Chlamydomonas reinhardtii is a model system for studying cilia, photosynthesis, and other core features of eukaryotes, and is also an emerging source of biofuels. Despite its importance to basic and applied biological research, the level and pattern of genetic variation in this haploid green alga has yet to be characterized on a genome-wide scale. To improve understanding of C. reinhardtii's genetic variability, we generated low coverage whole genome resequencing data for nearly all of the available isolates of this species, which were sampled from a number of sites in North America over the past ∼70 years. Based on the analysis of more than 62,000 single nucleotide polymorphisms, we identified two groups of isolates that represent geographical subpopulations of the species. We also found that measurements of genetic diversity were highly variable throughout the genome, in part due to technical factors. We studied the level and pattern of linkage disequilibrium (LD), and observed one chromosome that exhibits elevated LD. Furthermore, we detected widespread evidence of recombination across the genome, which implies that outcrossing occurs in natural populations of this species. In summary, our study provides multiple insights into the sequence diversity of C. reinhardtii that will be useful to future studies of natural genetic variation in this organism. Chlamydomonas reinhardtii is a haploid, unicellular eukaryote that is an important model system in biology  every 100 bases in the nuclear genome C. reinhardtii, the level and pattern of genetic diversity in this species has yet to be comprehensively assessed.Presently, about a dozen wild isolates of C. reinhardtii, we conducted a genome-wide analysis of its genetic diversity. We resequenced at low genomic coverage most of the available C. reinhardtii isolates, which originate from multiple states in the US , as well as a single field in Quebec .We obtained nearly all of the available natural isolates of this species . CC-125, CC-503, CC-1373, CC-2935, CC-2936, CC2937, and CC-2938 formed the first clade (‘Group I’), while CC-1952, CC-2290, CC-2342, CC-2343, CC-2344, CC-2931, and CC-2932 formed the second clade (‘Group II’). We found that CC-125 and CC-503 were nearly identical, as was expected given their common ancestry . PCA is a multivariate statistical approach that is often used to identify signatures of population structure in large-scale SNP genotyping datasets .We used additional methods to study the population structure among the isolates. Results from both Principal Components Analysis (PCA) and the program STRUCTURE K putative ancestral subpopulations to the genomes of individuals in a sample K = 2), and implementations of the program with K = 2 or K = 3 gave ancestry assignments for the strains that were consistent with the neighbor-joining tree and PCA . This suggests that the two populations may share a recent evolutionary origin. We also note that the geographic bounds of the C. reinhardtii populations may be temporally dynamic, as CC-125 and CC-1373, which were sampled from Massachusetts in 1945, were more closely related to recent isolates from Quebec than to recent isolates from the US , and 19.8% in 3′ UTRs. We used the identified SNPs to estimate C. reinhardtii's nucleotide diversity . We found that measures of sequence variation based on segregating sites (θW) π) π were generally ∼8% lower than estimates of θW (W observed in intergenic regions (0.0011) and the highest value of θW observed in introns (0.0028). Exons also showed reduced sequence variation relative to introns and UTRs (θW = 0.0015 for exons vs. θW = 0.0028 for introns and θW = 0.0026 for UTRs), although nucleotide diversity was ∼38% higher in exons than in intergenic regions.We determined the levels of genetic variation at different site classes in the es of θW Fig. 2, C. reinhardtii genome. To do this, we split the genome into equally sized and non-overlapping windows of 100 kb, and examined local levels of nucleotide diversity across the genome, as measured by θW (we note that other window sizes produced similar results). We first analyzed levels of nucleotide diversity across the genome in the two subpopulations described above. We found that local levels of genetic variation in the genome tended to be higher among the Group II strains, suggesting that this subpopulation has a larger effective population size than the Group I subpopulation . Because of our small sample size, we combined the data from the two subpopulations to increase our statistical power to detect unusual patterns of nucleotide diversity throughout the genome. When we did this, we observed substantial heterogeneity in levels of observed genetic variation .The effect of intergenic DNA on measurements of genetic diversity for Chromosome 15 may be a technical artifact. We assessed the completeness of the reference genome within each genomic window , and found substantial heterogeneity in the quality of the reference sequence Fig. 3C.−16), and explained ∼9.8% of the variance in nucleotide diversity in a simple linear regression. Thus, how intergenic DNA is distributed throughout the C. reinhardtii genome influences our observations of genetic diversity, although additional data are necessary to fully determine the contributions of biological and technical factors to this signature.Even though technical factors likely play a role in our measurements of genetic diversity on Chromosome 15, we cannot rule out an additional contribution by biological factors, such as negative selection on intergenic regions. For instance, the amount of data generated for Chromosome 15 was comparable to the other chromosomes Fig. 3D,C. reinhardtii. The allele frequency spectrum can provide information about the types of historical events that have influenced genetic diversity in a species, as well as the extent of natural selection on different site classes  is the nonrandom association of alleles at different sites in the genome. We sought to examine the rate at which LD decays at linked sites in the sample using the Fig. S3), suggesting a common cause for high LD on Chromosome 9 in the two subpopulations. The regions with high LD did not appear to be due to reduced local recombination. Chromosome 9, including the region with high LD, showed an above average level of recombination relative to the rest of the genome in a controlled cross of two C. reinhardtii strains . The occurrence of partial selective sweeps is another possible explanation for the presence of long and incompletely fixed haplotypes in these regions. Because the regions of high LD are large and span more than a megabase, it is difficult to speculate which gene or genes in these regions might have been the target of any selection.We also looked at the rate of decay of LD at the level of individual chromosomes. Most chromosomes exhibited rates of decay of LD that were similar to genome-wide values Fig. 5. C. reinhardtii, recombination occurs when two individuals of opposite mating types encounter each other, cross, and produce offspring. For any recombination event to be detectable in cross progeny, two mates must carry different alleles of markers that flank a recombination event. We examined the data for pairs of linked sites that exhibited evidence of recombination either at the whole species level or within the subpopulations. We found that a large number of SNP pairs distributed on each of the chromosomes showed evidence of recombination based on the segregation of all four possible combinations of two linked SNPs C. reinhardtii outcrosses in nature.Recombination is the primary factor that causes LD to break down. In a haploid species like r) is known for a species and an estimate of the population recombination rate (ρ = 2Ner) has been obtained for a population genetics sample from that species, one can estimate how many generations of outcrossing have contributed to the sample (Methods). Using the program LDHat C. reinhardtii is needed to accurately determine the rate of outcrossing in this species, our results suggest that a large number of mating events have taken place in natural populations of this organism.We attempted to estimate the amount of outcrossing that occurred during the evolutionary history of the isolates. To do this, we followed the example of Ruderfer et al. C. reinhardtii is a widely studied model system in biology, but the genetic diversity of this species is not well understood. To characterize the sequence variation of C. reinhardtii, we generated genome-wide resequencing data for most of the available isolates of this species. We found that nucleotide diversity in C. reinhardtii is ∼0.0021 per base. This value is slightly lower than in other unicellular, eukaryotic model systems, such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, which possess nucleotide diversities of ∼0.0057 C. reinhardtii has the raw material to serve as a system for studying natural genetic variation. In an attempt to address multiple basic questions about the population genetics of C. reinhardtii, we used the identified genetic variants to determine the level and pattern of sequence diversity across the examined isolates and their genomes. In the following subsections, we elaborate upon a number of previously unknown features of genetic variation in C. reinhardtii.C. reinhardtii, which we refer to as Groups I and II  of 0.0014 per base, which is ∼66% of the species-wide value. The Quebec isolates are not the only local population that exhibits genetic diversity in the data: CC-2931 and CC-2932, which were collected from the same field in North Carolina and are both in Group II, also possess a number of sequence differences. Considering just CC-2931 and CC-2932, we obtained an estimate of θW that was equal to 0.0013 . These results suggest that local populations of C. reinhardtii may provide valuable study systems for geneticists and evolutionary biologists.Our results also show that local populations of C. reinhardtii, particularly when all of the sequenced isolates were considered. Moreover, by examining the levels of LD on individual chromosomes, we were able to identify regions of the genome with unusually high LD. We found one chromosome – Chromosome 9 – that exhibited high LD, possibly due to the recent action of positive selection at loci on this chromosome. It is worth noting that high LD was found on Chromosome 9 in both Groups I and II, suggesting positive selection or some other causative factor has acted across a large geographic area that spans multiple subpopulations of this species.Linkage disequilibrium is a topic of interest in population genomics studies for multiple reasons: it can provide valuable insights into the history of a population, and is a central consideration for genotype-phenotype association mapping. We showed that LD decays rapidly throughout the genome and exhibits moderately low baseline levels in C. reinhardtii during its evolutionary history. Historical recombination has led to the randomization of linked sites and a decrease in the baseline level of LD throughout the genome. Indeed, we estimate that more than 1,000 generations of outcrossing have occurred during the evolutionary history of the examined isolates. Our results definitively show that C. reinhardtii undergoes sex in nature. This finding has ramifications for general understanding of the ecology of this species, and suggests that genome-wide association mapping could be a useful technique in C. reinhardtii if a larger number of isolates are collected.Part of the reason we could detect unusual signatures of LD is that extensive recombination has occurred in C. reinhardtii to study core features of eukaryotes, such as photosynthesis and the function and regulation of flagella, that cannot be examined in the other unicellular, eukaryotic model systems. However, multiple factors, including the availability of only small number of wild isolates and lack of a very closely related outgroup, presently limit population genomics and quantitative genetics research in this organism. Thus, our report represents only a starting point in the genome-wide characterization of C. reinhardtii's diversity. Indeed, a more thorough assessment of the population structure and sequence variation of C. reinhardtii is needed. This will require the collection of a larger panel of isolates, which will ideally be obtained from across a broader geographic range and with more intensive local sampling. We have already begun to generate such a resource, and are optimistic that it will provide deeper insights into the extent and genetic mechanisms of ongoing adaptation and functional diversity in this important model organism.Natural genetic variation may provide new opportunities for using http://www.chlamycollection.org/) using Qiagen Plant DNeasy kits. We constructed sequencing libraries using the Nextera library construction kit (Illumina/Epicentre Biotechnology). We conducted the sequencing using a mixture of 50 base ×50 base paired-end reads from the University of Southern California Epigenome Center and 93 base ×93 base paired-end reads from the Beijing Genomics Institute. Sequencing data was deposited in the NCBI Short Read Archive under Project Accession PRJNA169374. Reads were mapped to the Joint Genome Institute v4 assembly of the C. reinhardtii genome. The sampe function of BWA was used to align the reads to the genome, and variants were identified using SAMTOOLS and BCFTOOLS C. reinhardtii genome (Version 169) was obtained from Phytozome v4.3 (http://www.phytozome.net/) and used to assign sequenced sites to functional classes.DNA was extracted directly from samples of the isolates that were sent by the Chlamydomonas Resource Center (http://www.r-project.org/). To construct the neighbor-joining tree, we used the ape and apTreeshape packages; the princomp function was used to conduct PCA. We also used STRUCTURE v2.3.2 to examine the strains for population subdivision K values from 2 to 4 with a burn in of 25,000 Markov Chain Monte Carlo (MCMC) cycles and 25,000 subsequent MCMC cycles. 