Wild mushroom foraging has a long tradition, especially in the region of Central Europe. Wild mushrooms are a valuable food resource, as they provide nutritional benefits to the European population. They offer a relatively high content of protein and are traditionally used in many European cuisines as a substitute for meat. This is particularly true in times of crisis, such as wars and pandemics. The study presented in this paper shows that wild mushrooms can substitute around 0.2 percent of daily protein intake and contribute around 3% to the agricultural output of the Czech economy, which was selected as a representative for Central Europe. The calculated real price of wild mushrooms indicates their increasing popularity as a source of food protein in Central Europe, while their price seems to be independent of the quantity supplied. Wild mushrooms, also known as mycetes, are a diverse group of fungi that grow in the wild. They can be found in a variety of habitats, including forests, grasslands, and even urban environments. These fungi play a critical role in ecosystems, serving as decomposers of organic matter and providing a source of nutrition for other organisms.The number of species of higher fungi in Europe is around 10,000 ,3,4,5, wWild mushrooms are a valuable resource for humans. They have been used for centuries as a food source and for their medicinal properties. Some species, such as the prized truffle, enjoy high prices in the market. While some wild mushrooms are safe to eat, it is important to properly identify them before their consumption . In receWild mushrooms have long been considered a sustainable source of food due to their ability to grow in a variety of habitats and their low environmental impacts. According to , wild muFurthermore, wild mushrooms provide important nutritional benefits. They are a good source of protein, vitamins, and minerals and have been shown to have anti-inflammatory and antioxidant properties and high water content .Overall, it is important to determine the current role of wild mushrooms in protein supply in Central European countries and also look at the economic value of wild mushroom foraging. This is important because wild mushrooms can play an important role in times of crisis, provide necessary nutrition to the population, and contribute to the better health of the population. As mushrooms are becoming more popular as a marketable product within the agricultural sector, it is also necessary to look at the evolution of the prices of mushrooms. This means it is important to determine the real prices of wild mushrooms, provide predictions of wild mushroom prices for the next decade and, at the same time, calculate how much protein is supplied by wild mushrooms. This will help to describe factors that can influence wild mushroom prices over time. The uncertain political situation in the world and disease outbreak threats that may lead to decreases in protein supply and wild mushrooms may play an important role in food security.fomes fomentarius and two balls of common birch (Piptoporus betulinus) strung on a leather string. Perhaps they served as amulets or medicine. Wall paintings in a Chukotka cave with a completely realistic depiction of toadstools (Amanita sp.) are approximately the same age. They were most likely used by tribal shamans who induced ecstatic states with the help of hallucinogenic substances contained in toadstools [Prehistoric humans have long considered mushrooms a common dietary enhancement. Healers, priests, and magicians probably knew the intoxicating, hallucinogenic, and sometimes even fatal or, on the contrary, healing effects of mushrooms. Extracts from poisonous mushrooms could be used in warfare or hunting. The understanding of the role of mushrooms was based on the experiences of previous generations, determined to a large extent by geographical and social conditions . Clear eadstools . The oldOn the Art of Cooking in a recipe for “mushrooms growing on trees” [Boletus edulis) mushrooms. Cookbooks from the 15th−17th centuries contain recipes for the preparation of mushrooms. In these cookbooks, mushrooms are classified as foods that replaced meat during the fasting period [The culinary use of mushrooms is first mentioned in the literature of the Roman Empire. The oyster mushroom is mentioned by Marcus Apicius  in the book n trees” ,10. Moren trees” . MushrooPleurotus sp.), various types of champignons (Agaricus sp.), or shiitake (Lentinula edodes) [Today, the majority of edible mushrooms come from cultivated cultures such as oyster ( edodes) . For morAccording to Kalač and Svoboda , the amoMushrooms have higher protein content than, for example, vegetables, and thus, they are considered to be an important nutrition source for many Central Europeans who prefer non-animal-based sources of protein. In particular, wild mushrooms are rich in essential amino acids and other essential nutrients, making them a valuable addition to a balanced diet . MushrooWild mushrooms have been a source of food during famines in Europe for centuries. In the Middle Ages, for example, during times of scarcity, people would venture into the forest to gather wild mushrooms for sustenance .Amanita caesarea) during times of scarcity in ancient Rome. This mushroom, which is native to the Mediterranean region, was highly prized for its delicious flavour and was often used as a substitute for meat [One example of this can be seen in the use of Caesar’s mushroom  dough filled with, e.g., meat, eggs, potatoes, cabbage, mushrooms, cottage cheese, and other fillings .Mushrooms are an important source of protein, are a source of vitamins and minerals, and contain substances that can strengthen the immune system. Today, health-conscious consumers are increasingly looking for alternative ways to improve their diet. Mushrooms are part of a balanced diet because of their active substances, which, in the future, can prevent diseases such as diabetes, hypertension, atherosclerosis, and cancer .Oudemansiella mucida), from which the antifungal antibiotic mucidin, later manufactured under the name Mucidermin Spofa, was isolated [Mushrooms have been used since ancient times to treat various diseases. Mycotherapy is based on the foundations of traditional Chinese medicine. However, only very few higher fungi have penetrated official medicine. One of them is the porcelain slug  wrote that mushrooms are “a poison that gives pleasure”. Usually, mushrooms are harder to digest, and many of them also contain substances harmful to health. For centuries, people have come to terms with this fact in a difficult and painful way. The initial knowledge of mushrooms can only be assumed to have been acquired by shamans and tribal chiefs. Unintentional poisonings certainly occurred before people learned to identify mushrooms and passed on these experiences. One of the first known mushroom poisonings is that of the family of the poet Euripides (484–406 BC). The best-known ancient story, which has been elaborated several times in the literature, is about the assassination of the emperor Tiberius Claudius Nero Germanicus (54 AD), for whom his eunuch Halótus prepared a toadstool dressing for his meal. It seems that mushrooms were also used as poison to kill unwanted persons; for example, the deaths of Pope Clement VII, King Charles VI, and Prince Beronil of Naples are attributed to mushroom poisoning ,10,41. PIn addition, recently, some mushrooms were found to accumulate substances that have adverse effects on human health, especially mercury, lead, cadmium, and astatine. Therefore, it is necessary to collect edible mushrooms in non-contaminated places, outside of industrial areas, where it is certain that mushroom fruiting bodies will not contain these substances .Data regarding mushroom harvesting in the Czech Republic were taken from the publications of the Ministry of Agriculture of the Czech Republic, which are published every year under the name “Report on the State of Forests and Forestry in the Czech Republic” . The repThe original research used the same methodology every year. Data collection was performed within the so-called omnibus survey, a quantitative research method where the data are collected from many subjects during the same interview. The subjects were asked by trained interviewers during F2F (Face-to-Face) questioning. The process was realised through the CAPI method  .The total number of conducted interviews was around one thousand every year. Specifically, the average number of respondents was 1019, with the lowest value being 1000 and the highest value being 1035. A representative sample was achieved using quota sampling, which examined the following factors: gender, age, education, the size of the municipality of permanent residence, and the size of the region . To illuThe data were collected for the years 1994–2021. There were two primary variables prepared for further analysis: (1) the annual amount of foraged wild mushrooms in the Czech Republic in mil. kg and (2) the annual collection of mushrooms in the Czech Republic expressed in current prices, in mil. CZK.Statistical dependency was tested using regression analysis, which can be expressed using Equation (1):was used .As it is important to find out the influence of inflation, the real price of wild mushrooms must be calculated. For the purpose of the real price calculation, the Consumer Price Index (CPI) was used. The Consumer Price Index measures the price of a basket containing representative goods and services in the Czech Republic. The methodology of the Czech Statistical Office uses a basket of about 450 items . Based oBy setting the earliest year (1994) as the base year, all values in the following years were recalculated as real prices in 1994.Data on protein consumption were taken from the databases of ourworldindata.org , the WorThe free-of-charge collection of mushrooms is allowed on 2.5 m ha of forest area accessible to the public . The quaThe figure illustrates that there are significant interannual differences in the foraging of wild mushrooms as a result of several factors of both natural and socio-economic characteristics. These differences can be also described by using descriptive statistics, which are presented in The data show that the median and mean are very close to each other, which implies that the data on wild mushrooms foraged are evenly distributed from the lowest to the highest values. Positive skewness indicates that there are more observations to the right of the mean. This means that there are more years with higher-than-average amounts of foraged mushrooms. The standard deviation of 5.46 indicates that the typical amount of foraged mushrooms is approximately 17 to 27 tons.The amounts represented in (Boletus edulis), but they are the most dependent on the course of the weather. They require warm temperatures and plenty of moisture in forests. While wild mushroom foraging is unpredictable in its nature, it offers an irreplaceable, sustainable, and