Issue |
Knowl. Manag. Aquat. Ecosyst.
Number 426, 2025
Ecological, evolutionary and environmental implications of floating photovoltaicst
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Article Number | 2 | |
Number of page(s) | 12 | |
DOI | https://doi.org/10.1051/kmae/2024023 | |
Published online | 15 January 2025 |
Research Paper
Filling the gaps in the genetic diversity of Austropotamobius torrentium (Astacidae, Decapoda) from one of the few unexplored hot spots
1
Department of Hydroecology and Water Protection, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
2
Department of Genetic Research, Institute for Biological Research “Siniša Stanković”, National Institute of Republic of Serbia, University of Belgrade, 11108 Belgrade, Serbia
3
Faculty of Natural Sciences and Mathematics, University of Banja Luka, 78000, Banja Luka, Bosnia and Herzegovina
4
Institute of Zoology, Faculty of Biology, University of Belgrade, 11060 Belgrade, Serbia
* Corresponding author: katarinas@ibiss.bg.ac.rs
a Katarina Zorić and Vanja Bugarski-Stanojević contributed equally to this work.
Received:
18
September
2024
Accepted:
11
December
2024
Recent molecular analyses of the stone crayfish have revealed a high degree of genetic diversity. The greatest diversity is found in the western Balkans (Dinarides), where more than half of the known phylogroups exist in a relatively small geographical area, some of them having smaller distribution range than the others. While the Croatian and Slovenian parts of the Austropotamobius torrentium areal are well described, data from Bosnia and Herzegovina (BA) are lacking. Here we provide data from 13 different localities in the northwestern parts of BA. We analysed two mtDNA markers and the results revealed high genetic diversity with a total of 12 MT-COI and nine MT-16SrRNA haplotypes, with the majority of novel haplotypes. Both genes confirmed the presence of two known phylogroups and the discovery of a new group named VOJ. The CSE phylogroup was the most widespread and restricted to the Vrbas basin. The first detection of the BAN phylogroup in BA indicates its wider distribution and connects previously isolated findings from Croatia. The discovery of 18 unique haplotypes as well as a new phylogroup is of particular interest, but further studies are needed to clarify their exact relationship to other lineages.
Key words: native crayfish / mtDNA / Vrbas drainage / Una drainage / Bosnia and Herzegovina
© K. Zorić et al., Published by EDP Sciences 2025
This is an Open Access article distributed under the terms of the Creative Commons Attribution License CC-BY-ND (https://creativecommons.org/licenses/by-nd/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. If you remix, transform, or build upon the material, you may not distribute the modified material.
1 Introduction
The family of freshwater crayfish (Astacidae) comprises four genera: Austropotamobius, Astacus, Pontastacus, and Pacifastacus. Three genera are native to Europe, while the genus Pacifastacus, native to North America, was introduced into European waters. The current distribution of freshwater crayfish in Europe is the result of natural processes that took place from the Miocene to the Pleistocene (Klobučar et al., 2013; Pârvulescu et al., 2019; Lovrenčić et al., 2020a; Stanković et al., 2024). As a result of taxonomic revisions over the last 60 years (summarised in Crandall and De Grave, 2017) and recent molecular analyses (Blaha et al., 2021), seven species of freshwater crayfish have been identified as autochthonous to the European continent: Astacus astacus (Linnaeus, 1758), Astacus colchicus Kessler, 1876, Pontastacus pachypus (Rathke, 1837), Pontastacus leptodactylus (Eschscholtz, 1823), Austropotamobius fulcisianus (Ninni, 1886), Austropotamobius torrentium (Schrank, 1803), Austropotamobius pallipes (Lereboullet, 1858), and an additional endemic species Austropotamobius bihariensis Pârvulescu, 2019 with a narrow distribution range in Romania (Pârvulescu, 2019).
Austropotamobius torrentium is widespread species in central and southeastern Europe. The distribution area (Fig. 1) extends from Bulgaria in the east to Luxembourg and France in the west, from Germany and the Czech Republic in the north to Greece and Turkey in the south (Holdich, 2002; Kouba et al., 2014; Ion et al., 2024). Throughout its entire range, the species is classified as “Data Deficient” with a declining population status in the IUCN Red List of Threatened Species (Füreder et al., 2017). In addition, stone crayfish is listed in Annex II of the EU Habitats Directive 92/43/EEC (Council of Europe, 1992) as a species requiring special conservation measures (Souty-Grosset et al., 2006) and in Appendix III of the Bern Convention (Council of Europe, 1979). Although the assessment of the status of populations at European level was already carried out in 2010, no quantitative data on the rate of decline is available. The main threats identified were invasive species, habitat modification and degradation (habitat loss), pollution and eutrophication (Füreder et al., 2006; Berger and Füreder, 2013). In the Republic of Srpska (Bosnia and Herzegovina), the species is listed in the Regulation on Strictly Protected and Protected Species (Official Gazette of the Republic of Srpska No. 65/20) (Anonymous, 2020).
