis (PCA) of four different adaptive types of ground beetles: (a,b) head; (c,d) pronotum; (e,f) elytra. (a,c,e) represent scatterplot; (b,d,f) represent the energy map.</figcaption></figure>

From the perspective of the morphological variation of the head (Figure 3a) and pronotum (Figure 3c), it was found that the aphaenopsian had no overlap and could be distinguished well from anophthalmic and surface-dwelling carabid beetles. The semi-aphaenopsian carabid beetle type was between the aphaenopsian and anophthalmic types and was more similar to the anophthalmic type because of its larger overlaps. The latter was closer to the surface-dwelling carabid beetles. Regarding the morphological variation of the elytra (Figure 3e), these four types overlapped more overall. No significant differences were found between elytra of the semi-aphaenopsian, aphaenopsian, and anophthalmic types.

The energy map of the coordinate origin and the extreme points of the PCA scatterplot of carabid beetles' head, pronotum, and elytra (Figure 3b,d,f) show that the length/width ratio of the head and pronotum has a tendency to decrease significantly, while their lateral edges expand outward in the positive direction of PC1. The widest point of the head moves to the front, but the front edge of the pronotum tends to be wider than the rear edge. Elytra tend to be more slender and the scutellum appears to be narrower. The anterior of the edge side of the elytra has a tendency to undergo adduction, with the shoulders disappearing and the position of the hair pores moving to the distal end of the elytra. In the positive direction of PC2, the posterior edge of the head is sunken inward and the front and caudal
and after the conversion were 0.999995, 0.999992, and 1.000000, respectively, which met the requirements.

## 3.2. PCA of the Morphological Variation in Head, Pronotum and Elytra

Principal component analysis was performed on the morphological data of the head, pronotum, and elytra for 141 species of carabid beetles, with 96, 96, and 108 principal components being obtained, respectively. Among them, the first principal component (PC1) accounted for 89.86%, 84.95%, and 39.35% of the overall variance, while the second principal component (PC2) accounted for 3.94%, 7.75%, and 27.20%, respectively. Using PC1 and PC2, which affect the morphological variation, as the abscissa and ordinate, respectively, a scatterplot of the morphological variation was obtained, and a 90% equal frequency ellipse was constructed based on the cave adaptation type of these carabid beetles (Figure 3a,c,e).

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Principal component analysis (PCA) of four different adaptive types of ground beetles: (a,b) head; (c,d) pronotum; (e,f) elytra. (a,c,e) represent scatterplot; (b,d,f) represent the energy map.</figcaption></figure>

From the perspective of the morphological variation of the head (Figure 3a) and pronotum (Figure 3c), it was found that the aphaenopsian had no overlap and could be distinguished well from anophthalmic and surface-dwelling carabid beetles. The semi-aphaenopsian carabid beetle type was between the aphaenopsian and anophthalmic types and was more similar to the anophthalmic type because of its larger overlaps. The latter was closer to the surface-dwelling carabid beetles. Regarding the morphological variation of the elytra (Figure 3e), these four types overlapped more overall. No significant differences were found between elytra of the semi-aphaenopsian, aphaenopsian, and anophthalmic types.

The energy map of the coordinate origin and the extreme points of the PCA scatterplot of carabid beetles' head, pronotum, and elytra (Figure 3b,d,f) show that the length/width ratio of the head and pronotum has a tendency to decrease significantly, while their lateral edges expand outward in the positive direction of PC1. The widest point of the head moves to the front, but the front edge of the pronotum tends to be wider than the rear edge. Elytra tend to be more slender and the scutellum appears to be narrower. The anterior of the edge side of the elytra has a tendency to undergo adduction, with the shoulders disappearing and the position of the hair pores moving to the distal end of the elytra. In the positive direction of PC2, the posterior edge of the head is sunken inward and the front and caudal
corners of the pronotum tend to become acute from an obtuse angle. The first four elytra hair pores are more scattered and the last three are closer together.

### 3.3. CVA of the Morphological Variation in Head, Pronotum, and Elytra

According to the results of the PCA, CVA was used to analyze the morphological variation in the distances of the head, pronotum, and elytra among all the genera of carabid beetles. The results showed that the Mahalanobis distance and Procrustes distance between aphaenopsian and surface-dwelling types were largest when adaptability to a cave environment was used as the basis for grouping. For the head, pronotum, and elytra, the maximum Mahalanobis distance (Table 1) was 28.5719, 20.8313, and 20.7926, respectively, while the maximum Procrustes distance (Table 2) was 0.3429, 0.3258, and 0.1032, respectively.

<table><caption>**Table 1.** Mahalanobis distances from four adaptive types of ground beetles based on the head, pronotum, and elytra, respectively.</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>11.1596//8.9433//12.6030</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>13.9436//10.7282//15.3457</td><td>6.6324//7.6456//7.0232</td><td></td></tr><tr><td>Surface-dwelling</td><td>28.5719//20.8313//20.7926</td><td>23.0163//16.3761//18.8595</td><td>22.5005//16.7463//16.2172</td></tr></tbody></table>

<table><caption>**Table 2.** Procrustes distances from four adaptive types of ground beetles based on the head, pronotum, and elytra, respectively.</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>0.1319//0.1409//0.0516</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>0.2451//0.2342//0.0854</td><td>0.1227//0.0995//0.0485</td><td></td></tr><tr><td>Surface-dwelling</td><td>0.3429//0.3258//0.1032</td><td>0.2275//0.1981//0.0644</td><td>0.109//0.101//0.0589</td></tr></tbody></table>

The Mahalanobis distance (Table 3) and Procrustes distance (Table 3) of the head, pronotum, and elytra were tested to determine the significance of the differences. It was shown that the four different types (aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling carabid beetles) had high significant differences from each other (*p* < 0.01).

<table><caption>**Table 3.** *P*-values of the differences in Mahalanobis and Procrustes distances for the four adaptive types of ground beetles (10,000 permutation test, consistent for the head, pronotum, and elytra).</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>&lt;0.0001</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td></td></tr><tr><td>Surface-dwelling</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td></tr></tbody></table>

### 3.4. The Phenotypic Evolutionary Relationship between Cave Trechini Genera

Based on the Procrustes distance matrix of the average morphology among 53 genera of carabid beetles, a cluster analysis was performed to construct a morphological phenotypic tree, including the head, pronotum, elytra, and all three (Figure 4). The results revealed that there were differences between the four phenotypic trees, but the variation trend of the head and the integrated morphological phenotypic tree was relatively close. When the 53 genera branched for the first time, the aphaenopsian and semi-aphaenopsian genera clustered into a clade, while the anophthalmic and surface-dwelling genera of carabid beetles clustered into another clade.
corners of the pronotum tend to become acute from an obtuse angle. The first four elytra hair pores are more scattered and the last three are closer together.

### 3.3. CVA of the Morphological Variation in Head, Pronotum, and Elytra

According to the results of the PCA, CVA was used to analyze the morphological variation in the distances of the head, pronotum, and elytra among all the genera of carabid beetles. The results showed that the Mahalanobis distance and Procrustes distance between aphaenopsian and surface-dwelling types were largest when adaptability to a cave environment was used as the basis for grouping. For the head, pronotum, and elytra, the maximum Mahalanobis distance (Table 1) was 28.5719, 20.8313, and 20.7926, respectively, while the maximum Procrustes distance (Table 2) was 0.3429, 0.3258, and 0.1032, respectively.

<table><caption>**Table 1.** Mahalanobis distances from four adaptive types of ground beetles based on the head, pronotum, and elytra, respectively.</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>11.1596//8.9433//12.6030</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>13.9436//10.7282//15.3457</td><td>6.6324//7.6456//7.0232</td><td></td></tr><tr><td>Surface-dwelling</td><td>28.5719//20.8313//20.7926</td><td>23.0163//16.3761//18.8595</td><td>22.5005//16.7463//16.2172</td></tr></tbody></table>

<table><caption>**Table 2.** Procrustes distances from four adaptive types of ground beetles based on the head, pronotum, and elytra, respectively.</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>0.1319//0.1409//0.0516</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>0.2451//0.2342//0.0854</td><td>0.1227//0.0995//0.0485</td><td></td></tr><tr><td>Surface-dwelling</td><td>0.3429//0.3258//0.1032</td><td>0.2275//0.1981//0.0644</td><td>0.109//0.101//0.0589</td></tr></tbody></table>

The Mahalanobis distance (Table 3) and Procrustes distance (Table 3) of the head, pronotum, and elytra were tested to determine the significance of the differences. It was shown that the four different types (aphaenopsian, semi-aphaenopsian, anophthalmic, and surface-dwelling carabid beetles) had high significant differences from each other (*p* < 0.01).

