Zoological Systematics, 42(1): 90–101 (January 2017), DOI: 10.11865/zs.201708

ORIGINAL ARTICLE

Geometric morphometric analysis of Eysarcoris guttiger, E. annamita and E. ventralis (: )

Rongrong Li1, Hufang Zhang1, 2, Shengcai Li1 *, Ming Bai3 *

1College of Agronomy, Shanxi Agricultural University, Taigu 030801, China 2 Department of Biology, Xinzhou Teachers’ University, Xinzhou 034000, China 3Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China *Corresponding authors, E-mails: [email protected]; [email protected]

Abstract The genus Eysarcoris can be easily distinguished from other genera through the two spots in the basal angle of the scutellum. Nevertheless, Eysarcoris species show complex variances. Geometric morphometric methods have been increasingly applied to distinguish species and to define the boundary of genera among . In the present study, geometric morphometric approach was firstly employed to evaluate the shape variation of three characters (fore wing, hind wing and pygophore) of E. guttiger, E. annamita and E. ventralis using E. aeneus as outgroup to ascertain whether this approach is a reliable method for the of Eysarcoris. Analysis was conducted on the landmarks of the three characters of these species. Multivariate regression of procrustes coordinates against centroid size was conducted to test the presence of allometry. Principal component analysis (PCA), canonical variate analysis (CVA) and cluster analysis were utilized to describe variations in shapes among the studied species. For all of the three characters, though PCA analysis showed some overlap among species, p-values for procrustes distance and mahalanobis distance were all less than 0.0001. The distribution of the three studied species corresponds with their species status. This study demonstrates that the geometric morphometrics of both the fore wing and the hind wing might represent a possible tool for the identification of species within this genus.

Key words Geometric morphometrics, principal component analysis, canonical variate analysis, cluster analysis.

1 Introduction

Eysarcoris (Hemiptera: Pentatomidae) is a genus of small, mottled brown stink bugs found in Europe, Asia, Africa and Australia (Wood & Mcdonald, 1984). Since it was established by Hahn in 1834, there are 14 species (nine in China) recorded in the Palearctic and Oriental Regions of China (Hahn, 1834; Rider et al., 2002; Rider, 2006; Tsai & Rédei, 2009). Some species of this genus such as E. aeneus, E. ventralis and E. trimaculatus are main pests of rice in many rice-producing countries (Lee et al., 2009; Nasiruddin & Roy, 2012). Eysarcoris can be easily distinguished from other genera because of its basal angle feature in the scutellum with small, yellow, or pale smooth spots. Nevertheless, Eysarcoris species show complex variances. E. ventralis is highly similar to E. guttiger, but the former displays smaller levigate angular spots near each basal angle of the scutellum than the latter (Yang, 1962). However, the size of the spots in E. guttiger varies. Some specimens of E. guttiger have large spots, whereas others have very small or practically obsolete spots (Distant, 1902). E. annamita allied to E. guttiger, but can be differed by the

Special Issue: Geometric morphometrics: Current shape and future directions Received 27 December 2016, accepted 6 January 2017 Executive editor: Fuqiang Chen

