Genotoxic and mutagenic evaluation in Eisenia foetida annelids exposed to iron ore tailings from the region of Brumadinho, MG, Brazil.

Soils that have a disproportion of metallic elements due to anthropic activities endanger the terrestrial fauna. This study evaluated whether earthworms (Eisenia foetida) exposed to ore tailings from Brumadinho region presented a higher frequency of genotoxic and mutagenic damages than annelids from a reference area (control). The animals were exposed to substrates containing 0%, 25%, 50%, 75%, and 100% iron mining waste. The results indicated increased DNA damage (p < 0.05), detected by the comet assay at 25% and 50%. There was a three-fold increase in micronuclei in animals on the substrates with the highest concentrations (75% and 100%) [F = 3.095; p = 0.02]. The earthworms lost weight as the percentage of mining waste increased. We concluded that E. foetida presented DNA damage in the contaminated soils of Brumadinho. However, more research is fundamental, once the environmental disaster in Brumadinho was one of the biggest mining catastrophe in Brazil.

Circulating MicroRNAs as Specific Biomarkers in Atrial Fibrillation: A Meta-Analysis.

Atrial fibrillation (AF) is the most frequently occurring supraventricular arrhythmia. Although microRNAs (miRNAs) have been associated with AF pathogenesis, standard protocols for quantifying and selecting specific miRNAs for clinical use as biomarkers should be optimized. In this study, we evaluated the clinical application of miRNAs as biomarkers for the prognosis and diagnosis of AF. Literature searches were conducted on PubMed, Cochrane Library, and EMBASE. We included prospective or retrospective observational studies that had been published as of 14 February 2022; our main objective was to analyze the relationship between circulating miRNAs and AF. The data were extracted using the descriptors "Atrial fibrillation AND miRNA", "Atrial fibrillation AND diagnostic AND miRNA", and "Atrial fibrillation AND prognosis AND miRNA". No filters were applied for period delimitation, type of publication, or language. Studies using samples isolated from blood plasma and TaqMan and RT-qPCR for detecting and quantifying miRNAs were selected, and those that used atrial tissue samples were excluded. We identified 272 articles and excluded 102 duplicated articles. Two authors independently read the titles and abstracts of 170 out of 272 articles and selected 56 potential articles, 6 of which were selected for final review. Our analysis revealed a significant association between AF and miR-4798 [OR = 1.90 (95% CI 1.45-2.47)], AF and miRNA-133a [2.77 (2.73-2.82)], AF and miRNA-150 [3.77 (1.50-9.46); I2 = 70%], AF and miRNA-21 [2.23 (1.20-4.17); I2 = 99%], AF and hsa-miRNA4443 [2.32 (2.20-2.44)], and AF and miR-20a-5p [3.67 (1.42-9.49)]. The association between miRNAs and AF showed an OR of 2.51 [95% CI 1.99-3.16; I2 = 99%]. Our meta-analysis demonstrated that circulating miRNAs are potential biomarkers of AF, as they exhibit stable expression post-sample collection. In addition to regulating cellular processes, such as proliferation, differentiation, development, and cell death, miRNAs were found to be linked to arrhythmia development.

Toxicity evaluation of the combination of emerging pollutants with polyethylene microplastics in zebrafish: Perspective study of genotoxicity, mutagenicity, and redox unbalance.

Despite the toxicity of microplastics (MPs) in freshwater fish has been demonstrated in previous studies, their effects when mixed with other pollutants (organic and inorganic) are poorly understood. Thus, we aimed to test the hypothesis that the association of polyethylene MPs (PE-MPs) to a mix of emerging pollutants induces more adverse genotoxic, mutagenic, and redox unbalance effects in adult zebrafish (Danio rerio), after 15 days of exposure. Although the accumulation of MPs in animals was greater in animals exposed to PE-MPs alone, erythrocyte DNA damage (comet assay) and the frequency of erythrocytic nuclear abnormalities (ENAs) evidenced in zebrafish exposed to PE-MPs alone were as pronounced as those observed in animals exposed to the mix of pollutant (alone or in combination with MPs), which constitutes the big picture of the current study. Moreover, we noticed that such effects were associated with an imbalance between pro-and antioxidant metabolism in animals, whose activity of superoxide dismutase (SOD) and catalase (CAT) was assessed in different organs which were not sufficient to counterbalance the production of reactive oxygen species [hydrogen peroxide (H2O2)] and nitrogen [nitric oxide (NO)] evaluated. The principal component analysis (PCA) also revealed that while the antioxidant activity was more pronounced in the brain and liver of animals, the highest production of H2O2 was perceived in the gills and muscles, suggesting that the biochemical response of the animals was organ-dependent. Thus, the present study did not demonstrate antagonistic, synergistic, or additive effects on animals exposed to the combination between PE-MPs and a mix of pollutants in the zebrafish, which reinforces the theory that interactions between pollutants in aquatic ecosystems may be as complex as their effects on freshwater ichthyofauna.

