DNA barcoding has been proved successful to provide resolution beyond the boundaries of morphological information. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Hexacentrus species in China collections. In total, 83 specimens of five Hexacentrus species were barcoded using standard mitochondrial cytochrome c oxidase subunit I (COI) gene. Except for H. japonicus, barcode gaps were present in the remaining Hexacentrus species. Taxon ID tree generated seven BOLD’s barcode index numbers (BINs), four of which were in agreement with the morphological species. For H. japonicus, the maximum intraspecific divergence (4.43%) produced a minimal overlap (0.64%), and 19 specimens were divided into three different BINs. There may be cryptic species within the current H. japonicus. This study adds to a growing body of DNA barcodes that have become available for katydids, and shows that a DNA barcoding approach enables the identification of known Hexacentrus species with a very high resolution.

BOLD, China, DNA Barcoding, Hexacentrus , species delineation

DNA barcoding employs short, standardized gene regions (5' segment of mitochondrial cytochrome oxidase subunit I for animals) as an internal tag to enable metazoan species identification (Hebert et al. 2003). Schmidt et al. (2015) found that DNA barcoding largely supported 250 years of classical taxonomy for central European bees. Unlike distinct species, closely related species offer a great challenge for phylogeny reconstruction and species identification with DNA barcoding due to overlapping genetic variation (Dai et al. 2012). For example, Versteirt et al. (2015) found that DNA barcoding offered a reliable framework for mosquito species identification in Belgium except for some closely related species. Zhou et al. (2012) found that molecular identification with DNA barcoding supported most traditional morphological species of genus Ruspolia in China.

In this study, our objective is to assess the utility of DNA barcoding for closely related katydid species, belonging to the genus Hexacentrus (Serville, 1831) in China. Hexacentrus is mainly distributed in Australian, Afrotropical and Oriental realms. Hexacentrus is a particularly speciose genus, containing 24 known species (Eades et al. 2016). Hexacentrus was the single genus within Hexacentrinae, which has been reported in China according to “Orthoptera Species File” (Eades et al. 2016). Up to now, a total of six Hexacentrus species have been reported, including H. japonicus (Karny, 1907), H. unicolor (Serville, 1831), H. yunnaneus (Bey-Bienko, 1962), H. fuscipes (Matsumura & Shiraki, 1908), H. mundus (Walker, 1869) and H. expansus (Wang and Shi 2005). Due to the rather difficult morphological discrimination between Hexacentrus species, an interspecific molecular delineation was needed. To make Hexacentrus more accessible to the scientific community, the open access project “BHC” had been initiated in the Barcoding of Life Data systems (BOLD) (Ratnasingham and Hebert 2007). There are a few DNA barcoding study concentrated on Hexacentrus, and the number of barcode sequences was limited in BOLD. The goals of this study are as following: (i) it will allow scientists with molecular capability but insufficient knowledge of Hexacentrus taxonomy and systematics to recognize species and document the biodiversity of Hexacentrus. (ii) For Tettigoniidae taxonomists, it contributes to integrative taxonomic approaches, such as the elucidation of related species and clarification of problematic species groups, association of the sexes within one species, and the identification of new species (Gibbs 2009, 2011, Packer et al. 2009, Schmidt et al. 2015). To this end, we checked for the presence of species barcode gaps and cryptic diversity within species. BOLD’s barcode index number (BIN) analysis tool (Ratnasingham and Hebert 2013) was used to analyze Hexacentrus.

COI sequences were recovered from 86 of the 91 specimens that were analyzed with barcode compliant records from 83 specimens representing five species. Three records have no barcode compliant records because of low quality of trace file. A number of 80 barcodes belong to four previously identified species whereas three analyzed specimens were only identified to genus level because they are female; they probably are H. yunnaneus due to collection in Yunnan and separated from other specimens. The 658bp length sequences without indel (insertion/deletion) had full-length records. COI sequences were translated to amino acid sequences to check for stop codons and shifts in reading frame that might indicate the presence of nuclear mitochondrial copies (numts), but none were detected. Diagnostic character analysis was consistent with that of traditional external appearance discrimination. H. expansus only having one specimen was not analyzed, thus lacking diagnostic character.