90 © Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 91

abdomen beneath with the whole disk brassy-black (Bu & Zheng, 1997). Traditionally, the taxonomy of this group is mainly based on the structure of the male genitalia. However, in addition to comparisons of male genitalia and morphological descriptions, novel methods should be developed to identify the species. Geometric morphometrics, based on cartesian coordinates, can statistically analyze shape variation and its covariation with other variables (Slice, 2007). Unlike traditional morphometrics which measures distances between landmarks, this technique focuses on the information about the spatial relationships between the landmarks and tries to capture the morphological features objectively (Rohlf & Marcus 1993; Adam et al. 2004). This makes it relatively easy to draw informative pictures to illustrate the results (Zelditch et al., 2004). Male genitalia are the important taxonomic characteristic in Pentatomidae including Eysarcoris (Baker, 1931; Agarwal & Baijal, 1984; Hasan, 1991). Geometric morphometric analyses of the shapes of the male genitalia among populations of rice black bugs were established (Torres et al., 2013). Wings of insects are excellent material for geometric morphometric studies because they are essentially two dimensional (Klingenberg & Mclntyre, 1998; Pavlinov, 2001). Wings were first used as a characteristic for traditional classification in 1893 (Comstock, 1893; Kunkel, 2004). Since 1970’s, many other authors have used geometric morphometric techniques to analyze wing shapes and these studies indicated that it was a valuable characteristic for sibling species determination and genera delimitation among insects (Plowright & Stephen, 1973; De la Riva et al., 2001; Matias et al., 2001; Tofilski, 2008; Tüzün, 2009). In Hemiptera, wing geometry studies were mostly focused on Triatomine (Hemiptera: Reduviidae) (Matias et al., 2001; Gumiel et al., 2003; Campos et al., 2011; Sol Gaspe et al., 2013; Díaz et al., 2014), and the use of this effective approach in studying Eysarcoris has not been reported. In this present paper, the geometric morphometric technique was employed to quantify the morphological features of the wings and pygophore among the studied species of the genus Eysarcoris. The central aim of our study was to analyze the shape variation among E. guttiger, E. annamita and E. ventralis and test the possible use of wing shape patterns for the taxonomy of Eysarcoris.

2 Materials and methods

2.1 Insects

A total of 191 specimens of the 4 species of Eysarcoris were examined in this study. The localities and number of specimens of each species were provided in Table 1. Samples were collected mainly by manual pick-up or by using sweep nets from fields in China. All specimens were deposited in Shanxi Agricultural University and Nankai University in China.

2.2 Data acquisition

Fore wings were removed using dissecting needles and forceps, and mounted separately between microscope slides. Because the body of bugs in Eysarcoris is small and the hind wing is easily broken, we spread the hind wings on ethanol-dampened microscope slides instead of removing them from the body when taking photos. Pygophores were also removed with the use of dissecting needles and fixed with prepared fixative. Images of the three characters (fore wing, hind wings and pygophore) were obtained using a digital camera Olympus DP71 which was mounted on the ocular of stereomicroscope Olympus SZX16. The software tpsUtil Ver.1.26 (Rohlf, 2010) was used to convert the images into tps file. Curves and landmarks were digitized with tpsDig Ver.2.05 (Rohlf, 2006) and all semi-landmarks were converted to landmarks. For the fore wings, six landmarks and one curve were drawn. The curve represented the outline of the membrane and was resampled into 50 semi-landmarks. For the hind wings eight landmarks and one curve were drawn. The curve represented the outline of remigium and vannus of I and II and was resampled into 50 semi-landmarks. For the pygophore one curve were drawn. The curve was represented the right part of the dorsoposterior rim and ventroposterior rim of the pygophore and was resampled into 50 semi-landmarks (Fig. 1). All landmarks and curves were based on homologous or corresponding criteria.

2.3 Size variation and allometry

The raw coordinates of each landmark were scaled, translated, and rotated using the generalized least-squares procrustes superimposition method to remove the variance caused by the scale, position, and orientation (Bookstein, 1991). Size variation was examined using centroid size (the square root of the summed squared distance of each landmark from the 92 Li et al.

center of the form). Differences in size of the three characters among species were assessed using one-way ANOVA method implanted in Paleontological Statistics software (PAST) 3.06 (Hammer et al., 2001).

Figure 1. Landmarks and curves selection. A. Fore wing. B. Hind wing. C. Pygophore. © Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 93

To test for the presence of allometry, a multivariate regression of procrustes coordinates against centroid size on pooled within-group (pooled by species) variation, was conducted using MorphoJ 1.06d (Klingenberge, 2011). The significance of the allometry was calculated with a permutation test with 10,000 iterations.