Farmers exposed to pesticides have almost five times more DNA damage: a meta-analysis study.

We carried out a meta-analytical review of possible DNA damage resulting from occupational exposure to pesticides in farmers in the scientific literature. After the search, screening, and eligibility criteria steps, we included 42 studies to analyze random effect calculation. DNA damage in farmers occupationally exposed to pesticides represents an effect of SMD 4.63 [CI 95% 3.94-5.32; p <0.001]. We observed a high heterogeneity rate between the studies and an asymmetry of the bias analysis results. We performed a meta-regression on the parameters. The Olive Tail Moment (OTM) was the most effective comet assay parameter in the evaluated studies. The Damage Index (DI) was more conservative and highlighted the variability between studies caused by distinct methodologies that showed more significant effects and greater deviations. An analysis of confounding factors demonstrated a slight DNA damage in smokers who were occupationally exposed to pesticides compared to nonsmokers, indicating genotoxicity but smaller than the pesticide effect. The present study shows the greater risk that occupationally exposed rural workers have of developing related diseases due to pesticides' genotoxic effect.

Influence of genetic polymorphisms in glutathione-S-transferases gene in response to imatinib among Brazilian patients with chronic myeloid leukemia.

Polymorphism in metabolizing enzymes can influence drug response as well as the risk for adverse drug reactions. Nevertheless, there are still few studies analyzing the consequence of polymorphisms for the Glutathione-S-transferases (GST) gene to drug response in chronic myeloid leukemia (CML). This study reports, the influence of GSTP1*B and GSTT1/GSTM1null polymorphisms in response to imatinib in CML patients in a Brazilian population. One hundred thirty-nine CML patients from the Clinical Hospital of Goiânia, Goiás, Brazil, treated with imatinib were enrolled in this study. Genotyping of GSTT1 and GSTM1 genes deletions were performed by qPCR and of GSTP1 gene was performed by RFLP-PCR. The frequency of GSTP1*1B, GSTT1 and GSTM1null polymorphisms were determined for all patients. The influence of each patient's genotypes was analyzed with the patient's response to imatinib treatment. Brazilian CML patients revealed GSTT1 and GSTM1 genes deletions. GSTT1 deletion was found in 19.3% of patients and GSTM1 deletion in 48.7% of patients with CML. GSTT1/GSTM1 deletion was found in 11.7% in Brazilian CML patients. The "G allele" of GSTP1*B, is associated with later cytogenetic response in imatinib therapy. While, the gene presence combined with GG genotype (GSTM1 present/GSTPI-GG) conferred a tend to a later cytogenetic response to patients. GSTP1*B and GSTT1/GSTM1null polymorphisms influence treatment response in CML. Brazilian CML patients presenting GSTP1 AA/AG genotypes alone and in combination with GSTT1 null reach the cytogenetic response faster, while patients presenting GSTP1-GG and GSTMI positive genotypes may take longer to achieve cytogenetic response. As a result, it allows a better prognosis, with the use of an alternative therapy, other than reducing treatment cost.

