Butterflies (Lepidoptera: Papilionoidea) of Iguaçu National Park and surrounding areas in southern Brazil: a long-term survey, with six new records for the Brazilian fauna

Abstract Iguaçu National Park is the second largest (1852.62 km²) protected area in the Atlantic Forest domain and harbors the largest area of semideciduous seasonal forest in Brazil. In this study, we present 795 subspecies and 787 species of butterflies that occur in this protected area and its surrounding areas, collected over 15 years and ten months using different non-standardized sampling methods. We also searched for additional records in the literature, entomological collections, and citizen science platforms on the internet. Among the sampled taxa, six are recorded for the first time in Brazil: Emesis orichalceus Stichel, 1916, Theope p. pakitza Hall & Harvey, 1998 (Riodinidae), Elbella v. viriditas (Skinner, 1920), Apaustus gracilis ssp. n. (Hesperiidae), Deltaya sp. n. (Nymphalidae), and Symbiopsis sp. n. (Lycaenidae). Another six are listed as endangered in lists of butterflies of conservation concern. The records for some species significantly increase previously documented distributions.

Additionally, since butterflies are good bioindicators, responding quickly to environmental changes, well-documented inventories provide the base data that allow early detection of such changes through monitoring (Freitas et al. 2003, Freitas et al. 2006, Santos et al. 2016).
The Atlantic Forest is one of the most important hotspots of biodiversity in the world, and one of the most threatened Brazilian domains, with only ~11.7% of its original vegetation cover (Ribeiro et al. 2009).Its geographic extent in latitude (3°S to 31°S), longitude (35°W to 60°W) and elevation (0 to 3000 m) (Ribeiro et al. 2009) makes it a diverse domain, with a wide range of climate regimes and environmental heterogeneity.Among all Brazilian domains, the Atlantic Forest has the most representative butterfly inventories (Santos et al. 2008, Shirai et al. 2019), and its regional richness exceeds 2100 species (Brown Jr. & Freitas 2000).Despite these results, more information to better understand patterns of butterfly geographic distributions is needed (Francini et al. 2011, Iserhard et al. 2017).
Iguaçu National Park (hereafter Iguaçu NP) is a protected area in the Atlantic Forest domain that harbors the largest preserved area of semideciduous seasonal forest in Brazil (Urban 2002).Mielke (1968) was the first to publish a butterfly list for the region, with emphasis on Hesperiidae, and recorded 106 species in the municipality of Foz do Iguaçu.Decades later, a Rapid Ecological Assessment (REA), conducted to develop a management plan in this protected area, recorded 257 species of six families of butterflies (Mielke 1998).After that, Graciotim & Morais (2016) published a list with 69 fruit-feeding nymphalid species for this region.The authors sampled using Van Someren-Rydon traps placed along two trails, which represented the two principal phytophysiognomies in the park (Urban 2002, ICMBio 2018b).With further sampling, Santos et al. (2018) updated this list to 104 fruit-feeding nymphalid species.Considering the estimated richness of 700 butterfly species for Iguaçu NP (IBAMA 1999) and the 653 species recorded in Argentina's Iguazú National Park (Núñez-Bustos 2009), it appeared that Iguaçu NP was still under sampled.The purpose of this paper is to remedy this undersampling by producing an exhaustive list resulting from more than 15 years of sampling at Iguaçu NP and its surrounding areas, supplemented by records from the literature and from entomological collections.Our data are intended to contribute both to the taxonomy and ecology of Brazilian butterflies and to be used for conservation decisions in this emblematic National Park of the Atlantic Forest in Brazil.

