Integration of molecular, bioacoustical and morphological data reveals two new cryptic species of Pelodytes (Anura, Pelodytidae) from the Iberian Peninsula Author Díaz-Rodríguez, Jesús Author Gehara, Marcelo Author Márquez, Rafael Author Vences, Miguel Author Gonçalves, Helena Author Sequeira, Fernando Author Martínez-Solano, Iñigo Author Tejedo, Miguel text Zootaxa 2017 4243 1 1 41 journal article 36287 10.11646/zootaxa.4243.1.1 3fdbeb23-61b9-41cd-a579-ed768746cb33 1175-5326 398686 CE502CF7-6F19-43A2-AD79-15DBE777E28B Pelodytes atlanticus sp. nov. ( Fig. 11 ) Identity. Populations of this species have been considered as P. punctatus until now (for instance, Sanchiz et al. 2002 ). The species was referred to as Pelodytes sp. by Crespo et al. (2008) and van de Vliet et al. (2012) and as lineage A in Díaz-Rodríguez et al. (2015) . Diagnosis. Assigned to the genus Pelodytes based on high morphological similarity to P. punctatus , the type species of the genus; vertical pupil; network of dark lines in the skin of tadpoles; tadpoles with sinistral spiraculum; and molecular phylogenetic relationships. Of similar size as P. ibericus ; mean SVL 31.4 mm (maximum 39.2 mm ) in males, 34.7 mm ( 40.4 mm ) in females. Morphologically similar to the other three western Pelodytes species, but distinguished from P. hespericus sp. nov. and P. punctatus by smaller body size ( Table 3 ; Fig. 6 ), from P. ibericus by longer limbs and fewer B notes in advertisement calls ( Figs. 6–7 ), and from P. hespericus sp. nov. by more B notes in advertisement calls ( Fig. 7 ). Furthermore, distinguished from all other Pelodytes species by concordant differences in mtDNA and nDNA sequences. Holotype . EBD 34645 ( GLA 03 -JDR2015), adult male collected by Jesús Díaz-Rodríguez on 29 January 2015 at Mindelo Ornithological Reserve , Mindelo , Porto , Portugal . Geo. coord: 41.318539, -8.737131. Paratypes . EBD 34646 ( GLA 01 -JDR 2015) and EBD 34647 ( GLA 02 -JDR 2015), adult males collected on 29 January 2015 by same collector and at same locality as holotype . ZSM 1 /2011 collected from Nives, north of Portimão ; ZSM 1201/2012, collected from Barão de São Miguel; ZSM 194 /2016, male specimen collected near Vila do Bispo , Portugal ; and vouchers from Lisbon Natural Museum, collected on 1983 by E.G. Crespo : MB 110 collected on 1983 by E.G. Crespo from Cacém ; twelve specimens from the series MB 112, collected on 21 October 1982 by E.G. Crespo from Cascais; and ten specimens from the series MB 113 collected on 27 October 1987 by E.G. Crespo at Benfica. Etymology. The specific epithet atlanticus is a genitive adjective derived from the name of Titan Atlas who, according to the ancient greek mythology, lived beyond the strait of Gibraltar and refers to the species’ exclusive distribution along the Atlantic coast of Portugal . Description of the holotype . Adult male in excellent state of preservation. Some tissue removed ventrally from right thigh for molecular analysis. Measurements: SVL, 33.0; HW, 11.6; HL, 11.0; TD, 1.9; ED, 3.6; END, 2.7; NSD, 2.6; NND, 2.3; HAL, 9.0; FORL, 20.0; HIL, 52.6; FOL, 16.6; FOTL, 25.4; TIL, 16.5. Body relatively slender; head dorsally flattened, slightly wider than longer, wider than body; snout slightly pointed in dorsal view, rounded and flattened in lateral view; nostrils directed dorsolaterally, slightly protuberant, nearer to tip of snout than to eye; canthus rostralis indistinct, straight; loreal region very slightly concave, almost flat; eye moderately large, notably protuberant dorsally; interocular distance slightly larger than horizontal eye diameter, larger than internarial and eye-nostril distance; pupil vertical; tympanum externally visible, distinct, oval, wider vertically than horizontally, its horizontal diameter being 68% of vertical eye diameter, positioned near corner of mouth; supratympanic fold distinct in its anterodorsal part running from posterior corner of eye to level of forelimb insertion; tongue free posteriorly, of truncated shape, vomerine teeth distinct, in two patches between and at the same level as choanae; choanae moderately sized, ovoid. Premaxillary and maxillary teeth present. Arms slender, webbing almost absent, very small rudiments recognizable; comparative finger length I<II<IV<III, fingers with dermal fringes. No enlarged finger disks. One large, conical subarticular tubercle present proximally on each finger; three flattened metacarpal tubercles; inner and outer metacarpal tubercles of different size (outer slightly