Herpetology Notes, volume 11: 565-572 (2018) (published online on 29 July 2018)

Helminth communities of Pithecopus nordestinus (Anura: ) in forest remnants, Brazil

Priscila Almeida de Sena1,4, Breno Moura Conceição2, Paula Fonseca Silva2, Winny Gomes O Silva1, Wagner Berenguel Ferreira2, Valdemiro Amaro da Silva Júnior3, Geraldo Jorge Barbosa de Moura2,4,5 and Jaqueline Bianque de Oliveira1,4,5,*

Abstract. The helminth parasite communities of anurans are determined by environmental and host factors. For to describe the parasites and to investigate the host and environmental factors that can influence the composition and structure of the helminth fauna were studied 72 specimens of Pithecopus nordestinus (Caramaschi 2006) collected in two different areas in northeastern region of Brazil: Estação Ecológica do Tapacurá (EET), a remnant of Atlantic Rainforest in Pernambuco state (n=39), and Floresta Nacional do Araripe (FLONA), a remnant of Altitudinal Forest in Ceará state (n=33). Helminths of six taxa (five nematodes and one acanthocephalan) were identified, being the nematodes the most prevalent helminths (Cosmocercidae gen. sp., Cosmocerca parva, Cosmocercella phyllomedusae, Oxyascaris caudacutus and Rhabdias sp.), followed by acanthocephalans (Centrorhynchus sp.). The prevalence and the mean intensity of infection were higher in the from FLONA. Body size and sex of hosts did not influence the abundance of helminths, but the parasites abundance was different between the two study sites, being higher in FLONA than in EET. The two studied populations of P. nordestinus presented a higher parasite richness when compared to other studies with species from the Phyllomedusidae family, and the abundance of helminths was different between the two study areas, demonstrating that local characteristics as well as the host ecology and diet are crucial in host-parasite relationship in forested environments. The knowledge about the composition and structure of helminth fauna is important for the conservation of anurans and ecosystems where they live.

Keywords: anurans, parasites, Nematoda, Acanthocephala, Atlantic Rainforest, Altitudinal Forest, conservation

Introduction are excellent models for the parasite-host relationships studies, since they occupy a variety of 1 Laboratório de Parasitologia, Universidade Federal Rural de habitats, have different patterns of life cycle, different Pernambuco, Rua Dom Manoel de Medeiros SN, CEP 52171- reproductive strategies and occupy various positions in 900, Recife, Pernambuco, Brazil food webs (Koprivnikar et al., 2012). Although Brazil 2 Laboratório de Estudos Herpetológicos e Paleoherpetológicos, has the most diverse anurofauna (Segalla et al., 2014), Universidade Federal Rural de Pernambuco, Rua Dom Manoel studies on the helminth fauna associated with these de Medeiros SN, CEP 52171-900, Recife, Pernambuco, hosts are still scarce (Santos and Amato 2010a) and, Brazil 3 Laboratório de Patologia, Universidade Federal Rural de most of them, restricted to taxonomic descriptions and/ Pernambuco, Rua Dom Manoel de Medeiros SN, CEP 52171- or records of occurrence (Pinhão et al., 2009). 900, Recife, Pernambuco, Brazil The parasites may act as opportunistic or as primary 4 Programa de Pós-Graduação em Ciência Tropical, agents of diseases in anurans (Koprivnikar et al., 2012), Universidade Federal Rural de Pernambuco, Rua Dom Manoel influencing growth and reproduction rates (Campião de Medeiros SN, CEP 52171-900, Recife, Pernambuco, et al., 2009). Furthermore, they are considered as Brazil 5 bioindicators of environmental change, providing Programa de Pós-Graduação em Ecologia, Universidade Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros information on the ecosystems health and structure of SN, CEP 52171-900, Recife, Pernambuco, Brazil the food web (Campião et al., 2009; Koprivnikar et al., 6 Programa de Educação Tutorial (PET-Biologia), Universidade 2012). The body size, age, sex, diet and host behavior are Federal Rural de Pernambuco, Rua Dom Manoel de Medeiros important variables in the composition and structure of SN, CEP 52171-900, Recife, Pernambuco, Brazil parasite communities of anurans (Hamann et al., 2009; * Corresponding author. E-email: [email protected] 566 Priscila Almeida de Sena et al.

