The identification of the species of the ‘ Spilogona contractifrons species-group ’ and the ‘ Spilogona nitidicauda species-group ’ ( Diptera , Muscidae ) based on morphological and molecular analysis

Muscid species of the ‘Spilogona contractifrons species-group’ (Spilogona alticola (Malloch, 1920), S. arctica (Zetterstedt, 1838), S. contractifrons (Zetterstedt, 1838), S. orthosurstyla Xue & Tian, 1988) and of the ‘Spilogona nitidicauda species-group’ (S. nitidicauda (Schnabl, 1911), S. hissarensis Hennig, 1959, S. imitatrix (Malloch, 1921), S. platyfrons Sorokina, 2018) are notoriously difficult to distinguish. In this paper, their morphological features are analysed, images of the male head, frons and abdomen of all the species are given, and the male terminalia are figured. The study of extensive material has shown that all the morphologically recognised species in each of these groups are valid species. An identification key is provided for both groups of species. To confirm the morphological differences, genetic differences in the cytochrome oxidase I gene of flies of the ‘Spilogona contractifrons speciesgroup’ and of the ‘Spilogona nitidicauda species-group’ were analysed. It is shown that members of both groups of species have not only distinguishing morphological characters but also fixed substitutions in the DNA sequences. Since a low interspecific polymorphism is known in the Muscidae Latreille, 1802, the revealed genetic distances confirm the existence of separate species or subspecies in each of the groups studied.


Introduction
The genus Spilogona Schnabl, 1911 is one of the most speciose genera of the Muscidae Latreille, 1802 and is dominant both in the number of species and in the number of individuals in cold habitats, such as the high-mountain and latitudinal tundra (Sorokina 2012a(Sorokina , 2012b(Sorokina , 2013a(Sorokina , 2013b(Sorokina , 2017;;Sorokina & Khruleva 2012).This genus has been most comprehensively studied in the Nearctic Region, in particular in the territory of northern Canada and Alaska (Huckett 1965).The number of arctic species of Spilogona known from the Nearctic Region is 88.The tundra zones of the Palaearctic Region have not been studied for a long time, and accordingly the number of known species of Spilogona for these territories is significantly less (66) (Hennig 1959a(Hennig , 1959b(Hennig , 1959c)).Recent papers on this genus have increased the number of Palaearctic species to 90, whilst the number of Nearctic species has decreased to 86 (Sorokina 2018).
It should be noted that the changes in the number of species of Spilogona is due not only to the description of new species, but also to the synonymization of some Nearctic and Palaearctic species.The reason for the latter is that most species of Spilogona have a circumpolar or arctic-montane distribution, but this fact was not previously taken into account by researchers when describing new species.Moreover, the genus Spilogona consists of small to medium-sized, dark-coloured muscids covered in greyish to blackish-brown dusting, frequently revealing paired dark markings, which makes them very similar to each other.Furthermore, the high number of species encountered in arctic situations in combination with an often poor state of preservation of specimens collected in traps, preserved in alcohol, etc., make species identification of samples of Spilogona a difficult task even for the specialist.There are therefore still are many problems in clarifying the taxonomic status of the various known species of Spilogona.This paper is dedicated to flies of the 'Spilogona contractifrons species-group' and the 'Spilogona nitidicauda species-group'.Members of these species groups are some of the most dominant in the tundra zone.Like other species of the genus, adults of these groups of species are predaceous on other small and soft-bodied insects close to running water.One of their important roles in nature is their known activity as predators on the adults and larvae of biting black flies (Simuliidae) and on other small insects (Werner & Pont 2006a, 2006b).However, adults of these species live at the same time and in the same biotopes and have very similar morphological characters, and this causes difficulty with their identification and therefore confusion in the results of field research.Moreover, there are different opinions as to the number of existing species in each of these groups of species.
The 'Spilogona contractifrons species-group' includes four morphologically very similar species: S. contractifrons (Zetterstedt, 1838), S. alticola (Malloch, 1920), S. arctica (Zetterstedt, 1838) and S. orthosurstyla Xue & Tian, 1988.The first three species are widely distributed, and consequently have given rise to discussion for many years.Opinions have varied about the presence of only two, or even one species, but not three.Collin (1930) recorded just two species, S. contractifrons and S. arctica from Greenland.Hennig (1959a: 288) and Pont (1986: 166) regarded S. arctica as a synonym of S. contractifrons.Spilogona alticola was previously known only from North America.In his monograph, Huckett (1965: 238) included all three as valid species but added, under the entry for S. contractifrons, "I am doubtful whether alticola Malloch can be regarded specifically as different to contractifrons".Later, Michelsen (2006: 118) reinstated S. arctica as a valid species.In a recent paper by the first author of the present paper, four species of this group of species were reported in the Altai Mountains (Sorokina 2018).
In the genus Spilogona, species limits are occasionally ambiguous and species identification based on morphological characters is difficult.In such cases, a molecular-genetic analysis can be applied.Species identification and species delimitation are more accurate when based not only on morphological but also on molecular-genetic characters.Unfortunately, there are very few papers devoted to the DNA barcoding of the Muscidae and most of them have used the sequence data to perform phylogenetic analyses (Savage et al. 2004;Schuehli et al. 2004Schuehli et al. , 2007;;Kutty et al. 2008Kutty et al. , 2014;;Haseyama et al. 2015).Nevertheless, there is one paper in which the relevance of DNA barcoding as a taxonomic tool in muscid fly identification has been established (Renaud et al. 2012).
The aim of this paper is to analyse genetic differences in the cytochrome oxidase I gene of flies of the 'Spilogona contractifrons species-group' and the 'Spilogona nitidicauda species-group', to confirm the morphological differences.

