Exploring the Diversity of Micropterigidae and Eriocraniidae (Lepidoptera) in South Italian Forests by DNA Barcoding

Abstract

Micropterigidae and Eriocraniidae are poorly investigated in Southern Europe. Despite the richness of the Italian territory, studies devoted to investigating their molecular diversity are lacking. Furthermore, knowledge concerning the diversity and species distribution of these families in southern Italian regions is very poor. The aim of this paper is to contribute to filling the gaps in knowledge on the diversity of Micropterigidae and Eriocraniidae in southern Italian regions by analysing, through DNA barcoding, the samples collected in this geographic area by the authors in recent years. In total, 37 specimens were submitted to DNA barcoding analysis. The sequences were subsequently identified by comparing them with those available in the Barcoding of Life Data Systems. The results provided the first record and the first sequences of the Eriocraniidae family for southern Italy, the first record in continental Italy of a formerly Sicilian endemic species, the first sequences of an endemic species, and the first sequence belonging either to a previously described but unsequenced species or to an undescribed species. In conclusion, DNA barcoding of these families in southern Italy provided very interesting insights, improving their molecular libraries and the knowledge on the fauna of this territory. Daytime studies can further improve these results, but addressing the gaps concerning immature stages should be the major goal of future research.

1. Introduction

Micropterigidae Herrich-Schäffer, 1855 is the only family belonging to the suborder Zeugloptera and is regarded as the sister group to all Lepidoptera, with adults characterised by functional mandibles [1]. Eriocraniidae Rebel, 1901 is the only family belonging to the infraorder Dacnonypha and is regarded as the sister group of the suborder Glossata, as adults possess a rudimentary functional proboscis [1]. Due to their systematic position, these families have been the preferred subject for studies on the Lepidoptera phylogeny [2,3,4], aimed at clarifying the evolution of the order. Despite their great phylogenetic distance, the adults of these families share some traits, such as the metallic colouration of wings, small dimensions, predominant diel activity, and univoltine life cycle.

The early stages of European Micropterigidae are little known. Eggs can be laid on the soil surface among vegetation, and the larvae feed on liverworts and fresh photosynthetic tissue of plants, though they may also consume decaying angiosperm leaves [5] or possibly on fungi within rotten logs or soil [6]. Unfortunately, several larvae are still undescribed. The few known pupae of European species develop within a robust cocoon [7]. The adults are diurnal and can be found in spring feeding on flowers of various trees, herbs or fern spores [7]. The family includes approximately 160 described species worldwide [8], distributed across all the major zoogeographical regions [1]. The European fauna comprises 51 species, most of which have a very restricted range. The Italian checklist [9] originally included 29 species, 2 of which were considered doubtful. After the revision of old records and the description of 4 new species, this number was updated to 32, including those from Sicily and Sardinia, which host the richest Micropterigidae fauna in Europe, with 19 endemics.

Eriocraniidae larvae are leaf miners on broadleaf trees. The final-instar larva leaves the mine and pupates on the soil within a silken cocoon [1]. Diurnal adults have a rudimentary proboscis adapted for ingesting water droplets. This family comprises approximately 29 species worldwide [8], exclusively distributed in the Holarctic region [1]. Ten species are present in Europe, seven of which have been recorded from Italy. None of them showed a restricted range.

The description of new species is much rarer in Eriocraniidae than in Micropterigidae. In fact, only two species of the former have been described in Europe after World War II, compared with 27 species of the latter (www.lepiforum.org accessed on 13 January 2025). The integration of molecular techniques in taxonomy contributed to accelerate species discoveries in the last years [10]. DNA barcoding, the most popular molecular technique for identifying cryptic species [11] and estimating intraspecific genetic variability [12], was employed for the description of the youngest Eriocraniidae species, Eriocrania marci Schmid, 2024, in the Alps, as well as for the description of some Micropterigidae [13].

