Morphology, songs and genetics identify two new cicada species from Morocco: Tettigettalna afroamissa sp. nov. and Berberigetta dimelodica gen. nov. & sp. nov. (Hemiptera: Cicadettini) Author Costa, Gonçalo João Author Nunes, Vera L. Author Marabuto, Eduardo Author Mendes, Raquel Author Laurentino, Telma G. Author Quartau, José Alberto Author Paulo, Octávio S. Author Simões, Paula Cristina text Zootaxa 2017 4237 3 517 544 journal article 36374 10.11646/zootaxa.4237.3.4 8902b86d-e42c-4dd7-b1ce-eba9a6a44ae6 1175-5326 344747 9E48300E-4F19-4C80-A834-8BF6D23E83EF Tettigettalna afroamissa sp. nov. Costa, Nunes, Marabuto, Mendes & Simões Material examined Paratypical series consist of 13 specimens (ten males and three females). Designated holotype is SP18_3779 (♂) and female paratype is SP18_3780 (♀). See Table 2 for additional information on the paratypical series, specimen IDs, collection sites and GPS data. See Figure 2 for images of male holotype, female paratype and for details of the male genitalia. Male morphology Head Head slightly less broad than pronotum; Supra-antennal plates nearly meeting the eye and produced into a pointed lobe; Postclypeus rounded to subquadrate in frontal view, rounded between top and sides in lateral view, transversely grooved towards distal ends; Rostrum brown, reaching the center of mid-trochanters (in rest). Antennae dark-brown, 7-segmented. Dorsal surface of head brown with front bearing a yellowish stripe extending to outer borders; Yellowish stripe at beginning of epicranial suture extending to pronotum. Eyes brown, three red ocelli. Postclypeus dark brown, with apical yellowish-brown spot extending to frons, grooves light-brown or yellowish. Supra-antennal plates dark-brown and yellowish-brown towards distal ends. Gena and lorum brown to dark-brown covered in long white pilosity. Anteclypeus brown to dark-brown with a lighter brownish fascia surrounding a central dark-brown spot. Thorax Pronotal collar slightly larger than head width, widened, sloping laterally and evenly rounded dorsally. Pronotal tooth present mid-laterally. Scutellum wider than long. Epimeral lobe not reaching operculum. Submedian sigillae well defined. Metanotum partly visible at dorsal midline not expanded over tymbals. Pronotum with an olive-green arrow shaped stripe at dorsal midline bordered with dark-brown in fresh specimens (in preserved specimens this fades away to light brown). Remainder of pronotum brown, with dark-brown markings bearing yellowish borders. Mesonotum on overall brown, with a lighter “crown-like” marking, lateral margins of mesonotum yellowish. Scutellum brown, with a longitudinal dark-brown fascia at midline expanding towards the ends, reaching metanotum. Sides of scutellum with a dense pilosity on lateral-anterior ends with a fading gradient of dark-brown to yellowish towards the posterior end with defined, longitudinal, slightly transverse grooves. Metanotum brown, with a dark-brown patch at dorsal midline. Ventral side of thorax brown. FIGURE 2. Body and male genitalia morphology of Tettigettalna afroamissa . A,B—Designated male holotype of T. afroamissa in dorsal and ventral views, respectively. Scale bar equals 10 mm; C, D—Designated female paratype of T. afroamissa in dorsal and ventral views, respectively. Scale bar equals 10 mm; E, F—Male paratype’s pygophore in in lateral and posterior views, respectively. Scale bar equals 500 µm. Photos taken on dry specimens; G, H—Aedeagus in upper and lateral views, respectively. Scale bars equal 200 µm. Photos taken of material preserved in Kaiser gelatin. Legs Profemur with three to four dark-brown erect spines. Primary spine clearly separated. Metatibiae with