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... Exploring Phenotypic and Neurocognitive Profiles in BCL11B Gene Mutations: A Case Study of a Family with Three Siblings Kevin 1 Medernach , 1Marian Background: BCL11B is a transcription factor gene that encodes a zinc finger domain, which is thought to play an important role in DNA binding, that is critical to the development of the nervous and immune system. Mutations in this gene are rare in the general population but associated with a range of neurodevelopmental abnormalities, intellectual disability, craniofacial dysmorphology, movement disorders, atopic disorders, and immune deficiencies. Most mutations in BCL11B arise de novo, with missense mutations often leading to the most severe neurodevelopmental defects. The findings of this poster aim to summarize current literature and to expand understanding regarding the phenotypic and neurodevelopmental abnormalities associated with mutations in the BCL11B gene. Methods: Literature review of previously published case reports was completed with the use of PubMed and Google Scholar. To date, 34 patient profiles were found and analyzed. An in-depth look at the phenotypic presentation of BCL11B mutations were gathered and summarized, including the known neurodevelopmental deficits seen in patients with BCL11B mutations. A paucity of information was previous published about neurocognitive functions and abilities in these individuals. We present a case series from one family with 3 full sibling cases (1 and 2: twins), who were evaluated for neurocognitive functioning across a broad battery. Testing looked at language skills, intelligence, executive functioning, learning and memory, attention, and visuospatial skills. 2 Chapman , BS, Leah PhD, Lia 1,2 Michelle Curtin , DO, FAAP Discussion: Case 2 Case 3 Referred for ASD Referred for ASD Referred for pre-surgical evaluation Language skills Visualconstructional reasoning Learning and Memory Working memory Processing speed Attention Inhibition Weaknesses Visual pattern detection and sequencing Strengths Weaknesses Language skills Language-based learning and memory Visual learning Working memory Processing speed Inhibition Strengths Weaknesses Visual memory Language skills Attention Language-based learning and memory Visual memory Cognitive flexibility Speeded naming Inhibition Math Calculation Verbal Fluency Visual learning Attention Processing speed Fluid reasoning Working memory Visual scanning Higher order problem solving Visual-motor integration Table 1: Neurocognitive profile of cases based on strength and weaknesses. 1 Intellectual Disability 74% 6 Speech Impairment and/or Language Delay 85% Gross Motor Delay 50% 4 2 25 13 Fine Motor Delay 18% Results: Unspecified Motor Delay 38% Total N =34 Autistic Features 18% 6 Normal Development 3% Cerebral Palsy 12% 17 Other Movement Disorders 6% 29 Figure 4: Developmental Features in Patients Harboring BCL11B Mutations. Cerebral Palsy Seizures Prominent Forehead Hypertelorism Hyper- or hypotonia Thin Upper Lip Movement Disorders Figure 1: Looking at facial dysmorphology in BCL11B mutations. Long Philtrum Dental Anomalies Pointed Chin Figure 3: Other features seen in BCL11B mutations. AIM 1: Through a review of current literature, 34 patient cases were analyzed, and developed a summary of the phenotypic and neurodevelopmental characteristics associated with BCL11B mutations. Most common phenotypic include: thin upper lip, long philtrum, eyebrow abnormalities, hypertelorism, and dental anomalies. The most common neurodevelopmental features included: intellectual disability, language and speech impairment, motor delays, and autistic features. AIM 2: Case series is the first to provide data on the neurocognitive abilities of 3 full siblings with BCL11B mutations with specific cognitive deficits and functioning. Individuals were referred for different indications and subsequently did not all receive the same assessment measures. All siblings had relative strengths in language skills. Case 1 and 2 had strengths in executive functioning with difficulties in social communication and interaction and restricted, repetitive behaviors. Case 3 had strength in math calculation that was not seen in case 1 or case 2 but overall more weaknesses (e.g. deficits in visual-spatial skills, executive functioning, speeded tasks). Case 3 testing revealed weaknesses in non-dominant hemisphere functions as well as subcortical functions which were not seen in case 1 and case 2. None of the siblings met criteria for intellectual disability vs. literature review. Next Steps: Craniosynostosis Figure 2: Picture of patients with BCL11B Mutations and associated features. PhD, & College of Osteopathic Medicine 2Wake Forest School of Medicine Case 1 Strengths 2 Thibodaux , Further assessment of neurocognitive profiles in individuals with BCL11B mutations with be important to gain a better understanding of the range of cognitive challenges that can be experienced. Case 3, who has the most severe deficits, is being referred for treatment with deep brain stimulation to address motor symptoms. The outcome of this procedure could have a remarkable impact on treatment and therapy options going forward for patients with BCL11B mutations. Citations: Spasticity  ...

... BACKGROUND The COVID-19 pandemic has accelerated the adoption of online learning across various academic disciplines, including medical education. This study aims to investigate how medical students' perceptions of online learning change before and after matriculation. A comparative thematic analysis was conducted at an osteopathic medical school in Indianapolis, IN, to explore the factors influencing these perceptions and the potential impact on students' educational experiences. The findings highlight the effects that one semester of medical school can have on changing the opinions of students regarding online learning. How Medical Students' Perceptions of Online Learning Change After Matriculation An overview of the qualitative process: MATERIALS & METHODS Survey Administration: A qualitative survey was administered to first-year medical students before and after one semester of medical school: Pre-medical school Post 1st semester Reading Responses: Wellbeing as a med student means getting enough sleep, feeling mental clarity, having time to talk and appreciate loved ones/friends, keeping in good physical shape, all well performing well on exams. The incoming first-year class at the Marian University College of Osteopathic Medicine (n=160) was invited to complete a qualitative online questionnaire at their orientation regarding their perceptions of wellness and how their well-being might be impacted by various factors throughout the program. This class is 70% female, 30% male with an age range of 21 to 37, with 81% between ages 21-24. Four responses were filtered out due to participants prior enrollment at a medical school. Six open-ended questions were asked about perceptions and experiences of mental health, well-being, and views on interventions such as non-mandatory lectures. Thematic analysis was conducted across all 156 responses. After completing the first semester five months later, first-years were asked to retake the survey. A code book was created after reading the responses. Then the codes were grouped into themes, and the two survey results were compared between the two time points. Initial Coding: Sleeping Mental health Time with friends and loved ones Physical health Doing well on exams Theming: Physical health Mental health Social Network Academics Thematic Results Question 5 Theme Pre-Medical Students (%) Post 1st Semester Medical Students (%) Control 87.10 75.48 "I love having a choice in the matter" 26.45 "[Online lectures] allow students to be flexible with their schedule" 23.22 "It allows students to save time and get more sleep if they need to one day " Balance Mental Health 40 17.42 Example Student Responses The Survey Questions administered in August and January: 1. What does medical student wellbeing mean to you? Can you give examples? 2. What factors do you think contribute to or affect medical student wellbeing? 3. In your experience, how have medical schools, health services and clinical placement settings changed student and trainee mental health and wellbeing? 4. What has Marian done that has affected your overall wellbeing? 5. What do you think about the idea and impact of medical students being encouraged to attend lecture in person but not required to? 6. Have you heard any positive or negative feedback from others about the way Marian helps with wellness? (Can you name the group (without identifying individuals)? Why do you think they felt this way?) RESULTS Participant responses were coded and grouped into the following eight themes: Spiritual Health, Physical Health, Mental Health, Financial Health, Academic Importance, Peer Support, Balance, and Control. When participants were asked what they think about the idea and impact of medical students being encouraged to attend lecture in person but not required to, 87% of the responses pre-matriculation were linked to Control, encompassing codes such as 'freedom of choice' and 'discipline'. Responses linked to Control remained elevated after matriculation. Balance was the next most mentioned theme from our results, with 40% occurrence rates in the pre-matriculation group. Students mentioned how the option of watching lectures on their own time gives them the flexibility to plan out their day and attend to other personal commitments. Responses related to Balance decreased to 26% after matriculation. Mental health was the third most prevalent theme from the prematriculation responses, with 17% of responses linked to this theme in the pre-matriculation group, and increased to 23% after completing one semester. Students responses mentioned that sometimes life happens. You might have a death in the family or you might get sick to the point that youre missing multiple days of school. With the option of watching lectures from home, students mentioned that this helps with their mental health, especially when theyre having an off day. Academics was also a prevalent theme among responses, encompassing codes such as the opportunity to pace yourself due to being able to pause and rewind lectures and accommodating different learning styles. Medical students looked forward to and ultimately enjoyed having non-mandatory lectures (Question 5). Students appreciated the control they found in having the choice over their schedule. In the responses students appreciated the flexibility of optional lectures and the money potentially saved by decreased travel. However, students did express that by not attending lectures there were missed opportunities to socialize with peers. FUTURE DIRECTIONS Meesam Zaheer, Madeline Schmiedeknecht, John Svendsen, Joseph Asper, Taylor Mihalic, Zachary Montgomery, Wyatt Reed, Asia Hudson, Julia Hum College of Osteopathic Medicine, Marian University, Indianapolis, IN, USA Further define Program Support and investigate what specific programs can help medical students maintain a sense of wellness. Compare first-year results to second years surveyed in the same academic year. Perform in-person interviews with medical students about their wellness to obtain first-person responses with more detail. Further delineate what would improve medical student wellness.  ...

... 9/5/2023 - Open Access Bitter melon extract suppresses metastatic breast cancer cells (MCF-7 cells) growth possibly by hindering glucose uptake Abhinav Kakuturu1*, Heeyun Choi1*, Leah G Noe1, Brianna N Scherer1, Bikram Sharma2, Bilon Khambu3, Bhupal P Bhetwal1 1Division of Biomedical Sciences, Marian University College of Osteopathic Medicine, Marian University - Indiana, Indianapolis, Indiana, United States 2Department of Biology, Ball State University, Muncie, Indiana, United States 3Department of Pathology and Laboratory Medicine, School of Medicine , Tulane University, New Orleans, Louisiana, United States To whom correspondence should be addressed: bpbhetwal@marian.edu *These authors contributed equally. Abstract Breast cancer is one of the most commonly diagnosed cancers among women, however the complete cure for metastatic breast cancer is lacking due to poor prognosis. There has been an increasing trend of dietary modifications including consumption of natural food for the prevention of cancer. One of the popular natural foods is bitter melon. Bitter melon grows in tropical and subtropical areas. Some of the beneficial effects of bitter melon towards disease including cancer have been reported at the whole body/organismal level. However, specific cellular mechanisms by which bitter melon exerts beneficial effects in breast cancer are lacking. In this study, we used a human metastatic breast cancer cell line, MCF-7 cell, to study if bitter melon alters glucose clearance from the culture medium. We co-cultured MCF-7 cells with bitter melon extract in the presence and absence of supplemented insulin and subsequently measured MCF-7 cells viability. In this study, we report a noble finding that bitter melon extract exerts cytotoxic effects on MCF-7 cells possibly via inhibition of glucose uptake. Our findings show that insulin rescues MCF-7 cells from the effects of bitter melon extract. 9/5/2023 - Open Access Figure 1. Insulin rescues human metastatic breast cancer cells (MCF-7 cells) from cytotoxic effects of bitter melon extract (BME): (A) MCF-7 cell viability determined by using Trypan blue assay. Different doses of bitter melon extract (BME) was used [0.5% to 10% (v/v)]. As mentioned in the description section, the bitter melon extract was obtained by grinding whole bitter melon (with seeds in the bitter melon). Cumulative data of average % viable cells (SD) with N=3. Average % significantly different from the no BME control cells (**P < 0.01, ***P < 0.001), one-way ANOVA. (B) Microscopic images of MCF-7 cells (cultured in low glucose DMEM medium) after washing with 1X PBS. (a) control cells with no BME added to the cell culture. (b), (c), (d), (e), and (f) are the MCF-7 cells co-cultured with different doses of BME(v/v %). Note that (f) contains some cell debris from dead cells that were not completely washed during PBS wash. These are not live cells. (g) Cells cocultured with insulin (50 ng/mL). (h) Cells co-cultured with 2% BME and insulin (50ng/mL). (C) Glucose concentration remaining in MCF-7 cell culture media. The cells were cultured in high glucose (450mg/dL) DMEM medium. Glucose remaining in the medium was measured by a glucometer after the 48 hours co-culturing with different doses of BME. The green bar represents cells co-cultured with BME (2%) and insulin (50ng/mL). Cumulative data of average amount of glucose in mg/dL (SD) with N=5. Average mg/dL significantly different (*P < 0.01), one-way ANOVA. (D) Cell viability of MCF-7 cells cultured in high glucose (450mg/dL) DMEM medium . Cell viability was determined by using MTT assay. Cells were co-cultured with 2% BME, insulin (200 ng/mL), or both 2% BME & insulin (50 or 100 or 200 ng/mL). Cumulative data of average % cell viability (SD) with N=3. Average % significantly different (*P < 0.01), one-way ANOVA. NS; Non significant. (E) Cell viability of MCF-7 cells cultured in low glucose (100mg/dL) DMEM medium. Cell viability was determined by using MTT assay. Cells were co-cultured with 2% BME, insulin (200 ng/mL), or both 2% BME & insulin (50 or 100 or 200 ng/mL). Cumulative data of average % cell viability (SD) with N=3., DMEM; Dulbeccos Modified Eagles Medium. 