Systematics of Spiny Predatory Katydids (Tettigoniidae: Listroscelidinae) from the Brazilian Atlantic Forest Based on Morphology and Molecular Data

Listroscelidinae (Orthoptera: Tettigoniidae) are insectivorous Pantropical katydids whose taxonomy presents a long history of controversy, with several genera incertae sedis. This work focused on species occurring in the Brazilian Atlantic Forest, one of the world's most threatened biomes. We examined material deposited in scientific collections and visited 15 conservation units from Rio de Janeiro to southern Bahia between November 2011 and January 2012, catching 104 specimens from 10 conservation units. Based on morphological and molecular data we redefined Listroscelidini, adding a new tribe, new genus and eight new species to the subfamily. Using morphological analysis, we redescribed and added new geographic records for six species, synonymized two species and built a provisional identification key for the Atlantic Forest Listroscelidinae. Molecular results suggest two new species and a new genus to be described, possibly by the fission of the genus Hamayulus. We also proposed a 500 bp region in the final portion of the COI to be used as a molecular barcode. Our data suggest that the Atlantic Forest Listroscelidinae are seriously endangered, because they occur in highly preserved forest remnants, show high rates of endemism and have a narrow geographic distribution. Based on our results, we suggest future collection efforts must take into account the molecular barcode data to accelerate species recognition.


Introduction
Listroscelidinae (Orthoptera: Tettigoniidae) are carnivorous (insectivorous) katydids [1] with a long and controversial taxonomic history. In 1891, Redtenbacher [2] placed these species in the tribe ''Listroscelini'', which he included in Conocephalinae, with Listroscelis Serville as the type genus. This tribe included a few genera now classified in two other subfamilies, Meconematinae and Hexacentrinae. In 1898, Saussure & Pictet [3] separated Listroscelis into ''Listroscelites'', but examined only L. arachnoides Redtenbacher, the type-species of Arachnoscelis, further described by Karny [4]. In 1906, Kirby [5] raised the tribe to the rank of subfamily and in 1915 Bruner [1] treated the group as a family and provided a taxonomic key for tropical American genera. In 1924, Karny [6] transferred a few genera to Meconematinae and in 1936 Zeuner [7,8] reevaluated Tettigoniidae, examining traditional characteristics of the head, wing venation and prothoracic tracheal apparatus. He proposed two closely related taxa: ''Conocephaloids'', including Listroscelidinae (treated as a subfamily), Salomoninae (including Agraeciinae) and Copiphorinae; and ''Tettigonioids'', including Tettigoniinae, Decticinae, Saginae, Mecopodinae and Phyllophorinae. In 1840, Zeuner [9] transferred Xiphidiopsis Redtenbacher, Phlugis Stål and Phlugiola Karny to Meconematinae but some doubts remained as he considered the differences between Meconematinae and Listroscelidinae to be only gradual. In 1979, Rentz [10] corrected the suprageneric name to Listroscelidinae, redefining it to include species with fastigium of vertex narrow and usually sulcated, fore and mid femora with robust spines and fore tibia with five to seven long spines.
The first column of Table 1 lists the current taxonomic classification of Listroscelidinae. Of the 22 known genera, 12 are organized into four tribes, while the remaining are incertae sedis. Of these, Arachnoscelis (incertae sedis in Table 1) was previously considered in Meconematinae [11,12]. Except for Neobarrettia Rehn that occurs in the Holarctic kingdom (southern North America) [13], the subfamily is Pantropical, with most species described in Neotropical, Oriental and Australotropical regions [14]. Listroscelidinae is highly diversified in Brazil with fourteen species in eight genera, six of which occur in the Atlantic Forest. The last taxonomic study was performed by Piza [15] in 1982.
Nothing is known about the natural history or distribution of the Brazilian species.
In this work, we aimed to revisit the classification of Listroscelidinae, focusing on species occurring in the Atlantic Forest, one of the world's most diverse and threatened biomes [22][23][24][25][26]. We did an extensive field expedition, visiting 15 protected forest areas to search for specimens of Listroscelidinae, which we identified, described or redescribed. We revised the taxonomy of Listroscelidinae and proposed phylogenetic hypotheses based on molecular markers for the studied species, in addition to proposing the use of a small mitochondrial sequence for molecular barcoding to accelerate future species recognition. Table 1. Taxonomic classification of Listroscelidinae.

Tribe
In most cases, we identified the components of the phallus with the terminology and abbreviations of Snodgrass [27], and the components of the male postabdomen with the abbreviations of Ingrisch [28]. Table 2 shows the abbreviations for measurements (in mm) and counting parts used in this work. In our descriptions, we provided measurements and ratios of holotype and allotype, and the range of variation for males and females of the whole type series or examined specimens. Some measurements were obtained from literature.
As with most katydids, Listroscelidinae are active at night; thus we collected them by active capture between 7:00 pm and 1:00 am with the aid of flashlights for at least three consecutive nights in each conservation unit. The specimens were photographed while alive and captured in transparent polyethylene vials of 500 ml with lids (pictures shown in Figs. S1, S2, S3, S4). Except for the individuals of Hamayulus gen. nov., which were completely preserved in alcohol, we dry-preserved the specimens in field and removed their intestines to better preserve their original surface color. For molecular procedures, we preserved one mid or hind leg of each individual in absolute ethanol and kept samples below 220uC.
Photographs of preserved specimens in lateral, dorsal and frontal views were taken using a Nikon D90 digital camera equipped with a Nikon AF-S 105 mm f/2.8 VR Micro-NIKKOR lens, except for specimens provided by the staff of the NMW. Specimens immersed in alcohol were photographed under a Zeiss Discovery V8 stereomicroscope equipped with a Zeiss MRc digital camera. Images of live specimens provided in the Supporting Information (Figs. S1, S2, S3, S4) were taken with a Nikon 4500.

Descriptions of new species and taxonomic review
For morphological analyses, we used specimens deposited in scientific collections and field-collected specimens that had been dried, pinned and labeled at the laboratory.
The specimens were examined and compared under a Zeiss Stemi 2000-C stereomicroscope or a Zeiss Discovery V8. Whenever possible, we dissected the phallus of at least one male of each genus. We created diagrams arranged for pictorial documentation and comparison between genera and species: one plate showing spines and tubercles on sternum, coxae and trochanters (Fig. 2); one showing external components of the phallus (Fig. 3) for genera comparisons; a plate of male postabdomens (Fig. 4); male and female subgenital plates (Fig. 5); and stridulatory files (Fig. 6) for species comparisons. For each examined species, we elaborated a plate showing habitus of male and female in dorsal, lateral and frontal views (in some cases we had individuals of only one gender). On each plate, images of lateral and dorsal views are shown at the same scale, but the scale varies between plates. Images of frontal view on all plates are at the same scale for purposes of comparison. Diagrams and plates were edited using Corel Draw 66. Descriptions and redescriptions include details of color patterns, important features for recognizing and characterizing Listroscelidinae [12].
We also examined specimens deposited in scientific collections, used to describe new species and for taxonomic revisions of this subfamily. Specimens were borrowed from UFES (one Listroscelis angustifrons (Piza) comb. nov.), MNRJ (three Megatympanon speculatum Piza, three L. carinata Karny and four Cerberodon viridis Perty) and IBB (M. speculatum). Type material of L. angustifrons comb. nov. and M. speculatum deposited in ESALQ were examined in a previous work [29]. We also had access to photographs of Listroscelis and Monocerophora species deposited at the NMW. We created four maps showing the distribution of species studied in the present work. These records were the result of our own field collections ( Fig. 1A-B) or obtained from literature.

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts were registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be accessed through any standard web browser by appending the LSID to the prefix ''http://zoobank. org/''. The LSID for this publication is: urn:lsid:zoobank.org: pub:FDC64475-A7BB-4B31-8D20-FE47224FE490*. The electronic edition of this work has been archived and is available from the following digital repositories: PubMed Central, LOCKSS and Orthoptera Species File Online (OSF).

