The family Axymyiidae includes four extant genera and nine species known from the Holarctic and Oriental regions, with only one species Mesaxymyia kerteszi (Duda, 1930) occurring in Europe. The genus Plesioaxymyia Sinclair, 2013 was first discovered in Alaska in 1962, but officially described only many years later. Up to now, the genus included one species Plesioaxymyia vespertina Sinclair, 2013, known from two female specimens from western North America. During the study of the Diptera fauna in Paanajarvi National Park (Northwest Russia), one female specimen of Plesioaxymyia, was found. It differs from the Nearctic P. vespertina by details of its female terminalia and is recognized as a new species, herein described as Plesioaxymyia imprevista sp. nov. The biology and geographic distribution of Plesioaxymyia is briefly discussed. The phylogenetic position of the genus, along with the family Axymyiidae is analyzed in the light of new molecular data, including sequences of three mitochondrial (ribosomal 12S and 16S and protein-encoding COI) and three nuclear genes (ribosomal 18S and 28S, protein-encoding CAD) for 72 terminal taxa. Axymyiidae is recovered as a monophyletic group with closest relatives in the infraorder Culicomorpha and Plesioaxymyia imprevista sp. nov. representing the sister taxon to all the other species of Axymyiidae included in the analysis.

Biology, distribution, lower Diptera, Nematocera, new species, Russia, systematics

The family Axymyiidae is often referred to as “enigmatic” in the literature (Wihlm and Courtney 2011; Blagoderov and Lukashevich 2013; Fitzgerald and Wood 2014). This small group of flies includes four extant genera and nine species known in the Holarctic and Oriental regions (Fitzgerald and Wood 2014). Additionally, eight species in four extinct genera have been described from the Jurassic or Early Cretaceous deposits of Asia (Blagoderov and Lukashevich 2013; Shi et al. 2013). Only one species, Mesaxymyia kerteszi (Duda, 1930), is so far known from Europe. This is an extremely rare taxon, with a few recent records from eastern Slovakia (Martinovský and Roháček 1993) and Northwest Russia (Polevoi et al. 2018).

As it is known so far, all immature records of Axymyiidae are associated with dead wood. Extremely specialized larvae live inside very wet, often submerged, logs of various tree species (Wood 1981; Krivosheina 2000). The systematic position of this family is still uncertain (see Blagoderov and Lukashevich 2013; Sinclair 2013; Fitzgerald and Wood 2014 for reviews). It is often placed in or as a sister group to Bibionomorpha, but close relationships with this infraorder are not always supported by reconstructions, based on molecular data. In the comprehensive study by Wiegmann et al. (2011), Axymyiidae were placed within Bibionomorpha sensu lato, as a sister group to Bibionomorpha sensu stricto, whereas in other studies this family was represented as a sister group to Culicomorpha (Bertone et al. 2008; Ševčík et al. 2016) or in an unresolved polytomy (Zhang et al. 2023).

During studies of the Diptera fauna in Paanajarvi National Park (Russia, Karelia), an unusual looking female specimen was collected, evidently belonging to the family Axymyiidae. Preliminary examination showed that it was not a representative of any previously known Palaearctic genus, but accords well with the genus Plesioaxymyia Sinclair, which includes one North American species, P. vespertina Sinclair, 2013. Being morphologically very similar, the Karelian specimen differs from P. vespertina by characters of its terminalia. Considering the general rarity of the family Axymyiidae and the evident importance of the new record we feel it necessary to describe the new species and not wait for additional material. Moreover, we could not miss an opportunity to re-evaluate the phylogenetic position of Plesioaxymyia and Axymyiidae as a whole, based on new molecular data for extant species.

The type specimen was collected in Paanajarvi National Park, located in the northwestern part of the Republic of Karelia, Russia (Fig. 1). The park was established in 1992, and presently occupies a territory of over 104,000 ha, mostly covered with natural coniferous forests (Bizhon and Systra 1996; Timofeeva and Kutenkov 2009). In 2021, we used three Malaise traps as part of an insect inventory program in the territory of the park. The Plesioaxymyia specimen was collected with a trap set in a patch of spruce- and pine-dominated forest of Vaccinium myrtillus type, in the vicinity of the abandoned village of Vartolambina. The operation period of the trap was from June 1–27, which corresponds to late spring. Willows and some early-season flowers (e.g., Tussilago) were in blossom, and remnants of snow still could be found in shady places.

Figure 1. 

Location of Paanajarvi National Park, Karelia, Russia.

The trap residue was initially kept in 70% alcohol, and one Axymyiidae specimen was recognized during a preliminary inspection in the laboratory. For a detailed study, the specimen was dried by xylol and amyl acetate baths (Achterberg 2009). Terminalia were detached and macerated in KOH for 24 hours, then neutralized in acetic acid, washed in 70% alcohol, and transferred to glycerine. Finally, terminalia were placed in glycerine vial and pinned together with the rest of the specimen. The holotype is stored in the collection of the Zoological institute, St. Petersburg, Russia (ZISP).

Images of the habitus and wing were taken with a Leica MZ 9.5 stereo microscope and those of the terminalia with a Leica DM1000 compound microscope, both supplied with a LOMO MC6.3 camera. Z-stacked image series were combined using Helicon Focus v. 8.2.0 software, and final plates prepared with GIMP. The morphological terminology follows Sinclair (2013). The distribution map was created using the online tool SimpleMappr, available at https://www.simplemappr.net/.

