Corresponding author: Milica Jovanović (
Academic editor: Yasen Mutafchiev
Glossiphoniid leeches are a diverse group and sometimes abundant elements of the aquatic fauna inhabiting various types of freshwater habitats. In this study, we sampled leeches of the genus
No ethical principles were violated when providing this study.
The authors declare no conflict of interests concerning this study.
Species of the family
Distribution and species boundaries of the leeches of the genus
At present, all of the European members of the genus
In this study, we applied a standard DNA barcoding marker, a fragment of the COI gene, to analyse specimens of the genus
Glossiphoniid leeches were collected from twenty-two sites in seven countries: Albania, Austria, Bosnia and Hercegovina, Germany, Kosovo, Montenegro and North Macedonia (Fig.
Morphological analysis of 33 individuals was performed using a stereomicroscope (Novex). Leeches were identified to species level according to
DNA analysis was conducted in the Central Research Laboratories of the NHM. Leeches fixed in 96% ethanol were stored at 4°C. Tissue samples from individuals (approx. 2 × 2 × 2 mm) were separated using sterile scalpels and tweezers. DNA was extracted with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The final volume of DNA solution was 40 µl.
The present study focused on the COI gene, which was amplified using a polymerase chain reaction (PCR). For all sampled leeches, a 708 bp section was amplified, which contains the standard DNA barcoding region. The universal primers LCO1490, 5′GGTCAACAAATCATAAAGATATTGG-3′ and HCO2198, 5′TAAACTTCAGGGTGACCAAAAAATCA-3′ (
Each reaction consisted of 0.5 units of TopTaq DNA polymerase (Qiagen), 2.5 µl 10× TopTaq PCR Buffer, 10 mM of each dNTP, 50 µM of each primer and 1 µl DNA template in a total reaction volume of 25 µl. The PCR cycling protocol included an initial denaturation at 94°C for 3 min, followed by 35 cycles of of denaturation at 94°C for 30 s, annealing for 30 s at 52°C and extension for 1 min at 72°C. The final step was an extension at 72°C for 10 min and a hold at 10°C.
The amplicons were checked by (1%) agarose gel electrophoresis. The QIAquick PCR Purification Kit (Qiagen) was employed to purify amplifications products. Sequencing was performed in both directions at Microsynth (Balgach, Switzerland) using the PCR primers.
Sequences (both strands) were checked and edited using BioEdit (
In order to assess the genetic differentiation of species within our dataset of 47
Morphological analysis of 29 specimens of the leech genus
The final alignment comprised 52 sequences and had a length of 658 nucleotide sites. Of the 33 COI sequences generated in the present study, all were included in the final dataset for the phylogenetic analysis.
Both the ML and NJ trees, based on COI sequences, were in agreement regarding the general topology. The ML tree is shown in Fig.
In both the NJ and ML trees,
For the fourth species sequenced in the present study,
Species from the two genera,
The mean K2P values between the morphologically determined species of
The highest mean intraspecific distances were observed within
For the ASAP analysis, the sequences of
Finally, the mPTP analysis grouped the
Morphological analysis of the examined leeches of the genus
In the course of the present study, only a single
Our results suggest that some specimens, represented by published sequences in our dataset, were probably misidentified. Sequences of two samples from one unnamed river in Croatia (
Our study revealed that
Concerning the species delimitation analyses, the different results of the approaches did not provide convincing conclusions. For example, the mPTP analysis combined
The obtained barcode gaps of 4–8% K2P in our dataset of COI sequences is comparable to an interspecific threshold of 5–7% distance that
Financial support was partly provided within the programme for Scientific & Technological Cooperation in the framework of the bilateral Agreement between the Government of the Republic of Austria and the Government of the Republic of Montenegro. Assistance given by Julia Schindelar and Bärbara Tautscher with the molecular genetic analysis was greatly appreciated. Dejan Dmitrović and Violeta Bërlajolli contributed this study with leech material from Bosnia & Herzegovina and Kosovo. We thank Joachim Händel for providing us with the photograph of
The authors declare no conflict of interests concerning this study.
Distribution map of localities where glossiphoniid leeches were collected. (red dots – present study records, blue dots – records from previous studies where coordinates are available). Note that each point may represent more than one species. Country codes of those countries, from which we had material, are indicated. The map was created using QGIS 2.8.11 software.
Photographs of selected leeches of
Maximum Likelihood tree of
Taxon names, locality information and accession numbers for the specimens used in phylogenetic analysis and distance estimations. Newly-sequenced taxa are shown in bold font. BOLD accession numbers are given for the sequences produced in the present study, while GenBank accession numbers are provided for published sequences.