DNA preparation, amplification and sequencing
We extracted the total DNA of mid or hind leg muscles of each individual using an adjusted standard phenol-chloroform protocol [30]. A 1300 bp fragment of the COI gene and a 1600 bp fragment of the rRNA 18S were amplified by touchdown PCR [31]. PCR was performed using 25 ml reactions with 1 unit GoTaq Flexi DNA Polymerase (Promega), 16 GoTaq Flexi Buffer (Promega), 0.1 mM dNTP, 1.5 mM MgCl 2 , 0.2 mM of each primer (Table S1), 20 to 100 ng DNA, and ultra-pure water. For COI gene amplification, the following parameters were used: 5 min at 94uC initial denaturation; 10 cycles of 1 min at 94uC, 1 min 45 s at 55 to 45uC (touchdown), and 1 min 45 s at 72uC; 30 cycles of 1 min at 94uC, 1 min 45 s at 45uC, and 1 min 45 s at 72uC; with a final extension at 72uC for 5 min. For 18S gene amplification parameters were as follows: 5 min at 94uC initial denaturation; 15 cycles of 1 min at 94uC, 1 min 45 s at 60 to 45uC (touchdown), and 1 min 45 s at 72uC; 25 cycles of 1 min at 94uC, 1 min 45 s at 45uC, and 1 min 45 s at 72uC; with a final extension at 72uC for 5 min. PCR products were inspected with 1.5% agarose gel electrophoresis using GelRed (Biotium) to confirm amplification and verify contamination. Amplicons were sequenced with DNA sequencer Applied Biosystems 37306l at the Macrogen Inc. (South Korea).

Sequence evaluation and alignment
The resulting chromatograms were evaluated with the program Consed [32]. We aligned both sequences using Muscle [33], implemented in Mega 5.0 [34]. Since COI is coding DNA, we aligned their putative amino acid sequences and reversed them to get the nucleotide sequences. Sequences showing double peaks in the chromatogram and unexpected stop codons or gaps of one or two base pairs into the aligned putative amino acid sequence were eliminated. It is possible that these sequences are fragments of mitochondrial DNA that migrated to the nucleus (numts) [35][36][37], a very common phenomenon in Orthoptera [38]. Sequences were submitted to GenBank after these analyses ( Table 3).

Analysis of molecular data
We analyzed both genes independently through Bayesian inference using the Markov Chain Monte Carlo (MCMC) method as implemented in MrBayes 3.1 [39,40]. We ran MrModeltest version 2.0 [41] with PAUP* version 4.0b10 [42] to choose the model parameters to be estimated by Bayesian analyses using the Akaike Information Criterion (AIC) [43]. Two simultaneous and independent runs, each containing one cold and three heated chains, were processed for 10 7 generations, each starting from a random tree (random topology and branch lengths) with random parameters. These parameters (topology, branch lengths and model parameters) were sampled every 1000 generations, resulting in 10 4 samples. After running the analyses, we burned-out 25% of the initial generations, checked for chain convergence (,0.01) and used the remaining topologies (7500) with their respective branch lengths to build a majority-rule consensus tree ( Fig. 7A-B).

DNA Barcodes
Sequences suitable for DNA Barcoding should be short, have low intraspecific divergence and high interspecific divergence [44]. Thus, we examined our COI alignment for a region that could fulfill these requirements and built a distance tree using Neighbor-Joining [45] with Kimura 2-parameters model [46]. We compared this tree to the tree generated by Bayesian Analysis based on complete COI sequences. Table 3 summarizes all species studied in this work , with the GenBank accession numbers for each gene, when applicable. We captured 104 individuals of Listroscelidinae in ten conservation units (Table 3, Fig. 1) in the Brazilian Atlantic Forest. Figure 22A-B shows records of species of all studied genera, except for Listroscelis species, which are shown in Fig. 22C-D. Using this material, we proposed a new tribe, recognized four genera (one new) and 14 morphospecies: eight new, four redescribed and two, Hamayulus sp. and Monocerophora sp., possibly new, but not described here due to the lack of adult males. In addition, we redescribed Listroscelis angustifrons comb. nov. and Megatympanon speculatum based on museum specimens. The second column of Table 1 summarizes the taxonomy proposed in this work. Figure 7A-B shows the gene trees based on COI and rRNA 18S, respectively.

Molecular analysis
Mitochondrial COI. The complete alignment of 48 COI gene sequences (Table 3) resulted in 1290 aligned sites, of which   540 are variable. The best fit model of sequence evolution using AIC was GTR + I + G. In Fig. 7A, after separating the outgroup (composed of other Tettigoniidae species) all sequences showed more differences between species, genera and tribes than within these taxa and all ancestor nodes were tightly defined (PP = 1.00), i.e., each taxon was represented by a well-defined cluster (Fig. 7A).
rRNA 18S. The complete alignment of 48 18S gene sequences (Table 3) resulted in 1615 aligned sites, of which only 11 were variable. The best fit model of sequence evolution using AIC was GTR + I. Among these sequences, several were identical, thus we chose to show a simplified 18S tree in Figure 7B. Unlike with COI, most taxa within Listroscelidinae were not well defined in this tree. After the separation of the outgroup, Listroscelidinae split into two poorly defined groups: the first containing the sequences of two species of Cerberodon (PP = 0.51) and the second containing the remaining sequences (PP = 0.76): a total of 36

DNA Barcodes
Through visual analysis, we determined the 59 portion of COI has higher interspecific than intraspecific variation. Thus, we delimited a fragment of 500 bp between sites 2394 and 2890 of the complete mitochondrial genome of Oxya chinensis (Thunberg) (Orthoptera: Acrididae, GenBank accession number: NC010219) (Fig. S5). The dendrogram obtained is similar to the one based on complete COI (Fig. 7A) and addresses the need to distinguish between species (Fig. S6).  fastigium sulcated at the longitudinal midline, (iii) sclerites of antennal sockets not in contact at midline, (iv) thoracic auditory spiracle completely exposed, oval and small, free from pronotum, (v) spines on each sternite stout, and spines on meso-and metasternum flattened, (vi) wings short, partially exposing the second abdominal tergite, (vii) femora with ventral spines devoid of minute spines between them, (viii) fore tibiae weakly curved, (ix) tympana with rounded openings, (x) outer edge of fore tibiae with one spur below the tympanum, (xi) male paraprocts comparatively modified, enlarged, (xii) phallus completely membranous.

