New and poorly known Holarctic species of Boletina Staeger, 1840 (Diptera, Mycetophilidae)

Abstract Background The genus Boletina is a species rich group of fungus gnats. Members of the genus are mainly known from temperate, boreal and arctic biomes. Phylogeny of the genus is still poorly resolved, dozens of species are insufficiently described and undescribed species are often discovered, especially from samples taken from the boreal zone. New information Four new species are described. Boletina valteri Salmela sp.n. (Finland), Boletina kullervoi Salmela sp.n. (Finland), B. hyperborea Salmela sp.n. (Finland, Norway, Sweden, Canada) and B. nuortti Salmela sp.n. (Finland). Boletina arctica Holmgren is redescribed and reported for the first time from the Canadian high arctic zone. Boletina borealis Zetterstedt and B. birulai Lundström are reported for the first time from Canada. Boletina subnitidula Sasakawa (syn. n.) is proposed as a junior synonym of B. pallidula Edwards.


Introduction
The genus Boletina Staeger, 1840 (Staeger 1840) belongs to the family Mycetophilidae and its subfamily Gnoristinae (Väisänen 1986, Rindal et al. 2009, or Gnoristini sensu Edwards 1925, Söli 1997. There are 137 recent Boletina species, of which 99 are Palaearctic, 11 Oriental and 34 Nearctic. Only seven species have a Holarctic range and 74 of the Palaearctic species are present in Europe (Suppl. material 1). It should be noted that the numbers presented above are expected to increase, as several Fennoscandian species await description ( Fig. 1) and in the Nearctic region the genus is very poorly known (Taber 2011, Taber 2013. Furthermore, 31 (23 %) out of all described species either are known only from female specimens or their descriptions are too deficient for an understanding of their morphology and thus allowing positive identification of the taxa (Suppl. material 1). In Europe the genus seems to have an increasing number of species toward higher latitudes, that is, there are more species in Fennoscandia than in either Central Europe or the Mediterranean region (Fig. 1). The boreal zone may harbor the highest number of Boletina species, but several species are also known to occur on the subarctic and Arctic zones (Lundström 1915, Zaitzev 1994, Polevoi 2013. It should be noted, however, that the number of conducted faunistic studies is higher in Fennoscandia than in southern Europe. Furthermore, some Boletina species display boreo-mountainous distribution pattern (see e.g. Kurina 2008), being absent from lowland areas; these both factors may obscure the latitudinal pattern presented here.
Species of the genus can be distinguished by the following morphological characteristics: wing membrane without macrotrichia; mediotergite bare; laterotergite haired or bare; Sc ending in C; Sc2 present or absent; R4 absent; Sc ending in C beyond level of base of crossvein ta; mouthparts shorter than height of head (modified from Söli et al. 2000, see also Saigusa 1968). In addition the male hypopygium has a pair of ventral lobes, here termed as sternal submedian appendages of gonocoxites, that have great value in species identification. Gonostyli may consist of one (e.g. all species described here) or two lobes (e.g. Boletina dubia (Meigen), B. landrocki Edwards, B. polaris Lundström). Among species with a single lobe of gonostylus, the gonostylus mostly has a setose and bulbose basal part (here termed as outer branch) and a narrower glabrous branch bearing a few strong apical spines or setae (inner branch). The cercus is conspicuous, bearing characteristic combs (i.e. stout darkened setae arranged in more or less regular rows or scattered on the dorsal surface). The aedaegal complex is more or less firmly attached to the sternal submedian appendage of gonocoxites via lateral branches or ventrodistal sclerites of the aedeagus (Sasakawa and Kimura 1974). Parameres are highly varied and mostly symmetrical (asymmetrical only in B. cornuta Zaitzev). However, the genus is paraphyletic (Martinsson et al. 2011), and monophyletic groups within the genus may deserve generic ranks of their own (see e.g. . Immature stages of the genus are extremely poorly known. In fact, notes on larval stages or rearing records are available for only nine species (Suppl. material 2), all of which are European species. Based on these records, the species are associated with decaying wood, soil, leaf litter and liverworts; interestingly only one species has been once reared from fungi (Suillus bovinus, Boletales) (Suppl. material 2). However, some of these records may represent pupation places, not real larval microhabitats (Martinsson et al. 2011). In the boreal zone, Boletina may be one of the most abundant groups of fungus gnats in entomological samples collected from forests (J. Salmela, pers.obs.). However, Boletina may also be abundant in treeless habitats such as mountains and mires (Edwards 1925, Jakovlev et al. 2014).
In the present paper we describe four newWe also tried to extract Boletina species (three from Finland, one from Fennoscandia and Canada). In addition, one new synonymous name is proposed and one species is reported for the first time from the Nearctic region.  Ševčík and Papp 2002, Parvu 2004, Schumann 2004, Kurina 2008, Ševčík and Vonička 2008, Bechev 2010 number is based on Kjaerandsen 2015, and unpublished records of 15 undescribed species (Kjaerandsen, Polevoi, Salmela & Söli, in prep.). The adjacent European countries were combined in order to reduce variation in their geographical areas. Latitudes represent rough midpoints of the respective groups of countries.