9/5/2023 - Open Access Description According to recent statistics, breast cancer has been one of the most commonly diagnosed cancers among women in the United States (Siegel, Miller, & Jemal, 2019). Numerous risk factors such as alcohol use, obesity, family history, hormonal therapy, diet, and age, etc. have been associated with breast cancer development (Akram, Iqbal, Daniyal, & Khan, 2017; Wood, Cuke, & Bedrosian, 2019). Even though several treatment plans have been developed, complete cure for metastatic breast cancer is lacking due to poor prognosis (Peart, 2017). Although various types of breast cancer cells have been used in research laboratories, MCF-7 cells are perhaps one of the most well studied cell lines (Jiang et al., 2016). The name of MCF-7 is derived from the Michigan Cancer Foundation, which were isolated from a 69-year-old woman with metastasis (Comsa, Cimpean, & Raica, 2015). MCF-7 cells are estrogen receptor (ER)-positive and progesterone receptor (PR)-positive cells with metastatic potential. The metastasis is developed by the secretion of vascular endothelial growth factor (VEGF-A) by MCF-7 cells and the VEGF induces migration and invasion of the cancer cells (Comsa et al., 2015). Using this cell line, researchers have found effective pharmacological targets to treat breast cancer. While there are some treatment options for breast cancer patients, strategies to prevent the disease altogether is the most effective way to deal with it, and one of the ways to prevent cancer is by regulating diet (De Cicco et al., 2019). Throughout the world, people have looked for and consumed natural diets due to health benefits they provide. A popular natural food is bitter melon. Bitter melon grows in tropical and subtropical areas such as Asia, mostly in India and Southeast Asia, Africa, the Caribbean, and California, Florida, and Texas in the United States (Pahlavani et al., 2019; Perez, Jayaprakasha, Crosby, & Patil, 2019; Raina, Kumar, & Agarwal, 2016). There are multiple ways to consume bitter melon: bitter melon juice or tea, salad, and stir-fry, and it has been used traditionally as folk medicine as well (Raina et al., 2016). For this reason, many researchers have paid attention to its health benefits, and they have investigated the effect of bitter melon on various cell functions using in vitro and in vivo study models. Researchers have found that BME inhibits the expression of cell cycle regulatory proteins such as cyclin B1 and D1, blocks G2-M transition of the cell cycle, and thus inducing apoptosis in cancer cells (Cao et al., 2015; Ray, Raychoudhuri, Steele, & Nerurkar, 2010). While anti-proliferative, proapoptotic, and autophagic effects of bitter melon were found in previous studies, these studies did not use the entire fruit of bitter melon when studying the anti-cancer effects of bitter melon on MCF-7 cells. Instead, deseeded bitter melon extract (BME) or any isolated compounds from bitter melon was used (Bai et al., 2016; Cao et al., 2015; Grossmann et al., 2009; Muhammad, Steele, Isbell, Philips, & Ray, 2017; Ray et al., 2010; Weng et al., 2013). In reality, people do not just eat isolated compounds of bitter melon or deseeded fruit; rather, they usually consume the entire fruit of bitter melon. Therefore, it is important to determine the effects of whole bitter melon extract on MCF-7 cells. We investigated the role of bitter melon extract from the entire fruit including seeds in this study. Furthermore, whether the effects of bitter melon on cellular functions is dose dependent has not been well established. We hypothesized that the juice extracted from whole bitter melon fruit (with seeds in the bitter melon) will exert cytotoxic effects on MCF-7 cells, similar to the effects observed from deseeded bitter melon or isolated compounds of bitter melon. To test this hypothesis, we co-cultured MCF-7 cells with increasing doses of BME followed by viability assays and microscopic imaging of cells. MCF-7 cells viability was decreased by BME in a dose dependent manner (Figure 1A). Microscopic imaging of cell culture revealed dose-dependent decline in the number of cells remaining in the culture dish (Figure 1B). The conditioned media from these cultures were separated and glucose concentration was measured in them. The cultures treated with the highest dose of BME that resulted in the least viability of MCF-7 cells (Figure 1A) had the highest concentration of remaining glucose in the conditioned media (Figure 1C). Reciprocally, the cultures treated with the lowest dose of BME that resulted in relatively the highest cell viability (Figure 1A) had the lowest concentration of remaining glucose in the medium (Figure 1C). These observations suggest that BME exerts cytotoxic effects on cells in a dose dependent manner. Furthermore, stronger cytotoxic effects on MCF-7 cells may lead to less number of remaining cells, which in turn likely results into less glucose clearance from the culture medium. Our findings in cancer cells is interesting compared to the effects of BME in glucose clearance by the healthy tissues (skeletal muscles and adipose tissues) reported by others (Ma, Yu, Xiao, & Wang, 2017; Nkambo, Anyama, & Onegi, 2013) (Shih, Lin, Lin, & Wu, 2009) (Roffey, Atwal, Johns, & Kubow, 2007). Both the in vitro and in vivo studies have reported that BME enhances glucose clearance from the extracellular fluid (culture medium or blood) by the healthy skeletal muscle cells or adipose cells. Thus, BME was reported to lower the blood glucose levels by increasing GLUT-4 transporter mediated downstream signaling pathways resulting in enhanced glucose clearance from blood. In the major insulin sensitive healthy tissues, insulin stimulates glucose uptake by enhancing mobilization of endocytosed GLUT-4 transporters to the cell membrane, which then facilitates glucose transport into the cells, a vital biochemical process for the metabolism & viability of mammalian cells. Thus, in our study, we wanted to test if enhancing glucose uptake by the MCF-7 cells would increase their viability under basal or stressed conditions. To the best of our knowledge, effects of altering glucose uptake by the MCF-7 cells on these cells viability have not been investigated. We used insulin to enhance glucose uptake by these cells and test if insulin supplementation will rescue BMEs cytotoxic effects in MCF-7 cell cultures. We 9/5/2023 - Open Access hypothesized that insulin supplementation will rescue BME induced toxicity on MCF-7 cells. To test this hypothesis, we cocultured MCF-7 cells with 2% BME & three different doses of insulin (50, 100, and 200 ng/mL). From the BME dose response experiments, 5% and 10% BME exerted such a strong cytotoxic effects that we observed almost zero viability of the MCF-7 cells in culture (Figure 1B). Thus, we decided to use a middle dose of BME (2%) for our rescue experiments. The higher dose of insulin alone (200 ng/mL), with no BME added, neither inhibited nor activated the cells cultured in high glucose medium (Figure 1D). However, we found that the insulin supplementation rescued the BME-induced cytotoxicity (Figure 1D) most likely by increasing glucose uptake by the cells. Among the three doses of insulin (50, 100, and 200 ng/mL), 50 ng/mL insulin rescued the cell toxicity close to normal viability. To our surprise, compared to 50 ng/mL insulin, higher doses of insulin (100, and 200 ng/mL) did not show a cumulative increase in the rescue effect (Figure 1D). Interestingly, at doses higher than 50 ng/mL, insulin appeared to show decreasing effects (Figure 1D). We do not know why higher doses of insulin become less effective in rescuing the cells from the BMEs cytotoxic effects. One possibility could be that maximal glucose uptake is achieved by the 50 ng/mL insulin. Furthermore, higher doses of insulin may exert suppressive effects on cells-a phenomenon named as insulin toxicity (Kolb, Kempf, Rohling, & Martin, 2020). It appears that when cells are exposed to higher doses of insulin, downregulation of insulin signaling occurs, which is not primarily due to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as a result of receptor dysfunction (Bertacca et al., 2005; Catalano et al., 2014). Bertacca and colleagues have reported that higher dose of insulin downregulates GLUT4 receptor expression on the cell surface (Bertacca et al., 2005). Downregulation of GLUT4 receptor is likely to add stress to the cells as they will not be efficient to take up glucose from the medium. Thus, the cells co-cultured with high insulin dose in a low glucose medium (100 mg/dL glucose compared to high glucose medium with 450 mg/dL glucose) are likely to experience double stress due to downregulation of GLUT4 receptors and low glucose environment. To further evaluate this possibility, we repeated the rescue experiments in low glucose DMEM medium in which glucose concentration is approximately five-fold lower. In the low glucose medium, concentration gradient of glucose between the culture medium and cell cytoplasm decreases. Thus, low glucose medium is likely to result into less glucose influx into the cells, which in turn can induce stress and thereby decrease viability in cells. We found that in contrast to the cells cultured in high glucose DMEM medium, cells cultured in low glucose DMEM medium lost rescue sensitivity to 200 ng/mL insulin. Moreover, higher dose of insulin alone (200 ng/mL), with no BME added, neither inhibited nor activated the cells cultured in low glucose medium (Figure 1E), similar to what we found in high glucose medium culture (Figure 1D). We do not know why 200 ng/mL insulin alone is neither beneficial nor harmful to cells. One of the limitations of this study is that we do not know if the BME (obtained from whole fruit with seeds in it) inhibits glucose uptake by the MCF-7 cells or glucose clearance from the medium is suppressed as a secondary effect of BMEs cytotoxicity on MCF-7 cells. Cancer cells express different subtypes of GLUT transporters (GLUT1, GLUT2, GLUT3, GLUT4 etc.) (Ancey, Contat, & Meylan, 2018). Previous studies have emphasized that ubiquitous GLUT-1 transporter plays major roles in glucose uptake in cancer cells (Shin & Koo, 2021). However, whether relative contributions of GLUT-1 and GLUT-4 transporters are different in MCF-7 cells under healthy versus stressful situations is not known. Our findings suggest that insulin dependent glucose uptake is critical to overcome stressors such as BME induced cytotoxicity. Our findings also suggest that the rescue effect of insulin is dependent on the glucose concentration in the media, particularly at higher doses of insulin (Figure 1D and Figure 1E). Studies have found overexpression of IGF-1 receptor in metastatic breast cancer cells compared to the normal cells (Bartucci, Morelli, Mauro, Ando, & Surmacz, 2001; Cullen et al., 1990; Surmacz, Guvakova, Nolan, Nicosia, & Sciacca, 1998). Furthermore, insulin and IGF-1 ligands can potentially cross stimulate their receptors (Griffeth, Bianda, & Nef, 2014). Therefore, enhanced glucose uptake could also be secondary to the signaling pathways associated with IGF-1 receptor stimulation and not necessarily via the well-established insulin-GLUT-4 pathway. Several of these possibilities could be investigated in the future. Methods Bitter Melon Extract (BME) Preparation Fresh bitter melons were purchased from an Asian grocery store. The whole bitter melon (with seeds in them) were washed and cut into small pieces to fit in the juice extractor (Black & Decker; Product # JE2400BD). The pieces of the entire fruits of bitter melons, seeds included, were used. The juice extract was centrifuged nine times for 15 minutes each at 5000 rpm, and then filter sterilized. BME aliquots were stored at - 80C. Cell culture & BME dose response Equal number of MCF-7 cells (1.0 x 105 viable cells/cm; Thermo Fisher Scientific, catalog # HTB-22) were plated in the 75 cm2 tissue culture treated culture flasks (Thermo Fisher Scientific, catalog # 156499) containing 30 mL medium (Millipore Sigma, DMEM-high glucose, catalog # D6429). The medium was supplemented with 10% Fetal Bovine Serum (Millipore 9/5/2023 - Open Access Sigma, catalog # F2442). Any other appropriate treatments such as BME, insulin etc were added to the medium just before adding the cell inoculum to the complete growth medium. Cells were incubated in an incubator (37C, 5% CO2, 0% O2) for 48 hours. After 48 hours, cultures were washed with 1X phosphate buffered saline (Millipore Sigma, catalog # TMS-012) followed by capturing of pictures using a microscope at the 40x magnification (Nikon, product # 25726). The glucose concentration remaining in the culture medium was also measured. Cell viability was also measured using Trypan Blue Exclusion assay. Glucose measurement After culturing cells for 48 hours, culture media were removed and centrifuged at 2000 rpm for 3 minutes, and then the media were transferred to sterile centrifuge tubes to measure the glucose levels from each tube using a glucometer (BioReactor Sciences, Model # GM100). Trypan blue exclusion assay After MCF-7 cells were cultured with the treatments for the required amount of time, the supernatant was collected from each culture flask. After supernatant was collected from each culture flask, the cells were washed with 1X PBS three times, and they were trypsinized from the bottom of the culture flasks with Trypsin-EDTA (0.25%), phenol red (catalog # 25200056). Trypsinized cells were combined with cells from the supernatant. The cells were resuspended in 1 mL DMEM (Millipore Sigma, DMEM-high glucose, catalog # D6429) and mixed softly with pipette. 20 L cells from the cell suspension were added to a cryovial. Equal parts of 0.4% trypan blue solution (VWR, CAS # 72-57-1) were added to the cryovial containing the cells to obtain a 1 to 1 dilution, and 9 L of the solution was pipetted from the cryovial and loaded into the hemocytometer chamber (Fisher Scientific, catalog # 02-671-55A). Then, cells were counted using a microscope at total magnification of 100X. Both total number of cells and blue-stained cells were counted from the four chamber of the hemocytometer, and number of bluestained cells were subtracted from the total number of cells to give the number of viable cells. After that, the average was taken from the number of viable cells, and then it was multiplied by 10,000 which represented the number of cells per mL and by a dilution factor, thus giving the number of viable cells per mL. MTT; 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay The cells were seeded at a concentration of 0.8 105 cells/mL into a 96-well plate. The cells were incubated (37 C, 5% CO2, 0% O2) with the medium alone or with the appropriate supplements (different doses of BME, different doses of insulin or both insulin and 2% BME) in the medium. After culturing cells for 48 hours, medium was aspirated from each well and 100 L fresh medium containing DMEM and 10% FBS was added to the wells. Then 10 L of MTT reagent (Millipore Sigma, catalog # 11465007001) was added to the wells. The contents of wells in the 96-well plate were mixed using a shaker followed by incubation of the plate (37 C, 5% CO2, 0% O2) for 4 hours. After 4 hours, 100 L of detergent (0.01N HCl with 10% sodium dodecyl sulfate) was added to each well and the plate was incubated in dark at room temperature for four hours. 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Cancer Res 70(5): 1925-31. PubMed ID: 20179194 Roffey BW, Atwal AS, Johns T, Kubow S. 2007. Water extracts from Momordica charantia increase glucose uptake and adiponectin secretion in 3T3-L1 adipose cells. J Ethnopharmacol 112(1): 77-84. PubMed ID: 17363205 Shih CC, Lin CH, Lin WL, Wu JB. 2009. Momordica charantia extract on insulin resistance and the skeletal muscle GLUT4 protein in fructose-fed rats. J Ethnopharmacol 123(1): 82-90. PubMed ID: 19429344 Shin E, Koo JS. 2021. Glucose Metabolism and Glucose Transporters in Breast Cancer. Front Cell Dev Biol 9: 728759. PubMed ID: 34552932 Siegel RL, Miller KD, Jemal A. 2019. Cancer statistics, 2019. CA Cancer J Clin 69(1): 7-34. PubMed ID: 30620402 Surmacz E, Guvakova MA, Nolan MK, Nicosia RF, Sciacca L. 1998. Type I insulin-like growth factor receptor function in breast cancer. Breast Cancer Res Treat 47(3): 255-67. PubMed ID: 9516080 9/5/2023 - Open Access Weng JR, Bai LY, Chiu CF, Hu JL, Chiu SJ, Wu CY. 2013. Cucurbitane Triterpenoid from Momordica charantia Induces Apoptosis and Autophagy in Breast Cancer Cells, in Part, through Peroxisome Proliferator-Activated Receptor Activation. Evid Based Complement Alternat Med 2013: 935675. PubMed ID: 23843889 Wood ME, Cuke M, Bedrosian I. 2019. Prevention Therapy for Breast Cancer: How Can We Do Better? Ann Surg Oncol 26(7): 1970-1972. PubMed ID: 30798445 Funding: This study was supported by the Faculty Research Development grants-Marian University College of Osteopathic Medicine to Dr. Bhupal P. Bhetwal. Author Contributions: Abhinav Kakuturu: data curation, formal analysis, investigation, methodology, writing - original draft. Heeyun Choi: data curation, formal analysis, investigation, methodology, writing - original draft. Leah G Noe: data curation, methodology. Brianna N Scherer: data curation, methodology. Bikram Sharma: formal analysis, writing - review editing. Bilon Khambu: formal analysis, writing - review editing. Bhupal P Bhetwal: conceptualization, investigation, funding acquisition, project administration, supervision, writing - original draft, writing - review editing. Reviewed By: Benjamin Spears History: Received August 17, 2023 Revision Received September 1, 2023 Accepted September 5, 2023 Published Online September 5, 2023 Indexed September 19, 2023 Copyright: 2023 by the authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kakuturu, A; Choi, H; Noe, LG; Scherer, BN; Sharma, B; Khambu, B; Bhetwal, BP (2023). Bitter melon extract suppresses metastatic breast cancer cells (MCF-7 cells) growth possibly by hindering glucose uptake. microPublication Biology. 10.17912/micropub.biology.000961  ...

