Entrepreneurship is a highly dynamic and important endeavor that spills over to economic, technological, and social canvas of a society in this rapidly changing globalized economy. The purpose of the present quantitative study is to investigate the associations among information and communication technologies, innovation, absorptive capacity, CEO’s temporal leadership, and competitive advantage by considering corporate entrepreneurship as a mediator. These factors have been incorporated because they play a predominant role to vie in a competitive environment for entrepreneurial success and economic growth. We used the response of 460 organizations, acquired on a Likert scale, to examine how antecedents of corporate entrepreneurship contribute toward competitive advantage. Structural equation modeling was employed to analyze the measurement and structural relationships including the mediation effects of corporate entrepreneurship. All the relationships with corporate entrepreneurship were found significant except the direct effect of absorptive capacity on competitive advantage. Hence, the results established corporate entrepreneurship as a mediator to predict competitive advantage partially by information and communication technologies (ICT) use, innovation, and temporal leadership. The findings also reveal that absorptive capacity reaps an entire competitive advantage only through corporate entrepreneurship. Practically, the study would be invaluable for organizations, entrepreneurs, and managers to capture a lot of opportunities in effectively managing scarce resources. The new economic and business setting caused by complex technological advances and an uncertain environment calls for faster and innovative response strategies to maintain competitive advantage. With the inability to perceive this departure, many projects failed, and consequently, organizations were unsuccessful to achieve their planned goals . RecentlCorporate entrepreneurship (CE) is a riposte for the survival and competitive success of business entities in this current situation . EntreprCorporate entrepreneurship is the ability of an organization to explore and exploit profitable opportunities without being inhibited by limitations of resources, rules, and regulations, as well as managerial decisions . It may ICT use can be defined as a “diverse set of technological tools and resources used to create, disseminate, store, and manage information” , p. 1. TOne of the other entrepreneurial constituents is absorptive capacity which can be defined as “the organization’s relative ability to develop a set of organizational routines and strategic processes through which it acquires, assimilates, transforms and exploits knowledge acquired from outside the organization in order to create value” , p. 3037allocation of temporal resources, scheduling, and temporal synchronization . These resources, once transformed into competitive advantage, would help face the global challenges.In the contemporary competitive world, entrepreneurs operate their business in a technology-rich environment. Entrepreneurs must also perform their activities earnestly while using tools of computing, online communication, and cooperation . ICT useHowever, investment in ICT should not be made in isolation, but it must be aligned with goals, missions, strategies, and directions of the organization. It must also be adopted according to users’ requirements, and ICT jobs must be well determined . Tang et1:HICT use has a significant impact on corporate entrepreneurship.Innovative organizations preemptively use innovation strategies for making business models, services, and new products and, hence, build a strong relationship between innovation and entrepreneurship to outperform in competitive markets. Due to the fast growing progress in technology and science, product innovation has become an overriding concern for those firms who are struggling to achieve competitive advantage . For fir2:HInnovation has a significant impact on corporate entrepreneurship.Firms with absorptive capacity increase their performance through access of external knowledge and show their willingness to reciprocate toward the external environment by innovative ways . AbsorptConsidering the abovementioned discussion, we may propose the following hypothesis:3:HAbsorptive capacity has a significant impact on corporate entrepreneurship.Temporal leadership, managed by leaders to meet deadlines, is an important factor for entrepreneurship because it acts as a coordinator between work, various time frames, and member contributions . CEOs’ t4:HCEO’s temporal leadership has a significant impact on corporate entrepreneurship.The success of entrepreneurship is associated with unique knowledge, skills of executives, and experience of entrepreneurs . Experie5:HCorporate entrepreneurship has a significant impact on competitive advantage.Information and communication technologies is the most significant element for economic development, and its extraordinary functions have brought fundamental changes for the development of research and education. Corporate entrepreneurship enabled by innovation and ICT aligns a firm’s strategies and resources because it constitutes dimensions that are vital for an organization to attain competitive advantage . Organiz6:HICT has a significant impact on competitive advantage.Due to multitudinous changes in the global world, corporate entrepreneurship starts new activities for organizations, follows new innovation processes, and takes interest in departing from the daily unchanging process for exploring, creating, and chasing new profitable opportunities . In orde7:HInnovation has a significant impact on competitive advantage.Absorptive capacity can integrate internal and external knowledge for the firms to be employed to develop new products and services. In order to increase absorptive capacity, global enterprises make use of digital platforms to combine internal and external knowledge . An orga8:HAbsorptive capacity has a significant impact on competitive advantage.The behavior of temporal leadership determines how teams respond effectively to time pressure. It supports members of the team in managing time, planning work, and attaining competitive advantage . So, the9:HCEO’s temporal leadership has a significant impact on competitive advantage.10:HCorporate Entrepreneurship as a Mediator.Since the industry is changing from traditional to economy-based as part of globalization, corporate entrepreneurship plays an important role in attaining a higher level of competitive advantage . The com10–A:HCorporate entrepreneurship mediates the relationship between ICT use and competitive advantage.10–B:HCorporate entrepreneurship mediates the relationship between innovation and competitive advantage.10–C:HCorporate entrepreneurship mediates the relationship between absorptive capacity, CEO’s temporal leadership, and competitive advantage.10–D:HCorporate entrepreneurship mediates the relationship between ICT use, innovation, absorptive capacity, CEO’s temporal leadership, and competitive advantage.Based upon the foregoing hypotheses, the research model is described in The target population for this study comprises both middle- and upper-level employees working in Lahore-based IT sector of Pakistan. Being deductive in nature, the research employs a quantitative approach including a well-structured questionnaire. A convenient sampling process was used for collection of data from 460 firms through emails and direct meetings. The representative employees had knowledge about the use of ICT and orientation of innovation and were well-experienced about how to take advantage of absorptive capacity through entrepreneurial behavior in their firms. Although 600 questionnaires were distributed to the IT firms, only 495 of them were received and only 460 were found complete in all respects. Thus, the eventual response rate was computed to be 76.66%.The sample size is consistent with the recommendations by It can be observed from the gender-wise frequency analysis of the data that 90.4% of the sample comprised male employees, while 9.6% of the respondents were female. Hence, the dominant majority of the respondents were male in this data. As per the age frequency, 55 of the respondents, i.e., 11.96% of the total sample, belonged to the age group of 21–30 years, while in the age bracket of 31–40 years, there were 286 respondents equivalent to 62.17%. In the age cohort of 41–50 years, the number of respondents was 113, which is 24.57% of the total respondents, whereas 6 respondents belonged to the age group greater than 51 years (1.3%). The survey also collected data about work experience of the respondents. There were 90 respondents who had work experience of 1 to 10 years (19.57%), and there were 332 of the respondents who possessed work experience of 11–20 years, i.e., 72.17%. However, in the other experience category of 20 years and above, there were 38 respondents making up 8.26% of the total sample size. The designation of the respondents is another aspect of demographics which indicates that a total of 430 respondents belong to middle management making up 93.5% of the total sample. The remaining 30 respondents were part of the upper management (6.52%).This study is a correlational design to examine the relationships among ICT use, innovation, temporal leadership, absorptive capacity, and corporate entrepreneurship to explore the potential causal impact of each of these factors on competitive advantage. As this research is deductive and quantitative in nature, it utilizes well-structured measurement scales made up of items denoting the respondents’ thoughts and opinions about ICT use, innovation, temporal leadership, absorptive capacity, corporate entrepreneurship, and competitive advantage in their businesses. All items computing these attitudinal variables used the five-point Likert scale response format . Information and communication technology (ICT use), used as the independent variable, was measured by a four-item scale developed by 2/DF) and root mean square error approximation (RMSEA).Structural equation modeling has been used with the help of AMOS 24 for testing the proposed hypotheses empirically. SEM has two elements: the first is confirmatory factor analysis (CFA), which is used to measure the validity of a model comprising unobserved and observed variables, and second component is path analysis that is used to fit the structural model with the latent variables . In the post hoc Harman’s single factor test  = 0.262, incremental fit index (IFI) = 0.284, TLI = 0.247, RMR = 0.103, and RMSEA = 0.150]. We also employed the common latent factor (CLF) test. The standardized regression weights of the model with and without CLF were juxtaposed, and the deviations less than 25% gave credence to non-existence of CMV  may be present in the data collected , NFI, IFI, comparative fit index (CFI), GFI, and RMSEA. In order to achieve a model’s suitability, the value of relative CMIN must be less than 5.0  with minimum criteria of cutoff as AVE > 0.5 . As showthan 0.5 , therebydf = 1.845, NFI = 0.867, TLI = 0.929, CFI = 0.934, GFI = 0.835, and RMSEA = 0.047).The regression estimates conducted through AMOS 24, shown in 1 corroborate that information and communication technologies is positively related to corporate entrepreneurship. The values of the estimates are 0.259, the standard error is 0.051, p-value is significant at 0.000 level, while the critical ratio of ICT use on corporate entrepreneurship is 5.121. The results for H2  demonstrate that innovation has a significant and positive impact on corporate entrepreneurship. The findings for hypothesis H3  reveal that absorptive capacity significantly affects corporate entrepreneurship. In our analysis , the results for hypothesis H3 establish that temporal leadership has also a significant