Despite the richness of the Italian territory, only occasional taxonomic and faunistic research has been conducted on Micropterigidae and Eriocraniidae, and no extensive study has been carried out on their molecular diversity. Furthermore, knowledge concerning their diversity and species distribution in the southern Italian regions of the Italian Peninsula, i.e., Campania, Puglia, Basilicata and Calabria, is mostly limited to the original descriptions of Micropterix garganoensis Heath, 1960 from Puglia, M. hartigi Heath, 1981 from Basilicata, and M. italica Heath, 1981 from Campania. To the best of our knowledge, additional data can be found in very few papers: Baldizzone and Scalercio [14] found Micropterix myrtetella Zeller, 1850 in Calabria; Zeller-Lukashort et al. [7] found M. allionella (Fabricius, 1794) in Basilicata, not in Campania as reported in their paper; Kurz et al. [15] recorded M. hartigi in Basilicata and Puglia.

The aim of this paper is to contribute to filling the gaps in knowledge on the diversity of Micropterigidae and Eriocraniidae in southern Italian regions by analysing the samples collected in this geographic area by the authors during last years through DNA barcoding. As a results, we expect (1) to improve molecular libraries for species identification by providing sequences of species not yet barcoded or previously barcoded from different geographic areas, (2) to refine faunistic knowledge of the southern Italian fauna, and (3) to identify cases in need of further taxonomic studies.

2. Materials and Methods

The examined material was collected in the Calabria region, the southernmost tip of the Peninsular Italy, mainly during surveys conducted in well-preserved mountain forest habitats and in highly fragmented forests of the plains, with only occasional daytime collection. Specifically, the surveys in which we collected Eriocraniidae involved maple forests and suburban areas, whereas Micropterigidae were collected in silver fir forests, olive groves with remnants of Mediterranean shrubs and riparian forests, fragmented thermophilous oak woodlots, beech–pine mixed forests and beech forests [16,17,18]. Further details on the sampled areas can be gathered in the cited literature.

Standardised samplings were performed utilising UV LED light traps, primarily targeting macromoths, but some voucher specimens of morphospecies belonging to microlepidoptera were also retained for future studies. Some specimens were occasionally collected during the daytime. The sampled Eriocraniidae and Micropterigidae specimens were successively submitted to DNA barcode analysis using SMRT sequencing on PacBio Sequel and SPRI DNA extraction at the Centre for Biodiversity Genomics, Canada. A total of 37 specimens, 32 Micropterigidae and 5 Eriocraniidae, were analysed and stored in the Lepidoptera collection of the Research Centre for Forestry and Wood, Rende, Italy. The sequences were identified by comparison with those available in the Barcoding of Life Data Systems (BOLDs) [19] using the “Full DB” search option available on the sequence page. Identification was provided along with a percentage of similarity to deposited sequences. The specimens were named after a careful screening of the tree-based identification (BOLD TaxonID tree), carried out on trees built using the Kimura 2 parameter as a distance model and considering the hundred most similar sequences. The recovered sequences were automatically assigned to a Barcode Index Number (BIN) [20], an online framework that algorithmically clusters barcode sequences, which in most cases correspond to a species [21,22,23]. Additionally, the Nearest Neighbour search was performed to identify the most similar taxonomic unit forming a BIN, further enhancing the taxonomic assessment of our specimens. All the data concerning the studied material are deposited in BOLD and available in the public dataset DS-PRIMITA Micropterigidae and Eriocraniidae of Southern Italy.

Finally, a cross-check of wing patterns and genitalia was performed for all the specimens, and a list of taxa resulting from molecular and morphological analyses was provided.

3. Results

Only 17 out of 32 Micropterigidae were successfully barcoded, recovering 644–658 bp long sequences clustering within five different BINs. One sample was contaminated, while no sequences were obtained for the remaining.

The most common species was Micropterix hartigi with Micropterix rothenbachii Frey, 1856 as the Nearest Neighbour (BOLD:ADK4986) (

Table 1. Summarised results of the samples analysed by DNA barcoding and morphology. BIN: Barcode Index Number; n: number of successfully and, in parenthesis, unsuccessfully barcoded specimens; N_BOLD: number of public records in BOLD (27 December 2024); NNdist: distance from the Nearest Neighbour; NA: not applicable.