<table><caption>**Table 3.** *P*-values of the differences in Mahalanobis and Procrustes distances for the four adaptive types of ground beetles (10,000 permutation test, consistent for the head, pronotum, and elytra).</caption><thead><tr><th></th><th>Aphaenopsian</th><th>Semi-Aphaenopsian</th><th>Anophthalmic</th></tr></thead><tbody><tr><td>Semi-aphaenopsian</td><td>&lt;0.0001</td><td></td><td></td></tr><tr><td>Anophthalmic</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td></td></tr><tr><td>Surface-dwelling</td><td>&lt;0.0001</td><td>&lt;0.0001</td><td>&lt;0.0001</td></tr></tbody></table>

### 3.4. The Phenotypic Evolutionary Relationship between Cave Trechini Genera

Based on the Procrustes distance matrix of the average morphology among 53 genera of carabid beetles, a cluster analysis was performed to construct a morphological phenotypic tree, including the head, pronotum, elytra, and all three (Figure 4). The results revealed that there were differences between the four phenotypic trees, but the variation trend of the head and the integrated morphological phenotypic tree was relatively close. When the 53 genera branched for the first time, the aphaenopsian and semi-aphaenopsian genera clustered into a clade, while the anophthalmic and surface-dwelling genera of carabid beetles clustered into another clade.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Phenotypic tree of ground beetle genera based on the Procrustes distance. (a) Head, (b) pronotum, (c) elytra, and (d) integrated morphology. Anphaenopsian: red letters; semi-anphaenopsian: green letters; anophthalmic: blue letters; surface-dwelling: purple letters.</figcaption></figure>

In the head phenotypic tree (Figure 4a), *Sidublemus* was the first to be differentiated into a single branch. *Dongodytes*, *Sinaphaenops*, *Giraffaphaenops*, and *Pilosaphaenops* were all found to be closely related. However, *Shuangheaphaenops*, *Uenotrechus*, *Xuedytes*, *Yanzaphaenops*, and *Minimaphaenops* were mixed together with mostly semi-aphaenopsian genera carabid beetles. From the integrated phenotypic tree (Figure 4d), it could be seen that *Giraffaphaenops* and *Xuedytes* were the earliest to differentiate, and they were located far from other genera. The relationship between *Yanzaphaenops* of the aphaenopsian and semi-aphaenopsian group was relatively close, while *Wanhuaphaenops* of the anophthalmic group had a close relationship to those of the aphaenopsian group.

In the pronotum phenotypic tree (Figure 4b), all the aphaenopsian group except for *Yanzaphaenops* was combined into a clade with *Wanhuaphaenops*, *Shenaphaenops*, and *Huoyanodytes*. The remaining three types of carabids genera were clustered together, while about 1/3 of the anophthalmic type were grouped with surface-dwelling carabid beetles in another clade. The result of the elytra phenotypic tree (Figure 4c) showed that *Dianotrechus* was the first to be differentiated into a single branch. The aphaenopsian group and a small part of the semi-aphaenopsian group were clustered into a clade. Among them, *Dongodytes* is closely related to *Xuedytes*, but the same highly specialized *Sinaphaenops* was far away from the other genera in the aphaenopsian group. Part of the semi-aphaenopsian and anophthalmic groups were grouped together with surface-dwelling carabids.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Phenotypic tree of ground beetle genera based on the Procrustes distance. (a) Head, (b) pronotum, (c) elytra, and (d) integrated morphology. Anphaenopsian: red letters; semi-anphaenopsian: green letters; anophthalmic: blue letters; surface-dwelling: purple letters.</figcaption></figure>

In the head phenotypic tree (Figure 4a), *Sidublemus* was the first to be differentiated into a single branch. *Dongodytes*, *Sinaphaenops*, *Giraffaphaenops*, and *Pilosaphaenops* were all found to be closely related. However, *Shuangheaphaenops*, *Uenotrechus*, *Xuedytes*, *Yanzaphaenops*, and *Minimaphaenops* were mixed together with mostly semi-aphaenopsian genera carabid beetles. From the integrated phenotypic tree (Figure 4d), it could be seen that *Giraffaphaenops* and *Xuedytes* were the earliest to differentiate, and they were located far from other genera. The relationship between *Yanzaphaenops* of the aphaenopsian and semi-aphaenopsian group was relatively close, while *Wanhuaphaenops* of the anophthalmic group had a close relationship to those of the aphaenopsian group.

In the pronotum phenotypic tree (Figure 4b), all the aphaenopsian group except for *Yanzaphaenops* was combined into a clade with *Wanhuaphaenops*, *Shenaphaenops*, and *Huoyanodytes*. The remaining three types of carabids genera were clustered together, while about 1/3 of the anophthalmic type were grouped with surface-dwelling carabid beetles in another clade. The result of the elytra phenotypic tree (Figure 4c) showed that *Dianotrechus* was the first to be differentiated into a single branch. The aphaenopsian group and a small part of the semi-aphaenopsian group were clustered into a clade. Among them, *Dongodytes* is closely related to *Xuedytes*, but the same highly specialized *Sinaphaenops* was far away from the other genera in the aphaenopsian group. Part of the semi-aphaenopsian and anophthalmic groups were grouped together with surface-dwelling carabids.
# 4. Discussion

## 4.1. Morphological Variation Direction of Cave-Adapted Trechine Beetles

The highly specialized morphological characteristics of cave-dwelling ground beetles have long attracted the attention of researchers [50]. Most previous studies in this area have focused on changes in the morphology of cave-dwelling ground beetles after their long-term adaptation to cave life [51–53]. The present research is the first to attempt to study the morphological adaptation and variation direction of cave-dwelling ground beetles using geometric morphological analysis.

In the extreme environment of caves, animals often show the adaptive characteristics of convergent evolution due to environmental pressure [54,55]. Luo et al. [56,57] found that the cave-dwelling *S. wangorum* shows a distinct head posterior constriction and elongated pronotum combined with long and slender legs. Our results showed that the more slender their body is the higher the degree to which the ground beetles had adapted to the cave environment. This is mainly manifested in the fact that the widest point of the head gradually moves to the front, while the anterior edge of the pronotum tends to be narrower than the posterior edge in surface-dwelling compared to aphaenopsian carabids. Surprisingly, there is little available information concerning the elytra vitiation of cavernicolous carabids or other beetles [58]. We found that the position of the hair pores gradually moved towards the edge of the elytra, except for the scutellum, with the elytra becoming slenderer in cave carabids. One of the reasons why elytra is slenderer is a consequence of reducing or the disappearance of hind wings (also known as humeral calli) [59], and this situation is more distinct among the cave-dwelling species, especially the highly specialized ones.

In addition, aphaenopsian species mostly wander on stalactite walls or roofs in complete darkness [60], while semi-aphaenopsian species run on low rock walls or along the ground [61]. Anophthalmic species often live under small rocks or under damp dead wood in caves, and their habits are relatively close to those of surface-dwelling species [62–64]. It is speculated that the extension of the head and pronotum of cave-adapted ground beetles effectively increases the flexibility of the head, which may help this species to find prey in caves where food is scarce [65]. In contrast, surface-dwelling carabids may face great survival challenges [66,67] and their strong bodies will help them to fight and escape when faced with threats.

## 4.2. Geometric Morphology Analysis to Judge the Phylogeny of Cavernicolous Carabids

The molecular phylogeny of cave Trechini in China was analyzed based on two mitochondrial and two nuclear genes [68]. The preliminary study showed that the Chinese cave Trechini of Carabidae does not form a monophyletic lineage but rather is composed of four main independent evolutionary clades, each of which contains at least one highly convergent troglomorphic species.

In our study, certain differences exist in the morphological phenotypic trees of the head, pronotum, and elytra based on the Procrustes distance of carabids. For example, typical aphaenopsian genera—such as *Dongodytes*, *Giraffaphaenops*, *Sinaphaenops*, and *Xuedytes*—show extreme morphological specialization, but they are not clustered into same clade phylogenetically (Figure 4) [69]. Moreover, the semi-aphaenopsian genera of *Shenaphaenops* and *Huoyanodytes* and the anophthalmic type of *Wanhuaphaenops* are more closely related to the aphaenopsian type. A similar situation was also found in the Pyrenean subterranean Trechini, where the phylogenetic relationship between species of the same morphological type was not found to be close [70]. It may therefore be inferred that various Trechini lineages were settled multiple times independently in caves and underwent parallel morphological changes.

Furthermore, we did not classify the subgenus as an independent taxon in the present study, but the morphological differences between some subgenera in the same genus are relatively considerable. These differences may have a certain impact on the results of overall average shape.
# 4. Discussion

## 4.1. *Morphological Variation Direction of Cave-Adapted Trechine Beetles*

The highly specialized morphological characteristics of cave-dwelling ground beetles have long attracted the attention of researchers [50]. Most previous studies in this area have focused on changes in the morphology of cave-dwelling ground beetles after their long-term adaptation to cave life [51–53]. The present research is the first to attempt to study the morphological adaptation and variation direction of cave-dwelling ground beetles using geometric morphological analysis.