relation with corporate entrepreneurship.The results in p-value = 0.000, SE = 0.085, and CR = 4.074) on competitive advantage (H6) shows that ICT use significantly influences competitive advantage. Similarly, innovation has shown a significant positive impact on competitive advantage (H7) . The value of the direct effect of absorptive for hypothesis H8 on competitive advantage is −0.003 and the p-value is insignificant (0.967), which indicates an insignificant relationship between absorptive capacity and competitive advantage. The results also suggest a significant relationship between temporal leadership and competitive advantage—hypothesis H9 . Finally, the result for hypothesis H5 also confirms that corporate entrepreneurship has a significant direct impact on competitive advantage . With reference to H10, there is an indirect effect of ICT use, innovation, absorptive capacity, and temporal leadership on competitive advantage through corporate entrepreneurship as a mediator. The significance values of the indirect relationships were determined in AMOS through bootstrapping procedure based on 2,000 bootstrap samples. The direct effects of ICT use, innovation, absorptive capacity, and temporal leadership on competitive advantage are 0.345, 0.133, −0.003, and 0.144, respectively, with p-values less than 0.05 except for the relationship of absorptive capacity. On the other hand, the corresponding indirect effects of ICT use, innovation, absorptive capacity, and temporal leadership on competitive advantage are 0.084, 0.061, 0.034, and 0.055 with p-values less than 0.05 showing that corporate entrepreneurship mediates all the relationships as reported in The regression analysis has also computed estimated direct effects of predictors on competitive advantage. The estimated regression weight of ICT use  are supported by 2) show a positive and significant relationship . This result is corroborated by the findings of 3  reveal that absorptive capacity is significantly and positively related to corporate entrepreneurship. The finding is similar to the conclusion made by 4 , 5). The empirical positive results  are ratified by the findings of previous research work  are consistent with the relevant previous studies. The results suggest that all these variables show positive and significant impact on competitive advantage, except absorptive capacity that does not impact the competitive advantage directly. Managing resources of ICT for enhancing the firm performance and attaining competitive advantage requires a firm culture, which may help in identifying, making, and assessing these opportunities , innovation (H10–B), and CEO’s temporal leadership (H10–C) with competitive advantage to the extent of 20, 31, and 28%, respectively. On the other hand, CE fully mediates between absorptive capacity and competitive advantage (H10–D) achieving the magnitude of 92%. It implies that absorptive capacity can be exploited to achieve competitive advantage meaningfully only through venturing entrepreneurship at the corporate level. Thus, we confirm that ICT, innovation, absorptive capacity, and temporal leadership coupled with corporate entrepreneurship help develop entrepreneurial activities, and their upshots attain more competitive advantage and ambitious goals.On the other hand, all the indirect effects are significant and positive, as reported in In this study, we emphasize the pivotal role played by strategic resources such as information and communication technologies, innovation, absorptive capacity, temporal leadership, and corporate entrepreneurship to better seize the opportunities in enhancing the firms’ competitiveness. At the practical level, this study has implications for managers, entrepreneurs, innovation adopters, and technology suppliers to better understand and transform dynamic capabilities and value creation resources into competitive advantage, which may further help them face global challenges. Our results have significance for policy makers as well, who may formulate policies that foster a culture of corporate entrepreneurship, and provide facilitating conditions such as entrepreneurial education, training, and an enabling environment to better exploit the opportunities offered by ICT, absorptive capacity, temporal leadership, and innovation. It may also enable firms to identify their strengths and weaknesses for increasing long-term competitiveness and profitability. Firms that have absorptive capacity can interact with the external environment innovatively to access external knowledge necessary for generating new product ideas to help attain additional competitive advantage. Consequently, this would produce a ripple effect in the form of jobs creation, greater exports, reduced imports, and growth in national GDP. In a nutshell, we suggest that corporate entrepreneurship channelizes the parameters required to reap the advantages of opportunities for an organization.On the social front, this study can improve the quality of life of the poor by offering people with equal opportunities who face difficulties. Organizations can foster their personnel’s entrepreneurial skills through trainings, workshops, mentoring, and motivation to further strengthen the organization. By adopting corporate entrepreneurship, managers can increase job opportunities by creating new markets with its impact on human resources and long-term competitive advantage. The growth and exports of any country considerably depend on companies’ competitiveness that are operating in the country. Consequently, countries can benefit from the competitiveness and innovative activities of the companies by implementing polices that incentivize them.Today’s information age and globalized environment reveal contemporary challenges that cannot be underrated. Despite these challenges, many opportunities can be gained through the proper use of resources for sustaining competitive advantage. ICT, innovation, absorptive capacity, and temporal leadership are strategic resources of an organization that play their role to achieve competitive advantage. However, these potential benefits can be realized in an environment fostered by entrepreneurial spirit. This paper asserts that ICT, absorptive capacity, temporal leadership, and innovation have a positive impact on competitiveness of an organization if opportunities are managed within the culture of an organization through corporate entrepreneurship. In this way, firms not only can maintain their present competitive advantage but also can cope with challenges and threats by exploiting new opportunities. The results are established on SEM analysis conducted on data collected from 460 IT firms through attitudinal measures of scale. According to the regression results, ICT use, innovation, absorptive capacity, and CEO’s temporal leadership show significant direct and indirect impacts on competitive advantage through corporate entrepreneurship. These results are consistent with past studies conducted by various researchers e.g., .The study has several implications for managers, entrepreneurs, innovation adopters, and technology suppliers to better understand and transform dynamic capabilities and value creation resources into competitive advantage, which may further help them face global challenges. Managers can make use of these significant elements to help maintain competitive advantage and for the creation of wealth, increase in sales, and growth of market share. It also has significance for policy makers, who may formulate policies that foster a culture of corporate entrepreneurship and provide facilitating conditions such as entrepreneurial education, training, and an enabling environment to better exploit the opportunities offered by ICT, absorptive capacity temporal leadership, and innovation. It may also assist firms to identify their strengths and weaknesses for increasing long-term competitiveness and profitability. Firms that have absorptive capacity can interact with the external environment innovatively to access external knowledge necessary for generating new product ideas to help attain additional competitive advantage. Consequently, this would produce a ripple effect in the form of jobs creation, greater exports, reduced imports, and growth in national GDP.In spite of several contributions, the present study has some limitations that may turn out to be an opportunity for further research. First, the data were collected through convenient sampling in one district of Lahore, which limits the generalizability of the results. The prospective research should be performed using a more representative probabilistic sampling technique and collecting data from other IT hubs of the country. Likewise, the proposed model or its adaptation may be cross-checked for its reliability in other entrepreneurial sectors. Moreover, granted that common method bias was not a perceptible issue, we still emphasize on applying alternative solutions to address this concern. Another point for the prospective work is to test endogeneity to detect endogenous regressors through the Hausman test.The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The serotonin transporter (SERT) terminates serotonin signaling by rapid presynaptic reuptake. SERT activity is modulated by antidepressants, e.g., S-citalopram and imipramine, to alleviate symptoms of depression and anxiety. SERT crystal structures reveal two S-citalopram binding pockets in the central binding (S1) site and the extracellular vestibule (S2 site). In this study, our combined in vitro and in silico analysis indicates that the bound S-citalopram or imipramine in S1 is allosterically coupled to the ligand binding to S2 through altering protein conformations. Remarkably, SERT inhibitor Lu AF60097, the first high-affinity S2-ligand reported and characterized here, allosterically couples the ligand binding to S1 through a similar mechanism. The SERT inhibition by Lu AF60097 is demonstrated by the potentiated imipramine binding and increased hippocampal serotonin level in rats. Together, we reveal a S1-S2 coupling mechanism that will facilitate rational design of high-affinity SERT allosteric inhibitors. The serotonin transporter (SERT) terminates serotonin signaling and its activity is modulated by antidepressants. Here authors reveal the mechanistic details underlying the coupling between the two binding sites in SERT and a high-affinity ligand for the allosteric site. The serotonin transporter (SERT) is embedded in the presynaptic membrane and terminates 5-HT transmission by rapid reuptake of released 5-HT. SERT belongs, together with the transporters for the neurotransmitters dopamine, norepinephrine, γ-aminobutyric acid, and glycine, to the family of neurotransmitter:sodium symporters (NSSs) that all exploit the electrochemical potential stored in the Na+ gradient to translocate substrates against their concentration gradients4. Pharmacological inhibition of SERT is known to alleviate symptoms of depression and anxiety, the two most prevalent psychiatric disorders ranking among the top five leading causes of disability worldwide6. Specifically, the selective serotonin reuptake inhibitors (SSRIs), such as S-citalopram (S-CIT)   psychostimulants such as MDMA (ecstasy), ibogaine, cocaine, and amphetamine10.Serotonin (5-HT) transmission is involved in many basic brain functions, such as regulation of mood, sleep, appetite, and sexual driveIT) Fig. , sertralMI) Fig.  and amit11, drosophila dopamine transporter12 and two bacterial NSSs, LeuT13 and MhsT14, have revealed a conserved structural fold for NSSs with a primary ligand binding (S1) site located in the center of the transmembrane domain. Interestingly, the existence of an allosteric binding site in SERT was reported more than three decades ago15. The key observation was that certain SERT inhibitors could impede the dissociation of a pre-bound