Chionactis occipitalis and C. annulata. In addition, we recommend retention of two subspecific designations within C. annulata (C. a. annulata and C. a. klauberi) that reflect regional shifts in both genetic and phenotypic variation within the species. Our results highlight the difficultly in validating taxonomic boundaries within lineages that are evolving under a time-dependent, continuous process.Accurate delineation of lineage diversity is increasingly important, as species distributions are becoming more reduced and threatened. During the last century, the subspecies category was often used to denote phenotypic variation within a species range and to provide a framework for understanding lineage differentiation, often considered incipient speciation. While this category has largely fallen into disuse, previously recognized subspecies often serve as important units for conservation policy and management when other information is lacking. In this study, we evaluated phenotypic subspecies hypotheses within shovel-nosed snakes on the basis of genetic data and considered how evolutionary processes such as gene flow influenced possible incongruence between phenotypic and genetic patterns. We used both traditional phylogenetic and Bayesian clustering analyses to infer range-wide genetic structure and spatially explicit analyses to detect possible boundary locations of lineage contact. Multilocus analyses supported three historically isolated groups with low to moderate levels of contemporary gene exchange. Genetic data did not support phenotypic subspecies as exclusive groups, and we detected patterns of discordance in areas where three subspecies are presumed to be in contact. Based on genetic and phenotypic evidence, we suggested that species-level diversity is underestimated in this group and we proposed that two species be recognized,  As a result, the subspecies rank has largely fallen into disuse. However, within the field of conservation biology, historically described subspecies often serve as important units for conservation policy and management when other information is lacking Although scientific debates over species concepts persist, the notion that species exist as separately evolving lineages, presumably under various stages of speciation, is central to discussions related to intraspecific diversity. While most contemporary species concepts agree conceptually that species are separately evolving “metapopulation lineages” . While numerous studies have demonstrated that phenotypic subspecies often fail to correspond to monophyletic genetic lineages, the generality of this pattern across taxa remains controversial Multi-locus genetic surveys that examine larger portions of the genome have garnered broad support as a preferred strategy for studying lineage divergence and genetic differentiation Chionactis occipitalis) and evaluate whether the phenotypic subspecies correspond to genetic groups on the basis of multiple selectively neutral loci. Chionactis occipitalis is a small colubrid snake occupying the arid valley floors and bajadas of the Mojave and Sonoran deserts in southwestern North America. Four subspecies have been recognized on the basis of variation in banding pattern and coloration throughout the distribution C. o. occipitalis (Mojave shovel-nosed snake), C. o. annulata (Colorado shovel-nosed snake), C. o. talpina (Nevada shovel-nosed snake), and C. o. klauberi (Tucson shovel-nosed snake). Chionactis occipitalis klauberi is of particular conservation interest. This subspecies occurs along the eastern edge of the species range in south central Arizona and has experienced declines in population size and range over the past several decades due to alteration and loss of habitat through agricultural and urban development. These factors prompted a petition to list the subspecies as threatened or endangered under the U.S. Endangered Species Act C. o. klauberi has been elevated to “candidate status” (Priority 3) under the ESA C. occipitalis may hinder further management actions C. o. klauberi was described despite the assumption that it formed a broad zone of intergradation with C. o. annulata across Arizona  specimens display intermediate morphological characteristics between the two subspecies. As a result, various authors have treated the range of C. o. klauberi differently C. o. klauberi was never clearly delineated.In this study, we investigate the species-subspecies boundaries of Western shovel-nosed snake  sequence data from 81 snakes and variation in 14 morphological characters (excluding color pattern) from 1543 snakes from across the range of the species. Sequence data revealed a deep phylogenetic split within the species with substantial genetic structuring within the two main clades. Most relevant to this study, mtDNA haplotypes of individuals identified as C. o. klauberi were intermixed with haplotypes belonging to C. o. annulata. Broad morphological overlap was detected between the four subspecies, and only minor geographic structure was evident for C. o. klauberi. Taken together, Wood et al.C. o. klauberi may represent a “morphological endpoint of clinal variation without concordant phylogenetic distinction”, but encouraged further hypothesis testing with nuclear loci to evaluate the mtDNA gene tree patterns and possible ecological/adaptive differences that may exist despite non-exclusive mtDNA genetic variation.Previous research by Wood C. occipitalis, placing emphasis on evaluating the genetic distinctiveness between klauberi and the neighboring annulata phenotypes across the Colorado and Sonoran deserts. Our objectives addressed three gaps in the current knowledge. First, we tested whether the nuclear genome shows patterns of structure that are consistent with the ranges of the four recognized phenotypic subspecies. Second, we tested for intergradation between annulata and klauberi, and identified the geographic location of a contact zone. Third, we quantified the extent to which the nuclear genetic clusters within C. occipitalis are isolated. Finally, we provide information from multiple genetic and phenotypic datasets to suggest taxonomic recommendations consistent with lineage evolution within this group.Here, we build on this research using 11 nuclear microsatellite loci and additional mtDNA sequence data to further evaluate the genetic groups within Chionactis occipitalis from localities throughout California, Arizona, Nevada, Baja California and Sonora, Mexico. Sampled localities represent the full range of the species and include all formally recognized subspecies  for use as an outgroup taxon in phylogenetic analyses. We also included the 80 mtDNA sequences generated from our previous study  for 140 individuals of Microsatellite library construction was performed in the Sequencing and Genotyping Facility at Cornell University Core Laboratory Center (CLC) using standard development techniques modified from We tested 32 loci for variability using six individuals from Arizona and California collection sites. Of the 32 loci, we selected 11 that were variable, amplified consistently, and yielded reliable genotyping scores. We divided these loci into three groups and simultaneously amplified 3–4 loci within each group using a Qiagen multiplex PCR kit in 10 µL reactions containing 5 µL of Qiagen multiplex PCR Master Mix, 1 µL primer mix (containing 2 µM of each primer), 1 µL Q-solution and 2 µL of RNase-free water. Amplified products were run on an ABI 3100 and performed in the CSUPERB Microchemical Core Facility at San Diego State University. We used GENE-MARKER v1.90 (SoftGenetics) to edit the raw allelic data and score allele sizes. We used MICROCHECKER et al. Chionactis or closely related sister species, we did not use fossil evidence to calibrate our clock-based analyses. We instead employed fixed substitution rates for the ND1 and 16S rRNA flanking regions that were estimated for other squamates  awtyPp ≥ 0.95) to infer mtDNA relationships and evaluate support for phylogenetic lineages within C. occipitalis.We used BEAST v1.8.0 HO), expected heterozygosity (HE), number of alleles (NA), and allelic richness (AR). These genetic indices were calculated using GENALEX v6.41 Genetic diversity was assessed at each microsatellite locus and genetic cluster by estimating the observed heterozygosity  that maximizes the within-cluster Hardy-Weinberg and linkage equilibria.We used two Bayesian genotypic clustering analyses to infer discrete genetic clusters within the dataset at two different spatial scales . First, we conducted a range-wide analysis of population structure using structure analyses, the number of clusters (K) that putatively best explains the dataset is inferred from the posterior probability distribution of the data given the range of K specified by the user, P(X|K). We ran the admixture model with an uncorrelated frequency model. We inferred the number of clusters (K) by comparing the results from the mean lnP(D|K) score against the Kmax (i.e. where the lnP(D|K) curve plateaus) and the Δ K criterion structure analyses within each inferred cluster structure analyses, we estimated the probability of one through 10 clusters (K) using 1,000,000 iterations of the MCMC algorithm following a burn-in of 500,000 iterations, with each run replicated ten times to check for the consistency across the Markov chains.For the geneland analyses, all parameters  are co-estimated simultaneously by the MCMC algorithm. Once completed, the most probable number of clusters and their geographic boundaries are inferred from the highest average posterior probability density (PPD) of genetic clusters. We followed the recommendations given by Guillot et al. K  =  1–5 with 10,000,000 MCMC iterations and a thinning of 250. Coordinates from each individual were used to run the spatial model and the potential error for spatial coordinates was set at 3 m. Post-processing of the MCMC outputs with the highest PPD followed a burn-in of 10,000 in order to obtain posterior probabilities of cluster membership for each individual.For FST estimates and isolation-by-distance (IBD) analyses as secondary methods to summarize microsatellite variation and to identify significantly differentiated genetic clusters inferred from the assignment tests. Pairwise genetic distances among clusters were based on allele frequency differences using FST following â developed by Rousset geneland (see results) that span the geographic range of C. o. annulata and C. o. klauberi. Because clustering approaches can sometimes mistake IBD for hierarchical population structure geneland could be explained by IBD. We accomplished this by testing the association between genetic distances and cluster membership with geographic distances treated as a covariate. All IBD analyses were performed in IBDWS 3.21 We used emN), which is the effective rate at which genes enter a population, as a criterion for defining the relative strength of genetic isolation between the range-wide clusters identified using structureC. occipitalis to obtain a measure of migration on a scale of generations, which was based on demographic observations of a similar fossorial species, Chilomeniscus straminus . We used the state of the Markov chain from a long run using final priors to seed three separate runs , after a series of initial runs to determine the most appropriate search parameters that maximized mixing.We used the population migration rate , maximum migration prior value (m  =  3), and maximum time of population splitting (t  =  15). A total of 100,000 steps were retained with a step length of 20 and the first 150,000 steps discarded as burn-in. We ordered the relative strength of the 2emN estimates following recommendations of emN ≤ 1, moderate isolation when 1 < 2emN ≤ 5 and weak when 5 < 2emN ≤ 25.For each analysis, we used the following settings for the prior distributions on population parameters: 160 chains with a geometric heating scheme (g1  =  0.99 and g2  =  0.5), maximum scalars for theta . Individuals of clade B occurred farther south in the range of C. o. occipitalis and were restricted to the Coachella Valley in California. A second lineage, hereafter called the ‘Sonoran lineage,’ was composed of two geographically disjunct clades (clades C and D). Individuals of clade C were primarily located in the Sonoran Desert of central Arizona encompassing the range of C. o. klauberi and the northeastern range of C. o. annulata. Clade D encompassed the range of C. o. occipitalis within the southeastern portion of the Mojave Desert in California and Arizona. A third clade (clade E) was routinely placed as sister to the Sonoran lineage, but posterior probabilities supporting this relationship were weak (Pp < 0.50). This clade was located in the Sonoran Desert