How the COVID-19 pandemic affects transgender health care - A cross-sectional online survey in 63 upper-middle-income and high-income countries.

Background: Due to the COVID-19 pandemic, access to medical care is restricted for nearly all non-acute conditions. Due to their status as a vulnerable social group and the inherent need for transition-related treatments, transgender people are assumed to be affected particularly severely by the restrictions caused by the COVID-19 pandemic. Methods: As an ad hoc collaboration between researchers, clinicians and 23 community organizations, we developed a web-based survey in German that was translated into 26 languages. Participants were recruited via community sources, social media channels, and snowball sampling since May 2020. The present sample is based on the data collected until August 9, 2020. We assessed demographical data, health problems, risk factors, COVID-19 data (e.g., contact history), and the influence of the COVID-19 pandemic on access to transgender health care services. To identify factors associated with the experience of restrictions, we conducted multiple logistic regression analysis. Results: 5267 transgender people from 63 upper-middle-income and high-income countries participated in the study. Over 50% of the participants had risk factors for a severe course of a COVID-19 infection and were at a high risk of avoiding COVID-19 treatment due to the fear of mistreatment or discrimination. Access to transgender health care services was restricted for 50% of the participants. Male sex assigned at birth and a lower monthly income were significant predictors for the experience of restrictions to health care. 35.0% reported at least one mental health condition and 3.2% have attempted suicide since the beginning of the COVID-19 pandemic. Discussion: Transgender people suffer under the severity of the pandemic due to the intersections between their status as a vulnerable social group, their high number of medical risk factors, and their need for ongoing medical treatment. The COVID-19 pandemic can potentiate these vulnerabilities, add new challenges for transgender people, and, therefore, can lead to devastating consequences, like severe physical or mental health issues, self-harming behavior, and suicidality.

Measuring the Change in Soft Palate Length and Shape Following Maxillary Advancement: A Cohort Study in Patients with Orofacial Clefts.

OBJECTIVE: To evaluate a method of measuring the change in palatal length and shape following maxillary advancement using synchronous lateral videofluoroscopy and voice recording in order to understand how movement of the maxilla may affect VPI risk in patients with cleft lip and/or palate (CL/P). DESIGN: Retrospective cohort study of children with cleft lip and/or palate. SETTING: Single center, tertiary children's hospital. PARTICIPANTS: Patients with cleft lip and/or palate who underwent maxillary advancement between 2016-21 inclusive. INTERVENTIONS: Maxillary advancement surgery, including those who underwent concurrent mandibular procedures. MAIN OUTCOME MEASURES: The length of the soft palate and the genu angle were measured throughout palatal dynamic range. Pre- and post-operative measurements were compared using a one sided T-test, with subgroup analysis for patients with clinical VPI. RESULTS: Ten patients were examined. The mean distance of maxillary advancement was 10.5 mm. The average increase in pre-genu soft palate length was 2.8 mm in the resting position and 2.9 mm in the closed position. The genu angle decreased in the closed position by 16.3 degrees. CONCLUSIONS: The soft palate showed limited ability to lengthen following maxillary advancement and this may explain the risk of VPI. There was partial compensation by the muscle sling of the palate as demonstrated by a more acute post-operative genu angle and this suggests one reason for the variability of VPI reported. Future research is required to investigate how length and shape changes measured using this method can predict VPI risk.

Heavy metal tolerance in Scopelophila cataractae: Transcriptomic and epigenetic datasets.

Studying how different plant groups deal with heavy metal exposure is crucial to improve our understanding of the diversity of molecular mechanisms involved in plant stress response. Here, we used RNA sequencing (RNA-seq) and epigenotyping by sequencing (epiGBS) to assess gene expression and DNA methylation changes respectively in plants from four populations of the metallophyte moss Scopelophila cataractae treated with Cd or Cu in the laboratory. We built RNA-seq and epiGBS sequencing libraries from control and treated samples from each population and sequenced them using Illumina HiSeq 3000 (PE-150 bp) and Illumina HiSeq X-Ten System (PE-150 bp) respectively. For the RNA-seq data, we performed a read quality filter, mapped the reads to the de novo transcriptome created with Trinity, and estimated transcript abundance for each sample. For the epiGBS data, we used a custom pipeline (https://doi.org/10.5281/zenodo.7040291) to map the reads to a de novo reference genome and performed strand-specific nucleotide (single nucleotide polymorphisms, SNPs) and methylation (single cytosine methylation polymorphisms, SMPs) variant calling. We filtered out SNPs and SMPs with low coverage within (positions with <10 sequencing reads per sample) and across samples (positions with poor representation on the full set of samples). Finally, we performed pairwise comparisons between control and treated samples from each population and identified differentially expressed genes and differentially methylated cytosines associated to heavy metal exposure. We payed particular attention to the different responses of the more and the less tolerant populations of S. cataractae. These datasets could contribute to future comparative studies of abiotic stress response across plant groups.