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... 4.5 5.4 Article The Conservation Genetics of Iris lacustris (Dwarf Lake Iris), a Great Lakes Endemic James Isaac Cohen and Salomon Turgman-Cohen Special Issue Advances in Plant Reproductive Ecology and Conservation Biology Edited by Dr. Brenda Molano-Flores and Dr. James Cohen https://doi.org/10.3390/plants12132557 plants Article The Conservation Genetics of Iris lacustris (Dwarf Lake Iris), a Great Lakes Endemic James Isaac Cohen 1, * and Salomon Turgman-Cohen 2 1 2 * Department of Botany and Plant Ecology, Weber State University, 1415 Edvalson St., Dept. 2504, Ogden, UT 84408-2504, USA E.S. Witchger School of Engineering, Marian University, 3200 Cold Spring Road, Indianapolis, IN 46222-1997, USA; sturgmancohen@marian.edu Correspondence: jamescohen@weber.edu Abstract: Iris lacustris, a northern Great Lakes endemic, is a rare species known from 165 occurrences across Lakes Michigan and Huron in the United States and Canada. Due to multiple factors, including habitat loss, lack of seed dispersal, patterns of reproduction, and forest succession, the species is threatened. Early population genetic studies using isozymes and allozymes recovered no to limited genetic variation within the species. To better explore genetic variation across the geographic range of I. lacustris and to identify units for conservation, we used tunable Genotyping-by-Sequencing (tGBS) with 171 individuals across 24 populations from Michigan and Wisconsin, and because the species is polyploid, we filtered the single nucleotide polymorphism (SNP) matrices using polyRAD to recognize diploid and tetraploid loci. Based on multiple population genetic approaches, we resolved three to four population clusters that are geographically structured across the range of the species. The species migrated from west to east across its geographic range, and minimal genetic exchange has occurred among populations. Four units for conservation are recognized, but nine adaptive units were identified, providing evidence for local adaptation across the geographic range of the species. Population genetic analyses with all, diploid, and tetraploid loci recovered similar results, which suggests that methods may be robust to variation in ploidy level. Keywords: genotyping-by-sequencing; Iris; Lake Huron; Lake Michigan; polyploidy; polyRAD; rare plants; tGBS Citation: Cohen, J.I.; Turgman-Cohen, S. The Conservation Genetics of Iris lacustris (Dwarf Lake Iris), a Great Lakes Endemic. Plants 2023, 12, 2557. 1. Introduction https://doi.org/10.3390/ In 1818, Thomas Nuttall described a new species of crested Iris L., Iris lacustris Nutt., on the gravelly shores of calcareous islands of Lake Huron [1]. Since then, the recognized geographic range of the species has expanded to include the northern regions of Lakes Huron and Michigan in the United States and Canada. Presently, the species is known from 165 occurrences, with more than half in Michigan (89) and the others split between Wisconsin (36) and Ontario (40) [2]. Plants of I. lacustris grow less than 15 cm in height [3], and this feature provides the species with its common name, Dwarf Lake Iris. The species bears self-compatible flowers, with purple sepals and purple petals with yellow and white markings, that are visited by various species of bees [4]. Across its geographic range, I. lacustris frequently inhabits the understory of coniferous forests along the shore, although a small number of inland populations are known (Figure 1) [2,5,6]. These habitats have thin entisols, and the dominant tree species primarily include Thuja occidentalis L., Abies balsamea (L.) Miller, and Picea glauca (Moench) Voss. The species has become a well-known endemic plant of the Great Lakes and is so characteristic of the region that it was recognized as the state wildflower of Michigan [7]. plants12132557 Academic Editor: Hwan Su Yoon Received: 5 May 2023 Revised: 26 May 2023 Accepted: 3 July 2023 Published: 5 July 2023 Copyright: 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Plants 2023, 12, 2557. https://doi.org/10.3390/plants12132557 https://www.mdpi.com/journal/plants Plants 2023, 12, 2557 2 of 17 Figure 1. Map of locations sampled in present study. Dark gray entire lines denote division between East, Mid1, Mid2, and West clusters (also recognized as management units). The dashed gray line separates Mid1 and Mid2 populations, and Mid includes both groups of populations together. Light gray lines separate Wisconsin (USA), Michigan (USA), and Ontario (Canada). Scale bar is 100 km, with each section representing 50 km. In 1988, 170 years after I. lacustris was initially described, the species was listed as federally threatened [5]. The small number of populations and individuals is due to multiple factors, including the loss of shoreline habitat, fungal infection of fruits, lack of seed dispersal, and overgrowth of the forest canopy that restricted plant growth, flower production, and sexual reproduction. Plants of the species currently reproduce more by vegetative growth than germination from the myrmecochorous seeds [5]. Despite this low germination rate, seeds can remain viable in the seedbank for at least 15 years [5], a factor that could influence long-term population growth and genetic diversity, although mass germination and recruitment are rare [4]. The ecology of I. lacustris has been examined to a greater extent than the genetic diversity of the species. To date, only three studies have explored this topic: Simonich and Morgan [8] examined nine populations in Wisconsin, using 22 allozyme markers, Orick [9] investigated nine populations in Michigan, using 24 isozymes, and Hannan and Orick [10] examined nine populations in Michigan, using 18 isozymes. In two studies, researchers identified genetic homogeneity across the populations; however, Orick [9] found overall heterozygosity to be 3.7%. Hannan and Orick [10] also note gene silencing may have been possible in four loci. In contrast to the genetic diversity recognized in I. lacustris, Hannan and Orick [10] found that the sister species, I. cristata Aiton [11], which has a wider geographic range across eastern North America, was variable at 11 of 15 loci. These studies Plants 2023, 12, 2557 3 of 17 suggest that the genetic diversity of this rare species of Iris is quite limited. This genetic paucity is intriguing because I. lacustris and its sister are both putative tetraploids [10], and polyploid plant species tend to have greater genetic variation than diploid relatives, although selfing tends to be higher in polyploids [1214]. Importantly, the genetic diversity of the I. lacustris may have implications for the ability of the species to respond to the changing environment across its geographic range and for various conservation efforts. In order to investigate the population and conservation genetics of the species in a comprehensive manner, we examined multiple populations from across Michigan and Wisconsin, and we used tunable Genotyping-by-Sequencing (tGBS [15]), a method of reduced representation sequencing, to identify single nucleotide polymorphisms (SNPs) among the populations. The objectives of the present study are threefold: (1) identify genetic diversity and population structure and substructure across the range of I. lacustris, (2) explore patterns of migration, and (3) recognize population clusters for management of this rare species. Given the paucity of genetic diversity identified in previous studies, we hypothesized that there would be limited genetic variation across the species. 2. Results 2.1. DNA Sequencing and Polyploid Filtering Among the 171 individuals of 24 populations across the geographic range of I. lacustris in Michigan and Wisconsin (Figure 1, Table 1), 726,786,603 paired-end reads were sequenced, with a mean of 4,225,503 reads per sample. The consensus sequence included 1,335,996 scaffolds with 196,139,854 bp (N50 = 644,994, L50 = 145). The mean per sample alignment and unique alignment to the consensus sequences are 93.9% and 74.4%, respectively. For the MCR90 dataset, 125 reads were interrogated per SNP across 2,341,730 bases, with 4.8% missing data for the final dataset. For the MCR50 dataset, 31 reads were interrogated per SNP across 23,904,409 bases, with 31.4% missing data for the final dataset. The numbers of SNPs in the diploid and tetraploid datasets identified through analysis in polyRAD are in Table 2. Table 1. Population and sampling information and assignation of populations to clusters based on results of various population genomic analyses, including recognition of management and adaptive units, based on loci not under and under selection, respectively. Cluster, management unit, and adaptive unit assignation is based on population genetic analyses with fastStructure, discriminant analysis of principal components (DAPC), principal component analyses (PCA), and others described in the text. Populations Sampled Number of Individuals Sampled Four Population Clusters in Analyses Three Population Clusters in Analyses Management Units (All Loci) Management Units (Diploid and Tetraploid Loci) Adaptive Units MI1 MI2 MI3 MI4 MI5 MI6 MI7 MI8 MI9 MI10 MI11 MI12 MI13 MI14 10 3 8 7 7 14 8 4 3 5 1 2 3 13 East Mid1 Mid1 Mid1 Mid2 West Mid2 Mid2 Mid2 East Mid1 Mid1 Mid2 East East Mid Mid Mid Mid West Mid Mid Mid East Mid Mid Mid East 1 2 2 2 3 4 2 3 2 1 2 2 3 1 1 2 2 2 3 4 2 3 2 3 2 2 3 3 1 2 3 3 4 5 4 4 6 7 3 3 4 7 Plants 2023, 12, 2557 4 of 17 Table 1. Cont. Populations Sampled Number of Individuals Sampled Four Population Clusters in Analyses Three Population Clusters in Analyses Management Units (All Loci) Management Units (Diploid and Tetraploid Loci) Adaptive Units MI15 MI16 MI17 MI18 MI19 MI20 MI21 MI22 WI4 WI5 8 3 10 10 10 3 7 8 12 12 East West Mid2 East Mid2 Mid1 East Mid2 West West East West Mid East Mid Mid East Mid West West 1 4 2 1 2 2 1 3 4 4 1 2 2 1 2 2 3 3 2 2 7 5 6 1 6 2 1 4 8 9 Table 2. Information on six SNP (single nucleotide polymorphism) datasets examined including best K (cluster) value under various analyses. Dashes indicate analysis was not performed for dataset. STRUCTURESELECTOR results include MedMedK, MedMeanK, MaxMedK, and MaxMeanK, and, therefore, may have a range of best K values due to different results from these four metrics. DAPC is discriminant analysis of principal components, and for these analyses, best K value is determined via Bayesian Information Criterion. Additional information on identification of loci under selection and best K values in text. All Loci Loci under Selection Loci Not under Selection Dataset SNPs Loci under Selection STRUCTURESELECTOR DAPC STRUCTURESELECTOR DAPC MCR90 MCR90 diploid loci MCR90 tetraploid loci MCR50 MCR50 diploid loci MCR50 tetraploid loci 5354 2106 1382 344,509 50,134 82,237 401 29 21 65,075 4311 6939 6 45 45 57 34 34 9 7 6 4 23 23 1214 1113 910 8 13 10 7 9 STRUCTURESELECTOR DAPC 34 3 23 3 7 1 1 1 2.2. Population Genomics Across all datasets, observed heterozygosity slightly exceeds expected heterozygosity, and FIS values are, in general, negative (Table 3). Pairwise FST values vary from 0.10.45, and results are similar among datasets (Table 4). Based on various AMOVA results, most of the variation is within samples, followed by between the populations, regardless of the datasets and partitioning of the populations (Supplemental Table S1). Mantel tests for isolation-bydistance analyses identify all datasets as having spatial structure (Supplemental Figure S1) with p < 0.001 for analyses of individuals, but only MCR90 datasets had spatial structure for populations (p < 0.05). Results from analyses in fastSTRUCTURE, STRUCTURE, MavericK, and tess3r are similar. Based on the results from STRUCTURESELECTOR, the optimal K values were greater for all loci analyzed together than for either the diploid or tetraploid loci analyzed independently (Table 2, Supplemental Table S2). Similar clusters were recovered with the different datasets (Figure 2, Table 1), with a clear division between three groupseastern, western, and central populationsand multiple analyses resulted in the central population being divided into two distinct groups at K = 4 and/or 5 (Figure 1, Supplemental Figures S2S4), especially for all loci in fastSTRUCTURE and multiple datasets with STRUCTURE, MavericK, and tess3r. At K = 45, the two Wisconsin populations were often recovered with unique genomic signatures suggestive of admixture, and this is particularly the case with the MCR90 datasets. While the results of conStruct are similar to others, the three distinct groups identified are more opaque, with boundaries between the eastern and western populations overlapping to a larger extent than with the other analyses (Supplemental Figure S5); although, similar patterns can be recognized at K = 4 and 5 for the MCR90 all and diploid loci datasets. Among all Plants 2023, 12, 2557 5 of 17 methods, the three populations on Bois Blanc Island in Michigan (MI5, MI13, and MI22), in the northwestern geographic range of the species, also include some individuals that show signals of admixture between the eastern and central populations (Figure 2). Graphs of K values for all analyses are included in Supplementary Materials (Figures S6S16). Table 3. Observed, expected, and total heterozygosity (HO , HS , HT ) and fixation index (FIS ) for the three MCR90 datasets for each population. Sample sizes are less than five for MI2, MI8, MI9, MI11, MI12, MI13, MI16, MI20, which could impact calculated statistics. MCR90 All Loci HS HT Population HO MI1 0.0586 0.0516 MI2 MI3 MI4 MI5 MI6 MI7 MI8 MI9 MI10 MI11 MI12 MI13 MI14 MI15 MI16 MI17 MI18 MI19 MI20 MI21 MI22 WI4 WI5 0.0503 0.0472 0.0581 0.0558 0.0957 0.054 0.0655 0.0594 0.0612 0.0475 0.0522 0.0557 0.0535 0.0573 0.0961 0.0671 0.0639 0.0651 0.0467 0.0624 0.0543 0.1081 0.1033 0.0411 0.0307 0.0451 0.0532 0.0704 0.049 0.0563 0.0401 0.0477 0.0385 0.0447 0.0488 0.0551 0.0694 0.062 0.0567 0.0575 0.0343 0.054 0.0492 0.0946 0.085 MCR90 Diploid Loci HS HT FIS HO 0.0516 0.1365 0.064 0.0519 0.0411 0.0307 0.0451 0.0532 0.0704 0.049 0.0563 0.0401 0.0477 0.0385 0.0447 0.0488 0.0551 0.0694 0.062 0.0567 0.0575 0.0343 0.054 0.0492 0.0946 0.085 0.2224 0.5394 0.2873 0.047 0.3593 0.1021 0.1631 0.4814 0.2842 0.3578 0.2463 0.0981 0.0395 0.384 0.0827 0.1277 0.1324 0.3597 0.1557 0.1035 0.1424 0.2157 0.0538 0.0521 0.0651 0.052 0.1043 0.0519 0.0677 0.0586 0.0554 0.0527 0.0592 0.0508 0.0542 0.059 0.0956 0.0609 0.0672 0.0645 0.0481 0.0628 0.0464 0.1032 0.1015 0.0417 0.0322 0.0479 0.0428 0.0742 0.042 0.0573 0.0373 0.043 0.0411 0.0409 0.0448 0.0516 0.0661 0.0488 0.0542 0.0512 0.0351 0.0497 0.0382 0.0848 0.0775 MCR90 Tetraploid Loci HS HT FIS HO 0.0519 0.2325 0.0548 0.0462 0.0462 0.1875 FIS 0.0417 0.0322 0.0479 0.0428 0.0742 0.042 0.0573 0.0373 0.043 0.0411 0.0409 0.0448 0.0516 0.0661 0.0488 0.0542 0.0512 0.0351 0.0497 0.0382 0.0848 0.0775 0.2883 0.6191 0.3582 0.2161 0.4064 0.2372 0.1816 0.5714 0.289 0.4413 0.2434 0.2105 0.1434 0.4464 0.2478 0.2401 0.2604 0.3711 0.262 0.2135 0.2179 0.3102 0.0532 0.0474 0.0628 0.051 0.0933 0.0559 0.067 0.0673 0.0515 0.0499 0.0551 0.0543 0.0497 0.0589 0.0795 0.0647 0.0643 0.0591 0.0516 0.0637 0.049 0.0895 0.094 0.0431 0.0301 0.0497 0.041 0.0675 0.0449 0.0555 0.0442 0.0383 0.0433 0.0401 0.0415 0.0502 0.0541 0.0524 0.0534 0.0487 0.0365 0.0512 0.0397 0.0745 0.0734 0.0431 0.0301 0.0497 0.041 0.0675 0.0449 0.0555 0.0442 0.0383 0.0433 0.0401 0.0415 0.0502 0.0541 0.0524 0.0534 0.0487 0.0365 0.0512 0.0397 0.0745 0.0734 0.2329 0.5768 0.2624 0.2444 0.3826 0.2455 0.2074 0.5217 0.3455 0.2749 0.3551 0.1989 0.1724 0.4686 0.2357 0.2054 0.215 0.4123 0.2435 0.2343 0.201 0.2814 Figure 2. Structure