European Journal of Taxonomy 237: 1-25

http://dx.doi.org/10.5852/ejt.2016.237

This work is licensed under a Creative Commons Attribution 3.0 License.

ISSN 2118-9773

www. europeanj ournaloftaxonomy. eu

2016 • Acanski J. et al.

Research article

urn:lsid:zoobank.org:pub:26A6D035-B2D9-48B2-9350-AEB9D6299B22

Defining species boundaries in the Merodon avidus complex

(Diptera, Syrphidae) using integrative taxonomy,

with the description of a new species

Jelena ACANSKI >•*, Ante VUJIC 2 , Mihajla DJAN 37 , Dragana OBREHT VIDAKOVIC 4 7 ,

Gunilla STAHLS 5 & Snezana RADENKOVIC 6 7

1 University of Novi Sad, BioSense Institute - Research Institute for Information Technologies in

Biosystems, Trg Dr Zorana Bindica 1, 21000 Novi Sad, Serbia.

^Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad,

Trg Dositeja Obradovica 2, 21000 Novi Sad, Serbia.

5 Zoology Unit, Finnish Museum of Natural History, PO Box 17, 00014 University of Helsinki, Finland.

* Corresponding author: [email protected]

2 Email: [email protected]

3 Email: [email protected]

4 Email: dragana. [email protected]

5 Email: gunilla stah1s@he1sinki fi

6 Email: [email protected]

1 um:lsid:zoobank.org:author:F02841B0-AC6B-4FC4-A64A-D13A3355B079

2 urn:lsid:zoobank.org:author:A20D5863-CF18-4BF7-BB68-0DA75D34B7A8

3 urn:lsid:zoobank.org:author:BE29D9A9-CC69-416C-8F85-4B8AA99A6C71

4 urn:lsid:zoobank.org:author:75E52F0E-EEDC-4EFl-907B-9202DBDEAC9F

5 urn:lsid:zoobank.org:author:D19B 1262-503A-48E2-91AE-F99D0F2EA3 A3

6 urn:lsid:zoobank.org:author:26DF35D9-55FA-4485-8E8C-ClF90EFE1036

Abstract. Several recent studies have detected and described complexes of cryptic and sibling species in

the genus Merodon (Diptera, Syrphidae). One representative of these complexes is the Merodon avidus

complex that contains four sibling species, which have proven difficult to distinguish using traditional

morphological characters. In the present study, we use two geometric morphometric approaches, as

well as molecular characters of the 5’-end of the mtDNA COI gene, to delimit sibling taxa. Analyses

based on these data were used to strengthen species boundaries within the complex, and to validate the

status of a previously-recognized cryptic taxon from Lesvos Island (Greece), here described as Merodon

megavidus Vujic & Radenkovic sp. nov. Geometric morphometric results of both wing and surstylus

shape confirm the present classification for three sibling species— M. avidus (Rossi, 1790), M. moenium

Wiedemann in Meigen, 1822 and M. ibericus Vujic, 2015 —and, importantly, clearly discriminate the

newly-described taxon Merodon megavidus sp. nov. In addition to our geometric morphometric results,

supporting characters were obtained from molecular analyses of mtDNA COI sequences, which clearly

differentiated M. megavidus sp. nov. from the other members of the M. avidus complex. Molecular

analyses revealed that the earliest divergence of M. ibericus occurred around 800 ky BP, while the most

recent separation happened between M. avidus and M. moenium around 87 ky BP.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

Keywords. Merodon avidus complex, COI, geometric morphometry, wing, surstylus.

Acanski J., Vujic A., Djan M., Vidakovic D.O., Stahls G. & Radenkovic S. 2016. Defining species boundaries

in the Merodon avidus complex (Diptera, Syrphidae) using integrative taxonomy, with the description of a new

species. European Journal of Taxonomy 237: 1-25. http://dx.doi.org/10.5852/eit.2016.237

Introduction

The genus Merodon Meigen, 1803 (Diptera: Syrphidae: Merodontini) has become the largest European

ho verily genus due to several recent studies describing many new taxa (Marcos-Garcia et al. 2007; Popov

2010; Radenkovic et al. 2011; Vujic et al. 2007, 2012, 2013a, 2013b, 2015). A considerable number

(37 of 57 species in southeastern Europe) of the taxa are morphologically and/or genetically cryptic,

with restricted distributional ranges in particular mountain ranges or islands (Vujic et al. 2016). Larvae

of Merodon species are phytophagous on the underground storage organs of plants (Amaryllidaceae,

Iridaceae and Hyacinthaceae) (Hurkmans 1993; Andric et al. 2014). Larvae of Merodon avidus (Rossi,

1790) were recently found in the bulbs of Ornithogalum umbellatum L. (Hyacinthaceae) (Andric et

al. 2014). This host plant is an important foodstuff for adults, as well as for larval development and is

widespread in Europe, Southwestern Asia and North Africa. Distribution of this host plant is wider than,

but overlaps with, the range of the M. avidus complex (Andric et al. 2015; Popovic et al. 2015).

Taxa of the Merodon avidus complex have been the subject of many studies in the last decade due

to perceived taxonomic difficulties (Milankov et al. 2001, 2009; Stahls et al. 2009). The M. avidus

complex is characterized by a considerable morphological variability, especially in the coloration of the

antennae, thorax, abdomen and legs (Popovic et al. 2015). This colour variability has been explained

by the differential availability of trophic resources during the larval stage (Hurkmans 1993). Popovic

et al. (2015) provided justifications for the identification of three species from the M. avidus complex

based on new diagnostic morphological characters, records of the seasonal activity and geographical

distribution of bi-voltine adults, nuclear allozyme and mtDNA COI sequence analyses, and descriptions

of the ecological preferences of the taxa.