Restoration of native saltmarshes can reverse arthropod assemblages and trophic interactions changed by a plant invasion.

Plant invasions profoundly impact both natural and managed ecosystems, and removal of the invasive plants addresses only part of the problem of restoring impacted areas. The rehabilitation of diverse communities and their ecosystem functions following removal of invasive plants is an important goal of ecological restoration. Arthropod assemblages and trophic interactions are important indicators of the success of restoration, but they have largely been overlooked in saltmarshes. We determined how arthropod assemblages and trophic interactions changed with the invasion of the exotic plant Spartina alterniflora and with the restoration of the native plant Phragmites australis following Spartina removal in a Chinese saltmarsh. We investigated multiple biotic and abiotic variables to gain insight into the factors underlying the changes in arthropod assemblages and trophic structure. We found that although Spartina invasion had changed arthropod diversity, community structure, feeding-guild composition, and the diets of arthropod natural enemies in the saltmarsh, these changes could be reversed by the restoration of native Phragmites vegetation following removal of the invader. The variation in arthropod assemblages and trophic structure were critically associated with four biotic and abiotic variables (aboveground biomass, plant density, leaf N, and soil salinity). Our findings demonstrate the positive effects of controlling invasive plants on biodiversity and nutrient cycling and provide a foundation for assessing the efficacy of ecological restoration projects in saltmarshes.

The Safety and Efficacy of Apixaban (Eliquis) in 5017 Post-bariatric Patients with 95.3% Follow-up: a Multicenter Study.

BACKGROUND: Thromboprophylaxis in bariatric surgery is widely debated; however, few large articles evaluate treatment plans and their efficacy. Herein, we make the first large-scale report of the safety and efficacy of apixaban (Eliquis) for thrombus prevention following bariatric surgery. PURPOSE: To evaluate the safety and efficacy of apixaban following bariatric surgery. SETTING: Three private institutes, USA. MATERIALS AND METHODS: Data from 5017 consecutive bariatric patients that were placed on postoperative apixaban for thromboprophylaxis were used for retrospective analysis. The dose prescribed to patients was 2.5 mg PO BID for a total of 30 days starting on day 3 postoperatively. RESULTS: In total, of the 5017 patients, 59.7%, 31.2%, 4.4%, 2.5%, 1.8%, and 0.1% of the patients had undergone sleeve gastrectomy (SG), single-anastomosis duodeno-ileal bypass with SG (SADI-S), Roux-en-Y gastric bypass (RYGB), conversion from SG to SADI, small bowel reconstruction, and RYGB reversal, respectively. The 30-day follow-up rate was 95.3%. In total, 1.7% of patients experienced apixaban-related side effects. The most common side effects were menorrhagia and rash. Two (0.03%) side effects developed into Clavien-Dindo grade II complications. Overall, 10 (0.1%) patients experienced thromboembolic complications (five (0.09%) PVTs and five (0.09%) PEs). In each case, the protocol was not followed for extenuating circumstances. There were no deaths or thromboembolic events in cases where the protocol was able to be fully followed. CONCLUSIONS: In conclusion, 30 days of postoperative apixaban appears to be safe and effective with minimal side effects while preventing thromboembolic events.

Repeated Genetic and Adaptive Phenotypic Divergence across Tidal Elevation in a Foundation Plant Species.

