An overlooked case for a century: taxonomy and systematics of a new Iberian species of Eumerus Meigen, 1822 (Diptera, Syrphidae)

. The hoverfly genus Eumerus Meigen, 1822 (Eristalinae: Merodontini) comprises 250+ described species, of which 36 are reported from the Iberian Peninsula. The high species diversity linked to the low degree of morphological differentiation between some species, which is even lower in females, leads to a high taxonomic complexity in this genus. The aim of this work is to confirm the morphological and molecular validity of an undescribed species of Eumerus , which is widespread in the Iberian Peninsula. The new species is described and compared with similar species. The genitalia of the new species are similar to those of Eumerus clavatus Becker, 1923 and Eumerus uncipes Rondani, 1850, but also share some features with Eumerus nudus Loew, 1848. The COI-5’ barcode is provided for the new taxon and analysed together with those of other named Eumerus sequences/species publicly available online. In the light of the morphology and barcoding data, the systematic position of the new species is discussed.

The most recent checklist of Iberian hoverflies included 31 species of Eumerus from mainland Spain plus the Balearic Islands (Ricarte & Marcos-García 2017), of which only one was endemic to Spain, Eumerus hispanicus van der Goot, 1966.After 2017, four new species endemic to the Iberian Peninsula have been described: Eumerus azabense Ricarte & Marcos-García in Ricarte et al. (2018), Eumerus bifurcatus van Steenis & Hauser in Grković et al. (2019b), Eumerus gibbosus van Steenis, Hauser & van Zuijen, 2017 and Eumerus grallator Smit in Grković et al. (2019a).Moreover, the status of Eumerus truncatus Rondani, 1868 has recently been clarified and it has been reported, for the first time, from Spain as well (van Steenis et al. 2017).The above-mentioned recent discoveries of Eumerus suggest that the diversity and distribution of this genus in the Iberian Peninsula is still far from being fully understood.
In the framework of an ongoing revision of the Eumerus diversity in the Iberian Peninsula, the aim of this work is to describe a new Iberian species and explore its systematic position based on morphological and molecular evidence.

Material examined
The specimens of the new species examined (79 specimens: 36 ♂♂ and 43 ♀♀) have been collected during a wide time span, from 1908 to 2021, at different Iberian localities (one specimen from Portugal) and by various collectors.Fresh specimens (caught over the last three years) were collected with hand net and killed by freezing.Additionally, specimens of similar species were examined as well.Examined material is now deposited in the following collections: All specimens are databased in an internal excel table at CIBIO.A unique bar code label was assigned to each of the specimens belonging to the CEUA-CIBIO collection.A map representing the distribution of the examined specimens was produced with the software QGIS (QGIS Development Team 2020) (Fig. 1).

Morphological study
An initial diagnosis of the new species with a morphological description of the male holotype and the female are given.Then, phenotypic variation, species distribution and biology of the new species are indicated.To complete the new species description, the following measures were taken with the software Leica Application Suite X (LAS X) ® ver.3.0.4.16529:Body length (l), from the base of the antenna to the posterior margin of tergum IV (Fig. 4A); body width (w), from tegula to tegula; head height (hh), from the top of the ocellar triangle to gena base (Fig. 4C); head width (hw), from one eye external side to the other (Fig. 4D); basoflagellomere ratio (length : width), of which length (bl) is measured from the pedicel apex to the apical margin of the basoflagellomere and width (bw) from the arista base to the ventral margin of basoflagellomere (Fig. 4C); and wing length (wl), from tegula to the apical apex of the wing (Fig. 4B).Male genitalia were dissected and prepared following Ricarte et al. (2012).Photographs of the holotype, one female paratype and male genitalia were taken with a Leica DFC 450 camera attached to a Leica M205 C binocular microscope.Male genitalia were hand drawn from printed photographs and stored in glycerine in plastic microvials.Morphological terminology used in the adult description follows Thompson (1999), except for terms "fossette" and "notopleural sulcus" which follow Dozckal & Pape (2009), and terminology for male genitalia follows Doczkal (1996).

