Biodiversity Data Journal :
Research Article
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Corresponding author: Vladimir Pešić (vladopesic@gmail.com)
Academic editor: Fernando Jacinavicius
Received: 22 Nov 2021 | Accepted: 16 Dec 2021 | Published: 20 Dec 2021
© 2021 Vladimir Pešić, Andrzej Zawal, Ana Manović, Aleksandra Bańkowska, Milica Jovanović
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pešić V, Zawal A, Manović A, Bańkowska A, Jovanović M (2021) A DNA barcode library for the water mites of Montenegro. Biodiversity Data Journal 9: e78311. https://doi.org/10.3897/BDJ.9.e78311
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Water mites (Acari, Hydrachnidia) are a significant component of freshwater ecosystems inhabiting a wide range of aquatic habitats. This study provides a first comprehensive DNA barcode library for the water mites of Montenegro. DNA barcodes were analysed from 233 specimens of water mites morphologically assigned to 86 species from 28 genera and 15 families. In the course of the study, four species, i.e. Lebertia reticulata (Koenike, 1919), Atractides inflatipalpis K.Viets, 1950, A. latipes (Szalay, 1935) and Parabrachypoda montii (Maglio, 1924) were molecularly confirmed as new for Montenegro and three species, i.e. Protzia octopora Lundblad, 1954, Piona laminata (Thor, 1901) and Unionicola ypsilophora (Bonz, 1783) are new for the Balkan Peninsula. Results are analysed using the Barcode Index Number system (BIN) and the Refined Single Linkage (RESL) of BOLD. The BIN assigned sequences to 98 clusters, while the RESL reveal 103 operational taxonomic units (OTUs). Unique BINs were revealed for 72 species (83.7%), whereas twelve species (14%) were characterised by two BINs and two species (2.3%) with three BINs. Amongst the studied taxa, 14 species were found with a high intraspecific sequence divergences (˃ 2.2%), emphasising the need for additional comprehensive morphological and molecular analysis of these species.
DNA barcoding, COI, water mites, Montenegro, species delimitation
Hydrachnidia, also known as water mites, is a most diverse and abundant group of arachnids in freshwater habitats (
Traditional morphology often underestimates the true diversity of water mites and, in recent years, it has been successfully replaced by an integrative approach that combines both morphological characteristics and molecular data (
Of the Balkan countries, Montenegro is one of the best studied from the taxonomic and faunistic point of view (
Currently, 201 species of water mites have been reported for Montenegro (
The aim of the study is to develop and evaluate the first library of barcodes for water mites from Montenegro, targeting a COI fragment of ~ 658 bp. Taking advantage of publicly available DNA barcode reference libraries, such as the BOLD and the use of the universal Barcode Index Number (BIN), allows us to assess the molecular diversity of water mite species inhabiting the territory of Montenegro, as well as to explore their distribution patterns in Europe. Moreover, this approach will allow us to also identify problematic species groups both for traditional taxonomy and for DNA barcoding.
Water mites were collected by hand netting, sorted live in the field and immediately preserved in 96% ethanol (EtOH) for the molecular analysis. Water mites were collected from 54 sampling sites in Montenegro (Fig.
Molecular analysis were conducted in the Canadian Centre for DNA Barcoding (Guelph, Ontario, Canada; (CCDB; http://ccdb.ca/) and in the Department of Invertebrate Zoology and Hydrobiology (DIZH), University of Łódź, Poland. For the methods used for cytochrome c oxidase subunit I (COI) gene amplification in DIZH, see
In CCDB, the chromatograms were assembled into consensus sequences for each specimen and uploaded to BOLD. The taxonomic account, voucher specimen ID, collecting locality and voucher depositor were incorporated into the system for further analysis. Water mite sequences, obtained during this study, were grouped in the “MNHYD” (DNA barcode reference library of Montenegrin water mites) dataset. Detailed voucher information, taxonomic classifications, photos, DNA barcode sequences, primer pairs used and trace files (including their quality) were uploaded to the dataset “MNHYD” on the Barcode of Life Data Systems (BOLD; www.boldsystems.org).
The translation of the COI sequences into amino acids did not contain any stop codon positions and blasting the sequences confirmed the absence of contaminations. In cases of the four Unionicola ypsylophora mites, we amplified Anodonta exulcerata DNA instead of water mite DNA. These specimens were excluded from further analysis.