bar graphs from fastSTRUCTURE for the six datasets analyzed in the present study for K = 35. Individual ancestry denoted by color. Plants 2023, 12, 2557 6 of 17 Table 4. Pairwise FST values and heatmap for MCR90 all loci (below diagonal) and MCR90 diploid loci (above diagonal). Below the diagonal, red indicates lower values, and blue is for higher values. Above the diagonal, yellow is for lower values, and green is for higher values. Sample sizes are less than five for MI2, MI8, MI9, MI11, MI12, MI13, MI16, MI20, which could impact calculated statistics. MI1 MI2 MI3 MI4 MI5 MI6 MI7 MI8 MI9 MI10 MI11 MI12 MI13 MI14 MI15 MI16 MI17 MI18 MI19 MI20 MI21 MI22 WI4 WI5 MI1 0.29 0.36 0.37 0.26 0.41 0.32 0.31 0.42 0.20 0.30 0.33 0.21 0.15 0.17 0.43 0.28 0.12 0.33 0.29 0.09 0.19 0.27 0.33 MI2 0.17 0.26 0.26 0.29 0.39 0.35 0.38 0.43 0.34 0.24 0.25 0.26 0.31 0.32 0.44 0.25 0.33 0.30 0.01 0.34 0.22 0.26 0.32 MI3 0.20 0.12 0.01 0.30 0.37 0.37 0.41 0.44 0.44 0.05 0.07 0.37 0.38 0.38 0.44 0.22 0.38 0.28 0.29 0.39 0.28 0.23 0.24 MI4 0.21 0.12 0.01 0.31 0.39 0.37 0.40 0.38 0.42 0.07 0.03 0.33 0.39 0.39 0.43 0.25 0.40 0.31 0.25 0.40 0.30 0.27 0.28 MI5 0.16 0.16 0.19 0.19 0.31 0.12 0.07 0.32 0.23 0.21 0.26 0.16 0.24 0.24 0.32 0.17 0.29 0.22 0.27 0.26 0.09 0.22 0.26 MI6 0.25 0.24 0.21 0.24 0.20 0.35 0.34 0.40 0.38 0.31 0.35 0.33 0.40 0.39 0.19 0.32 0.41 0.35 0.36 0.39 0.33 0.30 0.27 MI7 0.18 0.16 0.19 0.18 0.08 0.23 0.14 0.34 0.32 0.29 0.34 0.27 0.30 0.29 0.37 0.21 0.33 0.25 0.33 0.31 0.17 0.28 0.30 MI8 0.16 0.18 0.20 0.19 0.04 0.19 0.08 0.39 0.29 0.30 0.36 0.23 0.28 0.26 0.34 0.22 0.32 0.25 0.38 0.30 0.15 0.24 0.27 MI9 0.22 0.24 0.26 0.20 0.20 0.22 0.19 0.18 0.45 0.38 0.41 0.44 0.42 0.41 0.42 0.18 0.42 0.27 0.45 0.42 0.34 0.27 0.27 MI10 0.14 0.20 0.27 0.25 0.12 0.24 0.19 0.15 0.29 0.38 0.41 0.22 0.04 0.09 0.43 0.27 0.17 0.32 0.35 0.13 0.17 0.22 0.30 MI11 0.12 0.07 0.00 0.08 0.12 0.14 0.11 0.04 0.18 0.19 0.01 0.28 0.33 0.30 0.30 0.13 0.32 0.21 0.24 0.32 0.21 0.12 0.16 MI12 0.17 0.11 0.05 0.03 0.18 0.19 0.18 0.14 0.25 0.25 0.03 0.31 0.36 0.34 0.37 0.20 0.35 0.26 0.28 0.36 0.26 0.19 0.21 MI13 0.13 0.13 0.22 0.19 0.08 0.20 0.16 0.10 0.28 0.13 0.12 0.17 0.18 0.19 0.39 0.21 0.23 0.26 0.29 0.21 0.04 0.20 0.26 MI14 0.11 0.16 0.21 0.22 0.12 0.24 0.15 0.14 0.24 0.03 0.14 0.19 0.09 0.04 0.42 0.27 0.14 0.31 0.31 0.09 0.15 0.24 0.32 MI15 0.09 0.15 0.20 0.20 0.12 0.23 0.15 0.12 0.21 0.06 0.10 0.15 0.09 0.03 0.39 0.26 0.15 0.30 0.31 0.13 0.17 0.23 0.30 MI16 0.26 0.27 0.28 0.26 0.23 0.13 0.25 0.20 0.25 0.28 0.13 0.21 0.25 0.26 0.23 0.28 0.41 0.34 0.42 0.41 0.36 0.25 0.21 MI17 0.18 0.16 0.13 0.14 0.12 0.20 0.12 0.11 0.10 0.20 0.05 0.13 0.15 0.18 0.16 0.20 0.29 0.11 0.22 0.27 0.16 0.20 0.23 MI18 0.05 0.19 0.22 0.23 0.17 0.25 0.20 0.17 0.24 0.13 0.14 0.18 0.14 0.10 0.09 0.25 0.20 0.33 0.32 0.05 0.22 0.25 0.33 MI19 0.20 0.17 0.14 0.15 0.14 0.20 0.14 0.13 0.12 0.21 0.06 0.13 0.16 0.19 0.18 0.20 0.07 0.21 0.28 0.32 0.21 0.25 0.27 MI20 0.16 0.00 0.14 0.10 0.16 0.21 0.14 0.16 0.25 0.21 0.07 0.14 0.15 0.15 0.13 0.26 0.14 0.18 0.15 0.33 0.21 0.21 0.28 MI21 0.05 0.19 0.22 0.23 0.16 0.24 0.18 0.16 0.25 0.09 0.14 0.20 0.14 0.07 0.08 0.26 0.18 0.04 0.20 0.18 0.18 0.24 0.31 MI22 0.15 0.11 0.18 0.18 0.06 0.22 0.11 0.09 0.24 0.12 0.12 0.18 0.02 0.09 0.11 0.27 0.13 0.16 0.15 0.10 0.14 0.22 0.26 WI4 0.15 0.16 0.13 0.16 0.14 0.19 0.15 0.12 0.13 0.14 0.02 0.09 0.12 0.14 0.12 0.14 0.13 0.15 0.15 0.12 0.14 0.15 0.24 WI5 0.19 0.20 0.16 0.19 0.15 0.20 0.18 0.15 0.14 0.19 0.07 0.13 0.15 0.19 0.17 0.14 0.16 0.21 0.17 0.16 0.19 0.18 0.15 - Plants 2023, 12, 2557 7 of 17 The results of principal components analysis (PCA) and discriminant analyses of principal components (DAPC) are similar to those that explicitly consider a priori population structure. With PCA, three to four clusters were recovered corresponding to the same ones from the population assignation analyses, and this was more evident with the MCR90 datasets compared to the MCR50 ones. In all analyses, three populationsMI6, MI16, and WI5were recognized as most distinct from the other populations. Across DAPC analyses, individuals from populations tended to cluster together, and this is similar to results from other methods. In general, DAPC analyses recover MI6, MI16, and WI5 as distinct units or as a cluster together, with the results for MCR50 all loci being the only exception. In analyses with this dataset, WI5 was included in a cluster distinct from the other two populations, but with WI4 and populations from Michigan. In some analyses, such as MCR50 and MCR90 diploid loci, the divided cluster of central populations was identified. The number of loci under selection in each dataset is in Table 2. Patterns of migration inferred from BA3-SNPs suggest that migration is minimal, regardless of the dataset analyzed, and that most individuals are from their original population (Figure 3). While this was certainly the case for all loci for MCR90, analyses with only the diploid loci for three or four population clusters (Table 1) provide evidence of greater rates of migration between adjacent populations (Figure 3). Migration directly between the eastern and western populations was negligible. The relationship among the four population clusters that was most supported by the results of DIYABC-RF and abcranger varies depending on the dataset analyzed. For all, diploid, and tetraploid loci, (West (Mid1 (Mid2, East))), (West (East (Mid1, Mid2))), and (West (Mid1 (Mid2, East))) are recovered as optimal, respectively, and (Mid2 (West (Mid2, East))) and (West (East (Mid1, Mid2))) are identified as close second choices for all and tetraploid datasets, respectively. The one constant among the three optimal trees is that the western population is recognized to have diverged prior to the mid and eastern populations, and this also is the case for one of the near-optimal trees (Supplemental Figure S17). Figure 3. Patterns of migration based on MCR90 all loci (AC) and MCR90 diploid loci (D,E) as tt resolved using BA3-SNPs. (A) All populations, (B,D) 4 populations, (C,E) 3 populations. Outermost circle denotes each population, and inner circle shows origin of migrants from each population. Lines connecting populations demonstrate patterns of migration. tt tt tt Plants 2023, 12, 2557 8 of 17 2.3. Conservation Units Based on the method of Funk et al. [16], evolutionarily significant units (ESUs) were identified using all loci, as described below, and the management units (MUs), which are based on fastSTRUCTURE, PCA, and DAPC analyses with loci not under selection, are quite similar. The largest difference between ESUs and MUs is that the two populations in Wisconsin may or may not be included with the other two western populations, MI6 and MI16, depending on the use of all loci or only diploid or tetraploid loci (Figure 4). The populations on Bois Blanc Island also have mixed ancestry based on these loci. The adaptive units, which are based on fastSTRUCTURE, PCA, and DAPC analyses with loci under selection, provide quite different results. Generally, among analyses, nine adaptive units are recognized, and these are structured based on geography (Figure 4, Supplemental Figure S18, Table 1). Figure 4. Structure bar graphs for MCR90 all loci and three MCR50 datasets for loci under and not under selection (adaptive units and management units, respectively). Individual ancestry denoted by color. Groups for each listed in Table 1, and best K values noted in Table 2. The results of the fastSTRUCTURE, PCA, and DAPC are similar, with one exception. Unlike analyses with fastSTRUCTURE and PCA, where individuals of the same population cluster together, with DAPC, some individuals of the same population are members of different clusters. This is likely due to the large number of clusters identified as optimal, which is particularly the case for MCR90 and MCR50 datasets with all loci. 3. Discussion 3.1. Population Structure and Genetic Diversity Based on the multiple datasets explored using various methodological approaches, three or four different population clusters were frequently recognized for I. lacustris across Michigan and Wisconsin. These clusters are structured geographically, with eastern, central, and western groups, and at higher K values, the central group is subdivided into two groups that are also geographically oriented (Figures 1 and 2). In the three prior studies that employed isozymes and allozymes to examine the population genetics of I. lacustris [810], no to limited genetic diversity was identified in the populations. Each study only investigated the genetic diversity of populations within one state, using markers available at the time, which likely led to the paucity of genetic diversity. In the present study, many more loci were examined, and individuals from across most of the geographic range of the species were analyzed together, which provides a more holistic approach to elucidating the genetic diversity of the species. These results demonstrate that our hypothesisa lack of genetic diversity among the specieswas incorrect. Across all studied populations, statistically significant isolation-by-distance is noted, and much of the genetic variation occurs within samples and among populations, with little variation within each population. These results are, on some level, unsurprising for a species that is not only clonal but also includes minimal sexual reproduction. Sampling issues, such as small numbers of individuals studied for some populations and potential Plants 2023, 12, 2557 9 of 17 collection of ramets, could also have contributed to limited within-population genetic diversity. Additionally, almost all populations have negative FIS values, a finding frequently occurring with clonal plants [17]. A similar result was recovered by Edgeloe et al. [18] for another clonal, polyploid species, Posidonia australis Hook.f. Despite the clonal growth in these polyploid species, the multiple gene copies may provide sufficient genetic diversity and potential so that rare species, such as I. lacustris, do not suffer the negative long-term impacts of vegetative reproduction and inbreeding. The changing climate will certainly be a test as to whether the genetic diversity harbored in each population will be appropriate to adapt to new conditions [19]. Among the identified clusters of populations, there are two notable areas: Bois Blanc Island in the eastern part of the sampled range and the four western populations. In Bois Blanc Island, the populations display mixed ancestry between the eastern and central populations, and these were results recovered with multiple datasets and analyses. This mixed ancestry could occur because of hybridization on the island itself with ancestors from both populations colonizing and interbreeding there. Alternatively, hybridization could have taken place on the mainland of the lower peninsula of Michigan, such as at MI7 or MI8, followed by colonization of the island. While the signature of mixed ancestry identified in the present study may suggest that hybridization is recent, given that the species reproduces clonally, the signature of (older) hybridization could remain for an extended period of time. It is useful to keep in mind that the island and nearby areas on the mainland are some of the more heavily sampled geographic regions in the present study. This greater sampling could hint at a similar pattern in other areas if individuals were sampled to a larger extent. It was not possible to include representatives from Ontario, Canada in the study, and future studies that add these will likely have greater context for the relationship of the central and eastern populations to those even farther east. The four populations in the western cluster (MI6, MI16, WI4, and WI5) are notable. While these populations form a cluster in most analyses (Figure 2), the two Wisconsin populations (WI4 and 5) differ from those in Michigan, and, in some analyses, from each other. While WI4 and WI5 are geographically close together on the Door Peninsula and tend to cluster together in some analyses, WI4 is sometimes resolved as sharing ancestry with the eastern populations, which is not the case for WI5. This could be due to the retention of ancestral polymorphism or the fact that the establishment of each of these populations differs. However, in analyses that account for both genetic and geographic data (i.e., tess3r and conStruct), both Wisconsin populations are distinct clusters and/or are usually allied with the other western populations. This is particularly the case for the diploid dataset. In another, well-known Great Lakes shoreline endemic, Cirsium pitcheri Torr. & A.Gray, a similar pattern was recovered. The populations from the Door Peninsula are also quite distinct from others on Lake Michigan [20], and the northern populations on the peninsula share more alleles with the populations in the Upper Peninsula of Michigan than with some of the populations on the southern part of the peninsula. MI6 and MI16 are intriguing populations of I. lacustris because they are situated inland, and this is not the case for the other sampled populations. While other populations can be found a short distance from the shoreline, these populations are ca. 30 km from the current boundary of Lake Michigan. These two populations are consistently recognized as genetically distinct from the other sampled populations, and these both likely became established during higher water level periods of Glacial Lake Algonquin ca. 12,500 years ago [21,22]. As water levels decreased during the time of Glacial Lake Chippewa and subsequently rose to current levels, these two populations became isolated in suitable habitat (e.g., conifer wetland) that allowed individuals of I. lacustris to persist, but without the opportunity to interbreed with other, coastal populations, resulting in their distinct genetic signature (Figure 2). Plants 2023, 12, 2557 10 of 17 3.2. Migration and Demography After deglaciation, I. lacustris migrated eastward from the western part of its range. This pattern provides evidence that MI6 and MI16 became established early in the colonization of the species during times of higher water levels and, therefore, are relicts rather than the result of inland dispersal. Additionally, the central and then eastern populations developed via migration across northern Lakes Michigan and Huron, and these populations may have retained some of the ancestral polymorphisms in the more western populations, such as WI4 and WI5. This west-to-east pattern suggests that the populations in Ontario are the most recently established, a hypothesis that can be tested during a future study. The pattern noted here for I. lacustris differs from that of C. pitcheri, which is hypothesized to have migrated from east to west [20]. Overall, rates of migration, as inferred with BA3-SNPs, among populations are minimal, a result recovered in other species of Iris on the Korean Peninsula [23] and a pattern that is not uncommon for narrow endemics [20]. This minimal migration is the case for all 24 populations studied as well as with three and four population clusters inferred (Figure 3). Although the species presently reproduces within populations, migration occurred and may have provided an infusion of new alleles, even if this was not a common occurrence. In C. pitcheri, Fant et al. [20] note that the changes in the water level of the Great Lakes shaped the geographic distribution of this endemic species, with lower water levels allowing for increased connection among populations. Lake level changes could also have impacted the geographic distribution of I. lacustris. This is particularly the case for the more inland populations, which could have become established ca. 4500 years ago during the most recent high water levels for the lake. Lower lake levels may have influenced colonization of the islands as well as migration across the northern regions of Lake Michigan and allowed for the exchange of individuals that currently would be more challenging. An alternative hypothesis for the present geographic distribution of the species also exists. Van Kley and Wujek [6] and Brotske [4] provide evidence that I. lacustris can inhabit a diversity of ecosystems and that changes in patterns of disturbance and forest succession following European colonization of the area reduced the suitable habitat for the species (e.g., more forests with more closed canopies). This has resulted in populations primarily being restricted to shorelines where habitat was appropriate. If this is the case, the inland populations, such as MI6, would still represent relicts of a prior time, but this would be due to remnant habitat availability based on adequate disturbance regimes and/or seral stages, not prior establishment during higher water levels of the Great Lakes and subsequent serendipitous survival. 