Mesozoic evolution of cicadas and their origins of vocalization and root feeding

Domain: earth_science
Published: 2024-01-08
Editorial outcome: accept

Abstract
Extant cicada (Hemiptera: Cicadoidea) includes widely distributed Cicadidae and relictual Tettigarctidae, with fossils ascribed to these two groups based on several distinct, minimally varying morphological differences that define their extant counterparts. However, directly assigning Mesozoic fossils to modern taxa may overlook the role of unique and transitional features provided by fossils in tracking their early evolutionary paths. Here, based on adult and nymphal fossils from mid-Cretaceous Kachin amber of Myanmar, we explore the phylogenetic relationships and morphological disparities of fossil and extant cicadoids. Our results suggest that Cicadidae and Tettigarctidae might have diverged at or by the Middle Jurassic, with morphological evolution possibly shaped by host plant changes. The discovery of tymbal structures and anatomical analysis of adult fossils indicate that mid-Cretaceous cicadas were silent as modern Tettigarctidae or could have produced faint tymbal-related sounds. The discovery of final-instar nymphal and exuviae cicadoid fossils with fossorial forelegs and piercing-sucking mouthparts indicates that they had most likely adopted a subterranean lifestyle by the mid-Cretaceous, occupying the ecological niche of underground feeding on root. Our study traces the morphological, behavioral, and ecological evolution of Cicadoidea from the Mesozoic, emphasizing their adaptive traits and interactions with their living environments.

Introduction
Cicadas refer to the superfamily Cicadoidea, which are comprised of two modern families, Cicadidae and Tettigarctidae, whose monophyly is supported by molecular and morphological phylogenetic analyses of living species1,2. Extant Cicadidae consists of slightly more than 3000 worldwide species2–7, while Tettigarctidae is thought to be a relict group containing only one living genus with two species in Australia1,8–10. Members of Cicadidae are well-known for producing some of the loudest sounds among insects through their tymbal mechanisms that consist of intricate organs producing sound through vibration of a ribbed membrane11–13. By contrast, Tettigarctidae is a clade that lacks production of loud sounds, and instead uses vibrational signals transmitted through the substrate for communication14. These two, considerably different, expressions of sound have led to speculation on the interpretation of tymbal structures associated with sound production and the evolution of their behaviours8,14. No previous work has explored the structural features and related behaviours associated with understanding the evolution of communications in cicada fossils.

Another remarkable feature of Cicadoidea is that their nymphs possess long-term and subterranean life habits, reflecting distinct ecological niches and survival strategies between their nymphal and adult stages. Cicada nymphs live underground, tunnelling through the substrate and feeding on xylem sap of roots, until the final instar emerges to undergo a final moult, subsequently emerging as a winged adult that achieves sexual maturity before its short-lived stage as an adult15–20. Cicada nymphs can live underground for up to 17 years17,18, with their life cycles producing significant effects on forest soils, microbial biomass, nutrient availability, predators, and host plants21–25. Immature and imaginal stages of individuals do not equally respond to the same evolutionary forces; therefore, different growth stages are of great significance in revealing different aspects of evolutionary mechanisms26–28. Consequently, nymphal fossils are necessary to illuminate the complete life cycles of ancient cicadas and their effects on terrestrial ecosystems, both below- and aboveground. Nevertheless, cicadoid nymphal fossils are rare; only five incomplete and early instar nymphal fossils have been reported from mid- to Late Cretaceous and Cenozoic amber and an opal deposit29–32. These records clearly are insufficient to reflect the true evolutionary and ecological significance of cicadas as important soil and arboreal herbivores in insect history.

The adult fossil record of Cicadoidea includes 55 genera33–40, which is largely represented by wing fragments, and consequently provides limited morphological information for early cicadoid lineages. Currently, all adult Mesozoic fossils are classified within Tettigarctidae, mainly based on forewing venation. Despite a few fossil sites that preserve entire bodies of cicadoids, the present categorisation disregards many significant traits of these early fossils due to an over-reliance on traditional classification criteria and the lack of knowledge of continuous spatiotemporal morphological variation within this superfamily. To date, there is no clearly reviewed molecular evidence for assessing the divergence time between Cicadidae and Tettigarctidae. We think selecting the earliest currently known fossils of these two extant families for molecular clock calibration is disputable.

Here, we report newly described adults, a final-instar nymph, and exuviae of Cicadoidea from mid-Cretaceous (~99 Ma) Kachin amber that originated in northern Myanmar (Fig. 1). We use morphological data from fossil and extant Cicadoidea to conduct phylogenetic and morphological disparity analyses, trying to clarify the phylogenetic relationships between Mesozoic fossil and extant cicadoids and to illuminate macroevolutionary changes in their body structure adaptations. We discover the membranous tymbal and tymbal muscles associated with cicadoid sound production in the fossil record, and report fossil final-instar nymphs with specialised forelegs and long piercing-sucking mouthparts, indicative of both fossorial and root-feeding behaviours. In sum, we provide a more comprehensive picture of the relationships, and ecological and evolutionary history of early Cicadoidea.

Results
Systematic palaeontology
Order Hemiptera Linnaeus, 1758
Suborder Cicadomorpha Evans, 1946
Clade Clypeata Quadri, 1963
Superfamily Cicadoidea Latreille, 1802

Stem cicadoids
1
Eunotalia gen. nov.
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Type species Eunotalia emeryi sp. nov. by monotypy.
Included species: Eunotalia emeryi sp. nov.
Etymology: The generic name is a compound form, from Classical Greek prefix: eu-, meaning ‘true’ or ‘good’, and notos, meaning ‘back’ or ‘dorsum’.
Diagnosis: Pronotum subhexagonal and enlarged, without a wrinkled collar, concealing all or part of mesonotum except scutellum, pronotal lateral angles pointed at about the middle length of pronotum. Paramedian fissure not distinct and lateral fissure absent, ambient fissure extending from both sides to the middle and almost intersecting the paramedian fissure sides, forming an acute angle. Forewing wide, costal area broad and with a fold. RA, RP, MA and MP forking into four, two, four and two branches, respectively. CuA2 about half the length of CuA1, following the nodal line but slightly separated and oriented backward, and terminating around nodal clave.
Description: as for the species.
Remarks: The new genus differs from other genera of Cicadoidea by unique combination of features described in the diagnosis.
Eunotalia emeryi gen. et sp. nov. (Figs. 1a, 2a, i and Supplementary Fig. 2).
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Etymology: The specific epithet is given in honour of Prof. David Emery from the University of Sydney, for his contribution to providing extant Tettigarctidae for anatomical study and his discussion of this research.
Material: Holotype: MGM2016–014, male. Silicified, description see in ref. 41. Deposited in the Myanmar Gems Museum, Nay Pyi Taw, Myanmar.
Diagnosis: As for the genus by monotypy.
Description: See Supplementary Note 1.1.
Remarks: Tymbal structure is identified in this fossil species (Fig. 2q). The abdomen is filled with mineral (Fig. 2y). Meracanthus has an enlarged base, and the then tapering and narrowing forward; it is short and does not reach tergites II (Fig. 2ae). The thorax lacks identified opercula. The male genital includes identified structures such as anal style, median lobe of uncus and style (Fig. 2al).

