Research Article |
Corresponding author: Bing-zhong Ren ( bzren@163.com ) Academic editor: Fernando Montealegre-Z
© 2015 Xue Zhang, Ming Wen, Junjiain Li, Hui Zhu, Yinliang Wang, Bing-zhong Ren.
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 X, Wen M, Li J, Zhu H, Wang Y, Ren B (2015) Acoustic, genetic and morphological variations within the katydid Gampsocleis sedakovii (Orthoptera, Tettigonioidea). ZooKeys 529: 105-121. https://doi.org/10.3897/zookeys.529.6043
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In an attempt to explain the variation within this species and clarify the subspecies classification, an analysis of the genetic, calling songs, and morphological variations within the species Gampsocleis sedakovii is presented from Inner Mongolia, China. Recordings were compared of the male calling songs and analysis performed of selected acoustic variables. This analysis is combined with sequencing of mtDNA - COI and examination of morphological traits to perform cluster analyses. The trees constructed from different datasets were structurally similar, bisecting the six geographical populations studied. Based on two large branches in the analysis, the species Gampsocleis sedakovii was partitioned into two subspecies, Gampsocleis sedakovii sedakovii (Fischer von Waldheim, 1846) and Gampsocleis sedakovii obscura (Walker, 1869). Comparing all the traits, the individual of Elunchun (ELC) was the intermediate type in this species according to the acoustic, genetic, and morphological characteristics. This study provides evidence for insect acoustic signal divergence and the process of subspeciation.
Acoustics, gene, morphology, subspecies, interim morphs
Acoustic signals are important in several social behaviors of insects, such as sexual selection (
Gampsocleis is a genus within Tettigoniidae, which includes sixteen species, eleven of which are found in China. Gampsocleis sedakovii (Fischer von Waldheim, 1846), a medium to large-sized, xerophilic, and slightly thermophilic katydid, is the most common and ubiquitous species distributed in northeast China. Individuals of G. sedakovii are generally classified into two subspecies, Gampsocleis sedakovii sedakovii (Fischer von Waldheim, 1846) and Gampsocleis sedakovii obscura (Walker, 1869), differing morphologically in body size and the proportions of forewings and the pronotum (
The individuals of both subspecies (G. s. sedakovii and G. s. obscura) are excellent singers, and males sing at any time throughout the day. The calling song of G. s. sedakovii was already reported in a previous study (
The ratio between forewing and pronotum of G. s. sedakovii is much higher than that of G. s. obscura, while the G. s. obscura looks stronger than G. s. sedakovii. An “interim form” was found, consisting of individuals which had an intermediate ratio of forewing and pronotum between the averages for G. s. obscura and G. s. sedakovii, raising the possibility that the division of the subspecies within G. sedakovii should be reconsidered (see also
Different insect species have different acoustic signals and these signals have been used as an invariable trait for the recognition of conspecifics and the discrimination of heterospecifics (
Wing polymorphism is common in insects, such as katydids (
In this study the differentiation of the individuals collected from six locations of Inner Mongolia were analyzed and compared. Acoustic, morphological, and genetic differences were examined carefully. The analysis of the variation in the acoustic structure of G. sedakovii from different geographical localities provided the basis for further explorations on the divergence on acoustic communication of this species and support the view that acoustic variation can promote the formation of subspecies.
In 2013, within 7 days, 40 adults were collected of Gampsocleis sedakovii from six localities in Inner Monglia, northeast China; individuals from CES (Chaersen), BYCG (Bayancuogang), JDM (Jiaodaomu), WCG (Wuchagou), SMJ (Shamajie), and ELC (Elunchun) were also used (Fig.
The number, geographic coordinates and total number of individuals sampled in acoustic analysis.
No. | Location | N | Longitude (E) | Latitude (N) |
---|---|---|---|---|
1 | CES | 8 | 121.9013° | 46.4005° |
2 | BYCG | 7 | 120.3006° | 49.2014° |
3 | JDM | 6 | 121.0001° | 50.5005° |
4 | WCG | 7 | 120.3021° | 46.8003° |
5 | SMJ | 6 | 122.1001° | 47.6014° |
6 | ELC | 6 | 122.4021° | 48.2011° |
Morphological structures (e.g., tegmina, pronotum, and body) were measured using 0.01 mm digital vernier calipers. The width of the stridulatory file teeth (WTSF) was measured under the scanning electron microscope (SEM) (JSM-6510LV, Hitachi Ltd, Tokyo, Japan), and the number of teeth in a stridulatory file (NTSF) were also counted under SEM. Forty individuals, whose songs had been recorded, were preserved in 70–95% ethanol solution for genetic analyses. Latitude, longitude, and sample number for each locality were also recorded (Table
High quality sound samples were selected from all call sequences of each individual for acoustic parameters measurement using the software Cool Edit (Cool Edit pro V2.1, Adobe Systems). To remove the low frequency oscillations, high-pass filtering was performed before analysis. The cutoff frequency was 200 Hz. The song traits of these two subspecies were automatically analyzed using Matlab program (Matlab 7.0, Mathworks). The spectral analyses were also produced in Matlab using the toll Pwelch and the number of FFT points was 1024. The other parameters were set as default. The selected song traits were pulse duration (PD), pulse interval (PI), pulse repetition rate (PRR), dominant frequency (DF), highest frequency (HF), and lowest frequency (LF).
