DNA barcode assessment and population structure of aphidophagous hoverfly Sphaerophoria scripta: Implications for conservation biological control

Abstract With the advent of integrated pest management, the conservation of indigenous populations of natural enemies of pest species has become a relevant practice, necessitating the accurate identification of beneficial species and the inspection of evolutionary mechanisms affecting the long‐time persistence of their populations. The long hoverfly, Sphaerophoria scripta, represents one of the most potent aphidophagous control agents due to a worldwide distribution and a favorable constellation of biological traits. Therefore, we assessed five European S. scripta populations by combining molecular (cytochrome c oxidase subunit I‐ COI, internal transcribed spacer 2‐ ITS2, and allozyme loci) and morphological (wing size and shape) characters. COI sequences retrieved in this study were conjointly analyzed with BOLD/GenBank sequences of the other Sphaerophoria species to evaluate whether COI possessed a sufficient diagnostic value as a DNA barcode marker to consistently delimit allospecific individuals. Additionally, the aforementioned characters were used to inspect the population structure of S. scripta in Europe using methods based on individual‐ and population‐based genetic differences, as well as geometric morphometrics of wing traits. The results indicate numerous shared COI haplotypes among different Sphaerophoria species, thus disqualifying this marker from being an adequate barcoding region in this genus. Conversely, the analyses of population structuring revealed high population connectivity across Europe, therefore indicating strong tolerance of S. scripta to environmental heterogeneity. The results imply a multilocus approach as the next step in molecular identification of different Sphaerophoria species, while confirming the status of S. scripta as a powerful biocontrol agent of economically relevant aphid pests.


