Research Article |
Corresponding author: Zhijun Zhou ( zhijunzhou@163.com ) Academic editor: Zhu-Qing He
© 2022 Yizheng Zhao, Hui Wang, Huimin Huang, Zhijun Zhou.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhao Y, Wang H, Huang H, Zhou Z (2022) A DNA barcode library for katydids, cave crickets, and leaf-rolling crickets (Tettigoniidae, Rhaphidophoridae and Gryllacrididae) from Zhejiang Province, China. ZooKeys 1123: 147-171. https://doi.org/10.3897/zookeys.1123.86704
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Barcode libraries are generally assembled with two main objectives in mind: specimen identification and species discovery/delimitation. In this study, the standard COI barcode region was sequenced from 681 specimens belonging to katydids (Tettigoniidae), cave crickets (Rhaphidophoridae), and leaf-rolling crickets (Gryllacrididae) from Zhejiang Province, China. Of these, four COI-5P sequences were excluded from subsequent analyses because they were likely NUMTs (nuclear mitochondrial pseudogenes). The final dataset consisted of 677 barcode sequences representing 90 putative species-level taxa. Automated cluster delineation using the Barcode of Life Data System (BOLD) revealed 118 BINs (Barcodes Index Numbers). Among these 90 species-level taxa, 68 corresponded with morphospecies, while the remaining 22 were identified based on reverse taxonomy using BIN assignment. Thirteen of these morphospecies were represented by a single barcode (so-called singletons), and each of 19 morphospecies were split into more than one BIN. The consensus delimitation scheme yielded 55 Molecular Operational Taxonomic Units (MOTUs). Only four morphospecies (Imax > DNN) failed to be recovered as monophyletic clades (i.e., Elimaea terminalis, Phyllomimus klapperichi, Sinochlora szechwanensis and Xizicus howardi), so it is speculated that these may be species complexes. Therefore, the diversity of katydids, cave crickets, and leaf-rolling crickets in Zhejiang Province is probably slightly higher than what current taxonomy would suggest.
Barcode Index Number, cryptic species, Ensifera, Orthoptera, species delimitation
Accurate specimen identification and species discovery are fundamental to taxonomic research and essential prerequisite for many fields of research such as ecology, biogeography, and conservation biology (
Cryptic species generally refer to highly genetically differentiated, but morphologically indistinguishable species (
Effective identification of a query specimen through DNA barcode sequence requires reliable reference libraries of known taxa. The process of assembling comprehensive and high-quality reference libraries of DNA barcodes allows the identification of newly collected specimens and accelerates taxonomic progress. The use of DNA barcoding for specimen identification and species discovery is greatly facilitated by the Barcode of Life Data System (BOLD, http://www.boldsystems.org).
Members of the suborder Ensifera diverged into grylloid (crickets) and non-grylloid (katydids) clades at the Triassic/Jurassic boundary (
Much research has been done on Zhejiang katydid and related ensiferan groups (
Collections were performed throughout Zhejiang Province, China in the period of 2011–2019. Collection information (Fig.
Total genomic DNA was extracted from hind legs of adults (N = 676) and nymphs (N = 5) using the Dneasy Blood and Tissue Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s specifications. The remainder of the specimen was retained as a voucher stored at the Katydids Lab of Hebei University, China. The COI barcode region was amplified with primers COBU (5´-TYT CAA CAA AYC AYA ARG ATA TTG G-3´) and COBL (5´-TAA ACT TCW GGR TGW CCA AAR AAT CA-3´) (
Forward and reverse sequences were trimmed, edited, and assembled to produce a consensus barcode sequence using SeqMan Pro (DNA star, Inc., Madison, Wisconsin, USA) for each specimen. All COI-5P barcode sequences were examined for potential stop codons using Editseq (DNA star, Inc., Madison, Wisconsin, USA). All sequences were aligned by employing MUSCLE (codons) algorithm (Edgar, 2004) with default parameters in MEGA ver. 7.0 (
In addition to BIN Discordance analysis, we also used other molecular delineation methods to delineate MOTUs. To minimize the risk of oversplitting (
The results of different species delimitation methods were pairwise compared. Firstly, match ratio [2×Nmatch/(NA+NB)] (
The COI-5P of 681 specimens of katydids, cave crickets, and leaf-rolling crickets were sequenced. Among these specimens, 601 (88.25%) specimens were identified to 69 morphospecies (formally described species that are typically defined by distinct morphological characters) and the remaining 80 specimens were only identified at genus level (Tables
The preliminary “BIN Discordance” analysis (using BOLD ver.4 on 28 Dec., 2021) revealed five cases of merging, where each of the five BINs included two species from different genera or higher taxonomic taxa (Table
Results of the internal BIN discordance report for the five BINs of 83 specimens. # sequences have been resubmitted, * possibly NUMT coamplification.
