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Article

Genetic and Morphological Characterization of the Invasive Corbicula Lineages in European Russia

1
N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Nikolsky Avenue 20, 163020 Arkhangelsk, Russia
2
Department of Biology, Ecology and Biotechnology, Northern (Arctic) Federal University, Northern Dvina Emb. 17, 163002 Arkhangelsk, Russia
3
A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Leninsky Avenue 33, 119071 Moscow, Russia
*
Author to whom correspondence should be addressed.
Water 2023, 15(18), 3226; https://doi.org/10.3390/w15183226
Submission received: 8 August 2023 / Revised: 1 September 2023 / Accepted: 8 September 2023 / Published: 11 September 2023
(This article belongs to the Special Issue Advanced Research on Freshwater Invertebrate Ecology and Systematics)

Abstract

:
Despite the full attention of malacologists to the study of Corbicula clams, stimulated mainly due to their high invasive potential and unique breeding system, studies based on an integrative taxonomic approach to identify various invasive lineages of this genus in European Russia are still limited. Our fieldwork was conducted in the Don, Volga, and Caspian Sea basins. In total, four distinct morphotypes belonging to the European forms R, Rlc, S, and “Int” of Corbicula clams were distinguished. According to our molecular genetic data, two Corbicula lineages, relevant to the nominal species Corbicula fluminea and C. fluminalis, can be defined. We observed a discrepancy between mtDNA haplotypes and morphological features for all individuals that were sampled from the Don and Kura Rivers. Identified mismatch may be due to the androgenetic reproduction of Corbicula that leads to cytonuclear inconsistencies. The 28S rRNA polymorphism in C. fluminea and C. fluminalis from studied localities was recorded. This occurrence is perhaps due to hybridization events between forms R, S, and Rlc. The biogeographic origins of Corbicula invasive lineages are discussed. Corbicula fluminalis (FW17, form S) has been detected for first time in the native range, namely in Azerbaijan, Tajikistan, Turkey, and Myanmar. We assume that the invasion of C. fluminalis into reservoirs and watercourses of the Caspian Sea basin (Republic of Dagestan and Stavropol Krai of Russia) was from Western Asia and Transcaucasia, and the introduction of C. fluminea to the Don and Volga River basins was from unspecified European countries.

1. Introduction

The Corbicula clams (Bivalvia: Cyrenidae) are native to Africa, Asia, Australia, and the Middle East [1]. However, these clams largely extended their range during the past century and are now widespread around the world [2]. In Europe, Corbicula was detected for the first time in the 1980s [3]. Currently, Corbicula clams are dispersed across all the main European countries, including Russia [4,5,6,7,8]. Invasive Corbicula clams were first discovered in European Russia in the Shura-Ozen River (Caspian Sea basin) in 2013 [9]. Later on, Corbicula clams were found in Agrakhan Bay, Kubyakinsky Bank, Lake Kuznechonok, Yuzbash-Sulak Collector, and Prisulak Canal of the Caspian Sea from 2017–2021 [10,11]. In 2015, Corbicula was found in a cooler pond of the Kostromskaya Power Station, Volga River basin (Caspian Sea drainage) [12] and in heated waste water of the Arkhangelsk Thermal Power Plant, Northern Dvina River basin (White Sea basin) [7]. In 2017, several Corbicula specimens were found in a heated channel of the Novocherkassk Hydroelectric Power Station (GRES) and in the Don River (Azov Sea drainage) [13,14,15].
The invasive lineages of Corbicula reproduce through androgenesis, which occurs after self-fertilization or outcrossing and herewith the oocyte is fertilized by an unreduced sperm [16,17,18,19]. The maternal nuclear DNA is then entirely extruded as two polar bodies and only the “male” pronucleus remains [16,18]. In addition, the sperm from one lineage is able to fertilize the eggs of another lineage (so-called “mitochondrial DNA capture”) and the nuclear genome of one lineage becomes associated with the mitochondria of another one, resulting in cytonuclear mismatches [16,17,18]. In some cases, “nuclear genome capture” may be observed, when part or the entire maternal nuclear DNA is not completely expelled from the egg, whereby the offspring inherits a hybrid genome [2,7,16,19]. Furthermore, such hybridization events could be a cause of genetic divergence between androgenetic species [2]. With respect to invasive Corbicula clams, the analysis of mitochondrial DNA alone may be biased. Therefore, an integrative taxonomic approach should be used, combining the analysis of nuclear and mitochondrial DNA sites, as well as the morphological and anatomical data [7,20,21,22,23].
Currently, five distinct invasive Corbicula lineages have been reported: (1) form A/R (COI mtDNA haplotype FW5), being widespread throughout East Asia, Europe, South and North America, and North Africa; (2) form B/Rlc (COI mtDNA haplotype FW1/FW4) that was found in East Asia, North America and Europe; (3) form C/S (COI mtDNA haplotype FW17) that was recorded in South America and Europe; and (4) a new form D that was described from North America [2,5,17,18,24].
Despite the close attention to the study of Corbicula clams, stimulated mainly due to their high invasive potential and unique breeding system [25,26], studies based on combining molecular genetics, conchological, and anatomical data for this genus in European Russia are still rare. Currently, only two populations have been covered by molecular genetic studies [7,27], and only COI gene fragments were analyzed for the population in the Volga River basin [27]. The objectives of the current study were: (1) to identify the invasive lineages of Corbicula in European Russia based on a comprehensive approach uniting molecular genetics, morphological, and anatomical data; (2) to evaluate the genetic diversity and possible vectors of introduction of Corbicula spp. to European Russia.

