The Mycetophila ruficollis Meigen (Diptera, Mycetophilidae) group in Europe: elucidating species delimitation with COI and ITS2 sequence data

Abstract European species of the Mycetophila ruficollis group are compared on the basis of morphology and sequences of mitochondrial cytochrome oxidase subunit one (COI) and the ITS2 region of nuclear ribosomal DNA. The study represents the first evaluation of morphology-based species delimitation of closely related fungus gnat species by applying molecular information. Detailed descriptions and illustrations of the male terminalia are presented along with a key for the identification of all nine European species of the group. Phylogenetic analyses of molecular data generally supported the morphological species discrimination. The barcoding region of COI superseded ITS2 rDNA in resolving species. In the COI barcoding region interspecific differences ranged from 2.9 to 10.6% and the intraspecific distance from 0.08 to 0.8%. Only COI data distinguished between the similar and closely related Mycetophila ichneumonea and Mycetophila uninotata of which the latter was observed to include cryptic species. The host range of some species is suggested to be narrower than previously considered and to depend on the forest type. Presented evidence indicates the importance of analysing sequence data of morphologically very similar mycetophages reared from identified host fungi for elucidating species delimitation as well as their geographic and host ranges. New country records, viz. Estonia for Mycetophila evanida, Georgia for Mycetophila ichneumonea, Mycetophila idonea and Mycetophila ruficollis, and Norway for Mycetophila strobli, widen the known distribution ranges of these species.


Introduction
forms and noted that M. ichneumonea may be polytypic consisting of 2-3 subspecies. This led us to suggest that the species-group may hide some undiscovered diversity, i.e. cryptic species with possibly different larval diet. One of the most important and frequently used set of characters for delimiting cryptic species is that obtained from DNA sequence analyses. In studies of fungus gnats, DNA sequence data are so far mostly used to clarify phylogenetic relationships of subfamilies and/or genera (e.g. Rindal et al. 2009, Martinsson et al. 2011, Ševčík et al. 2014) but also to associate sexes of one species (Kurina et al. 2011) and compensate for the deficiencies of the morphological component in species identification of otherwise obscure material . For delimiting species, molecular characters have thus far been incorporated only in the genus Neuratelia . In addition to the widely used 'DNA barcode' fragment (COI) (e.g. Hebert et al. 2003, Hajibabaei et al. 2006), ITS2 sequences have successfully been applied for species delimitation. The aim of this study was to 1) test morphological species delimitation by the application of molecular methods, 2) present a key to European species of the M. ruficollis group supplemented by modern illustrations of male terminalia, and 3) search for possible cryptic species. Because this group includes morphologically extremely similar species, they are frequently being identified only to species-group level even by specialists. Our intention was to provide also a reliable DNA reference dataset that could be used in further DNA based identification of fungus gnats.

Morphological analyses
The study is based on material collected throughout the Europe during 1984 to 2014 mostly by Malaise traps, light traps and sweepnetting. In addition, several specimens from Lebanon and Georgia are also included. A part of the Estonian material was reared from macrofungi. For that, fruit bodies were isolated into plastic containers and covered with nylon gauze, while peat was used as a pupation substrate. Containers were incubated in a lab facility and checked every other day while emerged adults were collected by an aspirator (see also Põldmaa et al. 2015). Altogether 116 male specimens of the M. ruficollis group were morphologically studied.
The majority of the included material was initially collected into 70 % ethyl alcohol and studied under stereomicroscopes Olympus SZ61or Leica S8APO. For detailed study of male terminalia they were detached and macerated in a solution of KOH followed by neutralization in acetic acid and washing in distilled water (for details see Kurina 2003). The remaining chitinous structure was thereafter separated to several anatomical units which were: 1) inserted into glycerine for study and preserved as glycerine preparations or 2) slide-mounted individually in Euparal between two pieces of coverslip allowing them to be studied from both sides under a compound microscope (for details see Hippa and Kurina 2012). The preservation method of each specimen is indicated in the material sections. The habitus photo was taken from a specimen in alcohol. All photos of male terminalia were taken from preparations in Euparal and combined by software LAS V.4.1.0. from multiple gradually focused images taken by a camera Leica DFC 450 attached to the compound microscope Leica DM 6000 B (see also Kurina and Oliveira 2013). Morphological terminology follows generally that of Søli et al. (2000) while several specific terms of male terminalia (see Table 1) are used according to Laštovka (1972). The term "bristle" is used for a seta that is significantly larger in length and diameter than surrounding setae (see also Merz and Haenni 2000). Detailed revised descriptions of male terminalia figured by Laštovka (1972) and Laštovka and Kidd (1975), supplemented by illustrations, are provided herein.

