Five new Palaearctic species of Docosia (Diptera: Mycetophilidae), with updated molecular phylogeny of the genus

. A new phylogenetic hypothesis is proposed for the relationships among the species within the genus Docosia Winnertz, 1863, based on a combined analysis of ﬁ ve DNA markers (28S, ITS2, COI, COII and CytB). Five new species are described, Docosia anatolica Šev č ík sp. nov. from Turkey, D. japonica Kurina sp. nov. from Japan, D. peloponnensis Šev č ík sp. nov. from Greece, D. svanetica Kurina sp. nov. from Caucasus and D. polyspina Kurina sp. nov. from the Russian Far East. New country records of the following species are presented: D. diutina Plassmann, 1996 (Turkey), D. ﬂ avicoxa Strobl, 1900 (Georgia), D. gilvipes (Haliday in Walker, 1856) (Georgia), D. kerkini Kurina & Šev č ík, 2011 (Bulgaria), D. moravica Landrock, 1916 (Georgia), D. pannonica Lastovka & Šev č ík 2006 (Georgia) and D. rameli Kurina & Šev č ík, 2011 (Slovakia).


Introduction
Fungus gnats of the genus Docosia Winnertz, 1863 represent a rather well-defi ned group within the family Mycetophilidae Newman, 1834 (Diptera Linnaeus, 1758), with a mostly dark body, unmarked wings (Fig. 1) and identifi cation characters present mainly on the male terminalia (Laštovka & Ševčík 2006).It has been demonstrated that characters used to separate species in the keys, other than on male terminalia, do not correspond to the relationships revealed by molecular methods (Ševčík et al. 2016a).According to recent molecular studies, the genus Docosia belongs to the subfamily Gnoristinae Edwards, 1925, although this subfamily appears as paraphyletic, with respect to Mycetophilinae (Rindal et al. 2009;Ševčík et al. 2013;Kaspřák et al. 2019).
The known distribution of Docosia includes the Holarctic Region, with three species reported from the Neotropical Region (Oliveira & Amorim 2011, 2014) and a single species from the Oriental Region (Ševčík 2010b).The common European species Docosia gilvipes was also recorded from the Republic of South Africa (Kurina & Ševčík 2012).Altogether, 77 extant species have been described so far from the Holarctic Region including 57 from the Palaearctic and 20 from the Nearctic Regions (reviewed by Zaitzev 2011;Kurina & Ševčík 2012;Taber 2012;2018;Ševčík et al. 2016a).Within the Palaearctic Region, besides the relatively well-studied Europe with 34 known species (Kurina & Ševčík 2012;Ševčík et al. 2016a), Central Asia has 11 species recorded (Kurina 2006;Zaitzev 2011;Kurina & Ševčík 2012), while the other areas are rather sporadically sampled and studied.The phylogenetic relationships among particular species of Docosia are still poorly known, as well as the species spectrum in less-studied areas.The fi rst attempt to reconstruct the phylogeny of several Central European species of Docosia was that by Ševčík et al. (2016a), based on three mitochondrial gene fragments and the nuclear ITS2 region.Here, we present a new hypothesis for the relationships among the species of Docosia, which is a result of an expanded dataset, also including the nuclear 28S gene region, and with more than twice as many species of Docosia included.
The opportunity is also taken here to describe fi ve new Palaearctic species, updating the number of Palaearctic species of Docosia to 62 (Ševčík & Laštovka 2008;Kurina & Ševčík 2011;Ševčík et al. 2016a), and to present new national records of several rare species.

