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Article

Genetic Diversity and Distribution of Haplotypes of Freshwater Eel in Baltic Lakeland Based on Mitochondrial DNA D-Loop and Cytochrome b Sequence Variation

by
Jeļena Oreha
1,*,
Aleksandra Morozova
1,
Alberts Garkajs
1,
Muza Kirjušina
1,
Inese Gavarāne
2,
Vladimir G. Kostousov
3,
Mihails Pupins
1 and
Nataļja Škute
1,*
1
Department of Ecology, Institute of Life Sciences and Technologies, Daugavpils University, Parādes Str. 1A, LV-5400 Daugavpils, Latvia
2
Department of Biodiversity, Institute of Life Sciences and Technologies, Daugavpils University, Parādes Str. 1A, LV-5400 Daugavpils, Latvia
3
Institute of Fisheries of the National Academy of Sciences of the Republic of Belarus, 220072 Minsk, Belarus
*
Authors to whom correspondence should be addressed.
Diversity 2024, 16(9), 522; https://doi.org/10.3390/d16090522
Submission received: 1 July 2024 / Revised: 3 August 2024 / Accepted: 19 August 2024 / Published: 31 August 2024
(This article belongs to the Section Biodiversity Conservation)
Browse Figures
Versions Notes

Abstract

:
The European eel (Anguilla anguilla (L.)) is a unique catadromous euryhaline fish and the only anguillid eel species classified as ‘critically endangered’. The Baltic Lakeland area contains naturally recruited and introduced eels. There is great uncertainty regarding a baseline for the location and number of naturally dispersed eels in the region. Little is known about the genetic structure of the European eel populations in Baltic countries. The estimation of population genetic structure is important for the efficient management of naturally recruited and introduced eels. Two mitochondrial regions were used to investigate the genetic structure within and between eel samples from 11 waterbodies. In this study, new, unique, and widely distributed haplotypes were revealed. The studied eel population in the Baltic Lakeland shows high genetic diversity, which is possibly a result of intensive restocking programs. Sequences characterized for Anguilla rostrata were revealed in both mitochondrial regions. Understanding the genetic structure of eel populations worldwide is crucial for conservation efforts. Eel restocking in waterbodies where natural migration is restricted contributes to diversity loss for the world gene pool of eels.

1. Introduction

The European eel (Anguilla anguilla (L.)) is a unique catadromous euryhaline fish characterized by a complex biological cycle involving marine, brackish, and freshwater habitats [1]. It is the only anguillid eel species classified as ‘critically endangered’ [2], and its stock is in a multi-decadal decline. In freshwater and coastal waters, eels are endangered due to a number of reasons: overfishing (illegal fishing and exporting), pollution, diseases, parasite infections, predation, obstacles to migration (hydropower turbines and pumping stations), and habitat loss [3]. All these potential factors can act simultaneously [4,5]. A range of normative acts has been adopted for eel restoration, such as Regulation EC 1100/2007 [6]. Since 2001, the International Council for the Exploration of the Sea (ICES) has recommended that eel catches be ‘reduced to the lowest possible level’ [7].
The Baltic Lakeland area contains naturally recruited and introduced eels. A large part of the eel stock in the Baltic region is likely to consist of introduced eels, mainly from France [6]. There is therefore great uncertainty regarding a baseline for where and how many naturally dispersed eels there could be in the region. The stocking eels caught elsewhere cannot go to spawn due to improper stocking in waterbodies that are not suitable for eel migration. Only four waterbodies in Latvia are freely accessible, allowing the natural migration of eels [8]. Natural recruitment in Northern Europe, which includes the Baltic States, was estimated to be very low. Little is known about the genetic structure of the European eel populations in Baltic countries.
Eels experience a broad range of intrinsic and extrinsic influences during their lives, which impact their population dynamics and genetic structure. Knowledge on anguillid genetics continues to grow, and research can be separated into studies on genetic species and hybridization status, spatio-temporal genetic structure, genetic variability–fitness correlations, and the application of genomics in eel aquaculture [9]. Assessing the genetic structure of the A. anguilla population in the Baltic Lakeland region is important for the effective management of naturally recruited and introduced eels, which was the main goal of this study. Two mtDNA regions were used to investigate the genetic structure within and between eel samples from different waterbodies.
We assumed that the eel population in the investigated area would have no clear genetic structure and possibly have high genetic diversity. The main reason for these assumptions was the existence of obstacles to migration (including the fragmentation of waterbodies), due to which genetic diversity in the studied waterbodies cannot be updated naturally. Another important factor which has a strong influence on eel genetic diversity is regular restocking, which theoretically can make genetic diversity levels high or very low.
Research about the genetic diversity of eel populations in waterbodies in Latvia and Belarus has just started, and this genetic knowledge is important for eel resource management.