Condições socioeconômicas, de saúde e hábitos de vida dos catadores de material reciclável / Socio-economic, health and lifestyle conditions of recycled materials collectors

O presente estudo objetivou analisar as condições socioeconômicas, de saúde e hábitos de vida dos catadores de material reciclável em duas cooperativas no município de Guanambi - Bahia, Brasil. Trata-se de uma pesquisa de natureza descritiva, analítica e exploratória e todos os catadores foram entrevistados por meio de um questionário semiestruturado. Houve predominância da faixa etária entre 30 a 58 anos (80%) (p = 0,007), pardos (55%) (p = 0,02), casados (45%) (p = 0,02), com 2 a 3 filhos (55%) (p = 0,03). Os catadores informaram que começaram a trabalhar com menos de 18 anos (85%) (p = 0,002), e recebem uma renda mensal de até 1 salário mínimo com o trabalho de reciclagem (75%) (p = 0,02). Em relação às condições de saúde, 75% afirmaram que possuem Equipamentos de Proteção Individual (EPI) (p = 0,02), no entanto 80% (p = 0,02) não utilizaram o EPI e não consideram que seu trabalho pode provocar problema de saúde (75%) (p = 0,02). Percebe-se que os catadores de material reciclável precisam intensificar o uso dos EPI's, visando minimizar os danos com a saúde, além de melhorias na renda e na qualidade de vida.

The social, economic, health and lifestyle conditions of collectors of recyclable material from two coops in Guanambi, Brazil, are analyzed through a descriptive, analytic and exploratory research. All collectors were interviewed by a half-structured questionnaire. Predominant age bracket lay between 30 and 58 years (80%) (p= 0.007), brown colored (55%) (p= 0.02), married (45%) (p= 0.02), with 2 - 3 children (55%) (p= 0.03). Collectors informed that they started work when they were under 18 years old and received a month wage of up to one minimum wage, by collecting recycling material (75%) (p= 0.02). With regard to health conditions, 75% stated that had individual protection equipment (EPI) (p= 0.02), but 80% (p= 0.02) failed to use it, insisting that their work does not cause health problems (75%) (p= 0.02). It should be underscored that collectors should improve their earnings and life quality and use their equipments to minimize health risks.

Evaluation of polymorphisms in repair and detoxification genes in alcohol drinkers and non-drinkers using capillary electrophoresis.

Alcohol use disorder (AUD) causes about 3.3 million deaths around the world each year. It is the primary risk factor for the global burden of diseases in American countries. Long-term abuse of alcohol induces numerous molecular and biochemical changes in tissues exposed to alcohol. The toxic effects of alcohol are mediated by DNA damage through various mechanisms, such as induction of oxidative damage, DNA adducts, crosslinks, and DNA strand breaks. The main aim of the current study was to compare the frequency of SNP polymorphisms in XRCC1 (rs7997782) and GSTP1 (rs1695) genes involved in DNA repair of single strand breaks (SSB) and xenobiotic detoxification between alcohol addicts and a control group comprised of non-drinkers. Genetic polymorphisms were identified following allelic specific PCR designed to generate the amplicons containing the variants. Then amplicons were sequenced, and sequences were aligned against the human genome reference deposited in GenBank using the CLC Sequence Viewer software (version 7.6.1). The GG homozygotes in rs1695 (GSTP1) were significantly (p = 0.023) 3.8x more frequent among those with AUD when compared to the control group. No SNP variation was observed in rs7997782 (XRCC1). rs1695 variant has been associated with susceptibility to various diseases, including those related to alcohol consumption.

Avaliação química, ecotoxicológica e genotoxicológica de águas de cavas de mineração a céu aberto / Chemical, ecotoxicological and genotoxicological evaluation of waters of open pit mine lakes

RESUMO Lagos artificiais formados em áreas de mineração a céu aberto são unidades complexas e se constituem em passivos ambientais emergentes advindos da indústria da mineração. Embora estejam aumentando em número em várias partes do globo, são um problema ambiental recente e ainda pouco estudado, especialmente no tocante à qualidade e à toxicidade de suas águas, que podem apresentar riscos ambientais preocupantes, dado seu potencial de contaminação. O presente trabalho descreve os resultados da análise sazonal de aspectos químicos, ecotoxicológicos e genotoxicológicos das águas de três cavas de mineração de ouro desativadas localizadas em Mara Rosa, Goiás, Brasil. Amostras de água foram coletadas em perfil em duas estações climáticas distintas - inverno e verão - e foram analisadas quimicamente para determinação de metais e ânions. Também foram desenvolvidos testes ecotoxicológicos e ensaios cometa com peixes da espécie Danio rerio. Os resultados indicaram que as concentrações das espécies químicas analisadas se mostraram predominantemente crescentes no sentido da superfície ao fundo e mais elevadas durante a estação seca. As águas do Lago Azul demonstram estar quimicamente comprometidas, pois são ácidas e ricas em analitos potencialmente tóxicos, como alumínio, cádmio, chumbo, cobre, manganês, níquel e zinco. Não foram identificadas alterações ecotoxicológicas significativas para nenhuma das amostras analisadas, todavia, em termos genotoxicológicos, o Lago Azul apresentou danos ao DNA a partir da concentração de 25% na estação seca e de 50% na estação chuvosa.

