Biodiversity Data Journal : Research Article
PDF
Research Article
A DNA barcode library for the water mites of Montenegro
expand article infoVladimir Pešić, Andrzej Zawal§, Ana Manović, Aleksandra Bańkowska|, Milica Jovanović
‡ Department of Biology, University of Montenegro, Podgorica, Montenegro
§ Institute of Marine and Environmental Sciences, Center of Molecular Biology and Biotechnology, University of Szczecin, Szczecin, Poland
| Institute of Biology, University of Szczecin, Szczecin, Poland
Open Access

Abstract

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.

Keywords

DNA barcoding, COI, water mites, Montenegro, species delimitation

Introduction

Hydrachnidia, also known as water mites, is a most diverse and abundant group of arachnids in freshwater habitats (Davids et al. 2007). With nearly 7,500 species grouped into 550 genera (Smit 2020), they inhabit a wide range of aquatic habitats, including lotic, lentic, interstitial and temporary waters. Water mites have a complex life cycle that includes two pupa-like resting stages, i.e. protonymph and tritonymph and three active stages: larva is almost always parasitic, deutonymphs and adults that are predators of minute invertebrates (Davids et al. 2007). Some recent studies have shown that water mites can be good indicators of ecosystem health, especially of groundwater-dependent ecosystems (Pešić et al. 2019b). However, their time-consuming taxonomic identification has been identified as a major constraint for more significant involvement in rapid assessment programmes (Weigand et al. 2019).

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 (Martin et al. 2010, Pešić et al. 2017, Fisher et al. 2017, Pešić et al. 2019a, Pešić et al. 2020d, Pešić and Smit 2020). This process has been enhanced by the formation of the comprehensive DNA barcode reference libraries, such as the BOLD System (https://www.boldsystems.org/) and GenBank (https://www.ncbi.nlm.nih.gov/). DNA barcodes have been proposed and successfully adopted for water mites as an efficient method for detecting previously overlooked and/or misidentified species (Martin et al. 2010, Pešić et al. 2017, Pešić et al. 2019). The significant increase in the number of studies using DNA barcodes in recent years, especially in some regions, has laid the foundations for building a comprehensive library of DNA barcodes at the national and/or regional level (e.g. Blattner et al. 2019).

Of the Balkan countries, Montenegro is one of the best studied from the taxonomic and faunistic point of view (Pešić et al. 2018). Water mite research began in 1903 when the Czech zoologist Karl Thon published the first list of 13 species (Thon 1903). For more than one century, a large number of papers on the Montenegrin water mites have been published (Musselius 1912, Viets 1936, Pešić 2001, Pešić 2002b, Pešić 2002d, Pešić 2002a, Pešić 2002c, Pešić 2003a, Pešić 2003c, Pešić and Gerecke 2003, Di Sabatino et al. 2003, Pešić 2003b, Pešić 2004b, Pešić 2004a, Smit and Pešić 2004, Baker et al. 2008, Pešić et al. 2010, Pešić et al. 2012, Pešić et al. 2017, Bańkowska et al. 2016, Pešić et al. 2018, Zawal and Pešić 2018, Pešić et al. 2019a, Pešić et al. 2019c, Pešić et al. 2020a, Pešić et al. 2020b, Pešić et al. 2020c, Pešić et al. 2020d, Zawal et al. 2020, Pešić and Smit 2020, Pešić et al. 2021b)

Currently, 201 species of water mites have been reported for Montenegro (Pešić et al. 2018, Pešić et al. 2019c, Pešić et al. 2020c, Pešić et al. 2020d, Pešić and Smit 2020). This number makes up about 50% of the species known from the Balkans, which is estimated at about 400 species (Pešić et al. 2018). This is still a small number for the area of such hydrogeological characteristics and the turbulent geological history as the Balkans. Therefore, there is no reason not to believe that the expected number of water mites in the Balkans is at least at the level of Central Europe which is home to approximately 745 species (Gerecke et al. 2016).

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.

Material and methods

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. 1) during several sampling campaigns from 2018-2020. Photos from each studied specimen were taken before mo­lecular work started. The photographs were made using a camera on a Samsung Galaxy smartphone.

Figure 1.  

Sampling sites from Montenegro. The green colour represents the Danube Basin (Black Sea) and the yellow colour represents the Adriatic Basin.

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 Pešić et al. (2017). In CCDB, the specimens were sequenced for the barcode region of COI using standard invertebrate DNA extraction (Ivanova et al. 2007), amplification (Ivanova and Grainger 2007a) and sequencing protocols (Ivanova and Grainger 2007b). The DNA extracts were archived in −80°C freezers at the Centre for Biodiversity Genomics (CBG; biodiversitygenomics.net) and the specimen vouchers were stored in 95% EtOH and returned to the first author for morphological examination. Some of these vouchers were dissected as described elsewhere (Davids et al. 2007) and slide-mounted in Faure’s medium, while the rest were transferred to Koenike’s medium and stored in the collection of the first author at the Department of Biology in Podgorica.

DNA barcode analysis

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) (Felsenstein 1985). The distribution of BINs was performed by the Barcode of Life Data System v.4 (accessed 15 November 2021). The two-phase BIN analysis system in the first phase applies a first threshold of 2.2% (that allows a rough differentiation between intraspecific and interspecific distances), followed by refinements through Markov clustering into the final BINs (Ratnasingham and Hebert 2013). BOLD ID and accession numbers for all specimens included in final dataset are given in Table 1.

Table 1.

Details on barcoded specimens from Montenegro.

