Research Article |
Corresponding author: Yani Duan ( duanyani@hotmail.com ) Academic editor: Mick Webb
© 2019 Hong Zhang, Yalin Zhang, Yani Duan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhang H, Zhang Y, Duan Y (2019) DNA barcoding of Deltocephalus Burmeister leafhoppers (Cicadellidae, Deltocephalinae, Deltocephalini) in China. ZooKeys 867: 55-71. https://doi.org/10.3897/zookeys.867.35058
|
We investigated the feasibility of using the DNA barcode region in identifying Deltocephalus from China. Sequences of the barcode region of the mitochondrial COI gene were obtained for 98 specimens (Deltocephalus vulgaris – 88, Deltocephalus pulicaris – 5, Deltocephalus uncinatus – 5). The average genetic distances among morphological and geographical groups of D. vulgaris ranged from 0.9% to 6.3% and among the three species of Deltocephalus ranged from 16.4% to 21.9% without overlap, which effectively reveals the existence of a “DNA barcoding gap”. It is important to assess the status of these morphological variants and explore the genetic variation among Chinese populations of D. vulgaris because the status of this species has led to taxonomic confusion because specimens representing two distinct morphological variants based on the form of the aedeagus are often encountered at a single locality. Forty-five haplotypes (D. vulgaris – 36, D. pulicaris – 5, D. uncinatus – 4) were defined to perform the phylogenetic analyses; they revealed no distinct lineages corresponding either to the two morphotypes of D. vulgaris or to geographical populations. Thus, there is no evidence that these variants represent genetically distinct species.
COI, genetic distance, morphological variant
China contains threatened biodiversity hotspots, including one spanning the Palearctic and Oriental regions and containing a high level of species diversity (
Deltocephalini feed on grasses and sedges and are diverse and abundant in grassland ecosystems. This group contains 73 genera and 613 species around the world. Deltocephalus, type genus of this tribe contains 62 species distributed in the Old World and New World. Some species of this genus can transmit pathogenic diseases to economically important plants and are important economic pests; therefore, tools are needed for their rapid and accurate identification. Four species are described from China, two of them transmit pathogenic diseases. Identification of leafhopper species in most genera now requires dissection and examination of the male genitalia. However, some taxonomically problematic species apparently exhibit substantial intraspecific variation in male genital structures, and this causes confusion among taxonomists. One such practical example is D. vulgaris, which has well-documented morphological differences in the shape of the aedeagus (Figs
Based on DNA barcoding of leafhoppers, 63 barcodes from 45 species in Japan (15 subfamilies and 37 genera without Deltocephalini) were analysed (
In this study, we studied 98 COI sequences from three species of Deltocephalus. DNA barcoding data were used to investigate genetic variation of Chinese populations of D. vulgaris and to determine whether the morphological variants previously identified in this species represent distinct lineages. Our specific aims were to test the feasibility of using DNA barcoding data for identification of species of Deltocephalus, to determine the levels of the genetic variation within D. vulgaris, and to preliminarily discuss its possible correlation with morphological variation and biogeographic patterns.