| INTRODUC TI ON
Modern agriculture heavily relies on systematic chemical (insecticide) and mechanical (intense soil tillage) treatments to yield high productivity, consequently affecting water quality and availability, the emission of greenhouse gases, and furthermore disrupting natural ecological and evolutionary processes by causing detrimental changes in the populations of natural pest enemies (Chabert & Sarthou, 2017). Therefore, the negative consequences of contemporary agricultural practices to environmental and human health call for science-based practices aimed at biological control of insect pests by natural enemies in agro-and forest ecosystems (Le Hesran, Ras, Wajnberg, & Beukeboom, 2019). The field of crop pest control saw the rise of integrated pest management (IPM), an ecosystem approach to crop production and protection that utilizes various management practices to efficiently grow high-quality crops while minimizing the use of chemical control agents and promoting their sustainable use (Gomez-Polo et al., 2014). One of the IPM strategies involves conservation biological control (CBC), conservation of natural enemies through manipulation of the environment. Unlike classical biological control which introduces exotic control species as agents of pest management, CBC instead involves a collection of practices which protect and promote the presence of naturally present enemies of pest species in a targeted ecosystem (Eilenberg, Hajek, & Lomer, 2001). The main advantage of CBC programs is that the beneficial species providing pest suppression service are already adapted to the habitat and to the target pest, which increases the effectiveness of the insect pest control (Gomez-Polo et al., 2014).
On the other hand, for CBC approaches to be successful, a thorough fundamental knowledge of the biology and ecology of the pest control agent is necessary. Concerning the link between natural enemy diversity and pest control (Jonsson, Kaartinen, & Straub, 2017), effectiveness of conservation biological control depends on the abundance of pest predators as well as the proper taxonomic identity of species in a predator assemblage (Moreno, Lewins, & Barbosa, 2010;Snyder, 2019). Hence, deeper understanding of the link between natural enemy biodiversity and biocontrol enhances the efficacy of conservation biological control in the management of sustainable agricultural systems (Straub, Finke, & Snyder, 2008). Furthermore, in the recent years, molecular markers have also been extensively used to elucidate various aspects of biology and ecology of beneficial species involved in biological control (MacDonald & Loxdale, 2004).
One such issue includes the phenomena of population structure, tightly woven to the evolutionary mechanism of gene flow and therefore informative about the species dispersal and migratory activities (Raymond, Plantegenest, & Vialatte, 2013). Indeed, to provide sustainable biological control in the local and regional habitat, understanding the landscape effect on population connectivity of biological enemies has essential input (Happe et al., 2019). In addition, in the context of pest suppression ecosystem service, population structure is relevant for preserving the genetic diversity of the biocontrol agents (Le Hesran et al., 2019), as well as for testing their tolerance to environmental variability (Liu, Xiaoqiang, et al., 2019).
Regarding the ecosystem service benefits that the Sphaerophoria hoverflies provide, they are an especially interesting group since predation at the larval stage and pollination at the adult stage allow a potential synergy of ecosystem services (Rader et al., 2020). Within the genus, long hoverfly, S. scripta (Linnaeus, 1758;Speight, 2017; Figure 1), has received particular attention as a potent biocontrol agent since it is globally distributed, characterized by intense gene flow (Raymond et al., 2013), and an abundant aphid predator constituting hoverfly larva assemblages in lettuce crops and pepper greenhouses (e.g., Pineda & Marcos-García, 2008), as well as in other herbaceous plants (Speight, 2017). Furthermore, by boasting high genetic diversity, great colonization ability, and probably high phenotypic plasticity, long hoverfly, along with Episyrphus balteatus (De Geer, 1776), is considered to possess an outstanding adaptive potential with no parallel in any known arthropod species providing the same ecosystem services in agroecosystems (Raymond et al., 2013).
Given that the effectiveness of CBC programs depends on the biological characteristics of the species used to suppress pest taxa F I G U R E 1 Sphaerophoria scripta, the long hoverfly (Diptera: Syrphidae) (Jonsson, Wratten, Landis, & Gurr, 2008), S. scripta represents a potentially powerful candidate agent due to its aforementioned traits.
Finally, the taxa of the hoverfly family are known to exhibit cryptic species diversity, which has been successfully revealed using DNA barcoding in several instances (Milankov, Ståhls, Stamenković, & Vujić, 2008;Ståhls et al., 2009;Šašić et al., 2016), but which has not been utilized in S. scripta characterization.
Since essential components of CBC involve fast and accurate identification of natural enemies, as well as the inspection of evolutionary mechanisms acting upon their populations and thereby affecting the quality of their biocontrol service, we addressed two issues of CBC in this paper: 1) the utility of DNA barcoding in the accurate identification of the taxa within the aphidophagous hoverfly genus, Sphaerophoria; and 2) the issue of population structure and population connectivity of polyphagous predator, S. scripta. We implemented several approaches using mitochondrial and nuclear genetic loci belonging to sequence and phenotypic molecular marker classes, as well as phenotypic morphological characters of adult wings.
Firstly, we used a DNA barcode approach (Hebert, Cywinska, Ball, & deWaard, 2003;Hebert, Ratnasingham, & deWaard, 2003) based on a combination of two markers frequently utilized together in insect cryptic diversity identification: a mitochondrial, cytochrome c oxidase subunit I gene (COI mtDNA) and a nuclear, internal transcribed spacer 2 locus of the ribosomal DNA cluster (ITS2 rDNA). The DNA barcode approach relies on the existence of a barcode gap-the scenario when the greatest intraspecies haplotype divergence is lower than the least interspecies haplotype divergence (Hebert, Cywinska, et al., 2003;Hebert, Ratnasingham, et al., 2003;Puillandre, Lambert, Brouillet, & Achaz, 2011). An implicit premise to this approach, however, is that the DNA barcode method can indeed successfully distinguish between different Sphaerophoria species, which was also assessed by analyzing sequences belonging to 20 different Sphaerophoria species.
Secondly, we used clustering methods operating upon individual genetic differences to determine the most likely number of populations (=panmictic units) within the sample. Apart from COI mtDNA and ITS2 rDNA, allozyme loci were also included in these analyses, with algorithms deciphering the number of populations working in two setting modes-with and without incorporating the geographic origin of the sampled S. scripta individuals. Additionally, different geographical samples of S. scripta can be a priori defined as distinct populations and the differences among them can be tested using population-based (=group-based) methods. This goal was reached using all the aforementioned COI mtDNA, ITS2 rDNA, and allozyme loci, but also using the methodology of geometric morphometrics of adult traits (wing size and shape) as a complement to molecular markers. We opted to ascertain whether dispersal and migration of S. scripta individuals keep geographically remote sites genetically cohesive or whether there was a presence of population stratification, thereby affecting the co-evolutionary dynamics of long hoverfly and the aphids it predates upon, and consequently the quality of biological pest control service it provides.  (Hippa, Nielsen, & van Steenis, 2001;det. Milankov V, Francuski Lj). In total, 154 individuals were used for geometric morphometric analyses, while a sample subset was used for the molecular analyses (Table 1). In particular, 94 S. scripta specimens were used in the analyses based on allozyme loci, 69 individuals for the analyses based on COI mtDNA, while 39 individuals were genotyped at ITS2 rDNA locus (Table 1).

