Review of the Merodon natans group with description of a new species, a key to the adults of known species of the natans lineage and first descriptions of some preimaginal stages

Merodon natans group (Diptera, Syrphidae) taxa are reviewed using an integrative taxonomic approach combining morphological, morphometric and molecular techniques. The approach substantiates recognition of the three species: M. calcaratus (Fabricius, 1794), M. natans (Fabricius, 1794) and M. pulveris Vujić & Radenković in Radenković et al. 2011, and reveals the existence of a new species, M. makrisi Vujić, Radenković & Tot sp. nov., which is described. It also highlights the existence of a series of natans group populations, especially on some of the Mediterranean islands, in the Levant and in the Afrotropical Region, for which more comprehensive data are required to clarify their status. A key is provided to the natans lineage species currently recognised, and preimaginal stages of some natans-group species are described for the first time. Redescriptions for M. calcaratus and M. natans are provided. A neotype is selected for M. natans. Lectotypes are designated for M. annulatus (Fabricius, 1794) and M. melancholicus (Fabricius, 1794). Merodon annulatus is recognised as a synonym of M. natans.


Introduction
The phytophagous hoverfly genus Merodon Meigen, 1803 (Diptera, Syrphidae) contains 234 species distributed in the Palaearctic and Afrotropical regions, and introduced into North America and New Zealand (Vujić et al. 2021). Adults feed on flowers, ingesting pollen and nectar, whereas their larvae feed on bulbs and other underground storage organs of geophytes (i.e., Asparagaceae, Amaryllidaceae and Iridaceae). Several species-specific associations between Merodon species and geophyte hostplants have been confirmed; e.g., through records of oviposition behavior (Reemer and Goudsmits 2004), direct bulb-feeding (Popov 2010;Djan et al. 2020), and captive rearing of larvae (Pehlivan and Akbulut 1991;Stepanenko and Popov 1997;van Eck 2016). However, the larval food-plants and natural history remain unknown for the great majority of Merodon species and the immature stages have been described for only nine species within this genus so far (Heiss 1938;Stuckenberg 1956;Ricarte et al. 2008Andrić et al. 2014;Preradović et al. 2018;Vujić et al. 2020a).
The Merodon natans lineage contains the M. natans group and M. segetum (Fabricius, 1794) as an individual taxon. The Merodon natans group contains medium sized species with distinct pollinose ornamentation, vittae and fasciae on the scutum, and terga 2-4 with broad pollinose fasciae. This group includes three already known species, M. calcaratus (Fabricius, 1794), M. natans (Fabricius, 1794) and M. pulveris Vujić & Radenković in Radenković et al. 2011, recently revised by Radenković et al. (2011). In addition to marked morphological and molecular (5'-end COI sequence) differences between M. natans and M. pulveris (Radenković et al. 2011), Arok et al. (2019 provided geometric morphometric evidence for these species delimitation based on wing parameters. In the present work, a fourth species belonging to this group is described. The overall morphological similarity of these four taxa and the availability of the mentioned datasets prompted us to use an integrative approach to decipher the taxonomy of this species group. The aims of the present study are: 1) to review specimens of this group deposited in several entomological institutions and private collections; 2) to define and describe a new taxon of the Merodon natans species group, with geographical distribution map of all species presented; 3) to infer the species diversity within this species group using molecular and geometric morphometric data; 4) to present the first data about the preimaginal morphology of some species of this group; and 5) to discuss biological data about the host plants of the M. natans group.
Morphological study of adults. External morphological features of adults and characters of the male terminalia were observed using a Nikon SMZ 745T (Nikon Corporation, Tokyo, Japan) stereomicroscope. For studying the male terminalia structure, specimens were first relaxed in a humidity chamber and then their terminalia were ex-tracted with a hook-tipped entomological pin. The terminalia were cleaned by boiling in a 10% solution of potassium hydroxide (KOH) for 3-5 minutes. This was followed by brief immersion in glacial acetic acid (CH 3 COOH) to neutralize the KOH, and then by immersion in ethanol (C 2 H 5 OH) to remove the acid. Terminalia were examined and dissected in several drops of glycerin under stereomicroscope and finally, were stored in plastic microvials and pinned with the source specimens. Measurements of specimen size were made with the same stereoscope using an eyepiece graticule. Body length was measured from the frontal prominence, excluding the antenna to the tip of the abdomen. The length of the basoflagellomere was measured from its base to the apex ( Fig. 2A:bl), and its width was measured in the widest part between its dorsal and ventral margins ( Fig. 2A:bw).
Morphological study of the immature stages. Puparia were cleaned before morphological analysis. To do so, they were immersed in water for several hours. Soil and debris were removed from the surface using pins and brushes, and puparia were placed in an ultrasonic cleaner for five minutes, until adherent material had fallen off the integument. The head skeleton was removed from the antero-ventral margin of the puparium using entomological pins, then soaked in 10% potassium hydroxide (KOH) and heated for 15 minutes in order to remove the remaining tissue. It was then soaked in acetic acid to neutralize the KOH, followed by 70% ethanol to eliminate the acid, for a few minutes each. The skeleton was then preserved and examined in glycerin. The larva was frozen (-20°C) and preserved in 70% ethanol; no special preparation for the analysis was conducted.
Morphological studies on the puparium were conducted using a Hitachi SEM (Scanning Electron Microscope) S3000N (Hitachi Ltd,Tokyo,Japan) at 20 kV at variable-pressure (or low vacuum) mode and a Leica DFC 320 digital camera attached to a Leica MZ16 stereomicroscope (Leica Microsystems, Wetzlar, Germany). A Leica M205C stereomicroscope (with Leica DFC450 camera) (Leica Microsystems, Wetzlar, Germany) was used for the examination of the larva and for a general view of the puparium. Olympus SZX16 (with Olympus U-TVO.5XC-3 camera) (Olympus Corporation, Tokyo, Japan) and Nikon SMZ 745T (with Nikon Coolpix D7100 digital camera) (Nikon Corporation, Tokyo, Japan) stereomicroscopes were used for the examination of the head skeletons. Puparium dimensions were measured using an eyepiece micrometer attached to the stereomicroscope. Maximum puparial length, including the posterior respiratory process (prp), and maximum puparial width were recorded.
Morphological terminology. The adult morphological terminology used in descriptions and drawings follows Thompson (1999), except for terms "fossette" and "sensory pit" from Doczkal and Pape (2009), "proepimeron" from McAlpine (1981) and those proposed in Marcos-García et al. (2007) for structures of the male terminalia.

