DNA Barcoding and Species Boundary Delimitation of Selected Species of Chinese Acridoidea (: )

Jianhua Huang1,2, Aibing Zhang3, Shaoli Mao1, Yuan Huang1* 1 College of Life Sciences, Shaanxi Normal University, Xi’an, People’s Republic of China, 2 College of Life Sciences, Guangxi Normal University, Guilin, People’s Republic of China, 3 College of Life Sciences, Capital Normal University, Beijing, People’s Republic of China

Abstract We tested the performance of DNA barcoding in Acridoidea and attempted to solve species boundary delimitation problems in selected groups using COI barcodes. Three analysis methods were applied to reconstruct the phylogeny. K2P distances were used to assess the overlap range between intraspecific variation and interspecific divergence. ‘‘Best match (BM)’’, ‘‘best close match (BCM)’’, ‘‘all species barcodes (ASB)’’ and ‘‘back-propagation neural networks (BP-based method)’’ were utilized to test the success rate of species identification. Phylogenetic species concept and network analysis were employed to delimitate the species boundary in eight selected species groups. The results demonstrated that the COI barcode region performed better in phylogenetic reconstruction at genus and species levels than at higher-levels, but showed a little improvement in resolving the higher-level relationships when the third base data or both first and third base data were excluded. Most overlaps and incorrect identifications may be due to imperfect , indicating the critical role of taxonomic revision in DNA barcoding study. Species boundary delimitation confirmed the presence of oversplitting in six species groups and suggested that each group should be treated as a single species.

Citation: Huang J, Zhang A, Mao S, Huang Y (2013) DNA Barcoding and Species Boundary Delimitation of Selected Species of Chinese Acridoidea (Orthoptera: Caelifera). PLoS ONE 8(12): e82400. doi:10.1371/journal.pone.0082400 Editor: Donald James Colgan, Australian Museum, Australia Received June 7, 2013; Accepted October 22, 2013; Published December 20, 2013 Copyright: ß 2013 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study is supported by National Natural Science Foundation of China (No. 30970346, 31172076, 31260523), the Fundamental Research Funds for the Central Universities (GK201001004), China Postdoctoral Science Foundation (20070421105) and Guangxi Natural Science Foundation (2010GXNSFA013070). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction Catantopidae [26–28], Oedipodidae [29–30], Arcypteridae [31], Melanoplinae [32–35], Podisminae [36], Oedipodinae [37–39], Acridoidea is the largest group in Orthoptera, with more than Gomphocerinae [39–41], Proctolabinae [42], and so on. Because 1000 species known from China to date. However, imperfect of the difference in taxon sampling, selection of genetic markers taxonomy may exist in many species groups, especially in those and analytical strategy, the results were frequently inconsistent to with reduced tegmina. Many brachypterous and apterous new some extent among different case studies and the phylogenies species have been described from China during the last thirty years remain unresolved in many taxa. based on minor differences which might in fact be normal DNA barcoding is a diagnostic technique in which short DNA intraspecific variations, and some of them are extremely close sequence(s) can be used for species identification [43]. It has geographically to their nearest relatives, resulting in a possible attracted extensive attention from taxonomists all over the world. oversplitting, i.e. morphotypes are inappropriately recognized as Using the standard 658-base fragment of the 59 end of the species [1]. Some efforts have been made to resolve these issues mitochondrial gene cytochrome c oxidase subunit I (COI, cox1) as based on morphological study [2–4]. Of course, cryptic species the barcode region for [44], the proponents of the may exist in some groups and additional evidence is needed to approach suggest that at least 98% of congeneric species pairs of distinguish them from known close relatives. animals can be correctly identified through DNA barcoding [45]. is the largest family in Acridoidea, including 24 Besides assigning specimens to known species, DNA barcoding subfamilies and some genera unassigned to any subfamily [5]. also aids to highlight potential cryptic, synonymous and extinct However, some family names, such as Catantopidae [6], species [46–49] as well as to match adults with immature Oedipodidae, Arcypteridae [7] and Gomphoceridae [8], have specimens [50]. However, a lot of factors can affect the success been defined from those subfamilies. Molecular evidence has long rate of DNA barcoding, for example, the selection of gene been used to resolve the phylogeny, evolutionary history and fragment(s) [51], control of numts [52–53], monophyly of the taxonomy of Acridoidea at different hierarchical levels. Besides known species [1], analytical methods [54–59], success rate of some researches that discussed the monophyly of Caelifera as well barcode amplification and sequencing [60], and so on. The as Acridoidea [9–21], there were also many investigations effectiveness of barcoding is critically dependent upon species exploring the phylogenies and origins of their subordinate delineation [1]. Funk and Omland [61] found that about 23% of categories, including Acrididae [22–23], Pamphagidae [24–25],

PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e82400 DNA Barcoding of Chinese Acridoidea surveyed metazoan species were genetically polyphyletic or information can be used to assign individuals to species correctly paraphyletic. Nearly all failures of species identification through most of the time. However, incompatibility occurred when using DNA barcoding in cowries could be attributed to imperfect external morphological characters to identify the specimens. taxonomy (overlumping or overspliting) or incomplete lineage Generally, the length of tegmina is the main character distin- sorting [1], indicating that comprehensive taxonomic revision guishing these two species from each other. C. italicus has tegmina (species boundary delimitation) is crucial in the DNA barcoding reaching or exceeding the apices of the hind femora, but C. studies. abbreviatus has tegmina much shorter and distinctly not reaching Although there are a lot of successful examples in most the apices of the hind femora [6]. When examining specimens orders using DNA barcoding to identify species [62–66], we have from all over China, we found that most specimens from localities found only four studies concerning DNA barcoding of Acridoidea, south of theYangtze River had their tegmina obviously not one with positive result [67], one with a failed result [68], and two reaching the apices of the hind femora and can be assigned to C. cases focused on the distribution patterns and coamplification of abbreviatus without any doubt. However, those from localities north numts and their effects on DNA barcoding [52–53]. Lo´pez et al of the Yangtze River had their tegmina reaching or distinctly [69] discussed the species boundary delimitation of threatened exceeding the apices of the hind femora, and should be assigned to species from the Canary Islands using both C. italicus. Therefore, either the distribution range of C. italicus mitochondrial COI and nuclear ITS2 sequences. should be extended to most provinces of north China from To test the performance of DNA barcoding in Acridoidea on a Xinjiang according to the morphological identification, or the larger scale, we examined patterns of CO1 divergence in 72 taxonomic status of C. abbreviatus represented by populations in species/subspecies of mostly from China (Schistocerca north China should be supported by additional evidence to clarify americana and Chorthippus parallelus are not distributed in China and the incompatibility between the implications of morphological their sequences were downloaded from GenBank). identification and geographical distribution ranges. Taxon sampling was mainly focused on the following eight For the remaining two groups, the minor morphological morphologically similar but taxonomically questionable species differences between species within each group have usually been groups: (1) Sinopodisma houshana+S. lushiensis+S. qinlingensis; (2) considered as normal variations among individuals from different Pedopodisma tsinlingensis+P. funiushana+P. wudangshanensis; (3) Fruh- geographical populations. storferiola kulinga+F. huayinensis; (4) Shirakiacris shirakii+F. yunkweiensis; In this study, the overlap range between intraspecific variation (5) Spathosternum prasiniferum prasiniferum+S. p. sinense; (6) Calliptamus and interspecific divergence was analyzed, the success rate of italicus+C. abbreviatus; (7) Oedaleus decorus+O. asiaticus; (8) Oedaleus species identification was tested using ‘‘best match (BM)’’, ‘‘best infernalis+O. manjius. close match (BCM)’’, ‘‘all species barcodes (ASB)’’ and ‘‘back- For the first four groups, there is nearly no morphological propagation neural networks (BP-based method)’’, and species difference found between species within each group, and the only boundary delimitations of the eight species group were discussed criterion that can be used to identify species is geographical based on the COI barcode sequences. distribution information. Each group should be regarded as the same species according to the extremely high morphological Materials and Methods similarity. Shirakiacris yunkweiensis was synonymized with S. shirakii [70], but this has not yet been accepted by Chinese acridologists, Taxon sampling although there is no additional evidence to support their decision A total of 466 sequences from different individuals representing to retain the species status of S. yunkweiensis [6]. Pedopodisma, a genus 4 superfamilies 5 families 49 genera and 72 species/subspecies endemic to China, was synonymized with Sinopodisma [71], but were analyzed. Of these sequences, 20 were downloaded from Storozhenko’s treatment has not been accepted by Chinese GenBank (http://www.ncbi.nlm.nih.gov/) (Table S1), either COI acridologists [6] because tegmina are completely absent in fragments completely overlapped with the 658-bp Folmer region Pedopodisma but distinct though reduced in Sinopodisma. [75] or extracted from the published complete mitochondrial Spathosternum sinense was originally described as an independent genome. At least five individuals from each population and as species [72], then reduced to a race of S. prasiniferum [73], and even many populations as possible of the widespread species were synonymized with the latter [74]. However, Grunshaw’s synon- sampled whenever the specimens were available (Table S2). ymy was not accepted by other orthopterists, and S. sinense has However, sometimes only one or two individuals per species (or been recognized as a subspecies of S. prasiniferum until now [5]. S. locality) could be collected. All specimens were preserved in 100% prasiniferum prasiniferum and S. p. sinense are usually distinguished ethanol and stored at room temperature. from each other by their tegmen length, the former with tegmina reaching or slightly exceeding the apices of hind femora and the DNA extraction, PCR amplification and sequencing latter usually only reaching the middle of the hind femora. They Whole genomic DNA was extracted from muscle tissue of the may be allopatric and it remains unclear whether there is an hind femur using a routine phenol/chloroform method [76]. overlap distributional zone. However, we did collect a few female Using the degenerated primer pair designed for Orthoptera [67], individuals with long tegmina from the population of S. p. sinense, COBU (59-TYTCAACAAAYCAYAARGATATTGG-39) and but never found males with this phenotype. It is not clear to which COBL (59-TAAACTTCWGGRTGWCCAAARAATCA-39), the species/subspecies, sinense or prasiniferum, these rare individuals 658-bp fragments were amplified from the 59 region of the with long tegmina but from the population of S. p. sinense should be cytochrome c oxidase 1 (COI) gene that has been adopted as the assigned. Whether these two taxa are species or subspecies requires standard barcode for members of the kingdom [44]. clarification. The 25 ml PCR reaction mixture contained 13.875 mlof Calliptamus italicus is widely distributed in Europe, north Africa, ultrapure water, 2.5 mlof106 PCR buffer (Mg2+ free), 2.5 mlof central and west Asia as well as northwest China (Xinjiang and MgCl2 (25 mM), 2 ml of dNTP (2.5 mM), 1.5 ml of each primer Qinghai Province). C. abbreviatus is distributed in north Asia, Korea (0.01 mM), 0.125 ml of TaKaRa r-Taq polymerase, and 1 mlof and most provinces of China [6]. It seems that there is nearly no DNA template. Amplifications were performed using a TaKaRa geographical overlap between these two species and geographical PCR Thermal Cycler. The cycling protocol consisted of an initial

PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e82400 DNA Barcoding of Chinese Acridoidea denaturation step at 95uC for 5 min, followed by 30–35 cycles of Network analysis denaturation at 94uC for 45 s, annealing at 48uC for 45 s and Traditional bifurcating trees are less powerful for resolving extension at 70uC for 1 min 30 s, and a final extension at 72uC for relationship among intraspecific populations and closely related 10 min and then held at 4uC. PCR products were used directly for species than haplotype networks which can provide significant sequencing after purification. Sequencing primers were the same inferences about evolutionary relationships [85–86]. Therefore, we as those for PCR amplification. Products were labeled using the constructed haplotype networks for the eight aforementioned BigDyeH Terminator v.3.1 Cycle Sequencing Kit (Applied species groups. The construction of haplotype networks was Biosystems, Inc.) and sequenced bidirectionally using an ABI implemented in TSC1.21 [87]. PRISMTM 3100-Avant Genetic Analyzer. Intraspecific variation, interspecific divergence and DNA Sequence assembly and alignment barcoding overlap The raw sequence files were assembled using the Staden Sequence divergences were calculated using the Kimura two Package [77]. The assembled sequences were aligned using Clustal parameter (K2P) distance model [88–89]. The calculation of the X [78], and both ends of the sequences matching the primer sequence divergences was implemented in MEGA4.1 [84]. From sequences were excised to remove artificial nucleotide similarity the sequence divergence data, the extent of DNA barcoding gap/ introduced by PCR amplification, resulting in the final length of overlap was then explored as typically done in barcoding studies 658 bp for both phylogenetic and barcoding analysis. COI [1]. nucleotide sequences were translated to amino acid sequences to check for stop codons and shifts in reading frame that might Species assignment with distance-based methods and indicate the presence of nuclear mitochondrial copies (numts) [68]. the neural network approach Haplotype nucleotide sequences are deposited in GenBank Distance-based methods of species assignments in conjunction (KC139803—KC140101, Table S3). Each haplotype was blasted with computer simulations are capable of determining the using MEGABLAST option against the nucleotide collection (nr/ statistical significance of species identification success rates. We nt), available on the NCBI website (http://blast.ncbi.nlm.nih.gov/ therefore performed the ‘‘best match (BM)’’ and ‘‘best close match Blast.cgi?CMD = Web&PAGE_TYPE = BlastHome). Only haplo- (BCM)’’ [90] for the species with more than three individuals types that blasted within the correct suborder with E-valu- sampled, utilizing ‘‘single-sequence-omission’’ or ‘‘leave-one-out’’ # es 1.00E-30 were included in this study [53]. simulation. The BCM identification protocol first identifies the best barcode match of a query, but only assigns the species name Phylogeny reconstruction of that barcode to the query if the barcode is sufficiently similar. To test the resolution of COI barcode sequences in recon- This approach requires a threshold similarity value that defines structing the phylogeny of grasshoppers at different taxonomic how similar a barcode match needs to be before it can be levels, we performed analyses in both parsimony and Bayesian identified. Such a value could be estimated for a given data set by frameworks with the following 4 species as outgroups: Yunnanites obtaining a frequency distribution of all intraspecific pairwise coriacea and Atractomorpha sinensis (Pyrgomorphoidea), Bennia multi- distances and determining the threshold distance below which spinata (Eumastacoidea) and Criotettix bispinosus (Tetrigoidea). 95% of all intraspecific distances are found. After the BCM Within the parsimony framework, the aligned sequence data analysis, a more rigorous application of the best close match were analyzed by using heuristic search algorithms with 100 strategy, all species barcodes (ASB), was implemented. Here random addition replicates implemented in PAUP 4.0 beta 10 win information from all intraspecies barcodes in the reference data set [79]. To assess support, we calculated standard bootstrap values was utilized instead of just focusing on the barcode that is most based on 100 replicates (100 random-addition TBR replicates similar to the query. A list of all barcodes sorted by similarity to the each). query using the same threshold as for BCM was assembled for Within the Bayesian framework, we analyzed the data sets by each query. Queries were considered a success when they were using the program MrBayes 3.1.2 [80] after selecting best-fit followed by all intraspecies barcodes as long as there were at least models of nucleotide evolution under the AIC criteria by using two barcodes for the species. With this approach, the identifier will MrModeltest 3.7 [81]. The analyses consisted of running four be more confident about assigning this species name to the query simultaneous chains for 20 million generations (TVM+I+G), than in cases where multiple species names are found on the list of sampling every 1000 generations. Two independent identical best matches. Indeed, a conservative identifier would probably Bayesian runs were performed to ensure convergence on similar only assign a species name if the query is followed by all known results and the nodal support was assessed by using the posterior barcodes for a particular species and insist that there are at least probability generated from a consensus tree of the trees sampled two intraspecies matches. The BM, BCM and ASB approaches after burn-in. The amount of burn-in is the number of samples are implemented in the computer program TaxonDNA (available that will be discarded at the start of the run. Tracer 1.5 (http:// at http://taxondna.sf.net/) [90]. beast.bio.ed.ac.uk) was used to view the point where the chain Taking both accuracy and precision into account, we calculated reached stationary and then the burn-in was determined by the an ad hoc distance threshold for our data set using a simple linear number of samples before this point. regression [91]. The query results were subdivided into (1) true To provide a profile for the setup of taxa and groups for positives (TP), (2) false positives (FP), (3) true negatives (TN), and calculating genetic distances, a neighbor-joining (NJ) tree of K2P (4) false negatives (FN) [91]. The performances of BCM were distances was created to provide a graphic representation of the quantified by calculating accuracy ((TP+TN)/total number of patterning of divergence between species [82] because of its strong queries) and precision (TP/(number of not-discarded queries)) as track record in the analysis of large species assemblages [83]. NJ well as the overall identification (ID) error ((FP+FN)/total number tree building with 1000 bootstrap replicates was implemented in of queries) and the relative ID error (FP/number of not-discarded MEGA4.1 [84]. queries). Variations in the proportions of TP, TN, FP, FN, accuracy, precision, overall and relative ID errors were quantified

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for 30 arbitrary K2P distance thresholds (THRK2P) ranging from placements of a few species (Fig. 1C). All of the eight species THRK2P = the largest query-best match K2P distance in a library groups we focused on formed monophyletic clades respectively, (all queries are accepted as being correctly identified, i.e. none is but individuals within each group, excluding groups of Spathos- discarded as in the BM criterion) to THRK2P = 0.00 (only identical ternum and Calliptamus, clustered neither by species nor populations sequences are accepted as being correctly identified, all the others (See the section on species boundary delimitation for the detailed are discarded). Relationships between relative ID errors and K2P clades). distance thresholds were investigated through linear regression. To explore the further implications of the COI barcode The regression equation was then used to infer an ad hoc distance fragment in reconstructing phylogeny at higher-levels, the data threshold corresponding to the K2P distance yielding a relative ID set was reanalyzed twice under MP framework with exclusion of error,0.05 (THRK2P_0.05). This ad hoc threshold corresponds to third base data or both first and third base data. The results the K2P distance at which 95% of the not-discarded queries are suggested that it seemed to have a little improvement in resolving expected to be correctly identified. The analysis was performed by higher-level relationships (Figs. S1, S2, S3). Although the higher- Dr. Gontran Sonet using a script in R by himself. level relationships in the topologies were still not strongly Back-propagation neural network-based species identification supported and the monophylies of Catantopidae, Oedipodidae, (BP-based method) is a recently proposed method for DNA Arcypteridae and Gomphoceridae were still not completely barcoding [92–93]. The BP-based method proved to be powerful supported, most members of Catantopidae and Oedipodidae did in species assignments via DNA sequences, especially for closely show a closer relationship within family than between families related species [93]. We performed BP analysis for the same data (Figs. S2, S3). set as we did BCM analysis. The data set was randomly divided into a reference data set and a query data set. The reference data Intraspecific variation, interspecific divergence and DNA set was used to train a BP-neural network model, while the query barcoding gap data set was used as a test data set. The ratio of reference Based on the neighbor-joining (NJ) tree of K2P distances, taxa sequences to query sequences was about 1:1 since increasing the or groups were set up to calculate the intraspecific variations and reference sequences did not significantly improve species identi- interspecific divergences. The results showed that variations within fication success rate for the COI barcode [60]. For all these population were mostly distinctly less or slightly larger than 1%, simulations, the learning rate was set to 0.2, moment value to 0.5 intraspecific variations between populations were usually less than and training goal to 0.00001, as implemented in the program 3% (Table S4), a putative threshold for species assignment BPSI2.0 [92]. proposed by previous study [44], with Sinopodisma lofaoshana as the single exception which had much higher intraspecific variation Results (5.06–5.56%, average 5.25%) between interpopulation individuals. Two populations of S. lofaoshana, one from Hengshan and the other Phylogeny reconstruction from Jiulianshan, were sampled; the variations within population Both parsimony and Bayesian analyses of our data set were less than 1% but those between populations were slightly consistently recovered several monophyletic clades above genus higher than 5%. Consequently, S. lofaoshana was divided into two levels (Fig. 1A, 1B). However, the relationships among these clades groups to calculate intergroup divergences. The results showed could not be resolved well, and none of the Catantopidae, that the divergence between Jiulianshan populations of S. lofaoshana Oedipodidae, Arcypteridae or Gomphoceridae were supported as and S. rostellocerca was as low as 2.17–3.12% (average 2.53%), monophyletic (Fig. S1). This result is consistent with earlier studies indicating a closer relationship between them than between the [13–15]. two groups of S. lofaoshana. Excepting the Spathosternum and Calliptamus groups, each of the The interspecific divergences within each of the six species species groups we focused on was comprised of species that did not groups abovementioned (Table S4) and 22.5% of interspecific form reciprocally monophyletic clades. However, each of the six divergences within a genus were less than 3%, 77.5% of them species groups usually formed a monophyletic clade most having more than 3%, and 68.19% of them more than 5% (Table S5), high posterior probability and/or bootstrap values. In addition, resulting in a total overlap of 5.55% (from 0.0% to 5.55%) Sinopodisma lofaoshana formed two distantly separated clades, with between intraspecific variations and interspecific divergences the clade of individuals from Jiulianshan population as a sister to (Fig. 2). the S. rostellocerca clade, and the clade of individuals from Hengshan population as a sister to the S. wulingshana clade. Traulia Species assignment through BCM and BP-based methods minuta included the single representative of T. szetschuanensis in the In the case of identification with the BCM method, we Bayesian tree but excluded it in the MP tree with a bootstrap value assembled a data set including 432 sequences belonging to 43 of less than 75%. Monophyletic clades of Paratonkinacris vittifemoralis species, with each species including at least three sequences to were recovered both within the Bayesian and parsimony meet the requirement for ‘‘all species barcodes (ASB)’’ analysis. frameworks with posterior probability of 0.73 and bootstrap The threshold value for the identification simulation was support of 92%. calculated from the data set. Using the 95th percentile of Congeneric species formed monophyletic clades most of time in intraspecific distances (2%) computed from pairwise summary as the case that two or more species were sampled from a genus. The the threshold for the BCM simulation, the correct identification exceptions were Sinopodisma into which the genus Tonkinacris fell, according to ‘‘best close match (BCM)’’ is 91.66%, but that and Chorthippus which is a very difficult large grasshopper genus according to ‘‘all species barcodes (ASB)’’ is only 60.64% (Table 1). and needs a comprehensive revision (Fig. 1A, 1B). The genus The 5.32% ambiguous and 2.77% incorrect identifications in Pedopodisma was recovered either as a sister to the Sinopodisma+- BCM were all caused by the sequences from the six questionable Tonkinacris clade in the MP tree or as a sister to the Fruhstorferiola species groups (Table S6). However, the much higher ambiguous clade in the Bayesian tree. identification in ASB analysis (38.88%) occurred also in query The NJ analysis recovered a topology similar to those from sequences of Sinopodisma lofaoshana and S. rostellocerca as well as in parsimony and Bayesian analyses, with small differences in those of the six questionable species groups.

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Figure 1. Phylogenetic analysis based on mtDNA COI in parsimony, Bayesian and neighbour-joining (K2P) frameworks. Subclades within a species with more than one individual sampledare collapsed. The number in parentheses indicates its bootstrap support values or posterior probabilities. Colored blocks of branches indicate the clades consistently recovered in parsimony, Bayesian and NJ analyses. Bootstrap values of ,75 and posterior probability of ,0.95 are not recorded on the tree. A. Cladogram from parsimony analysis. B. Cladogram from Bayesian analysis. C. Cladogram from NJ analysis. doi:10.1371/journal.pone.0082400.g001

Taking both accuracy and precision into account, we calculated Species boundary delimitations for selected species an ad hoc distance threshold for the data set using a simple linear groups regression [91]. Unfortunately, according to the analysis, we could Calliptamus italicus species group. Since there were some not obtain an estimated relative identification error lower than doubts on the relationship between Calliptamus italicus and C. 0.06 with the current data set (Fig. 3). According to the regression abbreviatus, we first assigned a species name to each sequence line, we would have to use a distance threshold of 20.02 to obtain according to the localities of materials under study, i.e. the an estimated relative identification error of 0.05, but a negative materials from Xinjiang and Qinghai were provisionally consid- threshold is impossible to apply. By applying a threshold distance ered as C. italicus, and those from other areas of China were of 0, we can get the lowest estimated relative identification error considered as C. abbreviatus. The results showed that C. bararus and possible with this data set, i.e. the intercept = 0.06. More restrictive individuals of C. italicus from Xinjiang and Europe (NC_011305) distance thresholds will improve precision to some extent, yet formed monophyletic clades in the NJ tree respectively, both with negatively affect accuracy due to the higher proportions of queries 100% bootstrap value. However, the individuals of C. italicus from discarded [91]. The use of more restrictive distance thresholds in Qinghai did not cluster with those from Xinjiang and Europe, but our data set scarcely improved precision, but heavily decreased the formed another monophyletic clade with the specimens of C. accuracy when the threshold used was less than 0.01 (Fig. 4). abbreviatus. Individuals in the clade of C. abbreviatus did not cluster Instead of using the leave-one-out simulation for BCM methods, by populations (Fig. 5A). we used randomly selected reference and query sequences for BP- Analysis with haplotype network led to a similar result. The based method to investigate the performance of the barcode. This network was divided into three separate clades, Clade A, Clade B strategy was employed due to the slow training process which and Clade C (Fig. 5B). Clade A was composed of haplotypes of C. hinders the utility of the BP-based method in large scale simulation italicus from Xinjiang and Europe, Clade B consisted of haplotypes studies. Using the same sequence data as in BCM analysis, we of C. barbarus. However, the haplotypes of C. italicus from Qinghai assembled a training data set with 230 barcode sequences and a (marked with green color) formed clade C together with those of C. query data set with 202 barcode sequences. As a result, 10 query abbreviatus, suggesting that the individuals sampled from Qinghai in sequences were identified incorrectly with extremely high prob- this study actually belong to C. abbreviatus. Nearly no haplotypes of ability, 1 identified incorrectly with low probability, and 3 C. abbreviatus from the same population formed a relatively identified correctly with low probability, resulting in a success independent subclade, and two haplotypes were found shared by rate of 94.55%. All incorrect or inaccurate identification occurred multiple populations, one shared by three populations (Siping, Jilin in the six questionable species groups (Table S7). Province; Tongliao, Inner Mongolia Autonomous Region; Qin’an, Gansu Province) and the other by five populations (Anji, Zhejiang Province; Zhuolu, Hebei Province; Zhidan, Shaanxi Province; Qin’an, Gansu Province; Xunhua, Qinghai Province) (Fig. 5B).