2. Materials and Methods

2.1. Sample Collection

European eel samples were collected as part of the sampling efforts of eel monitoring plans between 2014 and 2020. Samples of fish tissue (skeletal muscles or fins) were taken and stored at −80 °C. The material was collected from seven lakes in Latvia, namely, Lake (hereafter abbreviated as ‘L.’) Usma, L. Liepaja, L. Kisezers, L. Aluksne, L. Vaidava, L. Razna, and L. Siver, and from three lakes in Belarus, namely, L. Myadzyel, L. Svir, and L.Vialikija Svaksty, and one river, namely, Myadzyelka (Figure 1, Table 1).

2.2. DNA Extraction and Mitochondrial DNA Sequencing

Genomic DNA was extracted from muscle tissue or fins according to the salt extraction method of Aljanabi and Martinez [10], which was successfully used in genetic research of aquatic organisms [11,12]. The quality and quantity of DNA samples were determined using a DeNovix DS-11 spectrophotometer (DeNovix Inc. Wilmington, DE USA). The extracted DNA was stored at −20 °C until analysis. For the analysis, the DNA was diluted to a concentration of 10 ng/μL.
The primers Ang1 which enabled the amplification of a fragment of mtDNA (part of the tRNA for proline and part of the D-loop region) created for A. anguilla [13,14] were used. The oligonucleotide primer Ang1 sequences were as follows: forward primer: 5′-TCGGTTTTGTAATCCGAAGA-3′; reverse primer: 5′-CCAAATGCCAGTAATAGTTCATTTTA-3′. The primers Ang2 for part of the cyt b gene developed for A. anguilla [15] were used. The oligonucleotide primer Ang2 sequences were as follows: forward primer: 5′-CCTCCTTCTTCTTTATCTGCCT-3′; reverse primer: 5′-GTTTTCTAGTCAACCTGCTAATGG-3′.
Polymerase chain reaction (PCR) was performed using an ABI 9700 thermocycler with a total reaction volume of 10 μL, containing ddH2O, 10 × PCR buffer, 25 mM MgCl2, 2 mM dNTP mix, 3 pmol of each primer, 0.1 U Taq DNA polymerase, and 100 ng template DNA. Amplification started with an initial denaturation step for 5 min at 95 °C, followed by 35 cycles (denaturation for 45 s at 94 °C, annealing for 45 s at 56 °C, elongation for 1 min at 72 °C), and ended with a final elongation step for 5 min at 72 °C. The length of the amplified fragment was approximately 550 bp for D-loop and 520 bp for cyt b. The amplified product was evaluated electrophoretically for quality using 2% agarose gels and was purified using an EXS-500 ExS-Pure™ Enzymatic PCR purification kit (NimaGen B.V. Hogelandseweg, The Netherlands).
Samples were sequenced in both directions using a Big-Dye Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher, Waltham, MA USA) on an ABI 310 automated sequencer. Thirty-four cyt b sequences from the first step of our studies [16] and five new cyt b sequences were analyzed together. The received 39 cyt b and 35 D-loop sequences were edited and then aligned with the Clustal W algorithm [17] using MEGA v.11 software [18]. All received sequences are stored in the public domain database in GenBank; the registered sequences were given the accession numbers OR682679–OR682715, OR689393–OR689397, OR689359–OR689392.