Material and methods
The material used for DNA extraction and species descriptions was collected with Malaise traps, light traps or sweep nets at various localities in the years 2012-2019 (see Table 1).The voucher specimens are deposited in the following depositories: Most of the specimens were collected into 70% ethanol.The type specimens are stored in glycerine medium in a plastic pinned microvial, in ethyl alcohol, or mounted from alcohol, using the chemical method described by Vockeroth (1966), and double-mounted using minute pins.For every studied specimen, the preservation method is indicated in the material section.Illustrations of the terminalia were prepared using a U-DA drawing tube attached to a compound microscope Olympus CX31.The digital images of the general habitus and terminalia were combined using the software LAS ver.4.1.0.from multiple gradually focused images taken by a Leica DFC 450 camera attached to a Leica 205C stereo microscope or a Leica DM 6000 B compound microscope, respectively (see also Jürgenstein et al. 2015).Adobe Photoshop CS5 was used for editing the fi gures and compiling the plates.Before placing in glycerol, the specimens of several species were treated in proteinase K to extract DNA.The protocol of DNA extraction, amplifi cation, sequencing and phylogenetic analysis is described in Ševčík et al. (2016b).The primers used for the PCR amplifi cations of mitochondrial and nuclear gene fragments are listed in Ševčík et al. (2016a, 2016b).
As outgroup taxa we selected two representatives of the subfamily Gnoristinae, in concordance with the most recent molecular study (Kaspřák et al. 2019).The resulting phylogenetic tree (consensus tree) was visualized using Interactive Tree Of Life (iTOL; Letunic & Bork 2016).

Taxonomic treatments
Phylum

Diff erential diagnosis
By structure of the male terminalia, D. anatolica sp.nov.resembles D. juxtamontana Chandler, 2004 andD. incolamontis Chandler, 2004.All species have a bifi d gonostylus and posteroventral margin of gonocoxites with a prominent medial process surrounded by two small lobes bearing setae.These setae are apically ramifi ed in D. anatolica sp.nov., but with an unknown structure in the other two species.Apically modifi ed setae at the posteroventral margin of gonocoxites are frequent in Central-Asian species (cf.Kurina & Ševčík 2012) but they are also observable in other species, e.g., in the European D. pannonica Laštovka & Ševčík, 2006.Docosia anatolica sp.nov.has the ventral lobe of the gonostylus obtriangular, with spines at both posterior corners, while it is slender and furcated, with medial and apical spines in D. juxtamonta and irregularly crescent-shaped with two subapical spines in D. incolamontis.

Etymology
The species name refers to the type locality in Anatolia (Asia Minor).

H
. Blackish brown with numerous pale setae.Three ocelli, lateral ones almost touching compound eyes, separated from eye margins by less than half of their own diameter.Mouthparts light brownish.Palpus brownish yellow, basal segment and apical part of last palpomere darker.Scape, pedicel and all fl agellomeres dark brown.Flagellomeres cylindrical, fl agellomeres 1 and 2 about twice as long as broad, other fl agellomeres (3 to 14) about three times as long as broad.

T
. All parts of thorax blackish brown, with light setae.Scutellum with numerous setae and several submarginal pale bristles about twice as long as scutellum.Antepronotum and proepisternum with pale bristles and short darker setae.Laterotergite and other pleural parts bare.Haltere pale yellow.
L .All coxae yellow, with basal third darkened.Femora mostly yellow, fore and mid femur dark along ventral margin, more extensively in proximal third; similarly hind femur, which is brownish also around tip. Trochanters blackish brown.Fore tibia brownish yellow, apicomedially with simple semicircular tibial organ (anteroapical depressed area), without strong setae, only densely covered with fi ne setulae.Mid tibia darkened dorsally.Hind tibia mostly brownish yellow, with small black dot approximately in middle of dorsal surface.

W
. Hyaline, unmarked.Radial veins and r-m dark brown, other veins paler, basal parts of m faint, almost not traceable.Sc, Rs and basal third of cu-stem asetose, other veins setose.Costa reaches to about one third of distance between R 5 and M 1 .Sc ends in R before level of beginning of m-stem.Posterior fork begins before anterior fork, approximately at level of basal third of r-m.

A
. All dark brown.

T (Figs 2A-E, 7A
). Dark brown except lighter gonostyli and posterior margin of gonocoxites.Tergite 9 subrectangular, about 1.5 times as long as broad, slightly broadening posteriorly, posterior margin with medial incision.Posteroventral margin of gonocoxites with blunt lateral projections and prominent rounded process medioventrally, surrounded by two shorter processes bearing apically ramifi ed thick setae.Gonostylus bifi d.Ventral lobe of gonostylus obtriangular in ventral view, with dark tooth at both posterior corners.Dorsal lobe of gonostylus larger than ventral lobe, bent inwards and rounded apically, with large aggregation of setulae along ventral surface anteriorly, prominent subapical seta posteriorly and with about 10 setae posterolaterally.Cercus with 16 combs of retinacula.

