Comparative Morphology of Antennae in Cicadoidea (Insecta: Hemiptera), with Respect to Functional, Taxonomic and Phylogenetic Implications

Zoologischer Anzeiger 276 (2018) 57e70

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Reasearch paper Comparative morphology of antennae in Cicadoidea (Insecta: Hemiptera), with respect to functional, taxonomic and phylogenetic implications

* Xu Wang, Qinglong Li, Cong Wei

Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Entomological Museum, Northwest A&F University, Yangling, Shaanxi 712100, China article info abstract

Article history: The antennae of 25 cicada species were examined using light and scanning electron microscopies, with Received 14 April 2017 particular attention given to the structures and arrangement of antennal sensilla in phylogenic analysis. Received in revised form Eight morphologically different types of sensilla were observed: sensilla trichodea (subtypes I, II and III), 15 May 2018 sensilla basiconica, sensilla coeloconica (subtypes I, II and III), sensilla styloconica, foramen olfactorium, Accepted 15 May 2018 sensilla cavitata-peg (subtypes I and II), sensilla campaniformia and cuticular spines. The arrangement of Available online 23 August 2018 antennal sensilla tends to be a highly regular distribution model, i.e., sensilla trichodea and sensilla basiconica mainly spread in the scape and pedicel, while sensilla coeloconica, foramen olfactorium and/ Keywords: ﬂ Cicadomorpha or sensilla styloconica merely spread in the agellum. The overall topology of phylogeny of Cicadidae Cicadidae based on antennal characters using PAUP, WinClada and IQtree analyses is compatible with the classi- Antennal sensilla ﬁcation of previously related studies, but shows that the genus Karenia Distant, 1888 is more allied to the Phylogenic analysis members of Cicadinae, and that the tribe Cicadatrini Distant, 1905 is located in the subfamily Cica- Cicadatrini dettinae instead of Cicadinae, which is consistent with the results of a recent molecular phylogenetic Karenia study of Cicadidae. The results imply that the traditional grouping of Cicadinae and Cicadettinae based on the presence or absence of timbal covers is unsound, which also supports related previous analyses based on morphology of Cicadidae. Related genera and tribes in Cicadidae need to be further addressed by phylogenetic analysis based on more morphological features combined with molecular data. © 2018 Elsevier GmbH. All rights reserved.

1. Introduction Cicadidae comprises all extant species of cicadas, except the two species of Tettigarctidae. Representing approximately 3208 described species the super- Moulds (2005) traced the phylogenetic relationships among family Cicadoidea is one of the lineages of Hemiptera, which are subfamilies, tribes and genera in Cicadidae based on morphological well known for the loud calling songs generated by the male adults characters, and divided Cicadidae into three subfamilies, i.e., Cica- (Sanborn 2013). The higher classification of Cicadoidea has been dinae Latreille, 1802, Cicadettinae Buckton, 1889, and Tettigadinae based mainly on characters connected with the sound-production Distant, 1905. However, Sanborn (2013) use the name Tibicininae organs and related morphological structures (Duffels 1993). Two Distant, 1905 instead of Tettigadinae, because Tettigadinae is a ju- families, Tettigarctidae and Cicadidae, are currently recognised in nior synonym of Tibicininae, although the type genus Tibicina of Cicadoidea (Moulds 2005; Sanborn 2013). The Tettigarctidae com- Tibicininae had been removed to Tettigadinae in the revised clas- prises only one extant genus, Tettigarcta White, 1845 (including two sification of Moulds (2005). More recently, Marshall et al. (2018) species confined to Australia), and five genera known only from reconstructed a molecular phylogeny of the cicadas with a review fossils, which are incapable of sound production similar to the of tribe and subfamily level classification, and four subfamilies are calling songs produced by other cicadas (Moulds 1990). The proposed. i.e., Cicadinae, Cicadettinae, Tibicininae, and Tettigo- myiinae, Distant, 1905, in which the tribes Tibicinini and Tettigadini are included in Tibicininae, thus, no appeal is need to suppress Tibicininae. The relationships concerning consensus in some taxa at * Corresponding author. tribe, subtribe and genus level of Cicadidae have remained E-mail address: [email protected] (C. Wei). https://doi.org/10.1016/j.jcz.2018.05.003 0044-5231/© 2018 Elsevier GmbH. All rights reserved. 58 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 controversial. For example, Boulard (1973, 1976a, b) removed five 2.2.2. Scanning electron microscopy (SEM) genera that lack timbals to a newly erected the family Platypediidae The removed antennae were immersed in phosphate buffer Kato, 1932 from Tibicinidae Distant, 1905, in which Karenia Distant, solution (PBS 0.1 M, pH 7.2) in centrifuge tubes for about 2 h. After 1888 was included. However, Boulard (1988) disbanded Platype- cleaned by ultrasonic oscillation device in PBS for 1 min, they were diidae and replaced Karenia to Tibicininae. Later, Moulds (2005) transferred to 2.5% glutaraldehyde (PBS, 0.1 M, pH 7.2). After the attributed Karenia to Cicadettinae. However, this genus and other antennae were immersed in glutaraldehyde at 4 C for 12 h, they probably related taxa were actually not included in the morpho- were rinsed five times in PBS (0.1 M, pH 7.2), and then dehydrated logical phylogenetic analyses of Moulds (2005). Marshall et al. in a graded ethanol series and 100% acetone. Then, the samples (2018) removed Karenia to Cicadinae from Cicadettinae according underwent critical point drying and were sputter-coated with gold. to their molecular phylogenetic analysis, although the exact Examinations and photographs were taken under a JEOL JSM-6360 placement of Karenia, along with the position of the Dundubiini LV scanning electron microscope at 15 kV. taxa, is poorly supported. Another example is the controversy over the systematic position of the tribe Cicadatrini Distant, 1905. The 2.2.3. Choice of outgroup genus Mogannia Amyot and Audinet-Serville, 1843 had been put in Within the Cicadoidea, the Tettigarctidae is widely considered to the tribe Moganniini Distant, 1905, and Cicadatra Kolenati, 1857, have several synapomorphic character states, leading it to be the Psalmocharias Kirkaldy, 1908 and Nipponosemia Kato, 1925 (Nip- sister group of all other cicadas (Evans 1956, 1963; Moulds 1990, ponosemia is now a junior synonym of Vagitanus Distant, 1918 (Lee 2005). In this regard, T. crinita was chosen as the outgroup for the 2014) had been attributed to the tribe Cicadini Latreille, 1802 Cicadidae in our study. (Duffels and van der Laan 1985; Chou et al. 1997). Lee and Hayashi (2004) transferred Psalmocharias and Nipponosemia (¼Vagitanus) into the tribe Moganniini. Latter, Lee and Hill (2010) synonymized 2.2.4. Characters the tribe Moganniini Distant, 1905 with their newly erected tribe Forty-two adult characters (see below) of the 25 representative Cicadatrini Distant, 1905, and remained Cicadatrini in the subfamily species were derived from antennae. All multistate characters were Cicadinae. However, Chen et al. (2012) noted that the systematic treated as unordered because alternative arrangements were position of the Cicadatrini (represented by Mogannia) remains considered equally plausible. These multistate characters were questionable, because it shares more common morphological transformed into numerical attributes (e.g., 0, 1 and 2…) repre- characters with some groups in Cicadettinae. Marshall et al. (2018) senting the different character states upon which we established attributed Cicadatrini to Cicadettinae according to their molecular the set of data (see Table 2). phylogenetic analysis. Various previous studies indicated that antennal characters 0 Number of flagellar segments: (0) four articulated segments; could be informative in tracing phylogenetic relationships among (1) five articulated segments; (2) distinctly more than five insects. Faucheux et al. (2006) found that stylus-shaped sensillum segments, with distal segmentation(s) undistinguishable. chaeticum is a primitive sensillum which only occurs in some lower 1 Scape: (0) straight; (1) curved apically. Lepidoptera. Li et al. (2013) found that the traditional classification 2 Pedicel: (0) about as long as width, gradually slightly thinner at superfamily and family level in Coleoptera are well supported by apically; (1) distinctly longer than width, distinctly con- phylogenetic analyses based on antennal characters. However, stricted medially; (2) distinctly longer than width, distinctly antennal characters have never been applied in any phylogenetic thickened apically. analysis in Cicadoidea, although some studies have been conducted 3 Flagellum: (0) segments of flagellum in similar diameter and on a couple of cicadas (Klein et al. 1988). length, with each segment short stick-shaped; (1) sharply Herein, we investigate the antennal morphology of represen- tapered apically, with distal segment generally shorter than tatives in Cicadoidea with particular attention given to antennal previous ones; (2) gradually tapered apically, with distal sensilla. We mainly aim to reveal the relationships among related segment very long and much slender. taxa based on phylogenetic analysis using the data set of antennal 4 Flagellar segment 1: (0) stick-shaped, distinctly constricted character attributes. medially; (1) clavate, tapered apically and distinctly constricted subapically; (2) same-diametered of full length or 2. Materials and methods gradually tapered apically, with no distinct constriction. 5 Flagellar segment 2: (0) stick-shaped, generally thick; (1) 2.1. Materials petiole-shaped, generally long and thin. 6 Cuticle of flagellar segment 1: (0) cuticular of sensilla field Twenty-five cicada species were used in the antennal investi- with weak hexagonal squamae, cuticular of other field with gation. All specimens were from the collection of the Entomological distinct hexagonal grids; (1) cuticular with distinct uniform Museum, Northwest A&F University (NWAFU), Yangling, China. hexagonal squamae. Detailed information of 25 representatives of Cicadoidea investi- 7 Flagellar tip: (0) pointed; (1) blunt. gated in this study is provided in Table 1. 8 Length of scape and pedicel: (0) length of scape less than 0.6 time that of pedicel. (1) Length of scape more than 0.6 time 2.2. Methods that of pedicel. 9 Length and width of flagellum segment 1: (0) thin, much 2.2.1. Light microscopy longer, length more than 5X its width; (1) thick, relative The whole antennae of cicadas (at least 5 male individuals shorter, length less than 5X its width. except the species Tettigarcta crinita Distant, 1883 (one male) and 10 Length of flagellar segments 1 and 2: (0) segment 2 much Paharia oorschoti Wang, Duffels and Wei, 2017 (one male)) were longer than segment 1; (1) segment 2 about 0.7e1.0X that of removed from the antennal fovea, and cleaned in phosphate buffer segment 1; (2) segment 2 less than 0.7X that of segment 1. solution (PBS 0.1 M, pH 7.2). The gross morphology and detailed 11 Width of flagellar segments 1 and 2: (0) segment 2 much features of the antennae were observed by the SMZ168 stereo- thinner than segment 1; (1) flagellar segment 2 similar to or scopic zoom microscope (Motic, Xiamen, China). slightly thinner than segment 1. X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 59