Taxa

Voucher Code

BOLD Process

ID

BIN

Locality

Coordinates

Limnocharidae

Limnochares aquatica

31. CG2020_6_C10

DNAEC032-20

BOLD:ACS0438

Podgorica, Zeta River at Pričelje

42.5022N, 19.2225E

Hydryphantidae

Panisus michaeli

CCDB 38361 A04

DCDDJ004-21

BOLD:ADT7504

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

CCDB 38361 A05

DCDDJ005-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

CCDB 38361 A06

DCDDJ006-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

Trichothyas jadrankae

CCDB-38679-A08

DNCBD008-20

BOLD:AEF1286

Bar, Poseljanski stream at Poseljani

42.3095N, 19.0518E

Partnunia naprintua

CCDB 38361 A08

DCDDJ008-21

BOLD:AEL6734

Andrijevica, spring at Trešnjevik

42.7392N, 19.6933E

Protzia invalvaris

CCDB 38361 C11

DCDDJ035-21

BOLD:AEI2833

Kolašin, Bistrica stream

42.8054N, 19.4456E

CCDB38233 A08

DCCDB008-21

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

CCDB38233 A09

DCCDB009-21

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

CCDB38233 A10

DCCDB010-21

Kolaši, Kolašinska rijeka stream

42.8391N, 19.5749E

Protzia squamosa paucipora

CCDB 38361 A09

DCDDJ009-21

BOLD:AEL1015

Kolašin, spring on road to Trešnjevik

42.7405N, 19.6801E

CCDB 38361 A10

DCDDJ010-21

Kolašin, spring on road to Trešnjevik

42.7405N, 19.6801E

CCDB 38361 A11

DCDDJ011-21

Kolašin, spring on road to Trešnjevik

42.7405N, 19.6801E

Protzia octopora

CCDB38233 D09

DCCDB045-21

BOLD:AEI5747

Kolašin, Bistrica stream

42.9871N, 19.4338E

Protzia halberti

Hyd_MN_VP7

DNAEC081-20

BOLD:AED9646

Bijelo Polje, Lještanica stream

43.0631N, 19.5808E

3. CG2020_8_2

DNAEC002-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5808E

4. CG2020_1

DNAEC003-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5808E

5. CG2020_1_3

DNAEC004-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5808E

Protzia rotunda

6. M18_01_1_D10

DNAEC045-20

BOLD:AED8976

Žabljak, Sedlo, spring Studenac

43.0973N, 19.0702E

CCDB-3867-E04

DNCBD052-20

Bar, Međurječka rijeka stream

42.0363N, 19.2179E

CCDB-3867-E05

DNCBD053-20

Bar, Međurječka rijeka stream

42.0363N, 19.2179E

Protzia rugosa

6. CG2020_1_4

DNAEC005-20

BOLD:AEE010

Bijelo Polje, Lještanica stream

43.0631N, 19.5808E

7. CG2020_8 B6

DNAEC017-20

Berane, spring nr Mon. Djurdjevi Stupovi

42.8527N, 19.862E

CCDB38233 D05

DCCDB041-21

Mojkovac, Bistrica stream

42.9871N, 19.4338E

Hydrodromidae

Hydrodroma reinhardi

CCDB-3867-G04

DNCBD076-20

BOLD:AEF0798

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

Hydrodroma torrenticola

CCDB-3867-E06

DNCBD054-20

BOLD:AEF3799

Bar, Medjurječka rijeka stream

42.0363N, 19.2179E

Lebertiidae

Lebertia jadrensis

CCDB 38361 C09

DCDDJ033-21

BOLD:ADK0383

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

CCDB-3867-G08

DNCBD080-20

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

CCDB 38361 C08

DCDDJ032-21

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

CCDB-3867-F10

DNCBD070-20

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

Lebertia cuneifera

CCDB 38363 A01

SEPTA001-21

BOLD:ADV4392

Nikšić, spring “Babino sicelo”