The reference library for the molecular identification of water mites sequenced in this study was analysed using the Barcode Index Number system (BIN) (
Taxa |
Voucher Code |
BOLD Process ID |
BIN |
Locality |
Coordinates |
Limnocharidae |
|||||
Limnochares aquatica |
Podgorica, Zeta River at Pričelje |
|
|||
Hydryphantidae |
|||||
Panisus michaeli |
Kolašin, Lalevića Dolovi, spring #1 |
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|||
Kolašin, Lalevića Dolovi, spring #1 |
|
||||
Kolašin, Lalevića Dolovi, spring #1 |
|
||||
Trichothyas jadrankae |
Bar, Poseljanski stream at Poseljani |
|
|||
Partnunia naprintua |
Andrijevica, spring at Trešnjevik |
|
|||
Protzia invalvaris |
Kolašin, Bistrica stream |
|
|||
Kolašin, Kolašinska rijeka stream |
|
||||
Kolašin, Kolašinska rijeka stream |
|
||||
Kolaši, Kolašinska rijeka stream |
|
||||
Protzia squamosa paucipora |
Kolašin, spring on road to Trešnjevik |
|
|||
Kolašin, spring on road to Trešnjevik |
|
||||
Kolašin, spring on road to Trešnjevik |
|
||||
Protzia octopora |
Kolašin, Bistrica stream |
|
|||
Protzia halberti |
Bijelo Polje, Lještanica stream |
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Bijelo Polje, Lještanica stream |
|
||||
Bijelo Polje, Lještanica stream |
|
||||
Bijelo Polje, Lještanica stream |
|
||||
Protzia rotunda |
Žabljak, Sedlo, spring Studenac |
|
|||
Bar, Međurječka rijeka stream |
|
||||
Bar, Međurječka rijeka stream |
|
||||
Protzia rugosa |
Bijelo Polje, Lještanica stream |
|
|||
Berane, spring nr Mon. Djurdjevi Stupovi |
|
||||
Mojkovac, Bistrica stream |
|
||||
Hydrodromidae |
|||||
Hydrodroma reinhardi |
Podgorica, Cijevna River at Dinoša |
|
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Hydrodroma torrenticola |
Bar, Medjurječka rijeka stream |
|
|||
Lebertiidae |
|||||
Lebertia jadrensis |
Kolašin, Bistrica stream at Crkvine |
|
|||
Podgorica, Cijevna River at Dinoša |
|
||||
Kolašin, Bistrica stream at Crkvine |
|
||||
Danilovgrad, spring below the bridge |
|
||||
Lebertia cuneifera |
BOLD:ADV4392 |
Nikšić, spring “Babino sicelo” |
|
||
Lebertia variolata |
Bar, stream in Godinje Village |
|
|||
Bar, stream in Godinje Village |
|
||||
Bar, Rikavac stream above Old Bar |
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||||
Bar, Rikavac stream above Old Bar |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Bar, Medjurječka rijeka stream |
|
||||
Bar, Medjurječka rijeka stream |
|
||||
Lebertia natans |
Danilovgrad, spring below the bridge |
|
|||
Danilovgrad, spring below the bridge |
|
||||
Danilovgrad, spring below the bridge |
|
||||
Lebertia glabra |
Kolašin, Kolašinska rijeka stream |
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Kolašin, Bistrica stream at Crkvine |
|
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Kolašin, Bistrica stream at Crkvine |
|
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Mojkovac, Bistrica stream |
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Mojkovac, Bistrica stream |
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Kolašin, Kolašinska rijeka stream |
|
||||
Lebertia inaequalis |
Tuzi, Vitoja, pool |
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|||
Tuzi, Vitoja, pool |
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||||
Tuzi, Vitoja, pool |
|
||||
Bar, Skadar Lake at Murići |
|
||||
Bar, Skadar Lake at Murići |
