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.     

DNA fingerprinting data and the problem of non-independence among pairwise comparisons

Multilocus DNA fingerprinting is commonly used to assess genetic similarity within and between geographically disjunct populations. Typically, the proportion of DNA fingerprinting bands shared between two individuals ( S _XY) is calculated for all possible pairwise comparisons and the resulting data analyzed parametrically to test differences in mean band-sharing among groups. The degree to which covariation among interdependent S _XY values ( S _ab - S _bc) biases the analyses is often unknown. Here, we assess the extent of covariation in four DNA fingerprinting studies and evaluate the effectiveness of two corrective procedures, a permutation test and a subsampling routine using only independent pairwise comparisons drawn without replacement from the overall data. Covariation among interdependent S _XY values was significantly greater than zero in every data set examined, including those from a bee, a rodent, and two passerine birds. Permutation tests did not correct for interdependence and yielded significance values nearly identical to those derived from uncorrected parametric procedures. In contrast, the subsampling procedure yielded corrected estimates of the standard error that were two to four times larger than those derived parametrically. As a result, comparisons that were significant using parametric tests were either non-significant or only marginally significant with the subsampling routine. We conclude that interdependence among S _XY values poses a substantial obstacle to hypothesis testing that must be addressed in future studies.     

Temporal effective size estimates of a managed walleye Sander vitreus population and implications for genetic-based management

The goal of this research was to use the long-term fishery data set and DNA from archived scales of walleye Sander vitreus in Escanaba Lake, WI, U.S.A., to improve the understanding of the underlying mechanism(s) influencing genetic diversity in naturally recruiting populations. The introduced population of S. vitreus in Escanaba Lake has a low mean effective population size ( N _E) between 124·6 and 185·5 despite a mean census size ( N _C) of 4659 ( N _E/ N _C c. 0·04), suggesting an accelerated rate of genetic drift between 1952 and 2002. These values are smaller than the median N _E range of several studies suggesting typical N _E/ N _C ratios of 0·11–0·16 in a wide range of taxa. N _E increased steadily during the past two sampled decades (1992 and 2002) and was consistent with a lowering of the variance in S. vitreus reproductive success, possibly linked to a large, sustained exploitation (mean 28%) rate. Variance in reproductive success is one of the most important factors influencing N _E in species, like S. vitreus , which have a potential for large fecundities and large juvenile mortalities (type III survivorship). The N _B estimates across six sequential cohorts (age classes of S. vitreus , assayed from 1994 to 1999) was consistent with estimates of N _E reported for 1992–2002. These results, coupled with in-depth census and exploitation data, show that the genetic characteristics of Escanaba Lake S. vitreus have changed substantially and that management activities, such as supplemental stocking and harvest practices, have profoundly influenced the genetic dynamics of S. vitreus in this lake.     

Low genetic variability of the koala Phascolarctos cinereus in south-eastern Australia following a severe population bottleneck

Genotyping of koalas at CA-repeat microsatellite loci has revealed significant differences in the levels of allelic diversity ( A ) and expected heterozygosity ( H¯ _E) between populations from north-eastern and south-eastern Australia. In the 10 populations studied, allelic diversity ranged from 8.0 in the Nowendoc population to 1.7 in the Kangaroo Is. population, and values of H¯ _E ranged from 0.831 in the Nowendoc population to 0.331 in the Kangaroo Is. population. Data from pooled populations revealed koalas from the north-eastern region had significantly higher levels of allelic diversity ( A = 11.5 ± 1.4) than those from south-eastern Australia ( A = 5.3 ± 1.0). Furthermore significantly higher heterozygosity levels were found in the north-eastern ( H¯ _E= 0.851) vs. the south-eastern ( H¯ _E= 0.436) regions of Australia. Following a near-extinction bottleneck in the 1920s, mainland Victorian and Kangaroo Is. koalas have been involved in an extensive program of relocations. The source populations of the relocated animals were islands in Westernport Bay, which were founded by very few individuals in the late 1800s and early 1900s. The significantly lower levels of variation between south-eastern Australian populations suggests that human intervention has had a severe effect on levels of genetic diversity in this region, and this may have long-term genetic consequences.     