low-cost source of protein.It is calculated that, on average, 0.2 percent of the daily protein supply may come from wild mushrooms. Forest mushroom collection data refer to year-round collection. In the Czech Republic, mushrooms are mainly collected in the summer and autumn months, mostly from July to October. However, some types of mushrooms are also found in the winter, for example, the increasingly popular fruiting bodies of oyster mushrooms. Czechs prefer porcini mushrooms As little is known about the potential value of wild mushroom foraging to the economy, the proportion of wild mushrooms foraged in the agricultural output was calculated. In The nominal price of mushrooms was collected together with the quantity of mushrooms in the survey for every year between 1994 and 2021. The price of mushrooms was adjusted for inflation using 1994 as the base year and is represented in The trend presented in Therefore, it is possible to use the model for the prediction of future prices of wild mushrooms. This can be used as one of the factors that may influence the availability of protein from wild mushrooms when not foraged for free. According to consumer behaviour theory, as supply increases, the price of a good should decrease. In order to find the relationship between the quantity of foraged mushrooms and the real price, which were assumed to be stationary, regression analysis was used. The results of the regression are presented in The results indicate that wild mushrooms do not fulfil the characteristics of a commodity , as there is no relationship between the foraged amount and the real price. A possible explanation could be that wild mushrooms are either imported or substituted in the cuisine with mushrooms that are grown and available in the Czech Republic see . The risWild mushrooms are an inseparable part of the Central European natural environment and have been collected for centuries as a unique source of food in times of both hunger and food scarcity, as well as a supplement in a diverse diet. Beranová  states tMushrooms are an important source of protein. Chang and Buswell  report nComparing the value of mushroom foraging, the lowest quantity of wild mushrooms foraged is in Denmark, where there is also a low yield of forest mushrooms due to natural conditions. The largest amounts of foraged wild mushrooms are in Sweden (up to 38%), Poland, and the Czech Republic . ResearcThe foraging of wild mushrooms has a long tradition in many parts of Europe. The popularity of wild mushroom foraging is specific to the region of Central Europe. Wild mushrooms are often used in European cuisines as a substitute for meat. Especially in times of crisis, wild mushrooms provide a nutritional source of food, especially protein. Wild mushrooms can be an important source of protein for several reasons. They are a good source of essential amino acids, which are the building blocks of protein. Additionally, wild mushrooms are low in calories and fat, making them a healthy protein option for people who are looking to maintain or lose weight. Another benefit of wild mushrooms as a protein source is that they are a sustainable and renewable resource. Unlike meat and dairy products, wild mushrooms can be harvested without harm to the environment. However, in many countries, the amounts of wild mushrooms that can be foraged are limited to mitigate environmental damage. Wild mushrooms also require less water and land to grow than many other protein sources while providing higher protein content in dry matter. The price of wild mushrooms can influence their availability. The increasing real price of mushrooms means that wild mushrooms are becoming more popular with consumers. Furthermore, wild mushrooms are often more expensive than cultivated mushrooms, which can make them less accessible to some consumers. Additionally, the harvesting of wild mushrooms can be labour-intensive, which can also drive up the cost. The conducted research shows that the real price of wild mushrooms has not been influenced by the supply of mushrooms, indicating that wild mushrooms do not possess characteristics of a commodity like other protein sources from crops such as wheat or oats. The overall contribution of wild mushrooms to the measure of agricultural output can be around three percent for the case of the Czech Republic and thus may constitute a significant source of income, particularly for rural areas.


Irrational prescription and its subsequent costs are a major challenge worldwide. Health systems must provide appropriate conditions for the implementation of national and international strategies to prevent irrational prescription. The aim of the present study was to determine the irrational surfactant prescription among neonates with respiratory distress and the resulting direct medical costs for private and public hospitals in Iran.This was a cross-sectional descriptive study performed retrospectively using data belonged to 846 patients. Initially, the data were extracted from the patients’ medical records and the information system of the Ministry of Health. The obtained data were then compared with the surfactant prescription guideline. Afterward, each neonatal surfactant prescription was evaluated based on the three filters listed in the guideline . Finally, chi-square and ANOVA tests were used to investigate the inter-variable relationships.The results showed that 37.47% of the prescriptions were irrational and the average costs of each irrational prescription was calculated as 274.37 dollars. It was estimated that irrational prescriptions account for about 53% of the total surfactant prescription cost. Among the selected provinces, Tehran and Ahvaz had the worst and the best performance, respectively. As well, public hospitals outperformed private hospitals in terms of the in drug selection, but they underperformed them in terms of the right dose determination.The results of the present study are considered as a warning to insurance organizations, in order to reduce unnecessary costs caused by these irrational prescriptions by developing new service purchase protocols. Our suggestion is the use of educational interventions to reduce irrational prescriptions due to drug selection as well as using computer alert approaches to reduce irrational prescriptions caused by wrong dose administration. Access to health services is one of the most important factors showing the medical progress in a country . AlthougWorld Health Organization (WHO) defined rational prescriptions as prescriptions in which the patient receives the right drug at the right dose and at the right time with the lowest possible cost, otherwise it is considered as irrational .In this regard, numerous factors such as physician to population ratio , 7, paymIrrational use of medicines poses a formidable challenge to health systems in many countries . Drugs aTo reduce these irrational prescriptions, 12 key strategies has been proposed by WHO for health care administrators. These strategies include the establishment of a multidisciplinary national body to coordinate policies, formulating clinical guidelines, selecting based on the list of essential drugs, setting up drug and therapeutics committees, promoting problem-based training in pharmacotherapy, continuing in-service medical education, promoting the supervision system, providing independent information, promoting public education about medicines, applying regulations, reserving governmental expenditure to equitable availability of drugs, eliminating perverse financial incentives .In recent years, according to unpublished data from the Ministry of Health of Iran as well as the interviews with policy makers in the field of neonatal health, the surfactant prescription in neonates is rapidly growing. Surfactant, which is used to treat respiratory distress diseases among neonates, is one of the vital and expensive drugs in Iranian pharmacopoeia. Respiratory distress diseases are known as one of the leading causes of neonatal death in developing countries , among wAccording to the directors of the Neonatal Health Department of the Ministry of Health of Iran, it is believed that the increase in surfactant prescription in recent years may possibly be due to irrational prescription and induced demand as the results of the implementation of HSRP. In recent years, many studies have been conducted on investigating various aspects of neonatal respiratory distress as well as the effects of various types of surfactants on it , 20. As A cross-sectional study was conducted in public and private hospitals in Iran during 2018 with the aim of determining the amount of irrational prescription and the resulting direct medical costs.The study population included all Iranian neonates who underwent surfactant therapy in 2018. In the present study, the cluster-stratified sampling method was used for selecting the cases. First, the provinces of Iran were divided into five main regions according to the classification of the Ministry of Interior , which was firstly developed by the Ministry of Health and Medical Education, was used as the instrument in the present study. The required data were extracted from the Iranian Maternal and Neonatal Network (IMAN Net) and the medical records in private and public hospitals.Based on this guideline, three steps of right drug, right dose, and the right time for a rational prescription were considered, and in this study, each one of the cases of surfactant prescription was investigated based on these three filters, respectively.Based on the prescribed indications, was surfactant drug a right decision for the neonates?Based on the surfactant type and the neonate weight, was the right dose prescribed for the neonate?Was the surfactant injected at the right time to the neonate?This index is a combination of the previous three components, which can be either in a verifiable (no missing data) or in an unverifiable (missing data) status. Moreover, the verifiable status of this index can be either rational or irrational, which were presented as the indexes of rational prescribing (IRP) and irrational prescribing (IIP) in the current study, respectively.When all three components of this index are rational, it means that all three components of drug selection, dose, and time are rational as well.When at least one of the three components of this index is irrational, it means that either the drug selection, dose or time is irrational.In the first step, we determined whether the patient was clinically eligible to undergo surfactant replacement therapy based on the four indications stated in the national guideline Table . ThereafEach indication consists of two variables, and an indication for one prescription was considered as rational only if both variables were rational. As well, if there was only one irrational variable, the related indication was considered as irrational for that prescription. Additionally, an indication was considered as unverifiable when