42.8043N, 19.2152E

Lebertia variolata

CCDB-3867-B05

DNCBD017-20

BOLD:ADK0996

Bar, stream in Godinje Village

42.2206N, 19.1118E

CCDB-3867-B07

DNCBD019-20

Bar, stream in Godinje Village

42.2206N, 19.1118E

CCDB-3867-D03

DNCBD039-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-D04

DNCBD040-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-D05

DNCBD041-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-D06

DNCBD042-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

16. M19_24_3_E7

DNAEC054-20

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

17. M19_24_3_E8

DNAEC055-20

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

Lebertia natans

CCDB38233 F03

DCCDB063-21

BOLD:AEF5684

Danilovgrad, spring below the bridge

42.5541N, 19.1057E

CCDB38233 F04

DCCDB064-21

Danilovgrad, spring below the bridge

42.5541N, 19.1057E

CCDB-3867-F06

DNCBD066-20

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

Lebertia glabra

CCDB38233 C04

DCCDB028-21

BOLD:AEI2925

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

CCDB38233 D03

DCCDB039-21

Kolašin, Bistrica stream at Crkvine

42.9871N, 19.4338E

CCDB38233 D04

DCCDB040-21

Kolašin, Bistrica stream at Crkvine

42.9871N, 19.4338E

CCDB38233 D01

DCCDB037-21

BOLD:ACS0595

Mojkovac, Bistrica stream

42.9871N, 19.4338E

CCDB38233 D02

DCCDB038-21

Mojkovac, Bistrica stream

42.9871N, 19.4338E

CCDB38233 C05

DCCDB029-21

BOLD:ACR9598

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

Lebertia inaequalis

CCDB-3867-C03

DNCBD027-20

BOLD:AEF5913

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB-3867-B11

DNCBD023-20

BOLD:ADF6223

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB-3867-C02

DNCBD026-20

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB 38363 B04

SEPTA016-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

CCDB 38363 B06

SEPTA018-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

CCDB 38363 B10

SEPTA022-21

Podgorica, Skadar Lake at Donja Plavnica

42.2724N, 19.2007E

CCDB 38363 B11

SEPTA023-21

Podgorica, Gornja Plavnica, river

42.2889N, 19.2108E

CCDB-3867-E12

DNCBD060-20

BOLD:AEF2742

Bar, Medjurjecka rijeka stream

42.0363N, 19.2179E

Lebertia insignis

CCDB38233 B12

DCCDB024-21

BOLD:AEB9107

Danilovgrad, River Zeta near Slap

42.6001N, 19.0656E

Lebertia maculosa

32. CG2020_1_C11

DNAEC033-20

BOLD:AED9197

Bijelo Polje, Lještanica stream

43.0631N, 19.5809E

33. CG2020_1_C12

DNAEC034-2

Bijelo Polje, Lještanica stream

43.0631N, 19.5809E

1. CG2020_8

DNAEC001-20

BOLD:AED9718

Berane, spring nr. Mon. Djurdjevi Stupovi

42.8527N, 19.862E

CCDB 38361 H01

DCDDJ085-21

Kolašin, spring at Monastir Morača

42.7668N, 19.3906E

Lebertia porosa

CCDB-3867-G09

DNCBD081-20

BOLD:ACS0974

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

CCDB 38363 C10

SEPTA034-21

Cetinje, River Crnojevića

42.3557N, 19.0228E

CCDB38233 A01

DCCDB001-21

BOLD:AED4662

Podgorica, spring Mareza

42.4801N, 19.1822E

7. CG2020_10

DNAEC006-20

Tuzi, Vitoja spring

42.3254N, 19.3628E

Lebertia reticulata

Hyd_MN_VP13

DNAEC086-20

BOLD:ADT9218

Šavnik, spring of Bukovica stream

43.0589N, 19.1103E

Hyd_MN_VP14

DNAEC087-20

Šavnik, spring of Bukovica stream

43.0589N, 19.1103E

CCDB 38363 A11

SEPTA011-21

Nikšić, spring Vukovo Vrelo

42.8574N, 18.9426E

Lebertia schechteli

9. CG2020

DNAEC008-20

BOLD:AED9612

Žabljak, Sedlo, spring Studenac

43.0973N, 19.0702E

10. CG2020_2_3

DNAEC009-20

Žabljak, Sedlo, spring Studenac

43.0973N, 19.0702E

Oxidae

Oxus angustipositus

CCDB 38361 C03

DCDDJ027-21

BOLD:AEB9099

Ulcinj, Šasko Lake

41.9768N, 19.3388E

CCDB-38679-A11

DNCBD011-20

Cetinje, Poseljanski stream, lower part

42.3057N, 19.0557E

CCDB 38363 B05

SEPTA017-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

CCDB 38363 B07

SEPTA019-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

Teutoniidae

Teutonia cometes

33. M19_20_3_F11

DNAEC068-20

BOLD:ACH7884

Podgorica, Mareza canal

42.479N, 19.1813E

Hyd_MN_VP5

DNAEC079-20

Danilovgrad, spring Svinjiška vrela

42.6384N, 19.0074E

Sperchontidae

Sperchon brevirostris

CCDB38233 D07

DCCDB043-21

BOLD:ACP6107

Mojkovac, Bistrica stream

42.9871N, 19.4338E

CCDB38233 D08

DCCDB044-21

BOLD:AED3857

Mojkovac, Bistrica stream

42.9871N, 19.4338E

CCDB38233 A11

DCCDB011-21

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

Sperchon clupeifer

Hyd_MN_VP11

DNAEC084-20

BOLD:AEE4061

Žabljak, Ljutica stream

43.1378N, 19.3023E

CCDB-3867-B04

DNCBD016-20

BOLD:ACS1100

Bar, stream in Godinje Village

42.2206N, 19.1118E

Sperchon hibernicus

CCDB-3867-D02

DNCBD038-20

BOLD:AEF3824

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

Sperchon hispidus

12. M19 29A 8_E3

DNAEC050-20

BOLD:AED3202

Danilovgrad, Zeta River at Spuž

42.5113N, 19.1982E

29. CG2020_7_C8C7

DNAEC030-20

Danilovgrad, Zeta River at Spuž

42.5113N, 19.1982E

Spechon denticulatus

10. CG2020_8 B8

DNAEC019-20

BOLD:AED8428

Berane, spring nr. Mon. Djurdjevi Stupovi

42.8527N, 19.862E

Sperchon papillosus

3. M19_12B_1_D7

DNAEC043-20

BOLD:AED2134

Budva, Lastva Grbaljska, stream

42.3103N, 18.8138E

Sperchon thienemanni

Hyd_MN_VP4

DNAEC078-20

BOLD:ADV4077

Šavnik, spring Kikov izvor near Boan

42.9465N, 19.1893E

Hyd_MN_VP10

DNAEC083-20

Žabljak, Sedlo, Studenac spring

43.0972N, 19.0702E

CCDB 38361 A03

DCDDJ003-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

CCDB 38363 A02

SEPTA002-21

Nikšić, Lukavica Mt., spring Babino Sicelo

42.8043N, 19.2152E

CCDB 38363 A04

SEPTA004-21

Nikšić, Lukavica Mt., spring Babino Sicelo

42.8043N, 19.2152E

CCDB 38363 A05

SEPTA005-21

Nikšić, Lukavica Mt., spring Babino Sicelo

42.8043N, 19.2152E

Sperchon violaceus

Hyd_MN_VP8

DNAEC088-20

BOLD:AAN0076

Žabljak, Mlinski potok stream

43.1494N, 19.0898E

27. M19_16A_3_F5

DNAEC062-

Kolašin, Biogradska River

42.8968N, 19.6047E

56. CG2020_1

DNAEC010-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5809E

57. CG2020_8

DNAEC011-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5809E

58. CG2020

DNAEC012-20

Bijelo Polje, Lještanica stream

43.0631N, 19.5809E

26. M19_16A_3_F4

DNAEC061-20

Kolašin, Biogradska River

42.8968N, 19.6047E

28. M19_16A_3_F6

DNAEC063-20

Kolašin, Biogradska River

42.8968N, 19.6047E

CCDB38233 D06

DCCDB042-21

Mojkovac, Bistrica stream

42.9871N, 19.4338E

CCDB38233 H10

DCCDB094-21

Mojkovac, spring in Bistrica Village

42.9862N, 19.4349E

CCDB38233 H11

DCCDB095-21

Mojkovac, spring in Bistrica Village

42.9862N, 19.4349E

Sperchonopsis verrucosa

CCDB 38361 B11

DCDDJ023-21

BOLD:AEK8297

Cetinje, spring “Smokov Vijenac”