2.3. Neutrality Test, Nucleotide Diversity, and Population Structure

The haplotype number (Nh), haplotype diversity (h), nucleotide diversity (π), number of polymorphic sites (S), number of mutations (ŋ), average number of nucleotide differences (k), Tajima’s D, and Fu’s Fs were calculated using DnaSP v6.12 [19] separately for sequences of the D-loop region and a part of the gene cyt b.
The haplotype networks of the D-loop region and the gene cyt b were constructed with the median joining (MJ) [20] algorithm, using the pegas package in R v 4.1.3 software [21,22]. The trees based on maximum likelihood (ML) (Tamura–Nei model) analysis for the gene cyt b and D-loop region with 1000 replicates were constructed using MEGA v.11 software [18] and visualized in FigTree v1.4.4 [23]. The map with the distribution of mtDNA cyt b haplotypes in Europe and North America was made using R v 4.1.3 software [22].
Sequences of the gene cytochrome b (further cyt b), including 16 A. rostrata sequences, 46 sequences of A. Anguilla, and 9 sequences of A. anguilla * A. rostrata hybrids, were retrieved from GenBank (Table S1), and 34 sequences of part of the gene cyt b from our recent research [16] and 5 new sequences from the present study were added to the analysis. Totally, 110 sequences of the gene cyt b of Atlantic eels were included to the study to evaluate haplotype distribution and the efficiency of the eel restocking program and reveal the migratory life histories of European eels (Table S1). All sequences were trimmed to 394 bp in order to investigate new data together with available data retrieved from GenBank.

3. Results

3.1. Variability in Part of Cyt b Gene

Altogether 19 haplotypes from 39 sequences were revealed for cyt b. The total number of aligned sites per sequence was 394. Diversity of cyt b haplotypes and its distribution across studied waterbodies in the Baltic Lakeland are shown in Figure 2. New analyzed sequences, three from individuals from L. Aluksne and one from L. Razna, belong to haplotypes which were described earlier [16]. Two haplotypes, namely II and III, are widely distributed in the studied waterbodies as well as in the North European region. One haplotype is quite rare and a new haplotype was revealed (XI and I, respectively). A sequence which belongs to the newly revealed haplotype is unique and was detected only in a sample from L. Aluksne. Out of the nineteen revealed haplotypes, eight were unique, that is, identical sequences were not found in GenBank. New sequences have been deposited in GenBank (Table S2).
The most widespread haplotypes in our research are II and III, which were detected in seven eel individuals from five waterbodies and in ten individuals from seven waterbodies, respectively (Figure 2). Revealed haplotype diversity was the highest in L. Svir, L. Siver, and in L. Kisezers. In these lakes, almost all identified haplotypes were different. A phylogenetic neighbor joining tree of sequences of part of the gene cyt b shows the relationship between investigated eel individuals (see Figure 3). The ML tree branched out into three main groups. The sample from L. Aluksnes detected as A. rostrata branched separately.

3.2. Distribution of mtDNA Cyt b Haplotypes in Europe and North America

Distributions of A. anguilla mtDNA cyt b haplotypes in Europe and North America are shown in Figure 4. Most of the haplotypes in the present study have been reported earlier for eels in their natural area of distribution. For instance, two of the widespread haplotypes were also reported for A. rostrata isolates from waterbodies in North America and Greenland. Four of the revealed haplotypes were reported for A. anguilla and A. rostrata hybrids in Greenland and Iceland [24,25,26]. One haplotype was detected in L. Aluksnes only, but that sequence has been reported in GenBank for twenty isolates as A. rostrata (American eel) from different waterbodies in North America [24] and for one A. rostrata isolate in Taiwan [26].