Table 1. Localities and numbers of the studied four species in China. Species Localities Latitude/Longitude Number E. annamita Xishuangbanna, Yunnan 22°0′37.31″N, 100°47′46.49″E 1♀ Dinghu Mountain, Guangdong 23°09′44.55″N, 112°32′59.07″E 1♀2♂ Daming Mountain, Guangxi 23°33′1.13″N, 108°21′48.99″E 3♀2♂ Dali, Yunnan 25°35′40.65″N, 100°13′43.15″E 1♀ Wuyanling, Zhejiang 27°42′15.89″N, 119°39′51.25″E 1♀ Yuelu Mountain, Hunan 28°11′31.02″N, 112°55′53.43″E 1♀ Shangrao County, Jiangxi 28°27′9.39″N, 117°54′16.77″E 1♂ Yanhe County, Guizhou 28°32′57.10″N, 108°28′50.91″E 11♀3♂ Mianning County, Sichuan 28°33′12.48″N, 102°10′30.49″E 1♀ Changde, Hunan 29°02′4.75″N, 111°41′34.53″E 2♀5♂ Qiyun Mountain, Jiangxi 29°49′0.33″N, 118°02′21.41″E 1♀1♂ Tianmu Mountain, Zhejiang 30°21′0.63″N,119°25′28.82″E 3♀2♂ Dujiangyan, Sichuan 31°0′13.37″N, 103°36′41.41″E 6♀3♂ E. ventralis Changsha, Hubei 28°13′52.99″N, 112°56′0.15″E 1♀ Jinhua, Zhejiang 29°16′11.67″ N 120°19′52.98″ E 4♂ Houhe Nature Reserve, Hubei 30°04′52.52″N, 110°37′14.59″E 1♀ Linyi, Shandong 35°06′18.14″N, 118°21′2.56″E 10♀2♂ Linfen, Shanxi 36°05′18.35″N, 111°30′48.13″E 1♂ Luo Mountain, Shandong 37°27′29.18″N, 120°28′28.22″E 1♂ Cangyan Mountain, Hebei 37°49′47.68″N, 114°09′44.39″E 1♀2♂ Yi County, Hebei 39°20′53.40″N, 115°29′32.00″E 1♂ Beijing 39°54′11.78″N, 116°24′3.31″E 1♀ Taian, Shandong 40°02′39.53″N, 117°24′5.77″E 3♀1♂ Baxian Mountain, Tianjin 40°10′55.48″N, 117°32′19.97″E 1♀ Xianren Island, Shandong 40°11′3.46″N, 121°59′31.62″E 4♀13♂ E. guttiger Dali, Yunnan 25°35′40.65″N, 100°13′43.15″E 1♂ Wuyunjie Nature Reserve, Hunan 27°42′15.89″N, 119°39′51.25″E 2♂ Jinhua, Zhejiang 29°16′11.67″ N 120°19′52.98″ E 1♂ Weishui Nature Reserve, Hubei 29°58′27.49″N, 111°33′13.90″E 1♀ Tianmu Mountain, Zhejiang 30°21′0.63″N,119°25′28.82″E 2♀1♂ Dujiangyan, Sichuan 31°0′13.37″N, 103°36′41.41″E 2♀2♂ Mianyang, Sichuan 31°28′11.35″N, 104°40′37.06″E 1♀ Wen County, Gansu 32°56′45.96″N, 104°40′50.66″E 3♀1♂ Baoji, Shannxi 34°21′48.17″N, 107°13′57.41″E 1♂ E. aeneus Jinhua, Zhejiang 29°16′11.67″ N 120°19′52.98″ E 1♀1♂ Qingfeng Mountain, Shannxi 34°03′22.65″ N 107°32′51.77″ E 1♀ Linyi, Shandong 35°06′18.14″ N 118°21′2.56″ E 16♀15♂ Pangquangou, Shanxi 37°49′21.78″ N 111°29′27.59″ E 19♀10♂ Luya Mountain, Shanxi 38°48′17.99″ N 112°05′54.97″ E 6♀6♂

2.4 Shape variation

The principal component analysis (PCA) was conducted to analyze shape variations of the three characters within and among groups using MorphoJ 1.06d (Klingenberge, 2011). A scatter plot of the first two principal components was generated 94 Li et al.

to summarize the distribution of individuals and the shape variations. Cluster analysis was performed in software PAST 3.06 (Hammer et al., 2001). Canonical variate analysis (CVA) was also conducted using MorphoJ 1.06d (Klingenberge, 2011) for analyzing and testing differences between groups. Procrustes distance and mahalanobis distance were calculated during the CVA analysis.

