﻿A new species of Svistella Gorochov, 1987 from Xizang, China (Orthoptera, Trigonidiidae, Trigonidiinae)

﻿Abstract The genus Svistella Gorochov, 1987 includes 10 species from Asia, with nine documented in China. In this study, a new species, Svistellayayun He, sp. nov., is described from Xizang, China. Morphologically, it resembles S.rufonotata (Chopard, 1932) but can be distinguished by a smaller inner tympanum, dark-brown setae on the 5th segment of the maxillary palp, and a rounded apex on the ectoparamere. To validate our morphological inferences and support the description of S.yayunsp. nov. as a new species, we performed a PCA based on bioacoustics parameters and molecular analysis. All Svistella species documented in China are distinguished by integrating their songs and DNA barcoding.


Introduction
The genus Svistella Gorochov, 1987 belongs to the family Trigonidiidae Saussure, 1877, with all 10 species endemic to Asia (Cigliano et al. 2023).Over the past 50 years, the number of Svistella species has increased significantly.Initially, it comprised two species: S. bifasciata (= Paratrigonidium bifasciatum Shiraki, 1911) and S. rufonotata (= Anaxipha rufonotata Chopard, 1932), with the former designated as the type species.In 1993, Anaxipha dubia Liu & Yin, 1993 was described from Yunnan, China.Subsequently, He et al. (2009) reassigned A. dubia to the genus Svistella and described three new species: S. tympanalis He, Li & Liu, 2009, S. anhuiensis He, Li & Liu, 2009, and S. fallax He, Li & Liu, 2009.A new addition, S. chekjawa Tan & Robillard, 2012, was revealed in Singapore by Tan and Robillard (2012).Lu et al. (2018a) compared the morphological characters and DNA barcoding of the genera Svistella and Paratrigonidium Brunner von Wattenwyl, 1893 species, and described a new species, S. fuscoterminata He & Liu, 2018, from Yunnan, China.Additional contributions have included the description of S. argentata Ma, Jing & Zhang, 2019by Ma et al. (2019), who also proposed S. tympanalis as a junior synonym of S. rufonotata.Li et al. (2021a) reported two additional species, S. wuyong He, 2021 andS. malu He, 2021, from Yunnan, China, based on morphological characteristics, calling-song analysis and molecular study (COI).Currently, nine Svistella species are known to occur in China (Cigliano et al. 2023) The divergence of cricket songs usually precedes visible morphological differences, making song variations a significant driving factor for the divergence of cricket species (Otte 1992).Many male cricket species produce songs by stridulating their forewings to attract mates (Alexander 1962;Desutter-Grandcolas 1997;Mhatre and Balakrishnan 2006).Some perspectives indicated that songs may function as a significant mechanism for pre-mating isolation among species and a valuable tool in inferring species boundaries (Lu et al. 2018b;Tan et al. 2023).The song features are stable parameters within the same species (Fulton 1928), which may serve as cues for species recognition (Walker and Carlysle 1975).Thus, many new species are often initially identified based on their songs (Walker and Funk 2014).However, the calling songs can be easily influenced by temperature (Walker and Cade 2003;Jang and Gerhardt 2007).Song analysis is often combined with morphological observations and molecular analysis to identify new species (Chen et al. 2019;Tian et al. 2019;Li et al. 2021b).
During entomological surveys conducted in 2023, we first noticed unique songs different from any known cricket species.Our morphological, bioacoustics, and molecular analyses placed those newly collected individuals within the genus Svistella.However, the new specimens are different from any known Svistella species.Here we describe a new species, Svistella yayun He, sp.nov.from Xizang, China, and all Chinese Svistella species are characterized by a combination of their morphology, songs, and DNA barcoding.

Sampling
We discovered this unknown species through its sounds during the night.Five individuals were collected from the wild and immediately preserved in 65% ethanol.After returning to the lab, a hind leg was preserved in anhydrous ethanol at −40 °C for molecular studies, and the remaining parts were preserved as dry specimens.

Song recording and analyses
We recorded songs by using a SONY PCM A10 (ICX-0471) recorder.Three song recordings of Svistella yayun He, sp.nov.and all song recordings of other Chinese Svistella species from Li et al. (2021a) were replayed on a computer and analyzed using the Cool Edit software.Svistella fallax was not included in our PCA, because the audio file was lost, and the peak frequency was not available.Since most song recordings are shorter than 1 minute, we analyze the number of echemes in a randomly captured 10-second fragment from each recording, repeating this process 10 times.Svistella anhuiensis, S. bifasciata, and S. argentata are regarded as continuous groups, and thus we define 10-second fragment of their song recordings as an echeme.We randomly select 10 echemes to analyze both their duration and the number of syllables in each.Bioacoustics characters-echeme interval, number of echemes per minute, number of syllables in each echeme, and peak frequency (Table 1)-are used in our PCA analysis.A principal component analysis (PCA) was performed in RStudio v. 2022.12.0 + 353 with PCAtools (Blighe and Lun 2023) based on collected bioacoustics parameters of the male calling songs (described below in song analysis).