Materials and methods
All studied Fennoscandian specimens were obtained from Malaise trap samples and are stored in 70 % ethanol. Material deposited in CNC is dry and pinned. The morphological terminology used here mainly follows Söli 1997. Terminology of some special parts of male genitalia is explained in the figures. The following acronyms for museums and collections are used in the text: ZMUT -Zoological Museum, University of Turku, Turku, Finland; CNC -Canadian National Collection of Insects, Arachnids and Nematodes, Ottawa, Canada; JES -Private collection of Jukka Salmela, Rovaniemi, Finland; TZS -Tromsø Museum, The Arctic University of Norway, Tromsø, Norway. All specimens in JES and ZMUT are stored in 70 % ethanol, kept in 2 ml plastic vials with a screw cap and a rubber o-ring seal. Hypopygia of some specimens are kept in separate 0,5 ml microvials in glycerol. We also tried to extract DNA barcode (see e.g. Mutanen et al. 2012), as most of our specimens were stored in ethanol. These were sent to the Biodiversity Institute of Ontario, University of Guelph, Canada, for DNA extraction and sequencing. Unfortunately our specimens yielded either no DNA or resulting sequences (n=2) were most likely contaminated. These two COI mtDNA fragments showed affinities to taxa that are quite unrelated to the species that they were supposed to represent, and the results are thus ignored here.

Diagnosis
A small Boletina species with unpatterned wings and a monochromatic abdomen. The cercus has three rows of stout setae, and the parameres are long and thin. The lobe of gonostylus is pointed. The new species is externally somewhat similar to B. pallidula Edwards, but that species has two rows of setae on cercus and shorter inner branch of gonostylus.

Etymology
The species is named after Valter Keltikangas , Finnish forest researcher and professor. In his famous novel "Seitsemän tuntia erämaata" (1977) he recollected his expedition to the vast mires and forests of Pomokaira, the type locality of the new species. The name of the new species is a genitive.

Distribution
European, so far only known from central Finnish Lapland.

Ecology
The type locality is a Salix swamp with meso-eutrophic groundwater seepages, surrounded by an old-growth boreal forest, dominated by spruce (Picea abies ssp. obovata), birch (Betula sp.), and bilberry (Vaccinium myrtillus) on the ground layer.

Taxon discussion
The new species seems to be quite isolated from other known species of the genus. It has relatively short antennal flagellomeres (length:width ratio of the 2nd flagellomere is 2), long and straight parameres and inconspicuous parameral apodemes. The parameres are also moveable in their bases. The basal, paired projections of the aedeagal complex are here considered as ejaculatory apodemes. Even more striking is the presence of an accessory structure lying close to sternal submedian appendages of gonocoxites. This structure is perhaps a part of aedeagal complex, a highly specialized organ derived from ventrodistal sclerites of aedeagus. The new species is somewhat similar to B. pallidula, but in addition to the diffrences in male genitalia given above, B. pallidula has lighter body coloration, slightly longer flagellomeres and is lacking vein Sc2.

Diagnosis
Medium-sized Boletina which is externally similar to B. borealis Zetterstedt, wings unpatterned. The new species has small, paired ventrodistal sclerites on the aedeagus, whereas B. borealis has larger sclerites. Parameres are short and widely separated, parameral apodemes having distinct lateral lobes.