However, difficulties in distinguishing the species of this complex based on morphological characters

remain, despite the numerous studies on the subject. Spring generations of Merodon avidus are very

similar to those of M. moenium Wiedemann in Meigen, 1822 based on external morphology, and, so,

are easily confused using existing diagnostic features (e.g., Milankov et al. 2001). Furthermore, it is

important to note that species of the M. avidus complex are not distinguishable by traditional visual

identification of the structures of male genitalia under a stereo microscope. Additionally, in their analysis

of COI barcodes of all Merodon species from Lesvos Island (Greece), Stahls et al. (2009) revealed the

presence of one cryptic taxon ( Merodon sp. nova 2, herein described as M. megavidus sp. nov.) within the

M. avidus complex, but did not present morphological diagnostic characters to enable characterization

of the new taxon.

In the present study, two different geometric morphometric approaches were applied to quantify wing

and surstylus shape variability among all hitherto described species of the Merodon avidus complex,

including the new fourth taxon M. megavidus sp. nov. Insect wing shape is highly heritable and

constitutes an important character for separating species (Birdsall etal. 2000). Geometric morphometric

analysis of the wing shape has been successfully used in taxonomic studies of multiple hoverfly taxa

(Francuski et al. 2009; Vujic et al. 2013b; Nedeljlcovic et al. 2013, 2015). The structure and/or shape of

parts of the male genitalia are very informative and thus useful for Syrphidae taxonomy and systematics

(e.g., Hippa & Stahls 2005). Differences in male genitalia structure (particularly the surstyli and

aedeagal parts) detected in morphological taxonomic studies of hoverflies have revealed them to be an

ACANSKI J. et al. , Species boundaries in the Merodon avidus complex

important mechanism of isolation between species (Rotheray & Gilbert 2011). However, differences in

genitalia structure between closely-related species can be very small, as shown for many hoverfly genera

(Dusek & Laska 1964; Hippa 1990; Nedeljlcovic et al. 2013, 2015; Vujic et al. 2013b, 2015). Geometric

morphometric analysis of surstylus shape can reveal subtle shape differences that are not detectable

or quantifiable by traditional visual examination (Mutanen & Pretorius 2007). This approach has only

recently been applied to the taxonomy of the Syrphidae, having been successfully implemented in the

genus Chrysotoxum Meigen, 1803 to distinguish four species (Nedeljkovic et al. 2013, 2015). The male

genitalia of Merodon species are sclerotized and rigid and thus stable structures, so they are well suited

for geometric morphometric analysis.

Our study had three objectives: (1) to further clarify the species borders of all taxa within the M. avidus

complex using integrative taxonomy (geometric morphometries of wings and male surstylus shape and

mtDNA COI sequences); (2) to estimate times of divergence among investigated taxa; and (3) to provide

descriptions and diagnostic characters of the new species. A typological (or morphological) species

concept is applied in this study, integrating all available data.

Material and methods

Studied material

A total of 444 specimens belonging to the Merodon avidus complex from Bulgaria, Croatia, Greece,

Italy, Montenegro, Morocco, Serbia, Spain and Turkey was analysed (Fig. 1; Appendix 1). Specimens

of M. avidus were sampled during spring, summer and autumn. All specimens were identified by Ante

Vujic and Snezana Radenkovic, and labelled using unique codes.

Fig. 1 . Map of population sampling locations of the Merodon avidus complex from the Western Palaearctic.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

Specimens detailed in the Results are in the collections of the following institutions:

FSUNS = Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Serbia

MZF = Finnish Museum of Natural History, Helsinki, F inl and

WML = World Museum Liverpool, UK

Numbers given alongside the abbreviations FSUNS, MZH, and WML in the text refer to unique

identifiers from the specimen database stored in the internal electronic database of the Faculty of

Sciences, University of Novi Sad.

For the description of the new species, the terminology follows McAlpine (1981) for non-genitalic

morphology and Marcos-Garcia et al. (2007) for morphology of the male terminalia.

Wing morphometry

Wings of all 444 available specimens were analysed using a landmark-based geometric morphometric

approach (Appendix 1). Population analysis was carried out for 418 specimens of 23 populations

(Appendix 1, marked with *). Populations with small sample sizes were excluded from the analysis

to avoid statistical errors. The left wing of each specimen was removed using micro-scissors and

mounted in Hoyer’s medium on a microscope slide. Eleven homologous landmarks were digitized at

vein intersections or terminations that could be reliably identified and best represented wing shape using

TpsDig 2.05 software (Rohlf 2006). Generalized least-squares Procrustes superimposition was first

applied to the landmark data to remove non-shape variation in terms of location, scale and orientation,

and also to superimpose the objects in a common coordinate system (Rohlf & Slice 1990; Zelditch et al.

2004). For the wing shape analysis, partial warp scores were calculated (Zelditch et al. 2004). Procrustes

superimposition and partial warps were computed using the free IMP software CoordGen7.14 and

CVAgen 7.14a (Sheets 2012). MorphoJ v.2.0. software (Klingenberg 2011) was used for visualization

of thin-plate spline deformation. Surstylus morphometry

Shape analysis of the posterior part of the left surstylus was carried out on 125 specimens of the M. avidus

complex using a semi-landmark geometric morphometric approach (Appendix 2). The posterior part of

the left surstylus (Fig. 2A: psl) was removed using a scalpel and placed on its side in glycerol on a

microscope slide, and a coverslip was placed on top of the surstylus to immobilize it. Due to a lack of

homologous anatomical loci, 30 semi-landmarks were digitized using the ‘resample curve by length’

option in TpsDig 2.05 software (Rohlf 2006). CoordGen 7.14 with an integrated Semiland module was

used for semi-landmark superimposition using a distance-minimizing protocol, which minimizes the

shape differences due to the arbitrary nature of semi-landmark positions along the curve (Bookstein

1997; Zelditch et al. 2004).