3 Results

3.1 Size variation and allometry

Multivariate regression of shape variables on centroid size was calculated. No allometry was found in the fore wing and pygophore (p= 0.1203 and p= 0.5761), and the null hypothesis of isometry was rejected (allometry was present) in hind wing (p= 0.0408). To remove allometry effects in hind wing, size-corrected shape variables (the residuals from the regression) was used in subsequent shape analysis. Wing size was largest in E. aeneus and smallest in E. guttiger. Pygophore size was largest in E. annamita (Fig. 2). For the fore wing, E. guttiger had significantly smaller centroid size (Tukey test, p<0.01) than the other species. For the pygophore, significant differences were observed between E. annamita and E.ventralis (Tukey test, p<0.05), E. guttiger and E. aeneus (Tukey test, p<0.05). For the hind wing, significant differences were observed between E. aeneus and E. annamita, E. annamita and E.ventralis, E. aeneus and E. guttiger, E. ventralis and E. guttiger (Tukey test, p<0.01) (Table 2).

Figure 2. Boxplot of the centroid sizes of the four Eysarcoris species. A. Fore wing. B. Hind wing. C. Pygophore.

Table 2. Tukey HSD for the CS among different species (p-values are above the diagonal; studentized range statistic q are below the diagonal). Forewing CS among different species E.aeneus E. annamita E. ventralis E.guttiger E. aeneus 0.9726 0.9474 0.0001381 E. annamita 0.6134 0.9996 0.0001416 E. ventralis 0.773 0.1596 0.0001443 E. guttiger 7.971 7.358 7.198 Hindwing CS among different species E. aeneus 0.0001757 0.9536 0.0001378 E. annamita 6.624 0.000468 0.866 E. ventralis 0.7387 5.885 0.00015 E. guttiger 7.719 1.095 6.98 Pygophore CS among different species E. aeneus 0.7686 0.1669 0.4092 E. annamita 1.369 0.02139 0.07501 E. ventralis 3.02 4.389 0.9428 E. guttiger 2.226 3.595 0.7936 © Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 95

3.2 Shape variation

The shape variations of all of the three characters (fore wing, hind wing and pygophore) were shown by the first two principal components of PCA (70.154%, 57.883% and 72.572%, respectively) (Fig. 3). For the fore wing, shape changes were observed in the displacement of the landmarks and the length/width ratio of the membrane. For the hind wing, shape changes were observed in the relative position of the eight landmarks and the curvature of the remigium and vannus of I and II. The main shape change of the pygophore was in the length/width ratio of the dorsoposterior and ventroposterior rims. Based on dendrograms for the three characters, all samples of E. annamita, E. ventralis, E. guttiger and E. aeneus were clustered into four distinctive groups (Fig. 4). Canonical variate analysis revealed differences in all of the three characters among the four species. The first two discriminant factors (CV1 and CV2, see Fig. 5) explained 95.860%, 82.197% and 94.249% of the fore wing, hind wing and pygophore shape variation, respectively. The studied species were well separated from each other along the first and second discriminant factors. Mahalanobis distance and procrustes distance showed significant differences among all of the species analyzed (Table 3).

