Description of Uniphylus gen. nov., a new genus of Carcharodini (Lepidoptera, Hesperiidae, Pyrginae) for Staphylus evemerus Godman & Salvin

: Staphylus evemerus Godman & Salvin, 1896 is a species with a unique set of morphological characters within Carcharodini. Also, mitochondrial cytochrome oxidase subunit 1 (COI) sequences analysis demonstrated a large genetic distance with other related genera of the tribe. Therefore, this paper aims to describe a new genus for this species, which is named as Uniphylus gen. nov. Besides the morphological redescription of the male of Uniphylus evemerus (Godman & Salvin, 1896) new comb., the description of the female is provided for the first time, as well as an updated distributional map with all records known so far for this species.


INTRODUCTION
Carcharodini is a tribe of Pyrginae (Hesperiidae, Lepidoptera) that includes approximately 300 species distributed in 35 genera, four of which are present in the Old World (Carcharodus Hübner, [1819], Spialia Swinhoe, 1912, Gomalia Moore, 1879and Muschampia Tutt, 1906), while the others occur in the New World regions (Warren et al. 2009, Cong et al. 2019, Li et al. 2019, Zhang et al. 2020a).Carcharodini is a sister-group of Pyrgini, with the divergence time occurring in the Eocene around 42 Mya, together with the other two tribes of Pyrginae, Achlyodini and Erynnini (Li et al. 2019).Though the monophyly of Carcharodini is strongly recovered in molecular analysis performed in above cited studies, no morphological synapomorphy has been recognized so far.Despite this, Warren et al. (2009) partially characterized the males of Carcharodini to possess an asymmetrical pattern in the genitalia morphology, known as "Nisoniades-type" valva (sensu Evans 1953).Most of the female genitalia of Carcharodini, as it is for many other groups of Lepidoptera, are not described.However, Siewert et al. (2016) illustrated and described the female genitalia of Sophista as asymmetrical as well.Concerning the immature stages, Carcharodini larval foodplants include a variety of dicots, although Cyclosemia herennius (Stoll, 1782) is the only New World Carcharodini known to feed on monocots (Warren et al. 2009).
When studying some Carcharodini species, it was noted that the Central American species Staphylus evemerus Godman & Salvin, 1896 is morphologically distinct from other species of Staphylus Godman & Salvin, 1896, with larger wings, forewings with inner margin concave and a unique male and female genitalia pattern.The morphological combination of characters of S. evemerus also does not fit into any of the known Carcharodini genera.To test a generic taxonomical hypothesis for S. evemerus, COIsequences of this species and 20 other of Staphylus were obtained.A large genetic distance was observed between S. evemerus and other Staphylus, corroborating the recognition of a new genus.Therefore, the aim of this paper is to describe a new Carcharodini genus to include S. evemerus, and to provide additional taxonomical notes on this species.