ABSTRACT Artificial lakes formed in open pit mining areas are complex units and constitute emerging environmental liabilities arising from the mining industry. Although they are increasing in number in several parts of the world, they still form a recent and little studied environmental problem, especially regarding the quality and toxicity of its waters, which may represent worrying environmental risks. This study describes the results of the seasonal analysis of chemical, ecotoxicological and genotoxicological aspects of the waters of three disused gold mining pit lakes located in Mara Rosa, Goiás, Brazil. Samples were collected in profile in two different climatic seasons, winter and summer, and were chemically analyzed to determine their load of metals and anions. Ecotoxicological Tests and Comet Assays were also developed with fish of the Danio rerio species. The results indicated that the concentrations of the chemical species analyzed were predominantly increased in the surface-bottom direction and higher during the dry season. Lago Azul waters have been shown to be chemically more compromised as they are acidic and rich in potentially toxic analytes such as aluminum, cadmium, lead, copper, manganese, nickel, and zinc. No significant ecotoxicological alterations were identified for any of the analyzed samples, although, in genotoxicological terms, Lago Azul presented DNA damage from concentrations of 25% in the dry season and 50% in the wet season.

Cytogenetics of two hylid frogs from Brazilian Cerrado.

Cytogenetic data can be useful for taxonomic and phylogenetic studies, as well as to provide information about chromosome evolution. Therefore, it may help design conservation priorities for some threatened species, such as anurans. Herein, we describe the karyotypes of Scinax constrictus and Ololygon centralis, native endemic species from the Brazilian Cerrado. Chromosome preparations for both species were stained with Giemsa for morphological analyses and then impregnated by the Ag-NOR method for localization of the nucleolar organizer region (NOR). Both species had 24 chromosomes, as confirmed by meiotic analyses, which showed 12 bivalents. Chromosome morphologies presented the same pattern for Scinax and Ololygon compared to species already karyotyped in both genera. The NOR was interstitially located in the long arm of pair 7 in S. constrictus, whereas in O. centralis it was found near the centromere in the long arm of pair 1, thus diverging from what is commonly found for other Ololygon species. Therefore, we provide the first description of the karyotype of O. centralis and the first report of the localization of the NOR for the karyotype of both species. Our study increases the cytogenetic knowledge in species of the genera Scinax and Ololygon, and provide support for further studies on the taxonomy, ecology, and evolution of hylid anurans.

Small de novo CNVs as biomarkers of parental exposure to low doses of ionizing radiation of caesium-137.

The radiological accident in Goiania in 1987 caused a trail of human contamination, animal, plant and environmental by a radionuclide. Exposure to ionizing radiation results in different types of DNA lesions. The mutagenic effects of ionizing radiation on the germline are special concern because they can endures for several generations, leading to an increase in the rate of mutations in children of irradiated parents. Thus, to evaluate the biological mechanisms of ionizing radiation in somatic and germline cells, with consequent determination of the rate mutations, is extremely important for the estimation of genetic risks. Recently it was established that Chromosomal Microarray Analysis is an important tool for detecting wide spectra of gains or losses in the human genome. Here we present the results of the effect of accidental exposure to low doses of ionizing radiation on the formation of CNVs in the progeny of a human population accidentally exposed to Caesium-137 during the radiological accident in Goiânia, Brazil.

Validation of Comet assay in Oregon-R and Wild type strains of Drosophila melanogaster exposed to a natural radioactive environment in Brazilian semiarid region.