3.3. Variability in Part of D-Loop Region

A total of 38 individuals were sequenced and the total number of sites was 393, the number of polymorphic sites was 73 and the number of conserved sites was 320. Genetic diversity statistics and neutrality tests for the D-loop region are shown in Table 2. Haplotype diversity (Hd) for all 37 sequences was calculated to be 0.991 +/− 0.009 SD and nucleotide diversity π was 0.031 +/− 0.0032 SD. The average number of nucleotide differences k = 12.14. In total, 84 point mutations were noted and 36 were parsimony informative sites. In spite of high haplotype diversity, low nucleotide diversity values suggested small differences between haplotypes. Both Tajima’s D and Fu’s Fs statistics were negative (−1.46 and −16.03) and statistically significant (**, p < 0.05), indicating an excess of rare nucleotide site variants in comparison to what would be expected under a neutral model of evolution.
Altogether, 32 haplotypes of the D-loop region were revealed. A summary of haplotype variation is given in Table S2. Diversity of haplotypes and their distribution across studied waterbodies are shown in Figure 5. All revealed haplotypes are unique (identical sequences in GenBank have not been found). Almost all haplotypes (28) are rare in the studied waterbodies and were revealed for one eel individual each. Haplotype I was found in eel individuals from three lakes (namely, L. Liepaja, L. Svir, and L. Vialikija Svaksty). Haplotypes II, XX, and V were revealed in two eel individuals each. Sequences of haplotype VIII were identified in GenBank as Anguilla rostrata with similarity from 96.3 up to 98.77 percent; similarity with A. anguilla was shown below 95 percent. Altogether, 37 sequences of part of the D-loop region have been deposited in GenBank.
The phylogenetic tree of revealed sequences of part of the D-loop region shows the relationship between investigated eel individuals (see Figure 6). The ML tree branched out into three main groups. The sample from L. Aluksnes detected as A. rostrata branched out separately.