BIN	n	NBOLD	Taxon	Faunistic Novelty	NNdist (p-dist)	Nearest Neighbour
BOLD:AAB3765	5 (0)	49	Dyseriocrania subpurpurella	New for Southern Italy	5.37	D. griseocapitella
BOLD:AAK6449	8 (5)	4	Micropterix hartigi	New for Calabria	2.45	M. rothenbachii
BOLD:AFS8746	4 (4)	0	Micropterix cassinella	New for Calabria	3.21	M. erctella
BOLD:AAN2269	2 (3)	1	Micropterix zangheriella	New for Calabria	1.12	M. rothenbachii
BOLD:ABZ4016	2 (0)	2	Micropterixtrinacriella	New for continental Italy	1.12	M. myrtetella
BOLD:ADK6125	1 (0)	0	Micropterix sp.	NA	1.64	M. schaefferi
NA	0 (3)	NA	Micropterix aruncella	New for Calabria	NA	NA
NA	0 (2)	NA	Micropterix sp.	NA	NA	NA

). A great genetic diversity was observed, with each specimen showing a different haplotype except for one pair and relatively high intra-BIN variability (average p-dist = 0.71; max p-dist = 1.44). The available Italian sequences are interspersed among the Calabrian ones (Figure 1a).

Four specimens of Micropterix cassinella Kurz, Kurz and Zeller, 2010 were barcoded for the first time and dissected to link the taxonomy to the sequence with certitude (Figure 2), with Micropterix erctella Walsingham, 1919 as the Nearest Neighbour (BOLD:ACJ9836). In this species, great genetic diversity was also observed, recovering three haplotypes, one of which was very divergent (Figure 1b). The intra-BIN average distances are similar to those observed for the previous species (average p-dist = 0.83; max p-dist = 1.61).

Two specimens were identified as Micropterix zangheriella Heath, 1963, with Micropterix rothenbachii as the Nearest Neighbour (BOLD:ADK4986) (Table 1). The intra-BIN average distance was 0.21 (p-dist), and the maximum distance was 0.32 (Figure 1a).

Two specimens belonged to Micropterix trinacriella Kurz, Zeller-Lukashort and Kurz, 1997, with Micropterix myrtetella as the Nearest Neighbour (BOLD:AFS9900). The intra-BIN average distance was 0.76 (p-dist), and maximum distance was 1.28, which is higher than the NNdist (Table 1). The high intra-BIN variation is due to the high dissimilarity between the Sicilian haplotype and the two observed Calabrian haplotypes, which differed by 1.26% and 1.55%, while the genetic divergence between the Calabrian ones was 0.78% (Figure 1c).

Only one barcoded Micropterix Hübner, 1825 female remained unnamed because the BIN was new to BOLD, and the female genitalia were not diagnostic. Its Nearest Neighbour was Micropterix schaefferi Heath, 1975 (BOLD:AAF7082) at a p-dist of 1.64 (Figure 1d).

Dissection of the genitalia showed that the unsuccessfully barcoded specimens belonged to the same species mentioned above, except for Micropterix aruncella (Scopoli, 1763), for which no sequences were obtained from three analysed specimens (Table 1). Two more females for which barcoding failed remained unidentified.

All the Eriocraniidae were successfully barcoded, recovering 652–655 bp long sequences. They belonged to Dyseriocrania subpurpurella (Haworth, 1828), with Dyseriocrania griseocapitella (Walsingham, 1898) as the Nearest Neighbour (BOLD:AAD3058) (Table 1). The intra-BIN average distance was 0.66 (p-dist), and the maximum distance was 2.25. One specimen was collected in the Pollino Massif and three in the Crati Valley. Three haplotypes were recovered with a maximum dissimilarity of 1.38% between them. The Pollino specimen shared the same haplotype with five specimens collected in Finland, Austria and the Italian Alps, while the specimens collected in the Crati Valley belonged to two different haplotypes and composed the basal dichotomy in the BIN cluster (Figure 1). They showed a dissimilarity ranging from 1.08% to 1.38% with the Pollino haplotype and a maximum dissimilarity of 0.61% between them.

4. Discussion

In this paper, we provide the first survey on the molecular diversity of Micropterigidae and Eriocraniidae in Southern Italy, demonstrating how field surveys can still provide valuable insights into the diversity and taxonomy of these families, even in Europe, probably the best-investigated continent in the world. In detail, we provide the first record and the first sequences of the Eriocraniidae family for Southern Italy, the first record in continental Italy of a formerly Sicilian endemic, the first sequences of an endemic species confirmed by genitalia morphology, and the first sequence belonging to either a previously described but unsequenced species or an undescribed species.