Molecular techniques
Based on the preliminary morphological determination, at least one male specimen from every species from each locality was allocated for DNA sequencing. For that, after detaching terminalia, the rest of the abdomen or a leg was placed in the lysis buffer, preserving the rest of the specimen. DNA was extracted by incubating the material overnight at 56 °C in 10X Reaction Buffer B (Solis Biodyne, Tartu, Estonia) with the addition of 2.5 μl (20 mg/ml) proteinase K (Fermentas, Lithuania). After 15 min at 98 °C the material was centrifuged and DNA solution pipetted into a new tube.
In 66 specimens, the 658 bp barcode region at the 5' end of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced with primers Lep-F1 and Lep-R1 (Hebert et al. 2004) or LCO1490 and HCO2198 (Folmer et al. 1994). In most of the specimens an additional 790 bp, following the barcoding region, were amplified and sequenced with primers C1-J-2195 and TL2-N-3014 (Simon et al. 1994). In 33 specimens, the second fragment of the internal transcribed spacer region (ITS2) was amplified and sequenced using primers ITS2A and ITS2B (Beebe and Saul 1995). PCR was performed in a total volume of 25μl, with the reaction mixture containing 1X HOT FIREPol®Blend Master Mix Ready to Load (Solis BioDyne, Tartu, Estonia), 10 pmol of primers and 20-80 ng of DNA. PCR was carried out in an Eppendorf Mastercycler epigradient thermocycler (Eppendorf AG, Hamburg, Germany). The initial denaturation at 95 °C for 15 min was followed by 35 cycles of 30 s at 95 °C, 30 s at 45-60 °C (depending on primers) and 1 min at 72 °C, followed by a final extension at 72 °C for 10 min. PCR products were visualised on a 1.2% agarose gel, and the remaining PCR product was purified with fast alkaline phosphatase and exonuclease I (Thermo Scientific, Pittsburgh, USA). DNA sequencing was performed at Macrogen Europe (Amsterdam, Netherlands) or at the Estonian Biocentre (Tartu, Estonia). All sequences obtained in this study were deposited in GenBank under accession numbers KR997602-KR997703.

Phylogenetic analyses
The sequences were edited and assembled with Sequencher 5.1 (Gene Codes, Ann Arbor, MI, USA), aligned with Mafft 6 online version (Katoh and Toh 2008) and edited manually using GeneDoc 2.6.0.3. Phylogenetic analyses were performed on the separate and combined COI and ITS2 datasets. Four sequences of Mycetophila fungorum were used as an outgroup in all analyses. Bayesian analyses were performed on all three datasets using MrBayes at the Cipres website (http://www.phylo.org) with default settings of the online version and invgamma model. Each analysis was run for 10 mln generations, of which every 1000th was sampled. The first 25% of sampled trees were discarded as burn-in. Posterior probabilities were calculated from remaining 7500 trees. Parsimony analyses were performed in PAUP (Swofford 2003). During the 1000 random searches 50 best trees were kept in each search that were all used for additional swapping. This was done until the limits of available computer memory was reached. The most parsimonious trees obtained were used to calculate the strict consensus. 1000 bootstrap replications were performed to assess branch support. Intra-and interspecific distance variance was calculated in MEGA6 (Tamura et al. 2013) using Kimura 2-parameter model (see e.g. Waugh 2007, Õunap andViidalepp 2009).

