Phylogeography of European grayling, <Emphasis Type="Italic">Thymallus thymallus</Emphasis> (Actinopterygii,Salmonidae), within the Northern Adriatic basin: evidence for native and exotic mitochondrial DNA lineages

Phylogeographic information on European grayling, Thymallus thymallus, is still fragmentary for the Northern Adriatic basin. In this article, we provide complete mitochondrial DNA control region sequence data of 456 grayling specimens from 21 sampling sites across distinct river drainages. Thirty-seven haplotypes were resolved and clustered into Danubian, Atlantic and Adriatic lineages. The latter clade, composed of 16 new haplotypes, was identified in 12 out of 17 Adriatic sampling sites and reached frequencies of 0.97 within single water courses of the Adige and the Po drainages. However, native Adriatic haplotypes were accompanied by Danubian and/or Atlantic variants in all cases. A positive correlation between hatchery haplotype frequency and annual stocking input pointed to a direct effect of stocking intensity on the genetic architecture of wild populations, although natural trans-basin colonisation may have additionally complicated the situation. However, both the extent and patterns of introgression between native and foreign strains, as well as microgeographic population structure within the Adriatic lineage will be clarified by future molecular surveys, based on nuclear genetic markers. Until then, conservation management must include an immediate cessation of stocking of commercial grayling stocks, as well as the prohibition of grayling translocation, even at the intra-drainage level.     

Genetic introgression between wild and stocked salmonids and the prospects for using molecular markers in population rehabilitation: the case of the Adriatic grayling (Thymallus thymallus L. 1785)

In the north Adriatic basin, a morphologically and genetically distinct lineage of grayling is found, designated as the Adriatic grayling. In Slovenia, the Adriatic grayling is restricted to the Soca river system, where it is critically endangered. The most pertinent threat is stocking with non-native, highly divergent Sava (Danubian) drainage stock, and this activity has been going on for more than four decades. The present study was designed to characterise the genetic structure of the Adriatic grayling in Slovenia, with particular emphasis on estimating the degree of introgression with non-indigenous stocked grayling. We analysed polymorphism at 154 microsatellite loci in samples representing grayling from the Adriatic and Danubian drainage stock. A relatively high number (12) of alleles, diagnostic for the Adriatic grayling, were identified. However, a correspondence analysis based on individual multilocus genotypes also revealed that there is no distinctive Adriatic group but rather a dispersed multitude of individuals that cannot be unambiguously distinguished from the more homogenous Danubian population. A Bayesian analysis of individual admixture coefficients confirmed this pattern and revealed extensive introgression between the Adriatic grayling and stocked grayling of Danubian origin. Average individual admixture coefficients showed that only between 50 and 60% of the original gene pools remained, and only few non-introgressed indigenous individuals could be identified. Microsatellite-based individual admixture analysis appear to be an important tool for identifying remaining non-introgressed indigenous individuals that could be used for restoring the original populations.     

Effects of stocking on the genetic diversity of brown trout populations of the Adriatic and Danubian drainages in Switzerland

This study focuses on genetic variation of brown trout Salmo trutta populations of the Adriatic and Danubian drainages in Switzerland. The allozyme and other protein loci data show a major replacement of native stocks from the Adriatic drainages by introduced hatchery trout of Atlantic basin origin. In most samples, diagnostic alleles for the Adriatic form of Salmo trutta f. fario and for the marbled trout Salmo trutta marmoratus are found at very low frequencies (f<0.15). Taking into account previous genetic studies on brown trout of this basin, the Danubian samples are not heavily contaminated with foreign alleles. The results are consistent with records of local stocking activities which account in part for the high introgression rates of Atlantic alleles into local populations of the Adriatic drainages. In addition, introgression is enhanced by a decrease of natural reproduction which is caused by a deterioration of trout habitats through human activities. Furthermore, a third mechanism is proposed that may contribute to the high introgression rates observed: if Atlantic trout are introduced, the reproductive barriers between the two native forms, marbled trout and Adriatic fario respectively, break down. Atlantic trout apparently hybridize with both native forms and generate gene flow between them. In some parts of Adriatic drainages in Switzerland, the patterns of introgression and hybridization are further complicated by introduction of trout from the Danubian system. Alleles of the marbled trout are also found in the samples of the Danubian drainage system. These are due to stocking activities across the watershed.     

