First Morpho-Functional Assessment of Immature Stages of Pelecocera Species (Diptera: Syrphidae) Feeding on False Truffles

Simple Summary The scarce genus of Pelecocera Meigen, 1822 (Diptera: Syrphidae) from the Holarctic Region has 14 species described. The taxonomic diagnosis of the immature stages of Pelecocera has not been performed; however, Pelecocera (Chamaesyrphus) japonica (Shiraki, 1956) larvae were found feeding on Rhizopogon roseolus in Japan. Following the findings in Japan, larvae of Pelecocera were collected in Denmark. We here report the first morphological description of the immature stages of Pelecocera (Chamaesyrphus) lugubris and Pelecocera (Pelecocera) tricincta, as well as specific data on their breeding sites. Larvae of both species were collected feeding on Rhizopogon luteolus in Denmark. The morphology of immature stages of P. lugubris and P. tricincta was studied by using both a scanning electron microscope and a stereomicroscope. A taxonomic diagnosis of the immature stages of Pelecocera and a taxonomic key are provided to separate them from the larvae of other genera. Abstract With 14 species, Pelecocera Meigen, 1822 is a scarce and small genus of hoverflies (Diptera: Syrphidae: Rhingiini) from the Holarctic Region. Apart from the finding of larvae of Pelecocera (Chamaesyrphus) japonica (Shiraki, 1956) in fungi in Japan, the larval biology of these hoverflies is virtually unknown. The early stages of all Pelecocera species are undescribed. The adults of Pelecocera (Pelecocera) tricincta Meigen, 1822 and Pelecocera (Chamaesyrphus) lugubris Perris, 1839 are found in Palearctic conifer forests with sand dunes. We here report the first morphological evidence of the immature stages of Pelecocera (P. lugubris and P. tricincta), as well as specific data on their breeding sites. Larvae of both species were collected feeding on the hypogean basidiomycete Rhizopogon luteolus Fr. & Nordholm, 1817 in Denmark in 2021. The first larval stage and second larval stage of P. tricincta, the third larval stage of P. lugubris, the anterior respiratory process, and the posterior respiratory process of the puparia of these two species were analyzed and studied using stereomicroscope and scanning electron microscope techniques. The chaetotaxy of the puparium of each species is also described and illustrated. A taxonomic diagnosis of the larvae of the genus Pelecocera is proposed to separate them from the larvae of other genera of the tribe.

Pelecocera (Pelecocera) tricincta Meigen, 1822 and Pelecocera (Chamaesyrphus) lugubris Perris, 1839 are two of the eight species occurring in Europe [4].However, the number of species in Europe remains unclear because a worldwide revision of the genus is needed to resolve all the taxonomic doubts that exist at the species level.Pelecocera tricincta can be found from the Iberian Peninsula to Siberia, including the Caucasus [12], while P. lugubris is present from Portugal to Scandinavia [4].The distribution of range of both species might be wider than currently known, especially that of P. lugubris, due to inaccurate knowledge of their adult phenology and inconspicuous presence in the field.
The adults of Pelecocera can be distinguished from other genera by their narrow black thorax, black with yellow abdominal spots, small size (<10 mm), thickness and position of the arista (hair-like and near the base of the basoflagellomere in Pelecocera (Chamaesyrphus) and thick and near the tip of the basoflagellomere in Pelecocera (Pelecocera), bare metasternum, and straight vein R 4+5 and crossvein r-m before the middle of the cell dm [1,13].
Due to the almost total absence of data on the larval morphology, there is no taxonomic diagnosis of the immature stages of Pelecocera, and there are no keys to separate larvae of this genus from those of other hoverfly genera.Only the morphological sculpture of the egg of Pelecocera (Chamaesyrphus) lusitanica (Mik, 1898) and P. tricincta was described by Kuznetzov [14].
This study aims to provide the first evidence of the larval morphology of Pelecocera hoverflies worldwide, as well as further data on their breeding sites in Europe from the findings of two species, P. tricincta and P. lugubris.In addition, a taxonomic diagnosis for the larvae of the Pelecocera species and a comparison between the immature stages of related genera are provided.

