High acoustic diversity and behavioral complexity of katydids in the Mesozoic soundscape.

Acoustic communication has played a key role in the evolution of a wide variety of vertebrates and insects. However, the reconstruction of ancient acoustic signals is challenging due to the extreme rarity of fossilized organs. Here, we report the earliest tympanal ears and sound-producing system (stridulatory apparatus) found in exceptionally preserved Mesozoic katydids. We present a database of the stridulatory apparatus and wing morphology of Mesozoic katydids and further calculate their probable singing frequencies and analyze the evolution of their acoustic communication. Our suite of analyses demonstrates that katydids evolved complex acoustic communication including mating signals, intermale communication, and directional hearing, at least by the Middle Jurassic. Additionally, katydids evolved a high diversity of singing frequencies including high-frequency musical calls, accompanied by acoustic niche partitioning at least by the Late Triassic, suggesting that acoustic communication might have been an important driver in the early radiation of these insects. The Early-Middle Jurassic katydid transition from Haglidae- to Prophalangopsidae-dominated faunas coincided with the diversification of derived mammalian clades and improvement of hearing in early mammals, supporting the hypothesis of the acoustic coevolution of mammals and katydids. Our findings not only highlight the ecological significance of insects in the Mesozoic soundscape but also contribute to our understanding of how acoustic communication has influenced animal evolution.

Multielectrode array use in insect auditory neuroscience to unravel the spatio-temporal response pattern in the prothoracic ganglion of Mecopoda elongata.

Mechanoreceptors in hearing organs transduce sound-induced mechanical responses into neuronal signals, which are further processed and forwarded to the brain along a chain of neurons in the auditory pathway. Bushcrickets (katydids) have their ears in the front leg tibia, and the first synaptic integration of sound-induced neuronal signals takes place in the primary auditory neuropil of the prothoracic ganglion. By combining intracellular recordings of the receptor activity in the ear, extracellular multichannel array recordings on top of the prothoracic ganglion and hook electrode recordings at the neck connective, we mapped the timing of neuronal responses to tonal sound stimuli along the auditory pathway from the ears towards the brain. The use of the multielectrode array allows the observation of spatio-temporal patterns of neuronal responses within the prothoracic ganglion. By eliminating the sensory input from one ear, we investigated the impact of contralateral projecting interneurons in the prothoracic ganglion and added to previous research on the functional importance of contralateral inhibition for binaural processing. Furthermore, our data analysis demonstrates changes in the signal integration processes at the synaptic level indicated by a long-lasting increase in the local field potential amplitude. We hypothesize that this persistent increase of the local field potential amplitude is important for the processing of complex signals, such as the conspecific song.

DNA barcoding of the genus Gampsocleis (Orthoptera, Tettigoniidae) from China.

DNA barcoding is a useful addition to the traditional morphology-based taxonomy. A ca. 650 bp fragment of the 5' end of mitochondrial cytochrome c oxidase subunit I (hereafter COI-5P) DNA barcoding was sued as a practical tool for Gampsocleis species identification. DNA barcodes from 889 specimens belonging to 8 putative Gampsocleis species was analyzed, including 687 newly generated DNA barcodes. These barcode sequences were clustered/grouped into Operational Taxonomic Units (OTUs) using the criteria of five algorithms, namely Barcode Index Number (BIN) System, Assemble Species by Automatic Partitioning (ASAP), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP). The Taxon ID Tree grouped sequences of morphospecies and almost all MOTUs in distinct nonoverlapping clusters. Both long- and short-winged Gampsocleis species are reciprocally monophyletic in the Taxon ID Tree. In BOLD, 889 barcode sequences are assigned to 17 BINs. The algorithms ASAP, jMOTU, bPTP and GMYC clustered the barcode sequences into 6, 13, 10, and 23 MOTUs, respectively. BIN, ASAP, and bPTP algorithm placed three long-winged species, G. sedakovii, G. sinensis and G. ussuriensis within the same MOTU. All species delimitation algorithms split two short-winged species,G. fletcheri and G. gratiosa into at least two MOTUs each, except for ASAP algorithm. More detailed molecular and morphological integrative studies are required to clarify the status of these MOTUs in the future.