Statistical analyses

Principal component analysis (PCA) was used to test variability of wing and surstylus shape without a

priori defining groups. Multivariate analysis of variance (MANOVA) and Fisher LSD post-hoc tests were

used to test the variability connected with shape differences between species. Additionally, canonical

variates analysis (CVA) and discriminant function analysis (DA) were used to test significance in wing

and surstylus shape differences, to produce distance matrices and to graphically present results. The

phenetic relationships among taxa were determined by UPGMA analysis based on squared Mahalanobis

distances computed from the DA applied to wing variables. All statistical analyses were conducted using

Statistica for Windows (StatSoft Inc. 2015: version 12).

Molecular analysis

The 5’-end of the cytochrome c oxidase 1 (COI) gene (‘barcode’ region) was amplified for three

specimens of M. megavidus sp. nov. from Lesvos Island, Greece. DNA was extracted from a leg of each

ACANSKI J. et al. , Species boundaries in the Merodon avidus complex

specimen using a slightly modified SDS Extraction Protocol (Chen et al. 2010). Genomic DNA vouchers

are conserved at the Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad

(AU827, AU885, AU886) and the Finnish Museum of Natural History, Helsinki (MZH S532). DNA

barcodes were amplified with the forward primer LCO (^’-GCTCAACAAATCATAAAGATATTGGo ’)

and the reverse primer HCO (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994).

PCR amplifications were carried out in 20 pi reaction volumes using the following mix of components:

1 x PCR buffer (Thermo Scientific), 2.5 mM MgCl 0 , 0.1 mM of each nucleotide, 1 U Taq polymerase

(Thermo Scientific), 2 pmol of each primer and 50 ng template DNA. PCR conditions were: initial

denaturation for 2 min at 95°C; 30 s denaturation at 94°, 30 s annealing at 49°C, 2 min extension at 72°C/30

cycles; and the final extension for 8 min at 72°C. Obtained amplicons were purified using the Exo-Sap

purification protocol (Thermo Scientific). Forward sequencing using the PCR primer was conducted at

the Sequencing Service Laboratory of the Finnish Institute for Molecular Medicine, Hels ink i, F inl and

(FIMM). In order to confirm the taxonomic status of M. megavidus sp. nov. specimens, we created

a dataset consisting of 22 DNA barcode sequences, 18 of which represented GenBank-accessioned

sequences of M. avidus , M. moenium and M. ibericus Vujic, 2015 (6 sequences per species, accession

numbers indicated in Fig. 6, and one sequence for M. megavidus sp. nov. (generated for the Stahls et

al. 2009 study), in addition to three newly-generated M. megavidus sp. nov. DNA barcode sequences in

this study). Sequences of Eumerus sulcitibius Rondani, 1868 (Ace. No. KT157875) and Platynochaetus

setosus (Fabricius, 1794) (Ace. No. KM224512) (both Merodontini) were used as outgroups. Sequence

alignment was conducted using the Clustal W algorithm (Thompson et al. 1994), as implemented in

BioEdit (Hall 1999), and final modifications were done by hand. The total length of the dataset after

alignment and trimming was 634 nucleotides. We constructed a Neighbour-joining (NJ) tree, and

phylogenetic trees using Maximum Likelihood (ML) and Maximum Parsimony (MP) analyses. The NJ

and ML trees were constructed using MEGA version 6 (Tamura et al. 2013) using a Tamura-3 parameter

model of nucleotide substitution with Gamma correction for among-site variation in substitution rates

(estimated in the same software). MP analysis was done using NONA (Goloboff 1999), with the aid

of Winclada (Nixon 2002), using the heuristic search algorithm and 1000 random addition replicates

(mult x 1000); holding 100 trees per round (hold/100), max trees set to 100000; and applying TBR

branch swapping. Bootstrap values were calculated for each tree using 1000 replicates. The genetic

relationships among species were also tested using a Median-Joining (MJ) network (Bandelt et al. 1999)

generated with Network v4.6.1.3 (available from http://www.fluxus-engineering.com/sharenet.htm l by

applying the default settings (e = 0 and the variable sites weighted equally = 10), with additional post¬

processing with the maximum parsimony (MP) option.

Pairwise Ost values among species of the Merodon avidus complex were calculated, together with

an exact test of population differentiation, using ARLEQUIN 3.5.1.2 (Excoffier & Lischer 2010).

Furthermore, we calculated the genetic distance between defined species using MEGA version 6 (Tamura

et al. 2013) in order to estimate times of divergence between genetic clusters based on uncorrected

p-distances divided by the pairwise evolutionary rate/MYR as described in Prohl et al. (2010). We used

the pairwise sequence divergence of the COI gene, relative to the mutational rate of 2.3% per million

years, as estimated for various arthropod taxa (Brower 1994).

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

Results

Description of new species

Family Syrphidae Latreille, 1802

Subfamily Eristalinae Newman 1834

Tribe Merodontini (Edwards, 1915)

Genus Merodon Meigen, 1803

Merodon megavidus Vujic & Radenkovic sp. nov.

um:lsid:zoobank.org:act:B2016D80-A7ED-4958-AE91-EE5E6A8C7C51

Figs 2C-D, 3-5

Diagnosis

Medium- to large-sized species (13-18mm); blackmesoscutum with four white microtrichose longitudinal

stripes; tapering orange and black abdomen with white, transverse, microtrichose bands on tergites

2-4 (exceptionally without bands on tergite 2); tarsi reddish-orange dorsally; hind femur medium wide

and slightly curved (Fig. 3C-D), with very short pile posteroventrally. Merodon megavidus Vujic &

Radenkovic sp. nov. belongs to the avidus complex (male genitalia in all species identical in shape, as

on Fig. 2). Merodon megavidus sp. nov. can be separated from the other members of the complex by

larger size, golden body pile, bright orange colour of the pale parts of legs and extremely short pile on

hind femur (Fig. 3C-D). These characteristics contrast with other species from the complex, which have

yellow to grayish pale body pile and longer pile on the hind femur (Fig. 3A-B).