of western Arizona, partially encompassed the ranges of C. o. annulata and annulata-klauberi ‘intergrades’, and was geographically nested in between clade C and D. Finally, the southernmost lineage, hereafter called the ‘Colorado lineage,’ occupied the southwestern portion of the range of C. o. annulata and was distributed in the lower Colorado River subdivision of the Sonoran Desert in California and Baja California (clade F), and southwestern Arizona and Sonora, Mexico (clade G).The mtDNA Bayesian analyses yielded a well-resolved phylogeny. Most notably, C. occipitalis occurred in the Pliocene to early Pleistocene , and observed heterozygosity for each locus ranged from 0.419 and 0.812, with an average value of 0.665 for all loci . There wstructure analyses strongly indicated a sharp plateau at Kmax =  3 as an approximate estimate for K under the admixture model. This result was corroborated by the Δ K criterion . Unlike the mtDNA patterns, C. o. occipitalis from the eastern Mojave Desert in California and Arizona were grouped with other geographically proximate individuals of C. o. occipitalis rather than clustering with individuals of C. o. annulata in the Sonoran Desert as inferred using the mtDNA data (see mtDNA clade D). The Colorado cluster encompassed the range of C. o. annulata in California, Arizona, and Mexico and portions of the previously reported intergrade zone. Finally, the Sonoran cluster occupied the presumed range of C. o. klauberi, portions of the previously reported intergrade zone, and a small portion of the range of C. o. annulata in west-central Arizona. Admixture among the Colorado and Sonoran clusters was apparent in the vicinity of the reported intergrade zone. Individual snakes sampled throughout this region tended to exhibit lower probability assignments  and lowest pairwise comparison was between the Mojave and Colorado clusters (FST  =  0.054). We detected a significant isolation-by-distance pattern for all collection sites combined  and for the three geneland clusters across Arizona . However, partial Mantel tests revealed a significant association between clusters and genetic distance  after correcting for the geographical distances. This result suggests allele frequency differences between clusters are not due to geographic distance alone.We used pairwise tructure . The higstructure analyses (emN) occurring from the Sonoran cluster to the Mojave cluster compared to the opposite direction. Strong to moderate genetic isolation was detected between the Colorado and Sonoran clusters, and higher gene migration was inferred from the Sonoran cluster into the Colorado cluster. We detected negligible variation among 2emN values estimated between the Mojave and Colorado clusters, where gene migration was strongly restricted in both directions.The IMa2 genetic isolation estimates detected asymmetric rates of gene migration between clusters obtained from analyses . ModeratChionactis occipitalis. This significantly expanded geographic sampling and the addition of nuclear loci, allowed us to better characterize the genetic structure within C. occipitalis. Our study did not find support for C. occipitalis subspecies as exclusive genetic groups. Patterns of discordance between the phenotypic and genotypic boundaries were detected in areas where C. o. occipitalis, C. o. annulata, and C. o. klauberi are presumed to be in contact. The only exception to this pattern was C. o. talpina, which was entirely nested within C. o. occipitalis on the basis of both mtDNA and microsatellite datasets.In this study, we used both mitochondrial DNA and 11 microsatellite loci to further assess whether patterns of population genetic structure are concordant with the spatial structuring of phenotypic variation that originally led to the subspecies descriptions within Differences in the spatial distribution of genetic and phenotypic variation are not unexpected when the boundaries of divergent lineages are parapatric. For instance, even though lineages may have diverged to the point of having some diagnosable differences (both phenotypic and genetic), these lineages may not be sufficiently reproductively isolated from each other to prevent gene flow in secondary contact. Although gene flow is often a homogenizing force C. occipitalis, there were a large proportion of individuals within C. o. occipitalis, C. o. annulata, and C. o. klauberi that were assigned to the Mojave, Colorado, and Sonoran clusters  with C. o. annulata along the northwestern region of the Salton Sea at the Imperial-Riverside county line. Assignment probabilities of C. o. annulata individuals within the northern Colorado Desert are consistent with some genetic admixture in this region. Although our gene migration estimates indicate strong isolation between Mojave  and Colorado (annulata) clusters, these estimates are greater than zero suggesting that complete isolation between them has not been achieved.These results highlight the difficultly in delimiting taxonomic boundaries within lineages that are evolving under a time-dependent, continuous process  clusters , respectC. o. klauberi and C. o. annulata were not genetically exclusive. The cluster containing all samples of C. o. klauberi  also included samples within the putative intergrade zone and a few C. o. annulata from northern La Paz County in Arizona. The key morphological characteristic used to diagnose C. o. klauberi, dark maculations infused within the red secondary bands that contact dorsally C. o. klauberi. He further noted several localities with “klauberi influences”, in particular “the Wickenburg area” and “east of Aguila” in northwestern Maricopa County, that corroborate well with the inferred cluster boundaries . The multilocus perspective presented here (mtDNA & microsatellites), provided evidence for at least three divergent groups that have broadly concordant boundaries with three of the phenotypically recognized subspecies . Because taxonomy should be consistent with the evolutionary history of a group, it is possible that these genetic groups represent independent evolutionary lineages and therefore constitute distinct species  klauberi exhibited the most discordance between the spatial distribution of genetic and phenotypic patterns, but this discordance may simply reflect the recency of its divergence , asymmetric gene flow along the zone of contact, and ambiguous delineation of the western phenotypic boundary. Therefore, some discordance within this geographic region would be expected. Nonetheless, we feel that the current phenotypic and genetic data available for klauberi at this time allows neither definitive acceptance nor rejection of species status for the lineage. Therefore, we propose the most advantageous and conservative strategy is to recognize two species in the group that conform to the divergent and phenotypically distinct Mojave and Colorado-Sonoran clusters: the Mojave shovel-nosed snake, Chionactis occipitalis  C. annulata  C. occipitalis possess more brown primary crossbands and no secondary red crossbands, while C. annulata have fewer black primary crossbands and possess red-to-orange secondary crossbands annulata and klauberi as subspecific designations within C. annulata in keeping with the genetic structure that was recovered across Arizona, while placing talpina in synonomy with C. occipitalis. In this framework, maintenance of the subspecific nomenclature within C. annulata provides putative species hypotheses that can be examined in more detail, and provides important taxonomic handles for conservation policy and wildlife management agencies.Previous authors C. occipitalis mtDNA haplotypes from the eastern Mojave Desert (mtDNA clade D) were nested within the mtDNA Sonoran lineage, but the microsatellite data assigned these same samples with high probability to all other C. occipitalis samples within the Mojave Desert. In the same way, haplotypes of C. a. annulata from west-central Arizona (clade E) formed a well-supported mtDNA clade, but could not be grouped with confidence to either the Colorado or Sonoran lineages. However, nuclear genotypes of these same samples were assigned to the genetic cluster containing the majority of all other C. a. annulata samples. Thus, it appears that phylogenetic relationships based on mtDNA for clades D and E are misleading and suffer from introgression at lineage boundaries.Because the genetic structure results differed slightly between marker types (mtDNA vs microsatellites), it is important to note where they differ and what these data reveal about subdivision within this species complex. Phylogenetic analysis of the mtDNA sequencing data revealed strong support for regionally distinctive lineages that sorted by desert basins , with some overlap of lineages extending across desert boundaries. Likewise, our range-wide assignment tests based on microsatellite data confirmed the presence of at least three geographically cohesive clusters. The spatial discordance among gene tree lineage and cluster boundaries was narrow and occurred along two zones of contact. First, et al.Wood et al.C. occipitalis and C. annulata is defined by an abrupt transition in elevation, temperature, total rainfall, vegetation and terrain features known as the Mojave and Sonoran ecotone The southwestern deserts of North America are marked by phylogeographical breaks that result in distinctive genetic groups within species occupying this region. By comparing the phylogeographical signature of co-distributed taxa, Wood Chionactis occipitalis and C. annulata. For now, we recommend retention of the names annulata and klauberi as subspecific designations within C. annulata that conform to the patterns of genetic structure , while placing C. o. talpina in synomony with C. occipitalis. Whether additional species should be recognized within C. annulata will depend on future characterization of morphological and genetic variation within the zone of contact.We have used data from a combination of mtDNA sequences and 11 microsatellite loci to study the genetic structure and phylogeography of Western shovel-nosed snakes. We evaluated whether phenotypic subspecies definitions were supported by genetic data, and did not find exact spatial congruence with the original subspecies hypotheses. Despite these results, both mitochondrial and nuclear data sets corroborated similar divergence patterns for at least three groups that are in differential contact along common distributional boundaries with low to moderate levels of gene exchange. Based on the available evidence, we suggested that species-level diversity is underestimated in this group. We proposed that at least two species be recognized, C. a. annulata and C. a. klauberi, continued recognition of each may assist in preserving the potential for future evolutionary change within this species. Further, these results may be particularly valuable for regional conservation planning in south-central Arizona. In addition to recovering unique genetic variation primarily within the range of C. a. klauberi, we were also able to better quantify the geographic extent of genetic intergradation with C. a. annulata. Instead of existing as a gradual cline over a broad area, the zone of intergradation was narrower and existed as steep shifts in allele frequencies. Gene flow between divergent lineages can create novel variation that can facilitate adaptive evolution Because species require genetic diversity to respond to changing environmental conditions, conservation strategies aimed at protecting regional genetic groups can maximize the species' evolutionary potential and resilience Figure S1Mojave lineage pruned from the full mtDNA phylogeny, shown in the upper left. Numbers at nodes represent posterior probability support values for individual clades.(PDF)Click here for additional data file.Figure S2Sonoran lineage pruned from the full mtDNA phylogeny, shown in the upper left. Numbers at nodes represent posterior probability support values for individual clades.(PDF)Click here for additional data file.Figure S3Colorado lineage pruned from the full mtDNA phylogeny, shown in the upper left. Numbers at nodes represent posterior probability support values for individual clades.(PDF)Click here for additional data file.Figure S4Results from the mean lnP(D|Kmax and the Δ ) score against the KK criterion of Evanno et al. [72].(PDF)Click here for additional data file.Figure S5Posterior density of the number of clusters (Kgeneland.) from the MCMC analysis of genetic structure across Arizona using (PDF)Click here for additional data file.Table S1Tissue samples of Chionactis occipitalis used in this study along with locality information, Dryad Digital Repository, doi:10.5061/dryad.77rf2.(XLSX)Click here for additional data file.