42.254N, 18.9902E

46. M19_16B_1_G10

DNAEC040-20

BOLD:ACS9705

Kolašin, Biogradska River

42.8968N, 19.6047E

Torrenticolidae

Monatractides madritensis

CCDB-3867-G11

DNCBD083-20

BOLD:AED3803

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

44. M19_12B_3_G8

DNAEC075-20

Budva, Lastva Grbaljska, first order stream

42.3103N, 18.8138E

CCDB-3867-B01

DNCBD013-20

BOLD:AEL3852

Bar, stream in Godinje Village

42.2206N, 19.1118E

Monatractides stadleri

CCDB38233 C03

DCCDB027-21

BOLD:AED3802

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

45. M19_129_3_G9

DNAEC076-20

Budva, Lastva Grbaljska, first order stream

42.3103N, 18.8138E

Torrenticola amplexa

CCDB-3867-F08

DNCBD068-20

BOLD:ACR0665

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

CCDB-3867-F09

DNCBD069-20

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

CCDB38233 G04

DCCDB076-21

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

Torrenticola brevirostris

42. M19_29A_5_G6

DNAEC073-20

BOLD:AED9586

Danilovgrad, Zeta River at Spuž

42.5113N, 19.1982E

CCDB 38363 C12

SEPTA036-21

Podgorica, Morača River in Podgorica

42.4368N, 19.2559E

Torrenticola dudichi

CCDB38233 D11

DCCDB047-21

BOLD:AED7520

Mojkovac, Bistrica stream

42.9871N, 19.4338E

43. M19_16A_4_G7

DNAEC074-20

Kolašin, Biogradska rijeka stream

42.8968N, 19.6047E

Torrenticola laskai

CCDB-3867-G06

DNCBD078-20

BOLD:AEF5471

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

CCDB-3867-B10

DNCBD022-20

Kolašin, Tara River near Mateševo

42.7898N, 19.5374E

CCDB-3867-E11

DNCBD059-20

BOLD:AED2306

Bar, Međurječka rijeka stream

42.0363N, 19.2179E

Torrenticola lukai

CCDB 38361 C12

DCDDJ036-21

BOLD:ACH9685

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

Torrenticola meridionalis

CCDB 38361 D02

DCDDJ038-21

BOLD:AED7519

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

CCDB-3867-G02

DNCBD074-20

Bar, Orahovštica River

42.2476N, 19.0798E

CCDB-3867-G01

DNCBD073-20

Bar, Orahovštica River

42.2476N, 19.0798E

CCDB-3867-B09

DNCBD021-20

Kolašin, River Drcka near Mateševo

42.7619N, 19.5549E

CCDB-3867-E01

DNCBD049-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-E03

DNCBD051-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB 38361 D01

DCDDJ037-21

BOLD:AEI3402

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

CCDB 38361 B08

DCDDJ020-21

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

CCDB38233 B10

DCCDB022-21

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

CCDB38233 D12

DCCDB048-21

Mojkovac, Bistrica stream

42.9871N, 19.4338E

Torrenticola similis

CCDB 38361 B09

DCDDJ021-21

BOLD:AEK9661

Kolašin, Bistrica stream at Crkvine

42.8054N, 19.4456E

Torrenticola barsica

CCDB-3867-E09

DNCBD057-20

BOLD:AEF1219

Bar, Međurječka rijeka stream

42.0363N, 19.2179E

CCDB-3867-F04

DNCBD064-20

Bar, Međurječka rijeka stream

42.0363N, 19.2179E

Torrenticola elliptica

CDB38233 B11

DCCDB023-21

BOLD:AEI9183

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

Torrenticola ungeri

19. M19_24_6_E10

DNAEC057-20

BOLD:AED2307

Bar, Međurječka rijeka stream

42.0226N, 19.22E

20. M19_24_6_E11

DNAEC058-20

Bar, Međurječka rijeka stream

42.0226N, 19.22E

CCDB-3867-D08

DNCBD044-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-G07

DNCBD079-20

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

Pseudotorrenticola rhynchota

CCDB-3867-B02

DNCBD014-20

BOLD:AEF1632

Bar, stream in Godinje Village

42.2206N, 19.1118E

CCDB-3867-B03

DNCBD015-20

Bar, stream in Godinje Village

42.2206N, 19.1118E

Limnesiidae

Limnesia undulata

CCDB-3867-C05

DNCBD029-20

BOLD:AAX5286

Tuzi, Vitoja, pools

42.324N, 19.3637E

CCDB 38363 C03

SEPTA027-21

Tuzi, Skadar Lake at Podhum

42.3139N, 19.3534E

Hygrobatidae

Atractides fluviatilis

CCDB-3867-G10

DNCBD082-20

BOLD:AEF1143

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

Atractides fissus

CCDB38233 B03

DCCDB015-21

BOLD:AEI1811

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

CCDB38233 D10

DCCDB046-21

Mojkovac, Bistrica stream

42.9871N, 19.4338E

Atractides anae

1. CG2020_8 B3

DNAEC014-20

BOLD:AED1201

Berane, spring nr. Mon. Djurdjevi Stupovi

42.8527N, 19.862E

Atractides inflatipalpis

29. M19_24_4_F7

DNAEC064-20

BOLD:AED3549

Bar, Međurječka rijeka stream

42.0226N, 19.22E

Atractides inflatipes

CCDB-3867-G03

DNCBD075-20

BOLD:AEF1144

Bar, Orahovštica stream

42.2476N, 19.0798E

Atractides fonticolus

CCDB38233 B09

DCCDB021-21

BOLD:AEI8720

Podgorica, Pričelje, spring Studenac

42.4835N, 19.2429E

CCDB38233 B08

DCCDB020-21

Podgorica, Pričelje, spring Studenac

42.4835N, 19.2429E

Atractides gibberipalpis

CCDB 38361 C07

DCDDJ031-21

BOLD:AEK7766

Mojkovac, Bistrica stream

42.8054N, 19.4456E

CCDB38233 B02

DCCDB014-21

BOLD:AEI3946

Kolašin, Kolašinska rijeka stream

42.8391N, 19.5749E

