# Genetic Diversity of Freshwater Leeches in Lake Gusinoe (Eastern Siberia, Russia)

Authors: Irina A. Kaygorodova; Nadezhda Mandzyak; Ekaterina Petryaeva; Nikolay M. Pronin
Journal: The Scientific World Journal
 (2014)

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## Abstract

The study of leeches from Lake Gusinoe and its adjacent area offered us the possibility to determine species diversity. As a result, an updated species list of the Gusinoe Hirudinea fauna (Annelida, Clitellata) has been compiled. There are two orders and three families of leeches in the Gusinoe area: order Rhynchobdellida (families Glossiphoniidae and Piscicolidae) and order Arhynchobdellida (family Erpobdellidae). In total, 6 leech species belonging to 6 genera have been identified. Of these, 3 taxa belonging to the family Glossiphoniidae (Alboglossiphonia heteroclita f. papillosa, Hemiclepsis marginata, and Helobdella stagnalis) and representatives of 3 unidentified species (Glossiphonia sp., Piscicola sp., and Erpobdella sp.) have been recorded. The checklist gives a contemporary overview of the species composition of leeches and information on their hosts or substrates. The validity of morphological identification of each taxon has been verified by phylogenetic approach with a molecular marker adopted for a DNA barcoding of most invertebrates.


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## Body

## 1. Introduction

Lake Gusinoe located 60 km from the famous Lake Baikal is one of the largest freshwater bodies in the Baikal basin and the largest in the Trans-Baikal region. The origin of this lake goes back to the period of 1740–1749 according to the data collected by the exiled Decembrist Nikolay Bestuzhev. The lake formed as a result of breakthrough of the Temnik River runoff (a tributary of the Selenga) towards the Lake Gusinoe depression and the partial filling of the depression via Lake Tsaidam by Tsagan-Ghol channel [1]. To date, the water area of Lake Gusinoe is 164 km2, the volume of water mass is 2.4 km3, the length is about 24.8 km, the width is from 5 to 8.5 km, the coastline is 62 km, and maximal depth is up to 28 m averaging 15 m [2]. The catchment area of the Gusinoe basin is 924 km2. It has a well-developed fluvial net. In total, there are 72 rivers and creeks with a total length of 312 km [3]. The average density of the river network basin is 0.34 km/km2, which is comparable with its great neighbour such as Lake Baikal. The longest tributary of the lake is the Zagustay River. Its length and catchment area are 44 km and 382 km2, respectively (Figure 1).Figure 1
Geographical location of the study region with indication of the main sampling sites in Lake Gusinoe, Lake Tsaidam, and Zagustay River.Taxonomic and ecological diversity of the lake biota has been studied insufficiently and unsystematically. Thus, only preliminary information on the existence of 6 Hirudinea species (Erpobdella octoculata, Glossiphonia complanata, G. heteroclita, Helobdella stagnalis, Hemiclepsis marginata, andPiscicola geometra) was available without the description of their biology and ecology [4]. Furthermore, the species identification was highly doubtful and needed clarification. Meanwhile, leeches are an important part of aquatic biota not only as an element in the trophic level, but also as parasites of other hydrobionts. Interest to Hirudinea sp. has been increased in recent years because of its possible relationship to transmission of bacterial and viral infections [5–9], as well as hematozoa including trematodes, cestodes, and nematodes [10] and parasitic flagellates [11–13], which are considered to be pathogenic organisms for aquatic animals. Moreover, ulceration, hemorrhage, and inflammation associated with leech attachment sites weaken the host undoubtedly and may predispose hosts to bacterial infections.Sequencing of particular mitochondrial genes in animals, such as Folmer’s fragment of cytochromec oxidase subunit I (COI) gene, can yield phylogenetic information and aid in the identification of species. For leeches, DNA barcoding could be particularly useful, as their identification using standard taxonomic techniques can often be ambiguous [14]. In this study, COI sequences of leech species were obtained for specimens collected in different sites of Lake Gusinoe and in eastern Ukraine, resulting in the addition of 14 leech sequences to the GenBank database. Using COI sequences of closely related species that already existed in GenBank, phylogenetic interrelationships were traced out. Genetic diversity was revealed and evaluated. Morphological data were used to corroborate DNA identification.


## 2. Material and Methods

### 2.1. Sample Collection

Biological material was collected during expeditions to Lake Gusinoe in 2012-2013 (Figure1). Additional samples were collected in the adjacent water bodies, the Zagustay River flowing into the lake in its northern part and Lake Tsaidam adjoining to the southeastern shore of Lake Gusinoe and connecting with the latter by a channel.To collect leeches, we have inspected various underwater objects (macrophytes, rotten wood, driftwood, snags, stones, etc.) to which hirudinids could be attached. In most cases, piscine leeches were collected directly from the fish caught with different fishing tackles such as fishing net and hydrobiological net or were found on aquatic vegetation (like water milfoil, pondweed, and waterweed).The leech specimens were directly fixed in 80% ethanol. Alcoholised leech tissues retain flexibility, making investigation of annulation and allocation of genital openings easier. Moreover, such material is suitable for further molecular analysis.Morphological analysis was performed using a stereomicroscope MSP-2 var. 2 (LOMO). [15–17]. Reference specimens were deposited in the zoological collection of the Laboratory of Molecular Systematics, Limnological Institute.