Etymology

The name megavidus refers to the large size (Greek word me gas means “large”) and great similarity with

Merodon avidus.

Type material

Holotype

GREECE: S, Lesvos, Petrounta, 26 Jul. 2015, leg. A. Vujic and S. Radenkovic (FSUNS 10132).

Fig. 2. Merodon megavidus Vujic & Radenkovic sp. nov., male genitalia. A. Epandrium, lateral view.

B. Left surstylus, anterior view. C. Hypandrium, lateral view. Abbreviations: psl = posterior surstylus lobe;

asl = anterior surstylus lobe; c = cercus; ae = aedeagus; ea = ejaculatory apodeme. Scale bar = 0.5 mm.

ACANSKI J. et ah. Species boundaries in the Memdon avidus complex

Para types

GREECE: Lesvos: 1 ?, (WML Misc 38). Agiassos: 1 3 , 1 Jul, 2007, leg. M. Hull (WML H723); 1 ?,

8 Jun. 2003, leg. G. Stahls (FSTJNS 04385); 1 $, 8 Jun. 2003, leg. G. Stahls (MZH); 1 3 , 16 Jun. 2004,

leg. M. Hull (WML Hu-93); 3 33 , 19 Jun. 2003, leg. M. Hull (WML Hu70, Hu-78, Hu-81); 1 3 ,

20 Apr. 2004, leg. M. Hull (WML Hu-92); 4 33, 20 Jun. 2004, leg. M. Hull (WML Hu-77, Hu-94-96);

5 33 , 22 Jun. 2004, leg. M. Hull (WML Hu-76, Hu-97-100); 2 33 , 22 Jun. 2004, leg, M. Hull (FSUSN

04390, 04391); 2 33 , 22 Jun. 2009, leg. M. Hull (WML H1432, H1433); 1 ?, 23 Jun. 1999, leg. M.

Hull (FSUNS 04392); 3 33 , 23 Jun. 2003, leg. M. Hull (WML Hu-69, Hu-79, Hu-80). Vatoussa: 16

33 , 10$?, 26 Jul. 2015, leg. A. Vujic and S. Radenkovic (FSUNS 10133-10151,10159, 10160,10162,

10163, 10243, 10253, 10256); 1 3 , 1-4 Jun. 2012, leg. Nakas (FSUNS C94). Plomari: 2 33 , 14 Jul.

2004, (FSUNS 02325, 03966); 1 3 , 14. Jul. 2004, leg. H. Dahm (S532).

Fig. 3. Hind leg, lateral view. A-B. Merodon avidus (Rossi, 1790). A. 3. B ?. — C-D. M. megavidus

Vujic & Radenkovic sp. nov. C. 3 - D. ?. Scale bar = 1 mm.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

Description

Male (Figs 2C, 3, 4A, 5A)

Head (Fig. 4A). Antenna (Fig. 4A) orange, first fiagellomere 1.8-2.0 times as long as wide, 2.0 times

longer than pedicel, concave, apex acute; arista: second, third and basal part of fourth flagellomeres

pale, fourth fiagellomere dark brown in apical 2 /3 and thickened basally, 1.4 times longer than first

fiagellomere; with short, dense microtrichia. Face and frons black, covered with long golden pile and

silver, dense microtrichia. Oral margin shiny black, except for the lateral microtrichose areas (Fig. 4A).

Vertical triangle isosceles, shiny black except in front of the anterior ocellus that has pale microtrichia,

covered with long orange pile except for black pile on the ocellar triangle. Ocellar triangle equilateral.

Eye contiguity about 12 ommatidia long. Vertical triangle: eye contiguity: ocellar triangle = 1.5 : 0.7 :

1. Eye pile dense, white. Occiput with orange pile, along the eye margin with dense white microtrichia

and posteriorly with metallic, bluish-greenish lustre.

Thorax. Mesoscutum and scutellum black with bronze lustre, covered with relatively long, dense, erect

golden pile. Side of mesoscutum above wing-base with a patch of black pile. Mesoscutum with two lateral

and two submedian, longitudinal, white microtrichose stripes. Proepimeron, posterior anepisternum,

anteroventral and posterodorsal part of katepisternum, anepimeron, metasternum and katatergite with

long golden pile and grey-green microtrichia. Wing hyaline, with dense microtrichia; veins dark brown

except for light brown C, Sc and Rl. Calypter pale yellow. Haltere with light brown pedicel and yellow

capitulum. Legs orange, except for the black basal 3 A of the front- and mid-femora. Pile on legs golden.

Hind femur (Fig. 3C) moderately thickened and curved, about 3.6 times as long as deep. Pile on hind

femur very short.

Abdomen (Fig. 5A). Dark with white microtrichose bands, tapering, 1.4 times longer than mesonotum

(including scutellum). Tergites orange and reddish except for black tergite 1 and central parts of tergites

2-3 (and 4) (Fig. 5A); orange-reddish parts of variable size on tergite 3 and 4, laterally and along

microtrichose bands. Tergites 2-A each with a pair of white microtrichose marks (exceptionally absent

only on tergite 2); tergites 3-4 with wide, oblique bands (Fig. 5A). Pile on tergites golden. Sternites

translucent, orange to brown towards the tip of the abdomen, covered with long yellow pile.