MATERIALS AND METHODS
The material used in this study is from the following public and private collections: DZUP -Coleção Entomológica Pe.Jesus de Santiago Moure, Universidade Federal do Paraná, Curitiba, Paraná, Brazil; MGCL -McGuire Center for Lepidoptera and Biodiversity, Florida Museum of Natural History, University of Florida, Gainesville, Florida, United States; NHMUK -The Natural History Museum, London, United Kingdom; OM -Olaf Hermann Hendrik Mielke collection, Curitiba, Paraná, Brazil.Eight specimens of Uniphylus evemerus new comb.were found in these institutions.
For the molecular analysis, two legs (meso and metathoracic from the same side) from 22 skippers of the internal group and related Carcharodini genera (Table I) were detached and sent to the Canadian Center for DNA Barcoding (CCDB) for DNA extraction, amplification, and sequencing of the mitochondrial cytochrome oxidase subunit 1 (COI) using standardized methods (Hebert et al. 2003).The obtained sequences were included in GenBank with accession numbers available in table I.These specimens were identified through analysis of their genitalia and comparison with original descriptions and other important taxonomical studies, such as Evans (1953) andSteinhauser (1989).COI sequences available in GenBank (NCBI 2020) of other Carcharodini species were also included in the analysis (Janzen et al. 2011, Pratt et al. 2015, Lavinia et al. 2017, Pfeiler 2018, Attiná et al. 2021, Earl et al. 2021, Santos et al. 2022).The chosen species for the analysis were selected based on the recent phylogenetic hypothesis for Carcharodini (Cong et al. 2019 andZhang et al. 2022a).For this analysis, a total of 42 specimens from 39 species belonging to ten Carcharodini genera were used (Table I).
The molecular alignment for COI sequences was performed using the software Muscle in MEGA7 (Kumar et al. 2016).The pairwise genetic distances (%) was also calculated in MEGA7, and the frequency distribution of genetic divergence was plotted using pairwise values.The optimal model of nucleotide evolution was selected by ModelFinder (Kalyaanamoorthy et al. 2017) using the AICc criterion, resulting in GTR+F+I+G4.Maximum likelihood analysis was conducted using the software IQ-Tree (Nguyen et al. 2015) implemented in the PhyloSuit (Zhang et al. 2020c).For the Maximum Likelihood analysis, the ultra-fast bootstrap approximation method -UFBoot (5,000 bootstrap repetitions) was used (Minh et al. 2013), together with the SH-aLRT test (1,000 repetitions) (Guindon et al. 2010).The tree obtained was visualized and initially edited in the software FigTree v. 1.4.3 (Rambaut 20061.4.3 (Rambaut -2009)), and later in the software Inkscape 1.0.1 (Harrington et al. 2004(Harrington et al. -2005)).The tree was rooted at Viola minor (Hayward, 1933).Nodes were considered supported when both SH-aLRT ≥ 80 and UFBoot ≥ 95.
Abdomens of the studied specimens were detached and soaked in a heated 10% potassium hydroxide solution (KOH) for 5-10 minutes, and posteriorly dissected for the study of the genitalia.Dissected genitalia were stored in vials with glycerin, labeled accordingly, and photographed using a photo stacking process associated with a Leica® Application Suite Version 4.12.0 stereomicroscope.In the "examined material" section, the symbol "*" indicates that the genitalia of the specimens were dissected and analyzed.The general terminology follows Evans (1953), and the genitalia morphology follows Carneiro et al. (2013).The following abbreviations were used throughout the paper: DW (dorsal wing), FW (forewing), DFW (dorsal forewing), VFW (ventral forewing), HW (hindwing), DHW (dorsal hindwing), and VHW (ventral hindwing).
A taxonomical catalogue including all publications that mention the species is provided, following Mielke (2005).The full references cited in the catalogue are available in Lamas (2023).A distributional map was produced with SimpleMappr (Shorthouse 2010) from the label data of the specimens analyzed and literature records, consulting the geographic coordinates of the specific locality on Google Earth.In the cases where no specific locality was given, coordinates from the central region of the state were used.

Molecular data
A Maximum Likelihood analysis using the mitochondrial gene COI supported the monophyly of Uniphylus gen.nov.(ultrafast bootstrap support [ubs] = 100; SH-aLRT support = 100) (Fig. 1).The Uniphylus gen.nov.clade is sister to Staphylus, and these two forms a clade with Pholisora (Fig. 1).Interspecific genetic distance ranged from 9% to 14% (Supplementary Material -Table SI, Fig. 2); the genetic distance between Uniphylus gen.nov.and Staphylus was 10.9%, and between Uniphylus gen.nov.and Pholisora was 11%.Based on the sequences of the mitochondrial gene COI, in conjunct with the morphological differences, these results support the description of new genus to include Staphylus evemerus.predominantly on the internal margin.Fringe brown.Abdomen: Brown with scarce yellow scales dorsally and ventrally.Genitalia: Tegumen slightly longer than wide, with one central constricted region, proximal portion rounded at the proximal margin and larger than the distal margin.Ventral arms of the tegumen narrow and fused with dorsal projection of saccus, assuming that the boundaries between these structures are located at the angle between them.Saccus triangular, short, rounded proximally.Uncus conical-shaped, enlarged at the base, and ventrally with a large concavity that becomes a longitudinal opening towards the narrower apex, bearing some short hair-like scales dorsally.Gnathos developed as two short, sclerotized plates bearing fine microtrichia, fused ventrally by a thinner region bearing microtrichia.Valva longer than wide; sacculus slightly longer than wide, rectangle-shaped, as a fold into the valva; harpe about five times longer than wide, except at the base where it surpasses the dorsal margin of ampulla as a narrow conical process curved outwardly; the distal part of the harpe as a finger-like structure slightly curved upwardly, with upper margin subtly indented; ampulla very narrow, with upper margin concave; costa very thin following the general shape of the valve.Sacculus, harpe, and ampulla bear thin setae throughout their extensions, internally and externally.Aedeagus cylindrical, short, about half the length of valva; without spines and vesica without cornuti.Fultura superior absent.Fultura inferior developed, thin, crescent moonshaped in anterior view.

