Notes on the distribution of the genus Pseudopaludicola Miranda-Ribeiro , 1926 ( Anura : Leptodactylidae ) in Paraguay

Four species of Pseudopaludicola, Pseudopalud­ icola boliviana, P. falcipes, P. mystacalis and P. ternetzi, are usually cited for Paraguay. However, after analyzing 407 specimens assigned to this genus in herpetological collections of the country, we conclude that there are no specimens of P. falcipes in Paraguayan collections or vouchers cited in the literature, and almost all individuals referred to P. ternetzi are most probably P. ameghini. At the same time, a recently described species, P. motorz­ inho, is recorded for the first time in the country. Information on the distribution of these and the remaining species of Pseudopaludicola in Paraguay (P. boliviana and P. mystacalis) is provided.


INTRODUCTION
Pseudopaludicola Miranda Ribeiro, 1926 currently contains 21 species, which occur mainly in the lowlands of Argentina, Bolivia, Brazil, Colombia, Guyana, Suriname, Peru, Paraguay, Uruguay and Venezuela (Frost 2016). The genus includes very small (<30 mm snoutvent length), cryptic and highly polychromatic species (Lynch 1971;Haddad and Cardoso 1987;Pansonato et al. 2013;Carvalho et al. 2015aCarvalho et al. , 2015b. The genus is diagnosed by the presence of a hypertrophied antebrachial tubercle, epicoracoid cartilages slightly (or not) superposed, posterolateral process of the hyoid outlined or absent (Lynch 1989;Lobo 1995), and by ribosomal RNA sequences . The conservative morphology of the taxa leads to the use of acoustic, chromosomal and molecular data to address taxonomic studies Carvalho et al. 2015aCarvalho et al. , 2015b.
In Paraguay, four species of Pseudopaludicola are usually reported: P. falcipes (Hensel, 1867), P. mystacalis (Cope, 1887), P. boliviana Parker, 1927 andP. ternetzi Miranda-Ribeiro, 1937 (see Weiler et al. 2013 and references therein). The occurrence of P. falcipes in Paraguay is widely cited in the literature (Freiberg 1942;Cei 1980;Gallardo 1987;Langone 1994;Aquino et al. 1996;Brusquetti and Lavilla 2006;Lavilla and Brusquetti 2010;Weiler et al. 2013 andMotte et al. 2015). More recently, the distribution of this species in Paraguay was challenged by Langone et al. (2015), based on the absence of material of this species from Paraguay in some collections of Argentina, Brazil and Uruguay. However, these authors did not analyze material from Paraguayan collections. In turn, Pseudopaludicola ternetzi was cited by Lobo (1995) from the departments of Amambay, Concepción, Misiones, Caaguazú, Central, and San Pedro, including under this name the putative sample cited by McDiarmid and Foster (1987) as P. ameghini. Brusquetti and Lavilla (2006), extended the distribution of P. ternetzi to the departments of Boquerón and Presidente Hayes. However, the revalidation of Pseudopaludicola ameghini (Cope, 1887) from the synonymy of P. ternetzi by Pansonato et al. (2013) generates doubts about the identification of these Paraguayan populations. Of the remaining two species cited for the country, Pseudopaludicola mystacalis was reported only from Itapúa Department by Lobo (1995), and Pseudopaludicola boliviana was first cited for Asunción (capital district) by Parker (1935). Later, Lobo (1994b)  Tongue with small pigments at the base. Slender body, antebrachial tubercle developed; small, conical tubercle on the outer edge of heels. Toes slightly dilated distally, with T-shaped finger tips (Parker 1927;Lobo 1995, Cardozo andSuarez 2012). In addition, the presence of minute tubercles on upper eyelids (suggested by Lynch 1989) is variable ( Figure 2). 2n = 20 chromosomes (Cardozo et al. 2016).
one of them, Pseudopaludicola motorzinho Pansonato, Veiga-Menoncello, Mudrek, Jansen, Recco-Pimentel, Martins & Strüssmann, 2016, with distribution records in the states of Mato Grosso and Mato Grosso do Sul (Brazil), and in the department Santa Cruz (Bolivia), near the Paraguay border, so we consider its presence possible in Paraguay. Faced with these expectations (the absence of P. falcipes and the presence of P. motorzinho in the Paraguayan batrachofauna), the main goals of this contribution are to provide an update of the distribution of the genus Pseudopaludicola in Paraguay, to investigate the presence or absence of P. falcipes in the country, and to reanalyze all the available material assigned to Pseudopaludicola in Paraguayan collections.

