Variability of the key features and revision of a group of closely related species of grassflies (Diptera, Chloropidae, Meromyza)

Abstract The following external morphological features of members of the genus Meromyza Mg. have been analyzed: the ratio of the height of frontal triangle to the length of the head; the presence of black setae on the lower surface of genae; the degree of manifestation of ocellus spot; the ratio of the length of mesonotum to scutellum; the length and color of the mid strip of the mesonotum and its degree of manifestation on the scutellum; and the thickness of hind femurs. Additionally, the size and shape of anterior and posterior processes of the postgonite, and the type of attachment of the posterior process have been investigated. The authors have determined the range of variability of key features applicable to the reliable identification of closely related species of grass flies in the “meigeni” species group of the genus Meromyza, as well as the usefulness of CO1 mtDNA sequences for this purpose. The authors propose to re-instate the name M. laeta Meigen, 1838 (instead of using the name M. meigeni Nartshuk, 2006), to include seven species into the cluster “meigeni”, and to substitute the name of the cluster “meigeni” with the name “variegata”.


Introduction
The high variability of key external morphological features of Meromyza flies (Diptera, Chloropidae, Meromyza Meigen, 1830) has been noted by many authors (Nartshuk and Fedoseeva 2010). High variability in the color of palpi and mid stripe of the mesonotum, and shape of ocellus and occipital spots, etc. make it difficult to precisely identify specimens. Before features of the genital apparatus became key to the identification of Meromyza flies, only six species of this genus had been described, and species synonymy was highly disputed (Fedoseeva 1960). To date, Meromyza includes more than 90 species, and the identification key is based on a combination of external morphological features, and specific features of male genital apparatus (Nartshuk and Fedoseeva 2010). Recently, it was shown that the precision of species identification can be verified using molecular-genetic analysis of CO1 mtDNA gene (Safonkin et al. 2016). The results of genetic analysis combined with the shape of the anterior process of the postgonite made it possible to divide the genus into eight clusters, which were named after the species closest to the hypothetical ancestor (Safonkin et al. 2016). The flies of the "meigeni" cluster are populous throughout Europe. Excessive variability of external features in females of the "meigeni" cluster makes species identification based on female features very difficult. Despite the similarity of male anterior and posterior processes of the postgonite, the degree of their variability and its usefulness for species identification has not been previously studied. Upon our findings, the larvae of Meromyza variegata Meigen, 1830 from this cluster damage oat shoots, which necessitates the correct identification of adults.
The aim of this study was to determine the variability of key features of "meigeni" cluster flies, to select features unique for valid identification of the species, and to reexamine the naming of this cluster.

Material and methods
Collection sites of the material. Meromyza species were acquired from the collection of the Severtsov Institute of Ecology and Evolution (Moscow, Russia), and comprised material collected in different years in the Moscow, Tver and Tula regions of the Russian Federation, as well as the Brest region of the Republic of Belarus. In 2018, we also collected flies from the Czech Republic in the Pilsen Region (June 20, 49°75'82"N, 13°15'61"E), Jihlava (June 23, 49°39'66"N, 15°59'96"E), Brno (June 24, 49°23'01"N, 16°53'33"E) and Olomouc (June 25,49°63'40"N,17°34'35"E). We used M. bohemica Fed. as a model for the analysis of population variability of morphological features in the "meigeni"cluster.
External key features. Based on our own and published data, we analyzed face profile, the shape of the 3rd antennal segment, the length of the head, the color of arista and palpi, the height and width of the frontal triangle and their ratio, the ratio of the length of the head to the height of frontal triangle, the ratio of the genae height to the height of the 3rd antennal segment, the wrinkledness of the apex of frontal triangle, the shape and size of the ocellus spot, the presence of black setae on the lower surface of genae, the parafacial angle; the pattern of the occiput; the length of mesonotum and scutellum and their ratio, the length of the mid stripe of mesonotum, the color and length of mesonotal stripes, the presence of a stripe on scutellum, the color of the abdomen, the thickness of hind femurs, and body length.
Postgonites. The shape of the postgonite was studied using images acquired with a CamScan MV 2300 scanning electron microscope (Czech Republic). Measurements of the lateral surface of the postgonites for 80 Meromyza specimens were carried out using images acquired by a Keyence VHV-1000 light microscope (Japan), with an integrated data analysis program and with standard settings used throughout the study. We investigated a shape and an area of the anterior process of the postgonite, and the type of attachment of the posterior process. Measurements were recorded in micrometers. To prepare the samples, we extracted the postgonites from the abdomen of flies and affixed them onto paper.
We performed molecular-genetic analysis based on the nucleotide sequences of CO1 mtDNA locus previously obtained and deposited by us in GenBank (Safonkin et al. 2016). We deposited new nucleotide sequences of CO1 mtDNA from Meromyza bohemica Fedoseeva, 1962, M. femorata Macquart, 1835and M. rufa Fedoseeva, 1962 in GenBank with accession numbers MN 037808-MN 037814. The construction of the phylogenetic tree with new nucleotide sequences and analysis of phylogenetic relations were performed using the MEGA5 (Tamura et al. 2011) software package. Statistical data analysis was performed using Statistica 10.