Natural radiation of geological origin is a common phenomenon in Brazil, a country where radioactive agents such as uranium may be often found. As an unstable atom, uranium undergoes radioactive decay with the generation of a series of decay by-products, including radon, which may be highly genotoxic and trigger several pathological processes, among which cancer. Because it is a gas, radon may move freely between cracks and gaps in the ground, seeping upwards into the buildings and in the environment. In this study, two Drosophila melanogaster Meigen (Diptera, Drosophilidae) strains called Oregon-R and Wild (collected in a non-radioactive environment) were exposed to atmospheric radiation in the Lajes Pintadas city, in the semiarid zone of northeastern Brazil. After six days of environmental exposure, the organisms presented genetic damage significantly higher than that of the negative control group. The genotoxic effects observed reinforce the findings of other studies carried out in the same region, which warn about the environmental risks related to natural radioactivity occurrence. The results also validate the use of the Comet assay in hemocytes of D. melanogaster as a sensitive test to detect genotoxicity caused by natural radiation, and the use of a recently collected D. melanogaster strain in the environmental of radon.

Genotoxic and Cytotoxic Effects of Antiretroviral Combinations in Mice Bone Marrow.

Commonly used guidelines for the management of human immunodeficiency virus (HIV) infection (highly active antiretroviral therapy, HAART) include drug combinations such as tenofovir disoproxil fumarate (TDF) + lamivudine (3TC) and combivir [zidovudine (AZT) + 3TC] + efavirenz (EFV). These combinations may enhance the genotoxic effects induced by such drugs individually, since the therapy requires lifelong adherence and the drugs have unknown effects during treatment. Thus, the evaluation of the benefits and risks of HAART is of great importance. In order to assess the cytotoxic and genotoxic potential of three concentrations of each of the antiretroviral combinations TDF + 3TC (800 + 400, 1600 + 800, and 3200 + 1600 mg/kg body weight, BW) and combivir + EFV (200 + 100 + 400, 400 + 200 + 800, and 800 + 400 + 1600 mg/kg BW) after two exposure periods (24 h and 48 h), in the present study the in vivo comet assay (single-cell gel electrophoresis) and the mouse bone marrow micronucleus test were used. Neither TDF + 3TC nor combivir + EFV induced DNA damage at any concentrations tested after 24 h or 48 h using the comet assay. After 24 h, both combinations increased the micronucleus frequency at all concentrations tested. After 48 h, combivir + EFV increased the micronucleated polychromatic erythrocyte (MNPCE) frequency at the two highest concentrations tested. Polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE) ratio was high for both combinations, suggesting that they can be mitogenic. Since genotoxicity may be related to carcinogenesis, it is necessary to conduct further studies to verify the long-term mutagenic effects of these drugs.

Genotoxic, Cytotoxic, Antigenotoxic, and Anticytotoxic Effects of Sulfonamide Chalcone Using the Ames Test and the Mouse Bone Marrow Micronucleus Test.

Chalcones present several biological activities and sulfonamide chalcone derivatives have shown important biological applications, including antitumor activity. In this study, genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of the sulfonamide chalcone N-{4-[3-(4-nitrophenyl)prop-2-enoyl]phenyl} benzenesulfonamide (CPN) were assessed using the Salmonella typhimurium reverse mutation test (Ames test) and the mouse bone marrow micronucleus test. The results showed that CPN caused a small increase in the number of histidine revertant colonies in S. typhimurium strains TA98 and TA100, but not statistically significant (p > 0.05). The antimutagenicity test showed that CPN significantly decreased the number of His+ revertants in strain TA98 at all doses tested (p < 0.05), whereas in strain TA100 this occurred only at doses higher than 50 µg/plate (p < 0.05). The results of the micronucleus test indicated that CPN significantly increased the frequency of micronucleated polychromatic erythrocytes (MNPCE) at 24 h and 48 h, revealing a genotoxic effect of this compound. Also, a significant decrease in polychromatic/normochromatic erythrocyte ratio (PCE/NCE) was observed at the higher doses of CPN at 24 h and 48 h (p < 0.05), indicating its cytotoxic action. CPN co-administered with mitomycin C (MMC) significantly decreased the frequency of MNPCE at almost all doses tested at 24 h (p < 0.05), showing its antigenotoxic activity, and also presented a small decrease in MNPCE at 48 h (p > 0.05). Additionally, CPN co-administered with MMC significantly increased PCE/NCE ratio at all doses tested, demonstrating its anticytotoxic effect. In summary, CPN presented genotoxic, cytotoxic, antigenotoxic, and anticytotoxic properties.