25,000 SNPs were randomly chosen for use in the STRUCTURE analysis. We ran STRUCTURE three times for each K value, with the runs producing nearly identical results.The neighbor-joining analysis and PCA were conducted in R (http://en.wikipedia.org).Values of Spearman's ρ were computed using the cor.test function in R with method equal to ‘spearman.’ Latitudes of sampling were obtained from the Wikipedia pages for the cities in which strains were reportedly sampled  was estimated using the C. reinhardtii marker data and genetic map reported in http://ldhat.sourceforge.net; e parameter in ρ as the effective number of generations of outcrossing that have contributed to the sample e =  ρ/2r, where r was set at 1×10−7 Morgans per base.A standard formula for computing the Figure S1Principal component 3 is correlated with latitude of sampling. Strains are colored by their subpopulation.(PNG)Click here for additional data file.Figure S2Levels of nucleotide diversity across the genome measured independently for the two subpopulations. Values for Group I strains are shown with darker colors, while values for Group II strains are shown with lighter colors.(PNG)Click here for additional data file.Figure S3Linkage disequilibrium on Chromosome 9. Chromosome-wide r2 values are plotted for Chromosome 9 in the full isolate panel, Group I, and Group II in A, B, and C, respectively. A legend depicting the color scheme for the r2 values is in the lower right quadrant. Data is plotted according to the physical order of SNPs on the chromosome (shown by the line y = x), with locations of SNPs on the chromosome indicated by small lines that connect the LD plots to the y = x line. The length of the chromosome is indicated in the plots. Red lines are used to show regions that have consistently high haplotype structure in the subpopulations.(PNG)Click here for additional data file.Figure S4Local recombination rate throughout the genome, as estimated from a cross. Local recombination rates were estimated based on data from a cross of two C. reinhardtii strains (PNG)Click here for additional data file.Table S1SNPs detected on each chromosome by functional category.(XLS)Click here for additional data file.


Glossina pallidipes, a vector of human and animal African trypanosomiasis in eastern Africa. This colony is the source for Sterile Insect Technique (SIT) programs in East Africa. The source population of this colony is unclear and its genetic diversity has not previously been evaluated and compared to field populations.The IAEA colony is the only one available for mass rearing of We examined the genetic variation within and between the IAEA colony and its potential source populations in north Zimbabwe and the Kenya/Uganda border at 9 microsatellites loci to retrace the demographic history of the IAEA colony. We performed classical population genetics analyses and also combined historical and genetic data in a quantitative analysis using Approximate Bayesian Computation (ABC). There is no evidence of introgression from the north Zimbabwean population into the IAEA colony. Moreover, the ABC analyses revealed that the foundation and establishment of the colony was associated with a genetic bottleneck that has resulted in a loss of 35.7% of alleles and 54% of expected heterozygosity compared to its source population. Also, we show that tsetse control carried out in the 1990's is likely reduced the effective population size of the Kenya/Uganda border population.G. pallidipes biology.All the analyses indicate that the area of origin of the IAEA colony is the Kenya/Uganda border and that a genetic bottleneck was associated with the foundation and establishment of the colony. Genetic diversity associated with traits that are important for SIT may potentially have been lost during this genetic bottleneck which could lead to a suboptimal competitiveness of the colony males in the field. The genetic diversity of the colony is lower than that of field populations and so, studies using colony flies should be interpreted with caution when drawing general conclusions about  Glossina pallidipes, a vector of human African trypanosomiasis and arguably the main vector of animal African trypanosomiasis in eastern Africa. This colony is the main one used for basic research on this species and is intended to be used for Sterile Insect Technique (SIT) programs for control of field populations. The origins of this colony are not clear and the extent to which it is genetically representative of the species is unknown. Using population genetics analyses to compare the current colony with two potential source populations we have shown that the colony is from the Kenya/Uganda border and that its foundation and establishment in the laboratory were associated with a genetic bottleneck, i.e. reduction of genetic variation due to increased genetic drift in a population of reduced size. As a consequence, the genetic diversity of the colony is lower than that of G. pallidipes field populations.There is only one mass reared laboratory colony of  Cochliomyia hominivorax, a veterinary pest, in North and Central America Tsetse flies are vectors of African trypanosomes, parasites that cause human and animal African trypanosomiases. Tsetse control is one of the main methods used to combat the disease To be successful, SIT programmes need to overcome a number of potential genetic difficulties. Firstly, genetic and phenotypic differentiation can cause mating barriers between wild populations which can make SIT less effective depending on the geographical origin of the sterile insects Glossina austeni from Unguja Island (Zanzibar), Tanzania through an area-wide integrated tsetse eradication project terminated by a phase of SIT Glossina pallidipes, a vector of human African trypanosomiasis G. pallidipes males are about to be performed SIT has proven useful in tsetse control, the most striking example being the eradication of G. pallidipes colony is one of the very few laboratory colonies of this species worldwide and the only one with which mass rearing has been achieved to date. This is the reason why it has been used to start mass rearing in all the SIT Facilities that are currently rearing G. pallidipesG. pallidipes colony is generally considered to have been established from the laboratory colony of the University of Amsterdam, the Netherlands which was in turn established from wild pupae collected from Lugala, Uganda in 1975 G. pallidipes was initiated from pupae kindly donated by the Tsetse Research Laboratory, Bristol, England.” and this is believed to have been the start of the current IAEA colony G. pallidipes sent to IAEA in 1987 may have originated from northern Zimbabwe G. pallidipes IAEA colony and cannot rule out the possibility of admixture between colonies of Zimbabwean and Ugandan origin. This lack of clarity is potentially important because of the high genetic differentiation between G. pallidipes populations from Uganda and Zimbabwe The IAEA Molecular markers and population genetics can be used to reconstruct the demographic history of populations, thereby providing insight into aspects of population establishment and colonisation such as founder effect, population bottleneck and demographic expansion We examined here the genetic variation within and between the IAEA colony and its potential source populations in north Zimbabwe and the Kenya/Uganda border to retrace the demographic history of the IAEA colony. We performed classical population genetics analyses and also combined historical information and microsatellite data in a quantitative analysis of the genetic variation using ABC methods. We addressed the following specific questions: (i) Does the IAEA colony result from an admixture between the two potential source populations or originate from a single source? (ii) What is the level of genetic drift associated with laboratory colonisation and the rearing of the colony between its foundation and the present?G. pallidipes were obtained from the IAEA Seibersdorf colony and from its potential source populations , we tested whether the microsatellite locus GmmF10 could be amplified in flies collected in Busia as this marker can be amplified in G. pallidipes but not in G. fuscipesDistinguishing between G. pallidipes using multiplex PCR as described in www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer. The size estimation of amplified microsatellites was performed using GeneMarker v2.2.0 (SoftGenetics). The Excel Macro Autobin v0.9 utobin were then used within GeneMarker to automatically bin the alleles. Each peak was then checked manually.15 polymorphic microsatellites loci previously described in the literature http://dx.doi.org/10.5061/dryad.bt612Microsatellite data are available from the Dryad Digital Repository: Na) and the mean expected heterozygosity (H) eneclass 2 ver. 2.0.h FIS was estimated with Genepoponthe WebNa values between samples, allelic richness (AR) was estimated on the basis of minimum sample size with Fstat 2.9.3.2 AR and H between samples was assessed with the nonparametric Friedman and Wilcoxon sign rank tests (with the locus as a repetition unit). Deviation from Hardy–Weinberg equilibrium (HWE) was assessed with the probability test approach, using Genepoponthe Web.Genetic variation within samples was assessed using the mean number of alleles per locus  of clusters, set between 1 and 5, with a burn-in of 2×105 iterations followed by 106 iterations. The admixture model of ancestry together with the correlated allele frequencies model were used a priori on the origin  of each individuals, i.e. individuals were clustered only on the basis of their multilocus genotypes. Default values were maintained for all other parameters. K was estimated as the value leading to the highest likelihood for the data P(X|K) and with the ΔK statistics of Evanno et al. tructure Harvester Web v0.6.93 The second method allowed the concomitant assignment of individuals and inference of potential admixture. This clustering approach, implemented in SG. pallidipes IAEA colony and field populations under study. Microsatellite data were combined with prior information on the history and demography of those populations. Analyses were performed with Diyabc v 1.0.4.46 6 datasets were simulated for each competing scenario using parameter values drawn from prior distributions and assuming equiprobability of each scenario a priori. The simulated datasets had the same characteristics  as the observed dataset.We applied an Approximate Bayesian Computation (ABC) approach to infer the demographic history of the FSTi assigned to population j (Li→j) and the maximum likelihood estimates for admixture proportions Genetic variation was summarised using a set of summary statistics traditionally used in ABC for each population and each population pair In analysis 1  we focusG. pallidipes population temporarily In analysis 2  we focusThe IAEA colony demography and origin were examined in analysis 3, taking into account the scenarios selected in analysis 1 and 2. The IAEA colony was considered to originate from a single source, Busia or Rukomeshi, or from an admixture between both. Each of those three scenarios were considered with or without the possibility of a bottleneck associated with the laboratory establishment of the IAEA colony, giving a total of 6 competing scenarios . The anaFor all the ABC analyses performed, posterior probabilities of the competing scenarios were estimated by polychotomous logistic regression ABC analyses were performed on simulated pseudo-observed datasets (PODs) to evaluate the ability of our ABC analysis 3 to select the true scenario. For each of