3.3. Subsetting Diploid and Tetraploid Loci In the present study, polyRAD [24] was used to create datasets of diploid and tetraploid loci, and these were analyzed alongside a dataset of all loci for the MCR90 and MCR50 datasets. In general, analyses of all six datasets produced fairly similar results (Figure 2, Tables 3 and 4). fastSTRUCTURE analyses of MCR90 and MCR50 datasets of all loci resulted in the identification of a cluster of six populations in the central part of the sampled population of I. lacustris (MI2, MI3, MI4, MI11, MI12, and MI20) that was not recovered with the diploid or tetraploid datasets, although hints of this cluster can be seen in the MCR90 2N dataset at K = 5. This cluster is identified in all of the datasets with loci under selection as either one or two clusters (Figure 2) and with the MCR90 datasets analyzed with STRUCTURE [25] and MavericK [26]. The similar results among the datasets, regardless of ploidy, may provide some evidence that not disentangling diploid and tetraploid loci from all loci may not lead to spurious results using SNP data for population genomics [27]. This statement should be treated with skepticism because it is based only on one, empirical, study. Others who have used polyRAD to subset their datasets and identify diploid loci to use for population genomics [28,29], which is a practice aligned with assumptions of common methods [28], have not explored the use of all loci and/or tetraploid loci in comparison to only ones that Plants 2023, 12, 2557 11 of 17 segregate as diploids. It would be useful for additional studies on the population genomics of polyploid species to examine data employing all, diploid, and tetraploid (and higher) loci to determine if similar or divergent results are recovered. At the same time, the results presented herein may provide some level of confidence for researchers investigating the population genomics of species of unknown ploidy that use all loci identified via tGBS, and similar reduced-representation methods may not yield incongruent results. 3.4. Conservation Genetics of I. lacustris The evolutionarily significant units (ESUs) were described above with all loci used for population genomic analyses, and the management units (MUs), which were determined using only loci not under selection, are similar, but not identical to the ESUs; however, the differences are minor (Figure 4). Given the similar ESUs and MUs, the management of the populations of I. lacustris could be geographically clustered into three to four units. However, the results of the use of the loci under selection to resolve adaptive units (AUs) differ from those of ESUs and MUs (Supplemental Figure S18). The AUs provide evidence of local adaptation, so managing only three or four MUs would not necessarily ensure that all of the genetic diversity of the species is appropriately protected. A total of nine AUs are recognized (Table 1), and while these are also geographically clustered, the AUs are much smaller than are the ESUs and MUs (Figure 4). This local adaptation is, on some level, unsurprising, because even though the species is generally restricted to the same type of habitat presently (i.e., shorelines), climatic, soil, and vegetation differences occur across the geographic range of the species. Indeed, I. lacustris inhabits three of the landscape ecology regions of Michigan and multiple districts and subdistricts within each region [30,31]. Van Kley and Wujek [6] also recognized four soil types, four vegetation types, and pH variation across the species range. Given that the species primarily reproduces asexually, this can lead to a loss of genetic variation over time as a limited number of successful genotypes dominates each particular climatesoilvegetation combination. Consequently, the seemingly same type of habitat in a geographically distinct area may result in local adaptations to the specific region and ecosystem and contribute to outbreeding depression, limiting successful offspring from infrequent interpopulation crosses. 4. Materials and Methods 4.1. Plant Material During the summers of 2019 and 2020, leaf material of 171 individuals of I. lacustris was collected from 24 locations in Michigan and Wisconsin (Figure 1) and dried in silica gel. The number of individuals per population ranged from 1 to 12, depending on the suitability of the population for collection. Most individual plants were collected at least 3 m from each other to maximize the possibility of sampling genets, not ramets. Latitude and longitude were recorded for each specimen. 4.2. DNA Sequencing Leaf material was sent to data2bio (www.data2bio.com, accessed on 1 May 2023) for DNA isolation and tunable Genotyping-by-Sequencing (tGBS) to recognize single nucleotide polymorphisms (SNPs) across the populations. Using the restriction enzyme Bsp1286I, paired-end tGBS libraries were created [15] and subsequently sequenced with an Illumina HiSeq X (Illumina Inc., San Diego, CA, USA). Based on all sequence data, consensus reference sequences were generated with CD-HIT-454 [32] after sequencing depth was normalized to 50, and sequencing errors were corrected using Fiona [33]. Lowquality reads were discarded (PHRED quality < 15 and error rates 3%) and trimmed, and GSNAP [34] was employed to map reads to the reference sequences based on the following parameters: 2 mismatches per 36 bp and less than five total per 75 bp for tails. SNPs were identified based on the following criteria: two most common alleles supported by at least 30% of the aligned bases, at least five unique reads, the sum of the one or two most Plants 2023, 12, 2557 12 of 17 common alleles covering at least 80% of the aligned reads, and no polymorphisms in the first or last three base pairs of each read. From the SNPs, two datasets were created: MCR90 with up to 10% missing data and MCR50 with up to 50% missing data. 4.3. Polyploidy Filtering Because I. lacustris is a putative polyploid and many population genetic methods assume that species are (at most) diploid, polyRAD [24] was used to identify and filter loci that are diploid and tetraploid. The MCR90 and MCR50 datasets were filtered using the IteratePopStruct command to identify genotypes, and then the Hind /HE statistic [24,35] was employed to recognize diploid loci with Hind /HE < 0.5 and tetraploid loci with Hind /HE > 0.75. Datasets were created for each set of loci (Table 2). The number of SNPs in the diploid and tetraploid datasets does not equal the value in the initial datasets because of filtering with polyRAD. 4.4. Population Genomics Observed and expected heterozygosity measurements and F-statistics were calculated with hierfstat [36,37], and AMOVA was conducted with poppr [38]. All 24 populations were examined, as were the populations divided into three and four geographic clusters, which are based on the optimal K values from preliminary analyses in fastSTRUCTURE (Table 2) and patterns of population structure from STRUCTURE and MavericK. fastSTRUCTURE [39] was employed to identify population structure, including the optimal number of clusters (K), and for these analyses, K = 124 were analyzed for the six SNP datasets, using Structure_threader [40], on the Kettering University High-Performance Computing Cluster (KUHPC). Ten replicates were run for each K, with a convergence criterion of 0.000001, a simple prior, and 100 test sets for cross-validation. The CLUMPAK main pipeline, which includes CLUMPP [41] and DISTRUCT [42], was employed to organize, cluster, and visualize the results of independent fastSTRUCTURE analyses, via 10,000 permutations of the LargeKGreedy algorithm [43]. To identify the optimal K value(s), the marginal likelihood that maximizes model complexity from fastSTRUCTURE and the MedMedK, MedMeanK, MaxMedK, and MaxMeanK values determined by STRUCTURESELECTOR [44,45] were examined. These latter four metrics are useful for uneven sampling and are based on recognizing the number of clusters that include, at minimum, one subpopulation. Differences among these metrics are the result of the arithmetic mean or median used and the median or maximum number of clusters identified [45]. For comparison, and given potential variation in ploidy at loci [27], STRUCTURE [25] and MavericK [26] were also used, with Structure_threader, for analyses with the three MCR90 datasets. With STRUCTURE, the following parameters were used with K = 124: 1,000,000 steps and 500,000 burnin, with alpha and lambda of 1, and with or without admixture. Ten replicates were run for each K. CLUMPAK and STRUCTURESELECTOR were also used for STRUCTURE analyses, with the best K also determined via the method of Evanno et al. [46] and Ln Pr (X|K). MavericK analyses were run for K = 112 with five replicates per K, without admixture, using the following parameters for each replicate: 50,000 steps and 5000 burnin for Markov Chain Monte Carlo (MCMC) sampling and an alpha of 1500 steps and 5000 burnin, with 50 rungs, for thermodynamic integration (TI) sampling, and 100 expectation-maximization repeats. With MavericK, graphs were visualized with R [47], and the optimal K value was determined using TI. To explicitly include geographical data along with SNPs to investigate patterns of population genetics, tess3r [48] and conStruct [49] were used, and all datasets were analyzed with the former, but only the three MCR90 datasets with the latter. For tess3r, the alternating projected least squares method was undertaken for K = 124 for MCR90 and K = 112 for MCR50 datasets. Results for each K were visualized with bar graphs and maps in R [47], and the optimal K value was identified using the cross-validation plot for each dataset. For conStruct cross-validation, analyses were conducted with five replicates, for K = 18, using 10,000 MCMC iterations sampled every 1000 iterations and a training proportion of 0.50.8, Plants 2023, 12, 2557 13 of 17 depending on the dataset. Subsequently, analyses with K = 35 were conducted, with five replicates, using one chain run for 100,000 MCMC iterations sampled every 1000 iterations and with the spatial model. In addition to analyses for explicit population structure, all datasets were analyzed with principal component analyses (PCA), correspondence analyses (CA), and discriminant analyses of principal components (DAPC) in adegenet [50], principal coordinate analyses (PCoA) in hierfstat [36,37], and isolation-by-distance (IBD) analyses in adegenet using separate Mantel tests for population and individuals, with 999 simulations for the Mantel test. For DAPC for each dataset, the Bayesian Information Criterion (BIC) was used to identify the optimal number of clusters, and cross-validation was employed to explore the most appropriate number of PCs to retain for analysis. Loci under selection were determined with BayeScan [51] using 100,000 iterations, a burnin of 50,000 iterations, a thinning interval of 10, and a sample size of 5000, and for each analysis, 20 pilot runs were conducted, each with 5000 steps. Loci under selection were visualized in R using FST values and a false discovery rate of 0.05. Demographic history and patterns of migration were explored using BA3-SNPs [52,53], DIYABC Random Forest (DIYABC-RF) [54], and abcranger [55], and only the three MCR90 datasets were used for these analyses, with the three and four aforementioned population clusters used (apart from all 24 populations investigated with MCR90 with BA3-SNPs). For BA3-SNPs, the datasets were each run for 50 million Markov Chain Monte Carlo (MCMC) iterations, with 20 million MCMC burnin iterations, and a sampling interval of 2500 iterations, and the initial parameters for allele frequencies, inbreeding coefficient, and migration rates were tuned to vary between 0.20.6. For DIYABC-RF, the optimal scenario for patterns of diversification were examined among all 15 arrangements of four bifurcating populations. For each scenario, population size was modelled to vary after populations split and one and two other times for when the second and first populations diverge (Supplemental Figure S17). For analyses, all genetic diversity, FST distances, Neis distances, and admixture estimates were selected, and the analyses were run for 15 million simulations with a batch size of 1000. Using the results of the training, a random forest analysis was conducted with abcranger [55] using 1000 trees to identify the number of trees supporting each model and to estimate the parameters of the model, with and without linear discriminant analysis, for partial least squares (PLS) estimation on the optimal model for each dataset. 4.5. Conservation Units Conservation and management units were identified following the three-step method of Funk et al. [16], in which (1) evolutionarily significant units (ESUs) are recognized using all loci, (2) management units (MUs) are delimited with non-outlier loci, and (3) adaptive groups are determined using outlier loci. For the three steps, fastSTRUCTURE [39], PCA, and DAPC were used [50]. The first step was described above for datasets with all loci, and the other two steps were conducted using the same parameters for the three analyses and were based on two datasets (loci under and not under selection as determined via BayeScan [51]) for each MCR50 dataset and the all loci dataset of MCR90 (Table 2). The optimal K value was identified using STRUCTURESELECTOR [44], the marginal likelihood that maximizes model complexity from fastSTRUCTURE [39], and the BIC for DAPC with adegenet [50]. Based on the results of these analyses, ESUs, MUs, and adaptive groups were identified (Supplemental Figure S18). 5. Conclusions The present study provides evidence of genomic variation and local adaptation across the geographic range of the species, which is novel given the negligible genetic diversity previously recovered for I. lacustris [810]. However, as Van Kley and Wujek [6] stated thirty years ago, Despite a preference for a somewhat disturbed habitat, Iris lacustris will not grow where the habitat has been destroyed by residential, resort, or industrial development. Plants 2023, 12, 2557 14 of 17 Therefore, the conservation genetic results are of limited value if management steps are not taken to ensure that individuals of I. lacustris have the opportunity to be successful in situ. This includes not only ensuring intermediate light conditions and limited litter [5,6], but also that as much genetic diversity across the entire geographic range of the species is conserved and managed appropriately. Indeed, given the local genetic diversity recognized among the nine adaptive units, it would be prudent to strive to conserve representatives from these areas. This is particularly important because the populations that are best able to adapt to the changing climate in the Great Lakes region is presently unknown [56]. Therefore, to ensure the longevity of this charismatic species, appropriate long-term management is necessary. Future work that includes the populations of I. lacustris from Ontario can extend the presented results to investigate the ways in which these populations relate to those in the United States. Given the international geographic range of the species, conservation efforts that are binational would be particularly useful. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12132557/s1, Figure S1. Results for Isolation-by-Distance (IBD) for the six datasets. The x-axis is geographic distance, and the y-axis is genetic distance. Figure S2. Structure bar graphs from STRUCTURE for the six datasets analyzed in the present study for K (clusters) = 35. Individual ancestry denoted by color. Populations are denoted below each graph. Figure S3. Structure bar graphs from MavericK, without admixture, for the three MCR90 datasets analyzed in the present study for K (clusters) = 35. Individual ancestry denoted by color. Populations are denoted below each graph. Figure S4. tess3r maps of population assignation for the six datasets analyzed in the present study for K (clusters) = 35. Individual ancestry denoted by color. Figure S5. Maps and bar graphs of population assignation for the three MCR90 datasets analyzed in the present study for K (clusters) = 35. Individual ancestry denoted by color. Figure S6. Results for best K from StructureSelector for analyses with fastStructure for (A) MCR90 all loci, (B) MCR90 diploid loci, and (C) MCR90 tetraploid loci. Figure S7. Results for best K from StructureSelector for analyses with fastStructure for (A) MCR50 all loci, (B) MCR50 diploid loci, and (C) MCR50 tetraploid loci. Figure S8. Results for best K from StructureSelector for analyses with Structure without admixture for (A) MCR90 all loci, (B) MCR90 diploid loci, and (C) MCR90 tetraploid loci. Figure S9. Results for best K from StructureSelector for analyses with Structure with admixture for (A) MCR90 all loci, (B) MCR90 diploid loci, and (C) MCR90 tetraploid loci. Figure S10. Results for best K from MavericK, based on thermodynamic integration (TI), for analyses without admixture (A) MCR90 all loci, (B) MCR90 diploid loci, and (C) MCR90 tetraploid loci. Figure S11. Results for cross-validation scores for tess3r analyses for (A) MCR90 all loci, (B) MCR90 diploid loci, (C) MCR90 tetraploid loci, (D), MCR50 all loci, (E) MCR50 diploid loci, and (F) MCR50 tetraploid loci. Figure S12. Results for cross-validation scores for conStruct validation analyses for (A) MCR90 all loci, (B) MCR90 diploid loci, and (C) MCR90 tetraploid loci to identify best K (clusters). Graphs with blue and green dots are for spatial and non-spatial models, respectively, and graph with only blue dots displays predictive accuracy for spatial model with confidence intervals. Figure S13. Results for Bayesian Information Criterion (BIC), to identify best K (clusters), from discriminant analysis of principal components (DAPC) for (A) MCR90 all loci, (B) MCR90 diploid loci, (C) MCR90 tetraploid loci, (D), MCR50 all loci, (E) MCR50 diploid loci, and (F) MCR50 tetraploid loci. Figure S14. Results from StructureSelector for best K (clusters) fastStructure analyses for loci under selection for (A) MCR90 all loci, (B) MCR50 all loci, (C) MCR50 diploid loci, and (D) MCR50 tetraploid loci. Figure S15. Results from StructureSelector for best K (clusters) fastStructure analyses for loci not under selection for (A) MCR90 all loci, (B) MCR50 all loci, (C) MCR50 diploid loci, and (D) MCR50 tetraploid loci. Figure S16. Results for Bayesian Information Criterion (BIC), to identify best K (clusters), from discriminant analysis of principal components (DAPC) analyses for (A) MCR90 all loci under selection, (B) MCR50 all loci under selection, (C) MCR50 diploid loci under selection, (D), MCR50 tetraploid loci under selection, (E) MCR90 all loci not under selection, (F) MCR50 all loci not under selection, (G) MCR50 diploid loci not under selection, (H), MCR50 tetraploid loci not under selection. Figure S17. 15 branching scenarios evaluated in DIYABC. Pop 1 is East, Pop 2 is Mid 1, Pop 3 is Mid 2, Pop 4 is West. See Table 1 for population assignation to each population. Change in color represents potential change in population size. Scenario 3 is optimal for all and tetraploid loci, and scenario 7 is optimal for diploid loci. Figure S18. Nine Adaptive Units recognized from population genetic Plants 2023, 12, 2557 15 of 17 analyses using loci under selection. Map of locations sampled in present study. Dark gray entire lines denote division between East, Mid1, Mid2, and West clusters (also recognized as Management Units). The dashed gray line separates Mid1 and Mid2 populations, and Mid includes both groups of populations together. Light gray lines separate Wisconsin (USA), Michigan (USA), and Ontario (Canada). Scale bar, in red, represents 50 kilometers. Table S1. AMOVA results for all datasets. Table S2. K values for the MCR90 datasets for STRUCTURE and Maverick. Author Contributions: Conceptualization, J.I.C. and S.T.-C.; methodology, J.I.C. and S.T.-C.; formal analysis, J.I.C.; data curation, J.I.C.; writingoriginal draft preparation, J.I.C.; writingreview and editing, J.I.C. and S.T.-C.; visualization, J.I.C. and S.T.-C.; funding acquisition, J.I.C. and S.T.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a Kettering University Faculty Research Fellowship, the Michigan Natural Features Inventory (MNFI), and Weber State University. The APC was funded by MDPI. Data Availability Statement: Data files in VCF format are available at the Dryad repository (https: //doi.org/10.5061/dryad.xwdbrv1jh). Acknowledgments: The authors thank R. Hackett who collected the vast majority of the leaf material, and R. Bowman collected leaf material from Wisconsin. R. Hackett, P. Higman, C. Tansy, J. Dingledine, and S. Hicks provided invaluable conversation about the Dwarf Lake Iris. R. Hacketts comments on a draft of the manuscript were very helpful. Three reviewers provided helpful comments to improve the manuscript. We appreciate L. Coffey and data2bio for tGBS sequencing. L. Clark provided helpful comments on using polyRAD. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Nuttall, T. The Genera of North American Plants: And a Catalogue of the Species, to the Year 1817; D. Heartt: Philadelphia, PA, USA, 1817; Volume 1. U.S. Fish and Wildlife Service. Status ReviewDwarf Lake Iris (Iris lacustris); East Lansing Field Office: East Lansing, MI, USA, 2022; p. 10. Voss, E.G. Michigan Flora, 3rd ed.; Cranbrook Institute of Science: Bloomfield Hills, MI, USA; University of Michigan Herbarium: Ann Arbor, MI, USA, 1972; Volume 1, p. 488. Brotske, V. Pollination, Seed Dispersal, Germination, and Seedling Survival in the Federally Threatened Dwarf Lake Iris (Iris Lacustris). 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... Journal of Gender, Culture and Society ISSN: 2754-3293 DOI: 10.32996/jgcs Journal Homepage: www.al-kindipublisher.com/index.php/jgcs JGCS AL-KINDI CENTER FOR RESEARCH AND DEVELOPMENT | RESEARCH ARTICLE De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? Najla Taslim1 and Arthur D. Canales2 1Assistant professor, Department of Pharmacology & Toxicology, Imam Abdulrahman bin Faisal University Dammam, PO box 1982, Saudi Arabia 31442 2Chair, Department of Theology & Philosophy, Professor of Pastoral Theology & Ministry, AAPC; Certified Pastoral Counselor, Marian University, 122 Oldenburg Hall 3200 Cold Springs Road Indianapolis, IN 46222, USA Corresponding Author: Najla Taslim, E-mail: ntakbar@iau.edu.sa | ABSTRACT Gender dysphoria [GD] or a sex and gender discord that leads to enormous psychological suffering in some variants of transgender people is a highly complex and debatable subject. Muslim culture, religious and ethical values pose more challenges to the acceptance of GD than the Western system. The purpose of this narrative review is to present and discuss GD misconceptions and contrasting Islamic views with a fresh perspective and a rational approach in light of current advancements in this field. Since there are no directives in the Holy Quran and Hadith on matters of GD, Muslims have been polarized regarding GD. The so-called liberal scholars corroborate the scientific fact and view GD patients as a medical condition, which necessitates contemporary therapeutic interventions. Conversely, the so-called conservative approaches regard GD a delusional thinking probably arising from a mental illness, or a spiritual immorality, which necessitates psychotherapy or zealous religious guidance. Due to the religious tensions, generalized unawareness, and scarcity of the quality multifaceted research, there is widespread misconceptions and hostility toward transgender people in Islamic culture. The critical appraisal of the Islamic debate on GD proves that despite its ambiguity and intricacies, GD remains a stark reality and merits resolution. Though conflicted on GD, Islamic jurists concur that Islamic laws founded on core principles of love, and respect for all dictate rational reasoning, and enlightened approach on obscure matters. Young Muslims can help reconcile GD conflict in Islamic societies by initiating intrafaith dialogues and engaging their religious scholars in clinical and patient-centered research to broaden their perspective. Collaborative efforts can prompt conservative Islamic scholars to re-negotiate their antagonistic stance on GD or SRT. Islamic leaders can also influence local governments and policy makers to develop policies to improve the welfare of long-ignored transgender people that deserve attention, empathy, and treatment on moral and religious grounds. | KEYWORDS Gender dysphoria, Islam, Muslims, Religious controversy, Muslim countries | ARTICLE INFORMATION ACCEPTED: 03 February 2023 PUBLISHED: 03 February 2023 DOI: 10.32996/jgcs.2023.3.1.2 1. Introduction Gender dysphoria (GD) is an exceptionally complicated and challenging medical condition. Typically, a GD patient is extremely disturbed about her/his given sex which defies their gender appearance (American Psychiatric Association, 2016). Patients suffering from GD are colloquially or most popularly termed transgender. Despite being identified and diagnosed in the mid-twentieth century, GD and its standard evidence-based treatments that entail gender affirming treatments or sex reassignment therapies (SRT) are still universally controversial and are riddled with opposing views and theories, particularly in religious domain all over the world (Coleman, 2012; Hembree,2011; Ishak & Haneef, 2014; Levine, 2018). Copyright: 2022 the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) 4.0 license (https://creativecommons.org/licenses/by/4.0/). Published by Al-Kindi Centre for Research and Development, London, United Kingdom. Page | 6 JGCS 3(1): 06-18 It is no exaggeration to say that the religious debates on GD have been far more polarizing than any other discipline and has served to complicate the matter way beyond the comprehension of a layperson. Liberal religious scholars of the Abrahamic faiths Judaism, Christianity, and Islam corroborate the scientific data which call for a therapeutic resolution of the misalignment of gender and sex through SRT by affirming the anguish, helplessness, and travails of the GD person (Alipour,2017; Ishak & Haneef,2014; Kariminia 2010). Conversely, conservative believers regard GD as a behavioral act of defiance to God by confused or deranged minded people (Roy, 2020; Wirth, 2015). Unfortunately, the conservative approach has profoundly tarnished the perception of GD patients in many religious societies. Due to the religious schism, scarcity of scientific research, and generalized unawareness, there is a profound misunderstanding of transgender issues in a vast majority of Muslim countries (Jami, 2005). Transgender behavior is mightily discouraged, ridiculed or subjected to threatening slurs or violence in most Muslim countries. Such stigmatization has had forced transgender people to live as outcasts and impoverished lives without family or legal support system (Akhtar & Bilour, 2020; Gibson et al., 2016; Saeed &Farooq, 2018; Shah et al., 2018). Only Turkey, Iran, and Egypt legally recognize GD as a medical condition and offer SRT as medical treatment (Mazen, 2017; Turan et al., 2015). While Indonesia and Pakistan have recently accepted trans identity with official mandate; there is no clarity on the allowance for SRT (The transgender person; Valashany & Janghorbani, 2018). The rest of the worlds Muslim countries are either negligent or critical of GD patients and their right for SRT. In our previous article, we addressed in detail, the reality of sex and gender incongruence, various categories of GD patients, their global status and current treatment options, as well as social and medical controversies on GD from a Muslim perspective (Taslim et al., 2021). This article presents and clarifies some prevalent myths about GD and transgender people in Muslim societies, followed by a discussion of contrasting Islamic religious views on GD and its medical treatment. Finally, the article evaluates the controversial religious points that Muslim young people seek, with a fresh perspective and a logical discussion. We hope that this article will persuade the younger generation of Muslims to find a pragmatic resolution to GD by reconciling the controversies, promoting interactive research and fanning out public awareness on this social matter. The term young people is used throughout this essay to refer to people who are 18-35 years of age. 2. Methods A non-systematic literature review was conducted through scientific databases including, PubMed, Web of science, Google Scholar and Scopus, to search English language articles featuring Christian, Jewish, or Muslim perspectives on GD or SRT. All selected articles were retrieved by two independent reviewers and were published between the periods of 1960 to 2020. 3. Gender Dysphoria (GD) and Misconceptions in Islamic societies The influence of religion in shaping a culture or societys perception, attitude, or reaction to a matter, pertaining to human needs in religious societies cannot be overemphasized. Like many other societies, Muslim societies strictly implement gender binary system, where males are supposed to be men and females to be women (Wong, 2012). Islamic codes of life are derived from the divinely text of the Holy Quran and Hadith or sayings of the Prophet Muhammed [peace be upon Him], which perfectly cater for most social, personal, communal or spiritual needs of humans (Fard & Babookhani, 2019). When both Islamic sources are silent on a matter or adopt a neutral stance, Muslims resort to their religious scholars for guidance. GD issue represents a unique and challenging issue in Islamic tradition for multiple reasons: 1. Islamic jurisprudence, that is, the Quran and the Hadith have not elaborated on the transgender issues or its treatment; 2. There are various interpretations from Muslim scholars on transgender mattersa; 3. There is a lack of quality research in Muslim countries except for Turkey and Iran. 4. There is no legal transgender support system in Muslim countries except for Iran. All of these above factors have contributed to a generalized unawareness and widespread misconceptions of transgenderism in many Muslim nations. Table 1 presents myths, which are widely prevalent regarding transgender people in Muslim culture, and thus, merit debunking. Page | 7 De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? Table 1: Prevalent myths on transgender people, GD and SRT and in Muslim culture versus reality. Myth #1. Transgender people are mentally ill or psychiatric patients Reality Mistaken. Transgender people are not considered psychiatric patients any more according to the scientific research of the last sixty to eighty years. Medical experts believe that transgender is an inherent condition and is not in a persons control (Bancroft & Marks, 1968; Green & Money,1969; Wright et al., 2018) Myth #2. Transgender folks are the same as intersex Reality False Invalid. Intersex people had developmental defects in their genital organs, which could manifest as ambiguous or immaturely developed genitalia (Witchel, 2018). While transgender people are males or females (carrying normal genitalia), with mismatched gender expression. Consequently, intersex have disorder of sexual development (DSD) while transgender folks have disorder of gender identity. However, some intersex can be transgender as well if they are assigned a wrong sex at birth (Mouriquand et al., 2016). Myth #3. Transgender people are the same as homosexual persons. Reality Incorrect. Transgender people and homosexuals are distinct phenomenon. Homosexuals are attracted to a person of their own sex. Mostly, homosexuals are cis-gender (those with no sex and gender dispute in mind; they are the general people) and have no DSD or gender identity. Typically, a trans woman [born a biological male] is sexually attracted to men but this should not be construed as homosexuality. Data show that the majority of transgender folks are heterosexuals because their sexuality is driven by their gender expression and not the natal sex (Fein et al., 2018; Nieder et al., 2011). Figure 1 artistically explains the difference between a homosexual and a transgender. Myth #4. Transgender people can never be treated. Reality Wrong. Ample scientific data support that transgender folks can be reasonably managed with SRT which can allow a person to live according to their gender feelings and blend in the society like a regular man or woman being (Coleman et al., 2012; Gridley et al., 2016; Joseph et al., 2017; Shumer et al., 2016). Myth #5. Transgender people can be and should be treated with counseling and psychiatric medications. Reality Erroneous. Psychotherapy has been ineffective to reverse GD. Behavioral therapies can deteriorate a trans persons mental well-being and can subject them to depression and anxiety. WPATH guidelines warns against all kinds of reversion therapies through spiritual or psychological means after receiving reports of suicidal death as a result of such therapies (Bancroft & Marks, 1968; Green & Money, 1969; Wright et al., 2018). Myth #6. Transgender treatment is strictly prohibited by Islam. Reality Fabrication. Islamic scholars are divided as proponents and opponents of the SRT for GD management based on the varying interpretations of the holy Quran. Islamic scholars in Iran and Egypt have sanctioned SRT as a human right of transgender people those who long to be normal (See detail under section "Views of Muslim scholars on GD") Page | 8 JGCS 3(1): 06-18 Myth #7. Transgender people are sinners and must be avoided. Reality Untrue. Transgender-guilt is an experience or phenomenon that comes over transgender folks because they feel their bodies are different. Does that alone constitute a sin? All human beings are created in imago Dei, in the image and likeness of God; therefore, a transgender person cannot be a sinner (Canales, 2018). Matter of fact, the way societies treat their most vulnerable and marginalized members makes them the victims and not necessarily sinners. Figure 1. A graphic display of an example to differentiate between a homosexual and a transgender person. If a biological male is homosexual, he will perceive himself as a man (gender is man) but will be attracted to a male. However, if a biological male is transgender, he thinks himself as a woman [gender is transgender woman] and is attracted to males which is a normal heterosexual behavior. However, if a trans woman is attracted to another woman that is described as a homosexual behavior (that is rare in reality). The same above scenario can be applied for a trans man [a biological female] who is mostly attracted to women. 