Evolutionary distinctness of grayling {Thymallus thymallus) inhabiting the Adriatic river system, as based on mtDNA variation

The northern part of the peri-Adriatic river system is inhabited by a native grayling population. In comparison with the Danubian population most of the Adriatic population is regarded as morphologically distinct and therefore recognized as racially differentiated. DNA sequence analysis of the 394-bp fragment of the mtDNA control region and 363-bp fragment of the cytochrome b gene region from grayling specimens of the Adriatic, Danubian and Atlantic river systems revealed nine composite mtDNA haplotypes. The genetic divergence of the Adriatic and the Danubian-Atlantic haplotypes ranged from 2.52 to 3.10% and from 3.38 to 3.92% in the cytochrome b gene and control region, respectively. On the basis of sequence data an attempt was made to elucidate the phylogeography of the Adriatic population. The molecular data indicate an ancient monophyletic origin and also confirm its distinct position within the Thymallus thymallus species, in spite of the fact that stocking with the Danubian type occurred in the Adriatic river system. Therefore the Adriatic population deserves special attention. As a first step in its preservation, its formal distinction by using the name Adriatic grayling is proposed.     

Genetic diversity and phylogenetic origin of brown trout Salmo trutta populations in eastern Balkans

The study focuses on the phylogenetic origin and genetic diversity of brown trout in the eastern part of the Balkan Peninsula. It further aims to reveal the impact of human-mediated transfers and stocking with non-indigenous trout on the populations in this area. For these purposes, mtDNA control region and microsatellite variation of 204 individuals from 16 populations were analysed. The results indicate that mtDNA haplotypes from the lower Danube basin and southern Black Sea basins differ substantially from a subclade of the Danubian lineage consisting of haplotypes found so far in the most of the Danube basin and in the Caspian and Aral Sea basins. Considering also the results of demographic analyses, this study evidences a complex evolutionary history of brown trout in the southern and western parts of the Black Sea basin. In the Aegean Sea basin, a high frequency of the central haplotype of Adriatic mtDNA lineage has been found. The other Adriatic lineage haplotypes found in this basin differ from the central haplotype by one mutational step only, indicating a recent evolution of the Adriatic lineage in the Aegean Sea basin. Substantial genetic differentiation among populations and basins was revealed. The hybridization with Atlantic brown trout was indicated in both sea basins, but especially in the Danube basin. Compared to other European regions, it can be inferred that the introgression of exogenous brown trout in the eastern Balkan populations is rather low.     

Life history and genetic characterisation of sea trout Salmo trutta in the Adriatic Sea