2. Materials and Methods

2.1. Study Area and Field Sampling

The field investigation was performed within several localities of European Russia: Don and Volga River basins, Shura-Ozen, Sulak, and Samur Rivers (Caspian Sea basin, Republic of Dagestan), and Kura River (Terek River basin, Stavropol Krai) in 2022 (Figure 1 and Figure 2). Corbicula individuals were collected using a hand net and dredge and were fixed in 96% ethanol. A total of 439 specimens were gathered. The materials are stored in the collection of the Russian Museum of Biodiversity Hotspots, N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia.

2.2. Morphological and Anatomical Studies

The Corbicula shells were measured using a caliper (with an accuracy of 0.1 mm): shell length (SL), height (SH), and width (SW) (Supplementary Table S1). A total of 439 specimens were measured in this study. The shell shape, sculpture, umbo position, shape of the hinge, and surface coloration of Corbicula clams were examined (see [7,8,20,21] for detail). Shell images were obtained using a Canon EOS 80D Digital Camera (Canon Inc., Tokyo, Japan). The ethanol-preserved individuals were dissected; their mantle, siphons, and gills were extracted for visual examination. The soft bodies and siphons were photographed using an Leica M165C stereomicroscope (Leica Microsystems, Wetzlar, Germany) with a FLEXACAM C1 digital camera (Leica Microsystems, Wetzlar, Germany). The morphotypes (A/R, B/Rlc, and C/S) were distinguished according to Pfenninger et al. [28], Korniushin [29], Marescaux et al. [30], Pigneur et al. [31], Bespalaya et al. [7], and Morhun et al. [32]. To assess the extent of conchological variation in Corbicula morphotypes, principal components analysis (PCA) was conducted, using the following measurements (SL, SH, and SW). In total, measurements of 196 adult clams were used. The statistical analysis was conducted using the software PAST v.3.0 [33]. The shell elongation (SEI) and shell inflation (SII) indices were calculated according to Bolotov et al. [34].

2.3. DNA Extraction, PCR Amplification, and DNA Sequencing

Here, we present original molecular data for 144 Corbicula specimens (Supplementary Table S2). Total DNA was extracted from 96% ethanol-preserved foot tissues using the NucleoSpin Tissue Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), following the manufacturer’s protocol. The performed molecular genetic analysis included amplification and sequencing of mtDNA COI and nuclear 28S rRNA markers. The primer composites are presented comprehensively in our articles [35,36]. The PCR mix contained approximately 100 ng of total cell DNA, 10 pmol of each primer, 200 µmol of each dNTP, 2.5 µL of PCR buffer (with 20 mmol MgCl2), 0.8 units Taq DNA polymerase (SibEnzyme Ltd., Novosibirsk, Russia), and H2O was added for a final volume of 25 µL. Temperature cycling was as follows: 95 °C (4 min), 36 cycles of 95 °C (50 s), 52 °C (50 s), 72 °C (50 s), and a final extension at 72 °C (5 min). The forward and reverse sequencing was performed on an automatic sequencer (ABI PRISM 3730, Applied Biosystems) using the ABI PRISM BigDye Terminator v.3.1 reagent kit. The resulting sequences were checked manually using the sequence alignment editor BioEdit v.7.2.5 [37].