Geometric morphometric analysis of adults
Geometric morphometric analysis of wing shape, based on measurements of the venation, was conducted using 285 specimens (209 specimens from Arok et al. (2019), marked with * in the Supplementary file 1:  Table S1). Specimens from Algeria, Bulgaria, Cyprus, Italy, Portugal and Tunisia were not included in population level analysis due to small sample size. Three separate geometric morphometric analyses were conducted. Two analyses for species level identification were carried out separately on males and females due to sexual dimorphism (Arok et al. 2019), and a third to explore phenotypic differentiation among populations.
The geometric morphometric analyses were based on right wings which were dissected using micro-scissors under Nikon SMZ 745T stereomicroscope and mounted on a microscopic slide using Hoyer's medium. Each wing was labeled with a unique code for the FSUNS collection, together with other data relevant to the specimens, and photographed using a Leica DFC320 camera attached to a Leica MZ16 stereomicroscope.
Eleven homologous landmarks, evenly distributed across the wing, were digitized using TpsDig 2.05 (Rohlf 2017a) (Fig. 1). Generalised least squares Procrustes superimposition on the raw coordinates was done using TpsRelw v1.68 (Rohlf 2017b) to minimize non-shape variations in location, scale and orientation of wings, and to superimpose the wings in a common coordinate system (Rohlf and Slice 1990;Zelditch et al. 2004).
To explore wing-shape variation among specimens without a priori defined groups, a principal component analysis (PCA) was carried out. Next, the discriminant function analysis (DA) and canonical variate analysis (CVA) were employed to analyse the shape differences among species and populations. Phenetic relationships among the species and populations were characterised using an unweighted pair group method with arithmetic mean cluster analysis (UPGMA) based on squared Mahalanobis distances computed from the discriminant function analysis. Superimposed outline drawings were  produced using MorphoJ version 2.0 (Klingenberg 2011) to visualise differences in mean wing shape among species. All statistical analyses were calculated using Statistica for Windows version 13 (TIBCO Software Inc. 2018).

Molecular analysis
Genomic DNA of 42 adult hoverfly specimens was obtained for the present study. For specimens processed at FSUNS, DNA was extracted from meso and metalegs using the sodium dodecyl sulfate (SDS) extraction protocol (Chen et al. 2010). The same protocol was applied for DNA extraction from the mid body part of a larva (specimen AU 1590). The DNA vouchers are deposited at FSUNS. For specimens deposited at ZFMK (identified as ZFMK-DIP numbers in the Supplementary file 1: Table  S1), the extraction protocol by Mengual et al. (2018) was followed. Detailed information on analysed specimens is provided in the Supplementary file 1: Table S1.
For sequences obtained at ZFMK, polymerase chain reaction (PCR) amplification protocol follows Rozo-Lopez and Mengual (2015). For sequences obtained at FSUNS, PCRs were carried out in 25μl reaction volumes. The PCR mixture contained 1x reaction buffer (Thermo Scientific, Vilnius, Lithuania), 2.5 mM MgCl2, 0.1 mM of each nucleotide, 1.25U Taq polymerase (Thermo Scientific, Vilnius, Lithuania), 5 pmol of each primer, and 50-100 ng of template DNA. The amplification conditions were as follows: 95°C for 2 min; 29 cycles of 94°C for 30 s, 49°C (for the 3' end of the COI gene) for 30 s or 50°C (for the 5' end of the COI gene and D2-3 region of the 28S rRNA gene), and 72°C for 2 min; with a final extension at 72°C for 8 min. We used C1-J-2183 (also known as Jerry) and TL2-N-3014 (also known as Pat) primer pair for amplification and sequencing of the 3'COI (Simon et al. 1994), LCO1490 and HCO2198 (Folmer et al. 1994) for the 5' COI, and F2 and 3DR (Belshaw et al. 2001) for the D2-3 region of the 28S rRNA gene. PCR products were purified using Exonuclease I and FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific, Vilnius, Lithuania) following the manufacturer's instructions. ZFMK specimens were sequenced bidirectionally at Macrogen Europe (Amsterdam, The Netherlands), while FSUNS specimens were sequenced in forward direction by the Sequencing Laboratory of the Finnish Institute for Molecular Medicine (Helsinki, Finland) and Macrogen Europe (Amsterdam, The Netherlands).

Correlation among wing shape, genetic, spatial differentiation
To test pairwise correlations between morphometric, genetic, and geographical distances among species, simple two-tailed Mantel tests were performed (Mantel 1967) with 10 000 permutations in PaSSaGe version 2 (Rosenberg and Anderson 2011). Morphometric distances were represented as a matrix of pairwise squared Mahalanobis distances, and genetic distances as a matrix of uncorrected p distances. Geographical distances were calculated as the minimum distance between two species using QGIS (Quantum GIS Development Team 2012).

Taxonomy
Species belonging to the Merodon natans lineage, including M. segetum, share the following characters: posterior side of mesocoxa with less than 10 pile; pile on anterior anepisternum reduced; anterior lobe of surstylus in its inner side well developed, oval, rounded, pilose, without curved distal prolongation (Figs 5A-G, 8A, B, 10A-D); basoflagellomere elongated, 1.75 to 2.4 times as long as wide, narrowed in apical third ( Fig. 2A Fig. 3) contains medium sized, black, short pilose species characterized by the following diagnostic features: scutum with distinct white pollinose ornamentation, vittae and fasciae (less developed in males of M. calcaratus); terga 2-4 with broad white pollinose fasciae, which may be interrupted in the middle; legs black, except reddish-brown tibiae darkened in the middle and reddish-brown first and second tarsomeres of pro-, meso-and metalegs (in M. calcaratus all tarsomeres of pro-, meso-and metalegs black); in some specimens the base and apex of femora yellowish.

