The genus Reisseronia Sieder, 1956 (Lepidoptera, Psychidae, Epichnopteriginae) comprises 18 species, which are usually distributed in a limited number of very small areas in Europe and the Middle East. The genus Reisseronia was first reported in Poland in 2005; after detailed investigations, the Polish populations were described as separate species, namely R. imielinella Malkiewicz, Sobczyk & Larysz, 2013 and R. annae Larysz, 2017. Later, another population of bagworm moths belonging to the genus Reisseronia was found in Komańcza (Bieszczady Mountains). We carried out the first genetic studies of these parthenogenetic species from Poland. Sequence analysis of the DNA barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) revealed slight differences between three Polish populations of the genus Reisseronia.

The genus Reisseronia Sieder, 1956 (Lepidoptera, Psychidae, Epichnopteriginae) comprises 18 species distributed throughout Europe and the Middle East (Turkey) (Sobczyk and Werno 2021). Until 2000, only nine species of this genus were known; the remaining nine have been described in recent years. The genus is characterised by brachypterous females and parthenogenetic reproduction, which has been reported in three species. The distribution is often endemic and restricted to very small areas (Sobczyk and Werno 2021).

The genus Reisseronia was reported for the first time in Poland in 2005, when several psychid larvae were collected in Imielin near Katowice in Upper Silesia (Larysz 2007). The collected specimens were classified as Reisseronia gertrudae Sieder, 1962. However, due to some differences in morphology in the adults, pupae and life history, the specimens were described as a separate species, namely R. imielinella Malkiewicz, Sobczyk & Larysz, 2013 (Malkiewicz et al. 2013) (Fig. 1a). In the following years, another population was found in the Janów district of Katowice (Upper Silesia). The specimens were described as a new species, R. annae Larysz 2017 (Fig. 1b), based on morphological differences in the imagines, pupae and female genitalia (Larysz 2017). Another population of an unidentified species of Reisseronia was found in 2017 in Komańcza (Bieszczady Mountains) (Fig. 1c). In all three cases, the occurrence was limited to very small areas and parthenogenesis was confirmed.

Figure 1. 

Head and thorax ventral view of females. a. Reisseronia imielinella (Imielin near Katowice); b. Reisseronia annae (Katowice); c. Reisseronia sp. (Komańcza, Bieszczady Mountains). Scale bar: 0.1 mm.

Due to the lack of molecular data on this genus from Poland, we decided to perform a molecular analysis based on DNA barcode sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. We used the resulting data to better understand the relationships within the Polish members of the genus Reisseronia and their relationships within the other members of this genus.

The comparison with other Reisseronia species revealed no distinct differences between R. imielinella and R. annae as well as R. gertrudae and population from Komańcza. A close relationship with Reisseronia tarnierella (Bruand, 1851) is also noticeable (Fig. 2).

Figure 2. 

An unrooted Neighbor-joining tree, generated under the Kimura 2-parameter model, for the species of genus Reisseronia. Branch lengths represent the number of substitutions per site as percentage. The specimens from Poland are shade in colour.

The pairwise genetic distances between Reisseronia species, generated using the Kimura two-parameter method, are presented in Suppl. material 1. The pairwise divergence between these two species and the population from Komańcza and from R. gertrudae is only 0.9%. Surprisingly, no differences were noted between the specimens from Komańcza and the R. gertrudae sequence. The distance to other members of the genus Reisseronia ranges from 3.4% for R. tarnierella (BOLD ID: POESE455-22) to 13.7% for two undetermined species from Greece (BOLD ID: PHLAI1016-14 and LEATC042-13) (Suppl. material 1).