|
||||
Podgorica, Skadar Lake at Donja Plavnica |
|
||||
Podgorica, Gornja Plavnica, river |
|
||||
Bar, Medjurjecka rijeka stream |
|
||||
Lebertia insignis |
Danilovgrad, River Zeta near Slap |
|
|||
Lebertia maculosa |
Bijelo Polje, Lještanica stream |
|
|||
Bijelo Polje, Lještanica stream |
|
||||
Berane, spring nr. Mon. Djurdjevi Stupovi |
|
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Kolašin, spring at Monastir Morača |
|
||||
Lebertia porosa |
Podgorica, Cijevna River at Dinoša |
|
|||
Cetinje, River Crnojevića |
|
||||
Podgorica, spring Mareza |
|
||||
Tuzi, Vitoja spring |
|
||||
Lebertia reticulata |
Šavnik, spring of Bukovica stream |
|
|||
Šavnik, spring of Bukovica stream |
|
||||
Nikšić, spring Vukovo Vrelo |
|
||||
Lebertia schechteli |
Žabljak, Sedlo, spring Studenac |
|
|||
Žabljak, Sedlo, spring Studenac |
|
||||
Oxidae |
|||||
Oxus angustipositus |
Ulcinj, Šasko Lake |
|
|||
Cetinje, Poseljanski stream, lower part |
|
||||
Bar, Skadar Lake at Murići |
|
||||
Bar, Skadar Lake at Murići |
|
||||
Teutoniidae |
|||||
Teutonia cometes |
Podgorica, Mareza canal |
|
|||
Danilovgrad, spring Svinjiška vrela |
|
||||
Sperchontidae |
|||||
Sperchon brevirostris |
Mojkovac, Bistrica stream |
|
|||
Mojkovac, Bistrica stream |
|
||||
Kolašin, Kolašinska rijeka stream |
|
||||
Sperchon clupeifer |
Žabljak, Ljutica stream |
|
|||
Bar, stream in Godinje Village |
|
||||
Sperchon hibernicus |
Bar, Rikavac stream above Old Bar |
|
|||
Sperchon hispidus |
Danilovgrad, Zeta River at Spuž |
|
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Danilovgrad, Zeta River at Spuž |
|
||||
Spechon denticulatus |
Berane, spring nr. Mon. Djurdjevi Stupovi |
|
|||
Sperchon papillosus |
Budva, Lastva Grbaljska, stream |
|
|||
Sperchon thienemanni |
Šavnik, spring Kikov izvor near Boan |
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Žabljak, Sedlo, Studenac spring |
|
||||
Kolašin, Lalevića Dolovi, spring #1 |
|
||||
Nikšić, Lukavica Mt., spring Babino Sicelo |
|
||||
Nikšić, Lukavica Mt., spring Babino Sicelo |
|
||||
Nikšić, Lukavica Mt., spring Babino Sicelo |
|
||||
Sperchon violaceus |
Žabljak, Mlinski potok stream |
|
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Kolašin, Biogradska River |
|
||||
Bijelo Polje, Lještanica stream |
|
||||
Bijelo Polje, Lještanica stream |
|
||||
Bijelo Polje, Lještanica stream |
|
||||
Kolašin, Biogradska River |
|
||||
Kolašin, Biogradska River |
|
||||
Mojkovac, Bistrica stream |
|
||||
Mojkovac, spring in Bistrica Village |
|
||||
Mojkovac, spring in Bistrica Village |
|
||||
Sperchonopsis verrucosa |
Cetinje, spring “Smokov Vijenac” |
|
|||
Kolašin, Biogradska River |
|
||||
Torrenticolidae |
|||||
Monatractides madritensis |
Podgorica, Cijevna River at Dinoša |
|
|||
Budva, Lastva Grbaljska, first order stream |
|
||||
Bar, stream in Godinje Village |
|
||||
Monatractides stadleri |
Bar, Rikavac stream above Old Bar |
|
|||
Budva, Lastva Grbaljska, first order stream |
|
||||
Torrenticola amplexa |
Danilovgrad, spring below the bridge |
|
|||
Danilovgrad, spring below the bridge |
|
||||
Danilovgrad, spring below the bridge |
|
||||
Torrenticola brevirostris |
Danilovgrad, Zeta River at Spuž |
|
|||
Podgorica, Morača River in Podgorica |
|
||||
Torrenticola dudichi |
Mojkovac, Bistrica stream |
|
|||
Kolašin, Biogradska rijeka stream |