Material and methods
The material of Spilogona used in this study, including type material, is deposited in the Siberian Zoological Museum of the Institute of Systematics and Ecology of Animals, Russian Academy of Sciences, Siberian Branch, Novosibirsk (SZMN), the Zoological Museum of the Moscow State University, Moscow (ZMUM), the Zoological Institute, Saint Petersburg (ZISP), and the Canadian National Collection of Insects, Ottawa (CNC).

Morphological analyses
Specimens were examined using an Altami PSO745-T microscope for external morphological characters.For the dissection of the male and female terminalia, the end of abdomen was removed and boiled in a 10% KOH solution for 15-20 s.After dissection and study, the abdomen and terminalia were washed and then stored in microvials of glycerine pinned directly underneath the specimens.Pictures were made with the camera Canon EOS 600D.Illustrations were made in ink and then edited using Adobe Photoshop CS.

Molecular-genetics analyses
The molecular-genetic analysis was based on the study of the COI gene, the most standardized short genetic marker in an organism's DNA that is used as a taxonomic DNA barcoding method (Ratnasingham & Hebert 2007).Total DNA was extracted from samples of Spilogona using the DIAtom™ DNA Prep kit (Isogen, Russia).Polymerase chain reaction (PCR) of the cytochrome c oxidase subunit I (COI) locus was conducted with an Encyclo PCR kit (Evrogen, Russia) using TY-J-1460 (Simon et al. 1994) and COIR (Shaikevich 2007) primers.The following amplification conditions were used: initial denaturation at 94°С (5 min); 35 cycles: denaturing at 94°С (30 s), annealing at 55°С (30 s), elongation at 72°С (40 s); and final elongation at 72°С (10 min).The PCR fragments were purified from agarose gel with a Clean-Up Extraction Kit (Evrogen, Russia) and were sequenced using the BigDye Termination kit 3.1 (Applied Biosystems, USA).All sequences were deposited in the GenBank under accessions numbers MH413049-MH413064.
Specimens for genetic analysis were taken from different populations in the territories of Russia.The institution code, the voucher and the GenBank accessions number of specimens are indicated within brackets.А total of 16 specimens of 6 species of Spilogona was examined:
The citation of some original labels is given in quotation marks, but interpretations of these data are given in square brackets.