Allele frequencies of fifteen STR loci in a population from Central Brazil.

Allele frequencies for 15 short tandem repeats included in Powerplex 16 Kit (Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818) were determined in a sample of 429 unrelated individuals from the population of Goiânia, Goias, Central Brazil. Determination of the allele frequencies as well as of several commonly used statistics in forensic and paternity testing were defined. The forensic parameters presented high values and the most polymorphic loci were Penta E, following FGA and D18S51. The exact test demonstrated that the fifteen loci analyzed in the population of Goiania have no deviation from Hardy-Weinberg equilibrium (P > 0.05).

Karyotype characterization and nucleolar organizer regions of marsupial species (Didelphidae) from areas of Cerrado and Atlantic Forest in Brazil

The karyotypes of 23 specimens belonging to 16 species from nine genera of Brazilian marsupials (family Didelphidae) were studied. The animals were collected in eight localities of Cerrado or Atlantic Forest biomes in the states of Goiás, Tocantins and São Paulo. The karyotypes were analyzed after conventional Giemsa staining and silver staining of the nucleolus organizer regions (Ag-NORs). New karyotypic data were obtained for Gracilinanus microtarsus (2n = 14, FN = 24),Marmosops paulensis (2n = 14, FN = 24), Micoreus paraguayanus (2n = 14, FN = 20) and Monodelphis rubida (2n = 18, FN = 32) and are discussed in detail. The karyotypes of G. microtarsus, M. paulensis and M. paraguayanus include three large pairs of submetacentrics (pairs 1, 2 and 3) and a medium-sized metacentric or submetacentric pair 4. Pairs 5 and 6 are small submetacentrics in G. microtarsus and M. paulensis and acrocentrics in M. paraguayanus. M. paulensis presented a single Ag-NOR in pair 6 (6p6p), while M. paraguayanus exhibited multiple Ag-NORs in pairs 5 and 6 (5pq5pq6p6p). There was variation in size and morphology of the sex chromosomes among these species. Monodelphis rubida presented a karyotype with 2n = 18 and FN = 32 composed of a large submetacentric pair 1, a medium-sized metacentric pair 2 and six pairs of submetacentrics (pairs 3 through 8). The X was a small acrocentric and the Y was dot-like. A single Ag-NOR bearing pair (5p5p) characterized M. rubida. Relevant karyotypic information was obtained for 19 specimens belonging to 12 species collected in areas sampled for the first time [Caluromys lanatus and C. philander (2n = 14, FN = 20), Gracilinanus emiliae (2n = 14, FN = 24), Marmosa murina, Metachirus nudicaudatus and Micoureus demerarae (2n = 14, FN = 20), Monodelphis americana (2n = 18, FN = 32) and M. domestica (2n = 18, FN = 20), and Didelphis marsupialis, Philander frenata, P. opossum and P. sp (2n = 22, FN = 20)]. Although the karyotypes...

Population structure of Eupemphix nattereri (Amphibia, Anura, Leiuperidae) from Central Brazil

This study reports on 156 specimens of the amphibian Eupemphix nattereri, a widely distributed leiuperid, obtained from 11 municipalities of central Brazil. The extent of genetic variation was quantified by determining the mean number of alleles per locus and the proportion of polymorphic loci. An analysis of molecular variance (AMOVA) was performed on the random amplified polymorphic DNA (RAPD) haplotypes. The genetic distances obtained by calculating pairwise phist among local samples were used to determine population relationships using the unweighted pair-group method (UPGMA) and non-metric multidimensional scaling (NMDS). The cophenetic correlation was calculated to confirm agreement between the genetic matrix and the unweighted pair group method with averages (UPGMA) dendrogram. To determine if genetic distances were correlated to geographical distances we constructed pairwise genetic distance and geographical distance matrices and compared them using the Mantel test. The AMOVA results indicated significant genetic differences (p < 0.001) between E. nattereri populations, representing 69.5 percent of the within population genetic diversity. The Mantel test showed no significant correlation (r = 0.03; p = 0.45) between the genetic and geographical distance matrices. Our findings indicate that the genetic variation of E. nattereri populations was randomly distributed in geographic space and that gene flow for this species is probably structured at spatial scales smaller than those between our samples.