Bioacoustics of Neotibicen similaris

Song. The following describes a single phrase of a free-flying male of Neotibicen similaris similaris recorded in the morning on 29 August 2008 in Nassau County, Florida , at the junction of Highway A1A and Lofton Creek, about 7 air miles west-southwest of Fernandina Beach, the approximate location of the original town of Fernandina (recording 08. US .FL.LCC.T04.WAV) and type locality. This recording will be deposited at the online repository BioAcoustica ( Baker et al., 2015 ) and at www.insectsingers.com ( Marshall and Hill, 2010 ). Much like N. similaris apalachicola , the song frequency ranges from around 3 to 19 kHz (approximately the limit of the microphone used) and contains the following three sections: (1) A leading section consisting of a uniform buzz which was audible in the field but is not clearly visible against the background sound in the recording. (2) A main phrase that alternates sharply between high-pitched echemes and low-pitched echemes or sets of echemes; as in apalachicola , the high-pitched echemes are also higher in amplitude. The main phrase contains two parts that differ in the rate of alternation and in the temporal pattern. For each cycle in part I ( 5.7 s in duration), a high-pitched echeme (main energy 8–13 kHz, duration ca. 0.013 s ) containing 4–5 pulses is immediately followed by two low-pitched echemes (main energy with peaks at about 4.5 and 6.8 kHz), about 0.016 s and 0.013 s in duration, with the high-pitched echeme seamlessly "slurring" into the first low echeme. The two low-pitched echemes contain shorter pulses that are more numerous and more difficult to resolve than those in the high-pitched echemes. For each cycle in part II of the main phrase, each high-pitched echeme is followed by just one low-pitched echeme. The overall rate of alternation is 14.7 cycles/second in part I and 21.6/ sec in part II; note that these values are somewhat slower than in many of our other recordings measured (see below) probably because of a cool morning temperature. Figure 4 shows an example phrase from a higherquality recording made at a different location. Song variation. Measurements of song characters from 15 phrases sampled from throughout the range of Neotibicen similaris similaris are given in Table 2 (see Supplementary Table 1 for source locations). Note that most song phrases from throughout the range, including at the holotype locality, exhibited isolated irregularly patterned oscillations in the transition from the alternating section of the main phrase to the trailing buzz. Distinguishing characters. Neotibicen similaris apalachicola is easily distinguished from N. similaris similaris by features of the male calling song, especially a song phrase containing a single, slow rate of alternation (2–3 cycles/sec) between low-pitched and high-pitched sound, sounding like the word "easy" being slowly repeated. N. similaris similaris contains almost the same sound frequencies but alternates very rapidly between low- and high-pitched echemes at more than ten times the rate and increases the rate part-way into the phrase, forming a clacking rattle. The song of N. similaris apalachicola is superficially similar to that shared by N. winnemanna (Davis) and N. pruinosus (Say) , which also oscillates between high- and low-pitched sound, but the latter two species produce sound that is mostly below 8 kHz in pitch and the fine-scale structure of the sound is entirely unlike that of apalachicola (unpublished data). The song of N. similaris similaris superficially resembles the pulsed call phrase of N. tibicen (L.), but the rate of oscillation in the latter is only about half that of similaris and the fine-scale structure of the song is again entirely different. Morphologically, Neotibicen similaris apalachicola cannot be consistently distinguished from the nominate subspecies, but on average it has a larger body size, a wider pronotal collar, darker forewing infuscation, and longer male opercula (more often extending beyond sternite IV). Although some character means are significantly different, the ranges show broad overlap in all cases ( Table 1 ). Subtle differences exist on average in the wing venation as well, with the forewing vein r-m between ulnar cell 2 and apical cell 3 more likely to be one-fourth or less of the length of forewing vein m between apical cell 4 and ulnar cell 2. We found no significant differences in the male genitalia