Merodon calcaratus (Fabricius, 1794)
Syrphus calcaratus Fabricius, 1794: 301. Diagnosis. Merodon calcaratus is a medium sized (8-11 mm) black species. It can be easily distinguished from other members of the M. natans group by its shorter antenna; curved dorsal margin of basoflagellomere; small fossette near apex of basoflagellomere (Fig. 2G, H); narrow metafemur (Fig. 4G, H); black tarsomeres; completely black tergum 2. Narrow, fingerlike posterior surstyle lobe of male genitalia ( Fig. 5A-G). Redescription. MALE. Head: Antenna: short, dark brown to blackish; basoflagellomere about 1.75 times as long as wide with curved dorsal margin; small fossette positioned near apex of basoflagellomere. Face: black, white pollinose; covered with long white pile as long as pedicel; ventral part of face and anteroventral part of gena black, shiny; frontal triangle black, white pollinose, covered with dense, long white pile as long as pedicel; eyes holoptic, covered with white pile as long as scape; vertical triangle isosceles black, shiny, except for anterior part to anterior ocellus and posterior part to posterior ocelli white pollinose; vertical triangle covered with intermixed long white and black pile as long as pile on frontal triangle; ocellar triangle equilateral; occiput blackish white pollinose covered with white pile as long as pile on vertical triangle. Thorax: Scutum black, with less developed white pollinose vittae; covered with yellow erect pile as long as pile on occiput, in some specimens black pile present on area between transverse suture and scutellum; area above wing base with short black bristles; scutellum black covered with long yellowish pile as long as pile on scutum; pleura black, white pollinose; dorsal part of anterior anepisternum, posterior anepisternum, anterior anepimeron, dorsomedial part of anepimeron with long, dense white pile as long as pile on scutum; long white pile on katepisternum broadly separated with bare area between; proepimeron and katatergum with some white pile. Legs: femora black, yellow only at apex; metafemur narrowed ( Fig. 4G), with serrated triangular lamina in its apical part; tibiae black, yellow only at base and apex; tarsomeres dorsally black, ventrally yellow; legs covered with mainly whitish pile, black pile present on apex of femora and on tarsomeres dorsally; tarsomeres ventrolaterally with bristle, which color varies from black to yellowish. Wing: hyaline, covered with microtrichia, except for some bare areas in first and second basal cells; stigma light yellow; wing veins dark brown; halter and calypter yellowish. Abdomen: Black, slightly tapering; terga 2-4 with white pollinose fasciae; pollinose fasciae on terga separated, not reaching lateral margin; terga 2-3 covered with black adpressed pile medially and white pile laterally; tergum 4 covered with intermixed black and white pile; sterna blackish covered with white erect pile in some specimens pile on sternum 4 shorter than those on sterna 2-3. Male genitalia: Epandrium: posterior surstyle lobe of male genitalia narrow, fingerlike; anterior surstyle lobe on inner side oval shaped, covered with dense, short white pile (Fig. 5A-G); cercus square-like to rounded; Hypandrium: as in other members of Merodon natans group, medially broaded. FEMALE: Similar to male, except for normal sexual dimorphism and by following character: well developed white pollinose vittae present on scutum (in males scutum with less developed white pollinose vittae).
Variability. Merodon calcaratus is characterized by its easily recognizable narrow and long posterior surstyle lobe which however, is highly variable in its shape amongst specimens from different geographical populations: very narrow in width in specimens from Morocco (Fig. 5A); broader in width, medioventrally with a protuberance in specimens from Algeria (Fig. 5B, D); in specimens from Tunis similar to specimens from Algeria, but narrower in width (Fig. 5C); in specimens from Spain apically rounded, similar to specimens from Morocco, but broader in width (Fig. 5G); the specimen from Kenya is unique, because of the asymmetry of the left (Fig. 5E) and the right posterior surstyle lobe (Fig. 5F). This vari- ability of surstyle lobe is unusual among most of the species of the genus Merodon, but shape of basoflagellomere connects all these populations in one taxon. Genetic data clearly connect populations from Spain and Morocco in spite of differences in the shape of surstyle lobe. Lack of genetic data and small number of specimens in African populations prevents use of an integrative approach. We decided to keep all these populations under the name M. calcaratus until future research.
Distribution. Merodon calcaratus occurs in the southern part of the Iberian Peninsula (Spain, Portugal), in North Africa (Morocco, Algeria (Haffaressas et al. 2017), Tunis, Libya) and one isolated record present in East Africa (Kenya) (Fig. 6).   Description. MALE (Fig. 7A). Head: Antenna: dark brown; basal part of arista light brown; in some specimens basoflagellomere ventrally light brown; basoflagellomere two times as long as wide with acute apex; large fossette extending from base of arista to apex of basoflagellomere (Fig. 2E); lunule dark brown. Face: Black, white pollinose; covered with long white pile as long as pedicel, except for medial vitta extending from base of antenna to lower part of face without long white pile; ventral part of face and anteroventral part of gena black, shiny; frontal triangle black, white pollinose, covered with dense long white pile as long as pedicel; eyes holoptic, covered with dense white pile; in specimens from Israel and Somalia eyes covered with scarce, white pile as long as scapus; eye contiguity about 10 ommatidia long; vertical triangle isosceles black, shiny except for anterior part to anterior ocellus and posterior part to posterior ocelli white pollinose; vertical triangle covered with long white pile as long as pile on frontal triangle and intermixed black pile on ocellar triangle; ocellar triangle equilateral; occiput white pollinose covered with white pile as long as pile on vertical triangle. Thorax: Scutum black golden-bronze lustered with five white pollinose vittae; covered with yellow erect pile as long as pile on occiput and short black adpressed pile between transverse suture and scutellum; short black bristles present on area beyond transverse suture and above wing base; scutellum black, covered with long white pile as long as pile on scutum; pleura black, white pollinose; dorsal part of anterior anepisternum, posterior anepisternum, anterior anepimeron, dorsomedial part of anepimeron with long dense white pile as long as pile on scutum; long white pile on katepisternum broadly separated with bare area between; proepimeron and katatergum with some white pile. Legs: Femora black, yellow only at base and apex, covered with white pile; metafemur swollen with serrated triangular lamina (Fig. 4E); tibiae of pro-, meso-and metaleg reddish-brown, medially darkened, covered with white pile; colour of basitarsomere, second and third tarsomeres of pro-, meso-and metaleg varies from yellow to dark brown; fourth and fifth tarsomeres always darkened -light to dark brown; tarsomeres mainly covered with white pile, intermixed with some black pile. Wing: Hyaline, covered with microtrichia except for some bare areas in first and second basal cells; stigma light yellow; wing veins dark brown, basally yellowish; halter and calypter yellow. Abdomen: Black with golden-bronze luster, slightly tapering; tergum 2 without antero-lateral reddish maculae; terga 2-4 with white pollinose fasciae; pollinose fascia on tergum 2 widely separated; lateral margin of abdomen blackish and white pollinose, posterior margin of terga 3-4 broadly yellowish; terga covered with long golden erect pile as long as pile on mesonotum and some short, black, adpressed pile may be present near posterior margin of terga 2-3; sternum 1 dark brown to blackish; colour of sterna 2-4 varies from dark brown to light brown; sterna covered with long, white, erect pile longer than pile on terga.    Diagnosis. Merodon natans is a medium sized species (8-12 mm), which can be easily distinguished from M. calcaratus by its longer antenna ( Fig. 2A, B), broader metafemur in both sexes (Fig. 4A One appropriately labelled type was located in J.C. Fabricius collection (ZMUC), but only a pin without any remaining parts of the specimen is present in the collection ("193.20"). Consequently, we decided to designate a neotype because the type material has been destroyed. Neotype Redescription. MALE (Fig. 11A  with white erect pile as long as pile on occiput and short, black adpressed pile on area between transverse suture and scutellum; short black bristles present above wing base; scutellum black, covered with long white pile as long as pile on scutum; pleura black, white pollinose; dorsal part of anterior anepisternum, posterior anepisternum, anterior anepimeron, dorsomedial part of anepimeron with long, dense white pile as long as pile on scutum; long white pile on katepisternum broadly separated with bare area between; proepimeron and katatergum with some white pile. Legs: Femora black, yellow only at base and apex, covered with white pile, some black pile presented on its apical part; metafemur swollen with serrated triangular lamina (Fig. 4A); tibiae yellow medially darkened, covered with white pile; colour of tarsomere dorsally varies from yellow to dark brown, ventrally yellowish; tarsomeres mainly covered with white pile, intermixed with some black pile. Wing: Hyaline, covered with microtrichia except for some bare areas in first and second basal cells; stigma light yellow; wing veins dark brown, basally yellowish; halter and calypter yellow. Abdomen: Black, slightly tapering; tergum 2 with anterolateral reddish maculae, in some specimens anterolateral reddish maculae on tergum 2 absent; terga 2-4 with white pollinose fasciae; pollinose fasciae on tergum 2 widely separated, on terga 3-4 in some specimens may merge; lateral margin of abdomen blackish covered with white pollinosity, in some specimens lateral margin of abdomen yellowish; posterior margin of terga 3-4 yellowish; terga covered with adpressed black and white pile, shorter than pile on scutum; white pile on lateral margin of abdomen longer than pile on terga; sternum 1 dark brown; colour of sterna 2-4 varies from dark brown to light brown; sterna covered with long, white erect pile longer than pile on terga. Male genitalia: Epandrium (Fig. 10A, B): posterior surstyle lobe rounded, ventral margin of posterior surstyle lobe without visible triangular prominence from lateral view, although it can be present in some specimens, but it is small and visible only from ventral view; anterior surstyle lobe on inner side oval shaped, covered with dense, short white pile. Hypandrium: as in other members of Merodon natans group, hypandrium broaded medially. FEMALE (Fig. 11B, D): Similar to male except for normal sexual dimorphism. Frons black covered with white pollinosity, except for black shiny vitta extending from lunule to anterior ocellus.