In the last 20 years, great efforts have been made to determine the genetic profile of freshwater crayfish populations in the southeastern Europe (Trontelj et al., 2005; Schubart and Huber, 2006; Klobučar et al., 2013; Pârvulescu, 2019; Lovrenčić et al., 2020a; Stanković et al., 2024). Based on nucleotide sequence polymorphism analyses of two mitochondrial genes − cytochrome c oxidase I (MT-COI) and MT-16SrRNA − and a nuclear ITS2 (Lovrenčić et al., 2020), a high genetic and haplotype diversity of the species has been reported, characterised by the existence of eight significantly divergent phylogroups. Six of them have a distribution range within the north-central Dinaric region (NCD), while the remaining two include populations from the southern Balkans (SB) and central and southeastern Europe (CSE) (Trontelj et al., 2005; Klobučar et al., 2013; Lovrenčić et al., 2020a; Stanković et al., 2024). Of particular interest for the present study are phylogroups belonging to the NCD lineages (situated in the northern and central Dinarides, in western and southern Croatia and the associated border areas in Slovenia and Bosnia and Herzegovina, Stanković et al., 2024) as well as CSE lineage (SE Alps, Slovenia and upper Rhine Basin (Trontelj et al., 2005) and also in central and eastern Bosnia and Herzegovina (Stanković et al., 2024)).
Mitochondrial DNA (mtDNA) is a marker commonly used for genetic reconstruction of population history, demography, biogeography and speciation and is recommended for taxonomic studies (Hebert et al., 2003). Here, we analyse the nucleotide sequence polymorphism of two mitochondrial DNA (mtDNA) genes, MT-16SrRNA and MT-COI of A. torrentium populations in the northwestern part of Bosnia and Herzegovina (BA), a central region of the Balkan Peninsula for which very few data are available. This approach has already proven successful in analysing the genetic polymorphism of A. torrentium (Trontelj et al., 2005; Schubart and Huber, 2006; Klobučar et al., 2013; Petrusek et al., 2017; Pârvulescu et al., 2019). In this study, we compare new BA samples with those imported from GenBank originating from other regions within the distribution range of A. torrentium with the aim of complementing the biodiversity data and filling a gap in the records of the genetic structure of this strictly protected species in the territory of BA (Anonymous, 2020) in the context of known mtDNA haplotypes and phylogroups from the surrounding regions.
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Fig. 1 Geographic distribution of investigated localities. Left: map of the investigated area that corresponds to blue square marked in the right map. Localities comprise two watercourses: Vrbas (red marks) − 1 − 4, 6 − 9, 12, and Una (green marks) − 5, 10, 11, 13. Locality numbers correspond to Table 1. |
2 Materials and methods
2.1 Sample collection, DNA extraction, and sequencing
In the period 2018–2021, a total of 36 crayfish samples from 13 different localities (Fig. 1, Tab. 1) were collected from the tributaries of the two rivers Vrbas and Una. The investigated habitats are located in a hilly region with beech forests at an altitude of 180‒846 m above sea level, with megalithal and macrolithal dominate in the riverbeds with the lower proportion of psammal. The crayfish were caught by hand or with baited LiNi traps (Westmann et al., 1978).
Total DNA was isolated from a small part of muscle tissue of a pereiopod segment preserved in 96% ethanol using the AccuPrep Genomic DNA Extraction Kit (Bioneer Corporation, Daejeon, Korea). The quantity and quality of all DNA extracts were measured using the NanoPhotometer N60/N50 (Impplen, GmbH) and visualised by 1% agarose gel electrophoresis. Polymerase chain reactions (PCRs) for the amplification of mtDNA gene fragments were performed with universal primers: MT-16SrRNA (16Sar, 16Sbr) and MT-COI (LCO-1490, HCO-2198) from Veith et al. (2003) and Folmer et al. (1994), respectively. The thermal PCR profiles were as follows: for MT-16SrRNA: initial denaturation (3 min at 94 °C), followed by 40 cycles (30s at 94 °C, 30s at 52 °C, 40s at 72 °C) and a final elongation of 5 min at 72 °C; for MT-COI: initial denaturation (3 min at 94 °C), followed by 40 cycles (30s at 94 °C, 45s at 48 °C, 1 min at 72 °C) and a final elongation of 5 min at 72 °C. PCRs were performed using 1U Perpetual TaqDNA Polymerase (EurX, Poland) and approximately 100ng of genomic DNA in a final volume of 25 μL. Sanger sequencing was performed in one direction by a third party (Macrogen Europe). As universal primers for MT-16SrRNA gene were used, special attention was paid to sterile conditions to avoid contamination.
GenBank IDs with sampling localities of a total of 36 samples collected between 2018 and 2021 and analysed in this study.
