The specimens were collected with various types of light traps (UV pan traps, Pennsylvania traps, and light over a white cloth) or Malaise traps as indicated in the material analyzed sections. The morphological terminology follows Quinteiro and Holzenthal (2017). Paired structures were referred to in the singular form in the descriptions. The genitalia were studied after removing the abdomens of the examined specimens and clearing them using 85% lactic acid, as outlined by Blahnik et al. (2007). The prepared genitalia samples were transferred to microvials with 80% ethanol, and later were placed in depression slides with a drop of glycerin and examined using a compound microscope (Olympus CX31) at 100–400X magnification. Photographs were taken using a Motic Camera (Moticam A5) attached to the microscope. The photographs were stacked using the Helicon Focus® software and used as templates for the illustrations, which were made by tracing the structures digitally using Adobe Illustrator® CS6. Material examined, including types of the new species, are deposited in the Museu de Entomologia, Universidade Federal de Viçosa (UFVB), supplementary material (File S1).

For the phylogenetic placement of the new species we modified the morphological matrix of Quinteiro and Almeida (2021). We included 10 additional species to the original matrix (Oecetis capixabasp. nov.; O. ruschiisp. nov.; O. cataguaHenriques-Oliveira et al., 2018; O. furcata Quinteiro & Calor, 2015; O. acanthostema Quinteiro & Calor, 2015; O. acarati Angrisano & Sganga, 2009; O. calori Quinteiro & Holzenthal, 2017; O. hastapulla Quinteiro & Holzenthal 2017; O. machaera Quinteiro & Holzenthal, 2017; and O. avara Banks, 1895), and an additional character corresponding to the asymmetric spine on the phallic apparatus. The final phylogenetic dataset comprised 67 taxa (59 ingroup taxa, 8 outgroup) with 63 morphological characters (Table 1) and sequences of cytochrome oxidase I (COI, 658 bp) available for 30 terminals (Table 2). The original morphological characters and character states (Quinteiro and Almeida 2021) were modified following Sereno’s (2007) recommendations. Inapplicable characters were coded as ‘–‘; when the character state was not clear or could not be assessed, it was coded as ‘?‘. Polymorphic characters were treated in the analysis as ‘?’. Multistate characters were treated as unordered. The original dataset matrix was edited using the software Mesquite 3.8 (Maddison and Maddison 2023).

The phylogeny was based on a probabilistic framework through Bayesian inference as implemented in MrBayes 3.2.7 (Ronquist et al. 2012). The morphological dataset was analyzed using the Mk model (Lewis 2001). To adjust the heterogeneity in rates of evolution across morphological characters, we adopted a homoplasy-based partitioning strategy described by Rosa et al. (2019), which was shown to outperform other approaches for modeling among character rate variation. In this approach the levels of character compatibility are estimated from the relative homoplasy calculated from the consistency index generated in an implied weights parsimony analysis. The parsimony analysis was performed in TNT version 1.6 (Goloboff and Morales 2023). Heuristics searches were performed through ‘Traditional Search’, with 10,000 replications, three trees saved per replication. In order to segregate the characters according to homoplasy intervals, the adjusted K function implemented in TNT 1.6 was applied, so that the weight ratio between the characters with no homoplasy and the ones with most homoplasy was equal to 10, resulting in a K = 15. Consensus tree of the morphological parsimony analysis is available as supplementary material (File S3). The adjusted values of homoplasy of each character generated in TNT ‘character scores’ were combined into more inclusive intervals resulting in seven partitions (Table 3). These morphological character partitions were then used in the Bayesian analyses following the parameters indicated in Rosa et al. (2019) (LSET rates = equal; PRSET ratepr = variable, brlenspr = unconstrained:exp(10); LINK shape).

The COI sequences available in the BOLD website (Barcode of life Database) were included to provide additional evidence about taxa relationship. We included in the phylogenetic analyses COI sequence fragments for 30 species (Table 2). Given the unavailability of COI data for the outgroup species Setodes obscurus, we opted to include this fragment from S. incertus. The sequences were aligned using MAFFT through the method L-INS-I (Katoh and Standley 2013), partitioned by codon position, and concatenated with the morphological dataset using SequenceMatrix 1.8 (Vaidya et al. 2011). The COI evolution models were estimated using J-ModelTest 2 (Darriba et al. 2012), the models GTR+G+I, HKY+G, GTR+G were selected for the first, second, and third codons positions, respectively.

The analyses were performed through the CIPRES gateway (Miller et al. 2010) for 5,000,000 generations, with samples taken every 100 generations in 2 parallel analyses and 4 Markov chains. The initial 25% generations were discarded as burn-in. We checked the convergence among the analyses in Tracer 1.7 (Rambaut et al. 2018) checking if the Effective Sample Sizes (ESS) were all > 200. The maximum credibility Bayesian tree was calculated in MrBayes with all compatible groups allowed (contype = allcompat). The branch statistical support was measured by the posterior probability values (PP). Branches with support above 90% are considered strongly supported (Zander 2004). Given current arguments on statistical significance (Amrhein et al., 2019; Hurlbert et al., 2019; Pike, 2019; Wasserstein et al., 2019), the logic, background knowledge, and experimental design should also be evaluated alongside PP to establish a conclusion and determine its certainty. The trees were visualized and edited in FigTree 1.4.3 (Rambaut 2016) and Winclada 1.89 (Nixon 2002), and the final phylogeny was edited in Adobe Illustrator® CS6. Character state reconstructions were based on parsimony and performed in Mesquite 3.8.