Table 1 Collection information of the 25 representatives of Cicadoidea.

Family/Subfamily Species Collecting location Collecting time

Tettigarctidae Tettigarcta crinita Distant, 1883 Snowy Mts Hwy, Australia 28-II-1981 Cicadadae Tibicininae Subpsaltria yangi Chen, 1943 Mt. Helan, Ningxia 20-VI-2011 Paharia oorschoti Wang, Duffels and Wei, 2017 Kurubas Gecidi, Turkiye 18-VI-1985 Cicadettinae Tibeta zenobia (Distant, 1912) Yadong, Xizang 20-VII-1980 Leptopsalta admirabilis Kato, 1927 Mt. Helan, Inner Mongolia 09-VIII-2012 Cicadetta shansiensis (Esaki and Ishihara, 1950) Zhouzhi, Shaanxi 15-V-2011 Curvicicada xinjiangensis Chou and Lu, 1997 Bu'erjin, Sinkiang 28-VI-1960 Huechys sanguinea (De Geer, 1773) Xing'an, Guangxi 11-VII-2007 Katoa neokanagana (Liu, 1940) Pingwu,Sichuan 09-VII-2013 Cicadinae Karenia caelatata Distant, 1890 Ningshan, Shaanxi 20-VII-2012 Mogannia hebes (Walker, 1858) Xing'an, Guangxi 12-VII-2006 Mogannia conica (Germar, 1830) Changjiang, Hainan 27-V-2011 Cicadatra gregoryi China, 1925 Yongshan, Yunnan 28-V-1980 Psalmocharias querula (Pallas, 1773) Shihezi, Sinkiang 17-VI-1957 Platypleura kaempferi (Fabricius, 1794) Yangling, Shaanxi 15-VI-2011 Terpnosia sp. Zhouzhi, Shaanxi 20-V-2011 Tanna yunnanensis (Lei and Chou, 1997) Mengla, Yunnan 11-VI-1994 Tanna sinensis (Ouchi, 1938) Wufeng, Hubei 13-VII-2008 Purana gigas (Kato, 1930) Mt. Lushan, Jiangxi 05-VIII-1984 Meimuna mongolica (Distant, 1881) Yangling, Shaanxi 08-VII-2011 Dundubia spiculata Noualhier, 1896 Jinghong, Yunnan 30-VIII-2010 Platylomia radha (Distant, 1881) Menghai, Yunnan 23-V-2011 Platylomia bocki (Distant, 1882) Menghai, Yunnan 25-V-2011 Hyalessa maculaticollis (Moschulsky, 1866) Shimen, Hunan 26-VII-2013 Cryptotympana atrata (Fabricius, 1775) Yangling, Shaanxi 20-VI-2011

Table 2 Character state matrix of 25 taxa in phylogenetic analyses.

00000 00000 11111 11111 22222 22222 33333 33333 44

01234 56789 01234 56789 01234 56789 01234 56789 01

Tettigarcta crinita 10020 00000 01001 01000 11111 00111 11100 00000 01 Subpsaltria yangi 01100 01110 11101 11010 11111 10110 01000 00011 11 Paharia oorschoti 01100 01110 11100 00000 11110 00110 00000 00011 01 Cicadetta shansiensis 11211 01110 21101 01111 00110 00000 11000 00001 00 Tibeta zenobia 11202 01110 21100 01111 00100 00000 11000 00000 00 Leptopsalta admirabilis 11211 01110 20101 01111 00100 00000 11000 01000 01 Curvicicada xinjiangensis 11212 00010 21111 01111 11100 00000 11000 00011 01 Katoa neokanagana 11221 11010 20001 01110 01100 00000 11100 00011 01 Huechys sanguinea 11221 11010 21001 01100 00100 00000 11000 00001 01 Mogannia hebes 11212 01110 21100 00011 11100 00000 11000 00000 00 Mogannia conica 11212 01110 21100 01100 11100 00000 11000 00000 00 Cicadatra gregoryi 11221 10000 10101 01111 00100 00000 11000 01100 01 Psalmocharias querula 11201 01110 21101 00011 01101 00000 11000 00000 00 Tanna yunnanensis 21222 10011 00010 01111 00100 00000 10000 01111 00 Tanna sinensis 21222 00011 00010 01100 11100 00000 10000 01010 00 Terpnosia sp. 21222 00011 01010 01111 00101 00000 10000 00011 00 Purana gigas. 21222 10010 01010 01100 11101 00000 10000 00010 00 Meimuna mongolica 21222 10010 20010 01111 11101 10001 10010 00010 00 Dundubia spiculata 21222 10000 10010 01111 11101 10000 11000 01111 00 Platylomia radha 21221 10000 10010 01111 11101 00000 11000 01011 00 Platylomia bocki 21221 10010 10010 01111 11100 00000 11000 01011 00 Platypleura kaempferi 11212 11010 10110 01111 01100 00000 10000 00010 01 Cryptotympana atrata 11222 00011 10110 01111 00100 00000 10000 00110 00 Hyalessa maculaticollis 21121 11010 10010 01111 11100 00000 10000 01010 00 Karenia caelatata 21221 10010 10011 01111 11101 01000 11110 00110 01