| Sampling and DNA barcode data set building
Additionally, for the analysis of the utility of the standard DNA barcode marker (5' end of COI mtDNA) for the species delimitation within the genus Sphaerophoria, apart from S. scripta sequences obtained de novo in this study, sequences deposited in BOLD and GenBank databases were also included. The databases were searched for Sphaerophoria genus 5' COI mtDNA sequences and only the sequences belonging to the taxa identified to the species level were included. The sequences were aligned with S. scripta COI mtDNA sequences from this paper in BioEdit 7.0.5.3 (Hall, 1999) to choose an overlapping fragment which would be used for the subsequent analysis of the barcode utility. After the exclusion of sequences whose length was too short and/or which contained ambiguous nucleotide positions, the total data set consisted of 278 COI mtDNA sequences (481 bp-long): 215 originating from BOLD database, 6 sequences downloaded from GenBank database, and 57 S. scripta COI mtDNA sequences from this paper (Table S1) ; Table S1). For a subset of these sequences (Table S2)

TA B L E 1
The sampling localities and sample sizes for different Sphaerophoria scripta analyses performed in this study TL2-N-3014 (alias Pat; 5'-TCCAATGCACTAATCTGCCATATTA-3') primer pair (Simon et al., 1994). ITS2 rDNA sequence of 426 bp was amplified using ITS2A (5'-TGTGAACTGCAGGACACAT-3')/ ITS2B (5'-TATGCTTAAATTCAGGGGGT-3') primer pair (Beebe & Saul, 1995 . These sequences were included in the analyses of the utility of 5' COI mtDNA as a DNA barcode, but not in the individual-and population-based genetic analyses which only encompassed longer COI sequences (Table 1). Multiple sequence alignments were performed using the ClustalW algorithm, after which the alignments were manually checked and edited when necessary. COI mtDNA sequences were checked for STOP codons in MEGA X 10.0.5 (Kumar, Stecher, Li, Knyaz, & Tamura, 2018) to inspect the presence of nuclear mitochondrial pseudogenes (NUMTs).
The number of COI mtDNA haplotypes, ITS2 rDNA alleles, and variable positions was determined in DAMBE6 (Xia, 2017). Due to the lack of variability of ITS2 rDNA sequences (see Results below), this data set was excluded from the subsequent intended analyses.

| COI mtDNA barcode analysis
To infer whether there was cryptic diversity within the S. scripta specimens identified using morphological characters, we implemented a DNA barcode approach. Since this method relies on the existence of a barcode gap, if certain haplotypes are shared between two or more species, the barcode gap is closed and the marker is deemed of insufficient diagnostic utility for the species identification. Therefore, median-joining haplotype networks were firstly constructed in

| Individual-based clustering analyses of Sphaerophoria scripta specimens
The number of populations within the sample was investigated using nonspatial and spatial Bayesian clustering approaches based on individual genetic differences and implemented on both allozyme and COI mtDNA data sets. The nonspatial approach was implemented in BAPS 6 (Corander & Tang, 2007). For the population mixture analysis, Clustering of individuals was selected for allozyme loci,  Miller, 2005) using 10,000 iterations.  Figure 3). Raw landmark coordinates were superimposed using a full Procrustes fit procedure (Dryden & Mardia, 1998) and the set of shape variables (a matrix of Procrustes coordinates) and centroid size (CS; Bookstein, 1991) were extracted.

| Population-based molecular analyses of genetic differentiation
Geometric morphometric and statistical analyses were done using MorphoJ (Klingenberg, 2011) and PAST 3.26 (Hammer, Harper, & Ryan, 2001). To evaluate differences in wing size between groups (populations, sexes), we performed a permutational analysis of variance (ANOVA) on CS followed by a multiple comparison test (Tukey's pairwise post hoc test). The presence of allometry was estimated with multivariate regression of Procrustes coordinates against CS.
The significance of the allometry was computed by a permutation test with 10,000 replicates. When statistically significant allometric effect was found, residuals from regression were used for the analysis of shape variation. Wing shape variation was analyzed using canonical variate analysis (CVA)/multivariate analysis of variance (MANOVA) with 10,000 permutations, while discriminant analysis (DA) was employed to calculate the percentages of correct classification, cross-validated by a leave-one-out (jackknifing) procedure.

| RE SULTS
Among 39 individuals of S. scripta from the five European localities, only two alleles of ITS2 rDNA were found; the majority of the sample was represented by an allele A (35 individuals), while an alternative allele B was registered in the remaining four individuals (Table S3). Due to the lack of variability, no further analyses on ITS2 rDNA data set were performed.