This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis fluoroquinolones in complex samples.An amperometric magneto-immunosensor (AMIS) for the detection of residues of fluoroquinolone antibiotics in milk samples is described for the first time. The immunosensor presented combines magnetic beads biomodified with an antibody with a broad recognition profile of fluoroquinolones, a haptenized enzyme and a magnetic graphite–epoxy composite (m-GEC) electrode. After the immunochemical reaction with specific enzyme tracer, the antibody biomodified magnetic beads are easily captured by an electrode made of graphite-epoxy composite containing a magnet, which also acts as transducer for the electrochemical detection. In spite of the complexity of milk, the use of magnetic beads allows elimination of potential interferences caused by the matrix components; hence the AMIS could perform quantitative measurements, directly in these samples, without any additional sample cleanup or extraction step. The immunosensor is able to detect up to seven different fluoroquinolones far below the MRLs defined by the UE for milk; for example ciprofloxacin is detected directly in milk with an IC In this sense, antibiotics are used for therapy, prevention and control of diseases, as well as growth promoters. The use of antibiotics is a determinant factor in the selection of resistance in bacteria and the dosage of antimicrobials is closely related to the rate of antimicrobial resistance developed in bacteria –3. DevelFluoroquinolones (FQs) are synthetic antibiotics with a broad spectrum of antimicrobial activity that have been extensively used in veterinary medicine to treat or prevent bacterial infections in food-producing animals, aquaculture, and also pets. Several FQs are available for therapy of animals in many countries; however, the usage of these antibiotics differs greatly as regards animal species, indications, label indications, and geographic spread . Becauseet al. have been developed an immunosensor for the detection of tetracyclines antibiotics in milk using magnetic beads. In this case, they used a homemade magnet holder block to capture the magnetic beads onto a disposable commercial screen-printed electrodes, achieving good detectability in milk diluted two times in buffer  + B, where A is the maximal signal, B the minimum signal, C the concentration producing 50% of the maximal signal, and D is the slope at the inflection point of the sigmoid curve. LOD values were obtained as 90% of A value, the maximal signal.2.8.Ca  and measured using the ELISA and m-ELISA, as described above. For all fluoroquinolones it was possible to build standard curves that fitted to the four-parameter equation mentioned above. The cross-reactivity values were calculated according to the equation: (IC50 Ciprofloxacin/IC50 tested compounds) × 100.Stock solutions of different fluoroquinolone antibiotics were prepared in 0.05 M NaOH at a concentration of 0.01 M. Standard solutions were prepared for each analyte in PBST2.9.For electrochemical readout (AMIS), only the magneto enzyme tracer format (m-ETf), comprising the reagents 11-HRP and Ab171-MB, was assessed. A schematic representation of the whole procedure is shown in Ca) were placed in the polypropylene tubes and mixed with the Ab171-MB suspension  and the solution of 11-HRP , in this specific order, and incubated for 30 min at RT with gentle stirring. Afterwards, the magnetic beads were washed with PBST (3 × 300 μL), resuspended in PBST (160 μL) and captured by dipping the m-GEC electrode into the solution in the tubes just before the amperometric measurement in order to prevent undesired desorption of the protein complexes  and the enzyme substrate . The response was determined by polarizing the working electrode surface (m-GEC) at −0.150 V. As well as for ELISA and m-ELISA, the standard curve was fitted to a four-parameter logistic equation according to the formula (see above).Ciprofloxacin standard solutions . Different dilutions of whole bovine milk were employed for generate ciprofloxacin standard curves. The curves obtained using ELISA, m-ELISA and AMIS procedures were compared with the curve prepared in PBST3.2O2 to H2O) at the same time that hydroquinone (HQ) is oxidized to quinone (Q), and subsequently reduced again by the electrode applied potential . The samples or standards of fluoroquinolones are mixed with the immunoreagents listed above, and after incubation, the immunocomplexes linked to the magnetic beads are separated from the free fraction of the enzyme tracer with the support of a magnetic rack and later on captured with the m-GEC see . The amptial see .3.1.50 and LOD below the MRLs established by the UE for several tissues [Recently, our group has developed generic antibodies against fluoroquinolones and an indirect competitive ELISA assay able to detect, at least, the most important fluoroquinolones used in the veterinary field with values of IC tissues . This ELf, coating antigen and ETf, enzyme tracer formats) The CAf ELISA assay, performed using plates coated with the competitor 11-BSA and Ab171-HRP in solution , showed a very good detectability for ciprofloxacin with an IC50 of 0.84 μg/L and a LOD of 0.06 μg/L. The enzyme tracer was evaluated using an ETf ELISA, using plates coated with the antifluoroquinolones antisera (As171) without previous purification . Again, a very good detectability was accomplished , demonstrating that there was no loss of activity after labeling of the specific antibody. On the other hand, the similarity found between both formats indicates that the assay is not dependent on the format.The yield of the bioconjugation for 11-HRP (63%) was higher than for the Ab171-HRP (47%), maybe due to a minor purification steps involved during the process. Both ELISA formats show a very close detectability in terms of IC3.2.The main advantages of the use of magnetic beads in electrochemical immunosensors are their easy manipulation, the increase in the surface area, faster assay kinetics, improved washing and separation steps, the possibility of renewing the surface of the electrode and perspectives for system automation and miniaturization ,12. In tThe yield of the biomodification was checked by testing the protein content from the supernatant before and after the bioconjugation using UV at 280 nm, by interpolation the value of supernatant into a calibration line built using the corresponding protein (Rabbit IgG and BSA). In both cases was higher than 80%.f was selected to continue with the development of the AIMS, using the Ab171-MB.As in the case of ELISA, the m-ELISA parameters found were similar for both formats. Since production of 11-HRP was more cost and time effective, the TE3.3.f. Calibration curves for fluoroquinolones regulated by the UE were built in PBSTCa buffer and were run using ELISA and m-ELISA. For both assays, the standard curves for all fluoroquinolones were fitted to the four-parameter equation mentioned above with very good correlation coefficients (≥0.985). 50, cross reactivity percentage, with respect to the ciprofloxacin and the MRLs values, fixed by UE for milk. Detectability were very similar for both assays and close to the indirect ELISA previously reported [50 for m-ELISA respect to the microplate-based ELISA except for the oxolinic acid. In all cases the IC50 values were lower than 10 μg/L and seven fluoroquinolones congeners were recognized at concentrations below to the MRLs established by the UE in milk.In order to prove that the generic pattern of fluoroquinolone recognition was maintained in the magneto amperometric immunosensor, the specifitity of the new immunoreagents was tested using the TEreported , with a 3.4.Ca buffer, and running them in ELISA and m-ELISA. The calibration curves obtained under such conditions were compared to that built in just PBSTCa buffer.The term matrix effect is defined as the combined effect of all components of the sample other than the analyte on the measurement of the quantity . Milk is50 remains constant with values of 0.87 μg/L for buffer and 0.92 μg/L for milk. Regarding m-ELISA assay, no matrix effect was observed, even in raw milk without any dilution. The calibration curves showed a perfect parallelism to the buffer curve  were mixed with the biomodified magnetic beads Ab171-MB and 11-HRP , incubated for 30 min and, captured with the m-GEC. Measurement of the immunocomplexes formed was performed in the electrochemical cell. The detectability of the AMIS in buffer, in term of its IC50 recorded was 0.74 μg/L, while the LOD, in term of the IC80, was 0.02 μg/L  were fully characterized by ELISA and m-ELISA. From two-dimensional experiments, optimum concentrations of Ab171-MB (0.25 mg/mL) and 11-HRP (0.5 μg/mL) were adjusted to obtain a final current of 7–10 μA after 30 min of immunochemical reaction in absence of analyte. Standard solutions of ciprofloxacin . The simplicity of the electrochemical magneto immunosensor presented here makes it suitable for rapid and cheap semiquantitative and quantitative on-site analysis of fluoroquinolones in milk by nonqualified personnel.An electrochemical magneto-immunosensor for the detection of fluoroquinolone antibiotics in milk has been developed. Time of the analysis has been decreased at half by the use of two new enzyme labeled immunoreagents, prepared by covalent binding of antibody and hapten for fluoroquinolones to the enzyme HRP using cyanuric chloride as crosslinker. In addition, magnetic beads have been biofunctionalized using fluoroquinonole specific antibodies and antigen bioconjugates. All immunoreagents, including magnetic beads, have been characterized by ELISA and m-ELISA using two formats, ET


Umbra krameri) is a Middle Danubian endemic fish species, which is characterised by isolated populations living mainly in artificial habitats in the centre of its range, in the Carpathian Basin. For their long term preservation, reliable information is needed about the structure of stocks and the level of isolation. The recent distribution pattern, and the population genetic structure within and among regions were investigated to designate the Evolutionary Significant, Conservation and Management Units  and to explore the conservation biological value of the shrinking populations. In total, eight microsatellite loci were studied in 404 specimens originating from eight regions. The results revealed a pronounced population structure, where strictly limited gene flow was detected among regions, as well as various strengths of connections within regions. Following the results of hierarchical structure analyses, two ESUs were supposed in the Carpathian Basin, corresponding to the Danube and Tisza catchments. Our results recommend designating the borders of CUs in an 80–90km range and 16 clusters should be set up as MUs for the 33 investigated populations. How these genetic findings can be used to better allocate conservation resources for the long term maintenance of the metapopulation structure of this threathened endemic fish is discussed.The European mudminnow ( Although large floodplain rivers and their associated habitats  represent a small fraction of the total land area, they nonetheless harbor an outstanding number of endangered and threatened species worldwide , 2. HoweHabitat degradation results in the erosion of genetic diversity , 9, in p2 of land was flooded at least periodically in the basin [2 floodplain area of the Tisza River , for example, approximately 4,400 km2 was permanently inundated [2. In the entire Carpathian Basin, the area of wetlands and backwaters directly connected to rivers was reduced to 700 km2 [Umbra krameri Walbaum, 1792), a Middle Danubian endemic fish species [Perccottus glenii Dybowsky 1877), has further worsened the situation, especially in the Tiszanian area [In Europe, up to 90% of the floodplain area is altered from its natural state . Althoughe basin . Howevernundated   to explore the recent distribution of mudminnow populations in the Hungarian part of the Carpathian Basin, the area inhabited by the majority of the known stocks; (ii) to reveal spatial patterns in the population genetic structure and among sites’ migratory rates; (iii) to assess the conservation biological value of extremely small populations; and (iv) to designate ESUs, CUs and MUs.o: 40/2013.