12 Length of flagellum and scape þ pedicel: (0) length of flagellum 17 Sensilla trichodea subtype II on pedicel: (0) absent; (1) present. more than 3X that of scape þ pedicel; (1) length of flagellum 18 Sensilla trichodea subtype III on scape: (0) absent; (1) less than 3X that of scape þ pedicel. present. 13 Sensilla on segment 2: (0) with a large quantity of sensilla of 19 Sensilla trichodea subtype III on flagellum: (0) absent; (1) high-density; (1) with a small quantity of sensilla of low- present. density. 20 Sensilla basiconica on scape: (0) absent; (1) present. 14 Sensilla trichodea subtype I on scape and pedicel: (0) absent; 21 Sensilla basiconica on pedicel: (0) absent; (1) present. (1) present. 22 Sensilla coeloconica subtype I on flagellar segment 1 & 2: (0) 15 Sensilla trichodea subtype I on flagellum: (0) absent; (1) absent; (1) present. present. 23 Sensilla coeloconica subtype I on the rest of flagellar segments: 16 Sensilla trichodea subtype II on scape: (0) absent; (1) present. (0) absent; (1) present. 60 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

24 Sensilla coeloconica subtype II on flagellar segment 1: (0) ab- pedicel is thinner and longer than the scape. The flagellum is the sent; (1) present. thinnest and longest among the three parts. The segmentations 25 Sensilla coeloconica subtype II on flagellar segment 2: (0) ab- among scape, pedicel, and flagellar segments 1 and 2 are articulated sent; (1) present. in all cicadas. Among some species with considerably long and thin 26 Sensilla coeloconica subtype II on the rest of flagellar segments: flagella, the constrictions of the cuticle between the distal segments (0) absent; (1) present. are extremely slight, merely defined as segmental boundaries. 27 Sensilla coeloconica subtype III on flagellar segment 1: (0) ab- However, in the species with relatively short flagellum, the seg- sent; (1) present. mentation between successive distal segments can be easily 28 Sensilla coeloconica subtype III on flagellar segment 2: (0) ab- distinguished. The typical posture of the whole antenna is straight sent; (1) present. forward. The apex of the flagellum is usually characterized like a 29 Sensilla coeloconica subtype III on the rest of flagellar segments: finger. The cuticular surfaces of antennae, especially of the first (0) absent; (1) present. flagellar segment, are generally characterized by plaid or squama- 30 Sensilla styloconica on flagellar segment 1: (0) absent; (1) ceous patterns. present. 31 Sensilla styloconica on flagellar segment 2: (0) absent; (1) 3.2. Antennal sensilla present. 32 Sensilla styloconica on the rest of flagellar segments: (0) absent; 3.2.1. Sensilla trichodea (STr) (1) present. Sensilla trichodea are merely identified on the ventrolateral side 33 Sensilla campaniformia on flagellar segment 2: (0) absent; (1) of the scape and pedicel of antennae in cicadas except Subpsaltria present. yangi Chen, 1943, as several long sensilla trichodea are identified on 34 Sensilla cavitata-peg subtype I on flagellar segment 1: (0) ab- the whole antennae. Sensilla trichodea are the most abundant sent; (1) present. sensilla on the scape and pedicel, accounting for more than 50% of 35 Sensilla cavitata-peg subtype I on flagellar segment 2: (0) ab- all sensilla (except in the species with no STr). They could sent; (1) present. morphologically be divided into three subtypes: STrI, STrII and 36 Sensilla cavitata-peg subtype II on flagellar segment 1: (0) ab- STrIII (see Fig. 1). sent; (1) present. STrI (140e380 mm in length) are distally tapering sensilla, visible 37 Sensilla cavitata-peg subtype II on flagellar segment 2: (0) ab- as long, strong, straight or slightly curved hairs (Figs. 2A; 3D; 4A, C, sent; (1) present. J, K). They are mostly inclined in the direction of the long axis of the 38 Foramen olfactorium on flagellar segment 1: (0) absent; (1) antennae. No pores or longitudinal grooves on the surface are present. viewed at high magnification. 39 Foramen olfactorium on flagellar segment 2: (0) absent; (1) STrII (150e280 mm in length) are very slender and sharp-tipped present. hairs, gradually tapering from the base to the tip. They are slightly 40 Sensilla styloconica on the rest of flagellar segments: (0) absent; curved hairs inclined in the direction of the long axis (Figs. 2A; 3B; (1) present. 4A, C, FeH, L; 5A2, B1, C, D, H, K; 6A2, B2,CeG). 41 Cuticular spines on scape: (0) absent; (1) present. STrIII (65e130 mm in length) are short and thick, prostrate hairs, nearly appressed. These sensilla are distinctly swollen in the mid- dle, with apex much blunt (Figs. 3A; 4B, C, E, JeL; 5A1, C, D, F1eF3, 2.2.5. Phylogenetic analysis H, I, K, K1; 6A1, B1, C, E, G, I). When viewed at high magnification, Data were analysed using the heuristic search parsimony algo- longitudinal grooves are recognized on the surface of some STrIII. rithms implemented with PAUP version 4.0 beta 10. Tree searches utilized the tree bisection reconnection algorithm (TBR) conducting 3.2.2. Sensilla basiconica (SBa) 1000 random addition searches (RAS) starting from random trees; Sensilla basiconica (50e200 mm in length) are mainly identified other settings were left at their default values. The set of shortest in the sensilla field on the ventrolateral side of the scape and trees were filtered using the Filter Trees option to eliminate trees pedicel. They are observed as long, strong, straight or slightly that were less resolved than other compatible trees in order to find curved pegs with a slightly blunt tip apically and a well-defined the most resolved trees. A phylogenetic tree was also constructed in socket basally. No longitudinal grooves and pores could be IQtree v1.4.1 (Lam-Tung et al. 2015) using the ORDERED model observed on the smooth-surfaced at high magnification (Figs. 2A; selected by the software with other parameters default. An ultrafast 3B, C, F, G; 4D; 5B2, E, F3, G, J; 6D, F, H, I). bootstrap (UFB) (Bui et al. 2013) of 1000 replications and the SH- aLRT test were used in the analysis to assess branch supports. 3.2.3. Sensilla coeloconica (SCo) Character distributions were examined using WinClada 1.0 (Nixon The first segment of the flagellum provides a main field of 2002). The illustrated cladograms were edited using Adobe illus- sensilla coeloconica on the ventrolateral side, and the second and trator CS 6.0. third segments of the flagellum bear a few sensilla coeloconica on The higher classification of Cicadidae follows that of Marshall the same side as the SCo on the first segment. They account for et al. (2018). more than 70% of all sensilla in the flagellum. Morphologically, they could be divided into three subtypes: SCoI, SCoII and SCoIII. 3. Results SCoI (Figs. 2BeD; 3D, H, I; 7AeH; 8AeD, F, G, A1; 9AeI, H1)are characterized as large blunt cones (15e22 mm in length) positioned 3.1. Gross morphology of antennae in the cuticular depressions. These cones stand almost at right angles and recess over the cuticular surface. Occasionally, two to The setaceous antenna of adult, inserting in the antennal fovea four cones are located in one depression. SCoI are the most in of the cranium at the side of the postclypeus near an anterior numbers, accounting for about 90% of all the SCo. The cones are tentorial pit, consists of a scape (Sc), a pedicel (Pd) and a several- smooth-walled and blunt-tipped. In some SCoI, one or several segmented and distally tapering flagellum (Fl). The scape is thick ecdysial pores could be observed at the tip or at the lateral side of and short, and partially concealed in the antennal fovea. The the cone. X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 61

3.2.5. Foramen olfactorium (FOl) Foramen olfactorium (2.0e4.0 mm in diameter) are scattered over the same areas of sensilla styloconica, i.e., on the ventrolateral sides of the first segment of the flagellum and occasionally, a few are visible in the second flagellar segment. They are recognized as small ostioles formed by invaginations of antennal epidermis, and in some FOl, the rims are slightly, asymmetrically intumescent (Figs. 2B; 3D, H; 7B, C, F, H; 8A, F; 9C, G).