BIN | Conflicting species | Taxonomic rank |
---|---|---|
ADE4649 | Diestramima austrosinensis (6) Conocephalus gladiatus DBTZC033-21# | family |
ACD8581 | Conocephalus gladiatus (17) Tegra novaehollandiae DBTZC057-21* | subfamily |
ACD7803 | Isopsera sulcate (4) Orophyllus montanus RBTC2009-18* | subfamily |
ACD7324 | Ducetia japonica (47) Sinochlora szechwanensis RBTC2050-18* | genus |
ADF2961 | Melaneremus laticeps (4)Phryganogryllacris DBTZC097-21* | genus |
Genetic distances for the resulting sequences were calculated in the BOLD System Distance Summary and Barcode Gap Analysis tools based on the K2P model. Table
BIN assignments and genetic divergence of 68 morphospecies. BIN, Barcode Index Number; N, number of barcodes per BIN; Imean, mean intraspecific distance; Imax, maximum intraspecific distance; DNN, distance to nearest neighbour; species in bold and labelled* Imax> DNN. Singletons are labeled as N/A and could not be evaluated.
Species | BIN (N) | I mean | I max | Nearest Neighbour | DNN |
---|---|---|---|---|---|
Gryllacrididae | |||||
Apotrechus bilobus | ADF4059 (3) | 0.31 | 0.46 | Eugryllacris elongata DBTZC100-21 | 14.88 |
Capnogryllacris melanocrania | ADF2751 (1) AEJ4972 (1) ADF2750 (2) AEJ9445 (2) | 3.21 | 5.01 | Eugryllacris elongata GRY018-16 | 16.4 |
Eugryllacris elongata | AEK0366 (1) ADF4811 (5) | 3.63 | 10.84 | Apotrechus bilobus DBTZC103-21 | 14.88 |
Homogryllacris anelytra | ADF3866 (3) | 0.83 | 1.09 | Phryganogryllacris xiai GRY040-16 | 17.42 |
Melaneremus fuscoterminatus * | ADF2959 (1) ADF2960 (1) | 14.73 | 14.73 | Melaneremus laticeps DBTZC101-21 | 3.78 |
Melaneremus laticeps | ADF2961 (4) | 0.08 | 0.15 | Melaneremus fuscoterminatus GRY049-16 | 3.78 |
Metriogryllacris permodesta | ADF4959 (1) | N/A | 0 | Phryganogryllacris xiai GRY040-16 | 18.4 |
Phryganogryllacris superangulata | ADF3568 (5) | 0.15 | 0.31 | Capnogryllacris melanocrania DBTZC078-21 | 18.92 |
Phryganogryllacris xiai | ADF3457 (1) | N/A | 0 | Homogryllacris anelytra DBTZC096-21 | 17.42 |
Rhaphidophoridae | |||||
Diestramima austrosinensis | ADE4649 (6) | 0.3 | 0.61 | Diestramima brevis DBTZC116-21 | 5.39 |
Diestramima brevis | AEJ2460 (5) | 0.37 | 0.93 | Diestramima austrosinensis DBTZC054-21 | 5.39 |
Gymnaetoides testaceus | AEJ5191 (3) | 1.45 | 2.18 | Tachycines meditationis DBTZC126-21 | 11.39 |
Microtachycines elongatus | AEJ2738 (2) | 0.62 | 0.62 | Tachycines meditationis DBTZC130-21 | 11.75 |
Tachycines meditationis | AEJ6894 (1) AEK0279 (2) AEJ9615 (4) | 1.77 | 3.31 | Gymnaetoides testaceus DBTZC123-21 | 11.39 |
Tettigoniidae | |||||
Atlanticus interval | ADE2184 (3) | 0.72 | 1.08 | Holochlora venusta RBTC2022-18 | 19.38 |
Conocephalus bidentatus | ADB6577 (1) | N/A | 0 | Conocephalus maculatus RBTC1645-16 | 18.31 |