both variables were unverifiable or one of the variables was unverifiable and the other one was rational. Afterward, by juxtaposing the variables of an indication, we determined the state of each indication for a specific prescription , apart from the status of the other indications.Next, by simultaneously evaluating all these four indications, we determined the number of patients who had irrational surfactant selection. The surfactant selection was considered as irrational when all the four indications emphasized the irrationality of a prescription. Moreover, the surfactant selection was considered as rational when at least one indication has confirmed the rationality of that prescription. Besides, other cases in which the combination of these indications was in an irrational, unverifiable state, they were all considered as unverifiable.In the second step, a “standard dose” was established based on the type of the prescribed surfactant and the neonate’s weight for each patient. This standard dose was then compared with the prescribed dose. If the dose of the three surfactants of Alveofact, BLES, and Survanta deviated the standard dose by a maximum of ± 0.50, the prescribed dose was considered as irrational in the present study as well. Since the minimum and maximum administered doses of Curosurf surfactant are provided in the guideline, the minimum and maximum standard doses were determined for each neonate. In addition, if the prescribed dose was in the “standard dose range” or even if it deviated the standard dose range up to two decimal digits, it was still considered as rational. These doses, which were regarded as irrational in terms of the standard stated in the national guideline, by the suggestion of the research team were considered as rational, which are listed as “rational doses without sensitivity " in the findings. Finally, if the data on the dose was missing, this component would be considered as unverifiable.In this step, it was determined whether the patient received the surfactant at the right time. In terms of the above-mentioned guideline, we considered the time periods less and more than 48 h of birth as rational and irrational, respectively.In this section, two types of the fixed and variable costs were identified for each prescription. Accordingly, the fixed cost includes medical consumables , which are the same for all types of prescriptions. Variable cost includes the price of surfactant vial in 2018, which was calculated to be varied according to the type of the prescribed surfactant. Accordingly, the related prices were determined based on the average price of the largest surfactant distributor company in Iran in 2018.In the present study, data analysis was conducted using Stata ver. 19. Quantitative variables were reported by mean and standard deviation, and qualitative variables were also reported by frequency and percentage. Finally, chi-square and ANOVA tests were used to investigate the inter-variable relationships.The total index of the prescribing and its sub-components is listed in Table The results show some significant differences between public and private centers in terms of the total index of prescribing, especially regarding the drug selection component Table .In Iran, according to IMAN Net 13% of all surfactant prescriptions in neonates is related to Tehran province. It was also found that the total index of prescription had the worst and best statuses in Tehran and Ahvaz provinces, respectively Table .The cost of one surfactant prescription depending on the type of surfactant varies from a minimum of 298.12 dollars (Curosurf) to a maximum of 385.80 dollars (Alveofact) in Iran (2018).A prescription was considered as irrational either when the drug selection or dose or time was irrational or when more than one of these factors were irrational. Table In Iran, a total of 11,284 neonates with respiratory distress disease underwent surfactant treatment. According to the results of the present study, it was estimated that in private and public hospitals, there were 1991 and 4021 irrational prescriptions in total, respectively. According to the average price of each type of surfactant prescription, the total direct medical cost of surfactant prescriptions for neonates with respiratory distress was estimated as 3,096,087.32 dollars and the direct medical cost due to irrational prescription in these neonates was obtained as 1,649,563.71 dollars. Approximately, 53% of these costs were related to irrational prescriptions. Notably, by taking the costs of Relative Value Units of surfactant prescription (RVUs) into account, the costs of irrational prescriptions reached 2,077,207.18 dollars  neonates and irrational for 317 (53.27%) neonates. In a similar study on 4182 prescriptions, Campino et al. (2009) has previously showed that the rate of prescription error in the NICU is 20.70% . MoreoveOur suggestion to reduce such errors is to use educational interventions because these interventions to a large extent can lead to some changes in physicians’ prescription behavior . As wellIn Iran, the private and public sectors both provide health care and treatment services; however, the public sector and specially the Ministry of Health play a more significant role in this regard . The pubThe results of this study show that the total index of prescribing, especially the component of drug selection, was significantly different between public and private centers. The drug selection was found to be irrational in 7.62% of the verifiable prescriptions in public hospitals, while this rate was 40.80% in private hospitals. Public hospitals seem to have more commitment to rational prescription indications. Other researchers have also confirmed that patients receive better treatment services in public centers compared to private ones according to clinical guidelines. In fact, since in the public sector, the educational level is better and the opportunities for training are more, therefore, higher rates of correct diagnosis and correct prescription are provided; also, this difference in the quality of care between the public and private sectors likely reflects financial incentive aspects . Of noteIn the present study, the index of irrational drug prescribing in public and private hospitals was obtained as 48.73% and 65.63%, respectively. Although the percentage of right drug selection was higher in public hospitals, they had lower percentage of right dose compared to private hospitals. Aronson et al. have stated that one of the most important cause of human errors in physicians is fatigue. Fatigue is known as one of the most important human causes of error among physicians . Since tThe total index of irrational drug prescribing was the highest in Tehran province (67.36%) and the lowest one was in Ahvaz province (39.47%). The results show that Tehran province has the weakest performance in surfactant prescription in neonates among the selected provinces that were investigated in the current study.The situation of organizations also is effective on the occurrence of errors. Although human factors often are the first cause of error, and the environment as a latent factor has a significant impact on the occurrence of error. It is expected that the higher the number of prescriptions, the higher the probability of the occurrence of error. ConsideThe direct cost of treatment due to irrational prescription surfactant among the selected hospitals of the present study was calculated as 111,217.63 dollars. Besides, the average cost of each irrational surfactant prescription was 274.37 dollars. It was estimated that the direct cost of treatment for all Iranian neonates with respiratory distress disease who underwent surfactant treatment was 3,096,087.32 dollars, of whom about 53% was due to irrational prescriptions.In this regard, the results of a study conducted in Nepal revealed that 20–52% of the total drug costs are due to irrational drug prescriptions and the average drug cost per prescription was calculated as 5.7 Nepalese rupees . As wellThe cost of irrational prescriptions is considered as a barrier that deprives people of health services  and chalOne of the main limitations of the present study was that, according to national standards, we expected medical records to be available electronically in hospitals. However, this was not the case in some of the hospitals, and the research team had to wait for the medical records to be scanned and sent; therefore, the study period was longer than we had anticipated. In addition, the quality of medical records may have been different in the investigated hospitals, which could affect the findings. Another limitation of this study is that the average length of stay (LOS) was not considered in the analysis, whereas a longer LOS can increase the possibility of any medication error. It should be noted that this research is based on a descriptive design and few controls have been used to identify statistical differences between regions. Also, in this research, without investigating the root causes of these irrational prescriptions, we have only determined the amount of these prescriptions; for future research, we strongly recommend investigating the root causes of these irrational prescriptions.The present study aimed to determine the irrational surfactant prescription among neonates and then to compare this index in private and public hospitals of the selected provinces in Iran. The results show that Tehran, which is the capital of Iran, has the worst performance in terms of surfactant prescription among the selected provinces. It was also found that although the percentage of right drug selection was higher in public hospitals, they had lower percentage of right dose compared to private hospitals. Finally, it was estimated that about 53% of the total cost of surfactant therapy in neonates was related to irrational prescriptions.It is suggested to form multidisciplinary specialized committees consisting of different stakeholders at the regional level, in order to evaluate the performance of medical centers more effectively, and formulate and implement appropriate policies and guidelines. It must be acknowledged that we can all make errors in any organization. Overall, several reasons exist for these errors and different solutions to prevent them. However, we must begin by recognizing that errors are undeniable and then take some steps to minimize them. The first step in reducing errors is identifying their extent. Thereafter, the root causes of the errors must be identified, and finally, preventive strategies must be applied. Considering the fact that surfactant is one of the most expensive drugs in Iranian Pharmacopoeia, the results of the present study are regarded as a necessity and a warning for insurance organizations to develop new protocols for purchasing services during the process of surfactant therapy in neonates, in order to prevent unnecessary burdens caused by the irrational prescription of this drug.