the 6 scenarios of the ABC analysis 3 , 100 PODiyabc we evaluated the ability of the selected scenario and of its parameters posterior distributions to generate simulated data that are similar to the observed data set 4 PODs using the scenario selected in the ABC analysis 3 and parameters values drawn from the posterior distributions of the parameters. Summary statistics distributions corresponding to those 104 PODs were then compared to the observed summary statistics. To reduce the bias introduced by the use of the same set of summary statistics for the ABC analysis and the model checking δμ)2 distance 4 PODs Using the “model checking” option in Diyabc to simulate the source population in 1975 under the scenario selected by the ABC analysis 3. To take into account the inter-simulation variation, we simulated 100 datasets under the scenario selected by the ABC analysis 3. In addition to the four samples  included in the ABC analysis 3, a fifth sample corresponding to the 1975 source population was simulated. This sample of the 1975 source population correspond to 26 multilocus genotypes at 9 microsatellite loci having the same characteristics as the ones used to produced the observed data. We simulated 26 multilocus genotypes to be able to directly compare the genetic diversity of the 1975 source population simulated and of the IAEA 2013 sample which is made up of 26 individuals  and could be the results of the presence of null alleles at this locus in Rukomeshi. Most of the analyses including the Structure analysis and the ABC analysis 3 were repeated after excluding locus Gpc26b from the dataset. The results obtained were very similar to the ones obtained with 9 loci (data not shown) and the deviation from HWE at locus Gpc26b was thus considered inconsequential for our inferences. No linkage disequilibrium was detected between loci in any sample.The complete dataset analysed  showed moderate polymorphism with an average of 6.556 (SD = 3.046) alleles per locus over all samples. The number of alleles ranged from 3 at locus GmmC17 to 11 at loci GpC101 and GmmA06. Fifty seven (96.6%) of the 59 alleles observed over all samples were present in the Rukomeshi sample. In Rukomeshi all loci were polymorphic whereas locus GpCAG133 was monomorphic in Busia and both IAEA samples and locus GmmC17 was monomorphic in IAEA 2013. Only the Rukomeshi sample was found to deviate from Hardy-Weinberg equilibrium  which waNa) was heterogeneous between samples, ranging from 2.2 [Allelic richness (AR) = 2.1] in the IAEA colony to 6.3 (AR = 5.9) in Rukomeshi (H) ranged from 0.19 in the IAEA colony to 0.56 in Rukomeshi  while they were no different in Busia and the IAEA samples . A different pattern was observed with H which was significantly higher in the field populations than in the IAEA colony  but was not different between the two field populations . The tests for mode shift of the distribution of allele frequency classes and for heterozygosity excess .Pairwise comparisons between samples showed highly significant genetic differentiation tests and large to very large i to each sample of the potential source population s (Li→s) and the results are indicated in Li→s values for Busia also indicate that this is a more likely source population of the IAEA colony than Rukomeshi.We also investigated the mean multilocus individual assignment likelihood of each IAEA sample tructure inferred the most likely number of genetic clusters (K) to be 2 or 3. The statistic ΔKK = 2 while the highest likelihood of the data was obtained at K = 3  formed a distinct cluster except for a few Busia individuals that clustered within the IAEA cluster or were admixed between the Busia and the IAEA cluster and vice and versa.In a third approach, Sat K = 3 . At K = 3  were rare in the IAEA colony  while the other two  had relatively high frequencies (0.04 to 0.29). However, the interpretation of these data must also take into account the total number of alleles in both Busia and Rukomeshi (23 and 57 respectively). Fisher's exact test shows that the number of private alleles from Busia observed into the IAEA colony (0/23) is not statistically different from the number of private alleles from Rukomeshi .In the ABC analysis 1 focused on Busia, the scenario with a strong bottleneck (scenario A) obtained a posterior probability of 0.997 . The conThose results were taken into account and the demography of Busia and Rukomeshi were modelled accordingly in the ABC analysis 3 . To idenNe, BusN. corresponds to Busia Ne before the bottleneck; the genetic data for the Busia sample did not contain any information about the Busia Ne after the genetic bottleneck (data not shown). All the posterior distributions of the Ne estimated substantially differ from the corresponding prior probability distributions  programs in Kenya, Tanzania and Ethiopia The Measurements of genetic variation between samples and Bayesian clustering clearly indicated a strong similarity between the colony and the Busia population and no introgression from Rukomeshi. The only counter-evidence is four private alleles from Rukomeshi (present in Rukomeshi and not in Busia) that were observed in the IAEA colony. This result lacked statistical significance however. A potential explanation for this observation is that the Busia population experienced a genetic bottleneck between the establishment of the IAEA colony and the collection of our Busia sample. Those four alleles could have been lost in the Busia population through this genetic bottleneck which would imply that, while those four alleles are private to Rukomeshi relatively to our Busia sample, they might not have been private to Rukomeshi relatively to the Busia population before the genetic bottleneck.The bottleneck in Busia is most likely to have been caused by tsetse control measures carried out in the area in the 1990's The ABC analysis gives a numerical prediction for the population size during the bottleneck. Our estimates revealed that the genetic drift associated with the foundation and establishment of the IAEA colony was strong with an effective number of individuals associated with the foundation and establishment of the colony around 27. This is consistent with the historical record that only 36 females were used to start the colony G. pallidipes but those studies were constrained by using isoenzyme markers and analytical approaches based largely on measurement of allele frequencies and heterozygosity To conclude, all the analyses indicate clearly that the area of origin of the IAEA colony is the Kenya/Uganda border and that a severe genetic bottleneck was associated with the foundation and establishment of the colony causing a marked loss of genetic diversity. These data contrast with earlier findings on colonies of Na and AR which, in isolation, seem to indicate no loss of diversity in the colony. These findings contrast however with the heterozygosity excess, the mode shift of the distribution of the allele frequency classes and the ABC analysis all of which indicate that the Busia population has experienced a severe genetic bottleneck. Using the ABC it was possible to take into account the demography of the Busia population between the foundation of the IAEA colony and the sampling of flies in Busia. The current study clearly demonstrates that model based inferences, such as ABC, are more powerful at detecting genetic bottlenecks compared to moment based methods such as the ones implemented in the program BottleneckThe data set analysed in the present study is a good illustration of the usefulness of model based inferences, such as the ABC, over more classical population genetics analyses. It is clear from our data that a direct comparison of the genetic diversity of laboratory colony with that of the sample collected from its source population resulted in similar values for ottleneck are able to detect recent genetic bottleneck 2Ne-4Ne generations before sampling. The bottleneck associated to the IAEA colony is likely to have occurred about 150 generations before sampling which potentially explains why the tests implemented in Bottleneck did not detect any bottleneck in the IAEA colony from contemporary samples. Moreover, Hoban et al. As indicated by Luikart et al. G. pallidipes could potentially have been lost during the strong genetic bottleneck that was associated with the IAEA colony foundation and establishment. For example, some genetic diversity associated with mating competitiveness could have been lost that would lead to a suboptimal mating competitiveness of the colony males in the field.The high level of genetic drift that occurred in the IAEA colony could have a negative impact on SIT because some of the wild-type characteristics of Theoretically, several things can be done to reduce genetic drift associated with rearing organisms in captivity. In a colony already established it is possible to limit genetic drift by reducing the variation in reproductive success by reducing the number of non-mated individuals and/or by homogenising the contribution of each family to the next generation. However, for SIT, that requires mass production of males for sterilization and release into to field, reducing the variation in reproductive success is not feasible. Indeed, SIT requires the production of tens of thousands of insects and, in such conditions, the work load associated with measures seeking the homogenisation of the contribution of each family to the next generation would be too demanding. Moreover, for tsetse SIT, the requirement to produce spare males to be sterilised goes against any procedure that would aim at reducing the number of non-mated individuals. Indeed, males for SIT are spared before mating  and this is made possible by the use of a skewed sex ratio at mating For colony established from scratch, it is obvious that maximising the number of founder individuals and/or providing a continuous input of individuals from the field will limit the genetic diversity loss compared to the field population of origin. However, this could also prevent laboratory establishment and mass rearing that requires a high level of adaptation of the reared organisms to the laboratory conditions G. pallidipes, many unsuccessful attempts have been made to raise small laboratory colonies G. pallidipes colonies reported in the literature, the IAEA colony was started from the smallest number of founding individuals while it is the only one that has reached mass rearing. Indeed, all the attempts to establish G. pallidipes colonies that are reported in the literature have been made with many more founding individuals In G. pallidipes field populations. When doing so it is important to take into account the fact that the Busia population likely experienced a genetic bottleneck, probably in the 1990's. In accordance with our inferences about this genetic bottleneck in Busia, Ouma et al. AR and 21% for H) lower than that of other G. pallidipes populations in Uganda and Kenya. Taking into account this discrepancy of genetic diversity between Busia and other field populations, we can conclude that the genetic diversity of the IAEA colony is on average around 55% (45% for AR and 63% for H) lower than that of other G. pallidipes populations in Uganda and Kenya. This is concordant with our comparison between the genetic diversity of the IAEA colony with the simulated Busia 1975 sample (AR and 66% for H) lower than in the Rukomeshi population.Combining our results with those of Ouma et al. 5 sample . The difG. pallidipes. It should be noted that no genetic diversity loss compared to field populations have been detected in other colonies of different species of tsetse  except in a G. p. gambiensis colony Insect laboratory colonies should be genetically similar to field populations for meaningful studies to be performed on different aspects of biology of a species Figure S1Successful amplification of locus GmmF10 in all the samples from Busia.(PDF)Click here for additional data file.Supplementary file S1G. pallidipes using multiplex PCR. Includes Table S4 , Table S5  and Table S6 .Evaluation of 15 microsatellite loci for their use in population genetics analyses of (DOCX)Click here for additional data file.Supplementary file S2Table S7  and Figure S2 (Demographic scenario considered to infer the demographic history of the Busia and Rukomeshi populations).Inferring the past demography of the Busia and the Rukomeshi populations using Approximate Bayesian computation (ABC). Includes (DOCX)Click here for additional data file.Table S1Characteristics of the 9 microsatellites loci used in this study.(DOCX)Click here for additional data file.Table S2G. pallidipes microsatellite loci used.Allele “bins” defined for the 9 (DOCX)Click here for additional data file.Table S3Summary statistics used in the different ABC analyses.(DOCX)Click here for additional data file.


