Miconia affinis across the Isthmus of Panama. Furthermore, we investigated the impact of geographic distance, forest cover, and elevation on genetic differentiation among populations using circuit theory and regression modeling within a landscape genetics framework. We report marked differences in historical and contemporary migration rates and moderately high levels of genetic differentiation in M. affinis populations across the Isthmus of Panama. Genetic differentiation increased significantly with elevation and geographic distance among populations; however, we did not find that forest cover enhanced or reduced genetic differentiation in the study region. Overall, our results reveal strong dispersal for M. affinis across human-altered landscapes, highlighting the potential use of this species for reforestation in tropical regions. Additionally, this study demonstrates the importance of considering topography when designing programs aimed at conserving genetic diversity within degraded tropical landscapes.The regeneration of disturbed forest is an essential part of tropical forest ecology, both with respect to natural disturbance regimes and large-scale human-mediated logging, grazing, and agriculture. Pioneer tree species are critical for facilitating the transition from deforested land to secondary forest because they stabilize terrain and enhance connectivity between forest fragments by increasing matrix permeability and initiating disperser community assembly. Despite the ecological importance of early successional species, little is known about their ability to maintain gene flow across deforested landscapes. Utilizing highly polymorphic microsatellite markers, we examined patterns of genetic diversity and differentiation for the pioneer understory tree  Forest ecosystem function is dependent on the colonization and establishment of native trees, which provide essential ecosystem services such as carbon sequestration, erosion control and the provisioning of food and nesting resources for humans and wildlife . HoweverReforestation implies the release of cultivated and translocated trees, which can have important genetic consequences for native populations such as alterations in their genetic diversity and gene flow dynamics and breakdowns of local adaptation –9. Thus The first and simplest hypothesis regarding gene flow across space is the isolation by distance pattern . The IBD pattern is characterized by a declining probability of genetic identity with geographic distance  and has Thus, a second potential factor influencing gene flow among tree populations is elevation. As in any ecological gradient, many species have an ecological optimum along elevational gradients ,21. For Finally, forest fragmentation is expected to reduce genetic diversity, increase genetic differentiation, and potentially increase inbreeding in tree populations ,33. HoweGiven the potentially major impacts of distance, elevation, and forest cover on plant gene flow, it is particularly urgent to investigate gene flow processes for tropical trees, as tropical regions exhibit substantial elevation gradients, greater proportions of animal-dispersed plant species, and rapid rates of deforestation. Specifically, species ranges in the tropics are frequently delimited by elevation, with distinct community assembly processes along elevation gradients  and poteMiconia affinis, a common early successional Neotropical tree that frequently colonizes forest gaps, riparian areas, and exposed hillsides  and pairwise explanatory variables . Geographic refers to log-transformed null resistance distance.Models are ranked based upon the difference between Akaike’s Information Criterion (AIC) in each individual model and the lowest AIC model (ΔAIC). Akaike’s weights (ω(DOCX)Click here for additional data file.S6 TableST / (1-GST)]. Geographic refers to log-transformed null resistance distance.Model averaged coefficients not overlapping with zero are indicated with asterisks. The genetic differentiation between populations was calculated as [G(DOCX)Click here for additional data file.S7 Tablei) for each model are listed. The genetic differentiation between populations was calculated as [DST / (1-DST)]. Geographic refers to log-transformed null resistance distance.Models are ranked based upon the difference between Akaike’s Information Criterion (AIC) in each individual model and the lowest AIC model (ΔAIC). Akaike’s weights (ω(DOCX)Click here for additional data file.S8 TableST / (1-DST)]. Geographic refers to log-transformed null resistance distance.Model averaged coefficients not overlapping with zero are indicated with asterisks. The genetic differentiation between populations was calculated as [D(DOCX)Click here for additional data file.S9 Tablei) provide the weight for each model. Elevation was computed as the Euclidean elevation distance matrix. Deforestation represents the percentage of deforested cover and was coded as a resistance variable. Geographic refers to null resistance distance.Models are ranked based upon the difference between Akaike’s Information Criterion (AIC) in each individual model and the lowest AIC model (ΔAIC). Akaike’s weights (ω(DOCX)Click here for additional data file.S10 TableST were used as estimates of genetic differentiation between M. affinis’ populations. Elevation was computed as the Euclidean elevation distance matrix. Deforestation refers to the percentage of deforested cover and was coded as a resistance variable. Geographic refers to null resistance distance. All explanatory variables are centered.Model averaged coefficients not overlapping with zero are indicated with asterisks. Population pairwise F(DOCX)Click here for additional data file.


Reichardia picroides, Scolymus hispanicus, Scandix pecten-veneris, Leontodon tuberosus, Cichorium spinosum, Sonchus asper ssp. glaucescens, Urospermum picroides, Prasium majus, Hypochoeris radicata, Centaurea raphanina ssp. raphanina and Anagallis arvensis were collected and identified from nine regions with distinct microclimate (Lassithi prefecture), and their genetic composition was studied by means of RAPD markers.The present work aimed to investigate the genetic structure of 11 edible herbs grown in the wild of eastern Crete that are becoming vulnerable due to habitat destruction and unregulated harvesting. Thirty three populations  of Fst values among regions were moderate to high, suggesting partial population fragmentation. Bayesian structure analysis revealed fine genetic composition and substantial admixture between species present in different regions, although clustering was mainly geographically related.A total of ten primers per population were used to detect genetic diversity and bootstrap analysis was conducted for clustering the samples. High levels of heterogeneity were revealed while the Analysis of Molecular Variance documented that variance was allocated mainly within populations and at a lesser extent among populations. High altitude regions, with little residential and agricultural development , were the areas where high biodiversity was detected. On the other hand, coastal regions had lower biodiversity, probably due to degradation of their habitat. Greece is a country where more than six thousand plant species are listed, from which almost 500 of them are indigenous. At the island of Crete alone about 2000 different taxa have been recorded . Local ein situ and/or ex situ) that are under threat, mainly because of over-harvesting from the wild (it is difficult to cultivate them and no proper guidelines exist) as well as by the agricultural intensification and urbanization. Recently, a directive from the Greek forestry department was issued for all regions in Crete prohibiting collecting from the wild of edible plants for marketing reasons, while restricted quantities are allowed for personal use, in order to promote the growth of natural populations.Another distinct feature of the Cretan vegetation is that many herbs, endemic or not, are edible and constitute an integral part of the everyday traditional diet. Several studies on wild edible herbs consumed in Crete even today have demonstrated their great nutritional value –6. Howevex situ collection, in situ preservation and possibly propagation of genetic material.To acquire a general overview of the genetic diversity of 11 edible wild species, 33 populations were collected from nine different locations of eastern Crete  totaling 268 individuals. Surveying morphological variation of wild species is a rather cumbersome and difficult task since little (if any) morphological descriptors have been developed and there is always the chance that collectors may remove specimens included in any genetic study. In the present study plants were collected for botanical identification as well as for DNA fingerprinting using RAPD markers. We aimed to address: a) the extent of genetic diversity of the sampled populations, b) the within and between population genetic diversity, and c) the genetic structure of species/populations related to their distribution. The ultimate purpose is to identify areas where species exhibit greater variability for S. hispanicus, 100% for L. tuberosus, 91.66% for C. raphanina ssp. raphanina, 90.90% for A. arvensis, 88.88% for R. picroides, 70% for U. picroides, 66.66% for H. radicata and 60% for P. majus). Mean heterozygosity (Table S. hispanicus (0.271 ± 0.017), C. raphanina Sm. ssp. raphanina (0.253 ± 0.013) and H. radicata (0.246 ± 0.016), while the lowest values were recorded for A. arvensis (0.182 ± 0.012), S. asper subsp. glaucescens (0.181 ± 0.009) and P. majus (0.140 ± 0.008).In total, 268 plants were identified and studied in their natural habitat Figure ; Table 1ty Table  was highS. hispanicus, 95%; U. picroides, 87%; R. picroides, 87%; C. spinosum, 85%). On the other hand, the lowest within population diversity was recorded for S. asper ssp. glaucescens (69%) that also had the highest Fst values (0.310) revealing high levels of divergence.High levels of genetic heterogeneity Table  were detS. pecten-veneris, C. spinosum, H. radicata, C. raphanina spp. raphanina, A. arvensis and S. asper spp. glaucescens a definite region-oriented clustering was recorded. Overall, extensive admixture was recorded among accessions of different populations and several subclusters were formed. On the contrary, other populations seemed to cluster together due to reduced genetic diversity among them. Results for each species are as follows:In general, samples were organized in small clusters and further divided in subgroups, showing moderate and high bootstrap values Figure . In seveFst values were recorded among populations and variation occurred almost exclusively within populations  as indicated by AMOVA and populations were fairly diverged . In this species it seems that variability mainly exists