three to four long fine spurs on inner side, and two smaller spurs on outer side with finely dispersed white pilosity. Apex of metatibia surrounded by smaller numerous brown spurs. Tarsal formula: 3-3-3. Legs generally brown in colour. Coxae and trochanters yellowish with a central dark-brown stripe, better defined on the hind legs. Femurs and tibiae brown with two dark-brown longitudinal fasciae. Profemurs with a swollen dark brown fascia surrounded by two yellowish/ light brown stripes, varying somewhat among individuals. Dark-brown border along the spines. Tarsi dark-brown on dorsal side, brown on ventral side. Protarsi darker in colour. Wings Forewing and hindwing with eight and six apical cells, respectively. Ulnar cell 3 angled towards radial cell; Forewing costa parallel-sided to radial cell; Pterostigma present. CuA vein weakly bowed; M+CuA meeting at basal cell with stems fused. Vein RA1 aligned closely with subcostal for its length. CuA1 divided by a crossvein with shorter proximal part. CuP and 1A unfused at their bases. Veins C and R+Sc close together. Outer forewing margin developed for its total length. Hindwing first cubital cell width at distal end much greater than second cubital cell. Hindwing anal lobe broad with 3A vein long and strongly curved at distal end. Hindwing RP and M veins fused at their base. Larger forewing proximal veins yellowish with smaller apical veins brown, same for hindwing. Forewing basal membrane yellow. Hindwing plaga yellow. Opercula More or less confluent with distal margin of tympanal cavity, well developed towards abdominal midline with sharply rounded apices facing midline. Opercula extending but not reaching posterior border of StII. Opercula distally yellow, dark-brown at base. Meracanthus triangular, following same colour pattern as opercula. Tymbals Tymbals lacking a tymbal cover. Five ribs, four of which arising from top of a large basal dome, covering about half the tymbal width, and expanding in width towards the posterior side. Fifth rib as an extension of basal dome more or less defined, varying between specimens. First and second anterior ribs, slender, with a transverse break at about halfway of basal dome. Tymbal plate light-grey, ribs and basal dome brownish-grey. Abdomen Abdomen with somewhat scattered white pilosity. T1 uniformly dark-brown; T2 uniformly darkbrown with a transversal stripe, slightly pointed towards posterior end of abdomen on each side; T3 to T7 darkbrown anteriorly becoming lighter on posterior side; T8 dark-brown. StI mainly dark-brown, yellow posterior margin; StII mainly dark-brown, with yellow lateral borders. StIII to StVI light brown, with a brown spot at midline, forming a well-defined stripe. StVII large, brown, as long as or slightly longer than StVIII; StVIII brown, densely covered in pilosity. Epipleurites brown with yellow posterior border. Genitalia ( Figures 2 E to 2H) Pygophore dark-brown on dorsal surface and brown on lateral sides. Pygophore distal shoulder not developed. Pygophore inner tooth absent. Upper lobe flat and moderately developed, distant from dorsal beak with a sharply rounded tip; Basal lobe present, moderately developed and rounded in lateral view. Dorsal beak present and part of chitinized pygophore. Claspers dark-brown, medium-sized, closely aligned ending on a rounded, sharp tip. Uncus brown, duck-bill shaped, small and flat, not dominant. Uncus lateral lobes absent. Aedeagus basal plate, in lateral view, with an undulated ventral surface skewed towards the proximal end; In ventral view, apically broad with a small constriction mid-ventrally expanding afterwards with a midgroove between two longitudinally expanded