Genetic analysis of fish genomes and populations: allozyme variation within and among Atlantic salmon from Downeast rivers of Maine

Analysis of genetic variation within and among samples of naturally produced Atlantic salmon ( n  = 372) from 7 Maine (U.S.A.) and one Canadian river were conducted based on 54 allozyme loci. Eight of the 54 loci proved polymorphic, and estimated heterozygosities ( H _S) based on all loci ranged from 0·012 to 0·026 (mean = 0·021, s . e . = 0·002). Only one of 56 tests revealed genotypic proportions that deviated significantly from Hardy–Weinberg expectations. Genetic distances ( D ) between samples ranged from 0·002 to 0·022. No obvious association existed between genetic and geographic distances. Cluster analysis of genetic distances revealed the Dennys River sample as the most differentiated when all samples were included in the analysis, though bootstrap support of the cluster analysis was generally weak. G ‐tests revealed significant differences in allele frequencies among samples at five of the polymorphic loci, and the G ‐value summed over all loci also indicated significant differences among samples. F _ST values indicated that 3·4% of the total genetic diversity was due to variability among samples, while 96·6% was due to variability within samples. These results indicate that the Atlantic salmon analyzed in this study had levels of genetic variability and differentiation among samples comparable to native populations from other areas collected across a similar geographic range.     

Allozyme Diversity in Populations of Cymbidium goeringii (Orchidaceae)

Abstract: Using 14 allozyme loci, we investigated levels of genetic diversity within populations, and degree of genetic divergence among 24 populations of Cymbidium goeringii (Orchidaceae) in Korea and Japan. Cymbidium goeringii maintains high levels of genetic diversity both at population (mean expected heterozygosity, H _e = 0.238) and species levels (0.260). Means of H _e found in 24 populations were not significantly different from each other. About 90 % of the total variation in the species is common to all populations (mean G _ST = 0.108). No unique allele was found in any population. The indirect estimate of gene flow based on the mean G _ST was high ( Nm = 2.06). Nei's genetic identities for pairs of populations had high values (mean = 0.974 [SD = 0.013]). The Mantel-Z test showed a significant correlation between genetic distance and geographic distance. However, the mean G _ST value between 17 populations in Korea and seven Japanese populations was relatively low (0.029), even though the land connection between the southern Korean peninsula and southern Japanese archipelagos has not existed since the middle Pleistocene. Large numbers of small seeds of C. goeringii might travel long distances by wind from populations to populations both in Korea and Japan, increasing genetic diversity within populations and maintaining low genetic differentiation among populations.     

Marked genetic structuring and extreme dispersal limitation in the Pyrenean brook newt Calotriton asper (Amphibia: Salamandridae) revealed by genome-wide AFLP but not mtDNA

Direct estimation of dispersal rates at large geographic scales can be technically and logistically challenging, especially in small animals of low vagility like amphibians. The use of molecular markers to reveal patterns of genetic structure provides an indirect way to infer dispersal rates and patterns of recent and historical gene flow among populations. Here, we use mitochondrial DNA (mtDNA) sequence data and genome-wide amplified fragment length polymorphism markers to examine population structure in the Pyrenean brook newt ( Calotriton asper ) across four main drainages in the French Pyrenees. mtDNA sequence data (2040 bp) revealed three phylogroups shallowly differentiated and with low genetic diversity. In sharp contrast, variation in 382 amplified fragment length polymorphism loci was high and revealed a clear pattern of isolation by distance consistent with long-term restriction of gene flow at three spatial scales: (i) among all four main drainages, (ii) between sites within drainages, and (iii) even between adjacent populations separated by less than 4 km. The high pairwise F _ST values between localities across numerous loci, together with the high frequency of fixed alleles in several populations, suggests a combination of marked geographic isolation, small population sizes and very limited dispersal in C. asper . The contrasting lack of variation detected in mtDNA sequence data is intriguing and underscores the importance of multilocus approaches to detect true patterns of gene flow in natural populations of amphibians.     