In the extreme environment of caves, animals often show the adaptive characteristics of convergent evolution due to environmental pressure [54,55]. Luo et al. [56,57] found that the cave-dwelling *S. wangorum* shows a distinct head posterior constriction and elongated pronotum combined with long and slender legs. Our results showed that the more slender their body is the higher the degree to which the ground beetles had adapted to the cave environment. This is mainly manifested in the fact that the widest point of the head gradually moves to the front, while the anterior edge of the pronotum tends to be narrower than the posterior edge in surface-dwelling compared to aphaenopsian carabids. Surprisingly, there is little available information concerning the elytra vitiation of cavernicolous carabids or other beetles [58]. We found that the position of the hair pores gradually moved towards the edge of the elytra, except for the scutellum, with the elytra becoming slenderer in cave carabids. One of the reasons why elytra is slenderer is a consequence of reducing or the disappearance of hind wings (also known as humeral calli) [59], and this situation is more distinct among the cave-dwelling species, especially the highly specialized ones.

In addition, aphaenopsian species mostly wander on stalactite walls or roofs in complete darkness [60], while semi-aphaenopsian species run on low rock walls or along the ground [61]. Anophthalmic species often live under small rocks or under damp dead wood in caves, and their habits are relatively close to those of surface-dwelling species [62–64]. It is speculated that the extension of the head and pronotum of cave-adapted ground beetles effectively increases the flexibility of the head, which may help this species to find prey in caves where food is scarce [65]. In contrast, surface-dwelling carabids may face great survival challenges [66,67] and their strong bodies will help them to fight and escape when faced with threats.

## 4.2. *Geometric Morphology Analysis to Judge the Phylogeny of Cavernicolous Carabids*

The molecular phylogeny of cave Trechini in China was analyzed based on two mitochondrial and two nuclear genes [68]. The preliminary study showed that the Chinese cave Trechini of Carabidae does not form a monophyletic lineage but rather is composed of four main independent evolutionary clades, each of which contains at least one highly convergent troglomorphic species.

In our study, certain differences exist in the morphological phenotypic trees of the head, pronotum, and elytra based on the Procrustes distance of carabids. For example, typical aphaenopsian genera—such as *Dongodytes*, *Giraffaphaenops*, *Sinaphaenops*, and *Xuedytes*—show extreme morphological specialization, but they are not clustered into same clade phylogenetically (Figure 4) [69]. Moreover, the semi-aphaenopsian genera of *Shenaphaenops* and *Huoyanodytes* and the anophthalmic type of *Wanhuaphaenops* are more closely related to the aphaenopsian type. A similar situation was also found in the Pyrenean subterranean Trechini, where the phylogenetic relationship between species of the same morphological type was not found to be close [70]. It may therefore be inferred that various Trechini lineages were settled multiple times independently in caves and underwent parallel morphological changes.

Furthermore, we did not classify the subgenus as an independent taxon in the present study, but the morphological differences between some subgenera in the same genus are relatively considerable. These differences may have a certain impact on the results of overall average shape.
Moreover, the length between the clade of *Giraffaphaenops* + *Xuedytes* and other genera is extensive in the integrated morphological phenotypic tree (Figure 4). This may have been caused by long periods of geographical isolation, or there may still be large gaps between these two genera and others. Future geometric morphometry of research for these groups could focus on adding the missing new genera and combining molecular phylogeny and biogeography for analysis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/insects12111002/s1, Figure S1: Photographs of samples, Landmark Data S1: Semi-landmarks of samples.

**Author Contributions:** Conceptualization, M.T., S.H., X.L. and W.L.; methodology, M.C.; software, M.C.; validation, W.G. and W.L.; formal analysis, M.C., W.G. and W.L.; investigation, M.T., S.H., M.C. and W.L.; resources, M.T., S.H., M.C. and W.L.; data curation, M.T., S.H., M.C., W.G. and W.L.; writing—original draft preparation, M.C., W.G. and W.L.; writing—review and editing, M.T., S.H., M.C., W.L., W.G. and X.L.; visualization, W.L. and W.G.; supervision, M.T. and W.L.; project administration, M.T. and W.L.; funding acquisition, M.T. and W.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 41871039.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We greatly appreciated Huang Zhengzhong (Institute pf Zoology, Chinese Academy of Science) and Ge Zhentai (South China Agricultural University), who provided help with using the geometric morphology analysis software. We would like to thank the caving team members of SCAU for their assistance with fieldwork.

**Conflicts of Interest:** The authors declare no conflict of interest.

## Appendix A

<table><caption>**Table A1.** Basal information of the studied specimens used in the geometric morphometric analysis.</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>1</td><td><i>Dongodytes</i> (Dongodytes) baxian Tian, 2011</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>2</td><td><i>D.</i> (D.) <i>elongatus</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>3</td><td><i>D.</i> (D.) <i>fowleri</i> Deuve, 1993</td><td>Guangxi, Bama County</td><td>aphaenopsian</td></tr><tr><td>4</td><td><i>D.</i> (D.) <i>giraffa</i> Uéno, 2005</td><td>Guangxi, Tian'e County</td><td>aphaenopsian</td></tr><tr><td>5</td><td><i>D.</i> (D.) <i>grandis</i> Uéno, 1998</td><td>Guangxi, Fengshan County</td><td>aphaenopsian</td></tr><tr><td>6</td><td><i>D.</i> (D.) <i>lani</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>7</td><td><i>D.</i> (D.) <i>tonywhitteni</i> Yang, Huang &amp; Tian, 2018</td><td>Guangxi, Bama County</td><td>aphaenopsian</td></tr><tr><td>8</td><td><i>D.</i> (D.) <i>troglodytes</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>9</td><td><i>D.</i> (Dongodytodes) brevipenis Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>10</td><td><i>D.</i> (D.) <i>deharvengi</i> Tian, 2011</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>11</td><td><i>D.</i> (D.) <i>inexpectatus</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>12</td><td><i>D.</i> (D.) <i>jinzhuensis</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>13</td><td><i>D.</i> (D.) <i>yaophilus</i> Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Dahu County</td><td>aphaenopsian</td></tr><tr><td>14</td><td><i>Giraffaphaenops clarkei</i> Deuve, 2002</td><td>Guangxi, Leye County</td><td>aphaenopsian</td></tr><tr><td>15</td><td><i>G.</i> <i>yangi</i> Tian &amp; Luo, 2015</td><td>Guangxi, Tianlin County</td><td>aphaenopsian</td></tr><tr><td>16</td><td><i>M.</i> (Enshiaphaenops) senecali Deuve, 2016</td><td>Hubei, Enshi Autonomous Prefecture</td><td>aphaenopsian</td></tr><tr><td>17</td><td><i>M.</i> (Minimaphaenops) lipsae Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>aphaenopsian</td></tr><tr><td>18</td><td><i>Pilosaphaenops pilosulus</i> Deuve &amp; Tian, 2008</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>19</td><td><i>P. whitteni</i> Tian, 2010</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>20</td><td><i>Shuangheaphaenops elegans</i> Tian, 2017</td><td>Guizhou, Suiyang County</td><td>aphaenopsian</td></tr><tr><td>21</td><td><i>Sinaphaenops</i> (Dongaphaenops) xuxiakei Deuve &amp; Tian, 2014</td><td>Guizhou, Pan County</td><td>aphaenopsian</td></tr><tr><td>22</td><td><i>S.</i> (Sinaphaenops) <i>banshanicus</i> Tian, Chen &amp; Tang, 2017</td><td>Guizhou, Guiding County</td><td>aphaenopsian</td></tr><tr><td>23</td><td><i>S.</i> (S.) <i>gracilior</i> Uéno &amp; Ran, 1998</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>24</td><td><i>S.</i> (S.) <i>lipoi</i> Chen, Huang &amp; Tian, 2020</td><td>Guizhou, Guiyang City</td><td>aphaenopsian</td></tr><tr><td>25</td><td><i>S.</i> (S.) <i>mirabilissimus</i> Uéno &amp; Wang, 1991</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>26</td><td><i>S.</i> (S.) <i>mochongensis</i> Tian &amp; Huang, 2015</td><td>Guizhou, Duyun City</td><td>aphaenopsian</td></tr><tr><td>27</td><td><i>S.</i> (S.) <i>orthogenys</i> Uéno, 2002</td><td>Guizhou, Sandu County</td><td>aphaenopsian</td></tr></tbody></table>
Moreover, the length between the clade of *Giraffaphaenops* + *Xuedytes* and other genera is extensive in the integrated morphological phenotypic tree (Figure 4). This may have been caused by long periods of geographical isolation, or there may still be large gaps between these two genera and others. Future geometric morphometry of research for these groups could focus on adding the missing new genera and combining molecular phylogeny and biogeography for analysis.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/insects12111002/s1, Figure S1: Photographs of samples, Landmark Data S1: Semi-landmarks of samples.