A total of 262 individuals from 22 populations across the entire range of the species were collected. Our results indicate that B. pleuraden comprises five well-supported mitochondrial lineages roughly corresponding to five geographical areas. The phylogeographic structure of B. pleuraden has been shaped primarily by the unique regional responses of the Yunnan Plateau to the rapid uplift of the Qinghai-Tibetan Plateau occurred c. 2.5 Mya (B phrase of Qingzang Movement) and climatic oscillation during middle Pleistocene (c. 0.64–0.36 Mya), rather than by the paleo-drainage systems. The present wide distribution of the species has resulted from recent population expansion (c. 0.053–0.025 Mya) from multiple refugia prior to the Last Glacial Maximum, corresponding to the scenario of “refugia within refugia”.Factors that determine genetic structure of species in southwestern China remain largely unknown. In this study, sequences of two mitochondrial genes (COI and cyt  Genetic diversity and population structure of species are affected not only by their life histories and ecological traits The Yungui Plateau is adjacent to the Qinghai-Tibetan Plateau (QTP) and it is one of the world's biodiversity hotspots Nanorana yunnanensisTerminalia franchetiiBabina pleuraden and N. yunnanensis have largely overlap distribution ranges N. yunnanensis may have the same impact on sympatric B. pleuraden and thus current genetic structure of B. pleuraden may also be associated with the paleo-drainage system.Contemporary drainage systems develop very well on the Yunnan Plateau . According to Clark et al. B. pleuraden during Quaternary climatic oscillation and its current distribution resulted from population expansion.Additionally, paleogeographical event is just one facet of the recent history of the earth, and Quaternary climatic oscillation has been proved to be equally important in shaping the genetic structure of many species. For regions that were glaciated and covered with continuous sheets of ice, species have been shown that they expanded from southern refugia after the Last Glacial Maximum (LGM) B. pleuraden) is endemic to southwestern China, and its distribution covers the Yungui Plateau as well as southwestern Sichuan Basin B. pleuraden an ideal model for investigating the effects of paleogeological  and paleoclimatic events on species in southwestern China.The Yunnan pond frog (c oxidase subunit I (COI) and cytochrome b (cyt b) to examine the phylogeographic pattern and genetic structure of B. pleuraden. Our specific objectives are to test for the following two hypotheses: i) Genetic structure of this standing-water dwelling frog is determined by the drainage history, and ii) There were multiple refugia rather than single refugium for B. pleuraden during Quaternary climatic oscillation and the species had experienced population expansion. Such information will not only shed light on the evolutionary history of this species, but also facilitate understanding of the historical and ongoing evolutionary driving forces for maintaining the extraordinarily high biodiversity of southwestern China.In this study, we use sequences of mitochondrial cytochrome This study did not require any ethical or institutional approvals according to “Law of People's Republic of China on the Protection of Wildlife” and “Regulations for the Implementation of the People's Republic of China on the Protection of terrestrial Wildlife” because this species is not protected by any law and all sampling was conducted outside protected areas. In accordance with the requirements of “Regulation for the Collection of Genetic Resources (HJ 628–2011)”, only toe-clip tissues were collected and stored in 99% ethanol immediately after removal, and then animals were released immediately after treating wounds with antiseptic.B. pleuraden . Additionally, haplotype network was constructed using TCS 1.21 DNA sequences were aligned using CLUSTAL X 1.83 Clinotarsus curtipes, Glandirana rugosa, Hylarana malabarica, Lithobates catesbeianus, Odorrana tormota, Odorrana ishikawae, Pelophylax chosenicus, Pelophylax kurtmuelleri, Pelophylax ridibundus, Rana chaochiaoensis and Staurois latopalmatus  was estimated using an uncorrelated lognormal relaxed molecular clock model as implemented in Beast 1.6.2 h) and nucleotide diversity (π) in Arlequin 3.1 CT) of the overall genetic variance. For this analysis, populations were grouped according to the mtDNA lineages recovered in phylogenetic analyses. Several other groupings were tested.Genetic diversity was estimated by haplotype diversity . The time at which the population expansion began (t) can be estimated from the relationship τ = 2utu is the estimated mutation rate of the sequences. Additionally, the hypothesis of population expansion was also tested using Fu's FS test D statistics To test for the hypothesis that the species had experienced population expansion, under which a unimodal and approximately Poisson distributed mismatch distribution is expected, mismatch analysis was performed with Arlequin. Under the null hypothesis of sudden expansion, the raggedness index quantifying the smoothness of the observed mismatch distribution S, the minimum number of sorting events required to explain the population subdivision S-value of ML genealogy and the S-values of the simulated genealogies.Coalescent simulations of genealogies constrained within models of population divergence provide a powerful means of assessing the fit of observed genetic patterns to different phylogeographic hypotheses Ne) for the simulations was estimated using the mutation parameter θ (Theta-W) calculated by DnaSP. We converted the theta to Ne using the equation θ = 2Neμ, with the estimated mean substitution rate. During coalescent simulations, the overall Ne was set to equal the empirical estimate, and the Ne of the refugia population was constrained to a size proportional to the overall Ne. Absolute time (years) was converted to coalescent time (generations), assuming a generation time of 2.5 years. TMRCA dates estimated from Beast were used to construct multiple refugia hypothesis (detailed in Results).Effective population size  were defined by 226 polymorphic sites when cyt b and COI sequences were combined. Of the 63 haplotypes, only six  were shared among different sampling sites from same regional group and no haplotype was shared among different regional groups (b and COI haplotypes (including two outgroups) have been submitted to GenBank: accession numbers HQ395289–HQ395353.The aligned sequences of cyt l groups . The seqb and COI genes (P = 0.087), so all further analyses was performed on the combined data. The HKY+G model was selected as the best-fit model of nucleotide substitution.The partition homogeneity test revealed no significant conflicting phylogenetic signals between cyt ML, MP, and BI analyses produced highly consistent tree topologies . Monophyc. 2.44 (95% HPD: 1.19–3.84) Mya. Subsequently, lineage B separated from lineages C, D, and E approximately 2.01 (95% HPD: 0.93–3.16) Mya, lineage C separated from lineages D and E approximately 0.64 (95% HPD: 0.28–1.01) Mya, and lineage D separated from lineage E approximately 0.36 (95% HPD: 0.16–0.58) Mya  Mya .CT = 0.95141, P<0.001) and was inferred to be the most probable geographical subdivision  revealed that grouping based on the five major lineages (A–E), which roughly corresponds to the five geographical regions , respectively, resulted in the highest value among group variation , corresponding to the five major lineages obtained by phylogenetic analyses . No alteθ values and the estimated mean rate of 0.036 substitutions per site per million years, the effective population size was calculated , with populations from northwestern and central Yunnan Plateau diverging into two further lineages (D and E)  and the value of FS was significantly negative (P<0.05), supporting the population expansion model within sub-lineage A2. Significantly negative values of FS and Tajima's D statistics were obtained for lineage E, also supporting that this lineage had undergone a sudden demographic expansion. For lineage C, both mismatch analysis and neutrality tests rejected the sudden demographic expansion hypothesis. Based on the estimated mean rate of 0.036 substitutions per site per million years, we calculated the demographic expansion of A2, D, and E to have occurred c. 0.025, 0.049, and 0.053 Mya, respectively  within leuraden , roughlyN. yunnanensis and B. pleuraden is habitat preference. Nanorana yunnanensis is associated with lotic environments B. pleuraden lives and breeds in lentic environments such as paddy fields, ditches, and ponds Genetic diversity and population structure of species can be affected by their life histories and ecological traits c. 2.44 Mya. This divergence event is consistent with the disruption of planation surface in the Yunnan Plateau occurred early Pleistocene Although geographically the Red River Fault locates between sites of lineage A and that of other lineages, it seems to have no contribution to the split between western and other lineages because its right-lateral slip started from 4.7 Mya N. yunnanensisT. franchetii, a plant endemic to the river valleys of Southwest China CT values were obviously lower when lineage B was grouped together with lineages D and/or E . With repeated range changes, surviving populations may pass through many adaptations and reorganizations, allowing their lineages to diverge and accumulate genetic differences Although the Xiaojiang Fault located between sites of lineage C and that of other lineages, it seems to have no contribution to the divergence of lineage C because its left-lateral slip started from 2–4 Mya 0.36 Mya . These tB. pleuraden revealed here is consistent with the scenario of “refugia within refugia” described by Gómez and Lunt Several recent studies supported southwestern China as a major glaciation refugium e.g. . MultiplB. pleuraden in northwestern, central and eastern Yunnan Plateau might have resulted from multiple independent expansion events. In addition, co-occurrence of A1 and A2 and higher nucleotide diversity (0.008324±0.004761) in MG suggest secondary contact and so population expansion. These expectations are supported by the mismatch analysis, which detected sign of population expansion within lineages A, D, and E with the exception of C. Contrary to the mismatch analysis, neutrality tests revealed no strong sign of demographic growth within lineages A and D , mtDNA haplotypes and their frequencies, as well as estimates of gene diversity and nucleotide diversity.(DOC)Click here for additional data file.Table S2Species and sequences incorporated into the analysis of divergence dating.(DOC)Click here for additional data file.Table S3Results of hierarchical analysis of molecular variance (AMOVA).(DOC)Click here for additional data file.