Santos and Amato 2010a; Toledo et al., 2013, 2015; specimens of P. nordestinus that were evaluated, Campião et al., 2014a, 2016), which act as definitive, which were deposited in the Coleção Herpetológica e intermediate and paratenic hosts of a wide variety of Paleoherpetológica of the Universidade Federal Rural helminths (Santos and Amato 2010a; Toledo et al., de Pernambuco: 39 were collected in September 2014 2013, 2015; Campião et al., 2009, 2014a, 2014b). Body in the Estação Ecológica do Tapacurá - EET, in the size is a determining factor for the composition of the municipality of São Lourenço da Mata, Pernambuco anuran parasite community, due to the greater amount of state, and 33 were collected in July 2012 in the Floresta food eaten and added extra and intracorporeal surface, Nacional do Araripe - FLONA, in the municipality of increasing the possibilities of oral and skin infection Jardim, state of Ceará. (Hamann et al., 2009; Santos and Amato 2010a; The EET (08°03’S/ 35°10’W) is a remnant of the Campião et al., 2016). On the other hand, environmental Atlantic Rainforest, consisting of a stationary forest characteristics can influence the helminth community of semi-deciduous evergreen type (Andrade-Lima 1957), anurans (Campião et al., 2012). with a total area of approximately 800 ha, of which Pithecopus nordestinus (Caramaschi 2006) (Anura: about 400 ha are made up of forested areas, surrounded Phyllomedusidae) was described in 2006 from a by sugarcane monoculture (Azevedo-Júnior 1990). It taxonomic revision of Phitecopus hyponcondrialis has an As’ climate type with average annual rainfall of (Daudin, 1800) by Caramaschi (2006). At this time, both 1300 mm per year and six months with less than 100 species were inserted in the genus , but mm. recent phylogenetic analysis placed all the Phyllomedusa The FLONA (7°22’5.12’’S/39°20’18.83’’O), a from the hypocondrialis group in a resurrected genus remnant of Altitudinal Forest, covers an area of around named Pithecopus (Duellman et al., 2016). 38,262 ha and 720 m altitude, between the municipalities of Barbalha, Crato, Jardim and Santana do Cariri, in the This anuran is widely distributed in Atlantic Forest state of Ceará (Muniz et al.,, 2015). Located at the top of morphoclimatic areas, including Caatinga enclaves the Chapada do Araripe, with a predominantly rainforest moist forests and Caatinga shrubland (Moura et al., vegetation, but with lots of babaçu palms (Orbignya 2015). It is arboreal, “sit-and-wait” predator and phalerata Mart) which features a secondary forest. It preferably occupying the herbaceous layer, where it lays has a tropical rainy climate, with annual rainfall around its eggs in leaves located on the water depth (Moura et 1000 mm with less than 30 mm of rainfall in the driest al., 2015). As regards the conservation status, this species months. is classified as DD (data deficient) by the International Union for Conservation of Nature (IUCN 2017) and as Collection and processing of parasites.—The snout- LC (Least Concern) in the Brazilian list of endangered vent length (SVL) of each animal was recorded (average species by the Ministério de Meio Ambiente e Recursos of 3 cm SVL) and sex determination (45 males and 27 Naturais Renováveis (Portaria MMA 444/14) and in females) was performed from the view of the gonads. the state list of endangered species of the Secretaria The animals had their stomachs, intestines, liver, gall Estadual de Meio Ambiente de Pernambuco (SEMA bladders, lungs, kidneys, bladders and body cavities Resolução 01-08/01/2015). systematically examined for the presence of parasites. The only record of parasites of this species is the The acanthocephalans were stained and cleared with description of the nematode Raillietnema minor carmine and eugenol, respectively, and nematodes were Freitas and Dobbin Jr, 1961 (Vicente et al., 1991) cleared with lactophenol. Identified parasites were when this species was still considered as Phyllomedusa deposited in the Parasite Collection of the Laboratório de hypocondrialis (Campião et al., 2014b). So this is the Parasitologia (CPLAPAR) of the Universidade Federal first effort to identify the parasite fauna of P. nordestinus. Rural de Pernambuco, state of Pernambuco, Brazil. Besides this, we hypothesized that helminth communities Samples of parasitized organs and/or tissues were fixed present differences between the two study environments in buffered 10% formalin and processed by standard (Atlantic Rainforest and Altitudinal Forest) and that histological techniques. host characteristics (size and sex) play an important role Data analysis.—The prevalence, mean intensity of in structuring parasitic communities. infection and the mean abundance were calculated as proposed by Bush et al., (1997). Materials and Methods Parasites abundance data were tested for normality by Origin of animals and study sites.—Seventy-two the Kolmogorov-Smirnov test. As the data did not show Helminth communities of Pithecopus nordestinus in forest remnants, Brazil 567