### 2.2. DNA Sequencing

Sequences of the mitochondrial cytochromec oxidase subunit I (COI) were newly generated from 12 specimens from the Lake Gusinoe area and two individuals ofGlossiphonia concolor were from Ukraine (Table 1).Table 1
Taxa, localities, information on host species (if known), and GenBank accession numbers for COI sequences of leeches used in phylogenetic analysis.
Species
Collection locality
Host species/substrate
GenBank accession number
Order Arhynchobdellida
Family Erpobdellidae
Dina krilata
Lake Ohrid, Albania
HM246629
Dina latestriata
Lake Prespa, Macedonia
HM246600
Dina lepinja
Lake Ohrid, Albania
HM246597
Dina lineata
Ligemii Bogovines, Macedonia
HM246611
Dina lyhnida
Lake Ohrid, Macedonia
HM246589
Dina ohridana
Lake Ohrid, Macedonia
HM24663
Dina svilesta
Lake Ohrid, Macedonia
HM246598
Erpobdella japonica
Korea
AF116026
Erpobdella japonica
Nagano, Japan
AB679654
Erpobdella mexicana
Fuentes Brotantes, Mexico
DQ235595
Erpobdella monostriata
Mecklenburg-Vorpommern, Germany
HM246601
Erpobdella montezuma
Arizona, USA
GQ368760
Erpobdella nigricollis
Mecklenburg-Vorpommern, Germany
HM246603
Erpobdella obscura
Ontario, Canada
AF003273
Erpobdella ochoterenai
Xochimilco, Mexico
DQ235596
Erpobdella octoculata
Lake Ohrid, Macedonia
HM246555
Erpobdella octoculata
Uzbekistan
HQ336344
Erpobdella punctata
Ontario, Canada
AF003275
Erpobdella sp.
Lake Gusinoe, Russia
Stones
KM095091
Erpobdellasp.
Zagustay River, Russia
Stones
KM095092
Erpobdella testacea
Mecklenburg-Vorpommern, Germany
HM246602
Erpobdella triannulata
Catemaco, Mexico
DQ235602
Erpobdella vilnensis
Mecklenburg-Vorpommern, Germany
HM246551
E. vilnensis
Germany
DQ009663
E. vilnensis
Sachsen-Anhalt, Germany
HM246585
Family Salifidae
B. weberi
San Jose, Costa Rica
HQ336339
Order Rhynchobdellida
Family Glossiphoniidae
Placobdella costata
Italy
AY962461
Theromyzon bifarium
North America
AY047330
Theromyzon tessulatum
?
AY047318
Theromyzon pallens
France
AF003279
Theromyzon rude
Ontario, Canada
AF003262
Hemiclepsis marginata
France
AF003259
Hemiclepsis marginata
Lake Gusinoe, Russia
Stones
KM095093
Hemiclepsis marginata
Lake Tsaidam, Russia
Esox lucius
KM095094
Helobdella modesta
Ohio, USA
AF329040
Helobdella stagnalis
Cotswolds, UK
AF329041
Helobdella stagnalis
France
AF116018
Helobdella stagnalis
Lake Gusinoe, Russia
Stones
KM095095
Helobdella stagnalis
Lake Gusinoe, Russia
Stones
KM095096
Glossiphonia concolor
Ukraine
KM095097
Glossiphonia concolor
Ukraine
KM095098
Glossiphonia concolor
Kila Arn, Sweden
AY962458
Glossiphonia elegans
North America
AF003258
Glossiphonia  verrucata
Rio Sadde, Italy
AY962459
Glossiphonia sp.
Lake Gusinoe, Russia
Macrophytes
KM095099
Glossiphonia complanata
United Kingdom
AY047321
Glossiphonia complanata
Mecklenburg-Vorpommern, Germany
HM246608
Glossiphonia complanata
Aff stream, Paimpont, France
AF003277
Alboglossiphonia quadrata
Namibia
AY962455
Alboglossiphonia  lata
I-Lan County, Taiwan
AY962454
Alboglossiphonia  weberi
Hawaii, USA
AY962453
Alboglossiphonia heteroclita
Michigan, USA
AF116016
Alboglossiphonia heteroclita
Lake Gusinoe, Russia
Stones
KM095100
Alboglossiphonia heteroclita
Lake Gusinoe, Russia
Stones
KM095101
Alboglossiphonia heteroclita
Zagustay River, Russia
Snag
KM095102
family Piscicolidae
subfamily Piscicolinae
Piscicolasp.
Lake Gusinoe, Russia
Perca fluviatilis
KM095103
Piscicolasp.
Lake Gusinoe, Russia
macrophytes
KM095104
Cystobranchus meyeri
Tennessee, USA
DQ414315
Cystobranchus salmositicus
British Columbia, Canada
DQ414316
Cystobranchus virginicus
North Carolina, USA
DQ414317
Cystobranchus respirans
Sava River, Slovenia
AY336021
Johanssonia arctica
Varangerfjord, Norway
Paralithodes camtschatica
AY336012
Johanssonia arctica
Newfoundland, Canada
DQ414320
Limnotrachelobdella okae
Nevelskoy Strait, Russia
Huso dauricus
AY336022
Caspiobdella fadejewi
Elz bei Riegel, Germany
AY336020
Piscicola milneri
Quebec, Canada
DQ414337
Piscicola geometra
Siversky Donets River, Ukraine
AY336015
Piscicola geometra
Neckar-Altarm, Germany
AY336014
Piscicola geometra
Eyang de la Musse,   France
AF003280
Subfamily Platybdellinae
Pontobdella muricata
Gulf of Piran, Slovenia
AY336029Total DNA was extracted from a small portion of the posterior sucker of the leeches according to a slightly modified method using cetavlon [18]. This tissue was selected in order to avoid host blood contamination. COI gene fragments were amplified with oligonucleotide primers universal for most invertebrates [19] using 1.5 μL of primers at 10 μM, 4 μL of dNTP-mix at 10 μM, 0.25 μL of Taq polymerase, 5 μL buffer 10x, 1-2 μL DNA sample, and MilliQ H2O for a total volume of 50 μL. Reaction mixtures were preheated to 94°C for 1 min followed by 30 cycles of denaturation at 94°C (30 s), annealing at 47°C (40 s), and extension at 72°C (80 s), and final extension at 72°C (7 min).PCR fragments were sequenced at the CJSC “Syntol” (Moscow, Russia). The homologous gene sequences of 44 related Hirudinea species available from previous studies were also included in the present analyses for comparative purposes. GenBank accession numbers of all representative species are provided in Table1.


### 2.3. Bioinformatic Analyses

The sequences were aligned with the ClustalW [20] using default parameters and then were verified by hand. The final dataset matrix included 70 terminals and 709 aligned nucleotide characters. Phylogenetic analyses were carried out using maximum likelihood (ML) method implemented in MEGA V6.06 [21]. All distance values among COI sequences were calculated in MEGA V6.06 using a model of molecular evolution that was considered to describe the substitution pattern the best. Pairwise distance analyses were conducted using the Tajima-Nei model [22]. The differences in the composition bias among sequences were considered in evolutionary comparisons [23]. The percentage of trees in which the associated taxa clustered together was estimated using 500 bootstrap replications. Initial tree for the heuristic search was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. Comparisons of nucleotide sequences to sequence databases and estimation of the statistical significance of matches, as well as the search for regions of local similarity among the homologous DNA fragments, were performed using the BLAST program [24, 25].


## 2.1. Sample Collection

Biological material was collected during expeditions to Lake Gusinoe in 2012-2013 (Figure1). Additional samples were collected in the adjacent water bodies, the Zagustay River flowing into the lake in its northern part and Lake Tsaidam adjoining to the southeastern shore of Lake Gusinoe and connecting with the latter by a channel.To collect leeches, we have inspected various underwater objects (macrophytes, rotten wood, driftwood, snags, stones, etc.) to which hirudinids could be attached. In most cases, piscine leeches were collected directly from the fish caught with different fishing tackles such as fishing net and hydrobiological net or were found on aquatic vegetation (like water milfoil, pondweed, and waterweed).The leech specimens were directly fixed in 80% ethanol. Alcoholised leech tissues retain flexibility, making investigation of annulation and allocation of genital openings easier. Moreover, such material is suitable for further molecular analysis.Morphological analysis was performed using a stereomicroscope MSP-2 var. 2 (LOMO). [15–17]. Reference specimens were deposited in the zoological collection of the Laboratory of Molecular Systematics, Limnological Institute.