Examined Material and Adult/Larva Identification
Fruiting bodies of R. luteolus with both species (P.lugubris (n = 8) and P. tricincta (n = 9)) were found on several locations in Denmark, such as Hvidbjerg Klitplantage (56.8619, 8.3325) and Svinkløv Klitplantage (57.1420, 9.3033) (Figure 1) by Leif Bloss Carstensen in September 2021.The "Klitplantage" is a dune plantation made to prevent sand drifts.Most of the trees present on both sites are conifers, especially Pinus mugo Turra, 1764 (Figure 2).The fruiting bodies were at ground level up to ten meters from pine trees.Only a few specimens of l1 were checked for larvae on site, and not all were infested.Some fruiting bodies and the sand underneath had over 30 larvae (Leif Bloss Carstensen, pers.com.).The larvae were reared in several small plastic containers with one or few fruiting bodies from the same location.The containers were stored in a cupboard in a partially shaped part of a carport at environmental temperature (−15 • C to 32 • C) (September 2021-September 2022).The containers were checked daily to record changes in the immature development.Three (one first stage (L1) and two second stage (L2)) larvae of P. tricincta and one third stage (L3) larva of P. lugubris were preserved in 70% alcohol, and the rest were reared for adult identification.Six larvae of P. tricincta and seven larvae of P. lugubris pupated and five adults of each species emerged from these puparia (Figure 3).The match between the larvae and the pupae was based on the features of the posterior respiratory process (PRP).Adults were identified using the taxonomic key of Lair et al. [4].Examined species were deposited at the CEUA-CIBIO collection, University of Alicante, Spain.
pupated and five adults of each species emerged from these puparia (Figure 3).The match between the larvae and the pupae was based on the features of the posterior respiratory process (PRP).Adults were identified using the taxonomic key of Lair et al. [4].Examined species were deposited at the CEUA-CIBIO collection, University of Alicante, Spain.pupated and five adults of each species emerged from these puparia (Figure 3).The match between the larvae and the pupae was based on the features of the posterior respiratory process (PRP).Adults were identified using the taxonomic key of Lair et al. [4].Examined species were deposited at the CEUA-CIBIO collection, University of Alicante, Spain.

Sample Preparation and Study
The pupae were cleaned in an ultrasonic bath for 10 minutes and brushed to remove any dirt.The head skeleton was removed from one L2 larva of P. tricincta by soaking it in hot 10% KOH for 5 minutes, and it was examined in glycerin.General features of the larva, puparium, and head skeleton were observed under a Leica M205 C binocular stereomicroscope (Leica Camera AG, Wetzlar, Germany).The asterisk (*) in the section 'Immature stages of Pelecocera: taxonomic diagnosis' indicates the most distinguishable characters.The length/width of the larva/puparium were measured at their maxima, with the width always in the abdomen (Figure 10).The measurements of pupal spiracles were length from the base to the apex, width at the maximum point in the middle, and space between the apices of the pupal spiracles (Figure 12E).For the PRP, the width was measured at the transverse ridge and the length above/below the transverse ridge.Photos were produced as stacks of individual images made with a camera (Leica DMC 5400, Leica Camera AG, Wetzlar, Germany) attached to a binocular stereomicroscope (Leica M205 C).Stacks were made in Leica Application Suite Las X ® , v.4.12.0,Leica Microsystems, Wetzlar, Germany.The drawings were made from printed photos.The colors on the head skeleton drawings indicate the level of sclerotization (lighter = less, dark = heavy).The distribution map of the collected Pelecocera larvae in Denmark was produced with the software QGis 3.32 [15].For a more detailed description of the anterior respiratory process (ARP), pupal spiracles, and PRP, a scanning electron microscope (SEM) was used.One puparium and one larva were mounted on aluminum stubs with double-sided adhesive carbon tape.The samples were imaged with a Jeol JSM-IT500HR SEM (JEOL Ltd., Tokyo, Japan) in variable pressure mode to be able to recover the material.

Sample Preparation and Study
The pupae were cleaned in an ultrasonic bath for 10 min and brushed to remove any dirt.The head skeleton was removed from one L2 larva of P. tricincta by soaking it in hot 10% KOH for 5 min, and it was examined in glycerin.General features of the larva, puparium, and head skeleton were observed under a Leica M205 C binocular stereomicroscope (Leica Camera AG, Wetzlar, Germany).The asterisk (*) in Section 3.4 indicates the most distinguishable characters.The length/width of the larva/puparium were measured at their maxima, with the width always in the abdomen (Figure 10).The measurements of pupal spiracles were length from the base to the apex, width at the maximum point in the middle, and space between the apices of the pupal spiracles (Figure 12E).For the PRP, the width was measured at the transverse ridge and the length above/below the transverse ridge.Photos were produced as stacks of individual images made with a camera (Leica DMC 5400, Leica Camera AG, Wetzlar, Germany) attached to a binocular stereomicroscope (Leica M205 C).Stacks were made in Leica Application Suite Las X ® , v.4.12.0,Leica Microsystems, Wetzlar, Germany.The drawings were made from printed photos.The colors on the head skeleton drawings indicate the level of sclerotization (lighter = less, dark = heavy).The distribution map of the collected Pelecocera larvae in Denmark was produced with the software QGis 3.32 [15].For a more detailed description of the anterior respiratory process (ARP), pupal spiracles, and PRP, a scanning electron microscope (SEM) was used.One puparium and one larva were mounted on aluminum stubs with double-sided adhesive carbon tape.The samples were imaged with a Jeol JSM-IT500HR SEM (JEOL Ltd., Tokyo, Japan) in variable pressure mode to be able to recover the material.