of the two subspecies of N. similaris . Both subspecies of Neotibicen similaris are easily distinguished from other USA Neotibicen species (see Table 3 ) by the recurved spines protruding from the uncus, which can be viewed without genitalic dissection if the pygofer is gently extruded with a pin while the specimen is soft. In addition, N. similaris possesses a partly to entirely black pronotal collar (generally present only in N. tibicen , N. lyricen and N. similaris ), a dark pronotum (green in N. tibicen ), and clear wing membranes (suffused with brown in N. lyricen ). Aberrant specimens of other eastern Neotibicen that usually have green pronotal collars, including linnei (Smith and Grossbeck) , winnemanna (Davis) , robinsonianus (Davis) and davisi , either have bright green markings on the mesothorax (the first three species) or are small, with rounded opercula and a wide head ( N. davisi ). Both subspecies of N. similaris are also clearly distinguished from all other USA cicadas by song. Pronotal Male Mesothorax Wing suffused collar color genitalia pattern color with brown distally? N. similaris similaris and N. s. apalachicola black recurved spine dull green/brown no N. tibicen black no spine green/black slightly N. lyricen lyricen black no spine green/brown slightly (more in l. virescens ) N. linnei , N. winnemanna green no spine green/black slightly in some N. robinsonianus dark green no spine green/brown no N. davisi green/brown no spine green/brown no Ecology and behavior of Neotibicen similaris subspecies. Calendar dates for our records of adults and emerging cicadas of Neotibicen similaris ranged from 2 July to 28 September . Other sources show N. similaris active in Florida from mid-June until late October ( Sanborn et al. 2008 ; Walker 2000 ). Males sing mainly in bright sunshine. The average time of observation of singing cicadas in our study was 12:30 PM, with singing beginning around 8:30 AM and only rarely extending after 6:30 PM. We observed almost no dusk singing, compared to some species like N. linnei which are often active around sunset. Males of N. similaris apalachicola commonly flew to a new singing station after each song, especially in the morning hours, while males of N. similaris similaris appeared more likely to sing several song phrases from one location. When more than one song phrase was sung from a single perch, males of both subspecies produced a continuous low buzz between phrases, as in other Neotibicen cicadas. Interestingly, although some cicada species use vertical movements of the abdomen to alter song pitch (e.g., Magicicada septendecim , see Allard 1937 ), no such movement was observed when the holotype male of N. similaris apalachicola sang in a cage. Most males heard singing did so from very high stations (almost all over 6m , the maximum reach of our net poles, and most were much higher). Like all Neotibicen spp., mature males utter a loud alarm call when disturbed or handled.

One male in the FAC collection with the label data "PA: BERKS Co.//Douglasville// 31-VIII-83 // F.W.Skillman" was examined for this study. The uncus exhibits the unmistakable inward curving double spines. The only Berks County in the United States is in Pennsylvania. We listened for cicadas around the specified location on the afternoon of 18 September 2009 , under cool but sunny conditions, but we heard no Neotibicen similaris songs. Additional searching would be worthwhile because this record considerably expands the published range. It is possible that the specimen was mislabeled or collected in Berks County after having been moved there as an egg or a nymph on a transplanted tree (e.g., Chilcote & Stehr 1984 ). Hybridization. Putative hybrid songs combining characteristics of Neotibicen similaris similaris and N. similaris apalachicola were observed in many locations where the subspecies come into contact and apparently interbreed. Examples are shown in Figure 6 and vary from songs more resembling subspecies similaris ( Fig. 6 a), to those more resembling apalachicola ( Fig. 6 b, c), to some with the characters dramatically shifting within the song ( Fig. 6 d). Fig. 6 e shows how the detailed structure of the song in Fig. 6 d includes elements of both apalachicola (the long high-pitched echeme — compare to Fig. 3 c) and similaris (alternating short echemes of high and low pitch, compare to Fig. 4 d). Note that, while it was our impression in the field that consecutive