Genetic and morphological variation in a common European cyprinid, Leuciscus cephalus within and across Central European drainages

Genetic variability and differentiation of the common European cyprinid Leusciscus cephalus was investigated within and across the drainages of the rivers Rhine, Elbe and Danube using starch gel electrophoresis and morphological characters. Eleven of 24 presumptive enzyme loci showed some allozymic variation. Compared to other freshwater fish, genetic variability within all sites was high ( H _e=0·074–0·113). This was explained by the wide geographical and ecological range of the chub. In contrast, genetic and morphological differentiation between sites and drainages was low (F_ST=0·15), which may be due to the high dispersal ability of the chub and translocations of spawn by waterfowl. The phylogeographic structure of Leuciscus cephalus in north-eastern Bavaria showed low congruence with Cottus gobio populations sampled in the same area.     

Allozyme Variation and the Genetic Structure of Populations of the Rare Sedge Carex misera (Cyperaceae)

Abstract Carex misera is a rare sedge, endemic to rocky outcrops and mountain summits within the southern Appalachian Mountains from northern Georgia to northern North Carolina. We assessed allozyme diversity for 406 individuals from nine populations over most of the geographic range. Twenty-seven putative loci were assayed and eight (30%) were found to be polymorphic. Nei's gene diversity statistics ( H _T =0.043, H _S =0.019, G _ST =0.551) indicated low levels of variation but relatively highly differentiated populations, suggesting little gene flow. Significant deviations from genotypic expectations under Hardy-Weinberg equilibrium, high positive fixation indices, and the existence of small genetic neighborhoods within populations suggest that at least some inbreeding occurs. Cluster analysis of Nei's genetic identity statistics and principal component analysis of allele frequency data showed high similarity among the six southern populations with the two northern populations more differentiated from them and from each other. These results suggest that preservation of the northern populations is necessary to conserve the already low levels of genetic diversity within the species.     

Subtle population structuring within a highly vagile marine invertebrate, the veined squid Loligo forbesi, demonstrated with microsatellite DNA markers

Microsatellite DNA markers were applied for the first time in a population genetic study of a cephalopod and compared with previous estimates of genetic differentiation obtained using allozyme and mitochondrial DNA (mtDNA) markers. Levels of genetic variation detected with microsatellites were much higher than found with previous markers (mean number of alleles per locus=10.6, mean expected heterozygosity ( H _E)=0.79; allozyme H _E=0.08; mtDNA restriction fragment length polymorphism (RFLP) H _E=0.16). In agreement with previous studies, microsatellites demonstrated genetic uniformity across the population occupying the European shelf seas of the North East Atlantic, and extreme genetic differentiation of the Azores population ( R _ST/ F _ST=0.252/0.245; allozyme F _ST=0.536; mtDNA F _ST=0.789). In contrast to other markers, microsatellites detected more subtle, and significant, levels of differentiation between the populations of the North East Atlantic offshore banks (Rockall and Faroes) and the shelf population ( R _ST=0.048 and 0.057). Breakdown of extensive gene flow among these populations is indicated, with hydrographic (water depth) and hydrodynamic (isolating current regimes) factors suggested as possible barriers to migration. The demonstration of genetic subdivision in an abundant, highly mobile marine invertebrate has implications for the interpretation of dispersal and population dynamics, and consequent management, of such a commercially exploited species. Relative levels of differentiation indicated by the three different marker systems, and the use of measures of differentiation (assuming different mutation models), are discussed.     

Widespread gene flow and high genetic variability in populations of water voles Arvicola terrestris in patchy habitats

Theory predicts that the impact of gene flow on the genetic structure of populations in patchy habitats depends on its scale and the demographic attributes of demes (e.g. local colony sizes and timing of reproduction), but empirical evidence is scarce. We inferred the impact of gene flow on genetic structure among populations of water voles Arvicola terrestris that differed in average colony sizes, population turnover and degree of patchiness. Colonies typically consisted of few reproducing adults and several juveniles. Twelve polymorphic microsatellite DNA loci were examined. Levels of individual genetic variability in all areas were high ( H _O= 0.69–0.78). Assignments of juveniles to parents revealed frequent dispersal over long distances. The populations showed negative F _IS values among juveniles, F _IS values around zero among adults, high F _ST values among colonies for juveniles, and moderate, often insignificant, F _ST values for parents. We inferred that excess heterozygosity within colonies reflected the few individuals dispersing from a large area to form discrete breeding colonies. Thus pre-breeding dispersal followed by rapid reproduction results in a seasonal increase in differentiation due to local family groups. Genetic variation was as high in low-density populations in patchy habitats as in populations in continuous habitats used for comparison. In contrast to most theoretical predictions, we found that populations living in patchy habitats can maintain high levels of genetic variability when only a few adults contribute to breeding in each colony, when the variance of reproductive success among colonies is likely to be low, and when dispersal between colonies exceeds nearest-neighbour distances.     