radiolabeled ligand19. The most potent impedance being by S-CIT on [3H]S-CIT dissociation, however, only displays a low potency (IC50 is ~5 µM), and in spite of intensive investigations21 no other compound has shown to possess higher potency22. Based on computational modeling and experimental binding studies, we previously located the low-affinity second binding site for S-CIT and clomipramine to the extracellular vestibule (EV), the entry pathway toward the S1 site21. Interestingly, in one of the recent hSERT crystal structures (PDB 5I73), two S-CIT molecules are bound to the protein: one in the S1 site (denoted as S1:S-CIT) and another bound to a binding site in the EV—the S2 site (denoted as S2:S-CIT) ~13 Å above the S1 site23. This is consistent with previous findings that the EV of LeuT harbors a S2 site capable of binding ligands26. The comparison of the 5I73 structure to the structure bound with only one S-CIT in S1 (PDB 5I71) shows that they have identical conformations in the EV, and it is not clear whether ligand bindings in the S1 and S2 sites allosterically interact with each other through modulation of any specific structural motif28, which may result in conformational changes.The structures of human SERT30 resulting in fewer side effects. In addition, compared with competitive inhibitors to the endogenous ligand, they may retain some of the functions of the target proteins. Indeed, several well-known allosteric modulators possess novel pharmacologic properties such as use-dependency , or activity modulation of the endogenous ligand , or to perform asymmetric signaling as in metabotropic glutamate receptor complexes31, all providing advantageous therapeutic potentials over orthosteric modulators. For S-CIT, it has been proposed that its allosteric binding in SERT contributes to its higher efficacy and faster onset observed in clinical trials as compared with racemic citalopram36. However, the low affinity of S-CIT to S2 relative to S1 hampers the possibility to reveal the specific therapeutic potentials in targeting this site. Thus, a high affinity and selective S2-bound ligand would facilitate not only a thorough mechanistic understanding of allosteric communications between the S2 and S1 sites, but also a proper evaluation of the therapeutic potential of allosteric modulation in SERT.Allosteric modulators can potentially possess higher selectivity due to the divergence of the binding sites among homologous proteinsS)-1-(4-fluorophenyl)-1-piperidin-1-yl)propyl)-1,3-dihydroisobenzofuran-5-carboxamide), in vitro, in silico, and in vivo.Here, we provide mechanistic evidence for allosteric modulations between the S1 and S2 sites in SERT by showing that the effects of ligand binding to the S1 site allosterically propagate through altered conformation of a structural motif between the S1 and S2 sites. In the context of these findings, we report the identification and characterizations of a high-affinity S2-bound inhibitor for SERT, Lu AF60097 . Accordingly, we added 25 nM of either [3H]S-CIT or [3H]IMI to membranes of COS-7 cells transiently expressing hSERT wild type (WT), and then measured the dissociation rates of the two ligands in the presence of increasing concentrations of S- or R-CIT (0.4 µM–1 mM) that should occupy the S2 site at high concentrations (S2:S-CIT and S2:R-CIT). The increase in S2-occupancy by either S- or R-CIT results in a dose-dependent inhibition of dissociation by the S1-bound radioligand. The concentration of a S2-bound ligand causing 50% decrease in the dissociation of a S1-bound radioligand (IC50) is used as a measure to reflect the inhibitory  potency for S2 binding (see “Methods”). We found that the allosteric potency of S2:S-CIT was 29-fold higher in the presence of S1:[3H]S-CIT than in the presence of S1:[3H]IMI  to that of the 5I71 structure. When comparing the two S-CIT bound SERT crystal structures, 5I71 and 5I73, we found that the EV space occupied by the S2:S-CIT in 5I73 is filled by a dodecane in 5I71   simulations at microsecond scale using the ts3 and WT SERT models (see “Methods”) in complexes with different combinations of S1- and S2-bound ligands  allosteric potency on inhibiting the S1:[3H]S-CIT dissociation  inhibition of the S1:IMI as the radioligand, [3H]IMI was not displaced by Lu AF60097, possibly because Lu AF60097 has a high S2 affinity in the presence of S1:IMI and locks [3H]IMI in the S1 site , suggesting a combined competitive and non-competitive mechanism. Thus, together with the [3H]S-CIT equilibrium binding results, we found that Lu AF60097 possesses a S1-binding-based competitive component as well, when the S1 site is not occupied by IMI.To examine whether the inhibition of 5-HT uptake is due to a blockade by a competitive or a non-competitive action by Lu AF60097, we performed IMI dissociation in SERT, though with low potency39.The presence of an allosteric site in SERT has been known for more than three decades3H]IMI, to the same extent as when [3H]S-CIT is bound to S1. Together, the results suggest that the configuration of the Thr497-Phe335 motif is sensitive to the identity of the S1-bound ligand and plays a critical role in the allosteric communication between the S1 and S2 sites.Here, we show at atomistic detail that the S1 and S2 binding sites in SERT are allosterically coupled to each other. By combining extensive (~125 µs) MD simulations of various conditions and site-directed mutagenesis, we show that the impact of ligand binding to the S1 site propagates through the Thr497-Phe335 motif to alter the configuration of the S2 site. In particular, the propensity of Glu494 in the S2 site to form a salt bridge with the S2-bound ligands is differentially affected by S1:S-CIT and S1:IMI. Consistent with this prediction, removal of the negative charge of Glu494 (E494Q) results in similar S2 affinities for S-CIT in the presence of either S1:IMI or S1:S-CIT, while the same impact on the S2 affinities of R-CIT were observed as well  is able to elicit a marked increase of extracellular 5-HT levels in the microdialysis analysis, suggesting that the compound is also capable of targeting SERT in vivo. This we further substantiated by showing that co-administration of IMI and Lu AF60097 in vitro Fig.  and in vThe human SERT was cloned into the pUbi1z vector using the NotI and XbaI. Mutations herein were generated using the QuickChange method  or ordered through GeneArt. All mutations were confirmed by DNA sequencing. The used primers wereL406E, CGCAGGTCCCAGCCTCGAGTTCATCACGTATGCAGA486E, CTTTTGGAGGGGAGTACGTGGTGAAGE494K, GAAGCTGCTGGAGAAGTACGCCACGGGGF556L, CATTTGCAGTTTACTCATGAGCCCGCCACF556R, CATCATTTGCAGTAGACTGATGAGCCCGOnly the sense primers are shown. The complimentary antisense primers were also used.R(1×) and incubated for 20 min for micelle formation. The mixture was added to 10 ml DMEM 1885 medium containing 4 million COS-7 cells. After 5 h, 25 ml DMEM1885 with antibiotic was added. After 48 h. the cells were harvested with PBS + 5 mM EDTA, washed, and lysed with 1 ultrasound burst (Branson Sonifier with microtip) in membrane buffer . Membranes were pelleted at 4900 × g and resuspended in membrane buffer containing 0.3 M sucrose and stored at −80 °C until used.Membranes were prepared from COS-7 cells , after they were transient transfected with hSERT WT or mutants using the Lipo2000 transfection protocol (Invitrogen): 2 µg SERT plasmid and 6 µL Lipofectamine were mixed each with 250 µL Opti-Mem3H]IMI and [3H]S-CIT dissociation rate assay 25 nM [3H]IMI (40–50 Ci/mmol) or [3H]S-CIT (85 Ci/mmol) were added to membranes incubated for 30 min at 0 °C. Dissociation was initiated by 12× dilution of membrane aliquots with membrane buffer containing 0.1 µM paroxetine and the indicated concentrations of allosteric inhibitor. Note: 0.1 µM paroxetine has no allosteric effect on the dissociation of [3H]IMI and [3H]S-CIT. The dissociation was assessed at seven time points (5–10–15–20–30–50–70 min) and stopped by rapid filtration of the samples through GF/B filters using a Tomtec cell harvester and washed for 20 s with ice-cold 0.2 M NaCl. Experiments were performed in a water bath at a temperature where t½ for control dissociation (i.e. dissociation without drug) were set to ~15 min. All experiments were performed in at least three independent experiments.[3H]S-CIT and [3H]IMI competitive binding experiments were performed on membrane preparations of COS-7 cells transiently transfected with SERT WT. Membrane aliquots were mixed with membrane buffer containing 5–10 nM of either [3H]S-CIT or [3H]IMI and Lu AF60097 in the indicated concentrations to a final volume of 300 µL. The binding experiments were incubated 1 h at room temperature and subsequently filtered, washed and counted as described under ‘[3H]IMI and [3H]S-CIT dissociation rate assay’. Non-specific binding was determined by adding 5 µM paroxetine.[3H]5-HT uptake experiments were performed using 5-hydroxytryptamine (25–30 Ci/mmol). COS-7 cells, transfected with SERT WT or SERT mutants were seeded in 24-well dishes (105 cells/well) coated with poly-ornithine. The seeded cell number were adjusted to achieve an uptake level of maximally 10% of total added [3H]5-HT. The uptake assays were carried out 2 days after transfection. Prior to the experiments the cells were washed once in 800 µL uptake buffer  at room temperature. Drugs tested for inhibition of uptake were added to cells in indicated concentrations 30 min prior to addition of 0.3 µCi [3H]5-HT. After incubating 3 min (WT) or 5 min (mutants) the cells were washed twice with 500 µL ice-cold uptake buffer, lysed in 250 µL of 1% SDS and left for 1 h at 37 °C. All samples were transferred to 24-well counting plates, and 500 µL Opti-phase Hi Safe 3 scintillation fluid was added followed by counting in a Wallac Tri-Lux beta-scintillation counter. Nonspecific uptake was determined in the presence of 1 µM paroxetine. All determinations were performed in triplicate.) at indicated unlabeled ligand concentrations were calculated and expressed relative to the dissociation rate constant without the presence of unlabeled ligand (kbuffer). The allosteric potency was determined as the drug concentration that impairs the dissociation rate by 50% compared with dissociation in buffer. IC50 values were calculated from concentration effect curves of normalized dissociation ratio (k[drug]/kbuffer) versus log[drug] and are shown as mean values calculated from means of pIC50 and the SE interval from the pIC50 ± S.E. All data were analyzed by linear or nonlinear regression analysis using Prism 7.0 .The allosteric potency was calculated as previously describedFurther information on research design is available in the Supplementary InformationPeer Review FileSupplementary Movie 1Supplementary Movie 2Reporting SummaryDescription of Additional Supplementary Files