Stem cicadids
Cretotettigarcta ref. 34
Type species. Cretotettigarcta burmensis34.
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Hpanraais35, syn. nov.
Type species Hpanraais problematicus35.
Included species: Cretotettigarcta bumensis34, Cretotettigarcta problematica35 comb. nov., and Cretotettigarcta shcherbakovi sp. nov.
Material: Holotype: NIGP167304, male, deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences; Nanjing, China.
Diagnosis (adapted from ref. 34): Pronotal lateral fissure present but weak; pronotum with wrinkled pronotal collar, almost the half length of the pronotum, lateral collar well-developed; pronotum concealed the part of mesonotum besides scutellum. The length of exposed mesonotum (except scutellum) no more than the half the length of pronotum; mesonotum with scutellum extending to around the end of abdomen. Apex of profemur with a robust lateral tooth-like spine; the upper medial part of protibia with a tooth-like spine; meso and metatarsi preserved two pairs of lateral spines, the second pair stronger than the first pair. Claw with fan-like arolium, arolium centre eminence with a two-petal ornamentation. Forewing with relatively short RA and close to the nodal line; CuA with a strong arc bending shape at the base; M and CuA not fused or connected with short vein; CuA2 sinuous and more than half the length of CuA1.
Description: as in ref. 34.
Remarks: The mesoscutellum of Cretotettigarcta extending to the end of abdomen which is distinguished from other cicadoid genera.
Cretotettigarcta problematica35 comb. nov. (Figs. 1b, 2c, k and Supplementary Fig. 3).
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Material: Holotype: NIGP168934, only one forewing. New material here: NIGP201895. Both are deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences; Nanjing, China.
Diagnosis (adapted from ref. 35): Male body length ~25.2 mm, width ~11.3 mm; the forewing length ~16.8 mm, width ~5.7 mm. Pronotal collar developed, spread out laterally, and lateral angle pointed. Separation of M closed to the separation of RP from R at the wing length. Crossvein m-cu connected CuA1 and MP. Upper medial side of protibia with a relatively sharp spine.
Description: See Supplementary Note 1.2.
Remarks: New material of C. problematicus comb. nov. fits the revised diagnosis of Cretotettigarcta, with preserved body structures and long mesoscutellum.
Cretotettigarcta shcherbakovi sp. nov. (Figs. 1c, 2d, l and Supplementary Fig. 4).
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Etymology: The specific epithet is given in honour of Prof. Dmitry E. Shcherbakov from Paleontological Institute, Russian Academy of Sciences, for thanks to his contributions to the research of fossil Tettigarctidae.
Material: Holotype: NIGP201896, male, deposited in the Nanjing Institute of Geology and Palaeontology of the Chinese Academy of Sciences; Nanjing, China.
Diagnosis: Male body length ~13.6 mm, width ~5.9 mm; the forewing length ~11.1 mm, width ~3.9 mm. Distance between the compound eyes approaching the 1/2 diameter of the compound eyes. Pronotal lateral angle blunt, concave in the middle of pronotal posterior margin not distinct. Crossvein m-cu connected between CuA1 and forking point of MP. Upper medial side of protibia with a relatively blunt spine.
Description: See Supplementary Note 1.3.
Remarks: Crossvein m-cu connects to CuA1 and forking point of MP is distinguished in the new species from the other known species in this genus. The tymbal structure is identified in this fossil species (Fig. 2t). The meracanthus is not preserved completely, but it exhibits an enlarged base (Fig. 2l). The abdomen is deformed.

Vetuprosbole34
Typespecies: Vetuprosbole parallelica34, by monotypy.
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Included species: Vetuprosbole parallelica34.
Material: Holotype: NIGP168021, gender unknown. New material here: NIGP201897, female (Figs. 1d, 2e, m and Supplementary Fig. 5). Both are deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences; Nanjing, China.
Diagnosis (adapted from ref. 34): Pronotum concealing small part of mesonotum beside scutellum, with wrinkled collar. Pronotal lateral fissure relatively weak. Length of exposed part of mesonotum (except scutellum) slightly shorter than the length of pronotum. Mesonotum with scutellum extending near abdominal tergite II. Metatarsus preserved two pairs of lateral spines, and second pair of spines more developed than the first pair. Claw developed with fan-like arolium. Forewing RA forking into three branches and RA1 next to Sc and split before the margin. CuA long and sinuous, more than 2/3 length of CuA2. CuP fused a short segment with 1A at the basal part.
Description: as for the species of a new specimen. See Supplementary Note 1.4.
Remarks: New features found in the new material of V. parallelica include a wrinkled collar that is less than half the length of the pronotum, a larger exposed mesonotum with a scutellum, and CuP fused to a segment with 1A. These features allow it to be distinguished from all other known genera of Cicadoidea. The meracanthus of V. parallelica is triangular and reaches tergites II (Supplementary Fig. 5c). The thorax lacks identified opercula. The female genital includes identified structures such as gonocoxite XIX, ovipositor and ovipositor sheath.

Pranwanna gen. nov.
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Type species Pranwanna xiai sp. nov., by monotypy.
Included species: Pranwanna xiai sp. nov.
Etymology: The generic name, pranwanna, is from the Jingpho language spoken in Kachin State of Myanmar, meaning ‘primitive’.
Diagnosis: Pronotum sub-trapezoidal; front edge forms an open, neck-like feature; the middle of posterior margin concave, resembling W-shape of the entire posterior margin; wrinkled collar half-length of pronotum. Paramedian and lateral fissures well-developed. Pronotum concealing part of mesonotum. Length of exposed mesonotum slightly shorter than the length of pronotal collar; mesonotum with inflated scutellum, rounded distally, extending near abdominal tergite, scutoscutellar sulcus and base of lateral ridge prominent; two dark brown bands on the surface of scutellum in male specimen, and the bands widened gradually from the centre to both sides; female not observed the band pattern on scutellum. No developed and dilated opercula. Forewing with separation of M and the separation of RP from Sc+R around the nodal; MA forking into two branches and MP single; common stem of M+CuA long; CuA short and straight. 1A fused a short segment with CuP. 2A absent. Nodal line relatively weak. Both male and females specimen possessing tymbal; male with robust tymbal muscles and developed abdominal cavity.
Description: as for the species.
Remarks: Pranwanna gen. nov. is the smallest cicadoid in the fossil record so far. The distinct lateral fissure; scutellum distal aspect rounded and blunt, 2A absent, with long common stem of M+CuA distinguish the new taxon from other known genera of Mesozoic cicadoids.
Pranwanna xiai sp. nov. (Figs. 1e–h, 2e, f, n, o and Supplementary Figs. 6, 7)
urn:lsid:zoobank.org:pub:8596E387-5FC6-4BA7-BBCA-B959D3BEC5E5
Etymology: The specific epithet is given in honour of Mr. Fangyuan Xia from the Lingpoge Amber Museum for his contribution to discovery the holotype and allotype specimens.
Material: Holotype: LYU–BC2001, male. Allotype: LYU–BC2002, female. Paratype: LYU–BC2003, male. Type specimens are deposited in the Linyi University, Linyi, China.
Diagnosis: As for the genus by monotypy.
Description: See Supplementary Note 1.5.
Remarks: Tymbal and tymbal muscles are identified in this fossil species (Fig. 2u, v, aa, ab). The abdomen has a cavity and preserves malpighian tubules. (Supplementary Fig. 7). Meracanthus is quite long in male, beyond half the length of tergites II of abdomen; meracanthus in female reaches approximately tergite II of abdomen (Fig. 2ah, ai). The thorax lacks identified opercula (Fig. 2ah, ai). The male genital includes identified structures such as anal style, aedeagus, upper lobe of pygofer, basal lobe of pygofer and style (Fig. 2ao). The female genital includes identified structures such as anal style, gonocoxite XIX and ovipositor (Fig. 2ap).