Atractides inflatus

14. M19_12_4_E5

DNAEC052-20

BOLD:ACB4677

Budva, Lastva Grbaljska, first order stream

42.3103N, 18.8138E

Atractides nodipalpis

CCDB-3867-F07

DNCBD067-20

BOLD:ACR0209

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

41. M19_29A_1_G5

DNAEC072-20

Danilovgrad, Zeta River at Spuž

42.5113N, 19.1982E

CCDB-3867-F05

DNCBD065-20

BOLD:AED3547

Danilovgrad, spring below the bridge

42.5542N, 19.1059E

40. M19_29A_1_G4

DNAEC071-20

Danilovgrad, Zeta River at Spuž

42.5113N, 19.1982E

Atractides pennatus

CCDB-3867-F11

DNCBD071-20

BOLD:ADF7007

Bar, Orahovštica stream

42.2476N, 19.0798E

CCDB-38679-A09

DNCBD009-20

Bar, Poseljani, Poseljanski stream

42.3057N, 19.0557E

25. CG2020_9_C6

DNAEC028-20

Podgorica, Mareza spring

42.4801N, 19.1821E

23. CG2020_9_C5

DNAEC027-20

Podgorica, Mareza spring

42.4801N, 19.1821E

3. CG2020_2 B4

DNAEC015-20

Žabljak, Sedlo, Studenac spring

43.0973N, 19.0702E

32. M19_23_1_F10

DNAEC067-20

Nikšić, Vidrovan, Vukovo Vrelo spring

42.8575N, 18.9414E

31. M19_23_1_F9

DNAEC066-20

Nikšić, Vidrovan, Vukovo Vrelo spring

42.8575N, 18.9414E

4. M19_22_1 D8

DNAEC042-20

Nikšić, spring in Miločani Village

42.8265N, 18.9018E

CCDB 38363 C01

SEPTA025-21

Budva, spring Smokov Vijenac

42.2346N, 18.907E

CCDB 38363 B12

SEPTA024-21

Budva, spring Smokov Vijenac

42.2346N, 18.907E

Atractides robustus

CCDB-3867-D12

DNCBD048-20

BOLD:ADZ9348

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-D11

DNCBD047-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB-3867-D10

DNCBD046-20

Bar, Rikavac stream above Old Bar

42.1001N, 19.1432E

CCDB 38361 H02

DCDDJ086-21

Kolašin, spring nr. Monastir Morača

42.7668N, 19.3906E

CCDB38233 B01

DCCDB013-21

Kolašin, Kolašinska Rijeka stream

42.8391N, 19.5749E

Atractides latipes

18. M19_08B_7_E9

DNAEC056-20

BOLD:AED4000

Podgorica, River Cijevna at Trgaja

42.3964N, 19.3798E

Atractides stankovici

CCDB38233 C08

DCCDB032-21

BOLD:AED3550

Dnilovgrad, River Zeta near Slap

42.6001N, 19.0656E

CCDB38233 C07

DCCDB031-21

Danilovgrad, River Zeta near Slap

42.6001N, 19.0656E

13. CG2020_4 B10

DNAEC020-20

Podgorica, Mareza canal

42.479N, 19.1813E

14. CG2020_4 B11

DNAEC021-20

Podgorica, Mareza canal

42.479N, 19.1813E

Hygrobates calliger

CCDB 38361 C06

DCDDJ030-21

BOLD:AEL5782

Kolašin, Crkvine, Bistrica stream

42.8054N, 19.4456E

CCDB-38679-A04

DNCBD004-20

BOLD:AEF4261

Bar, Poseljanski stream at Poseljani

42.3095N, 19.0518E

CCDB-38679-A03

DNCBD003-20

Bar, Poseljanski stream at Poseljani

42.3095N, 19.0518E

Hygrobates foreli

Hyd_MN_VP6

DNAEC080-20

BOLD:AEE3281

Žabljak, Mlinski potok stream

43.1494N, 19.0898E

Hygrobates lacrima

27. CG2020_3_C7

DNAEC029-20

BOLD:AED2490

Kolašin, Tara River near Mateševo

42.7897N, 19.5383E

Hygrobates limnocrenicus

13. M19_20_5_E4

DNAEC051-20

BOLD:AED2489

Podgorica, Mareza canal

42.479N, 19.1813E

Hygrobates longipalpis

CCDB-3867-C07

DNCBD031-20

BOLD:ACR9783

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB-3867-C09

DNCBD033-20

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB-38679-A10

DNCBD010-20

Bar, Poseljani, Poseljanski stream

42.3057N, 19.0557E

CCDB 38363 C04

SEPTA028-21

Tuzi, Skadar Lake at Podhum

42.3139N, 19.3534E

Hygrobates mediterraneus

7. M19_24_2_D11

DNAEC046-20

BOLD:AED2190

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

8. M19_24_2_D12

DNAEC047-20

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

36. M19_24_1_G1

DNAEC070-20

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

CCDB-3867-F01

DNCBD061-20

Bar, Medjurječka rijeka stream

42.0363N, 19.2179E

Hygrobates norvegicus

Hyd_MN_VP3

DNAEC077-20

BOLD:ACH7323

Šavnik, spring Kikov izvor near Boan

42.9465N, 19.1893E

CCDB 38361 A01

DCDDJ001-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

CCDB 38361 A02

DCDDJ002-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

CCDB 38361 A07

DCDDJ007-21

Kolašin, Lalevića Dolovi, spring #1

42.899N, 19.631E

Unionicolidae

Neumania imitata

15. M19_29C_2_E6

DNAEC053-20

BOLD:AED4073

Danilovgrad, River Zeta at Spuž

42.5113N, 19.1982E

Neumania limosa

CCDB-3867-C10

DNCBD034-20

BOLD:AEF5902

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB-3867-C01

DNCBD025-20

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB38233 G06

DCCDB078-21

Tuzi, Vitoja, pool

42.324N, 19.3637E

Unionicola minor

CCDB-3867-G12

DNCBD084-20

BOLD:AEF4865

Ulcinj, Šasko Lake

41.9768N, 19.3389E

CCDB 38361 C02

DCDDJ026-21

Ulcinj, Šasko Lake

41.9768N, 19.3389E

CCDB 38361 C05

DCDDJ029-21

Ulcinj, Šasko Lake

41.9768N, 19.3389E

CCDB 38363 B09

SEPTA021-21

Tuzi, Vitoja, pool

42.324N, 19.3637E

CCDB 38361 C04

DCDDJ028-21

BOLD:AAU0335

Ulcinj, Šasko Lake

41.9768N, 19.3389E

Unionicola ypsilophora

CCDB 38363 D04

SEPTA040-21

Cetinje, River Cnojevica (Anodonta exulcerata)

