The genus Setina Schrank, 1802 (Erebidae, Arctiinae) is taxonomically complex due to pronounced intraspecific and interpopulation variability, which may indicate relatively recent or ongoing speciation processes. As a consequence, the male genitalia are remarkably uniform throughout the genus and exhibit few species-specific characters. The most informative diagnostic features are found in the structures of the valvae and the architecture of the vesica. In females, the main distinguishing characters involve the shape and size of the ductus bursae and the appendix bursae. Species of Setina are small to medium-sized and display clear sexual dimorphism in the shape and size of wings, the length and width of the abdomen, and the configuration of the antennae. The forewings are typically yellowish with dark dots or lines, whereas the hindwings are usually paler and bear a marginal row of dark spots, which may be partly reduced or absent. Members of the genus inhabit sandy and rocky slopes and subxerothermic grasslands, and may occur at elevations exceeding 3000 m in the Alps. Adults are on the wing from late spring to early autumn (de Freina and Witt 1987; Leraut 2018).

The genus Setina is currently considered to comprise six species in Europe. Reflecting the high variability within the genus, numerous synonyms and fourteen subspecies were listed by Witt and Ronkay (2011): three subspecies of Setina irrorella (Linnaeus, 1758); two of Setina flavicans (Geyer, 1836); three of Setina alpestris Zeller, 1865; four of Setina aurita (Esper, 1787), in its current sense; two of Setina roscida (Denis & Schiffermüller, 1775); and the Iberian endemic Setina cantabrica de Freina & Witt, 1985. Earlier, Trawöger (1991), in a study on hybridization within the genus, recognized up to eight subspecies of the so-called S. aurita, although he distinguished only three sympatric species: S. irrorella, S. aurita and S. roscida. Subsequently, Parenzan and Scalercio (2001) proposed a taxonomic grouping based on male genital morphology and ecological traits. They recognized the S. irrorella group—comprising S. irrorella, S. alpestris and S. aurita—characterized by a spiny clasper, and the S. roscida group—comprising S. roscida, S. cantabrica and S. flavicans—distinguished by a concave clasper. Later, Leraut (2006) treated S. flavicans as a subspecies of S. irrorella, S. alpestris as a subspecies of S. ramosa (Fabricius, 1793), and S. cantabrica as a subspecies of S. roscida. More recently, Leraut (2018) noted that populations of most Setina species in Western-central Europe remain poorly known and that the nomenclature of several taxa is still unsettled. He reviewed various taxonomic arrangements proposed by earlier authors, including Rougeot and Viette (1978), Burmann and Tarmann (1985), de Freina and Witt (1987), Leraut (2006) and Witt and Ronkay (2011). In addition, Leraut (2018), following observations already noted by Burmann (1955), suggested that the considerable morphological variability observed in the genus may partly reflect phenotypic plasticity related to elevation, as some species—particularly S. irrorella—occur from sea level to more than 3,600 m. In that same work, Leraut (2018) proposed the use of the name S. ramosa in place of the commonly used S. aurita, arguing that the latter is a junior homonym of Noctua aurita, now Trigonephra aurita (Fabricius, 1787) (Noctuidae: Oncocnemidinae). However, this interpretation overlooked that Setina ramosa (Fabricius, 1793) was originally described in the genus Bombyx and therefore constitutes a more recent primary homonym of Bombyx ramosa Esper, 1786, currently recognized as Calliergis ramosa (Esper, 1786). Finally, Leraut (2018) considered S. irrorella, S. roscida, S. ramosa and S. alpestris as valid species.

The present investigation was prompted by results obtained during an effort to DNA-barcode all macroheteroceran Lepidoptera species occurring in the Iberian Peninsula., This work revealed that Iberian individuals of the genus Setina share barcode haplotypes with populations from Central Europe. In this study, we analyze mitochondrial COI sequences together with the geographical distribution of the identified lineages to evaluate differences among European population groups and to provide new insights into their taxonomic relationships. Our research aims to examine all taxa within a broader European context, avoiding interpretations centered on particular regions—such as Central Europe on the one hand and south-western Europe on the other—which in the past have sometimes led to the over- or underestimating of the taxonomic status of certain taxa.

The genus Setina is a species-group with a great inter-individual and inter-population variability, whose species often show a similar type and range of intra- and interspecific variation in external features, and with the male genitalia very uniform throughout the genus. Furthermore, the alleged morphological specific features are usually only slightly different. Only According to Witt and Ronkay (2011), differential features are found in the female genitalia, where the shape and size of the specialized ductus bursae and the appendix bursae define the species-rank taxa. Thus, relatively high degree of hybridization in sympatric species cannot be ruled out. In fact, hybridization may partly explain the variation in external features observed (Burmann, 1955; Burmann & Tarmann, 1985).

Leraut (2018) analyzed extensive samples of European populations of Setina species and concluded that, according to the study of their biology, as well as that of the external characters and the genitalia, the genus Setina in Europe was separated into four species, named S. roscida, S. aurita, S. irrorella and S. alpestris, which tend to vary by constituting more or less locally homogeneous populations, with crosses between species that could lead to hybridization and introgression in the contact zones.