4. Views of Muslim Scholars on GD Religious authorities of the Scripture (the Abrahamic religions) the Judaism, Christianity and Islam are identically split as conservatives/traditional (opponents) and liberals/progressive (proponents) on their verdict of GD and SRT based on their theological beliefs, interpretation of doctrines and judicial application of those principles on transgender affairs (Alipour,2017; Ishak & Haneef, 2014). Conservatives denounce and oppose the transgender theory and its SRT; while the liberals advocate for transgender rights and affirm SRT as a valid therapeutic plan to relieve GD (Kariminia, 2010; Roy, 2020; Wirth, 2015). The root cause of the dispute is the question whether humans should be recognized by their anatomy (gender/body) or psychology [mind/soul]. For conservatives, the body is prioritized. For liberals, the mind, and soul are primary. Below is a GD-driven debate in Muslim traditions. Lists of conservative and liberal references from Jewish/Christian sources are presented below in Table 2 for interested readers. Evidently, these sources feature mixed and inconclusive messages on the trans issue. Page | 9 De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? Table 2. Biblical quotations used by conservations and liberals for and against transgender people. Conservative/Traditional References Liberal/Progressive References No one whose testicles are crushed or whose male member is cut off shall not enter the assembly of the LORD. (Bible: Deuteronomy 23: 12). For thus say the LORD: To the eunuchs he who keep my Sabbaths, who choose the things that please me and hold fast my covenant, I will give, in my house and within my walls, a monument and a name better than sons and daughters; I give them an everlasting name that shall not be cut off (Bible: Isaiah 56: 45). A woman shall not wear a mans apparel, nor shall a man put on a womans garment; for whoever does such things is abhorrent to the LORD God (Bible: Deuter-onomy 22: 5). For there are eunuchs who had been so from birth, and there are eunuchs who had been made eunuchs by others, and there are eunuchs who had made themselves eunuchs for the sake of the kingdom of heaven. Let anyone accept this who can (Bible: Mathew 19: 12). Do not judge by appearances, but judge with right judgment (Bible:John 7:24). 4.1 Conservative/Traditional Views Traditional Muslim scholars have maintained a denunciation tone against the SRT since inception (Ishak & Haneef, 2014; Roy, 2020; Wirth, 2015). From their perspective, God has destined humans to be male or female and any deviation goes against Gods creative nature (Bible, 1989: Genesis 1: 2728; Quran: 42:49-50]. Traditionalists maintain that excising out someones healthy tissues and replacing themwith artificial ones is a desecration of Gods creation. Another cogent conservative argument is that superficially modifying someones physical appearance does not transform ones XX and XY chromosomes; therefore, a persons sex inherently remains the same as one was born (Ishak & Haneef, 2014; Kalbasi & Deleer, 2016; Widdows, 2002). Only acceptable surgeries are life-saving or restorative surgeries such as for intersex (Zainuddin & Mahdy, 2017). Conventional views on transgender people are strengthened by the following references from the Quran or Hadith. And I [Satan] will mislead them, and I will arouse in them [sinful] desires, and I will command them so they will slit the ears of cattle, and I will command them so they will change the creation of Allah." And whoever takes Satan as an ally instead of Allah has certainly sustained a clear loss. (Quran: 4:119). To Allah belongs the dominion of the heavens and the earth; He creates what he wills. He gives to whom He wills female [children], and He gives to whom He wills males. Or grants them a mix of males and females, and causes whomever He pleases to be barren. He is All- Knowing, All-Powerful (Quran: 42:49-50). Prophet Muhammad [pbuh] was also quoted as saying, God cursed the males who appear like women and the females who appear like men (Karim, 1994, Vol. 1, p. 613). Besides, the Prophet has declared that Harm shall neither be inflicted nor reciprocated (Ibn Majah, Vol. 2, p. 60). Fundamentally, the above represents the conventional wisdom of traditional Muslim understanding on altering Gods creation for humans. 4.2 Liberal/Progressive Views Progressive Muslim scholars demonstrate remarkable tolerance and acceptance of transgender people as a sexual minority and call for their medical treatment (Alipour 2017, Kariminia 2010, Khomeini 1964; Zaharin & Pallotta-Chiarolli, 2020). The former Page | 10 JGCS 3(1): 06-18 Iranian supreme leader - the late Grand ayatollah Imam Khomeini had issued a comprehensive and historic Islamic decree/edict or fatwa on GD in 1970s and allowed SRT as a basic treatment of transgender people (Khomeini, 1964 ). Imam Khomeini rebutted the above conservative views on the following grounds: Treating an innately behavioral disordered state of a person or his distress is not akin to tampering with Gods creation but is a medical necessity to alleviate a persons suffering and it is important to restore a patients natural appearance in line with this soul [gender] (Kariminia, 2010). The Prophet Muhammads [pbuh] condemnation of cross-dressing was for imposters and not for the natural transgender persons per se. The Prophet was furious and issued this denouncement when a male impersonating a transgender woman gained access to womens private quarters (Ibn hajer, 1985; Sarcheshmehpour et al., 2018). If transgender people are not allowed to rectify their bodies they will fulfill their carnal desires by way of homosexuality, [which is absolutely forbidden in Islam] (Kariminia, 2010). The Prophet Mohammad [pbuh] stated God has not created ailments except that He has anticipated by His will a cure for it. (Ibn majah). And, the scientifically proven cure for GD is SRT. This was the very first fatwa on religious grounds from any religious scholars of the Abrahamic faith. Figure 2 shows the systematic approach Imam Khomeini followed within the Islamic theoretical and juridical framework to deliver his historic decree (Zaharin & Pallotta-Chiarolli, 2020). Presently, many other prominent Iranian ayatollahs or marajah such as Al-Sinaei, and the currently ruling Seyyed Ali Khamenei etc. fully support this fatwa as a valid provision for GD treatment while Al-sistani allows conditionally permission for SRT on a case-by-case basis (Kalbasi & Deleer 2016). Figure 2. Imam Khomeinis methodical pathway to issuing his historic fatwa supporting the transgender right of treatment. Tantawi and Al-Azhar objectively evaluated Khomeinis approach and consented to its authenticity, by standards of Islamic laws. They however, adopted the Sunni method of Ijtihad that is, their opinion (ray) and analogy (qiyas). (Alipour, 2017). *Principle of permissibility in Shia jurisprudence implies that that if an action cannot be clearly regarded as being forbidden or permissible in Islam, it is permitted and lawful (Alipour, 2017). ** The Islamic principles are amenable to circumstances like a swine-flesh is allowed to be eaten when a person is desperate from hunger, eating is permitted during the fasting month for travelers or sick persons. (2: 173; 2: 185) Although Imam Khomeinis redeeming fatwa initially sparked the spate of criticism from conservative Islamic jurists, this fatwa prevailed in Iran where it started a boom of SRT (Kalbasi & Deleer 2016). Currently, Iran ranks second in the world to performing Page | 11 De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? SRT on national and international clients (Bardford, 2008). Iranian transgender people have had been entitled to full government support for the sex transition until the countrys financial affairs were deteriorated by the international economic sanctions (Bardford, 2008, Saeidzadeh, 2016). Imam Khomeini had also written a comprehensive book featuring the social rights and legal responsibilities of the newly converted women/men in light of Islamic jurisprudence (Khomeini 1964). The English translation of his book is underway in an Islamic seminary in Qum, Iran. Egyptian and Sunni Muslim scholars Mufti Al-Tantawi and Sheikh Al-Azhar have also corroborated with Khomeinis fatwa, after much research and deliberation. Hence, SRT was sanctioned in Egypt in 2007 (Ataman, 2011, Ishak & Haneef, 2014, Mazen, 2017). Undoubtedly, Khomeinis fatwa has served as a catalysts and religious permit for moderate transgender Muslims across the globes who intend to undergo SRT. Below are excerpts from Imam Khomeini and Mufti Tantawis fatwa: In the Name of God. Sex-reassignment surgery is not prohibited in sharia law (to treat GD) if reliable medical doctors recommend it. Inshallah you will be safe and hopefully the people whom you had mentioned might take care of your situation. (Khomeinis fatwa cited in Alipour, 2017) To sum up: It is permissible to perform the operation in order to reveal what was hidden of male or female organs. Indeed, it is obligatory to do so on the grounds that it must be considered a treatment, when a trustworthy doctor advises it. It is, however, not permissible to do it at the mere wish to change sex from woman to man, or vice versa. (Tantawis fatwa cited in Alipour, 2017.) Taken together, it is evident that progressive Islamic scholars approach to SRT is spiritually-inspired and is a contextual derivation of the Scripture backed by evidences from contemporary science as distinct from that of the conservatives which closely focused on the literalist approach or fundamentalist method prioritized by natal sex. 5. Discussion and Rational Assessment Considering the above religious discussion and all medical and ethical controversies on GD that have been discussed in a previous article, there is no easy way to understand the dynamics of GD (Taslim et al., 2021). However, there is little doubt that transgender people worldwide face hatred, discrimination, repulsion, and violence due to their out of the norm gender behavior. Rationally, the atypical human behavior or the misaligned physical appearance should be rectified to restore their human dignity and social respect. However, the dispute is on what constitutes the right treatment for GD? As shown in Figure 3, the majority of stakeholders agree that the scientific evidence-based SRT are the valid treatment options for GD patient except for conservative branch who vehemently oppose (Ishak & Haneef, 2014; Kalbasi & Deleer, 2016). The diagram will help transgender people discern for themselves the support they have in the medical, scientific, and religious communities. Figure 3. A depiction of varied positions on SRT For conservatives, GD is a product of delusional thinking, moral deprivation, impaired reasoning, spiritual corruption, and defiance to God (Roy, 2020; Wirth 2015). Therefore, conservatives maintain that GD should be reformed by administering conversion therapy, religious counseling, nurturing spirituality, gender appropriate training, or psychotherapy. Conversely, scientific data Page | 12 JGCS 3(1): 06-18 maintains that none of the above modalities, as well as psychiatric medicines have ever proven successful in the last hundred years (Bancroft & Marks, 1968; Green & Money, 1969; Wright et al., 2018). This clearly calls for more research and investigation for those who disregard the SRT. Below, is a logical appraisal of the major arguments against GD or SRT. 5.1 GD is an Unreal Condition and a Forbidden Behavior in Islam Due to its unknown origin, many conservative and bioethicists express skepticism over GD (Ishak and Haneef, 2014; Levine, 2018; Roy, 2020; Wirth, 2015). Nevertheless, the Holy Quran testifies the existential presence of effeminate men and quotes them while commanding Muslim women to observe modesty and wear a formal headdress or veil in the presence of unrelated men. Interestingly, Muslim women are exempted to observe such a dress code when around effeminate men or men with no desire for women (Quran: 24:31). Unlike many other social-sexual acts such as, adultery, fornication, masturbation, and homosexuality, which are repeatedly defined and explained at numerous instance in Quran, the transgender behavior is not denounced at all in Quran (Quran: 7: 80-84; 24:2-9; 24:23; 5: 38-39). Similarly, the Hadith on the condemnation of a cross-dresser is absolutely misquoted. Many Islamic scholars wrote that the Prophets wrath was directed at a male who disguised himself as a transgender woman and committed a repulsive and explicit unethical act and entered the female quarters (Kugle, 2010; Sarcheshmehpour et al., 2018). This Hadith has been mentioned in many authentic and famously available Islamic books b such as Ibn Majah, Al-Bukhari, Al-Tirmidhi, Ibn Hanbal, and Abu Dawud. On another account, the Prophet was recorded to have saved the life of a transgender person when others wanted to kill him as quoted in Sunan Abu-Dawud, Book 41, Number 4910. Hence, it cannot be claimed that the God despises a transgender person due to their gender selection. Transgender feelings are real, if transgender feelings were unreal, then GD patients would not endure humiliation, mistreatment, face stigma, and prejudice for their entire lives. GD is a real psychological condition and a medical problem. GD is emotional and physical; it deserves serious attention and treatment. Moral and religious leaders should be acting with empathy and compassion by starting to treat transgender people with openness and affirmation (Canales, 2018). 