Taxonomy
Included taxa. Hamayulus gen. nov. This new tribe is proposed based on the morphological differences with the current recognized tribes, and supported by molecular data provided here (Fig. 7A). Description. Head. Fastigium triangular, shorter than the first antennomere of the flagellum, dorsally flattened and bearing a linear sulcus at the longitudinal midline. Eyes oval and frontally protruding, each inserted next to the upper edge of the subjacent antennal socket. Head twice as long as wide; appearing globose in frontal view, mainly due to the protruding vertex. Frons triangular, with a conspicuous ocellus. Surface of clypeus, genae    (Table 3). Bold numbers on the left side of each species name correspond to the collection site of each specimen (Fig. 1 Fig. 2A). Meso-and metasternum with flattened spines. Legs. Each coxa bearing one ventral spine, acute or rounded, at the basal and another at the distal portion, ventrally ( Fig. 2A). Legs slender and elongated; hind femora longer than the body. Fore and mid femora without a ventral longitudinal furrow. Both ventral margins of femora armed with spines. Fore tibiae straight, each with a spine at the outer side of the dorsum, below the tympanum. Hind tibiae with spines on the dorsal and ventral margins. Genicular lobes of legs ending in an acute tip. Fore tibiae with tympanal openings located dorsally; sclerites completely inflated; openings about three times as long as wide. Male postabdomen. Paraprocts elongated (Fig. 4A). Subgenital plate wide; apical portion emarginated (Fig. 5A). Phallus membranous, devoid of titillator (Fig. 3A). Female postabdomen. Subgenital plate three times longer than wide; apical portion triangular, without emargination (Fig. 5B). Ovipositor shorter than the length of the body, mostly straight; upper valve as wide as lower valve; apex of ovipositor acute (Fig. 8D). This new genus includes H. rufomaculatus sp. nov. and a second species that is known only from immatures (one female and seven males) and therefore not described here. This second Hamayulus species was recognized based on molecular data ( Fig. 7A-B).
Diagnosis. This species can be distinguished by the following combination of characters: (i) body mostly greenish, (ii) tegmina short, covering only the meso-and metanotum, (iii) stridulatory file and apical portion of each tegmen with a red mark, (iv) last two abdominal tergites with a red mark at the midline of about onethird the length of the tergite, (v) male paraprocts modified as an elongated structure with an acute tip.
Description Head. Antennal scape twice as long as pedicel. Antennae inserted at the middle of the face in frontal view. Frons subtriangular and slightly inflated. Clypeus short, about one-quarter the length of the labrum. Mandibles with blackish cutting edge. Wings. Tegmina with stridulatory file as shown in Fig. 6A. Legs. Femora bearing thick bristles over the entire dorsal surface, ventrally with spines decreasing in length from base to apex. Fore tibiae with nine spines in male and five in female. Tarsi with the fourth tarsomere as long as the first three together. Male postabdomen. Cerci cylindrical (Fig. 4A), width decreasing from middle to apex; apical portion narrower, with tubercles and more bristles than basal portion; tip rounded. Styli short, about one-quarter the length of the subgenital plate (Fig. 5A). Phallus as shown in Fig. 3A. Female postabdomen. Ovipositor slightly shorter than abdomen (Fig. 8D). Coloration. Body mostly greenish when alive ( Fig. S1; after death specimens were stored in alcohol and lost the natural color). Male cerci reddish. Ovipositor with a blackish stripe at the outer lateral surface. Alive, individuals with eyes, abdominal sternites and lateral portion of last two abdominal tergites yellowish. Sclerites of tympana and tegmina reddish. Females are devoid of reddish spots at the abdominal apex (Fig.  S1).
Variation. Measurements of males (n = 4, including the Hamayulus sp. ( Figure 22A) The distinction of Hamayulus sp. was based in molecular results ( Fig. 7A-B). The sampled individuals are all immature and, therefore, morphological identification is not possible at this moment.
Specimens examined. Diagnosis. The genera belonging to this tribe share the following combination of features: (i) body usually robust, (ii) fastigium laterally compressed, narrower and shorter than the first antennomere of the flagellum, usually sulcated (projecting upward and not sulcated in Monocerophora), (iii) antennal sockets usually not in contact at midline, (iv) thoracic auditory spiracle completely exposed, oval and enlarged, free from pronotum, (v) pro-and mesosternum with slender spines, usually flattened at the metasternum, (vi) tegmina covering at least the first two abdominal tergites, (vii) ventral portion of femora with large spines usually interspaced with minute ones, (viii) fore tibiae notably curved, (ix) tympana with elongated and wide openings, with (x) two dorsal rounded pits below each tympanal opening, one at the inner and another at the outer edge of the tibia and, (xi) male paraprocts semitriangular, wide, concealing the membranous portion of the phallus, with lateral outer corners of each plate with a down curved spine (arrows in Figure 4), (xii) titillator usually consisting of a longitudinal sclerite attached to the dorsal lobe of the phallus, with two projections protruding outwards.
Cerberodon Perty, 1832 (Figures 9, 10, 22B, S2) Type species: Cerberodon viridis Perty, 1832 Redescription. Head. Eyes globose and frontally prominent, inserted separately from the ventral margin of antennal sockets. Antennal sockets separated by half the maximum width of the eyes. Head globose and wide in frontal view, due to the protruding vertex; about as wide as long in dorsal view. Frons subtriangular, with a tiny rounded projection in the middle of the longitudinal midline, where the inconspicuous ocellus is located (ocellus with the same color as the frons). Face, genae and basal portion of clypeus strongly wrinkled, with a brain-like appearance. Mandibles robust, with elongated apex; in male, the apical portion of the left mandible is strongly angulated, elongated and curved upwards. Mandibles bear a basal process at the cutting edge. Maxillary and labial palpi greenish. Thorax. Pronotum with anterior margin slightly curved inward, posterior margin almost straight. Prozona with a transverse furrow extending to the lateral lobe, reaching the anterior margin. Mesozona with a straight transverse furrow which is strongly curved forward on the lateral lobes; in lateral view this furrow forms a strong dorsal depression. Metazona with a transverse furrow extending to the lateral lobe, reaching the lower margin. Lateral lobes straight, except for their posterior portions; posterior margin slightly slanted; posterior corners rounded, without sinus humeralis. Each sternite with two stout spines, the ones on the metasternum flattened (Fig. 2B). Legs. Wings fully developed or shortened; tegmina devoid of distinctive marks. Coxae usually with an acute or rounded spine on the basal portion (Fig. 2B). Trochanters usually with an acute spine on the apical portion. Legs robust and short; hind femora shorter than body length. Fore and mid femora with a wide longitudinal ventral furrow. Both ventral margins of femora armed with long spines interspersed with more delicate ones. Fore tibiae slightly curved inwards, with a small spur or pit (when the spur is lost) dorsally, below and close to each tympanal opening. Mid tibiae with four to seven spines. Hind tibiae with spines on their dorsal and ventral margins. Genicular lobes of all legs ending in an acute spine. Tympanal openings located at the outer edge of tibiae; entire region of the tympana slightly inflated, with elongated tympanal openings, the width one-eleventh the length. Male postabdomen. Cerci bent in a somewhat acute angle, ending in a hook-like acuminate tip ( Fig. 4B-C); apical portion as long as basal portion, with tubercles and long bristles. Supraanal plate triangular or rounded. Subgenital plate wide; apical portion with a V-shaped emargination (Fig. 5C, E). Styli short, about onequarter the length of the plate or shorter. Phallus with prominent titillator, visible from dorsal and lateral views ( Fig. 3C-D), projected outwards, ending in a rough lobe. The titillator is composed by two independent bars with only their bases attached to the dorsal lobe; at the midline, the titillator is close to two transversal secondary sclerites that form a kind of bridge, but are not fused (Fig. 3B). Female postabdomen. Subgenital plate triangular or subtriangular, with a V-shaped emargination of about one-quarter the length of the plate at the longitudinal midline ( Fig. 5D Diagnosis. This species can be distinguished from C. portokalipes by the following combination of characters: (i) tegmina longer than abdomen, (ii) fore tibiae with ventral surface blackish.
Redescription. (Based on images of type material and examination of additional specimens). Head. Frons subtriangular and protruding (Fig. 9C, F). Mandibles with outer margin wrinkled. Wings. Tegmina with stridulatory file as shown in Fig. 6B. Male postabdomen. Cerci with basal portion wider than apical portion, strongly curved inwards and ending in an acute tip (Fig. 4B). Subgenital plate with a deep V-shaped emargination shorter than half the length of the plate (Fig. 5C). Phallus as shown in Fig. 3C-D. Female postabdomen. Subgenital plate with a V-shaped emargination (Fig. 5D) of about one-quarter the length of the plate. Ovipositor as long as abdomen (Fig. 9D). Coloration. Body mostly light greenish and dark brownish. Head dorsally greenish. Fastigium dark brownish. Scape and pedicel ventrally chestnut-colored and dorsally greenish; flagellum light brownish. Eyes light brownish, with cuticle around them blackish. Frons dark brownish. Alive, surfaces described as dark brownish or chestnut-colored are dark reddish (face, anterior and posterior margin of pronotum, spines on legs, mouthparts in ventral view, areas at sternum, abdominal spiracles, abdominal sternites, dorsal and ventral margin of ovipositor). Eyes pinkish frontally and whitish dorsally; labrum pinkish, lighter than after dead. Face, genae and clypeus dark brownish. Mandibles light brownish with cutting edge dark. Labrum brownish. Mouthparts in ventral view dark brownish. Pronotum greenish. Tegmina with primary and secondary veins greenish, surface between veins dark brownish, anal and stridulatory area dark brownish. Legs mostly greenish with spines dark brownish; coxae and trochanters chestnut-colored ventrally, with remaining surface greenish. Fore legs with ventral surface of femora and surface close to spines of tibiae blackish. Tarsi and tarsal claws dark brownish. Sternum greenish, with some chestnut-colored spots. Tergites greenish; sternites in male light reddish, and in female whitish ( Fig. S2A-F).
Listroscelis Serville, 1831 ( Figures 11-18, 22C-D, S3) Type species. Listroscelis armata Serville, 1831 Redescription. Head. Eyes globose and protruding, not touching the antennal sockets. Apex of antennal sockets in dorsal view shorter than half the length of eyes. Head elongated, width in dorsal view about twice as long as wide; vertex barely visible in frontal view, not protruding. Frons subtriangular or oval, with a rounded projection at middle, where the defined or undefined ocellus is located. Face with surface smooth or slightly wrinkled. Mandibles with apex elongated and curved inwards, with a basal process at the cutting edge, symmetric or asymmetric in males; when asymmetric, the left mandible has its lateral portion elongated and curved inward (different from Cerberodon species, in which the apex of the left mandible is elongated and is devoid of lateral projection). Thorax. Pronotum with anterior and posterior margins straight or slightly concave. Prozona with a transverse furrow extending to the lateral lobes, not reaching the lower margin. Mesozona with a transverse furrow, extending to the lateral lobes, sometimes reaching the lower margin. Metazona with a straight transverse furrow extending laterally to the lateral lobes, almost reaching the posterior margin. Lateral lobes with lower margin almost straight; posterior margin slightly oblique, with rounded corners and without sinus humeralis. Each sternite with two spines with rounded tips; prosternal spines comparatively slender; metasternal spines flattened or slender (Fig. 2C). Wings. Wings fully developed. Tegmina with a bright yellowish spot at the base. Legs. Hind coxae with ventral margin usually bearing one basal and one distal spine, the tip of the spines can be acute or rounded (Fig. 2C). Legs slender. Hind femora as long as the body or slightly longer. Fore and mid femora with a wide longitudinal furrow along the ventral surface. All femora armed with long stout  spines interspersed or not with tiny spines. Fore tibiae with surface close to spines blackish, and a small rounded pit dorsally, below and close to each tympanal opening. Mid tibiae with four to six ventral spines. Hind tibiae with several spines on the dorsal and ventral margins. Genicular lobes of all legs ending in an acute tip. Tympanal openings located at the dorsal surface of the tibiae, the width of each opening about one-sixth the length; ear region conspicuously inflated. Male postabdomen. Tergite X unmodified or modified, elongated or bearing lobes, emarginated or not; when elongated, it covers epiproct and paraprocts (Fig. 4D, G, J). Cerci with bent apex, or slightly curved inward or downward Paraprocts triangular and concealing the phallus. Phallus with an entire titillator composed by a transversal bridge which arises from the basal portion of the dorsal lobe, and with two outward projections with rounded tips (Fig. 3E-F). Female postabdomen. Subgenital plate sometimes emarginated (Fig. 5H, K, M, P, R, S). Ovipositor slightly upcurved; upper valve about three times as wide as lower valve; apex of ovipositor acute (Figs. 11D, 13D, 14D, 16D, 17D, 18A).