Cloning and sequencing of mitochondrial DNA control region within the genus Gampsocleis was previously conducted by Zhang, who found that G. sedakovii haplotypes clustered into two distinct clades. Total genomic DNA was extracted from the hind femur muscles of 18 insects (selected from the samples obtained the acoustic data). DNA was extracted by a standard phenol-chloroform-isoamyl alcohol (PCI) extraction with slight modification (
DNA sequences were aligned using the multiple-sequence program Clustal x 1.8 with parameters setting to default (
Acoustic and stridulatory files characteristics of G. sedakovii, obtained from specimens collected from different locations, were tested by cluster analysis using R Programming Language, respectively. Six traits were used in acoustic cluster analysis, including both aspects of time domain and frequency domain features: PD, PI, PRR, DF, HF, and LF. WL, NTSF, WTSF, LP, BL and WL/LP were contained in this analysis for morphological cluster.
Acoustic parameters measured are shown in Table
Time-domain and frequency-domain features of Gampsocleis sedakovii from six geographic populations.
Location | PD (ms) | PI (ms) | PRR | DF (kHz) | HF (kHz) | LF (kHz) | GAN |
---|---|---|---|---|---|---|---|
CES | 20.4 ± 0.00 | 13.1 ± 0.00 | 0.028 ± 0.00 | 8.1 ± 0.10 | 23.7 ± 0.28 | 5.9 ± 0.09 | KT283620 ~ KT283622 |
BYCG | 13.3 ± 0.00 | 14.1 ± 0.00 | 0.036 ± 0.00 | 12.0 ± 0.19 | 21.0 ± 0.10 | 7.1 ± 0.09 | KT283617 ~ KT283619 |
JDM | 11.8± 0.00 | 10.8 ± 0.00 | 0.044 ± 0.00 | 10.8 ± 0.04 | 22.1 ± 0.07 | 4.9 ± 0.04 | KT283614 ~ KT283616 |
WCG | 20.1 ± 0.00 | 12.1 ± 0.00 | 0.031 ± 0.00 | 10.6 ± 0.03 | 19.3 ± 0.15 | 5.1 ± 0.06 | KT283605 ~ KT283607 |
SMJ | 9.4 ± 0.00 | 9.2 ± 0.00 | 0.054 ± 0.00 | 11.1 ± 0.06 | 19.6 ± 0.07 | 4.8 ± 0.05 | KT283611 ~ KT283613 |
ELC | 10.4 ± 0.00 | 10.0 ± 0.00 | 0.049 ± 0.00 | 8.3 ± 0.26 | 19.8±0. 23 | 5.1±0.06 | KT283608 ~ KT283610 |
Analysis of variance tables for the analysis of calling song and morphological traits for male Gampsocleis sedakovii among six geographic populations.
Mean Square | d.f. | F | Sig. | |
---|---|---|---|---|
PD | 0.001 | 5 | 188.344 | <0.001 |
PI | 0.000 | 5 | 61.899 | <0.001 |
DF | 50.170 | 5 | 88.193 | <0.001 |
HF | 113.971 | 5 | 1123.716 | <0.001 |
LF | 22.599 | 5 | 127.105 | <0.001 |
WL | 351.056 | 5 | 1129.041 | <0.001 |
WTSF | 1041.250 | 5 | 6.818 | <0.001 |
NTSF | 9.289 | 5 | 1.268 | 0.381 |
LP | 98.797 | 5 | 2964.154 | <0.001 |
BL | 1532.304 | 5 | 12162.586 | <0.001 |
The proximity matrix of analysis of distance of these geographical populations.