Phylogeny and morphological disparity
Here, we perform morphological phylogenetic analyses of the Cicadoidea, integrating both fossils and extant taxa. The data matrix was analysed using the maximum parsimony method and Bayesian inference. We synthesised morphological information from both published extant and fossil species and supplemented this with micro-CT scan anatomical data from six newly reported adult amber fossils and four extant specimens included in this study. This examination resulted in a total of 81 morphological characters that were used to reconstruct the phylogenetic trees (Supplementary Note 2, Supplementary Data 1 and Supplementary Fig. 8). In this study, we primarily aim to establish phylogenetic relationships between the extant cicadoids and fossil groups discovered in the Middle Jurassic Daohugou deposit, Inner Mongolia, China, and in mid-Cretaceous Kachin amber in Northern Myanmar. The reason behind our selection of fossils from these two localities for phylogenetic reconstruction is attributed to the discovery of a relatively rich and morphologically complete collection of Cicadoidea fossils. These fossils, preserving both winged and non-winged body structures, enable us to establish a precise morphological correspondence between the winged and non-winged parts within the same species.

The main results from maximum parsimony and Bayesian inference are relatively congruent (Supplementary Fig. 8). In both analyses, Eunotalia gen. nov. was recovered as the stem group of the cicadoids. Tianyuprosbole zhengi was recovered as the sister-group of the extant tettigarctids. Shuraboprosbole, Sanmai, and Macrotettigarcta from Daohugou, as well as Cretotettigarcta, Vetuprosbole, and Pranwanna gen. nov. from Kachin amber were recovered into a large clade with extant cicadids. In the results of maximum parsimony, Shuraboprosbole, Sanmai, and Macrotettigarcta cluster as one clade, although this is not strongly supported. In the results of Bayesian inference, these genera are shown to constitute a paraphyletic group. The groups, Cretotettigarcta, Vetuprosbole, and Pranwanna gen. nov., were recovered closer to the extant cicadids. Pranwanna gen. nov. was recovered nearest to the clade of extant cicadids. The clearly relationship between Cretotettigarcta and Vetuprosbole was not strongly supported in both analyses. Considering that any evolutionary clade can be divided into a stem group and a crown group in theory42, and to better illustrate the relationships between the fossils and extant taxa, we provide a simplified schematic of the phylogenetic results in Fig. 3a.

To facilitate the identification of small changes in morphological structures, we also quantified specific body morphology using continuous or categorical variable features data, separately considering aspects such as non-winged adult body structures, wing venation and outline features, and key nymphal leg characters. Different data analysis protocols, including a principal component analysis (PCA), non-metric multidimensional scaling (NMDS), and a principal coordinate analysis (PcoA), have been utilised to perform morphological disparity analysis of the body structures43–49.

Through morphological observations, we have discovered that some homologous non-wing body features present different results across various species. However, these characters have often been overlooked in previous identifications, such as the distance between the compound eyes, the length of the pronotum, the collar of the pronotum, the position of the anterior lateral angles of the pronotum relative to pronotum length, the exposed length of the mesonotum and the terminal morphology of the mesonotum. We selected ten landmarks to quantify the relative positions of these structures (Supplementary Fig. 9 and Supplementary Table 1). Figure 3b shows the PCA results of the morphospace for the dorsal features of the head and thorax, obtained through geometric morphometrics. The first three principal components account for 63.2%, 14.9% and 6.6% of the variation, with the cumulative variation explaining 84.7% of the total shape variance of dorsal features of the head and thorax, whose support these features could reflect the main pattern of variation in differences of the dorsal profile of the head and thorax (Fig. 3b, Supplementary Tables 2 and 3).

Because forewing morphology plays a crucial role in the taxonomic study of insect fossils, analysing the morphospace of the forewing assists in visualisation of morphological similarities, facilitating the analysis of morphological differences and comparisons with phylogenetic outcomes. Figure 3c illustrates the result of morphological disparity obtained by quantifying the forewing features within the Cicadoidea. To ensure an unweighted and comprehensive selection of features, we chose 32 characters, including as many forewing classificational features as possible (Supplementary Note 3 and Supplementary Data 2). We employed a method that describes discrete data to quantify forewing characters and utilised NMDS for data analysis. The stress value i.e. 0.1272 indicates that the result has a good reliability level. The similarity among forewings can be judged by the distance among the scatter points to some extent (Fig. 3c). The forewings of Mesozoic cicadoid fossils exhibit a higher similarity to extant crown tettigarctids. The overall similarity of the forewings of modern tettigarctids is closer to those of crown cicadids than most Mesozoic cicadoids are to crown cicadids. Pranwanna gen. nov shows a greater resemblance to the forewings of the crown cicadids than other known Mesozoic groups. The morphospace of forewing morphology in the Mesozoic is greater than that in the Cenozoic, which is, in turn, greater than that of modern groups. Mesozoic groups, particularly the stem groups of the Cicadoidea, are located on the far left of Fig. 3c. Following that, the stem cicadids appears, with the crown cicadids situated on the far right of Fig. 3c. The distribution of morphological similarity is consistent with phylogenetic relationships and temporal evolution, indicating that the evolution of wing morphology has a certain directionality.

From the Mesozoic fossil groups to modern crown groups, a morphological evolutionary trend in wing profile also seems present. Therefore, we selected nine landmarks to calibrate the wing profile individually and conducted a PCA (Supplementary Figs. 10a, 11 and Supplementary Table 4). The results support the major variation pattern of forewing profile difference to a certain extent (Supplementary Fig. 10a and Supplementary Tables 5, 6). The morphological changes in forewing profiles from the Mesozoic groups to modern crown groups are mainly reflected in changes in the curvature of the wing profile, which overall is transformed from a rectangular ellipse to a triangle (Supplementary Fig. 11).

Moreover, to further assist in understanding the morphological changes, we attempted to gather and compare statistics on body length and width, forewing length and width, length of the prothoracic and exposed mesothoracic notum, distance between the two compound eyes, width of head, width of the base of the labium, and width of the rostrum from both fossil and extant groups. The statistical trend shows that, wing length and width, body length and wing length and body length and width had proportional relationships (Supplementary Fig. 10b, c). From the analysis of measurement data from both fossil and modern species, we found that overall body length does not show a pattern of increase from the Mesozoic to the modern cicadoids. The results indicate that the body length and wing length of both fossil cicadas and extant cicadas fall within a relatively stable range, specifically between 5.45–64.6 mm and 5.6–78.5 mm (Supplementary Fig. 10b, c and Source Data), respectively, although the body width of many compressed fossils cannot be accurately measured due to preservation issues. However, both statistical analysis and morphological observations indicate that crown group cicadas generally display a tendency towards wider bodies, evident in features such as increased head width, greater distances between compound eyes (Supplementary Fig. 10d and Figs. 2a–h, 3b), wider clypeus (Supplementary Fig. 10e and Fig. 2i–p), and the appearance of a central groove in the postclypeus (Fig. 2p). From the discovery of stem groups to modern crown groups of Cicadoidea, there is a noticeable overall macroevolutionary trend of increased exposure of the mesonotum and a shortening of the pronotum (Supplementary Fig. 10f and Figs. 2a–h, 3b).

Morphological analysis of nymphal fossils
The five nymphal specimens (Fig. 1i–m) are certainly placed in Cicadoidea, based on their well-developed capsular compound eyes, prominent postclypeus, long piercing-sucking mouthparts, and specialised fossorial forelegs with developed femoral combs. The distinctive features of each specimen are sufficient to classify these individuals into five species (Supplementary Note 4 for descriptions and Supplementary Data 3 for a comparative summary). However, it is a challenge to clearly connect fossil adults to nymphs in the separated amber pieces. To facilitate the correspondence of these nymphal fossils with adult fossils in a future study, we provisionally categorise them as undetermined Cicadoidea species 1 (NIGP201898, Fig. 1i), species 2 (MGM2016–0.17, Fig. 1j), species 3 (LYU–BC2004, Fig. 1k), species 4 (NIGP201900, Fig. 1l), and species 5 (NIGP201901, Fig. 1m). Species 1 is a final-instar nymph (Supplementary Fig. 12) and spp. 2 to 5 are exuviae retaining features of the final instar nymphs (Supplementary Figs. 13–15).