**Author Contributions:** Conceptualization, M.T., S.H., X.L. and W.L.; methodology, M.C.; software, M.C.; validation, W.G. and W.L.; formal analysis, M.C., W.G. and W.L.; investigation, M.T., S.H., M.C. and W.L.; resources, M.T., S.H., M.C. and W.L.; data curation, M.T., S.H., M.C., W.G. and W.L.; writing—original draft preparation, M.C., W.G. and W.L.; writing—review and editing, M.T., S.H., M.C., W.L., W.G. and X.L.; visualization, W.L. and W.G.; supervision, M.T. and W.L.; project administration, M.T. and W.L.; funding acquisition, M.T. and W.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 41871039.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We greatly appreciated Huang Zhengzhong (Institute pf Zoology, Chinese Academy of Science) and Ge Zhentai (South China Agricultural University), who provided help with using the geometric morphology analysis software. We would like to thank the caving team members of SCAU for their assistance with fieldwork.

**Conflicts of Interest:** The authors declare no conflict of interest.

## Appendix A

<table><caption>**Table A1.** Basal information of the studied specimens used in the geometric morphometric analysis.</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>1</td><td>Dongodytes (Dongodytes) baxian Tian, 2011</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>2</td><td>D. (D.) elongatus Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>3</td><td>D. (D.) fowleri Deuve, 1993</td><td>Guangxi, Bama County</td><td>aphaenopsian</td></tr><tr><td>4</td><td>D. (D.) giraffa Uéno, 2005</td><td>Guangxi, Tian'e County</td><td>aphaenopsian</td></tr><tr><td>5</td><td>D. (D.) grandis Uéno, 1998</td><td>Guangxi, Fengshan County</td><td>aphaenopsian</td></tr><tr><td>6</td><td>D. (D.) lani Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>7</td><td>D. (D.) tonywhitteni Yang, Huang &amp; Tian, 2018</td><td>Guangxi, Bama County</td><td>aphaenopsian</td></tr><tr><td>8</td><td>D. (D.) troglodytes Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>9</td><td>D. (Dongodytodes) brevipenis Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>10</td><td>D. (D.) deharvengi Tian, 2011</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>11</td><td>D. (D.) inexpectatus Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>12</td><td>D. (D.) jinzhuensis Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>13</td><td>D. (D.) yaophilus Tian, Yin &amp; Huang, 2014</td><td>Guangxi, Dahu County</td><td>aphaenopsian</td></tr><tr><td>14</td><td>Giraffaphaenops clarkei Deuve, 2002</td><td>Guangxi, Leye County</td><td>aphaenopsian</td></tr><tr><td>15</td><td>G. yangi Tian &amp; Luo, 2015</td><td>Guangxi, Tianlin County</td><td>aphaenopsian</td></tr><tr><td>16</td><td>M. (Enshiaphaenops) senecali Deuve, 2016</td><td>Hubei, Enshi Autonomous Prefecture</td><td>aphaenopsian</td></tr><tr><td>17</td><td>M. (Minimaphaenops) lipsae Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>aphaenopsian</td></tr><tr><td>18</td><td>Pilosaphaenops pilosulus Deuve &amp; Tian, 2008</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>19</td><td>P. whitteni Tian, 2010</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>20</td><td>Shuangheaphaenops elegans Tian, 2017</td><td>Guizhou, Suiyang County</td><td>aphaenopsian</td></tr><tr><td>21</td><td>Sinaphaenops (Dongaphaenops) xuxiakei Deuve &amp; Tian, 2014</td><td>Guizhou, Pan County</td><td>aphaenopsian</td></tr><tr><td>22</td><td>S. (Sinaphaenops) banshanicus Tian, Chen &amp; Tang, 2017</td><td>Guizhou, Guiding County</td><td>aphaenopsian</td></tr><tr><td>23</td><td>S. (S.) gracilior Uéno &amp; Ran, 1998</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>24</td><td>S. (S.) lipoi Chen, Huang &amp; Tian, 2020</td><td>Guizhou, Guiyang City</td><td>aphaenopsian</td></tr><tr><td>25</td><td>S. (S.) mirabilissimus Uéno &amp; Wang, 1991</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>26</td><td>S. (S.) mochongensis Tian &amp; Huang, 2015</td><td>Guizhou, Duyun City</td><td>aphaenopsian</td></tr><tr><td>27</td><td>S. (S.) orthogenys Uéno, 2002</td><td>Guizhou, Sandu County</td><td>aphaenopsian</td></tr></tbody></table>
<table><caption>Table A1. *Cont.*</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>28</td><td>S. (S.) wangorum Uéno & Ran, 1998</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>29</td><td>S. (S.) yaolinensis Tian, Chen & Yang, 2017</td><td>Guizhou, Duyun City</td><td>aphaenopsian</td></tr><tr><td>30</td><td>S. (Thaumastaphaenops) bidraconis Uéno, 2002</td><td>Guizhou, Ziyun County</td><td>aphaenopsian</td></tr><tr><td>31</td><td>S. (T.) pulcherrimus Magrini, Vanni & Zanon, 1997</td><td>Guizhou, Ziyun County</td><td>aphaenopsian</td></tr><tr><td>32</td><td>Uenotrechus deuvei Tian & Chen, 2017</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>33</td><td>U. gejianbangi Tian & Wei, 2017</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>34</td><td>U. liboensis Deuve & Tian, 1999</td><td>Guizhou, Maolan County</td><td>aphaenopsian</td></tr><tr><td>35</td><td>U. nandanensis Deuve & Tian, 2010</td><td>Guizhou, Nandan County</td><td>aphaenopsian</td></tr><tr><td>36</td><td>Xuedytes bellus Tian & Huang, 2017</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>37</td><td>Yanzaphaenops hirundinis Uéno, 2005</td><td>Hubei, Shennongjia</td><td>aphaenopsian</td></tr><tr><td>38</td><td>Aspidaphaenops dudou Tian & Huang, 2018</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>39</td><td>A. masakii Uéno, 2006</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>40</td><td>A. reflexus Uéno, 2006</td><td>Guizhou, Ceheng County</td><td>semi-aphaenopsian</td></tr><tr><td>41</td><td>A. volatidraconis Uéno, 2006</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>42</td><td>A. xiongda Tian & Huang, 2018</td><td>Guizhou, Anlong County</td><td>semi-aphaenopsian</td></tr><tr><td>43</td><td>Boreaphaenops angustus Uéno, 2002</td><td>Hubei, Shennongjia</td><td>semi-aphaenopsian</td></tr><tr><td>44</td><td>Guiaphaenops deuvei Tian, Feng & Wei, 2017</td><td>Guangxi, Lingyun County</td><td>semi-aphaenopsian</td></tr><tr><td>45</td><td>G. lingyunensis Deuve, 2002</td><td>Guangxi, Lingyun County</td><td>semi-aphaenopsian</td></tr><tr><td>46</td><td>Guizhaphaenops (Guizhaphaenops) giganteus Uéno, 2000</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>47</td><td>G. (G.) lipsorum Deuve, 1999</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>48</td><td>G. (G.) pouillyi Deuve & Queinnec, 2014</td><td>Guizhou, Pan County</td><td>semi-aphaenopsian</td></tr><tr><td>49</td><td>G. (G.) striatus Uéno, 2000</td><td>Guizhou, Liupanshui City</td><td>semi-aphaenopsian</td></tr><tr><td>50</td><td>G. (G.) zhijinensis Uéno & Ran, 2004</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>51</td><td>G. (G.) zorzini Vigna Taglianti, 1997</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>52</td><td>G. (Semiaphaenops) lipsorum zunyiensis Deuve & Tian, 2018</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>53</td><td>G. (S.) martii Deuve, 2001</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>54</td><td>G. (S.) yudongensis Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>55</td><td>Huoyanodytes tujiaphilus Tian & Huang, 2016</td><td>Hunan, Longshan County</td><td>semi-aphaenopsian</td></tr><tr><td>56</td><td>Jiangxiaphaenops longiceps Uéno & Clarke, 2007</td><td>Jiangxi, Shangrao City</td><td>semi-aphaenopsian</td></tr><tr><td>57</td><td>Luoxiaotrechus deuvei Tian & Yin, 2013</td><td>Hunan, You County</td><td>semi-aphaenopsian</td></tr><tr><td>58</td><td>L. yini Tian & Huang, 2015</td><td>Jiangxi, Pingxiang City</td><td>semi-aphaenopsian</td></tr><tr><td>59</td><td>Plesioaphaenops annae Deuve & Tian, 2011</td><td>Guangxi, Longlin County</td><td>semi-aphaenopsian</td></tr><tr><td>60</td><td>Shenaphaenops humeralis Uéno, 1999</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>61</td><td>Shiqianaphaenops majusculus Uéno, 1999</td><td>Guizhou, Shiqian County</td><td>semi-aphaenopsian</td></tr><tr><td>62</td><td>Toshiaphaenops globipennis Uéno, 1999</td><td>Hubei, Xianfeng County</td><td>semi-aphaenopsian</td></tr><tr><td>63</td><td>T. ovicollis Uéno, 1999</td><td>Hunan, Longshan County</td><td>semi-aphaenopsian</td></tr><tr><td>64</td><td>Zhijinaphaenops gravidulus Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>65</td><td>Z. haozhicus Deuve & Tian, 2018</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>66</td><td>Z. jingliae Deuve & Tian, 2015</td><td>Guizhou, Xifeng County</td><td>semi-aphaenopsian</td></tr><tr><td>67</td><td>Z. lii Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>68</td><td>Z. liuae