Controlled devitrification of vitreous samples for high-resolution single-particle cryo-EM reduces beam-induced motion in the critical early frames of a movie stack by a factor of approximately four. −2 of an exposure by a factor of ∼4, substantially enhancing the contribution of the initial, minimally damaged frames to a structure. A 3D apoferritin map reconstructed from the first frames of 20 000 particle images of devitrified samples resolved undamaged side chains. Devitrification of frozen-hydrated specimens helps to overcome beam-induced specimen motion in single-particle cryo-EM, as a further step towards realizing the full potential of cryo-EM for high-resolution structure determination.As cryo-EM approaches the physical resolution limits imposed by electron optics and radiation damage, it becomes increasingly urgent to address the issues that impede high-resolution structure determination of biological specimens. One of the persistent problems has been beam-induced movement, which occurs when the specimen is irradiated with high-energy electrons. Beam-induced movement results in image blurring and loss of high-resolution information. It is particularly severe for biological samples in unsupported thin films of vitreous water. By controlled devitrification of conventionally plunge-frozen samples, the suspended film of vitrified water was converted into cubic ice, a polycrystalline, mechanically stable solid. It is shown that compared with vitrified samples, devitrification reduces beam-induced movement in the first 5 e Å Vitrified grids were clipped in Krios autoloader cartridges. For devitrification, clipped grids were placed into the milled wells of an aluminium plate of a home-built grid heater  cooled to around −190°C with liquid nitrogen. The top plate was gradually warmed to −120°C. After 10 min at this temperature, the plate was cooled to −190°C  and the clipped grids were transferred to a storage box under liquid nitrogen. In other devitrification experiments, grids were placed directly onto the metal plate of the grid heater for 5–7 s at a temperature of −100 to −110°C before manual immersion in liquid nitrogen. Grids were handled and mounted in a humidity-controlled atmosphere (∼20% relative humidity at 21°C). Both devitrification procedures gave similar results.Quantifoil R2/2 400 mesh copper grids were pre-treated by washing in trichloromethane for >2 h. The grids were glow-discharged twice before 3 µl of sample solution was applied and plunge-frozen using a Vitrobot Mark IV. Initial experiments were performed with alcohol oxidase as a stable, soluble test sample that was readily available in our laboratory, at a concentration of ∼0.7 mg ml2.2.EPU automatic acquisition software at a transmitted fluence per frame of 1.1–1.3 e Å−2. Defocus was in the range 0.6–2.5 µm. The energy filter was set to a slit width of 20 eV. Four micrographs were acquired per foil hole.Micrographs were collected on a Titan Krios electron microscope equipped with a Gatan K2 camera with energy filter in counting mode at a pixel size of 1.077 Å. 40-frame movies were collected with a total exposure time of 8 s using the 2.3.RELION-3 .Micrographs were pre-processed in rson 2003. Average2.4.MotionCor2 in RELION-3 as before, but the corrected movie stacks were written out. Frames 1–20 were extracted individually from the corrected movie stacks. From these single frames, 20 000 randomly chosen, fully processed particles were extracted. For each frame two half-maps were reconstructed, applying previously assigned orientation and octahedral symmetry in relion_reconstruct. The half-maps were then masked as for the full data set, and sharpened by the RELION post-processing procedure. Maps were drawn with ChimeraX  by raising its temperature to roughly 20–30°C above the glass-transition point near −137°C. The grids were then transferred into the autoloader of a Titan Krios electron microscope. Control grids of vitrified but otherwise identical samples were loaded and examined in separate sessions. Grid squares screened in the electron microscope for successful devitrification fell into one of three categories.(i) The vitreous water film in a number of adjacent grid squares had devitrified. Devitrified areas were distinguished from vitreous squares by tilting the stage at ∼6400× magnification, which caused the ice reflections to move in the direction perpendicular to the tilt axis. If the ice layer was very thin and the reflections were weak, the holes were checked at a magnification of 130 000× as used for image acquisition. At this magnification the cubic ice lattice was easily visible by eye and crystallinity was evident in the image transform Fig. 1.(ii) Other squares on the same grid appeared to be covered with contaminating atmospheric ice crystals Fig. 2. Dependi(iii) Some grids appeared to be unchanged compared with grids that had not been placed onto the heater plate and none of the grid squares had devitrified.Images of horse apoferritin or alcohol oxidase on plunge-frozen carbon-coated copper grids were devitrified on a nitrogen-cooled metal plate ]. The resolution of apoferritin maps in vitreous water ranged from 2.83 to 2.52 Å, with up to 51 200 particles, depending on the sample [Fig. 4b)]. FSC curves for vitreous and devitrified apoferritin specimens are shown in Supplementary Fig. S4. The preferred orientation of alcohol oxidase particles on the grid ] indicated an initial drift of 0.64 Å per frame (∼1.2 e Å−2) in cubic ice and 2.28 Å in vitreous water, which subsided to 0.46 Å (devitrified) and 1.01 Å (vitrified) per frame after ∼5 e Å−2. Average particle trajectories of apoferritin [Fig. 5b)] indicated an initial drift of 0.50 Å (ApoF devitrified 1) and 0.66 Å (ApoF devitrfied 2) per frame, which decreased to 0.30 and 0.33 Å, respectively, after ∼5 e Å−2. On the devitrified apoferritin grids, movement in the second phase was more erratic. In the vitreous apoferritin sample (ApoF vitreous 1–7) the initial drift ranged from 1.64 to 2.59 Å per frame and decreased to 0.69–1.4 Å after ∼5 e Å−2. Devitrification thus reduced the drift by a factor of ∼4 in the initial frames and by a factor of ∼2 in subsequent frames. The relative per-frame B factors of the early frames were correspondingly better for devitrified specimens  of each movie stack in the ApoF devitrified 1 data set achieved a resolution of 3.1 Å, the highest of all single-frame reconstructions. In the ApoF devitrified 2 data set frame 4 was the best, but both were very much better than any of the seven vitreous samples. The resolution of first-frame (fluence of 1.15–1.23 e Å−2) reconstructions of apoferritin in vitreous water ranged from 3.6 to 4.5 Å and was thus substantially worse than for devitrified samples. In movie stacks of vitreous samples, frames 4–7 were consistently better than earlier or later frames.The map reconstructed from the first frames  B factors  from devitrified grids are consistently better than those from vitrified samples. This is clearly demonstrated by Figs. 7Many factors influence the quality of an electron cryo-micrograph, and therefore great care has to be taken when comparing micrographs from different grids acquired in separate microscope sessions. Different ice thicknesses, grid handling, electron-optical settings or different image-acquisition routines can all affect the quality of the resulting high-resolution density map. Even though we attempted to keep all these parameters constant, the quality of individual grids, grid squares and image stacks inevitably varies. This is demonstrated by the resolution achieved with horse apoferritin on seven different vitrified samples, which ranged from 2.52 to 2.83 Å with 51 200 particles Fig. 4. Presumaet al., 2016et al., 2020The behavior of vitreous water upon warming is difficult to control and devitrification on an EM grid can be erratic. Occasionally, grids did not devitrify even though they were kept on the grid heater for minutes, presumably owing to small differences in local surface temperature. Often, large contaminating ice crystals formed on the grid squares, most likely from atmospheric water, even though all experiments were conducted in the same way at constant, low humidity in an atmosphere of cold, dry nitrogen gas. On these grid squares water within the foil holes remained vitreous. Surprisingly, we never found contaminating ice and clean cubic ice on the same grid square. At present it is not clear why some grid squares do not contaminate and warm up sufficiently for the formation of cubic ice. It is most likely that this is due to local thermal gradients that vary from grid to grid. Another complication arises from the requirement that for high-resolution imaging areas of successful devitrification need to coincide with areas of suitable ice thickness. New approaches to the preparation of better cryo-EM specimens (Razinkov Ice contamination remained problematic when the grid was warmed up in liquid ethane or in the vacuum of a microscope column. Attempts to devitrify grids in freshly prepared liquid ethane at a controlled temperature above the glass-transition temperature of water resulted in ice contamination that was more subtle than in a dry nitrogen atmosphere but was evident at low magnification. The contamination resembled ‘leopardskin ice’ Fig. 2, which oIn principle, it should be possible to avoid ice contamination when grids are devitrified in the high vacuum of an electron microscope, and this has worked in previous experiments (Cyrklaff & Kühlbrandt, 1994et al., 1996et al., 2019To take full advantage of the improved stability of the cubic ice lattice, it would be desirable to devitrify specimens on all-gold grids (Russo & Passmore, 2014et al., 2020With the radiation-sensitive biological samples studied by cryo-EM, it is particularly important to extract as much information as possible from the precious early frames of a movie stack that are recorded before the sample has suffered extensive radiation damage. By transforming vitreous water to mechanically more stable cubic ice, stress trapped in the specimen during rapid vitrification is released prior to electron irradiation and this reduces beam-induced motion in early frames. The novel all-gold hexAuFoil specimen supports that reduce beam-induced motion to less than 1 Å r.m.s.d. appear to resolve this long-standing issue (Naydenova 10.1107/S2052252520016243/pw5016sup1.pdfSupplementary Figures. DOI:


Decades of studies have established that nuclear lamin polymers form the nuclear lamina, a protein meshwork that supports the nuclear envelope structure and tethers heterochromatin to the nuclear periphery. Much less is known about unpolymerized nuclear lamins in the nuclear interior, some of which are now known to undergo specific phosphorylation. A recent finding that phosphorylated lamins bind gene enhancer regions offers a new hypothesis that lamin phosphorylation may influence transcriptional regulation in the nuclear interior. In this review, we discuss the regulation, localization, and functions of phosphorylated lamins. We summarize kinases that phosphorylate lamins in a variety of biological contexts. Our discussion extends to laminopathies, a spectrum of degenerative disorders caused by lamin gene mutations, such as cardiomyopathies and progeria. We compare the prevailing hypothesis for laminopathy pathogenesis based on lamins’ function at the nuclear lamina with an emerging hypothesis based on phosphorylated lamins’ function in the nuclear interior. The nuclear lamina is a protein meshwork that covers the nuclear side of the inner nuclear membrane in animal cells. Nuclear lamins are a class of intermediate filament proteins and constitute the nuclear lamina by polymerizing and assembling into filaments. The nuclear lamina provides structural integrity to the nucleus and serves as a scaffold for interphase chromosomes by tethering heterochromatin domains to the nuclear periphery . In addiLMNA in humans. B-type lamins include Lamin B1 encoded by LMNB1 and Lamin B2 encoded by LMNB2 in humans. The A-type lamin gene arose in vertebrate evolution from the ancestral B-type lamin genes, which are conserved across metazoans [There are two nuclear lamin types, A-type and B-type . A-type etazoans . Each laetazoans ,16. A-tyetazoans . In contetazoans ,22. The Nuclear lamins have also been observed in the interior of the nucleus of interphase cells ,23–25. NPhosphorylation of nuclear-peripheral lamins provides the mechanistic basis for nuclear lamina disassembly during the mitosis phase of the cell cycle. Nuclear lamin phosphorylation causes lamin depolymerization at the onset of mitosis for nuclear envelope breakdown . At the In this review, we summarize the current understanding of molecular features, localization, regulation, and functions of phosphorylated nuclear lamins. We distinguish the various cellular pathways through which lamins are phosphorylated. We discuss our