A review of the early stages and host plants of the genera Eumerus and Merodon (Diptera: Syrphidae), with new data on four species

The genera Eumerus and Merodon (Diptera: Syrphidae) have a high taxonomic diversity (300+ species altogether), but life histories of most species are unknown. In addition, these hoverfly genera are recognised to be pests (ornamental plants and vegetable crops). In this paper, early stages of four hoverfly species are described, Eumerus hungaricus Szilády, 1940, Eumerus nudus Loew, 1848 and Merodon geniculatus Strobl, 1909, from Spain, and Eumerus strigatus Walker, 1859, from California, USA. Larvae of E. nudus were obtained from swollen roots of Asphodelus cerasiferus J. Gay. Larvae of E. hungaricus were found in bulbs of Narcissus confusus Pugsley. The host plant of the examined specimen of Eumerus strigatus is unknown. Larvae of M. geniculatus were reared from bulbs of different species of Narcissus L. Scanning electron microscope imaging was used to study and illustrate the anterior respiratory processes, pupal spiracles and posterior respiratory processes of the new early stages. A compilation of all available information on the early stages and host plants of Eumerus (21 spp.) and Merodon (15 spp) is provided, as well as an identification key to all known larvae/puparia of these genera. Eumerus elavarensis Séguy, 1961 is proposed as a new synonym of E. hungaricus and first data of this species are reported from Austria, Bulgaria, Spain and Turkey. In Eumerus, larvae are alleged to rely on the previous presence of decay organisms, but in the larvae of E. nudus the sclerotisation and size of the mandibular hooks suggest that this larva can generate decay from intact plant tissue.


Introduction
As a biodiversity hotspot, and with many unique species, the Mediterranean is an area important for conservation [1]. Wild species of animals and plants have adapted their life cycles to the characteristic water deficit during summer and many plant species have developed underground storage organs (bulbs, tubers or swollen roots), for example Amaryllidaceae, Xhantorrhoeaceae (incl. Asphodelaceae) or Hyacinthaceae plants [2,3]. These are PLOS  The general aim of the present study is to understand better the biology and functional morphology of Eumerus and Merodon larvae by studying the early stages of a Merodon species and three Eumerus species and providing data on their host plants. All available information on the early stages and host plants of these two hoverfly genera is compiled and systematically presented. In addition, an up-to-date key to the known puparia of Eumerus and Merodon is provided to facilitate the identification of larvae found both in natural and agricultural conditions.

Materials and methods
Fieldwork to search for early stages of Eumerus and Merodon in underground storage organs of geophytes took place in different localities of Spain. In La Font Roja Natural Park, Alicante, South-Eastern Spain, Eumerus larvae were collected in the swollen roots of bulbs of Asphodelus cerasiferus J. Gay (Xanthorrhoeaceae) in 2009-2010. In Sierra de Béjar, Salamanca, Central-Western Spain, Eumerus larvae were found in bulbs of Narcissus confusus Pugsley (Amayllidaceae) in 2010. In Sierra de Mariola Natural Park, Alicante, South-Eastern Spain, larvae and puparia of Merodon geniculatus Strobl, 1909 were obtained from bulbs of different Narcissus species in 2010. In all cases, bulbs were dug out to be checked for signs of larval feeding, tunnels or decomposed tissues. Non-attacked bulbs were buried again to facilitate their regeneration. The Narcissus species were identified by Dr Segundo Ríos (University of Alicante). The studied puparium (+ emerged female) of E. strigatus originates from an unknown host plant in California and it was stored in the California Department of Food and Agriculture, USA (CDFA). All required permits and approvals were obtained for the field work from the authorities of the visited protected areas (La Font Roja Natural Park and Sierra de Mariola Natural Park). No protected insect species was sampled.
Larvae were transported to the laboratory and reared in plastic boxes with mesh at the top and with their original host plant. Boxes were kept in a chamber under controlled conditions, at 20˚C, 65-85% humidity and without light. Boxes were inspected daily to find puparia, which were transferred individually to Petri dishes until adult emergence. When possible, the dates were recorded of the finding of a larva/puparium in the field, puparium formation and adult emergence. Emerged adults were identified using Stackelberg [41], Vujić and Šimić [42] and Speight & Garrigue [12], for Eumerus, and Marcos-García et al. [32] for M. geniculatus. The E. strigatus female was also confirmed genetically with DNA sequences of COI.