Photosynthetic organisms have colonized a wide variety of habitats in the terrestrial, freshwater and marine biomes. These habitats are characterized by significant differences in key physiological factors, including light intensity and spectral distribution, nutrients and water availability, temperature. The result is a conspicuously complex process that depends on the coordinated activity of several sub-processes whose adaptation underpins the optimisation of the photosynthetic apparatus to the specific habitat. The limited range of techniques available to measure photosynthesis in the field provides only partial insights into the operation and regulation of photosynthesis. The investigation of the diversity of adaptive strategies that have evolved as part of the adaptation of photosynthesis has largely been restricted to laboratory studies made on organisms grown and measured under stable, controlled conditions. This was, and still is, necessary in order to design reproducible, straightforward experiments addressing the fundamental mechanisms underlying the function and regulation of photosynthesis that, however, still eludes complete understanding even under the relatively controlled growth conditions.Nonetheless, in recent years, there has been a growing interest in improving the understanding of the mechanisms that allow photosynthetic organisms to respond to the fluctuating conditions of the natural environments These fluctuations can be substantial, occurring on different timescales, ranging from the short term (in the sub-second range) to the long term . Given the variety of adaptive strategies utilised by photosynthetic organism to thrive in specific habitats, the response mechanisms that have evolved to deal with environmental fluctuations are expected to be as diverse and complex.The papers collected in this Research Topic reflect the variety of approaches employed to address the mechanism of response of photosynthetic organisms to fluctuating light conditions.Gjindali et al. review both experimental and modelling approaches to address and describe short- and long-term responses to light fluctuations in land plants. A second review by Wang et al. discusses the limitations occurring under very dim light, in some species of vascular plants which generally are considered to have adapted to higher photon fluxes.Ye et al. demonstrate the benefits of mathematical descriptions that do not rely on asymptotic extrapolations to describe the light-saturation curves of net photosynthesis, carbon and oxygen fixation in land plants. Hommel et al. present a comparative study of short-term fluctuation and rapid responses of the photosynthetic apparatus to light-dark and spectral transitions, highlighting both similarities, as well as differences, in these responses in the model vascular plant Arabidopsis thaliana.Moreover, Tula et al. used the recombinant expression of flavodiiron (flv) proteins from Synechocystis sp.PCC6803, a recently discovered alternative electron sinks in cyanobacteria, algae and mosses , employ strategies previously considered exclusive to high light conditions.Further contributions to the Research Topic use unicellular cyanobacteria to investigate the acclimation to light changes at subcellular and molecular level. The study of On the whole, this Research Topic highlights the broad range of strategies that photosynthetic organisms may employ to adapt to ambient fluctuations, and how these strategies may not only depend on given taxa, but being highly heterogeneous down to leaf or cellular level.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Train distance estimation in a turnout area is an important task for the autonomous driving of urban railway transit, since this function can assist trains in sensing the positions of other trains within the turnout area and prevent potential collision accidents. However, because of large incident angles on object surfaces and far distances, Lidar or stereo vision cannot provide satisfactory precision for such scenarios. In this paper, we propose a method for train distance estimation in a turnout area based on monocular vision: firstly, the side windows of trains in turnout areas are detected by instance segmentation based on YOLOv8; secondly, the vertical directions, the upper edges and lower edges of side windows of the train are extracted by feature extraction; finally, the distance to the target train is calculated with an appropriated pinhole camera model. The proposed method is validated by practical data captured from Hong Kong Metro Tsuen Wan Line. A dataset of 2477 images is built to train the instance segmentation neural network, and the network is able to attain an MIoU of 92.43% and a MPA of 97.47% for segmentation. The accuracy of train distance estimation is then evaluated in four typical turnout area scenarios with ground truth data from on-board Lidar. The experiment results indicate that the proposed method achieves a mean RMSE of 0.9523 m for train distance estimation in four typical turnout area scenarios, which is sufficient for determining the occupancy of crossover in turnout areas. In recent years, the development of visual sensors and detection algorithms has brought new opportunities for the autonomous driving of urban rail transit trains ,2,3. By Turnout is a vital component of railway systems, mainly positioned in areas such as yards and terminal loops , where tFor distance estimation, Lidar is generally considered to be the most suitable sensor for its far measuring range and high accuracy. The basic theory of Lidar is to calculate the distance to objects by emitting and receiving laser pulses and measuring their return time. Due to its high precision and extended range , Lidar iApart from Lidar, camera vision is another suitable choice for distance measurement in the field of autonomous driving. While cameras cannot directly acquire distance information like Lidar, they indirectly acquire distance information by distance estimation algorithms. Unlike the active ranging of Lidar which involves emitting and receiving laser pulses, cameras passively capture natural light reflected from the detected objects’ surfaces, thus they can reliably acquire information of color, texture, and light intensity of targets . While cIn most cases, object distance estimation in computer vision is achieved by using stereo vision, and the depths are estimated by triangulation of feature points. which are extracted and matched from two stereo images . For shoIn order to overcome the problems of stereo vision in the estimation of relatively far distances, a number of authors have proposed solutions based on monocular vision. Unlike stereo vision, monocular distance measurement relies solely on images captured by a single camera, the relative distance of objects is inferred by analyzing the information of object size, shape, perspective relationships, and combining this with the camera’s intrinsic and extrinsic parameters. This approach often requires on, Ref.  proposeson, Ref.  proposeson, Ref.  proposeson, Ref. .(1)A monocular vision-based distance estimation framework is designed to achieve the detection of trains in turnout areas. According to the authors’ knowledge, this problem has not been solved with other sensors or strategies, such as Lidar and stereo vision.(2)An instance segmentation strategy is proposed for train side window detection. By treating multiple side windows in each train carriage as separate entities, the segmentation can be achieved robustly and accurately even in poor illuminance conditions.(3)A geometric feature extraction strategy is proposed to obtain the quantitative representation of train side window contours, thus the further scale-based distance estimation could be achieved with an acceptable accuracy in practical usage.Comparing to detection with bounding box, instance segmentation provides pixel-wise contour of the detected object, therefore improves the accuracy of target size estimation ,24. MoreThe remainder of this paper is structured as follows: In this paper, we propose a method of distance estimation of trains in turnout areas based on monocular vision. The main idea is to extract features of known dimensions by instance segmentation, and estimate the distance to the target train with an approximated pinhole camera model, as shown in The proposed method consists of two main parts: instance segmentation of train side windows and distance estimation based on geometric features.Visible light cameras are sensitive to environmental light interference. To adapt scenarios with different illuminance conditions in urban rail transit, especially for those in tunnels with no extra illumination, in this paper, we select the side window of the train as the object to calculate the scale factor of distance estimation. The main reasons are two-fold. Firstly, the side window of the train has a rectangular shape, and the width and height of the windows are constant values. Secondly, the side window of train remains bright even in tunnels with no extra illuminance, thus it can always be detected according to the contrast with the dark background, as shown in The segmentation of an independent side window is not an easy task, since the contour of an independent window is not clear when it is captured from a far distance, as shown in Instance segmentation goes beyond semantic segmentation by not only assigning each pixel a semantic label, but also distinguishing the boundaries of individual object instances. It provides unique identifiers for each instance of the same class, accurately outlining the contours of each object and highlighting their independent presence in the image. This is useful when more than one train carriages show in the captured image of on-board camera. In this paper, we choose YOLOv8 as the neural network model for instance segmentation. YOLO (You Only Look Once) is a popular object detection and image segmentation model, which was developed by Joseph Redmon and Ali Farhadi at the University of Washington . As a cuAfter training with the appropriate dataset, the model can provide pixel-wise regions of side windows of target trains.With the segmented regions of side window, the geometric features should be extracted to estimate the scale factor. Since the side windows of train have rectangular shape, their width and height both provide scale information in monocular distance estimation, theoretically. However, due to potential obstructions in the train operating environment, such as tunnels walls, it is quite difficult to detect the entire lateral length of a train carriage’s window in a randomly given frame of image. Therefore, the horizontal dimension of the window is not an appropriate geometric feature for distance estimation. On the other hand, the vertical dimension of the window is less susceptible to obstruction and is better suited as a geometric feature for distance estimation.Therefore, in this paper, we choose the vertical height of side windows as the key to calculate the scale factor and estimate the distance to the target train. The workflow after instance segmentation is as shown in To obtain the pixel height of the side window in the image, the vertical direction of the side window is needed. We here calculate the vertical direction in 3 steps.(1)By performing minimum bounding box fitting on the instance segmentation regions, a rough estimation of the width and height of the windows is obtained, as depicted with red rectangles in (2)By locally applying Otsu’s thresholding and Canny edge detection in the instance segmentation regions, the contour edges of individual side windows within each train carriage are obtained, as shown in (3)By employing Hough line detection to the contour edges, a bunch of straight lines can be extracted. Considering the dimensions and main directions of the fitted bounding boxes, the lines for vertical edges of side windows are then determined, and by computing the directional average of the detected vertical lines, the optimal estimation for the vertical dimension of the side windows is obtained, as depicted with green lines in To estimate the distance of the whole target train side window, upper and lower edges of side windows are needed. However, the edges of the side window areas detected by instance segmentation are often curved rather than straight lines, which may cause errors for distance estimation. Therefore, to obtain well-aligned upper and lower edges of side windows, we here use linear fitting to solve this problem in 3 steps.(1)At the center of the minimum bounding box obtained from instance segmentation, auxiliary lines are constructed perpendicular to the side window’s vertical direction. Along the auxiliary lines, multiple sampling points are selected at a certain interval, as depicted with the khaki color in (2)Starting from these sampling points, extension lines are drawn in both the positive and negative directions along the side windows’ vertical directions. The intersection points of these extension lines with the instance segmentation contours are then calculated as the estimation for the upper and lower edges of the side window, as depicted with a yellow color in (3)Linear fitting is separately applied to the sets of points representing the upper and lower edges, resulting in linear estimation for the upper and lower edges of the side window, as depicted with blue lines in The basic idea of the monocular distance estimation is to estimate the scale factor from f, for a piece of train side window with a height of H, it can be projected to the image sensor plane with a pixel height of h. We denote the distances from the upper and lower edges to the camera optical center C as D is significantly larger than H, we can obtain an approximation that D in depth direction and do not care about altitude direction, we can simply calculate the distance by using similar triangles, as Equation .The network training was performed utilizing an Nvidia RTX3070 GPU. Taking into consideration the 8 GB VRAM and the size of the model, we configured the batch size as 8 for 500 epochs. Other detailed information of the training configuration is as shown in To enhance the network’s adaptability, we also employed data augmentation during the training process. This involved adjustments to the original dataset such as HSV  adjustments, translation range, scale range, etc. The details of data augmentation are as shown in The tendency of losses, precision, recall and mAPs are as shown in For the train side windows in the four typical scenarios, the segmentation results are as shown in For scene 1, the results in For scene 2, the results in For scene 3, the results in For scene 4, the results in i, and i, respectively. Based on the results from the test set, the instance segmentation network model trained by us achieved a MIoU of 92.43% and a MPA of 97.47%.For further quantitative evaluation of instance segmentation performance, two popular indicators, the Mean Intersection-over-Union (MIoU) metric and the mean pixel accuracy (MPA) metric were used in this paper, as shown in Equations  and 3),,3),(2)MThe above-mentioned results show that the proposed side window segmentation strategy works with an acceptable accuracy even in scenarios of bad illumination conditions. Although the segmented contours of the side windows are not as smooth as the annotation, they still keep regular shapes and dimensions, which can provide sufficient geometric information for the subsequent process of scale calculation.For performance evaluation of train distance estimation, ground truth distance to the target train is the best choice. Even though the train body cannot provide a returned point cloud due to large incidence angles on object surfaces, there are other cases in which the train head provides returned point clouds. By this consideration, we overlay the point cloud frames in which the train head or tail is still detectable and thus obtained the ground truth position of the train body in the four scenes, as shown in For Based on the above-mentioned experiment configuration and ground truth values, distance estimation experiments were implemented. To maintain generality, we selected sampling points at a one pixel interval within the window area and calculated distances by combining the corresponding window pixel height at each sampling point. The results of train position estimation of the four typical scenes are as shown in For quantitative evaluation, we use RMSE (root mean square error) to evaluate the error of distance estimation of train side windows, as shown in Since the original intention of distance estimation of the target train is to determine the occupancy of crossover, it should be accurate enough to distinguish whether the target train is on a crossover. Since the mean RMSE of distance estimation in four typical scenarios is 0.9523 m and less than the standard gauge of 1.435 m, the proposed train distance estimation framework is considered to be accurate enough for determining crossover occupancy.In this paper, we propose a framework of distance estimation of trains in turnout areas based on monocular vision. The side windows of target train are segmented based on YOLOv8 neural network, then the geometric features are extracted for distance estimation. For validation and evaluation, we implement experiments on the Hong Kong Metro Tsuen Wan Line, a dataset that includes 2477 images that are built for instance segmentation model training, and distance estimation experiments are implemented in four typical turnout area scenarios. According to the results, the proposed method can estimate the distance of trains in turnout areas where Lidar fails to obtain a point cloud, and the accuracy is enough for determining crossover occupancy. Furthermore, the proposed method works well in scenes of bad illumination and scenes where only a part of the target train is visible.However, this paper also has several limitations. Firstly, limited by the quantity of dataset annotation, in this paper we only studied the instance segmentation based on a single image frame. The future work will focus on the enhancement of instance segmentation by exploring a neural network with a temporal context. Secondly, this paper only utilized data from the tunnel section of the Tsuen Wan Line in Hong Kong. The future work will consider data from more urban rail transit lines and scenarios to improve the method’s generalization and robustness. Finally, this paper only utilized side windows as geometric features for scale calculation, future work will also integrate more features to improve the accuracy and robustness of distance estimation.