Results
Species differences of the cluster "meigeni". Based on the analyzed features (see methodology), most features either do not differ, or their dimensional boundaries overlap. We determined that the following features are the most applicable for species identification (Table 1) Males differ in the structure and size of the postgonites. The difference in the area of the anterior process of the postgonite is statistically significant in most species (Table  2). The species also differ in shape of the anterior and posterior processes of the postgonite, and by the type of attachment of the posterior process to the anterior one, and by the line of attachment of the posterior process relative to the line of attachment of the anterior process of the postgonite to the hypandria.
The population variability of key features was analyzed in M. bohemica Fed. as the model species. The first feature was the black setae on the lower surface of genae; a 10% and 15% variability in number of individuals with more than five setae was observed in the same population, and among studied populations, respectively. Length of the body, mm 3-3,5 3.5-4.5 4.5-5 3-3.5 3-3.5 3.5-4 4-5 The second feature was the length of the mid stripe of the mesonotum. In most adult flies, the length of the stripe was ¾ the length of the mesonotum. The largest proportion of males and females with extreme length ratios (2/3 and 1) were found in the eastern population.
The third feature was the color of the mid stripe of the mesonotum. Red is the most common color, with greater number of specimens with dark red or brown stripes found in the central and western populations.
The phylogenetic tree based on the CO1 mtDNA of previously obtained nucleotide sequences (Safonkin et al. 2016) and new ones from M. bohemica, M. femorata   and M. rufa puts the sequences from these species into the cluster composed of M. meigeni, M. mosquensis, M. variegata; M. meigeni and M. variegata are the most close to a hypothetical haplotype of the cluster, also we can easily see that other species of this cluster divide from M. variegata (Fig. 1).