Larvae were described from their third larval stage, which was distinguished from other stages by having two differentiated discs on the first abdominal segment dorsally [43]. Larvae were preserved in 70% ethanol after immersion in cold water and boiling for about 4 minutes, with the purpose of fixation. For their study, puparia were cleaned with a fine paint brush after soaking in distilled water for 24h to soften materials covering the specimen; before cleaning, puparia were individualised in Eppendorf tubes with water to be treated in an ultrasonic bath at 50Hz for individual periods of 5 min, up to 25 minutes in total (individual periods of ultrasounds lasted 5 min in order to avoid pupal spiracles to be detached from the puparium). Once prepared for examination, larvae and puparia were studied with a stereo microscope.
For description of early stages, body size was measured as the length from the anterior margin of the prothorax to the anus in ventral view. Height and width of early stages were measured at their maxima. For Eumerus, the size of the posterior respiratory process (PRP) was measured as the distance between the transverse ridge and the centre of the spiracular plate (a) and expressed as a proportion of the width at the transverse ridge level (b). For Merodon, dorso-ventral height at the base of the PRP (c) was expressed as a proportion of the width at the base (d) [4]. Measurements were made with a LEICA M205C stereo microscope and the software Leica Application Suitie v.4.8. To describe the ornamentation of the anterior respiratory processes (ARP), pupal spiracles and PRP, photos were made with a HITACHI S-3000N scanning electron microscope (SEM). Head skeletons were obtained from the antero-ventral margin of emerged puparia. All puparia were soaked in a solution of KOH for 30 minutes and the head skeleton was removed with pins. Head skeletons were preserved in glycerine and studied in glycerine or 70% ethanol. Morphological terminology of early stages follows Hartley [43] and Rotheray [44], except for that of the head skeleton that follows Hartley [45], Roberts [46] and Rotheray & Gilbert [47]. Morphological terminology of adults follows Thompson [48]. Species distribution follows Speight [6] and locality data of the material examined in the present paper.
Thorax: ARP 0.1 mm long by 0.07 mm width, cylindrical in shape, slightly tapered towards the apex and curved to the centre of the body, light brown in colour, apex with two linear spiracular openings (Fig 2A); mesothoracic prolegs absent.
Abdomen: first abdominal segment with pupal spiracles 0.29 mm long, separated by 6× their length, bearing on the dorsal surface irregularly-spaced, round-shaped tubercles ( Fig  3A); each tubercle with 4-5 linear spiracular openings, arranged radially ( Fig 4A); pupal spiracle surface shiny and almost smooth with irregular fine and shallow marks, granulated at the apex ( Fig 3A); prolegs present bearing groups of small hooks lacking conspicuous planta; anal segment elongate, bearing three pairs of lappets, the first pair virtually absent, the second inconspicuous, divided into two projections, and the third well developed; PRP: inclined upward from the transverse ridge to the apex; transverse ridge conspicuous; a = mean 0.41mm (range 0.37-0.43); b = mean 0.43mm (range 0.41-0.43); a/b = 0.95 (n = 3); PRP shiny and brown in colour, transverse ridge conspicuous; PRP with fine transversal marks below the ridge and shallow punctures above; the final part of the PRP, after the punctured area, is smooth and curved, smaller in diameter than the rest of the PRP; PRP asymmetric, especially in the section from the transverse ridge to the PRP apex ( Fig 5A); spiracular plates with three pairs of curved spiracular openings, with four pairs of setae around the margin of the plate (in the examined specimens, all setae were broken) ( Fig 6A). Taxonomic notes: E. hungaricus was described from males collected in central Hungary [49]. The type material of E. hungaricus is destroyed [50], but Szilády illustrated in the original description the metaleg of his new species. The metaleg of E. hungaricus is rather characteristic within the genus; the femur is swollen and has long pile ventrally (length of longest pile same as maximum width of femur) and the posterior side of tibia has a conspicuous bump bearing pile. The lateral margins of tergites 3 rd and 4 th are adorned with conspicuous long setae, similar to Eumerus pulchellus. Doesburg [50] redescribed the male of E. hungaricus and described the female for the first time. Doesburg [50]   the neotype is, or immediately upon publication has become, the property of a recognized scientific or educational institution, cited by name, that maintains a research collection, with