Amphiprion mccullochi  and its more widespread sister species, A. akindynos. A mitochondrial DNA (mtDNA) phylogram showed reciprocal monophyly was lacking for the two species, with two supported groups, each containing representatives of both species, but no shared haplotypes and up to 12 species, but not location-specific management units (MUs). Population genetic analyses suggested evolutionary connectivity among samples of each species (mtDNA), while ecological connectivity was only evident among populations of the endemic, A. mccullochi. This suggests higher dispersal between endemic anemonefish populations at both evolutionary and ecological timeframes, despite separation by hundreds of kilometers. The complex mtDNA structure results from historical hybridization and introgression in the evolutionary past of these species, validated by msat analyses . Both species had high genetic diversities . While high gd and connectivity reduce extinction risk, identifying and protecting populations implicated in generating reticulate structure among these species should be a conservation priority.Endemic species on islands are considered at risk of extinction for several reasons, including limited dispersal abilities, small population sizes, and low genetic diversity. We used mitochondrial DNA (D-Loop) and 17 microsatellite loci to explore the evolutionary relationship between an endemic anemonefish,  Although the genus has a broad Indo–Pacific distribution, more than 25% of species are endemic to isolated islands or have peripheral populations at these remote locations  and its more widespread sister species, A. akindynos, found on the Great Barrier Reef, New Caledonia and the subtropical east coast of Australia. The endemic McCulloch's anemonefish is of particular conservation concern because it has three characteristics known to elevate the risk of extinction—a very small geographic range  shared with A. akindynos, its sister species, on the eastern Australian coast ? (2) How many management units  What is the contemporary relationship of connectivity between and within species, based on msat DNA? (4) What are the genetic diversities of these sister species and do they suggest resilience or susceptibility to extinction and environmental change? Identifying MUs, including the direction of connectivity between these units, is essential information to ascertain best practice management and maximize biodiversity conservation of these important southernmost coral reefs. We discuss the implications of the underlying genetic structure to extinction risk and the conservation of these remote populations.This study addressed the following specific questions: (1) What is the evolutionary relationship between the sister species, A. mccullochi) has the smallest geographic range of any of the 28 species of anemonefish and is endemic to three isolated, oceanic locations more than 600 km off the east Australian coastline . It is only found living in close association with its host anemone, E. quadricolor, and occurs at depths between 2 and 45 m  population (n = 20) and a southern peripheral, One Tree Island (OTI) population (n = 24), spanning 1200 km. The geographic ranges of the two sister species are separated by at least 600 km of deep open ocean habitat.Finclips from 60  n = 30;  and from3 of tissue using standard chelex-proteinase K digestion extraction procedures (2. The noncoding regions of the mtDNA were selectively amplified by polymerase chain reaction (PCR) using the following primers for the damselfish Acanthachromis polyacanthus: dLoop F (5′-CATATATGTRTTATCAACATTA-3′) and CR-E H16498R (5′-CCTGAAGTAGGAACCAGATG-3′) (A. mccullochi and A. akindynos). The resulting phylogeographic tree was poorly resolved and provided no resolution. Thus, only D-loop results are presented in this study to identify haplotypic differences. We also genotyped both A. mccullochi and A. akindynos using 17 microsatellite markers developed for A. mccullochi  . PrimersGATG-3′)  on 10 incullochi . Six mulA. clarkii (DQ343928.1), A. clarkii , A. chrysopterus (DQ343927.1), A. latezonatus (DQ343933.1), and A. leucokranos (DQ343934.1). All A. akindynos sequences (DQ250449.1 to DQ250492.1) from A. mccullochi sequences based on the close genetic relationship between these two species .Forward sequences were automatically aligned using the plugin CLUSTAL W in Geneious Pro 4.7 , conserv species . One GBRA. mccullochi were parsimony informative. The transition (ts): transversion (tv) substitution ratio was approximately 6:1. There were a total of 65 variable sites excluding five single base indels. The nucleotide composition was AT biased with 71.11% AT:28.89% GC (A. mccullochi Dloop), 71.60% AT:28.40% GC (A. akindynos Dloop), and 71.36% AT:28.64% GC (combined A. mccullochi and A. akindynos Dloop), which is consistent with fish mitochondrial DNA . The best outgroup rooted ML tree from GARLI was selected to reconstruct the evolutionary history with bootstrap values for each clade from all four analyses, if present  Bay present . A minim present  to ident present .Fs parameter for population stasis ; however, for A. akindynos selective neutrality of the mtDNA was accepted , while population stasis was rejected , suggesting a spatial expansion by A. akindynos. Following 90,000 re-samplings of the data, the F-statistics  were determined using an analysis of molecular variance (AMOVA) (h) and nucleotide diversities (%π) of the data were interpreted based on Fst comparisons (measured in Arlequin ver. 3.5) initially identified no differences between locations for either species, which informed further inter- and intraspecific analyses between clades . We also evaluated population stasis using Fu's n stasis  and Tajin stasis , both of (AMOVA)  to detec (AMOVA) . Haplotyn clades . Isolatin clades .GENEPOP 4.0  was usedF-statistics  were determined using an AMOVA  was also used to determine contemporary gene flow between populations as per http://www.Rproject.org). We retained 75 principal components (PCs) comprising 95% of the total genetic information as predictors for DA.Given contrasting results found with mtDNA and msat DNA, we specifically tested for interbreeding using two programs: (1) NEWHYBRIDS , which iA. mccullochi, whereas the apparently older Group 2 contains 70% of all sampled A. akindynos represented.The best outgroup rooted ML phylogram and the MST identified two strongly supported evolutionary groups and this was supported by all additional phylogenetic analyses . It appeA. mccullochi MUs; LI—OTI in five of the seven A. akindynos MUs. When samples were structured by MU irrespective of sampling location (MU1–12) during population genetic analyses, pairwise Fst values indicating significant genetic differentiation were obtained between all but two MU pairs . Exceptions were MU5–6  and MU5–9 . Lack of significance among these MU-pairs, despite very large pairwise Fst values may result from smaller sample sizes in these three clades than in the remaining nine clades (see st = 0.894 (P < 0.001); however, this only explained 10.56% of the variation, while 49.56% and 39.88% of the variation occurred among groups (1 vs. 2) and among populations within groups, respectively  and four to eight (MU5–12) were in Group 2. Haplotype sharing was observed between locations within species: MR—LHI in all five ades see . AMOVA rFst values for a number of interspecies and mixed-species comparisons, consistent with the phylogenetic signals described above. There was no mtDNA differentiation between sample locations (LHI vs. MR or OTI vs. LI) within either species . Given this and disregarding species identity, to compare fish from offshore to fish from continental shelf locations (OTI or LI vs. LHI or MR), all combinations of comparisons showed significant pairwise mtDNA Fst values . The more statistically rigorous AMOVA of samples structured by region (LHI and MR vs. OTI and LI) confirmed mtDNA genetic partitioning with more than two-thirds (68.31%) of the genetic variation within locations, Φst = 0.317 (P < 0.0001), and less than one-third of the variation detected among regions , which was not significant, Φct = 0.327 (P = 0.341). Further, none of the variation occurred among locations within regions , Φsc = –0.014 (P = 0.596). There was no IBD based on mtDNA from all locations sampled, using a Mantel test of pairwise geographic (km) and genetic (Fst) distance between locations .We further examined levels of gene flow between locations and/or species using pairwise Fs parameter for population stasis . In contrast, population stasis was rejected for A. akindynos , suggesting that this species has undergone spatial expansion. This was confirmed by both Bayesian and mismatch distribution analyses (data not shown). When mtDNA data of specimens from all locations, regardless of species partition, were considered collectively (MR vs. LHI vs. OTI vs. LI), total mtDNA suggested selective neutrality  and appeared to be either evolutionarily stable or to represent admixture of previously differentiated lineages.Both Fu's n stasis  and Tajin stasis  acceptedFst values were largely consistent with mtDNA results: There was no genetic differentiation between A. mccullochi populations, LHI-MR , but significant differentiation existed between A. akindynos populations, LI and OTI . Importantly, the more statistically rigorous AMOVA of samples structured by region (LHI and MR vs. OTI and LI) identified msat genetic partitioning within locations, accounting for 94.14% of the genetic variation; Φst = 0.06 (P < 0.001). Only 5.01% of the variation occurred among regions , but this was not significant, Φct = 0.05 (P = 0.337), and almost none (0.85%) of the variation, albeit significant, occurred among locations within regions , Φsc = 0.009  distance between all sampled locations, regardless of species .Msat pairwise K = 2 (A. mccullochi populations (MR and LHI) appeared to have overlapping genotypic profiles, as per mtDNA, while A. akindynos populations (OTI and LI on the GBR) were genetically distinct from each other , nucleotide (%π), and genotypic (gd) diversities for each sampled location . Total haplotype, nucleotide, and genotypic diversity were also high when species were combined , but nucleotide diversity was at least one-third (%π = 0.11–1.76) that of the aforementioned values for each species  there are no shared mtDNA haplotypes between present day populations of the two species; demographic geneflow is very limited between the species, as measured by msat DNA and Migrate-n; (3) there is population genetic partitioning between the two species, evident from both mtDNA and msatDNA, even if treated as populations rather than species and; (4) msat genotypes of almost 97% of individuals were assigned back to the population they were collected from and there was strong and total partitioning between species following DAPC analysis of msat DNA. Together, this