Molecular study
DNA was extracted both from one mesoleg and one metaleg of five paratypes (2 males and 3 females) and one male of Eumerus nudus Loew, 1848 using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) following the manufacture's protocol for animal tissues.PCR amplifications of the 5' end region of the Cytochrome c oxidase subunit I (COI-5') gene were performed with the universal primers LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994).All amplifications were carried out in a total volume of 25 µl containing 1 × of Buffer reaction, 0.4 mM of dNTPs, 0.2 µM of each primer, 2 mM of MgCl 2 and 1 unit of NZYTaq II DNA polymerase.Thermocycler conditions followed Grković et al. (2019b).PCR products were visualized with an electrophoresis process in a 1% agarose gel and sequenced at Macrogen Inc. (Macrogen, Spain).
The COI-5' barcode sequences obtained were edited by eye with the program Sequencher ver.5.4.6 (Gene Codes Corporation 2017).Then, COI-5' sequences for each species of Eumerus available at the public repositories GenBank and BOLD (Ratnasingham & Hebert 2007) were downloaded (see Table 1).We excluded barcodes of Eumerus figurans Walker, 1859 and Eumerus superbus Shannon, 1927 as the pictures of the specimens assigned to them do not appear to correspond to these species.
First alignment was performed automatically with MAFFT online service (Katoh et al. 2017) which was checked by eye with the program AliView ver.1.25 (Larsson 2014).The final matrix had a length of 658 bp.We employed DAMBE7 (Xia 2018) to determine levels of variation for the first and third codon positions.We carried out a Neighbor-Joining (NJ) and a Maximum Likelihood (ML) analyses in MEGA7 (Kumar et al. 2016)

Diagnosis
Medium sized (6.5-9.5 mm) black species (Fig. 2), slightly larger than E. strigatus, which can be distinguished from similar species by the black pilosity on vertical triangle (Fig. 3), the absence of a baso-ventral ridge in the metatibia (Fig. 5B), the shape of sternum IV with a pair of black spinose valves (Fig. 6) and the general morphology of male genitalia (Fig. 7).