Included species. This genus includes L. angustifrons (Piza, 1960)  Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) surface of body dark brownish and greenish, (ii) face, genae and clypeus slightly wrinkled (but not brain-like), (iii) left mandible of male slightly elongated inward, (iv) each tegmen with a yellowish spot of about one-eighth the tegmen length, (v) male tergite X elongated, its width decreasing abruptly from middle to apex, which has a deep median emargination.
Redescription (based on images of type material and examination of additional specimens). It is unknown how many specimens were measured by Karny [50], males (n = 2 syntypes): Mandibles with apical portion elongated and acuminate, in male the left mandible being more elongated than the right one (Fig. 11C, F). Wings. Tegmina with stridulatory file as shown in Fig. 6D. Male postabdomen. Cerci of male elongated, forcepsshaped (Fig. 4D), with tip curved downward. Subgenital plate with an emargination leading to a V-shaped cut that is less than half the length of the plate (Fig. 5G). Styli about one-third the length of the plate. Female postabdomen. Subgenital plate as shown in Fig. 5H. Ovipositor as long as abdomen (Fig. 11D). Coloration. Body mostly dark brownish and dark greenish. Dorsal surface of head dark brownish. Fastigium with dorsal portion yellowish and sides brownish. Eyes dark brownish; dorsal inner surface with a dark yellowish oval area. Sclerites of antennal sockets blackish; antennal scape and pedicel brownish; flagellum dark brownish. Frons dark brownish. Face and clypeus dark brownish. Labrum with basal portion light yellowish and remaining surface dark brownish. Mandibles, maxillary and labial palpi dark brownish. Pronotum dark brownish; lateral lobes without distinct marks. Epimeron with the surface close to the prothoracic spiracle light brownish. Tegmina dark brownish, with primary and secondary veins as well as surface between them brownish. Hind wings light brownish. Legs with coloration pattern as follows: coxae and trochanters dark brownish, femora dark greenish, tibiae dark brownish. Additionally, fore femora have a dark brownish stripe at the lower margin of the lateral portion; in females this stripe is weaker. Femoral and tibial spines dark brownish. Surface surrounding tympana dark brownish. Tarsi dark brownish, the third-one darker; claws dark brownish with darker tips. Alive, some parts are aquamarine blue, as follows: ventral portion of legs, pleura close to all coxae, abdominal sternites, pleurites, subgenital plate and cerci. Parts which are light greenish: dorsal and lateral area of legs, coxae, trochanters, and basal portion of the ovipositor. Femora with a whitish spot at the base of each spine. Abdominal spiracles whitish. Tergites somewhat light purple ( Fig.  S3A-C).
Variation. Type material data not included. Measurements of male (n = The species is here transferred to Listroscelis. It cannot remain in Cerberodon because species of that genus have strongly wrinkled face, genae and basal portion of the clypeus, with a conspicuous brain-like appearance (Figs. 9C, F, 10C, F), while in L. angustifrons comb. nov. they are smooth or slightly wrinkled (Fig. 12B). Each tegmen of Listroscelis angustifrons comb. nov. has a bright spot on the basal portion (Fig. 12A), similar to other Listroscelis species (e.g. Figs. 11A, D, 13A, D, 16A, D), while species of Cerberodon are devoid of such spots at tegmina (Figs. 9A, D,  Figs. 10A, D). In L. angustifrons comb. nov. the titillator is a single, undivided sclerite, a feature common to all examined Listroscelis species (Fig. 3E), while in Cerberodon the titillator has two separated sclerites (Fig. 3C).
Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) surface of body mostly chestnut-colored and blackish, (ii) surface of face, genae and clypeus slightly wrinkled, not brain-like, (iii) left mandible of male elongated and held beyond the frontal plane of the head, tip upcurved, (iv) each tegmen with a yellowish spot of about one-tenth its length, (v) male tergite X unmodified.
Distribution. The type-locality is ''Espírito Santo'', but the exact locality is unknown. The examined specimens are registered from Alagados do Itabapoana and Domingos Martins, in Espírito Santo (Fig. 22C). These are the first accurate records of geographic distribution for the species. Two males collected from Espírito Santo (Museum of Vienna) were erroneously identified by Redtenbacher as L. atrata. The correct identification is L. angustifrons comb. nov. The species is most similar to L. atrata and L. magnomaculata sp. nov.
Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) surface of body mostly dark chestnut-colored and light and dark greenish, (ii) face, genae and clypeus with transverse wrinkles arranged horizontally, parallel to the vertex, (iii) left mandible of male with pre-apical portion elongated and bent upward, (iv) base of each tegmen with a yellowish spot of about one-quarter the length of tegmen, (v) male tergite X unmodified.
Variation  Distribution. The specimens were sampled in RPPN Serra Bonita, locality 2 (Figs. 1B, 22D). Individuals of this species were abundant in the field, unlike other species of the genus.
Diagnosis. This species can be distinguished from the other Listroscelis by the following combination of features: (i) surface of body light greenish with few light brownish areas, (ii) face, genae and clypeus smooth, (iii) mandibles symmetric, (iv) each tegmen with yellowish spot about one-twelfth its length, (v) male tergite X elongated, its width decreasing abruptly from middle to apex; posterior margin with a deep oval-shaped emargination at middle.  (Fig. 14C, F). Face somewhat smooth, barely wrinkled. Wings. Tegmina with stridulatory file as shown in Fig. 6G. Male postabdomen. Cerci with apical portion slightly curved downwards (Fig. 4G). Subgenital plate wide, its posterior margin with a V-shaped emargination at middle (Fig. 5L). Styli of about one-fifth the length of the plate. Female postabdomen. Subgenital plate as shown in Fig. 5M. Ovipositor a bit longer than abdomen (Fig. 14D). Coloration. Body mostly light greenish and light brownish. Dorsal surface of head light greenish. Fastigium dorsally whitish and laterally brownish. Sclerites of antennal sockets blackish; antennal scape and pedicel blackish; flagellum dark brownish. Eyes dark brownish with diffuse blackish spots. Face light brownish; sides light greenish. Clypeus and labrum light brownish. Mandibles with basal portion and sides light brownish; remaining surface, including cutting edge, blackish. Ventral portion of mouthparts light brownish. Maxillary and labial palpi greenish. Pronotum light greenish, lateral lobes without distinctive marks. Sternites and spines light greenish. Tegmina, including primary and secondary veins, light greenish; surface between veins dark brownish. Legs, including coxae and trochanters, light greenish; spines of femora and tibiae light brownish. Ear region light brownish. Tarsi light greenish, the claws light greenish with dark brownish tips. Abdominal tergites and sternites light greenish. Alive, individuals had more vivid greenish coloration (Fig. S3G-H). Distribution. The specimens were sampled in Reserva Biológica (ReBio) de Sooretama, locality 9 (Figs. 1B, 22D). We have also seen images from a specimen deposited in the Museum of Vienna, collected at ''Reserva do Rio Doce'' in Espírito Santo, near the type-locality.
Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) body mostly dark to light brownish and light greenish, (ii) face, genae and clypeus almost smooth, slightly wrinkled, (iii) mandibles symmetric, (iv) each tegmen with a yellowish spot of about onesixteenth its length, (v) male tergite X unmodified.
Description (holotype =).  16-18; sHTv, inner margin 9-12, outer 11. Head. Frons triangular, with well-defined ocellus (Fig. 15C). Wings. Tegmina with stridulatory file as shown in Fig. 6H. Male postabdomen. Cerci with apical portion bend inward (Fig. 4H). Subgenital plate with a deep V-shaped emargination (Fig. 5N) Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) surface of body dark and light brownish, (ii) face, genae and clypeus smooth in females and slightly wrinkled in males, (iii) left mandible of male bent before apex and with tip projecting upward, (iv) each tegmen with a yellowish spot of about oneeleventh its length, (v) male tergite X unmodified.
Description  (Fig. 16C, F). Face slightly wrinkled in male and smooth in female. Wings. Tegmina with stridulatory file as shown in Fig. 6I. Male postabdomen. Cerci stout, with tip strongly curved inwards (Fig. 4I). Subgenital plate with a V-shaped emargination at midline (Fig. 5O); styli of about half the length of the plate. Female postabdomen. Subgenital plate as shown in Fig. 5P. Ovipositor slightly longer than abdomen (Fig. 16D). Coloration. Surface of body mostly dark and light brownish. Dorsal surface of head dark brownish, with a whitish stripe extending from the tip of the fastigium to the posterior margin of head. Fastigium with lateral portions dark brownish. Eyes dark brownish with diffuse darker spots. Sclerites of antennal sockets blackish. Antennal scape and pedicel in male somewhat blackish; pedicel with the inner area yellowish, in female dark brownish with diffuse blackish spots. Flagellum light brownish. Frons and face dark brownish. Clypeus in male whitish with yellowish margin, in female with basal portion close to the angles blackish; labrum blackish with basal portion yellowish. Mandibles blackish. Ventral surface of mouthparts yellowish. Maxillary and labial palpi light greenish. Pronotum dark brownish; each lateral lobe with a blackish stripe from the lower to the posterior margin. Epimeron with surface close to the prothoracic spiracle light brownish; prothoracic spiracle with blackish margins. Thoracic sternites and spines yellowish. Tegmina light brownish with primary veins light brownish, secondary veins light greenish, surface between veins light brownish. Legs with coxae and trochanters yellowish and with diffuse dark brownish spots; mid and hind coxae and trochanters darker than those of fore legs. Femora and tibiae dark brownish. Apical portion of femora darker. Femoral spines dark brownish. Hind femora with lower margin blackish. Tibial spines light brownish. Tarsi dark brownish; claws dark brownish with blackish tips. Abdominal tergites light brownish and sternites dark brownish. Alive, specimens with abdominal tergites and sternites light greenish; whitish stripe at the dorsal surface of head more vivid (Fig. S3I-J). Distribution. The specimens were sampled in P.E. Serra do Brigadeiro, locality 10 (Figs. 1B, 22D).
Diagnosis. This species can be distinguished from the other Listroscelis species by the following combination of features: (i) body mostly dark and light brownish, (ii) face, genae and clypeus mostly smooth, with few wrinkles, (iii) mandibles symmetric, (iv) each tegmen with a yellowish spot of about one-tenth its length, (v) male tergite X wide, with an oval-shaped emargination of about one-quarter the length of the plate at middle.  (Fig. 17C, F). Face mostly smooth, with few wrinkles. Wings. Tegmina with stridulatory file as shown in Fig. 6J. Male postabdomen. Cerci stout, apical portion narrowed and curved downwards (Fig. 4J). Subgenital plate wide, with a V-shaped emargination (Fig. 5Q). Styli of about one-fifth the length of the plate. Female postabdomen. Subgenital plate as shown in Fig. 5R. Ovipositor a bit longer than abdomen (Fig. 17D). Coloration. Body mostly light and dark brownish. Dorsal surface of head dark brownish. Fastigium yellowish dorsally and light brownish laterally. Eyes dark brownish with diffuse darker spots. Sclerites of antennal sockets blackish; antennal scape and pedicel light brownish with indistinct dark brownish spots; flagellum dark brownish. Clypeus yellowish, labrum light brownish with dark, almost black apical portion. Mandibles blackish. Ventral surface of mouthparts light brownish. Maxillary and labial palpi yellowish. Pronotum dark brownish; lateral lobes without distinctive marks. Sternum and spines light brownish. Epimeron close to the prothoracic spiracle light brownish; prothoracic spiracle with blackish margin. Tegmina light brownish; primary veins dark brownish, secondary veins light brownish, surface between veins dark brownish. Hind wings light brownish. Legs, including coxae and trochanters, light brownish; femora darker than tibiae; femoral spines dark brownish; tibial spines dark brownish with lighter tips. Ear region dark brownish. Tarsi light brownish, the third one darker; claws light brownish with darker tip. Abdominal tergites and sternites light brownish. Alive, they have almost the same colors as preserved specimens (Fig. S3K).