Factors affecting the distribution of genetic variability in the guppy, Poecilia reticulata

Previous studies on Trinidadian guppies have shown an apparent association of genetic variability, expressed as mean heterozygosity (Ho) at allozyme loci, with river order: fish from lowland sites exhibit higher mean Ho than their upland counterparts. Detailed genetic and behavioural observations were undertaken in the present study by repeated sampling throughout two river courses to examine population heterogeneity. Results show that the predation regime mediated mating behaviour of upland and lowland populations in the wild is not as divergent as previous laboratory observations have suggested. Furthermore, genetic variation at the eight loci variable in Trinidadian populations (p₁₀₀=0·13–O·88, H₀=O·03–0·23) appears to bear little association with predation regime, but is related to sub-structuring of populations by habitat features and geographic isolation. We therefore conclude that the observed patterns of genetic variability arise primarily as a consequence of stochastic as opposed to deterministic factors.     

Genetic variation at allozyme and RAPD loci in sessile oak Quercus petraea (Matt.) Liebl.: the role of history and geography

The nuclear genetic variation within and among 21 populations of sessile oak was estimated at 31 RAPD loci in conjunction with previous estimates of variation at eight allozyme loci. The aim of the study was to assess the relative role of isolation-by-distance and postglacial history on patterns of nuclear variation. Because of its small effective population size and maternal transmission, the chloroplast genome is a good marker of population history. Both kinds of nuclear variation (RAPD and allozyme) were therefore compared, first, to the geographical distances among populations and, secondly, to chloroplast DNA restriction polymorphism in the same populations. Multiple Mantel tests were used for this purpose. Although RAPDs revealed less genetic diversity than allozymes, levels of genetic differentiation ( G _ST) were identical. The standard genetic distance calculated at all RAPD loci was correlated with geographical distances but not with the genetic distance calculated from chloroplast DNA data. Conversely, allozyme variation was correlated with chloroplast DNA variation, but not with geography. Possibly, divergent selection at two allozyme loci during the glacial period could explain this pattern. Because of its greater number of loci assayed, RAPDs probably provided a less biased picture of the relative role of geography and history.     

Mitochondrial DNA phylogeography of Glyptothorax fokiensis and Glyptothorax hainanensis in Asia

Whole mitochondrial DNA cytochrome b sequences in 62 fish from 13 locations in Southeast China identified two major clades corresponding to two allopatric taxa, Glyptothorax fokiensis fokiensis and Glyptothorax fokiensis hainanensis . Reciprocal monophyly and a molecular clock separation between these two taxa of 2·3 million years indicate these taxa should be elevated to species. Mismatch distributions and Fu's F _S statistic suggest that both G. fokiensis and G. hainanensis have experienced recent population expansions. Analysis of molecular variance indicates that most of the genetic variation resides among populations within both species, with Φ _ST= 0·645 for G. fokiensis and 0·801 for G. hainanensis , suggesting restricted gene flow among populations. Significant correlations between the geographic and the genetic distances provide support for the importance of geographic isolations between populations. Nested clade analysis also confirms low levels of genetic exchanges between the two major groups and between populations within each group. The phylogeographical pattern among populations of Glyptothorax in East Asia can be attributed to historical fragmentations, demographic expansions and occasional long-distance dispersals stimulated by tectonic activity and Ice Age climate changes.     

