Corresponding author: Halil Ibrahimi (
Academic editor: R. Holzenthal
In this paper we describe
Ibrahimi H, Vitecek S, Previšić A, Kučinić M, Waringer J, Graf W, Balint M, Keresztes L, Pauls SU (2016)
The genus
The Sharr Mountains represent the border area of three countries, i.e., the Republic of Kosovo, Macedonia, and a small portion extending into north-eastern Albania. This region is characterized by substantial forest ecosystems, diverse geomorphological and hydrological features, and high numbers of endemic and relict species. The name of this mountain range appears in antiquity as “Scardus” “Scordus” or “Scodrus” (
Due to the lack of systematic inventories, biodiversity data for the Sharr Mountains in all three countries are incomplete and are mostly limited to several plant groups, or large mammals. Data for reptiles, amphibians, small mammals, fish, and particularly insects are scarce and outdated (
In an ongoing project on the caddisfly fauna of Kosovo (e.g.,
We collected adult caddisflies with entomological nets and handpicking from the riparian vegetation near the streams, and nocturnal light trapping in the vicinity of the streams. Nocturnal light trapping followed Department of Biology, Faculty of Mathematics and Natural Sciences, University of Prishtina “Hasan Prishtina”, Prishtinë, Republic of Kosovo Croatian Natural History Museum, Zagreb, Croatia (coll. Kučinić- Biologiezentrum des Oberösterreichischen Landesmuseums, Linz, Austria
Morphological characteristics of male terminalia were examined in cleared specimens. Specimens were cleared using either the Qiagen Blood and Tissue Kit for DNA-extraction according to the manufacturer’s recommendation and subsequent KOH-treatment (
Whole genomic DNA was extracted from the abdomen or the thorax of adult or larval specimens using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. Standard PCR procedures and primers were used to amplify three mitochondrial gene regions (mtCOI5-P, mtCOI3-P, 16SrDNA) and three nuclear gene regions (CADH, WG, 28SnrDNA) (Table
PCR primers and PCR cycling conditions.
Fragment | Primers & Primer Concentration | PCR Cycling conditions | Taq Kit | Additional Reagents | |
---|---|---|---|---|---|
mtCOI5-P | HCO2198 & LCO1490 ( |
0.25 µM | 5'95°C, 5 x (30"95°C, 1'44°C, 1'72°C), 15x (30"95°C, 30"48°C, 1'72°C), 20 x (30"95°C, 30"50°C, 1' + (10'’ * n) 72°C) | peqGOLDHotTaq | - |
mtCOI3-P | Jerry & S20 ( |
0.25 µM | 5'95°C, 35 x (45"95°C, 30"45°C, 45"72°C), 5'72°C | peqGOLDHotTaq | - |
16SrDNA | Lepto-F &Lepto-R ( |
0.75 µM | 3'95°C, 35 x (30"95°C, 30"52°C, 40"72°C), 5'72°C | peqGOLDHotTaq | 4 mg BSA |
|
WGbDrrev (5'-accctctcccgcarcacttgag) &WGbDrfwd (5'-cttgctggatgcgtctgcc)1 | 0.5 µM | 5'95°C, 35 x (45"95°C, 45"60°C, 90"72°C), 7'72°C | QiagenHotstarTaq plus Master mix | - |
CADH | 1028r-ino &743nF-ino ( |
0.25 µM | 5'95°C, 35 x (45"95°C, 30"50°C, 45"72°C), 5'72°C | peqGOLDHotTaq | - |
28SnrDNA | D1-3up1 (5'-CGAGTAGCGGCGAGCGAACGGA) & D3-TRIC-DN (5'-ATTCCCCTGACTTCGACCTGA)2 | 0.25µM | 3'95°C, 35 x (45"95°C, 45"60°C, 60"72°C), 5'72°C | peqGOLDHotTaq | 2 mg BSA, 5% DMSO |
1: unpublished primer sequence by M. Bálint 2: unpublished primer sequence by K. Kjer
Sequences were edited in Geneious R6 (
Substitution models used in phylogenetic analysis.
