Studies have consistently indicated that the majority of individuals meeting the US Prevention Services Task Force guidelines for genetic testing have not had genetic counseling or testing. Despite increased availability and lower costs of multiplex cancer gene panels, there remains a gap in genetics services that has not been addressed by the current care delivery models. Lower cost of DNA sequencing with online patient-initiated ordering could increase test availability, but the ideal quantity and delivery method of patient education is not known. We hypothesized that online genetic education and testing with access to board certified genetic counselors could improve access to genetic testing while maintaining test quality and clinical utility. The MAGENTA (MAking GENetic Testing Accessible) trial is a nationwide randomized study designed to compare the effectiveness of online genetic education with pre- and post-test telephone genetic counseling to three potentially more accessible alternative approaches: online genetic education with optional telephone counseling, online genetic education with required pre-test telephone genetic counseling, and online genetic education with required post-test telephone genetic counseling.3000 women nationwide will undergo genetic testing for 19 hereditary cancer genes. This is a randomized four-arm non-inferiority study with equal randomization. The four study arms were selected to independently assess the delivery of genetic information both before and after genetic testing (pre-test and post-test) by either requiring telephone genetic counseling or providing only online education with optional telephone counseling. Patients have post-test telephone counseling when testing positive for a pathogenic inherited mutation in all four arms. Surveys measuring psychological, behavioral and cognitive state are completed online at baseline, 3 months, 12 months and 24 months post-results disclosure. The primary study outcome is cancer-risk distress at 3 months post-result disclosure.This trial will assess the use of a genetic service model using online access and electronic education, while evaluating the need for personal pre- and post-test genetic counseling. Data from this study may lead to increased options for delivery of genetic testing and possibly increase access to genetic testing. Identifying more individuals with inherited cancer susceptibility will allow targeted cancer prevention.NCT02993068 .Clinicaltrials.gov:  BRCA1 and BRCA2 allows for prevention and early detection.Rapid developments in cancer genetics over the past decade have changed the landscape of genetic testing for hereditary cancer. The availability of next generation sequencing, coupled with the Supreme Court decision invalidating patents on human genes, has led to substantially decreased cost of germline testing and has created a shift from single syndrome or gene by gene testing to multiplex gene panel testing. Identification of individuals who carry a mutation in cancer predisposition genes such as However, studies have consistently indicated that the majority of individuals meeting the United States Prevention Services Task Force (USPSTF) guidelines for genetic testing have not had genetic counseling or testing . ChilderVarious service delivery models (SDMs) have been explored and adopted to increase access to cancer genetic testing services. The traditional SDM for cancer genetic testing employs in-person pre-test and post-test genetic counseling. Telephone genetic counseling has been shown to be non-inferior to in-person counseling and is now a widely accepted SDM with the potential to provide services to patients who live far from genetics specialists , 5. OnliOnline access to physician ordered genetic testing for clinically actionable hereditary cancer genes has the potential to increase access to genetic testing while maintaining test quality and clinical utility. Smit and colleagues found that individuals believe online communication is an acceptable mode of receiving medical information, and that it is important to have a health professional available to discuss questions and content as needed . One stuThe availability of online genetic education detailing the benefits and limitations of hereditary cancer testing with access to board certified genetic counselors offers a promising alternative that may overcome barriers faced by current SDMs. Such a model could allow both genetics and non-genetics clinicians to order genetic testing while providing patients with detailed online information regarding hereditary cancer testing, overcoming the major barriers of physician coordinated testing, including limitations of physician time and knowledge. Remote access testing may eliminate the major barriers of in-person counseling, including time and money spent on travel, while increasing flexibility and accessibility, which continue to be challenges of both in-person counseling and telephone counseling. Remote access genetic testing via the use of an online genetic services platform allows individuals to access information using the internet with the ability to revisit if they choose, avoiding the need for appointments and allowing for patient-driven and convenient genetic services. Electronic copies of genetic test results may be shared easily with family members and medical providers. For individuals found to have a pathogenic mutation, online platforms facilitate easy information sharing, and the option of online access to testing services may promote the uptake of cascade testing in blood relatives.One question in this proposed SDM is how much personalized counseling is needed both pre-test and post-test to optimize patient outcomes. Does required personalized counseling reduce patient distress and improve understanding or does it add unnecessary cost and time and reduce likelihood of test completion? Is pre- or post-test counseling more important and if counseling is provided as an option, how many patients will utilize telephone counseling, and can we identify patient subsets who have greater benefit from personalized counseling? To address these considerations, we are conducting a prospective study to evaluate different amount of personalized genetic counseling within an online genetic testing model to determine less counseling is non-inferior to our current accepted SDM at delivering hereditary cancer risk information. The purpose of this paper is to describe the design of the MAGENTA study, a prospective randomized trial of online genetic education vs telephone genetic counseling for hereditary cancer genetic testing.ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MSH2, MSH6, MLH1, NBN, PALB2, PMS2, PTEN, RAD51C, RAD51D, TP53, and STK11. Our primary hypothesis is that the outcomes for subjects receiving one of the three alternative approaches utilizing genetic education online will not be inferior to those of participants receiving a current acceptable standard of telephone genetic counseling both pre- and post-testing [The MAGENTA (MAking GENetic Testing Accessible) trial is designed to compare the effectiveness of pre- and post-test telephone counseling to three potentially more accessible alternatives for individuals undergoing analysis of 19 hereditary breast and ovarian cancer (OC) genes. The genes included are: -testing , 5.After online eligibility screening and electronic study consent , randomization into one of four study arms is performed automatically using a web-based data management platform . The fouThe MAGENTA study’s target population is women that may be at increased risk to develop ovarian cancer based on their personal or family history of cancer or due to a known cancer susceptibility gene mutation in the family. Women must be 30 years of age or older and must have at least one ovary, as we hope to identify women who will be able to take actionable measures to reduce ovarian cancer risk. As a safety precaution, all participants are required to identify a local healthcare provider with whom their genetic test results will be shared. Complete eligibility criteria are outlined in Table Recruiting efforts are being carried out through clinical settings, advocacy groups, and various social media platforms. The different recruitment strategies were designed to capture a heterogeneous group of women, which will allow us to assess whether we improve access to genetic testing in diverse patient populations stratified for race and ethnicity, as well as, socioeconomic, educational and geographical backgrounds.http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm436811.pdf).Given the size and large geographic area  for which we are recruiting and enrolling participants, the use of electronic informed consent (IC) became a vital component to the study. Electronic informed consent is defined by the FDA as the use of electronic systems and processes that may employ multiple electronic media, such as text, graphics and videos, to convey information related to the study and obtain and document informed consent (http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm436811.pdf).Our biggest challenge was ensuring that the person signing the IC is the same as the person completing the study procedures. We accomplished this by requiring all participants to create a unique username and password that must be used to confirm their identity before signing the IC and before completing each study survey. To ensure that consent is truly informed, we created a series of comprehension questions that the participant must answer correctly before being allowed to sign the IC. The IC was delivered to study participants by email using the survey tools in REDCap. All other aspects of the FDA draft guidance were followed, including allowing the participant the ability to print a copy of the IC and call the study coordinator for questions , genetics knowledge, cognitive outcomes , and behavioral outcomes. Surveys are completed at baseline, 3 months, 12 months and 24 months post-results disclosure. The specific measures and time-points are summarized in Table The primary study outcome is cancer-risk distress at 3 months post-result disclosure, as measured by the Impact of Events Scale (IES) , 19.All participants receive online genetic education, including a 5-min pre-test educational video and an online results report. The video provides an explanation of hereditary cancer, inheritance patterns, associated cancer risks, and an overview of the possible results. Participants randomized to receive pre-test telephone counseling are prompted to schedule their telephone appointments online and will complete telephone counseling in addition to the online genetic education. The usual content of pre- and post-test genetic counseling for hereditary cancer risk has been well described , 21. FolWe employ a non-inferiority design with a non-inferiority margin of 4 points, as suggested by Schwarz et al. (2014), for the difference in IES score between the control arm and each of the other three study arms at 3 months post-result disclosure.n = 4400) of those who consent to participate will complete the baseline questionnaire and then be randomized. Of these, we will enroll around 750 subjects with a known familial mutation into the cascade group, with the remaining 3650 subjects, who have an increased likelihood of carrying a deleterious mutation in an ovarian cancer predisposition gene based on personal and family history of disease, as the group of interest for our primary analysis. We expect that 65% (n = 2375) of the 3650 participants with personal and/or family history of disease will follow through with providing a saliva sample for analysis, with 75% of those subjects completing the study through the 3 month assessment (n = 1780), which equates to about 450 participants on each of the four study arms.We are enrolling 5200 eligible women with a goal of 3000 completing genetic testing. We expect that around 85%  is more than 6% less than it is for participants who were randomized to receive telephone genetic counseling before testing (Arms C and D), and that the completion rate for the participants receiving only online genetic education after testing (Arms A and D) is more than 6% less than it is for participants who were randomized to receive telephone genetic counseling after testing (Arms B and D). This 6% non-inferiority margin is more conservative than the 7.5% margin used by Schwartz et al. [In addition to testing our primary and secondary hypotheses, we will summarize for each study arm the key outcomes related to cognition, affect and behavior, and the changes in scores from baseline. We will also use regression models to model the scores (and changes in the scores from baseline) as a function of study arm, risk group, and other covariates.BRCA1, BRCA2, and Lynch syndrome. The discussions surrounding population-based screening for hereditary cancer genes has highlighted a challenge regarding the ability to provide this service on a large scale in an efficient and effective manner [In-person genetic counseling is likely to remain standard practice in large cancer centers and academic institutions. However, alternative service delivery models for genetic counseling must be further explored to find an optimal balance of clinical quality with increased access and financial sustainability. This is especially important given the largely debated topic of population screening for e manner . DetermiThe nationwide MAGENTA trial is the first study to assess the use of a genetic service model using online access and electronic education for population based testing in individuals at elevated risk of ovarian cancer, while evaluating the need for personal pre and post-test genetic counseling. Innovative service delivery models are essential to decreasing barriers to genetic testing, particularly among underserved populations. Identifying more individuals with inherited cancer susceptibility will allow targeted prevention and ultimately save lives.


Piaractus mesopotamicus) is a Neotropical fish with remarkable productive performance for aquaculture. Knowledge of genetic resources in Neotropical fish is essential for their applications in breeding programs. The aim of this study was to characterize the genetic diversity of seven farmed populations of pacu which will constitute the basis for a broodstock foundation for coming breeding programs in Brazil. Analysis of one wild population (Paraná River) was used as a reference to compare genetic parameters in the farmed populations. The analyses were performed using 32 single-nucleotide polymorphisms (SNP) and 8 simple sequence repeat (SSR) markers. No significant differences in genetic diversity between populations estimated through the number of alleles and allelic richness, observed heterozygosity, expected heterozygosity, and minimum allele frequency were detected (p > 0.05). Low genetic diversity was observed in all farmed stocks and the wild population. Moreover, we detected low genetic structure when comparing farmed and wild populations for SNPs  and SSRs . Analysis of molecular variance (AMOVA) demonstrated that genetic variation was mostly within populations. Kinship analysis showed that most fish farms included related individuals at a proportion of at least 25%. Our results suggest that the basal broodstock for pacu breeding programs should be founded with individuals from different fish farms for higher genetic diversity and to avoid inbreeding risks.The pacu ( Piaractus mesopotamicus) is a Characiform fish with a wide natural distribution throughout La Plata basin which covers an area over five South American countries: Brazil, Uruguay, Bolivia, Paraguay, and Argentina. Wild populations of pacu are threatened by overfishing since this species is considered to have high commercial value, with large-scale catches occurring by industrial and recreational fisheries [The pacu  and 8 SSRs to assess the genetic diversity, structure, and kinship as a support to designing a suitable pre-breeding program for this species.In this study, seven farmed stocks and one wild population of This study was conducted in strict accordance with the recommendations of the National Council for Control of Animal Experimentation (CONCEA)  and was approved by the Animal Use Ethics Committee (CEUA), protocol no. 22.255/15. The present study was performed under authorization no. 33435-1 issued through the Chico Mendes Institute for the Conservation of Biodiversity, Brazilian Ministry for the Environment (ICMBio). Fin clips were collected from each fish under benzocaine anesthesia and all efforts were made to minimize suffering. Fin samples were stored in 95% ethanol at −20 °C.Individuals of seven different broodstocks from fish farms in Brazil (São Paulo State) were collected (FF1–FF7) . Markers were genotyped in a 3730XL DNA analyzer (Applied Biosystems) in Macrogen (Korea). Allele scoring was performed with the Peak Scanner program (Applied Biosystems) using the GeneScan 500 LIZ Size Standard.SSR genotyping was performed using eight microsatellite markers  in two Multiplex PCR reactions according to Posner  and prevSNP genotyping was carried out using 32 markers obtained from the liver transcriptome . Analysinull) and allelic dropout in microsatellite loci were tested using MICRO-CHECKER 2.2.3 [a), number of private alleles (Np), and observed (Hobs) and expected (Hexp) heterozygosity were estimated using CERVUS 3.0 [p < 0.05) were performed using GENEPOP 4.0.11 [r) and inbreeding coefficient (FIS) were estimated using FSTAT 2.9.3.2 software [The presence of null alleles  was estimated by the linkage disequilibrium method in NeESTIMATOR V2.01 [The effective population size   using STThe partitioning of variation at different hierarchical levels was calculated by analysis of molecular variance (AMOVA) in ARLEQUIN version 3.5.2.2  using 10xy) [xy < 0.125) corresponding to unrelated individuals; intermediate values 0.125 ≤ rxy ≤ 0.375 were considered half-siblings; and rxy ˃ 0.375 were considered full siblings [Kinship coefficients (rxy)  and potexy) . Parentasiblings .p values of HWE and FIS values are shown