Table 1. Prevalence (%), mean intensity (MI ± SD), mean abundance (MA ± SD), richness and mean richness of the helminth parasites of Pithecopus nordestinus (Caramaschi, 2006) in forest remnants of Northeastern Brazil, Estação Ecológica do Tapacurá (ETT), Pernambuco state and Floresta Nacional do Araripe (FLONA), Ceará state.

Parameters General± SD FLONA± SD ETT± SD Prevalence (%) 63.9 90.9 41.0 Mean intensity 8.2 ± 7.2 10.6 ± 9.8 5.5 ± 5.6 Mean abundance 6.9 ± 7.2 11.3 ± 9.8 3.1 ± 5.6 Richness 6 5 4 Mean richness 0.8±0.7 1.2±0.6 0.8±0.8 Males (n=45) Prevalence (%) 66.7 92.0 35.0 Mean intensity 8.1±6.9 8.7±5.6 5.9±4.3 Mean abundance 6.5±6.9 9.8±5.6 2.4±4.3 Richness 6 5 3 Mean richness 0.8±0.7 1.1±0.5 0.5±0.8 Females (n=27) Prevalence (%) 59.2 87.5 47.4 Mean intensity 8.4±11.3 12.7±12.4 5.4±4.6 Mean abundance 7.5±11.3 15.9±12.4 3.9±4.6 Richness 5 4 4 Mean richness 0.9±0.8 1.4±0.7 0.7±0.8

(SD) standard deviation

a normal distribution, the Mann-Whitney U test was the hosts were parasitized by one (43.1%) or two species used to assess differences between the total abundance (20.8%) of helminths. of parasites species and of the most abundant species In FLONA, 372 helminths were identified belonging depending on origin (FLONA and EET) and sex (male to five taxa (Table 3) and the animals were infected by and female). Spearman correlation was used to verify one (66.7%) or two species (24.2%), while in ETT were the correlation between the intensity of infection and collected 122 helminths belonging to four taxa (Table the host’s size and to check the correlation between 3) and the infected hosts showed one (23.1%) or two the parasites richness and the host size. All analyzes species (17.9%) of helminths. were performed using Statistica Software 8.0, with Cosmocercidae were the most prevalent and abundant a significance level of 0.05, according to Zar (2010) parasites, but it was not possible to identify at the genus recommendations. level due to the absence of males in the samples. The encysted cystacanths could not be identified at the Results genus level because in most of them the proboscis was A total of 494 helminths belonging to six taxa (five partially retracted. adult nematodes and one acanthocephalan cystacanth) The parasites abundance was different between the were identified in examined specimens. Data of two study sites (Mann-Whitney test, p=0.0000), being prevalence, abundance, intensity and helminth richness higher in FLONA than in EET (Table 1). However, there are indicated in table 1. was no difference of the parasitic abundance in relation The identified helminths were: Cosmocercidae gen. to sex (Mann-Whitney test, p=0.6983, n=27, 7.5±11.3 sp., cistacanths of Centrorhynchus sp. Van Cleave, 1916, for females, n=45, 6.5±6.9 for males) and no correlation Cosmocerca parva Travassos, 1925, Cosmocercella was observed between abundance and host size (n=72; phyllomedusae Baker and Vaucher, 1983, Oxyascaris Spearman, p=0.7761; r=0.034). caudacutus Baker and Vaucher, 1984 and Rhabdias Cosmocercidae gen. sp. showed a greater abundance sp. Stiles and Hassal, 1905. Data of prevalence, mean (Mann-Whitney test, p=0.0000) in FLONA than in EET intensity, mean abundance, the site of infection animal (Table 3). There was no difference in the abundance of origin of these parasites are presented in table 2. Overall, Cosmocercidae in relation to sex (Mann-Whitney test, 568 Priscila Almeida de Sena et al.