Distance summary and Barcode Gap analysis

Mean intraspecies divergence was 1.32% (ranged between 0.57% and 2.43%), and maximum intraspecies divergence 4.43% was observed in H. japonicus (Table 1). When correcting for the uneven sample sizes of species, the within-species divergence decreased from 1.32% to 1.23%. Between Hexacentrus species, the average K2P genetic distance was 12.54%, whereas minimum genetic distance only 3.79% (Table 1).

Table 1.

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Mean and maximum intraspecific and nearest neighbor (NN) distance for all specimens.

Species	Mean intraspecific distance	Max intraspecific distance	Nearest neighbor	Distance to NN
Hexacentrus expansus	N/A	N/A	Hexacentrus unicolor	13.19
Hexacentrus japonicus	2.43	4.43	Hexacentrus mundus	3.79
Hexacentrus mundus	0.57	0.93	Hexacentrus japonicus	3.79
Hexacentrus sp.	0.72	1.08	Hexacentrus unicolor	9.72
Hexacentrus unicolor	1.19	3.79	Hexacentrus sp.	9.72

Singleton species (H. expansus) were excluded from barcode gap analysis. Except for H. japonicus, barcode gap was present in the remaining Hexacentrus species (Fig. 1). Although the maximum intraspecific divergence of H. unicolor was more than 2% (ranged between 0 and 3.79%), but still less than minimum interspecific between H. unicolor and its NN (Nearest Neighbor) Hexacentrus spp. However, the maximum intraspecific divergence of H. japonicus (4.43%) produced a minimal overlap (0.64%).

Figure 1. 

Barcode gap plot showed the distance to the nearest neighbor (NN) vs. the maximum intraspecific distance Kimura-2-parameter (K2P) for 83 specimens. Dots above the 1:1 line indicated the presence of a barcode gap.

Taxon ID tree analysis

The taxon ID tree was divided into seven clades represented by different BINs (Fig. 2). Hexacentrus unicolor, H. mundus and H. expansus were composed well-supported monophyletic groups, which were fully congruent with the morphological species. Hexacentrus japonicus were divided into three different BINs. Although no morphological differences were observed among these three BINs, there might be cryptic species within the current H. japonicus.

Figure 2. 

Taxon ID tree revealed seven well-differentiated haplogroups. Process ID, location, and BINs were shown in the tree. The clusters with a blue box indicated there may be two new putative ‘cryptic species’ within H. japonicus. The clade with a black box indicated the specimen had more mutation within H. unicolor.

The present study evaluated the efficacy of using DNA barcodes for the identification of Hexacentrus in China and provided a group of sequences associated with the identified species. Using these DNA barcoding, not only can one delineate the boundaries between species, but also assign taxonomic status to unknown specimens from known species.

Hexacentrus unicolor was controversial, and H. plantaris (Burmeister, 1838) and Tedla sellata (Walker, 1869) were considered as its synonyms. H. unicolor is distributed in south of the Yangtze River. In this study, however, one specimen was collected from Henan. In fact, molecular data support H. unicolor as a single group, with all specimens sharing one BIN (BOLD: ACD 7247). The specimen with black box (Fig. 2) came from Guizhou, and had more mutations compared to the other H. unicolor specimens. We cannot be completely certain that this phenomenon was due to geographic isolation rather than sequencing or calibrating errors. Hexacentrus japonicus was closely related to H. unicolor, and they are widely distributed in the southwest, central and south areas of China. The tegmina of male H. japonicas was short and broad (about 2.75–3.00 times), whereas H. unicolor was long and narrow (about 2.95–3.30 times) as long as broad (Wang and Shi 2005). Nevertheless, the analyses revealed that H. japonicus ranged to Hebei and Liaoning. Interestingly, H. japonicus contained three BINs (BOLD: ACX 8110, BOLD: ACD 8277, BOLD: ACD 8278) with high intraspecific distance (> 2%). This group included 20 specimens, in which no morphological differences were found. Therefore it is necessary to clarify the status of this species complex as it may include more species than currently recognized. Hexacentrus mundus was only recorded in Guangxi and Yunnan. In this study, four specimens from Hainan were also identified as H. mundus because only 2–3 larger teeth in the middle part of stridulatory file, however there are 6–7 large teeth in H. japonicas and H. unicolor (Wang and Shi 2005). Thus H. mundus’s distribution was enlarged. All specimens of H. mundus were assigned as 1 BIN, which clearly confirm a consistency between molecular and morphological analyses. Hexacentrus expansus, due to the obviously inflated male tegmina, was easy to identify only by the morphological method. BIN assignments also revealed that H. expansus was a separated clade with only a single male available for analysis. The specimens from Yunnan almost certainly represent the ‘true’ H. yunnaneus because the type location of this species (Hekou, Yunnan) was in close proximity. Hence, further specimens are needed to be analyzed, especially male material.