Deuve & Tian, 2015</td><td>Guizhou, Xifeng County</td><td>semi-aphaenopsian</td></tr><tr><td>69</td><td>Z. multisetifer Deuve & Tian, 2018</td><td>Guizhou, Bijie City</td><td>semi-aphaenopsian</td></tr><tr><td>70</td><td>Z. pubescens Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>712</td><td>Z. wenganicus Deuve & Tian, 2018</td><td>Guizhou, Wengan County</td><td>semi-aphaenopsian</td></tr><tr><td>72</td><td>Z. zunyicus Deuve & Tian, 2018</td><td>Guizhou, Zhunyi City</td><td>semi-aphaenopsian</td></tr><tr><td>73</td><td>Bathytrechus rueci Uéno, 2005</td><td>Guangxi, Leye County</td><td>anophthalmic</td></tr><tr><td>74</td><td>Cathaiaphaenops (Amygdalotrechus) amplipennis Uéno, 2000</td><td>Hubei, Xianfeng County</td><td>anophthalmic</td></tr><tr><td>75</td><td>C. (A.) chuandongziensis Deuve, 1999</td><td>Hubei, Banqiao Town</td><td>anophthalmic</td></tr><tr><td>76</td><td>C. (A.) cychroides Deuve & Tian, 2016</td><td>Hubei, Lichuan City</td><td>anophthalmic</td></tr><tr><td>77</td><td>C. (A.) draconis Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>anophthalmic</td></tr><tr><td>78</td><td>C. (A.) enshiensis Deuve & Tian, 2016</td><td>Hubei, Banqiao Town</td><td>anophthalmic</td></tr><tr><td>79</td><td>C. (A.) lagredeae Deuve, 2016</td><td>Hubei, Enshi Autonomous Prefecture</td><td>anophthalmic</td></tr><tr><td>80</td><td>C. (A.) lynchae Deuve & Tian, 2008</td><td>Hubei, Jianshi County</td><td>anophthalmic</td></tr><tr><td>81</td><td>C. (A.) vignataglianti Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>anophthalmic</td></tr><tr><td>82</td><td>C. (Cathaiaphaenops) delprati Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>83</td><td>Cimmeritodes (Cimmeritodes) huangi Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>84</td><td>C. (Dianocimmerites) crassifemoralis Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>anophthalmic</td></tr><tr><td>85</td><td>C. (Shimenrites) shimenensis Deuve & Tian, 2017</td><td>Hunan, Shimen County</td><td>anophthalmic</td></tr><tr><td>86</td><td>C. (Xiangcimmerites) zhongfangensis Deuve & Tian, 2016</td><td>Hunan, Zhongfang County</td><td>anophthalmic</td></tr><tr><td>87</td><td>C. (Zhecimmerites) parvus Tian & Li, 2016</td><td>Anhui, Chaohu City</td><td>anophthalmic</td></tr><tr><td>88</td><td>C. (Z.) zhejiangensis Deuve & Tian, 2015</td><td>Zhejiang, Changshan County</td><td>anophthalmic</td></tr><tr><td>89</td><td>Deuveaphaenops (Deuveaphaenops) qimenxicus Tian & Huang, 2017</td><td>Chongqing, Wulong District</td><td>anophthalmic</td></tr><tr><td>90</td><td>D. (Furongius) gelaophilus Tian & Huang, 2017</td><td>Guizhou, Zhunyi City</td><td>anophthalmic</td></tr><tr><td>91</td><td>Dianotrechus gueorguievi Tian, 2016</td><td>Yunnan, Anning City</td><td>anophthalmic</td></tr><tr><td>92</td><td>Dongoblemus kemadongicus Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>anophthalmic</td></tr><tr><td>93</td><td>Graciliblemus lipingensis Deuve & Tian, 2016</td><td>Guizhou, Liping County</td><td>anophthalmic</td></tr><tr><td>94</td><td>Jiulongotrechus pubescens Tian, Huang & Wang, 2015</td><td>Guizhou, Tongren City</td><td>anophthalmic</td></tr><tr><td>95</td><td>Junaphaenops tumidipennis Uéno, 1997</td><td>Yunnan, Kunming City</td><td>anophthalmic</td></tr><tr><td>96</td><td>Libotrechus duanensis Lin & Tian, 2014</td><td>Guangxi, Dahu County</td><td>anophthalmic</td></tr><tr><td>97</td><td>L. nishikawai Uéno, 1998</td><td>Guizhou, Libo County</td><td>anophthalmic</td></tr><tr><td>98</td><td>Microblemus rieae Uéno, 2007</td><td>Zhejiang, Jinghua City</td><td>anophthalmic</td></tr><tr><td>99</td><td>Oodinotrechus (Oodinotrechus) kishimotoi Uéno, 1998</td><td>Guizhou, Libo County</td><td>anophthalmic</td></tr></tbody></table>
<table><caption>Table A1. *Cont.*</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>28</td><td>S. (S.) wangorum Uéno & Ran, 1998</td><td>Guizhou, Libo County</td><td>aphaenopsian</td></tr><tr><td>29</td><td>S. (S.) yaolinensis Tian, Chen & Yang, 2017</td><td>Guizhou, Duyun City</td><td>aphaenopsian</td></tr><tr><td>30</td><td>S. (Thaumastaphaenops) bidraconis Uéno, 2002</td><td>Guizhou, Ziyun County</td><td>aphaenopsian</td></tr><tr><td>31</td><td>S. (T.) pulcherrimus Magrini, Vanni & Zanon, 1997</td><td>Guizhou, Ziyun County</td><td>aphaenopsian</td></tr><tr><td>32</td><td>Uenotrechus deuvei Tian & Chen, 2017</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>33</td><td>U. gejianbangi Tian & Wei, 2017</td><td>Guangxi, Huanjiang County</td><td>aphaenopsian</td></tr><tr><td>34</td><td>U. liboensis Deuve & Tian, 1999</td><td>Guizhou, Maolan County</td><td>aphaenopsian</td></tr><tr><td>35</td><td>U. nandanensis Deuve & Tian, 2010</td><td>Guizhou, Nandan County</td><td>aphaenopsian</td></tr><tr><td>36</td><td>Xuedytes bellus Tian & Huang, 2017</td><td>Guangxi, Du'an County</td><td>aphaenopsian</td></tr><tr><td>37</td><td>Yanzaphaenops hirundinis Uéno, 2005</td><td>Hubei, Shennongjia</td><td>aphaenopsian</td></tr><tr><td>38</td><td>Aspidaphaenops dudou Tian & Huang, 2018</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>39</td><td>A. masakii Uéno, 2006</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>40</td><td>A. reflexus Uéno, 2006</td><td>Guizhou, Ceheng County</td><td>semi-aphaenopsian</td></tr><tr><td>41</td><td>A. volatidraconis Uéno, 2006</td><td>Guizhou, Xingyi County</td><td>semi-aphaenopsian</td></tr><tr><td>42</td><td>A. xiongda Tian & Huang, 2018</td><td>Guizhou, Anlong County</td><td>semi-aphaenopsian</td></tr><tr><td>43</td><td>Boreaphaenops angustus Uéno, 2002</td><td>Hubei, Shennongjia</td><td>semi-aphaenopsian</td></tr><tr><td>44</td><td>Guiaphaenops deuvei Tian, Feng & Wei, 2017</td><td>Guangxi, Lingyun County</td><td>semi-aphaenopsian</td></tr><tr><td>45</td><td>G. lingyunensis Deuve, 2002</td><td>Guangxi, Lingyun County</td><td>semi-aphaenopsian</td></tr><tr><td>46</td><td>Guizhaphaenops (Guizhaphaenops) giganteus Uéno, 2000</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>47</td><td>G. (G.) lipsorum Deuve, 1999</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>48</td><td>G. (G.) pouillyi Deuve & Queinnec, 2014</td><td>Guizhou, Pan County</td><td>semi-aphaenopsian</td></tr><tr><td>49</td><td>G. (G.) striatus Uéno, 2000</td><td>Guizhou, Liupanshui City</td><td>semi-aphaenopsian</td></tr><tr><td>50</td><td>G. (G.) zhijinensis Uéno & Ran, 2004</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>51</td><td>G. (G.) zorzini Vigna Taglianti, 1997</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>52</td><td>G. (Semiaphaenops) lipsorum zunyiensis Deuve & Tian, 2018</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>53</td><td>G. (S.) martii Deuve, 2001</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>54</td><td>G. (S.) yudongensis Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>semi-aphaenopsian</td></tr><tr><td>55</td><td>Huoyanodytes tujiaphilus Tian & Huang, 2016</td><td>Hunan, Longshan County</td><td>semi-aphaenopsian</td></tr><tr><td>56</td><td>Jiangxiaphaenops longiceps Uéno & Clarke, 2007</td><td>Jiangxi, Shangrao City</td><td>semi-aphaenopsian</td></tr><tr><td>57</td><td>Luoxiaotrechus deuvei Tian & Yin, 2013</td><td>Hunan, You County</td><td>semi-aphaenopsian</td></tr><tr><td>58</td><td>L. yini Tian & Huang, 2015</td><td>Jiangxi, Pingxiang City</td><td>semi-aphaenopsian</td></tr><tr><td>59</td><td>Plesioaphaenops annae Deuve & Tian, 2011</td><td>Guangxi, Longlin County</td><td>semi-aphaenopsian</td></tr><tr><td>60</td><td>Shenaphaenops humeralis Uéno, 1999</td><td>Guangxi, Shuicheng County</td><td>semi-aphaenopsian</td></tr><tr><td>61</td><td>Shiqianaphaenops majusculus Uéno, 1999</td><td>Guizhou, Shiqian County</td><td>semi-aphaenopsian</td></tr><tr><td>62</td><td>Toshiaphaenops globipennis Uéno, 1999</td><td>Hubei, Xianfeng County</td><td>semi-aphaenopsian</td></tr><tr><td>63</td><td>T. ovicollis Uéno, 1999</td><td>Hunan, Longshan County</td><td>semi-aphaenopsian</td></tr><tr><td>64</td><td>Zhijinaphaenops gravidulus Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>65</td><td>Z. haozhicus Deuve & Tian, 2018</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>66</td><td>Z. jingliae Deuve & Tian, 2015</td><td>Guizhou, Xifeng County</td><td>semi-aphaenopsian</td></tr><tr><td>67</td><td>Z. lii Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>68</td><td>Z. liuae Deuve & Tian, 2015</td><td>Guizhou, Xifeng County</td><td>semi-aphaenopsian</td></tr><tr><td>69</td><td>Z. multisetifer Deuve & Tian, 2018</td><td>Guizhou, Bijie