Molecular methods principally follow those described in Ševčík et al. (2016). A total of 72 terminal taxa are included in the dataset (Appendix 1). Most of the specimens used in this study were collected by Malaise traps during the years 2000–2022. Some sequences were taken from the GenBank database. The material used in the molecular study was stored in ethanol (70% to 96%) or pinned. For DNA extraction, we used a NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s protocol. PCRs (total volume = 20 μl) were performed using specific primers for sequences of three mitochondrial (ribosomal 12S and 16S and protein-encoding COI) and three nuclear genes (ribosomal 18S and 28S, protein-encoding CAD). The primers used for PCR amplifications and sequencing are listed in Table 1.

All amplified products were purified using a Gel/PCR DNA Fragments Extraction Kit (Geneaid, New Taipei City, Taiwan) following the manufacturer’s protocol. Purified products were sequenced by Macrogen Europe (Netherlands) or Eurofins Genomics (Germany). The sequences were assembled and edited in Sequencher v. 5.0 (Gene Codes Corporation, Ann Arbor, MI, USA) or SeqTrace v. 0.9.0 (Stucky 2012). GenBank accession numbers for all the sequences are listed in the Appendix 1. All sequences were checked with the NCBI database using the BLAST and in single-gene trees to avoid possible contamination or other inappropriate results.

Alignments of all genes were created using MAFFT v. 7 on the MAFFT server (http://mafft.cbrc.jp/alignment/server/). The resulting alignments were visually inspected and manually refined in BioEdit v. 7.2.5 (Hall 1999) when necessary. All unreliably aligned regions of rRNA genes were removed in the program GBLOCKS v. 0.91b (Castresana 2000); with conditions set as follows: allow smaller blocks, allow gap positions within the final blocks, allow less strict flanking positions and do not allow many contiguous non-conserved positions, or in ClipKIT v. 2.2.2 (Steenwyk et al. 2020) using the -gappy option with a threshold of 0.7. The third positions of COI gene were excluded from the subsequent analyses using software DAMBE (Xia and Xie 2001). The alignments were concatenated using FASconCAT v. 1.0 (Kück and Longo 2014). The final data matrix consisted of 5419 characters: 12S – 301 bp, 16S – 287 bp, 18S – 1985 bp, 28S – 1005 bp, COI – 436 bp (third positions removed) and CAD – 1405 bp.

The final concatenated dataset was partitioned by gene and codon position and subsequently analysed using the maximum likelihood (ML) method. Analyses were conducted using IQ-TREE v. 1 (Nguyen et al. 2015) on the IQ-TREE web server (Trifinopoulos et al. 2016). Best-fitting substitution models were chosen automatically by the IQ-TREE software: 12S – TVM+F+I+G4; 16S – TVM+F+I+G4; 18S – GTR+F+I+G4; 28S – TVM+F+I+G4; CAD_1 – TN+F+I+G4; CAD_2 – SYM+I+G4; CAD_3 – GTR+F+I+G4; COI_1 – TIM2+F+I+G4; COI_2 – TIM3+F+I+G4; without free-rate heterogeneity. Branch supports were evaluated using 1000 ultrafast bootstrap (Hoang et al. 2018). All other settings were left as default. The node support values are given in the form of ultrafast bootstrap (= ufboot). The resulting phylogenetic tree (consensus trees) was visualized using the Interactive Tree of Life (iTOL v. 7.0; Letunic and Bork 2024). A species of Mecoptera, Boreus hyemalis (Linnaeus, 1767), was used as a root.

Our reconstruction of the molecular phylogeny is the first to incorporate representatives of all extant genera of Axymyiidae, although this still represents only approximately half of the known species. Axymyiids constitute a relatively isolated group, with the Culicomorpha families representing its closest evolutionary relatives. Both groups share an aquatic or semi-aquatic larval habitat. The sister group relation of Axymyiidae to Culicomorpha is in agreement with the findings of Bertone et al. (2008) and Ševčík et al. (2016) and is now evidenced with much better (ufboot = 84) support. While this may not yet be considered as strongly reliable, it is assumed that it could be even better supported if more taxa were included.

The current, unexpected placement of the species in relation to the Axymyiidae genera may be an artefact caused by the incomplete DNA data for Mesaxymyia kerteszi and Axymyia furcata. However, new molecular data provide additional background for the necessity of a revised generic classification of Axymyiidae. It is likely that such a revision will not be possible until details of the morphology of preimaginal stages and, preferably, also genetic data on all extant species are available.

We express our sincere gratitude to the staff of Paanajarvi National Park, and especially to Anastasiya Protasova for their assistance during the field studies. We also highly appreciate help from Elena Lukashevich (Moscow, Russia) and Andrei Przhiboro (St. Petersburg, Russia) with some important literature sources on the family Axymyiidae. The field research in Taiwan was carried out in cooperation with the National Chung Hsing University, Taichung, and the National Museum of Natural Science, Taichung. We are especially grateful to Sheng-Feng Lin and Michal Tkoč for their help with the field work in Taiwan. Peter Chandler (Melksham, UK), Bradley Sinclair (Ottawa, Canada) and Scott Fitzgerald (Corvallis, USA) kindly read the manuscript and provided useful comments.