The present study provides a DNA barcode library for the world Zygaenidae (Lepidoptera). This study reports 1031 sequence data of the COI gene DNA barcodes for more than 240 species in four of the five subfamilies of the family Zygaenidae. This is about 20% of the world Zygaenidae species. Our results demonstrate the specificity of the COI gene sequences at the species level in most of the studied Zygaenidae and agree with already established taxonomic opinions. The study confirms the effectiveness of DNA barcoding as a tool for determination of most Zygaenidae species. However, some of the results are contradictory. Some cases of shared barcodes have been found, as well as cases of deep intraspecific sequence divergence in species that are well separated by morphological and biological characters. These cases are discussed in detail. Overall, when combined with morphological and biochemical data, as well as biological and ecological observations, DNA barcoding results can be a useful support for taxonomic decisions.

Zygaenidae Latreille, 1809, is a family of Lepidoptera well known for the biochemical properties of its species, capable of synthesizing hydrogen cyanide used as a defensive mechanism. Zygaenids, commonly known as burnet, forester and smoky moths, are typically day-flying insects. The family encompasses about 1200 species distributed worldwide, of which several are known as pests. Many species have restricted distributions and represent very sensitive ecological indicators, often used, along with butterflies, as an important umbrella group for ecological evaluations (Nazarov and Efetov 1993; Schmitt 2003; Tarmann 2009).

The taxonomy of these moths is generally well established; it is based on the comparison of morphological characters (habitus and genitalia morphology), but noticeably also in a large part on the integration of biological and ecological characters rarely available (or only available to a lesser extent) in other families of moths. A global taxonomic system was proposed by Alberti (1954, 1958–1959) in a comprehensive revision of the world’s fauna. This system has been improved during the past 60 years (Naumann and Tremewan 1984; Tarmann 1984, 2004; Efetov and Tarmann 1999, 2012; Hofmann and Tremewan 1996; Yen 2003), based on an exceptional variety of characters such as larval morphology (the chaetotaxy of larvae (Efetov and Hayashi 2008), microstructures of the integument (Efetov and Tarmann 2017a)), head morphology (including biometry), characters in the structure of the antennae, wings, legs, scales, abdomen (e.g. coremata, lateral ‘glands’), special habits of larvae (e.g. leaf mining, boring or free feeding), cocoon construction, special calling and mating habits (Efetov 1996a, 1998a, 1998b, 1999; Efetov and Tarmann 2013b, 2017b; Efetov et al. 2011b; Efetov and Knyazev 2014; Knyazev et al. 2015), pheromones (Subchev et al. 1998, 2013, 2016; Efetov et al. 2010b, 2014b, 2014c, 2015b, 2016, 2018; Razov et al. 2017; Cengiz Can et al. 2018), mimicry, the examination of the karyotypes (Efetov et al. 2004, 2015a), protein electrophoresis results, biochemical analyses combined with the toxicity of the Zygaenidae and the study of antigen properties of haemolymph proteins (monoclonal immunosystematics) (Efetov 2005). This refined system, however, still retains some unsolved questions, especially with respect to phylogenetic relationships. The evolution of the family has been partly investigated over the past decade through the use of molecules, morphology or a combination of both (Efetov 2006, 2012b; Efetov and Savchuk 2009; Efetov et al. 2011a, 2010b, 2014b, 2014c, 2015b, 2016; Subchev et al. 2013, 2016; Efetov and Tarmann 2013b; Mollet 2015), although genetic studies at intraspecific and interspecific levels have been generally limited to species of economic importance (Schmitt and Seitz 2004). The distinction of closely related species often relies on the use of very precise sets of characters and can be highly challenging for non-experts, precluding a broader use of these insects as ecological indicators.

The family Zygaenidae has a worldwide distribution and is divided into five subfamilies: Inouelinae Efetov and Tarmann, 2017 (Oriental), Procridinae Boisduval, 1828 (Holarctic, Afrotropical, Oriental, Australian, Neotropical), Chalcosiinae Walker, 1865 (Palaearctic, Oriental), Callizygaeninae Alberti, 1954 (Oriental), and Zygaeninae Latreille, 1809 (Palaearctic, Oriental, Afrotropical) (Alberti 1954, 1958–1959; Tarmann 1984, 1994, 2004; Efetov and Tarmann 2012, 2017a; Hofmann and Tremewan 1996; Efetov et al. 2014a; Yen 2003).

Several molecular phylogenetic studies of Zygaenidae were published by O. Niehuis et al. (2006a, 2006b, 2007). Selected morphological and biological characters were investigated together with RNA secondary structure variations (Niehuis et al. 2006a) or by the analysis of several mitochondrial and nuclear markers (Niehuis et al. 2006b, 2007). These studies are mainly restricted to species of the subfamily Zygaeninae (genus Zygaena Fabricius, 1775) (Niehuis et al. 2006a, 2007). Only a few specimens of Procridinae, Callizygaeninae and Chalcosiinae were included (Niehuis et al. 2006b) while Inouelinae are completely absent.