within populations  behaves as a cross-pollinator and that some individuals appear as self-pollinators. They can grow up quickly as self-pollinators in order to colonize an area and then behave as cross-pollinators resulting in a buildup of diversity [For all species studied, genetic variability among populations was evident to a lesser or greater extent. For each species, the corresponding dendrogram showed a trend for population clustering according to geographic origin, which is consistent with other studies . Furtherdistance . Howeverdistance . Moreoveulations –16. Hypoiversity . Honnay  [et al.  and Orti [et al.  refer toiversity . In the iversity , 20). ThA. arvensis and S. asper ssp. glaucescens, indicating population fragmentation that could lead to limited gene flow [Low genetic variability was found for ene flow . In isolene flow –25. Furtene flow  and selfene flow . Moreoveene flow . On the ene flow , 28, leaene flow .Cicer arietinum [Lens taxa [Poa annua [R. picroides, L. tuberosus). These observations show the dynamics of populations in various regions as well as the differentiation of areas concerning their biodiversity for all species of indigenous edible plants.Heterozygosity varies greatly in other annual taxa examined e.g., He = 0.004 in rietinum , He = 0.ens taxa , or He =oa annua . This pooa annua , althougoa annua . It is boa annua –36. Nybooa annua , have ilin situ conservation of genetic resources and the selection of protected areas. The regions of Kefala, Agrilos, Tziritis and Ziros presented important biodiversity for most of the species investigated, while the areas of Mochlos, Kastelas, Limenaria and Richitis had fewer species occurrences with reduced variability. The areas of Kefala, Agrilos, Tourloti and Ziros are located in relatively high altitude and have restricted residential development, therefore most species appear to have great heterogeneity. In the region of Kefala, the species S. hispanicus, L. tuberosus, and C. raphanina Sm. subsp. raphanina exhibit broad genetic diversity. In Agrilos great variability is documented for S. asper subsp. glaucescens, U. picroides, H. radicata and A. arvensis, while in Ziros for R. picroides, S. pecten-veneris, and P. majus. Cichorium spinosum was only found in coastal areas showing high diversity in the Richtis region. Finally at Tourloti, S. pectin and P. majus had the highest heterogeneity.The data presented could facilitate a decision for the The above findings could be explained mainly on the basis of land use and the overall human activity in the regions. The areas with the lowest diversity include mainly cultivated land (olive trees and intensified vegetable cultivation) and large settlements with tourism development. The low levels of diversity in agricultural land could be caused by the pressure due to the cultivation techniques  and may be associated with an increase in inbreeding as the number of plants in a population decline since the only place where they reproduce is at the margins of cultivated land . FurtherIn contrast, regions showing greater plant biodiversity are clearly less residential , with less intensified agriculture production . Furthermore, these areas are usually difficult to access, crossed by rugged rural dirt roads and only approached by shepherds and residents for seasonal works, with fallow land and limited ovine presence. Unfortunately, during the last years the Ziros plateau is becoming more accessible (paved roads) and more residential, while the grazing sheep/goat populations have increased due to internal migration from the west of Crete .C. spinosum and S. hispanicus). This in turn has increased collection of edible plants from the wild for commercial purposes since collectors have a market with high retail prices. The unmonitored collection, which does not take place in the traditional way , could lead to the decrease or even the extinction of a species.The species examined in the current study could provide an insight to the capacity of eastern Crete genetic resources for edible plants with potential economic value. The increased interest of consumers already has overcome the narrow base of the island, and has resulted in the commercial cultivation of some species , which in turn damages the biodiversity of the region.http://filotis.itia.ntua.gr/biotopes/c/A00050056/) is characterized as degraded/slow degrading, due to disturbances in the ecological balance caused by fires, tourism development and overgrazing, and must be prioritized as a preserved area. Kefala and Agrilos  are also designated as degraded/slow degrading. Given the fact that the recent land use changes and residential development apparently can affect the genetic structure of the Cretan edible plant communities, regions should be closely monitored over the coming years and develop conservation policies.The condition for the species examined is considered threatening. Furthermore, Ziros  are reported in Figure Reichardia picroides) was collected at Ziros and was the 42th sample acquired.Global Positioning System (GPS) data of eastern Crete areas and plants of the present study  and the lysis buffer I, following manufacturer’s instructions. DNA concentration and quality was calculated spectrophotometrically  and confirmed with 1% agarose electrophoresis using standard λ-phage molecular weights. More than 30 random decamer primers (Operon) were used for the initial screening of samples, from which nine or ten primers, depending on the species, were retained for their ability to produce unambiguous polymorphic products among genotypes (Table http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). An allele-based estimate was used to calculate mean heterozygosity over loci among all members of the population using GenAlEx 6.4 [DNA fragments were scored as present (1)/absent (0) for each reaction and were assembled in a binary data matrix table. Genetic similarities and bootstrap analysis were performed using the FreeTree program . DendrogAlEx 6.4 . Genotyphttp://taylor0.biology.ucla.edu/structureHarvester).A Bayesian model-based clustering approach for identification of the genetic structure in the edible herb germplasm was performed using STRUCTURE 2.3.4 . The pro


Dreissena polymorpha, Pallas, 1771) is one of the most invasive species of freshwater bivalves, due to a combination of biological and anthropogenic factors. Once this species has been introduced to a new area, individuals form dense aggregations that are very difficult to remove, leading to many adverse socioeconomic and ecological consequences. In this study, we identified, tested, and validated a new set of polymorphic microsatellite loci  using a Massive Parallel Sequencing (MPS) platform. After several pruning steps, 93 SSRs could potentially be amplified. Out of these SSRs, 14 were polymorphic, producing a polymorphic yield of 15.05%. These 14 polymorphic microsatellites were fully validated in a first approximation of the genetic population structure of D. polymorpha in the Iberian Peninsula. Based on this polymorphic yield, we propose a criterion for establishing the number of SSRs that require validation in similar species, depending on the final use of the markers. These results could be used to optimize MPS approaches in the development of microsatellites as genetic markers, which would reduce the cost of this process.The zebra mussel ( Dreissena polymorpha, Pallas, 1771) is a successful invasive bivalve that is native to the brackish and fresh waters that drain into seas of the Ponto-Caspian region  [Dreissena polymorpha has several biological attributes that facilitate the success of its invasions, including rapid growth with early sexual maturity, dispersal by larvae, unspecific food preference, and gregarious behavior [The zebra mussel (ov Seas) . Dreissebehavior . The invbehavior .D. polymorpha is of public interest because of the associated major economic and ecological damage caused by this species. Once individuals are introduced to a new area, high-density aggregations [D. polymorpha include the inability usage of freshwater superficies for recreational purposes and the blockage of artificial water-conducts, among others. The latter creates serious working problems to industries and water supply systems, as well as to the watering and refrigeration systems of hydroelectric, thermic, and nuclear structures [D. polymorpha species in Europe [The freshwater invasion of egations  have manegations . The socructures . Understructures . The mitn Europe –9, revean Europe –11.D. polymorpha across Europe and North America, only a few microsatellites have been described for this species [Microsatellites , also kn species –24 and t species , 25.de novo. One of the causes of having limited number of microsatellite loci is consequence of the high cost and labor-intensive approaches required in traditional methodologies of microsatellites isolation [For most non-model species, few or none microsatellite are described, and they must be isolated solation –27, but solation , even insolation .D. polymorpha employed traditional hybridization probes. The implementation of new microsatellites by MPS methods may provide new markers for assessing the dispersion capacity and invasion routes of D. polymorpha, which would help with the establishment of management strategies to control the invasive potential of this species [D. polymorpha. This process involved establishing procedures to set up and implement a protocol for the identification and validation of new markers for application in successive non-model species and organisms. The second objective was to determine the number of microsatellite markers needed for several genetic applications. The newly developed markers are expected to be used in subsequent studies focusing on the population genetic structure of D. polymorpha, to determine its invasion history and dispersal routes through Europe. This information could potentially help predict possible new areas of invasion, which would allow the implementation of preventative management to prevent accidental introduction.All of the microsatellites identified in  species . Thus, tD. polymorpha individuals by two phenol-chloroform and ethanol precipitations, as described in [Total DNA was isolated separately from three ribed in . RNase tD. polymorpha.DNA was sequenced using a 454 GS FLX platform using 1/8 of a plate. Reads were assembled using GS DE NOVO ASSEMBLER software, version 2.5.3.  . Two strWe used TANDEM REPEATS FINDER (TRF) software version 4.04  to identTandem repeat structures identified by TRF were pruned to obtain the Potential Amplifiable Loci (PAL), after removing structures composed of less than five tandem repetitions and regions with less than 30 base pairs in each of the flanking sequences and with positive primer annealing positions. In addition, we considered two neighboring SSRs separated by less than 100 nucleotides as the same locus (a compound microsatellite).et al. [PCR primers were designed  using PRet al. .D. polymorpha individuals were collected from six representative locations where they had been introduced in north-eastern Spain . DNA samples (25–100 ng) were added to each PCR mix. The thermal cycles consisted of an initial denaturing step of 3 min at 94°C, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 50°C or 60°C for 90 s, and extension at 72°C for 90 s, and a final extension period of 10 min at 72°C. Negative controls were included in all PCR runs to confirm that there was no cross-contamination. PCR products were read in a 3130 Genetic Analyzer (Applied Biosystems).The PCR mix was prepared in a final volume of 20 μL, containing 1X Buffer (BIOLINE), 1.5 μM MgClD. polymorpha individuals from four different locations separated in range of 167 and 562 km. If these initial PCR conditions failed or unspecific results were obtained, a second attempt was made using the same samples, but increasing the annealing temperature to 60°C. Successfully validated loci were genotyped in 48 D. polymorpha individuals to assess polymorphism and allele richness.Validation analysis of PALs was conducted in two steps . First, Ho) and expected (Hs) heterozygosities were assessed for each new polymorphic locus for conformance to Hardy-Weinberg and Linkage disequilibrium expectations by the exact test implemented in GENEPOP 4.0 software [PIC) index to estimate the information values of each microsatellite marker [We evaluated the presence of null alleles in each locus using MICRO-CHECKER software, version 2.2.3 . The obssoftware . We calce marker . MicrosaD. polymorpha in the Iberian Peninsula. We analyzed 48 D. polymorpha individuals from six representative locations where they are present in north-eastern Spain (HWE) of genotypic distributions was measured at each site by the exact probability test [A), the estimated expected heterozygosity (Hs), and allelic richness (Ar) from allele frequencies using FSTAT 2.9.3 [FST) [K) was estimated among all sampled collections using the Bayesian Markov Chain Monte Carlo approaching method of STRUCTURE software version 2.3.2 [K (1 to 6) were repeated five times. A burn-in period of 20,000 steps, followed by 100,000 Monte Carlo replicates, was simulated with the model of independent allele frequencies. The optimal K value was selected following the recommendations of Pritchard et al. [D. polymorpha collections provided by the genetic results were estimated by Analyses of Molecular Variance (AMOVA) conducted in ARLEQUIN 3.5 [We also tested the 14 new polymorphic microsatellites in the preliminary characterization of the genetic structure of rn Spain . The Harity test  using GEity test . Benjamiity test  False DiAT 2.9.3 . In addi3 [FST)  and signon 2.3.2 . Runs fod et al. , accordiQUIN 3.5 . In the SSR structures as possible, we selected the second assembly with the modified parameters “Large or Complex Genome” and “Heterozygotic Mode,” because it generated a higher N50 value with a greater number of long contigs of the same sequence quality.The 454 GS FLX run generated 110,593 reads , with a total of 36,554,817 bases . GS DE NTRF software identified a total of 299 single SSR structures. After removing homopolymers, 288 SSRs were identified with an overall estimated density of 0.18 SSRs/Kb. The SSRs were distributed in 6.60%, 37.85%, 39.58%, and 15.97% of di-, tri-, tetra-, and pentanucleotides respectively  of the 14 microsatellite loci  to 13 (Dp31). Estimates of gene diversity and the PIC index varied among loci. The lowest value of expected heterozygosity was obtained for Dp2 (Hs = 0.20), while the highest value was obtained for Dp1 (Hs = 0.83). The lowest PIC index was in Dp68 (PIC = 0.19), while the highest was in Dp31 (PIC = 0.83) (Dp1 and Dp2) presented significant deviations from Hardy-Weinberg equilibrium at the 5% significance level, and all loci appeared to be unlinked, according to the lack of gametic disequilibria in each location  . Only twlocation .D. polymorpha in the Iberian Peninsula. All 48 individuals collected from the six locations were used, with a sample size of eight individuals from each sampling site  and genetic diversity (Hs) in the Llobregat River  compared to the Ebro River basin . The lowest number of total alleles (A) across all 14 loci also corresponded to the Llobregat River (A = 43) in comparison to the Ebro River (A = 46–50)  .FST) among locations was FST = 0.044. None of the pairwise FST values were significant after FDR correction . Following the recommendations of Pritchard et al. [K = 1). Finally, the AMOVA test revealed similar genetic variance between the tested river basins (3.20%) and among locations within river basins (3.89%). Furthermore, genetic variance was not significant (P = 0.062) in the comparison between the two river basins.Estimated average genetic differentiation  compared to the average number of SSR structures identified using hybridization methodologies  (This study is the first to use MPS platforms to discover new SSRs in = 18.52) . However= 18.52) . Yet, th= 18.52) .D. polymorpha. This value is situated in the lower range of 50–1,500 microsatellites/Mb expected in eukaryotes [D. polymorpha  [Microsatellite isolation and validation in bivalve species is challenging for several reasons. For instance, we found about180 microsatellites per Mb in karyotes . This ph 1.6 Gb) –48, comb 1.6 Gb) , 49. In  1.6 Gb) .A = 4.79) and mean gene diversity (Hs = 0.56) compared to the value obtained by previous studies analyzing the population genetic structure of D. polymorpha  compared to the sample sizes used by previous studies . The pr(n = 309  and n = [n = 386 ). This det al. [et al. [D. polymorpha, a median of seven microsatellites is required  obtained in this study, we could estimate the number of initial SSR markers that would require validation in mollusk species depending on the desired genetic study . Thus, fD. polymorpha individuals in the Iberian Peninsula belong to the same genetic population supporting a previous preliminary genetics study using a single microsatellite marker [D. polymorpha could be caused by a combination of a free-swimming larval dispersion and human mediated, particularly in ballast waters in vessels. Nevertheless, further analysis with a larger sample size from more locations, and even including some of the previously described microsatellites [A preliminary analysis on the genetic structure  of these microsatellites in the Iberian Peninsula failed to show any population structure, but demonstrated the usefulness of these new markers. Our results suggest that all e marker . Since ie marker , the exptellites –24, is cD. polymorpha, which, added to those developed by Naish and Gosling, Feldheim et al, and Thomas et al [In summary, this study identified and validated 14 new polymorphic microsatellites in as et al –24, incrD. polymorpha at six locations in Spain using all 14 new microsatellite markers. Our results showed that all six locations represent the same genetic population; however, further analysis with a larger sample size from more locations, along with more microsatellites, is crucial to confirm this result. In conclusion, this study demonstrates the usefulness of these new markers for population genetic analyses in D. polymorpha.In addition, we conducted a preliminary study of the genetic structure of D. polymorpha were submitted to the NCBI Sequence Read Archive under accession number SRX803533. De novo assembly was also submitted in DDBJ/EMBL/GenBank Whole Genome Shotgun project when contigs size was longer than 500 nucleotides under accession number JWHF00000000. Monomorphic microsatellite sequences were submitted in Genbank with accession numbers KP274952–KP275018, and polymorphic microsatellite sequences with accession numbers JQ812984–JQ812997.The 454 GS FLX reads of S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Galápagos Archipelago. Understanding the phylogeographic history and population genetics of invasive species on the Archipelago is an important step in predicting future spread and designing effective management strategies. In this study, we describe the invasion pathway of Rattus rattus across the Galápagos using microsatellite data, coupled with historical knowledge. Microsatellite genotypes were generated for 581 R. rattus sampled from 15 islands in the archipelago. The genetic data suggest that there are at least three genetic lineages of R. rattus present on the Galápagos Islands. The spatial distributions of these lineages correspond to the main centers of human settlement in the archipelago. There was limited admixture among these three lineages, and these finding coupled with low rates of gene flow among island populations suggests that interisland movement of R. rattus is rare. The low migration among islands recorded for the species will have a positive impact on future eradication efforts.Human activity has facilitated the introduction of a number of alien mammal species to the  Rattus  are major vertebrate invaders and have been introduced to vast areas outside their native ranges due to their small size, adaptability, and commensal association with humans  is among the world's worst invasive species , Norway rat (R. norvegicus), and the House mouse (Mus musculus). From historical records and the detailed record of human activities on the islands, the sequence of introductions of these three species to the Galápagos Islands can be hypothesized. The sources of the initial colonizers and exact routes of dispersal across the islands for each species, however, remain to be clarified.Alien invasive species represent an ever‐growing threat to many global ecological systems. Members of the genus R. rattus has invaded the largest number of islands (36) in the archipelago and the species now occurs in a wide range of human and nonhuman associated habitats  group, and Isla Santa Cruz–Isla Baltra group. The Isla Pinzón population was morphologically very distinct from all other island populations and was aligned with the Isla Floreana–Isla Isabela–Isla San Cristóbal group only by the allozyme data  and dispersal patterns of this species across the Galápagos Islands.The evolution of taxa native to the Galápagos Archipelago has received much recent attention , while several islands like Isla Sombrero Chino are much smaller  from multiple localities on larger islands and single trapping areas on small islands. Nonetheless, in this study, we refer to all individuals sampled from a particular island as members of the same population. Because smaller islands are often spatially associated with larger islands and as such would be expected to have elevated interisland rates of migrations, smaller landmasses were assigned to larger islands  