The legs have the most distinctive identifying features of these fossil nymphs (Supplementary Figs. 12, 14, 15, 16). Species 1, 4 and 5 share several characteristics with extant Cicadidae, including the profemur with an accessory tooth, and the secondary apical tooth of tibia that are small, obtuse, and no longer than 1/2 length of apical tibial tooth (Supplementary Fig. 14a–i). Species 2 and 3 show the typical features of extant Tettigarctidae, including the secondary apical tooth of tibia of the profemur are much longer and more robust, resembling a giant pair of pliers, and no less than 1/2 the length of apical tibial tooth separated from the apical tooth by a certain distance (Supplementary Fig. 14k–p). The unique features of species 2 and 3 namely, include two lateral spines on the outer surface of the profemur, two rows of spines on both sides of the mesotibia, a dominant and prong-like spine among apical spines on the mesotibia and metatibia, and a metatarsus with lateral spines. The examined fossil present four or five teeth of the femoral comb. Final-instar nymphs of extant Tettigarctidae display three teeth of the femoral comb whereas the final-instar nymph of extant Cicadidae display three to nine teeth of the femoral comb50 (Supplementary Data 3). Most species of Cicadidae display six to nine comb teeth, which seem to be a derived character of Cicadidae. The terminal teeth of the femoral comb in extant cicadoids obviously are aggregated into a large flat plate; while this feature is not evident in the current fossils, it seems to indicate distinctive digging abilities. Additionally, other features including the reach of the mouthparts, the diameter and shape of last antennal article, and development of the point of the tibial blade, as detailed in Supplementary Data 3, also are noteworthy. These nymphal traits varied among fossil and living species, which could be useful for future taxonomic and phylogenetic analyses.

We conducted a morphological disparity analysis, a principal coordinates analysis (PCoA), on the leg features of cicada nymphs (Fig. 3d). To prevent discrepancies resulting from structural deformations in the captured images, we quantified the leg features using descriptive discrete data (Supplementary Table 7). PCoA results display the morphological disparity between nymphs from mid-Cretaceous taxa to extant taxa. These results show that the morphological similarities of the nymph species 1, 4 and 5 are closer to crown cicadids, and that the morphological similarities of nymphal species 2 and 3 are closer to crown tettigarctids when compared with crown cicadids (Fig. 3d and Supplementary Table 8).

We have also compared the five nymphs that have been found in the fossil record so far (Supplementary Fig. 16). Because both living Cicadidae and Tettigarctidae possess an accessory femur tooth (Fig. 3d and Supplementary Fig. 14q–t), it’s not a reliable characteristic to classify fossils into these two families. As a result, using this trait for classification makes it unreliable to place the earlier instar nymphs found in Kachin amber31, late Eocene Baltic amber29, and mid-Miocene Dominican amber31 within the crown cicadid clade. The micro-CT data of the early Pliocene nymph from Indonesia shows that the intermediate and first teeth are developed on the femoral comb32, indicating that this specimen does not represent a first instar nymph51. The nymphal leg fragments found in Late Cretaceous New Jersey amber, shown in a line sketch30, exhibit a developed femoral comb that displays four teeth, indicating that they belong to a final-instar nymph and further confirming the generally low number of comb teeth in early cicada nymphs. Other features of this specimen12, such as two confirmed stout spines on the outer face of the femur and a large secondary apical tooth on the tibia that is separated from the tibial apical tooth by a certain distance, show similarities with spp. 2 and 3. Enlarged pretarsus is found in the nymphs of Mosozoic fossil spp. 1–3 and extant tettigarctids34, 52, but not yet in extant cicadids and nymphal sp. 4 fossil. Also associated with adults, an arolium and empodium are present in Mesozoic adult fossils and extant Tettigarctidae respectively, while extant Cicadidae lacks these structures1, 8. We infer that the enlarged pretarsus in the nymphs is manifested as an arolium in the adults, which is a plesiomorphic feature of Cicadoidea. The empodium is an apomorphy of extant Tettigarctidae. Overall, most of the nymphal features of extant Cicadidae and Tettigarctidae have existed since the mid-Cretaceous.

Methods
Materials
The amber specimens, including six new adults and five nymphs, described here originated from the Hukawng Valley in Tanaing Township, Myitkyina District of Kachin State, in Myanmar. The geographical coordinates of the site are ~26°15′N, 96°34′E. The age of these ambers is 98.79±0.62 Ma or early Cenomanian of the earliest Late Cretaceous87. Specimens MGM2016–014 and MGM2016–017 are deposited in the Myanmar Gems Museum in Nay Pyi Taw, Myanmar. Specimens NIGP201895, NIGP201896, NIGP201897, NIGP201898, NIGP201900 and NIGP201901 are deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (NIGPAS), in Nanjing, China. Specimens LYU–BC2001, LYU–BC2002, LYU–BC2003 and LYU–BC2004 are deposited in the Linyi University, Linyi, China. Amber specimens were collected in 2013–2016, before the Myanmar army closed the Kachin amber mines in November 2017. The fossils were acquired in full compliance with the laws of Myanmar. All authors declare that the ambers reported in this study are not involved in armed conflict and ethnic strife in Myanmar.

The specimens of extant Tettigarcta were collected in Tasmania and Victoria, Australia and provided by Prof. David Emery from University of Sydney. The specimens of extant Hyalessa maculaticollis, Platypleura kaempferi and Cryptotympana atrata were collected from Nanjing, Jiangsu Province, China. The descriptive terminology for the adult structures is mainly based on refs. 1, 8; and for the nymphal structures, the descriptive terminology is mainly based on ref. 88 and follow the changed terminology of forewings in this study (Supplementary Note 5 and Supplementary Fig. 19) based on new anatomical observations and a reconciled terminology. The most significant alterations are the discovery of a short RA1 vein of the forewing near the nodal line. The structure name, point of blade of tibia (pbt), is changed to secondary apical tooth of tibia (sapt). For other interpretations please see Supplementary Note 4.

Optical microscopy
Photographs were taken using a Zeiss AXIO Zoom V16 stereomicroscope system at the State Key Laboratory of Paleobiology and Stratigraphy, NIGPAS, and at the Institute of Geosciences, University of Bonn, Germany, and using a Keyence VHX-7000 microscope at the Institute of Geology and Paleontology, Charles University, Czech Republic. Each image was digitally stacked with 10–60 individual focal planes, using the software Helicon Focus 7 for better illustration of the 3-dimensional (3D) characteristics.

X-ray micro-computed tomography (Micro-CT)
Specimens MGM2016–017, NIGP201896, NIGP201897, LYU–BC2001, T. crinita (male), T. crinita (female), Pl. kaempferi (male), Pl. kaempferi (female) were scanned using a 3D X-ray microscope (3D-XRM) (Zeiss Xradia 520 Versa) at the State Key Laboratory of Paleobiology and Stratigraphy, NIGPAS. Images were generated using CCD-based 0.4× and 4× (only LYU–BC2001) objectives, at an X-ray voltage of 50 kV (power 4 W), with voxel sizes of 16.357, 15.519, 20.317, 4.1036, 41.149, 49.601, 35.513, 45.092 μm, respectively. Specimens, LYU–BC2002, NIGP201895, and NIGP0011 were scanned using a phoenix v|tome|x micro-CT at the Institute of Geosciences, University of Bonn. Images of LYU-BC2002, NIGP201895, and NIGP0011 were generated by a CCD-based 40x (only LYU–BC2002) and 20× objective and at an X-ray voltage of 100 kV (120 mA), 90 kV (130 mA), 80 kV (80 mA) with the voxel size 5.1, 9.9, and 9.9 μm, respectively. Data for all specimens were collected over 360°, and the number of images ranged from 1301 to 2051. The exposure time for each projection is between 1.5 s and 5 s. Volume data processing was performed using the software Avizo 8.1 (FEI Visualization Sciences Group, Burlington, MA) and VGStudio Max (version 3.0, Volume Graphics, Heidelberg, Germany).