|
||||
Torrenticola laskai |
Podgorica, Cijevna River at Dinoša |
|
|||
Kolašin, Tara River near Mateševo |
|
||||
Bar, Međurječka rijeka stream |
|
||||
Torrenticola lukai |
Kolašin, Bistrica stream at Crkvine |
|
|||
Torrenticola meridionalis |
Kolašin, Bistrica stream at Crkvine |
|
|||
Bar, Orahovštica River |
|
||||
Bar, Orahovštica River |
|
||||
Kolašin, River Drcka near Mateševo |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Kolašin, Bistrica stream at Crkvine |
|
||||
Kolašin, Bistrica stream at Crkvine |
|
||||
Kolašin, Kolašinska rijeka stream |
|
||||
Mojkovac, Bistrica stream |
|
||||
Torrenticola similis |
Kolašin, Bistrica stream at Crkvine |
|
|||
Torrenticola barsica |
Bar, Međurječka rijeka stream |
|
|||
Bar, Međurječka rijeka stream |
|
||||
Torrenticola elliptica |
Kolašin, Kolašinska rijeka stream |
|
|||
Torrenticola ungeri |
Bar, Međurječka rijeka stream |
|
|||
Bar, Međurječka rijeka stream |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Podgorica, Cijevna River at Dinoša |
|
||||
Pseudotorrenticola rhynchota |
Bar, stream in Godinje Village |
|
|||
Bar, stream in Godinje Village |
|
||||
Limnesiidae |
|||||
Limnesia undulata |
Tuzi, Vitoja, pools |
|
|||
Tuzi, Skadar Lake at Podhum |
|
||||
Hygrobatidae |
|||||
Atractides fluviatilis |
Podgorica, Cijevna River at Dinoša |
|
|||
Atractides fissus |
Kolašin, Kolašinska rijeka stream |
|
|||
Mojkovac, Bistrica stream |
|
||||
Atractides anae |
Berane, spring nr. Mon. Djurdjevi Stupovi |
|
|||
Atractides inflatipalpis |
Bar, Međurječka rijeka stream |
|
|||
Atractides inflatipes |
Bar, Orahovštica stream |
|
|||
Atractides fonticolus |
Podgorica, Pričelje, spring Studenac |
|
|||
Podgorica, Pričelje, spring Studenac |
|
||||
Atractides gibberipalpis |
Mojkovac, Bistrica stream |
|
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Kolašin, Kolašinska rijeka stream |
|
||||
Atractides inflatus |
Budva, Lastva Grbaljska, first order stream |
|
|||
Atractides nodipalpis |
Danilovgrad, spring below the bridge |
|
|||
Danilovgrad, Zeta River at Spuž |
|
||||
Danilovgrad, spring below the bridge |
|
||||
Danilovgrad, Zeta River at Spuž |
|
||||
Atractides pennatus |
Bar, Orahovštica stream |
|
|||
Bar, Poseljani, Poseljanski stream |
|
||||
Podgorica, Mareza spring |
|
||||
Podgorica, Mareza spring |
|
||||
Žabljak, Sedlo, Studenac spring |
|
||||
Nikšić, Vidrovan, Vukovo Vrelo spring |
|
||||
Nikšić, Vidrovan, Vukovo Vrelo spring |
|
||||
Nikšić, spring in Miločani Village |
|
||||
Budva, spring Smokov Vijenac |
|
||||
Budva, spring Smokov Vijenac |
|
||||
Atractides robustus |
Bar, Rikavac stream above Old Bar |
|
|||
Bar, Rikavac stream above Old Bar |
|
||||
Bar, Rikavac stream above Old Bar |
|
||||
Kolašin, spring nr. Monastir Morača |
|
||||
Kolašin, Kolašinska Rijeka stream |
|
||||
Atractides latipes |
Podgorica, River Cijevna at Trgaja |
|
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Atractides stankovici |
Dnilovgrad, River Zeta near Slap |
|
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Danilovgrad, River Zeta near Slap |
|
||||
Podgorica, Mareza canal |
|
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Podgorica, Mareza canal |
|
||||
Hygrobates calliger |
Kolašin, Crkvine, Bistrica stream |
|
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Bar, Poseljanski stream at Poseljani |