Table 2. Number of parasites collected (N), prevalence (%), mean intensity (MI±SD), mean abundance (MA±SD), the site of infection (SI) animal origin (AO) and amplitude (A) of the helminth parasites of Pithecopus nordestinus (Caramaschi, 2006) in forest remnants of Northeastern Brazil, Estação Ecológica do Tapacurá (ETT), Pernambuco state and Floresta Nacional do Araripe (FLONA), Ceará state.  3DUDVLWHV 1 3 0,“6' 0$“6' 6, $2   $FDQWKRFHSKDOD        &HQWURUK\QFKLGDH9DQ&OHDYH   “ “ VWRPDFK ((7   1HPDWRGD        &RVPRFHUFLGDHJHQVS   “ “ VPDOODQGODUJH ((7DQG  LQWHVWLQH )/21$   &RVPRFHUFDSDUYD7UDYDVVRV   “ “ VPDOODQGODUJH ((7DQG  LQWHVWLQH )/21$  &RVPRFHUFHOODSK\OORPHGXVDH%DFKHU 9DXFKHU   “ “ ODUJHLQWHVWLQH ((7DQG  )/21$   2[\DVFDULVFDXGDFXWXV%DNHU 9DXFKHU   “ “ VWRPDFKDQG ((7DQG  VPDOOLQWHVWLQH )/21$  5KDEGLDVVS6WLOHV +DVVDO     ERG\FDYLW\ )/21$   7RWDO        6' VWDQGDUGGHYLDWLRQ       

p=0.2592, n=27, 4.7±9.6 for females, n=45, 4.9±6.2 and Hamann 2008; Hamann et al., 2009; Pinhão et al., for males). The Cosmocercidae abundance was also 2009; Campião et al., 2012; Toledo et al., 2015). In this not associated with the host size (n=72; Spearman, work, the abundance of parasites was not associated p=0.9032; r= -0014). with host size and showed no difference in relation to The comparison of P. nordestinus parasitic richness the sex of the studied animals, which was also reported with other species of the family Phyllomedusidae in by Santos and Amato (2010a) for Rhinella fernandezae South America (Table 4) showed that P. nordestinus has Gallardo, 1957. This may be a result of the analyzed a greater helminths richness. type series due to its small variation in SVL, making None of the parasitized animals presented macroscopic it impossible to register any mathematical relationship lesions associated with parasites and histopathological between the data. Besides this, anurans immunity should analysis revealed no inflammatory reactions in the be considered as a factor that can limit the parasitic organs and/or tissues parasitized. richness and abundance of parasites (Koprivnikar et al., 2012). The relationship between body size and the Discussion richness and abundance of parasites was demonstrated The helminth fauna associated with P. nordestinus in in Leptodactylus latrans (Steffen, 1815) (Toledo et al., Atlantic Rainforest and Altitudinal Forest is composed 2015), L. podicipinus (Cope, 1862) and L. chaquensis of generalist species, with the exception of Cos. Cei, 1950 (Campião et al., 2009, 2016). phyllomedusae, as registered other studies with anurans The populations of P. nordestinus of this study in South America (Pinhão et al., 2009; Santos and Amato showed a higher parasite richness when compared to 2010a; Toledo et al., 2013; Campião et al., 2014b). The other studies with species from the Phyllomedusidae parasitism by Cos. phyllomedusae has been recorded family, despite the variation in the number of specimens only in P. hypocondrialis (Baker, 1987), P. palliata, P. evaluated, which might be associated with geographic tomopterna and P. vaillanti (Bursey et al., 2001). Several differences (Pinhão et al., 2009; Toledo et al., 2015) intrinsic aspects to the anurans are very important to and biomes (Koprivnikar et al., 2012). Although Aho determining the parasitic richness, favoring the success (1990) highlights that the parasitic richness itself is of transmission by monoxenous and heteroxenous not a determining parameter to infer the environmental parasites (Campião et al., 2016) founded in this study. conditions, the study of helminth components enables The abundance of parasites was higher in animals from the understanding of the correlation between the FLONA, which can be attributed to the factors specific parasites, their hosts and the environment (Poulin, 2007; to the locality (Aho 1990; Kehr et al., 2000; González Campião et al., 2012; Koprivnikar et al., 2012). Local Helminth communities of Pithecopus nordestinus in forest remnants, Brazil 569

Table 3. Number of parasites collected (N), frequency of occurrence (F), prevalence (%), mean intensity (MI ± SD) and mean abundance (MA ± SD) of the helminth parasites of Pithecopus nordestinus (Caramaschi, 2006) in forest remnants of Northeastern Brazil, Estação Ecológica do Tapacurá (ETT), Pernambuco state and Floresta Nacional do Araripe (FLONA), Ceará state.