1. The brown trout Salmo trutta is characterised by both anadromous (sea trout) and resident populations, naturally occurring in Atlantic and Ponto-Caspian rivers. Sea trout are currently considered absent from rivers of the Mediterranean area, probably because of the non-optimal chemical–physical characteristics of the Mediterranean Sea. However, the occasional bycatch of smoltified S. trutta in the Adriatic Sea is well known among fishermen and the biological explanation of this phenomenon is still controversial. The aim of this study was to compare the genetic diversity of freshwater and marine brown trout to try to understand the factors underlying the presence of putative anadromous brown trout in the Adriatic Sea.
2. In this study, we analysed the genetic diversity of: (1) wild brown trout collected from the Esino River (central Italy); (2) a domestic strain of brown trout used for stocking the study area; and (3) a sample of Adriatic sea trout collected near the outlet of the Esino River. Together with genetic analysis, we carried out scale analysis in order to track the freshwater/marine stages of the life cycle in the sea trout samples. The genetic characterisation was carried out by polymerase chain reaction–restriction fragment length polymorphism analysis of the mtDNA fragment ND-5/6 and the nuclear locus LDH-C1* and by genotyping 15 microsatellite loci. The genetic polymorphism obtained was used to investigate intra- and inter-population genetic diversity, rates of genetic introgression between wild and domestic samples and the origin of sea trout specimens by using assignment tests.
3. Our genetic analyses demonstrated that the sea trout analysed in this study are from the domestic strain of Atlantic origin used in central Italy for stocking activities. The level of genetic introgression between native and domestic samples is high in the Esino River. The populations more resilient to introgressive hybridisation appeared to be those living in the portion of the river network dominated by carbonate rocks. Assignment tests (GeneClass) suggest the existence of a link between stocking efforts and the freshwater origin of the sea trout. In addition, data obtained from the analysis of scales, size measurement, and sex determination showed a pattern of smolt age, size, and sex ratio very similar to those observed in other anadromous populations.
4. In conclusion, the present study highlighted that sea trout from the central Adriatic Sea originated from brown trout of Atlantic origin inhabiting the Esino River. Their seaward migratory behaviour could represent a consequence of an active migration instead of a passive displacement by water flow. Our results also showed that traditional stocking practices represent a negative activity for the conservation of the last Mediterranean native S. trutta populations.

     

Genetic assessment of spatiotemporal evolutionary relationships and stocking effects in grayling (Thymallus thymallus, Salmonidae)

Domestically reared introduced (or escaped) individuals can have detrimental genetic effects on the indigenous populations into which they are released. Consequently, numerous studies have attempted to estimate whether non-native specimens have contributed to the gene pool of wild populations. So far, the key limiting factor of such studies has been their lack of appropriate baseline genetic material. Here, microsatellite DNA analyses of historical scale samples and contemporary wild and introduced populations were used to assess spatiotemporal population structure and stocking effects among endangered Lake Saimaa (eastern Finland) grayling ( Thymallus thymallus , Salmonidae). Significant decreases in genetic differentiation were detected between wild and introduced populations since the commencement of stocking in 1986. Accordingly, up to 15% of the contemporary wild grayling were confidently identified to be of pure hatchery origin, and recent hybridization between the hatchery and indigenous individuals appeared likely. Despite these clear genetic imprints of stocking, the contemporary populations exhibited evolutionary relationships congruent with the sampling locations, and up to 73% of contemporary individuals were identified to be of pure indigenous origin. The use of historical baseline material should prove efficient for monitoring gene flow between domesticated and wild populations in other species also, e.g. salmonids, game animals and plants.     

Determinants of hierarchical genetic structure in Atlantic salmon populations: environmental factors vs. anthropogenic influences

Disentangling the effects of natural environmental features and anthropogenic factors on the genetic structure of endangered populations is an important challenge for conservation biology. Here, we investigated the combined influences of major environmental features and stocking with non‐native fish on the genetic structure and local adaptation of Atlantic salmon (Salmo salar) populations. We used 17 microsatellite loci to genotype 975 individuals originating from 34 French rivers. Bayesian analyses revealed a hierarchical genetic structure into five geographically distinct clusters. Coastal distance, geological substrate and river length were strong predictors of population structure. Gene flow was higher among rivers with similar geologies, suggesting local adaptation to geological substrate. The effect of river length was mainly owing to one highly differentiated population that has the farthest spawning grounds off the river mouth (up to 900 km) and the largest fish, suggesting local adaptation to river length. We detected high levels of admixture in stocked populations but also in neighbouring ones, implying large‐scale impacts of stocking through dispersal of non‐native individuals. However, we found relatively few admixed individuals suggesting a lower fitness of stocked fish and/or some reproductive isolation between wild and stocked individuals. When excluding stocked populations, genetic structure increased as did its correlation with environmental factors. This study overall indicates that geological substrate and river length are major environmental factors influencing gene flow and potential local adaptation among Atlantic salmon populations but that stocking with non‐native individuals may ultimately disrupt these natural patterns of gene flow among locally adapted populations.     