F I G U R E 3
The positions of 16 landmarks used for wing size and shape geometric morphometric analysis of Sphaerophoria scripta

| Allozyme variation
Out of the eight analyzed enzyme loci, four loci were polymorphic.
Up to three alleles were registered per population for the loci Idh-1, Mdh-2, and Me, while up to four alleles were registered per population for the locus Ao (Table S4). All the sampled individuals were homozygous for all the analyzed enzyme loci.

| COI mtDNA barcode assessment
The analysis of 66 COI mtDNA sequences (1,209 bp-long) sampled from the five European localities retrieved 33 haplotypes (H1-H33; Figure 4,  Figure 4). The haplotypes within this mitochondrial haplogroup were registered in all the sampled localities, apart from Germany. The range of p distances was 0.08%-0.50% within the major haplogroup, 0.08%-0.33% for the minor haplogroup, and 0.33%-0.83% between the haplogroups (Table S1), therefore indicating the absence of the barcode gap since the upper range of p distances within the groups (0.50%) was greater than the lower range between the groups (0.33%). However, when the COI mtDNA sequences were aligned against a complete mitochondrial sequence of Episyrphus balteatus, the haplotypes of the minor haplogroup were characterized by a diagnostic combination of six substitutions: 1,783 T, 1,978 G, 2,495 G, 2,500 T, 2,734 C, and 2,761 G ( Figure S1). shared between two or more allospecific individuals: h8, h16, h17, h27, and h32 were common for two respective species, the most frequent haplotype h1 was shared among three species, h20 was retrieved from individuals belonging to four different species, while h18 was present in five different Sphaerophoria species (Table S5).
Since the existence of barcode gaps depends on the condition of the greatest intraspecies divergence being lower than the smallest interspecies divergence, when two species share certain haplotypes (the lowest interspecies divergence: p = 0%; see

| Individual-based genetic clustering analyses
For S. scripta COI mtDNA data set, BAPS retrieved three as the most probable number of clusters. The majority of the haplotypes (19/33) were grouped together in cluster K1, while haplotypes H27-H33, belonging to the minor COI mtDNA haplogroup, were recognized as a separate genetic cluster (K3). The remaining haplotypes were grouped together in a cluster K2 which included two separate subgroups-K2a, whose haplotypes (H5, H23, H24, H26) were grouped by having a common 2,761 G substitution, and K2b, whose haplotypes (H2, H19, H25) shared a 2,788 T substitution ( Figure S1). The populations sampled in the Netherlands, Germany, and Slovenia had high average population membership probabilities to cluster K1 (>80%, Concerning the allozyme loci, Bayesian clustering method implemented in BAPS retrieved five as the most probable number of clusters. However, neither of the geographic samples was particularly differentiated as belonging to any of the genetic clusters (the highest average membership probability to any of the clusters was lower than 45%; Table S6). Furthermore, STRUCTURE retrieved four and five as the most probable number of homogenous genetic groups within the total sample using the methods of Evano et al. (2005) and Pritchard et al. (2000), respectively ( Figure 6). Once again, regardless of the value of K, neither of the geographic samples had significantly high mean population membership coefficient to the retrieved genetic clusters (threshold value is usually taken at 80%, but no locality had membership coefficient higher than 46%; Table S7). Therefore, all the geographic samples were genetically admixed. The inclusion of spatial data in Geneland indicated four as the most probable number of clusters, with Slovenia, Bosnia and Herzegovina, and Greece representing discrete samples, while the northernmost samples-the Netherlands and Germany, were grouped together. Finally, Mantel's test results showed no statistically relevant correlation between genetic and geographic distances (r = .03, p = .15).

| Population-based molecular analyses of genetic differentiation
The analysis of molecular variance based on COI mtDNA found no significant Φ ST value for the S. scripta geographic samples (p = .74;

| Wing geometric morphometrics
Prior to wing shape variation analysis, multivariate regression of shape variables on CS was performed for a total sample, as well as for each sex separately. In an overall sample, 7.2% (p < .0001) of shape variation was explained by size, while it was 4.0% (p = .05) in females and 10.2% (p < .0001) in males. Thus, due to the presence of allometry, regression residuals were used for further analysis of shape variation.