Taxonomic analysis of 'Spilogona contractifrons species-group'
Flies of this group of species can be distinguished as follows: scutum with 3 post dc setae, mid tibia without pv and with 1 ad seta, fore tibia with 1 p seta, hind femur with a row of pv setae, mid femur with anterior preapical seta, haltere yellow, facial edge not projecting beyond level of profrons, prementum shining, scutellum without downwardly-directed preapical setulae on upper border of declivities, abdomen plump, katepisternal setae arranged in a more or less equilateral triangle like Coenosia Meigen, 1826.
Four species of this group are currently recognised: the widespread S. alticola, S. arctica, S. contractifrons, and the East Asian S. orthosurstyla.Research for the current paper has included a detailed study of extensive material from the territory of Russia, from both tundra zones and mountain areas, and personal study of the bulk of Huckett's collection deposited in the Canadian National Collection, Ottawa, Canada.
The study of extensive material has confirmed the existence of four species in this group.Despite the difficulties in the recognition of species of this group, there are several helpful diagnostic characters: the shape of male sternite 5, the shape of male terminalia, and the colour of the scutum (Figs 1-2, Table 1).The colour of the scutum can be variable in different populations but the shape of the male sternite 5 is constant and is clearly visible in dry specimens (Fig. 1 G-I).

Remark
In view of the distribution of other species of this group, we can assume that this species also lives in the arctic zone.

Molecular-genetic analysis of 'Spilogona contractifrons species-group'
Since in our collection there is only one specimen of S. orthosurstyla and it is not in good condition, the molecular analysis was performed only on the other three species of this group.
Genetic distances between species are shown in the ML tree (Fig. 3) and in Table 2. Regardless of the geographic origin of the specimens, all three studied species in this group have identical COI gene sequences within the species.At the same time, at least five nucleotide substitutions (single nucleotide polymorphisms (SNPs)) vary between S. alticola and S. contractifrons.Spilogona arctica differs from S. alticola with 13 SNP and from S. contractifrons with 12 SNP; one substitution results in amino acid change (M112L).
The genetic distance between S. alticola and S. contractifrons is 0.6%, and between each of these species and S. arctica is 1.8%.All three species formed a distinct cluster on the ML tree with high (98-99%) statistical support in which the associated taxa clustered together (Fig. 3).
The following results were obtained after comparing the sequences of the studied specimens with the annotated sequences in Genbank.Spilogona arctica from the Altai, Taimyr and from the Nenets AO is identical with S. arctica from Sweden.All of them are distinguished by a single substitution of C438T from S. arctica from Canada and Greenland (Supplementary file).Spilogona contractifrons from the Altai, Nenets AO, Kurgan and Naryan-Mar is identical in the COI gene with S. contractifrons from Norway and Sweden.But S. alticola from the Altai clustered with S. contractifrons from Canada (Fig. 3).

Taxonomic analysis of 'Spilogona nitidicauda species-group'
Flies of this group of species can be distinguished as follows: scutum with 4 post dc setae, anepisternum with interspatial setae, facial edge not projecting beyond level of profrons, hind femur without pv setae, scutellum without downwardly-directed preapical setulae on upper border of declivities, abdomen flattened with large trapezoidal marks on tergite 3.
Four species of this group are currently recognised: S. nitidicauda, S. hissarensis, S. platyfrons and S. imitatrix.Spilogona hissarensis was studied only from the original type-series.The other species of this group were studied with material from the territory of Russia and from Huckett's collection deposited in the Canadian National Collection, Ottawa, Canada.
Despite the difficulty in distinguishing the species of this group, there are several helpful diagnostic characters: the width of frons and parafacial, presence of pollen on the prementum, the colour of the scutum and abdomen, the shape of male terminalia (Figs 4-5).Additional diagnostic characters are given in Table 3.The species of this group can be separated by the following couples:

Remark
Mountain species, but probably living in the arctic tundra too.