in The parameters of genetic variability for pacu populations determined by 8 SSR and 32 SNP markers are shown in For SSR loci, a total of 44 alleles were detected in the analyzed populations. The average number of alleles per locus was 3.84 ± 1.16, with numbers ranging between 2 and 7. Allelic richness ranged from 2.000 to 6.140, with average population values ranging between 3.331 ± 0.868 (FF1) and 3.975 ± 1.189 (FF7). Private alleles were detected in individuals belonging to FF2, FF4, FF7, and WILD.Monomorphic SNPs were found more frequently in FF2 (six monomorphic SNPs) and FF4 (four monomorphic SNPs). By contrast, FF1, WILD, FF6, and FF7 showed only polymorphic SNPs and the highest MAF average values . On the other hand, FF3 (0.269 ± 0.138) was the fish farms with the lowest MAF average values.obs was 0.502 ± 0.219 for SSRs and 0.391 ± 0.173 for SNPs. When the average Hexp was estimated, 0.525 ± 0.170 was found for SSRs and 0.379 ± 0.133 for SNPs. The genetic diversity values observed in the fish farms  were not significantly different from the WILD values , which showed Ar = 3.420 ± 1.0005, Hobs = 0.441 ± 0.213, and Hexp = 0.434 ± 0.214 for SSRs and MAF = 0.275 ± 0.132, Hobs = 0.385 ± 0.171, and Hexp = 0.370 ± 0.130 for SNPs (The average Hfor SNPs .p = 0.001), even when populations were subjected to global tests for all loci. By contrast, some SSR loci departed from HWE after Bonferroni correction (p = 0.006), such as locus Pm4  and locus Pm5 (FF4). Null alleles were detected in the locus Pm4  and locus Pm11 (FF2) (p < 0.05) when applying global tests for SSRs. However, only FF4 presented significant Hardy–Weinberg disequilibrium after Bonferroni correction (p < 0.006).All SNP loci were in accordance with HWE after Bonferroni correction (11 (FF2) . NeitherFIS values varied similarly between SSR (−0.113 and 0.212) and SNP loci (−0.082 to 0.056). However, no significant deviations from zero of FIS values were found in the fish farms when Bonferroni correction was applied for SNPs (p = 0.001) and SSRs (p = 0.006). Additionally, FIS values were not considered significantly different from zero over all loci for both markers (p > 0.05), presenting low 95% confidence interval values for SSRs (−0.022 to 0.135) and SNPs (−0.075 to 0.011) when bootstrapping over loci analyses were performed.Overall population p < 0.001). For SSR markers, LD was detected in FF4 (5 pairs), FF2 (2), FF1 (1), FF5 (1), and WILD (1) (p < 0.01). However, in both markers, the observed LD was not homogeneous among populations or loci , FF7 (5), WILD (4), FF5 (2), FF3 (1), and FF4 (1)  and in WILD for SNPs (0.02) and the highest values in FF4 for SSRs (0.07) and in FF1 for SNPs (0.22). Evidence of recent reductions in population size was found in all fish farms (M-ratio < 0.68) by considering SSR analysis . N8 in FF6 . The ratanalysis  characteAfter the detection of reduced effective population sizes, kinship evaluation showed that most of the fish farms had related individuals  in a proportion of at least 25%, as estimated from both marker sets , with siFST values suggested low genetic differentiation among populations when using SSRs  or SNPs . Pairwise FST values were also calculated for fish farms and significant differentiation was found between most pairs when considering SNP and SSR sets   and ranged between −0.002 and 0.204. The highest significant FST values (p < 0.006) were observed between WILD and FF4 (FST = 0.204) and FF2 and FF4 (FST = 0.143). Conversely, the lowest genetic differentiation was detected between FF3 and FF6 . Regarding SNPs, the highest genetic differentiation (p < 0.001) was between FF2 and FF4 (FST = 0.146). Conversely, WILD and FF7 (FST = 0.032) registered the lowest genetic differentiation (p < 0.001).Global ctively) . Low to FST analyses.To evaluate the level of admixture among samples, Bayesian model-based clustering analyses were performed based on the ΔK distribution by examining SSR and SNP loci. The selection of the estimated number of clusters in the dataset was based on the number of analyzed fish farms (K = 1 to 8). According to the analysis based on the Evanno method , the hypp < 0.001) and 91.1% by SSRs (p < 0.0001). The estimated variation between groups (FCT) was only 2.2% (p = 0.01) for SNPs and 3.9% (p = 0.02) for SSRs. In addition, the variation among populations and within groups (FSC) presented 5.3% of the genetic variation (p < 0.001) for SNPs and 5.0% (p < 0.0001) for SSRs.Despite the genetic structure among populations being confirmed by STRUCTURE analysis, AMOVA analysis based on groups supported by the results of STRUCTURE showed that the higher percentage of genetic variation was assigned to differentiation within populations: 92.4% by SNPs . Therefore, our hypothesis is that the WILD population may be threatened due to the high level of exploitation imposed by commercial and/or recreational fishing, since the region where the WILD population was collected is a famous fishing spot in Brazil [Considering SSR loci, the analysis of genetic diversity parameters estimated on pacu broodstocks showed low values for the number of alleles (ranging around 4) and heterozygosity (ranging around 0.500). It was expected that cultivated stocks would present low genetic diversity values, as herein observed; these low values characterize populations with genetic drift events due to low effective population sizes and, consequently, recent bottleneck effects/founder events, similar to other studies in a related Neotropical species, chypomus . Howeverchypomus  that repn Brazil . Moreoven Brazil ,42 whichobs and Hexp) and MAF indicated low genetic variability in the farmed populations studied. This hypothesis was based on similar values found in WILD and sampled fish farms, considering that wild populations of pacu have no expressive heterozygosity values when using microsatellite loci. In addition, the diversity values herein observed were similar to those in other fish studies involving SNP analyses [Although there is no comparative research of the genetic diversity of pacu populations using SNP markers, the values of heterozygosity  must receive special attention, since our results will serve to find a base population of pacu for upcoming breeding programs.In general, farmed stocks have the tendency to show reduced genetic variability over generations due to artificial selection and a reduced number of breeders in the initial base population ,46,47,48Kinship analysis has been an essential tool in genetic pre-breeding programs of fish to reduce inbreeding rates by directing the mating of unrelated individuals ,50,51,52Impacts related to insufficient individuals used in hatchery productions and their interference with the genetic diversity of cultivated populations have been studied for important species cultivated worldwide, such as Atlantic salmon . SimilarFST estimates indicating genetic differentiation (driven by genetic drift and isolation), as detected in FF4, similar to in previous studies carried out in other related species such as P. brachypomus [FST values suggested significant genetic differentiation of 0.067 by SNPs and 0.080 by SSRs , indicating low differentiation between the farmed stocks. This may be explained using two hypotheses: (i) stock foundation based on sharing of breeders among fish farms, resulting in genetic similarities between broodstocks, and/or (ii) stock foundation in the fish farms based on the capture of wild breeders, which are characterized as belonging to a panmictic unit due to the lack of genetic structure in natural populations [In the present study, our initial hypothesis was based on the fact that pacu farmed stocks did not have gene flow because the broodstocks are geographically isolated and producers frequently do not perform exchange of breeders among the fish farms. Therefore, we would expect higher values of chypomus . Howeverulations ,17, partThe STRUCTURE analysis revealed differences between SNPs and SSRs, showing three and two clusters for the pacu stocks, respectively. In this study, the dataset of SNPs originated from the pacu liver transcriptome and these SNPs were annotated mostly in genes related to productive traits, including SNPs classified as non-synonymous mutations . TherefoThe results of this study are aimed to provide initial knowledge about the genetic profile of pacu stocks in different fish farms, considering the importance of pacu to South America aquaculture and the necessity to offer subsidies for the development of its production. The results provide information relevant to one of the most important cultivated Neotropical species. The genetic variability and differentiation of stocks and fish farm profiles considering the risks of inbreeding and the necessity of directed matings of the stocks should be known in order to take appropriate actions for the creation of the base population. In conclusion, the SNP and SSR sets showed their applicability in a pre-breeding program, particularly in delineating the formation of the best families in terms of genetic variability and genetic structure.