Going with the flow - how a stream insect, Pteronarcys californica, exploits local flows to increase oxygen availability.

For insects, life in water is challenging because oxygen supply is typically low compared with in air. Oxygen limitation may occur when oxygen levels or water flows are low or when warm temperatures stimulate metabolic demand for oxygen. A potential mechanism for mitigating oxygen shortages is behavior - moving to cooler, more oxygenated or faster flowing microhabitats. Whether stream insects can make meaningful choices, however, depends on: (i) how temperature, oxygen and flow vary at microspatial scales and (ii) the ability of insects to sense and exploit that variation. To assess the extent of microspatial variation in conditions, we measured temperature, oxygen saturation and flow velocity within riffles of two streams in Montana, USA. In the lab, we then examined preferences of nymphs of the stonefly Pteronarcys californica to experimental gradients based on field-measured values. Temperature and oxygen level varied only slightly within stream riffles. By contrast, flow velocity was highly heterogeneous, often varying by more than 125 cm s-1 within riffles and 44 cm s-1 around individual cobbles. Exploiting micro-variation in flow may thus be the most reliable option for altering rates of oxygen transport. In support of this prediction, P. californica showed little ability to exploit gradients in temperature and oxygen but readily exploited micro-variation in flow - consistently choosing higher flows when conditions were warm or hypoxic. These behaviors may help stream insects mitigate low-oxygen stress from climate change and other anthropogenic disturbances.

Exaggerated mandibles are correlated with enhanced foraging efficacy in male Auckland tree weta.

Sexual selection has driven the evolution of weaponry for males to fight rivals to gain access to females. Although weapons are predicted to increase males' reproductive success, they are also expected to incur costs and may impair functional activities, including foraging. Using feeding assays, we tested whether the enlarged mandibles of Auckland tree weta (Hemideina thoracica) impact feeding activity (the total volume of biomass consumed, bite rate, and number of foraging visits) and foraging behaviour (time spent moving, feeding, or stationary). We predicted that increased head capsule size in male weta would hinder their foraging efficacy. However, we found that weta with longer heads fed at a faster rate and spent less time foraging than weta with smaller heads, regardless of sex. Contrary to expectations that weapons impede functional activities, our results demonstrate that exaggerated traits can improve feeding performance and may offer benefits other than increased mating success.

Molecular and biochemical changes in Locusta migratoria (Orthoptera: Acrididae) infected with Paranosema locustae.

Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen's life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Temperature preferences drive additive biotic homogenization of Orthoptera assemblages.

The degradation of natural habitats is causing ongoing homogenization of biological communities and declines in terrestrial insect biodiversity, particularly in agricultural landscapes. Orthoptera are focal species of nature conservation and experienced significant diversity losses over the past decades. However, the causes underlying these changes are not yet fully understood. We analysed changes in Orthoptera assemblages surveyed in 1988, 2004 and 2019 on 198 plots distributed across four major grassland types in Central Europe. We demonstrated compositional differences in Orthoptera assemblages found in wet, dry and mesic grasslands, as well as ruderal habitats decreased, indicating biotic homogenization. However, mean α-diversity of Orthoptera assemblages increased over the study period. We detected increasing numbers of species with preferences for higher temperatures in mesic and wet grasslands. By analysing the temperature, moisture and vegetation preferences of Orthoptera, we found that additive homogenization was driven by a loss of species adapted to extremely dry and nitrogen-poor habitats and a parallel spread of species preferring warmer macroclimates.

Soil Invertebrates Generate Microplastics From Polystyrene Foam Debris.