Molecular-genetic analysis of 'Spilogona nitidicauda species-group'
Since only the old type specimens of S. hissarensis are known, the molecular analysis was performed on the other three species of this group.
DNA variability is higher within taxa of the 'Spilogona nitidicauda species-group' in comparison with taxa of the 'Spilogona contractifrons species-group'.Intraspecies DNA variability was found in S. nitidicauda and S. platyfrons from different localities.The isolate SN38 of Spilogona nitidicauda from the Altai (Muzdy-Bulak lake) differs from the isolates SN40 and SN41 of S. nitidicauda from Chukotka AO (Beringovskii) by a synonymous substitution of C186T.Both isolates from Chukotka have identical DNA.The SP32 of S. platyfrons from the Shapshalsky ridge (Altai) differs from the SP33 of S. platyfrons from the Krasnaya Mountain (Altai) by substitutions of T636C (Supplementary file).Spilogona nitidicauda can be distinguished from S. platyfrons by 3-4 SNPs.Spilogona imitatrix differs from S. platyfrons with 6 SNP, and from S. nitidicauda with 7 SNP.
The genetic distance between S. nitidicauda and S. platyfrons is 0.4%, between S. platyfrons and S. imitatrix is 1.2%, and between S. nitidicauda and S. imitatrix is 1.6%.All three species formed distinct clusters on the ML tree, but the differences between S. nitidicauda and S. platyfrons are insignificant statistically.
The following results were obtained after comparing the sequences of the studied specimens with the annotated sequences in GenBank.Unfortunately, there were no DNA sequences of S. nitidicauda and S. platyfrons in GenBank nor in the BOLD database, so comparison with our results was impossible.But there were many DNA sequences of S. imitatrix from Canada.
Spilogona imitatrix from the Altai is identical with several individuals of S. imitatrix from Canada (KR615453 and KR622696).However, several individuals from GenBank identified as S. imitatrix have a very variable DNA, as can be seen on the ML tree (Fig. 3).Such a high polymorphism may be due to errors in morphological identification.