suggests that geneflow between species occurred in the evolutionary past but is either no longer occurring, or is happening at a level not detectable in this study.We argue that the high level of mtDNA genetic partitioning observed—while not identifying species-specific lineages or location-specific MUs—is most consistent with an evolutionary history of at least two reticulate events between es e.g., , includiA. akindynos  and at least seven MUs were identified in this study, with at least two c scales  and intec scales .A. akindynos and A. mccullochi, which strengthens our suggestion of evolutionary hybridization as current day populations are not mixing. Likewise, LI and OTI A. akindynos populations were genetically distinct which is consistent with A. mccullochi populations. The difference between species in the level of gene flow between populations probably represents different geographic distances between the sample populations (McCulloch populations were 160 km apart and the akindynos sample populations were 1200 km apart). This gene flow between populations is promising for A. mccullochi since it facilitates recolonization if one population was to go locally extinct. However, only two locations in each species were sampled and further samples of A. akindynos from the Great Barrier Reef, southern reefs , and New Caledonia, as well as samples of A. mccullochi from Elizabeth Reef will be needed to fully quantify gene flow between all populations of these species.Microsatellite loci detected very limited geneflow between the sister species h almost double and π close to an order of magnitude greater than the cut-off defined by h and π >0.5). Similarly, high levels of genetic diversity for msatDNA were also found in both species. This suggests that populations of both species are either large and stable with long evolutionary histories, or that there has been secondary contact between differentiated lineages . The high genetic diversity of A. mccullochi appears to have arisen through complex reticulate evolution involving historical (but not contemporary) hybridization. High genetic diversity and population connectivity may reduce extinction risk in A. mccullochi, but identifying and protecting populations implicated in generating the complex reticulate structure among this species should be a conservation priority. Conserving A. mccullochi would best be achieved by protecting each of its MUs and by minimizing threats to population size, such as habitat loss  and collection for the aquarium trade.Conserving genetic diversity is considered a priority by the IUCN , and eve


Dicamptodon tenebrosus) in two core regions  versus the species' northern peripheral region  where the species is listed as threatened. Coalescent-based demographic simulations were consistent with a pattern of post-glacial range expansion, with both ancestral and current estimates of effective population size being much larger within the core region relative to the periphery. However, contrary to predictions of recent human-induced population decline in the less genetically diverse peripheral region, there was no genetic signature of population size change. Effects of current demographic processes on genetic structure were evident using a resistance-based landscape genetics approach. Among core populations, genetic structure was best explained by length of the growing season and isolation by resistance (i.e. a ‘flat’ landscape), but at the periphery, topography (slope and elevation) had the greatest influence on genetic structure. Although reduced genetic variation at the range periphery of D. tenebrosus appears to be largely the result of biogeographical history rather than recent impacts, our analyses suggest that inherent landscape features act to alter dispersal pathways uniquely in different parts of the species' geographic range, with implications for habitat management.With predicted decreases in genetic diversity and greater genetic differentiation at range peripheries relative to their cores, it can be difficult to distinguish between the roles of current disturbance versus historic processes in shaping contemporary genetic patterns. To address this problem, we test for differences in historic demography and landscape genetic structure of coastal giant salamanders ( Processes structuring genetic diversity across species' ranges are complex, particularly as populations can vary in connectivity across heterogeneous or fragmented landscapes, or be influenced by geographically variable biogeographical histories that shape current genetic variation eN) and population connectivity at the periphery Peripheral populations generally occur in marginal habitats or areas that are climatically unfavourable, which may limit further expansion and result in genetically isolated populations that have an increased risk of local extinction Hence, disentangling the effects of anthropogenic disturbance versus historical biogeographical processes across species' ranges will help to avoid bias in conservation strategies based on a single study area Dicamptodon tenebrosus) as our focal species, we test whether historical or recent factors have affected population genetic structure at the species' northern periphery versus two core localities using a combination of coalescent modelling and a landscape genetics approach.Amphibians are highly suited to the study of landscape genetic patterns due to their limited dispersal capacity and sensitivity to fine-scale landscape structure D. tenebrosus, which was formed by post-Pleistocene range expansion from the Columbia River Valley in Washington State, USA, up to the northern range limit, which is delineated by the Fraser River in British Columbia eN and genetic variation at range margins D. tenebrosus over the last 100 years are a likely driver of fragmentation effects Our sample regions are located within the northern clade of D. tenebrosus is listed as nationally ‘Threatened’  in Canada and is on the Provincial ‘Red List’ in British Columbia, primarily due to impacts of forest harvest and urban encroachment Although not currently a species of concern in the United States, eN in core and peripheral regions. Current influences on genetic structure are examined using a multiple pathway approach based on circuit theory that identifies how gene flow is limited by landscape resistance in terms of topographical, climatic and land cover features Using a coalescent demographic simulation D. tenebrosusNe, and if so, (2) historical range-expansion processes will have shaped current genetic patterns when the peripheral region shows a stable historic-to-recent population size compared with the core region, which is predicted to show an expansion signature  and the Washington State University Institutional Animal Care and Use Committee.D. tenebrosus) occurs in small streams from sea level to 1830 m elevation in the Pacific Northwest coniferous forests of the United States and south-western Canada 2) in the Chilliwack River watershed of British Columbia. Dicamptodon tenebrosus is highly sedentary The coastal giant salamander : Willapa Hills (WH), (area sampled ∼50 km2), South Cascades (SC) (area sampled ∼40 km2)  (2) was taken from each individual and preserved in 95% ethanol for DNA extraction as described in Steele et al. ∼40 km2) . These s Canada) . For WH  Canada) . A samplDNA extractions were performed using a standard phenol-chloroform ethanol precipitation protocol Individuals with missing genetic data at three or more loci were excluded from the dataset. Individuals from each stream were screened for genetic relatedness in the program Colony 2.0 ST between regions and sites was calculated in Microsatellite Analyser 4.05 (MSA) K = 1–6 were performed with a 50 000 burn in and 1000000 Markov Chain Monte Carlo (MCMC) iterations, under a model of admixture and correlated allele frequencies. The number of genetic clusters (K) was determined using the method of Evanno et al. K method Genetic differentiation between the three study regions  was examined using six loci that were in HW equilibrium across all regions, which excluded D05, D17 and D25 (not in HW equilibrium for WH). Allelic richness within regions was calculated in FSTAT 2.9 We examined whether there were historic or recent changes in effective population sizes and the timing of these changes in core versus peripheral regions, using MSVAR 1.3 0N), ancestral population size at the time of demographic change (1N), and the time in generations since population size change T = aT/0N (aT = number of generations since the beginning of the expansion/decline) D. tenebrosus, yet we used a conservative generation time estimate of 12.5 years for simulations of ‘time since population change’, based on a predicted maximum life-span of 20 yrs r = 0N/1N) to determine population size changes where r = 1 indicates stability, r>1 indicates expansion, and r<1 indicates decline in the effective population size 8 updates and a thinning interval of 10 000 so that 20 000 estimated parameter sets were derived from the posterior distribution The parameters of interest for the current study were: current effective population size  while in CV convergence was supported for 0N (1.23) and 1N (1.10), but less so for T (1.87). The last half of each chain was used to make a combined consensus chain of 50 000 data points, and summary statistics of the marginal posterior distributions for N0, N1 and T were estimated as the mean, 0.025 and 0.975 quantiles.We processed the output from MSVAR 1.3 using the program BOA 1.1.4 for R version 2.3.1 Girod et al. ST) among streams within regions was calculated with Bonferroni correction using MSA. We compared the performance of two different measures of genetic distance between sample sites: G′ST and Dps . G′ST is a standardized measure that is appropriate for examining genetic differentiation between datasets with different numbers of loci, and among loci with different levels of variation ST to be <1 ps between sample sites  because this measure avoids equilibrium assumptions of G′ST and is sensitive to genetic differences while controlling for low variation in allele frequencies among populations Genetic differentiation  and each region was clipped with a minimum buffer of 500 m surrounding all sample sites to minimise ‘edge effects’ associated with calculating resistance values (as suggested by ArcGIS software version 9.3.1 (ESRI) was used to parameterise nine landscape variables, which pertain to topographic characteristics , habitat permeability , or temperature and precipitation  (described in http://www.geobase.ca/geobase/en/index.html (Canadian Council on Geomatics). For WH and SC, land cover and canopy cover data were from the 2001 National Land Cover dataset, stream data were from the National Hydrography Dataset (USGS 1999) and elevation, slope and heat load were derived from USGS digital elevation models. Frost-free