4. Discussion

Investigation of Atlantic eel populations is common around the world and shows that they are unstable and fragile under different factors. In the past five decades the European eel (A. Anguilla) population has experienced up to a 90% decline in recruitment in some parts of its distribution range [7,27]. One future strategy for sustainability of freshwater eels is to provide the genetic data of this species. For haplotype diversity, the wide use of the conservative gene cyt b can reveal more recent events in population and the highly variable D-loop region can reveal ongoing evolution [10,14,15,25,28].
Little is known about the genetic structure of the European eel populations in Baltic countries. There were studies of mtDNA D-loop sequences [10,14] and sequences of the gene cyt b [15] of European eels caught in Lithuanian territorial waters. The results of the studies indicate that although A. anguilla is considered a panmictic species the genetic structure of its population indicated by mtDNA markers could be characterized as a genetic mosaic. By analyzing the sequences of the cyt b gene of mtDNA for individuals caught in locations throughout the Baltic Lakeland (in inland waters of Latvia and Belarus), the population genetic structure of A. anguilla in the area was determined [16].
Research on the genetic diversity of eel populations in waterbodies of Latvia and Belarus has just begun, and this genetic knowledge is essential for eel resource management.
Inland waters in Latvia and Belarus contain both naturally recruited and introduced eels. After 1990, the species was recorded in fishing haul statistics from sixteen Latvian lakes, but only four of these waterbodies are freely accessible to natural migration of the species [8]. Natural eel migration from waterbodies in Belarus to the Curonian Lagoon in the Baltic Sea is possible via the Neman River [28]. Restocking of eels in Latvian waterbodies has continued. However, eel restocking in Belarusian waterbodies has been restricted since 2008 in accordance with Regulation EC 1100/2007.
Assessing genetic parameters is an important factor in making decisions regarding the development and implementation of conservation strategies for the European eel. In the present research, haplotype diversity was high and nucleotide diversity was low in all investigated eels across all sampling sites for both the sequenced cyt b gene and in the D-loop region, indicating small differences between haplotypes. Eels, being highly migratory and long-lived species, may have complex population structures and multiple distinct subpopulations [29]. This can lead to the accumulation of diverse haplotypes over time due to migration, selective pressures, and restocking. On the other hand, population bottlenecks or founder effects could cause a decrease in nucleotide diversity by limiting the number of unique alleles that are inherited in the population. High haplotype diversity and low nucleotide diversity in the European eel population suggest a population bottleneck followed by rapid population growth and the accumulation of mutations [30]. This was shown by the cyt b gene for European eel populations from waterbodies in Ireland, Italy, Morocco, Sweden, and the UK [27]. Later studies also demonstrated similar patterns in naturally recruited and introduced eels from inland waterbodies in Lithuania, the Curonian Lagoon, and the Gulf of Riga [15]. In our case, these patterns could be the result of restocking, which has been carried out regularly in lakes, while natural eel migration is only possible from four waterbodies in Latvia [8]. The median joining network of cyt b haplotypes (Figure 2) indicates an ancestral haplotype (II). Haplotypes I, III, VI, VII, and XVIII, which differ by one transition from haplotype II, form five small haplogroups. The median joining network of D-loop region haplotypes (Figure 5) shows similarities with cyt b haplotype grouping, with haplotype I possibly being the most common ancestral type. All identified haplotype sequences are unique, with no analogous sequences found in GenBank.
The haplotypes, which have end positions in the network and differ from nearby haplotypes in one or two transitions, could show recent emergence. Similar assignments of haplotypes are also shown in other eel population research [15,28]. Thus, all of this points to a bottleneck effect that occurred in the past in eel populations, as confirmed not only by genetic data but also by the documented decline in eel populations over the past five decades [7,27].
Figure 4 shows the distribution of cyt b haplotypes worldwide. For this analysis, 35 cyt b sequences from the present study were used, including only identical sequences retrieved from GenBank (see Table S1 for sequences). The revealed sequences are identified as belonging to European eel and its hybrids with American eel in Latvian and Belarusian waterbodies. During the research a significant number of unique haplotypes were detected, indicating that the eel population in part of the Baltic Lakeland is unique. Given that only four waterbodies in Latvia are freely accessible for migration (as mentioned above), nearly all of these unique haplotypes may be lost from the global eel gene pool.