proper facilities for preserving name-bearing types, and that makes them accessible for study" but such a statement has never been published. In fact, Doesburg's neotype was deposited in his own collection. Furthermore, it is questionable if a neotype was needed for the "purpose of clarifying the taxonomic status" (Article 75.3.1.), because the identity of the species was never in doubt (despite the type specimen being destroyed), because of the detailed description and the drawings. E. hungaricus is just a rarely collected species and Séguy likely did not check all the west Palaearctic Eumerus descriptions when describing E. elaverensis in 1961, and therefore overlooked E. hungaricus. Despite us not agreeing with the validity of Doesburg's neotype designation, we agree that the specimens Doesburg [50] studied are conspecific with Szilády's E. hungaricus. One of us (Martin Hauser) examined a specimen of E. hungaricus from Doesburg's series (labelled as Neo-paratype), as well as three (2 males and 1 female) syntypes of E. elaverensis, which was described from France [52]. The examined neo-paratype of E. hungaricus and the two male syntypes of E. elaverensis shared the metatibia morphology, the long hairs at the lateral margins of abdomen (this character is shared by a very small group of Eumerus species) and the yellow apex of the 4 th tergite. For these and other morphological similarities, we consider all these specimens to be conspecific and we propose E. elaverensis as a junior synonym of E. hungaricus.
Head skeleton: (Fig 1B): mandibular hooks sclerotised and mandibular lobes fleshy and fused with the mandibles, with dorsal cornu tapering towards the apex, fin shaped, in profile view; mandibular hooks 0.6mm long, with accessory teeth and, in apical view, separated at apex by the same distance than basal width.
Abdomen: integument spiculated, without setae; sensilla with at least two setae each; prolegs on abdominal segments 1-6 bearing two parallel rows of crochets (anterior row with 4-5 crochets; posterior row with 2-4 smaller crochets); anal segment elongate (2.20 mm long, about 1.2× longer than the 6 th abdominal segment), with ventral part longer than dorsal and then segment oriented upward in appearance; three pairs of conspicuous lappets; middle lappets divided into two separate conic projections, 1 st and 2 nd conic, 3 rd elongated, longer than the other pairs of lappets; PRP: a = 0.46mm (0.39-0.56); b = 0.65mm (0.59-0.72); a/ b = 0.74 (n = 4); dark brown, shiny, sub-elliptical in cross section; below transverse ridge strongly striated longitudinally, finely transversally, above coriaceous and smoother towards the apex ( Fig 5B); spiracular plate with three pairs of ω-shaped spiracular openings and four pairs of multibranched setae around the margin of the plate. Spiracular scars in a pair of rounded depressions (Fig 6B).
Head skeleton: see under larva description (Fig 1B). Thorax: ARP 0.11mm long by 0.07mm width, cylindrical in shape, slightly swollen to the apex, yellowish to dark brown in colour, apex with two openings (Fig 2B); mesothoracic prolegs absent.
Species distribution: from Spain to the former Yugoslavia and Turkey, through Southern France and Italy (also in Sicily); Romania; Northern Africa: Morocco, Algeria and Tunisia.
Head skeleton: (Fig 1C): mandibular hooks sclerotised, not massive, with accessory teeth present; dorsal cornu tapering posteriorly in profile view, little sclerotised, almost entirely translucent; lips coated in setae and mandibular lobes with conspicuous ridges present after removal of the head skeleton from the puparium.
Thorax: ARP 0.1mm long, width of 0.72mm and height of 0.04mm, oval in shape and light brown in colour, apex with a groove separating 2 linear spiracular openings ( Fig 2C); mesothoracic prolegs absent.
Abdomen: integument smooth, bearing transversal rows of small hooks along the body; first abdominal segment with 0.34mm long pupal spiracles, separated by 4.5× their length, bearing irregularly-spaced, round-shaped tubercles ( Fig 3C); each tubercle with 5-6 spiracular openings, arranged radially ( Fig 4C); pupal spiracle surface between tubercles almost smooth but granulated at the apex ( Fig 3C); prolegs present on the first six abdominal segments bearing two rows of crochets; anal segment elongate, with three pairs of lappets bearing sensilla, middle ones divided into two smaller projections than those of the other pairs; PRP: a = 0.4mm; b = 0.47mm; a/b = 0.85 (n = 1); below ridge, fine transversal striations with some diagonal wrinkles; immediately above ridge with bulges, diminishing towards the apex until smooth (Fig 5C); spiracular openings U-shaped, with 4 pairs of linear and divided setae around the margin of the spiracular plate (Fig 6C).