Euclidean Distance | ||||||
---|---|---|---|---|---|---|
CES | BYCG | JDM | WCG | SMJ | ELC | |
CES | .000 | 5.925 | 3.520 | 14.312 | 14.889 | 13.455 |
BYCG | 5.925 | .000 | 3.772 | 11.254 | 11.715 | 11.261 |
JDM | 3.520 | 3.772 | .000 | 13.255 | 13.751 | 12.876 |
WCG | 14.312 | 11.254 | 13.255 | .000 | .950 | 2.436 |
SMJ | 14.889 | 11.715 | 13.751 | .950 | .000 | 3.088 |
ELC | 13.455 | 11.261 | 12.876 | 2.436 | 3.088 | .000 |
SEMs as used to determine if the stridulatory files of G. sedakovii from specimens of different localities were similar to each other. They were claviform and the teeth in the middle section were wider than those located at both ends of the file (Fig.
In this part of observation, all six morphological traits, except for the number of teeth of a stridulatory file, had significant differences among the other five morphological parameters across the individuals captured from six locations (Table
Morphological characteristics of specimens from the different sampling sites.
Location | CES | BYCG | JDM | WCG | SMJ | ELC |
---|---|---|---|---|---|---|
NTSF | 116.7 ± 0.41 | 115.1 ± 0.49 | 115.5 ± 0.55 | 115.6 ± 0.47 | 115.3 ± 0.54 | 115.5 ± 0.49 |
WL (mm) | 34.0 ± 0.12 | 34.0 ± 0.12 | 34.0 ± 0.12 | 27.1 ± 0.06 | 27.3 ± 0.06 | 28.3 ± 0.05 |
WTSF (µm) | 93.0 ± 3.07 | 96.0 ± 1.16 | 93.5 ± 3.08 | 104.5 ± 0.62 | 105.2 ± 0.68 | 103.9 ± 0.44 |
LP (mm) | 7.8 ± 0.02 | 6.8 ± 0.01 | 8.7 ± 0.02 | 8.3 ± 0.01 | 8.6 ± 0.02 | 8.5 ± 0.01 |
BL (mm) | 29.1 ± 0.03 | 24.1 ± 0.04 | 31.5 ± 0.03 | 28.5 ± 0.01 | 33.0 ± 0.03 | 31.6 ± 0.03 |
WL/LP | 4.1 ~ 4.4 | 4.8 ~ 5.2 | 3.7 ~ 4.1 | 3.1 ~ 3.2 | 3.1 ~ 3.3 | 3.3 ~ 3.6 |
Based on the sequence of partial mtDNA (COI), individuals from six locations distinctly formed two separate clades in the NJ analysis. One clade consisted of the individuals from CES, BYCG, and JDM, while the individuals of the other three sites were grouped together (Fig.
Based on five song traits and six morphological parameters, individuals from the six regions were clustered, based on acoustic traits and morphological parameters respectively, and it was found that the cluster results were consistent with each other. Both cluster results of acoustic signals and morphological features showed there were two main clades among these samples. Specifically, individuals from CES, BYCG, and JDM grouped together and composed one branch. The other branch consisted of the individuals from SMJ, WCG, and ELC (Figs
In this study molecular, acoustic, and morphological differentiation has been analyzed in G. sedakovii collected from six sampling sites. By genetic analysis, the individuals from different geographical populations grouped into two clades. This was consistent with the results from the analysis of calling songs and morphological characteristics. For G. sedakovii, the morphological features were used to support traditional taxonomy. However, using only morphological traits led to different conclusions and using genetic data,
In contrast with these results, the description of the songs of G. s. sedakovii, previously made by
Evolutionary studies of selected orthopteran taxa have improved our knowledge of the role that insect songs play in speciation (
In the study of Apis cerana, the discovery of the new species showed that the classification of subspecies need not be based on differences in geographical region (
In other animal groups, such as frogs (
In summary, this study shows that there are two lineages within the species G. sedakovii. This conclusion supports the existing classification with two subspecies. Further examination, including samples from more geographical populations, will be needed for a more robust assessment of phylogenetic analysis.
Two large groups within species G. sedakovii were discovered by performing genetic, morphological, and acoustic analysis. Our data justifies the existing classification of G. sedakovii into two subspecies, G. s. sedakovii and G. s. obscura. We found the calling songs differed with geographical distribution, suggesting that acoustic variation might play an important role in the formation of new subspecies.
This work is supported by Natural Science Foundation of China (No. 31172133; 31400345; 31501890), Natural Science Foundation of Jilin Province (No. 20150520072JH; 20150101068JC) and the Fundamental Research Funds for the Central Universities (No. 2412015KJ017; 2412015KJ015). We are extremely grateful to the members of our laboratory for collecting materials. This article is based upon the work supported by the Center Lab, School of Life Sciences, Northeast Normal University, Changchun, China.