|
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Bar, Poseljanski stream at Poseljani |
|
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Hygrobates foreli |
Žabljak, Mlinski potok stream |
|
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Hygrobates lacrima |
Kolašin, Tara River near Mateševo |
|
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Hygrobates limnocrenicus |
Podgorica, Mareza canal |
|
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Hygrobates longipalpis |
Tuzi, Vitoja, pool |
|
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Tuzi, Vitoja, pool |
|
||||
Bar, Poseljani, Poseljanski stream |
|
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Tuzi, Skadar Lake at Podhum |
|
||||
Hygrobates mediterraneus |
Bar, Medjurječka rijeka stream |
|
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Bar, Medjurječka rijeka stream |
|
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Bar, Medjurječka rijeka stream |
|
||||
Bar, Medjurječka rijeka stream |
|
||||
Hygrobates norvegicus |
Šavnik, spring Kikov izvor near Boan |
|
|||
Kolašin, Lalevića Dolovi, spring #1 |
|
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Kolašin, Lalevića Dolovi, spring #1 |
|
||||
Kolašin, Lalevića Dolovi, spring #1 |
|
||||
Unionicolidae |
|||||
Neumania imitata |
Danilovgrad, River Zeta at Spuž |
|
|||
Neumania limosa |
Tuzi, Vitoja, pool |
|
|||
Tuzi, Vitoja, pool |
|
||||
Tuzi, Vitoja, pool |
|
||||
Unionicola minor |
Ulcinj, Šasko Lake |
|
|||
Ulcinj, Šasko Lake |
|
||||
Ulcinj, Šasko Lake |
|
||||
Tuzi, Vitoja, pool |
|
||||
Ulcinj, Šasko Lake |
|
||||
Unionicola ypsilophora |
Cetinje, River Cnojevica (Anodonta exulcerata) |
|
|||
Piona damkoehleri |
Danilovgrad, Moromiš pond |
|
|||
Danilovgrad, Moromiš pond |
|
||||
Danilovgrad, Moromiš pond |
|
||||
Danilovgrad, Moromiš pond |
|
||||
Danilovgrad, Moromiš pond |
|
||||
Piona laminata |
Danilovgrad, Moromiš pond |
|
|||
Piona disparilis |
Šavnik, spring of Bukovica stream, pool |
|
|||
Nikšić, Vukovo Vrelo spring, pool |
|
||||
Nikšić, Vukovo Vrelo spring, pool |
|
||||
Typhis torris |
Tuzi, Vitoja, pool |
|
|||
Typhis ornatus |
Danilovgrad, Moromiš pond |
|
|||
Danilovgrad, Moromiš pond |
|
||||
Wettinidae |
|||||
Wettina lacustris |
Podgorica, Mareza canal |
|
|||
Mideopsidae |
|||||
Mideopsis milankovici |
Bar, Medjurječka rijeka stream |
|
|||
Mideopsis roztoczensis |
Cetinje, Poseljanski stream |
|
|||
Podgorica, Cijevna River at Dinoša |
|
||||
Danilovgrad, Zeta River at Spuž |
|
||||
Danilovgrad, Zeta River at Spuž |
|
||||
Danilovgrad, Zeta River at Vranjske Njive |
|
||||
Athienemanniidae |
|||||
Mundamella germanica |
Danilovgrad, Spuž, spring near Zeta River |
|
|||
Aturidae |
|||||
Hexaxonopsis serrata |
Bar, Skadar Lake at Murići |
|
|||
Bar, Skadar Lake at Murići |
|
||||
Bar, Skadar Lake at Murići |
|
||||
Parabrachypoda montii |
Podgorica, Mareza canal |
|
|||
Woolastokia rotundifrons |
Šavnik, Tušina River at Boan |
|
|||
Šavnik, Tušina River at Boan |
|
||||
Arrenuridae |
|||||
Arrenurus cylindratus |
Podgorica, Mareza canal |
|
|||
Arrenurus refractarioulus |
Nikšić, Lukavica Mt., pools |
|
All obtained BINs were inspected for concordance using BOLD Workbench. The Refined Single Linkage (RESL) algorithm was used to assign water mite barcodes to Operational Taxonomic Units (OTUs).