Biology. The preferred environment of the species is semi-arid, sandy calcareous grasslands with scattered
Variability. The posterior surstyle lobe in males of M. natans varies in shape, as in the case of M. calcaratus, but is less distinct. In the Montenegro (Boka Kotorska) population of M. natans and in a single specimen from Spain, the posterior surstyle lobe is narrower (Fig. 10B).
Distribution. Merodon natans occurs in most countries of southern Europe (Spain, Italy, Croatia, Serbia, Bulgaria, North Macedonia, Montenegro, Greece) and part of western Europe (France) (Fig. 6). According to Marcos-García et al. (2007) (Speight 2020). Adults prefer open areas with tall herbs and scrub (Fig. 11A, B, E), within dry woodland; a secretive species, as easily collected by use of Malaise traps as by direct observation (Speight 2020). Flowers visited include: Prospero autumnale (Fig. 11C,  D), Foeniculum sp., Mentha sp., Solidago sp., Drimia maritima. The flight period is in Spring (April/May) and Autumn (end August/beginning October). The puparium, described here, is found in the host plant Prospero autumnale in Serbia (Fig. 11C, D).  nov., but differs by the following characters of the male genitalia: ventral margin of posterior surstyle lobe with distinct triangular prominence ( Fig. 10C:v), in M. natans without visible triangular prominence from lateral view (Fig. 10A, B), it can be present in some specimens, but it is small and visible only from ventral view (Fig. 10A, B); posterior surstyle lobe broader and shorter (Fig. 10C

Distribution. Merodon pulveris inhabits the Anatolian
Peninsula, the eastern Mediterranean islands (Lesvos, Samos, Rhodes) and Cyprus, but is absent from southern and western Europe as opposed to M. natans (Fig. 6).
Biology. Preferred environments (Fig. 12C) are open areas in Eastern European maquis on limestone near coniferous forests with large populations of its host plant Pros-  (Fig. 12A). Flight period is in spring (April-May) and autumn (late September/mid October). The larva described here was found in Prospero autumnale on Lesvos island in Greece.