2.4. Sequence Alignment and Phylogeographic Analyses

The sequences were aligned using the MUSCLE algorithm of MEGA7 [38]. Each new sequence was checked for similar sequences from GenBank through the nucleotide Basic Local Alignment Search Tool, BLASTn [39]. The phylogeographic analyses were executed based on a median joining network approach using Network v.4.6.1.3 software with default settings [40]. The dataset contains 318 COI sequences, including 202 sequences obtained from NCBI GenBank (Supplementary Tables S2 and S3). We followed [41] in using the term “lineage” for designation of the elementary taxonomic units within Corbicula spp. The 28S rRNA polymorphisms in selected Corbicula species were identified with the Toggle Conversed Sites tool of MEGA11 at a 50% level [7,42].

3. Results

3.1. Shell Morphology and Anatomy of Clams

In total, four distinct morphotypes of Corbicula clams were distinguished: European forms R, Rlc, and S [28,30], as well as an “intermediate morphotype” (“Int”), which displayed an intermediate morphology between Rlc and S (Table 1). The average shell length ± SD of adult Corbicula relating to the R morphotype was 15.8 ± 4.5 mm (min–max 10.2–26.0 mm, N = 22), that of the S morphotype was 14.2 ± 2.8 (min–max 10.0–23.4 mm, N = 118), and that of the Rlc morphotype was 12.9 ± 3.9 (min–max 10.0–19.3 mm, N = 17).
The average shell length ±SD of adult specimens of the “intermediate morphotype” was 13.1 ± 1.5 mm (min–max 10.0–15.5 mm, N = 39). The current study presents the first report on records of C. fluminalis from the Samur and Kura Rivers. The main morphological characteristics of the Corbicula lineages are presented in Table S1 and Figure 3. The anatomical structures of siphons are described in Table 1.
Morphometrically, Corbicula lineages clearly differ from each other by the shell inflation index (Kruskal–Wallis test: H = 93.6, df = 3, N = 196, p < 0.0001; post hoc comparisons given in Supplementary Table S4) and the shell elongation index (Kruskal–Wallis test: H = 60.73, df = 3, N = 196, p < 0.0001; post hoc comparisons given in Supplementary Table S5).
At the same time, the PCA of shell variation (SL, SH, and SW) demonstrates that there is no difference between clams belonging to the R, Rlc, and S morphotypes of Corbicula, because all individuals form a single group in the multivariate space (Figure 4). The “intermediate morphotype” is morphologically different from the R, Rlc, and S morphotypes and forms a relatively separate group in the space of principal components PC1 and PC2 (Figure 4).

3.2. Mitochondrial DNA Sequences

We obtained 144 new COI sequences from specimens sampled from the rivers of European Russia during this study (Supplementary Table S2). The COI haplotype analyses allowed us to define two distant genetic lineages (Figure 5). The first lineage apparently matches to C. fluminea (=Corbicula clade FW5; =Corbicula European form R [2,41]). It was found by us in the Don and Volga River basins. The second lineage genetically belongs to another clade, C. fluminalis (=Corbicula European form S; = Corbicula clade FW17 [2,41]); it was registered from the Shura-Ozen, Sulak, and Samur Rivers and Kura River drainage (Figure 5). In addition, the invasive C. fluminalis (FW17, form S) has been detected for the first time in its native range: Azerbaijan (Southwest Asia), Tajikistan (Central Asia), Turkey (Western Asia), and Myanmar (Southeast Asia) (Figure 5).
We discovered an incongruity between mtDNA haplotypes and morphotypes from the Don and Kura River basins (Table 1). The specimens that share the FW5 haplotype correspond to the morphotype Rlc, based on morphological and anatomical features. The specimens having the “intermediate morphotype” by the shell features show the FW17 haplotype of the mtDNA (Table 1).