3.2.6. Sensilla cavitata-peg (SCa) A small number of sensilla cavitata-peg are scattered over the first and second flagellar segments. Morphologically, they could be distinguished into two subtypes: SCaI and SCaII. SCaI (Fig. 8HeJ) are visible as raised cuticular collars, on which one small and slightly raised dome is located. Generally, these cavitata-pegs are gregariously located in one amorphous cuticular depression. At high magnification the collars are unsmooth-walled with slight latitudinal grooves and small pores, but some of them are covered with several protuberances. SCaII (Figs. 7I; 8B, E; 9F, F1, F2) are recognized as small, stout and hollow cones, in which a small and stout peg is deposited. These sensilla are solitarily or gregariously located in one cuticular depression (5.5e14.0 mm in diameter).

3.2.7. Sensilla campaniformia (SC) Sensilla campaniformia are merely visible on the apical several ﬂagellar segments. They are identiﬁed as small cuticular domes about 5.5e8.5 mm in diameter, and usually one pore situates at the center of the dome (Figs. 7H1, 9E1).

3.2.8. Cuticular spines (CS) Cuticular spines (about 3.5 mm in length) are only identiﬁed on a centralized ﬁeld of the scape distally in large quantities with a high density. These cuticular spines are very short with pointed ends. They are visible as bristles formed by the prominences of the antennal cuticle (Figs. 3G; 4G, I, K; 5K1).

Fig. 1. Antennae of Cicadoidea. (A) Tettigarcta crinite. (B) Subpsaltria yangi. (C) Paharia 3.3. Distributions of sensilla in cicada antennae oorschoti (D) Tettigetta shansiensis. (E) Tibeta zenobia. (F) Kosemia admirabilis. (G) Huechys sanguinea. (H) Katoa neokanagana. (I) Psalmocharias querula. (J) Cicadatra gregoryi. (K) Mogannia hebes. (L) Mogannia conica. (M) Curvicicada xinjiangensis. (N) Antennal sensilla are generally distributed in cicada antennae in Karenia caelatata. (O) Terpnosia sp. (P) Aetanna yunnanensis. (Q) Tanna sinensis. (R) regularity. The long sensilla, such as sensilla trichodea and sensilla Purana gigas. (S) Dundubia spiculata. (T) Platylomia bocki. (U) Platylomia radha. (V) basiconica, are merely in the ventrolateral sides of the scape and Meimuna mongolica. (W) Platypleura kaempferi. (X) Hyalessa maculaticollis. (Y) Cryp- the pedicel, except the case that a few sensilla trichodea were totympana atrata. AS, Antennal socket; Sc, Scape; Pd, Pedicel; Fl, Flagellum. Scale bars: observed on the flagellum of S. yangi. Sensilla coeloconica, foramen (I, K, L) ¼ 0.25 mm, (E, N, Q, SeU, X) ¼ 1 mm, (AeD, FeH, J, MeP, R, V, W, Y) ¼ 0.5 mm. olfactorium and/or sensilla styloconica are mainly in the ventrolateral side of the first flagellar segment, and a small number of SCoII (Fig. 2C; 3E; 7D, H; 9D) are observed as small, unsmooth- these sensilla are in the second and/or third flagellar segments. A walled nails in the cuticular depressions (3.5e8.0 mm in diameter). schematic distribution pattern of sensilla in cicada antennae is SCoIII (Figs. 2BeD; 3J) are recognized as small and slightly blunt- provided in Fig. 10. tipped pegs deposited at the bottom of large, vertical-precipiced depressions (5.5e8.5 mm in diameter). SCoII and SCoIII are merely 3.4. Phylogenetic analyses limited in a few species, which account for about 10% of the SCo. The nails of SCoII are scraggy with unsmooth cuticle, and usually Different morphological states of each character are corre- recess below the cuticular surface. The rim of SCoII depressions is spondingly transferred into numerical attributes to establish the generally constricted. The pegs of SCoIII are considerably stout and data set (see Table 2), upon which our phylogenetic study is based. slightly blunt-tipped, and their depressions are generally very deep PAUP analysis produced 1000 most parsimonious trees, with a and columniform. length of 132 steps, a consistency index (CI) of 0.33, a retention index (RI) of 0.61, and a rescaled consistency index (RCI) of 0.20. In 3.2.4. Sensilla styloconica (SSt) the strict consensus tree (Fig. 11), the subfamily Tibicininae is cor- Sensilla styloconica (7.5e10.0 mm in diameter) are mainly scat- responding to Clade 1, consisting of P. oorschoti and S. yangi. The tered over the ventral surface of the first flagellar segment and Clade 2 consists of representatives which are currently classified usually, a few also present on the second flagellar segment. They are into Cicadinae and Cicadettinae. However, Katoa neokanagana (Liu, recognized as stout cones, inserting in small depressions. The rim of 1940) and Huechys sanguinea (Degeer, 1773), currently belonging to SSt depressions are considerably inflated symmetrically with Cicadettinae, are clustered into Clade 3 which is isolated from other smooth surface (Figs. 2C; 7A, DeF, G, H; 8AeD, F, G, A1; 9A, B, DeI). genera of Cicadettinae within Clade 4. In Clade 5, Mogannia hebes 62 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

Fig. 2. Antennal sensilla of Tettigarcta crinita (Tettigarctidae). (A) Antennal sensilla on the scape. (BeD) Antennal sensilla on the ﬂagellum. STrI, Sensilla trichodea subtype I; STrII, Sensilla trichodea subtype II; SBa, Sensilla basiconica; SCoI, Sensilla coeloconica subtype I; SCoII, Sensilla coeloconica subtype II; SCoIII, Sensilla coeloconica subtype III; SSt, Sensilla styloconica; FOl, Foramen olfactorium. Scale bars, (A) ¼ 100 mm; (B, C) ¼ 20 mm, (D) ¼ 10 mm.

