New species and new records of black fungus gnats (Diptera: Sciaridae) from the Viidumäe Nature Reserve, Estonia

An inventory of Sciaridae (Diptera: Sciaroidea) from a eutrophic fen and a spring brook in Viidumäe Nature Reserve (Estonia, Saaremaa Island) recorded a total of 60 species, of which 57 are new records for Estonia, including two that are new to science and described herein as Cratyna (Diversicratyna) palustricola sp. nov. (Estonia) and Sciara bryophila sp. nov. (Estonia, Finland). This has raised the number of Sciaridae known from Estonia from 6 to 63.


Introduction
Black fungus gnats (Diptera Linnaeus, 1758: Sciaridae Billberg, 1820) are lower Diptera that inhabit most terrestrial habitats, breeding in, for example, decaying wood, leaf detritus and fruiting bodies of fungi (e.g., Menzel & Mohrig 2000;Vilkamaa & Komonen 2001), and some species seem to be confi ned to peat bogs or other moist environments (e.g., Rudzinski 1993;Heller 1998;Salmela & Vilkamaa 2005). In general, the Sciaridae is a very poorly known family, and undescribed species regularly appear in samples from various parts of the Holarctic region (e.g., Hippa et al. 2003Hippa et al. , 2010Vilkamaa et al. 2013a;Wu et al. 2013;Shi et al. 2014;Heller et al. 2015), even from relatively well-studied Central Europe (Menzel et al. 2003a;. A small number of species may be pests in greenhouses or mushroom farms (Menzel et al. 2003b). However, the biology and distribution of most species of the family are insuffi ciently known.

Material and methods
Two study sites were selected in Viidumäe Nature Reserve (Estonia, Saaremaa Island, 58.2966° N, 22.0863° E): a eutrophic fen (Kanna) and a spring brook (Nakimetsa). The Kanna site is an open fen infl uenced by lime deposits, with a moderate fl ow of ground water, and occasional dwarf Scots pines (Pinus sylvestris L.). The fen is surrounded by pine-dominated heath forest. The Nakimetsa site is a calcareous spring brook near the Viidumäe esker. The brook fl ows through a pine forest, almost undisturbed, with occasional broadleaf trees. Spring water values for pH (7.86-8.07) and conductivity (23.5-41.4 mS / cm) indicate lime-rich conditions (T. Talvi, pers. com.).
The material was collected using two Malaise traps at each site. Ethylene glycol was used as a preservative in the traps and the material was fi nally preserved in 70% ethanol. The traps were in situ from mid-April to mid-November 2002, and were emptied at monthly intervals. Sciarids were sorted out from the material and slide-mounted in Euparal. Only males were preserved and studied.
The examined material, including types, is deposited in the following collections: MZH = Zoological Museum, Finnish Museum of Natural History, Helsinki, Finland LMM = Regional Museum of Lapland, Rovaniemi, Finland SDEI = Senckenberg Deutsches Entomologisches Institut, Müncheberg, Germany A 658 bp fragment of cytochrome c oxidase subunit I (COI) was sequenced from two specimens of Sciara bryophila sp. nov. The specimens were placed in 96% ethanol in a 96-well lysis microplate and dispatched to the Canadian Centre for DNA Barcoding, Biodiversity Institute of Ontario, where DNA was extracted by a non-destructive method and sequenced using standard protocols and primers (deWaard et al. 2008). The new sequences are deposited in GenBank under accession numbers KY200864 and KY200865.

Etymology
The name, a noun in apposition, is derived from the Latin words ʻpalustrisʼ (ʻmarshyʼ) and ʻ-colaʼ (ʻinhabitantʼ), referring to the habitat of the species.