Etymology
This species is named after the Spanish entomologist Juan Gil Collado for his important contribution to the knowledge of the Iberian hoverflies (Gil-Collado 1930).The specific epithet 'colladoi' refers to his second surname and should be treated as a noun in the genitive case.
Head.Eye contiguity virtually of the same length (0.31 mm) as the height of the frontal triangle (0.33 mm).Eye pilosity consisting of rather short, scarce and pale pile, specially conspicuous on the ventral part of eye.Face and frontal triangle (including lunules) densely and white pollinose, covered with white pile (Fig. 3A).Vertical triangle black and with long, erect, black pile (Fig. 3C), pollinose at its anterior apex and with two extremely reduced pollinose spots which are located behind posterior ocelli.Ocellar triangle isosceles.Occiput black and purple iridescent (under artificial white lightning), with pile shorter than those on the vertical triangle; occiput white pollinose along the eye margin.Scape and pedicel dark brown; pedicel with black pile which are longer on the ventral side.Basoflagellomere almost square (ratio length:width = 1:1.1),convex dorsally and slightly concave ventrally, dark brown except baso-ventrally, with a lunulate black fossette at the distal margin.Pedicel and basoflagellomere  sparsely white pollinose (pollinosity more obvious in dorsal view and under artificial white lighting).Arista black (Fig. 3E).
tHorax.Mesonotum and lateral sides of the thorax black.Anterior and posterior parts of the scutum covered with rather short and white pile, in the medial part white pile intermixed with short and semireclined black pile; scutum with two medial white pollinose vittae extending over the anterior two thirds of the scutum and with a small circular grey pollinose spot posterior to transverse suture; lateral margins of the scutum with green metallic reflections.Notopleural sulcus absent.Disc of scutellum with white pile slightly longer than those on the scutum; posterior margin of scutellum with teeth-like protuberances, each bearing a long black pilis apically.Posterior anepisternum, anterior anepimeron and katepisternum on its postero-lateral area densely long white pilose.Katatergum with discrete bunch of yellow setae.Pleuron grey pollinose except the posterior anepisternum postero-laterally, anterior anepimeron centrally, ventral margin of posterior anepimeron and the dorsal margin of katepisternum.Femora black, basis and apices light brown.Basal half of pro-and mesotibia light brown and apical half black.Basal third of metatibia light brown and apical two thirds black.Dorsal surface of all tarsi black, ventrally yellowish brown.Posterior side of pro-and mesofemora white pilose.Metafemur densely white pilose, ventrally with pile of different lengths including some which are nearly twice as long as the reminder.Metafemur with an anterior row of 6 spinae and a posterior row of 14 spinae curved towards the posterior side of the metafemur (Fig. 5A).Metatibia flattened, without a baso-ventral ridge (Fig. 5B).Wing membrane extensively microtrichose; posterior margin of the wing with dense, short and brown pile; margin of ventral calypter with rather long and yellow pile; margin of dorsal calypter with pile shorter than those of ventral calypter; halter yellow.abdoMen.Terga I-IV black.Lateral margin of tergum I grey pollinose and white pilose.Terga II-IV densely white pilose on their lateral margins (pile on anterior corners of tergum II rather long); terga II-IV each with a pair of slightly curved, white pollinose maculae, terga covered with rather short, reclined and white pile.Anterior margins of terga III-IV black pilose.Posterior margin of tergum IV light brown with a marginal line of black pile along it.Sternum IV with two quadrangular, straight projections on the posterior margin; central area of the sternum IV black pilose and protruding in a rounded, nose-like expansion; sternum IV with two black spinose valves under the medial area (Fig. 6).
Male Genitalia.Epandrium with a simple posterior surstylar lobe convex anteriorly and straight posteriorly (Fig. 7B).Cercus with long pile.Anterior surstylar lobe with two large hyaline lobes and a small third accessory lobe in between (Fig. 7B-C).Hypandrium with a prominence at the base which shows a small indentation (Fig. 7A).

Female
Same as the male, besides the sexual dimorphism, except for the following characters: eye pilosity more abundant; basoflagellomere 1.0-1.2× wider than long and larger than in male (Fig. 3D, F); black macula at the distal margin of the basoflagellomere more noticeable (Fig. 3F); white pollinose spots behind the posterior ocelli larger than in male; frons white pollinose along eye margins but grey on its central area (Fig. 3B).

Variation
Based on the examined material, E. colladoi Ricarte & Aguado-Aranda sp.nov.shows intraspecific variation in body length (♂ = 6.43-8.91 mm, n = 21; ♀ = 7.69-9.58mm, n = 15), length of frontal triangle in males (0.28-0.38 mm), length of eye contiguity in males (0.24-0.41 mm), the presence or absence of two reduced white pollinose spots behind posterior ocelli (in males), the inclination of the spinae on the ventral side of the metafemur (from almost not inclined at all to an inclination of 90°) and the extent of the coloration on the metatibia (from light brown to black).

Distribution
The new species has been recorded from different Spanish and Portuguese localities, all in the Mediterranean region of the Iberian Peninsula (Fig. 1).

Biology
The flight period of E. colladoi Ricarte & Aguado-Aranda sp.nov.runs from late May to October.Adults can be found in a wide altitudinal range (80-1200 m) mainly associated with mountain areas.The predominant vegetation in these areas are woodlands of pine (Pinus L.) or oak (Quercus L.) where some adults have been sampled feeding on inflorescences of Thapsia villosa L. (Apiaceae).