3.3. Nuclear DNA Sequence Polymorphism

We received 28S rRNA sequences from 56 individuals, including 28 individuals of C. fluminea (Müller, 1774) (=Corbicula clade FW5, European form R) and 28 individuals of C. fluminalis (Müller, 1774) (=Corbicula clade FW17; =Corbicula European form S) (Table 1). The 28S rRNA polymorphism of some individuals of Corbicula was detected: those from Shura-Ozen, Sulak, and Samur Rivers (Caspian Sea basin) at 50%, from Kura River (Terek River basin) at 60%, from Don and Volga River basins at 50% (Table 2). Thus, 18 genotyped specimens of C. fluminea and 19 genotyped specimens of C. fluminalis displayed polymorphic chromatograph profiles. Relying on the visual examination of sequences, we assume that specimens of C. fluminalis and C. fluminea from investigated sites could comprise elements of nuclear genomes occurring from several other Corbicula lineages (Table 2).
Then, we collated our 28S rRNA gene sequences with those received from GenBank according to Bespalaya et al. [7,20]. It is established that four individuals of C. fluminalis from Kura River have three variants of 28S rRNA: the first shows 99.76% similarity (with one nucleotide substitution) to that of C. fluminea from the USA [43] (GenBank accession No. KC429490); the second shows 99.61% similarity (with two nucleotide substitutions) to C. fluminea from South Korea [21] (GenBank accession No. MZ427291); the third shows 99.09% similarity (with six nucleotide substitutions) to C. fluminea from South Korea [21] (GenBank accession No. MZ427291; Table 2).
The 28S rRNA gene sequences from Shura-Ozen, Sulak, and Samur Rivers (Republic of Dagestan) (Figure 1, No. 2) were identical to that from Kura River (Stavropol Krai) (Figure 1, No. 1), but their combinations in each individual were different (Table 2).
In samples from the Don and Volga River basins, ten individuals of C. fluminea have 28S rRNA identical to that of C. fluminea from the USA [43] (GenBank accession No. KC429490); and ten individuals have 28S rRNA identical to C. fluminea from northern European Russia (Northern Dvina River basin) [7] (GenBank accession No. KX347247, KX347249, KX347250, MG757364, and MG757366, Table 2).

4. Discussion

4.1. Genetic and Morphological Analysis of Corbicula Lineages from European Russia

In general, two Corbicula lineages were discovered in the studied area: C. fluminea (=Corbicula clade FW5) was found in the Don and Volga River basins, and C. fluminalis (=Corbicula clade FW17) in the Caspian Sea basin (Shura-Ozen, Sulak, and Samur Rivers, Kura River basin) (Figure 4, Table 1). At the same time, we have recognized four distinct morphotypes of Corbicula clams based on morphological and anatomical data, i.e., R, Rlc, S, and “Int” [28,30] (Table 1). We recorded an incongruity between mtDNA haplotypes and morphological features for all the clams that were collected from the Don and Kura River basins (Stavropol Krai) (Table 1). Specimens from the Don River having the FW5 haplotype correspond to the morphotype Rlc based on morphological and anatomical features. Besides that, specimens from the Kura River have the “intermediate morphotype” (Rlc + S) by the shell features and show the FW17 haplotype of the mtDNA. Interestingly, the European Rlc form belongs to the same mitochondrial lineage as the American Form B, but the B morphotype has a deep purple inner shell surface in contrast to the Rlc morphotype [2,17,31,44]. Identified mismatches are possibly due to the specificity of Corbicula reproduction through androgenesis that may lead to cytonuclear inconsistencies [7,16,20,21]. According to a number of studies, cytonuclear mismatches usually have been detected in places in which different lineages are in coexistence [2,6,7,17,18,31,41,45]. In our research, we found lineages living in sympatry in the Don River basin, namely Rlc and R, and in the Caspian Sea basin (Republic of Dagestan), namely S, R, and Rlc [11]. In the Volga and Kura River basins, we found only individuals with R and “Int” morphotypes, respectively. It was established that the mixed “cytonuclear genotype” can rapidly spread and become fixed in a population [16]. Consequently, populations discovered by us could have been formed as a result of the invasion of androgenic hybrid Corbicula lineages. In addition, the possibility that we were unable to discover other lineages of Corbicula in the Volga and Kura River basins due to their low numbers cannot be excluded. For example, in a heated channel of the Arkhangelsk TPP, we found two lineages of Corbicula. Of these, the Rlc form was rare with a mean density of 0.6 ind./m2 [7]. Moreover, Vastrade et al. [41] expressed a hypothesis that nuclear captures may also occur within a single lineage. Presumably, all points of view regarding the formation of populations in the Kura and Volga Rivers, described above, are not mutually exclusive, and in reality, several scenarios could take place.