(II.14.). However, since the studied species is strictly protected in Hungary, any procedure to be applied in connection with it is controlled by the order of government No: 348/2006.(XII.23.), and is subject to authorisation from the National Inspectorate for Environment, Nature and Water. The collection and storage of mudminnow tissue samples was authorized by this bureau . During the sampling procedure, all efforts were made to minimize suffering. Field studies did not involve other fish species that were endangered according to the IUCN Red List of Threatened Species v. 2014.3 (www.iucnredlist.org).This study was carried out following relevant national and international guidelines pertaining to the care and welfare of fish. Our surveys  are not qualified as animal tests by the operative Hungarian law , using 10–20 mg of tissue, as per the manufacturer’s instructions. Quality and quantity of the extracted DNA were verified using a NanoDrop 2000c Spectrophotometer .Studied areas and sites were selected based on the last published distribution data of mudminnow . SpecimeNine microsatellite markers previously developed by Winkler and Weiss  were invUmbra krameri microsatellites UkrTet2 (GenBank: FJ228219.1) and UkrTet3 (GenBank: FJ228220.1) sequences are identical, the reverse complements of each other. The DNA strand direction was mistakenly reversed for these. Data analysis of 156 fish samples (the first 14 populations) further strengthened this hypothesis. Allele numbers (n = 18) and allele frequencies, allele specific Fst values (Fst = 0.156) and the proportion of null alleles (prop. = 0.040372339) were equal for the UkrTet2 and UkrTet3 loci. Consequently, the UkrTet2 marker was left out of further statistical analyses.Microsatellite markers  and primFst values, were calculated using GenAlEx 6.5 [o, He), fixation index (F) and list the private alleles for each population.Fisher’s exact test of linkage disequilibrium and tests for deviations from Hardy-Weinberg equilibrium were conducted using GENEPOP 4.2.2  for eachAlEx 6.5 . SimilarFst values were corrected using the ENA procedure of Chapuis and Estoup [Frt–H0: individuals are shuffled among regions, Fsr–H0: individuals are shuffled within regions, Fst–H0: populations are shuffled among regions, Fis–H0: individuals are shuffled within populations, and Fit–H0: individuals are shuffled in the whole sample).The Lynch & Ritland  estimatod Estoup , 54. Gend Estoup  estimatod Estoup , and pred Estoup  was usedGenetic population structure was inferred using the hierarchical approach  of the SFst-calculation. Mutation rates among loci were estimated from the data. Migration was estimated for the original regional groups designated by catchment basin, and based on the results of hierarchical STRUCTURE analyses at the three levels.Migration rate estimation was performed by MIGRATE-N 3.2.15 , 64, usiFst data and pairwise straight line geographic distances (GGD) with 999 randomisations for the whole dataset and at drainage basin levels. On the individual level, two kinds of spatial autocorrelation [Spatial genetic structure was assessed at the population level using Mantel tests , throughrelation , 67 comprelation . Positivrelation . All of relation .As a result of our broad field investigations, European mudminnow was observed at more than 40 sampling sites from the Middle Danubian catchment . A totalFis values (>0.08) at all loci among the 404 samples. These results suggest strong genetic substructure caused by geographical isolation and/or non-random mating in subgroups. Characteristics of each microsatellite locus used in our work are provided in Fst values ranged between 0.148 and 0.482. Chi-Square Tests for Hardy-Weinberg Equilibrium for each loci at each population showed significant values in 17 cases , and we calculated high ases see .A total of 136 alleles were observed for the eight loci used in the analysis. The whole raw dataset used for further analyses is indicated in As MICROCHECKER analyses proved the presence of false homozygotes  in some cases , therefore null allele correction was made prior to further analyses see .13 of the detected 136 alleles were unique to a single location . The freFst values ranged between -0.01 and 0.476 (mean: 0.195), and only five of the 528 pairwise comparisons  did not show significant differences (Pairwise genetic distances ranged between 0.198 and 0.821 (mean: 0.621). Pairwise ferences .Mean within-population pairwise correlation relatedness values, “r”, ranged between 0.063 and 0.651, and were significant in all cases . These vThe PCoA plot based on pairwise population genetic distances  showed cis, showed significant within- and among-regional differentiation and population level differences.AMOVA analysis showed that among-population and among-regional differences represented 12.8% and 7.8% of the total variation respectively, while within-population differences accounted for 78.9% of the total genetic variance. Results of the regional F-statistics, except FBayesian hierarchical clustering separated Danubian and Tiszaian mudminnow stocks in the first step . The onlBayesian cross-validation showed that 98.3%, 89.6%, and 71.8% of the individuals were grouped correctly on drainage basin, region and population levels respectively . Most ofResult of the cross validation procedure showed negligible linkage between the Danubian and Tisza drainage basins. Only three specimens  were misclassified between the two drainage basins. Within a drainage basin, the percentage of misclassified cases is higher; five specimens of the total 156 , and 13 specimens of the 248  were classified to other regions on the Tisza and Danube drainage basins respectively. The highest number of misclassified cases was found between the Middle-Hungarian and Hanság-Szigetköz regions (E and F regions) where 4.9% and 10% of the stocks were misclassified.Population assignment results from GeneClass2 strongly support the structure revealed on the three hierarchical levels identified by Bayesian STRUCTURE analyses. 97.8, 96.0 and 94.6% of the individuals were classified correctly on the three hierarchical levels respectively see: . The thiThe between-region (A-H) migration ranged between 0.28 and 2.31 individuals per generation. Mutation-scaled immigration rate is less than one individual per generation in most cases, therefore negligible gene flow is assumed among the eight studied regions in the Carpathian Basin, especially in the case of regions C and H.Migration computations between the two clusters  showed that the Danube system received 8.19 (Θ = 2.4941 and M = 3.286) and released 7.24 (Θ = 2.5105 and M = 2.887) migrating individuals per generation to the Tisza drainage basin alone.On the second level of STRUCTURE analysis, the immigration rate (M) between the four clusters Figs  and 6 foFst and GGD semimatrices . The inverse was found for the two drainage basins, where the genetic differences showed significant correlations both for the Tisza  and the Danubian drainage basins  as well. Therefore, population level IBD can be detected only within the Danubian and Tisza drainage basins  [The field surveys of the current work have provided new information about the present day distribution of the European mudminnow at the centre of its range. In the “C” and “F” regions, stable and large assemblages were found, contrary to earlier communication which noted that the mudminnow was extremely rare and had been extirpated from most habitats in these regions . At the  glenii) , Europea glenii) . At presIn almost all regions, the largest assemblages were found in artificial habitats. Of the 33 sampling sites, only seven were in a near-pristine state . ConsequThis study presents the first data on the population genetic structure of threatened and endemic European mudminnow for the centre of its distribution range. In general, remarkable genetic differentiation was found among the 33 populations originating from eight regions. The spatial structure revealed seems to be quite fixed with negligible migration among the studied regions . The genSome of the studied populations showed complete isolation and a high level of relatedness Figs  and 5. PAlthough European mudminnow has a restricted dispersal ability , and hisNannoperca obscura Klunzinger, 1872), and found similarly high levels of isolation, and designed CUs in very similar ranges. These results therefore show that preferred habitat type and dispersion abilities may have major roles in the formulation of these features rather than the geographic range  or the taxonomic position  of the studied species.According to data from the literature , muFst computation showed significant differentiation in most cases, none of the small stocks differed considerably from their neighbouring populations . These separate entities need special attention to prevent substantial genetic diversity loss. The ee e.g., ).The investigations reported here reveal unexpectedly high genetic diversity of this endemic fish species, despite its declining and fragmented populations. The studied stocks show high levels of genetic variability, and this pattern seems to have been only slightly influenced by anthropogenic impacts  so far. Continued habitat loss, along with the invasion of non-native competitors, strongly threaten this habitat specialist, endemic fish. Climate change increases the probability and severity of dry periods in the area, and represents a high risk for European mudminnow with its shrunken and fragmented population structure . ConsequS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Cichlasoma urophthalmus (Mayan cichlid). Our cytochrome b data supported an introduction from Guatemala, while our microsatellite data suggested movement of Mayan Cichlids from the upper Yucatán Peninsula to Guatemala and introductions from Guatemala and Belize to Florida. The mismatch between mitochondrial and nuclear genomes suggests admixture of a female lineage from Guatemala, where all individuals were fixed for the mitochondrial haplotype found in the introduced population, and a more diverse but also relatively small number of individuals from Belize. The Florida cytochrome b haplotype appears to be absent from Belize (0 out of 136 fish screened from Belize had this haplotype). Genetic structure within the Florida population was minimal, indicating a panmictic population, while Mexican and Central American samples displayed more genetic subdivision. Individuals from the Upper Yucatán Peninsula and the Petén region of Guatemala were more