located off the west coast of Ecuador in the eastern Pacific Ocean is recognized as a World Heritage Site and a biodiversity hotspot . Permits were provided by the Parque Nacional Galápagos. Field methods followed guidelines for use of wild mammals in research  using published primers , 0.1 μL AmpliTaq® DNA polymerase Applied Biosystems, Foster City, California, United States, 2.0 μL 10× reaction buffer, 1.5 μL of 8 mmol/L dNTPs mixture, 1.0 μL of each 10 μmol/L primer, 1.0 μL betaine, and 11.4 μL DEPC H2O for a final reaction volume of 20 μL. Positive and negative controls were used during PCR to ensure accuracy of results within and across PCRs and microsatellite size calls. Following optimization, the thermal profile consisted of the following parameters: one cycle at 95°C for 5 min, followed by 35 cycles at 95°C for 20 sec, locus‐specific annealing temperature for 30 sec, 72°C for 90 sec with an increase of 5 sec/cycle, and a final extension of 72°C for 10 min. Amplified fragments were diluted as necessary and run with ILS 600 size standard  in HiDi formamide on an SCE 9610 . Chromatograms were analyzed using GenoSpectrum software  with both automated peak detection and manual inspection. Allele size data were exported from GenoSpectrum and then binned and scored using a custom PERL script as well as manual correction in Microsoft Excel.DNA was extracted using DNeasy tissue kits , and DNA was stored in elution buffer at −20°C. Successful extraction was verified by visualization on a 1% agarose gel, and extracts were diluted 1:10 for subsequent amplifications. Ten loci were amplified in FSTAT 2.9 (Goudet HO), average expected heterozygosity (HE), allelic richness (AR), and the fixation index (F).FreeNA  correction, implemented in FreeNA. This method corrects for the bias introduced by the presence of null alleles  in GenAIEx. Isolation by distance was tested using Mantel tests; the pairwise geographic distance matrix (in km) was calculated using the latitude and longitude coordinates of the center of each island  measures and geographic distance was assessed with 10,000 random permutations in GenAIEx. The hierarchical genetic differentiation  was statistically examined using an analysis of molecular variance (AMOVA) in GenAIEx.Genetic differentiation among island populations of K) ranging from 1 to 10. Each run was performed under the admixture model with correlated allelic frequencies, using 100,000 Markov chain Monte Carlo iterations and a burn‐in of 10,000 iterations, which was sufficient for likelihood stabilization. The optimal number of clusters was assessed using the Evanno et al.  tree representing the relationships among sampling localities was constructed using the PHYLIP package v. 3.69 , indicating substantial population subdivision across the islands. Original pairwise FST estimates (uncorrected) all fall within the 95% CI of the ENA‐corrected estimates (data not shown). Thus, the effects of any null alleles present in the R. rattus data set are likely minimal and all further analysis was performed using data from all loci.The mean null allele frequencies for five loci  exceeded 10%. The presence of null alleles in data can be problematic for calculations of genetic variability as they can decrease within‐population variance and so inflate FR. rattus population, reflected in the high allelic richness (which corrects for the biasing effects of sample size variation) estimated  is comparable although historical records suggest that these islands were colonized at least 200 years apart. There was also no evidence for an increase in genetic diversity with increasing size of island, with many smaller islands having populations that contain allelic richness comparable with populations sampled from larger landmasses – for example; although the number of R. rattus collected from Isla Isabela and Isla Sombrero Chino differ, these two islands have comparable levels of observed and expected heterozygosities and allelic richness despite a considerable difference in island size . The AMOVA revealed that most of the genetic variance could be explained by within population differences .Pairwise es Table  were allK) identified in STRUCTURE analyses using the Evanno et al.  and when island populations are assigned to the clusters suggested by the STRUCTURE analysis (P = 1.0). This suggests that the genetic structure among the island populations of this invasive rodent species results from a combination of migration and genetic drift. There was, however, considerable variation in the contribution of gene flow and genetic drift among island populations of R. rattus , suggesting that genetic drift has played a much greater role in the demographic history of this population .The migration‐drift model was favored by 2MOD, both when individuals were assigned to islands . Migration rates exceeding 1% were observed between the R. rattus population of Isla Santa Cruz and populations on the geographically proximate islands of Isla Seymour Norte , Isla Baltra , Islote Venecia , Isla Eden , and Islote Punta Bowditch . Similarly, migration rates higher than 1% were observed among the Santiago R. rattus population and populations on Isla Sombrero Chino , Isla San Cristóbal , and Isla Bartolomé . Low migration rates with 95% CI with lower bounds including zero were also observed when R. rattus individuals were assigned to clusters suggested by STRUCTURE analysis.Multiple runs of BayesAss yielded consistently low estimates  of contemporary gene flow indicating limited migration among most of the island populations of R. rattus present on the islands. The levels of heterozygosity for the R. rattus across the Galápagos (R. rattus: HO = 0.12–0.45) are within the lower range observed in other archipelago populations  separate colonization events from different source populations. The geographic patterning of the genetic variation for this species is in agreement with the colonization pattern described by Patton et al. (R. rattus on the geographically proximate islands of Isla Sombrero Chino, Rocas Bainbridge, and Isla Bartolomé to this first wave of colonization. A second independent introduction of R. rattus to the archipelago likely occurred on Isla Floreana associated with the establishment of Villamil's settlement in 1832. The spread of R. rattus individuals belonging to this genetic grouping from the source population to the islands of Isla Isabela and Isla San Cristóbal was likely through human‐mediated dispersal. It is then likely that humans facilitated the dispersal of R. rattus to Islote Marielas from Isla Isabela. The most recent introduction of R. rattus occurred on the islands of Isla Baltra and Isla Santa Cruz, with individuals belonging to this latter genetic cluster also colonizing the adjacent islands of Isla Seymour Norte, Islote Venecia, Isla Eden, and Islote Punte Bowditch. The distinctiveness of the R. rattus on the Isla Pinzón as suggested by Patton et al. (R. rattus with populations on Isla Floreana, Islote Marielas, Isla Isabela, and Isla San Cristóbal. The PCoA plots, NJ topology, and percolation network for R. rattus, however, did not closely associate the Isla Pinzón population with the Isla Floreana genetic grouping. This genetic differentiation coupled with previously reported morphological distinctiveness of this population (Patton et al. R. rattus on Isla Pinzón may have been established by a fourth distinct colonization event. Alternatively, members of the Isla Floreana genetic grouping may have initially seeded the population on Isla Pinzón, but genetic drift coupled to an extended period of isolation could be responsible for the genetic differentiation of the R. rattus population on this island. Although there are fewer historical records documenting the introduction and spread of the other invasive rodents, M. musculus and R. norvegicus, across the archipelago, it seems likely that the phylogeographic pattern of these species on the islands has also been shaped by multiple independent colonization events.The genetic structuring observed in the microsatellite data for n et al. . Historin et al.  was agaiThe biodiversity on several islands in the archipelago have already benefited from eradication of other invasive mammal species (Carrion et al. None declared.http://datadryad.org under DRYAD Repository entry doi: 10.5061/dryad.1s88tMicrosatellite genotypes are available for download at


This is a contribution to the history of scientific advance in the past 70 years concerning the identification of genetic information, its molecular structure, the identification of its functions and the molecular mechanisms of its evolution. Particular attention is thereby given to horizontal gene transfer among microorganisms, as well as to biosafety considerations with regard to beneficial applications of acquired scientific knowledge. The discovery of horizontal gene transfer is related to the introduction of experimental microbial genetics some 70 years ago. Soon thereafter, medical microbiology identified the raising problem of increasing antibiotic resistance of pathogenic bacteria. This medical problem stimulated research in bacterial genetics which revealed that horizontal gene transfer is involved in some of the genetic variations causing resistance to antibiotics.As a contribution to the history of scientific investigations, we trace here a sequence of steps of conceptual and experimental approaches to understand microbial evolution at the molecular level. This shall allow us to extrapolate to generally valid laws of nature guiding biological evolution by self-organization. We will also discuss implications of the acquired scientific knowledge.i.e., to carry out genetic experiments with bacteria as well as with bacteriophages.It is in the first half of the 20th century that microbiologists became aware that bacterial isolates and bacterial viruses (bacteriophages) under study could spontaneously produce phenotypic variants. This property offered the chance to investigate recombination between different mutants and between different microbial strains, pneumococcal bacteria has identified two forms: smooth (S) cells which are virulent, and rough (R) cells which are not virulent as seen upon infection of mice in laboratory tests. Using this experimental system, Griffith [Early work with Griffith  infectedGriffith  identifiGriffith  showed tGriffith . These aEscherichia coli strains [− recipient bacteria. This allows a copy of the replicating F to enter into the partner cell. From its integrated Hfr state, the F transfer normally also transfers neighboring parts of the donor chromosome. This kind of experiments led to the conclusion that the E. coli genome is one large circular DNA molecule. Occasionally, F undergoes unprecise excision from the Hfr chromosome. It thereby becomes autonomous again and carries in addition