Fragment | unpartitioned | codon position 1 | codon position 2 | codon position 3 |
---|---|---|---|---|
mtCOI5-P | GTR+G+I | TN93+G | TN93+G | HKY |
mtCOI3-P | GTR+G+I | TN93+G+I | K2+G | HKY |
16SrDNA | T92+G | - | - | - |
|
T92+G | T92 | JC+G | JC |
CADH | T92+G+I | HKY+G | TN93 | T92 |
28SnrDNA | T92+G+I | - | - | - |
To examine species delineation and association of morphologically similar species of Western Balkan
Collection data of specimens and length of partial gene sequences used in phylogenetic inference. Abbreviations: Speciment ID, unique study-specific specimen identifier; BOLD ID, BOLD process ID – a unique Barcode of Life Database-specific specimen identifier. Numbers in square parentheses after fragment length indicate number of missing positions. Collectors: – Andela Ćukusić – Ana Previšić – Boštjan Surina – Dejan Dmitrović – Goran Šukalo – Halil Ibrahimi – Iva Mihoci – Mladen Kučinić – Vladimir Krpać – Wolfram Graf
Specimen ID | BOLD ID | 28SnrDNA | COI-5P | CADH | COI-3P | 16Sr-DNA | Wnt1 | Collectors | Coll. date | Latitude (N) | Longitude (E) | Elevation | Taxon |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
fAns0101L |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 360[0n] | 0 |
|
09.vi.2013 |
|
|
1888 |
|
fDar0106M |
|
923[84n] | 658[0n] | 848[0n] | 541[0n] | 360[0n] | 346[0n] | 02.vi.2013 |
|
|
1920 |
|
|
fDar0107M |
|
1040[0n] | 658[0n] | 848[0n] | 541[0n] | 360[0n] | 346[0n] | 02.vi.2013 |
|
|
1920 |
|
|
fDda0204M |
|
1038[0n] | 658[0n] | 0 | 541[0n] | 360[0n] | 346[0n] | 07.vi.2013 |
|
|
980 |
|
|
fDda0208M |
|
1036[2n] | 658[0n] | 848[0n] | 541[0n] | 360[0n] | 346[0n] | 07.vi.2013 |
|
|
980 |
|
|
fDdd0801M |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] |
|
10.vii.2013 |
|
|
960 |
|
fDdd0802F |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] |
|
10.vii.2013 |
|
|
960 |
|
fDds0110M |
|
1038[0n] | 658[0n] | 848[0n] | 474[0n] | 360[0n] | 346[0n] | 29.v.2013 |
|
||||
fDds0111M |
|
1038[0n] | 658[0n] | 848[0n] | 0 | 360[0n] | 346[0n] | 29.v.2013 |
|
||||
fDil0109M |
|
1038[0n] | 658[0n] | 847[1n] | 541[0n] | 360[0n] | 346[0n] | 06.vi.2013 |
|
|
1830 |
|
|
fDju0103M |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] | 04.vi.2013 |
|
|
1175 |
|
|
fDju0104M |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] | 04.vi.2013 |
|
|
1175 |
|
|
fDke0105M |
|
1038[0n] | 658[0n] | 847[1n] | 541[0n] | 362[0n] | 346[0n] | 13.ix.2013 |
|
|
2010 |
|
|
fDke0106M |
|
1036[1n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] | 13.ix.2013 |
|
|
2010 |
|
|
fDkr0101M |
|
1037[1n] | 658[0n] | 848[0n] | 541[0n] | 362[0n] | 346[0n] |
|
30.v.2009 |
|
|
|
|
fDkr0102M |
|
0 | 658[0n] | 0 | 541[0n] | 362[0n] | 346[0n] |
|
30.v.2009 |
|
|
|
|
fDpc0106M |
|
1038[0n] | 658[0n] | 847[1n] | 0 | 360[0n] | 346[0n] | 31.v.2012 |
|
|
1279 |
|
|
fDpe0105M |
|
1038[0n] | 658[0n] | 848[0n] | 541[0n] | 360[0n] | 346[0n] | 28.vii.2012 |
|
|
2300 |
|
|
fDpe0106F |
|
1038[0n] | 658[0n] | 845[3n] | 541[0n] | 327[0n] | 346[0n] | 28.vii.2012 |
|
|
2300 |
|
|
fMelaus0101M |
|
1038[0n] | 658[0n] | 842[6n] | 541[0n] | 361[0n] | 0 |
|
20.x.2013 |
|
|
|
|
fMelaus0102F |
|
1038[0n] | 658[0n] | 843[5n] | 0 | 361[0n] | 0 |
|
20.x.2013 |
|
|
|
|
fDsp4403F |
|
1002[0n] | 658[0n] | 850[0n] | 541[0n] | 360[0n] | 345[0n] |
|
21.v.2014 |
|
|
1558 | |
fDsp4402M |
|
1002[0n] | 454[0n] | 848[2n] | 541[0n] | 360[0n] | 345[0n] |
|
21.v.2014 |
|
|
1558 | |
fDsp4401M |
|
1002[0n] | 658[0n] | 849[1n] | 541[0n] | 360[0n] | 345[0n] |
|
21.v.2014 |
|
|
1558 | |
fDsp4501M |
|
1038[0n] | 658[0n] | 0 | 542[0n] | 362[0n] | 345[0n] | 01.x.2014 |
|
|
393 |
|
|
fDsp4502L |
|
1037[0n] | 658[0n] | 850[0n] | 542[0n] | 362[0n] | 345[0n] | 19.x.2014 |
|
|
456 |
|
To assess potential conflicts or incongruence among gene fragments, B/MCMCMC single gene analyses were conducted in MrBayes 3.2 (
Bayesian inference of the concatenated dataset (mtCOI5-P + mtCOI3-P + 16SrDNA + CADH + WG + 28SnrDNA) was performed in MrBayes 3.2, implementing the respective substitution models. Four parallel runs with twelve chains each were carried out (10×106 generations, sampling every 5000th generation). Analytical parameters were examined as stated above. A majority clade credibility tree was estimated based on trees sampled by MrBayes after discarding the first 600 trees of each run as burn-in.
Republic of Kosovo, Sharr Mountains.
Males of the new species are most similar to
Females of the new species are most similar to
Male genitalia of
Male genitalia of
lateral lobe (
Female genitalia of
Female genitalia of
The species epithet
During our field survey in the Sharr Mountains we found
In a B/MCMCMC phylogeny based on partial sequence data from six loci, monophyly of
Results of phylogenetic inference. B/MCMC species tree analysis for nine
The combination of the gene fragments mtCOI3-P, 16SrDNA, and WG was previously demonstrated to successfully resolve phylogenetic relationships of
Data on the ecology of species closely related to
There are currently about 30 stonefly (
The biodiversity of the Sharr Mountains is threatened by illegal logging, water extraction from springs, expansion of touristic activities and several other anthropogenic factors (
The description of
The fieldwork in Kosovo was partially financed by the Ministry of Education, Science and Technology of the Republic of Kosovo through the project “Identification of rare aquatic insects in some spring areas in Kosovo”, Project holder Halil Ibrahimi, and United Nations Development Program through the project “Conservation of Biodiversity and Sustainable Land Use Management in Dragash”, Project managers Maria Elena Zuniga Barrientos and Halil Ibrahimi. The fieldwork in Albania and molecular analysis were done within the project “The Austrian Science Fund