Results
Based on the morphology, mainly that of male terminalia, the studied material was identified to belong to all nine species of the M. ruficollis group known from Europe. Deviation in morphological characters of some specimens suggested that these might represent additional undescribed species. Phylogenetic analyses, based on molecular data, recognised seven well supported clades, corresponding to the morphologically distinguished species. Fresh material, suitable for molecular analyses, was not available for two European species: M. sepulta and M. suffusala. The phylogenies led to reconsideration of morphology-based identification in several specimens. Consequently, the colouration and dimensions of the gnats' body were realised to be variable but the characters of male terminalia mostly constant within a species.
The interspecific genetic distance among species of the M. ruficollis group was calculated for the COI barcoding region, including 647 bp (Table 2). Interspecific differences ranged from 2.9% (between M. ichneumonea and M. uninotata) to 10.6% (between M. evanida and M. idonea), with the mean interspecific distance of 8.1%. The intraspecific distance ranged from 0.08 to 0.8% except for 2.3% in M. uninotata. The second region of COI, following the barcoding region, distinguished the seven analysed species based on 4.0-9.3% of interspecific variation (data not shown).
The COI datamatrix comprised 66 sequences and 1432 characters (1108 constant, 287 parsimony informative). The ITS2 datamatrix comprised 37 sequences and 584 characters of which 535 were constant and 47 parsimony informative. The combined COI and ITS2 datamatrix comprised 37 sequences and 2016 characters (1662 constant, 331 parsimony informative). When comparing the ITS2 and COI regions in  (Fig. 4) distinguished six strongly supported species (bootstrap support 94% to 100%, posterior probabilities 0.94-1.0) in the ingroup. However, M. uninotata appeared paraphyletic with three strongly sup- ported lineages. The analyses of COI data also support close relationship between M. ichneumonea and M. uninotata as well as M. evanida and M. britannica. By contrast, the ITS2 trees (not shown) were much less resolved, with four well-supported groups recognised. The ITS2 phylogenies did not distinguish M. ichneumonea from M. uninotata with one M. strobli specimen from Italy also placed in this clade. The consensus of most parsimonious (Fig. 5) as well as the Bayesian trees calculated from 37 combined COI and ITS2 sequences were well resolved and with higher support values for most of the clades than obtained in analyses of individual gene regions. The main difference was observed in the three lineages of M. uninotata forming a monophyletic group, yet receiving only low support.
Members of the M. ruficollis species group were selected from the material of adults reared from mushrooms collected from Estonia during 1988-1990(Kurina 1991 and extensive sampling in five pine dominated boreal forests in 2011  as well as scattered localities in the following years. Among the >11 000 adults reared from the pine forest material most gnats from the M. ruficollis species group were sequenced and identified as belonging to M. ichneumonea. Among the 100 fungal species, this gnat emerged only from 12 fruit bodies of Lactarius and 4 fruit bodies of Cortinarius. Only one specimen, reared from Rhodocollybia butyracea, represented M. ruficollis. The study presents new species records for Estonia (M. evanida), Georgia (M. ichneumonea, M. idonea, M. ruficollis) and Norway (M. strobli).