DNA barcoding, as one effective tool in insect taxonomy, had been already applied widely. It can rapidly acquire molecular data, simplifying species classification and identification. Yet, DNA barcodes has been argued to be unreliable for consistent species identification by many authors due to a number of drawbacks (DeSalle et al. 2005; Will et al. 2005; Rubinoff et al. 2006; Ebach 2011). Recent speciation, incomplete lineage sorting, interspecific hybridization and infection by endosymbiotic bacteria such as Wolbachia (Funk and Omland 2003) may all interfere with the performance of DNA barcoding in insects (Virgilio et al. 2010). In this context, most of the species can be amplified successfully; however, five specimens cannot be translated and three specimens only had one represented trace file, which cannot meet the DNA barcoding standards. The deep inspection of trace files indicated that most of these failures arose from co-amplification of the bacterial endosymbiont Wolbachia, which disturbed normal interpretation of trace file. It had been estimated that Wolbachia is present in two-thirds of all insect species (Hilgenboecker et al. 2008). There was no reason to doubt the absence of Wolbachia in Hexacentrus. Zhou et al. (2014) found that the nuclear sequence of mitochondrial (numts) reported in Mecopoda niponensis may form a separate clade. The same case was reported in Podisma pedestris (Bensasson et al. 2000). But in this study, the clades in H. japonicus were not caused by numts for all sequences analyzed without translation early termination, base indel, frameshift mutations. On the other hand, geographic isolation was also rejected, for only the specimens from Yunnan included three BINs in this group. The most probable reason was the existence of cryptic species compared to other Hexacentrus species.

Species boundaries were hard to delimit only based on morphologyas, and analyses including additional sources of information such as molecular data, biogeography, behavior and ecology has been called integrative taxonomy which has been shown to be very useful (Dayrat 2005). We are convinced that DNA barcoding can promote the Hexacentrus species identification. Our study showed that all known Hexacentrus species could be delimitating rapidly through DNA barcoding in China, except for H. japonicus. Hexacentrus japonicus was problematic when using BOLD analysis. Finally, regardless of the promising results, the incorporation of nuclear genes is valuable for species delimitation and might strengthen the results, as they are independent of the maternal inherited mitochondrial genes.

The ideal situation would be that each species was represented by sequence from its type material, particularly the holotype. Type specimens were also dried specimens and DNA degraded at different level, so not only amplification was difficult, but also the damage of specimens can’t be neglected. Recently, Prosser et al. (2016) successfully obtained sequences from century-old type specimens using next-generation sequencing (NGS). We believed that DNA barcoding is useful in revealing cryptic biodiversity, potentially facilitating traditional taxonomy in future.

We are grateful to the following people for collecting specimens and providing valuable comments during the manuscript preparation: Qiong Song; Ji–Yuan Feng; Zhi–Lin Chen; Bao–Jie Du; Xun Bian. This study was supported by National Natural Science Foundation of China (No. 31471985), and Excellent Youth Scholars Program of Higher Education of Hebei Province (No. BJ2014006).