Assessing natural and disturbed population structure in European grayling Thymallus thymallus: melding phylogeographic,population genetic and jurisdictional perspectives for conservation planning

Natural and human‐mediated population structure of European grayling Thymallus thymallus was assessed in primarily Austrian reaches of the Danube River catchment. Data on phylogeographic structure based on mitochondrial DNA (mtDNA) were combined with variation found across 13 microsatellite loci to assess introgression stemming from stocking activities. Populations north of the Alps generally showed no signs of long‐term introgression from allochthonous stocks, although one population comprised two distinct genotypic groups, one of which may stem from recently stocked material. South of the Alps, levels of introgression with stocks from the north were extensive and only one of six populations is believed to represent a reference state. Allelic diversity and expected heterozygosity were positively correlated with mtDNA admixture supporting more recent introgression and that there is little or no reproductive barrier between these two major lineages north and south of the Alps. A third unrelated mtDNA lineage is described from the Lafnitz, a tributary of the Raab drainage. The high lineage diversity in the upper Austrian Danube is not concordant with an existing model of T. thymallus evolutionarily significant units in Europe. Caveats in naming such units or following a strict hierarchical conservation unit structure for broadly distributed species with complex phylogeographic distributions stretching over various jurisdictions are discussed. The necessity of using both phylogeographic and population genetic approaches in evaluating the history and conservation value of populations in a conservation context is additionally highlighted.     

Impact of climate change and human‐mediated introgression on southern European Atlantic salmon populations

This study focuses on temporal changes in Atlantic salmon (Salmo salar) populations from the vulnerable periphery of the species range (northern Spain). Using microsatellite markers to assess population structuring and introgression of exogenous genes in four different temporal samples collected across 20 years, we have determined the relative weights of climate and stocking practices in shaping contemporary regional population genetic patterns. Climate, represented by the North Atlantic Oscillation Index, was identified as the main factor for determining the level of population genetic differentiation. Populations within the region have become homogenized through gene flow enhanced by straying of adult salmon from natal rivers and subsequent interchange of genes among rivers due to warmer temperatures. At the same time, and in line with documented changes in stock transfer strategies, evidence of genetic introgression from past stock transfers has decreased throughout the study period, becoming a secondary factor in erasing population structuring. The ability to disentangle the effects of climatic changes and anthropogenic factors (fisheries management practices) is essential for effective long‐term conservation of this iconic species. We emphasize the importance of evaluating all factors which may be linked to stocking practices in vulnerable species, particularly those sensitive to climate change.     

Alpine headwater streams as reservoirs of remnant populations of the Danubian clade of brown trout

1. The watercourses of Austria are mainly part of the Danube drainage system. Nonetheless, only few brown trout (Salmo trutta) populations of the Danubian phylogenetic lineage have been found in this region, not being introgressed by hatchery‐reared fish of the allochthonous Atlantic lineage. The present study was aimed at identifying further waterbodies that might harbour the non‐admixed gene pool of the Danubian clade trout. 2. In a first step, the complete mitochondrial DNA control region of 447 specimens from 20 sampling sites was sequenced to screen for the presence of specific haplotypes. In a second step, 332 individuals from 15 populations were analysed at 10 microsatellite DNA loci. 3. Although sampling concentrated on remote Alpine headwater streams, introgression of hatchery strains was more common than expected from existing stocking records. For seven populations, however, no evidence of recent hatchery trout introgression was found and it is suggested that they might represent the indigenous gene pool. 4. Populations showing no evidence of hatchery introgression displayed lower levels of intrapopulation variability and higher degrees of differentiation compared to purported admixed populations. 5. Our study demonstrates the necessity of combining different molecular markers to reveal the impact of introgression into wild populations, since at some sampling sites admixture would have remained undetected if only a single molecular technique was applied. 6. The identification and characterisation of non‐introgressed populations provides important information for setting conservation priorities and preventing further loss of genetic integrity of a unique element of the native fish fauna.     