Key to the species of the M. ruficollis group in Europe based on characters of male terminalia
The key is compiled on the basis of original data, Laštovka (1972), and Laštovka and Kidd (1975).
1 4 th palpal segment wider than the 3 rd and about twice as wide as 5 th , and about as long as the 5 th (Laštovka and Kidd 1975: Fig.1). Posterior margin of gonocoxite ventromedially undulating or with diminutive central prominence ...2 -4 th palpal segment about as wide as the 3 rd and only slightly wider than the 5 th , and distinctly shorter than the 5 th (Laštovka and Kidd 1975: Figs 7-15 Central spot of wing narrow and indistinct (sometimes almost absent). Dorsal branch of gonostylus with: lateral margin almost stright and distal posterior process subequal to proximal posterior process, both separated by wide and deep notch (Fig. 9)  Posterior margin of gonocoxite laterally from ventromedial convexity straight (Fig. 40). Posterior margin of dorsal branch of gonostylus proximally from medial bristle with 5-6 gradually diminishing strong bristles (Fig. 8)  Male terminalia. Posterior margin of gonocoxite slightly concave ventromedially, and with abrupt and blunt projections laterally. Posterior impression wide. Anterior impression with anteriorly evenly divergent wide arms. Ventral branch of gonostylus with narrow, short and asymmetrical posterior process which bears minute warts; ventral surface with 6-9 long bristles deviating from other setosity; spine 1 and spine 2 of almost equal height and width; spine 1 sharply pointed; spine 2 blunt; spines 3 and 4 smaller, pointed and close to each other. Dorsal branch of gonostylus abruptly narrowed beyond the medial bristle; lateral margin with shallow concavity or almost straight; distal posterior process very small, separated from proximal posterior process by a narrow but distinct notch, both processes apically rounded. Posterior margin proximally from medial bristle with 5 gradually diminishing bristles followed by 2 small setae. Basal angle slightly rounded, basal margin with few setae. Distal posterior process apically with small seta, proximal posterior process apically bare. Ejaculatory apodeme with semi-rounded or proximally truncated base and without rim. Aedeagal guides wide, apically widened, extending beyond aedeagus distally, lateral impressions wide. Aedeagal apodemes laterally angular.
Intraspecific variation. Laštovka and Kidd (1975) figured the ventromedial margin of the gonocoxite with a small convexity medially that was not observed in the studied material. They also described two different forms of the dorsal branch of the gonostylus: 1) slender and slightly narrowing beyond the medial bristle, and 2) shorter and abruptly narrowing beyond the medial bristle. The last character resembles that of M. ruficollis, M. laffooni Laštovka, 1972 and somewhat also M. suffusala. All studied specimens had the dorsal branch of the gonostylus slender, corresponding to the first form. Laštovka and Kidd (1975) found M. britannica to be most similar to M. evanida. In some studied specimens from Greece, the spine 2 on the ventral branch of the gonostylus is more massive, being longer than the spine 1. In Greek specimens the spine 2 in the ventral branch of the gonostylus is blunt while it is sharply pointed in Italian specimens.
Hosts and distribution. Mycetophila britannica has been earlier reared from Polyporus squamosus, Armillaria mellea, Hebeloma crustuliniforme, Russula nigricans, Hypholoma sp. and Lactarius resimus (Laštovka and Kidd 1975, Yakovlev 1994, Chandler 2010), while we have studied sweepnetted and trapped material only. Having been described from the British Isles, the species is widely distributed in Western Europe extending also to Norway and the Middle East (Kjaerandsen 2012, Chandler 2013. Except for a finding in Russian Karelia (Kjaerandsen 2012) it is not found in Eastern Europe. According to Chandler and Ribeiro (1995) and Chandler et al. (2005), M. britannica is common in the Mediterranean region including Morocco. Laštovka, 1972 Figs 8, 21, 22, 39, 40 Material. ESTONIA. 1♂, Jõgeva county, Pataste, 58°34'52,2"N, 26°46'42,3"E, 5.x-19. Male terminalia. Posterior margin of gonocoxite ventromedially with clear convexity, and with abrupt and blunt projections laterally. Posterior impression rather wide. Anterior impression with anteriorly evenly divergent narrow arms. Ventral branch of gonostylus with semicircular posterior process which bears minute warts and few setae; spine 1 slender, evenly tapering and sharply pointed; spine 2 shorter, thicker and rather blunt; spines 3 and 4 much smaller than spine 1, sharply pointed and close to each other. Dorsal branch of gonostylus steeply tapering; lateral margin with shallow concavity; distal posterior process about half as high as proximal posterior process, both separated by shallow concavity. Distal posterior process with few setae; proximal posterior process bare with 2-3 setae deviating from other setosity on its base. Posterior margin proximally from medial bristle with 6 gradually diminishing bristles; internal surface with one somewhat stronger seta next to the medial bristle. Basal angle right-angled, basal margin with setae. Ejaculatory apodeme with subquadrate base and very wide rim. Aedeagus widened apically, apical margin slightly convex. Aedeagal guides with wide lateral impressions; apically narrow and rounded, not extending upper margin of aedeagus. Aedeagal apodemes laterally angular.