Conocephalus gladiatus | ADE4649 (1) ACD8581 (17) | 1.06 | 2.18 | Conocephalus maculatus RBTC1645-16 | 16.97 |
Conocephalus maculatus | ACD2116 (1) ADB5579 (2) | 3.62 | 5.43 | Conocephalus gladiatus DBTZC032-21 | 16.97 |
Conocephalus melaenus | ACD4634 (20) | 0.1 | 0.31 | Conocephalus gladiatus DBTZC032-21 | 17.65 |
Deflorita deflorita | ADB3725 (14) | 0.79 | 2.67 | Hemielimaea chinensis RBTC2067-18 | 16.35 |
Ducetia japonica | ACD7324 (47) | 1.23 | 2.67 | Kuwayamaea brachyptera DBTZC001-21 | 14.3 |
Elimaea annamensis | ADE1944 (9) | 0.46 | 1.55 | Elimaea terminalis RBTC2046-18 | 6.98 |
Elimaea cheni | ADB3480 (13) | 0.09 | 0.46 | Elimaea nanpingensis DBTZC006-21 | 9.69 |
Elimaea nanpingensis | ADB3475 (12) | 0.06 | 0.17 | Elimaea cheni DBTZC026-21 | 9.69 |
Elimaea terminalis * | ADB3392 (3) ADB3394 (3) | 6.35 | 10.68 | Elimaea annamensis RBTC1668-16 | 6.98 |
Euconocephalus nasutus | ACD6726 (2) | 1.39 | 1.39 | Ruspolia dubia RBTC1561-16 | 14.22 |
Euxiphidiopsis capricercus | ADE2467 (1) | N/A | 0 | Gampsocleis sinensis RBTC1223-16 | 18.21 |
Gampsocleis sinensis | AAY1322 (58) | 0.87 | 2.03 | Euxiphidiopsis capricercus HLXX121-16 | 18.21 |
Grigoriora cheni | ADE0541 (7) | 0.79 | 1.39 | Sinocyrtaspis brachycerca PSM013-19 | 13.2 |
Hemielimaea chinensis | ADB3478 (16) AEJ5565 (2) ADE2233 (4) | 1.51 | 3.63 | Elimaea nanpingensis DBTZC006-21 | 14.64 |
Hexacentrus japonicus | ACD8277 (4) ADM2486 (4) | 1.53 | 2.66 | Hexacentrus unicolor BHC097-18 | 12.42 |
Hexacentrus unicolor | ACD7247 (36) | 0.65 | 2.03 | Hexacentrus japonicus BHC079-15 | 12.42 |
Holochlora japonica | ADE1373 (6) | 0.16 | 0.31 | Holochlora venusta RBTC2063-18 | 9.45 |
Holochlora venusta | ADB6143 (12) | 0.05 | 0.31 | Holochlora japonica RBTC1717-16 | 9.45 |
Isopsera denticulata | AEJ6400 (1) ADE1596 (5) ADB3788 (7) ACD5193 (9) | 5.96 | 9.72 | Deflorita deflorita RBTC216-16 | 17.88 |
Isopsera furcocerca | ADB4481 (5) | 0 | 0 | Paraxantia huangshanensis RBTC1295-16 | 17.96 |
Isopsera sulcate | ACD7803 (4) | 0.18 | 0.31 | Isopsera furcocerca RBTC196-16 | 19.17 |
Kuwayamaea brachyptera | AEJ7401 (1) AEK2062 (1) AEK1896 (3) | 1.81 | 2.82 | Ducetia japonica DBTZC015-21 | 14.3 |
Mecopoda niponensis | AAF0977 (1) ACD8152 (18) | 1.09 | 7.53 | Diestramima austrosinensis DBTZC054-21 | 15.02 |
Mirollia bispina | ADB4146 (3) | 0.61 | 0.77 | Mirollia bispinosa RBTC406-16 | 4.61 |
Mirollia bispinosa | ADB4148 (3) | 0.1 | 0.15 | Mirollia bispina RBTC237-16 | 4.61 |
Nigrimacula paraquadrinotata | ACD6675 (1) | N/A | 0 | Grigoriora cheni HLXX071-16 | 15.82 |
Palaeoagraecia ascenda | ACD8365 (4) | 0 | 0 | Mecopoda niponensis RBTC2086-18 | 16.8 |
Paraxantia huangshanensis | ADB6578 (1) | N/A | 0 | Nigrimacula paraquadrinotata HLXX059-16 | 16.76 |
Phaneroptera falcata | AAL2811 (2) | 0.31 | 0.31 | Kuwayamaea brachyptera DBTZC012-21 | 15.98 |