to its own genes some genes from its bacterial host. Such hybrid derivatives are called F’ and they are actually vectors for bacterial genes which can become transferred into recipient bacteria upon conjugation ..EschericF’-like conjugative plasmids have soon become known to act sometimes in the horizontal transfer of antibiotic resistance determinants. This can seriously contribute to the spreading of these determinants to pathogenic bacteria, in particular in the presence of antibiotics exerting a selective pressure.Salmonella bacteria [Salmonella phage P22 and for the E. coli phage P1. In contrast, in specialized transduction, the transducing viral particle contains a hybrid molecule with a part of the phage genes and some bacterial genes. In the late 1950’s I had the chance to identify for the first time such a hybrid genome in λgal derivatives of phage λ. This hybrid DNA molecule was still able to undergo autonomous replication, but instead of some of its viral coat genes, it carried genetic determinants for the fermentation of galactose picked up in the host chromosome [Transduction was first seen in studies on gene exchange between bacteria . In latebacteria ). Two diUpon genetic experimentation using several bacterial strains and their phages, a phenomenon called host-controlled modification was encountered by a few independent scientists. In the 1960’s we succeeded to unravel its molecular mechanisms . In brieIn this context, it might be relevant to briefly mention here that in the world of bacteria and their viruses, several other barriers are known to limit the rates of uptake of foreign genetic information. Among these are a lack of bacterial surface compatibility for conjugational pair formation or for the uptake of DNA molecules from the medium, bacteriophage host range specificities, a requirement for functional harmony between the invading genetic information with the recipient’s genome, and a rather unexpected microbial immunity, called CRISPR-Cas, acquired upon a previous infection .In view of the large size of DNA molecules carrying the genomic information of any organism, researchers reflected on possibilities to sort out particular genes and to multiply them in view of their structural and functional studies. Viral DNA molecules and F conjugative plasmids were envisaged to serve as autonomously replicating gene vectors, into which a relatively short DNA fragment could be spliced. Such hybrids might then serve for studies of both structural and functional properties of the inserted DNA fragment. This possibility became realistic when bacterial restriction enzymes became available around 1970 .Scientists involved in this work discussed measures to be taken in order to prevent undesirable effects of recombinant DNA molecules with yet unknown genetic information. Such conjectural effects might affect the health of the investigator, on the one hand, and they might also render possible an undesirable spreading of the inserted genetic information to other organisms after an accidental or a deliberate release into the environment, on the other hand . The ansBoth, already available data on spontaneous genetic variation and novel experimental results, in particular from microbial genetics, revealed a multitude of specific molecular mechanisms to contribute to the overall spontaneous genetic variation. We have classified these identified molecular mechanisms of genetic variation into three natural strategies of genetic variation . These aThese natural laws of genetic variation are solidly shown to apply to microorganisms. Fewer experimental data are at this time available for higher organisms, but reports on cross-species gene transfer , as wellExperimental observations show that in spontaneously occurring genetic variation specific gene products are often involved, on the one hand, and/or non-genetic properties of the non-living world, on the other hand. Some of the involved gene products act as variation generators, others as modulators of the rates of genetic variation, which provides to the living organisms a relatively high genetic stability required for maintenance of life . Many ofIn this self-organized biological evolution nature uses, besides the activities of evolution genes, a number of already mentioned non-genetic elements such as structural flexibilities (isomeric forms) and limited chemical stability of biological molecules, random encounter and environmental mutagens . Random encounter is of particular relevance for horizontal gene transfer.i.e., the universality of the genetic code, plays thereby an essential role [The natural strategy to occasionally accept foreign genetic information by horizontal gene transfer links all living organisms into a global system. Whereas Darwin had drawn the evolutionary tree linking different organisms at the bottom of the tree, we can now, in addition, interlink different branches of the tree with horizontal connectors in order to allow for horizontal transfer of individual genes . Being aial role ,18.etc.) to a healthy life of their hosts. This cohabitation is called microbiome and it can be maintained for long periods of time, whereby the microorganisms reside sometimes outside and in other cases inside of the cells of their host organism. Any such cohabitation must favor an occasional gene transfer between the involved organisms [In recent years it became obvious that all kinds of higher organisms, plants, animals and humans carry in and on their bodies a high number of microorganisms. These are only rarely pathogenic. They rather profit from a stable symbiosis. The higher organisms offer to bacteria good habitats, and bacteria contribute in a number of ways  can best contribute to conceptually understand the self-organized natural process of the slow but steady evolution of life to a rich biodiversity within a global system of interdependencies. This does not only concern the genomic information, but also compositional differences of appropriate habitats, including the availability of healthy nutrition.In our daily life we are used to observe and to understand relatively fast processes. However, both the evolution of life and cosmic evolution are extremely slow, long-term processes which we have difficulty to perceive with our sensory organs. This may be a possible reason why a number of people (including some life scientists) still remain anchored in a fundamentalistic world-view claiming that there is no evolution. From an updated scientific view-point, we understand that a fast evolution of life could be detrimental. We assume that in the long past of biological evolution, the evolution gene activities of variation generators and of modulators of the rates of genetic variation have become fine-tuned to work as they do today. Note that spontaneous genetic variation is contingent so that by far not all genetic variants show improved functions . Therefoi.e., the acquisition of genetic information that had been developed in other kinds of living organisms. By chance, a gene acquisition can provide to the receiving organism a welcome novel function that provides a selective advantage. An example is the acquisition of genetic information for antibiotic resistance by pathogenic bacteria. We see here a sharing in the evolutionary success of other organisms. Of course, horizontal gene transfer can, in other cases, also lead to a selective disadvantage by negative impacts on the functional harmony of the receiving organism.Let us start with horizontal gene transfer, The quality of local mutagenesis resides mainly in a possible stepwise improvement of an already existing biological function. Again, such mutations are contingent and not precisely targeted as a response to identified needs.Intragenomic DNA rearrangements offer possibilities to fuse functional segments of DNA sequences; this can occasionally result in novel activities. The fusion between two different functional domains may, by a rare chance, lead to a novel, welcome function. Or the fusion of a functional gene with an alternative control element for gene expression can influence the expression of the concerned gene.We have to be aware that in all these events, functional improvements can be a relatively rare consequence, while a selective disadvantage and in the extreme case lethality are more likely to result. The self-organizing biological evolution requires thereby a sacrifice from one individual in a large population undergoing a slow but steady evolution. Let us mention in this context that religious persons may encounter conceptual difficulties to accept that genetic adaptation to novel life conditions occurs with some contingency.in vitro construction of genomic segments. With regard to the formation of recombinant DNA molecules, this precautionary principle can validly contribute to the biosafety of the envisaged product of biosynthesis. Interesting examples of in vitro transfer of useful genetic information into another living organism allowing for a domesticated production and harvest of the relevant gene product were reported recently [As we have already outlined above, in the recombinant DNA technique selected DNA sequences are sorted out and spliced into other DNA molecules such as natural gene vectors. This and other procedures, such as site-directed mutagenesis, have become important contributions to the so-called synthetic biology that can serve for useful applications of fundamental scientific knowledge on biological functions. Insights into principles of natural strategies of genetic variation as outlined in recently . We can