Andrias davidianus, endemic to central and southern mainland in China, has suffered from sharp range and population size declines over the past three decades. However, the levels and patterns of genetic diversity of A. davidianus populations in wild remain poorly understood. Herein, we explore the levels and phylogeographic patterns of genetic diversity of wild‐caught A. davidianus using larvae and adult collection with the aid of sequence variation in (a) the mitochondrial DNA (mtDNA) fragments , (b) 19 whole mtDNA genomes, and (c) nuclear recombinase activating gene 2 . Phylogenetic analyses based on mtDNA datasets uncovered seven divergent mitochondrial clades (A–G), which likely originated in association with the uplifting of mountains during the Late Miocene, specific habitat requirements, barriers including mountains and drainages and lower dispersal ability. The distributions of clades were geographic partitioned and confined in neighboring regions. Furthermore, we discovered some mountains, rivers, and provinces harbored more than one clades. RAG2 analyses revealed no obvious geographic patterns among the five alleles detected. Our study depicts a relatively intact distribution map of A. davidianus clades in natural species range and provides important knowledge that can be used to improve monitoring programs and develop a conservation strategy for this critically endangered organism.Understanding genetic diversity patterns of endangered species is an important premise for biodiversity conservation. The critically endangered salamander  The Chi) Figure  and has ) Figure . HoweverA. davidianus  sequences and found some genetic divergence among the analyzed populations. In addition, they detected genetic signatures of human‐assisted translocations on patterns of A. davidianus population differentiation. Tao et al.  and mtDNA markers. Furthermore, they exemplified broad genetic mixing among dinstinct clades based on mtDNA and microsatellite data for more than 1,000 farm‐bred individuals. Nevertheless, the relatively small sample sizes  and restricted geographic coverage of these studies limit the inference that can be drawn from them  and nuclear DNA (nuDNA) utilizing a large number of wild‐caught animals sampled from area covering most of its native distribution range (http://maps.iucnredlist.org). Larvae and adult individuals were included in this study. The main aims were (a) to assess how many distinct genetic clades the A. davidianus consist of in the natural species ranges and where they occur, (b) to explore what factors have shaped the patterns of genetic variability and differentiation in A. davidianus, and (c) to offer recommendations for A. davidianus conservation.In this study, we characterized phylogeographic pattern and differentiation of 22.1The methods involving animals in this study adhered to the Laboratory Animal Management Principles of China. All experimental protocols were approved by the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences.A. davidianus   Liang, . We inve) Liang, . We coll) Liang, . Since 2) Liang, . Skin co) Liang,  when lar) Liang,  under th) Liang,  that are) Liang,  were cap) Liang, . All 320Oral mucosa cells or exfoliated skin cuticles were obtained from the majority of specimens, and caudal fin clips were also collected from a few individuals. Animals were released after tissue collection at the site of capture. All extracted tissues were immediately stored in 99.5% alcohol at −20°C for DNA extraction.b gene (Cytb) of 3′‐end to the control region (CCR) for all individuals. Furthermore, 19 complete mtDNA genomes were amplified from a subset of samples representing each CCR clade to obtain robust phylogenetic trees  was added to our analyses. Partial sequences from the nuclear recombinase activating gene 2  were also sequenced from a subset of samples representing each clade and most of the CCR haplotypes. A pair of primers . All amplifications were performed in 50 μl volume with an initial denaturation period of 3 min at 94°C, which was followed by 35 cycles of 94°C for 45 s, primer‐specific annealing temperatures of 52–55°C for 1 min, 72°C for 1 min, and a final extension of 72°C for 10 min. A negative control with no template DNA was included in each PCR run. The PCR products were purified and sequenced with the same primers. DNA sequencing was performed on an ABI3730 with an ABIPRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit .The DNA was extracted using the DNA Preparation Kit  according to the manufacturers instructions. The total DNA from the supernatant was purified using an Easy‐DNA Kit . A pair of primers  were initially edited using the DNASTAR multiple package , aligned using MUSCLE  and produced 68 ingroup haplotypes. For the 19 whole mtDNA genomes , we extracted 22 transfer RNAs (tRNA), 13 protein‐coding genes with ND6 adjusted to present the same reading direction as the other genes, and 2 ribosomal RNA genes (rRNA) from the genomes by eye. These 37 genes were combined to produce 15,684 bp concatenated sequences, which were aligned along with those of the two outgroup species.The E Edgar, , and theE Edgar, . HaplotyE Edgar,  based onhttp://hammerlab.biosci.arizona.edu/IMGC/IMGC.html; Woerner, Cox, & Hammer, Nuclear gene sequences containing more than one ambiguous site were resolved using PHASE 2.1.1  and Cryptobranchus alleganiensis (GenBank: GQ368662), were selected as outgroups because of their close relationships with A. davidianus. The phylogenetic relationships among mitochondrial haplotypes were reconstructed using Bayesian inference (BI) and maximum likelihood (ML) for the CCR sequences and mtDNA genome, respectively. We employed the best‐fit nucleotide substitution model for BI and ML analyses. For the CCR sequences, the best‐fit substitution model (GTR + I + G) was selected using the Akaike information criterion (AIC) in MrModeltest v2.3 , haplotype diversity (h), and nucleotide diversity (πθ and ωθ), was estimated using DnaSP 5.10. Comparing estimates of current (πθ) and historical (ωθ) genetic diversity can provide genetic signatures into population dynamics over recent evolutionary history  pattern for CCR sequences was performed with Alleles In Space (AIS)  was calculated in Arlequin by calculating pairwise ϕST values among populations with more than five samples. We employed 1,000 permutations to assess significance for AMOVA and ϕST calculations using CCR sequences based on K2P distance. The existence of phylogeographic structure was examined by calculating two genetic differentiation indices (GST and NST) in DnaSP  identified five haplotypes (A1–A5). The network of the RAG2 sequences did not show any strong geographic patterning  and nucleotide diversity (πθ = 0.230), respectively. However, there was considerable variation among different populations  and historical (ωθ) genetic diversity for each population indicated that 12 out of 23 populations showed a pattern of decline .The A. davidianus was highly geographically structured, with 66.11% of the genetic variation attributable to differentiation among populations, a result which was highly significant (p < 0.001). Hierarchical AMOVA . A comparison of the fixation indices NST and GST revealed