## 2.2. DNA Sequencing

Sequences of the mitochondrial cytochromec oxidase subunit I (COI) were newly generated from 12 specimens from the Lake Gusinoe area and two individuals ofGlossiphonia concolor were from Ukraine (Table 1).Table 1
Taxa, localities, information on host species (if known), and GenBank accession numbers for COI sequences of leeches used in phylogenetic analysis.
Species
Collection locality
Host species/substrate
GenBank accession number
Order Arhynchobdellida
Family Erpobdellidae
Dina krilata
Lake Ohrid, Albania
HM246629
Dina latestriata
Lake Prespa, Macedonia
HM246600
Dina lepinja
Lake Ohrid, Albania
HM246597
Dina lineata
Ligemii Bogovines, Macedonia
HM246611
Dina lyhnida
Lake Ohrid, Macedonia
HM246589
Dina ohridana
Lake Ohrid, Macedonia
HM24663
Dina svilesta
Lake Ohrid, Macedonia
HM246598
Erpobdella japonica
Korea
AF116026
Erpobdella japonica
Nagano, Japan
AB679654
Erpobdella mexicana
Fuentes Brotantes, Mexico
DQ235595
Erpobdella monostriata
Mecklenburg-Vorpommern, Germany
HM246601
Erpobdella montezuma
Arizona, USA
GQ368760
Erpobdella nigricollis
Mecklenburg-Vorpommern, Germany
HM246603
Erpobdella obscura
Ontario, Canada
AF003273
Erpobdella ochoterenai
Xochimilco, Mexico
DQ235596
Erpobdella octoculata
Lake Ohrid, Macedonia
HM246555
Erpobdella octoculata
Uzbekistan
HQ336344
Erpobdella punctata
Ontario, Canada
AF003275
Erpobdella sp.
Lake Gusinoe, Russia
Stones
KM095091
Erpobdellasp.
Zagustay River, Russia
Stones
KM095092
Erpobdella testacea
Mecklenburg-Vorpommern, Germany
HM246602
Erpobdella triannulata
Catemaco, Mexico
DQ235602
Erpobdella vilnensis
Mecklenburg-Vorpommern, Germany
HM246551
E. vilnensis
Germany
DQ009663
E. vilnensis
Sachsen-Anhalt, Germany
HM246585
Family Salifidae
B. weberi
San Jose, Costa Rica
HQ336339
Order Rhynchobdellida
Family Glossiphoniidae
Placobdella costata
Italy
AY962461
Theromyzon bifarium
North America
AY047330
Theromyzon tessulatum
?
AY047318
Theromyzon pallens
France
AF003279
Theromyzon rude
Ontario, Canada
AF003262
Hemiclepsis marginata
France
AF003259
Hemiclepsis marginata
Lake Gusinoe, Russia
Stones
KM095093
Hemiclepsis marginata
Lake Tsaidam, Russia
Esox lucius
KM095094
Helobdella modesta
Ohio, USA
AF329040
Helobdella stagnalis
Cotswolds, UK
AF329041
Helobdella stagnalis
France
AF116018
Helobdella stagnalis
Lake Gusinoe, Russia
Stones
KM095095
Helobdella stagnalis
Lake Gusinoe, Russia
Stones
KM095096
Glossiphonia concolor
Ukraine
KM095097
Glossiphonia concolor
Ukraine
KM095098
Glossiphonia concolor
Kila Arn, Sweden
AY962458
Glossiphonia elegans
North America
AF003258
Glossiphonia  verrucata
Rio Sadde, Italy
AY962459
Glossiphonia sp.
Lake Gusinoe, Russia
Macrophytes
KM095099
Glossiphonia complanata
United Kingdom
AY047321
Glossiphonia complanata
Mecklenburg-Vorpommern, Germany
HM246608
Glossiphonia complanata
Aff stream, Paimpont, France
AF003277
Alboglossiphonia quadrata
Namibia
AY962455
Alboglossiphonia  lata
I-Lan County, Taiwan
AY962454
Alboglossiphonia  weberi
Hawaii, USA
AY962453
Alboglossiphonia heteroclita
Michigan, USA
AF116016
Alboglossiphonia heteroclita
Lake Gusinoe, Russia
Stones
KM095100
Alboglossiphonia heteroclita
Lake Gusinoe, Russia
Stones
KM095101
Alboglossiphonia heteroclita
Zagustay River, Russia
Snag
KM095102
family Piscicolidae
subfamily Piscicolinae
Piscicolasp.
Lake Gusinoe, Russia
Perca fluviatilis
KM095103
Piscicolasp.
Lake Gusinoe, Russia
macrophytes
KM095104
Cystobranchus meyeri
Tennessee, USA
DQ414315
Cystobranchus salmositicus
British Columbia, Canada
DQ414316
Cystobranchus virginicus
North Carolina, USA
DQ414317
Cystobranchus respirans
Sava River, Slovenia
AY336021
Johanssonia arctica
Varangerfjord, Norway
Paralithodes camtschatica
AY336012
Johanssonia arctica
Newfoundland, Canada
DQ414320
Limnotrachelobdella okae
Nevelskoy Strait, Russia
Huso dauricus
AY336022
Caspiobdella fadejewi
Elz bei Riegel, Germany
AY336020
Piscicola milneri
Quebec, Canada
DQ414337
Piscicola geometra
Siversky Donets River, Ukraine
AY336015
Piscicola geometra
Neckar-Altarm, Germany
AY336014
Piscicola geometra
Eyang de la Musse,   France
AF003280
Subfamily Platybdellinae
Pontobdella muricata
Gulf of Piran, Slovenia
AY336029Total DNA was extracted from a small portion of the posterior sucker of the leeches according to a slightly modified method using cetavlon [18]. This tissue was selected in order to avoid host blood contamination. COI gene fragments were amplified with oligonucleotide primers universal for most invertebrates [19] using 1.5 μL of primers at 10 μM, 4 μL of dNTP-mix at 10 μM, 0.25 μL of Taq polymerase, 5 μL buffer 10x, 1-2 μL DNA sample, and MilliQ H2O for a total volume of 50 μL. Reaction mixtures were preheated to 94°C for 1 min followed by 30 cycles of denaturation at 94°C (30 s), annealing at 47°C (40 s), and extension at 72°C (80 s), and final extension at 72°C (7 min).PCR fragments were sequenced at the CJSC “Syntol” (Moscow, Russia). The homologous gene sequences of 44 related Hirudinea species available from previous studies were also included in the present analyses for comparative purposes. GenBank accession numbers of all representative species are provided in Table1.


## 2.3. Bioinformatic Analyses

The sequences were aligned with the ClustalW [20] using default parameters and then were verified by hand. The final dataset matrix included 70 terminals and 709 aligned nucleotide characters. Phylogenetic analyses were carried out using maximum likelihood (ML) method implemented in MEGA V6.06 [21]. All distance values among COI sequences were calculated in MEGA V6.06 using a model of molecular evolution that was considered to describe the substitution pattern the best. Pairwise distance analyses were conducted using the Tajima-Nei model [22]. The differences in the composition bias among sequences were considered in evolutionary comparisons [23]. The percentage of trees in which the associated taxa clustered together was estimated using 500 bootstrap replications. Initial tree for the heuristic search was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. Comparisons of nucleotide sequences to sequence databases and estimation of the statistical significance of matches, as well as the search for regions of local similarity among the homologous DNA fragments, were performed using the BLAST program [24, 25].