Molecular Evidence for Introgression and Loss of Genetic Variability in Salmo (trutta) macrostigma as a Result of Massive Restocking of Apennine Populations (Northern and Central Italy)

We assessed structural gene variation (allozymes and mtDNA) of brown trout to evaluate the genetic variability of Apennine stream populations (Northern and Central Italy) and the possibility of introgression by alien genomes after massive restocking with hatchery strains (Atlantic stocks). Genetic variability within and between Apennine populations was extremely low in our samples. Only two allozyme loci were polymorphic and mean hetero-zygosity was also reduced compared to other brown trout populations. Allelic frequencies determined for both loci were similar to the ones detected in the corresponding hatchery spawners. The reduction or total absence of the Mediterranean nuclear (LDH-5) and mitochondrial (16S rDNA) diagnostic markers suggests the domestic origin of most populations, and the introgression effects carried out by non-native genomes. From a taxonomic point of view, a clear differentiation emerges among basins placed on opposite sides of the Apennine chain (Tyrrhenian and Adriatic regions). In particular, the presence of Mediterranean genotypes and haplotypes characterizing Salmo (trutta) macrostigma is sporadic along the eastern Apennine side, adding additional doubts on the original presence and wide distribution of this salmonid along the Adriatic side of the mountain chain. In spite of conservation programs devoted to preservation of local genetic characteristics of S. t. macrostigma, massive restocking practices with hatchery strains obtained by a few spawners is the major cause of significant `founder effect' and `inbreeding depression' even in Apennine regions.     

Microsatellite genetic variation reveals extensive introgression between wild and introduced stocks, and a new evolutionary unit in French pike Esox lucius L.

The genetic variability of 11 French pike Esox lucius populations from rivers or extensive aquaculture ponds was analysed using seven microsatellite markers. The fish stocking of eastern European pike in French rivers is widespread, so five supplementary populations from this region were also analysed. Genetic variability within the French population was small, and similar to that previously found in Europe for this species. French populations from stocked rivers and extensive farms formed a genetically homogeneous group, and did not diverge from most eastern European samples, which may reflect either ancestral polymorphism or a complete introgression of French populations by introduced stocks. Bayesian analysis of the admixture between populations enabled identification of the French populations that were likely to be affected by introduced stocks (of local or foreign origin). Three genetically distinct evolutionary branches were identified: continental Europe, Danube basin, and south‐western France. The last is a new finding in the species which needs to be further explored by comparisons with wild populations at other sites.     

Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>) mitochondrial DNA polymorphism in the centre of the marble trout distribution area

The marble trout, a lineage of the Salmo trutta complex, is endemic to the Southern Alpine region. Although it is endangered throughout its entire distribution range, population genetic data were lacking for the central area, including the upper Etsch/Adige River system (South Tyrol, Northern Italy). A total of 672 Salmo trutta specimens, comprising phenotypic marble trout and phenotypic brown trout, from 20 sampling sites throughout South Tyrol were analysed by sequencing the complete mitochondrial DNA control region. Thirteen distinct haplotypes were identified, which clustered within three major genetic lineages: the Marmoratus (MA), the Atlantic (AT) and the Danubian (DA) lineage. 41.7% of the investigated individuals carried haplotypes of the MA lineage, 47.9% of the AT lineage and 10.4% of the DA lineage. It is noticeable that AT haplotypes were present at all sampling sites and no “pure” marble trout population with exclusively MA haplotypes was found. This points to a considerable impact of stocking with allochthonous brown trout, given that there is no evidence for natural colonisation by individuals of the AT lineage. However, our data indicate, for at least four localities, a limited gene flow between the native marble trout and hatchery-reared strains. Future conservation and rehabilitation measures will thus have to concentrate on the identification of remnant pure marble trout individuals from such mixed populations. Handling editor: C. Sturmbauer     

