The Kallahdenniemi recreational area of the city of Helsinki (60.1870N 25.1445E) is situated in eastern Helsinki (Finland). This sandy soil forested area is dominated by Scots pine (Pinus sylvestris). From this area, the hoverflies Pelecocera caledonica and Pelecocera tricincta have been repeatedly recorded (https://laji.fi/). During several visits to Kallahdenniemi in late August 2023, the mentioned hoverfly species were observed. In autumn 2023, multiple fungal fruit bodies (sporocarps) of Rhizopogon sp. were found (Figs 1, 2), occurring in association with both young and mature Scots pines. In the field, some Rhizopogon fruit bodies were inspected for the presence of hoverfly larvae by making a slit using a sharp-edged knife to expose the inner fungal tissue for visual inspection. Multiple both young and mature sporocarps of Rhizopogon were inspected in Kallahdenniemi and larvae identified as hoverfly larvae were observed in a few sporocarps (Fig. 3). On 17 September and 2 October 2023, altogether six mature sporocarps of brownish-yellow colour (all about 3-4 cm in diameter) were taken for rearing. Sporocarps were placed together in a plastic jar with tissue paper on the bottom and kept outdoors in the shade. The sporocarps decayed into a liquefied olive-brown soup within 2-3 weeks after collection. From the jar with the liquefied sporocarps, five second or third instar larvae were removed and placed individually in tubes for immediate DNA analysis. The plastic jar still contained at least 20 additional hoverfly larvae (smaller and larger) and was kept outdoors until the beginning of November when severe frost nights occurred. Then puparia were searched for in the organic debris (leaves, small twigs etc.) amongst the tissue paper and on the surface area of "the soup". The puparia which had developed pupal horns were removed from the jar and placed individually in Petri dishes for emergence of adult flies. Emerged flies were pinned and the empty puparia glued on cardboard.

Figure 1.  

Rhizopogon luteolus. Helsinki, Kallahdenniemi. Photo: Gunilla Ståhls.

Figure 2.  

Rhizopogon luteolus. Helsinki, Kallahdenniemi. Photo: Gunilla Ståhls.

Figure 3.  

Pelecocera spp. larva, lateral view. Photo: Elvira Rättel and Gunilla Ståhls.

DNA was extracted from each entire larva using the Phire™ Tissue Direct PCR aster Mix #F-170S kit (Thermo Scientific Baltics UAB, Vilnius, Lithuania) following the Dilution & Storage protocol with some modifications. The Phire™ Tissue Direct PCR aster Mix is designed to perform PCR directly from tissue samples with no prior DNA purification. The larva was placed in an Eppendorf tube in 40 µl of Dilution Buffer and 0.8 µl of DNA Release Additive was added. The tube was briefly vortexed and centrifuged and then: 1) incubated at room temperature for about 20 min, 2) placed in +56°C for 10 min and 3) placed in a pre-heated block at 98°C for 2 minutes (after this stage, the larvae were removed and put in individual tubes with about 25% ethanol) and finally centrifuged at 11,000 rpm for 1 min. Two µl of supernatant was used in a 25 µl PCR reaction using the PCR Master Mix solution provided with the kit. The mtDNA COI barcode was PCR-amplified using universal primers LCO1490 and HCO2198 (Folmer et al. 1994) or a shorter fragment using primers Beet (Simon et al. 1994) and HCO2198. Amplified PCR products were electrophoresed on 1.5% agarose gels. Successful amplifications were treated with Exo-SapIT (USB Affymetrix, Ohio, USA) prior to sequencing. The PCR primers were used for sequencing, which was outsourced to the Sequencing Service Laboratory of FIMM Genomics (www.fimm.fi). The sequences were edited for base-calling errors and assembled using Sequencher™ (version 5.0) (Gene Codes Corporation, Ann Arbor, MI, USA) and selected barcode sequences were submitted to GenBank.

From the newly-obtained COI barcodes, three full length barcodes were added to a COI barcode data matrix including 25 sequences of Pelecocera spp. mined from the NCBI GenBank database (www.ncbi.nih.gov). Tree-based identification (Hebert et al. 2003) was based on comparison of obtained COI barcodes with those available for European species and was with the Neighbor-Joining method (Saitou and Nei 1987) under the Kimura 2-parameter substitution model (Kimura 1980) using the software MEGA11 (Tamura et al. 2021). All positions with less than 95% site coverage were eliminated, i.e. fewer than 5% alignment gaps, missing data and ambiguous bases were allowed at any position (partial deletion option). The barcode sequence of Pseudopelecocera latifrons (Loew, 1856) (GenBank accession number PP446810) was included to root the tree. The identification of the obtained adult flies was confirmed using keys provided in Van Eck and Mengual (2021).

Stacked images of specimen external morphology were taken with a Canon EOS 40S digital camera using d-cell software v. 5.1. Images were combined using Zerene stacker software v. 2, based on 50-100 exposures of the subjects.