Detailed morphological, ecological and genetic analyses may discriminate closely related species which possess slight sequence divergence from their nearest neighbour. Molecular analyses enable initial taxonomic evaluation in such taxa, but there is no objective way to select the algorithm for species delimitation or input parameters that best recover actual species boundaries when using barcodes only. In different groups of invertebrate taxa, a sequence divergence in the barcode region lower than 2% is assumed by many authors to correspond to intraspecific differences, while higher values of divergence are related to interspecific variation and groups of sequences are assumed to correspond to distinct MOTUs (Hausmann et al. 2011; Zhang and Bu 2022). However, divergence between young sister-species may fall below the 2% threshold, while unusually variable species may exceed it (Hausmann et al. 2011; Zhang and Bu 2022).

Discrimination of differences affecting these species requires more algorithmic finesse, and the selection of an effective algorithm for MOTU recognition is necessary. In our study BIN and ASAP methods were selected to analyze the Setina sequences. Barcode Index Number (BIN) system is a consistent and reasonably stable registry for animal MOTUs, recognized through sequence variation in the barcode region. Concerning the species delimitation analyses by ASAP, Puillandre et al. (2021) assessed its power on real COI barcode data sets, noticing the ASAP potential to become a relevant tool to test hypothesis on maternal ancestry in a barcode-based integrative taxonomy process. ASAP identifies a taxonomic partition ranked with different scores that must be subsequently tested against other evidence in an integrative taxonomy framework, especially when single-locus data are used.

In our study, BOLD calculated ten different BINs for all taxa, of which S. roscida has a single BIN, while S. aurita and S. irrorella have multiple BINs, perhaps as a result of hybridization. Similarly, the ASAP method for the barcoding data obtained in our study indicated the existence of the same three main clusters of Setina distributed from the Iberian Peninsula to Russia. The variation in the BIN assignments and the several branches calculated by ASAP for S. irrorella and S. aurita within or without the main groups suggest the inability of sequence variation at COI to diagnose other species.

This variability was noticed initially by Toulgoët (1975), remarking the general conformation of the genitalia, wings, venation, pattern, tymbal organs and coloring as basically identical in all representatives of the genus, and caterpillars show no differences in general aspect and coloration. In relation to this variation, Trawöger (1991) explained the polymorphism of S. aurita and S. irrorella populations by the reciprocal gene flow between the two species, which produced specimens with typical traits of the other species. This variability was also recorded by Witt and Ronkay (2011), listing several subspecies in each of their recognized species: four subspecies for S. aurita, -S. a. aurita, S. a. imbuta (Hübner, 1803), S. a. pfisteri Burmann & Tarmann, 1985 and S. a. teriolensis (Burmann, 1955)-, three subspecies for Setina irrorella -S. i. irrorella, S. i. freyeri (Nickerl, 1845), S. i. mediterranea (Daniel, 1964), two subspecies for Setina flavicans -S. f. flavicans and S. f. pseudoirrorella de Freina & Witt, 1985-, two subspecies for Setina roscida -S. r. roscida and S. r. kuhlweini (Hübner, 1824)- and only Setina cantabrica was separated as a different species in the roscida species group. Additionally, these authors considered three subspecies of Setina alpestris: S. a. alpestris Zeller, 1865, S. a. wolfsbergeri (Burmann, 1975) and S. a. pseudokuhlweini (Vorbrodt, 1914).

The taxonomic status of all these nominal taxa has been discussed by different lepidopterists. S. roscida cantabrica and S. irrorella flavicans were previously examined by de Freina and Witt (1985) based on Setina individuals from Spain and southern France and they concluded that S. irrorella was not present in the Iberian Peninsula, being replaced by S. flavicans. In their opinion, individuals from the French Pyrenees and northern Spain could be distinguished as a new subspecies, S. flavicans pseudoirrorella. Witt and Ronkay (2011) pointed out the differences between the male genitalia of the species: S. flavicans can be distinguished from those of S. alpestris by the dorsally more flattened uncus, shorter and apically more curved digitus, the longer clasper and the more robust basal cornutus of the vesica; S. roscida-S. cantabrica pair of species by the dorsally less arched uncus, much broader valvae with longer and more acute digitus and the shorter, thicker clasper, and S. irrorella and S. ramosa by some slighter and more variable characteristics when large series of individuals are studied as the more robust, medially evenly thick and distally less curved uncus, the longer and apically more curved digitus, the longer, thicker clasper and the larger basal cornutus of the vesica. The female genitalia of S. flavicans are most similar to those of S. irrorella while the differences with S. alpestris, S. aurita and S. roscida are conspicuous. Setina flavicans is considered by de Freina and Witt (1987), Ylla et al. (2010) and Witt and Ronkay (2011) as a distinct species while Leraut (2006) and Leraut (2018) considered nominotypical S. flavicans to be conspecific with S. irrorella.