One of the major evolutionary transitions that led to DNA replacing RNA as the primary informational molecule in biological systems is still the subject of an intense debate in the scientific community. DNA polymerases are currently split into various families. Families A, B, and C are the most significant. In bacteria and some types of viruses, enzymes from families A and C predominate, whereas family B enzymes are more common in Archaea, Eukarya, and some types of viruses. A phylogenetic analysis of these three families of DNA polymerase was carried out. We assumed that reverse transcriptase was the ancestor of DNA polymerases. Our findings suggest that families A and C emerged and organized themselves when the earliest bacterial lineages had diverged, and that these earliest lineages had RNA genomes that were in transition—that is, the information was temporally stored in DNA molecules that were continuously being produced by reverse transcription. The origin of DNA and the apparatus for its replication in the mitochondrial ancestors may have occurred independently of DNA and the replication machinery of other bacterial lineages, according to these two alternate modes of genetic material replication. The family C enzymes emerged in a particular bacterial lineage before being passed to viral lineages, which must have functioned by disseminating this machinery to the other lineages of bacteria. Bacterial DNA viruses must have evolved at least twice independently, in addition to the requirement that DNA have arisen twice in bacterial lineages. We offer two possible scenarios based on what we know about bacterial DNA polymerases. One hypothesis contends that family A was initially produced and spread to the other lineages through viral lineages before being supplanted by the emergence of family C and acquisition at that position of the principal replicative polymerase. The evidence points to the independence of these events and suggests that the viral lineage’s acquisition of cellular replicative machinery was crucial for the establishment of a DNA genome in the other bacterial lineages, since these viral lineages may have served as a conduit for the machinery’s delivery to other bacterial lineages that diverged with the RNA genome. Our data suggest that family B initially established itself in viral lineages and was transferred to ancestral Archaea lineages before the group diversified; thus, the DNA genome must have emerged first in this cellular lineage. Our data point to multiple evolutionary steps in the origins of DNA polymerase, having started off at least twice in the bacterial lineage and once in the archaeal lineage. Given that viral lineages are implicated in a significant portion of the distribution of DNA replication equipment in both bacterial (families A and C) and Archaeal lineages (family A), our data point to a complex scenario. Several transitions that occurred throughout the origin and maturation of biological systems were crucial for the development of life as we know it today. Some of the events that enabled life to exist on our planet include the emergence of the first informational molecule, the structuring of the genetic code, the formation of the translation system, and the establishment of basal metabolism. Currently, it is practically a consensus in the scientific community that the first informational molecule to be fixed in biological systems was RNA ,2,3,4. DIn the present work, we assume that reverse transcriptases are the ancestors of DNA polymerases, and we try to explain how DNA polymerases evolved and became diverse in both viral and basal cellular lineages.In the present work, proteins from families A, B, and C of DNA-dependent DNA polymerases were analyzed. Sequences were obtained from GenBank in the Refseq database. For family A DNA-dependent DNA polymerases, 100 sequences were obtained—80 representing Bacteria and chloroplast/mitochondria and 20 representing viruses. For family B of DNA-dependent DNA polymerases, 72 sequences were obtained,—6 from eukaryotes, 20 from archaea, 6 from bacteria, and 20 from viruses. For family C of DNA-dependent DNA polymerases, 61 sequences were obtained—51 from bacteria and 10 from viruses. As an outgroup, 6 RNA-dependent DNA polymerase sequences were used. All used sequences are curated and available in the http://www.phylo.org/). The analysis first involved 100 ML searches, each starting from one randomized stepwise addition parsimony tree (command –f d), under a GTRGAMMA model. All other parameters were estimated by the software. To assess the reliability of the nodes, nonparametric bootstrapping replicates under the same model were computed, allowing the program to halt bootstrapping automatically with the autoMRE bootstrapping criterion. To plot the calculated bootstrap values on the branches, the command –fb was used. The original trees are available in the Alignments were generated using MAFFT version 7  accordinOne of the major evolutionary transitions that led to DNA replacing RNA as the primary informational molecule in biological systems is still the subject of a long debate in the scientific community ,6,7,8. AIn this sense, by examining the pattern of diversification to DNA-dependent DNA polymerases, we may infer the routes followed by cellular lineages during the process of transition from RNA to DNA as the major informational molecule.The family A DNA-dependent DNA polymerases are widely distributed in the Bacteria domain, and in mitochondria and chloroplasts. Like many polymerases, it has a general structure organized like a right hand, with palm, finger, and thumb structural domains . In bactThe emergence of family A DNA polymerases in mitochondria/chloroplast ancestors allowed the maturation and fixation of DNA as an informational molecule in this lineage. It should be noted that when we talk about the ancestors of mitochondria and chloroplasts, we are not referring directly to the more recent group that gave rise to these organelles, but to the ancestral lineage of bacteria that gave rise to the group that would later establish endosymbiosis. During this fixation process or after the maturation of DNA to being the main informational molecule in the ancestors of mitochondria, this machinery must have been transferred to viral lineages, which must have allowed the maturation of the first lineages of bacterial viruses with DNA as the informational molecule, and subsequently, the family A proteins spread to the rest of the groups of bacteria which still had RNA genomes.. In this context, it is worth mentioning that although the scientific community is widely aware that mitochondria and chloroplasts originated from bacterial lineages, when we observe the DNA replication pattern of bacteria and mitochondria/chloroplasts, we can identify significant differences. Although both systems need a primer for the polymerase to start its activity, in mitochondria and chloroplasts, the two DNA strands are replicated independently. Replication is initiated in the heavy strand in a single direction, and after reaching the origin of replication of the light strand, this strand replicates in the opposite direction . In bactFamily C of DNA-dependent DNA polymerases is widely distributed in Bacteria and some viral families. In these groups, this family of polymerases is responsible for genome replication in both continuous and discontinuous strands. As mentioned above, the bacterial genome replication model—bidirectional and replication of both strands at the same time—generates, on the discontinuous strand, a series of RNA and DNA heteroduplex regions, known as Okazaki fragments . At the This resemblance in patterns of diversification supports the hypothesis that the first bacterial lineages were generated with an RNA or transitional genome, as previously noted, and that DNA was merely a transient stage that was continually being recreated through reverse transcription ,8,18,19.We can offer two possible scenarios based on what we know about bacterial DNA polymerases. The first hypothesis contends that family A was initially produced and spread to the other lineages through viral lineages before being supplanted by the emergence of family C and the acquisition at that position of the principal replicative polymerase.In this scenario, due to the low progressivity of family A DNA polymerases, the acquisition of the family C polymerases allowed greater efficiency in genome replication, along with an increase in its size. Family A was co-opted for a secondary function involved in the resolution of the Okazaki fragments. In the second scenario, family C DNA polymerases were acquired first and spread via viral lineages to other bacterial lineages. In this scenario, in these initial lineages, Okazaki fragments could generate RNA and DNA hybrid points that could not be resolved by the replicative enzyme, and a reverse transcriptase could be used to mature these regions. Due to the family A polymerases’ ability to resolve these heteroduplex regions, the secondary acquisition of these enzymes allowed for the improvement of the replication system. It is worth mentioning that in family A DNA polymerases, residual activity of reverse transcription has already been described, which suggests that an ancestral vestige is maintained in this family, which must have enabled this family to replace a function performed by reverse transcriptase ,31. The Family B DNA-dependent DNA polymerases are widely distributed among organisms of the Archaea and Eukarya lineages, and in eukaryotes, they are further diversified into several groups . The DNAThis unique pattern of diversification shows that the emergence of DNA as an informational molecule in Archaea and Eukarya had a separate evolutionary trajectory from the evolutionary history of the maturation of the DNA in bacterial lineages. In this sense, we conceive a scenario in which the Archaeal lineage was initially established with an RNA genome, and that, prior to the diversification of the various groups, the conversion of this RNA genome to DNA took place via the acquisition of viral machinery for DNA replication. In this context, Archaeal viruses’ lineages acquired their DNA genome before cellular lineages. It should be noted that, in this scenario, DNA as an informational molecule must have emerged before this same process occurred in bacterial lineages. Thus, not only did DNA originate independently in basal cellular lineages, but it also occurred at distinct temporal stages.Many hypotheses have been proposed about the origin and diversification of viral lineages. Models for their emergence assume an origin from three scenarios: (i) virus first, (ii) cell reduction, and (iii) escape ,38,39,40On the other hand, when analyzing the Archaeal viral lineages, we observed a slightly different pattern, since the data suggest that these lineages acquired their DNA genome before the emergence of DNA in cellular lineages. In this context, viral lineages transferred their replicative machinery to basal Archaeal lineages before the initial diversification of this group. In this way, this transfer process at a very primitive stage for Archaeal lineages may have provided the maturation of DNA as an informational molecule before this process appeared in bacterial lineages. Altogether, our data suggest a complex scenario for the emergence of DNA in different lineages of organisms, with this process following at least three independent routes.The origin of DNA as an informational molecule