Population divergence in the amphicarpic species Amphicarpaea edgeworthii Benth. (Fabaceae): microsatellite markers and leaf morphology

Comparative analyses of the genetic differentiation in microsatellite markers ( F _ST) and leaf morphology characters ( Q _ST) of Amphicarpaea edgeworthii Benth. were conducted to gain insight into the roles of random processes and natural selection in the population divergence. Simple sequence repeat analyses on 498 individuals of 19 natural populations demonstrate that a significant genetic differentiation occurs among populations (mean F _ST = 0.578), and A. edgeworthii is a highly self-fertilized species (mean selfing rate s  = 0.989). The distribution pattern of genetic diversity in this species shows that central populations possess high genetic diversity (e.g. population WL with H _E = 0.673 and population JG with H _E = 0.663), whereas peripheral ones have a low H _E as in population JD (0.011). The morphological divergence of leaf shape was estimated by the elliptical Fourier analysis on the data from 11 natural and four common garden populations. Leaf morphology analyses indicate the morphological divergence does not show strong correlation with the genetic differentiation ( R  = 0.260, P  = 0.069). By comparing the 95% confidence interval of Q _ST with that of F _ST, Q _ST values for five out of 12 quantitative traits are significantly higher than the average F _ST value over eight microsatellite loci. The comparison of F _ST and Q _ST suggests that two kinds of traits can be driven by different evolutionary forces, and the population divergence in leaf morphology is shaped by local selections.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 505–516.     

Effects of brackish water on growth, feed conversion and energy absorption efficiency by juvenile euryhaline and freshwater stenohaline fishes

Among six species of juvenile fishes (<6 months old), stenohaline species (channel catfish Ictalurus punctatus and goldfish Carassius auratus ) had their highest specific growth rate ( G ) and most efficient food conversion ratio ( E _C) and energy absorption efficiency ( I _E) in fresh water. Three of the euryhaline species (rainbow trout Oncorhynchus mykiss , striped bass Morone saxatilis and Gulf sturgeon Acipenser oxyrinchus desotoi ) had higher G and had more efficient E _C and I _E in 3 and 9‰ salinities than in lower salinities (fresh water and 1‰). For brown trout Salmo trutta (age 3–4 months), 9‰ was above the optimum level for G and E C. However, I _E for brown trout was not significantly different at 3 and 9‰ salinities. Over the salinity range tested, channel catfish had the largest change in G , E _C and I _E, while changes for euryhaline species were relatively small. Although all species tested survived and grew in all treatments, salinities as low as 1‰ adversely affected the stenohaline species, and 9‰ adversely affected brown trout.     

Genetic diversity and migration patterns of the aquatic macrophyte Potamogeton malaianus in a potamo-lacustrine system

1. Previously, the Yangtze River connected thousands of shallow lakes which together formed a potamo-lacustrine system capable of sustaining a rich variety of submerged macrophytes.
2.  Potamogeton malaianus is one of the dominant submerged macrophytes in many lakes of this area. Genetic variation and population structure of P. malaianus populations from ten lakes in the potamo-lacustrine system were assessed using inter-simple sequence repeat markers.
3. Twelve primer combinations produced a total of 166 unambiguous bands of which 117 (70.5%) were polymorphic. Potamogeton malaianus exhibited a moderate level of population genetic diversity ( P _P = 70.5%, H _E = 0.163 and I =  0.255), as compared with that of plants in the same habitat and range. The main factors responsible for this moderate value were the plant's mixed breeding system (both sexual and asexual) and the hydrological connectivity among habitats.
4.  F statistics, calculated using different approaches, consistently revealed a moderate genetic differentiation among populations, contributing about 20% of total genetic diversity. An estimate of gene flow (using F _ST) suggested that gene flow played a more important role than genetic drift in the current population genetic structure of P. malaianus ( Nm  = 1.131).
5. The genetic diversity of P. malaianus did not increase downstream. A high level of linkage–disequilibrium at the whole population level suggested that metapopulation processes may affect genetic structure. The migration pattern of P. malaianus was best explained by a two-dimensional stepping stone model, indicating that bird-mediated dispersal could greatly influence gene movements among lakes.     