Distribution: Fennoscandia south to Iberia and the Mediterranean; much of Europe through into Turkey and Russia; from the Urals to the Pacific coast (Sakhalin); Japan; introduced to North America and recorded from both Canada and the USA; introduced also to both Australia and New Zealand.
Examined material: a puparium (+ emerged adult female) obtained from a larva found in an unknown host plant in California (USA) [CSCA].
Head skeleton: (Fig 1D): mandibular hooks heavily sclerotised with both dorsal and ventral cornua bar-shaped in profile view; mandibular hooks 0.60mm long without accessory teeth.
Thorax: ARP sclerotised, 0.19mm long by 0.07 mm wide, cylindrical in shape, blackishbrown in colour, with two linear spiracular openings at the apex ( Fig 2D); pupal spiracles 0.77mm long, separated by a distance of 3× their length; surface extensively reticulated with lines drawing cells that encircle the spiracular tubercles, smoother near the base and granulated towards the apex ( Fig 3D); each spiracle bearing numerous domed tubercles irregularly distributed but less dense on the margin facing the centre of the segment; each tubercle with 4-5 radially arranged spiracular openings; mesothoracic prolegs absent.
Species distribution: Southern France and the Iberian Peninsula, Italy, Southern parts of the former Yugoslavian countries, from Bulgaria to Greece and Turkey; North Africa (Algeria and Morocco); Mediterranean islands: Balearic Islands, Corsica, Sardinia and Malta.
Examined material: 1 larva obtained next from a Narcissus dubius bulb in El Preventori

Key to early stages of Eumerus and Merodon species (third stage larvae and puparia)
A key to all known larvae/puparia of Eumerus and Merodon species is provided to facilitate the identification of these genera based on early stages found both in natural and cultured situations. Keys were elaborated by examination of actual specimens and descriptions/diagnoses/illustrations published in the references provided in Table 1. Authors had not access to early stages of the species marked with an asterisk ( Ã ) in the keys.

Discussion
Morphologically, all four species of Syrphidae studied in the present paper possess wellsclerotised mandibular hooks of different sizes, those of Merodon being larger than those of Eumerus, and, within Eumerus, those of E. nudus being the largest (Fig 1). In addition, all three studied Eumerus species have accessory teeth that surely assist the mouth hooks in rasping and scrapping solid tissue. Nevertheless, head skeletons of all these and other Eumerus species also have pharyngeal ridges [31]. Such structures indicate the ability of these species to feed on the fluids, and most probably the fungi and bacteria, associated with decay, as suggested by other studies focused on saprophagous hoverflies dependent on decomposing plant material [99,100]. Eumerus larvae develop better in previously decayed material, suggesting their more saprophagous than phytophagous feeding regime [65,101]. However, the larva of at least E. nudus appears to be capable of generating decay in intact plant tissue by mechanically damaging it and increasing the surface area to be attacked by microorganisms causing decay. Similarly, the larvae of E. compertus and E. tricolor have large mandibular hooks for feeding on intact plant tissue of Cistanche sp and Tragopogon spp plants, respectively (see Table 1). This is also important information that must be considered when searching for early stages of Eumerus in the field, as Eumerus larvae could be infesting a wider range of habitats than Merodon, both intact and liquefied plant tissues. In contrast, M. geniculatus lacks mandibular lobes but has the large heavily sclerotised mandibular hooks of other known Merodon larvae, suggesting a strict diet of living plant tissue, ripping apart the flesh of the bulbs where it lives.