Study area
Iguaçu NP is a Brazilian protected area located in the state of Paraná, between the geographic coordinates of 25º05' and 25º41'S and 53º40' and 54º38'W (Datum WGS84) (Figure 1).The park area is 1852.62 km² with a 420 km perimeter.It encompasses six municipalities in the state of Paraná (Céu Azul, Capanema, Matelândia, Serranópolis do Iguaçu, São Miguel do Iguaçu and Foz do Iguaçu) and borders another four (Capitão Leônidas Marques, Lindoeste, Santa Tereza do Oeste and Santa Terezinha de Itaipu).The Iguaçu River separates Iguaçu NP in Brazil from its sister park in Argentina (Iguazú National Park).Five rivers, with their sources outside of the park, flow across the park to the Iguaçu River: Gonçalves Dias, São João, Represa Grande, Silva Jardim and Benjamin Constant.A sixth river, the Floriano River, is the only one situated almost entirely in the park.Geologically, Iguaçu NP is located on the third Paraná plateau, in the Paraná Basin.
The climate in the region is classified as humid subtropical (Cfa) according to Köppen, with mean temperatures below 18°C during the coldest month and above 22°C during the hottest month (Alvares et al. 2013).Annual precipitation varies from 1600 to 1800 mm, and the rains are concentrated mainly during hot months.The dry season is not well defined, and the frequency of frosts is low (Nitsche et al. 2019).According to Google Earth (http://earth.google.com), the elevation of the park varies from 120 m, at the Iguaçu River, to over 700 m at its northeastern extremity.The park is located in the Atlantic Forest domain and is composed of four phytophysiognomies (ICMBio 2018b): 1) the seasonal semideciduous forest (hereafter SSF), 2) the mixed ombrophilous forest (hereafter MOF), 3) the floodplain (hereafter FP), and 4) the open formations (hereafter OF).The SSF occupies 85% of the park area and has two subformations, the submontane (up to 400 m), and the montane (between 400 and 600 m).The MOF has a single subformation, the montane (above 600 m).The FP occurs in small areas, subject to extreme flooding conditions for long periods, and is generally occupied by herbaceous-shrub vegetation.The OF comprises all open vegetation formations of anthropogenic or natural origin (Urban 2002, ICMBio 2018b) (Figure 2).The surrounding areas of the park exhibit a heterogeneous landscape, characterized by forest fragments, extensive areas dedicated to agriculture and raising livestock, and urban areas (ICMBio 2018b).

Sampling
The faunal list is primarily the result of 15 years and ten months of sampling (September 2007 to July 2023) of adult butterflies by the first author, mainly using entomological nets, but in some situations, also using Van Someren-Rydon (hereafter VSR) traps and the Ahrenholz technique, which was especially useful for sampling Hesperiidae butterflies (Freitas et al. 2021b).A digital camera was also used to record some species.Some immature stages were found, which were then raised in the laboratory until emergence.
In the collection with entomological net, many trails and environments were sampled, in all four phytophysiognomies and throughout the altitudinal range (Table 1).Following Brown Jr. & Freitas (2000), special emphasis was given to sites with resources that attract butterflies, like flowering plants, plant exudates, sunny areas in the interior of the forest, mud puddles, fermented fruits, wet sand along trails or river margins, feces or urine from carnivores, and carcasses.Some sites were more intensively sampled, especially those in the municipality of Foz do Iguaçu.Field sampling was carried out at all seasons, at different times of the day, from 9:00 a.m. to 7:00 p.m., and in all microclimates or habitats within the park and in surrounding areas.
In March 2013, VSR traps baited with a mixture of fermented banana and sugarcane juice were installed on two trails in the Iguaçu NP to sample fruit-feeding butterflies, following a protocol adapted from Uehara-Prado et al. (2005).These trails represent the two principal phytophysiognomies present in the park: 1) an area of SSF, located in the municipality of Foz do Iguaçu, and 2) an area of MOF, located in the municipality of Céu Azul.On each trail, ten traps were placed in line (starting at least 50 m from the forest edge) and with about 20 m between adjacent traps, alternating understory (~1.5 m above the ground) and canopy (~10 m above the ground).The traps remained suspended for two consecutive days on each trail (10 hours of effective sampling per day), resulting in a total sampling effort of 400 trap/hours. Most

