The Effect of Recurrent Floods on Genetic Composition of Marble Trout Populations

A changing global climate can threaten the diversity of species and ecosystems. We explore the consequences of catastrophic disturbances in determining the evolutionary and demographic histories of secluded marble trout populations in Slovenian streams subjected to weather extremes, in particular recurrent flash floods and debris flows causing massive mortalities. Using microsatellite data, a pattern of extreme genetic differentiation was found among populations (global FST of 0.716), which exceeds the highest values reported in freshwater fish. All locations showed low levels of genetic diversity as evidenced by low heterozygosities and a mean of only 2 alleles per locus, with few or no rare alleles. Many loci showed a discontinuous allele distribution, with missing alleles across the allele size range, suggestive of a population contraction. Accordingly, bottleneck episodes were inferred for all samples with a reduction in population size of 3–4 orders of magnitude. The reduced level of genetic diversity observed in all populations implies a strong impact of genetic drift, and suggests that along with limited gene flow, genetic differentiation might have been exacerbated by recurrent mortalities likely caused by flash flood and debris flows. Due to its low evolutionary potential the species might fail to cope with an intensification and altered frequency of flash flood events predicted to occur with climate change

Translocation of stream-dwelling salmonids in headwaters: insights from a 15-year reintroduction experience

Translocation programs are a common strategy to increase the number of viable populations of threatened freshwater fishes. Yet, only in a minority of cases the success or failure of translocations has been assessed through a quantitative analysis of demographic traits, compensatory responses, lifehistories and population dynamics of the threatened species. A paradigmatic case a translocation program combining both management- and research-oriented activities is represented by the Marble Trout Conservation Program, which started in 1993 in the upper reaches of the Soca, Idirjca and Baca river basins (Slovenia) for the conservation of stream-dwelling marble trout Salmo marmoratus. In order to enhance the viability of the species, two new populations were created in 1996 by stocking 500 marble trout aged 1? in previously fishless streams (Gorska and Zakojska) within the core habitat of the species. The new populations have been systematically monitored for 15 years by individually tagging and sampling marble trout. Our analyses show that deterministic extinction of marble trout populations are unlikely and that highmagnitude environmental stochasticity (i.e., severe floods) is the only main cause of local population extinction, despite the high resilience to flood-induced massive mortalities exhibited by marble trout through compensatory mechanisms (e.g., relaxation of density-dependent body growth and survival at low densities). Fishless headwaters, probably characterized by a history of recurrent severe floods, should not be considered as candidate sites for the creation of new populations. Fewer individuals than originally reintroduced (i.e., 500 fish aged 1? in each stream) might be sufficient to establish viable populations, since compensatory mechanisms are likely to regulate population size around stream carrying capacity in a few years. Besides enhancing the species viability, translocation programs can provide an excellent framework for the estimation of ecological traits (e.g., life-histories, demography, population dynamics etc.), identify potential vulnerabilities and thus guide well-formed management actions for the threatened species

The rehabilitation of the Marble trout, Salmo marmoratus in the upper Soca River basin, Slovenia

1° Colloque Italo-slovène sur la truite marmorataInternational audienceFollowing repeated restocking of Brown trout (Salmo trutta) dating from 1906, the marble trout (Salmo marmoratus) seems to have disappeared as a result of genetic pollution (hyhridization) from the lower reaches of the' Soca River.in Slovenia. A project for rehabilitating the Marble trout has been undertaken. A priliminary exploratory stage (1993-1995) that resulted in the publication in 1996 of an Action Plan, written in English and Slovenian, suggested that the cause of the disappearance of the Marble trout was hybridization, established the validity of the project in the region in question and provided us with essential information (occurence of genetically pure populations) that could be used to define our future strategy. We chose genetic rehabilitation, i.e. the replacement of a population of introduced and introgressed fish by a population of genetically pure fish of the native species, rather than a programme of eradicating undesirable fish by chemical methods, which would have been incompatible with the region's context. Our overall strategy therefore had two main aims: to ensure the long-term survival of populations of pure Marble trout (species conservation) and to rehabilitate the genes of Marble trout in the hybridization zone until foreign genes have almost been eliminated. Undergoing studies of the phase two of the project are described and preliminary results are presented

Analyse Expérimentale de l'Hybridation entre la Truite Commune (Salmo trutta) et la Truite Marbrée (Salmo marmoratus), Espèce Menacée : Premiers Résultats