songs made by the same hybrid male resembled each other more than those of other males, we were unable to confidently track individual singing cicadas, and the degree of song variability in hybrids remains undocumented. In the southern sector of the range of subspecies apalachicola ( Fig. 5 c), hybrid songs were heard only in a zone approximately 20 km wide separating the core apalachicola area from the surrounding populations of true similaris . Farther north, in Alabama and Georgia , pure and hybrid populations of both subspecies are less coherently distributed ( Fig. 5 b); we consider the significance of this pattern in the Discussion. Because our sampling was done rapidly by car, with many sites only briefly checked, our data do not resolve detailed spatial patterns within the hybrid zone, but the region with hybridization was obviously limited relative to the distributions of the parental subspecies. At 388 out of 438 sites where we found Neotibicen similaris cicadas, only one subspecies was heard. At 21 sites, normal songs of one or both parental subspecies were heard together with songs exhibiting hybrid influence. At 24 sites, all songs heard showed signs of hybrid influence, although again most of these were only very briefly sampled. The US .AL.GRN (N. side of Greenfield) and US .AL.WFC (Jct. 33/131 E. of Clio) sites were especially notable for the large number of varied hybrid songs heard (see Supplementary Table 1 for location details). There was no significant evidence of coexistence without hybridization: At five sites we noted both parental subspecies present without hybrid songs, but these were samples of less than one minute's duration with hybrid songs recorded at nearby sites. Two specimens were collected at sites where hybrid songs were common, one from Stewart Co. , GA ( DNA voucher 08. US .GA.LMK.01) and one from Mitchell Co. , GA ( DNA voucher 08. US .GA.FLI.01, see Supplementary Table 1). These specimens are housed at the University of Connecticut . The alarm sound of the first male was recorded.

Description, holotype male ( Fig. 1 A–D ). An overall large and dark-colored Neotibicen , with black background color and muted brown and green patterning, except where covered in white wax underneath. Small golden or silvery hairs can be seen, where they have not been rubbed off, in various crevices on the dorsal surface and to a lesser extent ventrally. Head. Mostly black, with small brown patches extending between postclypeus and compound eyes and near ocelli. Compound eyes, viewed dorsally, as wide as or slightly wider than pronotal collar, dark brown or black, with a fringe of short hairs posteriorly. Underside of head covered in white wax except for center of the postclypeus. Postclypeus mostly black with a small brown spot at the top and along the anterior midline, with nine ridges and a central groove. Anteclypeus covered with wax except for brown central midline. Lorum covered with wax. Proboscis light brown at base tending darker towards tip, extending to midway between hind coxae. Thorax. Pronotum mostly dark brown. Median sulcus muted yellow-green, bordered with black triangleshaped patches widening anteriorly and with a yellowish brown mark on either side posteriorly, just above the pronotal collar. Pronotal collar black, extreme lateral edges dull green. Mesonotum mostly black, with a thin “crown” pattern varying brown to green, and with the central portion above the cruciform element combining the two inner sigilla to form a large black patch. Lateral color patches, to the outer sides of the lateral sigillas, dark rusty brown. Cruciform elevation light brown, with the center notch black; metanotum brown. Underside of pronotum and mesonotum covered in white wax, this wax at least partially covering the coxae, trochanters and femora. Legs. With trochanters pale greenish brown, femora mostly brown. Base of tibia pale green on mid and hind legs, pale brown on forelegs, all tips dark brown. Tarsi pale and dark brown with black tarsal claws. Foreleg primary spine angled but not lying flat, secondary spine larger and more erect. Wings. Slightly longer than body, mainly hyaline. Forewing with basal cell mostly yellow-green, clear near vein CuA, flaps grey, and with only the faintest hint of yellow infuscation otherwise in the membranes. Dark brown infuscations present at the veins joining forewing cells u1/a2 and u2/a3. Basal half of forewing costa green, distal costa