To fully understand microplastics' impact on soil ecosystems, one must recognize soil organisms as not just passively enduring their negative effects, but potentially contributing to microplastics' formation, distribution, and dynamics in soil. We investigated the ability of four soil invertebrates, the cricket Acheta domesticus L. (Orthoptera: Gryllidae), the isopod Oniscus asellus L. (Isopoda: Oniscidae), larvae of the beetle Zophobas morio Fabricius (Coleoptera: Tenebrionidae), and the snail Cornu aspersum Müller (Stylommatophora: Helicidae) to fragment macroscopic pieces of weathered or pristine polystyrene (PS) foam. We placed invertebrates into arenas with single PS foam pieces for 24 h, then collected and assessed the microplastic content of each invertebrate's fecal material, its cadaver, and the sand substrate of its arena via hydrogen peroxide digestion, filtration, and fluorescent staining. All taxa excreted PS particles, though snails only to a tiny extent. Beetle larvae produced significantly more microplastics than snails, and crickets and isopods fragmented the weathered PS foam pieces more than the pristine pieces, which they left untouched. A follow-up experiment with pristine PS foam assessed the effect of different treatments mimicking exposure to the elements on fragmentation by isopods. PS foam pieces soaked in a soil suspension were significantly more fragmented than untreated pieces or pieces exposed to UV light alone. These findings indicate that soil invertebrates may represent a source of microplastics to the environment in places polluted with PS foam trash, and that the condition of macroplastic debris likely affects its palatability to these organisms.

Evolution of Gene Arrangements in the Mitogenomes of Ensifera and Characterization of the Complete Mitogenome of Schizodactylus jimo.

Gene arrangement (relative location of genes) is another evolutionary marker of the mitogenome that can provide extensive information on the evolutionary mechanism. To explore the evolution of gene arrangements in the mitogenome of diversified Ensifera, we sequenced the mitogenome of the unique dune cricket species found in China and used it for phylogenetic analysis, in combination with 84 known Ensiferan mitogenomes. The mitogenome of Schizodactylus jimo is a 16,428-bp circular molecule that contains 37 genes. We identified eight types of gene arrangement in the 85 ensiferan mitogenomes. The gene location changes (i.e., gene translocation and duplication) were in three gene blocks: I-Q-M-ND2, rrnl-rns-V, and ND3-A-R-N-S-E-F. From the phylogenetic tree, we found that Schizodactylus jimo and most other species share a typical and ancient gene arrangement type (Type I), while Grylloidea has two types (Types II and III), and the other five types are rare and scattered in the phylogenetic tree. We deduced that the tandem replication-random loss model is the evolutionary mechanism of gene arrangements in Ensifera. Selection pressure analysis revealed that purifying selection dominated the evolution of the ensiferan mitochondrial genome. This study suggests that most gene rearrangements in the ensiferan mitogenome are rare accidental events.

Acoustically eavesdropping bat predators take longer to capture katydid prey signalling in aggregation.

Prey that are signalling in aggregation become more conspicuous with increasing numbers and tend to attract more predators. Such grouping may, however, benefit prey by lowering the risk of being captured because of the predator's difficulty in targeting individuals. Previous studies have investigated anti-predatory benefits of prey aggregation using visual predators, but it is unclear whether such benefits are gained in an auditory context. We investigated whether katydids of the genus Mecopoda gain protection from their acoustically eavesdropping bat predator Megaderma spasma when calling in aggregation. In a choice experiment, bats approached calls of prey aggregations more often than those of prey calling alone, indicating that prey calling in aggregation are at higher risk. In prey capture tasks, however, the average time taken and the number of flight passes made by bats before capturing a katydid were significantly higher for prey calling in aggregation than when calling alone, indicating that prey face lower predation risk when calling in aggregation. Another common anti-predatory strategy, calling from within vegetation, increased the time taken by bats to capture katydids calling alone but did not increase the time taken to capture prey calling from aggregations. The increased time taken to capture prey calling in aggregation compared with solitary calling prey offers an escape opportunity, thus providing prey that signal acoustically in aggregations with anti-predatory benefits. For bats, greater detectability of calling prey aggregations is offset by lower foraging efficiency, and this trade-off may shape predator foraging strategies in natural environments.

The Ander's organ: a mechanism for anti-predator ultrasound in a relict orthopteran.