genetically similar to each other than to fish from nearby sites and movement of Mayan Cichlids between these regions occurred thousands of generations ago, suggestive of pre-Columbian human transportation of Mayan Cichlids through this region. Mayan Cichlids present a rare example of cytonuclear disequilibrium and reduced genetic diversity in the introduced population that persists more than 30 years (at least 7–8 generations) after introduction. We suggest that hybridization occurred in ornamental fish farms in Florida and may contribute their establishment in the novel habitat. Hybridization prior to release may contribute to other successful invasions.The number and diversity of source populations may influence the genetic diversity of newly introduced populations and affect the likelihood of their establishment and spread. We used the cytochrome b mitochondrial gene and nuclear microsatellite loci to identify the sources of a successful invader in southern Florida, USA,  Biological invasions have resulted in species declines, extinction of native biota, and extensive financial costs post hoc assumptions about the genetic effects of introductions and demographic stochasticity; the challenge that unsampled populations might be the true source should also be considered Identification of sources and pathways of invasions has traditionally been accomplished by examining historical data such as dates of first discovery in introduced areas and importation records, or by molecular analyses of native and introduced populations Non-native species are typically assumed to be under strong selective pressure to adapt to their new environment, become established, and spread Cytonuclear disequilibrium, the nonrandom association of organellar haplotypes and nuclear alleles, has been documented for interspecific hybrids Cichlasoma urophthalmus (Mayan Cichlid) is found in freshwater and salt water along the Atlantic slope of Central America including southern Mexico, Belize, Guatemala, Honduras and Nicaragua At least 13 species of cichlids have become established in Florida, which possesses no native members of the family Cichlidae Successful establishment of a nonnative species depends on many factors and varies with species. Understanding the origin and method of introduction of nonnative species is necessary for developing effective ecosystem management strategies and for preventing future introductions. A reconstruction of invasion pathways is needed to understand the effects of diversity of introductions, the number of founder individuals, and the combination of historically separate genotypes on introduced populations. We used mitochondrial and nuclear molecular markers to identify the source(s) of Mayan Cichlids in Florida to determine whether this successful invader resulted from single or multiple introductions.This study was carried out in strict accordance with the recommendations in the Guidelines for The Use of Fishes in Research of The American Fisheries Society, the American Institute of Fisheries Research Biologists, and the American Society of Ichthyologists and Herpetologists We collected tissue samples from 670 individual Mayan Cichlids from 23 sites in Florida  and 53 sites within Mexico and Central America , including sites in Belize, Honduras, Guatemala and Nicaragua . Fish we5′-TGATGAAACTTCGGCTCCC-3′ and CytbRev5′-CTGTTAGTCCGGCGATAGG-3′. These primers were designed specifically for this study using primers designed by 5′-CACCAACCTCCTCTCCGC-3′ and CytbIntR5′-TGGAAGGCAAAGAATCGGG-3′.A portion of the cytochrome b mitochondrial gene was amplified using CytbForInitially, 47 fish from four sites in Florida, four sites in Mexico, two sites in Belize and one site in Honduras were sequenced for a portion of the cytochrome b gene (851 bp). These sequences revealed six haplotypes, two of which were found in 43 individuals. The two haplotypes differentiated between fish from Mexico and Central America and fish from Florida, hereafter referred to as the CA haplotype and the Fl haplotype respectively; on the basis of those results, we screened the remaining samples for those two haplotypes using restriction endonucleases. Cytochrome b was first amplified using Polymerase Chain Reaction (PCR). Positively amplified DNA was then digested with EcoRV at 37°C for one hour. EcoRV digestion resulted in two fragments if an individual displayed the Fl haplotype and one fragment if the CA haplotype was present. DNA fragments were then separated electrophoretically, stained with ethidium bromide and viewed under UV light. The remaining 623 samples were screened for the CA and Fl haplotypes.Specimens from 357 individuals from 29 sites in Florida, Mexico, Belize, Guatemala, Honduras and Nicaragua were analyzed using 17 recently developed microsatellite nuclear markers . The maximal pairwise difference between sequences was 6 and the tranversion:transition ratio was weighted as 2∶1; we therefore specified the weighted genetic distance (epsilon) as 120 and conducted a median-joining analysis To investigate the relationships between clades, haplotype networks were built using Network v. 4.6.11 and Network Publisher  and percentages of polymorphic loci were calculated for Florida, Upper Yucatán Peninsula, South of Yucatán Peninsula, Belize, Guatemala, Honduras, and Nicaragua using GenAlEx v.6.5 To detect evidence of a recent bottleneck or reduction in population size of Mayan Cichlids in Florida, we used the software Bottleneck v.1.2.02 http://www.bioportal.uio.no) Genetic relatedness of populations was assessed using Bayesian clustering in STRUCTURE v.2.3.4 ABC was used to test different introduction pathways of Mayan Cichlids into Florida using the microsatellite data and the program DIYABC We tested two groups of scenarios using the software DIYABC v. 2.0 4 – 1E3). The ‘one sample summary statistics’ used for each population were the mean number of alleles, the mean genetic diversity, mean size variance and, mean Garza-Williamson's M. The ‘two sample summary statistics’ used were compared between population pairs, and included Fst, mean index of classification , and (δμ)2 genetic distance For both sets of scenario analyses in DIYABC, we used broadly defined priors as no prior values were known for the parameters . We usedSix haplotypes were recovered from sequencing cytochrome b for 47 individuals; the remaining 623 specimens were screened for the CA and Fl haplotypes. The CA and Fl haplotypes differed by six bases within cytochrome b (Genbank accession numbers KM079191 and KM079192). The phylogenetic tree of cytochrome b haplotypes displayed two distinct clades. One clade contained only individuals from the native range, while the second clade contained all the sampled individuals from Florida, some of the individuals from five Mexican sites  and all sampled individuals from two sites in Guatemala (Lago Petén Itza and Laguna Macanche) . NetworkSeventeen loci were analyzed for 356 specimens from 29 sites in Florida, the upper Yucatán Peninsula and south of the Yucatán Peninsula in Mexico, Belize, Honduras, Nicaragua and the Petén region of Guatemala. The Belize population exhibited the highest number of effective alleles (6.56) while Florida had the lowest (2.42) . ObserveThe two clusters from Florida and Mexico and Central America were analyzed separately by running additional structure analyses. Within the native range grouping, the data were also divided into two clusters (ΔK = 1908.25); the first cluster contained individuals from Mexico and Guatemala while the second contained individuals from Belize, Honduras and Guatemala . Within Scenario testing analysis of the first group of scenarios showed the highest support for scenario 10, in which fish from an unsampled source were introduced to Mexico, then to both Guatemala and BHN, and then from Guatemala to Florida ; Table 3We observed that the nuclear genetic markers, microsatellites, and the mitochondrial gene, cytochrome b, supported different routes for introduction of Mayan Cichlids into Florida. The nonrandom association of mitochondrial and nuclear alleles, cytonuclear disequilibrium, is strong evidence of introductions of Mayan Cichlids to South Florida through fish from multiple origins Phylogenetic analysis and haplotype distribution of cytochrome b indicated an introduction of Mayan Cichlids into Florida from the Petén region of Guatemala or the upper Yucatán Peninsula of Mexico. All but one fish from Florida carried the same cytochrome b haplotype suggesting that either a small number of founders, or low female effective population size carrying the Fl haplotype, were introduced and quickly spread e.g. . The lonPeténia splendida; The first group of scenarios we tested using ABC supported a pathway whereby Mayan Cichlids were introduced from an unsampled source to Mexico, then to both Guatemala and the cluster of Belize-Honduras-Nicaragua, and then from Guatemala to Florida. Cytochrome b results also supported Guatemala as the introduction source of Mayan Cichlids in Florida because they shared the Fl haplotype. We grouped Belize with Honduras and Nicaragua for the first group of scenarios because of their genetic similarity indicated by the cluster analysis. However, because Belize is within the Usumacinta drainage, unlike Honduras and Nicaragua, and because there was some genetic similarity of individuals between Florida and Belize, we grouped Belize separately for the second set of scenario testing. We investigated whether the ‘unsampled population’ indicated by the most supported scenario from group 1 was representative of a population near the Ria Grijalva basin where the sister species of Mayan Cichlids  Gambusia yucatana where individuals from northern Yucatán Peninsula and Petén were morphometrically more similar than with nearby sites The Fl haplotype was found in all fish from Lago Petén, Laguna Macanche, Cenote Ya-Bal-Ha, and Cenote Xtoloc, and some fish from Ría Lagartos, Cenote Zaci, and Ría Celestun. Although these areas are all part of the Yucatán Division of the Usumacinta Drainage Mayan Cichlids have become established in southern Florida; they have spread and impacted their introduced environment, representing a case of a successful invader that resulted from multiple introductions. Unlike other studies, the introductions from distinct sources did not increase overall genetic diversity compared to the native range. Instead, it resulted in a genetic bottleneck which decreased overall genetic diversity and produced novel combinations of mitochondrial haplotypes and nuclear alleles. Introduction was followed by rapid population growth and dispersal throughout south Florida. This admixture between distinct Belize and Guatemala lineages, probably accomplished while in cultivation in ornamental fish farms, could have improved fitness and facilitated establishment and spread in Florida.Figure S1Rate ofchange of the likelihood distribution (mean± SD) from STRUCTURE analysis. Calculated as L′(K) = L(K)−L(K−1) (see K−1) see . The hig(TIF)Click here for additional data file.Table S1Location and number of Mayan cichlid samples collected at each site.(DOCX)Click here for additional data file.Table S2Scenarios 1–15 for group 1 and scenarios 1–9 for group 2 in DIYABC analyses. Scenarios show the hypothesized movement pathways for Mayan Cichlids (indicated by downward arrows). For all models, time (t) increases upward.(DOCX)Click here for additional data file.