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Figure 2. Distribution pattern of intraspecific variation and interspecific divergence. doi:10.1371/journal.pone.0082400.g002

Therefore, it is clear that C. italicus and C. abbreviatus are two each group except that the body size of Sinopodisma houshana is separate species. However, the tegmina length should not be slightly larger than those of S. lushiensis and S. qinlingensis. All regarded as distinguishing character between these two species materials were collected from the type locality of each species since wing-length polymorphism is quite common in Acrididae except for those of S. houshana, which has a much more widespread [94–95]. As for the distribution of C. italicus in Qinghai, study with distribution than S. lushiensis and S. qinlingensis. Therefore, there is larger scale sampling is needed to clarify it. Possibly, C. italicus is no problem in assigning a morphospecies name to sampled prevented from extending its distribution to Qinghai by the barrier individuals. of the Altai Mountains and Qilian Mountains. It is also impossible The topology of the NJ tree showed that species of S. houshana for C. italicus to spread from Xinjiang to neighboring provinces group were slightly structured genetically but no species formed such as Gansu and Inner Mongolia because of the barriers independent reciprocally-monophyletic clades (Fig. 6A). The imposed by the Altai Mountains and deserts. intraspecific variations and interspecific divergences formed a complete overlap, with the latter falling completely within the Sinopodisma houshana and Pedopodisma tsinlingensis range of the former (Table 2). The relationship among species groups within the Pedopodisma tsinlingensis group was even more confused; The species are extremely similar within each group. Nearly no sequences of the three species scattered messily in the NJ tree and morphological difference can be found among the species within no species showed conspicuous genetic structure. The intraspecific

Table 1. Success rate of identification through ‘‘Best Match’’, ‘‘Best Close Match’’ and ‘‘All Species Barcode’’.

Identification method Best Match Best Close Match All Species Barcodes

Correct identifications 397 (91.89%) 396 (91.66%) 262 (60.64%) Ambiguous identifications 23 (5.32%) 23 (5.32%) 168 (38.88%) Incorrect identifications 12 (2.77%) 12 (2.77%) 1 (0.23%) Sequences without any match closer than 2.0% ---- 1 (0.23%) 1 (0.23%)

doi:10.1371/journal.pone.0082400.t001

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Figure 3. Relative identification errors at thirty arbitrary distance thresholds in BCM analysis. doi:10.1371/journal.pone.0082400.g003 genetic variations and interspecific divergences formed a broad into three subclades, with subclade I composed of haplotypes of S. overlap of 2.17% (Table 3). qinlingensis, subclade II composed of haplotypes of S. lushiensis and In the networks, haplotypes of each group formed a separate one of S. qinlingensis, and subclade III composed of haplotypes of S. clade (Fig. 6B, 6C). The clade of S. houshana group can be divided houshana. Haplotypes of S. lushiensis had a minimum of one

Figure 4. Accuracy and precision of identification at thirty distance thresholds ranging from K2P = 0.0295 to K2P = 0.000 in BCM analysis. doi:10.1371/journal.pone.0082400.g004

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Figure 5. NJ tree subclade and haplotype network of three Calliptamus species. A. NJ tree subclade. Different colors of the edges indicate individuals from different populations, the green asterisks indicate the individuals from Xunhua county, Qinghai Province. B. Haplotype network. Different colors indicate haplotypes from different populations, the ovals filled with multiple colors indicate haplotypes shared by several populations, and the green color represents haplotypes from Qinghai Province. doi:10.1371/journal.pone.0082400.g005 mutational step to those of S. qinlingensis and at least five mutational each group as a single species since they have no conspicuous steps to those of S. houshana, indicating the much closer relationship difference in either morphological or molecular characters. of this species with S. qinlingensis than with S. houshana. The haplotypes of S. houshana formed a so-called independent subclade, Fruhstorferiola kulinga species group the haplotypes from Yingshan population have a minimum of five Fruhstorferiola kulinga and F. huayinensis are extremely similar to mutational steps and those from Mulanshan population have a each other, and the characters used to distinguish them have been minimum of six mutational steps to those of S. lushiensis. However, found to be substantially variable even within populations, so there was a minimum of seven mutational steps between geographical distribution was used as the main criterion for haplotypes from Yinshan and Mulanshan populations, indicating preliminary identification of the materials under study. In the NJ that both of them have a much closer relationship to S. lushiensis tree, the sampled individuals of these two species displayed weak than between themselves. The clade of the Pedopodisma tsinlingensis genetic structure to some extent, but did not form reciprocally group can be divided into two subclades, with subclade I monophyletic clades, with one individual of F. huayinensis falling containing haplotypes from all three species and subclade II into the clade of F. kulinga and two individuals of F. kulinga falling containing haplotypes only from P. wudangshanensis. One haplotype into the clade of F. huayinensis (Fig. 7A). The intraspecific variation was found shared by P. tsinlingensis and P. funiushana. Haplotypes of of F. kulinga and F. huayinensis were 0,2.97% and 0,1.85% P. wudangshanensis in subclade I had a minimum of three respectively, but the pairwise distances between the two species are mutational steps to P. funiushana and a minimum of four 0.15,2.96%, leading to a complete overlap, i.e. the interspecific mutational steps to P. tsinlingensis, but had a minimum of nine divergences fell completely within the range of intraspecific mutational steps to those of the conspecifics in subclade II. variation of F. kulinga. The haplotype network of these two species Therefore, the validities of S. lushiensis, S. qinlingensis, P. funiushana can be divided into two subclades, with subclade I containing only and P. wudangshanensis are questionable. It is reasonable to consider haplotypes from F. kulinga and subclade II containing mainly haplotypes from F. huayingensis but two from F. kulinga (Fig. 7B).

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Figure 6. NJ tree subclade and haplotype network of the genera Sinopodisma and Pedopodisma. A. NJ tree subclade. B. Haplotype network of the Sinopodisma houshana species group; red color indicates Yinshan population and pink indicates Mulanshan population of S. houshana. C. Haplotype network of the Pedopodisma tsinlingensis species group. doi:10.1371/journal.pone.0082400.g006

Since the present molecular data do not separate these two Shirakiacris shirakii species group species from each other, and the morphological characters used to There are different opinions on the validity of Shirakiacris distinguish them have been found to be unstable, we would like to yunkweiensis [6,70]. In this study, the limited samples provided treat them provisionally as a single species before further additional evidence on the relationship between these two species. phylogeographical research with much larger scale samples. In the NJ tree (Fig. 8A), one individual of S. shirakii fell into the clade of S. yunkweiensis. The ranges of intraspecific variation within

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Table 2. Intraspecific variation and interspecific divergence Table 3. Intraspecific variation and interspecific divergence of the Sinopodisma houshana group. of the Pedopodisma tsinlingensis group.

Species Intraspecific variation Interspecific divergence Intraspecific Species variation Interspecific divergence S. houshana S. lushiensis P. tsinlingensis P. funiushana S. houshana 0,2.17% P. tsinlingensis 0,0.15% S. lushiensis 0.15,0.61% 0.77,1.85% P. funiushana 0,0.76% 0,0.92% S. qinlingensis 0,0.61% 0.92,1.70% 0.15,1.07% S. wudangshanensis 0.15,2.17% 0.61,2.33 0.46,2.33% doi:10.1371/journal.pone.0082400.t002 doi:10.1371/journal.pone.0082400.t003 S. shirakii and S. yunkweiensis were 0,1.7% and 0,1.54% populations of Xinjiang and Gansu and eighteen individuals of O. respectively, and the interspecific pairwise distances were asiaticus from six populations were sampled in this study. They did 0.61,2.01%, leading to a overlap of as much as 1.09% between not form reciprocally monophyletic clades in the NJ tree (Fig. 10A). intraspecific variation and interspecific divergence. The haplotype The ranges of intraspecific variations within O. decorus and O. network can be divided into two subclades (Fig. 8B), with subclade asiaticus were 0,2.17% and 0,1.23% respectively, and the I containing haplotypes from S. shirakii and subclade II containing interspecific pairwise distances were 1.39,2.01%, leading to a haplotypes from S. yunkweiensis, but one haplotype of S. shirakii had complete overlap between intraspecific variations and interspecific a minimum of four mutational steps to S. yunkweiensis, much divergences. The haplotypes of O. decorus were connected to the smaller than the minimum of ten mutational steps to other central network of O. asiaticus through three subclades, and there haplotypes of S. shirakii. was a minimum of four mutational steps between the haplotypes of Since no results from any analysis supported the validity of S. the two species (Fig. 10B). yunkweiensis, it should be regarded as a junior synonym of S. shirakii Oedaleus infernalis can be distinguished from O. manjius mainly by [70]. its yellowish brown tibiae and the narrower dark transverse band of the hind wing. Forty-four individuals of O. infernalis from 11 populations and seven individuals of O. manjius from 2 populations Spathosternum prasiniferum species group were sampled in this study. However, they did not form While Spathosternum prasiniferum sinense and S. p. prasiniferum are reciprocally monophyletic clades in the NJ tree (Fig. 10A). The regarded at present as two subspecies of the same species, they intraspecific variations of O. infernalis and O. manjius ranged from 0 formed reciprocally monophyletic clades in the NJ tree (Fig. 9A) to 2.47% and 0 to 1.7% respectively, and the interspecific pairwise and there was a conspicuous gap of 0.93% (from 0.61% to 1.54%) distances from 0 to 2.63%, leading to a broad overlap between , , between intraspecific variations (0 0.3% and 0.15 0.61% intraspecific variations and interspecific divergences within this respectively; Table S4) and interspecific divergences (pairwise group. The haplotypes of O. manjius were connected to the network distances varying between 1.54,1.85%). The haplotype network of O. infernalis through three subclades, and one haplotype was displayed a similar relationship and there was a minimum of ten found shared by them (Fig. 10C). mutational steps between the nominal subspecies (Fig. 9B). Therefore, the minor morphological differences between the Interestingly, there were four female individuals with long tegmina nominal species within each group may be attributed to collected together with S. p. sinense from Guilin. Judged solely adaptation to different environments and each group should be according to morphology, these four females should be recognized actually considered as comprising a single widely-distributed as S. p. prasiniferum, but they formed a monophyletic clade in the species. Further phylogeographical study may reveal much more gene tree with the short-winged individuals from the same precise genetic structure pattern in these two groups, including the population (Fig. 9A). The pairwise distances between these four color pattern polymorphism that commonly exist in Acrididae long-winged individuals and short-winged individuals varied [94,96]. between 0,0.3%, indicating that they were much closer genetically to and may be aberrant individuals of S. p. sinense. Discussion Furthermore, there were haplotypes shared by long- and short- winged individuals from the same population. Performance of COI barcode sequence in reconstructing Therefore, S. p. sinense and S. p. prasiniferum are at least two higher-level phylogeny distinct subspecies since they have conspicuous morphological Although the primary aim of our study was to investigate DNA differences and molecular divergences. Considering the common barcoding, the phylogeny inferred from COI barcode sequence presence of polymorphism in wing length in Acrididae [94–95], sheds new light on the relationship among taxa within Acrididae. the relatively low genetic distances between the two subspecies Given that the genus Pedopodisma did not fall within Sinopodisma (1.54,1.85%) and the broad distribution of S. prasiniferum, data either in the MP tree or in the Bayesian tree, it likely be an from more populations will certainly facilitate making a categorical independent genus [6] rather than a junior synonym of Sinopodisma decision. Possibly, the taxonomic status of S. p. sinense could even [71]. Further study with thorough sampling will certainly facilitate be raised to species-level based on futher evidences from a better understanding of the relationship between Sinopodisma and phylogeographical study. Pedopodisma. Since the monophylies of most genera and well delineated species were supported by our data set, it seemed that Oedaleus decorus and Oedaleus infernalis species groups COI barcode region performed much better in phylogenetic Oedaleus decorus differs from O. asiaticus mainly in having the dark reconstruction at genus and species levels than at higher levels. It is transverse band of hind wing broader and not interrupted at the reasonable to assume that the success of species assignment may be first anal vein. Eight individuals of O. decorus from three higher where the reconstructed evolution of the gene reflects