DNA barcoding based on genetic variation of a 658 base pair (bp) fragment of the 5′ end of the mitochondrial COI gene is a widely used tool for species discrimination. However, the DNA barcoding thresholds for species delimitation are variable. On the one hand, many studies have determined that the threshold for K2P genetic distance between two species is 2–3% (Hebert et al. 2003; Hajibabaei et al. 2006; Ross et al. 2008; Strutzenberger et al. 2012; Zahiri et al. 2014). On the other hand, values lower than 2% are typical for intraspecific variation or young sister-species (Huemer and Hausmann 2009; Hausmann et al. 2011; Mutanen et al. 2012; Dincă et. 2013). There are also a number of reports suggesting that species identification based solely on COI barcoding may be inaccurate. This problem may be caused by mitochondrial genome introgression, which is also present in Lepidoptera (Jiggins 2003; Zakharov et al. 2009; Mutanen et al. 2016; Cong et al. 2017), or by the occurrence of nuclear mitochondrial pseudogenes (Song et al. 2008). Therefore, based on the DNA barcoding approach only (K2P 2% threshold), we are dealing with a single Reisseronia species in Poland, which belongs to the parthenogenetic species R. gertrudae (TL: Austria, South Styria, Kitzeck in Sausal), whose wider distribution was recently confirmed (Predovnik et al. 2020). Moreover, the differentiation of closely related species complexes based only on DNA barcode COI sequences can be ambiguous, as reported in some cases of lepidopterans (van Nieukerken et al. 2012; Alipanah et al. 2022).

The differences in the female genitalia, leg morphology, antennae, pupae and pupal head plates suggest the validity of their species status. Additionally, a lack of correlation between morphological variability and variability in mitochondrial DNA sequences has been found in many Lepidoptera (e.g. Kato and Yagi 2004; Vandewoestijne et al. 2004; Korb et al. 2016; Domagała and Lis 2022). In the case of butterflies and moths, attention is paid to the variability of the wing pattern, but it cannot be excluded that the variability of other anatomical features is not reflected in the mtDNA sequence – for example, intraspecific variation in genitalia is known in several lepidopteran species (e.g. Mutanen and Kaitala 2006; Goulson 2008).

The correct classification of bagworm moths can be difficult. In the case of males, morphological identification is in many cases based on the morphometrics of the genitalia and analysis of the shape of the wing scales, but the identification of females is more difficult due to the lack of wings (Chevasco et al. 2014). According to Chevasco et al. (2014), molecular methods are an effective way to delimit species of bagworm moths, but they should be combined with the analysis of geometric morphometrics of male genitalia. On the one hand, in the case of the Polish members of Reisseronia, parthenogenesis has been confirmed, and so far, only females are known. On the other hand, according to Mutanen et al. (2012), a compromise approach should be applied for species delimitation, including cases exceeding the DNA barcoding threshold (e.g., arbitrarily 2%) but also indicating differences in ecological and morphological characters or differences in an unrelated nuclear markers.

In conclusion, our preliminary study based on a small number of individuals showed slight differences in COI sequences among three Polish populations of Reisseronia. At present, on the one hand we cannot exclude that we have a single Reisseronia species in Poland (Reiseronia gertrudae). On the other hand, we are dealing with parthenogenetic populations where the analysis of maternally inherited COI may be ambiguous. We will refrain from making any taxonomic decisions until we carry out additional analyses in the future. Whole-genome analyses will ultimately provide a better understanding of the relationships between these species as well as other Reisseronia members.

We are grateful to David C. Lees and Théo Léger for their insightful comments, suggestions, proof reading on the submitted and revised manuscript, and help in improving the manuscript. We would like to thank anonymous reviewer for their comments on the manuscript. We are thankful to Adam Malkiewicz (University of Wrocław) for the loaning us the Reisseronia gertrudae specimen. The molecular analyses were carried out using equipment of MCBR UO (International Research and Development Center of the University of Opole, Poland), which was established as part of a project cofinanced by the European Union under the European Regional Development Fund, RPO WO 2014–2020, Action 1.2 Infrastructure for R&D. Agreement No. RPOP.01.02.00-16-0001/17-00 dated 31 January 2018.