Mycetophila evanida
Intraspecific variation. In some cases the base of the ejaculatory apodeme resembles M. idonea, because of having its basal margin somewhat concave. The holotype has an additional small spine on the ventral branch of the gonostylus close to spines 3 and 4 (cf. Laštovka (1972: fig. 12). In studied material this spine was not observed.
Hosts and distribution. Mycetophila evanida has been reared from species of Russula, Lactarius, and Tubaria (Yakovlev 1994). The species is widely distributed in Europe extending also to the Eastern Palearctic (Laštovka 1972, Chandler 2013, Jakovlev 2014). The material from Estonia represents a new country record. Male terminalia. Posterior margin of gonocoxite ventromedially with shallow convexity, and with abrupt and blunt projections laterally. Posterior impression with narrow base and well widened posterior part. Anterior impression with anteriorly evenly divergent narrow arms. Ventral branch of gonostylus with symmetrical semioval posterior process, which bears minute warts and few setae on ventral surface; spine 1 wide, sharply pointed; spine 2 similar or somewhat longer but more slender; spines 3 and 4 about twice as small, evenly tapering and with about equal distance between each other and spine 2. Ventral branch of gonostylus with 4-5 strong bristles deviating from other setosity laterally on ventral surface. Dorsal branch of gonostylus steeply tapering; lateral margin with deep concavity; proximal posterior process about twice as high as distal posterior process, both separated by deep concavity. Distal posterior process with apical small setula and with a basal strong seta deviating from other setosity; proximal posterior process apically rounded with 1-2 strong basal setae deviating from other setosity. Posterior margin proximally from medial bristle with 4-5 gradually diminishing bristles followed by 3-4 smaller setae; internal surface with a stronger seta next to the medial bristle. Basal angle clearly outlined, angular or somewhat rounded; basal margin with few setae. Ejaculatory apodeme with campanulate base and without rim. Aedeagus oval or cross shaped. Aedeagal guides extending over apical part of aedeagus; apically rounded and divided into two lamellae; with lateral impressions very small. Aedeagal apodemes laterally slightly angular or arched and pointed apically.

Mycetophila ichneumonea
Intraspecific variation. In some cases the lateral margin of the dorsal branch of the gonostylus is shallower, and the distal posterior process and the posterior margin of the dorsal branch of the gonostylus resemble those of M. uninotata. The spine 2 on the ventral branch of the gonostylus compared to the spine 1 can be more prominent than described by Laštovka (1972).

Mycetophila idonea
Intraspecific variation. In some specimens from Georgia and Poland the base of the ejaculatory apodeme is blunt, resembling that of M. strobli. In a few cases the spine 2 on the ventral branch of the gonostylus is slender.

Mycetophila ruficollis
Intraspecific variation. The combination of wide 4 th and considerably short 5 th palpal segments, the ventroapical margin of the gonocoxite without any medial projections and the apically abruptly narrowed dorsal branch of the gonostylus are unique among European species of the group.

Mycetophila sepulta
Male terminalia. Posterior margin of gonocoxite with clear convexity ventromedially, and with abrupt and blunt projections laterally. Posterior impression considerably narrow at base but well widening posteriorly. Anterior impression with anteriorly evenly divergent narrow arms. Ventral branch of gonostylus with posterior process narrow, asymmetrical and high, with minute warts; spine 1 very slender and sharply pointed; spine 2 somewhat wider than spine 1, but also pointed and of same length; spines 3 and 4 smaller, pointed and rather close to each other. Dorsal branch of gonostylus slightly tapering, somewhat constricted at the medial bristle; lateral margin without or with very shallow concavity; distal posterior process and proximal posterior process almost of same height, apically rounded, separated by a rather wide notch. Distal posterior process with apical seta, proximal posterior process subapically with 3 setae. Posterior margin proximally from medial bristle with 4-5 gradually diminishing bristles followed by 2-3 setae; internal surface with a stronger seta next to the medial bristle. Basal angle slightly rounded, basal margin with few setae. Ejaculatory apodeme with heart-shaped base and with very narrow or barely visible rim. Aedeagus apically concave. Aedeagal guides rather wide, apically widened, not extending beyond aedeagus distally, lateral impressions wide. Aedeagal apodemes laterally angular.