## 3. Results

### 3.1. Taxonomic Review and a Brief Description of Each Taxon

Collection of parasitic and nonparasitic leeches (Hirudinea, Clitellata) from 12 geographical sampling sites (Figure1) was performed during the two-year seasons in Lake Gusinoe and its catchment area. Morphological analysis revealed six leech species (Table 1: in bold) belonging to two orders (Rhynchobdellida, Arhynchobdellida), three families (Glossiphoniidae, Piscicolidae, and Erpobdellidae) and six genera (Helobdella,Hemiclepsis,Glossiphonia,Alboglossiphonia,Piscicola, andErpobdella). All six species were found in Lake Gusinoe, and four of them were also detected in Lake Tsaidam, and only two species were reported in the Zagustay River. A poor “catch” in the river was due to the occasional sampling in a limited part of the lower reach of the river.In the brief commentary, we include a concise description of each leech species with the emphasis on host-parasite relationship, zoogeographical and ecological characteristics, and occurrence of species within the area. Numerical evaluation of biodiversity could be useful for understanding of the importance of species number in terms of actual biodiversity of parasites.Phylum ANNELIDA Lamarck, 1809Class CLITELLATA Michaelsen, 1919Subclass HIRUDINEA Lamarck, 1818 (synonym Hirudinida)Order RHYNCHOBDELLEA Blanchard, 1894Family GLOSSIPHONIIDAE Vaillant, 1890GenusHemiclepsis Vejdowsky, 1884.(1)
Hemiclepsis marginata (Müller, 1774) is as follows.
Local host: Esociformes: Esocidae:Esox luciusLinnaeus, 1758. invertebrates.Locality: Lake Gusinoe, Lake Tsaidam.A common Palaearctic species: bloodsucker of fish, tadpoles, and amphipods. Representatives of this species were numerous in the littoral part of these lakes. They were found on stones and in washout from aquatic vegetation. Living leeches are green and brownish-green with a length of 10–12 mm and 2.5–3 mm in width. Alcohol fixed specimens rapidly lose their beautiful intravital colouring.GenusHelobdella Blanchard, 1876, is as follows.(2)
One considersHelobdella stagnalis (Linnaeus, 1758).
Local host: small invertebrates (oligochaetes, larvae of amphibiotic insects, molluscs, and young amphipods).Locality: Lake Gusinoe, Lake Tsaidam.This species is considered as one of the most common freshwater leeches in the world, a cosmopolite.H. stagnalis inhabits shallow coastal parts within Lake Gusinoe and Lake Tsaidam. This is a small but numerous and voracious species. Like all representatives of the family, it shows a touching concern for posterity, bears batch of eggs until their hatching, and then suckles an offspring until its self-dependence.GenusGlossiphonia Johnson, 1816, is as follows.(3)
One considersGlossiphoniasp.
Local host: Mollusca: Gastropoda.Locality: Lake Gusinoe.For the first time reported in Lake Gusinoe. These leeches were found in a pondweed tangle at a depth of 1.0–2.5 m. Representatives of this group have three pairs of eyes with typical location for the genus, and they are morphologically similar toG. verrucata. This leech has a larger body size, reaching 30 mm in length and therefore is the largest representative of the genus in Eurasia.GenusAlboglossiphonia Lukin, 1976, is as follows.(4)
Alboglossiphonia heteroclita (Linnaeus, 1761).A widespread Holarctic species. This benthic species preys on small invertebrates. There are two forms of this species, which differ in the amount of pigmentation on the dorsal side of the body:f. papillosa andf. striata. The latter was not found in the study area.A.
heteroclita f. papillosa (Braun, 1805).
Local host: Mollusca: Gastropoda.Locality: Lake Gusinoe, Lake Tsaidam, Zagustay River.This form is for the first time listed for Lake Gusinoe area. Small-sized glossiphoniids (around 10 mm in length and 3-4 mm in width) have transparent body and are characterized by median row of dark spots. Considerfamily Piscicolidae Johnston, 1865 (=Icthyobdellidae Leuckart, 1863);genusPiscicola de Blainville, 1818.(5)
Piscicolasp. is as follows.
Local host: Perciformes: Percidae:Perca fluviatilis Linnaeus, 1758.Locality: Lake Gusinoe.This leech has been detected for the first time in Lake Gusinoe area. Recently, it has been reported in Lake Baikal [26, 27]. This is a small-sized leech (length up to 8 mm) with a special body coloration differing from a widespread speciesPiscicola geometra. Within Lake Gusinoe, one specimen was found on a perch and another specimen was found in the washout from water milfoil sampled on the north part of the lake. ConsiderOrder Arhynchobdellida Blanchard, 1894;Suborder Erpobdelliformes Sawyer, 1986;Family Erpobdellidae Blanchard, 1894;GenusErpobdella de Blainville, 1818.(6)
Erpobdellasp. is as follows.
Locality: Lake Gusinoe, Lake Tsaidam, Zagustay River.This taxon was recently listed for Lake Baikal [27, 28].Erpobdella sp. is widespread within the area. Depending on the environmental conditions, this animal may be a predator of small invertebrates, necrophage, or detritophage. Leeches are of differing sizes. The largest specimen of 56 mm in length and up to 6 mm in width was caught in the mouth of the Zagustay River. Sexually mature individuals with the smallest body size were collected in the north of Lake Gusinoe. Their length and width of the body were 22–28 and 3-4 mm, correspondingly.