Phylogeny
We established a multifaceted morphological scheme of phylogenetic data analyses that included Cicadoidea fossils and revealed their phylogenetic placements. We performed phylogenetic analyses under parsimony and Bayesian optimality criteria setting a data matrix of 81 characters and 22 taxa based on an established classification status close to the family level1, 73, 89 (Fig. 3a and Supplementary Fig. 8, Supplementary Note 2 and Supplementary Data 1). The phylogenetic matrix was coded in the software Notepad++. For outgroups that constrained tree topology, we used Hylicellidae including Cycloscytina gobiensis, Cercopidae including Aphrophora pectoralis, Cercopis vulnerata, Procercopidae including Jurocercopis, grandis, Anthoscytina elegans, and Paranthoscytina xiai90–93. We chose the type genus of Cycloscytina for Hylicellidae (Hemiptera) as the root of the tree. The parsimony analysis was conducted in TNT (v.1.6)94, using the Traditional Search option, with memory to store 30,000 trees, 10,000 replicates, with 100 trees to save per replication, and using the tree bisection reconnection algorithm. Four trees were obtained and the strict consensus tree was used equal weighting parsimony95. Bootstrap analyses were performed using a traditional search and 10,000 replicates, with outputs saved as absolute frequencies. Obtained trees were viewed using WinClada 1.00.0896. Supplementary Fig. 8 displayed the unambig changes only, slow optimisation and fast optimisation results of the strict consensus tree97 in Winclada. The Bayesian analysis was conducted in MrBayes (v.3.2.7a)98 using a Markov chain Monte Carlo statistical model. The analysis was run for 5,000,000 generations with a tree sampling frequency of every 100 iterations. Burn-in for tree samples was set at 25%. Convergence of independent runs was assessed using average standard deviation of split frequencies (about 0.0017), and potential scale reduction factors (about one for all parameters).

Morphological disparity
We selected ten landmarks to quantify morphological characteristics (Fig. 3b, Supplementary Fig. 9 and Supplementary Tables 1–3) to do the the principal components analysis (PCA) of geometric morphometrics on thorax and head profiles in dorsal view. We included 55 genera, with 4 fossil genera and 51 extant genera, resulting in a dataset of 175 specimens for analysis (Source Data). For the analysis of non-metric multidimensional scaling (NMDS) of forewing, we employed 32 characteristics of forewings of 90 species from 69 genera of cicadoids (Fig. 3c; Supplementary Note 3 and Supplementary Data 2). For the PCA of forewing profile, we selected nine landmarks to quantified the forewing profile. We included 66 genera, with 30 fossil genera and 36 extant genera, resulting in a dataset of 238 specimens for analysis (Supplementary Figs. 10a, 11, Supplementary Tables 4–6, and Source Data). We selected nine characters of the nymphal forelegs of seven species including five fossils and two extant species represented by extant Tettigarctidae and Cicadidae for the principal co-ordinates analysis (PCoA) (Fig. 3d and Supplementary Tables 7, 8). Landmarks were collected by tpsDig232. The PCA of the landmark data from the dorsal profile was standardised in Past 3.15 by Procrustes analysis and then conducted in OriginPro 2021. NMDS analysis of the characteristics of forewings was conducted in Past 3.15 selected Raup-Crick similarity index. PCA of the landmarks of the forewing profile, and the PCoA of the characters of nymphal forelegs were conducted in Past 3.1599. The morphometric measurement data of cicadoid adults was collected by ImageJ100 (Supplementary Fig. 10b–f), mainly based on previous studies and other internet resources (please see in Source Data). Genera in all datasets involving adult specimens includes specimens from both extant Tettigarctidae and the five subfamilies of Cicadidae.