Mycetophila strobli
Male terminalia. Posterior margin of gonocoxite ventromedially with clear angular convexity, and with abrupt and blunt projections laterally. Posterior impression with narrow base and well widened posterior part. Anterior impression with divergent arms which are sinuate at anterior fourth. Ventral branch of gonostylus with asymmetrical posterior process which bears minute warts and few setae; spine 1 short and sharply pointed; spine 2 blunt, almost twice as high and thick as spine 1; spines 3 and 4 sharply pointed, close to each other and similar in size to spine 1. Ventral branch of gonostylus with a few strong bristles deviating from other setosity laterally on ventral surface. Dorsal branch of gonostylus slightly tapering, lateral margin with shallow concavity; proximal posterior process about three times as high as distal posterior process, both separated with a shallow concavity. Distal posterior process apically and subapically with few setae; proximal posterior process bare and apically rounded. Posterior margin proximally from medial bristle with 4-6 gradually diminishing bristles; internal surface with one somewhat stronger seta next to the medial bristle. Basal angle slightly rounded, basal margin bare. Ejaculatory apodeme with semi-oval base and wide rim. Aedeagus slightly widened apically and with apical margin convex. Aedeagal guides with wide lateral impressions; subapically constricted and apically rounded, not extending upper margin of aedeagus. Aedeagal apodemes laterally angular.
Intraspecific variation. Occasionally the spine 2 on the ventral branch of the gonostylus is somewhat slender and pointed.

Mycetophila suffusala
Male terminalia. Posterior margin of gonocoxite ventrally straight except for diminutive central prominence, which is somewhat sunken into the posterior impression, and with abrupt and blunt projections laterally. Posterior impression wide and uncompressed. Anterior impression with evenly divergent arms anteriorly. Ventral branch of gonostylus with posterior process wide and angular, with minute warts; ventral surface with long and slender bristles deviating from other setosity; spine 1 sharply pointed, about as wide as spine 2; spine 2 geniculate, blunt, about as high as spine 1; spines 3 and 4 smaller, sharply pointed, close to each other. Dorsal branch of gonostylus abruptly narrowed beyond the medial bristle; lateral margin with concavity; distal posterior process very shallow, almost unnoticeable; proximal posterior process high and massive, apically rounded. Distal posterior process with setae, proximal posterior process apically bare. Dorsal surface with an indistinct band of setae from base of posterior processes to basal angle; the setae near basal angle are deviating from other setosity. Posterior margin proximally from medial bristle with 10-13 gradually diminishing bristles; internal surface with a stronger seta next to the medial bristle. Basal angle slightly rounded, basal margin with few setae. Ejaculatory apodeme with rectangular base and without rim. Aedeagus obovoid, with apical concavity. Aedeagal guides wide, apically hooked, extending beyond aedeagus distally, lateral impressions wide. Aedeagal apodemes laterally angular.