Comparison of different methods to construct a core germplasm collection in woody perennial species with simple sequence repeat markers. A case study in cherimoya (Annona cherimola, Annonaceae), an underutilised subtropical fruit tree species

Although molecular markers are becoming the tool of choice to develop core collections in plants, the examples of their use in woody perennial species are very scarce. In this work, we used simple sequence repeat (SSR) marker data to develop a core collection in an underutilised subtropical fruit tree species, cherimoya ( Annona cherimola , Annonaceae), from an initial collection of 279 genotypes from different countries. We compared six alternative allocation methods to construct the core collection, four not based upon the similarity dendrogram [random sampling, maximisation strategy (M strategy) and simulated annealing algorithm maximising both genetic diversity and number of SSR alleles] and two based on dendrogram data (logarithmic strategy and stepwise clustering). The diversity maintained in each subset was compared with that present in the entire collection. The results obtained indicate that the use of SSRs together with the M strategy is the most efficient method to develop a core collection in cherimoya. In the best subset, with 40 accessions, all the SSR alleles present in the whole collection were recovered and no significant differences in frequency distribution of alleles for any of the loci studied or in variability parameters ( H _O, H _E) were recorded between the core and the whole collection.     

Some morphometric and biochemical features of ready-to-migrate silver and pre-migratory yellow stages of the shortfin eel of south-eastern Australian waters

The mean total length ( L _T), mass and age of ready to migrate female silver shortfin eels Anguilla australis from the Hopkins River estuary and the mouth of the Merri River in south-eastern Australia, were 83·2 ± 1·2 cm, 1051 ± 51 g, and 17·2 ± 1·79 years, respectively. The eye index ( I _E) of the silver shortfin eels was < 5·2 (mean 7·64 ± 0·29) and differed significantly from that of the yellow shortfin eels collected from two other sites. The I _E increased with L _T (mm) and was related by log I _E= 2·656 log L _T6·925. The per cent moisture, protein and ash content of the liver of silver shortfin eels was significantly lower than in yellow shortfin eels, but lipid content was significantly higher in the former (35·5 ± 2·0%). The mean mass μg mg lipid ⁻) of saturates (230·4 ± 2·6 v . 181·7 ±2·6), monoenes (367·4 ± 6·3 v . 290·8 ± 8·9) and PUFA (177·3 ± 5·3 v . 159·7 ± 4·6) in muscle was significantly higher, and the great majority of individual fatty acids was found also in higher quantities in silver shortfin eels. In the liver, the PUFA found in the highest quantity was 22:6n-3, except in shortfin eels from Hopkins River estuary, and the amount of 18:2n-6 in the liver of silver shortfin eels was significantly higher than that in yellow shortfin eels but the reverse was true of 20:4n-6. In both muscle and liver tissues the saturate 16:0 and the monoene 18:ln-9 collectively accounted for >50% of all the fatty acids in the lipid.     

The patterns of genetic diversity in Carpentaria acuminata (Arecaceae), and rainforest history in northern Australia

Carpentaria acuminata occurs in monsoon rainforest and is endemic to the Northern Territory, Australia. The genetic diversity of C. acuminata populations was surveyed across the geographical range of the species using isozyme analysis. Genetic diversity within C. acuminata populations ( H _E = 0.143) was typical of rainforest species and woody angiosperms generally. Genetic diversity was not correlated with rainforest patch size. However, there was significant heterogeneity among populations ( F _ST = 0.379), with infrequent effective gene flow among populations ( Nm = 0.39). Genetic diversity was negatively correlated with increasing distance between neighbouring C. acuminata populations, but geographical distance was not a good predictor of genetic similarity. C. acuminata is a favoured food of mobile frugivores such as Torres Strait pigeons and flying foxes. The decreased diversity with decreasing density of populations indicated that seed dispersal by frugivores has been important for the maintenance of diversity in this species. Populations known to have originated on relatively young, Holocene landforms were not necessarily genetically depauperate. Gene flow by pollen is apparently limited because C. acuminata populations are significantly inbred regardless of genetic diversity ( F = 0.641). The distribution and diversity of rare alleles, i.e. those occurring in few populations, is consistent with the theory of rainforest contraction during the Pleistocene.     