Table 3. Shapes difference in the three characters among the four species, mahalanobis distance (left) and procrustes distance (right) (p-values are above the diagonal; distances between species are below the diagonal). Forewing shape divergence among different species E. aeneus E. annamita E. ventralis E. guttiger E. aeneus E. annamita E. ventralis E. guttiger E. aeneus <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 E. annamita 27.9793 <0.0001 <0.0001 0.0357 <0.0001 <0.0001 E. ventralis 99.4832 106.9988 <0.0001 0.0533 0.0579 <0.0001 E. guttiger 46.6554 44.4746 117.8375 0.0521 0.0621 0.0698 Hindwing shape divergence among different species E. aeneus <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 E. annamita 37.8745 <0.0001 <0.0001 0.0412 <0.0001 <0.0001 E. ventralis 81.8433 108.3623 <0.0001 0.0375 0.0615 <0.0001 E. guttiger 89.8751 73.0292 159.7600 0.0456 0.0360 0.0565 Pygophore shape divergence among difference species E. aeneus <0.0001 0.0001 0.0001 <0.0001 0.0001 0.0002 E. annamita 12.1708 0.0014 0.0042 1.1254 0.0019 0.0041 E. ventralis 21.7788 23.4708 0.0038 0.0959 0.1587 0.0032 E. guttiger 15.1996 17.7918 21.2184 0.0703 0.1363 0.1117

4 Disscussion

Geometric morphometrics, which has greater statistical power with the use of Cartesian coordinates of the landmarks (Rohlf & Marcus, 1993), is aimed at comparing shapes (Pavlinov, 2001) and has been demonstrated to be useful for identification at individual level (Baylac et al., 2003, Dujardin et al., 2003, Sadeghi et al., 2009). CVA analysis reveals that the distribution of the studied species corresponds with their species status. Moreover, significant shape divergences of the three characters were observed among the studied taxa. This finding indicated that the morphological boundaries of the species based on the sample specimens were distinctly separated from each other. The analyses of all the three characters gave similar results. In principle, difference in wing venation may be attributable to environmental factors (Villemant et al., 2007). In feature, the relationship between wing shape variance and environmental parameters should be studied. Phenetic similarities displayed by our results were consistent with traditional classification of the species based on all the three characters. Compared to traditional classification methods which are based on qualitative analysis, we provided quantitative method for classifying them. We hereby conclude that geometric morphometrics can be used as a reliable method for species delimitation of Eysarcoris. The taxonomical value of the three characters (fore wing, hind wing and pygophore) we studied here was not equivalent. According to our results, the pygophore provides the best characteristics for identification of the studied species. The geometric morphometrics of fore wing was very efficient in detecting variations among species and was used 96 Li et al.

Figure 3. PCA analysis. A. Fore wing. B. Hind wing. C. Pygophore.

© Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 97

Figure 4. Cluster analysis. A. Fore wing. B. Hind wing. C. Pygophore.

98 Li et al.

Figure 5. CVA analysis. A. Fore wing. B. Hind wing. C. Pygophore.

© Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 99

widely in the taxonomy of Triatoma (Gumiel et al., 2003; Campos et al., 2011). Landmarks for these studies were mostly chosen from the membranous part of the hemelytra. For some species, however, venation patterns of the membranous part were not always fixed (Torres et al., 2013). Our results suggest that the use of landmarks from clavus and semi-landmarks from membrane are diagnostic among the species of Eysarcoris. The shape of hind wing was widely used in studying Coleoptera (Bai et al., 2011, 2012), Hymenoptera (Aytekin et al., 2007), Lepidoptera (Barão et al., 2014) and Zygoptera (Sadeghi et al., 2009), but rarely used in studying Hemiptera. Compared to the fore wing, the venations of the hind wing appeared relatively stable. It can be used as a valid characteristic for species delimitation based on this study. Geometric morphometric analysis is a useful technique that enabled us to solve complex taxonomy questions by using discrete characteristics (Bai et al., 2014). In the present study, we, for the first time, applied this approach to analyze shape variation among the four species of Eysarcoris, and demonstrate that it is a reliable method for the taxonomy of this genus. The current classification of Eysarcoris is mainly based on phenotypical similarities, even though the taxonomic characteristics usually display significant overlap among sibling species. In future studies we hope to apply this approach with more characteristics to analyze shape variation among other species of Eysarcoris and to explore the relationship among them.