Plasmodium falciparum in Asembo, western Kenya showed that the parasite maintained overall genetic stability 5 years after insecticide-treated bed net (ITN) introduction in 1997. This study investigates further the genetic diversity of P. falciparum 10 years after initial ITN introduction in the same study area and compares this with two other neighbouring areas, where ITNs were introduced in 1998 (Gem) and 2004 (Karemo).An initial study of genetic diversity of From a cross-sectional survey conducted in 2007, 235 smear-positive blood samples collected from children ≤15-year-old in the original study area and two comparison areas were genotyped employing eight neutral microsatellites. Differences in multiple infections, allele frequency, parasite genetic diversity and parasite population structure between the three areas were assessed. Further, molecular data reported previously (1996 and 2001) were compared to the 2007 results in the original study area Asembo.A) declined with time in the original study area Asembo (from 95.9 %-2001 to 87.7 %-2007). In the neighbouring areas, MA was lower in the site where ITNs were introduced in 1998 (Gem 83.7 %) compared to where they were introduced in 2004 (Karemo 96.7 %) in 2007. Overall mean allele count (MAC ~ 2.65) and overall unbiased heterozygosity (He ~ 0.77) remained unchanged in 1996, 2001 and 2007 in Asembo and was the same level across the two neighbouring areas in 2007. Overall parasite population differentiation remained low over time and in the three areas at FST < 0.04. Both pairwise and multilocus linkage disequilibrium showed limited to no significant association between alleles in Asembo  and between three areas.Overall proportion of multiple infections  contains supplementary material, which is available to authorized users. Insecticide-treated bed nets (ITNs), including long-lasting insecticide-treated bed nets (LLINs), are an important tool for malaria control . In westITNs reduce malaria morbidity by killing or deterring mosquito vectors, thereby reducing the number of infective bites on human hosts . To be oPlasmodium falciparum maintained overall high genetic diversity and stability after 5 years of high ITN use [Anopheles gambiae [P. falciparum genetic diversity and population structure [A previous study on the effects of transmission reduction by ITNs on parasite population structure using neutral MS markers in western Kenya, showed that  ITN use  even in  gambiae –14. Clin gambiae , 15, 16.tructure –19.P. falciparum in western Kenya [P. falciparum with numerous reproductive units have been shown to contribute to extensive heterogeneity of the parasite with correspondingly limited or no genetic differentiation across different regions in high transmission areas in Africa [P. falciparum maintains a clonal structure with significant linkage disequilibrium (LD) in some high-transmission areas, indicating there are other factors influencing genetic diversity and population structure [Previous studies on genetic diversity over space and time conducted by others in Kenya reported limited time or geographical area effects on gene allelic frequencies of rn Kenya . Althougn Africa –24. Howetructure , 26. TheP. falciparum maintained overall high genetic diversity but with locus-specific variation, which contributed to differences in population sub-structure [P. falciparum in samples from 2007 in Asembo and the data were compared to that from two neighbouring areas where ITNs were first introduced in 1998  and 2004 , respectively. Assessing differences in parasite genetic diversity between Asembo, Gem and Karemo would inform on different area effects based on different ITN coverage and/or usage, and the possible role of migration of parasites between the areas. Further, comparison of parasite diversity within Asembo in the 1996, 2001 and 2007 surveys would show any possible temporal effects of ITN application on parasite population in the same locality with decline in entomology inoculation rate (EIR) and malaria prevalence. The same eight single copy neutral microsatellite (MS) markers used previously were employed in this study [P. falciparum. Assessments of changes between time points and between areas on P. falciparum population were quantified based on multiplicity of infection, allele frequency, unbiased heterozygosity, linkage disequilibrium (LD) and genetic differentiation.The initial assessment of effects of ITNs on parasite genetic diversity on samples collected in 1996 and 2001 in the original study area, Asembo (ITN introduction in 1997) in Rarieda sub-county, Siaya county of western Kenya, showed tructure . The curis study  to assesThis was a follow-up study in Siaya county of western Kenya where a two-phase, community-based, ITN trial was conducted from 1996 to 2001 –5, 11. TThis study examines parasite diversity in samples collected from a cross-sectional survey conducted in 2007 in the Asembo and compares the results with those from Gem and Karemo areas. The geographic relationship of the three areas is shown in Fig. From the 2007 cross-sectional survey, a total of 235 smear-positive samples collected from children ≤15-year-old from Asembo (n = 56), Gem (n = 87) and Karemo (n = 92) were used for genetic analysis of parasites. For Asembo, the molecular data from 69 and 74 smear-positive samples collected in 1996 and 2001, respectively, from children ≤5-year-old and reported earlier were also included for further temporal comparison . Dried bThe study was approved by the Ethical Review Committee of Kenya Medical Research Institute, Nairobi, Kenya, the Institutional Review Board of Michigan State University, East Lansing, MI, USA and the Institutional Review Board of CDC, Atlanta, GA, USA.P. falciparum parasites was assessed by scoring eight single copy neutral MS loci located on different chromosomes for all the samples as reported previously [P. falciparum (Pfg377) and two MS linked to genes of asexual stage antigens under possible natural immune selection (EBP and P195) were used. All MS scoring in base length and peak height, and quantification of multiple alleles used the same method as described previously [The genetic diversity of eviously . The seleviously , 29, 30.eviously . BrieflyA), while the highest number of allele count detected by any of the MS comprised the mean allele counts (MAC). Differences in both MA and MAC between time points or between areas were assessed using Pearson’s Chi square and one way analysis of variance (ANOVA). Conversely, only the predominant allele in each locus was used to analyse all other parameters of genetic diversity and population structure, including unbiased heterozygosity (He) and allele richness calculated as the average number of alleles per locus, LD and genetic differentiation (FST) [He), sampling variance of He, was calculated as described previously [p-levels obtained from z absolute values from the standard error (SE) of sampling variance. The LD measures the degree of association between gene pairs or among gene loci (structured population when LD is significant) assuming a null hypothesis of no association in random genetic recombination (population admixture when LD is insignificant). Pairwise LD, measuring the degree of association between MS, was obtained using ARLEQUIN [d) and the variance expected (Ve) at LD, assuming a null hypothesis (Ho) derived from 10,000 simulated data sets: Vd = Ve. Genetic differentiation (FST) was tested by the Fisher’s exact test using the GenePop Program [ST is a measure of the sum of genetic variability within and between parasite populations based on differences in allele frequencies. Categorization for FST was defined as no differentiation or low differentiation (FST < 0.05), moderate differentiation (≥0.05 FST < 0.15) and great differentiation (FST ≥ 0.15) as described previously and applied previously [All microsatellite raw data were managed using the Excel Microsatellite Tool Kit  and conson (FST) . Multiplon (FST) . Unbiaseeviously  with p-lARLEQUIN . Multilo program . Multilo Program . The FSTeviously , 37.He, but there were some differences in LD and FST between parasite populations from the three time points. For brevity, therefore, temporal data presented below focused on comparison of the parameters of multiple infection and genetic diversity only between 2001 and 2007 while comparison of LD and FST of parasite populations among 1996, 2001 and 2007 surveys are presented.Since the Asembo 2007 survey comprised children up to 15 years of age, initial data were stratified by age (≤5- and >5-year-old) and tested for differences in parasite genetic diversity  by any of the eight MS in the three study areas was over 80 %, a reflection of a highly polyclonal P. falciparum parasite population. In the different area analyses, the overall MA was significantly higher in Karemo at 96.7 % compared to Asembo (87.7 %) and Gem (83.7 %) (p = 0.01). In contrast, the overall mean allele counts (MAC) were similar (p = 0.53) at 2.76 (Asembo), 2.55 (Gem) and 2.68 (Karemo). For individual MS, only P195 showed significantly higher MA and MAC in Karemo compared to both Asembo and Gem (p = 0.01). This pattern was reversed for both Pfg377 and PfPK2 where both MA and MAC were significantly lower in Karemo compared to both Asembo and Gem as shown in Table Overall proportion of infections with more than one allele (M) p = 0.0. In contA dropped from 95.9 % in 2001 to 87.7 % in 2007 (p = 0.03), but the reduction in the overall MAC (from 3.1 in 2001 to 2.8 in 2007) was not statistically significant. At individual MS, there were significant decreases in MA and MAC from 2001 to 2007 at the P195 , EBP , and TAA109 , respectively. No other significant changes, including Pfg377 MS, were observed between the two time periods as shown in Table For the temporal effect analysis within Asembo, the overall MP. falciparum population in the three study areas. Allele numbers per locus ranged from a low of five for the Pfg377 locus in Asembo and Karemo to a high of 19 for the Poly-α locus in Karemo. The overall He was approximately 0.8 in the three study areas as shown in Table He was significantly lower at P195 locus in Gem (He = 0.65) than in both Asembo (He = 0.74) and Karemo (He = 0.74). No significant difference in He for other individual MS markers between the areas was detected  for 16, 14 and 15 MS pairs in Asembo, Gem and Karemo, respectively. Of note, Pfg377, the MS flanking the gene relating to gametocyte maturation, had the least number of significant pairwise LD  in Gem and Karemo, respectively, while no significant LD was observed in Asembo. Conversely, the remaining MS had at least ten of the significant pairwise LD in all three areas as shown in Additional file Overall, results of 28 pairwise comparisons for each area showed that LD was significant  where 14 pairs of MS showed significant LD. However, despite these overall changes in the number of significant pairwise LD in the different time points, LD at Pfg377 locus again showed the least number with only four significant pairs in the three time points [P195 and ADL in 1996, P195 and EBP in the 2001 and no pairs in 2007 survey (d) of 1.268 was significantly higher than the variance expected (Ve) which was 1.133 (p = 0.03) with an index of association . When individual MS were analysed, only P195 MS showed moderate genetic differentiation (FST ≥ 0.05 < 0.15) between any two areas in 2007. All other individual MS showed low differentiation (FST < 0.05) that was not significant between the three study areas  compared to the 1996/2007 testing where FST was 0.026. Incidentally, as reported previously, FST was also low in the 1996 and 2001 testing at FST 0.027 [ST results show that over the three time points spanning 10 years there was only limited differences in allele frequencies resulting in insignificant parasite population differentiation in Asembo area. At the individual MS, P195 locus, as in the different areas analyses, showed consistently moderate differentiation for the paired time point comparisons between the years 1996, 2001 and 2007  dropped from 95.9 % in 2001 to 87.7 % in 2007. The MA levels were similar in Asembo (87.7 %) and Gem (83.7 %) but significantly higher in Karemo (96.7 %) in 2007. However, the overall mean allele count MAC remained unchanged at around 2.65 in the three areas and at the different time points. Further, after 10 years of sustained ITNs use (1997–2007), the genetic diversity measured by He remained unchanged at approximately the same level over time and in the three areas (He ~ 0.78). Additionally, there was low parasite population differentiation for the three areas (FST = 0.021) and over time (FST < 0.04). The only slight difference observed was that in Asembo there was less significant pairwise LD and insignificant multilocus LD in 2001 compared to 1996 (baseline) and 10 years later (2007).This study was aimed at assessing changes on He observed in the three study areas remained high. The similarly high He for P. falciparum using neutral MS markers was reported in Kombewa and other areas of western Kenya, including Kapsulu, Kodera, Rangwe, Ringa, and Rota villages in surrounding counties, although no data on ITN usage were presented [He, coupled with low overall genetic differentiation between areas in this study suggest the possible existence of vibrant reproductive units that maintain the high diversity within Plasmodium parasite pools. The