Species list
We identified the specimens using taxonomic keys and species guides (e.g., Brown Jr. 1992, Canals 2003, D'Abrera 1984, 1987, 1989, 1994, 1995, Tyler et al. 1994, Klimaitis et al. 2018).We also compared specimens to images of type specimens of American butterflies, available in the Butterflies of America database, on internet (Warren et al. 2016), and to species maintained in reference collections (DZUP/UFPR and ZUEC/Unicamp).For some individuals, we examined the genitalia and, more rarely, we used barcoding sequences to determine the species.We also consulted specialists to help us determine specific groups of butterflies (see "Acknowledgements").For recorded subspecies with sympatric distribution, we use an "x" to indicate that two phenotypes (and sometimes the putative hybrids) are present.
Besides sampling, we compiled data from the literature (Mielke 1968, Jenkins 1990, Casagrande & Mielke 1992, Anken 1994, D'Abrera 1995, Mielke 1998, Hall & Harvey 2002, Callaghan 2010, Lourido 2011, Graciotim & Morais 2016) for any of the ten municipalities in the study area, updating the nomenclature when necessary.We also examined scientific collections, where we found species from the study area that were not previously recorded for the park.These include DZUP at UFPR; two local collections in Foz do Iguaçu, the Table 1.Sampled sites in the present study and in Mielke (1998), ordered from North to South.Nearby points were treated as a single point.In the case of trails, the altitude presented is the simple arithmetic mean between its highest and lowest point.SSFs = Seasonal Semideciduous Forest, subformation submontana; SSFm = Seasonal Semideciduous Forest, subformation montana; MOF = Mixed Ombrophilous Forest; FP = Floodplain; OF = Open formations.indicates that phenotypes of two subspecies are present; superscript letters a = the first subspecies correspond to the predominant phenotype; b = very few specimens were collected to determine predominant phenotype; c = there is evidence suggesting that the two subspecies could in fact be two valid species; F = strictly fruit-feeding species (sensu Freitas et al. 2014).

Ecomuseum of Itaipu and the Museum of Zoology in the Uniamérica
Universitary Center; and two private collections, D.R. Dolibaina and A.D. Warren.We did not include species that could not be identified, as well as literature records for which we did not find voucher material or which may represent species misidentification.In addition, we searched for pictures of butterflies from any of the ten municipalities within and bordering Iguaçu NP on the citizen science platform iNaturalist (https://www.inaturalist.org),Google Images (https://www.google.com/imghp?hl=EN) and Flickr (https://www.flickr.com),but no additional records were obtained.

Species richness
The 787 species recorded at Iguaçu NP and its surrounding areas represent 22.2% of the 3549 butterfly species known in Brazil (Casagrande & Duarte 2022).Except for the family Hedylidae, which was not included in previous Papilionoidea inventories in the Atlantic Forest, the other 786 species make Iguaçu NP the richest known site in butterfly species among areas with Semideciduous Forest.The park is also among the three richest sites in butterfly species of Atlantic Forest domain, together with the municipality of Linhares, in the state of Espírito Santo (835 species) (Brown Jr. & Freitas 2000), and Itatiaia National Park, in the states of Rio de Janeiro and Minas Gerais (914 species) (Zikán & Zikán 1968).
Among the records that may be added in the future to Iguaçu NP are the taxa that have been recorded exclusively at Iguazú National Park (hereafter Iguazú NP), Yacutinga Private Reserve (hereafter Yacutinga PR) and Surucuá Private Reserve (hereafter Surucuá PR), Table 3. Species that are likely to occur in Iguaçu NP and may be included in future lists, based on records from neighboring protected areas in Argentina (Iguazú NP, Yacutinga PR and Surucuá PR) (Núñez-Bustos 2008, 2009, 2019, Klimaitis et al. 2018, Núñez-Bustos & Penco 2020, Rosa et al. 2021, Núñez-Bustos in preparation).Species richness for each family are provided within parenthesis.