F I G U R E 6
Population genetic structure analysis of the five European Sphaerophoria scripta populations using STRUCTURE software on allozyme loci. The most probable number of clusters varied between the two methods- Evano et al. (2005) and Pritchard et al. (2000), corresponding to K = 4 and K = 5, respectively. Regardless of the most probable number of homogenous genetic groups within the sample, both scenarios indicate a pattern of high genetic admixture. NLD-the Netherlands (the Hague), DEU-Germany (Berlin), SVN-Slovenia (Bled), BIH-Bosnia and Herzegovina (Banja Luka), GRC-Greece (Araxos) of landmarks 9-14 being moved toward either distal or proximal part of wing in males and females, respectively, contributed to intersexual wing shape differences (Figure 8). Due to significant differences between genders, further wing size and shape variation analyses were done for each sex separately.
Permutational MANOVA revealed that in females interpopulation differences in wing shape were not significant (F = 0.78, p = .84) despite populations being separated on CVA scatterplot (Figure 9).
Moreover, pairwise Procrustes distances ranged from 0.0068 to 0.0110 and were nonsignificant (p > .05) for all population pairs (Table 3). Percentage of correct classification was 87.2% (10.6% jackknifed). Contrary to females, among males significant differences in wing shape were found (F = 2.11, p < .001). Procrustes distances ranged from 0.0072 to 0.0143 and were significant for five population pairs (Table 3). Furthermore, scatterplot obtained from CVA showed that along the first CV that accounting for 42.9% of total shape variation males from BIH partially separated from males from NLD, DEU, and GRC, while along CV2 (25.5%) males from NLD and GRC clustered with no overlap (Figure 9). Percentage of correct classification was 74.8% (29.0% jackknifed). Deformation grids showed that displacement of all landmarks contributed to shape differences between males of analyzed populations ( Figure 10).

| The utility of the COI mtDNA in delimiting Sphaerophoria species
In this study, a mitochondrial, cytochrome c oxidase subunit I gene (COI mtDNA) was used to test its usefulness in determining the species borders within the Sphaerophoria genus of hoverflies. In particular, we utilized a partial, 481 bp-long fragment of the standard, 658 bp-long DNA barcoding marker (Folmer et al., 1994). The results of the analysis indicated that eight haplotypes (h1, h8, h16, h17, h18, h20, h27, and h32) were shared between two or more Sphaerophoria species (Table S5), with the consequent lowest interspecies p divergence of 0% rendering this barcode marker inadequate for the separation of different Sphaerophoria congeners. The inclusion of the longer fragments of the standard 5' COI mtDNA (Table S2)  and different regions of this marker across different insect orders, and finding that the applicability of the marker is not significantly affected by using its shorter fragments until the case of using 40 bp-long mini-barcodes. Apart from COI mtDNA used in this study, 18S rRNA gene was previously used to decipher the phylogenetic relationships within Syrphidae family (Mengual, Ståhls, & Rojo, 2008). Sphaerophoria scripta and S. philanthus could not be separated apart using this slowly evolving marker, and neither could S. contigua from S. macrogaster as they shared identical haplotypes. These species pairs could however be distinguished from S. rueppelii and S. loewi, albeit by up to two substitutions (Mengual et al., 2008). Therefore, the future delimitation of different species of Sphaerophoria genus should incorporate a multi-character approach, possibly using faster-evolving loci than barcodes due to the supposed recent divergence of the studied taxa, as evidenced in the lack of the barcode gap between them.
The lack of the utility of 5' COI mtDNA to species delimitation in Sphaerophoria genus has important implications for the first goal of this study-recognizing whether there was cryptic diversity within S. F I G U R E 8 Wing shape differentiation between females (white bars) and males (gray bars) of Sphaerophoria scripta obtained with DA. Wing shape changes (black lines) from consensus configuration of landmarks (gray lines) along the discriminant axis are shown using the wireframe graphs (numbers refer to landmarks shown in Figure  scripta. Namely, we retrieved two distinct haplogroups when analyzing the longer, 1,209 bp-long fragment of this gene. Although no barcode gap separated the members of the major and minor haplogroups (Table S1), the members of the minor haplogroup were distinguished by a specific combination of six substitutions. However, this does not represent a strong evidence of putative cryptic diversity within S. scripta given the disputed resolution of this marker. Furthermore, ITS2 rDNA alleles A and B were present among the members of both the major and minor haplogroups (Table S3). Therefore, rather than representing distinct taxa, the existence of two haplogroups oriented around two central haplotypes (H1 and H27) is indicative of the past population expansion from two previously separated sources (the dumbbell pattern of haplotype network, Avise, 2000).
This is also evident in the geographical codistribution of the members of both haplogroups, and also in the fact that the less frequent haplotypes to arise, but they are slow to be lost via genetic drift due to the large effective size. This is indeed supported by the conclusion of Raymond et al. (2013), where a large genetic diversity was also observed in S. scripta based on microsatellite loci as nuclear markers.
Summarily, we conclude that COI mtDNA is an inadequate barcoding marker for the Sphaerophoria genus.