Genetic structure within and among regional populations of the Eurasian badger (Meles meles) from Denmark and the Netherlands

The Eurasian badger Meles meles has a wide distribution area ranging from Japan to Ireland. In western Europe badger habitats are severely disturbed by anthropogenic factors, leading to fragmentation into subpopulations and formation of a metapopulation substructuring of once continuous panmictic populations. We have examined the genetic structure of Dutch and Danish badger populations on a relatively small scale (within countries) and a larger scale (between countries). The levels of genetic variation of populations were moderate and did not differ significantly among populations (overall H _O=0.30, overall H _E=0.34). Considerable genetic differentiation between the Dutch and Danish populations was found (overall F _ST=0.32, mean pairwise Dutch–Danish F _ST=0.42), indicating a large-scale substructuring of these western European badger populations. Further analysis showed that the Danish badger population can be substructured into three clusters [ P _{( k =3)}=0.99], but the Dutch populations cluster into one more or less panmictic population [ P _{( k =1)}=0.99] with little or no substructuring. The presence of migration barriers, such as roads, together with the peninsular geography of Denmark, may have led to this structuring of badger populations. In contrast, measures that improve migration and connection to other populations from neighboring countries may have prevented substructuring of the Dutch badger population.     

Introgression of the avian naked neck gene assisted by DNA fingerprints

Theoretical predictions suggest that DNA markers can be useful tools for genomic selection in gene introgression programmes. An experiment was carried out to evaluate the efficiency of using multi-locus DNA markers in an introgression programme designed to transfer the naked neck gene from a donor to a recipient chicken line. The donor line was a commercial egg layer chicken stock heterozygous at the naked neck locus (Na/na⁺), while the recipients were from a Cornish broiler line. These two lines differ markedly in their average body weight, a quantitative trait that can also represent the comprehensive differences between the genomes of the two lines involved. Three groups of naked neck BC₁ individuals were selected according to the following criteria: (i) low band-sharing with their donor grandsires evaluated by multi-locus DNA markers, (ii) high body weight at six weeks of age, and (iii) selection at random as a control group. Birds from each of these groups were mated at random to individuals from the heavier Cornish line to produce three groups of BC₂ individuals whose body weights were recorded weekly from three to seven weeks of age. Results indicated that BC₂ birds obtained from BC₁ parents selected for band-sharing levels and those selected for body weight, performed equally well at 4–7 weeks of age; both were 3.1–3.9% heavier than birds from the randomly selected group. The additional genome recovery of the heavier broiler line, obtained by DNA markers, was found to be in agreement with theoretically predicted values.     

Influence of parameter settings in automated scoring of AFLPs on population genetic analysis

The use of procedures for the automated scoring of amplified fragment length polymorphisms (AFLP) fragments has recently increased. Corresponding software does not only automatically score the presence or absence of AFLP fragments, but also allows an evaluation of how different settings of scoring parameters influence subsequent population genetic analyses. In this study, we used the automated scoring package rawgeno to evaluate how five scoring parameters influence the number of polymorphic bins and estimates of pairwise genetic differentiation between populations (F_st). Steps were implemented in r to automatically run the scoring process in rawgeno for a set of different parameter combinations. While we found the scoring parameters minimum bin width and minimum number of samples per bin to have only weak influence on pairwise F_st values, maximum bin width and bin reproducibility had much stronger effects. The minimum average bin fluorescence scoring parameter affected F_st values in an only moderate way. At a range of scoring parameters around the default settings of rawgeno , the number of polymorphic bins as well as pairwise F_st values stayed rather constant. This study thus shows the particularities of AFLP scoring, be it either manual or automatical, can have profound effects on subsequent population genetic analysis.     