AbstractMicrogeographic genetic divergence can create fine-scale trait variation. When such divergence occurs within foundation species, then it might impact community structure and ecosystem function and cause other cascading ecological effects. We tested for parallel microgeographic trait and genetic divergence in Spartina alterniflora, a foundation species that dominates salt marshes of the US Atlantic and Gulf coasts. Spartina is characterized by tall-form (1-2 m) plants at lower tidal elevations and short-form (<0.5 m) plants at higher tidal elevations, yet whether this trait variation reflects plastic and/or genetically differentiated responses to these environmental conditions remains unclear. In the greenhouse, seedlings raised from tall-form plants grew taller than those from short-form plants, indicating a heritable difference in height. When we reciprocally transplanted seedlings back into the field for a growing season, composite fitness (survivorship and seed production) and key plant traits (plant height and biomass allocation) differed interactively across origin and transplant zones in a manner indicative of local adaptation. Further, a survey of single nucleotide polymorphisms revealed repeated, independent genetic differentiation between tall- and short-form Spartina at five of six tested marshes across the native range. The observed parallel, microgeographic genetic differentiation in Spartina likely underpins marsh health and functioning and provides an underappreciated mechanism that might increase capacity of marshes to adapt to rising sea levels.

Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity.

PREMISE: The distribution of genetic diversity on the landscape has critical ecological and evolutionary implications. This may be especially the case on a local scale for foundation plant species because they create and define ecological communities, contributing disproportionately to ecosystem function. METHODS: We examined the distribution of genetic diversity and clones, which we defined first as unique multilocus genotypes (MLG), and then by grouping similar MLGs into multilocus lineages. We used 186 markers from inter-simple sequence repeats (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus chinensis. We examined the relationship between genetic and clonal diversities, their variation with patch size, and the effect of the number of markers used to evaluate genetic diversity and structure in this species. RESULTS: Every ramet had a unique MLG. Almost all patches consisted of individuals belonging to a single multilocus lineages. We confirmed this with a clustering algorithm to group related genotypes. The predominance of a single lineage within each patch could be the result of the accumulation of somatic mutations, limited dispersal, some sexual reproduction with partners mainly restricted to the same patch, or a combination of all three. CONCLUSIONS: We found strong genetic structure among patches of L. chinensis. Consistent with previous work on the species, the clustering of similar genotypes within patches suggests that clonal reproduction combined with somatic mutation, limited dispersal, and some degree of sexual reproduction among neighbors causes individuals within a patch to be more closely related than among patches.

Long-Term (> 6 Years) Outcomes of Duodenal Switch (DS) Versus Single-Anastomosis Duodeno-Ileostomy with Sleeve Gastrectomy (SADI-S): a Matched Cohort Study.

BACKGROUND: There are no comparative studies on the long-term outcomes after the primary traditional duodenal switch (DS) and single-anastomosis duodeno-ileostomy with sleeve gastrectomy (SADI-S). PURPOSE: This study aims to compare the long-term outcomes in a matched cohort. SETTING: This study took place in a single private institute, in the USA. MATERIALS AND METHODS: Data from 266 patients who underwent a primary laparoscopic traditional DS or primary laparoscopic SADI-S from September 2011 to December 2015 by four surgeons were used for a retrospective matched cohort. Data were obtained by matching every DS patient to a SADI-S patient of the same age, sex, and body mass index (BMI). In addition, only patients that were out at least 5 years and had a minimum 5-year follow-up were included in the study. RESULTS: The matched cohort included 30 DS and 30 SADI-S patients. There were no statistically significant differences in the preoperative characteristics and baseline comorbidities between both groups. The SADI-S patients had significantly shorter operative time and length of stay. The overall long-term complications, especially the long-term Clavien-Dindo grade IIIb complications, were significantly fewer with SADI-S. At 6 years, the DS patients had statistically higher %EWL; however, the ending BMIs were statistically similar between both groups. There were no significant differences in the long-term comorbidity and nutritional outcomes of both groups. The long-term failure rates were comparable. CONCLUSIONS: Most long-term outcomes of SADI-S were either similar or significantly better than DS. Part of the reasons could be the surgeon's learning curve and the small sample sizes of both groups.

Inheritance of DNA methylation differences in the mangrove Rhizophora mangle.