Discussion
The first attempt to divide 28 new species of Meromyza into four distinct groups based on the male's postgonite morphology was made by Hubicka (1970 Meigen, 1838, M. variegata, andM. stackelbergi Fedoseeva, 1967, were placed by this author into a separate group ("variegata"). Our comparative analysis of key features described by Hubicka (1970) Hubicka (1970) as key features of species in the "variegata" group, showed that these morphological features are also non-unique. This fact is confirmed by the most complete key feature tables (Fedoseeva 2003, Nartshuk andFedoseeva 2010), in which M. lolli is absent. In our opinion, five species from the "variegata" group described by Hubicka (1970) can be included in a separate cluster, previously designated by us as "meigeni" (Safonkin et al. 2016). The difficulty of identification of species of the considered group is confirmed in the case of M. lidiae Nartshuk, 1992. This species, according to Nartshuk (1992), is close to M. laeta. However, Nartshuk and Fedoseeva (2011) pointed out that M. lidiae is a junior synonym for M. variegata. Nartshuk (2006) described the new species M. meigeni based on the absence of the holotype M. laeta though pointing out, that M. meigeni may be identical to the previously described M. laeta (Nartshuk 2006, Nartshuk andFedoseeva 2011). In the key to species of the genus (Nartshuk and Fedoseeva 2010), M. laeta is replaced by M. meigeni. However, we think that the species-specific description presented by the aforementioned authors (shape of the aedeagus and anterior process of the postgonite) is not sufficiently reliable to consider M. meigeni as a new species. Despite the description of M. meigeni by Nartshuk as a species unique to Slovenia, the shape of the aedeagus and anterior process of the postgonite, the key features of this species, are similar to those in M. laeta which was described as a species by different authors (Meigen 1830, 1838, Fedoseeva 1960, Hubicka 1970 from numerous regions of Europe. In our opinion, it suggests possible regional variability of M. laeta for a number of key features. We propose to go back to the traditional name of the species previously defined as M. laeta (Meigen 1830, 1838).
Molecular-genetic analysis of the CO1 mtDNA gene revealed a concordance between the haplotype and size of the postgonite anterior process (Safonkin et al. 2016); the species closer to the hypothetical ancestors of the cluster demonstrate the largest size of the anterior process of the postgonite. As stated in the above paper, the cluster in question was named as "meigeni", since the Network (phylogenetic program) places M. meigeni closer to a hypothetical haplotype than M. variegata. The dendrogram displays M. meigeni and M. variegata as practically equidistant from the hypothetical haplotype (Fig. 1). Also, the area of the anterior process of the postgonite in M. variegata is significantly larger than in M. meigeni (Table 2). Based on the concordance between molecular phylogenetic results (Fig. 1) and the size of the postgonite anterior process, we propose to name the cluster in question as "variegata" instead of "meigeni". The cluster's name is taken from the name of the species closer to the ancestor haplotype and with the largest size of the anterior process of the postgonite.
Thus, it is possible to identify seven species in the "variegata" cluster according to a combination of external key features and the postgonite structure: M. rufa, M. bohemica, M. femorata, M. laeta, M. variegata, M. mosquensis, and M. elbergi. Based on the original description (Nartshuk 1992), M. zimzerla Nartshuk, 1992 can be also placed into the "variegata" cluster. Molecular analysis of the second part of the CO1 mtDNA gene confirms the identification of six selected species of the "variegata" cluster. Currently, there is no molecular analysis data for M. elbergi and M. zimzerla, though the key features of M. elbergi are close to those in species of the "variegata" cluster, but the original description of M. zimzerla puts this species close to M. variegata, one of the species with large inter-population variability. Nartshuk and Fedoseeva (2011) pointed out that M. variegata sensu Fedoseeva, 1960= M. zimzerla Nartshuk, 1992 Our comparative analysis showed a high degree of variability of external key features among species of the "variegata" cluster. The structure of the postgonite and, especially, the size of its anterior process is species specific in males, whereas the females cannot be reliably identified as particular species only by external morphology. For example, in the keys of Fedoseeva (2003) and Nartshuk and Fedoseeva (2010), the presence of a 'large number' of black setae on the lower surface of the genae is the main criterion in identification of M. bohemica. Based on our analysis of the populations of this species, only onefifth to one-third of female specimens can be identified by this character as M. bohemica.
The same is true of M. meigeni; the main diagnostic feature in the identification of this species is the mid stripe of the mesonotum which reaches the scutellum but does not pass through it. However, in 1.4-29% of individuals of M. bohemica populations the mid stripe reaches the shield, instead. Also, the color of the stripes of the mesonotum varies from light to dark among specimens of M. bohemica populations. Such an important feature for species identification of the "variegata" cluster as palp color, can vary considerably from light to dark. In addition, the species are divided into two groups based on the shape of the palpi. However, the analysis of the shape of the palp requires examination of the object from the same angle, which is not always feasible when using the dry specimens.

Conclusions
We suggest, that approaches to the identification of the "variegata" cluster species should include external morphological and dimensional features of both males and females. Additionally, the analysis of males by peculiarities of the postgonites (structure and size of the area of the anterior process of the postgonite) and the analysis of females by the CO1 mtDNA should be used. This will allow the reliable species identification. A similar approach should be used in the identification of adults in other clusters of the genus Meromyza.
Key to species included in the proposed cluster "variegata" Hind femurs heavily thickened, postgonite area, body size, height of frontal triangle, height of genae relative to the 3rd segment of the pedicel is large, the end of the projecting part of anterior process of postgonite with transverse folds, posterior process of the postgonite is adjacent to the anterior one from the back (Fig. 2C)  Color of the strips of mesonotum rusty-red, yellow-brown, sometimes brownish, small genae height relative to the 3rd segment of the pedicel and small length of the mesonotum. Wide posterior process of the postgonite (Fig. 2F)