Seventy‐five EST‐linked Atlantic salmon (Salmo salar L.) microsatellite markers and their cross‐amplification in five salmonid species

An Atlantic salmon (Salmo salar L.) expressed sequence tag (EST) database consisting of 58 146 ESTs was screened for microsatellite sequences. Subsequent development of 75 polymorphic EST‐associated microsatellite markers in this species is described together with cross‐species amplification results of 133 gene‐associated tandem repeat markers in five salmonid species (Salmo trutta, Oncorhynchus mykiss, Salvelinus aplinus, Thymallus thymallus, Coregonus lavaretus). The number of alleles among EST‐linked microsatellites in Atlantic salmon ranged from two to 41 with an average of 12 alleles per locus. Cross‐species amplification resulted in detection of a total of 111 polymorphic locus‐species combinations (12–32 loci per species).     

Stock Transfers in Spanish Brown Trout Populations: A Long-term Assessment

Synopsis Stocking of fish from other populations has been commonly employed for enhancement of wild brown trout, Salmo trutta, populations in north Spain. Young hatchery reared brown trout of central European origin were introduced into some Asturian rivers every year since 1984. Based on variation at the isozyme locus LDH-C1* and at the microsatellite locus BFRO 002, two genetic markers race-specific in Salmo trutta, we detected introgression of foreign genomes into native gene pools in some Spanish trout populations where only pure native individuals were present 10 years ago. We strongly suggest development of alternative management policies for conservation of Spanish natural brown trout populations without endangering the traditional recreational fisheries. Jorge I. Izquierdo, Ana G. F. Castillo: These two authors contributed equally to the article.     

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.     

Genetic differentiation and hybridization in two naturally occurring sympatric trout Salmo spp. forms from a small karstic lake

In this study, multiple molecular markers [genotyping of 12 nuclear microsatellite loci and the protein‐coding gene ldh‐c1* plus sequencing of the mitochondrial DNA (mtDNA) control region] were employed to investigate the genetic structure of the two trout forms, Salmo cettii and Salmo fibreni, inhabiting Lake Posta Fibreno, central Italy. The two forms were found to share a unique mtDNA haplotype, belonging to a widespread Mediterranean haplogroup (AD). Bayesian clustering analyses showed that these two forms correspond to well‐defined autochthonous gene pools. Genetic introgression between the two gene pools, however, was observed, whose frequency appears to correlate with the environmental features of the spawning sites. The interplay of selection for the spawning sites, philopatry and natural selection can be argued to maintain genetic differentiation despite the lack of complete reproductive isolation.     

Microsatellite DNA markers for the study of Atlantic salmon (Salmo salar) kinship,population structure,and mixed‐fishery analyses

Eleven microsatellite DNA loci were identified and characterized for Atlantic salmon (Salmo salar) collected from the Penobscot River, Maine, USA and the River Nith, Scotland, UK. The markers revealed high levels of genetic diversity (seven to 48 alleles per locus), heterozygosity (to 100%), and allelic heterogeneity (all comparisons). Considerable differentiation was observed as the genetic distance (chord) between the two collections was 0.680 and the pairwise F_ST, 0.12, was highly significant. These findings are consistent with patterns of continental‐level differentiation observed previously using an alternate suite of microsatellite loci. Locus‐by‐locus analyses of molecular variance suggested that most markers were suitable for delineating kinships and population genetic structure.     

Genetic assessment of Atlantic salmon Salmo salar and sea trout Salmo trutta stocking in a Baltic Sea river

Microsatellite DNA variation was used to assess the outcome of stocking Atlantic salmon Salmo salar and migratory trout Salmo trutta in River Sävarå, N Sweden. No information on pre‐stocking genetic composition of S. salar and S. trutta in River Sävarå was available. In 2 year‐classes of S. salar smolt, microsatellite data indicated that post‐stocking genetic composition differed markedly (F_ST= 0·048) from the main donor strain, Byskeälven S. salar, and from other Gulf of Bothnia S. salar stocks (F_ST 0·047 and 0·132). The STRUCTURE programme failed to detect any substructuring within Sävarå salmon. It was concluded that only minor introgression estimated to a proportion of 0·11 (95% CI 0·07–0·16) has occurred in S. salar. Salmo trutta showed overall low differentiation among populations with maximum F_ST of 0·03 making analysis more cumbersome than in S. salar. Still, the SävaråS. trutta deviated significantly from potential donor populations, and STRUCTURE software supported that majority of trout in Sävarå formed a distinct genetic population. Admixture was more extensive in S. trutta and estimated to 0·17 (95% CI 0·10–0·25).     