Diagnosis. It can be distinguished from the other Listroscelis species by the following combination of features: (i) surface of body dark and light brownish and light greenish, (ii) face, genae and clypeus smooth, (iii) each tegmen with a yellowish spot of about one-quarter its length.
Monocerophora Walker, 1869 (Figures 19, 20, 22B, S4) Type species. Monocerophora longispina (Burmeister, 1838) Redescription. Head. Eyes globose, located laterally, inserted at the level of the ventral margin of antennal sockets. Apex of antennal sockets as high as dorsal margin of eyes. Face apparently slender, due to the fastigium and frons that are protruding upward; vertex barely visible in frontal view; width sixtenths the length of the head. Frons long and triangular, usually exceeding the apex of sclerites of antennal sockets; lower portion of the frons with a defined ocellus. Face smooth. Mandibles robust, with tip elongated. Mandibles without a basal process at the cutting edge. Thorax. Pronotum dorsally with anterior and posterior margins slightly concave. Prozona with a transverse furrow extending to the lateral lobe, not reaching the lower margin. Anterior margin of the mesozona with a transverse furrow extending to the lateral lobe but not reaching the ventral margin. Metazona with a straight transversal furrow reaching the posterior margin of lateral lobes. Lateral lobes with ventral margin straight; posterior margin straight. Each sternite with two slender spines with rounded tips (Fig. 2D). Wings. Wings well-developed; tegmina without a bright spot. Legs. Coxae bearing acute or rounded spines on the ventral margin, one at the basal and another at the distal portion (Fig. 2D). Legs slender; hind femora as long as the body. Fore and mid femora ventrally sulcated with broad longitudinal furrow. Femora of all legs with both ventral margins armed with long spines. Fore tibiae with a small rounded pit below and close to each tympanal opening. Hind tibiae with several spines on the dorsal and the ventral margins. Genicular lobes of the legs ending in an acute tip. Tympanal openings located dorsally on the tibiae; openings fairly wide, the width onetenth the length; area surrounding ear region not inflated. Male postabdomen. Cerci slightly curved downward; tubercles and bristles covering the entire surface (Fig. 4K). Supraanal plate triangular. Subgenital plate elongated, with a shallow emargina-tion; styli as long as the plate (Fig. 5T). Phallus with a single titillator formed by a transversal bridge which arises from the basal portion of the dorsal lobe, and with two free projections oriented outward, with rounded tips (Fig. 3G-H). Female postabdomen. Female subgenital plate wide, with a short U-shaped emargination at the posterior margin (Fig. 5U, V). Ovipositor shorter than body length, mostly straight; upper valve about three times as wide as lower valve; apex of ovipositor acute (Figs. 19D,  20D).
This genus includes M. longispina (Burmeister, 1838), M. minax Walker, 1869, reinstated status and M. spinosa (Karny, 1907). Molecular results showed three clades defined by the COI gene (Fig. 7A): the first clade from southern Bahia (localities 1, 2 and 4, Fig. 1), corresponding to M. minax reinstated status; a second from Espírito Santo (ReBio de Sooretama, locality 9), where all specimens are immature, making species-level identification impossible (Monocerophora sp.) and the third from the border of Minas Gerais and Rio de Janeiro (PARNA do Itatiaia, locality 13), corresponding to M. spinosa. Although the type-locality of M. spinosa is the state of Espírito Santo, the precise locality is unknown. However, as the specimens collected in Espírito Santo are grouped in a different clade than those from Rio de Janeiro, at this time the best option is to consider them Monocerophora sp. Specimens from PARNA do Itatiaia are all females and morphologically similar to the described male M. spinosa. It is important to carry out additional surveys to confirm the identity and distribution of M. spinosa and Monocerophora sp.
Monocerophora minax Walker, 1869, reinstated status ( Figures 19, 22B, S4A-C) Redtenbacher [2] synonymized M. longispina and M. minax based on specimens from Rio de Janeiro. Males of M. minax bear conspicuous long spine-shaped frontal fastigium (in the frons), and in M. longispina the frons is only slightly longer than antennal sockets according to the original description. Additionally, specimens from Rio de Janeiro checked by Redtenbacher have a brown face, different from the blackish face of M. minax. As Monocephora minax was described based on a male, this is the first time that females are described and registered.
Diagnosis. This species can be distinguished from M. spinosa by the following combination of features: (i) fastigium projected forward, with a rounded tip, (ii) male with frons strongly projected upward, reaching the tip of the antennal pedicel; in female, frons is about one-third the length of antennal scape, (iii) face and mandibles blackish.
Redescription. (Based on images of the holotype of M. minax =). Holotype =: LT 33.80. Head. Face mostly smooth, central portion close to clypeal suture wrinkled. Male postabdomen. Cerci stout, the tip slightly curved inward. Coloration. Dorsal surface of head blackish. Sclerites of antennal sockets, antennal scape, pedicel and flagellum blackish. Eyes dark brownish with diffuse blackish spots. Face and clypeus blackish. Labrum with basal portion yellowish, the remaining blackish. Maxillary and labial palpi yellowish. Pronotum dark brownish. Tegmina mostly light brownish; primary and secondary veins light brownish; surface between veins darker; stridulatory area blackish. Fore and mid legs with the following coloration pattern: femora dark brownish with lighter apical portion, ventral portion and spines blackish; tibiae with basal portion yellowish, central portion light brownish, distal portion and spines blackish. Hind legs dark brownish; femora with a blackish stripe at the ventral margin, extending from base to middle; dorsal surface blackish. Tarsi dark brownish, the third one darker.
Observations on collected males and females. Head. Mandibles stouter in male than in female (Fig. 19C, F). Clypeus slightly wrinkled. Wings. Tegmina with stridulatory file as shown in Fig. 6K. Male postabdomen. Supraanal plate trapezoidal, with the posterior margin rounded (Fig. 4K). Cerci as shown in Fig. 4K. Subgenital plate twice as long as wide (Fig. 5T); styli slightly longer than the plate. Female postabdomen. Subgenital plate as shown in Fig. 5U. Ovipositor as long as abdomen (Fig. 19D). Coloration. Body mostly light to dark brownish and blackish. Fastigium laterally dark brownish. Eyes with inner dorsal surface light brownish. Clypeus with basal suture yellowish; basal portion blackish; disc and apical portion chestnut-colored; basal portion in the middle whitish. Pronotum with anterior, posterior and rear half of the lateral margins blackish. Sternum blackish, with furrows of meso-and metasternum whitish; spines dark brownish. Hind wings and veins yellowish. Tarsal claws light brownish with darker tips. First to third abdominal sternites in male light brownish, the remaining ones blackish; sternites dark brownish in females. Alive, specimens with lighter coloration. Eyes blackish, with inner dorsal surface lighter and separated by a yellowish stripe. Antennal scape with a longitudinal whitish stripe at the outer portion. Spiracle of the third thoracic segment yellowish, more conspicuous than the others. Costal margin of the tegmina greenish (Figs. S4A-C).
Monocerophora spinosa (Karny, 1907) Listroscelis spinosa Karny, 1907 ( Figures 20, 22B, S4D-F) Diagnosis. This species can be distinguished from M. minax reinstated status by the following combination of features: (i) fastigium triangular with rounded tip, and about one-third the length of the scape, surpassing the apex of sclerites of antennal sockets, (ii) frons triangular, as high as the antennal sockets, (iii) face dark brownish, and mandibles blackish.
Redescription. (Based on images of the holotype =). Measurements of the holotype =: TL 30.00; PL 9.00; TegL 50.50; FF 28.00; HF 32.00; HT 36.00. Head. Face smooth, as shown in Fig. 20C. Male postabdomen. Cerci slightly cylindrical and curved downward. Coloration. Body mostly light to dark brownish and blackish. Dorsal surface of head with median portion blackish, except for a fine light brownish line extending from the fastigium tip to the posterior portion of head, sides light brownish. sides and frontal portion blackish. Eyes light brownish with diffuse blackish spots; inner dorsal surface yellowish. Sclerites of antennal sockets, scape and pedicel blackish. Frons dark brownish, with a well-defined light yellowish ocellus. Face dark brownish. Mandibles blackish with basal portion dark brownish. Maxillary and labial palpi brownish. Pronotum dark brownish, with a blackish median stripe. Tegmina light brownish; primary and secondary veins light brownish, with surface between them dark brownish; coastal margin blackish. Fore and mid femora dark brownish, apical portion lighter. Femora with light brownish spines, each spine with a whitish spot at the outer basal portion. Hind femora with a dark brownish stripe at the outer margin. Tibiae light brownish, basal portion lighter. Fore and mid tibial spines with light brownish tips. Tarsi light brownish, the third tarsomere darker. Abdominal sternites dark brownish.
Observations on collected females. Female postabdomen. Subgenital plate as shown in Fig. 5V. Coloration. Body mostly light to dark brownish and blackish. Antennomeres of flagellum light brownish, distal margin of each antennomere with a dark brownish stripe. Clypeus dark brownish with whitish disc (Fig. 20F). Mouthparts ventrally yellowish, except for the light brownish labial palpi. Pronotum with a blackish mark on the posterior portion of lateral lobes. Sternum blackish; areas close to the furrows of the pro-and metasternum black. Hind wings light brownish, veins of the same color. Legs with coxae and trochanters dark brownish, with diffuse blackish spots. Fore coxae and trochanters darker. Abdominal sternites dark brownish and pleurites blackish. Alive, individuals (only females were observed) lighter. Eyes dorsally light yellowish. Disc of clypeus more vividly whitish. Tibiae reddish. Abdominal pleura light purple. Basal portion of ovipositor pinkish (Fig. S4E). Immature female as shown in Fig. S4F.
Distribution. The specimens were sampled in PARNA do Itatiaia, locality 13 (Figs. 1B, 22B). Individuals were captured mostly in Arecaceae plants. The accurate type locality of M. spinosa is unknown, the only available information being ''Espirito Santo''.
Comment. As M. spinosa was described based on a male, this is the first time that the female is described and registered.
( Figure 22B) The distinction of Monocerophora sp. was based on molecular data (Fig. 7). The sampled individuals are all immature and, therefore, morphological identification is not possible at this moment.
Terpandrini Gorochov, 1990 The classification of Megatympanon is controversial. The genus was originally described by Piza [51] as a Tympanophorinae. Riek [52] considered that the metasternal spines are typical of Listroscelidinae and Saginae, but because of the presence of at least one dorsal apical spur on hind tibiae, Megatympanon was transferred to Listroscelidinae (as ''Listroscelinae''). Gorochov [53] proposed a new tribe, named Terpandrini, inside Saginae and including Neobarrettia and Terpandrus Stål. Later, Rentz [54] proposed another new tribe also named ''Terpandrini'', based on Terpandrus and a few Australian genera, but inside Listroscelidinae. The first proposal for the name Terpandrini is the valid one, and so Gorochov is the author of the tribe. The inclusion of Terpandrus in Listroscelidinae, rather than in Saginae, however, is the most recent opinion on the position of the genus and consequently transfers the tribe it names to Listroscelidinae. Megatympanon is very similar to some Terpandrus species [54]. Regardless of subfamily assignment, the monospecific Megatympanon is the only known genus of Terpandrini in South America. As we have not observed specimens of other genera of Terpandrini, a diagnosis of this tribe is not provided. However, a redescription of the species is necessary and is provided here, for future discussion on this matter.
Megatympanon Piza, 1958 Type species. Megatympanon speculatum Piza, 1958 Description of Megatympanon is in Piza [51]. Megatympanon speculatum Piza, 1958 ( Figures 21, 22A) Diagnosis. This species can be distinguished from the remaining Listroscelidinae by the following combination of features: (i) sclerites of antennal sockets touching in midline, concealing the tip of fastigium, (ii) Thoracic auditory spiracle oval and enlarged, partially covered by the paranotum, free from pronotum, (iii) each sternite with two spine-shaped processes, (iv) each tegmen with identical stridulatory area, (v) phallus devoid of titillator. Fastigium laterally compressed, narrower and shorter than the first antennomere of the flagellum (Fig. 21C, F). Eyes globose and laterally protruding, inserted close to the outer edge of antennal sockets. Apex of antennal sockets in frontal view as high as eyes.