2.2 Genetic diversity and phylogenetic analysis
All sequences were visually examined with the FinchTV 1.4.0 chromatogram viewer (Geospiza Inc.), compared and analysed with BioEdit Ver. 7.2.5 (Hall, 1999). The presence of stop codons and chimeric sequences was examined and compared with the sequences currently available in Gen-Bank using Basic Local Alignment Search Tool (BLAST) analysis. They were then aligned using ClustalW, which is implemented in the MEGA Ver. XI software (Molecular Evolutionary Genetics Analysis across computing platforms) (Tamura et al., 2021).
For the phylogenetic analysis of MT-16SrRNA, the final dataset contained a total of 88 sequences, i.e., 33 from the sampled individuals (Tab. 1) and 55 imported from GenBank, NCBI (Tab. S1). For the phylogenetic MT-COI analysis, the final dataset comprised 179 sequences, i.e. 28 from the sampled individuals (Tab. 1) and 151 from GenBank (Tab. S1). Three species were used as outgroups for both datasets: Austropotamobius pallipes, Astacus astacus and Pacifastacus leniusculus.
Genetic diversity parameters (h- the number of haplotypes, Hd- haplotype (gene) diversity, Pi- nucleotide diversity, Eta- total number of mutations) were estimated using DnaSP Ver. 6 (Rozas et al., 2017).
The estimation of evolutionary divergence between groups of sequences was calculated using the programme MEGA XI. The evolutionary divergence over sequence pairs between all detected phylogroups (p-distance) was calculated and the number of base differences per site from averaging over all sequence pairs between the groups is shown. The codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (“pairwise deletion” option).
Prior to the phylogenetic analysis, a best-fit substitution model for the aligned sequences including the outgroups was performed using JModelTest v.2.1.4. (Darriba et al., 2012). The best-fit substitution model in the aligned sequences tested was HKY+I+G (with gamma distribution and invariant sites). Phylogenetic analysis was performed using two different approaches to test the strength of the tree topology: Bayesian inference (BI) analysis in MrBayes (Ronquist and Huelsenbeck, 2003) and the Maximum Likelihood (ML) in PhyML (Guindon et al., 2010). Trees were created using FigTree Ver. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/) and MEGA XI. The BI analyses originated with random starting trees and were run for 20 × 106 generations, sampling every 1000th generation with the burn-in value set to 500. Combined trees of the various runs, a consensus tree was created using the 50% majority rule with the Bayesian posterior probability values of the relevant branches. The haplotype network was calculated and graphically presented using NETWORK Software (Bandelt et al., 1999). It consists of nodes and links (nucleotide differences) connecting the nodes. The nodes are either sequences from the data set or median vectors (mv) − a hypothetical, often ancestral sequence required to connect existing sequences within the network with maximum parsimony.
3 Results
3.1 MT-16SrRNA gene nucleotide sequence comparison
The 33 sequences obtained were deposited in the GenBank database under the accession numbers OP963758−OP963790 (Tab. 1). The final MT-16SrRNA dataset analysed here comprised 88 sequences together with 55 imported ones (Tab. S1). The data file with excluded outgroups contains 85 sequences, 493 sites and a total number of 464 nucleotide positions (excluding sites with gaps/missing data). There are 67 polymorphic and 397 invariable sites. The total number of mutations is 75 and a number of parsimony informative sites is 48. The overall number of haplotypes is h = 60; haplotype (gene) diversity Hd = 0.9442 ± 0.02; nucleotide diversity (per site) Pi = 0.01669 ± 0.00158. Among 33 newly sampled BA individuals nine haplotypes were detected, of which seven were new/original haplotypes labelled ba1-ba7 and only two were detected earlier: h4 and h27 (Klobučar et al., 2013). The results are presented as the relatedness of all 60 haplotypes detected in this analysis with previously described phylogroups (Lovrenčić et al., 2020): CSE − Central and Southeastern Europe; SB − South Balkans; ZV − Zeleni Vir; LD − Lika and Dalmatia; KOR − Kordun; BAN − Banovina; GK − Gorski Kotar and ŽPB − Žumberak, Plitvice and Bjelolasica.
The haplotypes analysed here, belonging to the CSE phylogroup, are: haplotype h4, which is the most common in the Vrbas samples from this study and also in several imported sequences; and a group of five original haplotypes ba1 ‒ ba5, four of which are similar to haplotype h4 (with only one nucleotide change). Haplotypes h27 and ba7 belong to the BAN phylogroup and were found in the Una and Vrbas river basins. However, we also discovered a specific haplotype ba6–two samples from Vojskova in the Una river basin, which do not belong to any formerly determined phylogroups. Here they are regarded as a separate phylogroup VOJ.
The relationships between the haplotypes represented by a series of nucleotide changes between them, as well as the median vectors, are shown graphically in a Network Median Joining Haplotype Tree (Fig. 2a). All haplotypes from our sample are associated with previously defined phylogroups from the Balkans, CSE and BAN, with the exception of a new haplotype ba6 from Vojskova, Una (marked as VOJ), which does not belong to any known phylogroup. It is positioned between phylogroups BAN, SB and ŽPB, with five/six nucleotide substitutions and two/3 median vectors (symbolised by black mv dots in the tree). Between VOJ and ZV there is only one median vector, but with 11 nucleotide changes.