Etymology
The species is named after Kullervo, the son of Kalervo. Kullervo is a tragic character in Kalevala, the Finnish national epic. Kullervo is also the first symphony composed by Jean Sibelius (1892). The name of the new species is a genitive.

Distribution
European, so far only known from eastern Finnish Lapland.

Ecology
Both collecting sites are pristine boreal forests. The collecting site in Törmäoja was a stream valley with lush vegetation, groundwater seepages and coniferous forest on the valley slopes. At Värriö the habitat was similar with the valley surrounded by sparse pine and spruce forest.

Taxon discussion
The new species appears to be closest to B. borealis and related species, including B.

Notes
Boletina kullervoi sp.n. is characterized by the arching apices of the sternal submedian appendages of the gonocoxites. In ventral view the sternal submedian appendages appear to be truncated and slightly oblique, but in reality the apices are club-like, firmly attached to aedeagal complex. If one is trying to remove the aedeagal complex from the hypopygium, apices of sternal submedian appendages remain attached to the aedeagal complex. The ventrodistal sclerites of the ejaculatory apodeme are here termed as paired, because there are short apical and basal projections. However, further morphological study is needed to verify whether these appendages are homologous to similar structures among species such as B. borealis and B. hyperborea sp.n.

Etymology
Hyperborea (an noun in apposition) is taken from the Greek mythology, meaning the far north.

Distribution
Northern parts of Fennoscandia (Finland, Norway, Sweden) and Canada. In Fennoscandia the species have been observed from the northern boreal zone and from the subarctic ecoregion. In Canada it has been observed from the Ogilvie Mountains in the Yukon territory, which is in the subarctic ecoregion.

Ecology
The collecting sites are boreal forests and alpine wetlands. Joutenoja and Värriö are headwater streams, characterized by groundwater seepages and surrounded by coniferous forests. Toskaljärvi is an oroarctic, alpine sloping rich-fen, dominated by brown mosses.  (Fig. 9b). Sternal submedian appendages of gonocoxites apically rounded, short and separated by a Y-shaped cleft (Fig. 9a). Gonostylus appearing unibranched, but is divided on inner and outer branches (Fig. 10a, b). Outer branch rather inconspicuous, bearing thin and rather short mesal hairs. Inner branch short, mostly glabrous, with a few small subapical setae, two or three black apical spines and a longer hyaline seta. Aedeagal complex rather short and wide (Fig. 10c, d, e). Parameres monochromatic, short and stout, tapering apically, their apices not exceeding tip of aedeagus (Fig. 10d). Parameral apodemes very wide in lateral view (Fig. 10e). Ejaculatory apodeme consisting of two short and wide basal lobes. Aedeagus with short ventrodistal sclerites, apex of aedeagus down-curved, coated by a hyaline membrane (Fig. 10c, e). a b Figure 9.

Diagnosis
A medium-sized Boletina species similar to B. silvatica Dziedzicki, amongst other species. Apices of the sternal submedian appendages are small, rounded and separated by a Y-shaped cleft. The new species is lacking dorsal projections on the gonostylus, as typical for species such as B. silvatica, B. subtriangularis Polevoi & Hedmark and B. triangularis Polevoi. Parameres of the new species are also distinctly shorter than those of the three above mentioned species. Wings unpatterned.

Etymology
The name of the new species is derived from the River Nuortti, a large tributary of the River Tuuloma. Nuortti is derived from north Sami word (nuorti), meaning east; the River Nuortti flows in a NE direction from Finland to Russia. The name of the new species is a noun in apposition.

Distribution
European, so far only known from Törmäoja conservation area, eastern Finnish Lapland.

Ecology
The type locality is a birch dominated boreal forest, bilberry and cowberry (Vaccinium vitis-idaea) on the ground layer.