Identification key to Merodon natans lineage
1. Area of short black pile present on anterobasal ~1/4 of profemur, ~1/5 of mesofemur and ~1/6-1/7 of metafemur (Fig. 13A, B). Antenna with sensory pit on inner side of basoflagellomere large, at least 3 times as long as basal diameter of first aristomere (in females sensory pit smaller). Male genitalia: surstyle lobe with more or less straight ventral margin (Fig. 14A); hypandrium medially not broadened, with setulae in ventral view (Fig. 14B). Large species ( -Antenna longer, at least 2 times longer than broad ( Fig. 2A-F); basoflagellomere without curved dorsal margin ( Fig. 2A-F); fossette larger in size, its position medianly or near to base of basoflogellomere ( Fig. 2A-F Most morphological characters of the puparia of M. makrisi sp. nov. and M. natans appear to be very similar, however the single specimen of M. natans is damaged and morphological analysis using electron microscopy could not be performed. Therefore, although the follow-ing description refers to both species, most of the characters have been described for M. makrisi sp. nov., but the features that seem to be different between them have been emphasized. Puparium description (Merodon makrisi sp. nov. n=2; M. natans n=1): Dimensions and shape (Fig. 15A). Length × width: 11 mm × 5 mm in M. makrisi sp. nov., and 10 mm × 5 mm (ca., puparium damaged) in M. natans; brownish in colour; sub-cylindrical; rough integument with larval segmentation persisting as transverse folds and wrinkles; integumental vestiture well-developed, with small Mandibles with dark sclerotized hooks, without accessory teeth, fused to external heavily sclerotized mandibular lobes; dorsal cornu narrowed and tapered slightly downwards towards sharp apex, representing whole length of ventral cornu (in M. natans) and slightly exceeding it (in M. makrisi sp. nov.); dorsal bridge, vertical plate and intermediate sclerite apparently fused together and all highly sclerotized (in M. natans, vertical plate slightly less sclerotized); ventral cornu elongate and narrow in profile view, wider and more heavily sclerotized at posterior end, with cibarium located at base, forming grinding mill of (not so sclerotized) pestle and (more sclerotized) mortar construction, at posterior end of cibarium. -Puparial spiracles: Sclerotized, dark brown in colour, stout, cylindrical in shape, gradually tapered, slightly pointed at end (Figs 16A, 17B), apex with shallow indentation visible on ventral side in M. makrisi sp. nov. (Fig. 16B); length ≈ 0.3 mm, twice as long as broad; separated by distance of five times their length; whole ventral and lateral surfaces (except apex) covered with irregularly-spaced, oval-shaped domed tubercles; whole spiracular surface (between tubercles, at base, and at apex) reticulate with polygonal pattern (Fig. 16A, B), more irregular on ventral side, with polygons noticeably smaller in apical part; 5-7 radially-arranged spiracular openings on each tubercle in M. makrisi sp. nov. (Fig. 16B).

Third instar larva of Merodon pulveris
Larva dimensions and shape: Length: 9 mm, width: 3 mm; sub-cylindrical; roundly arched dorsally and slightly flattened ventrally; anterior end truncated, inclined ventrally; uniformly yellowish to light brown in colour; rough integument with segmentation as transverse wrinkles, anterior segments corrugated with conspicuous folds; integumental vestiture well-developed, with short slightly pointed and sclerotized yellowish-brown spicules; segmental sensilla all conspicuous, consisting of wider basal papilla bearing very long needle-like terminal setae (Fig. 18). -Head: Mandibles with black sclerotized hooks (Fig. 19A, B), with large accessory teeth on basal outer side of hooks, clearly visible in lateral view (Fig. 19B), and another pair of very small accessory teeth on inner side of hooks, visible in frontal view (Fig. 19A); mouthhooks projecting downwards along each side of mouth, fused to brownish-black sclerotized external mandibular lobes at base; well-developed and sclerotized antenno-maxillary organs (Fig. 19A, B), located on pair of fleshy rounded projections between mouth and dorsal surface of prothorax, consisting of 2 pairs of cylindrical to conical-shaped structures tipped with different types of sensilla, antennae with single antennal sensory cone and one small sensilla on top surface; dorsal lip smooth with-out ornamentation. -Thorax: Lateral lips flat and coated in long, clearly pointed and slightly sclerotized spicules; dorsal surface of prothorax with five longitudinal grooves, with conspicuous, clearly pointed and yellowish-brown spicules distributed on grooves, with dome-shaped, very aggregated spicules on folds; dorsal surface of prothorax with pair of anterior spiracles (Fig. 19B) about twice as long as broad at base, sclerotized, cylindrical in shape, reddish-brown in colour, bilobulated at apex and completely retractile within inverted integumental pockets; mesothoracic prolegs absent. -Abdomen: Primordia of pupal spiracles (Fig. 19C) present on dorsal surface of first abdominal segment (indicating third larval stage); pronounced segmental sensilla all bearing setae; locomotory organs poorly developed, visible as pairs of raised domes on abdominal segments, located between segmental sensilla number 8 and 9, lacking crochets, having same type of ventral ornamentation but more developed and less sclerotized; two different pairs of lappets (Fig.  19D, E) on anal segment, ventro-lateral pair represented by longer fleshy papilla with one sensillum bearing seta, dorso-lateral pair with poorly developed basal papilla, apically divided, bearing one sensillum with long seta on top of each division; prp dark reddish-black and shiny, short (but clearly visible from dorsal view, Figs 18B, 19D), in shape of truncated cone, with annular groove at base, slightly wider than long, with base slightly wider than apex; entirely coriaceous, lateral surface with longitudinal indentations, with small dents, conspicuously ornamented from base to apex, differently in its (slightly narrowed) apical third (below spiracular plate); outline of spiracular plate sub-elliptical and slightly irregular, with indentations, bearing groove in joining point of two parts of plate, on both dorsal and ventral sides, visible in polar   view; spiracular plate with 4 pairs of slightly curved and convoluted irregularly-shaped spiracular openings around two central scars; spiracular scars in pair of abrupt cavities, two sunken depressions in middle of spiracular plate; 4 pairs of very well-developed branched inter-spiracular setae emerging from edge of spiracular plate (Fig. 19E).

Molecular data
The concatenated 5'-end and 3'-end COI gene sequence matrix contains 61 sequences of 1,358 bp length. The total number of the variable positions is 429, while 331 are parsimony informative. As a result of MP analysis, we inferred a strict consensus tree (length=1,242, consistency index=0.45, retention index=0.78) of four equally parsimonious trees (see Supplementary file 2: Figure S1). ML and MP analyses resulted in similar tree topologies (Fig. 20, Supplementary file 2: Figure S1). The Merodon natans species group is resolved as monophyletic with high bootstrap support (99 and 97) on both MP and ML trees. Species clades of M. natans, M. pulveris, M. calcaratus and M. makrisi sp. nov. are also well supported, each with a bootstrap value of 100. Immature specimen AU1590 (larva) from Lesvos island belongs to the M. pulveris clade. Within M. pulveris two clades can be distinguished, one which corresponds to specimens from Cyprus and the second which contains specimens from Greek islands (Samos, Rhodes and Lesvos) and from Turkey (see Supplementary file 1: Table S1). Both these clades are well supported (bootstrap values: ML=100 and 87; MP=99). Genetic distances based on concatenated 5'-end and 3'-end COI gene sequences between species pairs within the M. natans species group are in the range from 5.1% between M. natans and M. pulveris to 6.4% between M. pulveris and M. makrisi sp. nov. (Table 1). The average p distance between the two M. pulveris clades is 2.2%.
The combined COI+28S rRNA gene sequence matrix contains 48 sequences. The total length is 1,957 bp and there are 515 variable positions from which 377 are parsimony informative. Merodon makrisi sp. nov. is present with only one specimen due to lower sequencing success of 28S rRNA gene compared to COI gene. MP analysis resulted in a strict consensus tree (length=1,358 bp, con-sistency index=0.48, retention index=0.76) of two equally parsimonious trees and had similar topology as ML tree (Fig. 21, Supplementary file 3: Figure S2). The M. natans species group is recovered as monophyletic, as well as the species M. natans, M. pulveris and M. calcaratus (bootstrap support value of 100). The single specimen of M. makrisi sp. nov. is resolved in a separate branch, clearly divergent from all other species of the M. natans group.