Female
Unknown.

Biology
Unknown.  has 5 spines with the anteriormost one at the apex of the gonostylus and two times as long as the other).

Docosia japonica
The posteroventral margin of the gonocoxites resembles somewhat D. selini Kurina, 2006 described from Kazakhstan and subsequently found in the Chimgan area in Uzbekistan, but both species can be well distinguished by the shape of the gonostylus.

Etymology
The species is named after its occurrence in Japan. .Length of wing 3.8 mm.

H
. Black with numerous pale setae.Three ocelli, lateral ones almost touching compound eyes, separated from eye margins by less than half of their own diameter.Mouthparts light brownish.Palpus with basal three segments brown, 4 th segment light brown, 5 th segment yellowish.Scape dark brown, pedicel and all fl agellomeres somewhat lighter.Flagellomeres cylindrical, about two times as long as broad.

T
. All parts of thorax blackish brown to black, with light setae.Scutum somewhat darker than lateral parts of thorax.Scutellum with numerous setae, marginal setae not arranged to distinct pairs.Antepronotum and proepisternum with pale bristles and short darker setae.Laterotergite and other pleural parts bare.Haltere pale yellow.
L .All coxae, trochanters, femora and tibiae dark brown.Tibial spurs yellow.Tarsi dark yellow but seem brownish because of dense setae.Fore tibia apicomedially with semicircular tibial organ (anteroapical depressed area), without strong setae, only densely covered with fi ne yellow setulae.

W
. Hyaline, unmarked.Radial veins and r-m dark brown, other veins paler, m-stem faint.Sc, Rs, m-stem, basal part of M 1 , bm-m and basal one third of cu-stem asetose, other veins setose.Costa reaches to about one third of distance between R 5 and M 1 .Sc ends in R at level of beginning of m-stem.Posterior fork begins well before anterior fork, approximately at level of basal fi fth of r-m.

A
. Dark brown with pale setae, fi rst three segments with tergites laterally and sternites wholly yellowish.

T (Figs 3A-E, 7B
). Brown, with gonostyli and posteroventral area of gonocoxites yellow.Tergite 9 subquadrate, about as long as broad, posterior margin straight, with submarginal row of prominent setae about twice as long as other vestiture on tergite.Posteroventral margin of gonocoxites with quadrate, posteriorly slightly widening medial process having lateral aggregations of dark setae, medially separated by stripe of light setae.Gonostylus simple, tapering, apical half bent medially, with (1) long and pointed medial spine at medial margin, (2) row of three more anterior, blunt spines at medial margin, about three times shorter than posterior spine, (3) medial surface bearing minute setae, and (4) lateral surface bearing about 15 fi ne setae medially.Cercus with 13 combs of retinacula.

Female
Unknown.

Biology
Unknown.

Diff erential diagnosis
This species is unique among the species of Docosia in having a simple gonostylus with a sickle-like posterior projection.

Etymology
The species name refers to the type locality.

H
. Blackish brown with numerous pale setae.Three ocelli, lateral ones almost touching compound eyes, separated from eye margins by less than half of their own diameter.Clypeus blackish, with setae pale.Mouthparts light brownish.Palpus brownish yellow, basal segment and apical part of last palpomere darker.Scape, pedicel and all fl agellomeres dark brown.Flagellomeres cylindrical, fl agellomeres 1 to 7 about twice as long as broad, distal fl agellomeres (8 to 14) slightly conical, 2.5 to 3 times as long as broad.

T
. All parts of thorax blackish brown, with light setae.Scutellum with numerous setae and several submarginal pale bristles about twice as long as scutellum.Antepronotum and proepisternum with pale bristles and short darker setae.Laterotergite and other pleural parts bare.Haltere pale yellow.
L .All coxae yellow, with basal third darkened.Femora mostly yellow, fore and mid femur dark along ventral margin, more extensively in proximal third; similarly hind femur, brownish also around tip.All trochanters blackish brown.Tibiae and tarsi yellow, tarsal segments seemingly brownish because of dense setulae.Fore tibia apicomedially with semicircular tibial organ (anteroapical depressed area), without strong setae, only densely covered with fi ne setulae.