5.2 Human Beings Cannot Alter the Gods Handiwork Muslim (and Christian) conservatives maintain the argument that God created humans with special bodily attributes as part of Gods plan to empower all peoples to carry out specifically designated roles and responsibilities. According to them, tampering with Gods handiwork of the human body or natal sex, and revising or editing Gods designed plans for humans, is tantamount to desecrating Gods universal order (Ishak and Haneef ,2014; Levine, 2018; Roy, 2020; Wirth, 2015). This type of myopic ideology implies that the scientific community should not treat inborn or congenital diseases and let the persons suffer the rest of their lives. Consequently, the loving God would never want anyone to suffer, rather God expects humans to utilize knowledge, reason, logic, and wisdom, and the best resources to find cure for all illnesses whether be it cancer, GD or something else. The Quranic verse I will command them so they will slit the ears of cattle, and I will command them so they will change the creation of Allah. (Quran: 4:11) - the quote conservatives present to bolster their claims to prohibit SRT, has no bearing on the transgender issues. First, it was revealed to condemn the pre-Islamic religious rituals and practices of pagan, wherein they will mutilate their animals to mark and reserve them for their deities. Second, the truth is that humans lives are dynamic, in this world, and built on principles of change. Countless times we alter the Gods creation on a regular basis; cut down trees to make furniture, harvest wheat to bake bread, and dig up the earth to produce oil. Naturally, some humans are different, unique, and do not always fit the precise criteria of norm. These people need therapy not admonishment or rejection from those who have no idea about the pain, struggle, and heartache that transgender people face daily. 5.3 Removing the Health Organs is Immoral and Sinful According to critics of SRT, tearing out healthy male, female organs is unwise and unacceptable and must be forbidden. This conventional position on mutilating the healthy body deems colossally appropriate and one should not be offered this option of SRT to help someone escape their mundane troubles, achieve their vain desires, or follow their fleeting passion. Prohibition against SRT sounds a genuine ruling for a non-GD patient. Hence, both permissive fatwa of Imam Khomeini and Sheikh Tantawi for SRT clearly state that the permissibility of SRT is reserved exclusively for those medically diagnosed with GD. Moreover, expert physicians must recommend SRT. Adversely, though, GD means persistent, aggravating feelings and a psycho-sexual torture where a person has to choose between keeping a healthy uterus or testes, and healthy brain. 5.4 Harm Should not be Inflicted on a Person Conservative Muslim scholars have criticized the legitimacy of SRT and labeled them as unnecessary surgeries or act of selfmutilation. Interestingly, all Islamic jurists unanimously approve SRT for intersex: people born with ambiguous genitalia, hormones, or chromosomes (Zainuddin & Mahdy, 2017). Traditionalists argue not altering Gods creation is not applicable in the case of intersex birth because intersex people will remain confused and stressed about their identity. Ironically, the intent of SRT for GD Page | 13 De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? person is the same, to match the physiology with the psychological, and to alleviate the stress of a human being. The only difference is that the intersex problem is visible and transgender peoples invisible. Since the entire Islamic creed is built on the existence of an invisible God, it might be ignorance to only believe in things seen or proven as distinct from unseen. In Islamic philosophy, humans are a body-soul complex; with body being the mortal and soul being the eternal entity (Razak & Haneef, 2017). While the body has its sanctity, real human values lie with the souls. Since physiological [body] needs are different from psychological needs of souls, conservative viewpoints to give precedence to bodys anatomy [sex] over inner or psychological feelings [gender identity] seem groundless. Data shows that there are numerous categories of transgender people and not all variants suffer from GD and yearn for SRT (Richards et al., 2016). With medical experts and some religious scholars advocating for SRT, a logical conclusion should be to let a transgender person decide for themselves. Again, the only person that can describe their inner turmoil, distress, and helpless desire to be normal is the transgender person themselves. No one should force another person to undergo SRT, nor should we deny someone the SRT who desire to be normal within their skins. 5.5 Sex Can Never be Changed because Chromosomes Remain the Same For those who argue the genotypic superiority over phenotypic characteristics and claim that transgender people can never have a real sex-change because their chromosome XX OR XY cannot be switched. This criticism is valid against SRT; however, we ask ourselves if chromosomes are the only markers to define humans, or identify human diseases? Humans share 99% chromosomal homology with chimpanzees, 90% with cats, and 99.9% with one and other humans. Still human characteristics, personality traits, and behavior are drastically different from other species or from other fellow humans (Auton et al., 2015; Gibbons et al., 2004). Some human diseases such as, Downs, Turners Syndromes, Autism, and cancer can be identified genetically but this is not true for all diseases. People are born blind, deaf, or with heart defect and yet their chromosomes are normal. Some variants of intersex carry XX or XY chromosomes, but still their genitalia are deformed (Lee et al., 2006). Despite the availability of cytogenetic testing, endocrinology profile, and ultrasound findings, the sex assignment even for intersex is still challenging for physicians. Experts in the field consider the gender identity of the child a crucial determinant in assigning sex to intersex individuals. Experts also recommend delaying the procedure until 18-month of age when the psychosexual behavior (gender) of a child is more evident (Shimada & Tohda, 2004; Stambough et al., 2019). Unmistakably, chromosomes do not have the answer to all human problems. Perhaps future science will discover some other definitive markers for sex and gender. The Quran does not elaborate whether karyotype or psychosexual behavior defines a human personhood. The Quran does emphasize, Verily the most honored of you in the sight of Allah is (the one who is) the most righteous of you (Quran: 49: 13). Certainly, God is not concerned with issues of gender or sexuality. Gods priorities are clearly being virtuous and upright. Consequently, no one should judge another person for her/his decision for SRT, only a transgender person knows their heart and can accurately follow her/his own conscience. 5.6 Transgender People Can Never Procreate after SRT It is true that transgender people are deprived of fertility after sex change surgeries. Transgender people cannot bear offspring with new bodies (Bizic et al., 2018). Nevertheless, the science does offer the route of cryopreservation of the oocytes, sperms or gonadal tissues (Wierckx et al., 2012). Moreover, with recent successes in regenerative medicine, we should be anticipating that an autologous uterus or testes etc. can be lab-grown and transplanted to trans people post-SRT (Becherucci et al., 2018; Iannaccone et al., 2018). Furthermore, child adoption can always be a viable option for transgender people and is highly meritorious in Islamic doctrine. It should be noted that multitude of cis-gender men and women also suffer infertility and remain childless due to some other biological complications. Similarly, there are millions of orphan children who lose their parents. Human lives are rarely perfect; and are created perfectly, imperfect. 5.7 Allowance for SRT Equates Contravention of Islamic Boundaries Conservatives, be it social, political, or religious, have always argued that giving precedence of the gender expression or identity over natal sex will allow manipulation of this rule to undermine the sanctity of Islamic and moral values. Under the pretext of being a transgender person, anybody can mingle with other men and women, transgress the boundaries of their privacy, and hoodwink the law and society for personal gains. Page | 14 JGCS 3(1): 06-18 There is no denying the fact that the misrepresentation of gender expression can raise some concerns in religious societies. These concerns should be seriously addressed and stringent legal laws, policies and measures should be in place to minimize any violation of the privacy of all humans. In defense of religious permission for SRT, first, it is aforementioned that the religious edict of Imam Khomeini lucidly specifies that the permission for SRT is for GD patients only implying that this act is forbidden in Islam otherwise. The religious repercussion for the misuse of this fatwa is amply clear to believers. Second, we discussed in a previous article that the GD and SRT entail rigorous and extensive process (Taslim et al., 2021). SRT is too consuming emotionally, it takes an exhaustive amount of time, and immense financial hardship for most people to be enticing for non-GD patients. Third, the fear of deception should not deprive the GD patients their right to be the correct gender that they feel they were meant to be since birth. 6. Reflection and Future Directions Although, the accurate prevalence of transgender people in the Muslim world is largely unknown, due to underreporting and lack of epidemiological studies. Thus far, only Iran Malaysia, and Pakistan have shared their highly conservative transgender population data (Akhtar, 2016; Goodman et al., 2019; Winter et al., 2016). By extrapolating the worldwide transgender population data [0.41.3%], the number of transgender people in the Muslim countries could be extrapolated to be in millions (Winter et al., 2016). For such a palpable transgender population, there is massive unawareness on GD in a vast majority of Muslim countries even among health care professionals. The major contributing factors are lack of active research, multi-level complexities of GD, minimal to no involvement of policy makers, and religious authorities in transgender affairs. Religious doctrines in Muslim countries and guidance from religious leaders are crucial determinant in shaping a societys behavior and perception in Islamic societies. The cooperation, participation, and profound understanding of the adversities of GD by religious leaders is monumental. We propose that future studies would be wise to invite Islamic law experts, psychologists, bioethicists and medical professionals to join forces to initiate research projects exploring the etiology, epidemiology, management, and prevention of GD issue. Islamic laws prohibiting SRT must be revised to find a justifiable cure for GD bearing in mind the welfare, consent, and the ultimate happiness of GD patients. Future work should engage Islamic leaders in GD patient care. Exposing religious leaders to the real-life experiences of GD patients in order to witness the psychological pain and sufferings of GD patients at firsthand can broaden their ministry perspective on this condition, which can make Muslim societies amenable to transgender people. A living example is Khomeinis fatwa that undergirds the entire Iranian and Egyptian legal systems to favor the transgender community and restore their human rights. It is narrated that Imam Khomeini personally met with the transgender woman, thoroughly listened to her account of her predicament, consulted medical experts, and then issued a fatwa, thereafter. It would be wise if other Muslim scholars follow suit Imam Khomeinis example to glean deeper insight into transgender situation. Collaborative cross-disciplinary research projects and medical care will enhance understanding on the reality of GD problem. Furthermore, intra-faith dialogues can pave the path to end discord among religious scholars of various denominations and SRT can be granted. Moreover, Islamic religious leaders can educate and disabuse the general public of existing myths about GD patient from Islamic perspective which can eradicate social injustices to transgender people. In addition, religious leaders have an influential voice in amending the legal systems of Islamic countries. There is no doubt that religious figures can make Islamic societies tolerable for transgender people, which will only empower human flourishing around the globe. 7. Limitations of the Study This study is confined to discussing and presenting GD debate within Islamic jurisprudence only whereas GD is a universal and acutely controversial subject in major religions like Judaism and Christianity. Religious ambiguity on GD is the major hurdle in public acceptance of GD reality. However, progressive scholars in both Judaism and Christianity are paving the way for spreading tolerance on GD by citing excellent examples and quotes from scriptures that excite love of humanity (Canale, 2018; Slomowitz, 2019). Their seminal literary work has not been mentioned in this article 8. Conclusion Despite their general consensus on Islams founding principles of respect, tolerance, empathy, justice, and kindness for all humans, the Islamic jurists have been split on the GD issue. The holy Islamic sources of religious guidance have neither prohibited transgender behavior nor denounced its medical treatment. Hence, liberal Shia and Sunni Muslim scholars in Iran and Egypt have legitimized SRT by adopting a progressive approach of logical and reason [ijtehad] in Islamic jurisprudence combined with Page | 15 De Novo Evaluation of Gender Dysphoria Misconceptions and Islamic Religious Perplexity: Is there any Chance of Reconciliation? advanced scientific knowledge. Turkey and Indonesia have also allowed SRT under civil law. Unfortunately, the conservative scholars in other Muslim countries have unsympathetically denounced or utterly ignored GD, but have refrained from suggesting a pragmatic alternative treatment for GD. Due to the religious schism, and low research output in transgender field, a vast majority of Muslims are oblivious to GD. Consequently, transgender population in Muslim countries is either suffering internally or living lives of rejection, poverty, and marginalization. These desperate humans certainly need attention, empathy and support. The religious prohibition on SRT is a major hurdle in the process to normalize the lives of GD patients and their acceptance in Islamic societies. Muslim religious leaders play an important role in shaping peoples perception and moral values. The young religious experts should be invited to intra-faith dialogue to collaborate with medical and scientific communities to engage in clinical care of GD patients to broaden their perspectives. The golden Islamic principles that command love, empathy and compassion for all favor flexibility, progressive thinking and negotiation on an obscure matter to bring peace and normality in a humans life. Moreover, Islamic religious leaders can also help end stigma to transgender people by urging the general population to respect transgender folks, honor their dignity, and support their rights of equal treatment. To a layperson, the mixed religious message implies that GD is indeed one of the mysteries of God. It is a so-called sin or no sin, maybe it depends, as the very popular Hadith in Islam reads, the consequence of your actions is determined by your intentions. Hopefully, Muslim young people, along with their religious leaders, and scientists can bring peace to the sufferings of long-ignore GD patients and transgender people by attempting to resolve the religious division and by promoting awareness on this important topic. It is important to note that there are two major denominations of Muslims: (a) Sunni Muslims, which are the majority and represent 85% of all Muslims and (b) Shia Muslims, which represent a minority of 10-15% of Muslims. The rest of the other minor branches of Muslims are derived from either one of these major branches. Although there is a consensus on the fundamental principles of Islam, the interpretation of religious sources sometimes varies dramatically within and between two denominations on matters of importance. a There are other traditional Muslim authors and scholars who write on this topic: Majah, Al-Bukhari, Al-Tirmidhi, Ibn Hanbal, and Abu Dawud. There Hadith books are readily and popularly available in Muslim countries. b Funding: Please add: This research received no external funding Conflicts of Interest: The authors declare no conflict of interest. ORCID iD (if any) : https://orcid.org/0000-0002-7152-3883 Publishers Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Contribution of Individual Authors: Najla Taslim: Conceptualize and drafted the article ; Arthur D. 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