Family Species
Papilionidae (1) Protesilaus telesilaus vitellus (Frühstorfer, 1907) Lycaenidae ( 9 Macrosoma hedylaria (Warren, 1894) three neighboring protected areas in Argentina (Núñez-Bustos 2008, 2009, 2019, Klimaitis et  2. Taxonomic composition Brown Jr. & Freitas (2000) suggested that Nymphalidae generally represent 25% to 29% of the total species in a well-sampled inventory in the Neotropical region.At Iguaçu NP, the Nymphalidae make up 27.2% of the butterfly fauna, in full accord with predictions.Since the Nymphalidae can be satisfactorily documented with low sampling effort and their proportion in the butterfly fauna is relatively consistent, the richness of this family is an accurate surrogate for the total butterfly species richness.Alternatively, Robbins et al. (1996) suggested that Neotropical faunas consist of approximately a third Papilionidae + Pieridae + Nymphalidae (255 species at Iguaçu NP), a third Lycaenidae + Riodinidae (192 species at Iguaçu NP), and a third Hesperiidae (339 species at Iguaçu NP).This prediction was not especially accurate at Iguaçu NP.Further, even though the proportional richness of Hesperiidae greatly exceeded a third, we consider it likely that most species to be discovered in the future will belong to this family.The reason is that they are mostly inconspicuous, small-sized and fast-flying species that make them difficult to sample (Brown Jr. 1992, Brown Jr. & Freitas 1999).
At greater detail, the higher species richness of Hesperiidae (43.1%) than Nymphalidae (27.2%) is consistent in well-detailed Neotropical inventories (Brown Jr. & Freitas 2000) and may be a good indicator of "representativity" of butterfly sampling in the region (Francini et al. 2011).Furthermore, the predominance of Lycaenidae over Riodinidae is another expected pattern in butterfly assemblages in the southern Atlantic Forest (Francini et al. 2011) and seems to be related to a positive association between Riodinidae richness and local mean temperature (Brown Jr. 2005).Assemblages in northern locations of the Atlantic Forest and in the Amazon Basin, accordingly, show greater riodinid species richness.
So far, there are no long-term butterfly inventories carried out in locations in the Atlantic Forest that include Hedylidae.Thus, we do not know the representativeness of this family in the total butterfly fauna in this domain.In the Brazilian butterfly fauna, Hedylidae represents 0.6% of the total (Lourido & Duarte 2023).Assuming that the same proportion applies to the Atlantic Forest, we could expect the occurrence of up to four additional species of this family in Iguaçu NP, suggesting that it may be underrepresented in the present inventory.
By using VSR traps, 27 species of fruit-feeding nymphalids were sampled (out of a total of 112 sampled from this guild), of which four were sampled exclusively by this method: Amphidecta pignerator simplicia Weymer, 1910, A. reynoldsi, Moneuptychia castrensis (Schaus, 1902), and Yphthimoides yphthima (Felder & Felder, [1867]).In the present study, VSR traps were used in a complementary way, and despite the low sampling effort employed in this method, it was effective in recording species that were not found in the active sampling with entomological nets.This may be related to the habits of these species (Freitas et al. 2021b).For example, among the species that were sampled exclusively by traps, A. pignerator simplicia and A. reynoldsi have crepuscular habits, while Y. yphthima was sampled in a canopy trap.
Among the recorded species, six were included in the Brazil Red Book of Threatened Species of Fauna (ICMBio 2018a) and/or in the Paraná State Red Book of Threatened Species of Fauna (Mielke & Casagrande 2004).These species are Zonia zonia diabo, Ochropyge ruficauda, Passova passova practa (Hesperiidae), Narope cyllarus (Nymphalidae), Alesa prema and Joiceya praeclarus (Riodinidae).Greve et al. (2013) suggested changing the conservation status of J. praeclarus to DD ("Data Deficient") due to the new record for Foz do Iguaçu during the inventory.This record increased the species geographic distribution, which was previously known only from the type locality, in Mato Grosso, Brazil.In the case of Passova passova practa, Dolibaina et al. (2010) suggested removing the species from the Red List of Paraná, because new records of it in the study area and the abundance of larvae on host plant suggest that the rarity of this species is an adult sampling artifact.Z. zonia diabo and J. praeclarus were recorded only in surrounding areas of Iguaçu NP, in small patches of secondary forest, which suggests that these species may be more environmentally tolerant than previously thought.However, even though these species have not yet been detected inside the Iguaçu NP, they certainly also occur there, reinforcing the importance of this protected area for its conservation, because populations that occur in small forest fragments tend to be much more unstable and subject to local extinctions by stochastic factors or anthropogenic disturbances (Brown Jr. 1992).
Six species had their known distribution significantly increased after the present study: Camissecla vesper, Ipidecla crepundia, Nesiostrymon tristis (Lycaenidae), Joiceya praeclarus, Theope p. pakitza (Riodinidae), and Eunica m. malvina (Nymphalidae).The three species of Lycaenidae had previously been recorded in Rio de Janeiro state (Duarte et al. 2010).Theope p. pakitza had been known only from a Peruvian male (Hall 1998), and its female is illustrated here for the first time (Figure 4E).In the case of Eunica m. malvina, we recorded a female in March 2016 landed on a high leaf of Luehea divaricata Mart.& Zucc.(Malvaceae) at the margin of the Iguaçu River.Otherwise, in the Atlantic Forest this species occurs in the states of Bahia, Espírito Santo, Rio de Janeiro, Minas Gerais and São Paulo Paulo (Jenkins 1990, Santos et al. 2018).In the case of the distributions previously documented for the riodinids J. praeclarus and T. p. pakitza, the increase was over 1000 km, showing that even in a well-studied group like butterflies, the Wallacean shortfall is still present.
For those species that were only found as literature records or without vouchers, these records were not included.For example, there is a citation of Caligo b. brasiliensis (C. Felder, 1862) in an unpublished study, which was mentioned in a former version of the management plan of Iguaçu NP (IBAMA 1999), but the species was not listed in the present study because the voucher specimen was not located.Also, all records for this species from iNaturalist were rejected because the available pictures did not correspond to natural observations in Foz do Iguaçu, but to individuals raised in captivity in the butterfly center of "Parque das Aves", a private institution neighboring Iguaçu NP.Furthermore, the record of Taygetis virgilia (Cramer, 1776) (Nymphalidae) cited in IBAMA (1999) was rejected because the voucher specimen could not be located at DZUP.However, there are records for this species in Iguazú National Park (Misiones, Argentina) (Núñez-Bustos 2009), so it is likely that future studies may record it in the Brazilian Park as well.Finally, four species of fruit-feeding nymphalids reported in Graciotim & Morais (2016) were also not included, because: 1) the records of Taygetis sylvia Bates, 1866 and Yphthimoides ochracea (Butler, 1867) are possible misidentifications; and 2) based on nomenclatural updates (Espeland et al. 2023), Hermeuptychia hermes (Fabricius, 1775) and H. fallax (Felder & Felder, 1862) probably refer to H. atalanta (Butler, 1867) and H. gisella (Hayward, 1957), respectively.