Dissecting the U,M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat

Goat grasses (Aegilops spp.) contributed to the evolution of bread wheat and are important sources of genes and alleles for modern wheat improvement. However, their use in alien introgression breeding is hindered by poor knowledge of their genome structure and a lack of molecular tools. The analysis of large and complex genomes may be simplified by dissecting them into single chromosomes via flow cytometric sorting. In some species this is not possible due to similarities in relative DNA content among chromosomes within a karyotype. This work describes the distribution of GAA and ACG microsatellite repeats on chromosomes of the U, M, S and C genomes of Aegilops, and the use of microsatellite probes to label the chromosomes in suspension by fluorescence in situ hybridization (FISHIS). Bivariate flow cytometric analysis of chromosome DAPI fluorescence and fluorescence of FITC‐labelled microsatellites made it possible to discriminate all chromosomes and sort them with negligible contamination by other chromosomes. DNA of purified chromosomes was used as a template for polymerase chain reation (PCR) using Conserved Orthologous Set (COS) markers with known positions on wheat A, B and D genomes. Wheat–Aegilops macrosyntenic comparisons using COS markers revealed significant rearrangements in the U and C genomes, while the M and S genomes exhibited structure similar to wheat. Purified chromosome fractions provided an attractive resource to investigate the structure and evolution of the Aegilops genomes, and the COS markers assigned to Aegilops chromosomes will facilitate alien gene introgression into wheat.     

Isolation and cross‐familial amplification of 41 microsatellites for the brook charr (Salvelinus fontinalis)

The brook charr (Salvelinus fontinalis; Osteichthyes: Salmonidae) is a phenotypically diverse fish species inhabiting much of North America. But relatively few genetic diagnostic resources are available for this fish species. We isolated 41 microsatellites from S. fontinalis polymorphic in one or more species of salmonid fish. Thirty‐seven were polymorphic in brook charr, 15 in the congener Arctic charr (Salvelinus alpinus) and 14 in the lake charr (Salvelinus namaycush). Polymorphism was also relatively high in Oncorhynchus, where 21 loci were polymorphic in rainbow trout (Oncorhynchus mykiss) and 16 in cutthroat trout (Oncorhynchus clarkii) but only seven and four microsatellite loci were polymorphic in the more distantly related lake whitefish (Coregonus clupeaformis) and Atlantic salmon (Salmo salar), respectively. One duplicated locus (Sfo228Lav) was polymorphic at both duplicates in S. fontinalis.     

Introgression in the genus Salmo via allotriploids

Hybridization between sympatric species is not uncommon in the wild. Wild allotriploids (individuals with two chromosome sets from a species + one chromosome set from another species) are generally the result of a backcross between interspecific hybrids that produce unreduced gametes and one of the parental species. In animals, allotriploids are commonly sterile, except for some vertebrate species complexes in which allotriploids reproduce by parthenogenesis, gynogenesis and/or hybridogenesis, producing generally clonal or hemiclonal gametes; nuclear DNA introgression between hybridizing species is considered to be extremely rare. Employing species-specific molecular markers, we show genetic introgression between the chromosomally well-differentiated salmonids Atlantic salmon (2n = 58) and brown trout (2n = 80) through spontaneous bisexual reproduction of allotriploids leading to salmon-like offspring bearing some brown trout genes. Although introgression between these Salmo species can occur via allotriploids, we hypothesize that extinction of parental species can be discarded based on very low survival of allotriploid offspring.     

Bidirectional mitochondrial introgression between Korean cobitid fish mediated by hybridogenetic hybrids

Genomic introgression through interspecific hybridization has been observed in some species of the freshwater fish family Cobitidae. Within this family, a Cobitis hankugensis–Iksookimia longicorpa diploid–triploid hybrid species complex on the Korean peninsula is unique in displaying hybridogenesis, a unisexual reproduction mode that allows hybrids to mediate the transfer of mitochondrial DNA (but not nuclear DNA) between the two parent species. However, populations of the parental species in the wild have never been examined for the potential effect of introgression on their genomes. To address the genetic consequences of unisexual hybridization on the parental species, we examined genetic structure of the two parental species, C. hankugensis and I. longicorpa, in three independent natural habitats where they coexist with their hybrid complex using DNA sequence data of one mitochondrial gene and three nuclear genes. We found that mitochondrial introgression between the two species was extensive in all the examined localities, while there was no evidence of nuclear introgression across the species boundary. This result indicates that the hybridogenetic individuals mediate mitochondrial introgression from one species to the other, producing mito‐nuclear mosaic genomes such as C. hankugensis nuclear genomes associated with I. longicorpa mitochondrial DNA and the reverse. The direction and degree of introgression varied among the three localities, but the underlying mechanisms for this observation proved elusive. Introgression might depend on which species serves as the predominant sperm or ovum donor or the environmental conditions of the localities. The present study suggests that introgressive hybridization between pure C. hankugensis and I. longicorpa species is highly likely where the two species co‐occur with hybridogenetic individuals, but the consequence of introgression could be variable due to the history and environmental characteristics of particular populations across the parental species’ ranges.     