W
. Hyaline, unmarked.Radial veins and r-m dark brown, other veins paler, basal parts of m faint, almost not traceable.Sc, Rs and basal third of cu-stem asetose, other veins setose.Costa reaches to 0.45 of distance between R 5 and M 1 .Sc ends in R slightly beyond level of beginning of m-stem.Posterior fork begins before anterior fork, approximately at level of basal third of r-m.

A
. All dark brown.4A-E, 7D).Dark brown except lighter gonostyli and posterior margin of gonocoxites.Tergite 9 about as long as broad, slightly narrowing towards base, with slightly concave posterior margin.Posteroventral margin of gonocoxites without lateral projections, with distinct U-shaped medial incision and narrow digitiform medioventral process.Gonostylus simple, slightly bulging medially, with aggregation of small setae at mediobasal corner and with characteristic sickle-like posterior projection, with prominent basal setae at its ventral and dorsal sides.Cercus with 14 combs of retinacula.

Female
Unknown.

Biology
Unknown. ).Docosia polyspina sp.nov.markedly diff ers from the other species in having the gonostylus with three long spines at the medial margin and the internal fl ange of the posteroventral margin of the gonocoxites with about 10 simple blunt spines laterally on both sides.

Etymology
The name is combined from the Greek 'πολύ' (polú) 'many' and Latin 'spīna' 'spine', referring to the number of black spines on internal fl ange of the gonocoxites posteroventrally.Palpus yellowish, basal segment somewhat darker.Scape dark brown, pedicel and basal half of fi rst fl agellomere dark yellow, rest of fl agellomeres brown, lighter than scape.First three fl agellomeres cylindrical, about 1.5 times as long as broad, rest of fl agellomeres moniliform, about as long as broad.

T
. All parts of thorax blackish brown, with light setae.Scutellum with numerous setae and pair of submarginal pale bristles about twice as long as scutellum.Antepronotum and proepisternum with pale bristles and short darker setae.Laterotergite and other pleural parts bare.Haltere pale yellow.
L .All coxae yellow, with basal fi fth of fore-and midcoxae, and basal third of hind coxa darkened.Trochanters brownish.All femora and tibiae yellow.Fore tibia apicomedially with semicircular tibial organ (anteroapical depressed area), without strong setae, only densely covered with fi ne setulae.Tarsi seem brownish because of dense setae.

W
. Hyaline, unmarked.Radial veins and r-m dark brown, other veins paler, m-stem faint, almost not traceable.Sc, Rs, m-stem, bm-m and basal two thirds of cu-stem asetose, other veins setose.Costa reaches to about one third of distance between R 5 and M 1 .Sc ends in R at level of beginning of m-stem.Posterior fork begins well before anterior fork, approximately at level of basal fi fth of r-m.

A
. All dark brown with pale setae.

T (Figs 5A-E, 7C
).Light brown, with gonostyli slightly lighter.Tergite 9 widening posteriorly, about 1.3 times as long as broad, posterior margin slightly concave, anterior margin with V-shaped medial cleft.Posteroventral margin of gonocoxites with lateral extensions, covered with dense aggregation of fi ne setae, setae longer at posterior margin.Internal fl ange of posteroventral margin drawn out to large, posteriorly rounded medial process, with about 10 simple blunt spines laterally on both sides and about 10 setae medially.Gonostylus simple, tapering, with (1) row of three blunt medially directed spines at medial margin, about twice as long as spines on internal fl ange of posteroventral margin of gonocoxites, (2) two extremely long apically curved setae, clearly deviating from other setae of terminalia, and (3) about 15 setae on apical half ventrally.Cercus with 11 combs of retinacula.

Female
Unknown.