Both phylogenetic trees (for cyt b and D-loop) branched into three main groups (Figure 3 and Figure 6, respectively), and these groups do not correspond to specific lakes. The bootstrap values are generally quite low. Higher bootstrap values (>50) in both trees were revealed for nodes where sequences differ in a greater number of substitutions. The possible explanation for the lack of distinct geographic clades is gene flow and the possibility of migration among eels. Eels are known for their extensive migratory behavior, particularly during their spawning migrations. Unlike anadromous fishes, catadromous eels do not migrate to a freshwater site of previous occupancy (homing) or that is tied to a specific genetic lineage. European eels belong to the same panmictic population and distribute over a broad latitudinal range [31]. This behavior facilitates the exchange of genetic material across vast distances, thereby homogenizing the genetic differences between populations from different localities [32]. Earlier studies have shown that the temporal distance of recruiting juveniles of the European eel (Anguilla anguilla L.) was not correlated with genetic distance, and samples from similar sampling dates did not cluster together [33]. For A. anguilla elvers at the two studied Egyptian sites, it was also shown that samples from the Lake Burullus mouth and the Rosetta (Rachid) estuary also did not cluster together despite having several site-specific haplotypes [34].
However, considering that natural eel migration is possible in only four Latvian waterbodies [8], gene flow and migrations can only partly explain the phylogenetic links observed in the constructed trees. Historical and demographic events, such as population bottlenecks due to overfishing, disease, and migration obstacles, as well as past expansions from regular restocking, may also shape the current genetic links of eels in the studied lakes. These events can mix genetic lineages, leading to the homogenization of genetic structure. The trees likely reflect the phylogenetic relationship among eel individuals due to the mixing of genotypes from multiple years of restocking and the admixture of natural eel genotypes in waterbodies where natural migration is still possible. A sequence characteristic of A. rostrata was detected in Lake Aluksnes, both in the sequenced cyt b gene and the D-loop region (haplotype IX in Figure 2 and haplotype VIII in Figure 5, respectively), forming a separate branch in the trees shown in Figure 3 and Figure 6.
An identical sequence of a part of the cyt b gene has been reported in GenBank for 20 isolates as A. rostrata (American eel) from various waterbodies in the USA and Canada, as well as from one water body in Taiwan [24,35] (Figure 4).
The American eel (A. rostrata) is native to North America. However, in European waterbodies A. rostrata was exclusively found either among samples from stocked waters or in aquaculture farms [36]. The authors reject the possibility of A. rostrata and their hybrids migrating into single German rivers or lakes while the majority of the surrounding waters are inhabited exclusively by European eels.
In our case, Lake Aluksne is a waterbody that is not freely accessible to natural migrations [37], so the anthropogenic invasion may have resulted from incorrect restocking practices. Human activities, such as trade and aquaculture, can sometimes lead to the introduction of non-native species into new areas. Therefore, it is essential to monitor and manage these introductions to prevent potential ecological impacts. The impact of introduced species can be both positive and negative on native biota [38]. For example, the salmon parasite Gyrodactylus salaris was introduced to Norway with imported salmon smolt and rainbow trout (Oncorhynchus mykiss) fingerlings from Sweden in 1975. This parasite now causes annual losses estimated at NOK 200–250 million in Norwegian river systems [39]. However, the positive impacts of introducing fish species are often debatable [40]. Generally, most introduced fish species have negative effects on native species through predation, competition for resources, and the spread of parasites and diseases.
A. anguilla and A. rostrata are very similar species in terms of their biology. How could they coexist in common habitat? There is a study of A. rostrata introduced into a European river that aimed to estimate their competitive potential in a non-native habitat [41]. The results show that A. rostrata develops normally in European waters and successfully competes with the native European eel (Anguilla Anguilla). However, A. rostrata appears to be more susceptible to the Asian swimbladder nematode Anguillicola crassus than A. anguilla and could facilitate the further propagation of this parasite. Additionally, differences in fat content and gonad mass between two species reflect species-specific adaptations to spawning migration distances. So, that study indicates that A. rostrata is a potential competitor for the native fauna in European fresh waters. Marohn with coauthors [41] also suggest that it is unlikely that A. rostrata will establish self-sustaining populations in European waters due to its semelparous life cycle and the adaptation of A. rostrata larvae to shorter drift distances. Therefore, anthropogenic translocation is the main source of A. rostrata in Europe. Recent studies of eels from Lake Razna in Latvia revealed that 57% of analyzed individuals were infected with the swimbladder parasite A. crassus [42]. This underscores the need for genetic data on A. anguilla in Europe inland waters, particularly genetic data of restocking material before restocking is carried out.
We consider that future releases of A. rostrata into European waters must be strictly avoided so as not to further increase the pressure on A. anguilla and cause further deterioration of its stock situation. At least, eel restocking must be avoided in waterbodies where natural eel migration is still present.