Conclusion
Iguaçu NP and surrounding areas exhibit a heterogenous butterfly fauna with elements from different regions.Its species richness is high for a subtropical zone distant from the coast.Besides the three undescribed taxa recorded in this study, the number of other taxa originally described from specimens collected in the region, both in the Brazilian and Argentinian parks, is remarkable.Some examples are Ochropyge ruficauda, Atlides misma D'Abrera, 1995, Caeruleuptychia helena (Anken, 1994), and Agara pardalina yacutinga (Mielke & Casagrande, 2011).Furthermore, several unexpected records suggest that Iguaçu NP is a fertile field for new findings, such as the rediscoveries of Joiceya praeclarus and T. p. pakitza.The new records of these species represent a significant expansion in their geographic distributions, since J. praeclarus was known only from state of Mato Grosso in Brazil (over 1000 km) and had not been observed for 80 years, while T. p. pakitza was known only from Manú National Park in Peru (over 2000 km).
We recommend that future studies focus sampling efforts in MOF areas and their ecotone with SSF, which are the most preserved and least explored areas due to difficult accessibility.Considering the groups with high potential to represent new records for the park and its surroundings, we suggest that further studies aim at extensive collections using VSR traps for fruit-feeding Nymphalidae, the Ahrenholz technique for Hesperiidae, and light traps for mostly nocturnal Hedylidae.Due to the seasonality and short flight period of adults of certain species, some univoltine, these samplings must cover all months of the year, including during winter, in order to increase the chance of their detection.