Reconciling patterns of genetic variation with stream structure, earth history and biology in the Australian freshwater fish Craterocephalus stercusmuscarum (Atherinidae)

We examined the consequences of barriers, stream architecture and putative dispersal capability on levels of genetic differentiation among populations of the freshwater fish Craterocephalus stercusmuscarum. Seven polymorphic allozyme loci and sequences of a 498-bp fragment of the ATPase 6 mitochondrial DNA (mtDNA) gene were used to assess patterns of genetic variation among 16 populations from upland and lowland streams of five drainages in northern Queensland, Australia. Concordant patterns at both genetic markers revealed that there were significant levels of genetic subdivision among all populations, while an analysis of molecular variation showed that the distribution of genetic diversity was not consistent with contemporary drainage structure. There were reciprocally monophyletic mtDNA clades and fixed or large frequency differences at allozyme loci either side of instream barriers such as waterfalls. This implied barriers were effective in restricting gene flow between upland and lowland populations separated by waterfalls. However, there were two genetically distinct groups in upland areas, even within the same subcatchment, as well as high levels of genetic subdivision among lowland populations, suggesting barriers alone do not explain the patterns of genetic diversity. The data revealed a complex phylogeographic pattern, which we interpreted to be the result of one or more invasion events of independent lineages to different sections of each drainage, possibly mediated by well documented geomorphological changes. Our results highlight the importance of earth structure and history in shaping population genetic structure in stream organisms where dispersal capability may be limited, and reveal that the contemporary structure of drainages is not necessarily a good indicator of genetic relationships among populations.     

Genetic relationships among populations of wild resident, and wild and hatchery anadromous brook charr

To determine the genetic relationship of anadromous and resident life-history types within and among drainages, and compare several hatchery strains to their progenitor populations, brook charr Salvelinus fontinalis were examined for allozyme and mitochondrial DNA variation. Greater genetic similarity of sympatric anadromous and resident charr was found compared to similar life-history forms allopatrically, suggesting the two life-history types are not reproductively isolated. Low divergence among the mtDNA haplotypes suggests that the two life-history types are members of the same evolutionary lineage. Population differentiation from mtDNA data exceeded that from estimates based on allozymes. Genetic deviations from expectations suggest that the hatchery strains were derived from few individuals.     

Genetic differentiation in endemic Lobularia (Brassicaceae) in the Canary Islands

Patterns of genetic diversity were examined in five endemic subspecies of the Lobularia canariensis complex from six of the Canary Islands. The taxa are interfertile, insect-pollinated outbreeders with wind dispersal. Electrophoretic analysis revealed a high level of genetic polymorphism at ten loci coding soluble enzymes, with a mean of 2.38 alleles per locus, 73.7 % polymorphic loci, and a mean heterozygosity of 0.279. Excesses of homozygotes, indicating inbreeding, were observed in small populations. The average total diversity was high, F.,=0.518. Among-population diversity, F_ST=0.318, contributed more to the total diversity than within-population diversity, F_IS=0.222. Little geographic or taxonomic patterning of the allozyme variation was observed. The mean genetic identitity for pairwise comparisons of the 19 populations was 0.76, with a range of 0.51–0.96 and c. 17 % of the comparisons below 0.67 and c. 8 % above 0.90. The results contrast with the many cases of high genetic identities reported for populations of endemic plants on oceanic islands. High levels of allozyme divergence suggest a relatively old origin of the L. canariensis complex and a long period of isolation of some of the populations.     

Allozyme, mitochondrial-DNA, and morphometrie variability indicate cryptic species of anchovy (Engraulis encrasicolus)

Previous surveys of population structure in the Atlantic-Mediterranean anchovy Engraulis encrasicolus L. have reported heterogeneity in morphology, allozyme frequencies, and mitochondrial DNA haplotype frequencies at a regional scale. In particular, two stocks of anchovy have been detected in the Adriatic Sea. In this paper, the available data is reviewed with the aim to relate genetic variation to geography at the widest possible geographical scale, for investigating the evolutionary mechanisms underlying stock structure in anchovy. Correspondence analysis of allozyme frequencies (24 samples, three polymorphic loci) compiled from the literature indicates three distinct entities in the Mediterranean Sea. Open-sea or oceanic anchovy populations are genetically different from inshore-water populations within a region (Nei's ^ G _ST = 0.035–0.067), while broadscale geographical variation is weak for each of these two habitat-specific forms (^ G _ST = 0.005–0.006). Mitochondrial-DNA haplotype frequencies support the distinction between an inshore form and an oceanic form (^ G _ST = 0.067–0.107), with virtually no genetic differences among oceanic populations across the Gulf of Biscay, the western Mediterranean and the Ionian Sea (^ G _ST = −0.001). If natural selection on marker loci is unimportant, these results indicate the occurrence of two parapatric, genetically distinct, habitat-specific forms that are widely distributed throughout the Mediterranean Sea. Persistent allele and haplotype-frequency differences between these forms indicate reproductive isolation and the presence of an E. encrasicolus species complex in the Mediterranean. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society 75 : 261–269.     