Intraspecific variation.
Because of its larger size and details of the maxillary palpus, the species resembles M. ruficollis and by general structure of the male terminalia also M. britannica. However, the dark apical shade on the wing and details of the terminalia allow it to be safely distinguished.
Hosts and distribution. So far recorded only from Madeira and the Canary Islands (Chandler and Ribeiro 1995). Hosts unknown. Zetterstedt, 1852 Figs 14, 31, 32, 49, 50 Material. NORWAY. 2♂♂, Troms, Svensby, 69°40'01,2"N, 019°49'58,8"E, 18.vii. 2008, sweeping, O. Kurina leg. (IZBE0200113, IZBE0200072, in alcohol with terminalia in glycerine, abdomen used for DNA sequence: SJ303, SJ304). FINLAND. 3♂♂, Sodankylä, Paistipuolet, 75°319'15"N, 34°66'98,8"E, 1.vi-29.vi.2009, sweeping, J. Salmela leg. (IZBE0200114, IZBE0200115, IZBE0200116, in  Male terminalia. Posterior margin of gonocoxite with shallow convexity ventromedially, and with blunt and oblique projections laterally. Posterior impression very wide and compressed, with emarginated anterior margin. Anterior impression with anteriorly evenly divergent narrow arms. Ventral branch of gonostylus with asymmetrical, narrow and elongated posterior process with dense and long warts; spine 1 sharply pointed; spine 2 about the same size, pointed; spines 3 and 4 smaller, pointed, closer to each other than to spine 2. Dorsal branch of gonostylus steeply tapering; lateral margin with shallow concavity; distal posterior process and proximal posterior process about the same height, both separated by a deep notch. Distal posterior process with 1-2 apical small setae and one bigger subapical seta; proximal posterior process bare and angular. Posterior margin proximally from medial bristle with 3-4 bigger gradually diminishing bristles followed by 2-3 smaller setae; internal surface with a stronger seta next to the medial bristle. Basal angle almost right-angled; basal margin with few setae. Ejaculatory apodeme with campanulate base and without rim. Aedeagus mostly triangular-shaped, apically widened. Aedeagal guides: 1) with two lamellae, 2) with wide and shallow lateral impressions, and 3) apically rounded, not extending beyond aedeagus distally. Aedeagal apodemes laterally angular.    Intraspecific variation. The wide and compressed posterior impression of the gonocoxite, the distinct warts on the posterior process of the ventral branch of gonostylus and almost equal posterior processes of the dorsal branch of the gonostylus al-low the species to be safely distinguished. In Finnish material, spine 2 on the ventral branch of the gonostylus is more massive than described by Laštovka (1972). In some specimens the base of the ejaculatory apodeme and the lateral margin of the dorsal branch of the gonostylus resemble those of M. ichneumonea. Phylogenetic analysis based on molecular data revealed three different clades (Fig. 4) that cannot be distinguished based on morphology (see also Discussion).

Mycetophila uninotata
Hosts and distribution. Mycetophila uninotata has been reared from species of Collybia, Cortinarius and Lactarius (Yakovlev 1994). Widely distributed in Central and Northern Europe but seems to be absent in the Mediterranean (Chandler 2013). The distribution gap between Central and Eastern Europe (e.g. absence in Poland, Belarus, Ukraine) can be explained by insufficient collecting.