Our project “DNA barcoding of Zygaenidae moths” (ZYGMO) started in 2009 (Efetov et al. 2010) using the COI gene fragment proposed by Hebert et al. (2003a, 2003b) as a standard DNA barcode (Ratnasingham and Hebert 2007) with the goal to initiate a library of DNA barcode sequences for Zygaenidae species as a new tool for species identification in this family. Moreover, it was expected to confirm known species-complexes and possibly find new ones, as well as so far overlooked cryptic diversity. Each Zygaenidae barcode record in the Barcode of Life Data Systems (BOLD, http://www.boldsystems.org) (Ratnasingham and Hebert 2007) is accompanied by specimen images, detailed and geo-referenced collection data, complete taxonomic information and voucher repository data. These activities were undertaken in close cooperation between the Crimean Federal University (Crimea), the Tiroler Landesmuseen, Ferdinandeum (Austria) and the Biodiversity Institute of Ontario at the University of Guelph (Canada) under the framework of the “International Barcode of Life” (iBOL) project. There is another, smaller scale project focused on barcoding Zygaenidae and Papilionoidea, but this recent effort was geographically restricted to Switzerland (Litman et al. 2018).

In this paper, we conduct a critical analysis of our Zygaenidae DNA barcoding results. The current taxonomic system for studied species is discussed in light of the relevant results from our sequence analysis combined with traditionally used characters. Some principal remarks on barcoding and taxonomy as well as contradictory results and examples requiring special attention are listed and discussed below.

We obtained 1031 COI gene sequences for more than 240 described and undescribed species of Zygaenidae. The library comprises 975 public records from 60 countries, 247 BIN clusters. Complete specimen records, including images, specimen data and voucher information, GPS coordinates, applied primers, sequence and trace files, can be accessed in BOLD public dataset for ZYGMO (http://www.boldsystems.org/index.php/Public_SearchTerms).

Critical analysis of DNA barcode results in the light of traditionally applied species names demonstrates the specificity of sequences of this fragment of the COI gene at species level in the majority of the studied taxa (sequences longer than 550 bp). The mean intraspecific K2P divergence (within species) is 1.36%, interspecific (within genus) 7.44% and intergeneric (within family) 13.91%. So far, many species of Zygaenidae (especially Procridinae) could only be determined by examination of the genitalia structures. We found examples where the study of the COI gene sequence revealed misidentifications for specimens previously determined without dissection. Re-examination of these specimens based on the genitalia structure confirmed the barcoding result.

Several new Zygaenidae species were described during the last years by taking into account ZYGMO molecular data (Efetov 2012a; Efetov and Tarmann 2013a, 2014a, 2014b, 2016a, 2016b; Tarmann and Drouet 2015). In the discussion, as an example, we present in detail the application of COI gene investigation for the identification of Adscita (Procriterna) pligori Efetov, 2012.

Some cases of deep intraspecific sequence divergence as well as low interspecific divergence and shared barcodes within morphologically clearly distinguished species were found. At present, a significant number of the studied Zygaenidae species (nearly 15%) has shown deep intraspecific divergence – of more than 3%. Most of these cases are detected in Procridinae species, for example, in the subgenus Jordanita Verity, 1946, of the genus Jordanita. The maximum intraspecific distance in Jordanita (Jordanita) graeca (Jordan, 1907) is 5.72%, and in Jordanita (Jordanita) chloros (Hübner, 1813) it is 6.08%. On the other hand, the range of interspecific distance in this subgenus is very low, viz. 0.30–0.61% with barcode-sharing in morphologically well-separated species. Hence, the species of the subgenus Jordanita cannot be separated solely on the basis of DNA barcode data. Possible reasons for these results are discussed below.

An interesting result is that Zygaenoprocris khorassana (Alberti, 1939) is a distinct species from Z. chalcochlora Hampson, 1900, contrary to Efetov and Tarmann (1994), who had synonymized the two, and in support of Efetov and Tarmann (2012), who had reinstated Z. khorassana as a valid species.

Not long ago two new Zygaenoprocris species of the chalcochlora-group (Z. (Z.) efetovi Mollet & Tarmann, 2007, and Z. (Z.) hofmanni Mollet & Tarmann, 2007) were described, based on a different habitus to that of Z. (Z.) chalcochlora (the absence of shiny metallic scales) and slight differences in genitalia structures including larger papillae anales and shorter apophyses posteriores. The results of this study support these as distinct species.

Our DNA studies support high values of interspecific distances between species of the subgenus Zygaenoprocris with the mean distance being equal to 7.27%. Moreover, DNA barcode data show that Z. (Z.) chalcochlora possibly represents a species complex. The populations of Z. khorassana (with shiny metallic scales) in northern Iran are isolated from the populations of Z. chalcochlora in Pakistan (including the type locality of Z. chalcochlora) and Afghanistan (all specimens with small papillae anales and long apophyses posteriores). This information suggests that the above-mentioned characters of the papillae anales are much more important than we thought earlier.