The capacity to respond to environmental challenges ultimately relies on phenotypic variation which manifests from complex interactions of genetic and nongenetic mechanisms through development. While we know something about genetic variation and structure of many species of conservation importance, we know very little about the nongenetic contributions to variation. Rhizophora mangle is a foundation species that occurs in coastal estuarine habitats throughout the neotropics where it provides critical ecosystem functions and is potentially threatened by anthropogenic environmental changes. Several studies have documented landscape-level patterns of genetic variation in this species, but we know virtually nothing about the inheritance of nongenetic variation. To assess one type of nongenetic variation, we examined the patterns of DNA sequence and DNA methylation in maternal plants and offspring from natural populations of R. mangle from the Gulf Coast of Florida. We used a reduced representation bisulfite sequencing approach (epi-genotyping by sequencing; epiGBS) to address the following questions: (a) What are the levels of genetic and epigenetic diversity in natural populations of R. mangle? (b) How are genetic and epigenetic variation structured within and among populations? (c) How faithfully is epigenetic variation inherited? We found low genetic diversity but high epigenetic diversity from natural populations of maternal plants in the field. In addition, a large portion (up to ~25%) of epigenetic differences among offspring grown in common garden was explained by maternal family. Therefore, epigenetic variation could be an important source of response to challenging environments in the genetically depauperate populations of this foundation species.

Carbon and nitrogen pools and mobile fractions in surface soils across a mangrove saltmarsh ecotone.

In the subtropics, climate change is pushing woody mangrove forests into herbaceous saltmarshes, altering soil carbon (C) and nitrogen (N) pools, with implications for coastal wetland productivity and C and N exports. We quantified total C and N pools, and mobile fractions including extractable mineral N, extractable organic C and N, and active (aerobically mineralizable) C and N, in surface soils (top 7.6 cm) of adjacent mangrove (primarily Avicennia germinans) and saltmarsh (Juncus roemerianus) vegetation zones in tidal wetlands of west-central Florida (USA). We tested whether surface-soil accumulations of C, N, and their potentially mobile fractions are greater in mangrove than in saltmarsh owing to greater accumulations in the mangrove zone of soil organic matter (SOM) and fine mineral particles (C- and N-retaining soil constituents). Extractable organic fractions were 39-45% more concentrated in mangrove than in saltmarsh surface soil, and they scaled steeply and positively with SOM and fine mineral particle (silt + clay) concentrations, which themselves were likewise greater in mangrove soil. Elevation may drive this linkage. Mangrove locations were generally at lower elevations, which tended to have greater fine particle content in the surface soil. Active C and extractable mineral N were marginally (p < 0.1) greater in mangrove soil, while active N, total N, and total C showed no statistical differences between zones. Extractable organic C and N fractions composed greater shares of total C and N pools in mangrove than in saltmarsh surface soils, which is meaningful for ecosystem function, as persistent leaching of this fraction can perpetuate nutrient limitation. The active (mineralizable) C and N fractions we observed constituted a relatively small component of total C and N pools, suggesting that mangrove surface soils may export less C and N than would be expected from their large total C and N pools.

Epigenetic effects of parasites and pesticides on captive and wild nestling birds.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ectoparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we conducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identified a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontarget effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects.

Attitudes of transgender men and non-binary people to cervical screening: a cross-sectional mixed-methods study in the UK.

BACKGROUND: Transgender men and non-binary people assigned female at birth (TMNB) who have not had surgery to remove the cervix are recommended to undertake cervical screening with the same frequency as cisgender women, but evidence suggests that TMNB have lower odds of lifetime and up-to-date cervical screening uptake. AIM: To understand the attitudes towards and preferences for cervical screening among UK-based TMNB. DESIGN AND SETTING: Cross-sectional survey of TMNB at an NHS gender identity clinic (GIC) and an NHS sexual health service specialising in care of transgender people. METHOD: Recruitment was via email invitations to patients of the GIC and sexual health service. Inclusion criteria were: female sex assigned at birth; transgender man, masculine, or non-binary gender identity; aged ≥18 years; and UK resident. Quantitative results were analysed using descriptive statistics, and free-text comments were analysed thematically. RESULTS: In total there were 137 participants; 80% identified as transmasculine,18% as non-binary, and the remaining participants reported other noncisgender identities. Sixty-four participants (47%) were eligible for cervical screening and 37 (58%) of those had been screened. Only 34 (53%) of those eligible felt they had sufficient information about cervical screening. Just over half (n = 71/134, 53%) stated they would like the option to self-swab for high-risk human papillomavirus. Only half (n = 68/134, 51%) of participants were in favour of an automatic invitation for cervical screening. Thematic analysis identified a number of additional barriers to and facilitators of screening. CONCLUSION: TMNB have identified numerous potential areas for change that may improve cervical screening uptake and patient experience.

Epigenetics and the success of invasive plants.