Measurements
The sizes of the following body parts were measured on photographs by the ruler tool of Adobe Photoshop CC 2015.5.All the measurements are in millimeters (mm).

Terminology
Terminology used to describe the male genitalia follows Tan and Robillard (2012)

DNA extraction and amplification
The total genomic DNA was extracted from the muscles of a hind leg by Axy-Prep Genomic DNA Miniprep Kit (AXYGEN), according to the manufacturer's instructions.The fragments of the mitochondrial cytochrome c oxidase subunit I gene (COI, 658 bp) were sequenced.Primers COBU (TYTCAACAAAYCAYAAR-GATATTGG) and COBL (TAAACTTCWGGRTGWCCAAARAATCA) were used (Pan et al. 2006).GenBank accession numbers are shown in Table 2.

Molecular study
The COI sequences from newly reported species, along with 36 individuals and the outgroup Amusurgus genji obtained from GenBank, were aligned using the MUSCLE method in MEGA 11 (Tamura et al. 2021).A distance tree was constructed employing the neighbor-joining (NJ) method following Kimura 2-parameter (K2P) model, with 0.19 gamma parameter and 95% partial deletion.The bootstrap test was performed with 10000 replicates.To define species partitions and estimate the number of molecular operational taxonomic units (MOTUs), we used two DNA-based species delimitation methods: Automatic Barcode Gap Discovery (ABGD, Puillandre et al. 2012) and Assemble Species by Automatic Partitioning (ASAP, Puillandre et al. 2021).dark-brown setae on the 5 th segment of maxillary palp (Fig. 4A-F), an ectoparamere with rounded corner (Fig. 5A-C), and a smaller inner tympanum (Fig. 5D-G).Description.Male.Body size small.Head slightly wider than anterior margin of pronotum, occiput slightly convex (Figs 1A, 2A); frontal rostrum about as wide as first antennal segment, with two rows of setae extending to vertex (Fig. 2C); vertex not dorsally flattened; antennae long and pubescent; compound eyes slightly protruding forwards; 5 th segment of maxillary palpi triangular and swollen (Fig. 2D).Pronotum with setae, posterior margin widened; fore tibiae armed with two oval tympanums, with outer one bigger than inner one (Fig. 3A, B); hind tibiae bearing three pairs of dorsal spurs (Fig. 3C) and five apical spurs (two internal ones distinctly longer and three external ones shorter); tegmina barely reaching apex of abdomen.Cercus with long, thin hair.
Genitalia.Pseudepiphallus separated into two lateral parts joined by a straight sclerotized bridge.Pseudepiphallic lophi curved inwards with 3 or 4 forks apically.Posterior marginal area of endoparameron with minute teeth and short setae (Fig. 3E, F).
Coloration.Body brown; legs yellowish brown.Head orange and marked with five vertical red stripes extending to pronotum in dorsal view.Setae on the 5 th segment of maxillary palpi dark brown (Fig. 4D, E).Abdomen with 1 lateral red spot on both sides of each abdominal segment in female.Each hind femur with a dark-brown stripe near knees when alive (Fig. 1A, B) but disappearing after drying (Fig. 3D).Ovipositor dark brown; apical half darker than basal part.
Variation.A paratype (ECNU 4961) has seven dorsal spurs on the hind tibiae (four internal ones and three external ones), while all the other examined specimens bear six dorsal spurs on the hind tibiae.
Etymology.The specific epithet yayun is for the Chinese phonetic alphabet, 雅韵, which means "beautiful music".

Molecular study
In total, 39 COI sequences including our newly described S. yayun He, sp.nov., as well as the COI sequence of Amusurgus genji as the outgroup, were obtained.The results of the two molecular methods identified 11 putative species, which largely conform to the distance tree inferred from the NJ topology and are all consistent with separating S. yayun He, sp.nov.as a species (Fig. 6).
PCA results are shown in Fig. 8.The extracted components PC1 eigenvectors accounted for 57.90% of the variance, PC2 for 24.04%, PC3 for 15.05%, PC4 for 2.02%, and PC5 for 0.99%.Except for S. wuyong and S. malu, the remaining species can be clearly identified via the analysis of their calling songs.