Genome scan of hybridizing sunflowers from Texas (Helianthus annuus and H. debilis) reveals asymmetric patterns of introgression and small islands of genomic differentiation

Although the sexual transfer of genetic material between species (i.e. introgression) has been documented in many groups of plants and animals, genome‐wide patterns of introgression are poorly understood. Is most of the genome permeable to interspecific gene flow, or is introgression typically restricted to a handful of genomic regions? Here, we assess the genomic extent and direction of introgression between three sunflowers from the south‐central USA: the common sunflower, Helianthus annuus ssp. annuus; a near‐endemic to Texas, Helianthus debilis ssp. cucumerifolius; and their putative hybrid derivative, thought to have recently colonized Texas, H. annuus ssp. texanus. Analyses of variation at 88 genetically mapped microsatellite loci revealed that long‐term migration rates were high, genome‐wide and asymmetric, with higher migration rates from H. annuus texanus into the two parental taxa than vice versa. These results imply a longer history of intermittent contact between H. debilis and H. annuus than previously believed, and that H. annuus texanus may serve as a bridge for the transfer of alleles between its parental taxa. They also contradict recent theory suggesting that introgression should predominantly be in the direction of the colonizing species. As in previous studies of hybridizing sunflower species, regions of genetic differentiation appear small, whether estimated in terms of F_ST or unidirectional migration rates. Estimates of recent immigration and admixture were inconsistent, depending on the type of analysis. At the individual locus level, one marker showed striking asymmetry in migration rates, a pattern consistent with tight linkage to a Bateson–Dobzhansky–Muller incompatibility.     

Transatlantic secondary contact in Atlantic Salmon,comparing microsatellites,a single nucleotide polymorphism array and restriction‐site associated DNA sequencing for the resolution of complex spatial structure

Identification of discrete and unique assemblages of individuals or populations is central to the management of exploited species. Advances in population genomics provide new opportunities for re‐evaluating existing conservation units but comparisons among approaches remain rare. We compare the utility of RAD‐seq, a single nucleotide polymorphism (SNP) array and a microsatellite panel to resolve spatial structuring under a scenario of possible trans‐Atlantic secondary contact in a threatened Atlantic Salmon, Salmo salar, population in southern Newfoundland. Bayesian clustering indentified two large groups subdividing the existing conservation unit and multivariate analyses indicated significant similarity in spatial structuring among the three data sets. mtDNA alleles diagnostic for European ancestry displayed increased frequency in southeastern Newfoundland and were correlated with spatial structure in all marker types. Evidence consistent with introgression among these two groups was present in both SNP data sets but not the microsatellite data. Asymmetry in the degree of introgression was also apparent in SNP data sets with evidence of gene flow towards the east or European type. This work highlights the utility of RAD‐seq based approaches for the resolution of complex spatial patterns, resolves a region of trans‐Atlantic secondary contact in Atlantic Salmon in Newfoundland and demonstrates the utility of multiple marker comparisons in identifying dynamics of introgression.     

GENOME‐WIDE INVESTIGATION OF REPRODUCTIVE ISOLATION IN EXPERIMENTAL LINEAGES AND NATURAL SPECIES OF NEUROSPORA: IDENTIFYING CANDIDATE REGIONS BY MICROARRAY‐BASED GENOTYPING AND MAPPING

Inherent incompatibilities between genetic components from genomes of different species may cause intrinsic reproductive isolation. In evolution experiments designed to instigate speciation in laboratory populations of the filamentous fungus Neurospora, we previously discovered a pair of incompatibility loci (dfe and dma) that interact negatively to cause severe defects in sexual reproduction. Here we show that the dfe‐dma incompatibility also is a significant cause of genetic isolation between two naturally occurring species of Neurospora (N. crassa and N. intermedia). The strong incompatibility interaction has a simple genetic basis (two biallelic loci) and antagonistic epistasis occurs between heterospecific alleles only, consistent with the Dobzhansky–Muller model of genic incompatibility. We developed microarray‐based, restriction‐site associated DNA (RAD) markers that identified ～1500 polymorphisms between the genomes of the two species, and constructed the first interspecific physical map of Neurospora. With this new mapping resource, the approximate genomic locations of the incompatibility loci were determined using three different approaches: genome scanning, bulk‐segregant analyses, and introgression. These population, quantitative, and classical genetics methods concordantly identified two candidate regions, narrowing the search for each incompatibility locus to only ～2% of the nuclear genome. This study demonstrates how advances in high‐throughput, genome‐wide genotyping can be applied to mapping reproductive isolation genes and speciation research.     