(Walker, 1858), Mogannia conica (Germar, 1830), Psalmocharias Tibeta zenobia (Distant, 1912), Kosemia admirabilis (Kato, 1927) and querula (Pallas, 1773) and Cicadatra gregoryi China, 1925 (marked Tettigetta shansiensis (Esaki and Ishihara, 1950). In Clade 6, Curvi- with “*” in Fig. 11), formerly belonging to the tribe Cicadatrini of cicada xinjiangensis Chou and Lu, 1997 (marked with “D” in Fig. 11), Cicadinae, are clustered with the members of Cicadettinae, i.e., currently belonging to Cicadettinae, is isolated from other members

Fig. 3. Antennal sensilla of Tettigadinae in the classiﬁcation of Moulds (2005).(AeE) Subpsaltria yangi.(FeJ) Paharia oorschoti. STrI, Sensilla trichodea subtype I; STrII, Sensilla trichodea subtype II; STrIII, Sensilla trichodea subtype III; SBa, Sensilla basiconica; SCoI, Sensilla coeloconica subtype I; SCoII, Sensilla coeloconica subtype II; SCoIII, Sensilla coeloconica subtype III; FOl, Foramen olfactorium; CS, Cuticular spines; P, ecdysial pore. Scale bars, (A, B, C) ¼ 50 mm; (F) ¼ 100 mm; (D, G) ¼ 25 mm; (H) ¼ 15 mm; (I, J) ¼ 10 mm; (E) ¼ 5 mm. X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 63

Fig. 4. Antennal sensilla on the scape and pedicel of Cicadettinae in the classification of Moulds (2005). (A) Tettigetta shansiensis.(BeC) Tibeta zenobia.(DeE) Curvicicada xinjiangensis. (F, F1, G) Katoa neokanagana.(HeI) Huechys sanguinea. (J, K) Karenia caelatata. (L) Kosemia admirabilis. STrI, Sensilla trichodea subtype I; STrII, Sensilla trichodea subtype II; STrIII, Sensilla trichodea subtype III; SBa, Sensilla basiconica; CS, Cuticular spines. Scale bars, (K) ¼ 200 mm; (A, F, HeJ) ¼ 100 mm; (BeD, G, L) ¼ 50 mm; (E, F1) ¼ 25 mm. of Cicadettinae in Clade 5 but clustered with members of the subtype II on scape (char. 16: 0); and the other lineage in this clade is Cicadinae. split into Clade 3 and Clade 4. In Clade 3, C. gregoryi (marked with In the morphology tree from the program IQtree (Fig. 12), the “*” in Fig. 13) formerly belonging to Cicadinae clustered with some systematic relationship within Cicadidae is clearer than PAUP members of Cicadettinae. Clade 4 is characterised by flagellar analysis. However, M. hebes, M. conica, P. querula and C. gregoryi segment 1 same-diametered of full length or gradually tapered (marked with “*” in Fig. 12) of the tribe Cicadatrini also clustered apically, with no distinct constriction (char. 4: 2), and sensilla tri- with the representatives of Cicadettinae, which is the same as that chodea subtype I on scape and pedicel absent (char. 14: 0). In Clade 4, in PAUP analysis. C. xinjiangensis (marked with “#” in Fig. 12), T. zenobia, C. xinjiangensis (marked with “∧” and “#” respectively in currently belonging to the Cicadettinae, is also clustered with Fig. 13), currently belonging to Cicadettinae, clustered with the members of the Cicadinae. members of Cicadinae. The topological structure of Clade 5 is In the selected tree from WinClada analysis (Fig. 13), the sub- similar to that yielded by program PAUP (named Clade 6 in Fig. 11). family Tibicininae which consists of Paharia sp. and S. yangi is corresponding to Clade 1. The remaining taxa all clustered into 4. Discussion Clade 2, with the following synapomorphies: pedicel distinctly longer than width, distinctly thickened apically (char. 2: 2), sensilla 4.1. Morphological and functional aspects of antennal sensilla trichodea subtype III on flagellum present (char. 19: 1), sensilla basiconica on scape absent (char. 20: 0), sensilla coeloconica subtype I Sensilla trichodea (STr) are the most widespread and abundant on the rest of flagellar segments absent (char. 23: 0), sensilla coelo- sensilla in cicadas, which are visible as long hairs. These sensilla conica subtype III on flagellar segment 1 absent (char. 27: 0), sensilla have been reported to perform either or both mechano- and coeloconica subtype III on flagellar segment 2 absent (char. 28: 0), and chemo-sensory functions (Zacharuk 1985). In neotropical bugs, cuticular spines on scape absent (char. 41: 0). Clade 2 is split into two they were regarded as the main sensory receptors involved in the clades, which comprises all the representatives currently belonging chemical sense, as their smooth surface was covered with to the Cicadinae or Cicadettinae. Among which, P. querula is the first numerous pores throughout the wall (Silva et al. 2010). Some divergence in Clade 2 due to the absence of sensilla trichodea research indicated that STr were respond to sex pheromone, as 64 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

Fig. 5. Antennal sensilla on the scape and pedicel of Cicadinae in the classiﬁcation of Moulds (2005). (A1, A2, B1, B2) Tanna sinensis. (C) Aetanna yunnanensis. (D) Terpnosia sp. (E) Purana gigas. (F1, F2, F3) Dundubia spiculata.(GeH) Meimuna mongolica. (I) Mogannia hebes. (J) Mogannia conica. (K, K1) Cicadatra gregoryi. STrII, Sensilla trichodea subtype II; STrIII, Sensilla trichodea subtype III; SBa, Sensilla basiconica; CS, Cuticular spines. Scale bars, (A1, A2, E, F1, F2, F3) ¼ 100 mm; (B1, B2, C, D, GeK) ¼ 50 mm; (K1) ¼ 10 mm.

striking difference in the numbers was found between the two basiconica in cicadas is smooth, without any cuticular pores. sexes of Callosobruchus maculatus (Fabricius, 1775) (Faucheux et al. Whether sensilla basiconica function as sex-pheromone receptors 2006; Hu et al. 2009). However, some scholars argued that they in cicadas needs a further investigation. may be mechanoreceptors in perception of mechanosensory Sensilla cavitata-pegs (SCa) were argued as chemoreceptors or stimuli in many wasps (Pettersson et al. 2001; Roux et al. 2005; mechanoreceptors, thermoreceptors and hygroreceptors (Li et al. Onagbola and Fadamiro 2008). In cicadas, the smooth-surfaced STr, 2009). In M. hebes and M. conica, small pores observed on SCa without any pores, are much longer than other antennal sensilla, might suggest these sensilla function as chemoreceptors respond- indicating that they are more sensitive to receive mechanical ing to chemical stimuli. Sensilla styloconica (SSt) were previously stimuli. described in the cuterebrid Dermatobia hominis (Linnaeus, 1781) Sensilla coeloconica were previously described on the flagellum (de Fernandes et al. 2002), the leaf beetle Psylliodes chrysocephala of stink bugs Nezara viridula (Linnaeus, 1758) and Chrysocoris pur- (Linnaeus, 1758) (Bartlet et al. 1999) and the fly Lucilia cuprina purea (Westwood, 1837) (Brezot et al. 1997; Rani and Madhavendra (Wiedemann, 1830) (Sukontason et al. 2004), and they were sug- 2005), and they were reported as chemoreceptors related to air gested as mechano- and/or chemoreceptors (Zacharuk 1985; temperature changes (Ruchty et al. 2009) and to host plants' vol- Mitchell et al. 1999). However, the function of SSt is still in mystery. atile compounds (Pophf 1997). Klein et al. (1988) descripted the antennal sensilla, especially SCo on the flagellum of Magicicada 4.2. Phylogenetic implications of antennal morphology in cassini Fisher, 1852, with emphasis on their possible role in olfac- Cicadoidea tion. In our study, small pores were observed on some SCo, sug- gesting that SCo may be chemoreceptors in response to plant Although the gross morphology of antennae and antennal odour. sensilla are very similar in cicadas, the shape and segmentation of Sensilla basiconica (SBa) were suggested to be sex-pheromone antennae and, particularly, the arrangement of different types of receptors, because they had cuticular pores throughout their antennal sensilla among different taxa show some differences, walls (Silva et al. 2010), and had sexual dimorphism in numbers which may be informative in tracing the phylogenetic relationships (Merivee et al. 1998, 1999). However, the surface of sensilla of Cicadoidea. At the family/subfamily level, antennal sensilla X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 65

Fig. 6. Antennal sensilla on the scape and pedicel of Cicadinae in the classiﬁcation of Moulds (2005). (A1, A2) Platylomia radha. (B1, B2) Platylomia bocki. (C, D) Platypleura kaempferi. (E) Cryptotympana atrata.(F,G)Hyalessa maculaticollis. (H, I) Psalmocharias querula. STrII, Sensilla trichodea subtype II; STrIII, Sensilla trichodea subtype III; SBa, Sensilla basiconica. Scale bars, (A1, A2, F, G) ¼ 100 mm; (B1, B2, C, D, E, H, I) ¼ 50 mm.