4.2. The 28S rRNA Polymorphism

The 28S rRNA polymorphism in C. fluminea and C. fluminalis from studied localities is perhaps conditioned to hybridization events between forms A/R, C/S, and B/Rlc. In particular, the polymorphism discovered by us has probably happened from several hybridization actions followed by a “nuclear genome capture” between distinct Corbicula lineages. This phenomenon was described in detail in previous works [7,16,19,20,21]. A review of this body of literature shows that gene capture, considered initially as a rare event [19], is quite common in Corbicula populations [2,7,17,20,21,28,41]. For example, Vastrade et al. [41] revealed that most Corbicula individuals were heterozygous for 28S rRNA (84%). In our studies, the proportion of hybrid individuals varied from 50% to 60% depending on the population (Table 2). It is obvious that invasive Corbicula lineages originated as a result of androgenetic hybridization that is confirmed by the presence of a common 28S rRNA allele in invasive forms B, Rlc, and C/S and form A/R [6,16,41].

4.3. Dispersal Routes of the Invasive Corbicula Lineages to Aquatic Ecosystems of European Russia

Current molecular genetic studies indicate distinct geographic geneses for the invasive lineages of Corbicula [2,41]. In particular, the form A/R has originated in Japan, form B/Rlc is from the Asian mainland, and form C/S is from Africa [2,45]. Vastrade et al. [41] proposed an alternative hypothesis and suggested at least three distinct biogeographic sources of androgenetic lineages within the genus Corbicula, i.e., Southeast Asia as a source for the Vietnamese androgenetic Corbicula lineages, and Japan as a source for A/R, B, and Rlc forms. Furthermore, the authors suggested that the C/S form invaded South Africa and formed a population in this region. Hence, C. africana has probably originated from the C/S form rather than the opposite [41]. Until now, the FW17 (form C/S) invasive lineage has not been detected in the eastern part of the native range of Corbicula [2,5,41]. However, our COI haplotype network reveals that the COI sequences from Azerbaijan (Southwest Asia), Tajikistan (Central Asia), Turkey (Western Asia), and Myanmar (Southeast Asia) belong to C. fluminalis (=FW17; =form S). The sequences from Myanmar have four nucleotide substitutions from those from South America, Europe, Southwest Asia, and Central Asia (Figure 5). The sequences from Azerbaijan, Turkey, and Tajikistan were identical to those from South America and Europe or have only 1–2 nucleotide substitutions (Figure 5). Thus, the invasive lineage C. fluminalis (=FW17; =form S) has a wider distribution in Europe, South America, Southwest Asia, Southeast Asia, Western Asia, and Central Asia than it was thought previously. Taking into account that the COI sequences from the native range (Asia) analyzed by us were identical to samples from South America and Europe, we assume that invasion of this lineage has started from Asia, with a subsequent spread to Africa and South America, and then to Europe. This finding supports the hypothesis of Vastrade et al. [41] that the invasive lineage FW17 (form S) has probably expanded its range to South Africa. It cannot be excluded that the lineage FW17 may have also been introduced to the European subcontinent directly from the Asian populations. The COI sequences obtained by us from the Caspian Sea basin (Republic of Dagestan and Stavropol Krai of Russia) were identical to or have only 1–2 nucleotide substitutions compared to those from Europe, South America, northern European Russia, Azerbaijan, Tajikistan, and Turkey (Karasu River, Orontes River basin) (Figure 5). It should be noted that the type locality of C. fluminalis is situated in the Euphrates River, not far from the Orontes basin. Additionally, most individuals from the Caspian Sea basin share similar COI and 28S rRNA gene haplotypes, suggesting a possible common origin of these Corbicula lineages. Thus, we assume that the invasion of C. fluminalis (=form S; =Corbicula clade FW17) to reservoirs and watercourses of the Caspian Sea basin (Republic of Dagestan and Stavropol Krai) was from Western Asia and Transcaucasia. A similar hypothesis was expressed earlier by Nabozhenko M. and Nabozhenko S. [9] and by Voroshilova et al. [27]. All clams collected from the Don and Volga River basins share the single COI and 28S rRNA haplotypes that indicate a common origin of these Corbicula lineages. These specimens have an identical COI haplotype to sequences from South and North America, Europe, northern European Russia, China, and Japan. Consequently, we hypothesize that the introduction of C. fluminea (=Corbicula clade FW5) to the Don and Volga River basins was from unspecified European countries. Among the known dispersal mechanism of Corbicula clams, human activities such as transport of ballast water, construction of canals for various purposes, dams, irrigation and drainage systems, sand mining, and food resources, as well as natural passive transfer by migratory waterfowl and waterways, seem to be the most likely causes of global spreading of invasive lineages.