that NST was much larger than GST . The ϕST calculations (data not shown) showed that 83.3% of the pairwise population comparisons were statistically significant (p < 0.05).Nonhierarchical AMOVA Table  suggesteVA Table  of popul3.3The average estimates of the divergence time are shown in Figure 4A. davidianus populations and presents some conservation implications for this endangered species based on a wide geographic sampling and using multiple markers. In addition, we compared our study and Yan et al.  clade A; (b) clade B–C; (c) clade D–E; and (d) clade F–G. Our analyses showed clade D–E and clade F–G clustered together and clade B–C was supported to be sister to the two aforementioned major clades. This result contradicted the outcome demonstrated in Yan et al. , though seven clades were highly resolved. Three mtDNA genes employed in Yan et al.  was observed among the seven clades. The high levels of genetic diversity uncovered in  Routman  found thA. davidianus likely reflects long‐term isolation. The divergence between the major clades  was estimated to have occurred 6.38 to 11.05 Mya in the late Miocene during the intense uplift phase of the Qinghai‐Tibetan Plateau  and locality 31 (clade A) occur in close spatial proximity in Maoershan Mountains, these populations belong different mtDNA clades because they reside on different drainage systems opposite sides of the mountain system , and poor dispersal ability likely have influenced patterns and level of divergence among 00 m Fu, , the majRAG2 sequences uncovered high level of allele sharing among the seven clades. Compared to relatively obvious geographic structuring of the mtDNA clades, the distribution of the five RAG2 alleles displayed no obvious geographic structuring. Widespread and random distribution of alleles A1 and A2 in each population suggests that incomplete lineage sorting may explain the discrepancy between the topologies generated from mtDNA and nuDNA sequences.The analyses of 4.4A. davidianus face many threats from anthropogenic activities, such as habitat destruction, water pollution, and poaching  showed a pattern of genetic diversity decline. Thus, there are indications that at least some A. davidianus populations are losing genetic variation, and these trends should be followed up. Likewise, the population in locality 26 might be worth protection because of its genetic uniqueness of carrying allele A4. The highest priority for conservation should be allocated to the population in locality 31, which is the only known representative of the clade A and allele A3, which is also the indicated to be oldest of the seven detected mtDNA clades.The maintenance of genetic diversity is of critical importance for conserving the evolutionary potential of a given species  and D.Q.W (Grant Nos. LFBC0809).KU131040, KU131040–KU131048, KU131050–KU131059, KU131061–KU131066, KU131070–KU131071, KU131074–KU131075, KU131080–KU131082, KU131084, KU131090, KU131093, KU131096–KU131101, KU131105–KU131107, KU131110–KU131111, KU131113, KU131117–KU131122, KU131125, KU131131–KU131143, KU131145–KU131153, KU131163–KU131166, KU131168–KU131170, KU131172, KU131174–KU131179, KU131184–KU131187, KX503809–KX503810, and KX503816. Details regarding individual samples are available in Supporting Information Table S1 in Appendix DNA sequences have been deposited in GenBank under Accession numbers  Click here for additional data file.


Gyps coprotheres) drawn from across the southern African distribution of the species. Microsatellites are useful in quantifying genetic diversity at the population level. Populations of the endangered Cape vulture are currently monitored by conservation agencies and the data presented here can be used as an important baseline for future population genetic monitoring.Across the globe, vulture species are experiencing major population declines. A key factor for the long-term persistence of these endangered species is the maintenance of genetic diversity patterns within wild populations. The datasets presented in this descriptor includes microsatellite genotypes of 605 Cape vultures ( Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9896372 Gyps coprotheres) have shown a decline in the overall number of individuals in the wild and their global range is currently undergoing a significant reduction, with most breeding colonies found in South Africa5. A more rigorous approach is required to successfully stabilize and conserve this endangered vulture4. In order to ensure the long-term conservation of vulture populations, management practices should evaluate and maintain the amount and pattern of genetic diversity within current populations7.In recent years, Cape vultures . Archival museum samples (dried skin snips) were sourced from local South African museums  was used for all DNA extractions. The extraction protocol was modified for feather and archival samples to improve DNA yield. Samples were incubation with proteinase K for 48 hours in a shaking water bath (56 °C), the lysate was incubated in 70 °C B3 buffer for 45 minutes, the final volume of pre-warmed elution buffer (BE) was 80 μl. During the final elution step samples were incubated at 70 °C for 20 minutes followed by centrifuging and then reapplication of the solution onto the membrane. The samples were incubated again at 70 °C for an additional five minutes followed by the final centrifuging step.13 (Table TM Fast Multiplex PCR kit (KAPA Biosystems) following the manufacture’s protocol. All amplified products were analyzed using a 3130xL Genetic Analyzer housed at the Central Analytical Facility at Stellenbosch University, South Africa. The software GeneMarker v2.4.0 (Soft Genetics) was used for genotype scoring15.Thirteen microsatellite loci were selected from previous studiesGyps coprotheres genotyped using 13 microsatellite loci14 as well as the genotype scores for the 605 Gyps coprotheres individuals15. All associated metadata  is available in Supplementary Table The datasets are available on Zenodo and include the raw fragment analysis data for the 605 To ensure genotype data quality, all archival samples were re-amplified, and each locus was genotyped multiple times (up to five times) and compared for consistency. In addition, 20% of all feather, muscle and blood samples were re-amplified multiple times (up to five times) to verify the reliability of the data. Negative controls were including in each genetic analyzer run to check for contamination of reagents. When consistent genotypes were not generated the genotype scores was inputted as missing data.16 to ensure that duplicated genotypes were not included in the final data. This is particularly important in this study as genotypes were amplified from discarded feathers collected at colonies. Null alleles can be a problem in studies that use primers not designed for the study species and this can bias population structure analysis17. Uncorrected global FST were compared to FST values corrected using the excluding null alleles (ENA) method18 using a paired t-test. The paired t-tests were not significant  suggesting that these data are not affected by null alleles.We used identity analysis in the software Cervus v3.0.7Supplementary Table 2.Supplementary Table 1.


