Accurate species delimitation is essential for multiple disciplines such as ecology, evolution, conservation biology, among others (Adams et al. 2014). However, species delimitation between closely related species is often difficult since traditional taxonomy depends on the evolution of informative and consistent phenotypic characters (Bickford et al. 2007; Gokhman 2018; Matos-Maraví et al. 2019). Social wasps (Hymenoptera: Vespidae) exemplify this issue since they show different sibling species morphologically similar (Menezes et al. 2011; Buck et al. 2012; Lopes and Menezes 2017; Schmid-Egger et al. 2017). Hence, as in other taxonomic groups, the combination of different sources of biological information such as morphology and genetics is necessary to better understand the diversity of social wasps.

Pseudopolybia de Saussure, 1863 is a Neotropical genus of swarm-founding social wasps (Vespidae: Polistinae: Epiponini) comprising four species: Pseudopolybia compressa (de Saussure, 1854), Pseudopolybia difficilis (Ducke, 1905), Pseudopolybia langi Bequaert, 1944, and Pseudopolybia vespiceps (de Saussure, 1863). They are distinguished by the third segment of the labial palpi bearing a short, stout, curved bristle near its apex, and the number of palpal segments being six maxillary and four labial (Richards 1978; Andena et al. 2007). The genus distribution is from Nicaragua to southern Brazil, and they are found mainly in rainforests (Richards and Richards 1951; Richards 1978; West-Eberhard et al. 1995). Nests of P. compressa, P. difficilis, and P. vespiceps have been described (Wenzel 1998). They are usually arboreal with spherical or tapering envelope and the entrance hole is a simple hole at the lowest point of the envelope (Richards 1978).

Although there are only four described species within Pseudopolybia several taxonomic problems persist (e.g. Bequaert 1938; Richards 1978; Andena et al. 2007). For instance, P. compressa and P. vespiceps show striking color variation that was treated as different “morphs” (see Richards 1978). Thus, P. compressa included the morphs compressa (de Saussure), luctuosa (Smith), and laticincta (Ducke), while P. vespiceps comprised the “morphs” vespiceps (de Saussure) and testacea (Ducke). These names were first described as distinct species or varieties, but later reduced to varieties of their respective species (Bequaert 1938). Interestingly, the forms of P. compressa (e.g. laticincta and luctuosa) were found on the same nest from Ecuador (Ducke 1910). Hence, Andena et al. (2007) conclude that these “morphs” suggested by Richards (1978) “do not merit any treatment other than synonyms”.

In the present paper, we use an integrative taxonomic approach, i.e. analyzing the variation of mitochondrial genetic markers with molecular species delimitation methods, adult morphology, nest architecture, and male genitalia, to assess the taxonomic status of pinned museum specimens previously identified as P. compressa from Central-West and Northeast of Brazil. Based on multiple independent lines of evidence, we described a new Neotropical swarm-founding social wasp, Pseudopolybia cryptica sp. n.

Based on our integrative taxonomic approach, we can hypothesize that Pseudopolybia specimens collected in the Brazilian states of Mato Grosso and Bahia belong to a new species that we describe below. We consider several morphological characters including male genitalia (Fig. 1 and Table 1), nest architecture (Fig. 2), and two mitochondrial markers, COI and 16S, under a phylogenetic and molecular species delimitation approach (Fig. 3).

Our integrated evaluation of adult morphology, male genitalia, nest architecture, and results from molecular species delimitation support the new species P. cryptica and indicate a sister species relationship with P. compressa. From the morphological point of view, P. cryptica cannot be confused with any Pseudopolybia species, except for P. compressa (Fig. 5D). The blackish general color of P. cryptica is very similar to P. compressa, but P. cryptica does not present extensive yellow marks, mainly in the tergum I and II. Moreover, the metasoma of P. cryptica is entire brownish and not black like P. compressa. Despite P. compressa has color variants described as three “morphotypes” [P. compressa morph. compressa (de Saussure, 1854), P. compressa morph. luctuosa (Smith, 1857), and P. compressa morph. laticincta (Ducke, 1904)], we noticed that they are morphological different of P. cryptica. According to Smith (1857) and Richards (1978), the P. morph. luctuosa is smaller and black, with a few yellow marks, and it is very similar to the P. compressa morph. compressa and P. compressa morph. laticincta. Pseudopolybia cryptica is different, since it is darker than all other P. compressa morphs, and it is larger than the P. compressa morph. luctuosa. Additionally, the male genitalia was another important character in the diagnosis of this new species, since it has aedeagus with intermediate denticulation in apical portion and paramere with long and sparse bristles in the spine base as differences when compared with the male genitalia of P. compressa (Table 1).

Concerning our molecular data, the species delimitation analyses have been carried out with a clustering (ABGD) and tree-based (bPTP and mPTP) methods on two mitochondrial markers, COI and 16S. Empirical studies have shown that species delimitation methods based on single-locus data tend to under- and oversplit species (da Silva et al. 2017; Pentinsaari et al. 2017; Renner et al. 2017). Additionally, despite P. cryptica showed high genetic distance when compared with other congeners (e.g. more than 8% for COI), a species-delimitation decision considering genetic distance using only mitochondrial markers has to be taken with extreme caution (e.g. Mueller 2006; Waichert et al. 2019). First, in the Epiponini wasps queen number may vary from many to few during the colonial cycle (cyclic oligogyny) and such colony strategy can lead to differences between nuclear and mitochondrial genetic structure at microgeographical scales (Ross and Shoemaker 1997). Second, mitochondrial markers are supposed to be linked, non-recombining locus with maternal inheritance, and likely evolved under similar constraints. We are aware of the limitations caused by stochasticity of the coalescent process and gene flow for single-locus species delimitation methods as well as the use of only mitochondrial markers, but for our study, we take advantage of using these methods within an integrative taxonomic framework. For instance, our morphology-based analysis and the three molecular species delimitation methods with two mitochondrial markers consistently recognize P. cryptica as a distinct taxon and confirm that it cannot be assigned to any known described Pseudopolybia species including the three “morphotypes”.

Despite several studies performed molecular species delimitation approaches to identify Hymenoptera species (Fernández-Flores et al. 2013; Schwarzfeld and Sperling 2015; Williams et al. 2015; Hurtado-Burillo et al. 2017; Waichert et al. 2019; Brasero et al. 2020; Sabadini et al. 2020), molecular methods have not previously been applied to alpha-taxonomic problems in Epiponini wasps. Notably, based on morphological and molecular results for Synoeca species, Menezes et al. (2015) and Lopes and Menezes (2017) argued that the social wasp diversity in the Neotropical region may be underestimated due to morphological similarity of these insects, and hence they recommended the combination of morphology, population-level sampling, and genetics to systematic studies for the group. Our findings here reinforce the need of combining independent sources of biological data for the diversity study of social wasps.

A.S. and R.S.T.M. designed the study, funding acquisition, project administration, and led the writing of the manuscript. P.C.S.B. carried out the molecular laboratory work under supervision of R.S.T.M. S.M. collected samples. A.S., R.S.T.M., P.C.S.B., and M.A. performed the analyses. All authors gave final approval for publication.