5. Conclusions

Understanding the genetic structure of eel populations worldwide is crucial for conservation efforts. There is a crucial need for identifying genetic patterns of restocking recourse to detect and prevent potential threats for natural eel populations in advance. Regular restocking of eels into waterbodies where migration is restricted both into and out of these areas leads to a decrease in genetic diversity within the global eel population. Consequently, the gene pool of these individuals may be lost irreversibly from the global eel gene pool. We believe that the most beneficial solution would be to halt the eel restocking program entirely and focus on conserving and managing eel populations in their native habitats.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/d16090522/s1, Table S1: Sequences used for A. anguilla analysis of mtDNA cyt b haplotype distribution in Europe and North America [43,44,45,46,47,48]; Table S2: Summary of D-loop haplotype variation. Haplotypes are given in the first column, number of occurrences in the last column. Only variable sites, with sequence positions given above, are shown. Identity with the first sequence is denoted by a dot and substitution by a different base letter.

Author Contributions

Conceptualization, J.O., A.M. and N.Š.; methodology, J.O. and A.M.; validation, J.O. and A.M.; formal analysis, J.O., A.M. and A.G.; investigation, J.O. and A.M.; visualization, J.O. and A.M.; resources, M.K., I.G., M.P. and V.G.K.; writing—original draft preparation, J.O. and A.M.; supervision, N.Š.; writing—review and editing, all authors. All authors have read and agreed to the published version of the manuscript.