Genetic introgression between wild and stocked brown trout in the Douro River basin, Spain

The genetic diversity of Spanish brown trout is currently threatened by stocking with exogenous brown trout from Central and Northern Europe. In the Douro River basin 25% of the analysed populations in the present study showed introgression by genes of hatchery origin. The mean introgression estimated by the single locus approach ( S ) varied from 0 to 22% among populations, with a mean value of 3%. The hatchery allele markers were absent in populations where stocking ceased in 1993. However, the introgression effect was observed in all populations stocked until 1998. It seems that cessation of stocking is a good measure for restoring native populations. A thorough review of published and present data of genetic interactions between wild and stocked brown trout in Spanish rivers indicates different levels of introgression between basins. The absence of a clear geographical pattern in the introgression level suggests that ecological interactions and local stocking programmes may play an important role in stocking success. Finally, several guidelines are provided for conservation and management of native brown trout populations in Spanish rivers.     

Substantial genetic structure among stocked and native populations of the European grayling (<Emphasis Type="Italic">Thymallus thymallus</Emphasis>, Salmonidae) in the United Kingdom

While currently in a state of recovery in the United Kingdom (UK), the grayling (Thymallus thymallus) remains of conservation interest due to its historical decline, socio-economic value and the potential impact of hatchery-reared stock fish on the genetic structure and diversity of wild populations. However, little is known about the levels and distribution of genetic diversity among UK grayling populations. To this end, 27 UK populations of grayling were genotyped across 10 microsatellite loci and sequenced at the mtDNA D-Loop. All populations clustered into four higher-level groups: Northern England, Southern England, Wales, and group consisting of a mixture of native and introduced populations. Ten populations showed evidence of bottleneck or founder effects, and the effective population size (Ne) was low in all populations. In most cases, historical stocking records agreed with the genetic relationships revealed in the study. A D-Loop haplotype network supported the groupings observed in the nuclear data, while phylogenetic inference places the UK populations amongst Central European samples. The combined datasets demonstrate that many of the UK populations can be treated as separate Management Units and we recommend that to preserve population specific genetic diversity, that stocking should be an intervention of last resort. However, if stocking is deemed essential, brood stock should originate from the river to be stocked.     

Experience from Lipizzan horse and salmonid species endemic to the Adriatic river system. Examples for the application of molecular markers for preservation of biodiversity and management of animal genetic resources

Management of breeding- and free-living populations, traditionally based on phenotypic traits, relays more and more on availability of reliable information about the basic population genetic parameters as heterozygosity, mean number of alleles per locus, percentage of polymorphic loci, population structuring, genetic distances and others. Therefore, the application of molecular markers, revealing a great deal of phenotypically hidden information, becomes inevitable for population analysis. Conservation geneticists use this information for implementation of appropriate management policies. Application of molecular markers in Lipizzan horse breed, which is an example for a pedigreed breeding population, and in two endangered salmonid fish populations in Slovenia, are presented. In the Lipizzan horse breed, an insight in the population structure, overall heterozygosity, relationship between population parameters and phenotypic traits and reliability of pedigree data was gained by using molecular markers. Marble trout and Adriatic grayling were selected as examples for free-living populations, seriously endangered by human activity in the past. Development of informative molecular markers, their application and suggestions for appropriate conservation actions are described.     