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factors (high anuran richness, size and conservation behavior (low specificity of host) of the identified status of the fragment) and intrinsic ecology of the host nematodes (Anderson, 2000). can influence the parasite richness of anurans (Aho, Arboreal anurans have low vagility and their foraging 1990; Kehr et al., 2000; González and Hamann 2008; strategy sit and wait type reduces the possibility of Hamann et al., 2009; Pinhão et al., 2009; Schotthoefer et infection by nematodes (Aho, 1990), whose infective al., 2011; Campião et al., 2012; Koprivnikar et al., 2012; stages develop in the soil, such as Cosmocercidae Toledo et al., 2015; Campião et al., 2016), which may (including C. parva e Cos. phyllomedusae), Oxyascaris explain the higher richness and prevalence of helminths sp. e Rhabdias sp. (Anderson, 2000), which we identified in animals from FLONA than in EET. in this study. Nematodes of the Cosmocercidae family Pithecopus nordestinus is an arboreal species, which had the highest prevalence, mean intensity and mean primarily occupies the lower vertical stratum of the abundance, which has also been reported in studies forest (Moura et al., 2015), allowing greater interaction with L. podicipinus (Campião et al., 2009) and R. with the community of terrestrial winged and arboreal fernandezae (Santos and Amato 2010a), corroborating prey. Like most anurans, P. nordestinus is generalist the generalist behavior of these nematodes (Bursey et and opportunistic “sit-and-wait” predator and their diet al., 2001). Perhaps, the contact with the ground (even consists mainly of different groups of insects (Filho et in short periods) and the intake of its particles at the al., 2015) and arachnids (Santos et al., 2004; Caldas et time of foraging may favor infection by these parasites. al., 2015), which enables the heteroxenous helminth Unfortunately, only few Cosmocercidae could be infections, such as acanthocephalans which were identified (C. parva and Cos. phyllomedusae) due to the founded in the present study. absence of males in the samples, which was also reported According to Aho (1990), parasitism by nematodes in other studies in Brazil (Pinhão et al.,, 2009; Campião in arboreal anurans is infrequent, contrary to what et al., 2012; Toledo et al., 2013). This makes it difficult was observed in this study, where the nematodes were to know the true parasite richness of P. nordestinus, the most common helminths. Nematodes are also the pointing to the need for the use of molecular biology most common in all families of anurans that have been tools to help identify this large group of parasites. studied in South America (Campião et al., 2014b). A Cosmocercella phyllomedusae is considered a greater richness of nematodes may be related both to the specific parasite of arboreal anurans of Phyllomedusa monoxenous cycle of most species as with the generalist genus along with Cos. minor and Neocosmocercella 570 Priscila Almeida de Sena et al.

Table 4. Helminth species richness in species of Phyllomedusidae from South America.

Host N Richness Locality Reference Pithecopus nordestinus (Caramaschi, 2006) 72 6 Brazil This study Phyllomedusa atelopoides Duellman, Cadle, 3 2 Peru Bursey et al. (2001) and Cannatella, 1988 Phyllomedusa azurea Cope, 1862 36 1 Argentina Lunaschi and Drago (2010) Phyllomedusa burmeisteri Boulenger, 1882 NR 1 Brazil Vicente et al. (1991) Pithecopus hypocondrialis (Cope, 1862) 1 1 Paraguay McAllister et al. (2010a) (formely Phyllomedusa hypocondrialis) Pithecopus hypocondrialis (Cope, 1862) 1 1 Guiana McAllister et al. (2010b) Pithecopus hypocondrialis (Cope, 1862) NR 1 Brazil Freitas and Dobbin (1961) Phyllomedusa palliata Peters, 1873 5 3 Peru Bursey et al. (2001) (Peters, 1873) Phyllomedusa tomopterna Boulenger, 1882 7 4 Peru Bursey et al. (2001) Phyllomedusa vaillanti Boulenger, 1882 7 3 Peru Bursey et al. (2001) (N) sample number; (NR) not registered