Geometric morphometric analysis
Wing shape variation among specimens was quantified using PCA, which produced 18 principal components (PCs) in total within both males and females. First two principal components (PCs) described 35% of wingshape variation among male specimens and 36% among female specimens. In the space defined by the first two PCs groupings of conspecific specimens is clearly noticeable within both males and females (Fig. 22). In both cases, PC1 depicts wing shape differences between M. natans and M. pulveris, whereas PC2 differentiates M. calcaratus from M. natans and M. pulveris (Fig. 22).
Discriminant analysis provided evidence for highly significant wing shape differences among all species pairs (P < 0.    veris (Fig. 23A). Within females, CV1 with 64% of total wing shape variation points out the difference between M. natans and M. calcaratus and CV2 with 36% of total wing shape variation depicts the separation of M. pulveris from M. natans and M. calcaratus (Fig. 24A).
Phenogram based on squared Mahalanobis distances showed different phenetic relationships within males and females (Figs 23E,24E). In males, M. natans and M. calcaratus had the most similar wing shape (Fig. 23E), whereas in females phenetic relationships were consistent with our molecular results, with M. natans and M. pulveris having the most similar wing shape (Fig. 24E).
Pairwise differences in average wing shape were visualised using superimposed outline drawings which allows recognition of wing regions that are contributing to the species discrimination (Figs 23B-D, 24B-D). Within males, differences between species pair M. natans and M. calcaratus were associated with most prominent landmark displacements in the central and distal parts of their wings (Fig. 23B). Differences in wing shape between M. pulveris and M. calcaratus males were most obvious in distal part of the wing (Fig. 23C), while the differences between M. natans and M. pulveris were associated with landmark shifts in the proximal part of the wing (Fig. 23D). Contrary to males, wing shape differences between females of M. natans and M. calcaratus were mainly in the distal part (Fig. 24B). Major differences in wing shape between females of M. pulveris and M. calcaratus were found in the central and distal parts of the wing (Fig. 24C). Differences in wing shape between females of M. pulveris and M. natans were associated with landmark displacements in the central and proximal parts of wings (Fig. 24D).

Population-level analysis
Wing shape variation among populations was measured using discriminant and canonical variate analysis. DA showed correct classification for 81.85% of the specimens. Out of 270 specimens, 49 are misclassified, 46 into other conspecific populations, and only three as other species. Based on the UPGMA cluster analysis constructed with the Mahalanobis square distances, M. natans populations were the closest to each other, forming a cluster. Within this cluster, the population from Greece, Crete had the most distinct wing shape (Fig. 25B). The second cluster consists of two isolated branches, first with two western Mediterranean populations of M. calcaratus, and second one with conspecific populations of M. pulveris (Fig. 25B). According to topology, within M. pulveris branch specimens from Greece, Lesvos have the most distinct wing shape.
CVA produced eight significant CV axes, from which the first two were describing 72% of total shape variation (Fig. 25A). In the scatter plot described with these two CV axes, all conspecific populations were grouped together following the pattern of species delimitation results. First CV with 45% of total shape variation indicates separation of M. pulveris populations from M. natans populations, while CV2 with 27% of shape variation clearly separated populations of M. calcaratus from M. natans and M. pulveris (Fig. 25A).

Correlation among wing shape, genetic and spatial differentiation
Simple two-tailed Mantel tests revealed that geographical distance was not significantly correlated with wing shape differentiation nor with genetic differentiation among M. natans, M. pulveris and M. calcaratus (wing -geo graphy: p=0.63474, r=0.76691; genetic-geography: p=0.14519, r=0.97433). Differences between the species were exaggerated 3-fold to make them more visible E UPGMA phenogram constructed using squared Mahalanobis distances of wing shape.