Fig. 4. Merodon megavidus Vujic & Radenkovic sp. nov., head, antero-lateral view. A. $. B. §. Scale

bar = 1 mm

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

Male genitalia (Fig. 2). Similar to all species of the M. avidus complex. Anterior lobe of surstylus broad

and hairy (Fig. 2A-B); posterior lobe of surstylus ellipsoidal at ventral margin (Fig. 2A-B); cercus

rectangular, without prominences (Fig. 2A). Hypandrium elongate and sickle-shaped, without lateral

projections (Fig. 2C); lingula long (Fig. 2C).

Female (Figs 2D, 4B, 5B)

Similar to the male except for typical sexual dimorphism and for the following characteristics:

first flagellomere broader and longer; frons with two wide (about 0.34 width of frons) lateral silver

microtrichose longitudinal stripes; frons in the widest part about 0.25 width of head; white microtrichose

longitudinal stripes on mesoscutum more visible; broad stripe of black pile between wing bases; tergites

predominately red except for tergite 1 and darkened parts of tergites 2^1 in some specimens (Fig. 5B);

white, microtrichose, transverse bands on tergites 3M (Fig. 5B); tergites 2-3 with black pile on dark

parts; white microtrichose bands solely with pale pile.

Remarks

This species was mentioned as Merodon sp. nova 2 in Stahls et al. (2009) and Ricarte et al. (2012).

Stahls et al. (2009) assumed it to be a distinct species based on the different COI barcode sequences

Fig. 5. Merodon megavidus Vujic & Radenlcovic sp. nov., abdomen, dorsal view. A. $. B. $. Scale bar =

1 mm

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

obtained from two specimens morphologically similar to M. avidus taken from Lesvos Island. In the key

prepared as supporting material for Stahls et al. (2009), M. avidus and M. sp. nova 2 key out together,

without any morphological differences being described.

Distribution and habitat data

Lesvos Island (Greece). Maquis shrubland.

Species delimitation

Molecular data

Merodon megavidus sp. nov. clearly differs from other members of M. avidus complex based on our

barcoding fragment of COI. All conducted phylogenetic analyses resulted in similar tree topologies

(Figs 6-7; Appendix 3). Sequences from the Merodon avidus complex formed three separate clusters:

one cluster represented M. ibericus , a second comprised all M. megavidus sp. nov. sequences with

highly-significant bootstrap values (100), the third grouped sequences of M. avidus and M. moenium

together. All four M. megavidus sp. nov. sequences were identical, defining one haplotype unique to the

species. The number of mutational steps between M. megavidus sp. nov. and M. avidus is 14, with 13

and 22 mutational steps between M. megavidus sp. nov. and M. moenium and M. ibericus , respectively

(Fig. 8).

Our UPGMA tree based on genetic distances among species revealed genetic relationships among four

taxa of the Merodon avidus complex that support and strengthen the branch positions in wing and

surstylus phenograms described below (see Fig. 9).

Significant pairwise genetic divergence (<J)st value) was detected in each pairwise comparison between

M. megavidus sp. nov. and each of the other species M. ibericus , M. avidus and M. moenium (0.420, 0.492

and 0.529, respectively). Sequence divergence (uncorrected p distance) of the COI gene was used to

Platynochaetus setosus (KM224512)

H2H

Eumerus sulcitibius (KT157875)

100

Merodon ibericus (KT731119)

Merodon ibericus (KT731107)

Merodon ibericus (KT731116)

Merodon ibericus (KT731117)

Merodon ibericus (KT731111)

Merodon ibericus (KT731118)

100

— Merodon megavidus sp. nov. (KX017461)

— Merodon megavidus sp. nov. (KX017462)

— Merodon megavidus sp. nov. (KX017463)

— Merodon megavidus sp. nov (KX017464)

— Merodon avidus (KT731106)

— Merodon moenium (KT731122)

— Merodon moenium (KT731123)

Merodon moenium (KT731124)

— Merodon moenium (KT731132)

Merodon avidus (KT731127)

Merodon avidus (KT731126)

— Merodon avidus (KT731125)

Fig. 6. Maximum parsimony strict consensus tree based on DNA barcode COI sequences. Length 136

steps, Consistency Index (Cl) = 93, Retention Index (RI) = 95. Filled circles denote unique changes,

open circles non-unique.

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

Table 1. Below diagonal - pairwise p distances; above diagonal - estimated divergence times in years.

M. megavidus sp. nov.

M. ibericus

M. avidus

M. moenium

M. megavidus sp. nov.

—

826.000

521.000

478.000

M. ibericus

0.038

—

800.000

760.000

M. avidus

0.024

0.037

—

87.000

M. moenium

0.022

0.0035

0.004

assess relative divergence times between the four Merodon taxa (Table 1), indicating an initial separation

of M. ibericus from the rest of the complex around 800 ky BP. Divergence between M. megavidus

sp. nov. and M. avidns/M. moenium occurred around 500 ky BP. The most recent separation happened

between M. avidus and M. moenium , around 87 ky BP.