brown, ventrally with a black internal border. Vein CuP in forewing green, M and CuA in forewing and CuA and CuP in hindwing greenish brown, otherwise veins mostly black. Hindwing with veins 2A and 3A edged in brownish grey, flaps white. Abdomen. Tergites glossy black, with bright white wax spots on tergite II on either side above the timbal cover and centrally. Timbal covers black. Underside of abdomen with white wax coating the lateral edges of the sternites and the opercula, especially the lateral and basal edges. Sternites mostly black, with posterior margins of sternites III–VII medium brown, especially laterally. Sternite VIII forming an upright V-shape in posterior view. Opercula pale brownish green, overlapping for more than half their length. Opercula slightly pointed at tips, reaching the 4th sternite, lateral edges bowed inward slightly where they meet the timbal covers. Genitalia ( Figs. 1B, 1D , 2 A). Pygofer black, becoming brown towards base, in ventral view widest at about mid length, with widest width at base less than width of posterior margin of sternite VI; dorsal beak forming a sharp spine; distal shoulders weakly developed and rounded; basal lobes well-developed and visible in lateral view, in ventral view straight with rounded apices, not quite reaching to uncus. Median lobe of uncus black, broadest at base and narrowing evenly to a blunt apex that is almost divided by a deep dorsal suture; laterally with a large pair of black spines either side directed dorsally, their tips just visible dorsally (easily seen laterally). Aedeagus a sclerotized tube with a slightly flared, unsclerotized apex, at rest reaching just beyond the paired uncal spines. Anal styles dark brown. Song. The following describes a single recorded phrase of the holotype male numbered 08.US.FL.TRA.03 found in voucher recording 08.US.FL.TRA.03.T02.WAV which will be deposited at the online repository BioAcoustica ( Baker et al. , 2015 ) and at www.insectsingers.com ( Marshall and Hill, 2010 ). The holotype song phrase consists of approximately 20 s of timbal sound with a frequency range of approximately 2–19 kHz (approximately the limit of the microphone used), containing the following three sections: (1) a leading section consisting of a uniform buzz of increasing intensity approximately 1–2 s long (noted in the field, the holotype recording begins just after this section); (2) a main phrase that alternates sharply and seamlessly between shorter, higher amplitude, high-pitched echemes (0.08–0.13 s each) with sound frequencies mainly from 9–13 kHz and longer, lower amplitude, low-pitched echemes (0.35–0.38 s each) with sound frequencies mainly from 5.5–9.0 kHz (plus a secondary peak near 2.7 kHz) — the single low-pitched echeme could also be described as a series of seamlessly repeated four-pulse echemes; (3) a trailing buzz with sound energy mainly below 9 kHz. The rate of alternation between high- and low-pitched echemes in the main phrase is 2.1 cycles per second. Details of the waveform structures for the latter two parts of the song are as follows: Section (2) above, the main phrase, contains pulses repeated at about 370/sec in the shorter high-pitched echemes and pulses repeated at about 625/sec in the longer low-pitched echemes, the latter visibly grouped into fours based on amplitude patterns. Section (3) contains two-pulse echemes produced at about 240/sec and sometimes alternating in amplitude. In the holotype male phase, a subtle “rattle” lasting about 0.175 s appears near the beginning of the trailing section, suggesting the song pattern of the main phrase of N. similaris similaris (see below). The holotype recording is slightly distorted due to the short distance between the specimen and the microphone, which causes an exaggeration and smearing of the frequency spectrum at values over ca. 19 kHz; the important features of the song remain visible. The amplitude decreases at about 2.5 s into the track because the recorder gain was decreased. This recording was made on 21 July 2008 in the rear of an open car following a playback stimulus; ambient temperature was not recorded. Figure 3 shows an example phrase from a higher-quality recording made at a different location. Morphological variation in male paratypes . Most paratype specimens were not individually recorded singing and some were not collected at the type locality. Subspecies identification in these cases was made on the basis that only Neotibicen