Maximum likelihood (ML) and Bayesian (BI) inference yielded similar phylogenetic tree topologies; therefore, only the ML tree is presented in Figure 2b. The phylogenetic tree inferred from the MT-16SrRNA dataset illustrates that phylogroups CSE and SB are evolutionarily youngest, with the dominant haplotype h4 together with ba1 ‒ ba5 in CSE. Next to them is phylogroup BAN, comprising haplotypes h27 and ba7 and then phylogroups LD and ZV. The newly discovered phylogroup VOJ is positioned between ZV and ŽPB. The evolutionarily oldest is the phylogroup GK, placed in the base of the tree.
The estimates of evolutionary divergence over sequence pairs (p-distances) between all detected phylogroups are shown in Table 2. This analysis included 85 nucleotide sequences and a total of 497 positions in the final dataset. Among the lowest observed p-distances values, besides ŽPB and KOR are those between the new phylogroup VOJ (haplotype ba6) and ŽPB/ BAN phylogroups.
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Fig. 2 Phylogenetic and haplotype network trees constructed using MT-16SrRNA dataset. a: Network Median Joining haplotype tree; red − Vrbas and green − Una. Haplotype node size is related to a sample size. Black dots (mv)-median vector. Haplotype ratio is distributed by phylogroups: CSE- central and south-eastern Europe; SB- south Balkan; ZV − Zeleni Vir; LD − Lika and Dalmatia; KOR- Kordun; BAN − Banovina; GK − Gorski Kotar and ŽPB − Žumberak, Plitvice and Bjelolasica; VOJ* −Vojskova. b: ML phylogenetic tree with bootstrap values of both methods, ML and BI, indicated in the nodes respectively. Dash indicates values <50. New samples from this study are marked with circles that correspond to phylogroup colors. |
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Fig. 2 (Continued). |
The estimates of Evolutionary Divergence over Sequence Pairs between Groups (p-distance) calculated from the MT-16SrRNA dataset. The number of base differences per site from averaging over all sequence pairs between groups are shown. Standard error estimate(s) are shown above the diagonal.
3.2 MT-COI gene nucleotide sequence comparison
The final MT-COI gene dataset comprises 28 obtained sequences deposited in the GenBank database under accession numbers OQ048651 − OQ048659 and OQ04861–OQ04879 (Tab. 1) and 151 imported sequences listed in Table S1. The final dataset without outgroups contains 642 sites with 322 nucleotide positions (excluding sites with gaps/missing data) with 171 polymorphic sites and a total number of mutations, Eta = 119. The total number of haplotypes is h = 134; haplotype diversity, Hd = 0.9932 ± 0.0023; and nucleotide diversity (per site) Pi = 0.05591 ± 0.00267. Among the 28 newly sampled individuals from BA, 12 haplotypes were identified, of which 11 are new/original and only one h41 (Klobučar et al., 2013) was previously discovered.
The estimates of evolutionary divergence over sequence pairs (p-distances) between all detected phylogroups are shown in Table 3. This analysis included 176 nucleotide sequences and a total of 636 positions in the final dataset. These values are higher than those calculated from MT-16SrRNA dataset, with the lowest distance provided among CSE and SB.
Most haplotypes are associated with CSE phylogroup: the most abundant is ba1 and the others are ba2, ba3, ba5‒ba9, all of which were sampled in the Vrbas river basin (Fig. 3a). These are all new haplotypes. The newly discovered haplotypes ba4 (Dževerov potok, Vrbas basin) and ba11 (Subotica, Una basin) as well as h41 from the Vrbas basin, shared with an imported sample JF293445 from Badnjevice, Croatia, are all linked to BAN phylogroup. MT-COI gene analysis confirms the specific position of haplotype ba10 (Vojskova, Una river basin), i.e. it does not belong to any earlier described phylogroup. It is located between the SB and ZV groups, but is more distantly related to them compared to the MT-16SrRNA gene analysis.
Similar to the MT-16SrRNA dataset, maximum likelihood (ML) and Bayesian (BI) inference from the MT-COI gene data yielded similar tree topologies, which is why only the ML tree is shown in Figure 3b. The phylogenetic tree shows phylogroups CSE and SB as evolutionarily youngest, with high significance values in the branch nodes. Phylogroup BAN is clustered with these groups, also with high significance. The new phylogroup VOJ with the haplotype ba10 (Vojskova, Una) is placed among Dinaric karst lineages, between BAN and ŽPB and at the base of the phylogenetic ML tree are the phylogroups ZV and GK as the evolutionarily oldest.
The estimates of Evolutionary Divergence between Groups of Sequences (p-distance) calculated from MT-COI dataset. The number of base differences per site from estimation of net average between groups of sequences are shown. Standard error estimate(s) are shown above the diagonal.