### 3.2. COI Phylogeny

The final dataset matrix of 70 aligned COI nucleotide sequences in length of 709 base pairs was compiled for phylogenetic analysis. The alignment includes 14 newly generated sequences in this study and 56 other ones closely related to them. All GenBank accession numbers are listed in Table1. For a reconstruction of phylogenetic interrelationships, one must choose assumptions for modelling of molecular evolution, which are the most suitable for a particular dataset. The evolutionary distance between a pair of sequences usually is measured by the number of nucleotide substitutions occurring between them. The test of the best DNA model for estimating distances was performed in MEGA [21]. As a result, 24 models were suggested. For each model, AICc value (Akaike Information Criterion, corrected), maximum likelihood value (lnL), and the number of parameters (including branch lengths) were also presented. The Tamura-Nei gamma distance with the gamma model taking into account the different rates of substitution between nucleotides and the inequality of nucleotide frequencies (TN93+G+I) was selected for our dataset, since models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. The Tamura-Nei model [29] corrects for multiple hits, taking into account the differences in substitution rate between nucleotides and the inequality of nucleotide frequencies. It distinguishes between transitional substitution rates between purines and transversional substitution rates between pyrimidines. Nonuniformity of evolutionary rates among sites was additionally modelled by using a discrete gamma distribution (+G, parameter = 0.6761) with 5-rate categories and by assuming that a certain fraction of sites are evolutionarily invariable ([+I], 45.2390% sites). The percentage of trees, in which the associated taxa clustered together, was evaluated using bootstrap analysis. Initial tree for the heuristic search was obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. The phylogeny was inferred by using the maximum likelihood method based on the chosen model. The tree with the highest log likelihood (−10362.4467) is shown (Figure 2). The tree has a stable topology and a significant statistical reliability of the main branch nodes. The major nodes discussed in the text have the bootstrap value higher than 90%.Figure 2
ML-tree based on COI barcoding locus of selected leech species. Lineages of the leeches from the Gusinoe area are highlighted in red. Numbers next to the branches indicate bootstrap values. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 70 nucleotide sequences. There were a total of 709 positions in the final dataset.The species from the Lake Gusinoe area are clustered in six different lineages according to their generic belonging.The unclassified jawless leechGlossiphonia sp. from Lake Gusinoe forms a separate branch within the species of the genusGlossiphonia that corroborates an independent taxonomical status of this species. Genetic distances betweenGlossiphonia sp. and its congeners are above 9.0%, with maximal difference of 12.3% relative toG. verrucata (Table 2).Table 2
Estimates of evolutionary divergence (%) over sequence pairs between availableGlossiphonia species including specimen from Lake Gusinoe.
G. complanata
G. concolor
G. concolor
G. verrucata
G. elegans
G. complanata
G. concolor
7.8
G. concolor
10.1
9.0
G. verrucata
10.9
9.6
8.5
G. elegans
10.7
9.6
12.3
12.3
Glossiphoniasp.
9.0
10.1
11.8
12.3
11.6Three SiberianAlboglossiphonia heteroclita fall into the common clade with other representatives of the genus. The genetic distances of 13.3% and 13.8% between lineages within the clade distinguish studied samples fromA. lata andA. weberi, correspondingly. There are 11.3% of substitutions accumulated between COI sequences of North Americanand SiberianA. heteroclita. The genetic variation within the group of Siberian congeners is about 0.7%.The representatives ofHemiclepsis marginata from France and from the Gusinoe area are grouped together and their COI sequences differ insignificantly (0.6%) that supports the monospecificity of the genus.The representatives of the Holarctic speciesHelobdella stagnalis form a single lineage with 100% bootstrap support and 0.7% genetic variation within the group regardless of geographical distance of their populations. There is 1.1% genetic distance between European and Siberian specimens. The closest to “stagnalis” clade isH. modesta from Ohio, differing from their European and Siberian congeners in 8.6 and 8.4%, correspondingly.Two piscine leech parasites from Lake Gusinoe are grouped in the same cluster with various forms of the family Piscicolidae (Figure2). Within the cluster,Piscicola sp. appeared more closely related toP. milneri, with these two lineages having a genetic distance of 2.3%. BetweenPiscicola sp. andP. geometra, there are 7.0% of substitutions in their nucleotide sequences.Unclassified macrophagous leeches from the Gusinoe area in accordance with their generic belonging clustered within the Erpobdellidae, in close relation toE. japonica andE. octoculata. The percentage of base substitutions from averaging over all sequence pairs between availableErpobdella species groups is shown in Table 3. The resulted genetic distances vary from 4.1% (punctata/montezuma) to 20.0% (japonica/punctata). Specimens ofErpobdella sp. from Lake Gusinoe and the Zagustay River are the most distant fromE. punctata (18.1%),E. triannulata (16.7%), andE. mexicana (16.7%) and, on the contrary, are more closely related toE. japonica from Korea (0.7%),E. japonica from Japan (10.8%), andE. vilnensis (10.7%). All the rest congeners includingE. octoculata differ genetically from the leeches of Lake Gusinoe by more than 12.1% (Table 3).Table 3
Estimates of evolutionary divergence (%) over sequence pairs betweenErpobdella species including group of unidentified leeches from the Gusinoe area.
E. obscura
E. montezuma
E. punctata
E. triannulata
E. mexicana
E. ochoterinai
E. vilnensis
E. nigricollis
E. monosriata
E. testacea
E. octoculata
E. japonica
E. japonica
E. obscura
E. montezuma
15.8
E. punctata
16.7
4.1
E. triannulata
15.2
15.6
16.7
E. mexicana
16.1
17.2
17.7
14.5
E. ochoterinai
13.4
16.1
17.2
14.9
11.6
E. vilnensis
12.1
14.4
15.3
13.5
13.6
12.0
E. nigricollis
14.7
17.4
16.3
14.0
14.7
16.3
11.4
E. monosriata
14.5
16.7
17.0
15.6
16.7
17.9
12.6
10.8
E. testacea
14.7
16.7
18.4
16.1
14.7
16.7
14.0
12.7
14.5
E. octoculata
14.0
16.5
18.2
14.8
16.3
16.4
12.9
12.7
15.4
13.9
E. japonica
14.3
19.8
20.0
17.2
17.0
15.8
13.9
14.7
17.2
15.6
14.1
E.japonica
14.9
17.0
18.8
17.0
17.0
16.1
11.4
12.3
14.2
15.4
13.0
10.6
Erpobdellasp.
14.5
16.3
18.1
16.7
16.7
15.4
10.7
12.1
14.3
14.7
12.8
10.8
0.7


## 3.1. Taxonomic Review and a Brief Description of Each Taxon

Collection of parasitic and nonparasitic leeches (Hirudinea, Clitellata) from 12 geographical sampling sites (Figure1) was performed during the two-year seasons in Lake Gusinoe and its catchment area. Morphological analysis revealed six leech species (Table 1: in bold) belonging to two orders (Rhynchobdellida, Arhynchobdellida), three families (Glossiphoniidae, Piscicolidae, and Erpobdellidae) and six genera (Helobdella,Hemiclepsis,Glossiphonia,Alboglossiphonia,Piscicola, andErpobdella). All six species were found in Lake Gusinoe, and four of them were also detected in Lake Tsaidam, and only two species were reported in the Zagustay River. A poor “catch” in the river was due to the occasional sampling in a limited part of the lower reach of the river.In the brief commentary, we include a concise description of each leech species with the emphasis on host-parasite relationship, zoogeographical and ecological characteristics, and occurrence of species within the area. Numerical evaluation of biodiversity could be useful for understanding of the importance of species number in terms of actual biodiversity of parasites.Phylum ANNELIDA Lamarck, 1809Class CLITELLATA Michaelsen, 1919Subclass HIRUDINEA Lamarck, 1818 (synonym Hirudinida)Order RHYNCHOBDELLEA Blanchard, 1894Family GLOSSIPHONIIDAE Vaillant, 1890GenusHemiclepsis Vejdowsky, 1884.(1)
Hemiclepsis marginata (Müller, 1774) is as follows.
Local host: Esociformes: Esocidae:Esox luciusLinnaeus, 1758. invertebrates.Locality: Lake Gusinoe, Lake Tsaidam.A common Palaearctic species: bloodsucker of fish, tadpoles, and amphipods. Representatives of this species were numerous in the littoral part of these lakes. They were found on stones and in washout from aquatic vegetation. Living leeches are green and brownish-green with a length of 10–12 mm and 2.5–3 mm in width. Alcohol fixed specimens rapidly lose their beautiful intravital colouring.GenusHelobdella Blanchard, 1876, is as follows.(2)
One considersHelobdella stagnalis (Linnaeus, 1758).
Local host: small invertebrates (oligochaetes, larvae of amphibiotic insects, molluscs, and young amphipods).Locality: Lake Gusinoe, Lake Tsaidam.This species is considered as one of the most common freshwater leeches in the world, a cosmopolite.H. stagnalis inhabits shallow coastal parts within Lake Gusinoe and Lake Tsaidam. This is a small but numerous and voracious species. Like all representatives of the family, it shows a touching concern for posterity, bears batch of eggs until their hatching, and then suckles an offspring until its self-dependence.GenusGlossiphonia Johnson, 1816, is as follows.(3)
One considersGlossiphoniasp.
Local host: Mollusca: Gastropoda.Locality: Lake Gusinoe.For the first time reported in Lake Gusinoe. These leeches were found in a pondweed tangle at a depth of 1.0–2.5 m. Representatives of this group have three pairs of eyes with typical location for the genus, and they are morphologically similar toG. verrucata. This leech has a larger body size, reaching 30 mm in length and therefore is the largest representative of the genus in Eurasia.GenusAlboglossiphonia Lukin, 1976, is as follows.(4)
Alboglossiphonia heteroclita (Linnaeus, 1761).A widespread Holarctic species. This benthic species preys on small invertebrates. There are two forms of this species, which differ in the amount of pigmentation on the dorsal side of the body:f. papillosa andf. striata. The latter was not found in the study area.A.
heteroclita f. papillosa (Braun, 1805).
Local host: Mollusca: Gastropoda.Locality: Lake Gusinoe, Lake Tsaidam, Zagustay River.This form is for the first time listed for Lake Gusinoe area. Small-sized glossiphoniids (around 10 mm in length and 3-4 mm in width) have transparent body and are characterized by median row of dark spots. Considerfamily Piscicolidae Johnston, 1865 (=Icthyobdellidae Leuckart, 1863);genusPiscicola de Blainville, 1818.(5)
Piscicolasp. is as follows.
Local host: Perciformes: Percidae:Perca fluviatilis Linnaeus, 1758.Locality: Lake Gusinoe.This leech has been detected for the first time in Lake Gusinoe area. Recently, it has been reported in Lake Baikal [26, 27]. This is a small-sized leech (length up to 8 mm) with a special body coloration differing from a widespread speciesPiscicola geometra. Within Lake Gusinoe, one specimen was found on a perch and another specimen was found in the washout from water milfoil sampled on the north part of the lake. ConsiderOrder Arhynchobdellida Blanchard, 1894;Suborder Erpobdelliformes Sawyer, 1986;Family Erpobdellidae Blanchard, 1894;GenusErpobdella de Blainville, 1818.(6)
Erpobdellasp. is as follows.
Locality: Lake Gusinoe, Lake Tsaidam, Zagustay River.This taxon was recently listed for Lake Baikal [27, 28].Erpobdella sp. is widespread within the area. Depending on the environmental conditions, this animal may be a predator of small invertebrates, necrophage, or detritophage. Leeches are of differing sizes. The largest specimen of 56 mm in length and up to 6 mm in width was caught in the mouth of the Zagustay River. Sexually mature individuals with the smallest body size were collected in the north of Lake Gusinoe. Their length and width of the body were 22–28 and 3-4 mm, correspondingly.