similaris apalachicola songs were heard at and near those locations (see section on Distributions below). Many of the paratypes were preserved only one or two days post-ecdysis and so have only a light covering of wax and fine hairs coating much of the dorsal surface. Although the male paratypes are overall dark-colored, some have slightly brighter green and brown patterning, and in some the pronotal "crown" is more complete than in others. Many individuals have the veins in the basal half of the forewing and many hind wing veins green or brown (especially on faded specimens). Legs of some specimens are mostly pale green with the tip of each segment tending dark brown. Opercula vary considerably in size and degree of pointedness at the tip, many being almost triangular, some with the tip and lateral portions appearing flattened compared to the inner part which appears to bulge outwards slightly. In all male paratypes the opercula extend at least half-way down the 3rd sternite, and in most they reach at least to the anterior edge of the 4th sternite. The tip of the forewing at the tip of veins R1a and R1b is often lightly infuscated with black, this infuscation sometimes reaching to the apex and slightly around the wing margin. Size measurements are given in Table 1 . Song variation. Measurements of song characters from 13 phrases from throughout the range of Neotibicen similaris apalachicola are found in Table 2 (see Supplementary Table 1 for source locations). Occasional song phrases from throughout the range, including at the holotype locality, exhibited brief rattles of a small fraction of a second in duration in the transition from the alternating section of the song phrase to the trailing buzz. These features resemble the rapidly alternating section of the song of the nominate subspecies (see below). Females. Overall similar to males, but tending paler in color with more brown and green, especially ventrally, and with the basal half of the forewing veins usually green or brown (Supplementary Fig. 2 ). Ovipositor brown to dark brown, not extending beyond sheath. Abdominal sternites variable in color, commonly brown or greenish brown rather than black, sometimes with black centers, and with epipleurites brown to greenish brown. Ninth tergites varying from black adjacent to ovipositor to brown or tan laterally. Size measurements in mm for a sample of 13 female specimens are given in Table 1 . ZooBank registry. urn:lsid:zoobank.org:act:7E149853-51EB-4D61-A96F-B95E34E985C2 Character Neotibicen similaris apalachicola , n. subsp. (13♂, 13♀) ♂ Mean Min. Max. ♀ Mean Min. Max. Body length 35.1 32.2 37.7 30.9 29.2 33.8 Forewing length from tip to wing articulation 41.7* 38.1 43.4 41.0 38.5 43.3 Forewing width at widest point 13.3 12.6 14.0 12.8 12.2 14.0 Head width across eyes 14.4* 13.7 15.0 14.0* 13.6 14.9 Thorax width across pronotal collar including flanges 14.2** 12.9 15.1 14.1 13.6 14.9 Opercula length 11.6** 10.9 12.7 N/A N/A N/A Pronotal collar width at either lateral crown point 2.1** 1.8 2.4 2.0 1.8 2.3 Neotibicen similaris similaris (7♂, 4♀) Body length 33.4 29.8 35.4 30.5 29.7 31.0 Forewing length from tip to wing articulation 39.2* 35.6 42.5 39.9 38.0 41.5 Forewing width at widest point 12.8 11.7 13.8 12.9 12.1 13.3 Head width across eyes 13.6* 12.4 14.6 13.5* 13.2 13.8 Thorax width across pronotal collar including flanges 13.2** 12.3 14.3 13.5 12.8 14.2 Opercula length 10.2** 8.7 11.2 N/A N/A N/A Pronotal collar width at either lateral crown point 1.9** 1.6 2.0 1.9 1.8 2.0 Character Mean Min. Max. Neotibicen similaris apalachicola , n. subsp. (13♂) Main phrase length (s) 14.4** 9.5 19.3 Rate of alternation between high and low echemes (Hz) 2.3** 2.0 2.9 High pitched echeme duration (s) 0.12** 0.10 0.14 High pitched echeme dominant pitch (kHz) 9.9** 8.8 11.3 Low pitched echeme duration (s) 0.32** 0.24 0.39 Low pitched echeme dominant pitch (kHz) 6.8* 3.8 8.6 Neotibicen similaris similaris (15♂) Main phrase length (s) 10.7** 6.3 17.3 Length of part I of main phrase (s) 3.2 1.2 5.8 Rate of alternation between high and low echemes, part I (Hz) 21.7 14.9 24.8 Length of part II of main phrase (s) 7.4 4.6 11.9 Rate of alternation between high and low echemes, part II (Hz) 34.5** 21.9 41.1 High pitched echeme duration (s) 0.01** 0.008 0.015 High pitched echeme dominant pitch (kHz) 8.6** 7.9 9.6 Low pitched echeme duration (s) 0.02** 0.007 0.1 Low pitched echeme dominant pitch (kHz) 5.2* 2.4 7.9