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Figure 7. NJ tree Subclade and haplotype network of the genus Fruhstorferiola. A. NJ tree subclade. B. Haplotype network of the Fruhstorferiola kulinga group. F_kul indicates Fruhstorferiola kulinga (filled with grey color), F_hua indicates Fruhstroferiola huayinensis. doi:10.1371/journal.pone.0082400.g007 speciation events, particularly where closely related species are will certainly improve our understanding of the phylogeny of under study [97]. Acridoidea at higher-levels. Although the phylogeny at higher levels were not well resolved using COI barcode sequence, a little improvement was gained Efficacy of DNA barcoding in Acridoidea when the third base data or both first and third base data were The most important goal of DNA barcoding is to facilitate the excluded. This limited improvement may be due to: (1) the fact species discovery process by increasing the speed, objectivity, and that some groups may be paraphyletic or polyphyletic; (2) the bias efficiency of species identification. However, barcoding failure in taxon sampling; and (3) the use of the partial fragment of the associated with non-monophyly is very likely at the traditionally single COI gene. It is expected that combining COI barcode data recognized species level [1]. Such a mismatch of taxonomy and with other gene sequences (especially suitable nuclear genes), more genealogy was also observed in the New Zealand grasshopper thorough sampling and the exploration of new analytical strategies genus Sigaus [68]. In our data set, when the traditional species names were used, interspecific divergences extended at their lower

Figure 8. NJ tree subclade and haplotype network of the Shirakiacris shirakii group. A. NJ tree subclade. B. Haplotype network. doi:10.1371/journal.pone.0082400.g008

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Figure 9. NJ tree subclade and haplotype network of the Spathosternum prasiniferum species group. A. NJ tree subclade. The asterisks and pink color indicate the female individuals of S. prasiniferum sinense from Guilin with long tegmina. B. Haplotype network. S_pra represents S. prasiniferum prasiniferum and S_sin represents S. prasiniferum sinense. Black color indicates the haplotypes from the normal individuals of S. prasiniferum sinense with reduced tegmina. Grey color indicates the haplotypes from the female individuals of S. prasiniferum sinense from Guilin with long tegmina. doi:10.1371/journal.pone.0082400.g009 end well into the range of intraspecific variation (Fig. 2). Species While the average interspecific divergence between Spathosternum assignment through each of the ‘‘best close match (BCM)’’, ‘‘all prasiniferum prasiniferum and S. p. sinense is only 1.7%, they were species barcodes (ASB)’’ and ‘‘BP-based neural network’’ methods reciprocally monophyletic in the MP and NJ trees, and both have produced error rates of more than 5% (Tables 1). Since nearly all much constrained intraspecific variation (Table S4), giving rise to a incorrect identifications occurred in the sequences of the six distinctive gap between intraspecific variation and interspecific questionable species groups (Table S6, S7), and the species divergence. Furthermore, eight purely diagnostic positions, nearly boundary delimitation supported our presumption on the half of the total variable positions were found within the barcode relationships among species within each species group, this error sequence alignment (Fig. 11), and bases private to one of the two rate appeared to be mainly a result of imperfect taxonomy. subspecies existed in all the other variable positions, suggesting a Furthermore, no ad hoc threshold was available to obtain an conspicuous genetic divergence between these two taxa which may estimated relative identification error of 0.05. This result might be be very recently diverged species. Therefore, molecular data due to the high proportion of false positives caused by too high derived from much more extensive sampling should serve as genetic similarities among extremely close species. Such a high evidence to revive the species status of S. sinense and they can be genetic similarity can derive from either incomplete lineage sorting correctly identified most of time by using either morphological among newly diverged species or imperfect taxonomy. Therefore, characters (the length of tegmina) or the COI barcoding fragment it is expected that a much higher success rate could be obtained through a thoroughly sampled phylogeny. As for the four females when the revised species names are applied to the data set. It is of S. p. sinense with long tegmina from Guilin, their similarity to S. obvious that perfect taxonomy will greatly increase the success rate p. prasiniferum in tegmen length may be a result induced by of identification through DNA barcoding, and that taxonomic environmental factors. Such aberrant individuals occur extremely revision plays an important role in DNA barcoding studies [1]. rarely and have nearly no genetic difference from the abundant The single representative of Traulia szetschuanensis was resolved normal individuals with short tegmina. In such a case, it is DNA within Traulia minuta in Bayesian analysis (Fig. 1B) but outside of barcoding but not a traditional morphological approach that the latter in both the MP tree (Fig. 1A) and the NJ tree (Fig. 1C). It provides a more accurate identification for the aberrant individ- is possible that T. szetschuanensis will form a monophyletic clade in uals. Of course, geographical information will help us in phylogenetic trees when more individuals are sampled. The identifying such individuals if further study can support their divergence between them was 4%, much higher than the threshold allopatry. While the intraspecific variation of Sinopodisma lofaoshana of 3% and the 95th percentile distance threshold (2%), indicating exceeded the interspecific divergences of S. p. prasiniferum against S. that they can be correctly identified through DNA barcoding. p. sinense, they were in different parts of the tree. Although having a While Tonkinacris sinensis fell into the genus Sinopodisma in both of substantial impact during the discovery phase (i.e., in an the MP and Bayesian analyses, it was recovered as a monophyletic incompletely sampled group), such overlap will not affect clade that was the sister of the clade of Sinopodisma rostellocerca+S. identification of unknowns in a thoroughly sampled tree [1]. lofaoshana (Jiulianshan population) (Fig. 1A, 1B). It fell outside of Sinopodisma lofaoshana is the most difficult taxon in our data set. Sinopodisma in the NJ tree (Fig. 1C) and had an interspecific Two populations were sampled in this study and the result showed divergence of 5.89% from Sinopodisma rostellocerca and of 6.48% that the intraspecific variations between individuals of different from the Jiulianshan population of Sinopodisma lofaoshana. That is to populations (5.06–5.56%, average 5.25%) distinctly overlapped say, Tonkinacris sinensis can be correctly identified under a with the interspecific divergence between individuals of Jiulian- completely sampled phylogeny through DNA barcoding using shan population of S. lofaoshana and those of S. rostellocerca (2.17– either tree-based or threshold or other species identification 3.12%, average 2.53%). This overlap did not lead to any incorrect approaches. or ambiguous identification in either BCM or BP-based analysis because each population has more than one individual sampled

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Figure 10. NJ tree subclade and haplotype network of Oedaleus. A. NJ tree subclade. B. Haplotype network of the Oedaleus decorus group. O_dec indicates O. decorus and O_asi indicates O. asiaticus. C. Haplotype network of the Oedaleus infernalis group. O_inf indicates O. infernalis and O_man indicates O. manjius, the oval filled with white-black color indicates the haplotype shared by O. infernalis and O. manjius. doi:10.1371/journal.pone.0082400.g010 and the intraspecific variation within population was below 1% (0– In a word, the 91% of correct identification for the BCM method 0.92%). However, the ambiguous identification occurred when and the 94% for the BP-based method in Acridoidea are acceptable performing the ASB simulation because of the presence of the at the moment and consistent with the results reported in previous genetic distance overlap between these two species, suggesting that studies [58,90]. While mtDNA barcoding cannot offer good ASB, as a more rigorous barcoding method, can reveal much resolution at higher taxonomic levels, the completeness of the DNA more sensitively the presence of a possible taxonomic problem in barcode database against which unknown sequences are compared some groups. Both S. lofaoshana and S. rostellocerca have widespread will certainly increase the accuracy of species identification [99]. distributions in south China and appear to have an overlap zone. Although both populations of S lofaoshana formed monophyletic Implications of DNA barcoding in species boundary clades, further study with thorough sampling is needed to reveal delimitation the geographical genetic structures and to clarify its relationship to The accuracy of a threshold-based approach critically depends S. rostellocerca. upon the level of overlap between intraspecific variation and Nearly all erroneous identifications in the BCM and BP-based interspecific divergence across a phylogeny. However, the ranges analyses were caused by the six questionable species groups of both intraspecific variation and interspecific divergence will abovementioned. If it can be confirmed by further evidence that change in response to species delineation, thus leading to a inappropriate morphological classification does exist in these variation of the overlap [1]. Since many traditional species will groups, then a comprehensive revision, i.e. the synonymy for each appear to be genetically non-monophyletic because of imperfect group based on morphology and supported by other evidence, will taxonomy [61], it is necessary to make a comprehensive revision of lead to more accurate identification through DNA barcoding. In any group before or in combination with DNA barcoding study. any case, accurate taxon identification is extremely important for Nearly all traditional species were established based only on molecular studies [98]. morphological character sets, and many species, especially those

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Figure 11. Alignment of Spathosternum, showing the purely diagnostic positions (position 70, 151, 190, 247, 298, 316, 346, 548, marked with red boxes) and the total variable positions. S_sin313,321 indicate sequences of S. prasiniferum sinense (including the four long- winged individuals from the Guilin population)and S_pra322,326 indicate S. prasiniferum prasiniferum. doi:10.1371/journal.pone.0082400.g011 described much earlier, were not compared with relevant type found among species established by morphology alone, the specimens of closely-related species when newly described. Thus conclusion should be that only one species exists with morphological all species, especially if they are closely-related, should firstly be polymorphism, and synonymy should be proposed. Similarly, we revised to confirm the presence of morphological differences should re-examine morphology or move on to some other source of among them, and this will resolve directly to some extent the issue information once fixed DNA differences are observed among of oversplitting caused by the lack of type comparison [1]. aggregates in which morphological differences have not previously In this study, the barcoding sequence data set not only tested the been observed. This may show whether the DNA sequence success rate of identification but also provided additional data for divergences represent truly morphologically cryptic species or the morphologically questionable species groups. DNA barcoding genetic polymorphism within a single species. In other words, from supported our doubts about taxonomic accuracy in six species the barcoding perspectives, substantial sequence differentiation can groups and the validity of species in two groups, and facilitated a be explained as heteroplasmy or due to numts if it is not confirmed better understanding of the relationship among species within each with other sources of information. Conversely, even as few as one species group. Furthermore, ASB analysis also revealed the nucleotide of consistent differentiation can serve as a diagnostic particular relationship between Sinopodisma lofaoshana and S. character to identify and delineate a valid species if it is distinctly rostellocerca, which needs further study with more thorough different morphologically, or ecologically, or in other aspects from sampling. Therefore, DNA barcoding can not only increase the its closely related congeneric species [100]. Putative cryptic species speed and accuracy of species identification, but also help us detected by mtDNA barcoding merit closer investigation via highlight or resolve some taxonomic issues. analysis of, or more in-depth examination of, ecology and taxonomy As an identification tool, DNA barcoding should be used in [100–101]. Nuclear genetic data can be used as a check when conjunction with other information [52]. Species with distinct mtDNA barcoding reveals unexpected results, because deep genetic morphological differences observed should still be corroborated divisions found during mtDNA barcoding are not always reflected with other sources of data, including geographical, biological, in the nuclear genome in some groups [102–103]. ecological, reproductive, behavioral and DNA sequence informa- In conclusion, despite the problems of sampling size [104–105] tion [54]. If no additional non-morphological difference can be and the criticisms on methodological [106], theoretical [107] and

PLOS ONE | www.plosone.org 14 December 2013 | Volume 8 | Issue 12 | e82400 DNA Barcoding of Chinese Acridoidea empirical grounds [1,108–110], the prospect of DNA barcoding is (DOC) still promising if it is based on solid foundations of comprehensive Table S3 Cross reference list between GenBank acces- taxonomy. The exploration on new analytical methods sion numbers and numbers of vouchers sharing the [59,93,99,105,111–117] and the use of nuclear genes as additional same haplotype. effective DNA barcodes [60,103] will certainly promote the progress in DNA barcoding. (DOC) Table S4 ranges of intraspecific genetic variations. Supporting Information (DOC) Figure S1 MP tree infferred with complete base data. Table S5 Distribution of intraspecific variations and Members of Catantopidae are marked with red, those of interspecific divergences. Oedipodidae with deep blue, those of Arcypteridae with yellow, (DOC) those of Gomphoceridae with pink, those of Acrididae with green, Table S6 Sequences identified as ambiguous or incor- those of Pamphagidae with bright blue and other groups with black. rect in BCM analysis. (TIF) (DOC) Figure S2 MP tree infferred with exclusion of third base Table S7 Assignment of species through BP-based data. Members of Catantopidae are marked with red, those of method. Oedipodidae with deep blue, those of Arcypteridae with yellow, those of Gomphoceridae with pink, those of Acrididae with green, (DOC) those of Pamphagidae with bright blue and other groups with black. (TIF) Acknowledgments Figure S3 MP tree infferred with exclusion of both third We would like to thank Dr. Jerzy A. Lis for kindly sending us his and first base data. Members of Catantopidae are marked with publication, to thank Dr. Gontran Sonet for helping us calculate the ad hoc red, those of Oedipodidae with deep blue, those of Arcypteridae threshold. We are deeply indebted to Dr. Ioana C. Chintauan-Marquier and the other anonymous reviewers for their constructive academic with yellow, those of Gomphoceridae with pink, those of Acrididae suggestions and detailed grammatical polish on the manuscript. with green, those of Pamphagidae with bright blue and other groups with black. (TIF) Author Contributions Conceived and designed the experiments: JHH YH. Performed the Table S1 Sequences downloaded from NCBI. experiments: JHH SLM. Analyzed the data: JHH ABZ. Contributed (DOC) reagents/materials/analysis tools: JHH YH ABZ. Wrote the paper: JHH Table S2 Materials used in this study. ABZ YH.