![]() |
Fig. 3 Phylogenetic and haplotype network trees constructed using MT-COI dataset. a: Network Median Joining haplotype tree; red − Vrbas basin and green − Una. Haplotype node size is related to a sample size. Black dots (mv)-median vector. Haplotype ratio is distributed by phylogroups: CSE- central and south-eastern Europe; SB- south Balkan; ZV- Zeleni Vir; LD- Lika and Dalmatia; KOR- Kordun; BAN- Banovina; GK- Gorski Kotar; ŽPB- Žumberak, Plitvice and Bjelolasica; APU- Apuseni Mt.; Vojskova − VOJ*. b: ML phylogenetic tree with bootstrap values of both methods, ML and BI, indicated in the nodes. Dash indicates values <50. Some branches that contain only sequences imported from the GenBank are condensed. New samples from this study are marked with circles that correspond to phylogroup colors. |
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Fig. 3 (Continued). |
4 Discussion
The study area presented here is positioned in the middle of the distribution range of A. torrentium in central and southeastern Europe (Fig. 1). As the species is classified as “Data Deficient” with a declining population status in the IUCN Red List of Threatened Species (Füreder et al., 2017) and requires special conservation measures (Souty-Grosset et al., 2006) and is listed in Appendix III of the Bern Convention (Council of Europe, 1979), it was necessary to investigate the genetic status of samples from Bosnia and Herzegovina and their relationship with populations from the surrounding regions. The results of our study confirm the presence of high genetic diversity in stone crayfish from the Western Balkans. Eleven out of 12 MT-COI haplotypes and seven out of nine MT-16SrRNA haplotypes detected in this analysis were formerly unknown. The identified mtDNA phylogenetic tree topologies are generally consistent with previous data on the relationships between the known phylogroups (Trontelj et al., 2005; Klobučar et al., 2013;Petrusek et al., 2017; Pârvulescu et al., 2019; Lovrenčić et al., 2020a; Groza et al., 2021; Stanković et al., 2024), with GK representing the evolutionarily oldest and CSE and SB the youngest groups.
Given the high genetic diversity and the importance of the region for the diversification and evolution of this crustacean species, even smaller, understudied areas have the potential for some important additions to species knowledge (new phylogroups), as Lovrenčić et al. (2020a) have shown. It is worth mentioning that so far only two phylogroups − CSE and LD, have been confirmed from Bosnia and Herzegovina (Klobučar et al., 2013; Lovrenčić et al., 2020a). Our study shows the presence of another previously known phylogroup (BAN), but also an additional potentially new phylogroup (VOJ) belonging to Dinaric karst lineages, which could be of particular interest. Namely, in both genes MT-16SrRNA and MT-COI, the haplotypes ba6 and ba10 were found, respectively, which cannot be assigned to any known phylogroup. According to the phylogenetic tree topologies derived from both genes, the VOJ haplotypes belong to the evolutionarily older branches. These individuals were sampled from the Vojskova River (Una basin). The river harbours numerous streams and karst springs and exhibits a rather heterogeneous structure, while remaining relatively untouched by negative anthropogenic influences. The sampling site (the village of Donji Dubovik) is geographically the furthest from the other sites investigated. In addition, no stone crayfish (and no other astacids) were found during several field surveys in the immediate vicinity (personal communication with fishermen).
The BAN phylogroup was first discovered by Klobučar et al. (2013) and described with only 3 haplotypes from two isolated populations near the border between Croatia and Bosnia and Herzegovina (Banija/Banovina and Imotski). The data presented here confirm the broader occurrence and present two new MT-16SrRNA haplotypes (h27, distributed in the Una and Vrbas basins and ba7 in the Una basin) and a new MT-COI haplotype (ba11 in the Una basin). The haplotype h41 from the BAN phylogroup found in the Vrbas basin is shared with the Badnjevice locality in Croatia. Based on the available data, we suspect that in Bosnia and Herzegovina (at least in the Neretva and Bosna basins) an even greater presence of the BAN phylogroup is to be expected.
All other BA haplotypes belong to the CSE phylogroup. Five unique MT-16SrRNA haplotypes and one of the most common h4 haplotype are identified in the Vrbas basin, which is in common with a sample JN683358 from Žegovački potok, Serbia. Although the CSE phylogroup was the most abundant and widespread, it was restricted to the Vrbas basin. The reason for this could be the lower number of populations sampled in the Una basin (four) compared to the Vrbas basin (nine). As in the previous case, with further sampling we could probably assume that this widespread haplogroup is more abundant in the Una basin and perhaps also in the Sava, Bosna and Drina drainages.