Parentage testing in alpacas (Vicugna pacos) using semi-automated fluorescent multiplex PCRs with 10 microsatellite markers

The aim of this study was to assess and apply a microsatellite multiplex system for parentage determination in alpacas. An approach for parentage testing based on 10 microsatellites was evaluated in a population of 329 unrelated alpacas from different geographical zones in Perú. All microsatellite markers, which amplified in two multiplex reactions, were highly polymorphic with a mean of 14.5 alleles per locus (six to 28 alleles per locus) and an average expected heterozygosity ( H _E) of 0.8185 (range of 0.698–0.946). The total parentage exclusion probability was 0.999456 for excluding a candidate parent from parentage of an arbitrary offspring, given only the genotype of the offspring, and 0.999991 for excluding a candidate parent from parentage of an arbitrary offspring, given the genotype of the offspring and the other parent. In a case test of parentage assignment, the microsatellite panel assigned 38 (from 45 cases) offspring parentage to 10 sires with LOD scores ranging from 2.19 × 10⁺¹³ to 1.34 × 10⁺¹⁵ and Δ values ranging from 2.80 × 10⁺¹² to 1.34 × 10⁺¹⁵ with an estimated pedigree error rate of 15.5%. The performance of this multiplex panel of markers suggests that it will be useful in parentage testing of alpacas.     

Gaussian binning: a new kernel-based method for processing NMR spectroscopic data for metabolomics

In many metabolomics studies, NMR spectra are divided into bins of fixed width. This spectral quantification technique, known as uniform binning, is used to reduce the number of variables for pattern recognition techniques and to mitigate effects from variations in peak positions; however, shifts in peaks near the boundaries can cause dramatic quantitative changes in adjacent bins due to non-overlapping boundaries. Here we describe a new Gaussian binning method that incorporates overlapping bins to minimize these effects. A Gaussian kernel weights the signal contribution relative to distance from bin center, and the overlap between bins is controlled by the kernel standard deviation. Sensitivity to peak shift was assessed for a series of test spectra where the offset frequency was incremented in 0.5 Hz steps. For a 4 Hz shift within a bin width of 24 Hz, the error for uniform binning increased by 150%, while the error for Gaussian binning increased by 50%. Further, using a urinary metabolomics data set (from a toxicity study) and principal component analysis (PCA), we showed that the information content in the quantified features was equivalent for Gaussian and uniform binning methods. The separation between groups in the PCA scores plot, measured by the J ₂ quality metric, is as good or better for Gaussian binning versus uniform binning. The Gaussian method is shown to be robust in regards to peak shift, while still retaining the information needed by classification and multivariate statistical techniques for NMR-metabolomics data.     

Is the last glaciation the only relevant event for the present genetic population structure of the meadow brown butterfly Maniola jurtina (Lepidoptera: Nymphalidae)?

Phylogeographical studies are available for a considerable number of European species, but few analyses exist for temperate species with very large and fairly continuous populations that are also absent from Northern Europe. Therefore, we studied the butterfly Maniola jurtina as a model for this group. The species has two major genetic lineages (mean genetic distance between lineages: 0.033; F _CT: 0.052), most probably evolving in glacial differentiation centres in the western and eastern Mediterranean. The onset of this differentiation might have been the beginning of the last glacial stage maximum some 40 kyr bp . A hybrid zone between these two lineages exists in western Central Europe. No genetic substructures have been found within the two lineages ( F _SC: 0.017) and average genetic distances are very small. Therefore, it is highly probable that postglacial expansion was of the phalanx type. There is, at most, very limited differentiation at regional and local scales. However, the genetic diversity within populations is high (means: A : 2.68; H _E: 17.2%; P : 78%), as would be predicted for such a common species. Comparison of these results with a published allozyme analysis revealed a similar phylogeographical pattern, but lower genetic diversity in the latter. Morphological patterns of wings and genitalia show similar geographical patterns as allozyme data.   © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 419–431.     