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ROMIZ I11753 | Unnamed river, Croatia (CRO) |
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ROMIZ I11755 | Unnamed river, Croatia (CRO) |
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Rio Sadde, Italy (IT) |
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Chechuy River, Russia (RUS) |
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Lake near Meget, Russia (RUS) |
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BH1_1 | Krupa River near Vrbas, Bosnia and Hercegowina (BH) |
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This study |
MN1_1 | Karuč spring, Podgorica, Montenegro (MN) |
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This study |
MN1_2 | Karuč spring, Podgorica, Montenegro (MN) |
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This study |
MN2_1 | River Crnojevića, Cetinje, Montenegro (MN) |
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This study |
MN5_1 | Vitoja spring pool, Podgorica, Montenegro (MN) |
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This study |
MN6_1 | Dobro polje spring, Danilovgrad, Montenegro (MN) |
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This study |
MN7_1 | Mareza spring, Podgorica, Montenegro (MN) |
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This study |
MN8_1 | Karuč spring, Podgorica, Montenegro (MN) |
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This study |
AUS_Hir:2_1 | Kalte Wien, Vienna, Austria (AUS) |
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This study |
Gcomp1 | Stream from the lake Barschsee, Mecklenburg-Vorpommern, Germany, type locality (GER) |
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This study |
Gcomp2 | Stream from the lake Barschsee, Mecklenburg-Vorpommern, Germany, type locality(GER) |
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This study |
Gcomp3 | Small stream near Jesewitz, Saxony, Germany (GER) |
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This study |
Durance river, France (FR) |
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Europe (EU) |
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Creek, Mecklenburg-Vorpommern, |
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United Kingdom (UK) |
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ROMIZ I11750 | Korana river, Croatia (CRO) |
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ROMIZ I11749 | Korana river, Croatia (CRO) |
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ROMIZ I11748 | Korana river, Croatia (CRO) |
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ROMIZ I11717 | Sava river, Slovenia (SLO) |
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ROMIZ I11743 | Gacka river, Croatia (CRO) |
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MAC1_1 | St. Naum spring of Crni Drim, Ohrid Lake, North Macedonia (MAC) – type locality |
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This study |
MAC2_1 | Lagadin, Ohrid Lake, North Macedonia (MAC) |
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This study |
MAC2_2 | Lagadin, Ohrid Lake, North Macedonia (MAC) |
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This study |
MAC3_1 | Peštani, Ohrid Lake, North Macedonia (MAC) |
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This study |
MAC4_1 | Oteševo, Prespa Lake, North Macedonia (MAC) |
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This study |
ALB1a_d | Pogradec, Ohrid Lake, Albania (ALB) |
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This study |
ALB1a_1 | Pogradec, Ohrid Lake, Albania (ALB) |
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This study |
ALB3b_2 | Tushemisht, Ohrid Lake, Albania (ALB) |
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This study |
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Gconc1 | Krakower Obersee, Mecklenburg-Vorpommern, Germany (GER) |
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This study |
Kila river, Sweden (SWE) |
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Ukraine (UKR) |
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Gbalc1 | Toplla spring, Dečani, Kosovo (KOS) - type locality |
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This study |
Gbalc2 | Toplla spring, Dečani, Kosovo (KOS) - type locality |
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This study |
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Gnebu4 | Berliner Chausssee stream Nieplitz, Berlin, Germany -type locality |
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This study |
Yamalo-Nenets Autonomous Okrug, Russia (RUS) |
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KOS1_1 | Spring KS 40, Peje, Kosovo (KOS) |
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This study |
KOS1_2 | Spring KS 40, Peje, Kosovo (KOS) |
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This study |
BH3_1 | Banja Luka, Near castle, Bosnia & Herzegovina (BH) |
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This study |
Gnebu1 | Toplla spring, Dečani, Kosovo (KOS) |
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This study |
Gnebu3 | Toplla spring, Dečani, Kosovo (KOS) |
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This study |
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ROMIZ I11505 | Unknown pond, Nopiming, Manitoba, Canada (CAN) |
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Lake Bemidji, Beltrami County, Minnesota, (USA) |
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Lake Baikal, Russia (RUS) |
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MN4_1 | Oraška jama spring, Danilovgrad, Montenegro (MN) |
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This study |
MN9_1 | Crno oko spring, Podgorica, Montenegro (MN) |
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This study |
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BH3_2 | Near castle, Banja Luka, Bosnia & Herzegovina (BH) |
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This study |
MAC4_2 | Oteševo, Prespa Lake, North Macedonia (MAC) |
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This study |
Table S1.
Table
Interspecific mean K2P distances (below diagonal) and mean
File: oo_543534.docx