## 3.2. COI Phylogeny

The final dataset matrix of 70 aligned COI nucleotide sequences in length of 709 base pairs was compiled for phylogenetic analysis. The alignment includes 14 newly generated sequences in this study and 56 other ones closely related to them. All GenBank accession numbers are listed in Table1. For a reconstruction of phylogenetic interrelationships, one must choose assumptions for modelling of molecular evolution, which are the most suitable for a particular dataset. The evolutionary distance between a pair of sequences usually is measured by the number of nucleotide substitutions occurring between them. The test of the best DNA model for estimating distances was performed in MEGA [21]. As a result, 24 models were suggested. For each model, AICc value (Akaike Information Criterion, corrected), maximum likelihood value (lnL), and the number of parameters (including branch lengths) were also presented. The Tamura-Nei gamma distance with the gamma model taking into account the different rates of substitution between nucleotides and the inequality of nucleotide frequencies (TN93+G+I) was selected for our dataset, since models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. The Tamura-Nei model [29] corrects for multiple hits, taking into account the differences in substitution rate between nucleotides and the inequality of nucleotide frequencies. It distinguishes between transitional substitution rates between purines and transversional substitution rates between pyrimidines. Nonuniformity of evolutionary rates among sites was additionally modelled by using a discrete gamma distribution (+G, parameter = 0.6761) with 5-rate categories and by assuming that a certain fraction of sites are evolutionarily invariable ([+I], 45.2390% sites). The percentage of trees, in which the associated taxa clustered together, was evaluated using bootstrap analysis. Initial tree for the heuristic search was obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. The phylogeny was inferred by using the maximum likelihood method based on the chosen model. The tree with the highest log likelihood (−10362.4467) is shown (Figure 2). The tree has a stable topology and a significant statistical reliability of the main branch nodes. The major nodes discussed in the text have the bootstrap value higher than 90%.Figure 2
ML-tree based on COI barcoding locus of selected leech species. Lineages of the leeches from the Gusinoe area are highlighted in red. Numbers next to the branches indicate bootstrap values. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 70 nucleotide sequences. There were a total of 709 positions in the final dataset.The species from the Lake Gusinoe area are clustered in six different lineages according to their generic belonging.The unclassified jawless leechGlossiphonia sp. from Lake Gusinoe forms a separate branch within the species of the genusGlossiphonia that corroborates an independent taxonomical status of this species. Genetic distances betweenGlossiphonia sp. and its congeners are above 9.0%, with maximal difference of 12.3% relative toG. verrucata (Table 2).Table 2
Estimates of evolutionary divergence (%) over sequence pairs between availableGlossiphonia species including specimen from Lake Gusinoe.
G. complanata
G. concolor
G. concolor
G. verrucata
G. elegans
G. complanata
G. concolor
7.8
G. concolor
10.1
9.0
G. verrucata
10.9
9.6
8.5
G. elegans
10.7
9.6
12.3
12.3
Glossiphoniasp.
9.0
10.1
11.8
12.3
11.6Three SiberianAlboglossiphonia heteroclita fall into the common clade with other representatives of the genus. The genetic distances of 13.3% and 13.8% between lineages within the clade distinguish studied samples fromA. lata andA. weberi, correspondingly. There are 11.3% of substitutions accumulated between COI sequences of North Americanand SiberianA. heteroclita. The genetic variation within the group of Siberian congeners is about 0.7%.The representatives ofHemiclepsis marginata from France and from the Gusinoe area are grouped together and their COI sequences differ insignificantly (0.6%) that supports the monospecificity of the genus.The representatives of the Holarctic speciesHelobdella stagnalis form a single lineage with 100% bootstrap support and 0.7% genetic variation within the group regardless of geographical distance of their populations. There is 1.1% genetic distance between European and Siberian specimens. The closest to “stagnalis” clade isH. modesta from Ohio, differing from their European and Siberian congeners in 8.6 and 8.4%, correspondingly.Two piscine leech parasites from Lake Gusinoe are grouped in the same cluster with various forms of the family Piscicolidae (Figure2). Within the cluster,Piscicola sp. appeared more closely related toP. milneri, with these two lineages having a genetic distance of 2.3%. BetweenPiscicola sp. andP. geometra, there are 7.0% of substitutions in their nucleotide sequences.Unclassified macrophagous leeches from the Gusinoe area in accordance with their generic belonging clustered within the Erpobdellidae, in close relation toE. japonica andE. octoculata. The percentage of base substitutions from averaging over all sequence pairs between availableErpobdella species groups is shown in Table 3. The resulted genetic distances vary from 4.1% (punctata/montezuma) to 20.0% (japonica/punctata). Specimens ofErpobdella sp. from Lake Gusinoe and the Zagustay River are the most distant fromE. punctata (18.1%),E. triannulata (16.7%), andE. mexicana (16.7%) and, on the contrary, are more closely related toE. japonica from Korea (0.7%),E. japonica from Japan (10.8%), andE. vilnensis (10.7%). All the rest congeners includingE. octoculata differ genetically from the leeches of Lake Gusinoe by more than 12.1% (Table 3).Table 3
Estimates of evolutionary divergence (%) over sequence pairs betweenErpobdella species including group of unidentified leeches from the Gusinoe area.
E. obscura
E. montezuma
E. punctata
E. triannulata
E. mexicana
E. ochoterinai
E. vilnensis
E. nigricollis
E. monosriata
E. testacea
E. octoculata
E. japonica
E. japonica
E. obscura
E. montezuma
15.8
E. punctata
16.7
4.1
E. triannulata
15.2
15.6
16.7
E. mexicana
16.1
17.2
17.7
14.5
E. ochoterinai
13.4
16.1
17.2
14.9
11.6
E. vilnensis
12.1
14.4
15.3
13.5
13.6
12.0
E. nigricollis
14.7
17.4
16.3
14.0
14.7
16.3
11.4
E. monosriata
14.5
16.7
17.0
15.6
16.7
17.9
12.6
10.8
E. testacea
14.7
16.7
18.4
16.1
14.7
16.7
14.0
12.7
14.5
E. octoculata
14.0
16.5
18.2
14.8
16.3
16.4
12.9
12.7
15.4
13.9
E. japonica
14.3
19.8
20.0
17.2
17.0
15.8
13.9
14.7
17.2
15.6
14.1
E.japonica
14.9
17.0
18.8
17.0
17.0
16.1
11.4
12.3
14.2
15.4
13.0
10.6
Erpobdellasp.
14.5
16.3
18.1
16.7
16.7
15.4
10.7
12.1
14.3
14.7
12.8
10.8
0.7


