Aleš Snoj

Extreme genetic differentiation among the remnant populations of marble trout (Salmo marmoratus) in Slovenia

Populations of the marble trout (Salmo marmoratus) have declined critically due to introgression by brown trout (Salmo trutta) strains. In order to define strategies for long‐term conservation, we examined the genetic structure of the 8 known pure populations using 15 microsatellite loci. The analyses reveal extraordinarily strong genetic differentiation among populations separated by < 15 km, and extremely low levels of intrapopulation genetic variability. As natural recolonization seems highly unlikely, appropriate management and conservation strategies should comprise the reintroduction of pure populations from mixed stocks (translocation) to avoid further loss of genetic diversity.

Resolving taxonomic uncertainties using molecular systematics: Salmo dentex and the Balkan trout community

The genetic structure of Salmo dentex and its phylogenetic relations to sympatric salmonids in the Neretva and Skadar River basins were evaluated using mitochondrial DNA (mtDNA) control region, eight microsatellites, and somatolactin (SL) gene. In the Neretva River basin of Bosnia–Herzegovina, the results based on mtDNA analysis showed extensive haplotype sharing between S. marmoratus, S. dentex, and S. trutta, and were therefore not conclusive; however, F-statistics and assignment testing based on nuclear DNA markers indicated that S. dentex of the Neretva basin were grouped in a genetically unified cluster with S. marmoratus in the Neretva basin. Using the same analytical approach, S. dentex from the Skadar basin in Montenegro appeared to be genetically distinct from S. marmoratus in the same basin and indistinct from local S. trutta. Molecular data also indicated that S. dentex of the Neretva basin in Bosnia–Herzegovina are not closely related to S. dentex of the Skadar basin in Montenegro. Based on these results, we hypothesize S. dentex to be a particular life history form of S. marmoratus in the Neretva basin and of S. trutta in the Skadar basin. These results clearly demonstrate that S. dentex does not represent a monophyletic lineage and should not be considered a distinct species.

Genetic architecture of trout from Albania as revealed by mtDNA control region variation.

To determine the genetic architecture of trout in Albania, 87 individuals were collected from 19 riverine and lacustrine sites in Albania, FYROM and Greece. All individuals were analyzed for sequence variation in the mtDNA control region. Among fourteen haplotypes detected, four previously unpublished haplotypes, bearing a close relationship to haplotypes of the Adriatic and marmoratus lineages of Salmo trutta, were revealed. Ten previously described haplotypes, characteristic of S. ohridanus, S. letnica and the Adriatic and Mediterranean lineages of S. trutta, were also detected. Haplotypes detected in this study were placed in a well supported branch of S. ohridanus, and a cluster of Mediterranean – Adriatic – marmoratus haplotypes, which were further delimited into three subdivisions of Mediterranean, marmoratus, and a previously non-described formation of four Adriatic haplotypes (Balkan cluster). Haplotypes of the Balkan cluster and the other Adriatic haplotypes, do not represent a contiguous haplotype lineage and appear not to be closely related, indicating independent arrivals into the Adriatic drainage and suggesting successive colonization events. Despite the presence of marmoratus haplotypes in Albania, no marbled phenotype was found, confirming previously reported findings that there is no association between this phenotype and marmoratus haplotypes.

Phylogenetic origin of Salmo trutta L 1758 from Sicily, based on mitochondrial and nuclear DNA analyses

The phylogenetic status of brown trout Salmo trutta L 1758 in Sicily is uncertain as some reports describe these trout as S. macrostigma or S. cettii on one hand while other, contradictory reports imply a hatchery origin on the other. In order to clarify this situation, we performed sequence analysis of the mtDNA control region and restriction fragment analysis of the nuclear lactate dehydrogenase (LDH-C1*) gene. A single mitochondrial haplotype (At-s6) found previously in brown trout in Morocco, and two alleles at LDH-C1* (the ancestral*100, at a high frequency, and *90) were revealed. Our results suggest that Sicilian brown trout are native and that they probably colonized Sicily from west to east in an expansion, from the Atlantic Ocean basin, along the North-West African coast.

Regional structure despite limited mtDNA sequence diversity found in the endangered Huchen, Hucho hucho (Linnaeus, 1758)

We evaluate the hypothesis of no geographic structure in the Huchen (Hucho hucho), a large, predatory salmonid endemic to the Danube basin. Forty-seven individuals sampled from throughout the Huchen’s native range were genetically characterized. Extremely limited sequence diversity across 1,800 bases of mtDNA (the complete control region and partial NADH-1 subunit) evidenced by four closely related mtDNA haplotypes was found. Nonetheless, the geographic distribution of mtDNA repeats (5–10, 82-bp long copies per individual) as well as allelic diversity across two microsatellite loci indicated large-scale geographic structure between the north-western (Austria and Slovenia) distribution area and eastern (Slovakia and Ukraine) or southern (Bosnia-Herzegovina and Montenegro) sample sites. An extremely slow rate of substitution for the H. hucho mtDNA is considered along with the alternative hypotheses to explain the limited mtDNA diversity. Considering the regional genetic structure implied by our data, we advocate restrictions on the transport of brood fish or yearlings across the range of the species distribution and sale of Huchen across international boundaries. Future genetic analysis to support local conservation and monitoring efforts must focus on developing a high-resolution screen that may be applied to identify hatchery versus naturally reproduced individuals in the wild.