42.3546N, 19.0286E

Piona damkoehleri

CCDB 38361 B03

DCDDJ015-21

BOLD:AEK5107

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

CCDB 38361 B04

DCDDJ016-21

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

CCDB 38361 B05

DCDDJ017-21

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

CCDB 38361 B06

DCDDJ018-21

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

CCDB 38361 B07

DCDDJ019-21

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

Piona laminata

CCDB 38361 A12

DCDDJ012-21

BOLD:AEL3248

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

Piona disparilis

Hyd_MN_VP12

DNAEC085-20

BOLD:AEE3977

Šavnik, spring of Bukovica stream, pool

43.0589N, 19.1103E

CCDB 38363 A08

SEPTA008-21

Nikšić, Vukovo Vrelo spring, pool

42.8577N, 18.9416E

CCDB 38363 A09

SEPTA009-21

Nikšić, Vukovo Vrelo spring, pool

42.8577N, 18.9416E

Typhis torris

CCDB-3867-C08

DNCBD032-20

BOLD:AEF2208

Tuzi, Vitoja, pool

42.324N, 19.3637E

Typhis ornatus

CCDB 38361 B01

DCDDJ013-21

BOLD:ACS0401

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

CCDB 38361 B02

DCDDJ014-21

Danilovgrad, Moromiš pond

42.5322N, 19.1993E

Wettinidae

Wettina lacustris

30. M19_20_4_F8

DNAEC065-20

BOLD:ADL2726

Podgorica, Mareza canal

42.479N, 19.1813E

Mideopsidae

Mideopsis milankovici

22. M19_24_2_E12

DNAEC059-20

BOLD:AED2191

Bar, Medjurječka rijeka stream

42.0226N, 19.22E

Mideopsis roztoczensis

CCDB-38679-A02

DNCBD002-20

BOLD:ACI1492

Cetinje, Poseljanski stream

42.3095N, 19.0518E

CCDB-3867-G05

DNCBD077-20

Podgorica, Cijevna River at Dinoša

42.4057N, 19.3569E

CCDB38233 C12

DCCDB036-21

Danilovgrad, Zeta River at Spuž

42.5112N, 19.1991E

CCDB38233 C11

DCCDB035-21

Danilovgrad, Zeta River at Spuž

42.5112N, 19.1991E

CCDB 38363 D07

SEPTA043-21

Danilovgrad, Zeta River at Vranjske Njive

42.4683N, 19.2579E

Athienemanniidae

Mundamella germanica

1. KIA_20B_D6

DNAEC041-20

BOLD:AED6269

Danilovgrad, Spuž, spring near Zeta River

42.5113N, 19.1982E

Aturidae

Hexaxonopsis serrata

CCDB 38363 B01

SEPTA013-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

CCDB 38363 B02

SEPTA014-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

CCDB 38363 B03

SEPTA015-21

Bar, Skadar Lake at Murići

42.1637N, 19.2214E

Parabrachypoda montii

5. M19_20_6_D9

DNAEC044-20

BOLD:AED5455

Podgorica, Mareza canal

42.479N, 19.1813E

Woolastokia rotundifrons

10. M19_27_2_E1

DNAEC048-20

BOLD:AEE0289

Šavnik, Tušina River at Boan

42.9432N, 19.205E

11. M19_27_2_E2

DNAEC049-20

Šavnik, Tušina River at Boan

42.9432N, 19.205E

Arrenuridae

Arrenurus cylindratus

34. M19_20_1_F12

DNAEC069-20

BOLD:AED6864

Podgorica, Mareza canal

42.479N, 19.1813E

Arrenurus refractarioulus

CCDB 38363 A07

SEPTA007-21

Nikšić, Lukavica Mt., pools

42.8118N, 19.1872E

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 (Edgar 2004). Intra- and interspecific genetic distances were calculated, based on the Kimura 2-parameter model (K2P; Kimura 1980), using MEGA-X, version 10.1 (Kumar et al. 2018). The Neighbour-Joining (NJ) tree (edited in MEGA7, Kumar et al. 2016), based on K2P distances and pairwise deletion of missing data, was used to visualise similarity. The support for tree branches was calculated by the non-parametric bootstrap method (Felsenstein 1985) with 1000 replicates and shown next to the branches.

Results

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. Raupach et al. 2015).

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 1.

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 2.

Table 2.

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 3 showed a maximum interspecific divergence larger than 2%, resulting in these species in BOLD being spread over more than one BIN. Two species, Lebertia glabra Thor, 1897 and L. inaequalis appeared each with 3 BINs and twelve species, i.e. Lebertia maculosa Koenike, 1902, L. porosa Thor, 1900, Sperchon brevirostris Koenike, 1895, S. clupeifer Piersig, 1896, Sperchonopsis verrucosa (Protz, 1896), Monatractides madritensis (K. Viets, 1930), Torrenticola meridionalis, T. laskai Di Sabatino, 2009, Atractides gibberipalpis Piersig, 1898, A. nodipalpis Thor, 1899, Hygrobates calliger Piersig, 1896 and Unionicola minor (Soar, 1900), each with 2 BINs (Table 3). In total, unique BINs were revealed for 72 species (83.7%), two BINs for 12 species (14.0%) and three BINs for two species (2.3%).