Northern areas as refugia for temperate species under current climate warming: Atlantic salmon (Salmo salar L.) as a model in northern Europe

In this work, patterns of geographical genetic diversity in Atlantic salmon Salmo salar were studied across the whole Atlantic Arc; whether these patterns (and thus genetic population structure) were affected by water temperatures was also evaluated. Salmo salar populations were characterized using microsatellite loci and then analysed with reference to ocean surface temperature data from across the region. Analysis showed the presence of a latitudinal cline of genetic variability (higher in northern areas) and water temperatures (sea surface temperatures) determining genetic population structure (the latter in combination with genetic drift in southern populations). Under the current global change scenario, northern areas of Europe would constitute refugia for diversity in the future. This is effectively the inverse of what appears to have happened in glacial refugia during the last glacial maximum. From this perspective, the still abundant and large northern populations S. salar should be considered as precious as the small almost relict southern ones and given appropriate protection. Careful management of the species, coordinated across countries and latitudes, is needed in order to avoid its extinction in Europe.     

Hybridization between genetically modified Atlantic salmon and wild brown trout reveals novel ecological interactions

Interspecific hybridization is a route for transgenes from genetically modified (GM) animals to invade wild populations, yet the ecological effects and potential risks that may emerge from such hybridization are unknown. Through experimental crosses, we demonstrate transmission of a growth hormone transgene via hybridization between a candidate for commercial aquaculture production, GM Atlantic salmon (Salmo salar) and closely related wild brown trout (Salmo trutta). Transgenic hybrids were viable and grew more rapidly than transgenic salmon and other non-transgenic crosses in hatchery-like conditions. In stream mesocosms designed to more closely emulate natural conditions, transgenic hybrids appeared to express competitive dominance and suppressed the growth of transgenic and non-transgenic (wild-type) salmon by 82 and 54 per cent, respectively. To the best of our knowledge, this is the first demonstration of environmental impacts of hybridization between a GM animal and a closely related species. These results provide empirical evidence of the first steps towards introgression of foreign transgenes into the genomes of new species and contribute to the growing evidence that transgenic animals have complex and context-specific interactions with wild populations. We suggest that interspecific hybridization be explicitly considered when assessing the environmental consequences should transgenic animals escape to nature.     

Testing hypotheses for genealogical discordance in a rainforest lizard

Genealogical discordance, or when different genes tell distinct stories although they evolved under a shared history, often emerges from either coalescent stochasticity or introgression. In this study, we present a strong case of mito‐nuclear genealogical discordance in the Australian rainforest lizard species complex of Saproscincus basiliscus and S. lewisi. One of the lineages that comprises this complex, the Southern S. basiliscus lineage, is deeply divergent at the mitochondrial genome but shows markedly less divergence at the nuclear genome. By placing our results in a comparative context and reconstructing the lineages' demography via multilocus and coalescent‐based approximate Bayesian computation methods, we test hypotheses for how coalescent variance and introgression contribute to this pattern. These analyses suggest that the observed genealogical discordance likely results from introgression. Further, to generate such strong discordance, introgression probably acted in concert with other factors promoting asymmetric gene flow between the mitochondrial and nuclear genomes, such as selection or sex‐biased dispersal. This study offers a framework for testing sources of genealogical discordance and suggests that historical introgression can be an important force shaping the genetic diversity of species and their populations.     

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.     

Diversification of mitogenomes in three sympatric Altica flea beetles (Insecta,Chrysomelidae)

The Asian flea beetles Altica cirsicola, Altica fragariae and Altica viridicyanea are broadly sympatric and morphologically highly similar but feed on distantly related host plants. They have been suggested as a model for ecological speciation studies. However, their phylogeny and species limits remain uncertain. In this study, we added mitochondrial genomes from multiple individuals of each species to the growing database. Phylogenetic analyses based on 15 genes showed clear interspecific divergences of A. fragariae from the other species, but A. cirsicola and A. viridicyanea were not distinguishable by distance‐based or tree‐based methods of species delimitation due to non‐monophyly of mitogenomes relative to the morphologically defined entities, possibly affected by interspecific introgression. This was confirmed by wider sampling of mitochondrial COX1 (58 individuals) and the second internal transcribed spacer of nuclear ribosomal RNA cluster (ITS2; 68 individuals), which showed that ITS2, but not COX1, coincided with the morphological species limits. The full mitochondrial genomes are not able to shed further light on the species status, even with the most sensitive approach based on diagnostic characters, yet the whole mitogenome is useful to get improved estimates of intra‐ and interspecific variation, not affected by the stochastic error seen in individual genes.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.