A new species of Pseudopaludicola (Anura, Leiuperinae) from Espírito Santo, Brazil

Species Description

Synonyms. Pseudopaludicola aff. falcipes Almeida, Gasparini & Peloso (2011): 548 (listed) and Gasparini (2012): 15 (listed).

Holotype. CFBH 37715, an adult male collected on February 17, 2009 near Fazenda Jacuhy, municipality of Serra (20°13′41.4″S, 40°20′3.6″W, datum WGS 84; 2 m above sea level [asl]), Espírito Santo State, Brazil, by J.L. Gasparini and R.R. Zorzal (Figs. 1– 2).

Paratypes. CFBH 10817, 10821, 10823, 10825, 10827–8, 10832–3, 37679, 37718 (females); CFBH 10818, 10820, 10826, 10830–1, 10834–5, 37714–7, 37719 (males); CFBH 10819, 10822, 10824, 10829, 10836–7 (juveniles); and CFBH-T 2222–3; 6676–9 (adults preserved in 100% ethanol), with same data as holotype; LGE 20329 (male) collected by D. Cardozo and J.L. Gasparini on December 11 2017; 20330, 20332, 20334, 20336 (males), 20331, 20333, 20335 (females), 20337–8 (two adults males preserved in 100% ethanol) collected by D. Cardozo, J.L. Gasparini, and E. Gasparini on December 13, 2017. CFBH 39228–32 (males) collected in Restinga de Praia das Neves, municipality of Presidente Kennedy (21°14′41.53″S, 40°58′45.80″W; 6 m asl), Espírito Santo State, Brazil, by J.L. Gasparini and CFB Haddad; LGE 20340–1 (two adult males preserved in 100% ethanol) collected at Restinga de Praia das Neves, municipality of Presidente Kennedy (21°14′41.53″S, 40°58′45.80″W; 6 m asl), Espírito Santo State, Brazil, by D. Cardozo, J.L. Gasparini, and E. Gasparini on December 14, 2017.

Referred specimens. Four specimens collected near Parque Estadual Paulo César Vinha, Setiba, municipality of Guarapari (20°36′23.01″S, 40°25′13.27″W; 3 m asl), Espírito Santo State, Brasil: CFBH 4112 (female) collected on March 16, 2002 by J.L. Gasparini and R.C. Bianchi; CFBH 9967 (female), CFBH 9968 (male), collected on April 18, 2004 by A.F. Béda, P. Landgref Filho, and M. Uetanabaro; CFBH 33358 (female) collected on April 3, 2005 by J.L. Gasparini, P.L.V. Peloso, and H. Moura.

Diagnosis. The new species is assigned to Pseudopaludicola by its phylogenetic position and by the presence of a tubercle on the forearm, anterolateral processes of the hyoid absent, posterolateral processes of hyoid reduced, and epicoracoid cartilages slightly superposed. The new species is characterized by: (1) SVL 11.7–14.6 mm in males, 14.0–16.7 mm in females; (2) body slender; (3) fingertips knobbed with a central groove; (4) hindlimbs short; (5) abdominal fold complete; (6) arytenoid cartilages wide; (7) prepollex with base and two segments; (8) prehallux with base and one segment; (9) frontoparietal fontanelle partially exposed; (10) advertisement call with one note, composed of two isolated pulses per call; (11) dominant frequency ranging from 4380–4884 Hz; 12) diploid chromosome number of 22; and 13) Ag-NORs on 8q subterminal.

Comparison with other species. Pseudopaludicola restinga sp. nov. can be distinguished from all other recognized species of Pseudopaludicola by a combination of external morphological, osteological, acoustic, chromosomal characters, and 16S rDNA sequences.

The smaller SVL of males (11.7–14.6; n = 21, Table 2) in P. restinga sp. nov. clearly separates it from P. ameghini (14.1–19.3, Pansonato et al., 2013), P. giarettai (16.2–18.0, Carvalho, 2012), and P. ternetzi (16.0–18.6, Lobo, 1996). In addition, the slender body of P. restinga sp. nov. differentiates this species from P. ameghini and P. ternetzi that have robust bodies (Lobo, 1995; Lobo, 1996; Pansonato et al., 2013), and also from P. mineira which has a more globular aspect (Lobo, 1994; Lobo, 1995).