Table 3.

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

BOLD:ACS0595

12.52

Lebertia glabra

BOLD:ACS0595

20

0.64

1.36

NL, BG, ME, MK, IT, PL, SK

BOLD:AEJ3212

2.88

Lebertia glabra

BOLD:AEI925

3

0.64

0.96

ME

BOLD:ACO2179

12.02

2.

Lebertia inaequalis

BOLD:AEF5913

1

N/A

N/A

ME

BOLD:ADF6223

2.78

Lebertia inaequalis

BOLD:ADF6223

18

0.18

0.34

NL, PL, ME

BOLD:AEF5913

2.78

Lebertia inaequalis

BOLD:AEF2742

1

N/A

N/A

ME

BOLD:AEB4193

6.96

3.

Lebertia maculosa

BOLD:AED9718

3

1.27

1.6

ME, MK

BOLD:AED9197

2.76

Lebertia maculosa

BOLD:AED9197

2

0.16

0.16

ME

BOLD:AED9718

2.76

4.

Lebertia porosa

BOLD:ACS0974

133

0.81

2.37

NL, FR, ME, DE, GB, BG, IT, PL, SK, ES, CH

BOLD:AED4662

3.89

Lebertia porosa

BOLD:AED4662

12

0.14

0.85

ME

BOLD:ACS0974

3.89

5.

Sperchon brevirostris

BOLD:AED3857

3

0.32

0.48

ME, MK

BOLD:AEK3053

2.72

Sperchon brevirostris

BOLD:ACP6107

28

0.55

3.12

NO, DE, ME

BOLD:AED3857

7.53

6.

Sperchon clupeifer

BOLD:ACS1100

11

1.68

3.47

NL, DE, NO, MK, ME, RU

BOLD:AEE4061

8.7

Sperchon clupeifer

BOLD:AEE4061

1

N/A

N/A

ME

BOLD:ACS1100

8.7

7.

Sperchonopsis verrucosa

BOLD:AEK8297

1

N/A

N/A

ME, RO

BOLD:ACS0908

4.83

Sperchonopsis verrucosa

BOLD:ACS9705

9

0.29

0.97

NO, IT, ME

BOLD:ADU8190

9.83

8.

Monatractides madritensis

BOLD:AED3803

2

0.16

0.16

ME

BOLD:AEL3852

1.44

Monatractides madritensis

BOLD:AEL3852

2

0.64

0.64

ME, SR

BOLD:AED3803

1.44

9.

Torrenticola meridionalis

BOLD:AED7519

8

1.46

2.25

ME, MK

BOLD:AEI3402

6.57

Torrenticola meridionalis

BOLD:AEI3402

4

1.42

2.09

ME

BOLD:AEK9662

6.25

10.

Torrenticola laskai

BOLD:AEF5471

2

0.32

0.32

ME

BOLD:AED2306

2.17

Torrenticola laskai

BOLD:AED2306

4

0.82

1.34

RS, ME, RO

BOLD:AEF5471

2.17

11.

Atractides gibberipalpis

BOLD:AEK7766

1

N/A

N/A

ME

BOLD:AEI3946

4.81

Atractides gibberipalpis

BOLD:AEI3946

1

N/A

N/A

ME

BOLD:AEK7766

4.81

12.

Atractides nodipalpis

BOLD:ACR0209

41

0.59

3.05

NO, NL, GL, DE, ME, RS

BOLD:AED3548

13.3

Atractides nodipalpis

BOLD:AED3547

2

0

0

ME

BOLD:AAM4306

13.3

13.

Hygrobates calliger

BOLD:AEF4261

2

1.2

1.2

NO, ME

BOLD:AEK4720

16.18

Hygrobates calliger

BOLD:AEL5782

2

1.03

1.03

DE, ME

BOLD:AEK4720

14.61

14.

Unionicola minor

BOLD:AEF4865

3

0.59

0.7

ME

BOLD:ACI7165

17.02

Unionicola minor

BOLD:AAU0335

7

0.09

0.32

NO, NL, ME

BOLD:ACH3803

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 2).

Discussion

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 Pešić et al. (2018) and papers published thereafter (Pešić et al. 2019c, Pešić et al. 2020a, Pešić et al. 2020c, Pešić et al. 2020d, Pešić and Smit 2020). BOLD and RESL (OTU) analyses revealed 98 BINs and 103 OTUs, respectively, highlighting the high molecular diversity of the water mite fauna of Montenegro.

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 (Pešić et al. 2020a, Pešić et al. 2020c, Pešić et al. 2020d, Pešić and Smit 2020). All of these studies highlighted the importance of an integrated approach that combines the morphology-based taxonomy and DNA barcodes.

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 1). On the other hand, the intraspecific maximum distances between BINs within each of the other eleven species in the dataset were greater than 5% (Suppl. material 1).

Lebertia glabra, a species widely distributed in West Palaearctic (Di Sabatino et al. 2010) appeared in our dataset with 3 BINs. The first cluster (BIN:ACR9598) includes two specimens from Montenegro and The Netherlands; the second cluster (BIN:ACS0595) was more represented in BOLD and includes specimens from different parts of Europe - from The Netherlands and Poland to Montenegro, Italy and Macedonia. The third cluster (BOLD:AEI925) contained only specimens from Montenegro. The intraspecific K2P distances between all clusters ranged from 14.3 to 17.7% (Suppl. material 1).

Lebertia inaequalis, a species reported from the extended parts of the Palaearctic (Gerecke 2009, Di Sabatino et al. 2010), appeared in our dataset with 3 BINs, two of which each include only one specimen from Montenegro (BIN:AEF5913 and BIN:AEF2742, respectively). The third cluster (BIN:ADF6223), based on available records from BOLD, appears to be more widespread and contained specimens from The Netherlands, Poland and Montenegro. Intraspecific K2P distances between the latter cluster and BIN:AEF5913 was only 0.1%, while the distance from the second cluster (BIN:AEF2742) from Montenegro was rather large (17.3%; Suppl. material 1) highlighting the necessity of additional comprehensive morphological and molecu­lar analysis.