To fully utilize the characteristic climatic conditions in the southern region of China, a two-crop-a-year cultivation system technique for ‘Kyoho’ grape was developed during the past decade. After summer harvest in June, appropriate pruning and chemical treatments promote flowering and fruiting, which enables a second harvest in late December. Due to climatic differences between the two crop growing seasons, grape phenol and carotenoid metabolism differ greatly. The reported study analyzed the transcriptome of the carotenoid and phenylpropanoid/flavonoid pathways in grapes at four different stages during the two growing seasons. Compared with those in summer grapes, expression levels of the majority of genes involved in the carotenoid metabolic pathway in winter grapes were generally upregulated. This result was associated with lower rainfall and much more abundant sunlight during the second growing season. On the other hand, summer cropping strongly triggered the expression of upstream genes in the phenylpropanoid/flavonoid pathway at E-L 33 and E-L 35. Transcript levels of flavonoid 3’,5’-hydroxylase (F3’5’H), flavonoid 3’-hydroxylase (F3’H), flavonoid 3-hydroxylase (F3H) and glutathione S-transferase (GST) were upregulated in winter grapes at the mature stage. Together, these results might indicate that more flavonoids would be synthesized in ripe winter grapes during the mature stage of the second crop under much drier conditions, longer sunlight hours and lower temperature. These data provide a theoretical foundation for the secondary metabolism of berries grown under two-crop-a-year cultivation systems. The regions of southern China are considered to be suboptimal for grape cultivation because of extremely high temperature and concentrated rainfall . Fortunacis-epoxycarotenoid dioxygenase (NCED) is thought to be the key enzyme involved in ABA biosynthesis.Carotenoids are a family of C40 isoprenoid pigments that have critical functions in plants, including harvesting light during photosynthesis and providing cleavage products such as the well-known phytohormone abscisic acid (ABA) . The carPolyphenolic compounds play an important role in grape quality. Phenolic compounds containing flavonoids and nonflavonoids, anthocyanins, flavonols, and flavan-3-ols belong to flavonoids, whereas nonflavonoids include stilbenes, hydroxybenzoic and hydroxycinnamic acids. All of these compounds are derived from the phenylpropanoid/flavonoid pathway. The concentration of phenolic compounds in grapes depends on the variety and is also influenced by viticulture techniques and climatic conditions –11. The The climatic conditions in southern China constitute the foundation of two-crop-a-year cultivation development. This technique exploits the rich heat and sunlight during the second half of the year. Thus, the effects of climatic conditions on grape quality are emphasized in two-crop-a-year research. Many studies have shown that both phenolic compounds and carotenoid metabolism in grape berries are greatly influenced by climatic conditions –14. HighThe two-crop-a-year cultivation system has solved the problems of budbreak in the spring, flower bud differentiation during the second season and fruit maturation, but poor coloration and insufficient flavor of summer grapes as well as differences in grape quality between the two crops have not been explained thoroughly. Previous studies have shown that flavonoid composition, content and metabolism are often distinct between the two crops ,17. HoweVitis labrusca × Vitis vinifera L. cv. ‘Kyoho’ by RNA-seq. This paper provides new insights into the understanding of the mechanisms of secondary metabolism influenced by the growing season.Thus, the present research was designed to analyze the transcriptome of phenolic compounds and carotenoids in summer and winter grapes of This experiment was conducted during two growing seasons in 2016 on 4-year-old self-rooted ‘Kyoho’ grapevines in the vineyards of the Grape and Wine Research Institute, Guangxi Academy of Agricultural Sciences, located in Nanning, Guangxi Province . The vines in this vineyard were managed on a canopy frame with a single trunk and were planted in north-south-oriented rows spaced 2.0 m (between vines) × 6.0 m (between rows). Nutrition, pest, water and fertilizer management was carried out in accordance with uniform standards for two-crop-a-year as previously described .The key techniques of two-crop-a-year cultivation systems include dormancy breaking, rain shelter cultivation, soil management, irrigation, fertilizer, pruning, flower and fruit management, and disease and pest control. Two-crop-a-year cultivation systems involve two modes referred to as nonoverlap and overlap cultivation systems .In two-crop-a-year grape cultivation systems, the growing season for the first crop usually occurs from February to June, and that of the second crop occurs from August to December. The nonoverlap cultivation system is suitable for regions where the mean annual temperature > 20°C , and theGrape berries in three biological replicates were collected at four E-L stages : berriesAfter being transported to the laboratory, a subsample of 100 berries from each biological replicate was subjected to physiological measurements, including berry fresh weight, total soluble solids (TSS) content and titratable acidity (TA). The remaining berries were immediately frozen in liquid nitrogen and transported to the laboratory on dry ice for transcriptional analysis. The TSS concentrations in the juices were measured with a digital pocket handheld refractometer , and the TA was determined by titration with NaOH to the end point of pH 8.2 and was expressed as tartaric acid equivalents .A subsample of 50 berries was randomly selected from each biological replicate for RNA extraction. Transcriptome sequencing and data analysis were performed as described previously . HeatmapVvUbiquitin1 and VvActin were selected as two reference genes for qRT-PCR. The sequences of the specific primers used are provided in the supplementary data  - Ct (geometric mean of two reference genes) and Ct is the mean cycle threshold [To verify the reliability of the RNA-Seq-based transcript quantification, quantitative real-time PCR (qRT-PCR) was carried out using 14 genes related to the carotenoid and phenol pathways. The remnant RNAs from the RNA-seq experiment were used to synthesize cDNA, after which qRT-PCR was performed as previously described in –21. VvUbary data . All reahreshold . PearsonSignificant differences were determined when p < 0.05 according to independent t-tests. Partial least squares-discriminant analysis (PLS-DA) was performed with MetaboAnalyst 3.0, and statistical analysis was performed with SPSS  for Windows, version 20.0. Line graphs were constructed with Origin 8.0 software .It is well known that the synthesis and accumulation of grape carotenoids and phenolic compounds are strongly affected by environmental conditions such as temperature, sunlight and rainfall , 23. TabSamples that corresponded to the four stages of the E-L system  were meaBased on the FPKM values of the unigenes in each sample, the differentially expressed genes (DEGs) between two samples were screened . CompareO-methyltransferase , phenylalanine ammonialyase , NCED (VIT_19s0093g00550) and dihydroflavonol reductase . The GO annotations of the transcripts were supplemented in the supplementary data  pathway enrichment analysis revealed that 18 unigenes encoding 12 enzymes were involved in carotenoid and ABA metabolism  presented its maximum FPKM level at E-L 38. PSY (EC:2.5.1.32), Z-ISO, prolycopene isomerase , LBCY (EC:5.5.1.19) and lycopene epsilon-cyclase  were found to be involved in the upstream flux of carotenoid synthesis, and compared with that in the summer grapes, the expression of all the unigenes in the winter grapes was upregulated at the E-L 35, 37 and 38 stages. These results might suggest that the carotenoids contents in the winter grapes higher than summer grapes.The downstream flux of carotenoid metabolism in grape berries includes the synthesis of norisoprenoids, a group of potent flavor and aromatic compounds. In plants, CCD (EC:1.13.11.51) catalyzes the breakdown of carotenoids into volatile norisoprenoids , 25. TwoZEP and three annotated as NCED were identified. The ZEP unigenes showed similar expression profiles between the two crop growing seasons; compared with that at other stages, the expression of these unigenes at E-L 33 was upregulated. Young et al. [ZEPs from the green to harvest stages. In the present study, the three NCED transcripts exhibited different expression profiles. The NCED6 expression level increased gradually in both the summer and winter grapes throughout their development, and compared with that in the summer grapes, NCED6 expression in winter grapes was upregulated at E-L 38. Expression of the NCED1 and NCED2 transcripts peaked at E-L 33, and their expression levels in the summer grapes were higher than those in the winter grapes at E-L 33.In our study, one unigene annotated as g et al.  also repCompared with those in summer grapes, the expression levels of xanthosin dehydrogenase  and ABA glucosyltransferase  in winter grapes were upregulated throughout