Taxonomic remarks
Cratyna palustricola sp. nov. belongs to the subgenus Diversicratyna Menzel & Mohrig, 1998. The new species resembles Cratyna spiculosa (Rudzinski, 1993) and Cr. unispinula  in having a narrow gonostylus with a long apical tooth and a group of apical and / or subapical megasetae (cf. Rudzinski 1993: 286, fi gs 11-14; : 3, fi g. 3a-d). Cratyna palustricola sp. nov. and Cr. unispinula are similar in having the megasetae very narrow (thickened in Cr. spiculosa) and in having the maxillary palpus with three segments (two in Cr. spiculosa). Cratyna palustricola sp. nov. diff ers from Cr. unispinula in having a broader gonostylus, with its subapical megasetae more numerous (4-7 vs 2) and the basalmost ones placed nearly at the middle of the gonostylus. Furthermore, Cr. palustricola sp. nov. has a modifi ed long seta on the dorsal side of the gonostylus, not a megaseta as Cr. unispinula sometimes does (see Menzel & Mohrig 2000).
By its gonostylus, Cratyna (Diversicratyna) palustricola sp. nov. resembles Corynoptera salmelai Vilkamaa, Hippa & Heller, 2013 and Corynoptera spiciforceps Vilkamaa, Hippa & Heller, 2013. Cratyna palustricola is similar to C. spiciforceps in having the gonostylus evenly narrowed towards apex, whereas C. salmelai has it bulged medially. Cratyna palustricola is similar to C. spiciforceps and diff ers from C. salmelai in having the tegmen rounded apically and in having a sensory pit on the 1 st palpal segment, whereas C. salmelai has sharp apicolateral teeth on its tegmen and the 1 st palpal segment with a patch of sensilla. Furthemore, C. spiciforceps has an elongated seta medially on its gonostylus. Cratyna (Diversicratyna) palustricola sp. nov. diff ers from both species of Corynoptera in having 2 megasetae, not normal setae, on the dorsal side of the apical tooth of the gonostylus, confi rming its generic placement.

Etymology
The name, a Latin adjective, is derived from the Latinized Greek words ʻbryonʼ (ʻmossʼ) and ʻphiloʼ (ʻlovingʼ), referring to the mossy habitat of the species.
L . Pale brown, coxae darker. Coxal setae dark. Fore tibial organ with pale and fi ne vestiture forming large subtriangular patch. Fore tibial spur as long as tibial width. Hind tibia without spinose setae. Claws without teeth.

Taxonomic remarks
Sciara bryophila sp. nov. belongs to the Sciara humeralis group in the sense of Menzel & Mohrig (2000). These have a basically triangular gonostylus, strongly impressed and narrowed towards the apex, with strong megasetae (spines) which in some species are arranged in a long and narrow apical group on a common basal projection, and a basal, densely setose lobe. Although there is some variation between the specimens of Sciara bryophila sp. nov. from diff erent localities, for example, the specimens from northern Finland have stronger gonostylar megasetae than the central Finnish and Estonian specimens, we regard all as conspecifi c.
Sciara bryophila sp. nov. is similar to S. multispinulosa Mohrig & Kozánek, 1992 -described from North Korea in Mohrig et al. (1992: 19, fi g. 1a-c) and redescribed by Sutou et al. (2004: 185, fi g. 6a-b) on the basis of Japanese material -and S. kitakamiensis Sutou, 2004 described from Japan in Sutou et al. (2004: 186, fi g 7a-d). The mentioned three species have the gonostylus dorsally strongly impressed with a dorsobasal short lobe and a stronger ventral lobe with short, curved spine-like setae and the apex with strong megasetae, two of which are placed on a common basal body dorsally in the subapical part. Sciara bryophila sp. nov. is similar to S. kitakamiensis in having the ventral lobe of the gonostylus in the apical half of the gonostylus, and the gonostylus with only 9 strong megasetae, whereas S. multispinulosa has the ventral lobe at the middle of the gonostylus, and the gonostylus with 15-18 weak megasetae. Sciara bryophila sp. nov. is similar to S. multispinulosa and diff ers from S. kitakamiensis in having its gonostylus ventrally more impressed, its dorsal lobe more prominent and the dorsosubapical pair of setae much longer than their common base. Furthermore, S. bryophila sp. nov. diff ers from S. kitakamiensis in having the apical megasetae of the gonostylus separate, not placed in a common long basal projection, and in having the ventral lobe less apical in position and in having a few megasetae on the apical side of the ventral lobe (S. kitakamiensis has the ventral lobe nearly apical in position and has no megasetae on its dorsal side).