Redescription
Fastigium of frons triangular. Face, genae and clypeus smooth. Mandibles not robust and without modifications. Maxillary and labial palpi yellowish. Thorax. Pronotum with anterior margin straight; posterior margin strongly convex. Mesozona covering bases of tegmina dorsally. Prozona with a transverse curved furrow, extending to the lateral lobe but not reaching the ventral or anterior margin. Mesozona with a V-shaped transverse sulcus, extending to the lateral lobes, not reaching the ventral margins. Metazona with a transverse furrow extending laterally, not reaching the ventral margin. Lateral lobes with ventral margins curved, posterior margins oblique; corners rounded. Each sternite with two long and acute spines (Fig. 2E). Wings. Wings developed. Tegmina devoid of bright spot; stridulatory file as shown in Fig. 6L. Legs. Legs slender. Hind coxae with ventral margin bearing one basal acute spine (Fig. 2E). Hind trochanter with ventral margin bearing one apical rounded spine (Fig. 2E). Hind femora as long as body. Fore and mid femora with a broad longitudinal ventral furrow. Both ventral margins of femora armed with spines interspersed with thin spines. Fore tibiae straight, devoid of spur or pit below tympanal openings. Mid tibiae with seven to nine spines. Hind tibiae with spines on dorsal and ventral margins. Each genicular lobe with two acute spines. Tympanal openings located in the dorsal plane of fore tibiae; ear region not inflated; tympanal openings narrowed, and partly covered by lateral sclerites. Male postabdomen. Cerci cylindrical and curved inward, with acute tips (Fig. 4L). Supraanal plate short and subtriangular (Fig. 4L). Paraprocts short, triangular, without modifications. Subgenital plate wide; apical portion with a Vshaped emargination (Fig. 5W). Styli short, about one-quarter the length of the plate. Phallus devoid of titillators. Female postabdomen. Subgenital plate with a U-shaped cut emargination about one-third the length of the plate (Fig. 5X). Ovipositor shorter than abdomen, slightly upcurved; upper valve about four times as wide as lower valve; apex of ovipositor acute, lower valve with apical portion serrate ventrally (Fig. 21D). Coloration. There is no information on color of live individuals. Old specimens deposited in museums are yellowish, slightly greenish.
Variation. Measurements of male including holotype (n = 3): features in Listroscelidini. It is unknown whether this dimorphism is an ancestral condition retained by few living taxa or has independently arisen in different groups. Such features are also observed in Arachnoscelis, which has different developmental degrees of the mandibles. However, the way of development, as described by us, differs within Listroscelidini. Also, the mandible in Carliella, Cerberodon, Listroscelis and Hamayulus gen. nov.
(Hamayulini trib. nov.) bears a large, basal, ventral process, a state unique among Tettigoniidae [21], while Monocerophora's mandibles lacks this process, which may be a recent evolutionary loss. Fastigium. Listroscelidini, as well as the only North American listroscelidine Neobarrettia, have a compressed and narrowed fastigium. Monocerophora has an overdeveloped frons (as a result, in M. minax reinstated status, the frons is slightly directed upward), which has triangular or subtriangular shape; while in Cerberodon, Listroscelis and Isocarliella, the frons is a sulcated triangle, with the central portion protruding. In Terpandrini (Neobarretia and Megatympanon) and Arachnoscelis (incertae sedis), the fastigium is not markedly differentiated. In Terpandrini, the fastigium grows from the anterior portion of the occiput, between antennae (in dorsal view). In Hamayulus gen. nov. the fastigium is sulcated. Vertex. The vertex is remarkably protruded, mainly in Cerberodon (Listroscelidini), Neobarretia (Terpandrini) and Hamayulus gen. nov. (Hamayulini trib. nov.). It is related to the frontal or frontolateral position of eyes and possibly an adaptation to predatory habits. The position of eyes is similar to that observed in predatory Meconematinae, Saginae and Hexacentrinae.
Thorax. In Listroscelidini, the metazona is typically elevated from the sulcus, which is more evident in Monocerophora but also occurring in Neobarrettia (Terpandrini). In other Terpandrini, such as Megatympanon, Burnuia Rentz and Chlorobalius Tepper, the metazona is so elongated that it covers the basal dorsal portion of wings. Auditory Spiracle. The auditory spiracle of Listroscelidini is typical of Conocephaloid [8]: elongated and with a wide opening, free from pronotum and completely exposed; while in Megatympanon the spiracle is partially covered by the lateral lobes of pronotum. Sterna. Listroscelidinae are characterized by the presence of sternal spines. Within Listroscelidini, except for Monocerophora, the spines on the pro-and mesosternum are slender, and on the metasternum they are usually a bit flattened and similar to the ones of Neobarrettia (Terpandrini) species. In Monocerophora, all spines are slender with rounded tip; and in Megatympanon (Terpandrini), they are long and acute. Listroscelidini also have another type of armature at the ventral margin of coxae, each possessing one spine at the distal border and another at the proximal border, with different degrees of development. Sternal and coxal spines can be used for catching preys [13,56].
Legs. Only Listroscelidini have the fore tibiae notably curved with long movable spines, which are longer than the spines in Terpandrini species. Listroscelidini species also have two dorsal tiny spurs or rounded pits below each tympanum, one close to the inner and the other close to the outer tibial margin, which is an important characteristic of the tribe. Although living and preserved individuals are usually devoid of such dorsal spurs (we observed only one L. cohni sp. nov. with intact spurs), it is possible to observe their small attachment cavities (pits) and confirm their existence. Cohn (1957) reported that none of the Listroscelidinae checked by him (Cerberodon, Listroscelis and Macrometopon) bore intact dorsal spurs, which confirms that these structures are easily lost. In Hamayulus gen. nov., as in Decticini and Neobarrettia, there is only one spur (or cavity) close to the outer margin, while in Megatympanon there are neither spurs nor cavities. Cohn [13] stated that it is not clear whether the presence of two spurs below each tympanum is a derived and convergent or a more ancestral feature. In the latter case, it would have been retained in Listroscelidini and independently lost in other Listroscelidinae tribes. At least in Listroscelidini + Hamayulini trib. nov., the couple of spurs may be an ancestral condition. The ventral spines on all femora interspaced with minute spines is also a characteristic of Listroscelidini, but it is different in Monocerophora. Tympana. In most Listroscelidini, tympanal openings are elongated and wide, usually located at the dorsal surface of fore tibiae; while in Terpandrini, the openings are long, but narrow and barely visible. The tympana in Hamayulus gen. nov.are most similar to those of Arachnoscelis.
Postabdomen. Cerci, supraanal and subgenital plates are very variable among Listroscelidini species. Some patterns were found at genus level, e.g. Monocerophora and Cerberodon, but there are no apparent patterns in Listroscelis. Male paraprocts and phallus are remarkable shared characteristics: the paraprocts are wide, concealing the membranous portion of the phallus, and bear a tiny spine at the outer vertex; and the phallus consists of a conspicuous dorsal lobe and developed titillators. Cohn [13] dissected the phallus of a Macrometopon species, but did not describe its titillators. We decided to include Macrometopon in Listroscelidini because its single species has modified paraprocts, as in other species of the tribe, while in Terpadrini the paraprocts are simple. Ontogenetic differences. It is difficult to differentiate immature individuals of Listroscelis, Monocerophora and Cerberodon. While immature of Listroscelis and Monocerophora have a dark longitudinal midline at the pronotum, absent in adult Listroscelis (Figs. S3, S4), the asymmetric lengthened mandibles of Cerberodon and some species of Listroscelis are exclusive of adults. The lack of differentiation among immatures suggests a common origin of these genera (supporting their inclusion within the same tribe), while the differences among adults probably result from chronologic developmental differences. Curiously, the first characteristic cited, the dark longitudinal midline at the pronotum, is conserved in adults of other subfamilies of Tettigoniidae, such as Conocephalinae and Pseudophyllinae, as well as in Meconema Serville (Meconematinae), a genus that Zeuner [9] pointed out as a sister group of Listroscelis. This author suggested that the morphological differences between these genera are superficial and result only from different feeding habits.
Further taxonomic comments. The next logical step in the taxonomic revision of Listroscelidini is to review Terpandrini. As indicated, Neobarrettia is more similar in several features to Listroscelidini than to Terpandrini. In addition, it is the only North American genus of the tribe, while the others (except for the Brazilian Megatympanon) occur in Australia. The similarities between Megatympanon and Terpandrus (from Australia) were first exposed by Rentz [54].
Identification key to Listroscelidinae of the Brazilian Atlantic Forest 1 Sclerites of the antennal sockets in contact at midline. Tympanal openings narrow and concealed by lateral sclerites. Fore tibiae without a small pit or spur dorsally, below and close to each tympanal opening. Sternal spines ending in an acute tip. Paraprocts simple, triangular, devoid of spines… Megatympanon speculatum (Figs. 2E, 21) 19 Sclerites of the antennal sockets not in contact at midline. Tympanal openings wide, not concealed by lateral sclerites. Fore tibiae with a small pit or spur dorsally, below and close to each tympanal opening (two or only one at the outer side). Sternal spines ending in a rounded tip. Paraprocts modified… 2