A total of 98 specimens of Deltocephalus (D. vulgaris – 88, D. pulicaris – 5, D. uncinatus – 5) were collected with an insect sweep net in the daytime and by a light trap at night. Specimens were all collected directly into 95% or 100% ethanol and stored in -80 °C prior to study. The sample included D. vulgaris, D. uncinatus and D. pulicaris to facilitate comparison of inter- to intraspecific genetic variation in this group. Deltocephalus vulgaris specimens were divided into 11 groups based on their morphological differences and different geographical distributions in China (Table
Species | Group code | Sample size | Individual code | Haplotype | Locality | GenBank accession |
---|---|---|---|---|---|---|
D. vulgaris | YNA | 8 | YNA1 | Hap1 | Banhong Town, Yunnan Province | MK764780 |
YNA2 | Hap2 | Banhong Town, Yunnan Province | MK764781 | |||
YNA3 | Hap3 | Banhong Town, Yunnan Province | MK764782 | |||
YNA4 | Hap2 | Banhong Town, Yunnan Province | MK764783 | |||
YNA5 | Hap4 | Banhong Town, Yunnan Province | MK764784 | |||
YNA6 | Hap2 | Banhong Town, Yunnan Province | MK767485 | |||
YNA7 | Hap1 | Banhong Town, Yunnan Province | MK764786 | |||
YNA8 | Hap2 | Banhong Town, Yunnan Province | MK764787 | |||
YNB | 13 | YNB1 | Hap5 | Banhong Town, Yunnan Province | MK764788 | |
YNB2 | Hap1 | Banhong Town, Yunnan Province | MK764789 | |||
YNB3 | Hap5 | Banhong Town, Yunnan Province | MK764790 | |||
YNB4 | Hap5 | Banhong Town, Yunnan Province | MK764791 | |||
YNB5 | Hap5 | Banhong Town, Yunnan Province | MK764792 | |||
YNB6 | Hap5 | Banhong Town, Yunnan Province | MK764793 | |||
YNB7 | Hap5 | Banhong Town, Yunnan Province | MK764794 | |||
YNB8 | Hap6 | Banhong Town, Yunnan Province | MK764795 | |||
YNB9 | Hap7 | Banhong Town, Yunnan Province | MK764796 | |||
YNB10 | Hap8 | Banhong Town, Yunnan Province | MK764797 | |||
YNB11 | Hap5 | Banhong Town, Yunnan Province | MK764798 | |||
YNB12 | Hap5 | Banhong Town, Yunnan Province | MK764799 | |||
YNB13 | Hap5 | Banhong Town, Yunnan Province | MK764800 | |||
ZJA | 7 | ZJA1 | Hap9 | Lin’an County, Zhejiang Province | MK764801 | |
ZJA2 | Hap10 | Lin’an County, Zhejiang Province | MK764802 | |||
ZJA3 | Hap11 | Lin’an County, Zhejiang Province | MK764803 | |||
ZJA4 | Hap12 | Lin’an County, Zhejiang Province | MK764804 | |||
ZJA5 | Hap13 | Lin’an County, Zhejiang Province | MK764805 | |||
ZJA6 | Hap12 | Lin’an County, Zhejiang Province | MK764806 | |||
ZJA7 | Hap12 | Lin’an County, Zhejiang Province | MK764807 | |||
ZJB | 8 | ZJB1 | Hap14 | Kowloon Mountain, Zhejiang Province | MK764808 | |
ZJB2 | Hap10 | Kowloon Mountain, Zhejiang Province | MK764809 | |||
ZJB3 | Hap15 | Kowloon Mountain, Zhejiang Province | MK764810 | |||
ZJB4 | Hap12 | Kowloon Mountain, Zhejiang Province | MK764811 | |||
ZJB5 | Hap16 | Kowloon Mountain, Zhejiang Province | MK764812 | |||
ZJB6 | Hap17 | Kowloon Mountain, Zhejiang Province | MK764813 | |||
ZJB7 | Hap18 | Kowloon Mountain, Zhejiang Province | MK764814 | |||
ZJB8 | Hap19 | Kowloon Mountain, Zhejiang Province | MK764815 | |||
FJA | 7 | FJA1 | Hap20 | Shajian Town, Fujian Province | MK764816 | |
FJA2 | Hap20 | Shajian Town, Fujian Province | MK764817 | |||
FJA3 | Hap5 | Shajian Town, Fujian Province | MK764818 | |||
FJA4 | Hap21 | Shajian Town, Fujian Province | MK764819 | |||
FJA5 | Hap5 | Shajian Town, Fujian Province | MK764820 | |||
FJA6 | Hap20 | Shajian Town, Fujian Province | MK764821 | |||
FJA7 | Hap5 | Shajian Town, Fujian Province | MK764822 | |||
FJB | 7 | FJB1 | Hap22 | Shajian Town, Fujian Province | MK764823 | |
FJB2 | Hap20 | Shajian Town, Fujian Province | MK764824 | |||