Morphological Terminology
The terminology used for the description of larvae and puparia follows Rotheray [16].For each body segment, sensilla were numbered in the dorsoventral direction [17].A superscript ( A1. . . ) is used to indicate in which body segment a sensilla is located (e.g., 1 A1 -first sensilla of the first abdominal segment).The terminology used for the head skeleton follows Hartley [18].A compilation of abbreviations for morphological features used in this publication is shown in Table 1.Vermiform larva with the eighth abdominal segment (=anal segment) small and abruptly truncated with three pairs of well-developed lappets (Figure 4 and Figure 13).Prothorax with a pair of well-developed antenna-maxillary organs slightly sclerotized, mounted on a fleshy projection (Figure 6).Dorsal laterally of the prothorax with a pair of small, sclerotized ARP with wrinkled surface basally, smooth at the rest, and the apex with a tip with two spiracular openings (Figure 7).A pair of developed locomotory prominences without crochets in the mesothorax and from the first to the seventh abdominal segments (Figure 4B and Figure 13B).Outline of the PRP in dorsal view M-shaped PRP; tapering toward the apex in the lateral view but swollen above the transverse ridge (Figure 8).Spiracular plate with four pairs of long interspiracular setae, three pairs of spiracular openings, a pair of ecdysial scars, and a pair of perispiracular glands (Figure 9).Chaetotaxy (Figure 10): all observed sensilla-bearing setae.Prothorax: dorsal side with four pairs of sensilla (1 Pt -4 Pt ), lateral side with three pairs (5 Pt -7 Pt ) and ventral side with one pair (8 Pt ).Mesothorax: dorsally with three pairs of sensilla (1 Ms -3 Ms ), laterally with two pairs (4 Ms -5 Ms ), and ventrally with three pairs (6 Ms -8 Ms ).Metathorax: dorsally with three pairs (1 Mt -3 Mt ), laterally with two pairs (4 Mt -5 Mt ), and ventrally with three pairs (6 Mt -8 Mt ).Abdomen: from the first to the seventh abdominal segments dorsally with three pairs of sensilla (1 A1-7 -3 A1-7 ), laterally with four pairs (4 A1-7 -7 A1-7 ), and ventrally with three pairs (8 A1-7 -10 A1-7 ).Anal segment with two pairs of sensilla (1 A8 -2 A8 ) at the tip of the dorsal lappet; two pairs (3 A8 -4 A8 ) at the lateral side of the PRP; two pairs (5 A8 -6 A8 ) at the tip of the ventral PRP lappet; one pair (7 A8 ) at the tip of the ventral lappet; and three pairs (8 A8 -10 A8 ) ventrally.

L2 Larva (Figure
small and highly sclerotized with some parts less sclerotized (only appreciable at ventral view Figure 5B); dorsal cornu of same length as ventral cornu; pharyngeal ridges of same length as ventral cornu.PRP: yellowish, with a noticeable transverse ridge.small and highly sclerotized with some parts less sclerotized (only appreciable at ventral view Figure 5B); dorsal cornu of same length as ventral cornu; pharyngeal ridges of same length as ventral cornu.PRP: yellowish, with a noticeable transverse ridge.

Immature Stages of Pelecocera: Taxonomic Diagnosis
Mesothorax and first seven abdominal segments with well-developed crochetless locomotory prominences; outline of the PRP in the dorsal view M-shaped PRP* (Figure 8C); eighth abdominal segment small and abruptly truncated posteriorly.In Table 2, we can see a comparison between the considered sister groups of Pelecocera.

Immature Stages of Pelecocera: Taxonomic Diagnosis
Mesothorax and first seven abdominal segments with well-developed crochetless locomotory prominences; outline of the PRP in the dorsal view M-shaped PRP* (Figure 8C); eighth abdominal segment small and abruptly truncated posteriorly.In Table 2, we can see a comparison between the considered sister groups of Pelecocera.