The distance between specimens within one species sometimes exceeds the mean interspecific distance (1.36%). For example, we showed that all studied specimens of Zygaenoprocris (Molletia) duskei (Grum-Grshimailo, 1902) form one lineage on the ID Tree with the distance to nearest neighbour – Z. (M.) taftana (Alberti, 1939) – 4.74%, and to Z. (M.) persepolis (Alberti, 1938) – 5.72%. The distance between different specimens of Z. (M.) duskei kliri Keil, 2002, (Esfahan, Markazi) is 0.17%, the distance between specimens of the Z. (M.) duskei kermana (Alberti, 1967) (Kerman, Hormozgan) is 1.08%, while the distance between specimens of Z. (M.) duskei duskei (Grum-Grshimailo, 1902) (Sistan-e Baluchestan) is 3.93%.

It was known that the genus Illiberis (sensu lato) is a polyphyletic unit (Efetov 1996b, 1997, 1998a, 2010). The taxonomic status of some species-groups of this genus was revised (Efetov and Tarmann 2012). For example, the subgenera Hedina Alberti, 1954, and Zama Herrich-Schäffer, 1855, were raised to the generic level (Efetov and Tarmann 2012). This decision is supported by our DNA results (Fig. 1).

Figure 1. 

BOLD TaxonID Tree for some representatives of the genera Zama, Illiberis, Hedina, and Adscita (K2P, COI-5P, length > 550).

Some interesting findings were also made in the subfamily Zygaeninae. Since 2014 more than 80 COI sequences of specimens belonging to the minos-purpuralis complex from different localities have been studied. The COI results showed that the distances between Zygaena (Mesembrynus) minos persica Burgeff, 1926, from Iran and other barcode-sharing minos-purpuralis specimens are significant: 4.92% and 4.75% to the nearest Z. (M.) minos ([Denis and Schiffermüller], 1775) and Z. (M.) purpuralis (Brünnich, 1763) respectively. This is 2.5 times the distance of both Z. (M.) minos and Z. (M.) purpuralis to their nearest neighbour Z. (M.) erythrus (Hübner, 1806), which is 1.86%. The deep COI gene divergence of Z. (M.) minos persica supports the opinion that it may be a separate species (Nahirnić and Tarmann 2014).

Some other cases of barcode sharing (e.g. Chalcosia pectinicornis (Linnaeus, 1758) / Ch. phalaenaria (Guérin-Méneville, 1843); Zygaena (Mesembrynus) tamara Christoph, 1889 / Z. (M.) cuvieri Boisduval, 1828) and deep divergences (e. g. Zygaenoprocris (Molletia) persepolis (Alberti, 1938), Jordanita (Roccia) paupera (Christoph, 1887), some species of the subgenera Tremewania Efetov & Tarmann, 1999, and Jordanita of the genus Jordanita) were detected. Deep intraspecific divergences were also seen in Myrtartona rufiventris (Walker, 1854), Illiberis (Alterasvenia) ochracea Leech, 1898, Zygaenoprocris (Zygaenoprocris) chalcochlora, Triprocris cyanea Barnes & McDunnough, 1910, Gynautocera papilionaria Guérin-Méneville, 1831, Zygaena (Agrumenia) cocandica Erschoff, 1874, Z. (Zygaena) filipendulae (Linnaeus, 1758), Z. (Z.) lonicerae (Scheven, 1777). In these cases futher investigations including appropriate genetic markers are necessary.

Patterns of DNA barcode variation were examined in more than 240 species of the family Zygaenidae within the project “DNA barcoding of Zygaenidae moths” (ZYGMO), resulting in a DNA identification library available for most of the studied species. However, despite our efforts the major part of the Zygaenidae fauna is waiting to be DNA barcoded. As using DNA barcodes has proved itself as a quick and economical method for biodiversity investigations in this family, it is worthwhile to continue this effort.

Nevertheless, a significant number of cases of deep intraspecific sequence divergence as well as low interspecific divergence and shared barcodes within morphologically clearly separable Zygaenidae species were found. Further investigations within problematic groups should be undertaken, focused on the study of additional molecular markers.

The analysis of COI sequences provides additional data for a careful re-consideration of previously made decisions in Zygaenidae biogeography, systematics and taxonomy. Moreover, our results show that DNA barcoding results should always be discussed in combination with other data, viz. morphological, biological, ecological, biochemical etc.

We are indebted to Prof Dr F. Can (Turkey), Mr J.-M. Desse (France), Mr E. Drouet (France), Dr O. G. Gorbunov (Russia), Mrs E. Hayashi (Japan), Prof Dr P. Jakšić (Serbia), Mr Th. Keil (Germany), Mr J. Klír (Czech Republic), Dr E. Kokanova (Turkmenistan), Mr A. M. Kosenko (Ukraine), Dr C. Koshio (Japan), Mr B. Mollet (France), Ms A. Nahirnić (Serbia), Dr I. G. Pljushtch (Ukraine), Mr P. V. Ruchko (Russia), Dr E. V. Rutjan (Ukraine), and Mr A. N. Zamesov (Russia) for fruitful cooperation.

This research work was partly supported by the Project of Development Program of V. I. Vernadsky Crimean Federal University Network ‘Academic mobility of Young Scientists of Russia – AMMUR’ in 2017.