5. Conclusions

Here, we provide new and important insights into the invasive lineages of Corbicula clams in European Russia based on an integrative approach. According to our data, studied Corbicula populations comprise two androgenic invasive lineages, belonging to the biological species C. fluminea and C. fluminalis. We observed a discrepancy between mtDNA haplotypes and morphological features for all individuals that were sampled from the Don and Kura Rivers, which may be due to the androgenetic reproduction of Corbicula that leads to cytonuclear inconsistencies. The 28S rRNA polymorphism in C. fluminea and C. fluminalis from studied localities is perhaps due to hybridization events between forms R, S, and Rlc. It is obvious that the frequency of this process in Corbicula clams is clearly underestimated and remains poorly understood. Future studies should focus on the genetic diversity and cytogenetic characteristics of Corbicula clams both in their native range and beyond.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w15183226/s1, Table S1: Shell measurements of Corbicula clams from European Russia; Table S2: Information on newly sequenced specimens of Corbicula spp. from European Russia. Materials are from the collection of the RMBH, N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences (Arkhangelsk, Russia); Table S3: Information on additional COI sequences of Corbicula clams obtained from GenBank, including taxon, accession number, region, and data source; Table S4: The shell inflation index across the four Corbicula morphotypes. Kruskal–Wallis test statistic/p value: H (χ2) = 93.6; p ≤ 0.0001. Significant pairwise comparisons following Bonferroni correction; Table S5: The shell elongation index across the four Corbicula morphotypes. Kruskal–Wallis test statistic/p value: H (χ2) = 60.73; p ≤ 0.0001. Significant pairwise comparisons following Bonferroni correction.

Author Contributions

A.V.K. (Alexander V. Kropotin) and Y.V.B. conceptualized the paper. O.V.A., A.V.K. (Alexander V. Kropotin), I.S.K., O.V.T., D.M.P. and I.N.B. conducted field research and collected samples. A.S.A. created the maps. A.V.K. (Alexander V. Kropotin) and A.V.K. (Alexander V. Kondakov) performed the molecular genetic studies. O.V.A. created the figures. A.V.K. (Alexander V. Kropotin) and Y.V.B. wrote the paper, with input from all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Russian Science Foundation (project No. 21-14-00092 to Y.V.B., A.V.K., O.V.T. and A.V.K.—field sampling, molecular genetic and phylogeographic analyses of clams, and project No. 21-74-10155 to O.V.A., I.S.K. and A.S.A.—anatomical studies of clams, mapping and fieldwork) and by the Ministry of Science and Higher Education of the Russian Federation (project No. FUUW-2022-0039—shell morphometry and statistical analysis).

Data Availability Statement

The specimens are deposited in the Russian Museum of the Biodiversity Hotspots (RMBH) of N. Laverov Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences (Arkhangelsk, Russia). The new COI and 28S rRNA gene sequences obtained during this study are deposited in GenBank and presented in Supplementary Table S2.