lobes; Basal portion of basal plate directed forwards and away from thecal shaft; Basal plate ventral rib not apparent; Basal plate attached with a functional membranous “hinge”. Theca, in lateral view, curved into a gentle arc; Thecal pseudoparamers present, dorsal of theca, originating closer to theca than its base; Endothecal ventral support present; Thecal aperture upper diagonal in lateral view. Female morphology Females overall slightly darker than males. Pronotal posterior border light-brown. Mesonotal “crown-like” mark much more faded and smaller than males. Scutellum light-brown. Meso- and metatarsi lighter in colour, light-brown turning brown towards claws. Opercula almost reaching posterior border of StII but much smaller. T1 and T2 totally dark-brown. Abdominal ventral midline fascia dark-brown very well defined. StVII yellowish and split, with a light-brown groove on each side. Stigma dorsal beak dark-brown. Ovipositor brown with dark-brown tip. Body measurements for T. afroamissa males (n=10) Total length: 27.17 ± 1.25 mm ; Pronotal length: 2.79 ± 0.13 mm ; Mesonotal length: 4.35 ± 0.26 mm ; Forewing length: 21.26 ± 0.97 mm ; M+CuA length: 1.26 ± 0.19 mm . Female and additional body measurements can be found on Table 3 . Bioacoustics The male acoustic signals here described are based on the analysis of the calling song of six males recorded at T= 38–40 °C (see Figure 3 ). The typical calling song is composed by the repetition of a phrase subdivided into two parts: A—a first single, short echeme and B—a longer group composed of 9 ± 7.461 echemes (6–50, n=124) and the interval between parts A and B has a duration of 155±53 ms (112–539 ms; n=99). In 23.6% of the phrases part A was absent. We also report a single calling song with a continuous phrase without any apparent pauses. FIGURE 3. Tettigettalna afroamissa nov. sp. calling song profile with successive ampliation of recorded phrases. Mean frequency spectrum (1), oscillogram (2) and spectrogram (3). Calling song recorded on Afouzar, Middle Atlas, Morocco at 39- 40°C. Peak frequency of all calling songs is at 11.72 ± 0.79 kHz, maximum and minimum frequencies are 18.45 ± 1.74 kHz and 4.14 ± 0.44 kHz, respectively. Additional temporal and frequency-based variables are indicated in Table 4 . Because of the similarities in frequencies of parts A and B, these were grouped in the same analysis. Diagnosis T. afroamissa is morphologically similar to all other Tettigettalna spp. but presents some peculiarities, allowing for its ready separation from its closest relatives. With an average total body length of 27 mm , it seems to be the genus’ largest species (Mendes et al. 2014, Simões et al . 2014, Puissant & Sueur 2010 ). T. afroamissa shows unique colour traits: all examined specimens have a black stripe running across the entire length of the ventral surface of the abdomen and an olive-green arrow-shaped stripe in the pronotum midline, which, upon death, fades over time to a paler shade of green in dry specimens (see image S5 for a live male bearing the typical olive-green stripe on the pronotum). TABLE 3 . Descriptive statistics of morphological variables performeđ on samples of T. afroamissa anđ B. dimelodica . Bođy measurements are presenteđ as average ± SD in mm. T. afroamissa B. dimelodica Male (n=10) Female (n=3) Male (n=13) Female (n=1) Bođy region Cođe Mean ± SD Min%Max Mean ± SD Min%Max Mean ± SD Min%Max Heađ anđ thorax TL 27.31 ± 1.11 25.93%29.21 25.84 ± 1.16 24.72%27.03 16.99 ± 0.78 15.59%18.30 17.30 HL 2.05 ± 0.12 1.86%2.20 1.98 ± 0.06 1.93%2.02 1.47 ± 0.11 1.24%1.61 - HW 5.97 ± 0.21 5.72%6.29 5.64 ± 0.32 5.41%5.86 3.86 ± 0.15 3.56%4.13 - EO 0.77 ± 0.04 0.71%0.84 0.76 ± 0.03 0.73%0.78 0.54 ± 0.05 0.46%0.61 - OO 1.37 ± 0.05 1.3%1.46 1.34 ± 0.18 1.22%1.47 0.89 ± 0.04 0.81%0.96 - LrL 1.15 ± 0.11 1.01%1.37 1.05 ± 0.16 0.94%1.16 0.85 ± 0.08 0.76%1.04 - LiL 2.92 ± 0.14 2.66%3.10 2.62 ± 0.07 2.57%2.67 1.97 ± 0.11 1.70%2.15 - VW 2.91 ± 0.12 2.71%3.10 2.82 ± 0.25 2.64%2.99 1.91 ± 0.12 1.68%2.06 - FR 0.62 ± 0.05 0.55%0.69 0.6 ± 0.03 0.59%0.62 0.40 ± 0.06 0.31%0.52 - PC 2.36 ± 0.11 2.23%2.50 2.28 ± 0.01 2.28%2.29 1.54 ± 0.09 1.36%1.66 - PL 2.82 ± 0.09 2.67%2.95 2.66 ± 0.25 2.48%2.83 1.74 ± 0.15 1.46%1.98 - PW 6.71 ± 0.37 6.20%7.34 6.31 ± 0.43 6.01%6.62 4.31 ± 0.26 3.65%4.58 - ML 4.40 ± 0.19 4.15%4.68 4.12 ± 0.52 3.76%4.49 2.67 ± 0.11 2.43%2.85 - Abđomen OP 3.92 ± 0.17 3.64%4.17 1.76 ± 0.22 1.60%1.91 2.55 ± 0.16 2.09%2.73 - LS 1.62 ± 0.09 1.52%1.77 - - 1.32 ± 0.13 1.03%1.49 - TyL 1.53 ± 0.06 1.43%1.64 - - 0.99 ± 0.07 0.90%1.14 - TyW 2.84 ± 0.06 2.76%2.95 - - 1.93 ± 0.10 1.67%2.06 - Legs PF 3.20 ± 0.11 3.05%3.36 3.08 ± 0.06 3.04%3.13 2.10 ± 0.15 1.78%2.26 - Wings FwL 21.37 ± 0.85 20.16%22.84 20.28 ± 0.91 19.42%21.23 13.39 ± 0.54 12.42%14.27 13.59 FwW 7.50 ± 0.27 7.13%7.88 7.11 ± 0.48 6.67%7.62 5.37 ± 0.84 4.87%8.10 5.10 BCL 1.85 ± 0.14 1.68%2.06 1.72 ± 0.10 1.62%1.81 1.23 ± 0.11 1.09%1.42 1.24 McuA 1.26 ± 0.18 0.92%1.48 1.34 ± 0.21 1.10%1.50 1.21 ± 0.18 0.94%1.41 1.22 RCL 8.53 ± 0.36 7.93%9.16 8.32 ± 0.39 7.95%8.72 6.12 ± 0.36 5.20%6.59 6.24 afroamissa Phrase Part A Part B TABLE 4. Time anđ frequency baseđ parameters of the analyzeđ phrases of T. afroamissa . Frequency variables values are presenteđ in kHz. variables Mean±SD Min%Max n Mean±SD Min%Max n Mean±SD Min%Max n Duration (ms) 726 ± 582 314%3749 124 10 ± 4.5 5%27 97 720 ± 580 309%3733 124 Echeme đuration (ms) - - - Same as above 20.97 ± 8.26 5%43 1364 Echeme rate (echeme.s -1) - - - - - - 16.21 ± 1.73 10.88%19.42 1364 Cicada barbara 12.0 18.6 19.6 18.6 18.9 20.0 19.5 19.6 19.4 19.5 19.3 18.4 19.4 19.1 19.4 19.9 20.2 Cicada orni 13.3 17.2 19.3 19.3 19.1 20.0 19.7 19.5 19.4 20.6 18.4 19.3 19.2 18.9 18.7 19.0 18.7 Hilaphura varipes 21.5 19.6 11.4 12.4 11.2 11.7 12.4 12.3 12.0 12.0 11.2 10.9 11.3 11.4 10.9 12.9 9.8 Euryphara contentei 22.9 22.4 12.5 9.6 8.1 10.8 10.7 10.6 10.3 9.7 10.0 9.2 9.0 9.6 9.4 11.4 9.8 Tympanistalna gastrica 21.5 22.4 13.8 10.4 9.8 12.4 13.5 13.2 13.3 12.7 11.4 11.4 11.6 12.4 11.3 12.4 11.5 Tettigettacula baenai 21.9 22.2 12.3 8.7 10.7 10.4 10.6 10.6 10.3 9.6 11.0 9.0 8.6 9.0 8.9 11.5 9.7 Tettigettalna estrelae 23.4 23.3 12.9 11.7 13.7 11.3 7.7 7.4 7.8 5.3 9.2 5.8 5.1 5.4 4.8 9.5 11.8 Tettigettalna argentata 22.7 23.0 13.7 11.7 15.0 11.6 8.3 1.7 1.9 7.4 9.6 6.3 6.6 6.4 7.3 10.4 11.1 Tettigettalna mariae 22.8 22.7 13.7 11.6 14.7 11.6 8.0 1.8 1.2 7.1 9.4 6.1 6.2 6.1 6.9 10.8 10.7. Tettigettalna aneabi 22.7 22.6 13.3 11.2 14.8 11.2 8.4 1.9 1.2 7.3 8.8 5.9 6.7 6.0 7.2 10.4 10.0. Tettigettalna boulardi 22.8 24.2 13.2 10.5 14.0 10.3 5.6 7.9 7.6 7.8 9.2 5.6 4.3 5.4 4.9 9.8 11.6. Tettigettalna josei 22.5 21.1 12.2 10.7 12.5 11.9 10.0 10.4 10.2 9.5 10.0 8.0 8.7 7.9 8.5 9.6 10.0. T. helianthemi helianthemi 21.3 22.4 11.9 9.9 12.5 9.7 6.1 6.7 6.4 6.3 5.9 8.5 3.4 5.2 5.2 9.2 10.5. T. helianthemi galantei 22.6 22.3 12.4 9.6 12.7 9.2 5.3 7.0 6.6 7.1 4.5 9.4 3.6 5.0 4.3 9.0 11.0. Tettigettalna armandi 22.2 21.9 12.5 10.4 13.7 9.6 5.7 6.8 6.5 6.4 5.7 8.5 5.5 5.2 3.7 9.0 10.4. Tettigettalna defauti 22.7 21.6 11.9 10.1 12.3 9.5 5.0 7.8 7.3 7.7 5.2 9.1 5.5 4.5 3.9 8.3 10.9 Acoustic analysis enables easy and accurate identification of all Tetigettalna species. T. afroamissa is no exception. Its calling song is structurally different from all other Tettigettalna spp., although reminiscent of T. argentata (Olivier, 1790) and T. boulardi Puissant, 2010 .