Sequence comparisons were performed using MUSCLE alignment (
DNA barcodes of 233 specimens morphologically assigned to 86 species from 28 genera and 15 families of water mites from Montenegro were newly generated for this study. The specimens were collected through the “DNA-Eco” (DNA barcode reference library as a tool for sustainable management of freshwater ecosystems in the highly threatened Lake Skadar Basin) project. The current study develops the first COI barcode reference library of water mites for Montenegro with the focus on Skadar/Shkodra Lake catchment area.
Fragment lengths of the analysed DNA barcode fragments ranged from 201 to 658 (mean: 636.2) base pairs, including no stop codons, insertions or deletions. The DNA barcode region was characterised by a high AT-content: the mean sequence compositions were A = 30.82 ± 0.1252%, C = 20.39 ± 0.1222%, G = 14.91 ± 0.0709% and T = 33.88 ± 0.1253%. The obtained results are similar to those found in other arthropod studies (e.g.
The families Hygrobatidae Koch, 1842 and Lebertiidae Thor, 1900 are represented by the highest number of sequences (53 and 44, respectively). The opposite, the three families Arrenuridae Thor, 1900, Teutoniidae Koenike, 1910 and Limnesiidae Thor, 1900 are represented each with two sequences and the two familes Athienemanniidae K. Viets, 1922 and Wettinidae Cook, 1956 by the lowest number of sequences (each with one sequence). The most common genus was Lebertia Neuman, 1880, for which 44 barcode sequences (11 species) were generated, followed by Atractides Koch, 1837 (35 barcodes; 13 species), Torrenticola Piersig, 1896 and Sperchon Kramer, 1877 (29 and 26 barcodes, 10 and 8 species, respectively). Six genera were represented by a single specimen. The highest number of barcodes per species was reached for Atractides pennatus (K. Viets, 1922), Sperchon violaceus Walter, 1944 and Torrenticola meridionalis Di Sabatino and Cicolani, 1990 (each with 10 barcodes), followed by Lebertia inaequalis (Koch, 1837) and L. variolata Gerecke, 2009 (each with 8 barcodes) and Sperchon thienemanni Koenike, 1907 (6 barcodes). On the other hand, most species are represented by less than 5 DNA barcodes. Thirty-three species are represented by a single DNA barcode not allowing us to estimate the intraspecific distances. BOLD ID and accession numbers for all specimens included in final dataset are given in Table
The mean intrageneric K2P distance was 20.2 ± 0.0% (range 6.09-42.37%). The mean intraspecific nucleotide K2P distances were 2.43 ± 0.01% (ranging from 0% to 24.16%). The summary statistics showing significant changes of average K2P distances within the different taxonomic levels are given in Table
Summary table of K2P genetic distances within the different taxonomic levels derived from 233 analysed water mite specimens from Montenegro. The list of studied species is provided in Table 1. Deletion Method: Pairwise Deletion. Alignment: BOLD Aligner (Amino Acid based HMM).
Label |
n |
Taxa |
Comparisons |
Min Dist. (%) |
Mean Dist. (%) |
Max Dist. (%) |
SE Dist. (%) |
Within Species |
200 |
53 |
391 |
0.00 |
2.43 |
24.16 |
0.01 |
Within Genus |
207 |
14 |
2291 |
6.09 |
20.20 |
42.37 |
0.00 |
Within Family |
168 |
7 |
1054 |
16.17 |
37.14 |
63.16 |
0.01 |
The BIN and RESL (OTU) analyses assigned sequences to 98 BINs and 103 OTUs, respectively. Fifty BINs (159 records) were concordant (51%) and 48 BINs were represented by a single sequence (49%). At the time of publication of the dataset, fifty-five (56.1%) of these BINs (with 102 sequences) included sequences only from Montenegro, while the remaining BINs included sequences also from other countries.
Most of the morphologically-identified species show an intraspecific variation of less than 2%. However, the 14 taxa listed in Table
Species with intraspecific (ISD) maximum pairwise distances > 2.2% (p-dist.). Divergence values were calculated for all studied sequences, using the Nearest Neighbour Summary, implemented in the Barcode Gap Analysis tool provided by the Barcode of Life Data System (BOLD). BINs are based on the barcode analysis from 15 November 2021. Country codes (alpha-2 code): BG = Bulgaria, CH = Switzerland, DE = Germany, ES = Spain, FR = France, GB = United Kingdom, GL = Greenland, IT = Italy, NO = Norway, NL = Netherlands, ME = Montenegro, MK = North Macedonia, PL = Poland, RO = Romania, RS = Serbia, RU = Russia, SK = Slovakia. n = BIN member count.