International audienceExperimental survey of the hybridization between the Brown trout (Salmo trutta) and the threatened Marble trout (Salmo marmoratus) : Preliminary results. Populations of the marble trout (Salmo marmoratus), an endemic species of the Adriatic Basin, are critically declining due to introgression by brown trout (Salmo trutta) stocked strains. Hybrids between these two forms are fertile and presently predominant in most rivers of the species' range. A rehabilitation project was initiated in 1993 in the Soca and Idrijca river basins in Slovenia. The overall final objective of the project is to decrease the introgression rate in the upstream Soca basin. Our present study concerns a limited aspect of the overall project as it focuses on the hybridization mechanisms involved. Knowledge of these mechanisms is of the utmost importance if one wants to facilitate rehabilitation of the Marble trout. The mechanisms have been studied through experimentation in nature. Four experimental small rivers were selected in 1997 in Slovenia and emptied by electro fishing the following years: the Driselpoh, Stopnikarca, Prodarjeva and Baca streams. In 1999, we released 50% of marmoratus originating from the Soca basin and 50% of S. trutta from Switzerland. To facilitate later genetic analyses two pure populations of marble trout were crossed in order to increase the level of genetic variation at neutral marker level. Both strains were bred in a local fish farm. The fish were released as 1 + and marked with carlin tags. the main aim was to follow the demographic evolution of the populations, the reproduction features, to identify a possible interspecific competition and to define the genealogy of the offspring born in the experimental streams. The genetic analysis included seven variable microsatellite loci and we used the obtained multilocus genotypes to allocate offspring born in the river to a set of possible parents using a novel approach. A coalescence based method was used to estimate the effective population size (Ne) of each species as well as for hybrids. In the present study, we present the results obtained from the first cohort born in the Driselpoh stream. The main results are (i) a highly significant deficit of hybrids in the cohort born in the experimental stream, (ii) a low effective size during the first breeding and (iii) an apparent high heterosis effect in terms of size and weight of hybrids when compared to pure marble and brown trout offspring born in 2002

Conservation biology applied to fish: The example of a project for rehabilitating the marble trout (Salmo marmoratus) in Slovenia

ReviewInternational audienceThe conservation status of freshwater fish is of concern throughout the world, a third of all known species having become extinct or being endangered. Many of them have fragmented populations of small size whose future is in doubt. Conservation biology is the discipline that is used to study such populations, so that they can be managed in a way that will ensure their long-term survival. The history, development and future of conservation biology are described. Following repeated restocking of brown trout (Salmo trutta) dating from 1906, the marble trout (Salmo marmoratus) seems to have disappeared as a result of genetic pollution (hybridisation) from the lower reaches of the Soca River in Slovenia. A project for rehabilitating the marble trout has been undertaken and will serve as an example to illustrate an application of conservation biology to freshwater fish. A preliminary exploratory stage (1993-1995) that resulted in the publication in 1996 of an Action Plan, written in English and Slovenian, suggested that the cause of the disappearance of the marble trout was hybridisation, established the validity of the project in the region in question and provided us with essential information (occurrence of genetically pure populations) that could be used to define our future strategy. We chose genetic rehabilitation, i.e., the replacement of a population of introduced and introgressed fish by a population of genetically pure fish of the native species, rather than a programme of eradicating undesirable fish by chemical methods, which would have been incompatible with the region's context. Our overall strategy therefore had two main aims: to ensure the long-term survival of populations of pure marble trout (species conservation) and to rehabilitate the genes of marble trout in the hybridisation zone until foreign genes have almost been eliminated

The genetic diversity of native, stocked and hybrid populations of marble trout in the Soca river, Slovenia

International audienceThe marble trout (Salmo marmoratus) is an endangered species in Slovenia (and in Italy, Croatia and Albania) because of hybridization resulting from intensive stocking. Eleven populations of trout from the Soca river basin (Slovenia) were analysed using 31 presumptive enzyme loci. Three European control samples representing the main European genotypes of brown trout were also analysed. This analysis confirmed the existence of extensive hybridization in the main river, resulting in an equal mixture of alleles belonging to marble trout, and Danubian and Atlantic brown trout (Salmo trutta). Despite the high level of introgression observed in the main Soca River, nearly pure populations of marble trout were found in the headwaters of five tributaries. The five stations with pure marble trout will be the basis of a rehabilitation programme. Hardy–Weinberg and linkage disequilibria were observed and hypotheses are discussed to explain this. A scenario for the colonization of the Adriatic region is proposed

