Bioacoustics of Adenomera guarani

Adenomera guarani sp. nov. inhabits open areas that can easily be flooded by rainwater during the wet season. The species is abundant in partially urbanized areas, such as roadsides or gardens. Males call amidst grasses, outside water, during the dusk and early in the night. They can easily be detected by their vocalizations, but due to their small size and secretive habits, are very difficult to observe. At Parque Nacional Cerro Corá ( Amambay Department , Paraguay ), populations of A. guarani sp. nov. and A. diptyx were found at two sites less than 1 km from each other. Two males of A. guarani were found calling at dusk at the margins of a pond inside the forest, near Aquidabán stream, and A. diptyx males were calling on roadsides amidst grasses and at the margin of a stream inside the forest. Other species calling syntopically with A. guarani were Scinax fuscovarius (Lutz, 1925) , S. berthae (Barrio, 1962) , Pithecopus azureus (Cope, 1862) , Dendropsophus nanus (Boulenger, 1889) , D. minutus (Peters, 1872) , Leptodactylus elenae Heyer, 1978 , Rhinella diptycha (Cope, 1862) , and Physalaemus nattereri (Steindachner, 1863) . In Argentina , A. guarani was also found on roadsides associated with Copernicia alba Morong palms and Ludwigia sp. grassland, syntopically with S. acuminatus (Cope, 1862) , P. azureus , D. nanus and Boana raniceps (Cope, 1862) , and in banana plantations, under fallen leaves.

Description Holotype Body small, robust; head nearly as broad as long (HW/HL = 1.1); snout sub-elliptical dorsally and acuminate laterally; canthus rostralis round; loreal region slightly concave; eye dorsolateral, its diameter about 32% of head length; pupil horizontal, elliptical; palpebral membrane translucent, bordered in its upper edge by a black streak; tympanum distinct, its diameter about 63% of the eye diameter. Tympanic membrane translucent, with some melanophores on the central region; supratympanic fold well developed, extending from the back of the eye to arm insertion; nostrils anterolateral, closer to the tip of the snout than to the eye, flanged in almost all their extension, except on the inner edge; internarial distance about 26% of head width; triangular postcommissural gland; lower lip with a medial projection that fits into a notch in the upper lip; tongue ovoid; vocal sac subgular with a fold parallel to jaw extending to the forearm; maxillary and premaxillary teeth present; vomerine teeth located posterior to and between choanae, arranged in straight transverse rows. Dorsum glandular, with more conspicuous glands placed on the posterior region. A pair (dorsal and lateral) of glandular rows formed by small segments starting behind the eyes and running towards the posterior part of the dorsum; ventral surface of the body smooth, with a distinct discoidal fold (pectoral few distinctive in the specimen in conservation); an axillary gland behind each arm insertion. Arms short and robust; fingertips rounded, not expanded (character state A); relative finger length IV <II ≤ I <III; nuptial asperities absent; inner metacarpal tubercle oval and ½ smaller than the outer, rounded; palmar tubercles present; prominent, rounded and entire subarticular tubercles on fingers, those on the base of each finger the most noticeable. Shank slightly longer than the thigh; thigh length about 43% SVL; relative toe lengths I <II <V <III <IV, with rounded tip, neither expanded nor flattened (character state A); toes without webbing or lateral fringing; metatarsal tubercles oval, not keratinized; outer tubercle smaller than the inner; subarticular tubercles present; plantar and supernumerary tubercles absent; posterior part of dorsum and ventral surface of both heel and metatarsals with dispersed, small and white-tipped tubercles; distinct inner tarsal fold with a single row of small, white-tipped tubercles on the edge, running for about two-thirds of the tarsus length. Ventral surfaces of the legs smooth, except the ventral surface of thighs, which is areolate. Vent fold distinct on the upper anus. A cream, rounded and small paracloacal gland is present on each side of the vent. Measurements of the holotype (mm) SVL = 21.6, HL = 7.3, HW = 8.3, ED = 2.4, TD = 1.2, EN = 1.9, IOD = 2.2, IND = 2.2, UAL = 3.1, LAR = 4.3, HAL = 5.2, THL = 9.2, TIL = 9.7, TAL = 5.6 and FL = 11.2. Coloration of the holotype In life, dorsum medium grayish-brown, with a black mask-like patch on the interorbital region; chevron shaped mark on the scapular area, and several smaller spots on dorsum. Broad, light mid-dorsal stripe with diffuse borders, running from the chevron-shaped mark to the vent fold. Dorsal glandular row mainly highlighted in dark; lateral glandular row and paracloacal glands cream. Faded orange patches on elbows and heels. Distinct