Acknowledgments

The authors express their gratitude to Boris Levin and Dmitry Karabanov for the samples of Corbicula clams from Volga and Büyük Menderes River (Turkey).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Map of records of Asian Corbicula clams in European Russia. (1) Caspian Sea basin, Republic of Dagestan; (2) Kura River, Terek River basin, Stavropol Krai; (3) Don River basin, Rostov Oblast; (4) Volga River basin, Kostroma Oblast. The green dot indicates the locality of the Corbicula according to Bespalaya et al. [7]. The red dots (sampling areas) and the yellow dots (sampling sites) indicate where Corbicula samples were collected in this study.
Figure 1. Map of records of Asian Corbicula clams in European Russia. (1) Caspian Sea basin, Republic of Dagestan; (2) Kura River, Terek River basin, Stavropol Krai; (3) Don River basin, Rostov Oblast; (4) Volga River basin, Kostroma Oblast. The green dot indicates the locality of the Corbicula according to Bespalaya et al. [7]. The red dots (sampling areas) and the yellow dots (sampling sites) indicate where Corbicula samples were collected in this study.
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Figure 2. The habitat of Corbicula fluminalis in the Malyi Sulak River (Republic of Dagestan, Russia). Photos: O.V. Aksenova.
Figure 2. The habitat of Corbicula fluminalis in the Malyi Sulak River (Republic of Dagestan, Russia). Photos: O.V. Aksenova.
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Figure 3. Morphology and anatomy of Corbicula clams from European Russia: (A) specimen MCorb-131.1, C. fluminea (=lineage FW5; =Corbicula R form), Volgorechensk TPP, Volga River basin, Kostroma Oblast; (B) specimen MCorb-133.1, C. fluminea (=Corbicula lineage FW5; =Corbicula Rlc form), Don River basin, Rostov Oblast; (C) C. fluminea (= Corbicula R form), Don River basin, Rostov Oblast; (D) specimen MCorb-136.2, C. fluminalis (=Corbicula lineage FW17; =Corbicula form S; Caspian Sea basin, Republic of Dagestan); (E) specimen MCorb-141.1, C. fluminalis (=Corbicula lineage FW17; =Int form; Kura River, Terek River basin, Stavropol Krai). Scale bars—2 mm. Photos: O.V. Aksenova.
Figure 3. Morphology and anatomy of Corbicula clams from European Russia: (A) specimen MCorb-131.1, C. fluminea (=lineage FW5; =Corbicula R form), Volgorechensk TPP, Volga River basin, Kostroma Oblast; (B) specimen MCorb-133.1, C. fluminea (=Corbicula lineage FW5; =Corbicula Rlc form), Don River basin, Rostov Oblast; (C) C. fluminea (= Corbicula R form), Don River basin, Rostov Oblast; (D) specimen MCorb-136.2, C. fluminalis (=Corbicula lineage FW17; =Corbicula form S; Caspian Sea basin, Republic of Dagestan); (E) specimen MCorb-141.1, C. fluminalis (=Corbicula lineage FW17; =Int form; Kura River, Terek River basin, Stavropol Krai). Scale bars—2 mm. Photos: O.V. Aksenova.
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Figure 4. The principal components analysis (PCA) of the shell variation of R, Rlc, S, and “Int” forms of Corbicula clams from European Russia: R—green circles, Rlc—yellow circles, S—red circles, and “Int”—blue circles. Principal component PC1 explains 97.3% of the total variation, and PC2 explains 2.2% of it. Solid lines show 95% confidence ellipses.
Figure 4. The principal components analysis (PCA) of the shell variation of R, Rlc, S, and “Int” forms of Corbicula clams from European Russia: R—green circles, Rlc—yellow circles, S—red circles, and “Int”—blue circles. Principal component PC1 explains 97.3% of the total variation, and PC2 explains 2.2% of it. Solid lines show 95% confidence ellipses.
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Figure 5. Median-joining haplotype network of 318 COI sequences (length = 604 bp; Supplementary Table S2) of Corbicula species. Circles represent different haplotypes, with the size reflecting their frequency (the smallest = 1 sequence). Red dotted contours indicate putative species-level haplogroups. Numbers near branches indicate numbers of nucleotide substitutions per site.
Figure 5. Median-joining haplotype network of 318 COI sequences (length = 604 bp; Supplementary Table S2) of Corbicula species. Circles represent different haplotypes, with the size reflecting their frequency (the smallest = 1 sequence). Red dotted contours indicate putative species-level haplogroups. Numbers near branches indicate numbers of nucleotide substitutions per site.