Rainbow Trout in Europe: Introduction, Naturalization, and Impacts

The rainbow trout (Oncorhynchus mykiss) is probably the most widely introduced fish species in the world. Since the first translocation outside of the range of its natural distribution, the species has been introduced into at least 99 countries and has established reproducing populations in many different parts of the world. The present review aims to synthesize the existing information on these translocations, with special emphasis on self-sustaining populations in Europe, where continuous introductions have in general not led to naturalization. Our survey produced a list of more than 130 confirmed or potential self-sustaining populations across 16 European countries. The highest abundance of such populations was observed in the Alpine foothills of central Europe where naturalization is not limited to modified waters less suitable for native salmonids but also occurs commonly in pristine and near-natural waters. There is no consensus on the reasons for the absence of self-sustaining populations of rainbow trout across much of Europe, partly because knowledge of the mechanisms involved is limited, while the data collected here shed new light on the invasion biology of the species.

Evolutionary relationship between marble trout of the northern and the southern Adriatic basin.

Marble trout (Salmo marmoratus) populate two geographically separated areas in the northern and southern parts of the Adriatic Sea drainage. Although morphologically similar, each population is distinguished by a different set of unrelated mitochondrial haplotypes, suggesting that they have evolved from different ancestors. Due to a possible discordance between mitochondrial and species phylogeny, we performed phylogenetic analysis based on 22 nuclear loci. The results inferred from Maximum-likelihood and Bayesian Inference analysis revealed that northern and southern populations are closely related, forming a monophyletic group. This observation is concordant with the present marble trout classification, which considers both populations as conspecific. On the other hand, our findings are in marked contrast to those of previous mtDNA-based studies and highlight potential dangers of making phylogenetic inferences from mtDNA alone. Reasons for discordance between mtDNA and nDNA phylogeny are discussed with incomplete lineage sorting proposed as the most parsimonious explanation for mtDNA divergence in marble trout.

Molecular data suggest a multispecies complex of Phoxinus (Cyprinidae) in the Western Balkan Peninsula

This study aimed to evaluate the phylogeographic distribution of Phoxinus phoxinus sensu lato populations in the Balkan Peninsula using molecular methods. For the western Balkan Peninsula, two species (Danubian P. phoxinus and Adriatic P. lumaireul) have been suggested, but not confirmed by subsequent morphological studies or by genetic data. For the present study, more than 300 specimens were collected from the western Balkans. A partial sequence of the mt cyt b gene analysed using Bayesian and ML methods revealed several well-supported clades, with distances ranging from 4% to 11%. The clades were corroborated by RAG1 sequence analysis. Thus, the molecular analysis points to a multispecies complex of Phoxinus sp. with ranges of the clades extending north-west to south-east along the western Balkan Peninsula. In addition, the dispersion of fish through subterranean water connections in karst is indicated.

Environmental DNA in subterranean biology: range extension and taxonomic implications for Proteus

Europe’s obligate cave-dwelling amphibian Proteus anguinus inhabits subterranean waters of the north-western Balkan Peninsula. Because only fragments of its habitat are accessible to humans, this endangered salamander’s exact distribution has been difficult to establish. Here we introduce a quantitative real time polymerase chain reaction-based environmental DNA (eDNA) approach to detect the presence of Proteus using water samples collected from karst springs, wells or caves. In a survey conducted along the southern limit of its known range, we established a likely presence of Proteus at seven new sites, extending its range to Montenegro. Next, using specific molecular probes to discriminate the rare black morph of Proteus from the closely related white morph, we detected its eDNA at five new sites, thus more than doubling the known number of sites. In one of these we found both black and white Proteus eDNA together. This finding suggests that the two morphs may live in contact with each other in the same body of groundwater and that they may be reproductively isolated species. Our results show that the eDNA approach is suitable and efficient in addressing questions in biogeography, evolution, taxonomy and conservation of the cryptic subterranean fauna.

Fish migrate underground: the example of Delminichthys adspersus (Cyprinidae)

Complex aquatic systems of karst harbour a rich but little‐investigated biodiversity. In Croatia and Bosnia–Herzegovina karst, temporal springs are inhabited by a group of minnow‐like fishes that retreat to the associated ground water during dry seasons and spend several months underground. The most abundant species in this group is Delminichthys adspersus (Heckel 1843), which also has the most fragmented distribution range. To determine the population composition and dispersal patterns, and to detect potential underground migration, a large genetic data set comprising 544 specimens of D. adspersus covering most of its distribution area was analysed. Analysis of mitochondrial cytochrome b sequences (∼1000 bp) and eight microsatellite loci showed that D. adspersus comprises at least three subpopulations with gene flow occurring among them. Coalescent‐based analysis revealed a complex migration pattern, with several unidirectional dispersal paths, including between temporal springs that share no surface connection. The results of this study suggest the existence of recurrent underground migration of fish in a karst environment and demonstrate the complexity of its hydrological network. The findings are relevant to conservation strategies for endemic karst organisms and karst ecosystems as a whole.