Geometric morphometries - wing shape analysis

Principal component analysis conducted on 444 specimens of the M. avidus complex revealed six

principal components (PC) that together explained 63.9% of total wing shape variability. A MANOVA

with Fisher LSD post-hoc test showed that variability reflected shape changes among the investigated

r Merodon avidus (KT731106)

100

Eumerus sulcitibius (KT157875)

- Platynochaetus setosus (KM224512)

66 31 )

- Merodon moenium (KT731123)

Merodon moenium (KT731132)

I Merodon moenium (KT731124)

Merodon moenium (KT731124)

Merodon avidus (KT731127)

Merodon avidus (KT731125)

Merodon avidus (KT731126)

Merodon avidus (KT731125)

Merodon moenium (KT731124)

Merodon moenium (KT731122)

Merodon megavidus sp. nov. (KX017461)

Merodon megavidus sp. nov. (KX017462)

Merodon megavidus sp. nov. (KX017463)

Merodon megavidus sp. nov. (KX017464)

Merodon ibericus (KT731119)

Merodon ibericus (KT731107)

Merodon ibericus (KT731117)

r- Merodon ibericus (KT731116)

2 7 |j — Merodon ibericus (KT731111)

52 } Merodon ibericus (KT731118)

0.02

Fig. 7. Maximum Likelihood tree based on a 5’ fragment of COI mtDNA sequences from the Merodon

avidus complex. Bootstrap values (1000 replicates) are shown next to the branches. The tree is drawn to

scale, with branch lengths proportional to the number of substitutions per site.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

species in all six PCs (MANOVA: F lg P30 = 38.56; p < 0.00000). Further, DA showed that all species

differ highly significantly in wing shape (p < 0.00000), and correctly classified species with an overall

classification success of 90%. All specimens of M. megavidus sp. nov. were correctly classified, while

the lowest classification success was for M. ibericus (78%). Specimens belonging to M. avidus were

correctly classified with 89% and M. moenium with 93% certainty. Canonical analysis produced

three canonical axes (CV) related to wing shape differences (Fig. 10). CV1 separated M. avidus from

M. moenium with 58% of total variation (CV 1: Wilks’ Lambda = 0.145440; %2 = 832.8937; p < 0.00000),

while CV2 separated M. avidus and M. moenium from M. megavidus sp. nov. and M. ibericus with 27%

of total variation (CV2: Wilks’ Lambda = 0.388320; %2 = 408.6395; p < 0.00000) (Fig. 10A). Merodon

moenium and M. ibericus were clearly separated according to CV3 (CV3: Wilks’ Lambda = 0.698600;

%2 = 154.9484; p < 0.00000) (Fig. 10B). The phenogram constructed based on squared Mahalanobis

distances clearly depicts that M. avidus and M. moenium have more similar wing shapes than M. ibericus

and M. megavidus sp. nov. (Fig. 11). Differences in wing shape among species are depicted in Figure 12,

but have been exaggerated five-fold to make them more visible.

Additionally, the phenogram constructed based on squared Mahalanobis distances of wing shape showed

that all 26 analysed populations grouped according to species (Fig. 13).

Geometric morphometries - surstylus shape

PCA of surstylus shape revealed seven PC, of which the first six were connected with shape differences

among species (MANOVA: F 21333 = 5.280113; p < 0.00000). DA showed that all species differ highly

significantly in surstylus shape (p < 0.00000). All specimens ofM avidus and M. megavidus sp. nov. were

correctly classified (100%), while only two specimens ofM ibericus and M. moenium were misclassified

(97%). CVA found three CVs connected with shape change (Fig. 14). CV1 clearly separated M. avidus

from M. megavidus sp. nov., M. moenium and M. ibericus and represented 53% of total shape variability

(CV1: Wilks’ Lambda = 0.013392; %2 = 405.4285; p < 0.00000) (Fig. 14A). The second canonical axis

clearly separated M. megavidus sp. nov. from M. moenium and M. ibericus and was responsible for 25%

Fig. 8. Median-joining network of the mtDNA 5’-end of the COI gene. Circle sizes are proportional to

haplotype frequencies. Each branch represents one mutational step; if more than one mutational step is

present, it is denoted by the given number.

Merodon avidus

Merodon moenium

Merodon ibericus ■

Merodon megavidus sp. nov.

}

Fig. 9. UPGMA tree based on pairwise genetic distances for four species from the Merodon avidus

complex.

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

-1

-2

-3

-4 ■

-5 -

-8

• M. avidus

■ M. moenium

* M. ibericus

a M. megavidus sp. nov.

-6 -4 -2 0 2 4 6

CV2 (27%)

Fig. 10. Differences in wing shape among species of the M. avidus complex. A. Scatter plot of individual

scores of CV1 vs CV2. B. Scatter plot of individual scores of CV2 vs CVS.

Linkage Distance

Fig. 11. UPGMA phenogram constructed using the squared Mahalanobis distances of wing shape for

species of the M. avidus complex.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

of the variability (CV2: Wilks’ Lambda = 0.086702; %2 = 229.8565; p < 0.00000) (Fig. 14A). Merodon

moenium and M. ibericus were separated by CVS, with 18% of total shape variability (CVS: Wilks’

Lambda = 0.346752; %2 = 99.5597; p < 0.000305) (Fig. 14B). According to the phenogram constructed

based on squared Mahalanobis distances, M. megavidus sp. nov. has a more distinct surstylus shape,

while the surstyli of M. moenium and M. ibericus are the most similar (Fig. 15A). The main shape

differences among all three species lie in the posterior margin of the posterior part of the surstylus lobe

(Fig. 15A).

Discussion

Differentiation and taxonomic status of the cryptic species of the Merodon avidus complex has been

controversial for many years. An initial molecular analysis showed that M. avidus is a genetically and

geographically structured taxon with at least two cryptic species, which were designated as M. avidus

A and M. avidus B (Milankov et al. 2001). Further investigations based on the mtDNA COI marker

expanded on this by finding two additional cryptic species from the Iberian Peninsula (M bicolor Gil

Collado, 1930, now M. ibericus ) and Lesvos Island (Greece) (M sp. nova 2, now M. megavidus sp.

nov.) (Milankov et al. 2009, Stahls et al. 2009). In Milankov et al. (2009), wing shape analysis could not

distinguish taxa that could be differentiated by allozyme loci, even though these taxa differed in wing

size. Also, they found a great deal of similarity between allopatric metapopulation pairs of M. avidus

- M. ibericus

- M. moenium

Fig. 12. Thin-plate spline deformation grids showing wing shape differences between analysed species.