Systematic position of E. colladoi Ricarte & Aguado-Aranda sp. nov. based on DNA evidence
Both the NJ and ML trees had virtually identical topologies.In the ML-tree (Fig. 8), high node support values (> 70) were obtained for every species group but for the E. pulchellus group (see Introduction).All barcodes of E. colladoi Ricarte & Aguado-Aranda sp.nov.were similar to each other with just 1 base pair difference.Eumerus nudus is similar to E. colladoi sp.nov. in the general shape of the basoflagellomere, the pilosity of the scutum, the coloration of the posterior margin of tergum IV and shape of male genitalia (see Discussion).The morphological similarities between E. nudus and E. colladoi sp.nov.justified the incorporation of the E. nudus sequence in the molecular analyses.A COI-5' sequence of the E. nudus was generated as it had no representation in the consulted BOLD and GenBank databases.The barcode sequence of E. nudus grouped, as expected, with that of Eumerus alpinus Rondani, 1857 but they did not form a clade with E. colladoi sp.nov.In addition, the new species did not cluster with sequences of species of the E. clavatus group, which have even more similar male genitalia in comparison with E. colladoi sp.nov.together with a medium-sized body, a dark body coloration and an elongated and parallel-sided abdomen.The proportion of invariant sites for the first and third codon positions were 0.29 and 0.07, respectively.Level of variation is usually higher in the third codon position as it tends to have faster rate of evolution (Felsenstein 1978).