Interval (ms)	326 ± 116	186%906	94	-	-	-	51.20 ± 7.07	26%63 1340
Frequency variables	Peak frequency	Min frequency	Max frequency	Banđwiđth	Quartile 25	Quartile 50	Quartile 75	Quartile (75%-25%)
Mean ± SD Min%Max	11.72 ± 0.79 7.21%14.25	4.14 ± 0.44 3.93%8.81	18.45 ± 1.74 15.46%23.81	14.30 ± 1.87 7.68%19.87	9.93 ± 0.56 7.59%10.96	11.50 ± 0.48 10.03%12.75	12.82 ± 0.45 11.25%14.34	2.89 ± 0.55 1.41%4.69

TABLE 5. Mean pairwise genetic đistances (%) between the taxa consiđeređ for phylogenetic analysis: P-đistances in the upper điagonal anđ Kimura 2-parameter đistances in the lower điagonal. Highlighteđ values in bolđ belong to genus Tettigettalna . FIGURE 4. Bayesian inference phylogenetic tree of Cytochrome C oxidase subunit I mitochondrial DNA of T. afroamissa and B. dimelodica with other previous published taxa. Posterior probabilities are shown next to branch nodes. TET stands for Tettigettacula — Euryphara — Tympanistalna clade. Scale bar represents the number of estimated changes per branch length. C. barbara (Cba203) and C. orni (Cor298) were set as an outgroup. T. afroamissa and B. dimelodica taxa IDs are detailed on Table 2. Additional taxa details are included on supplementary information Table S1. Root was truncated with double dash totalling 0.6 changes per branch length. The song of T. afroamissa can be distinguished from T. argentata for it has higher echeme rate (t= 16.21 ± 1.73 echemes.s -1 vs t= 12.82 ± 1.49 echemes.s -1) and a shorter inter-echeme interval (t= 51.20 ± 7.07 ms vs t= 71.00 ± 13.00 ms) (Mendes et al. 2014). T. boulardi has a typical calling song with a short echeme (t= 200 ± 110 ms) followed by a long echeme (t= 2.17 ± 0.30 s ), whereas in T. afroamissa this initial echeme is even shorter (t= 10 ± 4.5 ms), followed by a succession of very short echemes (t=720 ± 580 ms), instead of a single one. Inter-phrase interval is also much shorter for T. afroamissa (t= 326 ± 116 ms) than for T. boulardi (t= 3270 ± 680 ms). For additional time and frequency measurements regarding T. boulardi see Puissant & Sueur (2010) . DNA barcoding Males from all sampled locations were sequenced for COI. Four haplotypes were recovered in a total of 10 sequences. The dataset includes one non-synonymous mutation and a total of 14 polymorphic sites, corresponding to a nucleotide diversity of π=0.1075. All T. afroamissa sequences grouped in a fully supported monophyletic clade ( Figure 4 ) and intraspecific pairwise distances (K2P) varied from 0.5 to 2.1 %. This clade clusters with remaining Tettigettalna spp. in an unresolved polytomy. Mean genetic distances among T. afroamissa and all other species of the genus are shown in Table 5 , and vary from 8.9% (with T. defauti ) to 11.9 % (with T. mariae ). Thus, the genetic distance associated with the fragment of COI used here, the “barcode gap”, is high enough to be used for DNA barcoding of T. afroamissa . FIGURE 5. Habitats of T. afroamissa (A-D) and B. dimelodica (D-F) in Morocco: Rif mountains near Chefchaouane (A), Bni Hadifa (B) and Taferka (C); Middle Atlas near Taza (D); Berkane (E) and El Hoceima (F). Specimens were captured in all locations but C (see supplementary Table, S2). Photos by VL Nunes. Habitat ( Figure 5 ) An arboreal species, inhabiting open Mediterranean-type woodland and tall scrubland. This species has been scored singing mainly on holm-oak trees ( Quercus rotundifolia ) and bushes such as Pistacia lentiscus and Cistus spp. but locally, in the Rif, it was found on pine trees ( Pinus spp.) ( Figure 5 B), Abies pinsapo var. marocana and Cedrus atlantica ( Figure 5 C) and almond trees ( Prunus dulcis ) . Distribution Northern Morocco , along the Rif Mountains and nearby Mediterranean coastline between Tetuan and Al Hoceima. Also found in the northern parts of the Middle Atlas, near Taza ( Figure 1 ). Not found near Ceuta or Tangier . Etymology Specific epithet formed by combining the suffix afro (pertaining to Africa) and the prefix amissa , feminine of the latin āmissus, meaning “having been lost” or “let go”. Literal translation would be “cicada (of the genus Tettigettalna ) left / lost in Africa” as this new species is the only Tettigettalna spp. known so far to occur in Africa, the remaining being European.