period and growing season precipitation for both CV, WH and SC regions were estimated based on a spline model by Rehfeldt Cell values for each landscape variable were converted directly into resistances based on expected linear predictions of suitability ST and Dps). Akaike's Information Criterion (AIC) was applied separately to each region to find the best landscape model for explaining genetic distance between sites To test the relative effects of landscape variables on genetic distance we used multiple matrix regressions using the R statistics package (2.11.0). This analysis included resistance matrices of all landscape variables with genetic distance as the dependent variable in both regions ; therefore we chose to use a model averaging approach. Model-averaged estimators often have a better measure of precision and reduced bias compared to estimators from just the selected best model After the exclusion of full-sibs , and individuals with >1/3 missing data , final sample sizes (n) were: CV, n = 387 (from 20 streams); WH, n = 213 (from 6 streams), and SC, n = 379 (from 13 streams) . The numST: WH vs. SC = 0.04; SC vs. CV = 0.09; WH vs. CV = 0.16; P<0.02 all comparisons)  , S6. AMOST = 0.04. Between SC and CV FST = 0.09, and for WH and CV FST = 0.16. All comparisons were significant after Bonferroni correction. Pairwise site FST comparisons were statistically different (P<0.05) after Bonferroni correction in 32.6% of comparisons for CV (ST (mean ±s.e.) between sites were moderate to low within regions . The peripheral region (CV) had lower allelic diversity in 7/9 loci  compared to the core regions  (Mean pairwise Euclidean distances (km ±s.e.) between sites for WH was 22.35 (±3.92), for SC was 9.5 (±0.75) and for CV was 17.6 (±0.8). Between core regions (WH+SC) pairwise Fs for CV , 37.5% fs for CV . Values r locus) . Allelicr = 0.948), so T (time since change) could not be inferred , which was ∼33% lower than in SC, 0N = 4286 . A slight historic population decline was detected in in SC (r = 0.802), estimated at approximately 849000 years ago  although two sites in SC (15.4%) showed evidence of a mode shift in allele frequencies . At the 2 = 0.00). Furthermore, all variables were highly correlated in WH . Variables with relative importance (RI) scores ≥0.60 were land cover   (2 = 0.20), followed closely by IBR+frost-free period  . A two-v ratios) . Only laed 0.99, , suggestps , but RI values ≥0.60 differed from SC, and included elevation, heat load index, and canopy cover   and RI values were ≥0.60 for slope, heat load index (0.71) and stream vs. other (1∶10 and 1∶100 ratios)  , and the = 0.13) . Two-var ratios) . RI was ad index . All othad index . Howeverad index .ps and G′ST. However the high correlation between these variables means that the additional effect of frost-free period over simple isolation by resistance should be interpreted cautiously. However in CV, the topographical variables elevation (Dps) and slope (G′ST) clearly performed better than IBR alone, with the effect of solar radiation (i.e heat load index) being another possible factor influencing landscape genetic structure in the peripheral region.To summarise the main landscape genetic findings, our results for SC consistently showed an effect of IBR+FFP for DD. tenebrosus. Historic range expansion effects appear to dominate current levels of genetic variation in both regions, with reduced Ne and genetic diversity at the periphery. Despite this, we reveal categorical differences in landscape effects on contemporary gene flow according to core or peripheral location, with stronger evidence for landscape-driven genetic structure at the periphery, in accordance with our hypotheses. Our results suggest that range-wide species conservation, may be better informed by concurrent analyses of historic demography and contemporary landscape genetic patterns that encompass more than one study area.By assessing only contemporary landscape genetic patterns, there is a risk of incorrectly attributing genetic patterns to recent landscape changes that are actually the result of historic biogeographical processes D. tenebrosus had a much smaller founding population than the core, which is in accordance with the previously documented northward range expansion D. tenebrosus conducted in the region eN, and the current stable population signature at the periphery. Smaller populations may be more prone to extinction and have reduced adaptive potential, which can inhibit or slow range expansion into new environments, resulting in a stable population signature In accordance with the ‘central-marginal’ hypothesis eN, potentially due to a loss of genetic diversity during range expansion D. tenebrosus within the early or mid-Pleistocene (1.7 mya ∼800000 ya) eN to be 31563 in the Columbia River Valley refugium. In the core, current landscape effects on genetic structure were evident within SC, but there were no effects within WH. The strong correlations between landscape variables in WH , indicating that census size does not equate to Ne/genetic diversity in these more productive populations.Notably, Dudaniec and Richardson Ascaphus truei) Anthropogenic effects on peripheral landscape genetic structure were not detected explicitly (e.g. via the variables canopy cover or land cover), though solar radiation, which is postively related to forest harvest, was among the top models. However, Dudaniec and Richardson D. tenebrosusD. tenebrosusEcologically dissimilar habitats within a species' range can select for variation in adaptive traits that are likely to reflect landscape resistance to dispersal and genetic patterns D. tenebrosus, it can be expected that metapopulation processes may drive reductions in genetic variation and increased differentiation between streams within regions, while genetic variation at the regional level does not likely decline at the same rate.The finding of greater genetic variation explained within streams than across regions (i.e. via AMOVA) may indicate non-equilibrium processes acting at different temporal and spatial scales, which can cause lower genetic differentiation between regions than that observed at lower hierarchical levels within regions (i.e. the stream level) From a spatial perspective, populations at ecological range limits may have a ‘patchier’ distribution, increasing pairwise genetic distances and landscape resistance. We acknowledge that the study area, and hence pairwise site distances were larger for CV than for SC and WH. However, previous studies suggest that a larger sample size and sampling effort is required in peripheral populations to capture the same proportion of genetic variation as in core populations due to stronger within-population spatial genetic structure D. tenebrosus, but patterns are not consistent across regions with respect to the underlying differences in core and peripheral landscape characteristics. Although additional replicates of core and peripheral regions may help to resolve these disparate regional patterns, this lack of consistency suggests that historical demographic processes strongly influence our observed landscape genetic patterns. Although geologically recent colonisation has evidently shaped the lower genetic diversity at the periphery, these historical effects may act to exacerbate population sensitivity to habitat fragmentation resulting from forest harvest. Therefore, interactions between regional topography and anthropogenic disturbances should be considered for the conservation of threatened D. tenebrosus populations, and potentially other co-occurring, stream-associated amphibians. Our study demonstrates that combining both coalescent and landscape genetic analyses can help to disentangle current from historical processes that influence contemporary patterns of spatial genetic variability.Our results suggest that historic demography influences location-specific landscape genetic processes in core and peripheral populations of Table S1Summary of collection sites (river drainage or label), site codes, final sample sizes (N) and geographical coordinates for British Columbia (BC) and Washington State (WA). Results from BOTTLENECK analyses are presented: ** indicates sites with significant heterozygote excess and a significant mode shift in allele frequency distribution; *indicates sites with just a significant mode shift in allele frequency distribution; † indicates sites with a significant heterozygote deficiency.(DOC)Click here for additional data file.Table S2Genetic diversity indices for each locus across all populations  and the number of populations (percentage in parentheses) not in Hardy-Weinberg equilibrium (HW) after correction for multiple comparisons (significance at the 0.05 level). Total sample sizes for each region are: Chilliwack Valley, n = 387, Willapa Hills, n = 213, South Cascades, n = 379. * locus excluded from analysis.(DOCX)Click here for additional data file.Table S3Parameter hyperpriors used for each simulation ran in MSVAR 1.3, for South Cascades and Chilliwack Valley datasets: ancestral population size (log N0), current population size (log N1), mutation rate (log u), and time since decline/expansion (log T).(DOCX)Click here for additional data file.Table S40), current population size (log N1), mutation rate (log u), and time since decline/expansion (log T). Starting values are the trial values for updating the parameters in the Metropolis-Hastings algorithm used in MSVAR. Locus D05 was excluded from the analysis for regional comparisons.Starting values for parameters for each locus used in all MSVAR simulations: ancestral population size (log N(DOCX)Click here for additional data file.Table S5D. tenebrosus between sampled streams in at the peripheral range in British Columbia, Canada: Chilliwack Valley (n = 387). Bold values were significantly different after Bonferroni correction.Pairwise Fst of (DOCX)Click here for additional data file.Table S6D. tenebrosus between sampled streams in Washington State, USA a) Willapa Hills, b) South Cascades. Bold values were significantly different after Bonferroni correction.Pairwise Fst of (DOCX)Click here for additional data file.Table S7Correlation matrix (Pearson's r) of landscape variables for the Willapa Hills (WH) core region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.Table S8Correlation matrix (Pearson's r) of landscape variables for the South Cascades (SC) core region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.Table S9Correlation matrix (Pearson's r) of landscape variables for the Chilliwack Valley (CV) peripheral region. STR10 = stream vs. terrestrial 1∶10, STR100 = stream vs. terrestrial 1∶100, LC10 = landcover 1∶10, CAN = canopy cover, FFP = frost free period, GSP = growing season precipitation, HLI = heat load index, IBR = isolation by resistance (flat), LC100 = landcover 1∶100, SLP = slope, ELEV = elevation.(DOCX)Click here for additional data file.