Taxon discussion
The new species is reminiscent of B. silvatica and related species, especially the shape of the aedeagus which indicates an affinity to B. nuortti sp.n. within this species group. However, species related to B. silvatica have a very deep sternal cleft of gonocoxites, whereas the cleft in B. nuortti sp.n. is not exceeding the half length of the gonocoxites. In addition, B. silvatica and related species possess a dorsal projection on gonostylus, a character absent on B. nuortti sp.n. Thirdly, parameres of the new species are relatively short; parameres of B. silvatica and related species greatly exceed the length of aedeagus (see e.g. Polevoi and Hedmark 2004).  (Fig. 11c). Gonocoxite apicomesally with two black spines (Fig. 11a). Inner branch of gonostylus pollex-like, bearing two long black apical setae and a black spine. Outer branch of gonostylus rectangular, widest subapically, with a modest apical peak (Fig. 11a, b). Mesal edge of gonostylus hairy. Parameres short and straight (Fig. 11c). Parameral apodemes + parameres resembling the letter L in lateral view (Fig. 11d). Aedeagus short and curved (Fig. 11d). Ejaculatory apodeme with basal projections. Gonocoxite internally with a narrow, pointed projection ( Fig. 11a). Close to this projection are two hyaline projections, bearing weak bristles; this latter projection is part of aedeagal complex.

Distribution
Boletina arctica has been recorded from Greenland (Holmgren 1872, Söli et al. 2015, Arctic Russia (Zaitzev 1994) and Akpatok Island, Hudson Strait, low arctic zone of Canada (Edwards 1933). Here we report the species from the high arctic zone, Axel Heiberg Island, Canada. Given the rarity of this species, B. arctica has been illustrated rather frequently in taxonomic literature. The first to illustrate this species was Rübsaamen (Rübsaamen 1898), but instead of B. arctica, he actually figured B. digitata Lundström (or some related species of this complex, see Suppl. material 1). Johannsen (Johannsen 1911) copied Rübsaamen's illustration of B. arctica and gave no additional occurrence data. Later Lundström (Lundström 1912) provided good figures, based on material loaned from ZMUC, Copenhagen, originally collected from Greenland. It is not clear whether a type specimen was examined, but most likely it was a non-type male, since the type material of B. arctica is not deposited in ZMUC ( http://www.zmuc.dk/EntoWeb/ collections-databaser/Diptera/Mycetophilidae%20all.htm). Hence, authors after Lundström have followed his interpretation of B. arctica. Edwards (Edwards 1933) was actually the first to report this species from North America (Akpatok Island), he illustrated Boletina arctica var. According to Edwards, his specimens were slightly different from a male specimen collected from Greenland. However, such differences on the outline of the gonostylus are due to aspect of the gonostylus to the viewer (Fig.  11b). Boletina arctica was also illustrated by Zaitzev (Zaitzev 1994) and recently by Söli et al. (Söli et al. 2015).
The species possesses some unique characters that have been misinterpreted in the literature. First of all, B. arctica has no sharply pointed parameres (cf. Edwards 1933, p. 612). Lundström, Edwards and Zaitzev have illustrated these sharply-pointed projections that actually stem from the inner lateral margin of the gonocoxite. Furthermore, close to these projections lie two spinose, hyaline projections that orginate from the aedeagal complex. The parameres themselves are inconspicuous, short and straight.

Distribution
Holarctic, here reported for the first time from Canada. The species was described from the arctic coast of Russia (New Siberian Islands and around Taimur, Lundström 1915), and was later rediscovered from arctic Russia (Dickson, New Siberian Islands, Zaitzev 1994). The species is also reported from Poland (Pommern, Landrock 1940) and Germany (Bavaria, Plassmann and Schacht 1999) and from Alaska in the USA (Hackman et al. 1988). However, the record from Germany is erroneous (misidentification, the male specimen is an undescribed species close to B. nigricoxa Staeger and B. cincticornis (Walker), det. J. Salmela) and the Polish record should also be re-examined and verified. Furthermore, the record of B. birulai larvae from Port Barrow, Alaska originally implicated in causing human myiasis to an entomologist (Casterline 1954), should be treated with caution. Larva of B. birulai has not been described and it remains unclear whether B. birulai or some other species was involved.

Taxon discussion
Boletina pallidula was described by Edwards (Edwards 1925) from the British Isles, and the male hypopygium has been later illustrated by Hutson et al. 1980 andZaitzev 1994. The species has a European range, including Fennoscandia, northern Russia, Central Europe, Bulgaria and Hungary (Chandler 2014). Sasakawa (Sasakawa 1994) described B. subnitidula from the Chiba prefekture in Japan based on material collected from Arisema serratum flowers. Sasakawa's description is very detailed, including illustrations on the male hypopygium, and hence we propose it as a junior synonym of B. pallidula. In his discussion on B. subnitidula Sasakawa (Sasakawa 1994, p