Genetic variation of European grayling (Thymallus thymallus) populations in the Western Balkans

In order to elucidate genetic composition of European grayling (Thymallus thymallus) populations in the Western Balkans, the partial mitochondrial DNA (mtDNA) control region was sequenced and 12 microsatellite loci genotyped in 14 populations originating from tributaries of the Adriatic and Danube drainages. Eleven mtDNA haplotypes were found, one confined to the Adriatic clade, one to the Alpine group and the rest to the ‘Balkan’ grayling phylogenetic clade. Haplotypes from the Balkan clade were confined to the Danube drainage and constituted two groups: northern group with haplotypes found in the Slovenian part of the Danube drainage, and southern group, consisting from Bosnia–Herzegovina and Montenegro. Substantial genetic distance between northern and southern groups of haplotypes (0.75–1.8%) and well supported divisions within the northern group indicate very structured grayling population within the studied Danube basin that most probably did not evolve due to vicariance but rather as a consequence of multiple colonization waves that might have occurred during the Pleistocene. Furthermore, genetic distance of ~4% between Adriatic and Danube populations’ haplotypes, suggest that their separation occurred in mid-Pliocene. These findings imply a complex colonization pattern of the Western Balkans drainages. Microsatellite data also confirm high genetic diversity in Western Balkans populations of grayling (on average 7.5 alleles per microsatellite locus and H _exp 0.58). Limited stocking activities were detected based on microsatellites and mtDNA data. Regarding current knowledge of grayling phylogeography appropriate management strategies were proposed to preserve unique, autochthonous grayling populations in Western Balkan.     

Identifying units for conservation using molecular systematics: the cautionary tale of the Karner blue butterfly

The federally endangered North American Karner blue butterfly (Lycaeides melissa samuelis) and the closely related Melissa blue butterfly (L. m. melissa) can be distinguished based on life history and morphology. Western populations of L. m. samuelis share mitochondrial haplotypes with L. m. melissa populations, while eastern populations of L. m. samuelis have divergent haplotypes. Here we test two hypotheses concerning the presence of L. m. melissa mitochondrial haplotypes in western L. m. samuelis populations: (i) mitochondrial introgression has occurred from L. m. melissa populations into western L. m. samuelis populations, or (ii) western populations of the nominal L. m. samuelis are more closely related to L. m. melissa than to eastern L. m. samuelis populations, yet are phenotypically similar to the latter. A Bayesian algorithm was used to cluster 190 L. melissa individuals based on 143 informative amplified fragment length polymorphism (AFLP) loci. This method clearly differentiated L. m. samuelis and L. m. melissa. Thus, genomic divergence was greater between western L. m. samuelis populations and L. m. melissa populations than it was between western and eastern populations of L. m. samuelis. This supports the hypothesis that the presence of L. m. melissa mitochondrial haplotypes in western L. m. samuelis populations is the result of mitochondrial introgression. These data provide valuable information for conservation and management plans for the endangered L. m. samuelis, and illustrate the risks of using data from a single locus for diagnosing significant units of biodiversity for conservation.     

Population structure, effective population size and adverse effects of stocking in the endangered Australian eastern freshwater cod Maccullochella ikei

Microsatellite markers were used to examine spatio-temporal genetic variation in the endangered eastern freshwater cod Maccullochella ikei in the Clarence River system, eastern Australia. High levels of population structure were detected. A model-based clustering analysis of multilocus genotypes identified four populations that were highly differentiated by F-statistics (F(ST) = 0·09 - 0·49; P < 0·05), suggesting fragmentation and restricted dispersal particularly among upstream sites. Hatchery breeding programmes were used to re-establish locally extirpated populations and to supplement remnant populations. Bayesian and frequency-based analyses of hatchery fingerling samples provided evidence for population admixture in the hatchery, with the majority of parental stock sourced from distinct upstream sites. Comparison between historical and contemporary wild-caught samples showed a significant loss of heterozygosity (21%) and allelic richness (24%) in the Mann and Nymboida Rivers since the commencement of stocking. Fragmentation may have been a causative factor; however, temporal shifts in allele frequencies suggest swamping with hatchery-produced M. ikei has contributed to the genetic decline in the largest wild population. This study demonstrates the importance of using information on genetic variation and population structure in the management of breeding and stocking programmes, particularly for threatened species.