Discussion
The analysis of genetic distances based on the differences in the cytochrome oxidase I gene in members of the 'Spilogona contractifrons species-group' and the 'Spilogona nitidicauda species-group' confirmed that there are not only morphological differences but also genetic differences between closely related species.
Our results with the DNA analysis of the COI gene have demonstrated that S. alticola, S. contractifrons and S. arctica are not synonymous because the genetic differences between individuals of these species persist in geographically distant locations (Fig. 6).The DNA of S. arctica is identical in individuals from different regions of Russia, Sweden, Greenland and Canada.Individuals of S. contractifrons from Russia are identical with S. contractifrons from Sweden and Norway, and all of them form a distinct cluster of "European" or "Palaearctic" S. contractifrons.It should be emphasised that the specimens of S. arctica and S. contractifrons from Russia were identical with specimens from the type localities (Sweden and Greenland) but show differences from specimens of these species from Canada.
In turn, specimens from Canada designated in GenBank as S. contractifrons were identical with specimens from the Altai identified by us as S. alticola.The latter species was described from specimens from USA (New Hampshire) and was later found in different areas of Northern Canada (Huckett 1965).The reasons for this result could be either the incorrect identification of the specimens deposited in GenBank, or a rejection of the existence of S. alticola species by Canadian researchers and a designation of such specimens as S. contractifrons.
In the 'Spilogona nitidicauda species-group' there are stable nucleotide differences between S. nitidicauda and S. platyfrons from different places and years of collection despite the morphological similarity.Although the genetic distance between S. nitidicauda and S. platyfrons is only 0.4%, S. imitatrix differs from them significantly.The DNA distance between S. imitatrix and S. nitidicauda is 1.6%, and that between S. imitatrix and S. platyfrons is 1.2% which demonstrates the separate species status of S. imitatrix.In Muscidae, ranges of 0.18% and 3.01% have been reported as averages for the means and maxima of COI intraspecific distances, respectively (Renaud et al. 2012).Limits of species with a distance to the nearest neighbour of < 2% in 137 species of Muscidae were supported by morphological characters and confirmed that the minimum interspecific distance in Muscidae ranges well below 2% for many species (Renaud et al. 2012).
Spilogona imitatrix was described from specimens from Labrador (Canada) and was later found in different regions of Canada and USA (Alaska).Recently this species was found in the Palaearctic Region for the first time, here known only in the Altai Mountains (Sorokina 2018).The DNA of S. imitatrix from the Altai (Terektinsky ridge) was identical with the DNA of several individuals of S. imitatrix from the Northwest Territories (Canada).There is a rather large number of the sequences of this species from Canada in GenBank.Several DNA sequences of S. imitatrix from Canada were randomly taken for genetic matching with the specimen of this species from the Altai.It transpired that one sequence (KR628150) clustered with S. platyfrons from the Altai, whilst the other sequences of Canadian specimens (# HM389123, # HM389124) differed from all of them by six nucleotide substitutions (Fig. 3).Such differences between DNA sequences of one species can be explained by errors in the initial morphological identification.This has been proved by the work of the first author of this paper (VSS) with the Muscidae collection in the CNC, where the following species were found under the name S. imitatrix, and vice versa: S. alticola, S. arctica, S. deflorata (Holmgren, 1872), S. incauta (Huckett, 1932), S. nitidicauda, S. trigonifera (Zetterstedt, 1838), S. sororcula (Zetterstedt, 1845), S. trilineata (Huckett, 1932) and another Spilogona sp.These misidentifications are not surprising, since most members of the genus Spilogona are difficult to distinguish, even for muscid specialists.Therefore, in addition to a genetic analysis it is important to have an idea of the most stable morphological characters in each group.According to our results with this genus, the following can be considered as the most stable morphological characters in Spilogona: presence of pv/v setae on mid tibia, presence of pv setae on hind femur, presence of dust on the prementum, shape of the sternite 5, presence interspatial seta(e) on anepisternum, number of post dc setae on the scutum (3 or 4), presence on the scutellum of downwardly-directed preapical setulae on upper border of declivities.The last character was proposed and used only by Huckett (1965) for the separation of species into groups in his key to the species of Spilogona, in addition to the other well-known characters.Other researchers (Hennig 1959a(Hennig , 1959b(Hennig , 1959c;;Gregor et al. 2002) used well-established characters for identification, specifically the number of post dc setae on the scutum (3 or 4), the elongation of the face, and the presence of pv setae on hind femur.The character suggested by Huckett (downwardlydirected preapical setulae on the upper border of declivities of scutellum) is quite difficult to see, which is probably why it has not been generally used by the authors of the keys.However, in our opinion, this is one of the important characters for the identification of the species of Spilogona.To demonstrate this, several outgroup species (S. aerea (Fallén, 1825), S. bifimbriata Huckett, 1965, S. baltica (Ringdahl, 1918) and S. tornensis (Ringdahl, 1926)) were included in the genetic analysis.These species have the same characters (3 and 4 post dc setae, mid tibia without v seta, hind femur with and without pv setae, face not projecting) as in the species groups analysed above, but, unlike them, these species have scutellum with downwardly-directed preapical setulae on the upper border of declivities.The results of the genetic analysis showed a distinct distance between the cluster of the 'Spilogona contractifrons species-group' and the 'Spilogona nitidicauda species-group' on the one hand, the members of which have scutellum without downwardly-directed preapical setulae on the upper border of declivities, and on the other hand the cluster of species having these setulae on the scutellum (Fig. 3).
The members of the 'Spilogona contractifrons species-group' and the 'Spilogona nitidicauda speciesgroup' thus have not only distinguishing morphological characters but also fixed substitutions in the DNA sequences.Since a low interspecific polymorphism is known in the Muscidae, the revealed genetic distances confirm the existence of separate species or subspecies in each of the groups studied.

Fig. 6 .
Fig. 6.Localities of the species of Spilogona Schnabl, 1911 used in the DNA analysis.Symbols denote the species, whilst the colour shows the same DNA sequences.A. 'S.contractifrons species-group'.B. 'S.nitidicauda species-group'.

Table 2 .
Estimates of evolutionary divergence between studied species of Spilogona Schnabl, 1911.The number of base substitutions per site from averaging over all sequence pairs between groups are shown.The analysis involved 16 nucleotide sequences.