FJB3 | Hap20 | Shajian Town, Fujian Province | MK764825 | |||
FJB4 | Hap20 | Shajian Town, Fujian Province | MK764826 | |||
FJB5 | Hap20 | Shajian Town, Fujian Province | MK764827 | |||
FJB6 | Hap8 | Shajian Town, Fujian Province | MK764828 | |||
FJB7 | Hap23 | Shajian Town, Fujian Province | MK764829 | |||
D. vulgaris | HNA | 9 | HNA1 | Hap24 | Jianfeng Mountain, Hainan Province | MK764830 |
HNA2 | Hap8 | Jianfeng Mountain, Hainan Province | MK764831 | |||
HNA3 | Hap8 | Jianfeng Mountain, Hainan Province | MK764832 | |||
HNA4 | Hap8 | Jianfeng Mountain, Hainan Province | MK764833 | |||
HNA5 | Hap8 | Jianfeng Mountain, Hainan Province | MK764834 | |||
HNA6 | Hap25 | Jianfeng Mountain, Hainan Province | MK764835 | |||
HNA7 | Hap8 | Jianfeng Mountain, Hainan Province | MK764836 | |||
HNA8 | Hap26 | Jianfeng Mountain, Hainan Province | MK764837 | |||
HNA9 | Hap27 | Jianfeng Mountain, Hainan Province | MK764838 | |||
HNB | 8 | HNB1 | Hap20 | Jianfeng Mountain, Hainan Province | MK764839 | |
HNB2 | Hap28 | Jianfeng Mountain, Hainan Province | MK764840 | |||
HNB3 | Hap29 | Jianfeng Mountain, Hainan Province | MK764841 | |||
HNB4 | Hap30 | Jianfeng Mountain, Hainan Province | MK764842 | |||
HNB5 | Hap8 | Jianfeng Mountain, Hainan Province | MK764843 | |||
HNB6 | Hap31 | Jianfeng Mountain, Hainan Province | MK764844 | |||
HNB7 | Hap8 | Jianfeng Mountain, Hainan Province | MK764845 | |||
HNB8 | Hap8 | Jianfeng Mountain, Hainan Province | MK764846 | |||
GDB | 9 | GDB1 | Hap32 | Patio Hill, Guangdong Province | MK764847 | |
GDB2 | Hap8 | Patio Hill, Guangdong Province | MK764848 | |||
GDB3 | Hap8 | Patio Hill, Guangdong Province | MK764849 | |||
GDB4 | Hap8 | Patio Hill, Guangdong Province | MK764850 | |||
GDB5 | Hap8 | Patio Hill, Guangdong Province | MK764851 | |||
GDB6 | Hap20 | Patio Hill, Guangdong Province | MK764852 | |||
GDB7 | Hap8 | Patio Hill, Guangdong Province | MK764853 | |||
GDB8 | Hap8 | Patio Hill, Guangdong Province | MK764854 | |||
GDB9 | Hap8 | Patio Hill, Guangdong Province | MK764855 | |||
GXA | 4 | GXA1 | Hap33 | Lingyun County, Guangxi Province | MK764856 | |
GXA2 | Hap1 | Lingyun County, Guangxi Province | MK764857 | |||
GXA3 | Hap34 | Lingyun County, Guangxi Province | MK764858 | |||
GXA4 | Hap20 | Lingyun County, Guangxi Province | MK764859 | |||
GXB | 8 | GXB1 | Hap35 | Shangsi County, Guangxi Province | MK764860 | |
GXB2 | Hap20 | Shangsi County, Guangxi Province | MK764861 | |||
GXB3 | Hap32 | Shangsi County, Guangxi Province | MK764862 | |||
GXB4 | Hap1 | Shangsi County, Guangxi Province | MK764863 | |||
GXB5 | Hap5 | Shangsi County, Guangxi Province | MK764864 | |||
GXB6 | Hap5 | Shangsi County, Guangxi Province | MK648065 | |||
GXB7 | Hap20 | Shangsi County, Guangxi Province | MK764866 | |||
GXB8 | Hap36 | Shangsi County, Guangxi Province | MK764867 | |||
D. pulicaris | XJ | 5 | XJ1 | Hap37 | Altay City, Xinjiang Province | MK764868 |
XJ2 | Hap38 | Altay City, Xinjiang Province | MK764869 | |||
XJ3 | Hap39 | Altay City, Xinjiang Province | MK764870 | |||
XJ4 | Hap40 | Altay City, Xinjiang Province | MK764871 | |||
XJ5 | Hap41 | Altay City, Xinjiang Province | MK764872 | |||
D. uncinatus | YN | 5 | YN1 | Hap42 | Menglong Town, Yunnan Province | MK764873 |
YN2 | Hap43 | Menglong Town, Yunnan Province | MK764874 | |||
YN3 | Hap43 | Menglong Town, Yunnan Province | MK764875 | |||
YN4 | Hap44 | Menglong Town, Yunnan Province | MK764876 | |||
YN5 | Hap45 | Menglong Town, Yunnan Province | MK764877 |
Morphological observations were made using an Olympus SZX10 stereoscopic microscope (Olympus Corporation, Tokyo, Japan). All photographs and drawings were modified with Adobe Photoshop CS.