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Figure S1. MP tree infferred with complete base data. Members of Catantopidae are marked with red, those of Oedipodidae with deep blue, those of Arcypteridae with yellow, those of Gomphoceridae with pink, those of Acrididae with green, those of Pamphagidae with bright blue and other groups with black.

Figure S2. MP tree infferred with exclusion of third base data. Members of Catantopidae are marked with red, those of Oedipodidae with deep blue, those of Arcypteridae with yellow, those of Gomphoceridae with pink, those of Acrididae with green, those of Pamphagidae with bright blue and other groups with black.

Figure S3. MP tree infferred with exclusion of both third and first base data. Members of Catantopidae are marked with red, those of Oedipodidae with deep blue, those of Arcypteridae with yellow, those of Gomphoceridae with pink, those of Acrididae with green, those of Pamphagidae with bright blue and other groups with black.

Table S1. Sequences downloaded from NCBI

Species Locality NCBI accession Reference Calliptamus italicus (Calliptaminae) Brje pri Komnu, Slovenia EU938373 ( NC_011305) Fenn, et al, 2008 Traulia minuta (Coptacridinae) Mengla, Yunnan, China FJ571149 Zhao, et al, 2010 Traulia szetschuanensis (Coptacridinae) Neixiang, Yunnan, China FJ571148 Zhao, et al, 2010 Meltripata chloronema (Coptacridinae) Mengla, Yunnan, China FJ571150 Zhao, et al, 2010 Choroedocus violaceipes (Eyprepocnemidinae) Jinggong,Yunnan, China FJ571158 Zhao, et al, 2010 Pedopodisma funiushana (Melanoplinae) Lushi, Henan, China FJ531686 Zhao, et al, 2010 Prumna arctica (Melanoplinae) Tahe, Helongjiang FJ531674 Zhao, et al, 2010 chinensis (Oxyinae) Chang’an, Xi’an, Shaanxi, China; NC_010219 Zhang & Huang, 2007 Schistocerca americana (Cyrtacanthacridinae) EU589056 Song, , et al, 2008. Schistocerca gregaria (Cyrtacanthacridinae) NC_013240 Erler, et al, 2010 Gastrimargus marmoratus (Oedipodinae) NC_011114 Ma, et al, 2009 Locusta migratoria (Oedipodinae) NC_001712 Flook, et al, 1995 Locusta migratoria migratoria (Oedipodinae) Buerjin, Xinjiang, China NC_011119 Direct submission Oedaleus asiaticus (Oedipodinae) NC_011115 Ma, et al, 2009 Chorthippus parallelus (Gomphocerinae) X95574, X95575 Szymura, et al, 1996 Chorthippus chinensis (Gomphocerinae) Jiugongshan, Hubei, China NC_011095 Liu & Huang, 2008 Acrida willemsei (Acridinae) Malaysia, Sabah, Croker Rng. NP, EU938372 (NC_011303) Fenn, et al, 2008 HQ station Rd. Phlaeoba albonema (Acridinae) Tianhetan, Guiyang, China NC_011827 Shi, et al, 2008 Atractomorpha sinensis (Pyrgomorphoidea) NC_011824 Ding, et al, 2007

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Ding FM, Shi HW, Huang Y (2007) Complete mitochondrial genome and secondary structures of lrRNA and srRNA of Atractomorpha sinensis (Orthoptera, Pyrgomorphidae). Zoological Research, 28：580－588 Erler S, Ferenz HJ, Moritz RFA, Kaatz HH (2010) Analysis of the mitochondrial genome of Schistocerca gregaria gregaria (Orthoptera: Acrididae). Biological Journal of Linnaeus Society of London, 99: 296—305. Fenn JD, Song H, Cameron SL, et al. (2008) A preliminary mitochondrial genome phylogeny of Orthoptera (Insecta) and approaches to maximizing phylogenetic signal found within mitochondrial genome data. Molecular Phylogenetics and Evolution, 49: 59—68. Flook PK, Rowell CH, Gellissen G (1995) The sequence, organization, and evolution of the Locusta migratoria mitochondrial genome. Journal of Molecular Evolution, 1995, 41: 928—941. Folmer O, Black M, Hoeh W, et al. (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3: 294-297. Liu Y, Huang Y (2008) Sequencing and analysis of complete mitochondrial genome of Chorthippus chinensis Tarb. Chinese Journal of Biochemistry and Molecular Biology, 24 (4): 329—335. Ma C, Liu CX, Yang PC, et al. (2009) The complete mitochondrial genomes of two band-winged grasshoppers, Gastrimargus marmoratus and Oedaleus asiaticus. BMC Genomics 2009, 10:156. doi:10.1186/1471-2164-10-156 Shi HW, Ding FM, Huang Y (2008) Complete Sequencing andAnalysis of mtDNA in Phlaeoba albonema Zheng. Chinese Journal of Biochemistry and Molecular Biology, 24 (7) :604～611 Song H, Buhay JE, Whiting MF, et al. (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proceedings of the National Academy of Science, USA, 105(36): 13486—13491. Szymura JM, Lunt DH, Hewitt GM (1996) The sequence and structure of the meadow grasshopper (Chorthippus parallelus) mitochondrial srRNA, ND2, COI, COII ATPase8 and 9 tRNA genes. Insect Molecular Biology, 5(2): 127—139. Zhao L, Zheng ZM, Huang Y, et al. (2010) Phylogeny of the Chinese Cantatopidae (Orthopter : Acridoidea) inferred from mitochondrial DNA sequences. Acta Scientiarum Naturalium Universitatis Sunyatseni, 49(4): 111—117. Zhang CY, Huang Y (2007). Complete mitochondrial genome of Oxya chinensis (Orthoptera: Acridoidea). Acta Biochimica et Biophysica Sinica, 40, 7–18.