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Table 1. The morphological and molecular characteristics of invasive Corbicula clams.
Table 1. The morphological and molecular characteristics of invasive Corbicula clams.
LocalitySpecies (Lineages, Forms)MorphotypeShell ShapeExternal Shell ColorationInternal Shell ColorationShell SculptureUmbo PositionAnatomy of SiphonsCOI HaplotypeHybridizing Lineages **
Volga River basinCorbicula fluminea (=Corbicula clade FW5; =Corbicula R form)RRound and broad, triangular Dark brownMatte, with
purple marks
Distinctly pronounced deep concentric ridges Slightly
shifted
Light yellow color with black spots in the siphonal papilla and dark pigmentation aroundFW5R × Rlc
Don River basinCorbicula fluminea (=Corbicula clade FW5)RlcOval triangularYellow or olive greenYellow white, with small
purple marks
Raised, regular concentric ribsCentrally locatedThe papillae are long, have different lengths, and orange coloration with black spots and dark coloration aroundFW5R × Rlc
C. fluminea (=R form) *ROval triangularDark brownWhite, with purple marksDistinctly pronounced deep concentric ridgesSlightly
shifted
N/AN/AN/A
Shura-Ozen Sulak, and Samur River basin (Republic of Dagestan)C. fluminalis (=Corbicula clade FW17; Corbicula form S)SNarrow, saddle-likeYellow or olive greenDeep purpleTight spaced
ridges
Significantly
prominent
The bases of each siphon and tentacle have a dark pigmentationFW17R × S
Kura River (Stavropol Krai)C. fluminalis (=Corbicula clade FW17; Corbicula form S)“Intermediate form” Rlc + SOval triangularYellow or olive greenDeep purpleRaised, regular
concentric ribs
Centrally locatedRather broad pigmented ring is not present. Siphonal papillae are pigmentedFW17Rlc × R × S
Notes: * Only empty shells were found; ** based on analysis of 28S rRNA gene sequences (Table 2); N/A—not available.
Table 2. 28S rRNA polymorphism in selected Corbicula species *.
Table 2. 28S rRNA polymorphism in selected Corbicula species *.
Species432433434438446485533549550570655726
C. fluminea (USA)-AGCACAGGTTT
C. fluminea (Arkhangelsk Oblast) ** (N = 5)-A/CA/GA/C··A/C··C/T··
C. fluminea (Kostroma Oblast) (N = 5)-···········
C. fluminea (Kostroma Oblast) ** (N = 5)-A/CA/GA/C··A/C··C/T··
C. fluminea (Rostov Oblast) (N = 5)-···········
C. fluminea (Rostov Oblast) ** (N = 5)-A/CA/GA/C··A/C··C/T··
C. fluminalis (Turkey) ** (N = 8)-CAA··CA/GG/TCG/T·
C. fluminalis (Dagestan) ** (N = 4)-····A······
C. fluminalis (Dagestan)** (N = 1)-CAA··C··C··
C. fluminalis (Dagestan) *** (N = 5)Var1-CAA··C··C··
Var2GCAA··CATCG·
C. fluminalis (Stavropol Krai) ** (N = 4)-····A······
C. fluminalis (Stavropol Krai) *** (N = 5)Var1-CAA··C··C··
Var2GCAA··CATCG·
C. fluminalis (Stavropol Krai) *** (N = 1)Var1-····A······
Var2GCAA··CATCG·
C. africana (South Africa)GCAAG·C··CG·
C. africana (South Africa)GCAA··C··C·C
Notes: *—Numbers in the top row refer to nucleotide positions within a condensed alignment of the sequences, letters are FASTA codes for nucleotides but with additional polymorphisms shown explicitly. Dots indicate a nucleotide matching the first sequence. Dashes indicate gaps after alignment of the sequences; **—Samples with two 28S rDNA variants of the same length; ***—Samples with two variants of 28S rRNA with different length.
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Kropotin, A.V.; Bespalaya, Y.V.; Aksenova, O.V.; Kondakov, A.V.; Aksenov, A.S.; Khrebtova, I.S.; Palatov, D.M.; Travina, O.V.; Bolotov, I.N. Genetic and Morphological Characterization of the Invasive Corbicula Lineages in European Russia. Water 2023, 15, 3226. https://doi.org/10.3390/w15183226

AMA Style

Kropotin AV, Bespalaya YV, Aksenova OV, Kondakov AV, Aksenov AS, Khrebtova IS, Palatov DM, Travina OV, Bolotov IN. Genetic and Morphological Characterization of the Invasive Corbicula Lineages in European Russia. Water. 2023; 15(18):3226. https://doi.org/10.3390/w15183226

Chicago/Turabian Style

Kropotin, Alexander V., Yulia V. Bespalaya, Olga V. Aksenova, Alexander V. Kondakov, Andrey S. Aksenov, Irina S. Khrebtova, Dmitry M. Palatov, Oksana V. Travina, and Ivan N. Bolotov. 2023. "Genetic and Morphological Characterization of the Invasive Corbicula Lineages in European Russia" Water 15, no. 18: 3226. https://doi.org/10.3390/w15183226

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