Fig. 7. Antennal sensilla on the ﬂagellum of Cicadettinae in the classiﬁcation of Moulds (2005). (A) Tettigetta shansiensis. (B) Tibeta zenobia. (C) Kosemia admirabilis. (D) Huechys sanguinea. (E, F) Katoa neokanagana. (G) Curvicicada xinjiangensis. (H, H1, I) Karenia caelatata. SCoI, Sensilla coeloconica subtype I; SCoII, Sensilla coeloconica subtype II; SSt, Sensilla styloconica; SCaII, Sensilla cavitata-peg subtype II; FOl, Foramen olfactorium; SC, Sensilla campaniformia. Scale bars, (B, F, H) ¼ 50 mm; (CeE) ¼ 25 mm; (A, G, I) ¼ 20 mm; (H1) ¼ 5 mm. 66 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

Fig. 8. Antennal sensilla on the ﬂagellum of Cicadinae in the classiﬁcation of Moulds (2005). (A, A1) Cryptotympana atrata. (B, C) Hyalessa maculaticollis.(DeE) Cicadatra gregoryi. (F) Psalmocharias querula.(GeH) Mogannia conica.(IeJ) Mogannia hebes. SCoI, Sensilla coeloconica subtype I; SSt, Sensilla styloconica; SCaI, Sensilla cavitata-peg subtype I; SCaII, Sensilla cavitata-peg subtype II; FOl, Foramen olfactorium. Scale bars, (A, D, G) ¼ 50 mm; (B) ¼ 25 mm; (A1 E, F) ¼ 20 mm; (C) ¼ 10 mm; (I) ¼ 5 mm, (H, J) ¼ 2.5 mm.

Fig. 9. Antennal sensilla on the ﬂagellum of Cicadinae in the classiﬁcation of Moulds (2005). (A) Aetanna yunnanensis. (B) Tanna sinensis. (C) Terpnosia sp. (D) Purana gigas. (E, E1) Meimuna mongolica. (F) Dundubia spiculata. (G) Platylomia radha. (H, H1) Platylomia bocki. (I) Platypleura kaempferi. SCoI, Sensilla coeloconica subtype I; SCoII, Sensilla coeloconica subtype II; SSt, Sensilla styloconica; SCaII, Sensilla cavitata-peg subtype II; FOl, Foramen olfactorium; SC, Sensilla campaniformia. Scale bars, (F, H, I) ¼ 50 mm; (A, E, E1, G) ¼ 25 mm; (D) ¼ 20 mm; (B, C) ¼ 15 mm; (F1) ¼ 5 mm; (F2) ¼ 2.5 mm. X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 67

2013; Lee 2014). The tribe Cicadatrini is currently placed in the subfamily Cicadinae (Sanborn 2013; Lee 2014). However, Chen et al. (2012) noted that Mogannia has the same morphological character attributes (Characters 24, 32, 37, 52, 61, 63, 65 in the investigation of Moulds (2005)) as in species of Cicadettinae, and that the Cicada- trini (represented by Mogannia) could be a member of the Cica- dettinae Similarly, Yang and Wei (2013) indicated that

Fig. 10. Distribution model of antennal sensilla in Cicadoidea. AS, Antennal socket; Nipponosemia (now a junior synonym of Vagitanus) of Cicadatrini Sc, Scape (partially concealed in the antennal socket); Pd, Pedicel; Fl, Flagellum. LS, also appeared to be more allied to the members of Cicadettinae Long sensilla such as sensilla trichodea and sensilla basiconica; SCo, Sensilla coelo- based on morphological characters. In the strict consensus analysis conica; FOl, Foramen olfactorium; SSt, Sensilla styloconica. of our study, Mogannia, Psalmocharias and Cicadatra (marked with “*” in Fig. 11) of the tribe Cicadatrini clustered with the represen- coeloconica subtype III (chars. 27, 28) are merely observed in spe- tatives of Cicadettinae (i.e., Tibeta, Leptopsalta and Tettigetta) cies of the family Tettigarctidae and the subfamily Tibicininae. The (Fig. 11). In the WinClada parsimonious tree (Fig. 13), the systematic scape in Tettigarctidae is distinctly curved apically (char. 1: 1), relationship within Cicadidae is clearer than yielded in the PAUP which is unique in Cicadoidea. The flagellum of Tibicininae species analysis. The cladogram is distinct classified into three lineages is four-segmented (char. 0: 0), and each segment is visible as a stick which are corresponding to three subfamilies of Cicadidae, i.e., (char. 3: 0). At the genus and species level, the sensilla cavitata-peg Tibicininae, Cicadettinae and Cicadinae. Psalmocharias, Mogannia subtype I are only distributed in the antennae in Mogannia (chars. and Cicadatra (both marked with “*” in Fig. 13) of Cicadatrini also 34, 35), which can be regarded as an autapomorphy of this genus. clustered with the representatives of Cicadettinae, which is the Sensilla trichodea subtype I in antennae of S. yangi were observed same as that in Fig. 11. The results indicate that Cicadatrini is more not only on the scape and pedicel, but also on the flagellum in S. allied to the group of Cicadettinae, which is identical to the result of yangi (char. 16: 1), as is different to other examined species (i.e., Marshall et al. (2018). merely observed on the scape and pedicel in other examined Regarding the systematic position of Karenia, this genus was species). initially placed in the subfamily Tibicininae, division Carinetaria by Regarding the systematic position of the tribe Cicadatrini, Lee Distant (1906). Boulard (1973, 1976a,b) took from the Tibicinidae and Hill (2010) synonymized the tribe Moganniini with their five genera that lack timbals, placing them as a new family Platy- newly erected tribe Cicadatrini which currently includes the genera pediidae in which the genus Karenia was included. But Boulard Cicadatra, Klapperichicen Dlabola, 1957, Psalmocharias, Shaoshia (1988) disbanded the family Platypediidae and replaced the Kar- Wei, Ahmed and Rizvi, 2010, Mogannia, Vagitanus, Emathia Stål, enia into Tibicininae. Duffels (1993) agreed with Boulard (1988) in 1866, Triglena Fieber, 1875 and Taungia Ollenbach, 1929 (Sanborn disbanding Platypediidae, “since the different genera probably lost

Fig. 11. Strict consensus tree of 1000 most parsimonious trees, showing current subfamily placements and proposed subfamily placements in this study. Numbered nodes (circled) relate to discussion in the text. Numbers above black bars relate to characters in Table 2. Numbers below nodes (bold print) are jackknife values from 1000 jackknife replications. Nodes lacking jackknife fall below 50%. 68 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

Fig. 12. Morphology tree of Cicadidae in the program IQtree. At each node, values denote ultrafast bootstrap (UFB) support.