recent observation suggesting that phosphorylated lamins act as transcriptional activators at enhancers in the nuclear interior. Finally, we extend our discussion to the ways in which laminopathy-causing mutations might influence lamin phosphorylation and the functions of phosphorylated lamins, offering new hypotheses for the pathogenesis of laminopathies.Nuclear lamins are composed of three structural domains: the short N-terminal head domain , the central rod domain (aa34-383 in Lamin A/C), and the C-terminal tail domain (aa384-646 in Lamin A)  Figure 1in vitro [in vivo [in vitro [in vivo [Phosphorylation of nuclear lamins reaches the highest level at the onset of the mitosis phase of the cell cycle to disassemble the lamin polymers ,43,45. Min vitro ,47 and i[in vivo . Evidenc[in vivo . Convers[in vivo . In addi[in vivo ,35,50 , two closely related kinases active under various extracellular stimuli. ERK1 and ERK2 phosphorylate Lamin A/C at Ser22 . Ser22 o in vivo . An inte in vivo ,86. In t in vivo ,86. Anot in vivo . Akt pho in vivo ,89. Akt patients . In addipatients .Several lamin kinases are known to promote extensive modulation of the nuclear lamina in interphase cells. PKC-δ phosphorylates B-type lamins for nuclear lamina disassembly during apoptosis ,92. DuriFinally, nuclear lamin phosphorylation can be catalyzed by viral kinases for virus egress. UL97, a CDK1-like kinase of human cytomegalovirus (HCMV), phosphorylates Lamin A/C at Ser22 . ConsistIn summary, a number of kinases and phosphatases operate on nuclear lamins within diverse biological contexts, sometimes at identical residues. It is plausible that these kinases and phosphatases regulate the equilibrium between polymer lamins and unpolymerized lamins in the interphase nuclei . What reD) for interactions between the C-terminal domain of Lamin A/C and DNA or nucleosomes [Nuclear lamins exhibit a high affinity to DNA and chromatin, with a nano-molar range dissociation constant , a premature aging syndrome caused by heterozygous utations . HGPS isrogerin’ . Progerirogerin’ . Progerirogerin’ . Supportrogerin’ ,140. Simrogerin’ .LMNA-related dilated cardiomyopathy patients [LMNA-related cardiomyopathy-patient iPSCs and identified genomic regions that lose heterochromatin [Recently, several groups, including ours, have conducted detailed analyses to define whether losses of heterochromatin are responsible for dysregulated gene expression in laminopathies. Our parallel analysis of gene expression, lamina–chromatin interaction, and histone modifications revealed that only a very small number of dysregulated genes are located within lost LADs in progeria-patient fibroblasts, although LAD losses do accompany losses of heterochromatin-associated histone modifications . Similarpatients . Howeverpatients . Berterohromatin . Again, hromatin . TogetheAn emerging new hypothesis, drawing upon evidence provided by recent reports on phosphorylated lamin activity, is that impaired functions of phosphorylated Lamin A/C in the nuclear interior underlie the pathogenesis of laminopathies. We therefore recently examined whether interactions between Ser22-phosphorylated Lamin C and enhancers are altered in the fibroblasts derived from progeria patients. This investigation led us to observe that a specific subset of enhancer-like elements either gain or lose interactions with Ser22-phosphorylated Lamin C in progeria . Consisthttp://www.umd.be/LMNA/). Second, pathogenic mutations may alter kinase-recognition motifs surrounding phosphorylation sites. While this scenario has not been explored in detail, Lin et al. predicted that pathogenic LMNA mutations decrease Lamin A/C phosphorylation overall in an in silico analysis [LMNA-related muscular-dystrophy patients only when mutations are located within the Ig fold [LMNA mutations or cells expressing mutant Lamin A that causes non-myopathic laminopathies [LMNA mutations render Ser458 accessible to Akt1 through a conformational change of the Ig fold domain [LMNA-wild-type HeLa cells [LMNA gene mutations [LMNA mutations could affect Lamin A/C phosphorylation. Overall, however, only a small number of studies have investigated the impact of the laminopathy mutations on lamin phosphorylation. Therefore, an unbiased survey of the phosphorylation state of Lamin A/C in tissues affected in laminopathies is warranted.There are multiple ways in which laminopathy mutations could affect nuclear lamin phosphorylation. First, laminopathy mutations could lead to nonsynonymous substitutions of the residues subject to phosphorylation. Such pathogenic substitutions in Lamin A/C include Thr10Ile associated with lipodystrophy , Ser22Leanalysis . Third,  Ig fold . In contopathies . The autd domain . HoweverLa cells . Finallyutations . Lin et utations . These sOur review catalogs an extensive repertoire of phosphorylation sites in nuclear lamins as well as kinases and phosphatases that regulate lamin phosphorylation (Supplementary Table 1). We recognize a wide variety of biological contexts that promote lamin phosphorylation during interphase in vivo, and a proteome-wide survey of lamin phosphorylation in normal and disease tissues. Finally, it should be noted that other post-translational modifications, such as sumoylation, acetylation, and ADP-ribosylation, have been reported for lamins but not studied as extensively as phosphorylation [Our review also clarifies a number of important questions that have been left unanswered. These questions concern the biological contexts in which nuclear lamin phosphorylation is regulated by specific kinases and phosphatases; the subcellular locations at which these kinases and phosphatases operate on lamins; the functions of phosphorylated nuclear lamins at chromatin and in other cellular processes; and the mechanisms by which laminopathy mutations affect lamin phosphorylation and the functions of phosphorylated lamins. Addressing these questions will require the development of new tools and techniques, such as phosphorylation-specific antibodies, manipulation of phosphorylation states rylation ,151. We Click here for additional data file.


The three large volcanoes in the Tharsis region of Mars: Arsia, Pavonis, and Ascraeus Montes all have fan-shaped deposits (FSDs) on their northern or western flanks consisting of a combination of parallel ridges, knobby/hummocky terrain, and a smooth, viscous flow-like unit. The FSDs are hypothesized to have formed in the Amazonian during a period of high spin-axis obliquity which redistributed polar ice to the equatorial Tharsis region resulting in thick (> 2 km), flowing ice deposits. Based on previous ice flow simulations and crater surveys, the ridges are interpreted to be recessional drop moraines formed as debris on the ice sheet surface was transported to the ice margin—forming a long ridge sequence over an extended (∼100 Myr) period of ice sheet retreat. We test this hypothesis using a high-resolution, thermomechanical ice sheet model assuming a lower ice loss rate (~ 0.5 mm/year) than prior work based on new experimental results of ice sublimation below a protective debris layer. Our ice flow simulation results, when combined with topographic observations from a long sequence of ridges located interior of the Pavonis FSD, show that the ridged units were more likely deposited during one or more periods of glacial advance (instead of retreat) when repetitive pulses (approx. 120 kyr periodicity) of ice accumulation during high obliquity produced kinematic waves which advected a large volume of surface debris to the ice margin. If ridge deposition does occur during glacial advance, it could explain the cyclic pattern of ridge spacing and would link the dominant, 120 kyr periodicity in obliquity to the time interval between adjacent ridges. By measuring the spacing between these ridges and applying this timescale, we constrain the velocity of glacial margin to be between 0.2 and 4 cm/Earth year—in close agreement with the numerical simulation. This re-interpretation of the FSD ridged unit suggests that the timescale of FSD formation (and perhaps the duration of the Amazonian high obliquity period) was shorter than previously reported. The fan-shaped deposits (FSDs) on the Tharsis volcanoes  Fig. a were fiIn this study, we investigate the influence of time-varying obliquity on the ice margin flow velocity, location, and shape using the thermomechanical Parallel Ice Sheet Model (PISM). Our simulation is compared with the observed spacing and cross-sectional area of ridges found within the Pavonis FSD in order to determine whether moraine deposition is more likely to occur during advance or retreat of the ice sheet. We first summarize the prior work regarding the FSD ridged unit and its geologic interpretation. Next, we describe the size and location of ridges at Pavonis Mons using CTX images and DEMs generated from CTX stereo pairs at both Pavonis and Arsia Montes. In the following section, we provide a description of the numerical model, its inputs, and assumptions. The final sections compare the simulation results with the observations, discuss the process(es) responsible for ridge emplacement, and summarize our findings.The arcuate, distal portion of the FSDs generally consists of a series of parallel ridges (∼1 km spacing) with variable heights ranging from 5 to 50 m based on MOLA laser altimetry observations conducted by Head et al.  at ArsiaGeologic mapping efforts in the Tharsis region have made use of crater abundances and superposition relationships to conclude that the FSD formed in the late Amazonian and were likely contemporaneous with the most recent volcanic activity on both the volcanic edifices and radial rift zones of Arsia, Pavonis, and Ascraeus Mons. Landforms such as tuyas and tiered, mounded, leveed and digitate lava flows, which are indicative of lava-ice interaction, have been found in all three FSDs  of retreat of the ice margin poses a conundrum regarding the Amazonian climate. In order to deposit a moraine, the ice margin needs to be roughly stationary while ice flow continues to advect ice and englacial/supraglacial debris to the ice margin. The location of the ice margin is a sensitive function of the aerial extent and rates of ablation and accumulation but is stationary when the total mass accumulated over the ice sheet is equal to the mass ablated (steady state). The conundrum arises from the observation that the moraines are so closely spaced (∼ 1 km) which accounts for a change of less than 1% of the length of the ice deposit assuming precipitation occurs on the slopes of the volcanic edifices as indicated in GCM results by Forget et al. . Such smSuperposition of remnant ice deposits (smooth unit) on ridges Fig.  and inteDuring retreat of the ice margin, relatively warm periods (during times of low obliquity) may temporarily enhance ice flow and stall the migration of the ice margin to result in a ridge.During glacial advance, the margin of the ice deposit may be slowed during times of high obliquity (cooler ice temperatures) which could allow even a low sublimation rate to maintain a temporarily fixed ice margin position—allowing debris to accumulate and a ridge to form.Renewed ice accumulation at the head of a sublimating ice deposit during high obliquity may produce a kinematic (velocity) wave which may collect surface debris and deposit a ridge upon reaching the ice margin.The steepness of the ice margin is expected to increase along with the ice viscosity during cold temperatures experienced during high obliquity at the equator which may cause slope failure of the surface lag and the formation of a ridge at the ice margin.The dry, cold Amazonian climate of Mars limits the scope of potential ridge-forming mechanisms to those which would operate under cold-based, dry glacial conditions. Given the dominance of obliquity on the Amazonian climate  responsible for