Table S2. Materials used in this study

Species Voucher number Locality, time and collector Acridoidea: Acrididae: Melanoplinae Sinopodisma rostellocerca gl0001-0002, 0014-0016 Yaoshan, Guilin, Guangxi, China; 27 July 2006; Jianhua Huang gl0003-0004, 0011-0013 Yong’an, Xing’an County, Guangxi, China; 4 July 2006; Jianhua Huang gl0005-0006 Huangsha, Lingui County, Guangxi, China; 13 August 2006; Jianhua Huang gl0007-0010 Gaozhai, Xing’an County, Guangxi, China; 6 August 2006; Jianhua Huang Sinopodisma lofaoshana gl0017, 0019-0022 Hengshan, Hunan, China; 28 August 2007; Jianhua Huang gl0026 Chukou, Zixing County, Hunan, China; 5 July 2005; Jianhua Huang gl0023-0025, 0027 Jiulianshan, Jiangxi, China; 21 July 2008; Fuming Shi Sinopodisma wulingshana gl0018, 0029-0034 Hengshan, Hunan, China; 29 August 2007; Jianhua Huang gl0035-0040 Daweishan, Liuyang, Hunan, China; 5 October 2007; Jianhua Huang gl0041-0046 Xingdoushan, Lichuan, Hubei, China; 3 August 2004; Yulin Zhong Sinopodisma qinlingensis gl0047-0052 Xunyangba, Ningshan, Shaanxi, China; 18 July 2008; Yuan Huang Sinopodisma lushiensis gl0053-0058 Shiziping, Lushi, Henan, China; 25 September 2008; Jianhua Huang Sinopodisma houshana gl0059-0064 Yingshan, Hubei, China; 26 August 2004; Yulin Zhong gl0065-0070 Mulanshan, Wuhan, Hubei, China; 29 September 2008; Jianhua Huang Pedopodisma wudangshanensis gl0071-0076 Wudangshan, Hubei, China; 27 July 2004; Yulin Zhong Pedopodisma tsinlingensis gl0077-0082 Nanwutai, Chang’an, Shaanxi, China; 7 September 2008; Jianhua Huang Pedopodisma funiushana gl0083-0088 Shiziping, Lushi, Henan, China; 25 September 2008; Jianhua Huang Fruhstorferiola tonkinensis gl0089-0094 Yong’an, Xing’an, Guangxi, China; 2 July 2006; Jianhua Huang Fruhstorferiola huayinensis gl0095-0100 Haopingshi, Taibaishan, Shaanxi, China; 13 July 2005; Zhaoqiang Qian gl0227-0231 Nanwutai, Chang’an, Shaanxi, China; 7 September 2008; Jianhua Huang gl0232-0234 Huayangchuan, Huayin, Shaanxi, China; 3 September 2008; Jianhua Huang gl0235-0240 Baiyunshan, Songxian, Henan, China; 17 August 2008; Jianhua Huang Fruhstorferiola kulinga gl0101-0106 Hengshan, Hunan, China; 29 August 2007; Jianhua Huang gl0107-0108 Jingshan, Jingzhou, Hubei, China; 10 October 2007; Fuming Shi gl0109-0112 Longmenhe, Xingshan, Hubei, China; 4 August 2003; Yulin Zhong gl0113-0115 Gaozhai, Xing’an, Guangxi, China; 9 August 2006; Jianhua Huang Emeiacris maculata gl0241-0246 Hengshan, Hunan, China; 29 August 2008; Jianhua Huang Paratonkinacris vittifemoralis gl0247-0251 Gaozhai, Xing’an, Guangxi, China; 8 July 2009; Jianhua Huang Ognevia longipennis gl0252-0256 Yangjiaping, Zhuolu, Hebei, China; 20 August 2005; Yuan Huang Zxj-4 Qianshan, Liaoning, China; 3 August 2007; Huimin Sun Zcy-2 Yuxian, Hebei, China; 21 August 2005; Yuan Huang Tonkinacris sinensis gl0257-0261 Yong’an, Xing’an, Guangxi, China; 2 July 2006; Jianhua Huang Prumna arctica gl0262-0266 Shibazhan, Heilongjiang, China; 1 September 2007; Huimin Sun Zxj-1 Tahe, Heilongjiang, China; 1 September 2007; Xiaojing Zhang Indopodisma kingdoni gl0267-0271 Chayu, Xizang, China; 1 October 2007; Fuming Shi Zubovskia koeppeni Qzy-M0709 Mianduhe, Yakeshi, Inner Mongolia, China; 21 July 2007; collector unknown Eyprepocnemidinae Shirakiacris shirakii gl0272, 0274-0276 Chang’an, Shaanxi, China; 1 November 2003; Rongsheng Lu gl0273 Yaoshan, Guilin, Guangxi, China; 27 July 2007; Jianhua Huang Lbp-LC12 Chang’an, Shaanxi, China; 1 November, 2001; collector unknown Shirakiacris yunkweiensis gl0277-0281 Xiachayu, Chayu, Xizang, China; 3 October 2007; Zhijun Zhou Lbp-LC13 Dali, Yunnan, China; 4 August 1997; collector unknown Choroedocus violaceipes Cch-M0806 Puer, Yunnan, China; 31 July 2007; Yuan Huang Calliptaminae Calliptamus abbreviatus gl0282-0287 Songbai, Shennongjia, Hubei, China; 31 July 2004; Yulin Zhong gl0342-0346 Siping, Jilin, China; 1 August 2007; Wei Ye gl0347-0351 Daqinggou, Tongliao, Inner Mongolia, China; 1 August 2007; Wei Ye gl0352 Tieling, Liaoning, China; 2 August 2007; Wei Ye gl0353 Anji, Zhejiang, China; 10 July 2006; Fangmei Ding gl0354-0356 Xincheng, Shanxi, China; 15 October 2006; Chenyan Zhang gl0357-0361 Yangjiaping, Zhuolu, Hebei, China; 20 August 2005; Yuan Huang gl0362-0366 Zhidan County, Shaanxi, China; 15 September 2004; Gang Chang gl0367-0371 Qin’an County, Gansu, China; 6 August 2006; Yuan Huang gl0372- gl0376 Xunhua, Qinghai, China; 25 July 2004; Jing Hu Lbp-LC17 Qin’an County, Gansu, China; 8 August 1999; collector unknown Zxj-8 Jiagedaqi, Heilongjiang, China; 6 September 2007; Huimin Sun Lhm-M0505 Yongjing, Gansu, China; 8 August 2001; Yuan Huang Calliptamus barbarus gl0288-0292 Xunhua, Qinghai, China; 25 July 2004; Jing Hu Calliptamus italicus gl0293- 0297 Mulei County, Xinjiang, China; 15 August 2005; collector unknown Zxj-9 Mulei County, Xinjiang, China; 13 August 2005; Xiaojing Zhang Catantopinae pinguis pinguis gl0298-0301 Zhashui, Shaanxi, China; 8 September 2002; collector unknown gl0302 Jiangkou, Ningshan, Shaanxi, China; 13 September 2008; Jianhua Huang Stenocatantops splendens gl0303-0307 Xiachayu, Chayu, Xizang, China; 3 October 2007; Zhijun Zhou Xenocatantops brachycerus gl0308-0312 Huashan, Huayin, Shaanxi, China; 2 September 2008; Jianhua Huang Lbp-LC14 Zhashui, Shaanxi, China; 7 September 2002; collector unknown Spathosterninae Spathosternum prasiniferum sinense gl0313-0317 Yaoshan, Guilin, Guangxi, China; 27 July 2006; Jianhua Huang gl0318-0321 Yaoshan, Guilin, Guangxi, China; 27 July 2006; Jianhua Huang (with tegmina reaching apex of hind femur, 4♀♀ from the same population as gl0313-0317) Spathosternum prasiniferum prasiniferum gl0322-0326 Jin’gu County, Yunnan, China; 31 July 2007; Yuan Huang Oxyinae Toacris yaoshanensis gl0327-0331 Dayaoshan, Jinxiu, Guangxi, China; 12 June 2006; Jianhua Huang Pseudoxya diminuta sl0318-0321 Liuwanshan, Yulin, Guangxi, China; 3 August 2006; Jia Li sl0322-0325 Mengla, Yunnan, China; 31 July 2007; Fuming Shi Oxya sp. sl0330-0332 Haikou, Hainan, China; 12 October 2003; Yuan Huang Oxya chinensis sl0333-0334 Shiziping, Lushi, Henan, China; 20 August 2008; Jianhua Huang Caryanda neoelegans Cch-M0807 Menglun, Yunnan, China; July 1998; Yuan Huang Coptacridinae Traulia minuta sl0311-0315 Caiyanghe, Simao, Yunnan, China; 28 July 2007; Yuan Huang Lgd-02 Caiyanghe, Simao, Yunnan, China; 28 July 2007; Yuan Huang Heiroglyphinae Hieroglyphus annulicornis Cch-M0805 Hangzhou, Zhejiang, China; July 2007; Fangmei Ding Habrocneminae Menglacris maculata Qzy-M0705 Caiyanghe, Puer, Yunnan, China; 28 July 2007; Yuan Huang Oedipodinae Trilophidia annulata gl0116-0120 Yangjiaping, Zhuolu, Hebei, China; 20 August 2005; Yuan Huang gl0121 Baoding, Hebei, China; 19 August 2005; Yuan Huang gl0122-0126 Tieling, Liaoning, China; 2 August 2007; Wei Ye gl0127-0129 Ya’an, Sichuan, China; 11 August 2007; Yuan Huang gl0130-0131 Qin’an, Gansu, China; 6 August 2006; Yuan Huang gl0132-0137 Jinan, Shandong, China; 23 September 2007; Yuan Huang gl0138 Jin’gu, Yunnan, China; 31 July 2007; Yuan Huang gl0139-0143 Menglun, Yunnan, China; 24 July 1998; Yuan Huang gl0144-0145 Lantian, Shaanxi, China; 23 September 2007; collector unknown gl0146-0149 Chang’an, Shaanxi, China; 2 November 2008; Jianhua Huang Wj-HC03 Weiqu, Chang’an, Shaanxi, China; 31 July 2002; Jing Wang Oedaleus decorus gl0150-0155 Mulei, Xinjiang, China; 23 August 2005; collector unknown Zxj-12 Xinyuan, Xinjiang, China; 15 August 2006; Xin Li Wj-HC05 Su'nan, Gansu, China; 26 July 2001; Yuan Huang Oedaleus asiaticus gl0168-0173 Xinbaerhuzuoqi, Inner Mongolia, China; 23 July 2007; Weigong Wang gl0174 Jinhekou, Yuxian, Hebei, China; 23 August 2005; collector unknown gl0175-0179 Qin’an, Gansu, China; 6 August 2006; Yuan Huang gl0180-0183 Xunhua, Qinhai, China; 25 July 2004; Jing Hu gl0184 Hulunbeier, Inner Mongolia; 26 July 2007; Chengying Pan Oedaleus manjius gl0156-0161 Yong’an, Xing’an, Guangxi, China; 4 July 2006; Jianhua Huang Wj-HC06 Zhashui, Shaanxi, China; 17 September 2002; collector unknown Oedaleus infernalis gl0162-0167 Qin’an, Gansu, China; 6 August 2006; Yuan Huang gl0185-0186 Zhashui, Shaanxi, China; 18 September 2002; collector unknown gl0187 Duqu, Chang’an, Shaanxi, China; 13 September 2003; collector unknown gl0188-0190 Xunyangba, Ningshan, Shaanxi, China; 19 September 2002; collector unknown gl0191-0196 Yangjiaping, Zhuolu, Hebei, China; 20 August 2005; Yuan Huang gl0197-0202 Jinan, Shandong, China; 23 September 2007; Yuan Huang gl0203-0208 Alukeerqian, Chifeng, Inner Mengolia, China; 3 August 2007; Wei Ye gl0209-0214 Siping, Jilin, China; 1 August 2007; Wei Ye gl0215-0220 Tieling, Liaoning, China; 3 August 2007; Wei Ye Wj-HC04 Linxia, Gansu, China; 12 August 2001; Yuan Huang Zxj-11a Dunhua, Jilin, China; 12 August 2007; Huimin Sun Zxj-11b Dunhua, Jilin, China; 12 August 2007; Xiaojing Zhang Oedaleus abruptus gl0337-0341 Sanlidian, Guilin, Guangxi, China; 17 September 2009; Jianhua Huang Aiolopus tamulus gl0221-0226 Chang’an, Shaanxi, China; 2 November 2008; Jianhua Huang gl0332-0336 Sanlidian, Guilin, Guangxi, China; 17 September 2009; Jianhua Huang Wj-HCO2 Haikou, Hainan, China; 12 October 2003; Yuan Huang Pternoscirta caliginosa sl0326 Jigongshan, Xinyang, Henan, China; 7 August 2009; Shaoli Mao sl0327-0329 Jiangkou, Ningshan, Shaanxi, China; 13 September 2008; Jianhua Huang Epacromius coerulipes Wsz-4 Qin’an, Guansu, China; May 2006; Yuan Huang Locusta migratoria manilensis Xll-2 Hubei, China; 5 August 2002; Yulin Zhong Gomphocerinae Ceracris nigricornis Xll-M0631 Shennongjia, Hubei, China; 19 August 2004; Yulin Zhong Omocestus haemorrhoidalis sl0335-0339 Huayangchuan, Huayin, Shaanxi, China; 3 September 2008; Jianhua Huang Euchorthippus unicolor sl0340-0344 Huayangchuan, Huayin, Shaanxi, China; 3 September 2008; Jianhua Huang Arcyptera coreana Ln-M0501 Taibaishan, Meixian, Shaanxi, China; 17 July 2002; Nian Liu Leuconemacris litangensis Gj-M0802 Litang, Sichuan, China; 9 August 2007; Yuan Huang Chorthippus brunneus huabeiensis Zxj-17 Beian, Heilongjiang, China; 27 August 2007; Huimin Sun Aeropus licenti Gj-M0803 Inner Mongolia, China; 19 July 2007; collector unknown Mongolotettix japonicus Zxj-30 Wudalianchi, Heilongjiang, China; 25 August 2007; Xin Li Ly-2 Yuxian, Hebei, China; 21 August 2005; Yunlin Zhong Pamphagidae Haplotropis neimongolensis Zxj-29 Jiagedaqi, Heilongjiang, China; 6 September 2007; Huimin Sun Haplotropis brunneriana Hj-M0502 Taibaishan, Meixian, Shaanxi, China; 17 July 2007; Jing Hu Sinotmethis amicus Gj-M0801 Wenquan, Xinjiang, China; 24 July 2005; collector unknown Pyrgomorphoidea Yunnanites coriacea Xll-M0632 Mengla, Yunnan, China; 21 July 2004; collector unknown Eumastacoidea Bennia multispinata Zxj-39 Yunnan, China; 21 July 2004; collector unknown Tetrigoidea Criotettix bispinosus Wsz-1 Beihai, Guangxi, China; February 2005; Shizhen Wei

Table S3 Cross reference list between GenBank accession numbers and numbers of vouchers sharing the same haplotype

