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

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.


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 km 2 , the volume of water mass is 2.4 km 3 , 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 km 2 . 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/km 2 , 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 km 2 , respectively ( Figure 1).
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, and Piscicola 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][6][7][8][9], as well as hematozoa including trematodes, cestodes, and nematodes Lake Gusinoe Figure 1: Geographical location of the study region with indication of the main sampling sites in Lake Gusinoe, Lake Tsaidam, and Zagustay River.
[10] and parasitic flagellates [11][12][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 cytochrome 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.

Sample Collection.
Biological material was collected during expeditions to Lake Gusinoe in 2012-2013 ( Figure 1). 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.

DNA Sequencing.
Sequences of the mitochondrial cytochrome oxidase subunit I (COI) were newly generated from 12 specimens from the Lake Gusinoe area and two individuals of Glossiphonia concolor were from Ukraine (Table 1).
Total 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]  The Scientific World Journal 3 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 Table 1.

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].

Taxonomic Review and a Brief Description of Each Taxon.
Collection of parasitic and nonparasitic leeches (Hirudinea, Clitellata) from 12 geographical sampling sites ( Figure 1) 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, and Erpobdella). 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. 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.
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.
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 to G. verrucata. This leech has a larger body size, reaching 30 mm in length and therefore is the largest representative of the genus in Eurasia.
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 and f. striata. The latter was not found in the study area.

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The Scientific World Journal  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 species Piscicola 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. Consider Order Arhynchobdellida Blanchard, 1894; Suborder Erpobdelliformes Sawyer, 1986; Family Erpobdellidae Blanchard, 1894; Genus Erpobdella de Blainville, 1818. (6) Erpobdella sp. is as follows.
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.  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 Table 1. 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%. The species from the Lake Gusinoe area are clustered in six different lineages according to their generic belonging.

COI
The unclassified jawless leech Glossiphonia sp. from Lake Gusinoe forms a separate branch within the species of the genus Glossiphonia that corroborates an independent taxonomical status of this species. Genetic distances between Glossiphonia sp. and its congeners are above 9.0%, with maximal difference of 12.3% relative to G. verrucata ( Table 2).
Three Siberian Alboglossiphonia 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 from A. lata and A. weberi, correspondingly. There are 11.3% of substitutions accumulated between COI sequences of North Americanand Siberian A. heteroclita. The genetic variation within the group of Siberian congeners is about 0.7%.
The representatives of Hemiclepsis 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 species Helobdella 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 is H. 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 (Figure 2). Within the cluster, Piscicola sp. appeared more closely related to P. milneri, with these two lineages having a genetic distance of 2.3%. Between Piscicola sp. and P. 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 to E. japonica and E. octoculata. The percentage of base substitutions from averaging over all sequence pairs between available Erpobdella species groups is shown in Table 3. The resulted genetic distances vary from 4.1% (punctata/montezuma) to 20.0% (japonica/punctata). Specimens of Erpobdella sp. from Lake Gusinoe and the Zagustay River are the most distant from E. punctata (18.1%), E. triannulata (16.7%), and E. mexicana (16.7%) and, on the contrary, are more closely related to E. japonica from Korea (0.7%), E. japonica from Japan (10.8%), and E. vilnensis (10.7%). All the rest congeners including E. octoculata differ genetically from the leeches of Lake Gusinoe by more than 12.1% (Table 3).

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 them Glossiphonia sp., Piscicola sp., and Erpobdella 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