Thresholds that require high sequence similarity among reads for assembly (stringent thresholds) as well as highly variable species may result in datasets in which divergent alleles are lost or divided into separate loci (‘over-splitting’), whereas liberal thresholds increase the risk of paralogous loci being combined into a single locus (‘under-splitting’). Comparisons among datasets or species are therefore potentially biased if different similarity thresholds are applied or if the species differ in levels of within-lineage genetic variation. We examine the impact of a range of similarity thresholds on assembly of empirical short read datasets from populations of four different non-model bird lineages (species or species pairs) with different levels of genetic divergence. We find that, in all species, stringent similarity thresholds result in fewer alleles per locus than more liberal thresholds, which appears to be the result of high levels of over-splitting. The frequency of putative under-splitting, conversely, is low at all thresholds. Inferred genetic distances between individuals, gene tree depths, and estimates of the ancestral mutation-scaled effective population size (θ) differ depending upon the similarity threshold applied. Relative differences in inferences across species differ even when the same threshold is applied, but may be dramatically different when datasets assembled under different thresholds are compared. These differences not only complicate comparisons across species, but also preclude the application of standard mutation rates for parameter calibration. We suggest some best practices for assembling short read data to maximize comparability, such as using more liberal thresholds and examining the impact of different thresholds on each dataset.Comparing inferences among datasets generated using short read sequencing may provide insight into the concerted impacts of divergence, gene flow and selection across organisms, but comparisons are complicated by biases introduced during dataset assembly. Sequence similarity thresholds allow the  With the proliferation of population-level datasets obtained using massively parallel sequencing technologies, there is increasing interest in studies that compare inferences from genomic datasets obtained from different species e.g.,  or from Sequence similarity provides the information necessary for assembling reads into orthologous loci . By settMany methods default to a stringent similarity threshold, often requiring 98–99% sequence similarity among reads for assembly e.g., . HoweverComparative phylogeographic and population genetics studies are particularly susceptible to biases resulting from similarity thresholds, particularly over-splitting. Different species often exhibit different levels of genetic diversity , and thiIn this study, we examine the effect of similarity thresholds on dataset assembly and phylogeographic inferences across four non-model bird lineages that vary in divergence. We sample two populations or species within each lineage and assemble a RAD-Seq dataset for each lineage at a series of similarity thresholds to assess the impact of different thresholds on the number of unique alleles observed within assembled loci. We investigate the effect of different similarity thresholds on estimates of standard population genetic and phylogeographic parameters within species and in comparisons across species.Rallus crepitans JF Gmelin, 1788) and King  rails, sister species of medium-sized water birds that interbreed in a narrow hybrid zone centered on a salinity gradient , a hummingbird endemic to Jamaica that comprises two subspecies (T. p. polytmus and T. p. scitulus) that differ primarily in bill coloration and interbreed in a narrow hybrid zone  and Baron’s  Spinetails are closely related, small insectivorous birds distributed along the Andes Mountains , a widespread insectivorous bird of lowland Neotropical forests that are separated by the Andes and differ in plumage, voice, and genetic markers  following the manufacturer’s protocol. We sent DNA extracts to the Cornell Institute of Genomic Diversity (IGD) to collect data using Genotyping by Sequencing, a RAD-Seq method . Brieflyde novo using the program denovo_map.pl. We used custom Python (https://github.com/mgharvey/misc_Python) to obtain sequence alignments of both alleles for each individual from the Stacks output files. Detailed settings are provided in the supplement  at all integer values from 93%  to 99% , reflecting the range of settings typically used for assembling intraspecific datasets. Assembly with similarity thresholds less stringent than 93% failed due to high computational demand in Stacks, but should not be necessary for the divergences examined here or for most other population-level studies. Reads with similarity values above the selected threshold clustered into assemblies, which we treated as independently segregating loci in downstream analyses. We disabled the use of secondary, more divergent reads for calling genotypes (Stacks setting -H) to prevent the assembly of reads that are less similar than the similarity threshold used for primary stacks. We set minimum depth per allele (Stacks setting -m) to ten, which provides a balance between the inclusion of singleton alleles  and the total size of the data matrix . We set We examined the number of unique alleles per locus across treatments to determine how different similarity thresholds affected each dataset. As an index of the frequency of under-splitting in each dataset, we calculated the number of loci containing individuals with more than two alleles. These loci were presumed to contain paralogous reads and were removed from further analysis. To assess the proportion of loci with putative over-split alleles, we mapped loci assembled under the more stringent thresholds (94–99%) to the set of loci assembled under the most liberal threshold (93%). This allowed us to detect instances in which multiple loci from the more stringent threshold mapped to the same locus from the liberal threshold. We used LASTZ  for mappp-distances and Jukes-Cantor corrected distances per unit sequence length at each locus. We measured distances between individuals by measuring the average distance between alleles in the first individual and those in the second individual. For loci containing variable sites, we also estimated stF between the two populations within each lineage using formula (3) of Over-splitting may reduce estimates of genetic distance between individuals or populations if they contain dissimilar alleles. Conversely, if over-splitting reduces the number of alleles within populations, this may reduce estimates of distance between populations. We calculated pairwise Over-splitting may also reduce average gene tree depth due to the loss of more variable loci owing to them being subdivided into two or more less variable loci. To reduce computation, we selected a random subsample of 1,000 loci for each lineage at each threshold for gene tree estimation. We selected the best-fit finite sites substitution model for each locus using mrAIC.pl  and condτ) as well as population standardized mutation rate parameters  for both daughter populations and an ancestral population. We set prior values using gamma distributions determined by a shape parameter (α) and scale parameter (β). Priors for both divergence time and population standardized mutation rate were set to α = 1 and β = 300. We ran analyses for a burn-in of 50,000 iterations and then sampled every other iteration for an additional 500,000 iterations.We used the 1,000 locus subsets from gene tree estimation to estimate ancestral and contemporary population sizes in each lineage at each similarity threshold using the coalescent model implemented in BP&P . Althougp < 2.20−16; Xenops contained, on average, 1.4 times as many alleles as Rallus when processed with a 99% similarity threshold, but 1.66 times as many alleles when processed with a 93% similarity threshold.After removing loci containing putative paralogous reads (see below), we recovered between 96,776 and 158,328 loci for the four lineages across the range of similarity thresholds examined . The simTrochilus exhibited roughly half the level of putative paralogy displayed in the other lineages  increased slightly with increasing similarity thresholds, but was less than 0.4% at all thresholds for all lineages . At all lineages . Dependilineages .p and Jukes-Cantor corrected genetic distances between individuals were reduced at more stringent similarity thresholds  showed no association with similarity thresholds  are widely applied currently to population-level studies e.g., , perhapsWe were unable to directly investigate the frequency of under-splitting and over-splitting in our datasets because we lack genome sequences for the non-model organisms examined. Our indirect measure of over-splitting may detect not just over-split loci, but also loci that are under-split in the assembly from the most liberal threshold but correctly separated in the assembly from the more stringent thresholds. This would be particularly likely if paralogy was common in the genomes under investigation or if very liberal similarity thresholds were examined. The frequency of under-splitting appears to be low enough in our datasets, however, that this effect would be minimal. Broad concordance between our results and prior investigations into over-splitting in systems with a genome for reference  suggest Results from our indirect measure of under-splitting are also broadly consistent with the low levels of under-splitting observed in prior work using reference genomes  and wereXenops minutus generally displayed the greatest allelic diversity and also the largest genetic distances between individuals, deepest gene trees, and highest θ values, which was perhaps not surprising given prior evidence of deep genetic divergences within this species  varies depending on the similarity threshold applied.Variance across the four study species in mean number of alleles and mean estimates of phylogeographic and population genetic parameters  of the two extant populations did not show an association with similarity threshold for any species. Circles and triangles are used to distinguish the two daughter populations from each species.Click here for additional data file.10.7717/peerj.895/supp-4Figure S4Mean values of population divergence time (tau) between the two extant populations did not show an association with similarity threshold for any species.Click here for additional data file.10.7717/peerj.895/supp-5Table S1Click here for additional data file.10.7717/peerj.895/supp-6Table S2Click here for additional data file.10.7717/peerj.895/supp-7Table S3Click here for additional data file.10.7717/peerj.895/supp-8Table S4Click here for additional data file.