Fig. 13. The selected parsimonious cladogram of Cicadidae in the program WinClada, with a length of 130, a consistency index of 0.33 and a retention index of 0.62. Unambiguous apomorphies mapped on branches, black circles indicate nonhomoplasious changes. X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70 69 their timbal organs independently”. Moulds (2005) attributed Boulard, M., 1976b. Sur une deuxieme cigale africaine depourvue d'appareil sonore e Karenia to Cicadettinae, but this genus and other probably related (Homoptera). Bull. Inst. Fond. Afri. Noire. 37, 629 636. Boulard, M., 1988. Taxonomie et nomenclature superieures des Cicadoidea. Histoire taxa were actually not included in his morphological phylogenetic problemes et solutions. Mus. Nat. Hist. Nat. 1, 1e89. analyses. Wei et al. (2009) and Thai and Yang (2012) followed Bui, Q.M., Minh, A.T.N., Arndt, H., 2013. Ultrafast approximation for phylogenetic e Moulds (2005), attributing this genus into Cicadettinae. Recently, bootstrap. Mol. Biol. Evol. 30, 1188 1195. Chen, X., Yang, M., Wei, C., 2012. Review of the cicada genus Mogannia Amyot & Marshall et al. (2018) removed Karenia to Cicadinae from Cica- Serville from China, with descriptions of three new species (Hemiptera: Cica- dettinae according to their molecular phylogenetic analysis. In our didae). Zootaxa 3568, 1e35. phylogenetic trees from PAUP, WinClada and IQtree analyses, Kar- Chou, I., Lei, Z., Li, L., Lu, X., Yao, W., 1997. The Cicadidae of China (Homoptera: e “D” e Cicadoidea). Tianze Press, Hong Kong, pp. 1 380 (in Chinese, English summary). enia (marked with in Figs. 11 13) clustered with the genus de Fernandes, F., Linardi, P.M., Chiarini-Garcia, H., 2002. Morphology of the antenna Meimuna first, and then clustered with other representatives of of Dermatobia hominis (Diptera: Cuterebridae) based on scanning electron mi- Cicadinae. Morphologically, the metanotum of Karenia is distinctly croscopy. J. Med. Entomol. 39 (1), 36e43. https://doi.org/10.1603/0022-2585- 39.1.36. concealed by the cruciform elevation at dorsal midline. This is the Distant, W.L., 1906. The Fauna of British India, Including Ceylon and Burma, vol. 3. same as other species in Cicadinae, but is different to members of Taylor and Francis, London, pp. 1e266. Cicadettinae whose metanotum is partly visible at dorsal midline Duffels, J.P., van der Laan, P.A., 1985. Catalogue of the Cicadoidea (Homoptera, (Moulds 2005). Furthermore, In Cicadettinae the uncus is duck-bill Auchenorhyncha) 1956-1980. Series Entomologica, vol. 34. Dr. W. Junk Pub- lishers, pp. 1e414. shaped and undeveloped, and the pair of claspers are well devel- Duffels, J.P., 1993. The systematic position of Moana expansa (Homoptera: Cicadi- oped; while in Cicadinae the uncus is well developed with uncal dae), with reference to sound organs and the higher classification of the su- e lobes much swollen and elongated, and the claspers are usually perfamily Cicadoidea. J. Nat. Hist. 27, 1223 1237. https://doi.org/10.1080/ 00222939300770731. degenerated or even disappeared (Moulds 2005). The strongly Evans, J.W., 1956. Palaeozoic and Mesozoic Hemiptera (Insecta). Aust. J. Zool. 4, swollen uncus in Karenia is similar to that in Cicadinae. In addition, 165e258. e the gross morphology of salivary glands of Karenia caelatata is Evans, J.W., 1963. The phylogeny of the Homoptera. Annu. Rev. Entomol. 8, 77 94. Faucheux, M.J., Kristensen, N.P., Yen, S.H., 2006. The antennae of neopseustid similar to those of species of Meimuna and Platylomia Stål, 1870 moths: morphology and phylogenetic implications, with special reference to (Zhong et al. 2012, 2013). Interestingly, Maccagnan and Martinelli the sensilla (Insecta, Lepidoptera, Neopseustidae). Zool. Anz. 245, 131e142. (2004, 2011) discovered that there were generally five apical https://doi.org/10.1016/j.jcz.2006.05.004. Hou, Z., Li, Q., Wei, C., 2014. Comparative morphometrics of final instar nymphs of spines on hind tibia in the last instar nymphs of species in Cicadi- three cicadas (Hemiptera: Cicadidae) in Guanzhong Plain, China. ZooKeys 425, nae, and Logan and Connolly (2005) and Maccagnan and Martinelli 33e50. https://doi.org/10.3897/zookeys.425.7897. (2011) found that there are four hind tibial spines apically in the last Hu, F., Zhang, G.N., Wang, J.J., 2009. Scanning electron microscopy studies of antennal sensilla of bruchid beetles, Callosobruchus chinensis (L.) and Callos- instar nymphs of species in Cicadettinae. The last instar nymphs of obruchus maculatus (F.) (Coleoptera: Bruchidae). Micron 40 (30), 320e326. K. caelatata also possess five apical spines on hind tibia (Hou et al. Klein, U., Bock, C., Kafka, W.A., Moore, T.E., 1988. Antennal sensilla of Magicicada 2014). These above-mentioned characteristics together with the cassini (Fisher) (Homoptera: Cicadidae): fine structure and electrophysiological e antennal characters investigated in the present research indicate evidence for olfaction. Int. J. Insect Morphol. Embryol. 17, 153 167. https://doi. org/10.1016/0020-7322(88)90009-8. that this genus is much closer to related taxa of Cicadinae. This is Lam-Tung, N., Heiko, A.S., Arndt, H., Bui, Q.M., 2015. IQ-TREE: a fast and effective also supported by Marshall et al. (2018). stochastic algorithm for estimating maximum likelihood phylogenies. Mol. Biol. e The aforementioned results indicate that the grouping of Cica- Evol. 32, 268 274. https://doi.org/10.1093/molbev/msu300. Lee, Y.J., 2014. Cicadas (Hemiptera: Cicadidae) of Laos, with the description of four dinae and Cicadettinae based on the presence or absence of timbal new genera and two new species. Ann. Soc. Entomol. FR. (N.S.) 50 (1), 59e81. covers is unsound. Our phylogenetic trees are partly incompatible Lee, Y.J., Hayashi, M., 2004. Taxonomic review of Cicadidae (Hemiptera, Auche- with the classification of Moulds (2005) and Marshall et al. (2018), norrhyncha) from Taiwan, part 3. Dundubiini (two other genera of Cicadina), e Moganiini, and Huechysini with a new genus and two new species. J. Asia Pac. e.g., Cicadettinae is not a monophyletic group (Figs. 11 13). How- Entomol. 7, 45e72. ever, the status of Karenia and Cicadatrini addressed in Marshall Lee, Y.J., Hill, K.B.R., 2010. Systematic revision of the genus Psithyristria Stål (Hem- et al. (2018) is supported by our phylogenetic analyses. Therefore, iptera: Cicadidae) with seven new species and a molecular phylogeny of the genus and higher taxa. Syst. Entomol. 35, 77e305. https://doi.org/10.1111/ despite the shortcomings, the phylogenetic analysis of Cicadidae j.1365-3113.2009.00509.x. based on antennal characters is informative for reconstructing the Li, Z.B., Yang, P., Peng, Y.Q., Yang, D.R., 2009. Antennal sensilla of female fig polli- phylogeny of cicadas. Given that antennal morphological characters nator Ceratosolen sp. and its ecological implication. Chin. Bull. Entomol. 46, 941e949. of limited taxa were used in the present study, the systematic status Li, X.M., Ren, G.D., Wang, X.P., 2013. Analysis of the taxonomic and phylogenetic of related taxa in Cicadidae needs to be further addressed using relationships of Mylabrini species (Coleoptera, Meloidae) based on antennal more morphological characters combined with molecular data. sensilla morphology. Chin. J. Appl. Entomol. 50, 242e252. Logan, D., Connolly, P., 2005. Cicadas from kiwifruit orchards in New Zealand and identification of their final instar exuviae (Cicadidae: Homoptera). N. Z. Ento- Acknowledgements mol. 28, 37e48. https://doi.org/10.1080/00779962.2005.9722684. Maccagnan, D.H.B., Martinelli, N.M., 2004. Descriçao~ das ninfas de Quesada gigas fi (Olivier) (Hemiptera: Cicadidae) associadas ao cafeeiro. Neotrop. Entomol. 33, This research was nancially supported by the National Natural 439e446. Science Foundation of China (Grant no. 31572302, 31772505). Maccagnan, D.H.B., Martinelli, N.M., 2011. Description and key to the fifth-instars of some Cicadas (Hemiptera: Cicadidae) associated with coffee plants in Brazil. Neotrop. Entomol. 40, 445e451. References Marshall, D.C., Moulds, M.S., Hill, K.B.R., Price, B.W., Wade, E.J., Owen, C.L., Goemans, G., Marathe, K., Sarkar, V., Cooley, J.R., Sanborn, A.F., Kunte, K., Bartlet, E., Romani, R., Williams, I.H., Isidoro, N., 1999. Functional anatomy of sen- Villet, M.H., Simon, C., 2018. A molecular phylogeny of the cicadas (Hemiptera: sory structures on the antennae of Psylliodes chrysocephala L. (Coleoptera: Cicadidae) with a review of tribe and subfamily level classification. Zootaxa Chrysomelidae). Int. J. Insect Morphol. Embryol. 28, 291e300. 4424 (1), 1e64. Brezot, P., Tauban, D., Renou, M., 1997. Sense organs on the antennal flagellum of the Merivee, E., Rahi, M., Bresciani, J., Ravn, H.P., Luik, A., 1998. Antennal sensilla of the green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomidae): sensillum click beetle, Limonius aeruginosus (Olivier) (Coleoptera: Elateridae). Int. J. In- types and numerical growth during the post-embryonic development. Int. J. sect Morphol. Embryol. 27, 311e318. http://doi.org/10.1016/S0020-7322(98) Insect Morphol. Embryol. 25, 427e441. https://doi.org/10.1016/S0020-7322(96) 00023-3. 00012-8. Merivee, E., Rahi, M., Luik, A., 1999. Antennal sensilla of the click beetle, Melanotus Boulard, M., 1973. Ydiellinae, sous-famille nouvelle de cigales Platypediidae: Cle des villosus (Geoffroy) (Coleoptera: Elateridae). Int. J. Insect Morphol. Embryol. 28, familles et sous-familles des Homopteres Cicadoidea. Ann. Societ e Entomolo- 41e51. http://doi.org/10.1016/S0020-7322(98)00032-4. gique France (N.S.) 9, 841e852. Mitchell, B.K., Itagaki, H., Rivet, M.P., 1999. Peripheral and central structure involved Boulard, M., 1976a. Un type nouveau d’appareil stridulant accessoire pour les in insect gustation. Microsc. Res. Tech. 47, 401e415. Cicadoidea. Revision de la classification superieure de la superfamille (Hom.). Moulds, M.S., 1990. Australian Cicadas. New South Wales University Press, Ken- J. Nat. Hist. 10, 399e407. https://doi.org/10.1080/00222937600770301. sington, p. 217 pp., 24 pls. 70 X. Wang et al. / Zoologischer Anzeiger 276 (2018) 57e70