Funding The project was supported by the National Natural Sciences Foundation of China (31440078, 31501876, 31501840), Shanxi Province Programs for Science and Technology Development (20150311010-7), the Research Equipment Development Project of Chinese Academy of Sciences (YZ201509), and the Graduate Innovation Project in Shanxi Province, China (2016BY067).

Acknowledgements This research was made possible through the assistance of many collaborators. We thank Prof. Wenjun Bu (Institute of Entomology, Nankai University) for the samples and identification of specimens. Thanks are given to Prof. Gongqin Sun (Department of Cell and Molecular Biology, University of Rhode Island) for English language revision. Thanks are given to Dr. Min Li (Department of Biology, Taiyuan Normal University) and Dr. Jiufeng Wei (College of Agronomy, Shanxi Agricultural University) for providing suggestions in modifying the manuscript.

References

Adam, D.C., Rohlf, F.J., Slice, D.E. 2004. Geometric morphometrics: ten years of progress following the “Revolution”. Italian Journal of Zoology, 71: 5–10. Agarwal, S.B., Baijal, H.N. 1984. Morphology of male genitalia of Scutellerinae and () with remarks on interrelationship within the family Pentatomidae. Journal of Entomological Research, 8: 53–60. Aytekin, M.A., Terzo, M., Rasmont, P., Çağatay, N. 2007. Landmark based geometric morphometric analysis of wing shape in sibiricobomubus vogt (Hymenoptera: Apidae: Bombus Latreille). Annales de la Société entomologique de France, 43(1): 95–102. Baker, A.D. 1931. A Study of the male genitalia of Canadian species of Pentatomidae. Canadian Journal of Research, 4: 148–220. Barão, K.R., Gonçalves, G.L., Mieleke, O.H.H., Kronforst, M.R., Moreira, G.R.P. 2014. Species boundaries in Philaethria butterflies: an integrative taxonomic analysis based on genitalia ultrastructure, wing geometric morphometrics, DNA sequences, and amplified fragment length polymorphisms. Zoological Journal of the Linnean Society, 170: 690–709. Bai, M., Beutel, R.G., Liu, W.G., Li, S., Zhang, M.N., Lu, Y.Y., Song, K.Q., Ren, D., Yang, X.K. 2014. Description of a new species of Glaresidae (Coleoptera: Scarabaeoidea) from the Jehol Biota of China with a geometric morphometric evaluation. Structure & Development, 72(3): 223–236. Bai, M., Beutel, R.G., Song, K.Q., Liu, W.G., Malqin, H., Li, S., Hu, X.Y., Yang, X.K. 2012. Evolutionary patterns of hind wing morphology in dung beetles (Coleoptera: Scarabaeinae). Arthropod Structure & Development, 41: 505–513. Bai, M., McCullough, E., Song, K.Q., Liu, W.G., Yang, X.K. 2011. Evolutionary constraints in hind wing shape in Chinese dung beetles (Coleoptera: Scarabaeinae). Plos one, 6(6): e21600. Baylac, M., Villemant, C., Simbolotti, G. 2003. Combining geometric morphometrics with pattern recognition for the investigation of species complex. Biological Journal of Linnean Society, 80(1): 89–98. Bookstein, F.L. 1991. Morphometric Tools for Landmark Data: Geometry and Biology. Cambridge University Press, Cambridge, 456 pp. Bu, W.J., Zheng, L.Y. 1997. Hemiptera: Tessaratomidae, Dinidoridae, Scutelleridae, Pentatomidae and Acanthosomatidae. In: Yang, X.K. (ed.). Insects of the Three Gorge Reservoir Area of Yangtze River. Chongqing Press, Chongqing. pp. 206–207. 100 Li et al.