Taxonomy
Female description (Figs.4c-d, 7).FW length.1.5 cm (n = 1).HW length.1.1 cm (n = 1).Genitalia.Tergum VIII quadrate with anterior apophysis; papilla analis somewhat quadrate, covered by setae and posterior apophysis slightly curved, longer than the length of papilla analis.Lamella antevaginalis as a very sclerotized plate, quadrate in ventral view, with an excavation in the middle of the distal margin, and another larger excavation in the proximal margin in the region where presumably is the ostium bursae.Presence of a slender membranous area separating the lamella antevaginalis from the lamella postvaginalis.Lamella postvaginalis as a narrow plate, with the distal margin and proximal margin nearly straight, bearing microtrichia.Ductus bursae membranous, about three times longer than corpus bursae.Corpus bursae rounded, membranous, about ¼ the length of ductus bursae.
The valva of the male genitalia is probably the most remarkable difference between U. evemerus new comb., and the other genera.While U. evemerus new comb.has a harpe developed as a finger-like structure, providing a unique aspect to the genitalia, in the other genera the valva is rectangular or ovoid (Fig. 8b, d, f, h).Regarding to the female genitalia, U. evemerus is the only Carcharodini species known to possess a developed anterior apophysis in the tergum VIII (Fig. 7b).

Etymology
The name Uniphylus is derived from two words: unicus, from the Latin for "only", an allusion to the unique morphology found in the type species of the genus, especially on the male genitalia morphology; and phylus from Staphylus, where the type species was included thus far.It is a noun in the genitive singular.The gender is masculine.   . 34.-DeVries, 1983, in Janzen. Costa Rican Nat. Hist., p. 676.-Bridges, 1988. Cat. Hesp. 1, p. 65;2, p. 57.-Bridges, 1994. Cat. Fam.-Group, Gen.-Group, Sp. Group SI Taxonomic history.Godman & Salvin (1896) described Staphylus evemerus.The species was transferred by Mabille (1903) to Bolla Mabille, 1903, andafter Shepard (1935) transferred it to Pholisora Scudder, 1872.Evans (1953) returned the species to Staphylus, and other authors followed this decision, mentioning the species in taxonomic comments, faunistic studies, and catalogs.

Uniphylus evemerus (Godman
Type.Staphylus evemerus Godman & Salvin, 1896 was described based on an unspecified number of specimens from Caché, Rio Sucio, Irazu (Costa Rica), collected by H. Rogers, and deposited in the Godman & Salvin's collection.A male specimen was found in the NHMUK (Fig. 3ac

Diagnosis and description
Same as the diagnosis and description of the genus.

Temporal distribution
Uniphylus evemerus (Godman & Salvin, 1896) new comb.has been recorded in the following months: February, June, July, and December.