Phylogeography and population history of the endangered golden sun moth (Synemon plana) revealed by allozymes and mitochondrial DNA analysis

A combination of allozyme and mitochondrial DNA markers were used to determine the contribution of recent and ancient causes of patterns of genetic variation within and among 46 populations of the endangered golden sun moth, Synemon plana. Allozyme analysis grouped the 46 populations into 5 major genetic clusters that corresponded closely with geographic location following a classic isolation-by-distance model. Phylogenetic analysis of 14 mtDNA haplotypes revealed two reciprocally monophyletic groups. One of these groups (containing 4 geographically distant populations) was clearly identified by allozyme analysis and represents a distinct evolutionary unit. The remaining 4 allozyme groups were not distinguishable by mtDNA analysis. The evidence suggests that the populations within these groups derived from a small founding population that underwent rapid demographic expansion in ancient times. This was followed by more recent population bottlenecks resulting from habitat fragmentation associated with the widespread introduction of agriculture into the region. The generally low levels of allozyme and nucleotide diversity within these populations support this hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Patterns of molecular genetic variation in Plantago major and P. intermedia in relation to ozone resistance

Patterns of molecular genetic variation were examined in seed collections of Plantago major and Plantago intermedia , used previously to investigate the variations in ozone (O₃) resistance of these species across Europe. Total genomic DNA was amplified with random primers (random amplied polymorphic DNA (RAPD) and inter- single sequence repeats (SSR)) to produce 73 genetic markers. In addition, allozyme and chloroplast variations were surveyed. Genetic markers were examined for association with O₃ resistance in 18 British populations of P. major as well as 27 continental European populations of P. major and P. intermedia . Two populations that exhibited increased resistance to O₃ following several years' exposure to high O₃ concentrations in the field showed decreased genetic variation over time. In addition, their genetic composition showed no drastic change, which suggests that the change in resistance to O₃ was probably the result of selection on genotypes already present in local populations (selection in situ ). It appears that selection for O₃ resistance may occur in independent populations, and also may involve a number of genetically determined traits. Consequently the finding that plants with similar degrees of O₃ resistance are not closely related was not unexpected. However, the finding of an association of several genetic markers with O₃ resistance merits further investigation.     

MITOCHONDRIAL DNA SEQUENCE VARIATION AMONG NATURAL POPULATIONS OF THE TRINIDAD GUPPY,POECILIA RETICULATA

Guppies were sampled from eight populations representing four river drainage basins in northern Trinidad, and from one population on the nearby island of Tobago. For each individual, a 465 base pair (bp) segment of the control region of the mitochondrial genome was sequenced. The resulting DNA sequences were subjected to sequence divergence calculations and the populations were linked by maximum parsimony analysis to determine their phylogenetic relationships. Mitochondrial DNA (mtDNA) sequence variation was found both within and between river drainages, correlated with the geographic features of northern Trinidad. The variation observed exists primarily between drainages, particularly between the Oropuche drainage and all other Trinidad drainages examined. Estimates of time of divergence between guppy populations of different drainages, based on mtDNA sequence variation, ranged from 100,000 to 200,000 for the most recently separated populations and from 600,000 to 1.2 million years between the Oropuche populations and all others examined. Examination of fish from northeastern South America will be required to determine whether these populations differentiated in their present locations or were the result of separate invasions of Trinidad from different Venezuelan sources. However, genetic isolation of these populations appears to predate the current physical separation of the island of Trinidad from the Venezuelan mainland.     

Population Genetic Structuring in <Emphasis Type="Italic">Acanthopagrus butcheri</Emphasis> (Pisces: Sparidae): Does Low Gene Flow Among Estuaries Apply to Both Sexes?