high diversity and limited genetic differentiation also suggest gene flow is likely to be a major factor in maintaining vast parasite pools in the geographic region. The negative FST results observed in this study further illustrate the extent of admixture and cross-breeding within parasite populations in the three areas. Gene flow due to human migration was reported previously as a contributing factor to a resilient Plasmodium parasite population in western Kenya [P. falciparum from hosts with a large pool of sub-microscopic asexual parasites and gametocytes predicted high uninterrupted transmission even with scaled-up LLIN coverage [Initial hypothesis of this study was that malaria transmission reduction, mainly by ITNs, would decrease parasite diversity. However, in spite of relative differences in duration of ITN implementation, use of ITNs and EIR between Karemo and Asembo , the overall resented , 38. Thern Kenya . Demograrn Kenya . In addicoverage . This roHe remained high and stable in 1996, 2001 [AC and the overall level of population differentiation (FST) remained low over the three time points. A notable change was observed only in 2001 in the LD parameters with less significant pairwise LD and insignificant multilocus LD compared to 1996 (baseline) and 10 years later (2007). It is possible that sudden changes in parasite population due to the initial transmission reduction by introduction of ITNs in 1997 could allow minor populations with different allele frequencies to become dominant which might result in admixture parasite population (insignificant LD) in 2001. It is also likely that sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) resistance contributed to malaria transmission [Within Asembo the overall 96, 2001  and 2007smission , resultismission . Other ssmission . Taken tsmission . Therefosmission .He was similar in the three areas and stable over time, there were differences in overall MA. In Karemo, where ITNs were introduced since 2004 (the shortest time) and with the lowest use at 20 %, MA was significantly higher at 96.7 % than in Asembo at 87.7 % (ITN introduction in 1997 with 51 % use), or Gem 83.7 % with ITN introduction in 1998 and 44 % use. Similarly, where temporal effects were assessed in Asembo, MA also significantly decreased from 2001 (95.9 %) to 2007 (87.7 %) after 10 years since introduction of ITNs. However, the temporal and area differences in MA were not substantial to affect the overall He, suggesting that multiple infections could be confounded by within-host competition among the parasite clones that are under selection by drug pressure, host immune pressure or parasites in different species of mosquito vector, all of which would be influenced by various malaria control measures. The results also suggest that measuring multiple infections could serve as an early indicator for change of malaria transmission.While overall In this study, there were also a few significant differences by different measures for individual MS markers, P195, Pfg377, EBP, PfPK2 and TAA109, among the three areas and/or between time points. Although the reasons for variations in EBP, PfPK2 and TAA109 loci between time points and among the areas are unclear, it is notable that P195 locus, the MS flanking gene encoding for asexual stage antigen under possible immune selection , consistThis study has a few limitations. The effect on parasite diversity was extrapolated based on neutral MS markers that may not fully capture the dynamism of the parasite population in the face of different control measures that include ITN use and drug pressure, which would further shape host immunity. Geographic proximity of the three study areas could have limited the ability to detect significant area divergences in the parasite populations. This is a cross sectional survey and while ITN coverage was high the actual use in the nights before sampling showed low usage  which limited dissecting the impact of ITNs on parasite diversity. The temporal comparison was also limited as only one area had at least 10 years of ITN use. Further studies on parasite genetic diversity/structure for longer periods and in wider geographical regions, as well as use of other unique and robust genetic markers of parasites , 49, wilP. falciparum over 10 years and across three different areas after scale-up of ITNs. The parasite resilience was reflected by a change in LD in Asembo at mid-point (5 years) but not at the 10-year time point. In addition to the gene flow between areas, other possible factors that might be attributed to the high and stable diversity of parasite population mainly are sub-microscopic infection and large gametocyte reservoir. Theoretically, a dramatic transmission reduction as a result of using multiple and intensive prevention and intervention measures can decrease parasite genetic diversity by creating a bottleneck effect on parasite population; for this to happen in western Kenya, such combined and intensive prevention and intervention measures must be sustained and cover wide geographic areas.This study has shown the overall high genetic diversity and stability of


By inadvertently collecting animals from different source populations, however, such efforts risk reintroducing individuals that have not been in genetic contact for significant periods of time. Using genetic analyses and 44 years of data from Camp Leakey, an orang-utan rehabilitation site on Borneo, we determined the minimum extent to which orang-utans representing non-native, geographically and reproductively isolated taxa were reintroduced into the surrounding wild population. We found two reintroduced females were from a non-native subspecies, and have since produced at least 22 hybridized and introgressed descendants to date, of which at least 15 are living. Given that Bornean orang-utan subspecies are thought to have diverged from a common ancestor around 176,000 years ago, with marked differentiation over the last 80,000 years, we highlight the need for further evaluation of the effects of hybridizing orang-utans of different taxa — particularly in light of the ~1500 displaced orang-utans awaiting urgent reintroduction. As endangered mammals are increasing in number in sanctuaries worldwide, we stress the need for re-examination of historical reintroductions, to assess the extent and effects of  Mustela nigripesLeontopithecus rosaliaCeratotherium simum cottoniChelonoidis hoodensis5Gorilla beringei beringei) in groups habituated to human observation, versus an annual 0.7%±0.06% decline in the number of unhabituated conspecificsLycaon pictus) under study in the Serengeti Ecosystem has been attributed to a rabies vaccination programme, which may have inadvertently compromised dogs’ immune responses through their immobilization, vaccination and radio collaringPopulations of large and endangered mammals are increasingly impacted by humans, and may live – even in wild or semi-wild settings – under intensive human management  to a remnant population of 50 Florida panthers (P. c. coryi) approximately doubled the estimated genetic diversity of the population, and was associated with a marked reduction in the frequency of deleterious traits associated with the breeding of related individuals, such as kinked tails, cryptorchidism and poor sperm quality1819Canis lupus), the arrival of a single wolf to a bottlenecked Scandinavian population originating from only two individuals was sufficient to dramatically increase genetic diversity (heterozygosity) and exponentially increase population growth2324The hybridization of genetically distant individuals may, under some circumstances, increase population fitness1516Despite this, the hybridization and introgression of representatives of genetically divergent lineages may have negative effects on a population’s overall fitness, as exemplified by the lack of fertility common in offspring resulting from the inter-breeding of different species262728303235c. 963 chimpanzees (Pan troglodytes), c. 72 bonobos (P. paniscus), c. 106 gorillas (Gorilla gorilla)  and c. 1516 orang-utans (Pongo spp.)Pongo pygmaeus) and Sumatra (Pongo abelii), with three geographically and reproductively isolated subspecies on Borneo: P. p. wurmbii in Central and southern West Kalimantan, P. p. morio in North and East Kalimantan and Sabah, and P. p. pygmaeus in northern West Kalimantan and Sarawak. Each Bornean subspecies is thought to have shared a common ancestor around 176,000 years ago, before radiating to different refugia following climatic fluctuations in the Pleistocene384038More than 2,650 great apes are confined in sanctuaries across the ranges of all four species: P. pygmaeus. Over 14 years, Galdikas and Brindamour rehabilitated and released more than 90 ex-captive or displaced orang-utans into the wild at Camp Leakey, on the southernmost promontory of Central Kalimantan44c. 470 kilometres from Tanjung Puting, and others from captivity in Java. In spite of this, YeagerIn 1971, however – when Galdikas and Brindamour established their pioneering research and reintroduction site, Camp Leakey, in Tanjung Puting National Park – all orang-utans were classified as a single species, Camp Leakey’s contemporary population comprises approximately 60 individuals, including 8 reintroduced female orang-utans. Two further reintroduced females have recently died. All 10 females reproduced; in one case, offspring are now into their fourth generation. As some paternities are known2, 1.38 μl 10 mM dNTP mix, 0.83 μl 10 mg/mL BSA, 0.134 μl each primer, 0.28 μl Taq polymerase (Life Technologies), 15.7 μl ddH2O and 2.75 μl DNA. Thermal cycling conditions were as follows: initial denaturation at 94 °C for 12 minutes; 40 cycles of 94 °C for 40 seconds, 61 °C for 30 seconds and 72 °C for 1 minute; and a final extension at 72 °C for 10 minutes. To avoid amplification of nuclear mitochondrial insertions (‘numts’), which have proved problematic in prior studies of hominoid mtDNA taxaFaecal samples were collected from orang-utans in and around the Camp Leakey study area, and genomic DNA extracted, as previously describedet al., 2001et al., 2010Sequences were manually corrected in MEGA 6.06Mitochondrial DNA control region sequences were generated from 10 presumed unrelated adult orang-utans  that were reintroduced to the wild at Camp Leakey in the 1970s and 1980s. An additional sequence was obtained from the first-generation offspring (Siswi) of a now-deceased reintroduced female (Siswoyo), and assumed to represent this female given that mtDNA is near-clonally maternally inherited. Of the 11 founders represented, five haplotypes – labelled A to E – were observed in the alignment. Of these, two (A and B) were identical to previously published sequences , while three  were novel (GenBank accession codes KU523975-KU523977). Haplotype C featured an insertion that was confirmed by sequencing an additional PCR product amplified from the same DNA extract. Haplotypes D and E each featured a transition, at different bases, when compared with published sequences. These transitions were consistent in all sequences generated from both PCR protocols.P. p. wurmbii. We found that nine orang-utan mtDNAs, comprising four haplotypes  did indeed fall in this clade. In addition, two females, Rani and Siswoyo, were found to share a haplotype (A) that clustered in the tree with mtDNAs derived from the subspecies P. p. pygmaeus from Northern West Kalimantan or Sarawak  observed a high number of shared private alleles between chimpanzees from the Gulf of Guinea (Pan troglodytes ellioti) and from Southern Cameroon (P. t. verus), suggesting recent introgression within a purported hybrid zoneet al. (2012) identified natural hybridization of anubis (Papio anubis) and yellow  baboons in the Amboseli basin in southern Kenya. This hybridization was notably asymmetric: more anubis baboons dispersed into, and were reproductively successful, within yellow baboon populations than vice-versa(Macaca mulatta) into long-tailed (M. fascicularis) macaques(Pan troglodytes verus) into central (P. t. troglodytes) and eastern (P. t. schweinfurthii) chimpanzeesPan troglodytes troglodytes) and eastern (P. t. schweinfurthii) chimpanzees, pointing out that the variation in these chimpanzees can be better described as following a pattern of increasing genetic differentiation with increasing distance38In spite of this, there is evidence in other primate taxa that hybridization occurs naturally along the borders of different species’ and subspecies’ ranges. Gonder Guidelines for the Reintroduction of Animals Born or Held in Captivity, the Association of Zoos and Aquariums suggested that the genotype of reintroduction candidates “should be the closest possible match” to that of the destination population, “so that subspecific distinctions can be maintained”71In the decades following the reintroductions at Camp Leakey, extensive guidelines have emerged to better facilitate wildlife reintroductions. Over time, these guidelines have placed more explicit focus on genetic considerations. In their et al.et al.In practice, however, these guidelines and legislation are challenging to follow or enforce. In many cases, it is simply too expensive to perform genetic testing: rescuing and accommodating displaced animals takes priority in the face of extremely limited funds. The origin of displaced animals might therefore be inferred as the region from which they were rescued .