Long-term benefits in animal breeding programs require that increases in genetic merit be balanced with the need to maintain diversity (lost due to inbreeding). This can be achieved by using optimal contribution selection. The availability of high-density DNA marker information enables the incorporation of genomic data into optimal contribution selection but this raises the question about how this information affects the balance between genetic merit and diversity.The effect of using genomic information in optimal contribution selection was examined based on simulated and real data on dairy bulls. We compared the genetic merit of selected animals at various levels of co-ancestry restrictions when using estimated breeding values based on parent average, genomic or progeny test information. Furthermore, we estimated the proportion of variation in estimated breeding values that is due to within-family differences.Optimal selection on genomic estimated breeding values increased genetic gain. Genetic merit was further increased using genomic rather than pedigree-based measures of co-ancestry under an inbreeding restriction policy. Using genomic instead of pedigree relationships to restrict inbreeding had a significant effect only when the population consisted of many large full-sib families; with a half-sib family structure, no difference was observed. In real data from dairy bulls, optimal contribution selection based on genomic estimated breeding values allowed for additional improvements in genetic merit at low to moderate inbreeding levels. Genomic estimated breeding values were more accurate and showed more within-family variation than parent average breeding values; for genomic estimated breeding values, 30 to 40% of the variation was due to within-family differences. Finally, there was no difference between constraining inbreeding via pedigree or genomic relationships in the real data.The use of genomic estimated breeding values increased genetic gain in optimal contribution selection. Genomic estimated breeding values were more accurate and showed more within-family variation, which led to higher genetic gains for the same restriction on inbreeding. Using genomic relationships to restrict inbreeding provided no additional gain, except in the case of very large full-sib families. Selection of livestock has led to increases in inbreeding over time, especially following the widespread use of artificial insemination and reproductive technologies. Inbreeding reduces the total number of heterozygotes in the population, hence reducing variation, which can cause a depression in fitness (inbreeding depression) and a decrease in selection response . SelectiEstimation of breeding values based on DNA marker information is now used in many livestock breeding programs . GenomicVarious methods have been developed to manage inbreeding in livestock breeding populations and all focus on managing inbreeding while increasing genetic gain -6. To atThe addition of genomic information to optimal contribution selection can have several impacts on the genetic gain achieved in a breeding program. First, EBV based on genomic information are generally more accurate than PA_EBV  and alloThe aims of this study were to test the effect of genomic information on the balance between genetic gain and inbreeding under optimal selection and to observe how much GEBV vary within a family in real data.Ne) of 1000. As described in Clark et al. [8 base pairs) were simulated with a mutation rate of 2.5.10-8 per site. The total number of single nucleotide polymorphisms (SNPs) segregating on the genome was approximately 1 670 000. Sixty thousand SNP markers and 6000 QTL were randomly selected from the SNP sequence for genomic analysis. Therefore, each SNP had a 3% chance of being used as a marker and a 0.03% chance of being used as a QTL. As in Meuwissen et al. [Genotype simulations were conducted using the Markovian Coalescence Simulator (MaCS)  to simulk et al. , 30 chron et al. , the addThe base haplotypes were randomly allocated to 500 male and 500 female base animals of a simulated population structure. Two subsequent generations received haplotypes via Mendelian inheritance, allowing recombination to occur according to genetic distance, i.e. 1% recombination per cM. Each generation in the simulated population contained 1000 animals . Twenty males were randomly selected in each generation and each male was randomly mated to 25 females. Each female had two offspring per generation . A second population was simulated with a full-sib family structure (full-sib population). This population included 25 males, each mated to two females and all females had 20 offspring each.All animals in each simulation were allocated breeding values and phenotypes. True breeding values (TBV) for each animal were determined using:βj is the additive effect of QTL (j) and Qkj is the QTL genotype at locus j, which was coded as 0, 1, or 2, as the number of copies of a given QTL allele that an individual (k) carries. Trait phenotypes were simulated by adding a random environmental effect drawn from a normal distribution with variance h2) of 0.3.where Selection of animals in the third generation was investigated. Phenotypes and SNP genotypes of the two previous generations of animals were used as the reference population . The selection candidates had no phenotypic records and their breeding values were estimated by BLUP, using either pedigree (PA_EBV) or genomic information (GEBV).As in Hayes et al. , we assuy is a vector of phenotypes, μ is the mean, 1n is a vector of 1 s, Z is a design matrix allocating records to breeding values, g is a vector of breeding values for animals in the reference set and the test set, and e is a vector of normal deviates with variance g) = GG is the genomic relationship matrix, and G was formed using the method described by VanRaden [g) = Aσa2 where A is the numerator relationship matrix based on pedigree and σa2 is the additive genetic variance. Variance components for both models were estimated with ASREML [where VanRaden . Pedigreh ASREML  and the x 'b, where x is a vector that relates to how much each selection candidate contributes to the next generation and b is a vector of EBV, using either genomic (GEBV), pedigree (PA_EBV) or true (TBV) breeding values. Rates of inbreeding were restricted by penalizing the average co-ancestry of the selection candidates to; c = λ x 'A x, where A is the (n x n) relationship matrix among the selection candidates based on pedigree and λ is the penalty factor applied to restrict inbreeding. Note that the level of inbreeding equals x 'A x / 2. Table x was determined. To evaluate the impact of restricting inbreeding using genomic relationships rather than pedigree relationships, A was substituted by G. The overall increase in genetic merit was measured as the increase in TBV and inbreeding was assessed based on the average genomic co-ancestry among selected individuals such that results from the alternate selection strategies were compared on an equal basis.Optimal contribution selection was undertaken to maximize the expected genetic value of future offspring while maintaining different levels of genetic diversity . The average genetic merit of the selected individuals (m) was maximized using the dynamic selection rule of Wray and Goddard ; m = x 'G to use in optimal contribution selection (as above). Note that PA_EBV were based on information before the bulls had progeny themselves and the GEBV were based on genomic information alone.Estimated breeding values and 50 k genotypes were obtained for 267 bulls from the Australian Dairy Holstein Improvement Scheme (ADHIS) and 140 bulls from the Livestock Improvement Corporation (LIC) in NZ. These EBV were for the 2007 cohort of Holstein bulls that obtained progeny test proofs in 2011. Breeding values  for milk, fat and protein yield were obtained from each organization. The average number of daughter records per sire was 90. Each dataset consisted primarily of a half-sib family structure but 30 bulls from the ADHIS dataset and 28 bulls from the LIC dataset shared one full sibling and the ADHIS dataset contained two families of four full siblings. The 50 k genotypes were used to form A) or genomic information (G). Genetic merit was maximized using either GEBV or PA_EBV. The overall genetic gain was measured as the increase in PT_EBV since it was the most accurate estimate of TBV and inbreeding was assessed based on the average genomic co-ancestry.Optimal contribution selection was performed (as above) for each dairy dataset, with the rate of inbreeding restricted by penalizing the average co-ancestry of the selection candidates using pedigree information  that remains is expected to be zero.Using GEBV as an estimate of genetic merit generally gave higher genetic gains than selection based on PA_EBV Figure . In the For the population that consisted of large full-sib families and when selection was on GEBV, the use of genomic relationships to constrain inbreeding resulted in greater genetic gain than the use of pedigree relationships Figure . In contTable Genomic breeding values for bulls in the ADHIS data set were more accurate than PA_EBV, which led to higher gains in merit when using GEBV as a measure of merit in optimal contribution selection Figure . ReducinThe use of genomic or pedigree-based relationships to restrict inbreeding resulted in similar genetic gains. However, the use of genomic relationships (compared to using pedigree relationships) increased the number of animals selected, although differences in merit (based on PT_BV) and average inbreeding were small or null for all traits measured, especially for the LIC dataset Figure . At low Table Genomic breeding values provided greater genetic gains than PA_EBV for the same rate of inbreeding. Using simulation, Nielsen et al.  previousAvendano et al.  suggesteSonesson et al.  noted thSimilar to the study by Schierenbeck et al. , we founTo achieve low inbreeding levels in a population that consists of many full-sib families, it will be necessary to select one animal from each family. When selection aims at increasing genetic gain, more than one animal per family may be needed. The within-family variation in GEBV makes it possible to use some within-family selection differential and the variation in relationship can allow for increases in genetic merit without compromising genetic diversity. This also occurs in populations that have a half-sib family structure but the additional variation in relationship that can be exploited is lower since variation in pedigree relationships and PA_EBV already exits. As in Pryce et al. , we obseThe amount of variation in EBV among family members may be important to determine how much gain can be obtained from using genomic relationships to manage inbreeding. Furthermore, within families, a positive correlation between GEBV and the genomic relationship may exist with the best selection candidate, which reduces the prospects for genomic relationships to create additional selection opportunities without reducing genetic gain.Genomic information can be used in livestock breeding programs to optimize genetic gain and inbreeding. Selection on GEBV increases genetic gain for a given rate of inbreeding because GEBV are more accurate and display more within-family variation than PA_EBV. Using genomic rather than pedigree-based relationships had limited impact on achieving additional genetic gain for a given rate of inbreeding in populations that consisted mainly of half-sib families (such as in dairy cattle), because the intra-class correlations among the EBV of relatives were already relatively low. However when the population consisted of large full-sib families, there was a clear benefit from using genomic relationships to control inbreeding, especially at moderate and low inbreeding constraints.The authors declared that they have no competing interests.SAC performed the simulation, analyses and drafted the manuscript. JHJW, JMH, BPK, and SAC conceived and designed the experiment. All authors have read and approved the final manuscript.