Lebertia porosa, a eurytopic and eurythermous species, often reported from standing waters and pools of streams across the Holarctic (Gerecke 2009, Di Sabatino et al. 2010), is currently in the process of being revised (R. Gerecke, pers. communication) using DNA barcodes. Stur (2017) showed that 18 specimens of L. porosa from Norway comprise 7 BINs with a mean intraspecific p-distance of 11.7% and maximum up to 18.5%. In our dataset, specimens, morphologically assigned to Lebertia porosa, were presented with two clusters. Based on the available records from BOLD, the first cluster (BIN:ACS0974) appeared to be well represented in the BOLD database with 133 records from different parts of Europe; the second cluster (BIN:AED4662) contained specimens only from Montenegro. In our study, specimens of the latter BIN were collected in large limnocrenic springs, such as Mareza and Vitoja, while specimens from the first cluster (BIN:ACS0974) were sampled in the lower reaches. The intraspecific K2P distance between these two L. porosa clusters in our dataset was estimated at 5.5% (Suppl. material 1).

Sperchon brevirostris, a species inhabiting low-and middle order streams in the study area (Pešić et al. 2010, Pešić et al. 2018), was represented in our material by two clusters. Based on the available records from BOLD, the first cluster (BIN:ACP6107) includes specimens from Norway, Germany and one specimen from Montenegro, while the second cluster (BOLD:AED3857) contained three specimens from Montenegro and North Macedonia. The K2P distance between these two clusters was 8.1% (Suppl. material 1). Similarly, S. clupeifer, a species frequently reported from Western Palaearctic (Di Sabatino et al. 2010), appeared with two clusters in our dataset. The first cluster (BIN:ACS1100) is well represented in BOLD and includes specimens from different part of Europe, while the second cluster (BIN:AEE4061) contained a single specimen from Montenegro. The intraspecific K2P distance between these two clusters in our dataset was estimated at 8.3% (Suppl. material 1).

Sperchonopsis verrucosa, a species often reported from the Holarctic Region (Gerecke et al. 2016), was represented in our study with two clusters. The first cluster (BIN:ACS9705) was more represented in BOLD and includes specimens from Norway, Italy and one specimen from Montenegro. The second cluster (BIN:AEK8297) includes two specimens from Montenegro and Romania. The intraspecific K2P distances between these two clusters was 11.2%, indicating the need for additional integrative analysis.

Torrenticola meridionalis, a species originally described from Italy, is widely distributed in Montenegro, inhabiting mainly low order streams (Pešić et al. 2018). It is morphologically closely related to T. elliptica which remains distinguishable in the male sex only, based on the stouter genital field. In our COI tree (Suppl. material 2), T. elliptica appeared as a sister clade to the clade that includes two clusters morphologically assigned to T. meridionalis (BIN:AEI3402 and BOLD:AED7519, respectively). The intraspecific K2P distances between T. elliptica and T. meridionalis clusters ranged from 8.6-9.0%. On the other hand, the K2P distance between T. meridionalis clusters in our dataset was estimated at 6.6%.

Atractides nodipalpis, a rhitrobiontic species, is the most frequently reported species of the genus in Europe (Gerecke et al. 2016). In our dataset, sequences of the specimens, morphologically assigned to the latter species, appeared as two clusters. Interestingly, specimens of both clusters were recorded syntopically. The first cluster (BIN:ACR0209) in the BOLD database was represented with 41 specimens from Norway (country of the type locality), The Netherlands, Montenegro and Russia, but also from Greenland. The second cluster (BIN:AED3547) includes two specimens from Montenegro. The intraspecific K2P distance between these two clusters was 18.8%, indicating the need for a comprehensive revision of this species complex.

Hygrobates calliger, a rhitrobiontic species widely distributed in the Palaearctic (Di Sabatino et al. 2010), was represented by two clusters in our dataset, each with two records in the BOLD database. The first cluster (BIN:AEF4261) includes specimens from Norway and Montenegro, while the second cluster (BIN:AEL5782) includes specimens from Germany and Montenegro. The intraspecific K2P distance between these two clusters was 20.9% (Suppl. material 1), suggesting the existence of possible hidden cryptic and/or pseudocryptic species.

The sequences of Atractides gibberipalpis, a rhitrobiontic species often reported from the Palaearctic (Pešić et al. 2021a), in our dataset were assigned to two different barcode clusters, each represented by a single specimen from Montenegro. The intraspecific K2P distance between these two clusters (BIN: BOLD:AEK7766 and BIN: BOLD:AEI3946, respectively) was estimated at 5% (Suppl. material 1).

Unionicola minor, a species widely distributed in Europe (Gerecke et al. 2016), was presented with two clusters in our dataset. Based on available data from BOLD, the first cluster (BIN:AAU0335) includes specimens from Norway and The Netherlands and one specimen from Lake Šasko in Montenegro. The second cluster (BIN:AAU0335) includes specimens only from Montenegro. The intraspecific K2P distances between these two clusters in our dataset was 23.8% (Suppl. material 1), suggesting the existence of cryptic (or pseudocryptic, see Pešić and Smit (2016) for a discussion about pseudocryptic speciation in water mites) species. Stålstedt et al. (2013) showed that the Swedish population of Unionicola minor consists of at least three cryptic species, emphasising the need for further research of the species in this complex.

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.

Acknowledgements

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.

Ethics and security

No ethical principles were violated when providing this study.

Conflicts of interest

The authors declare no conflict of interests concerning this study.

References

Supplementary materials

Suppl. material 1: Molecular distances 
Authors:  Vladimir Pešić, Andrzej Zawal, Ana Manović, Aleksandra Bańkowska, Milica Jovanović
Data type:  Molecular distances
Brief description: 

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.

Suppl. material 2: Compact Neighbour-Joining tree 
Authors:  Vladimir Pešić, Andrzej Zawal, Ana Manović, Aleksandra Bańkowska, Milica Jovanović
Data type:  Neighbour-joining tree
Brief description: 

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.