Figures preparation and artistic reconstruction
Figures were prepared in Adobe Photoshop CS6 and CorelDraw 2023. Line drawings were prepared in Procreate. The artistic reconstruction of Fig. 4 was prepared in Pixologic ZBrush, Maya and Adobe Photoshop CC.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents the first report of the presence of tymbal and tymbal muscles in a cicadoid fossil, and the presence of fossorial forelegs in a final-instar nymph fossil, allowing to suggest communication, digging and feeding behaviours in stem cicadoids. Still, the authors perfomed a phylogentic analysis based on morphology to test the position of new stem genera and species and the relationship between them and crown cicadoids selected species; they used characters from geometric morphometrics analysis and Non-metric Multidimensional Scaling (NMDS) to compare the morphology of head and notum, forelegs and wings venation between stem and crwon cicadoids, and finally they perfomed micro-computed tomographic scans to show a Cicadoidea morphological evolution.\n\nThe introduction is adequate with a good bibliographic review including recent papers. The methods adopted are suitable for the objectives of the paper, as mentioned before. The supplementary material is rich in information including figures and tables. My only suggestion for this section is to include the files of the character matrix and phylogenetic analysis.\n\nThe results and discussion sections have sentences wrongly allocated. I missed a better explanation of some figures in the results section, and I think the discussion section has phrases that should be in the results section.\n\nBelow are some suggestions and comments to improve the manuscript:\n68. I suggest changing “vocal expression” to “sounds expression” or “sounds communications” because the term “vocal” refers to having a voice.\n96. “Currently, all Mesozoic fossils are classified as Tettigarctidae, based principally on forewing venation”, but in Moulds (2018) table 1 (continued) page 446, Burmacicada protera Portinar & Kritsky, 2011 (Cicadidae) it is reported to Late Cretaceous.\n105. I suggest you report in this paragraph the analysis you performed (phylogentic, geometric morphometrics, NMDS) and for what purpose.\n122, 217. If you are stablishing a new genus and this one as a type genus, you have to determine the family-group level which this new genus belongs to and is the type, e.g. family, subfamily or tribe. For example, Cicadetta is the type genus of Cicadettinae (subfamily); Fidicina is the type genus of Fidicinini (tribe). Stem cicadoids is not a taxonomic name and therefore should not be considered to establish a type genus. If this determination is not possible because it is a fossil, then you should only establish as a new genus of Cicadoidea and not as a type genus. Please, see the article 64 of International Code on Zoological Nomenclature.\n114. I know the manuscript can be limited by length to be accepted in this journal, but my suggestion is, if possible, to include the full description of new genus and species in the main text, and not in supplementary material. The taxonomy is an important part of this study, and it is not practical for a reader see the descriptions truncated.\n133. I didn’t find the diagnosis of Eunotalia gen. nov. and E. emeryi sp. nov. in the main text or supplementary notes.\n150. I suggest adding a section name “included species” for each genus and list there the species included.\n213. Vetuprosbole parallelica isn’t in italic.\n256. …” nymphal fossils with adult fossils in a future study, we provisionally categorise them”. Change the point for a comma.\n265. I suggest repeating the figures related to the nymph species in this sentence.\n276. I think there is a reference for this sentence missing.\n312. Many parts in the discussion seem to be results. I suggest separating some sentences, e.g. “The results based on 81 characters (Fig. 3; Supplementary Fig. 13) demonstrate…” and include it in a section for Morphological analysis of adult fossils. Or change the name of the section to Results and Discussion.\n313. I think it is better to specify if the 10 specimens are adults.\n322. I suggest referring to the 81 characters with the Supplementary Note 4 together with the figures.\n334. “previous taxonomic treatments” is relative to stem or crown cicadoids?\n337. Fig. 3b – Change the B to lower case.\n391. Is this statistical analysis some part of the supplementary material? If so, it should be referenced here.\n400. “The distal aspect of the mesonotum in crown cicadids has a cruciform elevation, whereas the rest of cicadoid fossils have scutellum” I don’t understand this sentence since cruciform elevation and scutellum are synonyms (Moulds 2005, fig. 3, page 378).\n454. I didn’t understand the reference to Supplementary Figure 19 here.\n583. It is usual to mention the full name of the taxon at the first mention in the text.\n617. It would be interesting to mention the software where the characters were coded.\n623. The root should be stated.\nPhylogeny - I missed a better explanation of the results of the phylogenetic analysis and the characters in the final tree, e.g. which of the characters were recovered as synapomorphies and homoplastic? I didn’t find the results relating to the number of recovered trees and an explanation of how the implied weight analysis was performed as well as the calculation of k-values (e.g. SPR similarity) and which k-values were recovered.\nSupplementary Figure 19 – the meaning of the abbreviation “tcav” is missing in the caption.\nFigure 2. I suggest putting on the side of each species’ name the correspondent letter, furthermore the species referent to the letter h is missing in the caption.\nFigure 3a. When performing a phylogenetic analysis, it is not usual to use terminals with different ranks. I suggest changing within this figure the terminals represented by genera and subfamilies to species. Moreover, I didn’t find the information of which species were used representing the genera and subfamilies in the analysis, only some of them in the Supplementary Table 2, and in the lines 583 and 600. Besides, the meaning of the numbers in the internal nodes is missing to be informed in the caption. These numbers are not mentioned in the main text (results or discussion).\nFig. 3b, c, d. I missed a better explanation of what was found as results and what these figures represent.\nTatiana P. Ruschel", "reviewer_2": "The current contribution describes new adult and nymphal cicadoid insects from mid-Cretaceous Kachin amber in Myanmar, achieving a great detail of morphological understanding, presents a phylogeny of the group using both fossil and extant taxa, and provides a series or morphospace analyses using abundant data. The results significantly enrich the knowledge on the evolution of the group, identify tymbal structures and other associated structures such as muscles and a probably resonating cavity for the first time in the fossil record of these insects, allowing to make inferences about their possible sound production ability and communication. The nymphal cicadeoid fossils described —previously only known by an early-instar nymph also from Kachin amber and from a final-instar nymph drawn (yet not described) from slightly younger New Jersey amber (these works cited by the authors in Results), both with fossorial-type forelegs— have allowed the authors to infer and discuss the likely digging and underground habits of these immature insects by comparing with their modern counterparts.\nAlthough the text is generally well-written and makes a compelling case, it contains several language elements throughout that required improvement. I have tried to mend as many of these as I could, but please make an additional effort to polish this aspect.\nTaxa description and analyses are thorough and detailed. Figures are of excellent quality.\nPlease note that than I’m not an hemipterologist nor a native English speaker.\nI would like to highlight a few matters that I think are necessary to tackle before the present manuscript can be considered for publication. These, plus the language issues noted above, justify my decision of MINOR-MODERATE reviews for the present work.\n1) Need to tone down some claims by adding the proper degree of uncertainty.\nFirst, I would argue that the finding of nymphs with fossorial legs does not represent direct evidence of their habit and behaviour, since these are inferred through morphology and actualism, which my colleagues would agree that they are not devoid of problems. Therefore, I think adding a certain degree of caution and uncertainty is in order, both in abstract (introduction of “had most likely” in line 55) and discussion (“The specialized foreleg of in these fossil nymphs allows to infer their digging behaviour and subterranean lifestyle” rather than “The specialized foreleg of in these fossil nymphs demonstrates their digging behaviour and subterranean lifestyle”).\nAlso, line 525 reads “Cicada nymph fossils with fossorial forelegs here provide one of the few direct fossil records of root-feeding behaviour”. Similarly as the case above, I would argue that, since the authors are inferring root-feeding behaviour through the same structures that modern relatives having these fossorial habits possess, the present record does not represent a direct evidence of root-feeding behaviour but an indirect one.\nMoreover, the fact that all the material comes from a single locality/region from the mid-Cretaceous limits how general and global the conclusions can be, and so this aspect must be treated with caution. The most clear case of this is shown in the sentence around line 470 “species of Cicadoidea were comparatively silent for of* the Mesozoic compared with modern singing cicadas” (see context), which in my opinion has to be changed to “species of Cicadoidea may have been relatively silent during the Mesozoic compared with modern singing cicadas”.#\nOther instances in which the need to increase the degree of caution/uncertainty in the author’s statements is necessary from my poinf of view are provided in the annotated manuscript.\n2) Need to revise the format of the systematic section.\nI understand that a effort of condensing text and information has been made in order to save space in the ‘Systematic palaeontology’ section of Results, but in the process, there have been several alterations that affect the understandability of the section and its formal structure in my opinion.