Total genomic DNA was extracted from the whole abdomen of each leafhopper using the EasyPure Genomic DNA Kit (EE101; Transgen, Beijing, China) following the manufacturer’s instructions with the following modifications: abdomen incubated at 55 °C for about 20 hours, and with a nondestructive DNA extraction procedure to allow subsequent morphological observation. Genomic DNA extracts were stored in a freezer at -20 °C.
The barcode region (630bp) of the COI gene was amplified using primer combination (
The PCR products were examined using 1% agarose gel electrophoresis with ethidium bromide stain to check for successful amplification. The successful PCR products were sent to Beijing Tsingke Biotechnology Co., Ltd (China) for sequencing of both strands using the original PCR primers. All sequences collected in this study have been submitted to GenBank and accession numbers are shown in Table
The forward and reverse chromatograms were proofread and then assembled and edited using DNASTAR software (DNASTAR, Madison, Wisconsin, USA). Multiple sequence alignments were performed by CLUSTAL X 2.0.21 (
Our specimens from China included representatives of both previously reported morphotypes of the aedeagus of D. vulgaris. They also exhibited a range of more subtle variation in the curvature of the aedeagal shaft in lateral view. Under the current morphology-based concept, this species can nevertheless be identified by the colour pattern and the presence of a shallow apical notch on the aedeagus in posterior view.
The COI sequences are 630bp in length after alignment and trimming. Details of nucleotide composition are listed in Table
The average nucleotide composition of the COI sequences of Deltocephalus.
Group/Species | T (%) | C (%) | A (%) | G (%) | A+T (%) |
---|---|---|---|---|---|
YNA | 32.8 | 18.8 | 34.0 | 14.1 | 56.8 |
YNB | 33.1 | 18.3 | 33.5 | 15.1 | 66.6 |
ZJA | 32.8 | 19.0 | 34.2 | 14.4 | 67.0 |
ZJB | 32.9 | 18.9 | 34.1 | 14.1 | 67.0 |
FJA | 33.0 | 18.3 | 33.8 | 14.9 | 66.8 |
FJB | 33.0 | 18.4 | 34.1 | 14.5 | 67.1 |
HNA | 33.1 | 18.4 | 33.9 | 14.6 | 67.0 |
HNB | 33.0 | 18.3 | 34.0 | 14.7 | 67.0 |
GDB | 33.0 | 18.3 | 34.0 | 14.7 | 67.0 |
GXA | 33.0 | 18.4 | 33.9 | 14.7 | 66.9 |
GXB | 33.0 | 18.3 | 33.9 | 14.8 | 66.9 |
A total of A | 33.0 | 18.6 | 34.0 | 14.5 | 67.0 |
A total of B | 33.0 | 18.4 | 33.9 | 14.7 | 66.9 |
A total of A and B | 33.0 | 18.5 | 33.9 | 14.6 | 66.9 |
D. pulicaris | 33.7 | 20.9 | 30.6 | 14.8 | 64.3 |
D. uncinatus | 35.2 | 18.0 | 32.0 | 14.9 | 57.2 |
The results of haplotype sequences for the substitution saturation test indicate the value of Iss is smaller than Iss.c; namely, little substitutional saturation was detected, which is strongly informative for constructing phylogenetic trees.