Discussion
This study represents the first evaluation of morphology-based species delimitation of fungus gnats by applying DNA sequence data. Results of the analyses, based on molecular data obtained for seven out of the nine European species from the M. ruficollis group, mostly supported the morphological species delimitation outlined by Laštovka (1972) and Laštovka and Kidd (1975). The genetic distance between members of different species, calculated from the COI barcoding region exceeded 2.9%, with intraspecific distance remaining below 1%. Our results are thus in accordance with inter-and intraspecific variation in different insect orders, documented to be on average over 2% and under 1%, respectively (e.g. Hebert et al. 2004Hebert et al. , 2010. Only in M. uninotata the intraspecific distance was 2.3%, whereas three strongly supported clades were distinguished within this species in the phylogenetic trees. This evidence suggests that the current circumscription of M. uninotata includes two to three cryptic species. However, we were not able to find any morphological differences distinguishing these clades, each of which includes geographically distant material. More samples are needed to evaluate the genetic heterogeneity within M. uninotata. The barcoding region of COI provided a clear barcoding gap for the distinction of all species, except for those in the described subclade of M. ichneumonea and M. uninotata that seems to include several recently differentiated species. In general, both the inter-and intraspecific variation remained lower than observed in other groups of insects (Angélica et al. 2014, Wang et al. 2012, Schwarzfeld and Sperling 2014. The comparison of the three regions sequenced from two genes revealed that the variation in the barcoding region of COI was slightly higher than in the following stretch of comparable length. The latter has been widely used in taxonomic studies on fungus gnats, focusing on higher taxonomic levels (Rindal et al. 2009(Rindal et al. , Ševčík et al. 2013).
Here we showed its applicability also for species discrimination. By contrast, ITS2 rDNA that has become widely used in delimitation of insect taxa (e.g. Rokas et al. 2002, Schwarzfeld and Sperling 2014, Haarto and Ståhls 2014, included much less variation than each of the COI regions. Analyses of ITS2 rDNA data also resulted in lower phylogenetic resolution with some of the species remaining unresolved. Outperformance of COI over ITS2 has been observed also in Ichneumonidae (Schwarzfeld and Sperling 2014) and Mycetophilidae .
Identification of gnats in the M. ruficollis group on the basis of morphological characters is complicated due to considerable intraspecific, yet only limited interspecific variation, mostly observed only upon examination of male genitalia. It was found to be most difficult to distinguish M. strobli and M. uninotata from M. ichneumonea. Also Laštovka (1972) mentioned that M. uninotata is most similar to M. ichneumonea. The phylogenetic analyses revealed close relationship of M. uninotata and M. ichneumonea, with lowest distance between the COI barcodes of their members. The difficulties in identification are obviously caused by high variation within the current concept of M. uninotata, suggested here to comprise cryptic species. COI data revealed M. strobli as more distant, but it appeared indistinguishable from M. ichneumonea based on ITS2 sequences. The morphologically distinct members of geographically separated populations, observed in some species, were not distinguished in molecular analyses. For example, some specimens of M. idonea from Georgia and Poland differed morphologically from their conspecifics but appeared homogeneous in the sequence data. Our results suggest that several of the species in the M. ruficollis group have distinct host ranges. Thus far the larval stages of all European species in the group, except for M. suffusala, had been reported to feed from fruit bodies of a variety of mushrooms (Hackman and Meinander 1979, Yakovlev 1994, Chandler and Ribeiro 1995, Chandler 2010, Ševčík 2010, without preference for any fungal taxa. However, our data support the tendency apparent from literature records suggesting that gnats in the M. ruficollis group do not or only occasionally consume fruit bodies of the Boletales. Our results do not agree with the suggestion by Laštovka (1972) that polytypic M. ichneumonea consists of two to three subspecies. However, taken the difficulties in distinguishing this species from the M. uninotata complex, as reported here, it is possible that previous authors have had a mixture of species under consideration.
Combining morphological and molecular characters for identification of fungus gnats reared from identified fungal fruit bodies provides unique information of host use . Most of the fungus gnats from the M. ruficollis group reared during recent years in Estonia from more than 680 fruit bodies represented M. ichneumonea. While not of frequent occurrence, M. ichneumonea was reared mostly from fruit bodies of two phylogenetically distant genera: Lactarius and Cortinarius. Such 'disjunct host range' is typical for several phytophagous insects (e.g. Bernays andChapman 1994, Janz andNylin 1997) but has so far not been described for mycetophages. Molecular data confirmed the conspecificity of adults reared from these two host genera with no host-related variation observed in the ITS2 or COI sequences. Other species, obtained by rearing of adults, were generally feeding on hosts from other fungal genera. While larvae of M. ruficollis consumed mostly saprotrophic mushrooms, M. idonea was reared from a single fruit body of Amanita. Mycetophila strobli seemed to prefer species from the earliest diverged lineages in the genus Russula, recognised as the subgenus Compactae by most authors. Taken the extensive rearing experiments from diverse mushroom taxa collected from the pine-dominated boreal forests we consider the observed host use patterns to represent fungus gnats' specialisation in this habitat. Apparently, different fungi can serve as (preferred) hosts of members of the M. ruficollis group in other forest types.
Identification of closely related fungus gnats, as reported here in the M. ruficollis group, relies to large extent on a few morphological characters, mostly those of male terminalia. Blurred by intraspecific variation and the lack of such features in females, unambiguous identification is often impossible. Molecular data overcomes these obstacles and should be considered in species delimitation of fungus gnats. For that purpose, we advocate the use of the barcoding region of COI. Special value should be given to sequencing adults reared from identified fungi as these enable to elucidate host as well as geographic range of individual species of fungus gnats.