\n“Material” and “Description” subsections should always be directly under a properly introduced species name in which specimens are then listed, not under a genus heading without proper species introduction. The use of “Type genus” and “Type species” is problematic throughout the section. A species should be introduced after the “Type species” has been mentioned under a given genus, so the “Type species” line should not replace the formal introduction of a species (these are not redundant). I would also advise against merging etymology sections of new genera and their only species within (as with Eunotalia and Pranwanna). Lastly, a diagnosis is missing for the genus Eunotalia or its only species.\n3. One last matter that I would comment here relates to taphonomy and might impact, to a certain extent, the detailed reconstruction.\nRegarding the sentence “Fossils found in Kachin amber and a mass emergence of adult cicadas from the Jurassic Daohugou Lagerstatte demonstrate that they emerged from the soil and ascended to the tree canopy33,50“ in line 503, the fossils in Kachin amber could have also been trapped relatively close to the ground level or at the trunk level. The insects would not have necessarily had to reach the tree canopy to get trapped in resin, based on their preservation in amber. Thus, \"towards the tree canopy\" seems a more suitable expression.\nMoreover, roots are known to produce resin in abundance. Are there any taphonomic characteristics of the amber pieces where the cicadoids are preserved that allow to ascertain the aerial origin of the amber pieces and so that the nymphs were actually ascending to or towards the tree canopy? At least some of the amber pieces actually look cloudy due to abundant debris, which is not conclusive of “litter amber” but might be suggestive of it together with other signs (syninclusions, etc), which might be more indicative or resin entrapment close to the ground. Please consider this option.\nSimilarly, for the reconstruction it would be highly advisable to consider the option that fossils could have been trapped by resin fallen to the ground and capturing fauna therein (“litter amber”). Also, it is known that roots can produce resin in abundant quantities (for instance, see Alvarez-Parra et al, 2021, Dinosaur bonebed amber from an original swamp forest soil). Although the taphonomy of root resin production is poorly understood, and there is no evidence --to my knowledge-- yet of insect entrapment by subaerial resin (that produced by the roots), provided that the current reconstruction showcases the underground layer, not showing this aspect –yet tentatively-- would appear to be a missed chance due to the overall detail of the illustration.", "reviewer_3": "Nature communications\nGeneral comments- The authors performed micro-computed tomographic scans on 10 specimens from mid-Cretaceous Kachin amber and extant cicadoids By combining fossil and extant taxa, and using more detailed anatomical features revealed by micro CT results and morphologically based phylogenetic analysis; forewing and nymphal foreleg, revealed morphological evolutionary tendencies in Cicadoidea. This is a very interesting, original and important approach, however due to the nature of the studied material (fossil), important issues related to the M & M must be clarified. Please check some specific points below.\nGeometric morphometrics is a very sensitive technique. Unfortunately, I do not feel confident to evaluate whether the images obtained from specimens in the amber are precise enough for a geometric morphometric analysis. Any possible discrepancies in the position or in the condition of preservation of the analyzed insects could lead to misinterpretations and questionable information as basis for the new genera and species description.\nThe finding of the first evidence for vocalization associated tymbal structures in fossils, suggesting mid Cretaceous and some other interesting ecological aspects based on the analysis nymphs seems very robust and inedited. However, the scientific basses for the descriptions of new species and genera could be questionable.\nI presented below some brief suggestions for improving the manuscript but, unfortunately, I don’t feel comfortable to either recommend or reject this manuscript for publication.\nSpecific comments\nAbstract- Brief comments on the geographic area where the specimens where found.\nMain\nLines 76-77 In the sentence “live underground for as many years and tunnel through substrate using fossorial behaviors while feeding on xylem sap of roots from the first to fifth instars” and in the Lines 79-80 “ several weeks “. Terms like “many Years and several weeks” should be avoided. The following sentences in the manuscript could be synthesized including the precise information missing in the mentioned lines.\nMaterial and Methods\nIf possible, please provide the geographic coordinates from the sites where the analyzed samples were found.\nPlease comment whether the references cited for morphological nomenclatural were the same ones used for identification and comparisons for the new samples’ descriptions.\nI would suggest including the in text, the ten quantifiable morphological characteristics from the body dorsal profile of cicadoid species.\nIt is not clear whether the wing venation of the amber images could be precisely used for a geometric morphometrics analysis. The samples images in the amber are not defined by precision for analysis.\nPlease explore this point for a better description of the methods\nLegends- All legends must be self-explanatory. Please provide additional relevant information in the legends for all figs. For instance: geographic location, technique used, In the case of fig 1 geographic location, fig 2 the technique used for the images obtained; fig 3 number of species and specimens illustrated, please name the method used for the phylogenetic analysis etc; Fig 4 please provide basic information on the method used for the image reconstruction."}, "author_response": "Response: Thank you very much for your valuable feedback. We have carefully considered your comments and have reorganized some of the results and discussion. Please see the detailed answer below.\n\nBelow are some suggestions and comments to improve the manuscript:\nLine 68. I suggest changing “vocal expression” to “sounds expression” or “sounds communications” because the term “vocal” refers to having a voice.\nResponse: Agreed. Text modified the “vocal expressions” to “sounds expressions”.\nLine 96. Currently, all Mesozoic fossils are classified as Tettigarctidae, based principally on forewing venation’, but in Moulds (2018) table 1 (continued) page 446, Burmacicada protera Portinar & Kritsky, 2011 (Cicadidae) it is reported to Late Cretaceous.\nResponse: Thank you very much for pointing out this issue. Here we add “adult” between all and Mesozoic fossils, and modified this sentence and changed to“Currently, all adult Mesozoic cicadoid fossils are...”. Burmacicada protera Portinar & Kritsky, 2011 is thought to be a first-instar nymphal fossil and classified into Cicadidae. Please allow us to emphasize one additional point here. We think Burmacicada protera Portinar & Kritsky, 2011 as the nymphal fossil was classified into Cicadidae may be problematic. We compared the morphological structures of all nymphal fossil records with those of typical extant nymphs from the living species of Cicadidae and Tettigarctidae, and obtained the following results and conclusions “ Because both living Cicadidae and Tettigarctidae possess the accessoroy femur tooth (Supplementary Fig. 10q–t), its not a reliable characteristic to classify fossils into these two families. As a result, using this trait for classification makes it unreliable to place the earlier instar nymphs found in Kachin amber31, late Eocene Baltic amber29, and mid-Miocene Dominican amber31 within the crown cicadid clade.”. Please see line 426-431.\nLine 105. I suggest you report in this paragraph the analysis you performed (phylogentic, geometric morphometrics, NMDS) and for what purpose.\nResponse: Thank you very much for your suggestion. We add the contents “We conducted a study combining morphological characters of fossils and extant taxa of Cicadoidea to perform phylogenetic analysis and morphological disparity analysis for the first time. Our study revealed clearer phylogenetic relationships between fossil and extant groups of cicadoids, and shed light on the macroevolutionary of adaptive transformations in different body structures.” in this paragraph.\nLines 122, 217. If you are stablishing a new genus and this one as a type genus, you have to determine the family-group level which this new genus belongs to and is the type, e.g. family, subfamily or tribe. For example, Cicadetta is the type genus of Cicadettinae (subfamily); Fidicina is the type genus of Fidicinini (tribe). Stem cicadoids is not a taxonomic name and therefore should not be considered to establish a type genus. If this determination is not possible because it is a fossil, then you should only establish as a new genus of Cicadoidea and not as a type genus. Please, see the article 64 of International Code on Zoological Nomenclature.\nResponse: Thank you very much for pointing this out. We modified the establishment of the type genera to only establish the new genera in this study. We made modifications to lines 131 and 232, revising them to Eunotalia gen. nov. and Pranwanna gen. nov.\nLine 114. I know the manuscript can be limited by length to be accepted in this journal, but my suggestion is, if possible, to include the full description of new genus and species in the main text, and not in supplementary material. The taxonomy is an important part of this study, and it is not practical for a reader see the descriptions truncated.\nResponse: Thank you very much for your suggestion. We agree that the comprehensive description of the new genus and species is very important. However, compared to show complete description of the new genus and species based on the limited space, we would like to more emphasize the results obtained through the taxonomic work to reveal the phylogenetic relationships with extant taxa, key morphological macroevolution, and ecological behavioral analysis of fossils in this paper. All biological features that received relatively higher attention are mentioned in the main text and presented in the figures. Considering the different emphasis in research presentation, we have chosen to retain the complete description in the supplementary information for this study. We recognize the indispensable role of taxonomic work and specimen descriptions as the foundation for discussing morphological evolution, particularly in palaeontological research. Therefore, in our forthcoming study, we will further emphasize and showcase the taxonomic contents.