The results of genetic diversity and evolutionary divergence contribute to our assumption of a high genetic diversity of stone crayfish in this area. The total nucleotide diversity per site was high in our study, especially that of MT-COI. Both values are significantly higher than those reported in the literature (Klobučar et al., 2013). The values of evolutionary MT-16SrRNA divergence (average values of uncorrected (p) distances (in percent) between all phylogroups from the literature data (Klobučar et al., 2013) are highly consistent with the values from our study. The results from the MT-COI dataset yielded three to four times higher values for p-distances, genetic diversity parameters and haplotypes, which is to be expected considering that the MT-16SrRNA gene is more conserved. For this reason, some inconsistencies in the relationships between the phylogroups can be detected when the results from two genes are compared. The MT-COI evolutionary divergence found here between all analysed phylogroups is slightly lower than in the literature (Klobučar et al., 2013), but basically their relationships are the same. CSE and SB are the closest phylogroups with the lowest evolutionary divergence, followed by the divergence of BAN-SB and BAN-CSE. However, it is important to note that the values for the evolutionary distance between VOJ and all other phylogroups provided by both genes correspond to the values among all recognised lineages, i.e. phylogroups. This fact and the clear separation of the VOJ haplotypes in the Network Median Joining haplotype trees and the BI/ML phylogenetic trees resulting from both genes, are evidence that it is a separate new phylogroup. Nevertheless, the exact position of the VOJ phylogroup in the phylogenetic trees and its connection to other phylogroups cannot be determined with only two samples. The evolutionary divergence of the MT-16SrRNA gene of the new VOJ phylogroup shows a closer connection to ŽPB and BAN and is furthest away from phylogroups ZV and GK. The MT-COI dataset shows a slightly closer relationship of VOJ to BAN and SB. This suggests that a broader sample is required for further studies in this geographic region.
The three phylogroups ŽPB, LD and KOR occur in the close proximity to Bosnia and Herzegovina, in the border regions (Lovrenčić et al., 2020a), but were not detected in BA samples in this study. Given the geographical/hydrogeographical characteristics of the regions, we could expect that, if not all, at least some of these groups will be found in future studies in Bosnia and Herzegovina. Considering the limited dispersal potential of crustaceans in general (Kerby et al., 2005; Bubb et al., 2006) and the geographical/hydrogeographical aspects of the area (Karst Dinarides in Bosnia and Herzegovina) that favour speciation and diversification of species (Bănărescu, 2004; Marčić et al., 2011), the discovery of additional new phylogroups might not be far away.
Given the limited study area, which is in close proximity to a well-studied region in Croatia, the high genetic diversity in stone crayfish from the Western Balkans, which was also found in this study, was to be expected. Of particular interest however, is the discovery of original evolutionarily older haplotypes possibly belonging to a new phylogroup and its origin. VOJ could be a remnant population, diverged from BAN or SB. Following the Paratethys's continued retraction and the formation of modern river networks, including the Danube around 4.36 million years ago (de Leeuw et al., 2018), the SB lineage started to colonize the Danube basin, leading to the CSE lineage (Pârvulescu et al., 2019). This period could have isolated the VOJ lineage within its current karstic habitats, suggesting it originated from initial colonization attempts by BAN or SB.
A larger study area encompassing other main catchments of Bosnia and Herzegovina (Neretva, Bosna, and Drina) should shed more light on the status of this species in the region and could eventually modify the preserved topologies and phylogroup relationships.
Acknowledgements
The authors would like to thank two anonymous reviewers for their valuable suggestions that improved our manuscript. This research was funded by the Ministry of Science, Technological Development and Innovation of the Republic of Serbia − Grants No. 451-03-66/2024-03/200007, 451-03-65/2024-03/200178 and 451-03-66/2024-03/200178).
Supplementary Material
Table S1. List of MT-16S rRNA and MT-COI sequences imported from the GenBank. Access here
References
- Anonymous. 2020. The Rulebook on the proclamation and protection of strictly protected and protected wild species in Republic of Srpska (Official Gazette of RS, no. 65/20). [Google Scholar]
- Bănărescu PM. 2004. Distribution pattern of the aquatic fauna of the Balkan Peninsula. In: Griffiths HI, Kryštufek B, Reed JM, ed. Balkan biodiversity: pattern and process in the European hotspot. Dordrecht: Springer Netherlands, 203–217. [Google Scholar]