The use of acoustics in predator evasion is a widely reported phenomenon amongst invertebrate taxa, but the study of ultrasonic anti-predator acoustics is often limited to the prey of bats. Here, we describe the acoustic function and morphology of a unique stridulatory structure - the Ander's organ - in the relict orthopteran Cyphoderris monstrosa (Ensifera, Hagloidea). This species is one of just eight remaining members of the family Prophalangopsidae, a group with a fossil record of over 90 extinct species widespread during the Jurassic period. We reveal that the sound produced by this organ has the characteristics of a broadband ultrasonic anti-predator defence, with a peak frequency of 58±15.5 kHz and a bandwidth of 50 kHz (at 10 dB below peak). Evidence from sexual dimorphism, knowledge on hearing capabilities and assessment of local predators, suggests that the signal likely targets ground-dwelling predators. Additionally, we reveal a previously undescribed series of cavities underneath the organ that probably function as a mechanism for ultrasound amplification. Morphological structures homologous in both appearance and anatomical location to the Ander's organ are observed to varying degrees in 4 of the 7 other extant members of this family, with the remaining 3 yet to be assessed. Therefore, we suggest that such structures may either be more widely present in this ancient family than previously assumed, or have evolved to serve a key function in the long-term survival of these few species, allowing them to outlive their extinct counterparts.

Vibrational signalling, an underappreciated mode in cricket communication.

Signalling via substrate vibration represents one of the most ubiquitous and ancient modes of insect communication. In crickets (Grylloidea) and other taxa of tympanate Ensifera, production and detection of acoustic and vibrational signals are closely linked functionally and evolutionarily. Male stridulation produces both acoustic and vibrational signal components, the joint perception of which improves song recognition and female orientation towards the signaller. In addition to stridulation, vibrational signalling mainly through body tremulation and/or drumming with body parts on the substrate has long been known to be part of crickets' close-range communication, including courtship, mate guarding and aggression. Such signalling is typically exhibited by males, independently or in conjunction with stridulation, and occurs literally in all cricket lineages and species studied. It is further also part of the aggressive behaviour of females, and in a few cricket groups, females respond vibrationally to acoustic and/or vibrational signals from males. The characteristics and function of these signals have remained largely unexplored despite their prevalence. Moreover, the communication potential and also ubiquity of cricket vibrational signals are underappreciated, limiting our understanding of the function and evolution of the cricket signalling systems. By providing a concise review of the existing knowledge of cricket perception of vibrations and vibrational signalling behaviour, we critically comment on these views, discuss the communication value of the emitted signals and give some methodological advice respecting their registration and control. The review aims to increase awareness, understanding and research interest in this ancient and widespread signalling mode in cricket communication.

MtOrt: an empirical mitochondrial amino acid substitution model for evolutionary studies of Orthoptera insects.

BACKGROUND: Amino acid substitution models play an important role in inferring phylogenies from proteins. Although different amino acid substitution models have been proposed, only a few were estimated from mitochondrial protein sequences for specific taxa such as the mtArt model for Arthropoda. The increasing of mitochondrial genome data from broad Orthoptera taxa provides an opportunity to estimate the Orthoptera-specific mitochondrial amino acid empirical model. RESULTS: We sequenced complete mitochondrial genomes of 54 Orthoptera species, and estimated an amino acid substitution model (named mtOrt) by maximum likelihood method based on the 283 complete mitochondrial genomes available currently. The results indicated that there are obvious differences between mtOrt and the existing models, and the new model can better fit the Orthoptera mitochondrial protein datasets. Moreover, topologies of trees constructed using mtOrt and existing models are frequently different. MtOrt does indeed have an impact on likelihood improvement as well as tree topologies. The comparisons between the topologies of trees constructed using mtOrt and existing models show that the new model outperforms the existing models in inferring phylogenies from Orthoptera mitochondrial protein data. CONCLUSIONS: The new mitochondrial amino acid substitution model of Orthoptera shows obvious differences from the existing models, and outperforms the existing models in inferring phylogenies from Orthoptera mitochondrial protein sequences.

Equal fitness among alternative mating strategies in a harem polygynous insect.

Alternative mating strategies are widespread among animal taxa, with strategies controlled by a genetic polymorphism (Mendelian strategy) being rarer in nature than condition-dependent developmental strategies. Mendelian strategies are predicted to have equal average fitnesses and the proportion of offspring produced by a strategy should equal the equilibrium proportion of individuals representing the strategy in a population. Developmental strategies are not expected to produce offspring in equilibrium proportions; however, whether the alternative phenotypes should have equal average fitness is debated. The Wellington tree weta (Hemideina crassidens) (Orthoptera: Anostostomatidae) is a harem polygynous insect in which intense sexual competition has favoured the evolution of three alternative mating strategies that differ in weapon size and the ability to fight for control of harems. Here, we use molecular genotyping to test the hypothesis that the alternative strategies in this species are maintained by having equal relative fitness and that morphs produce offspring in equilibrium proportions. As expected, the average relative fitness of the three strategies did not significantly differ and the proportion of offspring produced by each morph is equal to the frequency of that morph in the population. Our results support the hypothesis that the alternative male morphs in H. crassidens represent Mendelian strategies.