DNA analyses
In the BOLD (Ratnasingham & Hebert 2007) database, the present barcodes cluster in a unique BIN (Barcode Index Number, BOLD:ADD2461), shared by no other specimens. The closest specimen in BOLD is 3.29% distant (K2P) from the sequenced type specimens, and the closest BIN (BOLD:ADD2588) is mainly composed of specimens belonging to S. humeralis Zetterstedt, 1851.

Ecology and distribution
The new species occurs in rich fens, that is, in peatlands dominated by brown mosses and characterised by pH values around 7 or even above. In Finland the species occurs from south boreal to north boreal zones, but in South Finland it is known only from one site (Toivakka). It is assumed that S. bryophila sp. nov. is a rare or relict-like species in the hemiboreal and south boreal zones, being more common in mid and north boreal zones, especially in areas with limestone or calcareous bedrock. There are no records from the subalpine zone and the species may be absent from the northernmost parts of Fennoscandia.

Faunistics
A relatively high number of new species and species new for the Estonian fauna were recorded from a rather small number of studied specimens (n = 225). Corynoptera saetistyla (24 specimens), C. verrucifera (Lengersdorf, 1952) (17 specimens), Cratyna nobilis (Winnertz, 1867) (17 specimens), Camptochaeta camptochaeta (Tuomikoski, 1960) (13 specimens) and Corynoptera postforcipata Rudzinski, 1993 (12 specimens) were most numerous. Most of the detected sciarid species are known from Central and / or Northern Europe, while many of them are common and widespread in the Palaearctic region. The sciarid species recorded in the Viidumäe Nature Reserve from April to November 2002 with four Malaise traps are listed in Table 1.

Discussion
A total of 225 individuals belonging to 60 species were identifi ed, of which Cratyna palustricola sp. nov. and Sciara bryophila sp. nov. are new to science. Of the 60 species collected in the Viidumäe Nature Reserve, only three were already known from other localities in Estonia: Corynoptera saetistyla, Corynoptera trepida and Cratyna uliginosa. Thus, 57 sciarid species are here recorded for the fi rst time from Estonia, including the two previously undescribed species (Table 1). The previously identifi ed species Chaetosciara estlandica, Leptosciarella brevipalpa and Dolichosciara nigrovittata were not found in our study, which may be due to the habitat structure of the collected areas and / or the trapping method used. Consequently, 63 sciarid species are currently known from Estonia. They are spread over 19 genera as follows: Bradysia Winnertz, 1867 (7 species); Camptochaeta Hippa & Vilkamaa, 1994 (3) Faunistically signifi cant are Camptochaeta sicilicula Hippa & Vilkamaa, 1994, Corynoptera marinae Mohrig & Mamaev, 1986, Corynoptera subtetrachaeta Komarova, 1995 and Dolichosciara saetosa (Lengersdorf, 1929). Only one individual of each of these rare species was captured in the Viidumäe Nature Reserve during an entire vegetation period. Furthermore, only a few specimens of these four species exist in collections, and they have been found in very few places in Europe.
Our results have revealed that the sciarid fauna of Estonia is very poorly known so far. The new faunistic records clearly refl ect the lack of previous studies rather than a large sampling eff ort in the present study. This is supported by the comparison of the previously known species inventory of the Baltic States with that of the Fennoscandian countries (Table 2). Currently only 63 species are known from Estonia, 28 from Latvia and 22 from Lithuania. The Fennoscandian fauna is much better studied (Norway = 143 species; Sweden = 299 species; Finland = 370 species). About 450 to 500 sciarid species are expected for the fauna of Fennoscandia and at least 300 for the Baltic States.  (Hippa & Vilkamaa, 1994) * 1 2 Corynoptera verrucifera (Lengersdorf, 1952) * 12 5 Cratyna (Cra.) uliginosa (Lengersdorf, 1929) 1 Cratyna (Div.) spiculosa (Rudzinski, 1993  The high number of identifi ed species, the two newly described species and the high proportion of species that were newly recorded for the Estonian fauna show that even relatively small fi eld studies can make a major contribution to biodiversity research. However, the discovery of new and faunistically interesting species shows also that mires, eutrophic fens and spring water-infl uenced habitats may provide new insights into sciarid ecology and taxonomy, and that they are worth protecting.   (2010) Latvia 10 28 Mohrig et al. (1985); Menzel (1992); Mohrig (1993); Mohrig & Menzel (1993, 1994; ;