Toward a marker-dense meiotic map of the potato genome: lessons from linkage group I

Segregation data were obtained for 1260 potato linkage group I-specific AFLP loci from a heterozygous diploid potato population. Analytical tools that identified potential typing errors and/or inconsistencies in the data and that assembled cosegregating markers into bins were applied. Bins contain multiple-marker data sets with an identical segregation pattern, which is defined as the bin signature. The bin signatures were used to construct a skeleton bin map that was based solely on observed recombination events. Markers that did not match any of the bin signatures exactly (and that were excluded from the calculation of the skeleton bin map) were placed on the map by maximum likelihood. The resulting maternal and paternal maps consisted of 95 and 101 bins, respectively. Markers derived from EcoRI/MseI, PstI/MseI, and SacI/MseI primer combinations showed different genetic distributions. Approximately three-fourths of the markers placed into a bin were considered to fit well on the basis of an estimated residual "error rate" of 0-3%. However, twice as many PstI-based markers fit badly, suggesting that parental PstI-site methylation patterns had changed in the population. Recombination frequencies were highly variable across the map. Inert, presumably centromeric, regions caused extensive marker clustering while recombination hotspots (or regions identical by descent) resulted in empty bins, despite the level of marker saturation.     

The bundlin pilin protein of enteropathogenic Escherichia coli is an N-acetyllactosamine-specific lectin

Synthetic N -acetyllactosamine (LacNAc) glycoside sequences coupled to BSA competitively inhibit enteropathogenic Escherichia coli (EPEC) localized adherence (LA) to human intestinal biopsy specimens and tissue culture cell monolayers. The LacNAc-specific adhesin appears to be associated with the bundle-forming pili (BFP) expressed by EPEC during the early stages of colonization. Herein, we report that recombinant bundlin inhibits EPEC LA to HEp-2 cells and binds to HEp-2 cells. Recombinant bundlin also binds, with millimolar association constants ( K _assoc), to synthetic LacNAc-Benzene and LacNAc-O(CH₂)₈CONH₂ glycosides as assessed in the gas phase by nanoelectrospray ionization mass spectrometry. Furthermore, LacNAc-BSA inhibits LA only of EPEC strains that express α bundlin alleles, suggesting putative locations for the LacNAc-binding pocket in the α bundlin monomer. Collectively, these results suggest that α bundlin possesses lectin-like properties that are responsible for LacNAc-specific initial adherence of α bundlin-expressing EPEC strains to host intestinal epithelial cells.     

Conservation Genetics of an Endangered Herb, Hanabusaya asiatica (Campanulaceae)

Abstract: Hanabusaya asiatica (Nakai) Nakai (Campanulaceae), a bee- pollinated, perennial herb, is restricted to the mountainous regions of the eastern-central Korean peninsula. Allozyme analyses for 348 individuals assessed the levels of genetic diversity for five populations. Spatial autocorrelation statistics were also used to examine the spatial distribution of allozyme polymorphisms. The species maintains high levels of allozyme diversity ( H _eS = 0.217) and it exhibits low allozyme differentiation among populations ( G _ST = 0.132) compared with other endemics (mean H _e = 0.096, G _ST = 0.248). There is an apparent pattern of isolation by distance among populations. These results suggest that H. asiatica is at a genetic equilibrium. A considerable deficit in numbers of heterozygotes suggests mating among relatives in populations. At least three populations of H. asiatica should be sampled or conserved to capture or maintain > 99 % of the genetic diversity in the species as a whole. Within local populations, individuals are distributed in a structured, isolation by distance, manner. Approximate genetic patch width in the populations of H. asiatica examined is 5 - 8 m. For conservation purposes, it is suggested that, in general, the sampling of H. asiatica should be conducted at intervals in order to efficiently sample the genetic diversity across an entire population.