City</td><td>semi-aphaenopsian</td></tr><tr><td>70</td><td>Z. pubescens Uéno & Ran, 2002</td><td>Guizhou, Zijin County</td><td>semi-aphaenopsian</td></tr><tr><td>712</td><td>Z. wenganicus Deuve & Tian, 2018</td><td>Guizhou, Wengan County</td><td>semi-aphaenopsian</td></tr><tr><td>72</td><td>Z. zunyicus Deuve & Tian, 2018</td><td>Guizhou, Zhunyi City</td><td>semi-aphaenopsian</td></tr><tr><td>73</td><td>Bathytrechus rucii Uéno, 2005</td><td>Guangxi, Leye County</td><td>anophthalmic</td></tr><tr><td>74</td><td>Cathaiaphaenops (Amygdalotrechus) amplipennis Uéno, 2000</td><td>Hubei, Xianfeng County</td><td>anophthalmic</td></tr><tr><td>75</td><td>C. (A.) chuandongziensis Deuve, 1999</td><td>Hubei, Banqiao Town</td><td>anophthalmic</td></tr><tr><td>76</td><td>C. (A.) cychroides Deuve & Tian, 2016</td><td>Hubei, Lichuan City</td><td>anophthalmic</td></tr><tr><td>77</td><td>C. (A.) draconis Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>anophthalmic</td></tr><tr><td>78</td><td>C. (A.) enshiensis Deuve & Tian, 2016</td><td>Hubei, Banqiao Town</td><td>anophthalmic</td></tr><tr><td>79</td><td>C. (A.) lagredeae Deuve, 2016</td><td>Hubei, Enshi Autonomous Prefecture</td><td>anophthalmic</td></tr><tr><td>80</td><td>C. (A.) lynchae Deuve & Tian, 2008</td><td>Hubei, Jianshi County</td><td>anophthalmic</td></tr><tr><td>81</td><td>C. (A.) vignataglianti Deuve, 1999</td><td>Chongqing, Fengjie County</td><td>anophthalmic</td></tr><tr><td>82</td><td>C. (Cathaiaphaenops) delprati Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>83</td><td>Cimmeritodes (Cimmeritodes) huangi Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>84</td><td>C. (Dianocimmerites) crassifemoralis Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>anophthalmic</td></tr><tr><td>85</td><td>C. (Shimenrites) shimenensis Deuve & Tian, 2017</td><td>Hunan, Shimen County</td><td>anophthalmic</td></tr><tr><td>86</td><td>C. (Xiangcimmerites) zhongfangensis Deuve & Tian, 2016</td><td>Hunan, Zhongfang County</td><td>anophthalmic</td></tr><tr><td>87</td><td>C. (Zhecimmerites) parvus Tian & Li, 2016</td><td>Anhui, Chaohu City</td><td>anophthalmic</td></tr><tr><td>88</td><td>C. (Z.) zhejiangensis Deuve & Tian, 2015</td><td>Zhejiang, Changshan County</td><td>anophthalmic</td></tr><tr><td>89</td><td>Deuveaphaenops (Deuveaphaenops) qimenxicus Tian & Huang, 2017</td><td>Chongqing, Wulong District</td><td>anophthalmic</td></tr><tr><td>90</td><td>D. (Furongius) gelaophilus Tian & Huang, 2017</td><td>Guizhou, Zhunyi City</td><td>anophthalmic</td></tr><tr><td>91</td><td>Dianotrechus gueorguievi Tian, 2016</td><td>Yunnan, Anning City</td><td>anophthalmic</td></tr><tr><td>92</td><td>Dongoblemus kemadongicus Deuve & Tian, 2016</td><td>Yunnan, Zhenxiong County</td><td>anophthalmic</td></tr><tr><td>93</td><td>Graciliblemus lipingensis Deuve & Tian, 2016</td><td>Guizhou, Liping County</td><td>anophthalmic</td></tr><tr><td>94</td><td>Jiulongotrechus pubescens Tian, Huang & Wang, 2015</td><td>Guizhou, Tongren City</td><td>anophthalmic</td></tr><tr><td>95</td><td>Junaphaenops tumidipennis Uéno, 1997</td><td>Yunnan, Kunming City</td><td>anophthalmic</td></tr><tr><td>96</td><td>Libotrechus duanensis Lin & Tian, 2014</td><td>Guangxi, Dahu County</td><td>anophthalmic</td></tr><tr><td>97</td><td>L. nishikawai Uéno, 1998</td><td>Guizhou, Libo County</td><td>anophthalmic</td></tr><tr><td>98</td><td>Microblemus rieae Uéno, 2007</td><td>Zhejiang, Jinghua City</td><td>anophthalmic</td></tr><tr><td>99</td><td>Oodinotrechus (Oodinotrechus) kishimotoi Uéno, 1998</td><td>Guizhou, Libo County</td><td>anophthalmic</td></tr></tbody></table>
<table><caption>Table A1. *Cont.*</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>100</td><td>O. (O.) liyoubangi Tian, 2014</td><td>Guangxi, Huanjiang County</td><td>anophthalmic</td></tr><tr><td>101</td><td>O. (Pingleotrechus) yinae Sun & Tian, 2015</td><td>Guangxi, Pingle County</td><td>anophthalmic</td></tr><tr><td>102</td><td>Qianaphaenops (Qianaphaenops) emersoni Tian & Clarke, 2012</td><td>Guizhou, Yanhe County</td><td>anophthalmic</td></tr><tr><td>103</td><td>Q. (Q.) longicornis Uéno, 2000</td><td>Guizhou, Fenggang County</td><td>anophthalmic</td></tr><tr><td>104</td><td>Q. (Q.) pilosus Uéno, 2000</td><td>Guizhou, Jiangkou County</td><td>anophthalmic</td></tr><tr><td>105</td><td>Q. (Q.) rotundicollis Uéno, 2000</td><td>Guizhou, Sinan County</td><td>anophthalmic</td></tr><tr><td>106</td><td>Q. (Q.) tenuis Uéno, 2000</td><td>Guizhou, Fenggang County</td><td>anophthalmic</td></tr><tr><td>107</td><td>Q. (Qiandongaphaenops) variabilis Tian, Huang & Wang, 2015</td><td>Guizhou, Cengong County</td><td>anophthalmic</td></tr><tr><td>108</td><td>Q. (Tiankengius) xigouicus Tian & Huang, 2018</td><td>Shanxi, Hanzhong City</td><td>anophthalmic</td></tr><tr><td>109</td><td>Qianotrechus (Qianotrechus) fani Uéno, 2003</td><td>Sichuan, Gulin County</td><td>anophthalmic</td></tr><tr><td>110</td><td>Q. (Q.) laevis Uéno, 2000</td><td>Guizhou, Zhengnan County</td><td>anophthalmic</td></tr><tr><td>111</td><td>Q. (Q.) magnicollis Uéno, 2000</td><td>Guizhou, Suiyang County</td><td>anophthalmic</td></tr><tr><td>112</td><td>Q. (Q.) tenuicollis cheni Uéno, 2003</td><td>Guizhou, Suiyang County</td><td>anophthalmic</td></tr><tr><td>113</td><td>Q. (Sanwangius) rowselli, Tian & Chen, 2019</td><td>Chongqing, Wulong District</td><td>anophthalmic</td></tr><tr><td>114</td><td>Satotrechus longlinensis Deuve & Tian, 2011</td><td>Guangxi, Longlin County</td><td>anophthalmic</td></tr><tr><td>115</td><td>S. rieae Uéno, 2006</td><td>Guizhou, Anlong County</td><td>anophthalmic</td></tr><tr><td>116</td><td>Shenoblemus minusculus Tian & Fang, 2020</td><td>Anhui, Huangshan City</td><td>anophthalmic</td></tr><tr><td>117</td><td>Shilinotrechus fusiformis Uéno, 2003</td><td>Yunnan, Shilin County</td><td>anophthalmic</td></tr><tr><td>118</td><td>S. intricatus Huang & Tian, 2015</td><td>Yunnan, Yiliang County</td><td>anophthalmic</td></tr><tr><td>119</td><td>Shuaphaenops parvicollis Uéno, 1999</td><td>Chongqing, Jinfoshan</td><td>anophthalmic</td></tr><tr><td>120</td><td>Sichuanotrechus albidraconis Uéno, 2006</td><td>Sichuan, Jiangyou City</td><td>anophthalmic</td></tr><tr><td>121</td><td>S. dakangensis Huang & Tian, 2015</td><td>Sichuan, Jiangyou City</td><td>anophthalmic</td></tr><tr><td>122</td><td>Sidublemus solidus Tian & Yin, 2013</td><td>Hunan, Guidong County</td><td>anophthalmic</td></tr><tr><td>123</td><td>Sinotroglodytes bedosae Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>124</td><td>S. hygrophilus Uéno, 2009</td><td>Hunan, Sangzhi County</td><td>anophthalmic</td></tr><tr><td>125</td><td>S. yanwangi Huang, Tian & Faille, 2020</td><td>Hubei, Yichang City</td><td>anophthalmic</td></tr><tr><td>126</td><td>Superbotrechus bennetti Deuve & Tian, 2009</td><td>Hubei, Yichang City</td><td>anophthalmic</td></tr><tr><td>127</td><td>Tianeotrechus trisetosus Tian & Tang, 2016</td><td>Guangxi, Tian'e County</td><td>anophthalmic</td></tr><tr><td>128</td><td>Tianzhuaphaenops jinshanensis Zhao & Tian, 2016</td><td>Guizhou, Tianzhu County</td><td>anophthalmic</td></tr><tr><td>129</td><td>Wanhuaphaenops zhangi Tian & Wang, 2016</td><td>Hunan, Chenzhou City</td><td>anophthalmic</td></tr><tr><td>130</td><td>Wanoblemus wui Tian & Fang, 2016</td><td>Anhui, Xuancheng City</td><td>anophthalmic</td></tr><tr><td>131</td><td>Wulongoblemus tsuiblemoides Uéno, 2007</td><td>Zhejiang, Jiangshan City</td><td>anophthalmic</td></tr><tr><td>132</td><td>Yunotrechus diannanensis Tian & Huang, 2014</td><td>Yunnan, Maguan County</td><td>anophthalmic</td></tr><tr><td>133</td><td>Agonotrechus spinangulus Belousov, Kabak & Liang, 2019</td><td>Sichuan, Muli County</td><td>surface-dwelling</td></tr><tr><td>134</td><td>Protrechiama crassipes Uéno, 1997</td><td>Sichuan, Meigu County</td><td>surface-dwelling</td></tr><tr><td>135</td><td>Sinotrechiama yunnanus Belousov, Kabak & Liang, 2019</td><td>Yunnan, Dayao County</td><td>surface-dwelling</td></tr><tr><td>136</td><td>Trechus aghiazicus Belousov & Kabak, 2019</td><td>Xinjiang, Zhaosu County</td><td>surface-dwelling</td></tr><tr><td>137</td><td>T. cratocephalus Belousov & Kabak, 2019</td><td>Xinjiang, Zhaosu County</td><td>surface-dwelling</td></tr><tr><td>138</td><td>T. saluki Belousov & Kabak, 2019</td><td>Xinjiang, Xinyuan County</td><td>surface-dwelling</td></tr><tr><td>139</td><td>T. torgaut Belousov & Kabak, 2019</td><td>Xinjiang, Hejing County</td><td>surface-dwelling</td></tr><tr><td>140</td><td>T. tsanmensis Belousov & Kabak, 2019</td><td>Xinjiang, Xinyuan County</td><td>surface-dwelling</td></tr></tbody></table>