Phaneroptera nigroantennata | ACD4406 (2) | 0.77 | 0.77 | Ducetia japonica RBTC397-16 | 14.49 |
Phyllomimus klapperichi | ADM7559 (1) ADB9999 (4) ADB4775 (6) | 10.3 | 17.47 | Ducetia japonica RBTC397-16 | 18.08 |
Pseudocosmetura fengyangshanensis | ADW0286 (1) | N/A | 0 | Sinocyrtaspis brachycerca PSM014-19 | 11.19 |
Pseudokuzicus pieli | ACD4648 (1) | N/A | 0 | Teratura megafurcula HLXX099-16 | 13.71 |
Pseudorhynchus concisus | ADB6233 (7) | 0.37 | 1.08 | Pyrgocorypha parva BOCON142-16 | 16.24 |
Pyrgocorypha parva | ADC0410 (3) | 0.51 | 0.77 | Pseudorhynchus concisus DBTZC059-21 | 16.24 |
Qinlingea brachystylata | ADB4056 (1) | N/A | 0 | Ruidocollaris truncatolobata RBTC1677-16 | 18.9 |
Ruidocollaris truncatolobata | ACD6433 (15) ADB6075 (5) | 2.2 | 5.85 | Ducetia japonica DBTZC015-21 | 16.25 |
Ruspolia dubia | ACD5503 (1) ADE5391 (3) | 1.09 | 1.55 | Euconocephalus nasutus RBTC1705-16 | 14.22 |
Ruspolia lineosa | ACD5257 (26) | 0.79 | 2.03 | Ruspolia dubia RBTC1649-16 | 15.55 |
Sinochlora longifissa | AEJ1447 (1) ADB3789 (34) | 1.1 | 3.81 | Sinochlora szechwanensis DBTZC067-21 | 5.68 |
Sinochlora sinensis | ACD4415 (1) | N/A | 0 | Sinochlora szechwanensis DBTZC038-21 | 5.93 |
Sinochlora szechwanensis* | ACI0121 (2) ADB3463 (4) | 4.61 | 8.71 | Sinochlora longifissa DBTZC039-21 | 5.68 |
Sinocyrtaspis brachycerca | ADX3437 (4) | 0.41 | 0.61 | Pseudocosmetura fengyangshanensis PSM017-19 | 11.19 |
Tegra novaehollandiae | ADB5353 (10) | 0.39 | 1.08 | Ducetia japonica RBTC249-16 | 17.85 |
Teratura megafurcula | ACD5306 (1) | N/A | 0 | Pseudokuzicus pieli RBTC411-16 | 13.71 |
Tettigonia chinensis | ACD6622 (8) | 0.32 | 0.77 | Hemielimaea chinensis DBTZC092-21 | 16.74 |
Xiphidiopsis gurneyi | ADE1670 (2) | 0 | 0 | Grigoriora cheni HLXX074-16 | 16.27 |
Xizicus biprocerus | ADE1374 (1) | N/A | 0 | Pseudokuzicus pieli RBTC411-16 | 14.68 |
Xizicus concavilaminus | ADB3332 (3) | 0.31 | 0.46 | Xizicus laminatus HLXX037-16 | 3.63 |
Xizicus howardi * | AEJ3139 (1) ADB5688 (10) ACD5539 (3) ADE3141 (4) | 6.13 | 21.64 | Xizicus laminatus HLXX037-16 | 3.31 |
Xizicus laminatus | ADB5868 (1) | N/A | 0 | Xizicus howardi RBTC1648-16 | 3.31 |
Xizicus szechwanensis | ADE0823 (2) ADB3348 (9) | 1.55 | 4.8 | Xizicus howardi DBTZC013-21 | 15.37 |
For the final dataset, 677 COI-5P records were assigned to 118 BINs that belong to 90 putative taxa. Among these, 68 corresponded to morphospecies, while another 22 belonged to a unique BIN that was currently only identified at genus level and highly likely to represent an unrecognized species. Of 68 morphospecies defined by morphology, a total of 49 contained only a single BIN, while 19 were represented by multiple BINs (Table
BIN assignments of 79 specimens identified only to genus level. BIN, Barcode Index Number; N, number of barcodes per BIN.