Differences between the species have been exaggerated five-fold to make them more visible.

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

A and M. avidus B; populations of M. avidus A from Macedonia and the Pannonian plain overlapped

with M. avidus B populations from Durmitor, Stara Planina and Kopaonilc in terms of wing shape. Only

a population of M. avidus A from Morinj (Montenegro) was clearly separated from other populations.

Subsequent analysis showed that this substantial overlap in wing shape was a result of incorrect species

identifications due to the morphological similarities between spring generations of M. avidus and

M. moenium (Popovic et al. 2015). Previously, it had been considered that M. moenium (M.. avidus B)

was a species distributed on mountainous regions of the Balkan Peninsula. However, it is now known

that while M. moenium is predominantly distributed in the continental parts of Europe, it also occurs in

some parts of the Mediterranean basin and the Black Sea coast, where it can occur sympatrically with

M. avidus. Further, allozyme analysis of morphologically different spring and autumn generations from

Umag (Croatia) clarified this misclassification issue by showing that both morphotypes belonged to

M. avidus (Popovic et al. 2015).

Our molecular analysis using the barcoding COI fragment for an additional three specimens from

Lesvos Island supports the presence of a morphologically cryptic taxon (M megavidus sp. nov.),

previously revealed by Stahls et al. (2009). All our phylogenetic analyses placed M. megavidus sp. nov.

as a separate independent cluster from M. ibericus and the M. avidus/M. moenium branch (Figs 6-9).

According to estimated divergence times, all diversification occurred in the Pleistocene (2.6 to 0.0117

MYA). This geological epoch was marked by repeated (at least 20) glacial and interglacial periods,

which influenced speciation and the distributions of many recent taxa in Europe (Julius & Kukla 1977;

Martinson et al. 1987; Perissoratis & Conispoliatis 2003). Recent studies have revealed many examples

of insect species that have altered their ranges and/or evolved as a response to repeated isolation during

glacial-interglacial cycles across three large peninsulas of Southern Europe (Hewitt 2001; Konstantinov

et al. 2009; Dapporto 2010; Nicholls et al. 2010; Zhu et al. 2013).

M. avidus

M. moenium

M. ibericus

M. megavidus sp. nov.

CRO-Umag

IT-Piemonte

GR-Pindos

IT-Pisa

GR-Drama

GR-Olympus

RS-Pcinja

GR-Lesvos

GR-Peloponnese

BU-Pirin

_ RS-Malinik

~ IT-Zuclo

RS-Tara

RS-Dubasnica

RS-StaraP

RS-Kopaonik

MN-Durmitor

RS-Djerdap

RS-Fruska Gora

_ RS-Malinik

MO-Atlas

_ ES-SNevada

GR--Lesvos

i_r

Linkage Distance

Fig. 13. UPGMA phenogram constructed using squared Mahalanobis distances of wing shape for

populations of species of the M. avidus complex.

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

The first mitochondrial diversification in the M. avidus complex took place in the Calabrian stage of the

Early Pleistocene (around 800 ky BP), when M. ibericus diverged from a co mm on ancestor. The Giinz-

Mindel interglacial corresponds to the approximate period when separation of M. megavidus sp. nov.

from the M. avidus/M. moenium lineage occurred. The most recent diversification, i.e., between M. avidus

and M. moenium, most likely took place at the end of the Riss-Wurm interglacial or the beginning of

the Wtirm glaciation period. The low species resolution ability of the 3’ and 5’ fragments of COI to

separate the M. avidus and M. moenium lineages (Milankov et al. 2009; Popovic et al. 2014, 2015) can

be explained by recent speciation and by the founder effect associated with postglacial recolonization

of northern Europe (Shikano et al. 2010). Both species share an identical haplotype of the COI 3’-end

(Popovic et al. 2014), although the 5’-end fragment haplotypes of DNA barcodes are not shared, they

give low support in cluster analyses (Popovic et al. 2015).

The most probable scenario for postglacial colonization of Europe is expansion of the M. avidus

and M. moenium lineages from the Balkan Peninsula into Central Europe (a “grasshopper” pattern

of colonization according to Hewitt 1999). The Pyrenees acted as a significant geographical barrier

preventing the dispersal of M. ibericus to other European areas.

Our geometric morphometric analyses show that all investigated species of the M. avidus complex

can be successfully discriminated based on wing and surstylus shape. The high overall success rate

of classification indicates that wings, and especially surstylus shape, have meaningful interspecific

discriminatory power. It is important to emphasize that, contrary to previous geometric morphometric

studies, we found highly significant differences in wing shape between M. avidus and M. moenium. The

phenogram based on squared Mahalanobis distances for wings is congruent with the UPGMA tree based

on genetic distances among species. Wing shape, as a highly heritable structure, has greater importance

in insect taxonomy than wing size (Birdsall et al. 2000), which has been confirmed by earlier studies of

hoverflies in which wing shape has been successfully used for identification and delimitation of species

(Vujic et al. 2013b; Nedeljkovic et al. 2013, 2015).

Additional evidence for species distinctiveness is provided by the highly significant differences in

surstylus shape between M. avidus, M. ibericus , M. megavidus sp. nov. and M. moenium. The main

differences in surstylus shape are connected to the posterior margin of the posterior part of the surstylus

A CV1 (53%)

-4

-2

2 4

CV2 (29%)

Fig. 14. Differences in the posterior part of the surstylus among species of the M. avidus complex.