Evolutionary rates of and selective constraints on the mitochondrial genomes of Orthoptera insects with different wing types.

Orthoptera is the most diverse order of polyneopterans, and the forewing and hindwing of its members exhibit extremely variability from full length to complete loss in many groups; thus, this order provides a good model for studying the effects of insect flight ability on the evolutionary constraints on and evolutionary rate of the mitochondrial genome. Based on a data set of mitochondrial genomes from 171 species, including 43 newly determined, we reconstructed Orthoptera phylogenetic relationships and estimated the divergence times of this group. The results supported Caelifera and Ensifera as two monophyletic groups, and revealed that Orthoptera originated in the Carboniferous (298.997 Mya). The date of divergence between the suborders Caelifera and Ensifera was 255.705 Mya, in the late Permian. The major lineages of Acrididae seemed to have radiated in the Cenozoic, and the six patterns of rearrangement of 171 Orthoptera mitogenomes mostly occurred in the Cretaceous and Cenozoic. Based on phylogenetic relationships and ancestral state reconstruction, we analysed the evolutionary selection pressure on and evolutionary rate of mitochondrial protein-coding genes (mPCGs). The results indicated that during approximately 300 Mya of evolution, these genes experienced purifying selection to maintain their function. Flightless orthopteran insects accumulated more non-synonymous mutations than flying species and experienced more relaxed evolutionary constraints. The different wing types had different evolutionary rates, and the mean evolutionary rate of Orthoptera mitochondrial mPCGs was 13.554 × 10-9 subs/s/y. The differences in selection pressures and evolutionary rates observed between the mitochondrial genomes suggested that functional constraints due to locomotion play an important role in the evolution of mitochondrial DNA in orthopteran insects with different wing types.

Material heterogeneity of male genitalia reduces genital damage in a bushcricket during sperm removal behaviour.

Sperm removal behaviour (SRB) is known in many animals, and male genital structures are often involved in the SRB, e.g. rubbing female genitalia vigorously. However, it remains unclear how those male genital structures function properly without severe genital damage during SRB. In the present study, we focused on the bushcricket Metaplastes ornatus and examined the biomechanics of male and female genital structures, involved in their SRB as a model case. During an initial phase of mating, males of this species thrust their subgenital plate with hook-like spurs and many microscopic spines into the female genital chamber. By moving the subgenital plate back-and-forth, males stimulate females, and this stimulation induces the ejection of sperm previously stored in females. We aimed to uncover the mechanics of the interaction between the subgenital plate and genital chamber during SRB. The genital morphology and its material composition were investigated using modern imaging and microscopy techniques. The obtained results showed a pronounced material heterogeneity in the subgenital plate and the genital chamber. The material heterogeneity was completely absent in that of a second bushcricket species, Poecilimon veluchianus, which does not exhibit SRB. Finite element simulations showed that the specific material heterogeneity can redistribute the stress in the subgenital plate of M. ornatus and, thereby, reduces stress concentration during SRB. This may explain why only a few examined males had a broken spur. We suggest that the observed structural features and material heterogeneity in M. ornatus are adaptations to their SRB.

On the tympanic membrane impedance of the katydid Copiphora gorgonensis (Insecta: Orthoptera: Tettigoniidae).

Katydids (bush-crickets) are endowed with tympanal ears located in their forelegs' tibiae. The tympana are backed by an air-filled tube, the acoustic trachea, which transfers the sound stimulus from a spiracular opening on the thorax to the internal side of the tympanic membranes (TM). In katydids the sound stimulus reaches both the external and internal side of the membranes, and the tympanal vibrations are then transferred to the hearing organ crista acustica (CA) that contains the fluid-immersed mechanoreceptors. Hence the tympana are principally involved in transmitting and converting airborne sound into fluid vibrations that stimulate the auditory sensilla. Consequently, what is the transmission power to the CA? Are the TM tuned to a certain frequency? To investigate this, the surface normal acoustic impedance of the TM is calculated using finite-element analysis in the katydid Copiphora gorgonensis. From this, the reflectance and transmittance are obtained at the TM. Based on the impedance results obtained from the pressure recordings at TM and the velocity field calculations in the AT, in the frequency range 5-40 kHz, it is concluded that the tympana have considerably higher transmission around 23 kHz, corresponding to the dominant frequency of the male pure-tone calling song in this species.