The distributional map was created in QGIS Firenze ver. 3.28 software, using shapefile vector layers from the Instituto Brasileiro de Geografia e Estatística (IBGE) and the Natural Earth (2023) raster data. The Terrestrial Ecosystems of the World layers used in the map are available from the World Wildlife Fund (WWF) (Olson et al. 2001). The distributional records showed in this map came from the examined material in this paper, data available at the Global Biodiversity Information Facility (GBIF), and published literature records (Denning 1951; Denning and Sykora 1966; Flint 1974, 1981; Angrisano and Sganga 2009; Quinteiro and Calor 2012, 2015; Quinteiro and Holzenthal 2017; Henriques-Oliveira et al. 2014, 2018, 2020; Bonfá-Neto et al. 2023, Moura and Quinteiro, 2023). Additional information from distributional records is available as supplementary material (File S2).

The maximum credibility Bayesian tree obtained from the morphological characters and COI is presented in Figure 1. To allow the observation of the character-states’ distribution on the resulting topology, the characters optimized under delayed transformations (DELTRAN) were displayed along the branches. The combined analysis changed some of the relationship among the species groups presented in the morphological phylogeny of Quinteiro and Almeida (2021), however most of the relationships between species groups have very low statistical support (<50%), reflecting the instability of these conclusions, emphasizing that caution should be used when considering these clades. The resulting tree recovered most species groups as monophyletic. The inconspicua and punctipennis were strongly supported, but other groups showed moderate (falicia group, PP = 76) to weak support (punctata group, PP = 61), and very low support (PP<20) to the avara and pratti groups. The splitting between the punctipennis group and other Oecetis remained as the first cladogenesis (PP = 98) but with the Austral species O. inscripta placed at a distinct lineage from the Neotropical species (PP = 42). In the Austral fauna, O. inscripta is placed in a specific group, the laustra group, characterized by the absence of any spine or paramere in the phallic apparatus (Wells 2004).

The avara group is indicated as monophyletic with very low support (PP = 16). The punctata group is placed in a distinct clade (PP = 61) and not within the avara group as in Quinteiro and Almeida (2021). The two floating species, O. rafaeli and O. silviae in the hypothesis of Quinteiro and Almeida (2021), are considered here as sister species (PP = 68). They are placed as a sister group of the avara + punctata groups (PP = 54).

The cladistic definition of the species groups according with the results are based on the following morphological characters: avara group: endotheca small (54:0), forewing fork I sessile (16:1), preanal appendage digitate (28:1) (PP = 16); inconspicua group: phallic apparatus asymmetrical (50:1), phallotremal sclerite curved (59:1), phallic apparatus round, inflated (52:1) (PP = 93); pratti group: mid leg femur with row of spines (1:1), forewing sectorial crossvein aligned (7:0), forewing apex acuminate (14:1), segment IX posterolateral margin setae absent (48:0) (PP = 10); punctata group: mid leg femur with row of spines (1:1), forewing fork I sessile (16:1), tergum X with irregular shape (33:2), inferior appendage ventral lobe small (42:0), inferior appendage quadrate, with thick setae (44:1) (PP = 61); punctipennis group: forewing fork V sessile (6:1), forewing apex acuminate (14:1), endotheca small (54:0) (PP = 98); falicia group: presence of dorsolateral process on the segment IX (23:1), tergum IX shorter than sternum IX (19:2), inferior appendage dorsal lobe triangular (37:3), and phallic apparatus elongate (51:1) (PP = 77).

Focusing on the falicia group (Fig. 2), the first cladogenesis shows a clade with Oecetis doesburgi and the afrotropical Oecetis akimi being the first to include typical characters of the group. The analysis shows a clade with North American, Central American and northern South American species (clade A), (O. hastapulla (O. arizonica, O. prolongata) (PP = 33), on which the most conspicuous synapomorphy is the inferior appendage distal lobe with apical incision (45:1). Within this clade, O. arizonica from southern USA and O. prolongata from Costa Rica are shown as strongly supported sister species (PP = 98). Oecetis testacea, from Europe and East Asia, was recovered as an independent and very autapomorphic lineage. The result also show a clade (PP = 64) formed by species primarily from the Atlantic Forest and some from Amazon and Central America, presenting an inferior appendage with ventral lobe (40:1), but without the dorsal lobe (36:0), foreleg tibia with apical spur (5:1) and midleg femur spines covering half podomere (2:1). The first cladogenesis of this clade are of species that do not have an asymmetric spine, i.e. the clade B (O. acarati (O. calori, O. fibra)) (PP = 31). The asymmetric spine on the phalloteca (62:1) appeared once among the analyzed species of the falicia group and defines what may be considered as an unresolved clade ((O. catagua, O. machaera), (O. capixabasp. nov., O. acanthostema), (O. ruschii (O. furcata, O. falicia))) (PP = 31). The clade C, O. capixabasp. nov., is grouped as sister of O. acanthostema (PP = 40) based on the characters: inferior appendage with spine-like setae (49:1), and phallotremal sclerite absent (60:0). The clade D, O. catagua and O. machaera, share the character dorsolateral process of segment IX straight (24:0) (PP = 45). Oecetis ruschiisp. nov. forms a clade with O. furcata + O. falicia (PP = 69) (clade E), which is based on the synapomorphies: dorsolateral process of segment IX forked (26:1), and inferior appendage ventral lobe triangular (41:2).