AbstractGymnotus  from different Brazilian hydrographic basins. Gymnotus sylvius presented a diploid number of 40 chromosomes (22m+12sm+6st), Gymnotus pantherinus presented 52 chromosomes (32m+18sm+2st), while Gymnotus inaequilabiatus (42m+10sm+2a)and Gymnotus cf. carapo (38m+12sm+4st) presented 54 chromosomes. The C-banding technique revealed centromeric marks in all chromosomes of all species. Besides that, conspicuous blocks of heterochromatin were found interstitially on the chromosomes of Gymnotus inaequilabiatus, Gymnotus cf. carapo,and Gymnotus pantherinus. All four species showed single nucleolus organizing regions confirmed by results obtained through Ag-NORs and FISH experiments using 18S rDNA probes, which showed the NORs localized on the first chromosome pair in Gymnotus inaequilabiatus, Gymnotus cf. carapo,and Gymnotus pantherinus, and on pair 2 in Gymnotus sylvius. CMA3 staining revealed additional unrelated NORs marks in Gymnotus sylvius and Gymnotus pantherinus. The 5S rDNA probes revealed signals on one pair in Gymnotus sylvius and two pairs in Gymnotus pantherinus; Gymnotus inaequilabiatus had about seventeen pairs marked, and Gymnotus cf. carapo had about fifteen pairs marked. It is considered that the high amount of heterochromatin PageBreakidentified in the chromosomes of Gymnotus inaequilabiatus and Gymnotus cf. carapo could have facilitated the dispersion of 5S rDNA in these species. Interstitial signals were detected on the first metacentric pair of Gymnotus sylvius by telomeric probes (TTAGGG)n indicating the possible occurrence of chromosomal fusions in this species. The present study reveals valuable cytotaxonomic markers for this group and allows a more precise evaluation of the processes involved in the karyotype differentiation and the interrelationships among different species of the genus Gymnotus.Conventional  and molecular  cytogenetic studies were carried out in specimens of ten distinct fish populations of the genus  Gymnotidae, Rhamphichthyidae, Hypopomidae, Sternopygidae, and Apteronotidae  and 5SB (5’-GCTGGTATGGCCGTAGC-3’) by PCR and the detection of hybridization signs was obtained with anti-digoxigenin-rhodamine (Roche Applied Science). The 5S probe in Gymnotus pantherinus and 18S rDNA probes in Gymnotus sylvius, Gymnotus cf. carapo and Gymnotus inaequilabiatus were labeled with biotin-16-dUTP (Roche Applied Science) by PCR and the detection of hybridization signs with avidin-FITC. Telomeric sites were identified with probes for sequences (TTAGGG)5 and (CCCTAA)5 labeled with digoxigenin 11-dUTP (Roche Applied Science) and the hybridization signs were detected with anti-digoxigenin-rhodamine. Chromosome morphology was determined according to arm relationships proposed by Molecular cytogenetic analysis involved the use of GC-specific fluorochrome Chromomycin A3 (CMA3)  and probACGGA-3’  and the TAGC-3’) . The 18SGymnotus fish species evidenced an expressive variation in the diploid number among the species, despite the conservative karyotypic feature among the representatives of the populations. Gymnotus sylvius presented 40 chromosomes n evidenced signals in the terminal position of all chromosomes in all populations analyzed  and Gymnotus cf. carapo (2n=54) from the ParanáPageBreak river basin. These authors suggested that the differences found in the diploid number of these species might have arisen from chromosomal rearrangements, mainly centric fusions. Notwithstanding these authors’ proposition, using whole chromosomes probes in the same sample analyzed by Gymnotus showed high genomic plasticity, and that the analyzed samples from the Amazon basin, usually denominated cryptic, were, in fact, distinct species.The differences related to the number and morphology of the chromosomes found among the species suggest the occurrence of structural and numerical rearrangements during the process of differentiation. Gymnotus sylvius. The presence of these ITSs in some chromosomes could be an indicative of recent centric fusion events, as previously discussed by Gymnotus sylvius and Gymnotus cf. carapo and Gymnotus carapo. These authors proposed that chromosomal fusion events would act as the most important mechanisms of karyotype evolution in this fish group. Further studies by Gymnotus sylvius and Gymnotus cf. carapo chromosomes, detected homologies among multiple chromosomes in these species, with a complete correspondence of bands, indicating a probable common ancestral origin.In the current study, the probes used for telomeric sequence (TTAGGG)n revealed signals of hybridization on the extremities of all chromosomes in all populations analyzed . HoweverGymnotus sylvius, as well as its absence in Gymnotus pantherinus, could be attributed to different factors, such as the occurrence of differences in the type of chromosomal rearrangements, the plasticity of the telomeric sequences or the divergence time of the species, which originated modifications in the sequences and possibly made them undetectable by the FISH technique. In a phylogenetic reorganization of the Gymnotiformes based on molecular and cytogenetic data, Gymnotus sylvius constitutes the most derived species amongst the representatives of Gymnotus, while Gymnotus pantherinus apparently differentiated much longer ago. The presence of the ITS in Gymnotus sylvius and its absence in Gymnotus pantherinus could be justified by the divergence time between these species. Furthermore, considering that a karyotype presenting 52 chromosomes would characterize the basal genomic group for Gymnotus , a species with the smallest numer modifications occurred in these sites, making it impossible to be detected by conventional FISH. Such situation is also proposed to occur in Gymnotus capanema (2n=34), a species with the smallest number of chromosomes within the genus Gymnotus with no ITS detected (Gymnotus sylvius would be caused by a loss of the telomere repetition, which could have facilitated events of chromosome fusion (The occurrence of ITS in some chromosomes of Gymnotus , it couldetected . Anothere fusion . This hyPageBreakThe identification of nucleolus organizer regions in the four species analyzed through silver nitrate staining and 18S rDNA probes revealed only one chromosome pair containing nucleolar sites and characterizing a simple NORs system, as previously cited (Gymnotus (Gymnotus, the motile and dynamic character of these sites was confirmed and permitted identification species-specific Ag-NORs marks, which led the authors to consider this feature as an interesting cytotaxonomic tool (ly cited . The polGymnotus . These rGymnotus . Withal,mic tool .Gymnotus sylvius and Gymnotus pantherinus,indicating the presence of additional GC-rich sequences. Gymnotus sylvius and Gymnotus pantherinus indicates heterochromatin heterogeneity between these species. The heterochromatin is mainly composed of repetitive DNA sequences, which is thought to evolve in parallel (Gymnotus could have originated such patterns of CMA3 staining.The use of CMA3 in metaphase chromosomes revealed additional marks to those identified in the ribosomal sites in parallel , resultiparallel . In thisGymnotus. Considering the great blocks of heterochromatin and the variation in the distribution pattern of 5S rDNA sites in Gymnotus inaequilabiatus, Gymnotus cf. carapo, and even in Gymnotus pantherinus, it can be considered that this situation might have favored the occurrence of structural rearrangements in the karyotype of these species, since heterochromatic areas are more propitious to breaks, and thus may facilitate the dispersion of this gene sequence. The absence of large heterochromatic blocks and the presence of 5S rDNA in a unique pair of chromosomes in Gymnotus sylvius could reinforce this hypothesis. Leporinus Spix, 1829 genus. Considering the high degree of dispersion of 5S rDNA in Gymnotus inaequilabiatus and Gymnotus cf. carapo species, it is possible that different classes of this ribosomal gene might be differentially distributed in the chromosomes of this species. On the other hand, in Gymnotus pantherinus and Gymnotus sylvius, the distinct classes of 5S rDNA could be co-located in the same chromosomes.The distribution patterns of 5S rDNA sequences showed a peculiar dispersion of these repetitive sites in the karyotypes of the species in the genus Rex3 retrotransposon type might be associated to the distribution and dispersion of 5S rDNA in the karyotypes of species in the genus Erythrinus Bloch and Schneider,PageBreak 1801. However, the existence of pseudogenes in the genome of Gymnotus inaequilabiatus and Gymnotus cf. carapo cannot be discarded, since the heterochromatin could have sequences similar to those of 5S rDNA, as suggested to occur in fish belonging to the genus Characidium (Centropyge Kaup, 1860 (Recent studies carried out by racidium  and Centup, 1860 .Gymnotus species, the results achieved in the current work revealed great differences in the chromosome structure in the species of this genus, indicating that all possible evolution ways passed through the differentiation process of chromosomes.Despite the marked karyotypic conservation found inside the populations of