larger than inner) and distinctly larger than central tubercle. Black nuptial pads on inner sides of first and second fingers, as one elongate patch on forearm, a second slightly bigger elongate patch on arm, and a small agglomeration on both sides of chest near axilla. Hindlimbs slender; tibiotarsal articulation reaches between eye and nostril when adpressed along body; inner metatarsal tubercle distinct but relatively small, outer metatarsal tubercle absent; proximal subarticular tubercles indistinct, remaining subarticular tubercles absent. Comparative toe length I<II<V<III<IV; third toe distinctly longer than fifth toe; toes with conspicuous dermal fringes to tip. Webbing between toes weakly expressed but clearly present and more than merely an extension of dermal fringes. Webbing formula difficult to determine due to absence of subarticular tubercles and presence of dermal fringes. Skin on the dorsal surface granular, with distinct warts; skin of throat, chest, and venter smooth except granular on posterior most part of belly; ventral surfaces of thighs and arms granular. Especially those warts that coincide with dark greenish spots on dorsum of slightly elongated shape and arranged in longitudinal rows. The anterodorsal part of supratympanic fold is continued into the inguinal region as a distinct row of small tubercles and ridges. Colour of the holotype : The skin colour is between dark gray to olive brown with drab green bands on the back and legs. The back is dotted with small round or oval prominences of olive colors. The ventral area, which is smooth or slightly rough, has a light colour from pale green to gray tones. Nuptial pads are grass green. Colour in life ( Fig. 11 ) is more clear with light greenish spots on the back and a design of a drab olive line from the eye to the insertion of anterior legs. FIGURE 11. (a,b) Dorsal and ventral views of preserved Pelodytes atlanticus sp. nov. holotype (male; specimen GLA-03). (c,d) Dorsolateral view of paratype (GLA-02) and holotype (GLA-03) in life. (e-i) dorsolateral, dorsal, and ventral view, as well as ventral view of hand and foot of male paratype ZSM 194/2016. Not to scale. Distribution. Pelodytes atlanticus is restricted to Portugal , comprising the central-eastern part of the country (Alto Alentejo), and coastal areas from Faro to Sagres in the Algarve, and from Cape of São Vicente to Cavado River, Esposende ( Crespo et al. 2008 ; Matos et al. 2010 ). The ecological range is similar to that of P. ibericus with hybrids occurring in a long and narrow contact zone between this species and P. atlanticus (van de Vliet et al. 2012 ; Díaz-Rodríguez et al. 2015 ). Natural history. Poorly known. Reproductive phenology is similar to P. ibericus and takes place early in the season, starting with the first rains at mid-autumn and extending into the winter. More detailed fieldwork is needed to determine the conservation status of populations in some regions (van de Vliet et al. 2012 ; Nunes et al. 2014 ). Given that suitable breeding habitats (i.e., temporary ponds in traditional Mediterranean farmland) are disappearing at an alarming rate in Portugal ( Beja & Alcazar 2003 ) affecting overall Mediterranean biodiversity ( Stoate et al. 2009 ), it is urgent to assess the conservation status of this species and possibly implement conservation measures. In the Natural Reserve of Paul do Boquilobo (central Portugal ), the introduction of the American crayfish ( Procambarus clarkii ) resulted in the extinction of local populations of P. atlanticus eight years later ( Cruz et al. 2008 ). Advertisement call and reproductive behavior. The call is similar to that of other western Pelodytes lineages, consisting of two types of notes ( A and B ). The number of B notes in a call is on average lower than in P. ibericus , but higher than in P. hespericus sp. nov. ( Fig. 7 ). Pargana (1998) found differences in call variables between Portuguese populations of P. atlanticus sp. nov. . from Lisboa , Alto Alentejo and Algarve, versus populations of P. ibericus from Baixo Alentejo and east Algarve. As with P. hespericus sp. nov. , call parameters were not related to the size or age of the male. Tadpole. We assessed morphological data in one tadpole in developmental stage 37 (field number ZCMV 14042 from Vila do Bispo, BL 25.9 mm , TL 49.4 mm ). The external morphology of this tadpole and one other specimen has a close similarity to those of P. punctatus , except the LTRF 4(2–4)/4(1–2) and 4(3–4)/5(1–3) ( Fig. 9c ).