\nLine 133. I didn’t find the diagnosis of Eunotalia gen. nov. and E. emeryi sp. nov. in the main text or supplementary notes.\nResponse: Thank you very much for pointing this out. We modified and added the contents to the section of diagnosis of Eunotalia gen. nov. and E. emeryi sp. nov. Initially, considering the word count and the fact that the specimen represents a single genus and species, we kept the key diagnostic features and comparisons in the \"Remarks\" section.\nLine 150 I suggest adding a section name “included species” for each genus and list there the species included.\nResponse: Thank you very much for your suggestion. We add included species of Cretotettigarcta. Please see line 179-181 “ The remaining fossil genera reported in this article consist of only one species each, which we have chosen not to add for now.”\nLine 213. Vetuprosbole parallelica isn’t in italic.\nResponse: Text modified. Thanks.\nLine 256 …”nymphal fossils with adult fossils in a future study, we provisionally categorise them”. Change the point for a comma.\nResponse: Text modified. Thanks.\nLine 265. I suggest repeating the figures related to the nymph species in this sentence.\nResponse: Text modified. Thanks.\nLine 276. I think there is a reference for this sentence missing.\nResponse: Thank you for pointing this out. Text modified to “ the final-instar nymph of extant Cicadidae display three to nine teeth of the femoral comb49 (Supplementary Table 1)”. We have added an article and supplementary information, which presents more of the comparative results we have summarized.\n49. Hou, Z., Li, Q., & Wei, C. Morphology and identification of the final instar nymphs of three cicadas (Hemiptera, Cicadidae) in Guanzhong Plain, China based on comparative morphometrics. ZooKeys 425, 33–50 (2014).\nLine 312. Many parts in the discussion seem to be results. I suggest separating some sentences, e.g. ‘The results based on 81 characters (Fig. 3; Supplementary Fig. 13) demonstrate…’ and include it in a section for Morphological analysis of adult fossils. Or change the name of the section to Results and Discussion.\nResponse: Thank you very much for your suggestions. We agree with your suggestion to place this part of the content in both the results and discussion sections, respectively.. Please see “Result” section 1.2 Phylogeny and morphological disparity and “Discussion” section 1. 1.Phylogenetic relationships, estimation of divergence time and morphological macroevoloution.\nLine 313. I think it is better to specify if the 10 specimens are adults.\nResponse: Text modified to “...and supplemented this with micro-CT scan anatomical data from six newly reported adult amber fossils and four extant specimens included in this study.”. Please lines 271-274. Thanks.\nLine 322. I suggest referring to the 81 characters with the Supplementary Note 4 together with the figures.\nResponse: Text modified to “This examination resulted in a total of 81 morphological data features that were used to reconstruct the phylogenetic tree (Supplementary Note 4, Table 2 and Fig. 13).” . Please lines 274-276. Thanks.\nLine334. previous taxonomic treatments’ is relative to stem or crown cicadoids?\nResponse “previous taxonomic treatments” refers to that all adult Mesozoic fossils are classified as Tettigarctidae.\nLine 337. Fig. 3b – Change the B to lower case.\nResponse: Text modified. Thanks.\nLine 391. Is this statistical analysis some part of the supplementary material? If so, it should be referenced here.\nResponse: Text modified. We added the supplementary information and modified to “... specifically between 5.45–49.5 mm and 5.6–76.0 mm (Supplementary Fig. 16a and b)”. Please see line 364-365.\nLine 400. “The distal aspect of the mesonotum in crown cicadids has a cruciform elevation, whereas the rest of cicadoid fossils have scutellum” I don’t understand this sentence since cruciform elevation and scutellum are synonyms (Moulds 2005, fig. 3, page 378).\nResponse: We believe that the cruciform elevation and scutellum are homologous structures, despite exhibiting morphological differences. The scutellum is predominantly an equilateral triangle shape in dorsal view, with (almost) no splitting at the distal end. The cruciform elevation is divided at the distal end, forming a cross-like shape in dorsal view. In the Mesozoic fossil records of Cicadoidea, this structure remains unsplit at the distal end, distinguishing it from the majority of extant Cicadidae species. When using these terms, we will consider their morphological variations and implications variations across time and space. Also because these terms represent homologous structures that we can analyze the macroevolutionary patterns of this structure appearing in fossils and extant taxa.\nLine 454. I didn’t understand the reference to Supplementary Figure 19 here.\nResponse: The citation positions of Supplementary Fig. 19 and Supplementary Fig. 18 should be swapped. Text modified to “Cicadidae instead of Tettigarctidae, which has undeveloped tymbal muscles and no resonant abdominal cavity14 (Supplementary Fig. 19). Since the internal organs, malpighian tubules, and muscles of Pr. xiai were preserved (Supplementary Fig. 18)...”. Figure 19 showcases light micrographs that compare the abdominal anatomical structures between Tettigarctidae and extant Cicadoidea, offering readers a more intuitive comprehension of the abdominal structures associated with tymbals in extant Cicadoidea.\nLine 583. It is usual to mention the full name of the taxon at the first mention in the text.\nResponse: Text modified. Thanks.\nLine 617. It would be interesting to mention the software where the characters were coded.\nResponse: Thank you for your suggestion. We added “the phylogenetic matrix was coded in software Notepad++”. Please see line 781.\nLine 623. The root should be stated.\nResponse: Thank you very much for pointing this out. We added “We chose the type genus Cycloscytina of Hylicellidae (Hemiptera) as the root of the tree”. Please see line 771.\nPhylogeny - I missed a better explanation of the results of the phylogenetic analysis and the characters in the final tree, e.g. which of the characters were recovered as synapomorphies and homoplastic? I didn’t find the results relating to the number of recovered trees and an explanation of how the implied weight analysis was performed as well as the calculation of k-values (e.g. SPR similarity) and which k-values were recovered.\nResponse: Thank you for your questions. We think the characters for the synapomorphies or homoplastic are relative. We dicussed certain structures in the discussion section. Please see lines 466-535 “The results combined from Fig. 3a and 3b suggest that Eunotalia emeryi gen. et sp. nov., as a stem cicadoid... “. We list the characters and provide phylogenetic reconstruction tree with characteristics, that is open for further discussion.\nThe k-valuesbefore we mentioned in this Methods section in line 776“...(IW) were used, with concavity values 3 and 12 respectively”. Now we modified the displayed phylogenetic results. We show the trees using equal weighting of characters, and show the unambig changes only, slow optimization (DELTRAN) and fast optimization (ACCTRAN) results of the strict consensus tree through the Winclada, which offers a more comprehensive view of the results. Please see in Supplementary Figure 13.\nSupplementary Figure 19 – the meaning of the abbreviation “tcav” is missing in the caption.\nResponse: Text modified to “Abbreviation: tcav, tymbal cavity ...”. Thanks.\nFigure 2. I suggest putting on the side of each species’ name the correspondent letter, furthermore the species referent to the letter h is missing in the caption.\nResponse: Thank you for your suggestion. We have made an attempt to add names beside the letter labels in Fig. 2. However, due to the small font size, they were difficult to discern and somewhat confusing. Therefore, we have decided to maintain the current style of the figure. The legend provides a clear explanation of the specific names. \"h\" represents Platypleura kaempferi (extant Cicadidae), while \"c\" and \"d\" represent a male and a female specimen of the species Pr. xiai, respectively. The modified text should read as \"..Pr. xiai (f and g).\".\nFigure 3a. When performing a phylogenetic analysis, it is not usual to use terminals with different ranks. I suggest changing within this figure the terminals represented by genera and subfamilies to species. Moreover, I didn’t find the information of which species were used representing the genera and subfamilies in the analysis, only some of them in the Supplementary Table 2, and in the lines 583 and 600. Besides, the meaning of the numbers in the internal nodes is missing to be informed in the caption. These numbers are not mentioned in the main text (results or discussion).\nResponse: Thank you very much for your suggestion. We modified and used the terminals in the same ranks for phylogenetic analysis. Please see the phylogenetic results in Supplementary Figure 13. Fig. 3a is the a schematic diagram of the phylogenetic relationships. As the phylogenetic placements of the obtained fossils does not fall within the crown cicadoids, and therefore doesn't involve determining the specific relationships between Mesozoic fossils and genera within Cicadidae, we represent the terminal taxa of the cicadoid crown groups in our study using a broader subfamily-level categorization. We also used the family names of outgroup and the genus names of reported cicadoid fossils in schematic diagram. We feel this might be more intuitive, so still keep the current form.\nThe numbers simply indicate and represent certain node positions. We have added descriptions of the meanings in the legend, which could potentially lead to further reflection, but this is not related to the results and discussions in the paper. To prevent any misinterpretation of the results, we have temporarily removed these nodes.\nFig. 3b, c, d. I missed a better explanation of what was found as results and what these figures represent.\nResponse: Referring to your previous suggestion, now we have separately introduced Fig. 3b, c, and d in the Results section. Please see 1.2 Phylogeny and morphological disparity. Figs. 3b and c please line 301-374. Fig. 3d please see the second paragraph of 2.1 Comparisons of extinct and extant species, line 415-422."}]

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