No. |
Species |
BIN |
n |
MeanISD |
MaxISD |
Country |
Nearest BIN/Species |
Distance to NN |
1. |
Lebertia glabra |
BOLD:ACR9598 |
2 |
0.8 |
0.8 |
ME, NL |
12.52 |
|
Lebertia glabra |
20 |
0.64 |
1.36 |
NL, BG, ME, MK, IT, PL, SK |
2.88 |
|||
Lebertia glabra |
BOLD:AEI925 |
3 |
0.64 |
0.96 |
ME |
12.02 |
||
2. |
Lebertia inaequalis |
1 |
N/A |
N/A |
ME |
2.78 |
||
Lebertia inaequalis |
18 |
0.18 |
0.34 |
NL, PL, ME |
2.78 |
|||
Lebertia inaequalis |
1 |
N/A |
N/A |
ME |
6.96 |
|||
3. |
Lebertia maculosa |
3 |
1.27 |
1.6 |
ME, MK |
2.76 |
||
Lebertia maculosa |
2 |
0.16 |
0.16 |
ME |
2.76 |
|||
4. |
Lebertia porosa |
133 |
0.81 |
2.37 |
NL, FR, ME, DE, GB, BG, IT, PL, SK, ES, CH |
3.89 |
||
Lebertia porosa |
12 |
0.14 |
0.85 |
ME |
3.89 |
|||
5. |
Sperchon brevirostris |
3 |
0.32 |
0.48 |
ME, MK |
2.72 |
||
Sperchon brevirostris |
28 |
0.55 |
3.12 |
NO, DE, ME |
7.53 |
|||
6. |
Sperchon clupeifer |
11 |
1.68 |
3.47 |
NL, DE, NO, MK, ME, RU |
8.7 |
||
Sperchon clupeifer |
1 |
N/A |
N/A |
ME |
8.7 |
|||
7. |
Sperchonopsis verrucosa |
1 |
N/A |
N/A |
ME, RO |
4.83 |
||
Sperchonopsis verrucosa |
9 |
0.29 |
0.97 |
NO, IT, ME |
9.83 |
|||
8. |
Monatractides madritensis |
2 |
0.16 |
0.16 |
ME |
1.44 |
||
Monatractides madritensis |
2 |
0.64 |
0.64 |
ME, SR |
1.44 |
|||
9. |
Torrenticola meridionalis |
8 |
1.46 |
2.25 |
ME, MK |
6.57 |
||
Torrenticola meridionalis |
4 |
1.42 |
2.09 |
ME |
6.25 |
|||
10. |
Torrenticola laskai |
2 |
0.32 |
0.32 |
ME |
2.17 |
||
Torrenticola laskai |
4 |
0.82 |
1.34 |
RS, ME, RO |
2.17 |
|||
11. |
Atractides gibberipalpis |
1 |
N/A |
N/A |
ME |
4.81 |
||
Atractides gibberipalpis |
1 |
N/A |
N/A |
ME |
4.81 |
|||
12. |
Atractides nodipalpis |
41 |
0.59 |
3.05 |
NO, NL, GL, DE, ME, RS |
13.3 |
||
Atractides nodipalpis |
2 |
0 |
0 |
ME |
13.3 |
|||
13. |
Hygrobates calliger |
2 |
1.2 |
1.2 |
NO, ME |
16.18 |
||
Hygrobates calliger |
2 |
1.03 |
1.03 |
DE, ME |
14.61 |
|||
14. |
Unionicola minor |
3 |
0.59 |
0.7 |
ME |
17.02 |
||
Unionicola minor |
7 |
0.09 |
0.32 |
NO, NL, ME |
16.03 |
The NJ analyses, based on K2P distances, revealed non-overlapping clusters with bootstrap support values > 95% for 50 species (58%) with more than one analysed specimen indicating a high congruence between BINs affiliation and morphological species identification. Moreover, specimens showing high intraspecific distances are also clearly separated into different clades. A more detailed topology of all analysed specimens is presented in the supporting information (Suppl. material
This study provides COI barcodes for 233 specimens representing 86 morphologically identified species of water mites from Montenegro. These represent 42.8% of Montenegrin water mite fauna, based on
Of the 86 species recorded in this study, 79 species were previously reported for Montenegro. DNA barcoding confirmed the presence of four species new for Montenegro, i.e. Lebertia reticulata (Koenike, 1919), Atractides inflatipalpis K.Viets, 1950, A. latipes (Szalay, 1935) and Parabrachypoda montii (Maglio, 1924). Three species, i.e. Protzia octopora Lundblad, 154, Piona laminata (Thor, 1901) and Unionicola ypsilophora (Bonz, 1783) are recorded for the first time for the Balkan Peninsula. Specimens of the latter species were found between the gill blades of mussels Anodonta exulcerata Clesin, 1876, whose identification was confirmed by molecular data.