- Bandelt HJ, Forster P, Röhl A. 1999. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16: 37–48. [CrossRef] [PubMed] [Google Scholar]
- Berger C, Füreder L. 2013. Linking species conservation management and legal species protection: a case study on stone crayfish. Freshw Crayfish 19: 161–175. [Google Scholar]
- Bláha M, Patoka J, Japoshvili B, Let M, Buřič M, Kouba A, Mumladze L. 2021. Genetic diversity, phylogenetic position and morphometric analysis of Astacus colchicus (Decapoda, Astacidae): a new insight into Eastern European crayfish fauna. Integr Zool 16: 368–378. [CrossRef] [PubMed] [Google Scholar]
- Bubb DH, Thom TJ, Lucas MC. 2006. Movement, dispersal and refuge use of co-occurring introduced and native crayfish. Freshw Biol 51: 1359–1368. [CrossRef] [Google Scholar]
- Council of Europe. 1979. Convention on the Conservation of European Wildlife and Natural Heritage, Appendix, III., Bern, Switzerland. [Google Scholar]
- Council of Europe. 1992. Council Directive 92/43/EEC on the conservation of natural habitats and of wild fauna and flora (Habitats Directive). [Google Scholar]
- Crandall KA, De Grave S. 2017. An updated classification of the freshwater crayfishes (Decapoda: Astacidea) of the world, with a complete species list. J Crust Biol 37: 615–653. [CrossRef] [Google Scholar]
- Darriba D, Taboada GL, Doallo R, Posada D. 2012. JModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9: 772. [CrossRef] [PubMed] [Google Scholar]
- de Leeuw A, Morton A, van Baak CGC, Vincent SJ. 2018. Timing of arrival of the Danube to the Black Sea: Provenance of sediments from DSDP site 380/380A. Terra Nova. 114–124. [Google Scholar]
- Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotech 3: 294–299. [Google Scholar]
- Füreder L, Edsman L, Holdich D, Kozak P, Machino Y, Pockl M, Renai B, Reynolds J, Schulz RW, Schulz HK. et al. 2006. Indigenous crayfish habitat and threats. In: Atlas of Crayfish in Europe, Souty-Grosset C, Holdich DM, Noel PY, Reynolds JD, Haffner P, ed. Museum National d'Histoire Naturelle, Paris, France, 25–47. [Google Scholar]
- Füreder L, Gherardi F, Souty-Grosset C. 2017. Austropotamobius torrentium (errata version published in 2017). The IUCN Red List of Threatened Species 2010: e.T2431A121724677. Accessed on 28 February 2023. [Google Scholar]
- Groza M-I, Cupșa D, Lovrenčić L, Maguire I 2021. First record of the stone crayfish in the Romanian lowlands. Knowl Manag Aquat Ecosyst 422: 27. [CrossRef] [EDP Sciences] [Google Scholar]
- Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59: 307–321. [CrossRef] [PubMed] [Google Scholar]
- Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41: 95–98. [Google Scholar]
- Hebert PDN, Cywinska A, Ball SL, DeWaard JR. 2003. Biological identifications through DNA barcodes. Proc R Soc B 270: 313–321. [CrossRef] [PubMed] [Google Scholar]
- Holdich DM. 2002. Distribution of crayfish in Europe and some adjoining countries. Bull Fr Pêche Piscic 367: 611–650. [CrossRef] [EDP Sciences] [Google Scholar]
- Ion MC, Bloomer CC, Bărăscu TI, Oficialdegui FJ, Shoobs NF, Williams BW, Scheers K, Clavero M, Grandjean F, Collas M, et al. 2024. World of Crayfish™: a web platform towards real-time global mapping of freshwater crayfish and their pathogens. PeerJ 12: e18229. [CrossRef] [PubMed] [Google Scholar]
- Kerby JL, Riley SP, Kats LB, Wilson P. 2005. Barriers and flow as limiting factors in the spread of an invasive crayfish (Procambarus clarkii) in southern California streams. Biol Conserv 126: 402–409. [CrossRef] [Google Scholar]
- Klobučar GI, Podnar M, Jelić M, Franjević D, Faller M, Štambuk A, Gottstein S, Simić V, Maguire I. 2013. Role of the Dinaric Karst (western Balkans) in shaping the phylogeographic structure of the threatened crayfish Austropotamobius torrentium. Freshw Biol 58: 1089–1105. [CrossRef] [Google Scholar]
- Kouba A, Petrusek A, Kozák P. 2014. Continental-wide distribution of crayfish species in Europe: Update and maps. Knowl Manag Aquat Ecosyst 413: 5. [Google Scholar]
- Lovrenčić L, Bonassin L, Boštjančić LL, Podnar M, Jelić M, Klobučar G, Jaklič M, Slavevska-Stamenković V, Hinić J, Maguire I 2020a. New insights into the genetic diversity of the stone crayfish: taxonomic and conservation implications. BMC Evol Biol 20: 1–20. [CrossRef] [PubMed] [Google Scholar]
- Lovrenčić L, Pavić V, Majnarić S, Abramović L, Jelić M, Maguire I. 2020b. Morphological diversity of the stone crayfish − traditional and geometric morphometric approach. Knowl Manag Aquat Ecosyst 421: 1. [Google Scholar]
- Marčić Z, Buj I, Duplić A, Ćaleta M, Mustafić P, Zanella D, Mrakovčić M. 2011. A new endemic cyprinid species from the Danube drainage. J Fish Biol 79: 418–430. [PubMed] [Google Scholar]
- Pârvulescu L. 2019. Introducing a new Austropotamobius crayfish species (Crustacea, Decapoda, Astacidae): a Miocene endemism of the Apuseni Mountains, Romania. Zool Anz 279: 94–102. [CrossRef] [Google Scholar]