Diff erential diagnosis
By the structure of the male terminalia, D. svanetica sp.nov.belongs to a group of Palaearctic species as defi ned for D. polyspina sp.nov.and resembles D. kerkini.Both species have a simple sickle-or crescent-shaped gonostylus with a row of medial spines and a posteroventral margin of gonocoxites with submedial humps, bearing a comb of short setae.The subsimilar combs of the setae at the posteroventral margins of the gonocoxites are represented in several Palaearctic species (e.g., D. dentata, D. lastovkai Chandler, 1994, D. melita Chandler et al., 2006), which have an otherwise diff erent structure of the gonostylus and aedeagal complex.Dcosia svanetica sp.nov.diff ers markedly from D. kerkini in the shape of the ninth tergite, which is subcircular, while it is apically very wide and broadly concave in D. kerkini.Moreover, D. svanetica sp.nov.has a gonostylus with a prominent apical and four smaller medial spines in spite of about 12 medial subequal spines in D. kerkini.The laterotergite is setose in D. svanetica, while it is bare in D. kerkini.

Etymology
The species name refers to the type locality in Svanetia, a historic province of Georgia, in the northwestern part of the country.

H
. Blackish brown with numerous pale setae.Three ocelli, the lateral ones almost touching compound eyes, separated from eye margins by less than half of their own diameter.Mouthparts light brownish.Palpus with two basal segments brownish and three apical segments yellow.Scape, pedicel and all fl agellomeres dark brown.Flagellomeres cylindrical, fl agellomeres 1 and 2 about 1.3 times as long as broad, other fl agellomeres 2-2.5 times as long as broad.

T
. All parts of thorax dark brown to blackish, with light setae.Scutellum with numerous pale setae and several submarginal pale bristles about twice as long as scutellum.Antepronotum and proepisternum with pale bristles and short darker setae.Laterotergite with 6-8 light bristles on posterior half, other pleural parts bare.Haltere pale yellow.
L .All coxae yellow, with basal fi fth of fore-and midcoxae, and basal third of hind coxa darkened.Trochanters brownish.Femora mostly yellow, fore and mid femora dark along ventral margin in proximal half, hind femur apically darkened.All tibiae yellow.Fore tibia apicomedially with semicircular tibial organ (anteroapical depressed area), without strong setae, only densely covered with fi ne setulae.Tarsi seem darker because of dense setae.

W
. Hyaline, unmarked.Radial veins and r-m dark brown, other veins paler, m-stem and basal parts of M 1 and M 2 faint, almost not traceable.Sc, Rs, bm-m, m-stem and basal third of cu-stem asetose, other veins setose.Costa reaches to about one third of the distance between R 5 and M 1 .Sc ends in R at level of beginning of m-stem.Posterior fork begins before anterior fork, approximately at level of basal third of r-m.

A
. Dark brown to blackish, with second and third segments light brown.6A-E, 7E).Dark brown except lighter gonostyli.Tergite 9 subcircular, about as long as broad, posterior margin slightly concave, anterior margin with wide V-shaped incision medially.

Female
Unknown.

Biology
Unknown.

Molecular phylogeny of Docosia
The phylogenetic tree for the concatenated dataset based on fi ve gene fragments is presented in Fig. 8.The genus Docosia was found to be monophyletic with a maximum ultrafast bootstrap support value (ufboot = 100).
Most of the relationships among the rest of the species included to this dataset are less supported (ufboot < 80), except for the group of six species related to D. nigra Landrock, 1928, which apparently constitute a monophyletic group (ufboot = 85).

New national records
The opportunity is taken here to record the following species as new to regional faunas.