Funding

The collection of material was partly funded within the European Social Fund project Nr. 2013/0016/1DP/1.1.1.2.0/13/ APIA/VIAA/055. The APC was funded by Latvian Council of Science project Nr. lzp-2021/1-0247.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The original data presented in the study are openly available in GenBank with accession numbers: OR689393–OR689397, OR682679–OR682715.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Sampling sites of studied European eels.
Figure 1. Sampling sites of studied European eels.
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Figure 2. Median joining haplotype network of 39 A. anguilla cyt b sequences. Each circle represents a haplotype and its size is proportional to haplotype frequency. Numbers indicate the name of haplotypes and colors represent locations.
Figure 2. Median joining haplotype network of 39 A. anguilla cyt b sequences. Each circle represents a haplotype and its size is proportional to haplotype frequency. Numbers indicate the name of haplotypes and colors represent locations.
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Figure 3. Maximum likelihood tree of cyt b sequences of investigated eel individuals. Bootstrap support >50 is shown next to the branching points. Leaf labels (sample code) for individual sequences are shown in Table 1. Colors represent locations (see Figure 2).
Figure 3. Maximum likelihood tree of cyt b sequences of investigated eel individuals. Bootstrap support >50 is shown next to the branching points. Leaf labels (sample code) for individual sequences are shown in Table 1. Colors represent locations (see Figure 2).
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Figure 4. Variability and distribution of mtDNA cyt b haplotypes in Europe and North America. Countries are marked with capital letters (GL: Greenland, CA: Canada, US: United States of America, MO: Morocco, ES: Spain, PT: Portugal, FR: France, IE: Ireland, SE: Sweden, LV: Latvia, BY: Belarus). Numbers and colors indicate the name of haplotypes.
Figure 4. Variability and distribution of mtDNA cyt b haplotypes in Europe and North America. Countries are marked with capital letters (GL: Greenland, CA: Canada, US: United States of America, MO: Morocco, ES: Spain, PT: Portugal, FR: France, IE: Ireland, SE: Sweden, LV: Latvia, BY: Belarus). Numbers and colors indicate the name of haplotypes.
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Figure 5. Median joining haplotype network of 37 A. anguilla D-loop sequences. Each circle represents a haplotype and its size is proportional to haplotype frequency. Numbers indicate the name of haplotypes and colors represent locations.
Figure 5. Median joining haplotype network of 37 A. anguilla D-loop sequences. Each circle represents a haplotype and its size is proportional to haplotype frequency. Numbers indicate the name of haplotypes and colors represent locations.
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Figure 6. Maximum likelihood tree of sequences of D-loop region. Bootstrap support >50 is shown next to the branching points. Leaf labels (sample code) for individual sequences are shown in Table 1. Colors represent locations (see Figure 5).
Figure 6. Maximum likelihood tree of sequences of D-loop region. Bootstrap support >50 is shown next to the branching points. Leaf labels (sample code) for individual sequences are shown in Table 1. Colors represent locations (see Figure 5).
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Table 1. Sampling details of eels (Anguilla anguilla) analyzed at cytochrome b (ncytb) and D-loop region (nD-loop).
Table 1. Sampling details of eels (Anguilla anguilla) analyzed at cytochrome b (ncytb) and D-loop region (nD-loop).
Sampling SitesCodeCountryLocationSampling YearnD-loopncytb
L. UsmaLUsmLatvia57°10′49″ N, 22°9′27″ E201444
L. LiepajaLLiepLatvia56°27′37″ N, 21°3′14″ E201433
L. KisezersLKisLatvia57°1′24″ N, 24°10′3″ E201435
L. AluksneLAlLatvia57°25′35.8″ N, 27°03′18.7″ E201442
L. VaidavaLVaidLatvia57°32′32.23″ N, 26°42′19.5″ E201822
L. RaznaLRazLatvia56°19′49″ N, 27°25′41″ E202041
L. SiverLSLatvia56°01′08.0″ N 27°19′54.6″ E202045
L. MyadzyelLMBelarus54°52′ N, 26°56′ E201933
L. SvirLSvBelarus54°48′16″ N, 26°28′27″ E201976
L.Vialikija SvakstyLVSBelarus54°58′0″ N, 26°34′53″ E201922
Myadzyelka RiverMuBelarus55°16′41″ N, 26°49′58″ E201922
Table 2. Genetic diversity statistics and neutrality tests for the D-loop and cyt b regions in Baltic Lakeland: the haplotype number (Nh), haplotype diversity (Hd), standard deviation (SD), nucleotide diversity (π), number of polymorphic sites (S), number of mutations (ŋ), average number of nucleotide differences (k), Tajima’s D, and Fu’ Fs statistics.
Table 2. Genetic diversity statistics and neutrality tests for the D-loop and cyt b regions in Baltic Lakeland: the haplotype number (Nh), haplotype diversity (Hd), standard deviation (SD), nucleotide diversity (π), number of polymorphic sites (S), number of mutations (ŋ), average number of nucleotide differences (k), Tajima’s D, and Fu’ Fs statistics.
RegionNhHd/SDkSŋπ/SDTajima’s DFu’s Fs
D-loop (393 sites)320.991 +/− 0.00912.1473840.031 +/− 0.0032−1.46 **−16.03 **
cyt b (394 sites)190.914 +/− 0.0322.9526270.0075 +/− 0.0017−1.94 *−10.39 *
*, p > 0.10—not significant, **, p < 0.05—significant.
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Oreha, J.; Morozova, A.; Garkajs, A.; Kirjušina, M.; Gavarāne, I.; Kostousov, V.G.; Pupins, M.; Škute, N. Genetic Diversity and Distribution of Haplotypes of Freshwater Eel in Baltic Lakeland Based on Mitochondrial DNA D-Loop and Cytochrome b Sequence Variation. Diversity 2024, 16, 522. https://doi.org/10.3390/d16090522

AMA Style

Oreha J, Morozova A, Garkajs A, Kirjušina M, Gavarāne I, Kostousov VG, Pupins M, Škute N. Genetic Diversity and Distribution of Haplotypes of Freshwater Eel in Baltic Lakeland Based on Mitochondrial DNA D-Loop and Cytochrome b Sequence Variation. Diversity. 2024; 16(9):522. https://doi.org/10.3390/d16090522

Chicago/Turabian Style

Oreha, Jeļena, Aleksandra Morozova, Alberts Garkajs, Muza Kirjušina, Inese Gavarāne, Vladimir G. Kostousov, Mihails Pupins, and Nataļja Škute. 2024. "Genetic Diversity and Distribution of Haplotypes of Freshwater Eel in Baltic Lakeland Based on Mitochondrial DNA D-Loop and Cytochrome b Sequence Variation" Diversity 16, no. 9: 522. https://doi.org/10.3390/d16090522

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