LEPTOCERIDAE Leach, 1815

Oecetis McLachlan, 1877

In this section we present new distributional records for Oecetis species in Brazil, new records for the country’s states are shown in bold.

The distribution of the species in clade A suggests that this clade is widespread from southern USA, Central America, and northern South America (Fig. 5 dot 3, 11 and 13). In the clade B, O. acarati is known from the southern Atlantic Forest (Argentina) and O. calori Quinteiro & Holzenthal 2017 from central Atlantic Forest (Brazil, Minas Gerais state) (Fig. 5 dot 2 and 4); O. fibra is widespread from the south to the central Atlantic Forest (Fig. 5 dot 9). On clade C, O. acanthostema is from northeastern Brazil Cerrado ecoregion and O. capixabasp. nov. from the Atlantic Forest (Brazil, Espírito Santo state) (Fig. 5 dot 1 and 5). For the clade D, O. catagua is from Cerrado and Atlantic Forest ecoregion (Brazil, Minas Gerais and Espírito Santo state) and O. machaera from Amazon forest (Brazil, Amazon state) (Fig. 5 dot 6 and 12). While for clade E, Oecetis ruschiisp. nov., and O. furcata have a close distribution both in northern Atlantic Forest (Bahia and Espírito Santo states in Brazil) (Fig. 5 dot 10 and 14), and O. falicia occurs in Central America (Panama) (Fig. 5 dot 8). This disjunct distribution between Oecetis falicia, O. ruschiisp. nov., and O. furcata suggests the possible existence of additional undescribed species within this lineage or a wider distribution range of the known species.

The historical connections between the Atlantic and Amazon Forests with their expansion over the dry vegetation (Cerrado ecoregion) has been advocated as a general hypothesis to the explanation of disjunct distributions of lineages inhabiting the two ecoregions, with an older connection occurring through a southern route during the Miocene, and a more recent connection in a route through the Northeast Region during the Pliocene and Pleistocene and associated with the Quaternary climate changes (Batalha-Filho et al. 2013; Ledo and Colli 2017). The cladogenesis between Oecetis catagua (from the Cerrado ecoregion) and O. machaera (from the Amazon Forest ecoregion), and the relationship of O. facilia (from Panama), Oecetis ruschiisp. nov. and O. furcata (from the Atlantic Forest ecoregion), may be explained by these ancient connections between the Atlantic and Amazon Forests, however, the limited information about species distribution (which is mostly from type locality only), and the unknown divergence times (which can be much older or much recent) restrict our conclusions about the biogeographical history of these species.

The asymmetric spiny process on the phallotheca is indicated to appear a single time within the falicia group and lost in O. furcata. An asymmetrical process evolved several times in different species groups as it is present also in the avara and punctata groups. The asymmetry in the phallic apparatus is also a synapomorphy of the inconspicua group, although the asymmetry in this group is not associated with the presence of a spiny process, but on the overall shape of the phallic apparatus.

Huber et al. (2007) provide a review of asymmetrical genitalia for several insect groups, including Trichoptera. Asymmetrical genitalia are reported in the majorTrichoptera subgroups and evolved multiple times convergently. The occurrence of asymmetric and symmetric individuals in a same species is also reported for Phylloicus Müller, 1880 (Prather 2003). Asymmetry in females with few exceptions is mostly not mentioned (Huber et al. 2007). Oecetis catagua has the asymmetric process on the phallic apparatus, and in the respective female there is no indication of asymmetry in the genitalia (Henriques-Oliveira et al. 2018). In insects, it seems that male asymmetries tend to evolve first, and female asymmetries evolve later, if they ever occur (Huber et al. 2007). Male position with torsion during copula is advocated as the most important aspect to explain insect genital asymmetry (Huber et al. 2007). However, there is not much detailed information about copulatory positions in Trichoptera. What is available suggests a final end-to-end position (Malicky 1973; Erman 1984) and no twist of the abdomen or even the phallus (Tobias 1972; Statzner 1974). Therefore, to better understand the role of Oecetis genitalia asymmetry, more in-depth research on copula and the functional interaction between the male and female genitalia is required.