Chersophilus duponti), an endangered songbird. An information-theoretic approach to model selection allowed us to address the importance of interactions between habitat variables, an aspect seldom considered in fragmentation studies, but which explained up to 65% of the variance in genetic parameters. Genetic diversity and inbreeding were influenced by the size of local populations depending on their degree of isolation, and genetic differentiation was positively related to isolation. We then identified a minimum local population of 19 male territories and a maximum distance of 30 km to the nearest population as thresholds from which genetic erosion becomes apparent. Our results alert on possibly misleading conclusions and suboptimal management recommendations when only additive effects are taken into account and encourage the use of most explanatory but easy-to-measure variables for the evaluation of genetic risks in conservation programmes.Size and isolation of local populations are main parameters of interest when assessing the genetic consequences of habitat fragmentation. However, their relative influence on the genetic erosion of local populations remains unclear. In this study, we first analysed how size and isolation of habitat patches influence the genetic variation of local populations of the Dupont's lark ( Human activities are threatening a large number of species worldwide through habitat loss and fragmentation Andren . ChangesTwo main microevolutionary processes influence genetic patterns in declining and fragmented populations: genetic drift and gene flow. These processes are determined by local population size and interpatch connectivity, parameters that are commonly taken into account in conservation planning under a metapopulation theory framework Hanski . While gCritical thresholds in habitat fragmentation have been defined as an abrupt and nonlinear change in some parameter across a small range of habitat loss (With and King Chersophilus duponti), a markedly sedentary steppe specialist passerine suffering from habitat loss and fragmentation in recent decades  of recently fragmented populations. Furthermore, we aimed to identify the thresholds of population size and isolation beyond which genetic erosion starts to accumulate to detectable levels. We focused on the endangered Dupont's lark  with GenePop on the Web (Raymond and Rousset AR) using the rarefaction method to correct for unequal sample sizes with Fstat 3.9.3 (Goudet FIS) and the average pairwise relatedness estimator (R). FIS was calculated with Genetix  with 2MOD . They were merged with their closest neighbour locality to increase sample sizes for analyses, yielding 33 local populations  and were grouped in the same genetic cluster , and both are generally good indicators of population sizes  and ecological and demographic attributes of each local population (i.e. population size and isolation). Population size (characterized by N.pop and area) and isolation  were fitted as explanatory variables. Following our hypotheses, we designed a priori five alternative models to assess the relationships between the explanatory variables and the eight response variables. The first three univariate models only included single explanatory variables  to assess their effects separately. The fourth model included area and isolation, while the fifth included area, isolation and the interaction between area and isolation. All models were tested twice alternatively using D.near and D.centre as isolation measures. We followed a model selection strategy using an information-theoretic approach (Johnson and Omland c) and relative weight of evidence for each model (wi) as the probability of model i being the best model for the observed data, given the set of candidate models. The most parsimonious model for each genetic index was selected based on a lower AICc and higher wi  and the ecological  conditions (Table S1). Top ranking models for AR, R and FST and He, FIS and DST. For simplicity, we include the formers in Supporting Information and report the latters and F in DST) and 65.3% (He) of the variation (Adj. R2) in the genetic parameters (Our exhaustive sampling of the whole Spanish (and thus European) Dupont's lark distribution captured a wide range of both the genetic (rameters .He) and 24% of the variance in accumulated inbreeding (F)  and F  and at 16 for DST . Therefore, populations with <16–19 males were prone to the loss of genetic diversity and to the accumulation of inbreeding and differentiation.Population size (N.pop) was retained as a statistically significant predictor in final models when using D.near as isolation measure and explained 22% of the variance in heterozygosity (ding (F) . MoreovePatch size (area) was not included as a single predictor but played a key role in several final models through its interaction with isolation (see below).FST, DST and F, respectively. On the other hand, D.near was selected instead of D.centre for AR, FST, DST and DST in 30 km to the nearest population .The distance to the centroid of all populations (D.centre) performed better as an isolation measure than the distance to the nearest population (D.near) for most genetic parameters . D.centrHe and AR), inbreeding (FIS) and relatedness (R)  and FIS . In all cases, genetic indexes varied differently with patch size depending on their degree of isolation, or conversely, they responded differently to an increase in isolation depending on their size. To better understand these interactions, we plotted the lowest and highest values of isolation found in our data, and the one where the effect of area did not promote a change in genetic indexes. Above or below this level of isolation, the relationships between genetic indexes and patch area changed, either increasing or decreasing. With low values of isolation (23 km to the centroid or 5.6 km to the nearest population), genetic diversity and inbreeding or relatedness decreased with patch size. Distances of 103–145 km to the centroid of all populations or at 19 km to the nearest population resulted in stable genetic indexes in all patch sizes. At the same time, patch sizes of 300, 375, 650 and 1800 ha made genetic indexes independent of their degree of isolation . Finally, larger values of isolation resulted in increasing genetic diversity and inbreeding with patch size.Models including the interaction between patch size and isolation explained the highest percentages of the observed variance in genetic diversity  size and isolation in explaining the observed variance in genetic parameters related to diversity, inbreeding and differentiation.Population size had a major influence on genetic indexes of Dupont's larks: genetic diversity increased and differentiation decreased with local population size, being the expected outcome of a less intense genetic drift in larger populations , panel AThe other parameter of interest, isolation, proved also of great importance for another genetic variable: genetic differentiation increased with isolation , panel BMost importantly, the interaction between patch size and isolation explained a much higher proportion of the variance in genetic diversity, inbreeding and relatedness than each of these variables alone, or when only additive effects were considered. Although the product of population size by isolation (captured in genetic models by the migration rate) is considered determinant for the spatial variance in gene frequency in population genetics theory, only a few empirical studies of wild populations have explicitly evaluated this interactive effect. Some studies considered fragmentation as the product of size and isolation, making it impossible to disentangle the relative effects of these interacting variables  in larger and less dense patches when these are highly isolated. These populations might therefore favour the maintenance of overall genetic diversity through the formation of spatially segregated groups of related individuals, although inbreeding and average relatedness will increase. Conversely, small isolated populations show less genetic diversity due to the combination of a lower effective population size and, probably, a longer time since isolation . Remarkably, this value is in fair accordance with the maximum dispersal distance of 20 km recorded for the Dupont's lark  should be targeted to prevent the loss of genetic diversity. Although these thresholds are relative to our power to detect changes in genetic parameters, which is a function of sampling size, genetic markers and the time since fragmentation started, these results raise concern over the situation of the species. Attending to this study, the majority of Spanish populations are currently below these threshold sizes; approximately 90% of the occupied patches are smaller than 1800 ha and half of them cover <300 ha (range 20–5000 ha). Regarding the isolation of populations, 73% of them are separated by more than 13 km and 54% by more than 19 km from the nearest population (range: 5.5–173 km). Consequently, many Dupont's lark populations may be prone to suffer an ‘extinction vortex’ and raise the possibility that genetic factors contributed to the decline in population's viability  should still help to improve management actions.Finally, our conceptual and methodological approach can be implemented at different spatial scales. Our study system covered the whole European distribution of an endangered species, but the same approach should work well for studies conducted at regional or metapopulation scales. We thus encourage the use of our approach  to desig