Data from: Genetic and life-history consequences of extreme climate events

Climate change is predicted to increase the frequency and intensity of extreme climate events. Tests on empirical data of theory-based predictions on the consequences of extreme climate events are thus necessary to understand the adaptive potential of species and the overarching risks associated with all aspects of climate change. We tested predictions on the genetic and life-history consequences of extreme climate events in two populations of marble trout Salmo marmoratus that have experienced severe demographic bottlenecks due to flash floods. We combined long-term field and genotyping data with pedigree reconstruction in a theory-based framework. Our results show that after flash floods, reproduction occurred at a younger age in one population. In both populations, we found the highest reproductive variance in the first cohort born after the floods due to a combination of fewer parents and higher early survival of offspring. A small number of parents allowed for demographic recovery after the floods, but the genetic bottleneck further reduced genetic diversity in both populations. Our results also elucidate some of the mechanisms responsible for a greater prevalence of faster life histories after the extreme event

Primena krioprezervacije sperme na gajenje i očuvanje salmonidnih vrsta: saradnja Slovenije i Mađarske

U svrhu očuvanja dve salmonidne vrste koje su autohtone u slivu reke Soče u Sloveniji, pastrmske glavatice (Salmo marmoratus) i lipljena Jadranskog porekla (Thymallus thymallus) primenjena je krioprezervacija sperme. Populacije ovih vrsta ozbiljno su ugrožene hibridizacijom i introgresijom sa alohtonim vrstama: potočnom pastrmkom (Salmo trutta m. fario) i lipljenom Dunavskog porekla koji su unešeni u sliv reke Soče u dvadesetom veku. Ribolovački klub Tolmin, koji upravlja jednim delom reke Soče, razvio je akcioni plan za očuvanje genetskih resursa i restauraciju autohtonih vrsta u njihova primarna staništa. Ovaj akcioni plan podrazumeva prestanak poribljavanja alohtonim salmonidnim vrstama koje mogu da naprave hibride sa lokalnim vrstama, identifikaciju postojećih “čistih” vrsta, stvaranje matica od onih jedinki koji pripadaju čistim vrstama, poribljavanje potomcima čistih vrsta, stvaranje “utočišta” za postojeće čiste vrste i stalni monitoring populacija riba u njihovim vodotokovima. Genetske analize ovih populacija izvršili su naučnici sa Odeljenja za Nauke o životinjama Univerziteta u Ljubljani. Krioprezervacija sperme Jadranskog lipljana i pastrmske glavatice čini sastavni deo akcionog plana za očuvanje od 2009. godine. Kada je reč o lipljanu, ne postoji više ni jedna čista populacija, stoga je cilj programa očuvanja povećanje udela Jadranskog genotipa kod matica. Sperma i uzorak peraja uzorkovani su od divljih mužjaka na mestu mresta. Sperma je krioprezervirana i sačuvana dok genetske analize nisu završene za svaki od uzoraka (2-3 nedelje). Krioprezervirana sperma koja je sadržala veću količinu Jadranskog genotipa nego što je to na početku definisano je otopljena i iskorišćena za fertilizaciju jaja takozvanih ‘Jadranskih’ ženki. Potomstvo iz jaja oplođenih krioprezerviranom spermom je odgajeno do matica i trenutno 70-80% matica lokalnog lipljana potiče iz krioprezervirane sperme. Kada je reč o pastrmskoj glavatici, krioprezervirana sperma se koristi za stvaranje ‘utočišta’. Sperma se sakuplja od divljih mužjaka čiste populacije pre sezone mresta (rani novembar) a zatim se krioprezervira. Sperma se čuva u tečnom azotu do sezone mresta (decembar-januar) kada dolazi do oplodnje jaja ženki koje pripadaju identičnoj populaciji. Oplodnja se obavlja krioprezerviranom supermom. Jaja u stadijumu očne mrlje se zatim nasade u veštačka gnezda u pripremljenom potoku koji predstavlja ‘utočište’. Stoga veliki broj mužjaka iz čiste populacije učestvuje u stvaranju novih populacija, dok u isto vreme nije potrebno ukloniti mužjake iz originalne populacije, a nasađivanje jaja u stadijumu očne mrlje obezbeđuje obeležavanje nove teritorije. Isti protokol se koristi za krioprezervaciju obe vrste: sperma se meša u odnosu 1:1 sa ekstenderom koji sadrži 200 mM glukoze, 40 mM KCl, 30 mM trisa (pH iznosi 8.0 sa cc. HCl) a metanol se koristi kao krioprotektant u finalnoj koncentraciji od 10% v/v . Cevčice od 0.5-ml se pune rastvorenom spermom koja se zatim zamrzava u pari tečnog azota na 3cm iznad nivoa azota u trajanju od 3 minuta. Pošto se sačuva u tečnom azotu, uzorci se tope u vodenoj kupatilu na 40 °C u trajanju od 13 sekundi. Ovako razvijeni protokol rezultira u 50-70% izmrešćenih jedinki kod obe vrste.Sperm cryopreservation was applied to the conservation efforts of two salmonid species autochthonous to the drainage of the Soča river in Slovenia, the marble trout (Salmo marmoratus) and the Adriatic lineage of the grayling (Thymallus thymallus). Populations of these species were seriously compromised by hybridization and introgression with allochthonous species: the brown trout (Salmo trutta m. fario) and the Danubian lineage of the grayling that were introduced to the Soča drainage during the 20th century. The Angling club of Tolmin, that manages a part of the Soča system has developed an action plan for the conservation of the genetic resources and restoration of the autochthonous species in their original habitats. This action plan includes the cessation of the stocking of any allochthonous salmonids that could hybridize with the local species, identification of existing pure populations, establishment of broodstocks of non-introgressed individuals, stocking the rivers with the progeny of the broodstocks, creation of „sanctuary” streams for the existing pure populations and continuous monitoring of fish populations in their watercourses. Genetic analyses of the populations are conducted by scientists from the Department of Animal Science of the University of Ljubljana. Cryopreservation of sperm from the Adriatic grayling and the marble trout has constituted an integral part of the conservation activities since 2009. In case of the grayling, no pure populations remain, thus, the objective of the conservation program is to increase the proportion of Adriatic genotype in the broodstock. Sperm and fin clips were collected from wild males on the spawning grounds. Sperm was cryopreserved and stored until the genetic analysis was completed on each sample (2-3 weeks). Cryopreserved sperm of individuals containing higher than a pre-defined proportion of Adriatic genotype was thawed and used for fertilization of eggs known „Adriatic” females. Progeny hatching from eggs fertilized with cryopreserved sperm was grown to broodstock and currently 70-80% of the local grayling broodstock originates from cryopreserved sperm. In case of the marble trout, sperm cryopreservation is used in the creation of „sanctuary” streams. Sperm is collected from wild males of a given pure population prior to the spawning season (early November) and cryopreserved. Sperm is stored in liquid nitrogen until the spawning season (December-January) when eggs of females from the identical population are fertilized with the cryopreserved sperm. Eyed eggs are then stocked into artificially created nests in the prepared „sanctuary” stream. Thus, a high number of males of the given pure population participates in the creation of the new population, removal of males from the original population is unnecessary, and stocking of eyed eggs ensures imprinting at the new location. The same protocol is used for cryopreservation of both species: sperm is mixed at a ratio of 1:1 with an extender containing 200 mM glucose, 40 mM KCl, 30 mM Tris (pH set to 8.0 with cc. HCl) and methanol is used as a cryoprotectant at 10% v/v final concentration. Diluted sperm is loaded into 0.5-ml straws and frozen in the vapor of liquid nitrogen at 3 cm above the level of nitrogen for 3 minutes. Following storage in liquid nitrogen, samples are thawed at a 40 °C water bath for 13 sec. The developed protocol results in 50-70% hatch in both species