Discussion
After more than a century since Litroscelidinae katydids were recognized as a suprageneric taxon, they remain barely known to science, which may be attributed to the difficulties in collecting these animals. In addition, some species are aggressive (e.g. Monocerophora and Cerberodon species) and others are very fast (e.g. Hamayulus spp.), making it difficult to observe and collect them in the field. In addition to the high number of individuals collected (104), our samples had approximately five females to each male, making it difficult to identify and describe the species, which are preferably identified by males. No males of L. itatiaia sp. nov., L. carinata sp. nov. or M. spinosa were collected.
Despite these difficulties, we identified 14 distinct morphospecies, 12 of which have been treated taxonomically here. We collected a maximum of three genera from each sampled site. Except for Listroscelis monnei sp. nov. and L. cohni sp. nov. (both collected in locality 4; see Figs. 1B, 22C-D), each genus was represented by a single species in the same sample site, indicating a high rate of endemism of Listroscelidini in Atlantic Forest remnants. In fact, all Listroscelidinae species sampled in this work have a narrow distribution and were primarily collected in wellpreserved forest remnants with low anthropogenic impacts, especially in areas close to water bodies, 50 cm to 2 m above the ground. The restricted distribution and high rate of endemism indicate that Listroscelidinae species may be at serious risk of extinction.
Our 18S tree contains the 14 morphospecies recognized here, while the COI tree contains only 12, since we found evidence for the presence of numts in many individuals and eliminated them to avoid confusion [37,57]. In order to circumvent these problems, we are investigating the specimens suspected of containing numts to quantify the numts and pursue a strategy for sequencing only mitochondrial sequences (Fialho & Yotoko, unpublished data).
Although limited by the smaller number of taxa, the COI tree shows evidence of 13 distinctive mitochondrial lineages, as shown in Fig. 7A. Moreover, these strains are well separated into genera and suggest two tribes within Listroscelidinae: Listroscelidini (composed by Cerberodon, Monocerophora and Listroscelis) and Hamayulini trib. nov. (composed of two species of Hamayulus gen. nov.). In turn, all sequences of Monocerophora and Listroscelis were identical in the 18S tree (Fig. 7B). The topology differences between genes used here were expected and resulted from inherent differences in mitochondrial and nuclear genomes. While mitochondrial DNA have higher rates of nucleotide substitution, which makes it an excellent tool to distinguish closely related species [58,59]; nuclear DNA evolves more slowly, making it useful for resolving deeper phylogenetic branches [60,61].
Beyond confirming our morphological findings, our molecular results gave us clues regarding a major division in Listroscelidinae. Both sequences revealed two significantly different lineages within Hamayulus gen. nov. (although both strains were collected in Bahia, most in locality 4, with only one exception, collected at site 3). That the sequences of 18S revealed differences between strains of Hamayulus gen. nov, while no differences were detected between Listroscelis and Monocerophora, indicates that the separation of these strains must be very ancient, and we are possibly dealing with two distinct genera. Unfortunately, we have collected only immature individuals of Hamayulus sp., therefore new sampling will be needed to test this hypothesis.
The COI tree allowed us to suggest Monocerophora be subdivided into four distinct mitochondrial lineages: M. spinosa (sampled at locality 13), two different lineages within M. minax reinstated status (the first sampled in localities 1 and 2 and the second in locality 4) and a lineage named Monocerophora sp. (sampled in locality 9). Neither Monocerophora sp. nor the second lineage of M. minax reinstated status were morphologically identified because all individuals were immature.
A different result was found in the L. fusca and L. itatiaia pair. In this case, the diagnosis based on morphology is clear, but the molecular differentiation is subtle. Again, these species were collected in different localities (10 and 13), suggesting geographic distribution should be taken into account in taxonomic studies of Listroscelidinae.
Our dendrogram (Fig. S6), based on the region chosen as the DNA Barcode (final portion of the COI, Fig. S5) showed significant differentiation between species [44], and is very similar to the phylogenetic hypothesis of Fig. 7A. This result indicates it is possible to identify known species of Listroscelidinae with one sequencing reaction using the primer COI_Orth_1R (Table S1).
Though powerful, DNA Barcoding should be used with caution. Morphological analyses are laborious and require extensive training. Our results suggest convergence of morphological and molecular results. Therefore, we recommend sequencing the DNA Barcode region of any new listroscelidine specimen, but if any evidence of numts is detected (see the checklist suggested by Cristiano et al. [37]), the sequence should be removed from the analysis and treated separately.
Taxonomy has much to gain from combining morphological and molecular approaches, provided they are done conscientiously, so that technical complications (such as the possibility of numts, for instance) do not affect the quality of final results. Such an approach can boost taxonomic studies within this group, allowing studies on the diversity and distribution of listroscelidine species in the Atlantic Forest and other biomes in a relatively short time.