Campos, R., Botto-Mahan, C., Coronado, X., Jaramillo, N., Panzera, F., Solari, A. 2011. Wing shape differentiation of Mepraia species (Hemiptera: Reduviidae). Infection, Genetics and Evolution, 11: 329–333. Comstock, J.H. 1893. Evolution and Taxonomy. An essay on the application of the theory of natural selection in the classifcation of and plants, illustrated by a study of the evolution of the wings of insects. Te Wilder Quarter-Century Book, Ithaca, New York. Available from http://snapper.bio.umass. edu/kunkel/comstock/eassy/ (accessed 12 October 2014). De la Riva, J., Le Pont, F., Ali, V., Matias, A., Mollinedo, S., Dujardin, J.P. 2001. Wing geometry as a tool for studying the Lutzomyia longipalpis (Diptera: Psychodidae) complex. Memórias do Instituto Oswaldo Cruz, 96(8): 1089–1094. Díaz, S., Panzera, F., Jaramillo-O, N., Pérez, R., Fernendez, R., Vallejo, G., Saldańa, A., Calzada, J.E., Triana, O., Gamez-Palacio, A. 2014. Genetic, Cytogenetic and Morphological Trends in the Evolution of the Rhodnius (Triatominae: Rhodniini) Trans-Andean Group. Plos one, 9(2): e87493. doi: 10.1371/journal.pone.0087493. Distant, W.L. 1902. The Fauna of British India, including Ceylon and Burma. Rhynchota. Vol. I. Heteroptera. Taylor and Francis, London. pp. 165–169. Dujardin, J.P., Le Pont, F., Baylac, M. 2003. Geographical versus interspecific differentiation of sand flies (Diptera: Psychodidae): a landmark data analysis. Bulletin of Entomological Research, 93: 87–90. doi: 10.1079/BER2002206. Gumiel, M., Catalá, S., Noireau, F., Rojas de Arias, A., García, A., Dujardin, J.P. 2003. Wing geometry in Triatoma infestans (Klug) and T. melanosoma Martinez, Olmedo & Carcavallo (Hemiptera: Reduviidae). Systematic Entomology, 28: 173–179. Hammer, Ø., Harper, D.A.T., Ryan, P.D. 2001. PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica, 4(1): 9. Hasan, S.A. 1991. A new species of the genus Eysarcoris Hahn (Heteroptera: Pentatomidae) from the Malayan subregion. Pakistan Journal of Scientific & Industrial Research, 34(9): 342–345. Hahn C.W. 1834. Die Wanzenartigen Insecten. Bd. 2. Nürnberg. 142 pp. Klingenberg, C.P. 2011. MorphoJ: an integrated software package for geometric morphometrics. Molecular Ecology Resources, 11(2): 353–357. Klingenberg, C., Mclntyre, G. 1998. Geometric morphometrics of developmental instability: analyzing patterns of fluctuating asymmetry with Procrustes methods. Evolution, 52: 1363–1375. doi: 10.2307/2411306. Kunkel, J.G. 2004. Wing discrimination projects. Available from http://marlin.bio.umass.edu/ biology/kenkel/ wing_discrim.html (accessed 12 October 2014). Lee, J.G., Hong, S.S., Kim, J.Y., Park, K.Y., Lim, J.W., Lee, J.H. 2009. Occurrence of stink bugs and pecky rice damage by stink bugs in paddy fields in Gyeonggi-do, Korea. Korean Journal of Applied Entomology, 48(1): 37–44. Matias, A., De la Riva, J.X., Torrez, M., Dujardin, J.P. 2001. Rhodnius robustus in Bolivia Identified by its Wings. Memóriasdo Instituto Oswaldo Cruz, 96(7): 947–950. Nasiruddin, M., Roy, R.C. 2012. Rice field insect pests during the rice growing seasons in two areas of Hathazari, Chittagong. Bangladesh Journal of Zoology, 40(1): 89–100. Pavlinov, I.Y. 2001. Geometric morphometrics, a new analytical approach to comparision of digitized images, In: Ryss, A.Y., Minter, D. (eds.). Information Technology in Biodiversity Research. Abstracts of the 2nd. International Symposium, St. Petersburg. pp. 41–90. Plowright, R.C., Stephen, W.P. 1973. Evolutionary relationships in northern European Bombus and Psithyrus species (Apidae: Hymenoptera). Canadian Entomologist, 105: 733–743. doi:10.4039/Ent105733-5. Rider, D.A. 2006. Family Pentatomidae. In: Aukema, B., Rieger, C. (eds.). Catalogue of the Heteroptera of the Palearctic Region. Vol. 5. The Netherlands Entomological Society, Amsterdam. pp. 298–302. Rider, D.A., Zheng, L.Y., Kerzhner, I.M. 2002. Checklist and nomenclatural notes on the Chinese Pentatomidae (Heteroptera). II. Pentatominae. Zoosystematica Rossica, 11(1): 135–153. Rohlf, F.J. 2006. Tps-DIG, Digitize Landmarks and Outlines, Version 2.05. [Software and Manual]. Department of Ecology and Evolution. State University of New York at Stony Brook, New York. Rohlf, F.J. 2010. Tps-UTIL, File Utility Program, Version 1.46. Department of Ecology and Evolution, State University of New Yorkat Stony Brook, New York. Rohlf, F.J., Marcus, L.F. 1993. A revolution in morphometrics. Trends in Ecology & Evolution, 8(4): 129–132. Sadeghi, S., Adriaens, D., Dumont, H.J. 2009. Geometric morphometric analysis of wing shape variation in ten European populations of Calopteryx splendens (Harris, 1782) (Zygoptera: Odonata). Odonatologia, 38(4): 343–360. Slice, D.E. 2007. Geometric morphometrics. Annual Review of Anthropology, 36: 261–281. doi:10.1146/annurev.anthro.34.081804. 120613. Sol Gaspe, M., Gurevitz, J.M., Gürtler, R.E., Dujardin, J.P. 2013. Origins of house reinfestation with Triatoma infestans after insecticide spraying in the Argentine Chaco using wing geometric morphometry. Infection, Genetics and Evolution, 17: 93–100. Tofilski, A. 2008. Using geometric morphometrics and standard morphometry to discriminate three honeybee subspecies. Apidologie, 39: 558–563. doi: 10.1051/apido:2008037. Torres, M.A.J., Ong, G.M.P., Joshi, R.C., Barrion, A.T., Sebastian, L.S., Demayo, C.G. 2013. Fore wing venation pattern and genital plate structure in a non-outbreak population of the Rice Black Bug (Scotinophara coarctataStål) from Lala, Lanao del Norte, Philippines. ABAH Bioflux, 5: 6–14. © Zoological Systematics, 42(1): 90–101 GM analysis of Eysarcoris 101

Tsai, J.F., Rédei, D. 2009. The identity of shield bugs described by Francis Walker from Taiwan (Hemiptera: Heteroptera: Pentatomidae). Zootaxa, 2152: 43–54. Tüzün, A. 2009. Significance of wing morphometry in distinguishing some of the Hymenoptera species. African Journal of Biotechnology, 8(14): 3353–3363. Villemant, C., Simbolotti, G., Kenis, M. 2007. Discrimination of Eubazus (Hymenoptera, Braconidae) sibling species using geometric morphometrics analysis of wing venation. Systematic Entomology, 32(4): 625–634. doi:10.1111/j.1365-3113.200 7.00389.x. Wood, I., McDonald, F.J.D. 1984. Revision of the Australian Eysarcoris group (Hemiptera: Pentatomidae). Journal of the Australian Entomological Society, 23: 253–264. Yang, W.Y. 1962. Economic insect fauna of China. Fasc. 2, Hemiptera: Pentatomidae. Science Press, Beijing, 91 pp. Zelditch, M.L., Swiderski, D.L., Sheets, H.D., Fink, W.L. 2004. Geometric Morphometrics for Biologists: A Primer. Elsevier Academic Press, New York and London.7 pp.