The knobbed toe tips with central grooves separate P. restinga sp. nov. from the species of the P. pusilla group (P. boliviana, P. ceratophyes, P. llanera, P. motorzinho, and P. pusilla), which have T-shaped toe tips (Lynch, 1989; Lobo, 1995; Cardozo & Suárez, 2012; Pansonato et al., 2016).

The presence of a complete abdominal fold differentiates P. restinga sp. nov. from P. falcipes, in which the abdominal fold is incomplete or absent (Lobo, 1994; Lobo, 1995), while the absence of tubercles on the eyelids separates the new species from P. hyleaustralis, which has minute tubercles on the eyelids (Pansonato et al., 2012).

When the hind limb is stretched anteriorly, the tibio-tarsal articulation reaches the anterior border of the eye in P. restinga sp. nov., whereas in P. jaredi, P. murundu, and P. saltica the tibio-tarsal articulation extends past the tip of the snout (Lobo, 1994; Lobo, 1995; Toledo, 2010; Andrade et al., 2016).

The vocal sac in males, with dispersed or aggregated small spots (see variation), separates P. restinga sp. nov. from P. atragula (areolate vocal sac with dark reticulation; Pansonato et al., 2014a). In addition, the developed vocal sac with a central fold (Fig. 2B) separates P. restinga sp. nov. from P. ibisoroca, which has a poorly developed vocal sac without folds (Pansonato et al., 2016).

The wide larynx, with arytenoid cartilages encompassing almost the entire region between the posteromedial processes of the hyoid (similar to P. mystacalis), separates P. restinga sp. nov. from P. ameghini, P. canga, P. ceratophyes, P. falcipes, P. giarettai, P. llanera, P. mineira, P. motorzinho, P. murundu, P. parnaiba, P. pocoto, P. pusilla, P. saltica, and P. ternetzi, in which the larynx is composed of two small oblong shell-shaped arytenoid cartilages that encompass less than half of the space between the posteromedial processes of the hyoid (for comparison see Fig. 1B, Lobo, 1995; Fig. 3C, Roberto, Cardozo & Ávila, 2013).

The advertisement call, composed of pulsed notes, separates P. restinga sp. nov. from all species with non-pulsed notes: P. canga (Giaretta & Kokubum, 2003), P. facureae (Andrade & Carvalho, 2013), P. giarettai (Carvalho, 2012), P. hyleaustralis (Pansonato et al., 2012), and P. parnaiba (Roberto, Cardozo & Ávila, 2013)). The call with isolated pulses, separates P. restinga sp. nov. from P. atragula, P. boliviana, P. ibisoroca, P. motorzinho and P. mystacalis, which have advertisement calls with concatenated pulses (Márquez, De la Riva & Bosch, 1995; Duré et al., 2004; Pansonato et al., 2013; Pansonato et al., 2014a; Pansonato et al., 2014b; Pansonato et al., 2016). In addition, P. restinga sp. nov. differs from the other species with isolated pulsed notes by having a lower number of pulses per note (two pulses per note): P. ameghini (three to six pulses per note; Pansonato et al., 2013), P. atragula (nine to 36 pulses per note; Pansonato et al., 2013); P. pocoto (three pulses per note; Magalhães et al., 2014); and P. ternetzi (three to six pulses per note; Cardozo & Toledo, 2013). Likewise, the variable advertisement calls of P. murundu (two to six pulses per note; Toledo et al., 2010), P. jaredi (two to seven pulses per note; Andrade et al., 2016), and P. saltica (one to four pulses per note; Pansonato et al., 2013) contrast with P. restinga sp. nov., in which the call is always composed of two pulses per note.

A karyotype with 2n = 22 chromosomes separates P. restinga sp. nov. from: P. ameghini, P. boliviana, P. ternetzi (2n = 20); P. atragula, P. canga, P. facureae, P. ibisoroca (2n = 18); and P. mystacalis (2n = 16) (Cardozo et al., 2016 and citations therein).