GenBank accession number number of vouchers sharing the GenBank accession number number of vouchers sharing the same haplotype same haplotype KC139803 gl0282, gl0283, gl0286 KC139953 gl0032 KC139804 gl0284 KC139954 gl0035 KC139805 gl0285 KC139955 gl0036 KC139806 gl0287 KC139956 gl0037 KC139807 gl0342, gl0343, gl0348, gl0350, KC139957 gl0038 Lbp-LC17 KC139808 gl0344 KC139958 gl0039 KC139809 gl0345 KC139959 gl0l040 KC139810 gl0346 KC139960 gl0041 KC139811 gl0347 KC139961 gl0042 KC139812 gl0349 KC139962 gl0043 KC139813 gl0351 KC139963 gl0044 KC139814 gl0352 KC139964 gl0045 KC139815 gl0354, gl0356 KC139965 gl0046 KC139816 gl0355 KC139966 gl0262 KC139817 gl0353, gl0357, gl0360, gl0361, gl0363, KC139967 gl0263 gl0365, gl0366, gl0370, gl0374, gl0375 KC139818 gl0358 KC139968 gl0264 KC139819 gl0359 KC139969 gl0265 KC139820 gl0362 KC139970 gl0266 KC139821 gl0364 KC139971 Zxj-1 KC139822 gl0367 KC139972 gl0257, gl0260 KC139823 gl0368, gl0369 KC139973 gl0258, gl0259, gl0261 KC139824 gl0371 KC139974 Qzy-M0709 KC139825 gl0372 KC139975 gl0298, gl0299, gl0300, gl0301 KC139826 gl0373, gl0376 KC139976 gl0302 KC139827 Zxj-8 KC139977 gl0303, gl0304, gl0305, gl0307 KC139828 Lhm-M0505 KC139978 gl0306 KC139829 gl0288, gl0289, gl0292 KC139979 gl0308 KC139830 gl0290, gl0291 KC139980 gl0309 KC139831 gl0293, Zxj-9 KC139981 gl0310 KC139832 gl0294, gl0297 KC139982 gl0311 KC139833 gl0295 KC139983 gl0312 KC139834 gl0296 KC139984 Lbp-LC14 KC139835 sl0311 KC139985 gl0313 KC139836 sl0312 KC139986 gl0314, gl0315, gl0316, gl0317, gl0319 KC139837 sl0313 KC139987 gl0318 KC139838 sl0314 KC139988 gl0320, gl0321 KC139839 sl0315 KC139989 gl0322 KC139840 Lgd-02 KC139990 gl0323 KC139841 gl0272, gl0276 KC139991 gl0324 KC139842 gl0273 KC139992 gl0325 KC139843 gl0274 KC139993 gl0326 KC139844 gl0275 KC139994 gl0327, gl0328, gl0329, gl0330, gl0331 KC139845 Lbp-LC12 KC139995 sl0318, sl0319 KC139846 gl0277 KC139996 sl0320 KC139847 gl0278, gl0279, gl0281 KC139997 sl0321 KC139848 gl0280 KC139998 sl0322 KC139849 Lbp-LC13 KC139999 sl0323 KC139850 Cch-M0806 KC140000 sl0330 KC139851 gl0241 KC140001 sl0331 KC139852 gl0242 KC140002 sl0332 KC139853 gl0243 KC140003 sl0333 KC139854 gl0244 KC140004 sl0334 KC139855 gl0245 KC140005 Cch-M0807 KC139856 gl0246 KC140006 Cch-M0805 KC139857 gl0247 KC140007 Qzy-M0705 KC139858 gl0248, gl0249 KC140008 gl0221, gl0223 KC139859 gl0250 KC140009 gl0222 KC139860 gl0251 KC140010 gl0224 KC139861 gl0095, gl0227, gl0231, gl0233 KC140011 gl0225 KC139862 gl0096 KC140012 gl0226, Wj-HCO2 KC139863 gl0097, gl0238 KC140013 gl0332, gl0333, gl0335, gl0336 KC139864 gl0098 KC140014 gl0334 KC139865 gl0099, gl0232, gl0234 KC140015 Wsz-4 KC139866 gl0100 KC140016 Xll-2 KC139867 gl0228 KC140017 gl0168 KC139868 gl0229 KC140018 gl0169 KC139869 gl0230 KC140019 gl0170 KC139870 gl0235 KC140020 gl0171 KC139871 gl0236 KC140021 gl0172, gl0174, gl0175, gl0176, gl0177, gl0178, gl0179, gl0180, gl0181 gl0182, gl0183 KC139872 gl0237, gl0239 KC140022 gl0173 KC139873 gl0240 KC140023 gl0184 KC139874 gl0101 KC140024 gl0150 KC139875 gl0102, gl0105, gl0106, gl0115 KC140025 gl0151, gl0154, gl0155 KC139876 gl0103 KC140026 gl0152 KC139877 gl0104 KC140027 gl0153 KC139878 gl0107 KC140028 Zxj-12 KC139879 gl0108 KC140029 Wj-HC05 KC139880 gl0109 KC140030 gl0162 KC139881 gl0110 KC140031 gl0163, gl0187, Wj-HC04 KC139882 gl0111 KC140032 gl0164, gl0185, gl0191, gl0193, gl0195, gl0196, gl0197, gl0206, gl0215, gl0217, gl0220, Zxj-11a, Zxj-11b KC139883 gl0112 KC140033 gl0165 KC139884 gl0113 KC140034 gl0166 KC139885 gl0114 KC140035 gl0167, gl0216 KC139886 gl0089 KC140036 gl0186 KC139887 gl0090 KC140037 gl0188, gl0190, gl0156, gl0159 KC139888 gl0091 KC140038 gl0189 KC139889 gl0092 KC140039 gl0192, gl0199, gl0201, gl0203, gl0205, gl0207, gl0208, gl0210, gl0211, gl0212, gl0213, gl0214 KC139890 gl0093, gl0094 KC140040 gl0194 KC139891 gl0267 KC140041 gl0198 KC139892 gl0268 KC140042 gl0200 KC139893 gl0269 KC140043 gl0202 KC139894 gl0270 KC140044 gl0204 KC139895 gl0271 KC140045 gl0209 KC139896 gl0252, gl0254, gl0255 KC140046 gl0218 KC139897 gl0253 KC140047 gl0219 KC139898 gl0256, Zxj-4 KC140048 gl0157 KC139899 Zcy-2 KC140049 gl0158, gl0160, gl0161 KC139900 gl0083 KC140050 Wj-HC06 KC139901 gl0087 KC140051 gl0337, gl0338, gl0341 KC139902 gl0088 KC140052 gl0339, gl0340 KC139903 gl0077, gl0080 KC140053 gl0116, gl0118 KC139904 gl0078, gl0079, gl0081, gl0082, gl0084, KC140054 gl0117, gl0133, gl0135, gl0136, gl0137 gl0085, gl0086 KC139905 gl0071 KC140055 gl0119 KC139906 gl0072 KC140056 gl0120 KC139907 gl0073 KC140057 gl0121, gl0124, gl0128, Wj-HC03 KC139908 gl0074 KC140058 gl0122 KC139909 gl0075 KC140059 gl0123 KC139910 gl0076 KC140060 gl0125 KC139911 gl0059, gl0063 KC140061 gl0126 KC139912 gl0060, gl0064 KC140062 gl0127, gl0129 KC139913 gl0061 KC140063 gl0130 KC139914 gl0062 KC140064 gl0131 KC139915 gl0065 KC140065 gl0132 KC139916 gl0066 KC140066 gl0134 KC139917 gl0067 KC140067 gl0138, gl0139 KC139918 gl0068 KC140068 gl0140 KC139919 gl0069 KC140069 gl0141 KC139920 gl0070 KC140070 gl0142 KC139921 gl0053 KC140071 gl0143 KC139922 gl0054 KC140072 gl0144 KC139923 gl0055 KC140073 gl0145 KC139924 gl0056 KC140074 gl0146 KC139925 gl0057 KC140075 gl0147 KC139926 gl0058 KC140076 gl0149 KC139927 gl0047, gl0051 KC140077 sl0326 KC139928 gl0048 KC140078 sl0327 KC139929 gl0049 KC140079 sl0328 KC139930 gl0050 KC140080 sl0329 KC139931 gl0052 KC140081 Gj-M0802 KC139932 gl0017, gl0019, gl0020 KC140082 sl0335 KC139933 gl0021, gl0022 KC140083 sl0336, sl0339 KC139934 gl0026 KC140084 sl0337 KC139935 gl0023 KC140085 sl0338 KC139936 gl0024 KC140086 sl0340 KC139937 gl0025 KC140087 sl0341 KC139938 gl0027 KC140088 sl0342 KC139939 gl0001, gl0016 KC140089 sl0343 KC139940 gl0002, gl0014, gl0015 KC140090 Ln-M0501 KC139941 gl0003, gl0012 KC140091 Xll-M0631 KC139942 gl0004, gl0011 KC140092 Zxj-17 KC139943 gl0005 KC140093 Gj-M0803 KC139944 gl0006 KC140094 Zxj-30 KC139945 gl0007, gl0009 KC140095 Ly-2 KC139946 gl0008 KC140096 Zxj-29 KC139947 gl0010 KC140097 Hj-M0502 KC139948 gl0013 KC140098 Gj-M0801 KC139949 gl0018 KC140099 Xll-M0632 KC139950 gl0029 KC140100 Zxj-39 KC139951 gl0030, gl0034 KC140101 Wsz-1 KC139952 gl0031, gl0033

Table S4. Ranges of intraspecific genetic variations

Species (number of Intraspecific variations Species (number of populations sampled) Intraspecific variations populations sampled) Calliptamus abbreviatus (13) 0~1.08% Prumna arcticus (2) 0.3~2.17% Calliptamus barbarus (1) 0~0.46% Tonkinacris sinensis (1) 0~0.3% Calliptamus italicus (2) 0~0.3% Diobolocatantops pingius (2) 0~0.3% Traulia minuta (1) 0~2.01% Stenocatantops splendens (1) 0~0.3% Shirakiacris shirakii (2) 0~1.7% Xenocatantops brachycerus (2) 0.15~1.07% Shirakiacris yunkweiensis (2) 0~1.54% Spathosternum prasiniferum sinense (1) 0~0.3% Shirakiacris shirakii+yunkweiensis (4) * 0~2.01% Spathosternum prasiniferum prasiniferum (1) 0.15~0.61% Emeiacris maculatus (1) 0~0. 61% Toacris yashanensis (1) 0 Paratonkinacris vittifemoralis (1) 0~1.38% Pseudoxya diminuta (2) 0~1.07% Fruhstorferiola huayinensis (4) 0~1.85% Oxya sp (1) 0.61~2.01% Fruhstorferiola kulinga (4) 0~2.97% Oxya chinensis (2) 0.15~0.46% Fruhstorferiola huayinensis+kulinga (8) * 0~2.97% Aiolopus tamulus (2) 0~2.01% Fruhstorferiola tonkinensis (1) 0~0.77% Locusta migratoria (1) 0~2.96% Indopodisma kindoni (1) 0.46~1.23% Oedaleus decorus (3) 0~2.17% Ognevia longipennis (3) 0~0.46% Oedaleus asiaticus (6) 0~1.23% Pedopodisma funiushana (1) 0~0.76% Oedaleus decorus +asiaticus (9) * 0~2.17% Pedopodisma tsinlingensis (1) 0~0.15% Oedaleus infernalis (11) 0~2.47% Pedopodisma wudangshanensis (1) 0.15~2.17% Oedaleus manjius (2) 0~1.7% Pedopodisma funiushana+tsinlingensis 0~2.33% Oedaleus infernalis +manjius (13) * 0~2.63% + wudangshanensis (3) * Sinopodisma houshana (2) 0~2.17% Oedaleus abrauptus (1) 0~0.15% Sinopodisma lushiensis (1) 0.15~0.61% Trilophidia annulata (11) 0~2.33% Sinopodisma qinlingensis (1) 0~0.61% Pternoscirta caliginosa (1) 0.15~1.07% Sinopodisma houshana+lushiensis+qinlingensis (4) * 0~2.17% Omocestus haemorrhoidalis (1) 0~0.92% Sinopodisma lofaoshana (2) 0~0.92% (within Euchorthippus unicolor (1) 0~0.61% population) 5.06~5.56% (between population) Sinopodisma rostellocerca (4) 0~2.49% Chorthippus parallelus (1) 1.85% Sinopodisma wulingshana (3) 0~2.32% Mongolotettix japonicus (2) 1.54% * The genetic distances between individuals of the same population are always less than 1%, and those between conspecific individuals from different populations of the same species usually less than 3%. The asterisk “*” indicates the species group which might be considered as the same species according to the morphological similarities.

Table S5. Distribution of intraspecific variations and interspecific divergences

Distribution of intraspecific variation Distribution of interspecific divergence within a genus Distance range frequency percentage cumulative Distance range frequency percentage cumulative <= 0.0% 422 10.75% 10.75% <= 0.0% 16 0.29% 0.29% 0.0% to 0.5% 1879 47.83% 58.58% 0.0% to 1.0% 555 10.26% 10.55% 0.5% to 1.0% 854 21.76% 80.34% 1.0% to 2.0% 475 8.77% 19.32% 1.0% to 1.5% 338 8.6% 88.94% 2.0% to 3.0% 172 3.18% 22.5% 1.5% to 2.0% 216 5.49% 94.43% 3.0% to 4.0% 121 2.24% 24.74% 2.0% to 2.5% 191 4.86% 99.29% 4.0% to 5.0% 382 7.05% 31.79% 2.5% to 3.0% 4 0.1% 99.39% 5.0% to 6.0% 1286 23.77% 55.56% 5.0% to 5.5% 20 0.51% 99.9% 6.0% to 7.0% 393 7.26% 62.82% 5.5% to 6.0% 4 0.1% 100% 7.0% to 8.0% 261 4.82% 67.64% 8.0% to 9.0% 1428 26.39% 94.03% 9.0% to 10.0% 191 3.54% 97.56% 10.0% to 11.0% 126 2.32% 99.88% 11.0% to 12.0% 4 0.08% 99.96% 12.0% to 13.0% 1 0.02% 99.98% 13.0% to 14.0% 1 0.02% 100%

Table S6 Sequences identified as ambiguous or incorrect in BCM analysis

Code of query sequence Morphospecies name Identification gl0240 Fruhstorferiola huayinensis Incorrect gl0107 Fruhstorferiola kulinga Incorrect gl0108 Fruhstorferiola kulinga Incorrect gl0109 Fruhstorferiola kulinga Incorrect gl0153 Oedaleus decorus Incorrect Wj-HC05 Oedaleus decorus Incorrect Zxj-12 Oedaleus decorus Incorrect gl0157 Oedaleus manjius Incorrect Wj-HC06 Oedaleus manjius Incorrect FJ531686 Pedopodisma funiushana Incorrect gl0273 Shirakiacris shirakii Incorrect gl0049 Sinopodisma qinlingensis Incorrect gl0100 Fruhstorferiola huayinensis Ambiguous gl0188 Oedaleus infernalis Ambiguous gl0190 Oedaleus infernalis Ambiguous gl0198 Oedaleus infernalis Ambiguous gl0202 Oedaleus infernalis Ambiguous gl0209 Oedaleus infernalis Ambiguous Wj-HC04 Oedaleus infernalis Ambiguous gl0156 Oedaleus manjius Ambiguous gl0159 Oedaleus manjius Ambiguous gl0083 Pedopodisma funiushana Ambiguous gl0084 Pedopodisma funiushana Ambiguous gl0085 Pedopodisma funiushana Ambiguous gl0086 Pedopodisma funiushana Ambiguous gl0087 Pedopodisma funiushana Ambiguous gl0088 Pedopodisma funiushana Ambiguous gl0078 Pedopodisma tsinlingensis Ambiguous gl0079 Pedopodisma tsinlingensis Ambiguous gl0081 Pedopodisma tsinlingensis Ambiguous gl0082 Pedopodisma tsinlingensis Ambiguous gl0280 Shirakiacris yunkweiensis Ambiguous gl0053 Sinopodisma lushiensis Ambiguous gl0058 Sinopodisma lushiensis Ambiguous gl0048 Sinopodisma qinlingensis Ambiguous

Table S7. Assignment of species through BP-based method.