Differences between the PRP lengths of Eumerus and Merodon, which is shorter in Merodon than in Eumerus, show that Eumerus is able to access air pockets within more liquefied materials while Merodon prevents its PRP being blocked by the decaying material left behind its larva in the excavated tunnels [47]. However, E. compertus and E. tricolor also have short PRP [59,86], probably adapted to live in the tunnels and cavities they produce in their host plants. Special attention must be focused on the PRP of E. hungaricus, which is asymmetric in all three studied specimens (Fig 5A), a remarkable feature not seen before in other known Eumerus species. Additionally, none of our four species, neither Eumerus nor Merodon, have mesothoracic prolegs. The similar E. obliquus and E. etnensis are the only described species which have mesothoracic prolegs [4,38].
Sampling methods for early stages of hoverflies living in underground storage organs of plants remain simple, and there is a need for innovation and a lot more effort in fieldwork. According to the current information on associations between Eumerus and Merodon and their host plants, their preference for geophytes makes the search for their host plants very complicated when plants are in their dormant state; even when the plant is visible, they do not always have symptoms of the presence of larvae inside their storage organs. It is very important to know the host plant of the hoverfly species being sought, as well as the development time in order to save time when digging for immature stages of hoverflies inside underground storage organs. This information about the host plants may be inferred by field observation of hoverfly behaviour during oviposition on plants. The knowledge on reproductive behaviour of both genera is still imprecise and biased. So far the majority of early stages found by different authors have been a result of extensive searches in similar plant biotypes. Our experience during field work tells us that an approach using adult behaviour in the wild, prior to the collection of early stages, helps greatly in finding early stages, although there is an important chance factor.
Other species of Eumerus, including E. nudus, have also been recently found in A. ramosus [12]. Plant species identification is important when studying insect-plant associations. Asphodelus cerasiferus distribution expands to the North of Spain whereas A. ramosus distribution is limited to the South and East of Spain. The taxonomic concept of A. cerasiferus includes some descriptions of A. ramosus non Linnaeus [102], so that those findings of the host plant of E. nudus of Speight and Garrigue from the western French Pyrenees [12] seem to belong to A. cerasiferus rather than A. ramosus E. hungaricus puparia were obtained in 2010 from wild bulbs of N. confusus (Amayllidaceae) in Sierra de Béjar (1000 m), in the mountain area of Salamanca province, situated in Central-Western Spain. E. strigatus is known to be a pest of different cultivated plants of commercial interest (see Table 1). M. geniculatus larvae were obtained from different species of Narcissus in 2010. From the Natural Park of Sierra de Mariola, Alicante (SE Spain), specimens of M. geniculatus were collected, as described above, from wild bulbs of N. dubius. However, from the Botanical Garden of Torretes (Ibi, Alicante, SE Spain), M. geniculatus specimens were taken from cultivated bulbs of Narcissus triandrus subsp. pallidulus (Graells) Rivas Goday, Narcissus rupicula Dufour and Narcissus tazetta L. As the specimens from the Botanical Garden of Torretes were taken from commercially obtained or exchanged bulbs from other botanical gardens, the relationships of this M. geniculatus with the bulbs where it was found is in doubt; specimens could have accidentally come inside previously bought infested bulbs or could have been infected right at the Botanical Garden of Torretes, according to the M. geniculatus distribution [32]. Another species of Merodon, M. equestris, is widely known for being a horticultural pest and M. geniculatus might behave as a pest too. In any case, the genus Merodon tends to be widely associated with Narcissus spp. along with other bulb plants containing toxic compounds as, for example, in the D. maritima fed on by M. luteihumerus [4]. Many more studies on larval biology, co-evolution or even about their ability to digest or eliminate the phytotoxins of these plant families are needed to elucidate the nutritional links between these hoverflies and their food plants.
All the information collated here starts to show the wide diversity of habitats and relationships Eumerus and Merodon establish with many different plants. Larvae of both Eumerus and Merodon seem to prefer underground storage organs of the families Xhantorrhoeaceae and Hyacinthaceae. Despite underground storage organs from monocot geophytes of these plant families being the main habitat for the early stages of both genera (Table 1), it is clear that species of Eumerus feed and live in both monocots and dicots, even in very different plants such as Orobancheaceae, Cactaceae, Euphorbiaceae or Asteraceae, but Merodon seems to only use monocot habitats. Although some Eumerus species appear to produce decay themselves in healthy parts of plants (e.g. E. nudus), the feeding regime of Eumerus larvae still remains clearly more saprophagous than phytophagous due to their morpho-functional adaptations and reported breeding sites, while Merodon is strictly phytophagous.