Discussion
In this study, the distance tree based on COI sequences shows that Svistella yayun sp.nov. is separate and distinct from other Svistella species and the reconstructed tree topology aligns with the earlier study using the same gene for eight species (Li et al. 2021a).Morphologically, S. yayun sp.nov. is similar to S. rufonotata, but it can be distinguished by the dark-brown setae on the 5 th segment of maxillary palp (light colored in S. rufonotata, Fig. 4), smaller inner tympanum with only 83.3-119.1 μm in long diameter (185.7-214.3μm in S. rufonotata, Fig. 5D-G) and rounded apex of the ectoparamere (relatively abrupt in S. rufonotata, Fig. 5A-C).Additionally, bioacoustics PCA unveils that the songs of S. yayun sp.nov.form a distinct cluster compared to S. rufonotata and all other previously described species.Collectively, our molecular, morphological, and bioacoustics analyses provide support for recognizing S. yayun sp.nov.as a new species.
Probably due to its small size, it is challenging to collect or observe S. yayun sp.nov. in the field without relying on its distinctive songs.In our prior experience, we have occasionally identified new orthopteran species in the field based on their unique songs (Liu et al. 2018;Chen et al. 2019;Tian et al. 2019;Li et al. 2021b).Despite the crucial role of song in the speciation and evolution of Orthoptera, bioacoustic data can significantly enhance our understanding of orthopteran taxonomy, particularly considering that the divergence in songs among species often precedes noticeable morphological differences (Otte 1992).This highlights the importance of incorporating bioacoustic data as a defining characteristic in studies of Orthoptera.
Discoveries of new Svistella and other new species of Trigonidiidae often reveal the influence of geographical barriers and communication signals on species isolation (Mendelson et al. 2004;Grace and Shaw 2011;Stamps and Shaw 2019;Li et al. 2021a).Most Svistella species have allopatric distributions, while parapatric Svistella species produce distinctive songs to attract females (Li et al. 2021a).This highlights the role of both geographical barriers and bioacoustic signals in isolating these species.Moreover, despite bioacoustic signals, chemical, and/or tactile cues may contribute to species' recognition to isolate different Svistella species in contact zones (Mullen et al. 2007).Similar patterns have been observed in other trigonidiid species, such as Laupala (Grace and Shaw 2011;Stamps and Shaw 2019).In line with recent studies, the discovery of S. yayun sp.nov.may underscore the association of geographical barriers, behavioral ecology, and Svistella speciation.

Figure 2 .
Figure 2. Svistella yayun He, sp.nov., male holotype, ECNU 4969, and female paratype, ECNU 4968 A habitus of male B habitus of female C male head and pronotum in dorsal view D male face in front view E female ovipositor in lateral view.Scale bars: 1 mm.

Figure 3 .
Figure 3. Svistella yayun He, sp.nov.A outer side of fore tibiae B inner side of fore tibia C hind tibiae in dorsal view D hind femora in lateral view E male genitalia in dorsal view F male genitalia in ventral view.All images are from holotype, ECNU 4969.Scale bars: 1 mm.

Figure 5 .
Figure 5. Male genitalia and right inner tympanum of Svistella rufonotata and S. yayun He, sp.nov.A male genitalia of S. yayun He, sp.nov. in ventral view, holotype, ECNU 4969 B, C male genitalia of S. rufonotata in ventral view from specimens ECNU 266 and ECNU 494, respectively D, E inner tympanum of S. yayun He, sp.nov.from a paratype ECNU 4961 and the holotype, ECNU 4969, respectively F, G inner tympanum of S. rufonotata from specimens ECNU 494 and ECNU 1756, respectively.Black arrows indicate the tip and a lateral process of ectoparamere.Scale bars: 0.5 mm.

Figure 6 .
Figure 6.Distance tree of Svistella species based on COI genes.Rooted by Amusurgus genji, the tree was constructed using the neighbor-joining (NJ) method with the Kimura 2-parameter model and a 0.19 gamma parameter.Topology supports of major nodes are indicated above branches by bootstrap value.Two DNA barcode-based (ABGD, ASAP) delimitation methods are represented by vertical bars in grey and blue, respectively, on the right side of the tree.

Figure 7 .
Figure 7. Spectrogram (upper) and oscillograms (lower) of male calling songs of Svistella yayun sp.nov.The red line in spectrogram indicates the peak frequency.

Figure 8 .
Figure 8. PCA figure description of Svistella species songs.Scatter plot of PC1 and PC2 of PCA based on bioacoustics measurements.Except for S. wuyong and S. malu, the remaining species can be clearly identified through the analysis of their calling songs.

Table 2 .
Collection information and COI GenBank accession number.
Type species.Svistella bifasciata(= Paratrigonidium bifasciatum Shiraki, 1911).Diagnosis.The new species is characterized as follows: small to medium body size for the genus; dark-brown setae on the 5 th segment of maxillary palp; small, inner tympanum; hind femora without black stripe; tegmina unicolor.It is morphologically similar to S. rufonotata but differs in having