in cellular lineages represents a huge evolutionary novelty, since it enabled greater stability in the storage of biological information, and it allowed an increase in the size of the genome of organisms. In the present work, the data suggest multiple origins for this molecule, having originated at least twice in the bacterial lineage and once in the Archaeal lineage. These data suggest a complex scenario, as it is suggested that viral lineages played an important role in the dissemination of DNA replication machinery in both bacterial and archaeal lineages. From the data presented, we can infer a scenario for the emergence of DNA as the main informational molecule in cellular lineages, as well as in viral lineages. Before suggesting a scenario for the origin of a DNA genome in basal cellular lineages, as well as for some viral lineages, we must delve into the scenario prior to this transitional event. In this context, we can think of three scenarios before the diversification of basal lineages: (i) the last common universal ancestor was a cellular organism with a DNA genome; (ii) the last common universal ancestor was cellularized and had an RNA genome, and (iii) the last common universal ancestor was not cellularized, thereby being a progenote with an RNA genome ,17,18,19The results of the present study allow us to suggest that cellular lineages emerged independently, even with an RNA genome. In this scenario, viral lineages emerged in parallel with cellular lineages. In the bacterial lineage, the emergence and maturation of DNA-dependent DNA polymerases, and of the DNA genome, occurred after initial diversification of this group, which occurred in at least two independent ways. In the case of family A, the transition process occurred in the ancestral lineages of mitochondria, wherein this machinery was transferred to viral lineages that spread to the rest of the bacterial lineages. In the case of family C, the replication machinery and DNA genome that emerged in another bacterial branch were transferred to a viral lineage and then spread to the rest of the bacterial lineages. Through this route of maturation of the DNA genome, the machinery emerged and established itself within cellular lineages. Viral lineages of bacteria inherited this characteristic of cellular lineages and acted as a dissemination system for the rest of the lineages that had not yet made the transition from an RNA genome to a DNA one. In the case of family B, the data suggest that the transition from an RNA genome to a DNA genome occurred first in the viral lineages and was transferred to the basal archaeal lineage before the diversification of this group. Thus, we suggest that DNA in Archaea arose as the main informational molecule before the same process occurred in bacterial lineages. In this context, we assume that all families of DNA-dependent DNA polymerases had an RNA-dependent DNA polymerase as their ancestral molecule through a process of diversification by radiation . This fact may explain in part why there is not any homology across the different families. As each family followed its own evolutionary path, the similarities in terms of sequence were lost, leaving just the structural similarities essential to this group of proteins’ functionalities. Although we assume the scenario described above, other scenarios were proposed and discussed by Leipe et al.  and by EAs a result, while the identification of the enzymes participating in certain processes may not directly point to the process’s origin, it may provide us with hints that help us build hypothetical scenarios. In this sense, creating many scenarios is legitimate for generating new hypotheses that need to be evaluated through improvements in data and analysis methodologies, even though they do not reflect a final decision on the matter.Clearly, the findings presented do not put an end to discussions concerning the origin and evolution of the DNA genome in cellular lineages; rather, they introduce a fresh scenario for discussion considering potential present-day and future evidence.


Protected areas (PA) are one of the primary tools for conserving and protecting biodiversity, but their goals have evolved overtime beyond nature conservation to include supporting human communities within and adjacent to the PA. UNESCO World Heritage Sites (WHS) offer a unique perspective on the success of PAs as they fall under three categories, cultural, natural, and mixed heritage sites. The nature of these categories encapsulates the inclusion of human communities in the goals of the WHS. To understand the impact and relationship the WHS has with its surrounding landscape, we assessed changes in three indicators, land use and land cover (LULC), human footprint (HF), and forest landscape integrity index (FLII), across three spatial scales, 1, 10, 100 km from the WHS boundary. We found that there is a conservation spillover effect at least within 1 km of the WHS boundary. In this buffer zone, HF was low and FLII was high. FLII was lower and HF was higher at larger spatial scales. The relationship between the WHS and its surrounding landscape is one reason to support the WHS network, however, management of PAs should be more explicit about this relationship as well as relationships between individual PAs. Protected areas (PA) are one of the primary tools for conserving and protecting biodiversity. As a conservation tool, over time the goals of PAs and conservation broadly have changed . Throughe.g., logging) within its boundaries, the result may be detrimental to ecosystem function within the border if left unchecked; however, the tradeoff is that local people can still have access to resources that were previously available to them. In this context, social ecological systems research has shown that some PAs designated for sustainable use , experienced more socioeconomic development that translated to empowerment of local people, fewer livelihood costs, and more cultural benefits  classification of PAs negatively or positively impact the surrounding landscape and to what spatial extent are those effects felt? As a unique subset of PAs, UNESCO World Heritage Sites provide a framework to understand the interactions between human and natural systems within and beyond PA boundaries. Moreover, international organizations from various sectors like the World Wildlife Fund, ICMM, and Globally Important Agricultural Heritage Systems, have commented on their role in conservation by explicitly mentioning preserving UNESCO World Heritage Sites or including them in their mission . In addiWHS are areas deemed to have universal value for their cultural, natural, or mixed heritage making them worthy of preservation for current and future generations to experience and learn from. The establishment of a heritage site is proposed by that community , suggestWe evaluated UNESCO World Heritage Sites (WHS) and their buffer zones of different scales at a global extent . We usedWe obtained primary WHS site location and PA data from the UNESCO World Heritage Site and the World Database on Protected Areas (WDPA) data downloads respectively . The oriIndicators used in the general literature to assess the impact of PAs include human appropriated net primary production HANPP,  and foreWe imported the WHS shapefile and indicator rasters into R. We used the st_buffer tool in the sf package  to creatWe observed the frequency distribution of land use and land cover to descriptively compare between scales, regions, and categories. For FLII and HF, we used linear models and AICc model selection  to deterAfter filtering WHS with the protected areas database, most of the remaining WHS are in Europe and North America, about 40% or 153 out of 390. The most common IUCN category is Not Applicable, about 28% of WHS or 110 out of 390. Of the WHS that do list their IUCN category, II is the most common with 74 of the 390. The remaining IUCN categories are as follows, V with 43, VI with 23, and IV with 17, Ia with seven, Ib with six, and III with three WHS . The mosThe most common LULC in the WHS varied as a function of distance , region,Patterns of LULC types across the three scales were different in the five regions. For example, of WHS in the Arab States, inside the border and beyond share bare areas and water bodies LULC types; however, the other types change with buffer distance. Within the PA boundary, mosaic vegetation was the only modal landcover type besides bare areas and water bodies . HoweverThe IUCN categories lend themselves to different frequencies of LULC types across spatial scales. For instance, around Ib, one of the stricter conservation IUCN categories, the most frequent LULC type was open needle leaved deciduous or evergreen forest. Interestingly, in this IUCN category, there was higher heterogeneity of LULC within the WHS than in the surrounding landscape . In Ia, Similar to the other variables, the three WHS categories illustrated varying patterns in LULC frequencies across the buffer distances . One notFor both HF and FLII mean and variance, the model that was supported best by the data was buffer distance along with the interaction between IUCN and WHS categories . As buffHF for both cultural and natural WHS was highest in IUCN category Ia , Table 2Our analysis illustrates our understanding of the evolution of PAs to include interactions between humans and nature at the global scale. Exploring these relationships is key to conservation success and development management plans that consider the impacts of decisions within and beyond the PA boundary and guide resource allocation and access . WHS offvs sustainable regulations. In this instance, the PA classification may be curtailing human pressures within the WHS but could be offsetting that development directly outside the border. Our global perspective also adds nuance to the discussion as land use and land cover patterns are heavily influenced by the biogeographical location of the WHS including climate and historical land use.The results of our analysis provide evidence of a spatial relationship between WHS and the surrounding landscape and suggest the potential positive effect the WHS classification can have on the surrounding landscape. For instance, there was a clear increase in HF at greater spatial extents and a decline in FLII. However, there was great variation in this pattern amongst the interactions between IUCN and WHS categories. For example, IUCN category VI had one of the highest FLII medians in all three WHS categories, suggesting better conservation success with sustainable use of natural resources within the site. Likewise, the descriptive patterns in LULC highlight how different conservation classifications and regions could influence land use within and beyond the WHS boundary, particularly when comparing the most common landcover experienced in those extents. For example, the surrounding landscapes of WHS categorized as Ia and VI have different compositions, highlighting the potential effect of strict The observed patterns in LULC associated with the WHS suggest testable relationships for future research. For example, a temporal analysis could test if the pattern of increasing cropland landcover beyond natural WHS is a consequence of lack of development within many natural WHS or if agriculture in the region has been misplaced due to WHS/PA establishment. This dichotomy is important to explore given the evidence that WHS/PA can serve as sources for food for local people . A lack In our subset of WHS