Discussion
If we compare the tree in Fig. 8 with the previous molecular phylogeny of Docosia by Ševčík et al. (2016a), it is evident that more extensive taxon sampling and the addition of one gene fragment have not increased the resolution substantially.Although we used a diff erent algorithm (IQ-Tree vs RAxML in the previous paper), the node support values are in both cases relatively low, indicating that new and more variable molecular markers should be tested to recover the interrelationships among particular species of Docosia.
Several pairs of closely related species can be recognized in the molecular tree (Fig. 8), although a close relationship between relevant species in a pair is not always clear from a morphological point of view.The fi rst example is the well-supported clade comprising Docosia fumosa and D. morionella.Although the former species is widely distributed, relatively well known, and its terminalia were fi gured, e.g., by Laštovka & Ševčík (2006), the latter species is extremely rare, with only several records available and with male terminalia fi gured by Chandler & Blasco-Zumeta (2001).It is diffi cult to fi nd distinct similarities between these two species.The laterotergite is pubescent in D. fumosa but bare in D. morionella, showing that pubescence of laterotergite is apparently a homoplastic character, evolved in parallel in diff erent lineages (see also Ševčík et al. 2016a).Concerning the colouration of the legs, both D. fumosa and D. morionella have a black hind femur, but this character state is also present in D. carbonaria and some other species.On the other hand, the male terminalia of D. morionella are rather similar to those of D. gilvipes, especially in the overall shape of the gonocoxites and the structure of the cerci, as noted by Chandler & Blasco-Zumeta (2001).However, D. gilvipes diff ers in having Sc setose and ending free.The latter character has been widely used to separate D. gilvipes from the other Palaearctic species.However, another very similar species, D. pseudogilvipes Kurina, 2008 recorded from the Italian Alps, has Sc bare and ending in R (Kurina 2008).Morover, other species considered as related to D. gilvipes, e.g., the Nearctic D. aceus Garrett, 1925(and probably also D. mcgrawi Taber, 2018) have Sc ending in R, while the recently described Neotropical species D. adusta Oliveira & Amorim, 2011 has Sc ending free and bare (Oliveira & Amorim 2011).Such a variation in wing venation shows that morphological characters other than on male terminalia should be used with caution in phylogenetic reconstructions of Docosia, and possibly also within other genera of fungus gnats.However, a careful case-by-case study is required before any general conclusion can be drawn.
Two species of the D. dichroa Loew, 1869 group are easily recognizable thanks to their rufous abdomen, a unique feature among all species of Docosia.We follow here the interpretation of these two species by Taber (2011Taber ( , 2012)), although Petr Laštovka in his unpublished material (original fi gures plus handmade notes available to the fi rst author) came to the conclusion that the species recently named by Taber (2011) as D. walpurga is actually true D. dichroa, and D. dichroa sensu Johannsen (1909) and Taber (2011Taber ( , 2012) ) needs a new name.However, we leave this issue here as open, until a re-examination of the type material is made (see also discussion in Taber 2018).
Another well-supported clade is D. carbonaria + D. rameli.Both species share the pubescence of the laterotergites, the similar shape of gonostylus and the overall structure of the posterior margin of the gonocoxites.Also, D. cephaloniae and D. lastovkai share many synapomorphies, mainly on the male terminalia, and several other species belong to this group of species as well (cf.Chandler et al. 2006).Both D. carbonaria and D. lastovkai apparently represent complexes of closely related species, especially in the Mediterranean region and eastern Europe, and other species may be discovered in the future.
Three southeastern European species (D. diutina, D. pannonica and D. rohaceki) represent a specifi c and well-defi ned group, based on their male terminalia.Especially the longitudinal tergite 9, distinct posterolateral processes of gonocoxites and the apically modifi ed (ramifying) setae at the posteroventral margin of the gonocoxites are very characteristic.These species also share some colour characteristics, e.g., a dark basal half of the hind coxa.Most of these features are also present in D. anatolica sp.nov.and D. nigra.All these species constitute a monophyletic group in the molecular tree (Fig. 8).In addition, several species not included in our molecular analysis certainly belong to this group, e.g., D. juxtamontana Chandler, 2004.

Table 1 (
continued on the next page).List of species included in the molecular analysis, with GenBank accession numbers.

Table 1
(continued).List of species included in the molecular analysis, with GenBank accession numbers.
By the structure of the male terminalia, D. japonica sp.nov.belongs to a group of Palaearctic species, as defi ned for D. polyspina Kurina sp.nov., and resembles D. kerkiniKurina & Ševčík, 2011, D. polyspina sp. nov.and D. svanetica Kurina sp.nov.All species have a simple sickle-or crescent-shaped gonostylus with a row of spines on the medial margin.However, unlike the other species, D. japonica sp.nov.has four medially situated spines with the posteriormost one three times as long as the anterior three (D.kerkini has 12 subequal spines, D. polyspina sp.nov.has 3 subequal spines and D. svanetica sp.nov.