Corresponding author: Olavi Kurina (
Academic editor: Vladimir Blagoderov
Both males and females of
Since the introduction of the ‘DNA barcoding’ approach (
The current study was initiated by finding both sexes of
All
The material has been deposited in IZBE (Institute of Agricultural and Environmental Science, Estonian University of Life Sciences, former Institute of Zoology and Botany) and all specimen data have been inserted into the database of Estonian animal collections (
The genomic DNA was extracted using a High Pure PCR Template Preparation Kit (Roche Diagnostics GmbH, Mannheim, Germany). Anterior segments of the abdomen that had been stored after genitalia dissection were crushed and used for the extraction. This process was carried out following the manufacturer’s instructions for extraction of genetic material from mammalian tissue.
A 762-bp fragment of cytochrome C oxidase subunit 1 (
In total, 9 specimens including three species of fungus gnats from the subfamily
Consensus sequences were created with the program Consed (
Bayesian phylogenetic analysis implementing the GTR+I model was performed using MrBayes 3.1 (
For determination of male material of
All specimens identified preliminarily as
Among the species used for phylogenetic analysis also
Details of specimens used for taxonomic study and molecular analysis
Voucher No | Species | Sex | Collecting site, collecting method and collector | Date | Method of preservation | GenBank acc. code for COI |
---|---|---|---|---|---|---|
IZBE0200002 | ♂ | Estonia, Palupõhja |
25.vii – 4. viii 2009 | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body dry mounted from ethanol | JN007851 | |
IZBE0200003 | ♀ | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body dry mounted from ethanol | JN007851 | |||
IZBE0200004 | ♂ | Greece, Central Macedonia, Kerkini lakes area, village Neo Petritsi, Sultanitsa site, |
12 – 18.v 2008 | In ethanol | ||
IZBE0200005 | ♂ | In ethanol | ||||
IZBE0200006 | ♀ | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body in ethanol | JN007850 | |||
IZBE0200007 | ♂ | 19 – 25. v 2008 | Abdomen used for DNA sequencing; rest of body and terminalia slide mounted | JN007850 | ||
IZBE0200008 | ♂ | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body in ethanol | JN007850 | |||
IZBE0200009 | ♂ | Slide mounted under 5 different coverslips | ||||
IZBE0200010 | ♂ | In ethanol | ||||
IZBE0200011 | ♂ | In ethanol | ||||
IZBE0200012 | ♂ | 25.v – 1. vi 2008 | Abdomen used for DNA sequencing; rest of body and terminalia slide mounted | JN007850 | ||
IZBE0200013 | ♂ | In ethanol | ||||
IZBE0200014 | ♀ | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body slide mounted | JN007850 | |||
IZBE0200015 | ♂ | Greece, Central Macedonia, Kerkini lakes area, village Neo Petritsi, Farfara site, |
16 – 22. vi 2008 | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body dry mounted from ethanol | JN007853 | |
IZBE0200016 | ♀ | Abdomen used for DNA sequencing; terminalia in glycerin; rest of body dry mounted from ethanol | JN007852 |
9♂♂ 2♀♀, for collecting data see
Female (
Length of body 4.65 – 4.94 mm (n=2).
Head brown with dark setae. Three equally sized ocelli in a triangular arrangement. Clypeus subrounded. Palpus with 5 light brown setose segments with ratios of 1: 1.17:1.58: 2.25:4.33. Mouthparts brownish. Antenna with 2+14 segments. Scape, pedicel and base of first flagellar segment light brown, rest of flagellomeres brown. Scape with sparse setae including dorsoapicals extending to middle of pedicel. Pedicel with sparse and all flagellomeres with dense setae. First flagellomere 3 times as long as wide, succeeding segments gradually shorter. Apical flagellomere cylindrical, about three times as long as wide.
Thorax brown. Scutum covered with pale setae including long lateral hairs. Lateral parts of thorax slightly paler than scutum. Antepronotum with numerous long pale hairs. Proepisternum with numerous shorter setae. Laterotergite and mediotergite with upward directed hairs. Anepimeron and metepisternum with short setae, anepisternum bare. Scutellum with setae not in distinct pairs.
Legs. Fore coxa light brown with hind margin and apical fourth yellow. Mid and hind coxae brown, apically slightly paler. All trochanters brown. All femora and tibiae yellow with apical brown markings. All tarsi dark brown. Tibiae with irregularly arranged setae but without distinct bristles. Fore tibia with a spur 0.18 of basitarsus length. Mid and hind tibiae both with two equal spurs, 0.19 and 0.21 of basitarsus length, respectively. Ratio of femur to tibia for fore, mid and hind legs: 1.00; 0.82; 0.81. Ratio of tibia to basitarsus for fore, mid and hind legs: 1.15; 1.57; 1.86.
Wing hyaline. Length of wing 4.00 – 4.23 mm (n=2). Ratio of length to width 2.83. Veins light brown, setose on both surfaces. Wing membrane with dense irregularly arranged microtrichia and with few macrotrichia in anal area and close to wing tip below of R1 and R5. C not produced beyond apex of R5, which is strongly sinuate. Sc ends in C at the level of beginning of medial fork. Sc2 situated at the level of middle of bM-Cu. M1basally obsolete. Cubital fork begins slightly before the base of r-m. Haltere pale with brownish knob. Both, stem and knob with short setae.
Abdomen brown with segments I–IV lighter. Terminalia (
Male (
Length of body 5.36 – 6.32, 5.65 mm (n=5). Length of wing 4.37 – 5.30, 4.76 mm (n=5), ratio of length to width 2.55 – 2.82, 2.64 (n=5). Coloration and other non-terminal characters similar to female except anepisternum, which has short setae on upper part. Terminalia brown. Gonostylus simple, without any additional lobes or spines, slender, tapering, curved medially and covered with short setae.
Bayesian phylogenetic tree (GTR+I model) of selected
The study was funded by Estonian Science Foundation grants 7558 and 8583, targeted financing projects SF0170160s08 and SF0180122s08, and by the European Union through the European Regional Development Fund (Center of Excellence FIBIR). OK is grateful to the European Commission’s Research Infrastructure for funding the study visit to MNHN via the SYNTHESYS programme (FR-TAF-5005) and is much obliged to the curator Dr. Christophe Daugeron (MNHN) for the opportunity to work with the collections in Paris. We thank Tuuli Reisberg (Estonian Biocentre, Tartu, Estonia) for technical assistance with sequencing. Peter Chandler (Melkshamn, United Kingdom) and Peter Kerr (Sacramento, USA) are acknowledged for their comments and suggestions to the manuscript.