depositing ridges by combining results from a thermomechanical ice sheet simulation with an analysis of the morphology of a series of ridges north of Pavonis Mons. Our model inputs assume ice accumulates in accord with GCM results at high obliquity and ice ablation occurs at a rate associated with the formation of a protective lag cover.Understanding the process of ridge formation is an essential component to decoding the Amazonian climate change record contained in the FSDs and for understanding how active the Martian water cycle was in the recent past.In contrast with ridges in the Arsia FSD, the Pavonis Mons ridges are generally wider, taller, are exposed over a larger portion of the FSD, and exhibit a quasi-periodic pattern in their spacing which make them an intriguing feature to study Figs.  and 3. AThe spacing between ∼ 40 ridges are measured using the CTX global mosaic (5.5 m/px) of the FSD interior Fig. a. DependThe mean spacing between ridges varies in a quasi-periodic fashion in which ridges are densely spaced in the southernmost portion of the FSD, become more widely spaced to the north, with this pattern repeating again Fig. b before °), asymmetric slopes in which the distal slope is steeper than the proximal (Price 1970).The amount of material within the ridges provides an additional constraint on the deposition timescale and debris layer thickness  and cross-sectional area provide useful information which can be combined with the ice sheet simulation results.To investigate the influence of obliquity on glacier dynamics and to constrain the ice flow velocity, we performed numerical simulations by adapting the open-source Parallel Ice Sheet Model (PISM)  with time  the simulation vertically integrates the shear strain rate given by Glen’s Law ) which gives:To calculate the horizontal velocity at elevation (h is the ice surface elevation and A(T∗) is a temperature-dependent ice softness parameter given by  and is also dependent on ice grain size despite terrestrial field evidence for n = 3 behavior even at low differential stress  ice sheets which generally result in differential stresses in excess of 100 kPa (see “Results and discussion” section) where n = 3 behavior tends to dominate , and apply a spatially and temporally constant basal heat flux of 30 mW m− 2 based on the estimated flux for Tharsis  DEM regridded to 500 m resolution. In order to reduce computational demand associated with a high spatial resolution, we use PISM in a flowline mode where flow occurs along a single axis (x-direction) which follows the steepest gradient down the northwest flank of Pavonis Mons and through the FSD  and low (30o at t = − 24.8 Myr) obliquity are shown in Fig. t = − 25.1 and − 24.8 Myr, respectively. Topographic profiles and instantaneous mass balance conditions at these times are given in panels b and c, while panels d and e give ice thickness profiles. The shaded region in Fig. Snapshots of the model during high  of ice exposed by a recent impact  based on repeat imagery (at 43.6Es) of buried, non-deforming ice using a water vapor diffusion equation modified from Chevrier et al.  in exchange with ice is given by the Clausius-Clapeyron equation:pt = 611 Pa and Tt = 273 K give the triple point of water and Hsubl. = 5.1058 × 104 J mol− 1 is the latent heat of sublimation. Combining Eqs.. In order to evaluate the timescale of the lag formation process and to determine the ice ablation rate in the model, we calculated the sublimation rate  while mid-latitude ice deposits have dust concentrations limited to < 10 vol%  is short compared with the few million years it takes ice to flow from the accumulation zone to the inner ridges of the FSD see “” sectionIdeally, we would hope to model the temporal and spatial variation in debris lag thickness within PISM as sublimation occurs and surface debris is advected , but this computationally and work-intensive sub-model is left as a topic for future research. For the purposes of this study, our focus is to provide PISM with a set of conditions representative of the Martian climate and debris layer during FSD formation which can provide a basis for further investigation into the moraine deposition process. Our mass balance parameterizations were chosen after exploring several different maximum accumulation and ablation rates to give an ice extent that reaches the interior set of ridges but does not flow beyond the extent of the FSD during the last 26 Myr of known obliquity variations.Constraining the age of the Pavonis FSD has been a difficult task as early crater counts using coarse (∼ 100 m/pixel) resolution THEMIS imagery failed to recognize that the thin, ridged unit of the FSD actually superpose fresh-looking impacts  is given by Ward  althougTeq):A is surface albedo and σ is the Stefan-Boltzmann constant. Variation in obliquity and eccentricity over the past 26 Myr determined by Laskar et al.  based on the shape of the logistic curve shown in Fig. The computed ice mass balance model input is shown in Fig. In order to determine when and where moraine deposition would be most likely from the PISM results, we saved ice surface velocities from the model every 2 kyr during the first 4 Myr of our simulation (which starts at − 26 Myr). We then imported these data into a Python script which tracked the advection of particles placed on the surface of the ice sheet using a 2D (time and space) linear interpolation.The location of particle deposition is at the base of Pavonis with all particles being placed at the − 100 km point on the flowline  and the number of model outputs used (every 2 kyr) pose a challenge to directly simulate ridge formation due to the generation of large file sizes and long computation times. Although temporal interpolation between outputs and spatial interpolation between model grid points is necessary, the particle-tracking analysis shown in Fig. Plots 13a–f show the density of particles on the ice surface and those that have been deposited on the substrate as blue and red histograms (500 m bin sizes), respectively. Between − 25.15 must be collected in order to deposit a ridge with a given cross-sectional area (A). Assuming that the ice margin is stagnant, and that both the lag layer and ridges are ice-free, this “debris collection distance” is:A consistency check on the kinematic wave deposition hypothesis comes from estimating the longitudinal distance (Dist) along the ice deposit which surface lag debris of thickness (L = 0.5 m) and a mean interior ridge area of 5000 m2  obliquity changes. This result is a departure from prior interpretations which suggested ridges were the result of longer term (~Myr) periods of near steady-state conditions. We revisit the potential ridge-forming mechanisms described in the introduction in the context of our simulation results:Ice sheet thinning results in the cessation of ice flow during ice margin retreat even during times of low obliquity (warm ice conditions) Fig. b, c. TheIce accumulation and, to a lesser extent, basal temperature changes affect the ice flow velocity in our simulations. During advance, the average ice sheet margin velocity is initially ~ 10 cm/year but slows to about 0.5 cm/year after a million years of flow Fig. . Ice surA pulsating advance of the ice margin results from several episodes of accumulation during short-lived periods of high obliquity associated with the 120 kyr periodicity in obliquity Fig. a. HoweveDespite this assumption, a thick, narrow pulse of surface debris does form at about − 20 Myr when a stagnant toe is reactivated by renewed accumulation producing a propagating velocity “step” Fig. c. In ourH)—given by:h/∂x) must be greater on Mars to compensate for the reduction in g. Identical simulations performed under Earth gravity resulted in a decrease in the ice margin slope from roughly 15° under Martian gravity to 10°  for the higher heat flux case. Therefore, the simulation presented here  of 120 kyr. Dividing the lengthscale by the timescale gives an estimate for the velocity of a thermal pulse through a Martian ice deposit  which is similar to modeled ice flow rates . In particular, the influence of the surface slope on ice temperature may help explain the larger size of ridges found within the Pavonis FSD. As ice advanced northward across the Pavonis FSD, ice temperatures at the toe may have varied markedly as the angle of incident sunlight changed with obliquity—resulting in more accentuated episodes of advance during high obliquity followed by a cold, stagnant margin during low obliquity. In this respect, PISM may be better suited to simulating the westward ice flow which occurred in the Arsia FSD where the margin slope orientation would have less influence on temperature which may account for the smaller, more closely spaced ridges found there.The surface temperature also plays a key role in determining the ice sublimation rate Eqs.  and 6. ADisregarding other factors (atmospheric deposition of volcanic ash or dust) that may influence the debris layer thickness, the factor of ~ 20 increase in particle concentration produced from accumulating surface debris in a kinematic wave suggests that the original sublimation lag thickness can be very roughly estimated by dividing the height of the ridge by 20. However, this “particle concentration factor” will likely change depending on location—increasing as the ice sheet becomes more extensive and debris can be collected over a larger area. Most interior ridges have heights of < 40 m suggesting an original surface debris thickness of < 2 m which would take less than 200 kyr to form from the sublimation of a 5 vol% dusty ice deposit Fig. a. Subseq− 2, deformation-induced strain heat, and obliquity-driven surface temperature variations while advection accounts for the horizontal heat transport. Horizontal ice flow rates are calculated using Glen’s Law (Eq. Thermomechanical ice sheet simulation of the Amazonian Pavonis ice sheet using obliquity inputs from the past 26 Myr provides important insights into the formation of a series of ridges in the interior of the Pavonis FSD. Our simulation assumes an obliquity-precipitation relationship given in Fig.  Law Eq.  under thThe reduced ablation rate used in our simulation is a key departure from previous Tharsis ice sheet simulations performed by Fastook et al. . The lowInstead, we propose that ridges are deposited during glacial advance when precipitation-induced perturbations to the surface ice velocity Fig. b concentWe conclude that initial sublimation of the uppermost portions of the ice deposit likely resulted in the formation of a debris lag which protected ice from further ablation—allowing it to slowly advance and deposit moraines as its flow velocity periodically fluctuated with obliquity. This new interpretation of the FSD ridge formation process provides an opportunity to further constrain the Amazonian climate history of Mars.Additional file 1: Figure S1. Obliquity History Five representative scenarios for the obliquity of Mars determined by Laskar et al. Additional file 2. Pavonis Simulation 20 mW/m2. Animated plots of the ice flow simulation which uses a basal heat flux of 20 mW/m2 showing the extent, thickness, basal stress, approximate heat flux, ice temperature, ice effective viscosity, and ice surface velocity.Additional file 3. Pavonis Simulation Earth Gravity 20 mW/m2. Animated plots of the ice flow simulation using Earth-like gravity and a basal heat flux of 20 mW/m2 showing the extent, thickness, basal stress, approximate heat flux, ice temperature, ice effective viscosity, and ice surface velocity.Additional file 4. Pavonis Simulation 30 mW/m2. Animated plots of the ice flow simulation presented in the text which uses a basal heat flux of 30 mW/m2 showing the extent, thickness, basal stress, approximate heat flux, ice temperature, ice effective viscosity, and ice surface velocity.