Response of Orthoptera assemblages to environmental change in a low-mountain range differs among grassland types.

Grasslands are among the most species-rich ecosystems in Europe. However, their biodiversity has become increasingly threatened by land-use and climate change. Here, we analyze Orthoptera assemblage shifts between 1996 and 2017 across three grassland types in the Black Forest (SW Germany) (N = 63): (i) formerly managed wet grasslands which have been frequently abandoned in recent decades (WET) (N = 15); (ii) common pastures which are still traditionally managed by rough grazing (COMMON) (N = 29), and (iii) mesic grasslands which have recently suffered from land-use intensification (MESIC) (N = 19). Both annual and summer temperatures increased during the study period. Orthoptera assemblages strongly responded to the altered environmental conditions in the grasslands. However, effects differed clearly among grassland types. Despite a strong increase in overall species richness in common pastures, neither the Community Farmland Index (CFI) nor the Community Temperature Index (CTI) had changed. In the two other grassland types, the CFI decreased and the CTI increased. The CFI - established here for Orthoptera - helped to disentangle the effects of climate and land-use change on Orthoptera assemblage composition. Based on our study, climate warming has led to biotic homogenization of the Orthoptera assemblages of wet grasslands affected by abandonment, and mesic grasslands affected by land-use intensification towards a dominance of more widespread species. In contrast, common pastures characterized by a high heterogeneity and low-intensity management were more resilient to the effects of climate warming.

Singleton molecular species delimitation based on COI-5P barcode sequences revealed high cryptic/undescribed diversity for Chinese katydids (Orthoptera: Tettigoniidae).

BACKGROUND: DNA barcoding has been developed as a useful tool for species discrimination. Several sequence-based species delimitation methods, such as Barcode Index Number (BIN), REfined Single Linkage (RESL), Automatic Barcode Gap Discovery (ABGD), a Java program uses an explicit, determinate algorithm to define Molecular Operational Taxonomic Unit (jMOTU), Generalized Mixed Yule Coalescent (GMYC), and Bayesian implementation of the Poisson Tree Processes model (bPTP), were used. Our aim was to estimate Chinese katydid biodiversity using standard DNA barcode cytochrome c oxidase subunit I (COI-5P) sequences. RESULTS: Detection of a barcoding gap by similarity-based analyses and clustering-base analyses indicated that 131 identified morphological species (morphospecies) were assigned to 196 BINs and were divided into four categories: (i) MATCH (83/131 = 64.89%), morphospecies were a perfect match between morphospecies and BINs (including 61 concordant BINs and 22 singleton BINs); (ii) MERGE (14/131 = 10.69%), morphospecies shared its unique BIN with other species; (iii) SPLIT (33/131 = 25.19%, when 22 singleton species were excluded, it rose to 33/109 = 30.28%), morphospecies were placed in more than one BIN; (iv) MIXTURE (4/131 = 5.34%), morphospecies showed a more complex partition involving both a merge and a split. Neighbor-joining (NJ) analyses showed that nearly all BINs and most morphospecies formed monophyletic cluster with little variation. The molecular operational taxonomic units (MOTUs) were defined considering only the more inclusive clades found by at least four of seven species delimitation methods. Our results robustly supported 61 of 109 (55.96%) morphospecies represented by more than one specimen, 159 of 213 (74.65%) concordant BINs, and 3 of 8 (37.5%) discordant BINs. CONCLUSIONS: Molecular species delimitation analyses generated a larger number of MOTUs compared with morphospecies. If these MOTU splits are proven to be true, Chinese katydids probably contain a seemingly large proportion of cryptic/undescribed taxa. Future amplification of additional molecular markers, particularly from the nuclear DNA, may be especially useful for specimens that were identified here as problematic taxa.