The average genetic distances among morphological and geographical groups of D. vulgaris ranged from 0.9% to 6.3% and among species of Deltocephalus ranged from 16.4% to 21.9% without overlap (Table
Kimura 2-parameter genetic distances between groups/species of Deltocephalus.
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|
YNA | ||||||||||||
YNB | 0.047 | |||||||||||
ZJA | 0.041 | 0.063 | ||||||||||
ZJB | 0.041 | 0.057 | 0.017 | |||||||||
FJA | 0.045 | 0.011 | 0.063 | 0.056 | ||||||||
FJB | 0.043 | 0.029 | 0.047 | 0.043 | 0.023 | |||||||
HNA | 0.042 | 0.031 | 0.049 | 0.046 | 0.026 | 0.031 | ||||||
HNB | 0.044 | 0.014 | 0.062 | 0.056 | 0.007 | 0.022 | 0.023 | |||||
GDB | 0.043 | 0.019 | 0.057 | 0.052 | 0.012 | 0.023 | 0.024 | 0.009 | ||||
GXA | 0.045 | 0.023 | 0.050 | 0.046 | 0.021 | 0.030 | 0.032 | 0.021 | 0.024 | |||
GXB | 0.045 | 0.022 | 0.055 | 0.052 | 0.019 | 0.031 | 0.032 | 0.020 | 0.023 | 0.028 | ||
D. pulicaris | 0.207 | 0.219 | 0.206 | 0.204 | 0.212 | 0.206 | 0.210 | 0.210 | 0.209 | 0.212 | 0.213 | |
D. uncinatus | 0.171 | 0.171 | 0.169 | 0.168 | 0.166 | 0.164 | 0.165 | 0.165 | 0.164 | 0.168 | 0.168 | 0.219 |
BI/ML tree of 45 COI haplotypes. The node support: BI posterior probabilities/ML bootstrap values. Posterior probabilities and bootstrap values under 0.5 and 50 are shown “-”. “?” means the positions of the different individual of D. vulgaris in ML tree is slightly different from those in BI tree.
DNA barcoding as a standardised method to provide rapid and accurate species demarcation and has been widely applied in identifying and delimiting taxa since it was first reported by
Differences in morphological characteristics, especially in male genitalia, have been the most reliable standard for discriminating among complex groups for many years. However, some cases of intraspecific variation in genital structures have been reported and these have led to uncertainty in the status of species and morphotypes.
Deltocephalus vulgaris, including 88 individuals in this study, and mainly representing two different forms of the aedeagus, were confirmed to be a single species grouped into a single clade with strongly support value in its phylogenetic trees (Figs
Our study shows a low intraspecific genetic distance between Guangdong and Hainan populations of D. vulgaris in southern China, suggesting that the Qiongzhou Strait (Fig.
In the present study, lack of apparent correlation between morphology and COI haplotype is consistent with the hypothesis that the observed morphological variation is intraspecific. Nevertheless, we acknowledge the possibility that two different leafhopper species may share the same, or similar, COI haplotype. Thus, study of other genes may, in the future, reveal higher levels of divergence between the two forms and support recognition of some morphological variants as separate species.
We express our sincere thanks to M. D. Webb, the Natural History Museum, London, UK and Dr C.H. Dietrich, Department of Entomology, University of Illinois, USA for reading the manuscript and making some suggestions. We also thank Dr J. R. Schrock, Emporia State University, USA for revising the manuscript. This study is supported by the National Natural Science Foundation of China (31000968), Anhui Provincial Natural Science Foundation (1608085MC55) and Anhui Provincial Colleges and Universities Natural Science Foundation (KJ2015A006).