O-allylation of polyols with readily available reagents and catalysts. Through exchanging the Lewis acid additives that recognize specific hydroxyls in a polyol substrate, our system managed to install a versatile allyl group to the target in a site-switchable manner. Our design demonstrates remarkable scope, and is amenable to the direct derivatization of various complex, bioactive natural products.Site-selective modification of complex molecules allows for rapid accesses to their analogues and derivatives, and, therefore, offers highly valuable opportunities to probe their functions. However, to selectively manipulate one out of many repeatedly occurring functional groups within a substrate represents a grand challenge in chemistry. Yet more demanding is to develop methods in which alterations to the reaction conditions lead to switching of the specific site of reaction. We report herein the development of a Pd/Lewis acid co-catalytic system that achieves not only site-selective, but site-switchable mono- O-allylation of polyols.Selective manipulation of one functional group, out of many repeatedly occurring in a substrate, represents a grand challenge in chemistry. Here, the authors report a Pd/Lewis acid cocatalytic system that achieves not only site-selective, but also site-switchable mono- The same kind of functional groups tend to undergo similar transformations, however, and differentiation typically only occurs due to subtle steric and electronic environments. Accordingly, to achieve high site-selectivity following a generalizable strategy remains a significant task in chemistry8. Even more demanding is the development of methods in which alterations to the reaction conditions result in switching of the specific site of reaction . Recently, systems that can override the intrinsic reactivity preferences and accomplish catalyst-controlled, site-switchable modification of complex polyols have emerged35. As eminent examples, in a series of landmark studies, the Miller group26, has identified several oligopeptide-based catalysts that enabled site-switchable modification of complex antibiotics, such as vancomycin, teicoplanin, and erythromycin. In these studies, the oligopeptide catalysts were designed as mimetics of the catalytic domains of enzymes. Kawabata et al.31 devised chiral pyridine derivatives that were used in the site-selective modification of C4-OH of glucopyranosides as well as natural products such as lanatoside C and avermectin B2a. The Tan group32 invented a pair of pseudoenantiomeric imidazole-based catalysts that allow site-divergent modification of polyols containing cis-1,2-diol moieties, including anticancer agent digitoxin. Nargony and coworkers33 employed chiral phosphoric acid catalysts to accomplish site-switchable glycosylation of 6-deoxy erythronolide. Our group34 reported the site-divergent O-propargylation of various monosaccharides as well as digitoxin employing a pair of chiral Cu-catalysts. These achievements notwithstanding, to develop general systems that can achieve catalyst-controlled, switchable site-selectivity still represents a challenge. In particular, methods capable of introducing a metabolically stable ether bond remain rather limited.Polyhydroxylated natural products, carbohydrates in particular, play critical roles in virtually all biological processes and they are essential components of many pharmaceuticals Fig. . Rapid aO-allylation of polyols by Pd/Lewis acid co-catalysis36. As a distinct feature of our system, the task of activating electrophiles and that of controlling site-selectivity were allocated, respectively, to the Pd-catalyst and the Lewis acid additive. Resembling the role of a gRNA in the Cas/gRNA37 system, the Lewis acid additive serves as a guide in our system, and determines the site of reaction in the modification of various polyols . More importantly, in this regime, switch of the reaction site could in principle be realized simply by tuning the properties  of the Lewis acid additives. Compared with approaches to achieve selectivity switch through modifying the whole catalytic complex  for each substrate, the advantage of our design lies in the greater ease and extent of changing the characters of Lewis acid additives.We were interested in the selective O-allylation of glucopyranoside 9 . Notably, the O’Doherty group41 reported the use of Ph2BOH to achieve Pd-catalyzed site-selective glycosylation. More intriguingly, the use of MgBu2 as an additive led to the preferential formation of the other isomer 9a, albeit with moderate selectivity (entry 7). Further optimization of reaction conditions revealed that simultaneous addition of MgBu2 with BINOL afforded 9a in dramatically improved site-selectivity and efficiency (entry 8). Thus, we identified two sets of conditions that install an allyl group onto the two different hydroxyl groups in 9 . These conditions employ an identical transition metal complex  and differ only in the oxophilic additives utilized [Mg(II) vs. B(III)]. Collectively, these results validated the proof of principle of a strategically distinct approach to achieve reagent-controlled, site-switchable modification of polyols.The validity of our design was first established in the site-divergent mono-e 9 Fig. . Compoun10), galactose (11–12), and fucose (13). All of these monosaccharides contain three contiguous secondary hydroxyls. In these cases, the allylation occurs at the C3-OH of the pyranose ring if an organoboron compound42 was used as additive (green arrows), but at the C2-OH when ZnEt2-prophenol complex43 was employed (orange arrows). To rationalize the switchable site-selectivity observed in these cases . The C2-OH, on the other hand, is most readily deprotonated by a base since it is closest to the electron-withdrawing anomeric center (cf. structure B).Following the above protocol, we proceeded to explore the generality of our strategy. We first examined this method in the site-selective modification of various monosaccharides Fig. , includi fucose 1. All of . 9 Fig. , we prop14 could be selectively alkylated in a controllable manner. As can be explained by the structures A and B shown above, the use of LiHMDS (orange arrow) and quinolin-8-ylboronic acid (green arrow) furnished the C2-OH allylated product 14a and the C3-OH allylated product 14b, respectively. The C4-OH allylated product 14c could be obtained by using 4-dimethylaminophenylboronic acid, which, through formation of a stable boronic ester (cf. structure C), deactivates the C2-OH and C3-OH45 and simultaneously enhances the reactivity of the C4-OH position.Each of the three hydroxyls in mannopyranoside 15 contains five free hydroxyl groups. The use of ZnEt2-prophenol complex directed the allyl group to the C2-OH of 15 with high selectivity (orange arrow). Switching the additive to phenylboronic acid altered the preferential reaction site to the C3-OH (green arrow). Moving to maltoside 16, we found the use of Taylor’s catalyst46  guided the installation of the allyl group to the C3-OH, while employment of Cu(OAc)2 steered the allyl group to the C2-OH instead. It is worth noting that synthetically versatile but chelating thioether groups present in 15 and 16 were well tolerated by our conditions. We further applied this method to the site-divergent modification of fructoside 17, which contains a pyranoside ring linked to a furanoside ring. We precisely delivered the allyl group to the C3-OH of the pyranoside ring using Taylor’s catalyst as an additive, or to the C3’-OH of the furanoside ring if applying Cu(OAc)2, instead. Here, we rationalize that Taylor’s catalyst complexes with the C3-OH of the pyranose ring (see structure E), but Cu(OAc)2 chelates47 with the C2-OH and the C3’-OH of 17 and activates the C3’-OH (cf. structure D). It is worth highlighting that, in this case, the selectivity switches between two hydroxyls that are six atoms apart. Therefore, it would be challenging to achieve a selectivity switch between these two hydroxyls through designing Pd-ligand complexes.We then showed this strategy could be adopted toward the site-switchable allylation of structurally more complex disaccharides Fig. . For exa18) is an ecdysteroid hormone that controls the metamorphosis and ecdysis of arthropods. This compound contains six free hydroxyls, three of which are secondary. Site-divergent modification of 18 could be achieved. If Taylor’s catalyst was employed as additive, the allylation occurred preferentially at the C2-OH (green arrow), whereas if Zn(II)-Prophenol complex was used, the allylation occurred at the C3-OH (orange arrow). Geniposide (19) is an iridoid glycoside that exhibits diverse biological activities. In addition to five free hydroxyls, this compound also contains a sensitive enol ether moiety and an enoate ester group. Employing our strategy, we could selectively install an allyl group to the C6-OH of the glucose ring employing Taylor’s catalyst, or to the C1’-OH with phenylboronic acid as an additive. Here, the site of reaction switches cleanly between two primary hydroxyls.We continued to apply our strategy to the site-switchable modification of polyhydroxylated natural products Fig. . 20-Hydr19) is a natural product that acts as an anti-inflammatory agent in the human body. The C2-OH of the glucopyranoside ring in 19 is intrinsically the most reactive hydroxyl and gets allylated preferentially in the absence of any additives (blue arrow). Intriguingly, applying Taylor’s catalyst as an additive in the standard reaction conditions delivered the allyl group to the C6-OH of 19 (green arrow), presumably via the intermediacy of the ate complex F. If the bulky, monovalent BPh3 was employed instead (orange arrow), the allylation occurs at the benzylic hydroxyl (C1’-OH) with high site-selectivity (cf. G). Thus, three individual hydroxyls within salicin could be selectively modified in a controllable fashion.Following the above principle, we were able to selectively modify up to three different hydroxyl groups within some complex polyols. For example, salicin (21) exhibits potent antiproliferative activities against several cancer cell lines at nanomolar concentrations49. This molecule contains eight free hydroxyls, including one primary, five secondary, and two tertiary ones. When no Lewis acid additive is used, the Pd-catalyzed allylation occurs at the secondary C1-OH to afford 21a with decent selectivity and efficiency (orange arrow). When we used methylboronic acid as an additive, the primary C19-OH was selectively modified to give product 21b in high yield (blue arrow). We hypothesize that methylboronic acid simultaneously complexed with the C1-OH, C5-OH and C19-OH of 21 (see structure H), and activated the least hindered primary hydroxyl in the Pd-catalyzed allylation reactions. If the oxaborole derivative 22 was employed instead, the site-selectivity changed again. This time the C3’-OH of the sugar ring was modified with high efficiency (green arrow). We assume that the bulky oxaborole 22 will preferentially form an ate complex with the vicinal diol moiety in the sugar ring (see structure I), which renders the equatorial C3’-OH most reactive. Lastly, the group that can be transferred by our method is not restricted to the parent allyl unit. For instance, cinnamyl groups bearing various substituents could be installed onto ouabain with good site-selectivities as well, if the corresponding electrophiles are employed , a fluorine53 atom (22c), or a pyridine54 ring (22d) onto the target molecule. Furthermore, a cross-metathesis reaction55 readily converted 20b to a dimeric structure 22e. Lastly, we prepared a conjugate of ouabain and glucose (24) from 21c and 23, taking advantage of the potent thiol-ene click reaction56. Therefore, when coupled with the synthetic versatility of alkenes, our site-switchable O-allylation reaction provides rapid and efficient accesses to derivatives and analogues of complex polyols.To further demonstrate the utility of this method as a tool for the late stage functionalization of naturally occurring polyols, we have performed the reactions shown in Fig. This study showcased the power of a dual catalytic strategy for site-switchable modification of target molecules, a key challenge in chemistry. This design allocates the task of