Moreover, species identification, based on molecular data conducted during this project, extended the list of Montenegrin water mites by description of several species new for science, i.e. Atractides anae Pešić, 2020, Hygrobates lacrima Pešić, 2020, H. limnocrenicus Pešić, 2020, H mediterraneus Pešić, 2020 and Mideopsis milankovici Pešić and Smit, 2020 (
Our study confirmed efficiency of DNA barcoding as a tool for the identification of water mites. In particular, 72 of the 86 morphologically-identified species exactly matched the BINs defined from BOLD. This result coincides with high identification efficiency rates through the BOLD Best Close Match analysis. Nevertheless, our data revealed also 14 species listed in Table 3 that showed high intraspecific distances (> 2.2%) suggesting possible cryptic and/or pseudocryptic diversification. Most of these possible cryptic and/or pseudocryptic species, as seen in Table 3, appear to be hidden within common species.
Three species, i.e. Lebertia maculosa, Monatractides madritensis and Torrenticola laskai appeared each with 2 BINs in our dataset. The intraspecific maximum distances between BINs within each of these species were below 3% (Suppl. material
Lebertia glabra, a species widely distributed in West Palaearctic (
Lebertia inaequalis, a species reported from the extended parts of the Palaearctic (
Lebertia porosa, a eurytopic and eurythermous species, often reported from standing waters and pools of streams across the Holarctic (
Sperchon brevirostris, a species inhabiting low-and middle order streams in the study area (
Sperchonopsis verrucosa, a species often reported from the Holarctic Region (
Torrenticola meridionalis, a species originally described from Italy, is widely distributed in Montenegro, inhabiting mainly low order streams (
Atractides nodipalpis, a rhitrobiontic species, is the most frequently reported species of the genus in Europe (
Hygrobates calliger, a rhitrobiontic species widely distributed in the Palaearctic (
The sequences of Atractides gibberipalpis, a rhitrobiontic species often reported from the Palaearctic (
Unionicola minor, a species widely distributed in Europe (
Taxonomic studies of the above species were outside the scope of this paper. Further studies with material from a wider geographical area, were needed to clarify taxonomy and elucidate the delimitation of the species in the above complexes. This process should be accompanied by sufficient barcode coverage to allow the detection of phylogeographic patterns and/or even the existence of possible overlooked cryptic species. The build-up of DNA barcode library for water mites of Montenegro represents a long-term task, aimed at improving molecular identification and inclusion of this group in environmental assessment programmes and, on the other hand, to stimulate further biodiversity research of this limnofaunistic group in Montenegro and the Balkans.
The authors are grateful to all those who helped during sample collections in Montenegro. This study is part of the “DNA-Eco” scientific project, supported by a grant of the Montenegrin Ministry of Science. The part of study was supported by Polish National Science Centre, Poland, grant no. 2017/27/N/NZ8/01568.
No ethical principles were violated when providing this study.
The authors declare no conflict of interests concerning this study.
Molecular distances, based on the Kimura 2-parameter model of the analysed specimens of water mites from Montenegro. BINs are based on the barcode analysis from 15 November 2021.
Compact Neighbour-Joining tree of all analysed water mite species based on Kimura 2-parameter distances. The tree was edited in MEGA7 (Kumar et al. 2016). Specimens are classified using ID numbers from BOLD and species name. BINs are based on the barcode analysis from 15 November 2021. Numbers next to nodes represent non-parametric bootstrap values (1,000 replicates, in %). The analyses involved all 233 COI nucleotide sequences.