Taxon | BIN (N) |
---|---|
Atlanticus | ADB5602 (1), ADB6974 (1), ADR7192 (1), ADE2402 (1), ADB3445 (2), ADE1821 (2), ADB3462 (3) |
Bulbistridulous | ADB3431 (1) |
Conanalus | ADB5687 (1) |
Elimaea | ADE1399 (1), ADM8940 (1), ADB3477 (1) |
Hexacentrus | ADB5446 (2) |
Kuwayamaea | ADB4962 (1), ADE2183 (1), ADE1620 (13), ADB6899 (27), ADB4961 (4), ADB5240 (4), ADB4960 (6) |
Phryganogryllacris | ADF3837 (4) |
Prohimerta | ADB4147 (1) |
The NJ tree was employed to assess support for detected BINs, not to reconstruct the phylogenic relationships. The NJ tree showed the majority of non-singleton species and BINs were recovered as monophyletic (Fig.
Distance class | n | Taxa | Comparisons | Min Dist (%) | Mean Dist (%) | Max Dist (%) |
---|---|---|---|---|---|---|
Intraspecific | 585 | 55 | 6407 | 0.00 | 1.44 | 21.49 |
Congeners | 314 | 13 | 2439 | 3.29 | 15.19 | 24.30 |
Confamilial | 598 | 3 | 139568 | 11.12 | 22.66 | 34.59 |
ASAP analysis identified 99 MOTUs with an asapscore of 9.00 (Fig.
Capnogryllacris melanocrania showed deep intraspecific divergence (Imax = 5.01%), and was split into four BINs (ADF2751, AEJ4972, ADF2750, AEJ9445), and these four BINs formed nearest-neighbour clusters. All species delimitation methods treated C. melanocrania as three MOTUs (ADF2750 and AEJ9445 were placed in a single MOTU, while ADF2751 and AEJ4972 were each placed in their own MOTU), except for ASAP which placed all specimens of C. melanocrania in two MOTUs. Eugryllacris elongata showed deep intraspecific divergence (Max Intra-Sp = 10.68%), and was split into two BINs (AEK0366, ADF4811). All species delimitation methods treated E. elongata AEK0366 and ADF4811 as two separate MOTUs. Melaneremus fuscoterminatus showed deep intraspecific divergence (Imax = 14.73%) and was split into two BINs (ADF2959 and ADF2960). The nearest neighbour of the M. laticeps ADF4959 clade was M. fuscoterminatus ADF2959, followed by M. fuscoterminatus ADF2960. All species delimitation methods suggested M. fuscoterminatus ADF2959 should be treated as a separate MOTUs. ASAP treated M. fuscoterminatus ADF2959 and M. laticeps ADF4959 as one MOTU. Conocephalus maculatus showed deep intraspecific divergence (Max Intra-Sp = 5.43%), and was split into two BINs (ACD2116, ADB5579). All species delimitation methods treated E. elongata ACD2116 and ADB5579 as two separate MOTUs. Sinochlora szechwanensis showed deep intraspecific divergence (Imax = 8.71%), while no barcode gap was present in S. szechwanensis and S. longifissa (Table
Calculation of match ratio, taxonomic index of congruence (Ctax), and relative taxonomic resolving power index (Rtax) for different species delimitation methods. The lower triangle shows Ctax, and the upper triangle shows Match ratio.