| Population structure of Sphaerophoria scripta in Europe
The remaining study goals included the inspection of the population's structure of S. scripta based on an integrative approach combining two classes of molecular markers (phenotypic-allozymes; and sequence-based COI mtDNA) and morphological phenotypic char-  (Bitner-Mathé & Klaczko, 1999) could, apart from differential selective regimes they experience, indeed be indicative of the restricted dispersal of male specimens.
However, this factor ultimately does not significantly contribute to F I G U R E 1 0 The wireframe graphs represent male wing shape changes (black lines) from consensus configuration of landmarks (gray lines) along the first two CVs. Numbers in the deformation grids refer to landmarks shown in Figure 3 the establishment of differentiated genetic clusters, as the homogenizing effect of the total gene flow was evident across the genomic loci which we analyzed.

| Implications for conservation biological control
In the light of the current global climate changes, as well as humanmediated habitat fragmentation, hoverflies represent a particularly noteworthy group of pest management interest as they possess a favorable configuration of biological traits which enables them rapid responses to changes in resource availability (Rader et al., 2020).
Given that conserving enemy biodiversity has a strong impact on pest abundance in agricultural ecosystems (Jonsson et al., 2017), Finally, by using a combination of several molecular and phenotypic markers we showed the absence of population structuring of the long hoverfly S. scripta across the five European localities. Our integrative study confirms a great dispersal capacity of this migratory species, which is compatible with the results from previous study (Raymond et al., 2013), where a different marker class was used (microsatellite loci). Hence, population connectivity observed for one of the main aphid biocontrol agents on cereal crops and orchards (e.g., Chabert & Sarthou, 2017) would significantly contribute to the pest management programs. In addition, by combining genetic-morphological approach we assessed a great evolutionary potential of this natural enemy species, which is crucial for maintaining sustain- by indicating a high tolerance to environmental variability, and by increasing colonization ability of species, which are highly favorable traits in the current context of global climate change and habitat fragmentation in agroecosystems (Liu, Xiaoqiang, et al., 2019;Raymond et al., 2013). Therefore, due to the specific traits of S. scripta (worldwide distribution, great genetic diversity, high phenotypic plasticity, intense gene flow, and population connectivity), this taxon represents a powerful agent of biological control. In the light of conservation biological control, it would therefore be of essential importance to protect its microhabitats within agroecosystems and local habitats that provide natural nurseries for natural enemy biodiversity (Gontijo, 2019;Snyder, 2019). For instance, it has been suggested that ungrazed patches in grazed grassland are valuable shelters for aphidophagous hoverflies (Speight, Good, & Sarthou, 2000). Likewise, cattle grazing and overall nitrogen enrichment of the soil represent effective strategies which could enhance the predator potential of S. scripta as plants which benefit from nitrogen-rich soil are particularly beneficial to aphids, therefore providing prey to the long hoverfly and bolstering its population connectivity by providing suitable patches between agroecosystems (Chabert & Sarthou, 2017;Happe et al., 2019).

ACK N OWLED G M ENTS
We thank the two anonymous reviewers for their constructive Syrphidae, Cerambycidae, and Lucanidae (Insecta)."

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.