Hybridization mechanisms between the endangered marble trout (Salmo marmoratus) and the brown trout (Salmo trutta) as revealed by in-stream experiments

International audiencePopulations of the marble trout (Salmo marmoratus) are critically declining due to introgression by brown trout (S. trutta) strains. Hybrids between the two forms are fertile and presently predominant in most rivers of the species' range. The involved hybridization mechanisms have been studied through two large scale in-stream experiments (Driselpoh and Stopnikarca) as 50% of each species have been stocked at the age of one year in fishless streams, each fish being individually marked. Diagnostic molecular markers were applied to test a partial reproductive isolation between the two species. Emphasis was put on survival and growth patterns of stocked fish (parental generation) as well as on fish hatched within the stream (F1 generation). No evidence of a partial reproductive barrier between the two species was observed. Survival of the parental generation depended on the year in both streams, as well as on the species in Stopnikarca, but was identical for both species in Driselpoh. In both streams survival of instream hatched individuals measured from 0+ to 2+ was lower for brown trout. In Driselpoh, F1 0+, 1+ and 2+ hybrids were larger than pure individuals. Larger hybrids were only observed in Stopnikarca when analyses focused on individuals in inter-specific competition suggesting that heterosis and stress effects may explain the observed size differences. Our results point out important ecological differences between marble and brown trout and have shown that hybridization can easily take place. The findings indicate that high F1 hybrid presence, survival and heterosis effects may impede marble trout rehabilitation in the area