DISCUSSION
Many genera of brown skippers, such as the Carcharodini, have been historically neglected in systematics studies.This is probably due to their lack of charismatic features that are found in other families of butterflies.It is not uncommon that taxonomic studies of Hesperiidae result in the description of new species and the reallocation of known species into different genera (e.g.Cong et al. 2019, Medeiros et al. 2019, 2020, Siewert et al. 2016, 2020, Zhang et al. 2022a, Lemes et al. 2023).
The use of genome analysis is revolutionizing the systematic studies of Hesperiidae (Cong et al. 2019, Zhang et al. 2022a, b).Ideally, sampling many loci for many taxa would be the best solution, however, this practice is still unsustainable for most research laboratories due to the expensive costs of the protocols (Talavera et al. 2021).Despite this, COI analyses are corroborating phylogenomic findings (Cong et al. 2019), showing utility in recovering higher level relationships.
The COI-tree presented here demonstrated that U. evemerus new comb.forms a sistergroup with Staphylus, and these two genera a larger clade including Pholisora (Fig. 1).However, it is important to emphasize that the inclusion of more species and more genes can change the relationships among clades.For example, Staphylus, with the new combination of S. evemerus (Godman & Salvin, 1896) in Uniphylus, includes now 47 species (Mielke 2005, Cong et al. 2019, Zhang et al. 2022a, b, Lemes et al. 2023), of which only 22 had their COI sequences analyzed in this study.Also, the inclusion of sequences of more species would likely increase the support values for the inner nodes on the COI-tree (Fig. 1).
In fact, the nuclear genome of Carcharodini suggests that Staphylus is a sister group with Hesperopsis, and Bolla is a sister group with Pholisora (Cong et al. 2019).The COI-tree of these same authors, on the other hand, suggests that Staphylus together with Hesperopsis forms a clade sister group with Bolla, which is recovered as paraphyletic, inside a larger clade including Pholisora.Therefore, the use of an integrative approach, using as much taxonomical tools as possible, is fundamental to understand the systematics of challenge groups, such as the "brown Carcharodini".Herein, it has been demonstrated that through molecular and morphology analysis, Uniphylus gen.nov. is supported as a strong taxonomic hypothesis.
The genetic distance of COI of U. evemerus new comb.compared to other Carcharodini genera is similar with the distance among genera in the tribe, ranging from 9% to 14% (Table SI).According to Cong et al. (2019), the genetic distance between clades of Hesperiidae genera is typically more than 10% in the COI.
Uniphylus evemerus (Godman & Salvin, 1896) new comb.has a unique combination of morphological characters that separates it from other genera.Godman & Salvin (1896) highlighted the distinctiveness of the male genitalia when compared to other Staphylus species, although these authors considered the species outwardly similar.Despite this, the strongest similarity between the wings of U. evemerus new.comb.with other Staphylus species is the brown ground color, present in most of the other genera of Carcharodini.
The concave inner margin of the FW of Uniphylus evemerus new comb.is morphologically closer to the FW of Incisus species, particularly I. incisus (Mabille, 1878) (Fig. 8e).In fact, Evans (1953, pages 95-96) had put these two species as closer species in his taxonomical key.However, the male and female genitalia pattern of U. evemerus new comb.is different than those of Incisus species (Lemes et al. in prep.).While males of Incisus species have a broader valva, U. evemerus new comb.has a narrow valva.In fact, U. evemerus new comb.has a unique genitalia pattern within Carcharodini.The very short aedeagus without spines or cornuti is another important characteristic.Although some studies recovered the male genitalia of some Carcharodini species as the "Nisoniades-type" valva (see Evans 1953, Warren et al. 2009, Siewert et al. 2016), this is not the case in any species of Bolla, Pholisora, Staphylus and Incisus, as well as it is not present in U. evemerus new comb.(Fig. 8b, d, f, h, j).
For the females, the lamella postvaginalis as a narrow plate and the lamella antivaginalis as a very sclerotized quadrate plate with an excavation in the middle of the distal margin, represent very different characteristics when compared to the same genitalia structures in other Staphylus, Incisus, and Bolla species (Mielke 1975, 1980, Steinhauser 1989, 1991, Steinhauser & Austin 1993, Austin 1997, Austin & Warren 2002, Lemes et al. in prep.).The presence of an anterior apophysis of U. evemerus new comb.is until now unknown from other females of Carcharodini.
Uniphylus gen.nov. is so far a monotypic genus, but the study of other genera of Carcharodini is encouraged, as this practice may reveal more species to be included in the genus.It is possible that the low number of specimens found in the visited collections is related to the drab wings ground color and the small size of Carcharodini species, which may be less interesting among Lepidoptera collectors, as suggested by Zacca et al. (2020).Fortunately, this Pyrginae tribe has recently received more attention by researchers, and other studies are currently in preparation to better elucidate their taxonomic diversity (Lemes et al. in prep.).

Figure 1 .
Figure 1.Relationship among Uniphylus evemerus (Godman & Salvin, 1896) new comb.and other genera of Carcharodini based on DNA barcode sequences obtained by Maximum likelihood inferred with IQTREE.Support values were considered sufficient when SH-aLRT ≥ 80 and UFBoot ≥ 95 at the same time ("*" represents sufficient support values).

Figure 9 .
Figure 9. Larva of the same individual of Uniphylus evemerus (Godman & Salvin, 1896) new comb.in the last instar (a, b, c).Note the presence of the parasitoid larvae of the microgastrine braconid Apanteles duniagarciae Fernández-Triana, 2014 visible through the sides of the caterpillar (see arrows in figure a).Also, note the cocoons of the parasitoid larvae that have emerged from the caterpillar (see arrows in figure c).Costa Rica, Guanacaste Province, Area de Conservacion Guanacaste, 27.VI.1997,Ruth Franco leg., voucher 97-SRNP-1457, Area de Conservacion Guanacaste Collection.Photos by Ruth Franco.

Table I .
Sequences of mitochondrial cytochrome oxidase subunit 1 (COI) used in this study.The depositary collections of the specimens used in this study and their respective accession numbers in GenBank are cited.Sequences of Uniphylus evemerus new.comb.bold highlighted.

Table I .
Continuation.