From the other species with 2n = 22 chromosomes, the Ag-NORs on 8q subterminal separates P. restinga sp. nov. from P. falcipes (8q pericentromeric; Fávero et al., 2011; Cardozo et al., 2016); P. mineira (4p pericentromeric; Duarte et al., 2010); and P. motorzinho (8q intersticial; Fávero et al., 2011), while the absence of sex chromosomes in P. restinga sp. nov. distinguishes it from P. saltica (XX/XY chromosome sex determination; Duarte et al., 2010). The new species shares the 2n = 22 diploid number and the location of NORs with P. jaredi, P. murundu, and P. saltica. However, Pair 8 in P. jaredi, P. murundu, and P. saltica are telocentric, FN = 42 (Duarte et al., 2010; Andrade et al., 2016), while in P. restinga sp. nov. the Pair 8 is subtelocentric; FN = 44.

Description of holotype. Head longer than wide, CI = 1.14. Snout acuminate in dorsal view, protruding beyond the jaw in lateral view. Nostrils nearer to the tip of the snout than to the eyes. Internarial distance slightly less than interorbital distance (Table 2). Vocal sac developed and with a central fold. Tympanum not visible. Undifferentiated canthus rostralis, loreal region flat. Tongue entire, oval, posteriorly free, base unpigmented. Premaxillary and maxillary teeth present. Body slender, skin smooth, with scarce small and flattened glandular warts on the flanks. An X-shaped glandular fold is present in the interscapular region. Ventral region whitish, with small groups of dark spots on the throat, abdomen, and thigh. Abdominal fold complete. Antebrachial tubercle evident at the lateral margin of the forearms. Inner and outer metacarpal tubercles elongated and rounded, respectively. Nuptial pad light brown, covering the inner region of the inner metacarpal tubercle. Length of fingers III < II < IV < V; fingers with developed distal subarticular tubercles; fingertips knobbed and with a central groove. Arm speckled dorsally. Hindlimb with striped dorsal pattern and two dark bands. Inner surface of thighs with light brown background color finely spotted with black. Inner metatarsal tubercle well developed, curved distally, outer metatarsal tubercle conical and perpendicular to the plane of the foot. Tarsal fold curved, with a thickening at the middle of the tarsus simulating a tubercle. Toes marginally fringed. Length of toes IV > III > V > II > I; toes with tips not expanded laterally; toe tips knobbed with central grooves.

Measurements of holotype (in millimeters). SVL 14.9; HL 4.8; HW 4.2; ED 1.7; IOD 1.3; IND 1.2; TL 7.3; THL 7.1; END 1.1; NSD 0.7; FL 7.9; LII 1.1; LIII 1.4.

Variation. The type series and the rest of the specimens identified herein as P. restinga sp. nov. exhibit little variation (Fig. 3). However, almost all specimens from Restinga de Praia das Neves possess brownish dorsolateral bands on a dark brown dorsal background, brownish thighs with dark brown crossed bands, a densely spotted belly, and an urostylar vertebral line (CFBH 39231–2) that sometimes reaches the tip of the snout (CFBH 39228). All other specimens, the urostylar vertebral line is absent and the skin of the dorsum varies from smooth to somewhat warty, with dispersed small glands (Figs. 3A–3B, 3D).

The ventral region is usually whitish with dispersed groups of tiny dark spots (in about 60% of the specimens), or dense spotted areas covering the entire venter except for the posterior region of the abdomen (Fig. 3C), a condition present in 20% of the specimens. Males have light brown nuptial pads covering most of the inner metacarpal tubercle and exceeding the size of the subarticular tubercle of Finger II (Fig. 2A).