activating electrophiles and that of controlling site-selectivity to, respectively, the Pd-catalyst and the Lewis acid additive. Thus, switch of reaction site was achieved simply through changing the identity of Lewis acid additives, a tactic that has not been systematically explored before. The allyl unit installed by this method could serve as an entry point to various other functional groups. We anticipate that the generality, immediate utility, and the operational ease of this method will inspire further exploration that ultimately leads to precise control of site-selectivity during modification of complex compounds.2-filled glovebox, nBu2Mg , (±)-BINOL  and THF (200 μL) were weighed into a screw capped vial  containing a stir bar. The mixture was stirred for 5 min, at which time compound 9  was added. The resulting solution was stirred for an additional 30 min.In a N2 , Pd2(dba)3•CHCl3 , PPh3 , and THF (200 μL) were added in sequence. The mixture was stirred for 10 min, and then transferred to Vial A. An additional portion of THF (2.6 mL) was added to Vial A so that [9] was adjusted to 0.05 M. Vial A was tightly capped, taken out of the glovebox, and stirred at −10 °C for 12 h (400 rpm). The reaction mixture was then concentrated in vacuo. An aliquot of the residue was taken for 1H NMR analysis, which indicated that products 9a and 9b were formed in a ratio of ca. 10:1. Flash chromatography (SiO2) using petroleum ether/EtOAc (6:1–3:1) as eluent afforded 9a as a yellow solid .To another vial, compound 2-filled glovebox, compound 9  and 2-(diphenylboryloxy)-ethanamine , and THF (500 μL) were weighed into a screw capped vial  containing a stir bar. The resulting solution was stirred for an additional 30 min.In a N2 , Pd2(dba)3•CHCl3 , PPh3 , and THF (200 μL) were added in sequence. The mixture was stirred for 10 min, and then transferred to Vial A. An additional portion of THF (0.3 mL) was added to Vial A so that [9] was adjusted to 0.2 M. Vial A was tightly capped, taken out of the glovebox, and stirred at 25 °C (outside temperature) for 12 h (400 rpm). The reaction mixture was then concentrated in vacuo. An aliquot of the residue was taken for 1H NMR analysis, which indicated that products 9a and 9b were formed in a ratio of 1:13. Flash chromatography (SiO2) using petroleum ether/EtOAc (3:1–1:1) as eluent afforded 9b as a pale yellow oil .To another vial, compound Supplementary Information


Antrophyum obovatum Baker were recently reported in one population on Jeju Island, Korea. In the present study, we surveyed more places to find new independent gametophyte populations of A. obovatum using the rbcL gene sequence-based DNA barcoding technique. We identified two new sites inhabited by independent gametophytes. Archegonia and juvenile sporophytes were independently observed in each location under slightly different environmental conditions. Consequently, in the case of this species, functional sporophyte production is likely suppressed by prezygotic and postzygotic sterility, depending on microenvironmental factors.Ferns have conspicuous sporophytes as the dominant phase in their life cycle; however, the gametophytes are completely separated from the sporophytes and supply their own nutrition, unlike in bryophytes and seed plants. Among the gametophytes, some maintain their populations in the gametophyte phase without progressing to sporophyte production and are known as independent gametophytes. Independent gametophytes of  All land plants generally have distinct gametophyte and sporophyte phases. In the life cycle of bryophytes, the gametophyte  is dominant in the alternation of generations, and the sporophyte  is attached to the gametophyte like an appendage. However, this has been altered in seed plants, and the gametophytes are less developed than the sporophytes and grow entirely inside the sporophyte. In the case of ferns, which belong to vascular plants but are not seed plants, there are also conspicuous sporophytes as the dominant phase (like in seed plants); however, they differ in that the gametophytes are completely separated from the sporophytes and supply their own nutrition.Nayar and Kaur  classifiIndependent gametophytes have mainly been discovered in temperate and continental regions ,19,20,21Antrophyum Kaulf is a member of the vittarioid ferns and comprises about 40 to 50 unclear species that are widespread in the tropics of both the Old and New World [Antrophyum obovatum Baker is mainly distributed in the tropical or subtropical areas of Asia, including the South Yangzi River in China, Taiwan, Vietnam, Thailand, Myanmar, India, Bhutan, Nepal, and the southern area of central Japan [A. obovatum was first reported on Jeju Island in Korea, and our previous research suggested that it was an obligate independent gametophyte based on molecular markers [A. obovatum extremely rare? (2) Is prezygotic sterility the only reason for the change from general gametophytes to independent gametophytes? (3) Are conspecific sporophytes absent on Jeju Island? Therefore, we surveyed more places to find new populations of this species on Jeju Island and carefully observed them to address these questions in detail.ew World . The morew World . Antrophal Japan . The gam markers . HoweverrbcL and matK for plant DNA barcode regions in terms of high universality and high variation, respectively [rbcL region also has high variation in ferns to identify nonhybridized gametophytes [rbcL regions to identify gametophytes and juvenile sporophytes in this study.As fern gametophytes are relatively simple and not differentiated into specialized organs compared with sporophytes , it is vectively , the rbctophytes  and has tophytes ,28,29,30tophytes ,32. For rbcL was successful in all samples of the 12 gametophytes and sporophytes. There was no variation in the rbcL sequences of the gametophytes and sporophytes among the three populations on Jeju Island, and they were completely identical to the A. obovatum sporophyte sequences collected from Japan and Taiwan.Polymerase chain reaction (PCR) amplification of A. obovatum because the condition of the first site was incongruent with the locations where the sporophytes of A. obovatum inhabit tree trunks or rocks [A. obovatum were newly found at two sites, but the environmental conditions of the sites were slightly different. Site A was a shady bunker built by stacked stones in the upper valley under dry conditions  nongemmiferous gametophytes without archegonia (under laboratory conditions), (2) gemmiferous gametophytes without archegonia (sites A and C), and (3) gemmiferous gametophytes with archegonia (site B). What makes these differences?From our observations, there were two differences between the gametophytes of us study , as wellboratory , which wboratory . It is pP. makinoi [Based on the observations of independent gametophytes of  makinoi , there w makinoi . As a re makinoi . Therefo makinoi ,35), thiCertain independent gametophytes have reproductive dysfunction that causes the inability to produce sporophytes, which might result from environmental or genetic factors ,17. BecaA. obovatum reside, site B is more humid than the others because it is surrounded by evergreen trees and has a permanent stream. Humidity affects the number and position of the archegonia. The total number of archegonia in Pteridium aquilinum cultured in high humidity (100%) was nearly four-fold higher than that in low humidity (90%), and the anomalous production of archegonia on the dorsal surface, which are normally produced on the ventral surface in nature, significantly appeared in high humidity conditions [Haplopteris flexuosa (Fee) E. H. Crane was observed at site B, and this is the only vittarioid sporophyte residing on Jeju Island. Our new findings of archegonia and juvenile sporophytes of A. obovatum were restricted to only one site, which is consistent with Kuo, Chen, Shinohara, Ebihara, Kudoh, Sato, Huang, and Chiou [A. obovatum at site B encounter a suitable microhabitat for fertilization, they might produce gametangia. As a result, juvenile sporophytes were observed only at site B.Among the three sites in which the gametophytes of nditions . Althougnd Chiou . When thA. obovatum were considered obligate gametophytes because they were first found in site C and the conspecific sporophytes of these had never been reported in Korea until that time. We wondered if they could not produce gametangia genetically or if the conspecific sporophytes did not actually occur on Jeju Island because no sexual organs were found in the individuals of site C. Fortunately, through successive investigation, we found two additional populations, one of which had archegonia-bearing individuals and then had juvenile sporophytes. Therefore, it was proved that the independent gametophytes of A. obovatum do not completely lose the ability to genetically produce gametangia.In our previous study , the gamVittaria appalachiana, classified as an obligate independent gametophyte by Rumsey and Sheffield [Trichomanes speciosum with independent gametophytes were classified as a facultative independent gametophyte by Rumsey and Sheffield [A. obovatum found in the present study were stunted in nature, and those translocated to the laboratory blanched and died after six months without any morphological changes.Juvenile sporophyte mortality associated with independent gametophytes has been previously reported. In the case of heffield , young sheffield . The juvheffield  and as aheffield , and theheffield . The juvH. flexuosa and archegonia at site B implied that this site is the most suitable place to grow vittarioid ferns compared with other sites on Jeju Island. Independent gametophytes at site B can produce gametangia, and fertilization rarely occurs in this population. However, environmental factors, such as subzero temperatures in winter, are not sufficient for sporophyte survival and might stunt young sporophytes. As a result, sporophytes might die before maturation, and this postzygotic sterility might keep the population as obligate independent gametophytes.As mentioned above, the three sites inhabited by independent gametophytes had slightly different environmental conditions. Site A was the outside wall of the bunker in a dry valley and was exposed to the sun for a couple of hours per day, while Site C was under shrubs located at the end of a valley connected with flatland. Both sites had higher light levels and relatively lower air humidity than site B, which was located in a valley with a stream and surrounded by evergreen trees. In addition, observations of A. obovatum were first found [We surveyed the valleys in southern Jeju Island several times during 2019 and 2020. As a result, we found two new gametophyte populations in Youngcheon-dong A,B and Ast found . During ® New . Voucher gametophyte and sporophyte specimens were deposited in the herbarium of Chungbuk National University and observed under an Olympus BX50 microscope .To observe their morphological characteristics, the gametophytes were washed with distilled water and mounted on slides with Entellan®PCR Premix  with 1 μM of each primer, 1 μM of DNA, and 18 μM of distilled water using Pteridaceae_rbcLF and Pteridaceae_rbcLR designed by Park, Kim, and Kim [Four thalli of the gametophytes from each population and one sporophyte from population B were washed using sterile distilled water several times to remove other bryophytes or soil particles. Each thallus and sporophyte was ground using a homogenizer for three cycles for 30 s at a speed of 3 m/s. DNA was extracted by following the manufacturer’s protocol for the DNeasy Plant Mini Kit . The PCR amplification of the rbcL region for 12 gametophytes and one sporophyte was performed with AccUPower and Kim . PCR conVoucher specimens were deposited in the herbarium of Chungbuk National University: Gametophyte: Andeok-myon, Seogwipo-si, Jeju-do, Korea. alt. 128 m, 5 December 2018, H.T. Kim, K. Lee & S.H. Park. CBNU2018-0387; Youngcheon-dong, Seogwipo-si, Jeju-do, Korea 298 m, 18 January 2020, S.H. Park. CBNU2020-0001; Youngcheon-dong, Seogwipo-si, Jeju-do, Korea alt. 304 m, 12 March 2020, H.T. Kim & S.H. Park. CBNU2020-0058; sporophyte: Youngcheon-dong, Seogwipo-si, Jeju-do, KOREA alt. 298 m, 18 January 2020, S.H. Park. CBNU2020-0002.