Biological invasions impose ecological and economic problems on a global scale, but also provide extraordinary opportunities for studying contemporary evolution. It is critical to understand the evolutionary processes that underly invasion success in order to successfully manage existing invaders, and to prevent future invasions. As successful invasive species sometimes are suspected to rapidly adjust to their new environments in spite of very low genetic diversity, we are obliged to re-evaluate genomic-level processes that translate into phenotypic diversity. In this paper, we review work that supports the idea that trait variation, within and among invasive populations, can be created through epigenetic or other non-genetic processes, particularly in clonal invaders where somatic changes can persist indefinitely. We consider several processes that have been implicated as adaptive in invasion success, focusing on various forms of 'genomic shock' resulting from exposure to environmental stress, hybridization and whole-genome duplication (polyploidy), and leading to various patterns of gene expression re-programming and epigenetic changes that contribute to phenotypic variation or even novelty. These mechanisms can contribute to transgressive phenotypes, including hybrid vigour and novel traits, and may thus help to understand the huge successes of some plant invaders, especially those that are genetically impoverished. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

A Matched Cohort Comparison of Long-term Outcomes of Roux-en-Y Gastric Bypass (RYGB) Versus Single-Anastomosis Duodeno-ileostomy with Sleeve Gastrectomy (SADI-S).

BACKGROUND: The long-term effectiveness of Roux-en-Y gastric bypass (RYGB) and single-anastomosis duodeno-ileostomy with sleeve gastrectomy (SADI-S) is unknown. PURPOSE: Compare the long-term outcomes. SETTING: Single private institute, USA. MATERIALS AND METHODS: Data from 1254 patients who underwent primary RYGB or SADI-S were used for a retrospective matched cohort. Data were obtained by matching every RYGB patient to a SADI-S patient of the same sex, body mass index (BMI), and weight. Only patients out 5 years and had at least one > 5-year follow-up visit were included. RESULTS: The matched cohort included 61 RYGB and 61 SADI-S patients. There was no statistical, demographic difference between the two groups. At 5 years, a 100% follow-up was available in each group. The intraoperative outcomes were significantly better with SADI-S. The 30-day readmission, reoperation, emergency department (ED) visits, and complication rates were statistically similar between the two groups. The long-term complication rates, Clavien-Dindo grade IIIb complications, and number of patients with more than one complication were significantly lower with SADI-S. Weight loss was significantly greater in the SADI-S group at 5 years. The long-term weight-loss failure rate was significantly higher in the RYGB group. The SADI-S procedure was associated with fewer reintervention through 6 years (14.7% patients vs. 39.3% patients, p = 0.001). Conversion or reversal of the procedure was required only in the RYGB group. There also was no significant difference in nutritional outcomes between the two procedures. CONCLUSIONS: This study showed that problems, including long-term complications, reinterventions, weight-loss failure, and conversion, were more often associated with RYGB than with SADI-S. The SADI-S may be considered one of the viable alternatives to RYGB.

Artificial selection for host resistance to tumour growth and subsequent cancer cell adaptations: an evolutionary arms race.

BACKGROUND: Cancer progression is governed by evolutionary dynamics in both the tumour population and its host. Since cancers die with the host, each new population of cancer cells must reinvent strategies to overcome the host's heritable defences. In contrast, host species evolve defence strategies over generations if tumour development limits procreation. METHODS: We investigate this "evolutionary arms race" through intentional breeding of immunodeficient SCID and immunocompetent Black/6 mice to evolve increased tumour suppression. Over 10 generations, we injected Lewis lung mouse carcinoma cells [LL/2-Luc-M38] and selectively bred the two individuals with the slowest tumour growth at day 11. Their male progeny were hosts in the subsequent round. RESULTS: The evolved SCID mice suppressed tumour growth through biomechanical restriction from increased mesenchymal proliferation, and the evolved Black/6 mice suppressed tumour growth by increasing immune-mediated killing of cancer cells. However, transcriptomic changes of multicellular tissue organisation and function genes allowed LL/2-Luc-M38 cells to adapt through increased matrix remodelling in SCID mice, and reduced angiogenesis, increased energy utilisation and accelerated proliferation in Black/6 mice. CONCLUSION: Host species can rapidly evolve both immunologic and non-immunologic tumour defences. However, cancer cell plasticity allows effective phenotypic and population-based counter strategies.