Osteology. Skull wider than long (Figs. 4A–4B). Maxillary arch complete. Alary processes of premaxillae directed dorsally, parallel to each other (frontal view). Premaxillae bearing 11 teeth and maxillae bearing 28 curved pedicellate teeth. Nasals narrow, ovoid, with irregular inner margins and ending in a posteriorly directed point. Nasals separated from each other and not overlapping the maxilla. In dorsal view, the nasals do not overlap the sphenethmoid. Anterior margin of the sphenethmoid not reaching the middle of the choanae; posterior margin W-shaped. In dorsal view, sphenethmoid short with a thin internasal septum. Frontoparietals with irregular inner margins, leaving the frontoparietal fontanelle partially visible. Exoccipitals separated by a wide mineralized band. Optic foramen large, ovoid, and located at the junction between the prootics and the sphenethmoid (exit of cranial nerves II, IV, and VI). Oculomotor foramen narrow in diameter, located posterior to the optic foramen (cranial nerve III). Prootic foramen oval, located posterior to the optic capsule (cranial nerve V). Jugular foramen with two holes (exit of cranial nerves IX and X). Pterygoids triradiate. Posterior rami of pterygoids longer than medial rami and separated from their respective quadrates; medial rami detached from the alae of the parasphenoid and supported by cartilage in the otic region of the skull. Anterior rami separated from the palatines. Quadratojugals reduced with evident anterior processes. Parasphenoid triradiate with the tip of the cultiform process being irregular. Parasphenoid alae perpendicular to the cultriform process. Neopalatines thin, overlapping the pars fascialis of the maxillae. Prevomers small, irregularly-shaped and without teeth. Squamosals with ventral rami wider at their ventral ends; zygomatic rami shorter than otic rami and with well-developed descending processes. Tympanic annuli cartilaginous, opened dorsally. Plectrum of uniform width. Operculum cartilaginous, rounded. Mandible with thin dentaries covering the lateral margins of Meckel’s cartilages. Inner part of Meckel’s cartilages covered by respective angular bones. Articular regions of the lower jaw cartilaginous, dentaries plain, without denticle-like structures.

Hyoid plate cartilaginous and with a mineralized median strip (Fig. 5A). Anteromedial processes of the hyoid short and divergent. Anterolateral process absent. Posterolateral processes barely developed. Posteromedial processes with cartilaginous ends united to the cricoid cartilage by ligaments. The larynx encompasses almost the entire region between the posteromedial processes of the hyoid.

Arytenoid cartilages of the larynx develop as two oblong shells. The cricoid cartilage forms a complete ring (Figs. 5B–5C) with short and thin bronchial process; undeveloped esophageal process; well-developed muscular processes; and poorly developed cardiac and articular processes.

Vertebral column with eight procoelous, non-imbricate presacral vertebrae (Fig. 6A). Cotylar facets of the atlas separated (type I of Lynch, 1971). Atlas body widest among the vertebrae. In dorsal view, the anterior margin of the atlas contacts Presacral II through a wide neural process. Anterior portion of the illium articulates with the ventral surface of the sacral diapophyses. Pubis cartilaginous. Sacral diapophyses narrow and not expanded. Urostyle with a well-developed dorsal spine, exit of spinal nerve X not visible. Urostylar articulation bicondylar.

Pectoral girdle arciferal (Fig. 6B), with the left epicoracoid slightly superimposed on the right, in ventral view. Clavicle thin, posterior margin concave, separated from the acromial region of the scapula by an anterior projection of the procoracoid. Scapula with poorly developed pars acromialis and pars glenoidalis. Anterior process of the suprascapula present. Omosternum mineralized, with slightly expanded terminal end. Sternum simple, not bifurcated, with osseous mesosternum and irregularly expanded cartilaginous xiphisternum.

Humerus with evident deltoid crest. Radius-ulna fused, leaving a narrow groove between them beginning at the union of the carpal elements and extending for one third of the length of both bones. Carpus composed of five elements: radiale, ulnare, Y-element, Distal Carpal V–IV–III, and Distal Carpal II. Metacarpals III, IV, and V, articulating with Distal Carpal V–IV–III; Metacarpal II with Distal Carpal II; base of prepollex articulating with the Y-element and Distal Carpal II. Prepollex with one osseous base and two mineralized segments. Phalangeal formula: 2–2–3–3. Toe tips knobbed with central groove (Fig. 6C).

Hindlimbs with the tibia-fibula fused at their distal ends. Tarsus composed of centrale, Distal Tarsal I, and Distal Tarsal II–III. Distal Tarsal I is the smallest and articulates with the other two elements. The centrale articulates with the prehallux base and Metatarsal I. Distal Tarsal II–III articulating with Metatarsals II and III, in contact with Metatarsal IV. Metatarsals IV and V articulate directly with the astragalus. The prehallux has one osseous base and one cartilaginous segment. Phalangeal formula: 2-2-3-4-3. Toe tips knobbed with central groove (Fig. 7D).