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<table><caption>Table A1. *Cont.*</caption><thead><tr><th>No.</th><th>Species</th><th>Locality</th><th>Adaptive Type</th></tr></thead><tbody><tr><td>100</td><td>O. (O.) liyoubangi Tian, 2014</td><td>Guangxi, Huanjiang County</td><td>anophthalmic</td></tr><tr><td>101</td><td>O. (Pingleotrechus) yinae Sun & Tian, 2015</td><td>Guangxi, Pingle County</td><td>anophthalmic</td></tr><tr><td>102</td><td>Qianaphaenops (Qianaphaenops) emersoni Tian & Clarke, 2012</td><td>Guizhou, Yanhe County</td><td>anophthalmic</td></tr><tr><td>103</td><td>Q. (Q.) longicornis Uéno, 2000</td><td>Guizhou, Fenggang County</td><td>anophthalmic</td></tr><tr><td>104</td><td>Q. (Q.) pilosus Uéno, 2000</td><td>Guizhou, Jiangkou County</td><td>anophthalmic</td></tr><tr><td>105</td><td>Q. (Q.) rotundicollis Uéno, 2000</td><td>Guizhou, Sinan County</td><td>anophthalmic</td></tr><tr><td>106</td><td>Q. (Q.) tenuis Uéno, 2000</td><td>Guizhou, Fenggang County</td><td>anophthalmic</td></tr><tr><td>107</td><td>Q. (Qiandongaphaenops) variabilis Tian, Huang & Wang, 2015</td><td>Guizhou, Cengong County</td><td>anophthalmic</td></tr><tr><td>108</td><td>Q. (Tiankengius) xigouicus Tian & Huang, 2018</td><td>Shanxi, Hanzhong City</td><td>anophthalmic</td></tr><tr><td>109</td><td>Qianotrechus (Qianotrechus) fani Uéno, 2003</td><td>Sichuan, Gulin County</td><td>anophthalmic</td></tr><tr><td>110</td><td>Q. (Q.) laevis Uéno, 2000</td><td>Guizhou, Zhengnan County</td><td>anophthalmic</td></tr><tr><td>111</td><td>Q. (Q.) magnicollis Uéno, 2000</td><td>Guizhou, Suiyang County</td><td>anophthalmic</td></tr><tr><td>112</td><td>Q. (Q.) tenuicollis cheni Uéno, 2003</td><td>Guizhou, Suiyang County</td><td>anophthalmic</td></tr><tr><td>113</td><td>Q. (Sanwangius) rowselli, Tian & Chen, 2019</td><td>Chongqing, Wulong District</td><td>anophthalmic</td></tr><tr><td>114</td><td>Satotrechus longlinensis Deuve & Tian, 2011</td><td>Guangxi, Longlin County</td><td>anophthalmic</td></tr><tr><td>115</td><td>S. rieae Uéno, 2006</td><td>Guizhou, Anlong County</td><td>anophthalmic</td></tr><tr><td>116</td><td>Shenoblemus minusculus Tian & Fang, 2020</td><td>Anhui, Huangshan City</td><td>anophthalmic</td></tr><tr><td>117</td><td>Shilinotrechus fusiformis Uéno, 2003</td><td>Yunnan, Shilin County</td><td>anophthalmic</td></tr><tr><td>118</td><td>S. intricatus Huang & Tian, 2015</td><td>Yunnan, Yiliang County</td><td>anophthalmic</td></tr><tr><td>119</td><td>Shuaphaenops parvicollis Uéno, 1999</td><td>Chongqing, Jinfoshan</td><td>anophthalmic</td></tr><tr><td>120</td><td>Sichuanotrechus albidraconis Uéno, 2006</td><td>Sichuan, Jiangyou City</td><td>anophthalmic</td></tr><tr><td>121</td><td>S. dakangensis Huang & Tian, 2015</td><td>Sichuan, Jiangyou City</td><td>anophthalmic</td></tr><tr><td>122</td><td>Sidublemus solidus Tian & Yin, 2013</td><td>Hunan, Guidong County</td><td>anophthalmic</td></tr><tr><td>123</td><td>Sinotroglodytes bedosae Deuve, 1996</td><td>Hunan, Longshan County</td><td>anophthalmic</td></tr><tr><td>124</td><td>S. hygrophilus Uéno, 2009</td><td>Hunan, Sangzhi County</td><td>anophthalmic</td></tr><tr><td>125</td><td>S. yanwangi Huang, Tian & Faille, 2020</td><td>Hubei, Yichang City</td><td>anophthalmic</td></tr><tr><td>126</td><td>Superbotrechus bennetti Deuve & Tian, 2009</td><td>Hubei, Yichang City</td><td>anophthalmic</td></tr><tr><td>127</td><td>Tianeotrechus trisetosus Tian & Tang, 2016</td><td>Guangxi, Tian'e County</td><td>anophthalmic</td></tr><tr><td>128</td><td>Tianzhuaphaenops jinshanensis Zhao & Tian, 2016</td><td>Guizhou, Tianzhu County</td><td>anophthalmic</td></tr><tr><td>129</td><td>Wanhuaphaenops zhangi Tian & Wang, 2016</td><td>Hunan, Chenzhou City</td><td>anophthalmic</td></tr><tr><td>130</td><td>Wanoblemus wui Tian & Fang, 2016</td><td>Anhui, Xuancheng City</td><td>anophthalmic</td></tr><tr><td>131</td><td>Wulongoblemus tsuiblemoides Uéno, 2007</td><td>Zhejiang, Jiangshan City</td><td>anophthalmic</td></tr><tr><td>132</td><td>Yunotrechus diannanensis Tian & Huang, 2014</td><td>Yunnan, Maguan County</td><td>anophthalmic</td></tr><tr><td>133</td><td>Agonotrechus spinangulus Belousov, Kabak & Liang, 2019</td><td>Sichuan, Muli County</td><td>surface-dwelling</td></tr><tr><td>134</td><td>Protrechiana crassipes Uéno, 1997</td><td>Sichuan, Meigu County</td><td>surface-dwelling</td></tr><tr><td>135</td><td>Sinotrechiana yunnanus Belousov, Kabak & Liang, 2019</td><td>Yunnan, Dayao County</td><td>surface-dwelling</td></tr><tr><td>136</td><td>Trechus aghiazicus Belousov & Kabak, 2019</td><td>Xinjiang, Zhaosu County</td><td>surface-dwelling</td></tr><tr><td>137</td><td>T. cratocephalus Belousov & Kabak, 2019</td><td>Xinjiang, Zhaosu County</td><td>surface-dwelling</td></tr><tr><td>138</td><td>T. saluki Belousov & Kabak, 2019</td><td>Xinjiang, Xinyuan County</td><td>surface-dwelling</td></tr><tr><td>139</td><td>T. torgaut Belousov & Kabak, 2019</td><td>Xinjiang, Hejing County</td><td>surface-dwelling</td></tr><tr><td>140</td><td>T. tsanmensis Belousov & Kabak, 2019</td><td>Xinjiang, Xinyuan County</td><td>surface-dwelling</td></tr></tbody></table>