MATERIALS AND METHODS
We analyzed all the specimens of the genus Pseudo paludicola housed at Museo Nacional de Historia Natural del Paraguay (MNHNP, San Lorenzo), in the herpetological collection of the Instituto de Investigación Biológica del Paraguay (IIBP-H, Asunción) and in the Colección Zoológica of the Facultad de Ciencias Exactas y Naturales (FACEN), Universidad Nacional de Asunción (CZCEN, San Lorenzo). The identifications were corroborated following the descriptions, diagnoses and redescriptions of each species (Hensel 1867;Cope 1887;Parker 1927;Miranda-Ribeiro 1937;Lobo 1994aLobo , 1995Lobo , 1996Pansonato et al. 2013Pansonato et al. , 2016. Localities contained in museum catalogs were georeferenced using gazetteers (Paynter 1989;Anonymous 1992), GPS records from collectors, estimations from maps and the information in Brusquetti and Lavilla (2006). We generated the map showing the distribution of Pseudopaludicola species in Paraguay using ArcGIS version 10.4. For ecoregions, we follow Dinerstein et al. (1995).   Pseudopaludicola motorzinho is morphologically cryptic in relation to P. boliviana, and the difference suggested by Pansonato et al. (2016) (absence of tubercles on upper eyelid) is not diagnostic (Figure 2). The identification of P. motorzinho from Estancia Pirá Potrero in Amambay Department was based on 13 specimens with 2n = 22 chromosomes, distinguishable from P. boliviana, whose karyotype is 2n = 20 chromosomes (Fávero et al 2011, as Pseudopaludicola sp.;Cardozo et al. 2016). (Cope, 1887) Slender body, smaller (SVL 10 to 17 mm) than P. ternetzi (Lobo 1996). Fingertips knobbed, head slightly elongated in ventral view. When hind leg is adpressed, heel goes beyond the eye, near the nostril. Dorsal coloration in preserved specimens brown, with few darker spots; some specimens with a vertebral line and two pale dorsolateral lines (Cope 1887;Lobo 1996), ventral coloration variable from immaculate to densely spotted. 2n = 16 chromosomes (Cardozo et al. 2016).

DISCUSSION
Although Pseudopaludicola falcipes has been the most cited species for the country, as noted above, no specimen of Pseudopaludicola with an absent or incomplete abdominal fold was recorded in any of the Paraguayan collections, which supports the suggestion of Langone et al. (2015) that this species does not occur in Paraguay. Furthermore, the specimen of P. falcipes from Bahía Negra (Motte et al. 2015), previously identified as P. boliviana by Brusquetti and Lavilla (2006) and Langone et al. (2015), was tentatively re-identified as P. motorzinho, while those referred by Brusquetti and Lavilla (2006) were re-identified mostly as P. mystacalis, plus nine specimens as P. boliviana and one as P. ameghini; three individuals from Boquerón and one from Alto Paraguay were re-identified as juveniles of a species of Physalaemus.
Pseudopaludicola ameghini was found in the departments of Amambay, Concepción, Misiones and Presidente Hayes, in transitional areas between Humid Chaco, Cerrado and Atlantic Forest ecoregions ( Figure 3B). The only record from Misiones (MNHNP 1209), isolated from the main distribution of the species, is doubtful and probably is a curatorial error in the transcription of collection data. The species is also distributed in Chapada dos Guimarães and Vila Bela da Santíssima Trindade, Mato Grosso, Brazil (Pansonato et al. 2013) and probably is in adjacent Bolivia (Frost 2016).
Based on the absence of a vertebral line in the examined specimens, we assigned the Paraguayan specimens, previously Pseudopaludicola ternetzi, to P. ameghini. In fact, the line, which might be present in specimens of P. ternetzi, is the only externally visible character suggested by Pansonato et al. (2013) to distinguish P. ameghini from P. ternetzi. However, as P. ameghini and P. ternetzi share similar external morphologies, advertisement calls with overlapping ranges (Pansonato et al. 2013;Cardozo and Toledo 2013), and chromosome number 2n=20 (Fávero et al. 2011;Cardozo et al. 2016, as P. ternetzi), we cannot discard the possibility that some of these specimens could be in fact P. ternetzi.
Pseudopaludicola boliviana is the most widely distributed species of the genus in the country ( Figure  3B), mainly in the Humid Chaco ecoregion, but is also   Motte et al. (2015) and those from Laguna General Díaz (MNHNP 3826-3827, 8217) are assignable to this species. Pseudopaludicola boliviana and P. motorzinho probably inhabit in syntopy in a wide area of their distribution, and using only the morphological features mentioned by Pansonato et al. (2016), it is almost impossible to assign specimens to either species. In Paraguay, P. motorzinho is probably associated with Chaco Seco and Cerrado ecoregions. Pseudopaludicola mystacalis is distributed in southern Paraguay (Humid Chaco), in the departments of Misiones and Ñeembucú, and western Itapúa ( Figure  3B). The species was first cited for Paraguay by Lobo (1995), based on individuals in the National Museum of Natural History, Smithsonian Institution (USNM 253526 to USNM 253528), this reference followed by Brusquetti and Lavilla (2006) (not included in Figure  3, because we were unable to examine the specimens). The general distribution of P. mystacalis also includes Argentina (Santa Fé and Entre Ríos provinces; Alcalde and Williams 2004), Bolivia (Beni and Santa Cruz departments; De la Riva et al. 2002) and Brazil (São Paulo, Goiás, Mato Grosso, Mato Grosso do Sul, Piauí, Maranhão and Pará states; Pansonato et al. 2014).