Moulds, M.S., 2005. An appraisal of the higher classification of cicadas (Hemiptera: sensilla in three stink bug species (Hemiptera: Pentatomidae). Micron 41, Cicadoidea) with special reference to the Australian fauna. Record Aust. Mus. 57, 289e300. http://doi.org/10.1016/j.micron.2009.11.009. 375e446. Sukontason, K., Sukontason, K.L., Piangjai, S., Boonchu, N., Chaiwong, T., Ngern- Nixon, K.C., 2002. WinClada Ver. 1.00.08. Published by the author, Ithaca, NY. klun, R., Sripakdee, D., Vogtsberger, R.C., Olson, J.K., 2004. Antennal sensilla Onagbola, E.O., Fadamiro, H.Y., 2008. Scanning electron microscopy studies of of some forensically important flies in families Calliphoridae, Sarcoph- antennal sensilla of Pteromalus cerealellae (Hymenoptera: Pteromalidae). agidae and Muscidae. Micron 35, 671e679. http://doi.org/10.1016/j.micron. Micron 39, 526e535. http://doi.org/10.1016/j.micron.2007.08.001. 2004.05.005. Pettersson, E.M., Hallberg, E., Birgersson, G., 2001. Evidence for the importance of odour- Thai, P.H., Yang, J.T., 2012. First record of the cicada genus Karenia Distant, 1888 receptionintheparasitoidRhopalicus tutela (Walker) (Hymenoptera: Pteromalidae). (Hemiptera: Cicadidae) from Vietnam, with description of a new species. J.Appl.Bio.125,293e301. https://doi.org/10.1046/j.1439-0418.2001.00550.x. Zootaxa 3153, 32e38. Pophf, B., 1997. Olfactory responses record from sensilla coeloconica of the silkmoth Wei, C., Tang, G., He, H., Zhang, Y., 2009. Review of the cicada genus Karenia Bombyx mori. Physiol. Entomol. 22, 239e248. https://doi.org/10.1111/j.1365- (Hemiptera: Cicadidae), with a description of one new species trapped by 3032.1997.tb01164.x. clapping hands and its entomogenous fungus. Syst. Biodivers. 7, 337e343. Rani, U.P., Madhavendra, S.S., 2005. External morphology of antennal and rostral https://doi.org/10.1017/S147720000999003X. sensilla in four hemipteran insects and their possible role in host plant selec- Yang, M., Wei, C., 2013. On the cicada genus Nipponosemia Kato, with description of tion. Int. J. Trop. Insect Sci. 25, 198e207. https://doi.org/10.1079/IJT200577. one new species from China (Hemiptera, Cicadidae). ZooKeys 293, 19e39. Ruchty, M., Romani, R., Kuebler, L.S., Ruschioni, S., Roces, F., Isidoro, N., https://doi.org/10.3897/zookeys.293.4649. Kleineidam, C.J., 2009. The thermo-sensitive sensilla coeloconica of leaf-cutting Zacharuk, R.Y., 1985. Antenna and sensilla. In: Kerkut, G.A., Gilbert, L.Y. (Eds.), ants (Atta volleneideri). Arthropod Struct. Dev. 38, 195e205. http://doi.org/10. Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 6. 1016/j.asd.2008.11.001. Pergamon, Oxford, pp. 1e69. Roux, O., van Baaren, J., Gers, C., Arvanitakis, L., Legal, L., 2005. Antennal structure Zhong, H., Wei, C., Zhang, Y., 2012. Gross morphology and ultrastructure of salivary and oviposition behavior of the Plutella xylostella specialist parasitoid: Cotesia glands of the mute cicada Karenia caelatata Distant (Hemiptera: Cicadoidea). plutellae. Microsc. Res. Tech. 68, 36e44. https://doi.org/10.1002/jemt.20220. Micron 45, 83e91. http://doi.org/10.1016/j.micron.2012.10.019. Sanborn, A.F., 2013. Catalogue of the Cicadoidea (Hemiptera: Auchenorrhyncha). Zhong, H., Zhang, Y., Wei, C., 2013. Salivary glands in Cicadidae (Hemiptera: Cica- Academic Press, 1001pp. doidea): comparative morphology, ultrastructure, and their phylogenetic sig- Silva, C.C.A., Capdeville, G., Moraes, M.C.B., Falcao, R., Solino, L.F., Laumann, R.A., nificance. Zoomorphology 132, 421e432. https://doi.org/10.1007/s00435-013- Silva, J.P., Borges, M., 2010. Morphology, distribution and abundance of antennal 0197-0.