Query sequence code Predicted species Probability #1 C_abb285 Calliptamus abbreviatus 0.942349 #2 C_abb286 Calliptamus abbreviatus 0.984127 #3 C_abb287 Calliptamus abbreviatus 0.984675 #4 C_abb345 Calliptamus abbreviatus 0.978831 #5 C_abb346 Calliptamus abbreviatus 0.989758 #6 C_abb349 Calliptamus abbreviatus 0.988526 #7 C_abb350 Calliptamus abbreviatus 0.990107 #8 C_abb351 Calliptamus abbreviatus 0.98952 #9 C_abb353 Calliptamus abbreviatus 0.988123 #10 C_abb356 Calliptamus abbreviatus 0.990981 #11 C_abb359 Calliptamus abbreviatus 0.988024 #12 C_abb360 Calliptamus abbreviatus 0.988123 #13 C_abb361 Calliptamus abbreviatus 0.988123 #14 C_abb365 Calliptamus abbreviatus 0.988123 #15 C_abb366 Calliptamus abbreviatus 0.988123 #16 C_abb369 Calliptamus abbreviatus 0.989089 #17 C_abb370 Calliptamus abbreviatus 0.988123 #18 C_abb371 Calliptamus abbreviatus 0.988459 #19 C_abb375 Calliptamus abbreviatus 0.988123 #20 C_abb376 Calliptamus abbreviatus 0.986713 #21 C_abb_Lhm Calliptamus abbreviatus 0.986894 #22 C_abb_zxj Calliptamus abbreviatus 0.989757 #23 C_bar291 Calliptamus barbarus 0.959281 #24 C_bar292 Calliptamus abbreviatus 0.965733 #25 C_ita297 Calliptamus italicus 0.975577 #26 C_ita_NC_011305 Calliptamus italicus 0.975563 #27 C_ita_zxj Calliptamus italicus 0.980694 #28 E_mac244 Emeiacris maculata 0.974642 #29 E_mac245 Emeiacris maculata 0.974519 #30 E_mac246 Emeiacris maculata 0.97235 #31 P_vit250 Paratonkinacris vittifemoralis 0.986012 #32 P_vit251 Paratonkinacris vittifemoralis 0.983906 #33 F_hua098 Fruhstorferiola huayinensis 0.511742 #34 F_hua099 Fruhstorferiola huayinensis 0.975892 #35 F_hua100 Fruhstorferiola huayinensis 0.49754 #36 F_hua230 Fruhstorferiola huayinensis 0.989397 #37 F_hua231 Fruhstorferiola huayinensis 0.988623 #38 F_hua233 Fruhstorferiola huayinensis 0.988623 #39 F_hua234 Fruhstorferiola huayinensis 0.975892 #40 F_hua238 Fruhstorferiola huayinensis 0.984397 #41 F_hua239 Fruhstorferiola huayinensis 0.981827 #42 F_hua240 Fruhstorferiola kulinga 0.985128 #43 F_kul104 Fruhstorferiola kulinga 0.988717 #44 F_kul105 Fruhstorferiola kulinga 0.988874 #45 F_kul106 Fruhstorferiola kulinga 0.988874 #46 F_kul108 Fruhstorferiola huayinensis 0.953346 #47 F_kul111 Fruhstorferiola kulinga 0.967439 #48 F_kul112 Fruhstorferiola kulinga 0.967786 #49 F_kul115 Fruhstorferiola kulinga 0.988874 #50 F_ton092 Fruhstorferiola tonkinensis 0.967645 #51 F_ton093 Fruhstorferiola tonkinensis 0.976889 #52 F_ton094 Fruhstorferiola tonkinensis 0.976889 #53 I_kin270 Indopodisma kingdoni 0.983393 #54 I_kin271 Indopodisma kingdoni 0.975119 #55 O_lon256 Ognevia longipennis 0.973263 #56 O_lon_zxj Ognevia longipennis 0.973263 #57 O_lon_zcy Ognevia longipennis 0.961679 #58 P_fun_084 Pedopodisma tsinlingensis 0.973709 #59 P_fun_085 Pedopodisma tsinlingensis 0.973709 #60 P_fun_086 Pedopodisma tsinlingensis 0.973709 #61 P_tsi_080 Pedopodisma tsinlingensis 0.972226 #62 P_tsi_081 Pedopodisma tsinlingensis 0.973709 #63 P_tsi_082 Pedopodisma tsinlingensis 0.973709 #64 P_wud_074 Pedopodisma funiusha 0.968561 #65 P_wud_075 Pedopodisma wudangshanensis 0.942986 #66 P_wud_076 Pedopodisma funiusha 0.892706 #67 S_hou_062 Sinopodisma houshana 0.952429 #68 S_hou_063 Sinopodisma houshana 0.971164 #69 S_hou_064 Sinopodisma houshana 0.976509 #70 S_hou_068 Sinopodisma houshana 0.978234 #71 S_hou_069 Sinopodisma houshana 0.979464 #72 S_hou_070 Sinopodisma houshana 0.975178 #73 S_lus_056 Sinopodisma lushiensis 0.957328 #74 S_lus_057 Sinopodisma lushiensis 0.952633 #75 S_lus_058 Sinopodisma lushiensis 0.536554 #76 S_qin_050 Sinopodisma qinlingensis 0.952128 #77 S_qin_051 Sinopodisma qinlingensis 0.979646 #78 S_qin_052 Sinopodisma qinlingensis 0.943538 #79 S_lof021 Sinopodisma lofaoshana 0.973749 #80 S_lof022 Sinopodisma lofaoshana 0.973749 #81 S_lof026 Sinopodisma lofaoshana 0.957353 #82 S_lof025 Sinopodisma lofaoshana 0.973094 #83 S_lof027 Sinopodisma lofaoshana 0.973583 #84 S_ros_006 Sinopodisma rostellocerca 0.973192 #85 S_ros_009 Sinopodisma rostellocerca 0.990078 #86 S_ros_010 Sinopodisma rostellocerca 0.983077 #87 S_ros_012 Sinopodisma rostellocerca 0.98374 #88 S_ros_013 Sinopodisma rostellocerca 0.982853 #89 S_ros_015 Sinopodisma rostellocerca 0.988624 #90 S_ros_016 Sinopodisma rostellocerca 0.989035 #91 S_wul_032 Sinopodisma wulingshana 0.933971 #92 S_wul_033 Sinopodisma wulingshana 0.981355 #93 S_wul_034 Sinopodisma wulingshana 0.98537 #94 S_wul_038 Sinopodisma wulingshana 0.977013 #95 S_wul_039 Sinopodisma wulingshana 0.943744 #96 S_wul_040 Sinopodisma wulingshana 0.955066 #97 S_wul_044 Sinopodisma wulingshana 0.984185 #98 S_wul_045 Sinopodisma wulingshana 0.955145 #99 S_wul_046 Sinopodisma wulingshana 0.969427 #100 P_arc265 Prumna arctica 0.972714 #101 P_arc266 Prumna arctica 0.95596 #102 P_arc_FJ531674 Prumna arctica 0.958999 #103 T_sin260 Tonkinacris sinensis 0.960067 #104 T_sin261 Tonkinacris sinensis 0.965169 #105 T_min_sl0315 Traulia minuta 0.970592 #106 T_min_Lgd Traulia minuta 0.975628 #107 T_min_FJ571149 Traulia minuta 0.968252 #108 S_shi275 Shirakiacris shirakii 0.956174 #109 S_shi276 Shirakiacris shirakii 0.960738 #110 S_shi_Lbp Shirakiacris shirakii 0.957122 #111 S_yun280 Shirakiacris shirakii 0.543701 #112 S_yun281 Shirakiacris yunkweiensis 0.968045 #113 S_yun_Lbp Shirakiacris shirakii 0.912641 #114 D_pin301 Diabolocatantops pinguis 0.967153 #115 D_pin302 Diabolocatantops pinguis 0.972652 #116 S_spl306 Stenocatantops splendens 0.947883 #117 S_spl307 Stenocatantops splendens 0.965141 #118X_bra311 Xenocatantops brachycerus 0.977701 #119X_bra312 Xenocatantops brachycerus 0.976412 #120X_bra_Lbp Xenocatantops brachycerus 0.976921 #121 S_sin316 Spathosternum prasiniferum sinense 0.974541 #122 S_sin317 Spathosternum prasiniferum sinense 0.974541 #123 S_sin320 Spathosternum prasiniferum sinense 0.972301 #124 S_sin321 Spathosternum prasiniferum sinense 0.972301 #125 S_pra325 Spathosternum prasiniferum prasiniferum 0.966416 #126 S_pra326 Spathosternum prasiniferum prasiniferum 0.972269 #127 T_yao330 Toacris yaoshanensis 0.963296 #128 T_yao331 Toacris yaoshanensis 0.963296 #129 P_dim_sl0320 Pseudoxya diminuta 0.988669 #130 P_dim_sl0321 Pseudoxya diminuta 0.987679 #131 P_dim_sl0324 Pseudoxya diminuta 0.987821 #132 P_dim_sl0325 Pseudoxya diminuta 0.987821 #133 O_chi_NC_010219 Oxya chinensis 0.970709 #134 A_tam224 Aiolopus tamulus 0.97805 #1351 A_tam225 Aiolopus tamulus 0.982141 #136 A_tam226 Aiolopus tamulus 0.970058 #137 A_tam335 Aiolopus tamulus 0.973667 #138 A_tam336 Aiolopus tamulus 0.973667 #139 A_tam_Wj Aiolopus tamulus 0.970058 #140 L_mig_Xll Locusta migratoria 0.955747 #141 O_asi171 Oedaleus asiaticus 0.968128 #142 O_asi172 Oedaleus asiaticus 0.98822 #143 O_asi173 Oedaleus asiaticus 0.95671 #144 O_asi177 Oedaleus asiaticus 0.98822 #145 O_asi178 Oedaleus asiaticus 0.98822 #146 O_asi179 Oedaleus asiaticus 0.98822 #147 O_asi182 Oedaleus asiaticus 0.98822 #148 O_asi183 Oedaleus asiaticus 0.98822 #149 O_dec153 Oedaleus asiaticus 0.836449 #150 O_dec154 Oedaleus decorus 0.95852 #151 O_dec155 Oedaleus decorus 0.95852 #152 O_dec_Wj Oedaleus asiaticus 0.810996 #153 O_inf165 Oedaleus infernalis 0.975717 #154 O_inf166 Oedaleus infernalis 0.97515 #155 O_inf167 Oedaleus infernalis 0.978689 #156 O_inf186 Oedaleus infernalis 0.913264 #157 O_inf189 Oedaleus infernalis 0.980189 #158 O_inf190 Oedaleus infernalis 0.980211 #159 O_inf194 Oedaleus infernalis 0.97929 #160 O_inf195 Oedaleus infernalis 0.979851 #161 O_inf196 Oedaleus infernalis 0.979851 #162 O_inf200 Oedaleus infernalis 0.871336 #163 O_inf201 Oedaleus infernalis 0.983077 #164 O_inf202 Oedaleus infernalis 0.967786 #165 O_inf206 Oedaleus infernalis 0.979851 #166 O_inf207 Oedaleus infernalis 0.983077 #167 O_inf208 Oedaleus infernalis 0.983077 #168 O_inf212 Oedaleus infernalis 0.983077 #169 O_inf213 Oedaleus infernalis 0.983077 #170 O_inf214 Oedaleus infernalis 0.983077 #171 O_inf218 Oedaleus infernalis 0.97312 #172 O_inf219 Oedaleus infernalis 0.967184 #173 O_inf220 Oedaleus infernalis 0.979851 #174 O_inf_Wj Oedaleus infernalis 0.949014 #175 O_man156 Oedaleus infernalis 0.980211 #176 O_man159 Oedaleus infernalis 0.980211 #177 O_man161 Oedaleus manjius 0.974857 #178 O_abr340 Oedaleus abruptus 0.970211 #179 O_abr341 Oedaleus abruptus 0.97189 #180 T_ann119 Trilophidia annulata 0.992431 #181 T_ann120 Trilophidia annulata 0.99051 #182 T_ann121 Trilophidia annulata 0.992201 #183 T_ann125 Trilophidia annulata 0.993663 #184 T_ann126 Trilophidia annulata 0.988524 #185 T_ann128 Trilophidia annulata 0.992201 #186 T_ann129 Trilophidia annulata 0.992064 #187 T_ann131 Trilophidia annulata 0.991696 #188 T_ann135 Trilophidia annulata 0.991248 #189 T_ann136 Trilophidia annulata 0.991248 #190 T_ann137 Trilophidia annulata 0.991248 #191 T_ann141 Trilophidia annulata 0.992877 #192 T_ann142 Trilophidia annulata 0.991527 #193 T_ann143 Trilophidia annulata 0.992305 #194 T_ann145 Trilophidia annulata 0.992925 #195 T_ann148 Trilophidia annulata 0.991248 #196 T_ann149 Trilophidia annulata 0.991992 #197 P_cal_sl0328 Pternoscirta caliginosa 0.976905 #198 P_cal_sl0329 Pternoscirta caliginosa 0.976517 #199 O_hae_sl0338 Omocestus haemorrhoidalis 0.971946 #200 O_hae_sl0339 Omocestus haemorrhoidalis 0.974353 #201 E_uni_sl0343 Euchorthippus unicolor 0.980456 #202 E_uni_sl0344 Euchorthippus unicolor 0.980456