paraguayaensis, and was recorded in P. hypocondrialis, in EET animals may be related to local differences in the P. palliata, P. tomopterna and P vaillanti in Paraguay components of helminth communities or hosts ecological and Peru (Baker and Vaucher, 1983; Bursey et al., 2001; characteristics such as aggregation of the population Mcallister et al., 2010a). This is therefore the first record and differences in consumer items of diet (González of Cos. phyllomedusae in Brazil and P. nordestinus is and Hamann, 2008; Hamann et al., 2009; Pinhão et recorded as a new host to this nematode. al., 2009). The prey identity, and their occurrence, may Besides of Cosmocercidae, Centrorhynchus sp. was be determinant for parasitism by acanthocephalans in also the most prevalent helminth. There is a great arboreal anurans, as suggested in this study. variation in the characteristics of the proboscis of the The parasitism by cystacanths has been recorded in genus Centrorhynchus, which makes identification several species of in South America (Torres and difficult (Moravec and Kaiser, 1995; Smales, 2007; Puga, 1996; Goldberg et al., 2009; Santos and Amato, Santos and Amato, 2010b). Studies with experimental 2010b; Campião et al., 2014a, 2014b). The presence infection and/or molecular biology could be used for the of encysted larvae in tissues indicates that these specific identification of cystacanths. Acanthocephalan acanthocephalans use P. nordestinus as paratenic host to adults are found in the intestine of vertebrates and reach its definitive host, probably birds (Falconiformes use terrestrial arthropods (Orthoptera, Isopoda and and Strigiformes) (Anderson, 2000; Smales, 2007; Coleoptera) as intermediate hosts to complete their Santos and Amato, 2010a, 2010b). Five species of cycle (Smales 2007; Santos and Amato 2010b). This Strigiformes and two Falconiformes species have been helminth was recorded only in EET animals, with recorded in EET (Lyra-Neves et al., 2012). Regarding high prevalence, mean intensity and mean abundance, the family Phyllomedusidae, unidentified cystacanths similar to that registered in Scinax nasicus Köhler of acanthocephalan and Centrorhynchus sp. have been and Böhme, 1996 (Hamann et al., 2009) and R. reported in Pithecopus palliates (Peters, 1873) and fernandezae (Santos and Amato, 2010a) associated Phyllomedusa sauvagii Boulenger, 1882 in Peru and to the predominant consumption of Coleoptera. The Paraguay, respectively (Bursey et al., 2001; Campião et parasitism by Centrorhynchus sp. can be associated to the al., 2014b). So this is the first record of P. nordestinus as consumption of Orthoptera and Coleoptera, which were a paratenic host for Centrorhynchus sp. found in the stomach contents of the infected animals The parasites may act as opportunistic or primary of this area (unpublished data). Although Orthoptera disease agents (Koprivnikar et al., 2012). Although insects have been found in the stomach contents of the there are reports that the infection by Rhabdias sp. animals from FLONA (unpublished data), parasitism by determine inflammatory processes, resulting in changes acanthocephalans was not detected. in the growth rate of several species (Koprivnikar et Differences in the results related to the acanthocephalans al., 2012), there is no information about the pathogenic Helminth communities of Pithecopus nordestinus in forest remnants, Brazil 571 potential of helminths identified in this study. This is a Campião, K.M., Delatorre, M., Rodrigues, R.B., Silva, R.J., knowledge gap that must be filled for the conservation Ferreira, V.L. (2012): The effect of local environmental variables of biodiversity and ecosystems (Daszak et al., 2000). on the helminth parasite communities of the pointedbelly frog Leptodactylus podicipinus from ponds in the Pantanal Wetlands. The results of this study show for the first time the Journal of Parasitology 98: 229-235. composition and structure of the helminth fauna of P. Campião, K.M., Silva, R.J., and V.L. Ferreira. (2014a): Helminth nordestinus. The abundance of parasites was different parasite communities of allopatric populations of the frog between the two study sites, demonstrating that local Leptodactylus podicipinus from Pantanal, Brazil. 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Accepted by Anamarija Zagar