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28. Benítez, H.A.; Lemic, D.; Bažok, R.; Gallardo-Araya, C.M. Evolutionary directional asymmetry and shape variation in *Diabrotica virgifera virgifera* (Coleoptera: Chrysomelidae): An example using hind wings. *Biol. J. Linn. Soc.* **2014**, *111*, 110–118. [CrossRef]

29. Roggero, A. Analysis of shape variation in *Phalops* Erichson genus (Coleoptera, Scarabaeoidea, Onthophagini). *Ital. J. Zool.* **2004**, *71*, 73–78. [CrossRef]

30. Macagno, A.L.M.; Pizzo, A.; Roggero, A.; Palestrini, C. Horn polyphenism and related head shape variation in a single-horned dung beetle: *Onthophagus (Palaeonthophagus) fracticornis* (Coleoptera: Scarabaeidae). *J. Zool. Syst. Evol. Res.* **2009**, *47*, 96–102. [CrossRef]

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32. Růžička, J.; Schneider, J.; Qubaiova, J.; Nishikawa, M. Revision of Palaearctic and Oriental Necrophila Kirby & Spence, part 2: Subgenus *Chrysosilpha* Portevin (Coleoptera: Silphidae). *Zootaxa* **2012**, *3360*, 33–58.

33. Zinetti, F.; Dapporto, L.; Vanni, S.; Magrini, P.; Bartolozzi, L.; Chelazzi, G.; Ciofi, C. Application of molecular genetics and geometric morphometrics to taxonomy and conservation of cave beetles in central Italy. *J. Insect Conserv.* **2013**, *17*, 921–932. [CrossRef]

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52. Moldovan, O.T.; Jalžić, B.; Erichsen, E. Adaptation of the mouthparts in some subterranean Cholevinae (Coleoptera, Leiodidae). *Natura Croatica* **2004**, *13*, 1–18.

53. White, W.; Culver, D.C. *Encyclopedia of Caves*; Academic Press: Amsterdam, The Nederland, 2012; p. 945.

54. Forsythe, T.G. The relationship between body form and habit in some Carabidae (Coleoptera). *J. Zool.* **1987**, *211*, 643–666. [CrossRef]

55. Pipan, T.; Culver, D.C. Shallow subterranean habitats. In *Encyclopedia of Caves*, 2nd ed.; White, W., Culver, D.C., Eds.; Academic Press: Amsterdam, The Nederland, 2012; pp. 683–690.

56. Liu, W.X.; Golovatch, S.I.; Wesner, T.; Tian, M.Y. Convergent Evolution of Unique Morphological Adaptations to a Subterranean Environment in Cave Millipedes (Diplopoda). *PLoS ONE* **2017**, *12*, e0170717. [CrossRef] [PubMed]

57. Luo, X.Z.; Wipfler, B.; Ribera, I.; Liang, H.B.; Tian, M.Y.; Ge, S.Q.; Beutel, R.G. The cephalic morphology of free-living and cave-dwelling species of trechine ground beetles from China (Coleoptera, Carabidae). *Org. Divers. Evol.* **2018**, *18*, 125–142. [CrossRef]

58. Luo, X.Z.; Wipfler, B.; Ribera, I.; Liang, H.B.; Tian, M.Y.; Ge, S.Q.; Beutel, R.G. The thoracic morphology of cave-dwelling and free-living ground beetles from China (Coleoptera, Carabidae, Trechinae). *Arthropod Struct. Dev.* **2018**, *47*, 662–674. [CrossRef]

59. Luo, X.Z.; Antunes-Carvalho, C.; Ribera, I.; Beutel, R.G. The thoracic morphology of the troglobiontic cholevine species *Troglocharinus ferreri* (Coleoptera, Leiodidae). *Arthropod Struct. Dev.* **2019**, *53*, 100900. [CrossRef] [PubMed]

60. Tian, M.Y. New records and new species of cave-dwelling trechine beetles from Mulun Nature Reserve, northern Guangxi, China (Insecta: Coleoptera: Carabidae: Trechinae). *Subterr. Biol.* **2019**, *7*, 69–73.

61. Tian, M.Y.; Huang, S.B. Contribution to the knowledge of the cavernicolous beetle genus *Aspidaphaenops* Uéno from Guizhou (Coleoptera: Carabidae: Trechinae). *Zootaxa* **2018**, *4422*, 244–258. [CrossRef]

62. Tian, M.Y. New records and a new species of the cavernicolous genus *Guiodytes* Tian, 2013 from Guangxi, China (Coleoptera: Carabidae: Scaritinae). *Zootaxa* **2014**, *3861*, 355–362. [CrossRef] [PubMed]

63. Lin, W.; Tian, M.Y. Supplemental notes on the genus *Libotrechus* Uéno, with description of a new species from Guangxi, southern China (Coleoptera: Carabidae: Trechinae). *Coleopts Bull.* **2014**, *68*, 429–433. [CrossRef]

64. Deuve, T.; He, L.; Tian, M.Y. Descriptions of the first semi-aphenopsian troglobiotic Patrobini and of a new anophthalmic cave-dwelling Trechini from central Sichuan, China (Coleoptera: Caraboidea). *Ann. Soc. Entomol. France* **2020**, *37*, 1–13. [CrossRef]

65. Pang, J.M.; Tian, M.Y. A remarkably modified species of the tribe Platynini (Coleoptera, Carabidae) from a limestone cave in Jiangxi Province, eastern China. *ZooKeys* **2014**, *382*, 1–12.

66. Wizen, G.; Gasith, A. An unprecedented role reversal: Ground beetle larvae (Coleoptera: Carabidae) lure amphibians and prey upon them. *PLoS ONE* **2011**, *6*, e25161. [CrossRef]

67. Sugiura, S.; Sato, T. Successful escape of bombardier beetles from predator digestive systems. *Biol. Lett.* **2018**, *14*, 20170647. [CrossRef]

68. Huang, S.B. Molecular Phylogeny of the Cavernicolous Trechine Beetles in China (Coleoptera: Carabidae). Master’s Thesis, South China Agricultural University, Guangzhou, China, 2016.

69. Huang, S.B.; Tian, M.Y.; Faille, A. A preliminary phylogeny of cave trechine beetles from China (Coleoptera: Carabidae: Trechini). *ARPHA Conf. Abstr.* **2020**, *3*, e51897. [CrossRef]

70. Faille, A.; Ribera, I.; Deharveng, L.; Bourdeau, C.; Garnery, L.; Deuve, T. A molecular phylogeny shows the single origin of the Pyrenean subterranean Trechini ground beetles (Coleoptera: Carabidae). *Mol. Phylogenet. Evol.* **2010**, *54*, 97–106. [CrossRef] [PubMed]