Immature Stages of Pelecocera: Taxonomic Diagnosis
Mesothorax and first seven abdominal segments with well-developed crochetless locomotory prominences; outline of the PRP in the dorsal view M-shaped PRP* (Figure 8C); eighth abdominal segment small and abruptly truncated posteriorly.In Table 2, we can see a comparison between the considered sister groups of Pelecocera.As a result of our study, the diagnosis of the larvae of Pelecocera will be added to the taxonomic key of syrphid larvae of Thompson and Rotheray [26] in step 10, and it will be modified as follows:

Discussion
Rotheray [27] mentions that all taxonomic keys for immature stages of hoverflies are provisional because there are many genera with undescribed larvae or some have few specimens available for description.In fact, in this work, we are adding the diagnosis of the genus Pelecocera to the general knowledge of the early stages of the hoverfly.According to the assessment of the characters states performed, the shape of the PRP appears to be one of the useful characters for this genus.
The immature stages of Pelecocera (Pelecocera) tricincta and Pelecocera (Chamaesyrphus) lugubris shared many features supporting the fact that they are two subgenera instead of two different genera as mentioned in Vujić et al. [7].Currently, the only way to distinguish these two subgenera is by the color of the PRP; that of P. tricincta is yellowish (Figure 8A), and that of P. lugubris is dark brown (Figure 8B).This feature must be taken with caution, as more immature stages of Pelecocera are found, and this difference may change.
Okada et al. [11] found the larvae of P. japonica feeding and developing in R. luteolus and R. roseolus.They did not describe the larvae but confirmed that Pelecocera larvae are true mycophagous, through a gut content analysis, which found undamaged spores of Rhizopogon.This is the same as observed with P. tricincta and P. lugubris, as both species were observed developing on fresh and rotten R. luteolus.This implies that the host fungi of the Pelecocera species probably are species of Rhizopogon, a genus of ectomycorrhizal basidiomycetes, which form hypogeous sporocarps commonly known as false truffles.
Rhizopogon is a worldwide fungus genus with over 100 species that are very specific to pine trees, especially those of the genus Pinus (Pinaceae) [28,29].For this reason, Rhizopogon has been used in many works related to applications and research in forestry [30,31].However, during the last decades, the R. roseolus has been declining due to the destruction of its coastal habitat in Japan [32].This is a very important problem for Pelecocera as it is very specific to this fungus genus, as was observed in Okada et al. [11] and in our work.For this reason, it is necessary to improve or develop projects for the conservation of the habitat of these fungi, because if they were to disappear, Pelecocera would also disappear.
Ferdinandea and Portevinia, with two stages and one immature stage described, respectively, are the sister groups of Pelecocera [7,9,12].Even though they are genetically related, larvae of these three genera do not share the same trophic habits and breeding sites: Ferdinandea larvae are saprophagous (feeding on tree sap) [27]; Portevinia larvae are phytophagous (feeding on Allium bulbs) [21], and Pelecocera larvae are mycophagous (feeding on Rhizopogon fungi).These differences in the ecology of these three genera show the importance of studying the larvae and not only the adults.Another difference is the morphology of the eighth abdominal segment, which is small and abruptly truncated (Figures 4B and 13B) in P. lugubris and P. tricincta, slightly truncated in Ferdinandea cuprea (Scopoli, 1763) and Ferdinandea fumipennis Kassebeer, 1999 [19,22,25], and flat in Portevinia maculata (Fallén, 1817) [21].These features are the most useful to differentiate these three genera.
Mycophagy is not very common in the immature stages of syrphids, with Cheilosia Meigen, 1833 being the genus with the most species with this food spectrum.Unfortunately, the head skeleton of the mycophagous species is not very well known.According to Rotheray and Gilbert [17], in the head skeleton of mycophages, the mandibular lobes are not fused with the mandibular apodemus, and the mandibles and mandibular lobes are slightly sclerotized.All these features can be observed in the head skeleton of Pelecocera, confirming that this genus feeds on fungi.Another feature that can be observed is the presence of pharyngeal ridges, which filter and concentrate the food to gain a higher nutritional value [33].Pharyngeal ridges are not exclusive to mycophages, as they can be found in the immature stages of saprophages (e.g., Eumerus Meigen, 1822) [34] and saproxylic hoverflies (e.g., Milesia Latreille, 1804) [35].
With the information provided in this work, it is hoped that more immature stages of other Pelecocera species can be found to better understand their larval biology.In addition, future work will be performed to find the egg, L1, and L2 of P. lugubris and L3 of P. tricincta to have more complete information of the morphology of all immature stages and thus be able to facilitate the precise diagnosis of the immature stages of the genus Pelecocera.

Table 1 .
Abbreviations used for morphological features of larvae/puparia.