In relation to Setina cantabricade Freina and Witt (1985) described it from the Picos de Europa in Asturias and, later on, Fernández-Vidal et al. (2003) extended its known range to north-west Spain, finding it in León, Lugo and Orense as well as in Asturias, where the species flew sympatrically in some places with S. flavicans. Corley (2013) studied some individuals from Portugal previously identified as S. flavicans, pointing out that their genitalia best match with those of S. roscida, leading to the conclusion that the placement of Leraut (2006) of S. cantabrica as a subspecies of S. roscida may well be correct although the Portuguese specimens (28 mm) are all larger than typicall S. roscida (20-24 mm). Subsequently, Leraut (2006) downgraded S. cantabrica to a subspecies of S. roscida based on the lack of significant differences in the male genitalia, although Witt and Ronkay (2011) considered that these differences are distinctive within the genus Setina. Leraut (2018) considered S. cantabrica as a subspecies of S. roscida considering the comments on non-significant differences in genitalia and within the variation between different subspecies of Leraut (2006) and Corley (2013).

The current distributions of Setina species are the result of multiple range shifts driven by past climatic changes, that profoundly influenced the biodiversity of Europe (for reviews, see e.g., Hewitt 1996, 1999, 2000, 2004; Taberlet et al. 1998; Schmitt 2007). Initially, S. roscida was considered a steppe species and S. aurita a rock dweller while S. irrorella, thought to be the probable ancestor of all, is the least specialized and the most widespread, inhabiting almost any biotope from sea level to 3,500 m above sea level. The Setina species, which allegedly migrated from the Eastern European steppes to the mountainous regions of Europe before the last Ice Age, were split up into numerous, mostly genetically isolated populations in the Alps, Pyrenees and Balkans. The ecologically uniform steppes of the east contrast with the conditions found at different altitudes in the valleys, mainly in the alpine region, which has led to the development of slight morphological divergence between populations. This has led to the description of numerous infraspecific taxa by authors because the mechanisms of genetic, ethological and ecological isolation have been not fully developed, allowing hybridization in areas of contact (Toulgoët 1975; de Freina and Witt 1984, 1985; Trawöger 1991, 1994; Parenzan and Scalercio 2001; Leraut 2018).

Distributional patterns are likely related to the isolation of populations in refugia that promoted genetic and phenotypic differentiation as a result of independent adaptation to local environments and genetic drift, with consequences for reproductive isolation between discrete refugial lineages and the creation of hybrid zones where diverged lineages came into secondary contact, especially in the Alpine regions (Capblancq et al. 2020; Augustijnen et al. 2022). The major lineages of Setina are represented by two widespread groups distributed throughout Europe namely S. irrorella from Spain to European parts of Russia and S. roscida from Iberian Peninsula to western and central Siberia, and S. aurita restricted to the entire Alps. Regarding Setina alpestris, generally characterized by the absence of the black suffusion on the underside of the forewings, it is often misidentified as S. ramosa, and its taxonomic status and distribution remains to be clarified, as also suggested by Leraut (2006). Trawöger (1994), comparing the variation between S. alpestris and S. aurita, suggested that the taxonomic status of S. alpestris cannot be recognized as a species despite the morphological and ethological differences and it could be considered as “semispecies” or species in “statu nascendi”.

All the species show a morphological pattern that could be considered evidence for similar ecological preferences or parallel histories during the Quaternary, suggesting that all groups share a recent common ancestor. This phylogeographic pattern has been suggested for other Lepidoptera (e.g., Agrodiaetus, Erebia, Melitaea, Parnassius, Chelis, Arctia), in which a marked role of climatic oscillations during the Pleistocene on population isolation and differentiation is postulated (Haubrich and Schmitt 2007; Todisco et al. 2012; Albre et al. 2008; Lenevea et al. 2009; Vila et al. 2010; Ortiz et al. 2016, 2023).

However, an increasing number of studies indicate that many endemic taxa inhabiting refugial regions are of Pleistocene origin and formed by allopatric fragmentation (e.g., Hewitt 1996; Ghanavi et al. 2025; Ortiz et al. 2016, 2023; Torrado-Blanco et al. 2024). The apparent absence of a clear lineage sorting between Setina species may be due relatively recent radiation. In some cases, they are described as distinct species and, in other cases, these taxa are considered as subspecies or lineages within species. The present occurrence of multiple morphs of species showing a sympatric and parapatric distribution is thought to be the result of a range expansion of S. irrorella and S. roscida since the last glaciation events, thanks to the high ecological plasticity of these species and the finding of many suitable environments. In addition, the presence of individuals with different phenotypes within the groups of particular species suggest hybridization processes between the species that cause a great morphological variability that can lead to misidentification.

We admit that the approach based on the barcode patterns of Setina species is of limited power for the resolution of their taxonomy as biospecies, but it is a contribution towards the understanding of the genus grouping the studied taxa into three species, Setina irrorella (Linnaeus, 1758), Setina roscida (Denis & Schiffermüller, 1775) and Setina aurita (Esper, 1787). However, this grouping should not be regarded as a phylogeny, before getting confirmation from a comprehensive molecular phylogenetic study. This morphological grouping should rather serve as a tool for species identification, regardless of the subspecies considered within each species or any new taxa that may be described.

As a final tale, we shall note that the specific name Setina aurita, as used here, should be reviewed, given the aforementioned homonymies.