## 4. Discussion

### 4.1. Species Diversity

At present, the occurrence of six species in the Lake Gusinoe area has been documented. This species diversity includes both widespread Holarctic and Palaearctic species and also new species from three families and six genera. Three species of the checklist have been reported for the first time in the region, of themGlossiphonia sp.,Piscicola sp., andErpobdella sp. These leeches were impracticable to determine, since the mosaic set of their morphological characters does not correspond to any known leech species description and currently available systematic keys. Most probably, the leeches with ambiguous species status,Glossiphonia sp.,Piscicola sp., andErpobdella sp., are potentially new species to science. At the same time,Piscicola geometra (Linnaeus, 1761),Erpobdella octoculata (Linnaeus, 1758), andGlossiphonia complanata (Malm, 1863) were excluded from the species list of Lake Gusinoe. Our precise morphological study of the collected material in the Gusinoe area did not detect these three species.The first study of Hirudinea was carried out in Lake Gusinoe in the early 1990s [4]. At that time, the species list had included the following species:Erpobdella octoculata,Glossiphonia complanata,G. heteroclita,Helobdella stagnalis,Hemiclepsis marginata, andPiscicola geometra. Until recently, it was assumed that freshwater environments on the vast territory of Siberia had been inhabited by widespread Palaearctic Hirudinea species only. However, the recent targeted studies of lakes and rivers of Eastern Siberia including the unique Lake Baikal [26–28, 30] disprove this hypothesis, since common speciesErpobdella octoculata,Glossiphonia complanata, andPiscicola geometra have not been found in the region.As for the leechG. heteroclita mentioned for Lake Gusinoe in the previous study [4], it is necessary to use a scientific binomial nameAlboglossiphonia heteroclita according to the currently valid systematics of the group. Moreover, the morphological examination allowed us to offer a more detailed determination of the leech taxonomic status, that is,Alboglossiphonia heteroclita f.papillosa, based on the presence of median row of dark spots on dorsal side, a key characteristic for this form.Thus, the updated checklist of leech fauna inhabiting Lake Gusinoe consists ofHelobdella stagnalis, Hemiclepsis marginata, Alboglossiphonia heteroclita f.striata, Glossiphonia sp.,Piscicola sp., andErpobdella sp.


### 4.2. Genetic Leech Diversity

DNA barcoding is a genomic method used to distinguish between various species of organisms [31].  Since the advent of DNA barcoding, there is widespread speculation that more than 2% of substitutions in marker fragment (COI) seem high enough to suggest the presence of multiple species lineages [31, 32]. Unfortunately, species for which a gene sequence has not been recorded cannot be identified by a database search [33]. The identification of a new species with DNA barcoding and those not included in a genomic repository must be accompanied by a standard taxonomy, geographical information, and other valid species delimitation attributes [34]. Our study has established such DNA barcodes for six species of Hirudinea from the Lake Gusinoe area.SiberianGlossiphonia sp. was found to be related phylogenetically to the relevant species group of the same genus (G. concolor,G. complanata, andG. verrucata, andG. elegans). Moreover, nucleotide sequences of SiberianGlossiphonia sp. achieved lower similarity values compared to the otherGlossiphonia species sequences obtained from leeches collected in different European countries (Table 1). Unexpectedly,Glossiphonia sp. was genetically far fromG. verrucata despite their close similarity in external morphology (Table 2). At the same time,G. concolor from Sweden was most likely attributed toG. concolor erroneously since COI sequences of the reliably identified specimens ofG. concolor from Ukraine (G. concolor 869 and 870, Figure 2) are located on a different branch of the phylogenetic tree. The high level of genetic distinctions, that is, 0.090 of substitutions per site in the barcoding fragment, leads us to consider Swedish and Ukranian “concolors” to be in fact two distinct taxa. We have to state that the nucleotide sequence AY962458 does not belong toG. concolor.The molecular data ofAlboglossiphonia heteroclita obtained from Siberia vary insignificantly within the group (0.7% variable sites). This is a strong support for their species identity. At the same time, 11.3% difference from North American cospecies is too high to be the same species. Consequently, the taxonomic status of leeches with a nucleotide sequence AF116016, which is also used as a DNA barcode, should be verified. Furthermore, there is only 1.0% genetic variation among AsianA. lata and AmericanA. weberi. This is a convincing evidence of the excessive taxonomic splitting, contrary to the previous conclusion on synonymy of these two species names in favour ofA. weberi since there are no clear morphological distinctions betweenlata andweberi as previously ascertained [15].Notwithstanding the fact thatHelobdella stagnalis andHemiclepsis marginata are among the most common leech species, prior to this study, there were only three COI sequences of European representatives available in the GenBank: twoH. stagnalis and oneH. marginata (Table 1). The genetic distances between geographically distant representatives of European and Siberian faunas (1.1% and 0.6%, resp.) correspond to a single species in each case.With regard to piscine leech parasites from Lake Gusinoe, their sequences are more relevant toP. milneri and not toP. geometra, contrary to the established opinion [4]. However, doubt about species belonging of the Siberian Piscicolidae is still left since the detected genetic distance of 2.3% does not allow them to be confidently attributed to a particular taxon. In this case, an advanced morphological examination of additional material is required.COI sequences ofErpobdella sp. from Lake Gusinoe and the Zagustay River are most genetically close toE. japonica from Korea, whereas they vary significantly relatively toE. japonica from Japan andE. vilnensis (Table 3). From the ML-tree (Figure 2), theErpobdella sp. is found to be a sister taxon ofE. japonica from Korea with a bootstrap value of 100 for this clade and ofE. japonica from Japan with a bootstrap support of 92. Genetic difference of 0.7% would allow us with certainty to attribute the unclassified Siberian samples to a certain species, if not a dubious species affiliation of the sampleE.japonica from Korea. The pairwise distance computed between the COI sequence ofE. japonica from Japan (AB679654) and the sequence ofE. japonica from Korea (AF116026) was 10.6% of substitutions (Table 3). A high score suggests a significant genetic separateness therefore belonging to distinct species. Since the type specimen for the original description ofE. japonica was collected in a Japanese lake [35], it is reasonable to recognize the taxonomic designation of the leech specimen (sequence AB679654) as valid, while the Korean sample (sequence AF116026) may be classified in “japonica” onlyde bene esse. Unfortunately, the determination of SiberianErpobdella up to species using DNA barcoding is still impossible because it is akin exactly to the incorrectly identifiedE.japonica.In conclusion, at least six leech species occurring in Lake Gusinoe have now acquired DNA barcodes. Our study has shown that a technique of DNA barcoding can be applied successfully to species identification even when morphological taxonomy cannot be employed. High values of genetic distances among species allow considering the fragment ofCOI as a suitable marker for study of inter- and intraspecific relationships in hirudinids. The use of species-specific DNA markers will significantly simplify the identification of Hirudinea species.