The sporocarps or fruit bodies were readily identified as Rhizopogon luteolus. The sporocarps of Rhizopogon luteolus are 1.5-5 cm in diameter, without stem, variable in shape being roundish, ovoid or of irregular shape (Grubisha et al. 2002). The fruit bodies are initially yellow in colour (Fig. 1), but turning brownish-yellow upon maturity (Fig. 2). The outer skin is tough and the inner tissue (gleba) is initially pale-yellow and solid, but becomes olive-brown and liquefied in mature and senescent fruit bodies. Rhizopogon luteolus occurs throughout most of mainland Europe, but is common only in sandy soil, pine-forested parts of northern Europe. In Finland, the species is recorded up to the 68° latitude and fruit bodies occur mainly in August and September (The Finnish Biodiversity Information Facility, https://laji.fi/observation/list?target=MX.236304).

MtDNA COI barcodes obtained from three out of the five tested larvae were used for tree-based identification. The COI barcode dataset used for tree-based identification included altogether 28 nucleotide sequences of Pelecocera spp. and Pseudopelecocera latifrons as root of the tree (GenBank accession numbers indicated in Fig. 4). The Neighbour-Joining analysis found the newly-obtained COI barcodes to be identical with those of the species of Pelecocera (Pelecocera) tricincta and Pelecocera (Chamaesyrphus) caledonica, respectively (Fig. 4). The COI barcode sequences for P. tricincta comprised 658 nucleotide and for P. caledonica 629 nucleotide positions and the DNA sequence matrix comprised 658 positions (inserting ? for the missing data). In the study of Lair et al. (2022), the authors noted that the COI barcodes of Pelecocera pruinosomaculata and P. scaevoides were resolved as intermixed in their tree, with a mean distance of 0.8% between the taxa. This study utilied part of the same sequences used by Lair et al. (2022) and uses the same taxon names and also recovers the COI barcodes of mentioned taxa as clustering together (Fig. 4).

Figure 4.  

Neighbour-Joining tree for mtDNA COI barcodes of Pelecocera spp.

Figure 5.  

Pelecocera caledonica, female head. Photo: Elvira Rättel and Gunilla Ståhls.

Figure 6.  

Pelecocera caledonica, empty puparium in dorsal view. Photo: Elvira Rättel and Gunilla Ståhls.

Each mature Rhizopogon luteolus sporocarp, which was taken from the field for rearing, contained some Pelecocera spp. larvae. The rearing process found that the pupae is the overwintering developmental stage. Okada et al. (2021) reported that the Pelecocera japonica larvae they found dwelled in the liquefied decaying inner tissue (gleba) of the specimens of Rhizopogon spp. The larvae of P. tricincta and P. caledonica here reported from Rhizopogon luteolus fruit bodies were likewise only found in liquefied fungal tissue of olive-brown colour and with an oily viscosity. The larvae were submerged in the liquefied fungal tissue with their posterior respiratory tube (prp) thrust out of the material. The liquefied material did not fill the inner space of the sporocarrp completely, leaving a non-filled small air space in the dorsal part (about 1/10^th of the gleba volume). This agrees with the description of Rotheray (1990) of Cheilosia larvae which develop in Boletaceae species. These larvae are also submerged in the decayed Boletaceae fungi of semi-liquid consistenc with the prp out of the decayed material.

The mouth parts of the inspected third instar larvae of the Pelecocera spp. in this study agree with structures indicated for fungus feeding Cheilosia larvae (Rotheray 1990). These structures are a cephalopharyngeal skeleton with a small dorsal mouth-hook not protruding from the mouth and fleshy mandibular lobes and a vestiture of the larvae consisting of rows of setae (Fig. 3). Images are provided for both Pelecocera tricincta (Fig. 7) and Pelecocera caledonica to illustrate the vestiture and other structures in the puparia (Fig. 6). Descriptions of the larvae and puparia for both Pelecocera caledonica and P. tricincta will be provided elsewhere (in prep.). Photos of the heads of the adult flies of the reared species are provided for morphological identification (Figs 5, 8).

Figure 7.  

Pelecocera tricincta, empty puparium in dorsal view. Photo: Elvira Rättel and Gunilla Ståhls.

Figure 8.  

Pelecocera tricincta, male head in anterior view. Photo: Elvira Rättel and Gunilla Ståhls.

Pennards (2020a) and Pennards (2020b) noted that Pelecocera caledonica and P. tricincta are broadly distributed in both northern and southern Europe. His Red List assessment indicated that potential conservation actions for the species should focus on the protection of the habitat where flies occur, but that the species are known to occur in both protected areas and in commercially harvested forests. I searched for False Truffles in Helsinki, Kallahdenniemi in September 2022, with no findings. Comparing monthly rainfalls of year 2022 and 2023, the monthly rainfalls were 2-3x higher for July, August and September in Helsinki in 2023 (Finnish Meteorological Institute https://www.ilmatieteenlaitos.fi/).

In September 2023, the Yellow False Truffles containing Pelecocera spp. larvae in the Kallahdenniemi recreational area were found immediately adjacent to footpaths between the public beach and car parking areas and about 10 sporocarps were observed in total in a small area with about 2 m radius. The occurrence of fungi in general is partly dependent on the amount of rainfall during the season, with the 2023 season receiving more rain in southern Finland and many False Truffles were obseved. The observed fruit bodies occurred in (late) autumn when the Kallahdenniemi area is less used by the public. It is not known to which extent human activities (e.g. trampling) could potentially affect the occurrence of the Rhizopogon fungi and any immediate conservation actions does, therefore, not seem warranted.