Integrative taxonomy
The Merodon natans lineage as an independent evolutionary clade was first established by Radenković et al. (2018b) and here we have confirmed its monophyly on both COI and COI +28S trees.
The integrative taxonomy approach using multiple data sources implemented in this study supports species delimitation based on morphological differences within the M. natans group. Thus, it has proved useful in taxonomy of the group, as it has been in many previous studies on the genus Merodon (e.g., Popović et al. 2014Popović et al. , 2015Ačanski et al. 2016;Šašić et al. 2016;Kočiš Tubić et al. 2018;Radenković et al. 2018a;Šašić Zorić et al. 2020;Vujić et al. 2020aVujić et al. , 2020bVujić et al. , 2020c. Morphological descriptions combined with molecular data resolved four species of the M. natans species group: M. calcaratus, M. pulveris, M. natans and M. makrisi sp. nov. The first three species are also supported by the results of geometric morphometry analysis. Additionally, species distribution data proved useful for discriminating among species as they are mostly allopatric, except on the island of Cyprus where both M. pulveris and M. makrisi sp. nov. can be found in sympatry. The four closely related species of the Merodon natans group have diagnostic morphological differences. Although M. calcaratus has exhibited high intraspecific variability, this species has diagnostic characters which make it morphologically the most divergent within the M. natans group. This is also supported by the M. calcaratus specimens resolved as sister to the other three species clades on the COI and COI+28S trees. Merodon makrisi sp. nov. is morphologically similar to both M. These three species are significantly divergent based on wing shape. In both males and females, the percentage of correct species classification in discriminant function analysis is excellent (males: 97.32%, females: 98.53%). This segregation is also noticeable in CVA scatterplots, as well as in clear conspecific clustering in population analysis, which clearly illustrates the division of these three species. In addition, based on Mantel tests there is no significant correlation of wing and genetic differentiation with geographical proximity among M. calcaratus, M. natans and M. pulveris. As mentioned above, an interesting wing-shape pattern among male specimens is observed. Although it would be expected due to morphological and molecular results, males of M. natans and M. pulveris do not have the most similar wing shape in the UPGMA phenogram. Similar findings were recorded between sympatric species from e.g., the M. aureus group: M. aureus and M. calidus Šašić Zorić, Ačanski (Vujic et al. 2020c). However, according to our findings M. natans and M. pulveris do not occur in sympatry, and we can only assume that the most different wing shape between males can be the result of sympatry or secondary contact in the past. Merodon makrisi sp. nov. was not included in the geometric morphometric analysis, but molecular data and morphological characters clearly delineate it from other taxa within the M. natans group. This species was not included in the geometric morphometric analysis due to an insufficient number of available specimens with intact and undamaged wings that were available for analysis. Considering that M. makrisi sp. nov. can be separated from other investigated species based on morphology, its exclusion from the geometric morphometric analysis does not affect the strength of the provided results.
Despite their adult morphological resemblance, COI genetic distance between M. natans and M. pulveris is relatively high (5.1%) and similar to values between more distant species pairs (5.3-6.4%). The specimens of M. pulveris from Cyprus, based on COI and COI+28S analyses, segregate as reciprocally monophyletic within the M. pulveris clade. This population is morphologically indistinguishable from other analysed M. pulveris populations despite a genetic distance value of 2.2%, while the data on geometric morphometry is limited due to a small sample size. Previous studies documented genetic distances between morphologically cryptic Merodon species mostly in the range 0.3-2.5% Popović et al. 2015;Šašić et al. 2016;Radenković et al. 2018a;Vujić et al. 2020c). Island speciation occurs often in the M. aureus species group, where endemic species are recorded on Andros, Crete, Cyprus, Naxos, Peloponnese and Rhodes Radenković et al. 2018a;Šašić Zorić et al. 2018). However, lack of additional data and relatively high genetic distance among the other species within the M. natans species group prevent us from any further conclusion about possible speciation on Cyprus. Furthermore, variation in morphological characters is present between populations of M. makrisi sp. nov. on the mainland and Cyprus. The Cyprus population of this species is characterized by stable morphological characters, while in populations from the mainland (Israel, Somalia) these features are variable. Thus, it is possible that the population of M. makrisi sp. nov. from Cyprus belongs to a distinct taxon, but no genetic data for mainland specimens of this species are available. Additional molecular and morphometric studies would certainly help to clarify the taxonomic status of populations of M. pulveris and M. makrisi sp. nov. from Cyprus (subspecies or small populations with limited variability).

Morphological characters of the immature stages
The shared character of the three species of the Merodon natans group examined in our study is the presence of two different pairs of lappets on the anal segment, the dorsolateral pair being apically divided. This trait was also reported in two species of the avidus-nigritarsis  (Fabricius, 1794)) were described as having three pairs of lappets with the middle one consisting of two projections (Ricarte et al. 2008. One of the most striking characters of the larva of M. pulveris is the possession of two sets of accessory teeth, a large pair on the basal outer side of the mandibular hooks and a small pair on the inner side of the hooks. Among all immature stages of Merodon species for which this character has been described, accessory teeth of the mandibular hooks were reported only for M. equestris and M. avidus larvae (Ricarte et al. 2008;Andrić et al. 2014), but only on the inner side of hooks. Nevertheless, accessory teeth were not recorded in puparia, either the ones examined here or those of other studied species including M. avidus. Furthermore, it has been suggested that there is mouthhook wear during larval development, making characters related to its shape deceptive in puparia (Preradović et al. 2018). The notable feature of the head skel-eton of M. makrisi sp. nov. is that the dorsal cornu slightly exceeds the ventral cornu in its length, whereas in M. natans both cornua are approximately the same length; in other species of the genus the dorsal cornu is usually more or less shorter than the ventral. Anterior spiracles of M. pulveris larva are bilobulated at the apex, the same shape as for the other two species of the M. natans group. The two spiracular openings at the apex of the anterior spiracle of M. makrisi sp. nov. and M. natans is a feature previously reported for the anterior respiratory process of M. geniculatus, M. avidus, M. opacus and M. hurkmansi, whereas M. luteihumerus and M. equestris exhibit 3-5 spiracular openings (Ricarte et al. 2008Andrić et al. 2014;Vujić et al. 2020a).
Shared characters of the three species examined in our study are the general shape and ornamentation of the posterior respiratory process (prp). The prp shape varies among Merodon species; for instance, very short cylinder (button-shaped) in M. avidus and M. opacus, barrel-shaped in M. calidus (puparium described as M. aureus in Preradović et al. (2018), redefined in Vujić et al. (2020c)) and markedly narrowed in the apical third in M. rufus Meigen, 1838(Andrić et al. 2014Preradović et al. 2018;Vujić et al. 2020a). In the M. natans group, the prp shape is a truncated cone, with a groove at the base. The prp being wider than long with base wider than apex is notable in M. makrisi sp. nov., however, these features are somewhat less pronounced in M. pulveris and even less in M. natans, with a slightly longer and more cylindrical prp. The lateral surface of the prp is entirely coriaceous, with small dents from base to apex and with slightly undulate longitudinal indentations in the basal part, somewhat resembling the prp of M. geniculatus (see figs 5D, 6D in Ricarte et al. (2017)). The part just below the spiracular plate is conspicuously ornamented in M. pulveris and M. natans, while it is smoother in M. makrisi sp. nov. Other species have different ornamentation of the prp surface; e.g., very smooth from base to apex in M. rufus (with small dents only in the area below spiracular plate) and with a network of small rounded protuberances in M. calidus (Preradović et al. 2018).
One of the problems in the morphological research of Merodon immature stages is the difficulty of finding specimens in the field, since the larval food-plants and the breeding and oviposition sites have not been recorded for most Merodon species (Hurkmans 1993;Rotheray 1993;Speight 2020). Another important handicap is the proper species identification of immature stages. Rearing phytophagous syrphid is not particularly complicated (Rotheray 1993) but can take months (Rotheray and Gilbert 2011). In our study, pupae of M. makrisi sp. nov. and M. natans were successfully reared. An identification of immature stages based on DNA COI barcode libraries generated for Merodon adults was suggested by Ståhls et al. (2009) and was successfully employed for larvae of M. avidus by Andrić et al. (2014), as well as for larva of M. pulveris in our study. Nevertheless, sequencing analysis is not possible in cases of rotten or parasitoidised specimens, as has been shown by Preradović et al. (2018). Furthermore, species-level illustrations are limited and a comprehensive identification key has been published only recently . The immature stages of only nine out of 234 species of Merodon have been described so far (Heiss 1938;Stuckenberg 1956;Ricarte et al. 2008Andrić et al. 2014;Preradović et al. 2018;Vujić et al. 2020a).