- Petrusek A, Filipova L, Kozubíková-Balcarová E, Grandjean F. 2017. High genetic variation of invasive signal crayfish in Europe reflects multiple introductions and secondary translocations. Freshw Sci 36: 838–850. [CrossRef] [Google Scholar]
- Ronquist F, Huelsenbeck JP. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinform 19: 1572–1574. [CrossRef] [PubMed] [Google Scholar]
- Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol 34: 3299–3302 [CrossRef] [PubMed] [Google Scholar]
- Schubart CD, Huber MGJ. 2006. Genetic comparisons of German populations of the stone crayfish, Austropotamobius torrentium (Crustacea: Astacidae). Bull Fr Pêche Piscic 380–381: 1019–1028. [Google Scholar]
- Souty-Grosset C, Holdich DM, Noël PY, Reynolds JD, Haffner P. 2006. Atlas of Crayfish in Europe, Muséum national d'Histoire naturelle, Paris. Patrim Nat 64: 187. [Google Scholar]
- Stanković D, Zorić K, Đuretanović S, Stamenković G, Ilić M, Marković V, Marić S. 2024. A new perspective on the molecular dating of the stone crayfish with an extended phylogeographic information on the species. Hydrobiologia. https://doi.org/10.1007/s10750-024-05613-3 [Google Scholar]
- Tamura K, Stecher G, Kumar S. 2021. MEGA 11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. https://doi.org/10.1093/molbev/msab120 [Google Scholar]
- Trontelj P, Machino Y, Sket B. 2005. Phylogenetic and phylogeographic relationships in the crayfish genus Austropotamobius inferred from mitochondrial COI gene sequences. Mol Phylogenet Evol 34: 212–226. [Google Scholar]
- Veith M, Kosuch J, Vences M. 2003. Climatic oscillations triggered post-Messinian speciation of Western Palearctic brown frogs (Amphibia, Ranidae). Mol Phylogenet Evol 26: 310–327. [CrossRef] [PubMed] [Google Scholar]
- Westman K, Pursiainen M, Vilkman R. 1978. A new folding trap model which prevents crayfish from escaping. Freshw. Crayfish 4: 235–242. [Google Scholar]
Cite this article as: Zorić K, Bugarski-Stanojević V, Roljić R, Nikolić V, Stamenković G, Ilić M, Marković V. 2025. Filling the gaps in the genetic diversity of Austropotamobius torrentium (Astacidae, Decapoda) from one of the few unexplored hot spots. Knowl. Manag. Aquat. Ecosyst., 426, 2
All Tables
GenBank IDs with sampling localities of a total of 36 samples collected between 2018 and 2021 and analysed in this study.
The estimates of Evolutionary Divergence over Sequence Pairs between Groups (p-distance) calculated from the MT-16SrRNA dataset. The number of base differences per site from averaging over all sequence pairs between groups are shown. Standard error estimate(s) are shown above the diagonal.
The estimates of Evolutionary Divergence between Groups of Sequences (p-distance) calculated from MT-COI dataset. The number of base differences per site from estimation of net average between groups of sequences are shown. Standard error estimate(s) are shown above the diagonal.
All Figures
![]() |
Fig. 1 Geographic distribution of investigated localities. Left: map of the investigated area that corresponds to blue square marked in the right map. Localities comprise two watercourses: Vrbas (red marks) − 1 − 4, 6 − 9, 12, and Una (green marks) − 5, 10, 11, 13. Locality numbers correspond to Table 1. |
In the text |
![]() |
Fig. 2 Phylogenetic and haplotype network trees constructed using MT-16SrRNA dataset. a: Network Median Joining haplotype tree; red − Vrbas and green − Una. Haplotype node size is related to a sample size. Black dots (mv)-median vector. Haplotype ratio is distributed by phylogroups: CSE- central and south-eastern Europe; SB- south Balkan; ZV − Zeleni Vir; LD − Lika and Dalmatia; KOR- Kordun; BAN − Banovina; GK − Gorski Kotar and ŽPB − Žumberak, Plitvice and Bjelolasica; VOJ* −Vojskova. b: ML phylogenetic tree with bootstrap values of both methods, ML and BI, indicated in the nodes respectively. Dash indicates values <50. New samples from this study are marked with circles that correspond to phylogroup colors. |
In the text |
![]() |
Fig. 2 (Continued). |
In the text |
![]() |
Fig. 3 Phylogenetic and haplotype network trees constructed using MT-COI dataset. a: Network Median Joining haplotype tree; red − Vrbas basin and green − Una. Haplotype node size is related to a sample size. Black dots (mv)-median vector. Haplotype ratio is distributed by phylogroups: CSE- central and south-eastern Europe; SB- south Balkan; ZV- Zeleni Vir; LD- Lika and Dalmatia; KOR- Kordun; BAN- Banovina; GK- Gorski Kotar; ŽPB- Žumberak, Plitvice and Bjelolasica; APU- Apuseni Mt.; Vojskova − VOJ*. b: ML phylogenetic tree with bootstrap values of both methods, ML and BI, indicated in the nodes. Dash indicates values <50. Some branches that contain only sequences imported from the GenBank are condensed. New samples from this study are marked with circles that correspond to phylogroup colors. |
In the text |
![]() |
Fig. 3 (Continued). |
In the text |
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