DNA barcoding failed in about 47% of our Micropterigidae samples, making this family one with a failure rate of 30.4% in BOLD. Their small size cannot explain this rate, as families composed of small species, such as Bucculatricidae and Gracillariidae, have significantly lower failure percentages, 5.3% and 7.2%, respectively, similar to those of the larger families such as Geometridae (9.2%) and Noctuidae (4.7%) (data from BOLD, accessed 13 January 2025). The presence of certain metabolites could likely interfere with DNA extraction and purification, significantly affecting the efficiency of DNA barcoding for this family.

Despite most species of Eriocraniidae and Micropterigidae having diurnal habits, they are sometimes attracted to light [7]. Most of our samples were collected using light traps, frequently set within dense forests. For example, during samplings carried out in a black pine–beech mixed forest of the Sila Massif, hundreds of Micropterix hartigi were collected with UV LED light traps (unpublished data).

Dyseriocrania subpurpurella has been reported from most European countries [24], but records are rarer in the south. In Italy, it was only recorded from the Alpine regions and Sicily [9], but the old Sicilian record concerning the form fastuosella Zeller, 1839 [25] has never been confirmed by new records. Our specimens were the first Eriocraniidae ever collected in Southern Italy. One specimen was collected in the Pollino Massif within an Acer-dominated forest at an elevation of 1280 m during a campaign devoted to the macromoth survey. One of these was collected on 17 April 2017 using a UV LED light trap. Another seven specimens, four of which were barcoded, were collected from late March to mid-April in a suburban area of the Crati Valley at about 200 m of elevation. These specimens were also collected at light during a long-term macromoth monitoring programme using a 160 W mercury vapour lamp [17]. This species was thus collected in very different habitats and with very different light sources. Interestingly, we found that the specimens collected in the Pollino Massif at the highest elevation shared the haplotype with some Finnish specimens collected at several thousand kilometres away, but they were clearly distinct from the other Calabrian specimens collected just a few tens of kilometres southward. The Pollino Massif (North Calabria) has been observed to be the southern range limit for haplotypes commonly found in Europe, as also for Thaumetopoea pityocampa (Denis & Schiffermüller, 1775) [26] and some butterflies [27,28,29], underlining the role of Central and Southern Calabria as one of Italy’s major glacial refugia [30,31,32].

Eriocraniidae showed a significantly lower differentiation rate across its range than Micropterigidae. In fact, none of the known European species can be considered endemic, as even the very recently described Eriocrania marci [33] was recorded in a large area of the Palaearctic region [26]. On the other hand, several Micropterigidae can be considered endemics [7], and most of them were found in Italy [34]. All the Micropterix species included in this paper are Italian endemic. Among them, the most interesting record concerns Micropterix trinacriella, previously considered a Sicilian endemic, now recorded for the first time in southern Italy. Our records demonstrated that M. trinacriella co-occurs in this region with its Nearest Neighbour, M. myrtetella, recently found in the Calabria region [14]. Unfortunately, both the collected specimens lacked genitalia due to a Dermestidae infestation during their stay on the spreading boards. Given the importance of this record, we will attempt to collect further specimens in the future to confirm their molecular identity through the morphology of the genitalia.

A morphological examination of the unsuccessfully barcoded specimens allowed us to recognise Micropterix aruncella as an additional species. The specimens belonged to the form nuraghella Amsel, 1936 in which silvery markings on the male forewings are absent, making it resemble Micropterix calthella (Linnaeus, 1761). The record of Tinea urticella Costa, 1834 for the Campania region, considered as a junior subjective synonym of M. calthella [7], could be attributed to the presence in the South of the previously undetected form of M. aruncella. In this case, it would become a junior subjective synonym of Micropterix aruncella. Only the examination of the male genitalia of the Tinea urticella type could clarify its taxonomy. One recovered BIN included one female that we could not attribute to any of barcoded species, suggesting that it belonged to a known species not yet barcoded or to a new, undescribed species. Further integrative taxonomic studies and, hopefully, the sampling of males may help to clarify this question.

5. Conclusions

DNA barcoding of Micropterigidae and Eriocraniidae in Southern Italy provided very interesting insights, improving their molecular libraries and expanding the knowledge of the region’s fauna. Daytime studies can further improve these results, but the need to fill the gaps concerning immature stages should be the primary goal of future research.