dark longitudinal bars on the dorsal region of lower arms, thighs, and shanks. Dark marks on lateral upper lip and behind nostrils. Posterior part of the supratympanic fold and the border of the upper eyelids highlighted with dark brown coloration. Body ventrally whitish, immaculate; gular region and ventral surface of thighs faded pink; lateral fold of the vocal sac black. In preservative, color patterns as in life, except for a duller coloration. Comparisons with congeners Adenomera guarani sp. nov. (robust body shape) is distinguished from three congeners with slender body shapes ( A diptyx , A. martinezi , and A. saci ). The body size of A. guarani (male SVL = 20.1–26.4 mm ) differs from that of large-sized species of the genus, especially A. glauciae (male SVL = 27.6–30.4 mm ) and A. lutzi (male SVL = 27.5–33.5 mm ). Adenomera guarani has a broad mid-dorsal stripe, whereas A. diptyx , A. martinezi , and A. saci have a vertebral pin stripe. Additionally, A. guarani is distinguished from A. diptyx by a mask-like pattern in the interorbital region (an interorbital bar in A. diptyx ). Adenomera guarani has toe tips unexpanded with a tapered point (character state A), whereas the following 14 species have moderately to fully expanded toe tips (states C–D, i.e., swollen, knob-shaped toe tips and toe discs, respectively): A. ajurauna , A. amicorum , A. andreae , A. chicomendesi , A. engelsi , A. gridipappi , A. guarayo , A. heyeri , A. inopinata , A. lutzi , A. marmorata , A. nana , A. simonstuarti , and A. tapajonica . Adenomera guarani differs from A. amicorum , A. aurantiaca , A. cotuba , A. glauciae , A . gridipappi , A. inopinata , A. kayapo , A. lutzi , A. phonotriccus , and A. tapajonica by the absence of an antebrachial tubercle. The exotrophic larva of A. guarani distinguishes the new species from almost all congeners, except A. saci and A. thomei ( Almeida & Angulo 2006 ; Carvalho & Giaretta 2013b ), as well as A. diptyx and A. martinezi (assumed based on calling habitat, but their tadpoles remain unknown; Carvalho & Giaretta 2013b ; T.R. Carvalho, field obs.). The tadpole of A. guarani has a labial tooth row formula (LTRF): 2(2)/3(1), whereas that of A. thomei has a LTRF: 2(2)/3 ( Almeida & Angulo 2006 ). Adenomera guarani sp. nov. can also be distinguished from congeners in advertisement call traits. The single-note call of A. guarani differs from the multi-note calls of A. amicorum , A. aurantiaca , A. cotuba , A. glauciae , A. gridipappi , A. inopinata , and A. simonstuarti . Among Adenomera with single-note calls, call notes of A. guarani are formed by incomplete pulses, whereas those of A. guarayo and A. phonotriccus are formed by complete pulses, and those of A. ajurauna , A. bokermanni , A. engelsi , A. kweti , A. lutzi , A. marmorata , A. nana , and A. saci are nonpulsed. From congeners also having single-note calls formed by incomplete pulses, A. guarani differs from A. andreae (24–68 per minute), A. araucaria (23–46 per minute), A. chicomendesi (5–29 per minute), A. heyeri (22–27 per minute), A. juikitam (30–52 per minute), A. kayapo (23–33 per minute), A. tapajonica (52–59 per minute), and A. thomei (10–23 per minute) by a higher note repetition rate (73–147 per minute; Table 3 ), and by a lower note rate than that of A. diptyx (176–299 per minute; Table 3 ). Adenomera guarani differs from A. coca (10–15; Angulo & Reichle 2008 ) and A. martinezi (15–21; Carvalho & Giaretta, 2013a ) by a lower pulse number per note (4–9; Table 3 ). Adenomera guarani cannot be consistently distinguished from the allopatric A. hylaedactyla in any of the analyzed call traits ( Carvalho et al. 2019a ). Still, these two species are segregated by distinct breeding habitats and reproductive modes. Adenomera guarani , endemic to the Humid Chaco and Pantanal Wetlands, has exotrophic larvae, whereas the Amazonian A. hylaedactyla has endotrophic larvae (Kokubum & Souza 2008; Carvalho et al. 2019a ). This is the most reliable character in the discrimination of the two species, in addition to their phylogenetic relationships not sharing an immediate common ancestor ( Fig. 2 ). Adenomera guarani is the sister taxon of A. diptyx , from which the new species is distinguished by morphological and acoustic traits. Variation ( Figs 10–12 ) Measurements of males and females are shown in Table 2 . Most specimens show small, white-tipped tubercles on the dorsal surface of the shank, heel, and the sole of the foot, but absent on the dorsal surface of thigh; tubercle density is variable and usually visible under magnification. The supratympanic fold in some specimens is barely visible (may be due to preservation methods). Paracloacal glands generally developed well, but sometimes only a colored spot. Axillary glands are conspicuous in recently fixed specimens ( Fig. 12B ). Postcommissural gland is variable in size and form. Inner metacarpal tubercle is ⅓ to ⅔ smaller than the outer. Palmar tubercles are variable in number (one or two by finger), varying even between the hands of the same specimen. Dorsal and lateral glandular rows have varying degrees of development (i.e., the number of individual glands and their extent along the body length). When well-developed (as observed in most specimens), the dorsal row extends from behind the eye and the lateral row runs from the posterodorsal region of the tympanum, almost overlapping with the supratympanic fold. Some specimens also have additional small glands posteriorly, sometimes a pair highlighting as a lumbar gland, variable in size and dark colored ( Fig. 12A ). A great variation of color patterns is exhibited in living Adenomera guarani sp. nov. ( Figs 10–11 ). Dorsal coloration is variable in shades of light to dark brown; even in the same individual exposed to different conditions of luminosity. Sometimes the broad mid-dorsal stripe is poorly evident ( Figs 10C, F–G , 11M ) and the mask pattern on the interorbital region is easier to distinguish in preserved specimens. Dark marks on the posterior surface of the dorsum variable in shape and number. The coloration of the glandular rows is homogeneous (dorsal row dark and lateral row light). The medial region of the paracloacal gland is always light-colored, but its borders bear dispersed dark-colored marks of variable size and shape. Color pattern on legs is variable from well-defined cross-bands to a splash of small and dark marks. In life, the ventral surface is usually whitish, but one individual (UNNEC 8357) showed a ventral coloration with black stains. Sexual dimorphism is evident. The snout of males in lateral view is shovel-shaped, with a well-developed, fleshy white ridge; in females, the snout is rounded and without ridges ( Fig. 12C–D ). Aside from the snout shape, nostrils are closer to the tip of the snout in females than in males. Only males have axillary glands. Paracloacal glands are more well-developed in males than in females (smaller or represented only by a pair of pale, small spots ( Fig. 12E–F ). Vocal sac in males with dark lateral sides ( Fig. 12E ). In life, the gular region of females is white ( Fig. 12H ). Advertisement call ( Fig. 7 , Table 3 ) We characterized the advertisement call of A. guarani sp. nov. based on 23 males from Argentina and Brazil (N = 399 quantified calls and 2423 quantified pulses; see Appendix 2 for locality data and voucher specimens). The call consists of single notes given at a repetition rate of 73 to 147 per minute (102± 18). Note duration varies from 45 to 98 (66± 11) ms, and the rise time is 1 to 59 % of note duration (10 ± 12). Notes are formed by 4 to 9 (6 ±1) incomplete pulses (i.e., not separated by silent gaps) emitted at a repetition rate of 60 to 146 per second (106 ± 16). The dominant frequency coincides with the second harmonic, ranging from 3984 to 4974 (4329 ± 165) Hz. Notes can have a modest or pronounced frequency modulation, either positive or negative, ranging from -345 to 603 (150± 112) Hz. Natural history Data about reproductive aspects from Brazilian and Argentinean populations were published by Zaracho & Kokubum (2017) under the name A. diptyx . In summary, Adenomera guarani sp. nov. lay eggs in a foam nest inside an underground chamber with the entrance partially closed, constructed by males near temporary pools and seasonally flooded areas. When the breeding microhabitat is flooded, embryos/tadpoles are washed out of the underground chamber and complete their development in the water. The larvae have functional mouthparts and a labial tooth row LTRF: 2(2)/3(1) ( Zaracho & Kokubum 2017 : fig. 2C). Diet and parasitism from Argentinean populations were provided also by Zaracho & Lamas (2008) and Zaracho et al. (2012a) , both under the name A. diptyx . Chromosome number The chromosome number 2n = 26 of A. guarani sp. nov. from Argentinean populations was reported by Zaracho & Hernando (2011) under the name of Adenomera diptyx .

Diagnosis Adenomera guarani sp. nov. is recognized within Adenomera by the following combination of character states: (1) body robust, (2) medium size (adult male SVL = 20.1–26.4 mm ); (3) light-colored, broad mid-dorsal stripe with diffuse borders, extending from above the vent to the scapular region; (4) a dark mask-like pattern formed by an inverted triangle in the interorbital region; (5) toe tips unexpanded with tapered point; (6) antebrachial tubercle absent; (7) exotrophic larvae; (8) labial tooth row formula (LTRF): 2(2)/3(1); (9) single-note advertisement call; (10) call notes formed by incomplete pulses; (11) relatively high note repetition rate (73–147 per minute); and (12) low pulse number (4–9 per note).