## 4.1. Species Diversity

At present, the occurrence of six species in the Lake Gusinoe area has been documented. This species diversity includes both widespread Holarctic and Palaearctic species and also new species from three families and six genera. Three species of the checklist have been reported for the first time in the region, of themGlossiphonia sp.,Piscicola sp., andErpobdella sp. These leeches were impracticable to determine, since the mosaic set of their morphological characters does not correspond to any known leech species description and currently available systematic keys. Most probably, the leeches with ambiguous species status,Glossiphonia sp.,Piscicola sp., andErpobdella sp., are potentially new species to science. At the same time,Piscicola geometra (Linnaeus, 1761),Erpobdella octoculata (Linnaeus, 1758), andGlossiphonia complanata (Malm, 1863) were excluded from the species list of Lake Gusinoe. Our precise morphological study of the collected material in the Gusinoe area did not detect these three species.The first study of Hirudinea was carried out in Lake Gusinoe in the early 1990s [4]. At that time, the species list had included the following species:Erpobdella octoculata,Glossiphonia complanata,G. heteroclita,Helobdella stagnalis,Hemiclepsis marginata, andPiscicola geometra. Until recently, it was assumed that freshwater environments on the vast territory of Siberia had been inhabited by widespread Palaearctic Hirudinea species only. However, the recent targeted studies of lakes and rivers of Eastern Siberia including the unique Lake Baikal [26–28, 30] disprove this hypothesis, since common speciesErpobdella octoculata,Glossiphonia complanata, andPiscicola geometra have not been found in the region.As for the leechG. heteroclita mentioned for Lake Gusinoe in the previous study [4], it is necessary to use a scientific binomial nameAlboglossiphonia heteroclita according to the currently valid systematics of the group. Moreover, the morphological examination allowed us to offer a more detailed determination of the leech taxonomic status, that is,Alboglossiphonia heteroclita f.papillosa, based on the presence of median row of dark spots on dorsal side, a key characteristic for this form.Thus, the updated checklist of leech fauna inhabiting Lake Gusinoe consists ofHelobdella stagnalis, Hemiclepsis marginata, Alboglossiphonia heteroclita f.striata, Glossiphonia sp.,Piscicola sp., andErpobdella sp.


## 4.2. Genetic Leech Diversity

DNA barcoding is a genomic method used to distinguish between various species of organisms [31].  Since the advent of DNA barcoding, there is widespread speculation that more than 2% of substitutions in marker fragment (COI) seem high enough to suggest the presence of multiple species lineages [31, 32]. Unfortunately, species for which a gene sequence has not been recorded cannot be identified by a database search [33]. The identification of a new species with DNA barcoding and those not included in a genomic repository must be accompanied by a standard taxonomy, geographical information, and other valid species delimitation attributes [34]. Our study has established such DNA barcodes for six species of Hirudinea from the Lake Gusinoe area.SiberianGlossiphonia sp. was found to be related phylogenetically to the relevant species group of the same genus (G. concolor,G. complanata, andG. verrucata, andG. elegans). Moreover, nucleotide sequences of SiberianGlossiphonia sp. achieved lower similarity values compared to the otherGlossiphonia species sequences obtained from leeches collected in different European countries (Table 1). Unexpectedly,Glossiphonia sp. was genetically far fromG. verrucata despite their close similarity in external morphology (Table 2). At the same time,G. concolor from Sweden was most likely attributed toG. concolor erroneously since COI sequences of the reliably identified specimens ofG. concolor from Ukraine (G. concolor 869 and 870, Figure 2) are located on a different branch of the phylogenetic tree. The high level of genetic distinctions, that is, 0.090 of substitutions per site in the barcoding fragment, leads us to consider Swedish and Ukranian “concolors” to be in fact two distinct taxa. We have to state that the nucleotide sequence AY962458 does not belong toG. concolor.The molecular data ofAlboglossiphonia heteroclita obtained from Siberia vary insignificantly within the group (0.7% variable sites). This is a strong support for their species identity. At the same time, 11.3% difference from North American cospecies is too high to be the same species. Consequently, the taxonomic status of leeches with a nucleotide sequence AF116016, which is also used as a DNA barcode, should be verified. Furthermore, there is only 1.0% genetic variation among AsianA. lata and AmericanA. weberi. This is a convincing evidence of the excessive taxonomic splitting, contrary to the previous conclusion on synonymy of these two species names in favour ofA. weberi since there are no clear morphological distinctions betweenlata andweberi as previously ascertained [15].Notwithstanding the fact thatHelobdella stagnalis andHemiclepsis marginata are among the most common leech species, prior to this study, there were only three COI sequences of European representatives available in the GenBank: twoH. stagnalis and oneH. marginata (Table 1). The genetic distances between geographically distant representatives of European and Siberian faunas (1.1% and 0.6%, resp.) correspond to a single species in each case.With regard to piscine leech parasites from Lake Gusinoe, their sequences are more relevant toP. milneri and not toP. geometra, contrary to the established opinion [4]. However, doubt about species belonging of the Siberian Piscicolidae is still left since the detected genetic distance of 2.3% does not allow them to be confidently attributed to a particular taxon. In this case, an advanced morphological examination of additional material is required.COI sequences ofErpobdella sp. from Lake Gusinoe and the Zagustay River are most genetically close toE. japonica from Korea, whereas they vary significantly relatively toE. japonica from Japan andE. vilnensis (Table 3). From the ML-tree (Figure 2), theErpobdella sp. is found to be a sister taxon ofE. japonica from Korea with a bootstrap value of 100 for this clade and ofE. japonica from Japan with a bootstrap support of 92. Genetic difference of 0.7% would allow us with certainty to attribute the unclassified Siberian samples to a certain species, if not a dubious species affiliation of the sampleE.japonica from Korea. The pairwise distance computed between the COI sequence ofE. japonica from Japan (AB679654) and the sequence ofE. japonica from Korea (AF116026) was 10.6% of substitutions (Table 3). A high score suggests a significant genetic separateness therefore belonging to distinct species. Since the type specimen for the original description ofE. japonica was collected in a Japanese lake [35], it is reasonable to recognize the taxonomic designation of the leech specimen (sequence AB679654) as valid, while the Korean sample (sequence AF116026) may be classified in “japonica” onlyde bene esse. Unfortunately, the determination of SiberianErpobdella up to species using DNA barcoding is still impossible because it is akin exactly to the incorrectly identifiedE.japonica.In conclusion, at least six leech species occurring in Lake Gusinoe have now acquired DNA barcodes. Our study has shown that a technique of DNA barcoding can be applied successfully to species identification even when morphological taxonomy cannot be employed. High values of genetic distances among species allow considering the fragment ofCOI as a suitable marker for study of inter- and intraspecific relationships in hirudinids. The use of species-specific DNA markers will significantly simplify the identification of Hirudinea species.



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*Source: 619127-2014-11-27.xml*