Long-term outcomes of primary single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S).

BACKGROUND: The long-term outcomes of primary single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) have never been reported in the literature. OBJECTIVES: The study aimed to evaluate the long-term outcomes after primary laparoscopic SADI-S (LSADI-S). SETTING: Single, private institute, United States. METHODS: Data from 750 patients who underwent a primary LSADI-S from June 2013 through November 2019 by 3 surgeons were retrospectively analyzed. RESULTS: Seven hundred fifty patients were included in the study. The mean age and preoperative body mass index were 49.3 ± 13.1 years and 50 ± 12.6 kg/m2, respectively. Follow-up was available on 109 patients (61%) at 5 years and on 87 patients (53%) at 6 years. Six patients did not have any follow-up. The average operative time and length of stay were 67.6 ± 27.4 minutes and 1.5 ± .8 days, respectively. The intraoperative, short-term, and long-term complication rates were 0%, 7.8%, 11.7%, respectively. The 30-day emergency room visit, readmission, and reoperation rates were .4%, 1.1%, and 1.1%, respectively. In total, there were 15 (2%) grade IIIb long-term complications unique to LSADI-S. Complete remission of type 2 diabetes was seen in 77% of the diabetic population. At 5 and 6 years, the mean change in body mass index was 17.5 ± 6.9 and 17.6 ± 6.4 kg/m2, respectively. The mortality rate was .5%. CONCLUSIONS: LSADI-S is effective in this retrospective review in achieving good initial weight loss and weight maintenance. Although our data show acceptable nutritional complications, questions still remain because of the retrospective nature of the study.

Comparisons of Natural and Cultivated Populations of Corydalis yanhusuo Indicate Divergent Patterns of Genetic and Epigenetic Variation.

Epigenetic variation may contribute to traits that are important in domestication, but how patterns of genetic and epigenetic variation differ between cultivated and wild plants remains poorly understood. In particular, we know little about how selection may shape epigenetic variation in natural and cultivated populations. In this study, we investigated 11 natural populations and 6 major cultivated populations using amplified fragment length polymorphism (AFLP) and methylation-sensitive AFLP (MS-AFLP or MSAP) markers to identify patterns of genetic and epigenetic diversity among Corydalis yanhusuo populations. We further explored correlations among genetic, epigenetic, alkaloidal, and climatic factors in natural and cultivated C. yanhusuo. We found support for a single origin for all cultivated populations, from a natural population which was differentiated from the other natural populations. The magnitude of F ST based on AFLP was significantly correlated with that for MSAP in pairwise comparisons in both natural and cultivated populations, suggesting a relationship between genetic and epigenetic variation in C. yanhusuo. This relationship was further supported by dbRDA (distance-based redundancy analyses) where some of the epigenetic variation could be explained by genetic variation in natural and cultivated populations. Genetic variation was slightly higher in natural than cultivated populations, and exceeded epigenetic variation in both types of populations. However, epigenetic differentiation exceeded that of genetic differentiation among cultivated populations, while the reverse was observed among natural populations. The differences between wild and cultivated plants may be partly due to processes inherent to cultivation and in particular the differences in mode of reproduction. The importance of epigenetic compared to genetic modifications is thought to vary depending on reproductive strategies, and C. yanhusuo usually reproduces sexually in natural environments, while the cultivated C. yanhusuo are propagated clonally. In addition, alkaloid content of C. yanhusuo varied across cultivated populations, and alkaloid content was significantly correlated to climatic variation, but also to genetic (6.89%) and even more so to epigenetic (14.09%) variation in cultivated populations. Our study demonstrates that epigenetic variation could be important in cultivation of C. yanhusuo and serve as a source of variation for response to environmental conditions.

Epigenetic Approaches in Non-Model Plants.

Reduced representation bisulfite sequencing is an emerging methodology for evolutionary and ecological genomics and epigenomics research because it provides a cost-effective, high-resolution tool for exploration and comparative analysis of DNA methylation and genetic variation. Here we describe how digestion of genomic plant DNA with restriction enzymes, subsequent bisulfite conversion of unmethylated cytosines, and final DNA sequencing allow for the examination of genome-wide genetic and epigenetic variation in plants without the need for a reference genome. We explain how the use of several combinations of barcoded adapters for the creation of highly multiplexed libraries allows the inclusion of up to 144 different samples/individuals in only one sequencing lane.