Figure 1 .
Figure 1.Map of Iguaçu National Park (in dark green) and its location in Brazil, with the main sampling sites; yellow circles = previous surveys by Mielke (1998); red triangles = sites surveyed in the present study.
Hesperiidae were deposited at the Entomological Collection Padre Jesus Santiago Moure in the Federal University of Paraná (DZUP/UFPR), and specimens of the other families were deposited at the Zoological Collection of the Museu de Diversidade Biológica in the University of Campinas (ZUEC/Unicamp).Some specimens of the genus Actinote were deposited at the Entomological Collection of the Museu de Zoologia e Paleontologia in the Federal University of Recôncavo da Bahia (MURB/UFRB).

Figure 2 .
Figure 2. Landscapes and environments at Iguaçu National Park: A) semideciduous seasonal forest, 200 m; B) mixed ombrophilous forest, over 700 m, with the presence of several individuals of Brazilian pine Araucaria angustifolia (Bertol.)Kuntze (Araucariaceae); C) stony beach on the banks of the Iguaçu River; D) Blue River; E) a dense patch of tree ferns (Cyatheaceae spp.);F) close view of forest understory with young juçara palm Euterpe edulis Mart.(Arecaceae).

Table 2 .
List of the butterflies and moth-butterflies (Lepidoptera: Papilionoidea) of Iguaçu National Park and surrounding areas.Species richness for each major taxa are provided within parentheses.* Taxa recorded by the first author solely through photographs of live specimens; ** records from collections or literature (see "Data Availability" for more details); # new records for Brazil; the "x"
(Rosa et al. 2021ustos & Penco 2020, Núñez- Bustos in preparation).Together, these protected areas in Argentina and the Iguaçu NP in Brazil, form a practically continuous forest, composed by the same vegetal formation (except by MOF, which does not occur in the Argentinian parks), separated by the Iguaçu River.As such, it is reasonable to expect that these areas share a similar fauna.At least 77 species have been recorded only in the Argentinian parks, but all are likely to occur at Iguaçu NP.These records include 1 Agrias claudina claudianus Staudinger,[1885]) in February 1987 at the Argentinian Falls, so it is possible that this species occurs in the Brazilian park as well.Among other species with possible occurrence in Iguaçu NP, we cite the Papilionidae Pterourus cleotas (Gray, 1832) and the Nymphalidae Splendeuptychia tupinamba Freitas, Huertas & Rosa 2021.P. cleotas has specimens deposited in DZUP/UFPR from Cascavel, a municipality with approximately the same elevation and a few kilometers from Santa Tereza do Oeste, in the Northern border of Iguaçu NP.The species S. tupinamba was recently described and recorded at Yacutinga PR (Misiones, Argentina)(Rosa et al. 2021). http://www.scielo.br/bnhttps://doi.org/10.1590/1676-0611-BN-2023-1487