Acanthopagrus butcheri completes its entire life history within estuaries and coastal lakes of southern Australia, although adults occasionally move between estuaries via the sea. Consequently, it is expected that populations of A. butcheri in different estuaries will be genetically distinct, with the magnitude of genetic divergence increasing with geographic isolation. However, previous genetic studies of A. butcheri from southeast Australia yielded conflicting results; allozyme variation exhibited minimal spatial structuring (θ = 0.012), whereas mitochondrial DNA distinguished the majority of populations analyzed (θ = 0.263) and genetic divergence was positively correlated with geographic isolation. This discrepancy could reflect high male gene flow, which impacts nuclear but not mitochondrial markers. Here we estimated allele frequencies at five nuclear microsatellite loci across 11 southeast Australian populations (595 individuals). Overall structuring of microsatellite variation was weaker (θ = 0.088) than that observed for mitochondrial DNA, but was able to distinguish a greater number of populations and was positively correlated with geographic distance. Therefore, we reject high male gene flow and invoke a stepping-stone model of infrequent gene flow among estuaries for both sexes. Likewise, management of A. butcheri within the study range should be conducted at the scale of individual or geographically proximate estuaries for both sexes. The lack of allozyme structuring in southeast Australia reflects either the large variance in structuring expected among loci under neutral conditions and the low number of allozymes surveyed or a recent colonization of estuaries such that some but not all nuclear loci have approached migration-drift equilibrium.     

Phylogeography of the plains killifish, Fundulus zebrinus

Drainage systems of the Great Plains and western Gulf Slope underwent substantial changes through diversions and stream captures during the Pleistocene, either as the result of the glacial advances or through independent geologic processes. The distributions of a variety of fishes that range across west-central North America, such as the plains killifish (Fundulus zebrinus), are thought to be the product of this Pleistocene influence. We examined the geographic pattern of genetic variation in F. zebrinus using three allozyme loci (n = 793), mitochondrial DNA restriction fragment length polymorphisms (RFLPs, n = 352), and sequencing of the mitochondrial cytochrome oxidase I (COI, n = 23) in an attempt to understand the roles of dispersal and vicariance. The phylogeographic patterns were concordant between the allozyme and mitochondrial data with the exception of the population in the North Canadian River. The populations fell into three geographic assemblages, which we designated as northern, central, and southern. A large phylogenetic break (average Roger's D = 0.702; average sequence divergence in RFLPs = 4.6%; average sequence divergence in COI = 5.5%) separated the northern/central and southern assemblages. The northern region was likely colonized sometime during the mid-Pleistocene. Fish in the Brazos and Pecos Rivers probably reached these drainages through stream captures of the Red River. The large phylogenetic break between the northern/central and southern clades supports previous attempts to recognize two species of plains killifish: F. zebrinus and F. kansae.     

SIGNIFICANT ROLE FOR HISTORICAL EFFECTS IN THE EVOLUTION OF REPRODUCTIVE ISOLATION: EVIDENCE FROM PATTERNS OF INTROGRESSION BETWEEN THE CYPRINID FISHES,LUXILUS CORNUTUS AND LUXILUS CHRYSOCEPHALUS

Samples of Luxilus cornutus, Luxilus chrysocephalus, and their hybrids were collected along hypothesized routes of dispersal from Pleistocene refugia to examine the significance of geographic variation in patterns of introgression between these species. Patterns of allozyme and mitochondrial DNA (mtDNA) variation were generally consistent with those from previous studies. Tests of Hardy-Weinberg equilibrium revealed significant deficiencies of heterozygotes in all samples, indicating some form of reproductive isolation. Mitochondrial DNAs of each species were not equally represented in F₁ hybrids; however, this bias was eliminated when the two largest samples were excluded from the analysis. Backcross hybrids exhibited biased mtDNA introgression, as samples from Lake Erie (eastern) and Lake Michigan (western) drainages showed significant excesses of mtDNAs from L. chrysocephalus and L. cornutus, respectively, relative to frequencies of diagnostic allozyme markers. The extent and direction of allozyme and mtDNA introgression was quantified by calculating isolation index values from morphologically “pure” individuals of each species from each locality. Analysis of variance of these measures identified limited introgression of allozyme variants with no geographic pattern, but significant differences in direction of mtDNA introgression between drainages (i.e., postglacial dispersal route). Association between patterns of mtDNA introgression and dispersal route across the latitudinal width of the contact zone is best explained by genetic divergence during past isolation of ancestral populations from these drainages. These results identify a significant role for historical effects in the evolution of reproductive isolation and the process of speciation.