Diversity and distribution
The four species of the Merodon natans group are mostly distributed within the Mediterranean Basin, but we report the first records of the presence of this species group in the Afrotropical Region, with the distributional range of two species reaching the easternmost parts of Sub-Saharan Africa. Merodon natans has been described geographically as a "Balkan" species, occurring throughout the Balkans and the eastern Mediterranean islands, except Dodecanese, North Aegean islands and Cyprus (Arok et al. 2019). Additional data in the present work confirm a broader distribution to the west, including records from Spain, Italy and France. Although there are records in the literature of M. natans from Gibraltar (Ebejer and Bensusan 2010) and Portugal (van Eck 2016), voucher specimens were examined by the authors in the work frame of this survey and identified as M. obscuritarsis Strobl in Czerny & Strobl, 1909. Merodon pulveris is characterized as having an "Anatolian" distribution, being found in western and southwestern Turkey, and on Dodecanese, North Aegean islands and Cyprus. A second species from the M. natans group occurring on the island of Cyprus is M. makrisi sp. nov., primarily distributed in Israel, but also with records from Somalia. The southernmost record of the M. natans group is one for M. calcaratus from Kenya. However, this species is mostly distributed along the northwestern (Mediterranean) coast of the African continent and on the Iberian Peninsula.
The regions with the highest diversity of Merodon taxa, such as the Mediterranean Basin, are characterized with flora rich in species of geophytes, as the bulbs and other underground storage organs of these plants are food sources for Merodon larvae (Ricarte et al. 2008. Therefore, the current geographical distributions of Merodon taxa are probably connected to areas with high diversity of this distinctive flora . Since the Afrotropical Region is rich in bulbous plants, it is assumed that the Merodon species diversity in this region is underestimated , with only 16 species (out of a total number of 234) described for this region to date (Vujić et al. 2021). The genus Merodon has been classified into over 20 monophyletic species groups (Vujić et al. 2021), with a vast majority of species present in the Palaearctic Region and only two species groups (prior to M. natans species group), M. aureus and M. desuturinus, known to have representatives in the Afrotropical Region as well . The M. desuturinus species group has been found to represent an important link between the Palaearctic and Afrotropical faunas, consisting of two clearly separate lineages, i.e., a Palaearctic and an Afrotropical lineage (the latter includ-ing M. melanocerus and M. planifacies subgroups and the species M. cuthbertsoni Curran, 1939, all from southern Africa) ). The results of our research show the presence of yet another Merodon species group in the Afrotropical Region, the M. natans species group, with the first published records of this genus in eastern Africa.
Out of the four species of the Merodon natans group, descriptions of immature stages are presented for three species: M. pulveris based on larva, and M. natans and M. makrisi sp. nov. based on puparia, all found in the bulbs of the same plant species, Prospero autumnale. With the small size of one such bulb in mind, it could be speculated that these larvae grow at a slow rate, but it is possible that larvae move from one bulb to another while they grow, since P. autumnale can be found in dense patches. However, in the locality where the puparium of M. makrisi sp. nov. was found, the loamy soil can be very dried out and hard, making it seemingly difficult to move from one bulb to another. On the other hand, bulbs of P. autumnale are found quite superficially, making it possible for the larva to migrate to another bulb over the ground. This plant had previously been recorded as a possible larval host-plant for M. natans and M. pulveris in Greece (Vujić et al. 2020d), and also for M. calcaratus in Portugal (van Eck 2016). Most of the known larval host-plants of Merodon species belong to the same plant family, Asparagaceae (Ricarte et al. 2008;Andrić et al. 2014;Preradović et al. 2018), although some are recorded within Amaryllidaceae (Heiss 1938;Ricarte et al. 2017;Popov and Mishustin pers. comm.) and Iridaceae (Stuckenberg 1956). In a recent study by Vujić et al. (2020a) it has been speculated that groups of related Merodon species might have the same host plant genus. Considering both our extant records and fitting distributions with Prospero Salisb. (WCSP 2019), M. natans species group could therefore be connected to this genus throughout most of its range. However, Prospero is not present in the Afrotropical Region, so larval development is most likely associated with other bulbous geophytes in this area, probably also from Asparagaceae subfamily Scilloideae (alternatively regarded as Hyacinthaceae). Moreover, it could be assumed that cases of aberrant distribution records may have been the result of plant introductions. More data are needed to obtain better supported conclusions about relations with food-plants. Furthermore, additional material and molecular analyses of Merodon populations from Somalia and Kenya will be useful to confirm their taxonomic identification or to reveal if they belong to a separate species. The male genitalia of M. calcaratus have very variable morphological features, even on an intrapopulation level. Nonetheless, all studied specimens are characterized with the same apomorphic characters, which clearly differentiate this taxon from other species of the M. natans group.

Authors' contributions
AV, TT, XM, AvanE, SRad performed the sampling; AV, TT, SRad conceived and designed the study; AV, TT, AAnd, JA, LjŠZ, CP-B, AAra, SV, MA, XM performed the experimental analysis; AV, TT, AAnd, JA, LjŠZ, CP-B participated in data analyses. AV, TT, AAnd, JA, LjŠZ, CP-B, AAra, MA, XM, AvanE, SRoj took part in draft preparation, contributed to discussions during preparation of the paper, as well as read and commented on. All authors approved the final version of the manuscript.

Acknowledgements
We are sincerely grateful to Martin Ebejer (Wales, United Kingdom) and Antonio Ricarte (Alicante, Spain) for sharing photos of Merodon natans from Gibraltar and Spain, which helped a lot to understand the distribution of this species. Christodoulos Makris (Limassol, Cyprus) collected many specimens in Cyprus, shared information on flower vis-