BIN | ASAP | jMOTU | sGMYC | mGMYC | bPTP -ML | bPTP-BI | |
---|---|---|---|---|---|---|---|
BIN | 0.76 | 0.82 | 0.79 | 0.73 | 0.87 | 0.86 | |
ASAP | 0.82 | 0.93 | 0.91 | 0.65 | 0.77 | 0.76 | |
jMOTU | 0.85 | 0.96 | 0.91 | 0.70 | 0.81 | 0.80 | |
sGMYC | 0.89 | 0.94 | 0.94 | 0.71 | 0.82 | 0.81 | |
mGMYC | 0.81 | 0.75 | 0.76 | 0.79 | 0.80 | 0.81 | |
bPTP-ML | 0.91 | 0.83 | 0.85 | 0.88 | 0.84 | 0.99 | |
bPTP-BI | 0.90 | 0.82 | 0.84 | 0.87 | 0.85 | 0.99 | |
Mean Ctax | 0.85 | 0.85 | 0.85 | 0.86 | 0.78 | 0.84 | 0.86 |
Rtax | 0.84 | 0.71 | 0.72 | 0.75 | 0.94 | 0.85 | 0.86 |
Species | 118 | 99 | 101 | 105 | 132 | 119 | 120 |
BOLD TaxonID Tree based on K2P distances and species delimitation results based on COI-5P sequences. The four barcode sequences marked by red are highly likely NUMTs and excluded from the species delimitation analyses. The MOTUs created by each delimitation algorithm are represented as squares on the right. The number within the rectangles indicates the number of MOTUs; no number indicates a single MOTU.
In the past several hundred years, species diagnostics have been traditionally based on morphological characterizations. Morphology-based specimen identification is time consuming and requires high levels of taxonomic expertise. Compared with traditional taxonomy, DNA barcoding is a fast and inexpensive method for species identification. Numerous studies have revealed cryptic species using DNA barcodes (
The utility of DNA barcoding heavily depends on the taxonomic coverage of an associated DNA barcode reference library. Barcode libraries are generally assembled with two main objectives in mind: specimen identification and aiding species discovery/delimitation (
BIN sharing between different species might be explained by mitochondrial introgression following hybridization, recent divergence with or without incomplete lineage sorting, inadequate taxonomy, misidentification (
Our analyses revealed 19 of 55 non-singleton morphospecies (34.55%) with multiple BINs. Most of these intraspecific BINs formed nearest-neighbour clusters to each other, reflecting the discrimination of geographical subclades within a currently recognized species. Previous studies have shown that BINs provide a very good reflection of classical taxonomy (
Applying multiple species delimitation methods to the same dataset can provide a more reliable picture of species-level clustering. We obtained more MOTUs based on both the distance-based species-delimitation (ASAP, jMOTU) and the phylogeny-based methods (GMYC and bPTP) than the number of morphospecies. Several species with deep intraspecific divergence were split into more than one MOTU, and most of these additional BINs formed nearest-neighbour subclusters on the NJ tree. It was worth exploring the large intraspecific genetic distances for the same species although they were clustered together. Inconsistencies in delimitation results occur frequently as the result of different species delimitation methods. The mGMYC analysis produced a considerably higher number of MOTUs than other methods. Rtax values ranged from 0.71 for ASAP to 0.94 for mGMYC (Table
Our DNA barcode library represents an important step for the molecular characterization of katydids, cave crickets, and leaf-rolling crickets in Zhejiang, China. Although some specimens still lack a Linnean name, their BIN assignments are treated as putative species in ecology, conservation biology and other biodiversity research (
The work of ZZ was supported by a grant of the Natural Science Foundation of Hebei Province (C2021201002).
List of species of the family Tettigoniidae, Rhaphidophoridae, and Gryllacrididae in Zhejiang Province, China
Data type: species data