Discussion
In the recent key to the species of Eumerus from Switzerland and surrounding parts of central Europe (Speight et al. 2021), E. colladoi Ricarte & Aguado-Aranda sp.nov.would key out as Eumerus amoenus Loew, 1848.However, the new species differs from E. amoenus in the eye pilosity, which is slightly longer and denser in E. colladoi sp.nov.; pollinosity found on the anterior apex of vertical triangle, which is sparser in E. amoenus; shape of basoflagellomere, which is slightly longer in E. amoenus; vertical triangle wholly covered with black pile in E. colladoi sp.nov.(except in the pollinose area of the anterior corner where a few yellow pile are present), black only on the ocellar triangle in E. amoenus; absence of a ridge on the baso-ventral side of the metatibia in E. colladoi sp.nov., a row of short reclined black spinae that is present in E. amoenus (Fig. 5C); shape of the sternum IV (Fig. 6), which is simple in E. amoenus; and the general morphology of the male genitalia (Fig. 7) (for E. amoenus genitalia, see Vujić & Šimić 1998).The fact that E. colladoi sp.nov.lacks a baso-ventral ridge in the metatibia is particularly interesting as this character state appears to be rare in the Iberian Eumerus (Martin Hauser in litt.).In addition, the new species was also run through the Stackelberg's (1961) key to the Palaearctic Eumerus and it keyed out to Eumerus chrysopygus Sack, 1941, which can easily be separated from E. colladoi sp.nov.by the eye pilosity, which is virtually absent in E. chrysopygus; pilosity of the scutum, which is longer and dark brown in E. chrysopygus but shorter and with white and black pile intermixed in E. colladoi sp.nov.; a ridge with a row of black setae on the baso-ventral side of metatibia, which is not present in E colladoi sp.nov.; length of metabasitarsus, which is shorter and much broader in E chrysopygus than in E. colladoi sp.nov.; pilosity of the posterior half of tergum IV, which is goldenyellow in E. chrysopygus and black in E. colladoi sp.nov.; and the sternum IV morphology, which in E. chrysopygus lacks the two spinose valves typical of E. colladoi sp.nov.(Fig. 6).
Eumerus colladoi Ricarte & Aguado-Aranda sp.nov. is also similar to the species of the E. clavatus group, Eumerus clavatus and Eumerus uncipes Rondani, 1850 (see Results) but the new species differs in the mesonotum pilosity (not golden-yellow but white intermixed with black pile posteriorly and medially), length of tergum IV (not twice the length of tergum III), external morphology of sternum III (lacking the prominence with long pile in its medial part) and the shape of sternum IV which is not plierlike shaped (see Speight et al. 2021: fig. 15).The male of E. colladoi sp.nov.can be easily distinguished from those of E. clavatus and E. uncipes by the shape of the metatibia, which is modified in these two species (see Speight et al. 2021 figs 16d, 17b), but simple in E. colladoi sp.nov.
The new species is easily differentiated from E. nudus (see Results) by the body size, which is normally larger in E. nudus; pile on the ventral side of the metafemur, which is clearly longer in E. nudus; the anterior row of spinae on the ventral side of the metafemur, which is on a gentle elevation in E. nudus and the general shape of sternum IV.Finally, females were also run through Stackelberg's key and keyed out to Eumerus kondarensis Stackelberg, 1952.Nevertheless, males of E. kondaresis have different genitalia (Stackelberg 1952) and the barcode analyses confirmed molecularly that the examined females belonged to the same species as the males of E. colladoi Ricarte & Aguado-Aranda sp.nov.These results prove that barcode sequence analysis is useful to match males and females in hoverfly species as sexual dimorphism and morphological similarity between females of different species make sometimes identification difficult.
Nevertheless, the obtained molecular trees must be cautiously taken when assessing species affiliations with groups or species relationships, as the species sampling is still too limited.Barcodes are only available for 43 species confirmed of the 250+ known valid species of Eumerus worldwide (Souba-Dols et al. 2020).Therefore, it is likely that the current systematic position of E. colladoi sp.nov.can change when barcodes of additional species of Eumerus are obtained.At the moment, the new species cannot be assigned to any of the defined groups of species and it would conform a monospecific group.Thus, a wider taxon sampling and the use of other informative markers are necessary to disentangle the actual phylogenetic relationships of E. colladoi sp.nov.with other species.
Eumerus colladoi Ricarte & Aguado-Aranda sp.nov. is distributed across the Iberian Peninsula (Fig. 1) but it has not been collected in its Eurosiberian Region, to the north (Rivas-Martínez et al. 2014).The fact that E. colladoi sp.nov.has never been reported from the Eurosiberian Region of the Iberian Peninsula (e.g., Marcos-García 1985a, 1985b, 1990;Ricarte et al. 2014) may indicate that it is absent from there.However, the new species may occur in parts of this Region as some records from close-by localities, for example Rodiezmo and Sena de Luna in the province of León show.On the other hand, the ubiquity of the new species suggests that the larvae depend on a widespread plant species in the Iberian Peninsula or they are polyphagous.Despite the availability of large amounts of data on the Syrphidae in Europe (Speight et al. 2020) and the number of recent publications on the taxonomy of Eumerus (e.g., Chroni et al. 2018;Grković et al. 2021;Speight et al. 2021), E. colladoi sp.nov.had not been unrevealed so far.So, it might be really absent from other parts of Europe.However, the generally poor knowledge of the hoverflies of North Africa (El-Hawagry & Gilbert 2019; Mebarkia et al. 2021) leads to a reasonable doubt about the occurrence of this new species in this part of the African continent, since other hoverfly species are present both in the Iberian Peninsula and North Africa, for example Mallota dusmeti Andréu, 1926(Pennards et al. 2021) and Merodon luteihumerus Marcos- García, Vujić & Mengual, 2007(Likov & Radenković 2021).At the moment, Eumerus colladoi sp.nov.must be considered as the sixth Iberian-endemic species of Eumerus, which now has 37 species recorded in the Iberian Peninsula.This region of Europe is home to a high diversity of Eumerus similar to that in other regions such as the Balkan Peninsula (Grković 2018).
CEUA-CIBIO = Colección Entomológica de la Universidad de Alicante, Research Institute CIBIO, University of Alicante, Spain MNCN = Museo Nacional de Ciencias Naturales, Madrid, Spain CSCA = California Department of Food and Agriculture, Sacramento, USA Coll. A. v. Eck = Private collection of André van Eck

Fig. 8 .
Fig. 8. Tree inferred using the Maximum Likelihood method.Bootstrap values > 50 are shown near nodes.Branch lengths measured in number of substitutions per site.

Table 1
(continued on next two pages).List of the species used in the molecular analyses.New sequences generated for this work are highlighted in bold.

Table 1 (
continued).List of the species used in the molecular analyses.New sequences generated for this work are highlighted in bold.

Table 1
(continued).List of the species used in the molecular analyses.New sequences generated for this work are highlighted in bold.