the whole period. Most transcripts of ABA 8’-hydroxylase  and BCH (EC:1.14.13.129) were expressed the most at E-L 33 for summer.To investigate the differences in phenylpropanoid/flavonoid biosynthesis pathway-related structural genes between the summer and winter grapes at four distinct developmental stages  during the 2016 growing season, we used RNA-seq to characterize the changes in gene expression at the transcript level . The resBotrytis cinerea infection [STS is a pivotal enzyme that catalyzes the biosynthesis of resveratrol and is known to be induced by ultraviolet (UV) irradiation and nfection . CompareThe hydroxylation pattern of flavonoids is known to be mediated by the enzymatic activity of F3’H (EC 1.14.13.21), F3H (EC:1.14.11.9) and flavonoid 3’,5’-hydroxylase , which catalyze the hydroxylation of naringenin and dihydrokaempferol at the 3’, 3’ and 3’,5’ positions of the B-ring, respectively –28. Our LAR and ANR in the winter grapes was upregulated at E-L 33.Furthermore, FLS is responsible for catalyzing the formation of dihydroflavonols into flavonols. Two FLS unigenes exhibited different variation trends between developmental stages of the two crops, and higher expression levels were found in winter grapes than in summer grapes at each stage. LAR and ANR are key regulators of flavan-3-ol and proanthocyanidin biosynthesis , and theO-glucosyltransferase (UFGT) can catalyze the formation of anthocyanin-3-O-glucosides, and 5GT is critical for the synthesis of anthocyanin-3,5-O-diglucosides [UFGT was higher at the later stages of grape maturity. Members of the GST family are believed to participate in vacuolar trafficking and the sequestration of anthocyanins from the endoplasmic reticulum to the vacuole [GST was significantly upregulated in winter grapes at the E-L 33, 35 and 38 stages.UDP-glucose: flavonoid 3-ucosides . In our  vacuole . In the V. quinquangularis Rehd. × V. vinifera L.), ‘Cabernet Sauvignon’ (V. vinifera L.), ‘Riesling’ (V. vinifera L.) [The metabolism of carotenoids and phenols in grapes is influenced by variety, environmental factors, developmental stage, and plant hormone regulation. In recent years, transcriptomic studies involving secondary metabolites that respond to internal and external factors have been prevalent ,31–34. Afera L.) , 35. ‘KyThe climatic conditions of summer and winter grapes differed greatly. During the first growing season, the temperature gradually increased from germination until harvest. However, the second crop showed the opposite results. During berry development, the number of sunshine hours was clearly greater for the winter grapes than for the summer grapes. Additionally, the rainfall during the summer growing season was much greater than that during the winter growing season. Therefore, the differences in climatic factors strongly affect the variation in quality-related indicators between the two crops. During the maturation of grapes, increased sugar contents are favored by sufficient sunlight . On the 13-norisoprenoids and ABA, and when they occur mainly as glycoconjugated forms, C13-norisoprenoids contribute to the characteristic aromas of many varieties [PSY is induced by light, water deficit, and hormones [PSY in the winter grapes was upregulated at the E-L 35, 37 and 38 stages as a result of drier climatic conditions and more sunlight during the winter season. In the present study, the transcriptional analysis of CCD genes in the two-crop-a-year grape berries revealed that, compared with that in the summer grapes, the expression of two unigenes in the winter grapes was upregulated. This observation was in agreement with the upregulated expression of CCD under much drier climatic conditions [NCED were similar between the summer and winter grapes, but the three transcripts showed different expression profiles during the different developmental stages. ABA8ox was predominantly expressed in the summer grapes at E-L 33, and ABA-GT expression peaked in the winter grapes at the same stage, indicating that ABA mainly broke down to PA in the summer grapes and was stored as ABA-GE in the winter grapes.Carotenoids are precursors of Carieties –39. The arieties . Phytoenarieties , 40; comnditions . ABA is nditions . The expLonger sunshine hours and stronger light intensity result in relatively greater amounts of carotenoids synthesized at the green stage and lower amounts synthesized at the harvest stage . Furtherp-coumaroyl-CoA; resveratrol is related to biotic and abiotic stresses in plants [Phenolic compounds, mainly phenolic acids, stilbenes and flavonoids, are among the most abundant secondary metabolites in grapes. Phenolic acids accumulate in grape berry skin and flesh, while stilbenes have been detected in the skin, fruit flesh and seeds , 42. Phen plants . In the LAR and DFR was highest at E-L 33 for both crops, and both genes were upregulated in the summer grapes. However, anthocyanins in winter ‘Kyoho’ grapes did not accumulate at the expense of flavan-3-ols in previous study [Flavan-3-ols, which share common upstream steps with both flavonols and anthocyanins, accumulated from E-L 27 (fruit set) until E-L 35 (veraison), after which they decreased . In the us study , even thF3’5’H, F3’H and F3H were upregulated in winter grapes at the E-L 37 and E-L 38 stages. These results also indicate that many more flavonoids accumulate in winter grapes than in summer grapes.F3’5’H, F3’H and F3H catalyze naringenin flavanones to form flavonoids containing flavonols, flavan-3-ols and anthocyanins . The expV. labrusca and V. vinifera, ‘Kyoho’ grapes contain 3-O-glucosides and 3,5-O-diglucosides in their skin [UFGT showed higher expression levels at some stages in the summer grapes than in the winter grapes, transcripts of GST were more abundant in the winter grapes than in the summer grapes at the E-L 33, 35 and 38 stages. Previous research has shown that, compared with summer grapes, winter grapes contain greater amounts of anthocyanins [As a hybrid of eir skin . Althougocyanins . High teocyanins –45. ThisGST was higher in the winter grapes than in the summer grapes. All of these factors could result in the production of less flavonoids and could be explained by extremely hot weather during the mature stage of the first crop. We anticipate that the present study will enrich theoretical research on two-crop-a-year cultivation systems and will facilitate additional investigations into the differences in grape berry quality between two crops grown during the same year.The two-crop-a-year cultivation system is based on unique weather conditions in southern China. This work investigated the transcriptomic dynamics of ‘Kyoho’, which was the first variety cultivated via this technique. These results mainly revealed differences in the transcriptional expression patterns of phenols and carotenoids in ‘Kyoho’ grapes during the four stages of grape berry development in a two-crop-a-year cultivation system. The results showed that the second crop produced winter grapes that were smaller than the summer grapes and had greater contents of TSS and TA. Compared with the summer grapes, the winter grapes overall showed higher expression levels of genes required for the synthesis of carotenoids, which could be attributed to lower rainfall and much more abundant sunlight during the second growing season. In addition, the expression of some genes involved in the upstream portion of the phenylpropanoid/flavonoid pathway was upregulated in the summer grapes at E-L 33 and E-L 35. Although the expression of some genes directly related to the synthesis of flavan-3-ols and anthocyanidins was upregulated in the summer grapes at E-L 37 and E-L 38, the expression of three hydroxylase genes and S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Oxalis alpina is a tetraploid, herbaceous species inhabiting the Sky Island region of the southwestern United States and northern Mexico. Our main objective in this study was to analyze the influence of climatic oscillations on the genetic structure of O. alpina and the impact of these oscillations on the evolutionary transition from tristylous to distylous reproductive systems. We used microsatellite markers and compared our results to a previous study using chloroplast genetic markers. The phylogeographic structure inferred by both markers was different, suggesting that intrinsic characteristics including the pollination system and seed dispersal have influenced patterns of gene flow. Microsatellites exhibited low genetic structure, showed no significant association between geographic and genetic distances, and all individual genotypes were assigned to two main groups. In contrast, chloroplast markers exhibited a strong association between geographic and genetic distance, had higher levels of genetic differentiation, and were assigned to five groups. Both types of DNA markers showed evidence of a northward expansion as a consequence of climate warming occurring in the last 10,000 years. The data from both types of markers support the hypothesis for several independent transitions from tristyly to distyly.Historical factors such as climatic oscillations during the Pleistocene epoch have dramatically impacted species distributions. Studies of the patterns of genetic structure in angiosperm species using molecular markers with different modes of inheritance contribute to a better understanding of potential differences in colonization and patterns of gene flow via pollen and seeds. These markers may also provide insights into the evolution of reproductive systems in plants.  