In the specimen examined, there are some osseous lateral projections at the anterior extremities of the metacarpal epiphysis and some phalanges. In the forelimb, in dorsal view, there is a small rounded sesamoid bone over Distal Carpal V–IV–III, embedded in the tendon of the muscle carpi ulnaris. In ventral view, the palmar sesamoid, embedded in the m. digitorum longus, has an irregular quadrangular shape. Glide sesamoids are also present in the flexor tendons at the junction of the proximal and medial phalanges of all fingers. Hind limb with osseous lateral projections at the epiphysis of some metatarsals and phalanges. Sesamoid graciella (at the joint between femur-tibia fibula, embedded at the tendon of the m. gracilis major), sesamoid cartilage (in the tendon of the m. plantaris profundus) and two ovoid plantar sesamoids of similar size are in contact with the tendons that form the aponeurosis plantaris. Additionally, near the distal end of the sacral diapophyses there are a couple of elongated sesamoid bones (sesamoid of the sacral vertebrae) of similar width, partially overlapping the diapophyses.

Advertisement call. Males of Pseudopaludicola restinga sp. nov. call during both day and night from along the margins of shallow temporary ponds, usually after heavy rainfall. The advertisement call (Fig. 7) consists of a repeated sequence of single notes, which are composed of two isolated pulses without frequency modulation. The length of each call is of 47 ms (39–58 ms), emitted at intervals of 83.26 ms (64–133 ms). Mean duration of the first pulse is 4.18 ms (3–5 ms) and of the second 5.27 ms (4–7 ms); mean interpulse interval is 38.62 ms (33–50 ms); mean rate of emission is 21.3 calls/s (17.2–25.6); and mean dominant frequency is 4592.32 Hz (4380–4884 Hz).

Cytogenetic analysis. Pseudopaludicola restinga sp. nov. has a diploid number of 22 chromosomes (2n = 2x = 22; FN = 44). The pairs 1–3, 5, 7, and 9–11 are metacentric; pairs 4 and 6 submetacentric, while pair 8 is subtelocentric (Fig. 8, Table 3). The C-banding pattern is distributed on all the centromeres of the chromosome complement, at the subterminal region of the long arm pair 8 and 9 (Fig. 9B). The NORs are located on 8q subterminal, in coincidence with the usually evident secondary constrictions; in all examined specimens no heteromorphic sex chromosomes were identified with the staining techniques used.

Etymology. The word “restinga” has an unclear origin in Brazilian Portuguese, but it is the proper name of the vegetation near the sea where the new species occurs: the restingas. Apparently, “res” comes from Latin, meaning “thing” and “tinga” comes from the Tupi indigenous language, meaning “white”, probably an allusion to the clear sandy soil of this formation. Here, restinga is used as a noun in apposition.

Distribution. Pseudopaludicola restinga sp. nov. is known from six municipalities in Espírito Santo State, Brazil (Fig. 9): Serra (type locality), Guarapari, Presidente Kennedy, Vitória (Reserva Ecológica Municipal Mata Paludosa), Vila Velha (Morada Interlagos and Vale Encantado lagoon), and Itapemirim (Lagoa das Sete Pontas or Lagoa Guanandy).

Natural history notes and conservation insights. This species can be found between dunes or at the borders of lagoons in wet or flooded places, mostly after rainfall events. The individuals were found vocalizing on the mud during the day. In addition, in the rainy season males also call in the evening and night. The restingas belong to the Atlantic Forest biome, a global biodiversity “hotspot” (Myers et al., 2000). In the restingas, the plants form paludous or herbaceous forests, but the constant disturbance of this habitat, induced by man, has led to the loss of most of their original area (Rocha et al., 2003). In concordance with the economic expansion of Brazil from 2000 to 2014, multiple infrastructure projects were developed on coastal areas of Espírito Santo, particularly port activity, and oil and gas exploitation. As a consequence, multiple pristine areas have been severely affected, including Restinga de Praia das Neves. The presence of a new anuran species in the restingas along the coastal areas of Espírito Santo contributes to the knowledge of the richness of vertebrate biodiversity of this fragile ecosystem, which requires conservation policies capable of maintaining and preserving the biological heritage of the region and of the country.