Conclusions
This is the first broad taxonomic and molecular work on Neotropical Listroscelidinae. Based on specimens collected in the Brazilian Atlantic Forest, as well as some specimens deposited in museums, we added a new tribe, a new genus and eight new species to the subfamily and redefined Listroscelidini. We redescribed and added new geographic records for six species, synonymized two species and built a provisional identification key for Listroscelidinae occurring in the Atlantic Forest. Only four Listroscelidinae genera remain unclassified: Arachnoscelis, with six described species from southern Central America and the westernmost portion of the southern Neotropics; the monospecific Liostethomimus Karny, described from southern Brazil (but the type-specimen is considered lost) and the monospecific Paralistroscelis Carl and Poecilomerus Karny, both occurring in Madagascar.
Our results suggest that the species of this subfamily, at least those restricted to the Atlantic Forest, are seriously endangered. This is because they have high rates of endemism, restricted distribution and occur mostly in highly preserved forest remnants. As such, it is important that future collection efforts take into account the use of barcodes in this article, in order to accelerate the identification of known species and the detection of possible new ones.  (Fig. S1) using the Neighborjoining method [45] with the substitution model Kimura 2parameter [46]. Values alongside internal nodes correspond to bootstrap values, calculated through 1000 replications. Vertical bars mean that a set of sequences belong to one species or genus, identified at right of each bar. Numbers besides species' names correspond to sampled localities (Fig. 1). Outgroup: Oxya chinensis (Thunberg) (GenBank: NC010219.1).

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Table S1 Primers sequences used in this work.

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Table S2 Geographic coordinates of the sampled conservation units. Locality numbers are the same shown in Fig. 1.