HistoriRegions with climatic oscillations that have impacted distribution of organisms represent natural laboratories for investigation of how historical processes such as bottlenecks, isolation, genetic drift, and natural selection have produced complex configurations of population structure and influenced the evolution of reproductive systems Boyd, . An areaOxalis alpina , the patterns of genetic structure are frequently dissimilar. On one hand, the dispersion of nuclear genes occurs via pollen and seeds, while the cytoplasmic genes are maternally inherited in most angiosperms, and thus, dispersal of cytoplasmic genes depends exclusively on seed movement Ennos, . Gene fla Figure .Oxalis alpina (Rose) Knuth  is a heterostylous, tetraploid, perennial herb inhabiting evergreen Madrean woodlands in the Sky Island region . Second, the incompatibility system includes different degrees of modification, with remarkable modifications in the northern ranges of the distribution. Weller et al.  analyze the influence of climatic oscillations on the genetic structure of O. alpina using microsatellite markers and propose phylogeographic scenarios to aid in understanding of the evolutionary processes behind the variation in the reproductive system of O. alpina and (2) compare the phylogeographic patterns inferred from the microsatellite data with previous findings from cpDNA data to reconstruct the historical scenarios that better explain the current patterns of genetic structure of O. alpina populations.Historical factors associated with range expansion and contraction during climatic oscillations in the Pleistocene Period might also have influenced the tristyly–distyly transition   was collected from 10–68 individuals per population , and 5–50 ng DNA template. An Applied Biosystems thermal cycler (GeneAmp PCR System 9700) and ABI PRISM 3130xl sequencer, respectively, were used for amplifications and to run microsatellite PCR reactions combined with an internal size standard Naurox , Nei's gene diversity corrected for sample size , number of alleles per locus (NA), and effective number of alleles per locus (NAe). SPAGeDi assumes polysomic inheritance, which is expected for low‐order autopolyploids such as Oxalis alpina. Average allelic richness (AR) was calculated for each population using the rarefaction method implemented in Allelic Diversity Analyzer (ADZE) while limiting sample size to 20  and cpDNA (haplotype diversity). The analyses were carried out using the program JMP .We used GenoDive  and an expected value (pRST) calculated after 5,000 allele size permutations using SPAGeDi version 1.3a , and 10 iterations for each k. Further analyses of the substructure within each main genetic cluster were analyzed using the same parameters as for the entire data set .To determine how many distinct population clusters were supported by the data, we used the software STRUCTURE 2.3.4  using the Bruvo distances for visualizing the genetic structure and relationships among samples through the R program POLYSAT   based on equations from Ennos . After the Sierra Ancha and White populations, the northern Pinal population exhibited the lowest levels of diversity for HE, while for the parameter NAe, the lowest level of diversity was found in the northern Pinaleño population; the lowest values for NA were found in Elenita and Buenos Aires, and for HO, the lowest value was found in Morse Canyon Chiricahua population. In contrast, the highest levels of genetic diversity for the Ho, HE parameter, and NAE parameters were found in the southwestern Santa Rita population, while Pinos Altos and Mariquita exhibited the highest level of diversity for the NA estimator (Table p = .051) relationship of latitude with the levels of genetic diversity estimated by HE, based on microsatellites markers (Table p = .03) on haplotype diversity. For both genetic markers, longitude and the interaction of latitude and longitude did not show any significant effect , and therefore, a total of 29 samples were removed from the analyses. The ratio of the number of genets to the number of ramets was from 0.73 to 0.99 . The RST parameter was marginally significantly higher than the pRST (0.28) (p = .053). The Bayesian individual assignments using STRUCTURE indicated that the most informative representation of the genetic structure of O. alpina corresponded to two groups  p = .05. The Bay) p = .05. The Bay) p = .05. The BayWe also examined the genetic structure within each cluster Figure . Four grr = −.04, p = .61). The popgraph software produced a population network with two subgraphs. Each subgraph included the same populations as the two clusters (I and II) detected by the Bayesian assignment .The neighbor‐joining phenogram recovered the Chiricahua cluster and the other three populations from cluster I with bootstrap values above 50. The remained clades were supported by very low bootstrap values . We also performed a Mantel test to compare the similarities of genetic distance matrices (based on FST) calculated from microsatellite and chloroplast sequence data. The correlation was not statistically significant . The value obtained for the pollen‐seed migration ratio was r = 5.62; we used GST = 0.73 for cpDNA, and GST = 0.26 and FIS = −0.0064 for microsatellite markers to calculate r.Regression analyses did not show any relationship between the microsatellite diversity parameters and cpDNA haplotype diversity , which suggests that the stepwise mutation model could be more suitable to explain the genetic differentiation among populations than gene flow. We should not interpret this result as evidence for phylogeographical pattern  because the result is only marginally significant. Oxalis alpina harbors two main genetic lineages, cluster I and cluster II, suggesting a latitudinal pattern of genetic structure. The negative effect of latitude on genetic diversity was detected from the covariance analysis.We found significant genetic structure for  Slatkin , unbiaseO. alpina was built using the GARP algorithm (Genetic Algorithm for Rule‐set Production)  predicted by niche modeling  than through microsatellite markers (GST = 0.26). Larger values for r, the ratio of migration between biparental (microsatellites) and maternally inherited markers . Therefore, gene flow via pollen and thus the mp/ms ratio (pollen and seed gene flow ratio) are smaller for O. alpina than the average found for angiosperms.Because dispersion by pollen frequently occurs over larger distances than by seeds, lower levels of genetic structure were expected for nuclear genes in comparison with cytoplasmic genes . Therefore, gene flow should be higher for nuclear markers carried by pollen than for organellar markers carried by seeds and may explain the detection of more genetically differentiated groups of populations using cpDNA than nuclear markers. Divergent patterns of genetic structure might also be explained by the more rapid coalescence of uniparentally inherited haploid alleles in mitochondrial or chloroplast DNA that have smaller effective population sizes than most nuclear loci  from the Chiricahua Mts in cluster I are genetically similar, which is also supported by the results from the Structure and NJ phenogram. These three populations are also located in close geographic proximity to each other, representing a remarkable example of the transition from tristyly to distyly. Weller et al.  found thWe did not find a significant association between genetic distances and mid‐styled morph frequency, indicating that distylous populations are genetically dissimilar, and therefore distyly probably originated from different tristylous populations. Additionally, genetic evidence from chloroplast data further suggests that distyly has evolved more than once , support that the route of colonization was probably from south to north, although the genetic signature of northern founder events inferred from microsatellites is not as clear as from cpDNA markers  occurred mainly in the southern ranges compared with the current distribution (Pérez‐Alquicira et al., Ionoxalis because of the South America origin of this section, and species have dispersed on several occasions to North America (Gardner et al., O. alpina. Moreover, because distylous populations are mainly located in northern ranges where founder events were frequent, genetic drift is likely to have influenced the evolutionary transition of tristyly–distyly system.Overall, our results indicate that microsatellite markers provide useful genetic information for tracing the possible routes of colonization of populations of None declared.All authors contributed to the design of the study and sample collection. JP‐A and OVT acquired sequencing and microsatellite data, respectively, and conducted data analyses. JP‐A, OVT, and SGW wrote the first draft of the manuscript, and all authors contributed critically to its revision and provided approval for the final submission.