Menopause results in a lack of regular menstrual cycles, leading to the reduction of estrogen production. On the other hand, ovarian androgen synthesis is still present at reduced levels and requires expression of several steroidogenic enzymes.This study was performed on 104 postmenopausal women hospitalized due to uterine leiomyomas, endometriosis, and/or a prolapsed uterus. Patients were divided into three groups depending on the time from menopause. Group A patients experienced menopause 1–5 years before enrollment in the study (42 women). Group B included women who had their last menstruation 5–10 years before the study (40 women). Group C consisted of 22 women who were more than 10 years past menopause. Hysterectomy or removal of the uterine corpus with adnexa was performed during laparotomy. We evaluated the expression of aromatase cytochrome P450 (CYP 19) and 17β-hydroxysteroid dehydrogenase (17β HSD) by employing immunohistochemistry.Activity of 17β-HSD and CYP19 was demonstrated in the cytoplasm of stromal cells of postmenopausal ovaries, epithelium cells coating the ovaries, vascular endothelial cells, and epithelial inclusion cysts. However, overall expression of both 17β-HSD and CYP 19 decreased with time after menopause.Demonstration of the activity of the key enzymes of ovarian steroidogenesis, CYP 19 and 17β-HSD, confirms steroidogenic activity in the ovaries of postmenopausal women. Nevertheless, ovarian steroidogenic activity decreases with time, and its significant decrease occurs 10 years after menopause. The key enzymes of ovarian steroidogenesis are hydroxylases and oxidases, which belong to a large family of cytochrome P450-4 that cThe most important step in women’s steroidogenesis is aromatisation of androgens, which leads to the generation of estrogens. This process is mediated by aromatase cytochrome P450 and is observed in the endoplasmic reticulum of granulosa cells in many steroidogenic and non-steroidogenic tissues and glands, including the ovaries, placenta, adipose tissue, hair, vascular smooth muscle, skin, liver, bones (osteoblasts), and blood vessels,8,9.2), androstendione (A) to estrone (E1), and 16α-hydroxylated dehydroepiandrosterone (DHEA) to estriol (E3)[1 and over 95% of the E2 circulating in serum are ovary-derived[The human gene encoding aromatase cytochrome P450 is located on the long arm of chromosome 15, region 21.2 (15q21.2) and is called CYP19. This name reflects enzymatic oxidation of the methyl group at C-19,4,10. Iniol (E3). In wome-derived. The res-derived,11.Unlike most of the enzymes involved in steroidogenesis, CYP 19 is expressed in target tissues with active steroidogenesis  as well as in other non-steroidogenic tissues-13. A nuThe regulation of estrogen activity depends on oxidation and reduction reactions mediated by isoforms of 17β-hydroxysteroid dehydrogenase (17β-HSD), which catalyze the conversion of less-active 17-keto steroids to more potent 17β-hydroxy steroids, and many isoforms of this enzyme have been identified, differing in expression profiles, regulatory mechanisms, and substrates. The scheme of ovarian steroidogenesis is presented in Figure Due to the recently widely discussed existence of postmenopausal ovarian steroidogenic activity and the influence of the ovaries on women’s postmenopausal life quality and overall life span we evaluated the immunoexpression of two key ovarian enzymes regulating steroidogenesis: CYP 19 and 17β-HSD in the ovaries of postmenopausal women. These observations are also essential for pre- and postmenopausal women with benign ovarian tumors to facilitate decision if the oophorectomy is necessary.This study extends our previous work on the morphological structure of the postmenopausal ovary,32 and iThe study group consisted of 104 postmenopausal women hospitalized due to uterine leiomyomas, endometriosis, and/or a prolapsed uterus between 2003 and 2008 at the Department of Reproduction and Urogynecology at the Pomeranian Medical University in Szczecin. All women subjects met the inclusion criteria and didn’t meet the exclusion criteria. The inclusion criteria were: i) 12 or more months since the last menstruation, ii) no postmenopausal hormonal therapy, iii) normal PAP smear and mammography, iv) normal blood pressure, v) no history of surgeries that could affect blood flow to the ovaries, and vi) no history of cancer or endocrine disorders such as thyroid disease or diabetes. The research plan was approved by the Bioethics Commission of the Pomeranian Medical University, and the research was supported by grant No. 2 PO5E-10527 from the Polish State Committee for Scientific Research.Patients who met the inclusion criteria were divided into three groups , depending on the time from menopause. Group A patients experienced menopause 1–5 years before being qualified for the study (42 women). Group B included women who had their last menstruation 5–10 years before the study (40 women). Group C consisted of 22 women who were at least 10 years post menopause. For ethical reasons, a control group was not studied.Hysterectomy or removal of the uterine corpus with adnexa was performed during laparotomy and the “wedge shape” samples of ovaries were obtained for histological analysis. To determine the expression of aromatase cytochrome P450 and 17β-HSD by IHC, samples were preserved in 4% buffered formalin solution and embedded in paraffin. Histological sections were placed in the pH 9.0 buffer and then boiled for 30 minutes in a water bath at 99°C. All sections were then incubated for 24 hours at 4°C in a humid chamber with MCA 2077S primary antibody  to detect CYP 19 and with HPA015307 primary antibody  to detect 17β-HSD.For anti-aromatase antibody detection, the ABC Staining System  was used, while for anti-17β-HSD primary antibody detection, labeled polymer from the Dako Real EnVision Detection System  was used. In both cases, to visualize the immunohistochemical reaction, 3,3’diaminobenzidine (DAB) was used. In the last step, sections were counterstained with Meyer’s hematoxylin. For the negative control, specific antibodies were omitted in the staining procedure. The slides were examined by employing light microscope (Olympus BX41).17β-HSD Figure  and CYP In group A, a strong intensity of both activities was demonstrated in epithelial inclusion cysts, epithelial cells coating the ovaries, and in the cytoplasm of stromal cells. The intensity of both reactions was strong and/or semi strong in vascular endothelial cells.In group B, strong immunoexpression of 17β-HSD was demonstrated in epithelial inclusion cysts and semi strong in ovarian stromal cells. Semi strong reaction was also observed in vascular endothelial cells, and in epithelium cells of the ovary. In the same group, semi strong or weak immunoexpression of CYP 19, was demonstrated in ovarian stromal cells, a semi strong reaction was observed in vascular endothelial cells, and epithelial inclusion cysts, while in and epithelium cells coating the ovary, the reaction was strong.In group C, the immunoexpression of 17β-HSD and CYP 19, in all studied ovarian structures  was weak or weak and semi strong in vascular endothelial cells.We observed that immunoexpression of both 17β-HSD and CYP 19 decreased with time after menopause Tables .The results presented here are the first concerning the immunoexpression of CYP 19 and17β-HSD in the human ovary of postmenopausal women. Our studies demonstrated CYP 19 and 17β-HSD expression in the ovarian surface epithelial cells, inclusion cysts, vascular endothelial cells, and stromal cells. The intensity of reaction decreased significantly with time after the last menstruation. The activity of key enzymes involved in gonadal steroidogenesis, such as 17β-HSD and CYP 19, provides strong evidence for the presence of the steroid biosynthesis in the postmenopausal ovaries. These results also indicate a reduction in the activity of gonadal steroidogenesis as a function of time elapsed from menopause. Thus, our results confirm the presence of relatively high concentrations of steroids in ovarian tissue homogenates and their reduction in postmenopausal women, especially in women 10 years after the last menstruation,12.The activity of key ovarian steroidogenesis enzymes in postmenopausal women are estimated with different methods, because the ovarian stromal cells represent a heterogeneous collection of cells-38.et al. showed that all enzymes necessary for androgen synthesis are present in the ovaries of postmenopausal women[The presence of enzymes of the aromatase cytochrome group was detected employing immunohistochemistry in the ovaries of postmenopausal women for the first time by Inkster and Brodie in 1991[Our current study demonstrates localization of two key enzymes involved in the steroidogenic biosynthesis pathway, CYP 19 and 17β-HSD, in postmenopausal ovaries, which indicates that the postmenopausal ovary acts as an important contributor to the synthesis of steroids destined for peripheral metabolism.Studies on the pathogenesis of diseases of the perimenopausal period identified aromatase activity not only in gonadal tissues but also in adipose tissue, muscles, mammary gland, and liver-41. More1, which is converted in the gonads and many peripheral tissues to E2. In transgenic mice, increased activity of 17β-HSD1 in the presence of E1 substrate leads to increased estrogen-dependent tumor growth. This was confirmed by administration of 17β-HSD1 inhibitor, which significantly reduced growth of this estrogen-dependent tumor[2 formation, despite the fact that its expression in peripheral tissues is low[Different types of 17β-HSD use various substrates. The most active type, 17β-HSD1, is highly complementary to estrogenic substrates, especially Ent tumor. The pres is low.A few studies demonstrated that genetic polymorphism of the steroidogenic enzymes in postmenopausal women’s ovaries cause dysfunction and may be a cause of increased risk of endometrial and breast cancers. Postmenopausal women with specific CYP19 genetic polymorphisms are at increased risk of breast cancer, and there were higher CYP19 and 17β-HSD activities in tumor cells than in normal cells. Moreover, CYP 19 activity was higher in women with breast and endometrial cancers than in healthy women-50, and On the one hand, it is known that postmenopausal women with preserved gonads have longer survival, higher life quality, and less-frequent cardiovascular complications, dementing illnesses, and osteoporotic complications. On the other hand, the presence of gonads increases the risk of ovarian and breast cancer. The U.S. data show that the proportion of deaths from cardiovascular diseases in postmenopausal women is much higher than from cancer, which is confirmed by data from the Central Statistical Office in Poland for the years 2009–2010. Our own data and data from the published literature also indicate that gonadal steroidogenic activity clearly decreases after 10 years from menopause, and in this period, preventive bilateral ovariectomy does not affect life quality and survival time but significantly reduces the risk of cancer.Our data support the conclusion that steroidogenesis, as evidenced by activities of the key ovarian steroidogenic enzymes, CYP 19 and 17β-HSD, is still active in the ovaries of postmenopausal women. This process decreases gradually after menopause and is significantly reduced after 10 years from the last menstruation. Therefore taking into consideration hormonal activity in postmenopausal ovaries, prophylactic, bilateral ovariectomy can be safely recommended in the first 10 years after the last menstruation.17βHSD: 17β-hydroxysteroid dehydrogenase; 17βHSD1: 17β-hydroxysteroid dehydrogenase type 1; 3βHSD: 3β-hydroxysteroid dehydrogenase; A: Androstendione; CYP19: Aromatase cytochrome P450; DAB: Diaminobenzidine; DHEA: Dehydroepiandrosterone; DHEAS: Dehydroepiandrosterone sulfate; E2: Estradiol; E3: Estriol; E1: Estrone; FSH: Follicle-stimulating Hormone; IHC: Immunohistochemistry; QRT: Querty Real Time.The authors all declare that they have no competing interests.AB: development of the research plan, material and data collection, performance of immunohistochemical analysis, and manuscript preparation. JB: help in performing immunohistochemical analysis and manuscript preparation. ML: help in research planning and supervision of the immunohistochemical analysis, help in manuscript preparation and correction. Bogdan Rumianowski: manuscript preparation and correction. SS-G: analysis of results and help in writing and correcting the manuscript. IR: help in writing and correcting the manuscript. AS: help in planning and supervision of the study. MZR: manuscript preparation and correction. All authors have read and approved the final manuscript.