A. Scatter plot of individual scores of CV1 vs CV2. B. Scatter plot of individual scores of CV2 vs CV3.

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

lobe, which is involved in gripping the female during copulation. It could be hypothesized that these

shape differences contribute to a mechanism of sexual isolation, especially since M. avidus has a distinct

surstylus shape compared to partly sympatric M. moenium. Although the exact mechanism of sexual

isolation in Syrphidae is not known, traditionally the shape of male genitalia is deemed a significant

mechanism of isolation between species (Rotheray & Gilbert 2011). Given that the morphology of

male genital structures is considered one of the fastest evolving traits in animal groups with internal

fertilization (Soto et al. 2013), and that insect genitalia are conspicuously variable even in closely

related taxa that are otherwise morphologically very similar (Hosken & Stockley 2004), we assert that

significant differences in surstylus shape is strong evidence for species delimitation. Recent studies have

found that natural and sexual selection (and their interaction) caused insect genital evolution (Hasson

et al. 2009; House et al. 2013), and that the degree of morphological differences in the structures of

genitalia is associated with geographic distance (Soto et al. 2013). Despite their recent speciation and

geographic proximity, M. avidus does not exhibit a similar surstylus shape to M. moenium. Other studies

have found that other factors, such as specific host plant relationships Soto (2012) or sexual selection

(Hosken & Stockley 2004), may promote further evolution in genital morphology.

Our population-level morphometric analysis of wing shape is in concordance with species delimitation

findings. All populations clustered according to species. Furthermore, in this analysis, M. megavidus

sp. nov. from Lesvos Island is distinct from all other species and/or populations, and it is important to

underline that this taxon was clearly differentiated from population(s) of M. avidus also from Lesvos

Island.

Fig. 15. Differences in the posterior part of the surstylus among species of the M. avidus complex.

A. UPGMA phenogram constructed using squared Mahalanobis distances. B. Thin-plate spline

deformation grids showing overall shape differences between analysed species.

Acknowledgements

We thank the curators of the several museums listed above in the “Studied material” section that

facilitated visits and loaned specimens for study. We thank John O'Brien for English revision. Financial

support was provided by the Serbian Ministry of Education, Science and Technological Development

(projects 01173002, III43002 and 011613001), the Provincial Secretariat for Science and Technological

Development (project ‘Genetic resources of agro-ecosystems in Vojvodina and sustainable agriculture’),

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

the Transnational Access to Research Infrastructures activity in the FP7 of the EC (ExpeER project, TA

visit ‘STEPS’), the FP7 EU project. Innosense, and National Strategic Reference Framework (NSRF)—

Research Funding Program: THALES.

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Manuscript received: 4 January 2016

Manuscript accepted: 23 May 2016

Published on: 14 October 2016

Topic editor: Gavin Broad

Desk editor: Kristiaan Hoedemakers

Printed versions of all papers are also deposited in the libraries of the institutes that are members of the

EJT consortium: Museum national d'Histoire naturelle, Paris, France; Botanic Garden Meise, Belgium;

Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United

Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of

Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands.

ACANSKI J. et al ., Species boundaries in the Merodon avidus complex

Appendix 1. List of specimens used for wing geometric morphometric analysis, by geographical area

and species.

Species

Country

Population

Bulgaria

Pirin Mts.*

Croatia

Umag*

Drama*

Lesvos island*

Greece

Olympus Mts.*

Peloponnesos*

Pindos Mts.*

M. avidus (Rossi, 1790)

Italy

Piemonte*

Pisa*

Montenegro

Durmitor Mts.

Djerdap gorge

Serbia

Malinik Mts. *

Pcinja*

Dubasnica Mts.

Turkey

Lake Baffa

Morocco

Atlas Mts.*

M. ibericus Vujic, 2015

Spain

Cadiz

Sierra Nevada Mts.*

Italy

Zuclo*

Castiglione Dei Pepoli

Montenegro

Durmitor Mts.*

Djerdap gorge*

M. moenium

Wiedemann in Meigen, 1822

Dubasnica Mts.*

Kopaonik Mts.*

Serbia

Stara Planina Mts.*

Fruska Gora*

Malinik Mts. *

Tara Mts.*

Vrsacke Planine

M. megavidus

Vujic & Radenkovic sp. nov.

Greece

Lesvos island*

Total

351

444

European Journal of Taxonomy 23 7 : 1-25 ( 2016 )

Appendix 2. List of specimens used for surstylus geometric morphometric analysis, by geographical

area and species.

Species

Country

Croatia

Greece

M. avidus (Rossi, 1790)

Italy

Serbia

Turkey

Morocco

M. ibericus Vujic, 2015

Spain

Montenegro

M. moenium Wiedemann in Meigen, 1822

Serbia

M. megavidus Vujic & Radenlcovic sp. nov.

Greece

Total

125

ACANSKI J. et al ., Species boundaries in the Merodon avidns complex

Appendix 3. Neighbor-joining tree based on a 5’ fragment of COI mtDNA sequences from the Merodon

avidus complex. Bootstrap values (1000 replicates) are shown next to the branches.

100

- Merodon avidus (KT731106)

- Merodon moenium (KT731123)

Merodon moenium (KT731132)

Merodon moenium (KT731124)

Merodon avidus (KT731127)

Merodon avidus (KT731125)

Merodon avidus (KT731126)

Merodon avidus (KT731125)

Merodon moenium (KT731122)

Merodon megavidus sp. nov. (KX017461)

Merodon megavidus sp. nov. (KX017462)

Merodon megavidus sp. nov. (KX017463)

Merodon megavidus sp. nov (KX017464)

100

Merodon ibericus (KT731119)

- Merodon ibericus (KT731 111)

- Merodon ibericus (KT731118)

_(- Merodon ibericus (KT731116)

Merodon ibericus (KT731107)

Merodon ibericus (KT731117)

Eumerus sulcitibius (KT157875)

Platynochaetus setosus (KM224512)

0.02