there are more natural WHS than there are cultural WHS, the inverse of the full WHS network. The different needs for cultural WHS than for natural suggest interesting questions. While there is clear value to a global analysis, it is still important to also consider the individual relationships a WHS has with the boarding landscape as these results may not be true for each WHS. In a case study analysis, the positive conservation spillover effect was not consistent at each site . For insBecause of the global nature of study, there were limitations in available datasets for indicators. High quality, fine grain, and easily accessible global data is lacking for environmental indicators. For instance, we had completed the analysis for human appropriated net primary production (HANPP), but then later decided to remove it from the study because the grain size was much larger than the other indicators relative to the size of our buffers. Even though it was removed from the study, HANPP showed similar patterns to FLII and HF with increasing averages as distance increased from the WHS boundary.In addition, better reporting on IUCN categories should be a priority for the UNESCO WHS convention for current and future WHS as the most common type in this study is Not Applicable. These categories are crucial to highlighting trends in the relationship between protection and environmental integrity of the PA itself and its neighboring landscape. These patterns may be the key to highlighting and implementing effective conservation methods and efforts of these culturally and naturally significant sites and their surrounding landscapes.The relationship between a PA and the bordering landscape is important for conservation success within the boundary and environmental integrity beyond. WHS, a global example of the evolution of the purpose of PAs, can have a positive conservation spillover effect into the surrounding landscape at least at the 1 km scale from the PA boundary. The scale of this effect is influenced by the distance from the WHS boundary and IUCN and WHS category type. However, studying the spatial and developmental context of each WHS before inscription is still needed to fully support conservation efforts.Understanding how PAs interact with and affect the surrounding landscapes is crucial to enhancing conservation efforts and meeting conservation goals globally. Landscape change outside the PA boundary can affect conservation methods and effectiveness within the PA itself and vice versa . The spa10.7717/peerj.15858/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15858/supp-2Supplemental Information 2Description of each IUCN category and the count and percent of these categories for the selected World Heritage study sites.Click here for additional data file.


Urban mobility and how people move in our towns and cities is garnering more attention, as solutions are sought to multiple challenges faced by residents; health and physical inactivity, climate change, air quality, urbanisation and accessibility. Traditional, siloed approaches limit impact and collaborative, systems approaches hold promise. However, systems approaches often remain theoretical and few practical applications of their added value have been demonstrated. This study illustrates how a systems approach can be used to underpin the development of a 9-step process to generate solutions for action on active mobility. The development of a systems map and a theory of change framework are key outputs of this 9-step process. The purpose of this paper is to describe how a systems map was developed in an Irish town utilising broad stakeholder engagement to map the variables that influence cycling in the town and to identify the leverage points for transformational interventions. The 2 emissions by sector  is located in the south east of Ireland and it's main urban centre is Kilkenny city with a population of 26,512 . It is t2. It is approximately 3 km wide and 4.5 km long with many destinations easily accessible by foot or by bike. The County Development Plan . The sampling method was unrestricted and self-selected (n = 30), final edits were made and the survey was circulated through social media platforms of the organisations represented in the original Smarter Travel committee.The community wide survey was conducted in June and July 2019. Ireland was re-opening following a full lockdown and moving into Phase 2 of Covid-19 restrictions; people were now allowed to travel within their county and meet up to six people from outside their household. Organised indoor events were not permitted and working from home was to continue where possible. For these reasons, the survey was hosted on the Survey Monkey platform, a non-probability (convenience) sample was generated . Additional items such as physical activity , bike an2.4.The stakeholder engagement was designed to elicit a greater understanding of the complex issues and the nuances that shape cycling patterns in a local context. Critically, a rich collection of experiences was gathered and diverse stakeholders were engaged early in the participatory planning of interventions. An in-depth qualitative study was conducted using several methods of data collection to ensure a broad and diverse engagement and to capture the voices that are often unheard. These methods included semi-structured interviews with key stakeholders, community workshops, targeted focus groups and reflective note taking following meetings and events throughout the engagement process. This approach enabled the researcher to understand the challenges faced by key stakeholders working at the intersection of transport and health. Throughout the process, stakeholders were encouraged to share potential solutions to mobility challenges, to identify possible leverage points and suggest interventions.For participation in the stakeholder engagement, respondents were contacted in advance, by email or phone, and invited to participate. Upon confirmation of attendance, follow up emails were sent to obtain informed consent and permission for recording, to describe the purpose of the study and present the interview or topic guide schedule. Interviews were recorded using a dictaphone or through the zoom platform. All interviews and workshops were transcribed verbatim, de-identified and imported into NVivo . Researcher notes and memos were also imported into NVivo.2.4.1.n = 7). An interview guide was developed based on the researcher's knowledge of the factors influencing cycling and the local context. The interview guide prompted respondents to describe their ideal city and possible mobility solutions, to discuss challenges to mobility and the necessary steps to help overcome these challenges. Interviewees were encouraged to share their unique personal and professional experiences of mobility and cycling in the city.Semi-structured, face-to-face interviews were conducted with key stakeholders in May and June of 2020. The set of stakeholders represented those in key decision maker roles at national and regional level, cycling advocates and city centre traders ; a forum for citizens involvement in decision making, the Chamber of Commerce, and Cycle Kilkenny; a group of organisations, including the local authority, working together to promote cycling in Kilkenny. The workshops moved to an online format on the zoom platform due to COVID-19 restrictions. Invitations were extended and permissions were sought through the three organisations. The workshops all followed a similar format. Firstly, information was presented on the demographic and travel trends, mode shares and the local policy context. Depending on the number of attendees, questions were then posed to all attendees or smaller groups with facilitators, with targeted probing of issues specific to the experiences and expertise of the participants. Issues arising varied greatly with the diverse stakeholders. Existing cyclists focused on the difficulties experienced moving through the city, cycling infrastructure deficiencies and the ideal cycling city. Non-cyclists discussed the barriers they faced to cycling, the forced car-dependency, the lack of alternative mobility choices and possible solutions. Participants were invited to use the chat function throughout the workshops. The smaller workshops were recorded and transcribed. Note-taking was used with break-out rooms and additional facilitators for the larger workshops.2.4.3.The online nature of the survey led to under representation of certain cohorts of the population. These were identified through a comparison of the respondent's data with the population demographics from the census. Typically, those missing or under-represented were the cohorts of the population that encounter limitations in how they engage with and move through the cityscape and those who traditionally face barriers to engagement. A targeted series of seven focus groups were conducted with the following participants: people with chronic health issues, people with an intellectual disability, people with physical disabilities, older adults, single parent families, members of the travelling community and members of migrant communities. The workshops and conversations focused on needs and experiences in regards to mobility, accessibility and public space. These workshops were conducted in partnership with the enterprise partner and analysed separately. The findings captured the experience of individuals and together the individual perspectives created a holistic picture and understanding of how people move through and interact with their city .2.4.4.The stakeholder engagement process was enriched by the embedment of the researcher alongside the newly formed active travel team. This enabled the researcher's involvement in, and attendance at planning and project meetings with city traders, elected representatives, strategic policy committees and transport authorities. A Sustainable Transport group was formed for this process with representatives from the local authorities, planning, transport and environment, and other stakeholders. Following three meetings, an application was made to Interreg Europe for a peer review process. This resulted in a two-day workshop with in-depth discussion with five experts in sustainable transport focusing on the mobility needs in Kilkenny. The Interreg Europe team facilitated the workshop with the peers, the local authority and other key stakeholders. Following this, a decision was made to extend the Sustainable Transport Group to a wider group of stakeholders and the local authority began the process of developing a Sustainable Urban Mobility Plan. Insights and understandings were gained throughout this process by the researcher. Further learnings were gained through attendance at a study visit with the Dutch Cycling Embassy undertaken by the Active Travel team and political representatives, meetings with the National Transport Authority and team meetings with the Active Travel team. Regular meetings and bike rides took place with cycling advocates. Over 50 workshops and meetings took place in the engagement process. Reflective notes were recorded during and after meetings and added to NVivo as memos. Prompts for note taking included discussions on challenges faced by the stakeholders, problem definition, and novel and innovative solutions.2.4.4.1.Several data analysis strategies were considered. The envisioned outcome required identifying, analysing and reporting themes to build the systems map which led the researcher to choose thematic analysis. This approach does not begin with a theory or hypothesis to be tested but instead adopts an inductive approach to data analysis . Respons2.5.A systems map was drafted by the researcher based on the findings of the literature review using the KUMU online platform. The initial draft of the systems map was adapted from the Global Action Plan on Physical Activity 2018–2030 (GAPPA). The GAPPA map sets out four strategic objectives; Create Active Societies, Create Active Systems, Create Active People, Create Active Environments. The factors identified from the literature as influencing cycling were added to the maps as elements. The first draft of the map was prepared prior to the stakeholder engagement and was presented to prompt discussion at the community workshops and meetings of the Sustainable Transport Group. The majority of issues arising throughout the community engagement process reflected the findings of the literature. However, following the thematic analysis of the qualitative research, the map was restructured and a fifth strategic objective; Create Safe Environments, was added. Leverage points, places within a complex system where a small shift in one thing can produce big changes in everything , were suggested throughout the engagement and recorded. Building the map was an iterative process, throughout the research, as the researcher worked alongside the Active Travel team and incorporated the learnings from planning and project meetings. The map is presented below in 3.This section presents the results of Steps 3, 4 and 5.3.1.Findings of the literature review informed the research design and initial draft of the systems map. Mobility trends were summarised and the detailed analysis is presented on the project website together with a review of the policy and planning context .3.2.•Adopt a systems approach to map the variables that influence cycling in Kilkenny City•Utilise broad stakeholder engagement to identify the leverage points for transformational interventions•Develop a theory of change framework to cultivate a cycling culture in KilkennyThe 9-step process was outlined to achieve these objectives.Through research team meetings and meetings with the research partners and other stakeholders, the following research objectives were formulated:The functional area of the research was defined as the two electoral areas in Kilkenny City and the electoral area of Kilkenny rural, covering the functional area of Kilkenny city.The stakeholders were identified as those already involved in the Cycle Kilkenny stakeholder group; Kilkenny Leader Partnership , Chamber of Commerce, Cycling Advocates, the Local Sports Partnership, Disability Access Group and transport representatives from Kilkenny County Council. Additional stakeholders were added so that the group was representative of the entire population. The stakeholder list was reviewed against the quintuple helix model , represe3.3.Over 50% of respondents lived in Kilkenny City, a further 17% lived within 5k of Kilkenny, a quarter of all respondents lived between 5 and 20k from Kilkenny and just over 5% live greater than 20k from Kilkenny City. Just over 60% of the respondents were female and 60% of all respondents were between 40 and 60 years of age. 90% of respondents owned a car and 66% had two or more cars. Approximately 12% did not have the use of a bicycle.Almost 90% felt that their community did not have a problem with crime or anti-social behaviour. The vast majority (88%) of the respondents felt that people in their community can be trusted and almost 70% felt that they knew their neighbours either moderately or extremely well.When asked about their community and cycling, 55% felt cycling was unsafe because of the traffic and almost half felt that there were no convenient routes for cycling. 82% of all respondents did not agree that cyclists were sufficiently separated from traffic. Five per cent had no interest in cycling, over 40% would like to cycle more and the same number again would like to cycle more if the conditions were right. The most frequently cited barrier was that people don't feel safe enough on the roads. The next three barriers were; not practical for carrying things, journeys are too far and multipurpose trips .When asked about their perceptions on cycling, over 90% of all respondents agreed or agreed strongly that people who cycle regularly improve both their health and well-being, that it would be better for the environment if more people cycled and Kilkenny would be a better place if more people cycled. The majority (96%) of all respondents believed that the Local Authority should be investing money in cycling. In spite of the concerns around safety, over half had positive experiences of cycling in Kilkenny but over 17% had never cycled in Kilkenny. The main motivators for cycling were to keep active, get fit or for health reasons and to spend time outdoors followed by environmental reasons.More than half (55%) of respondents wanted pedestrianised areas in the town centre. Outdoor public seating was also widely looked for. Almost 30% of respondents wanted to see better access for people with disabilities. The biggest changes that respondents would like to see in the town centre were more street markets, less empty shops and more art/recreational or leisure facilities and more small specialised shops. The two most sought-after design features were spaces for outside dining and provision of cycling lanes. Almost 90% of people felt that space should be reallocated in the town centre to prioritise pedestrians and cyclists and 85% felt there were sufficient open spaces in their community. An infographic see  above su3.4.A thematic analysis was conducted of the transcripts from semi-structured interviews with seven key stakeholders, three community workshops, and notes and memos from over 50 meetings. The findings of the focus groups were also included in the analysis. The main themes and related subthemes emerging from the inductive thematic analysis are presented in 3.5.The final systems map is presented in 4.The systems approach adopted for this research allowed for a clear understanding to be gained of the process of change for large, complex systems, specific to cycling in Irish towns. This approach enabled an examination of the interrelationships in the system from multiple perspectives . A broad4.1.The elements presented under this objective, represent the variables that underpin the system that will allow transformational change to occur. Specifically, there were seven elements which included; policy leadership and governance, integrated transport and land-use patterns, sustained finance mechanisms, multi-sectoral engagement, citizen engagement, advocacy and integrated data systems. Over the last number of decades, our transport systems have been designed in response to the growth in car ownership to facilitate increasing car use resulting in greater allocation of space in urban centres to private cars and the creation of hostile environments for pedestrians and cyclists. This has induced greater car use and car dependency and by favouring the car, urban sprawl and dispersed population patterns have ensued, resulting in reduced active modes . The tra4.2.Under these two objectives, seventeen elements were identified and the perception of lack of safety was to the fore across all engagements. This is consistent with narratives from other car-dependent countries. Without the provision of safe, segregated cycling infrastructure, cycling is only for “the strong and the fearless”, and perhaps “the enthused and confident” . The des4.3.These two strategic objectives contain elements that relate to cultivating a cycling culture in the wider society and under-served population groups and settings. The private car has traditionally been the first choice in Kilkenny with high car ownership and modal share. The lack of alternative mobility solutions was cited frequently in the community engagement and the visibility of other transport options is poor. Cycling is regarded as a crucial element of a multi-faceted solution that views cycling not as a standalone transport mode but as a key piece in the transport offering that is highly dependent on the allocation of space and integration with public transport, park and pedal, micro-mobility and other last mile solutions in smaller cities and towns . LeveragThe elements under Creating Active People were derived from a strong viewpoint that vulnerable road users were not catered for in a car orientated city, resulting in a loss of independent journeys and a disparity in access to social, health, education and employment opportunities. These inequalities associated with access to transport have been reported elsewhere in Ireland . To over4.4.Although, it was not a specific objective of the process, the five steps that culminated in the development of a draft systems maps helped create a vision for a cycling city in Kilkenny. The vision is a city that integrates a strategic cycle network with multi-modal provision whilst retaining the rich culture and heritage of the medieval city. The riparian, green spaces provide opportunities for accessible, connective corridors in the city, encouraging active engagement with nature. This vision, if it comes to fruition, will result in a healthier, more liveable city with greater quality of life for all citizens.The use of a participatory systems approach in this research, allowed for an enhanced engagement process and a detailed mapping of all the variables that influence cycling in Kilkenny and in similar small cities. Subsequently, the mapped variables informed the process of identifying leverage points and involving a broad group of stakeholders in solution generation. In this way, complex problems can be disaggregated, assigned to and analysed by the stakeholders with the expertise, experiences and potential to intervene. The systems approach has resulted in the foundations for an overarching vision, engaged stakeholders and a reservoir of resources to establish a platform to bring about transformational change in the mobility system. A limitation of the systems approach is the greater investment required in the planning stages. Broad stakeholder engagement necessitates additional co-ordination and results in longer preparatory phases. However, the ensuing actions are embedded in local organisations, and are systemic and transformational.The next steps of this research will focus on utilising this platform to operationalise the systems map. Casual loops will be identified in the systems map. Stocks and accumulations in the system such as car/bike ownership, existing infrastructure and ongoing investment, will be analysed. Interventions will be co-designed with stakeholders and mapped onto a Theory of Change Framework.5.The paper described how a systems map was developed in a small Irish city utilising broad stakeholder engagement to map the variables that influence cycling in the town and to identify the leverage points for transformational interventions. This work adds to the development of systems approaches to tackle complex problems as it synthesises the scientific evidence, a participatory systems approach and the pragmatic findings of the embedded researcher working alongside an active travel team. The systems map will form the basis of a framework for cultivating a cycling culture in small cities and will take it beyond a theoretical framework to an organic, live process.