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The research was carried out on the basis of direct measurements in the surroundings of four selected working coal-fired power plants and four working coking plants. The samples of suspended dust PM10, respirable fraction PM2.5 and submicron particulate matter PM1 were collected in the surroundings of power generation facilities and in the surroundings of coking plants.Location of measurement pointsThe location of the measurement points was selected in southern Poland, around the selected four working coal-fired power plants and four working coking plants. The sampling points in the surroundings of the power plant (P1, P2, P3 and P4) and the coking plant (K1, K2, K3 and K4) were located at the distance of approximately 2 km to the north-east from the respective object (Fig. 1).Figure 1Location of the sampling sites (the map was generated based on data from the BDL18 website).Full size imageThe location of the measurement points was a compromise, taking into account the representativeness of the receptor, the possibility to connect the testing equipment and the consent of the property owners. To eliminate the impact of a heating season, and especially that of low emissions, presented in the studies by19, the measurement sessions were carried out only in the summer season. The samples of particulate matter were collected on a weekly basis, with 4 sessions at one site. The methodology applied in this work is presented in20,21. The location of measurement sites:

point P1: 50° 08′ 37.87″ N; 18° 32′ 15.76″ (Golejów—a suburban district of Rybnik in the Śląskie Voivodeship, in the vicinity of a working power plant with a capacity of 1775 MW; population:

2 300);

point P2: 50° 45′ 35.41″ N; 17° 56′ 20.43″ E (Świerkle—a rural area in the Opolskie Voivodeship (Dobrzeń Wielki commune) near a working power plant with a capacity of 1,492 MW; population: 520);

point P3: 50° 12′ 33.46″ N; 19° 28′ 28.77″ E (Czyżówka—rural area in the Małopolskie Voivodeship (commune of Trzebinia) near a working power plant with a capacity of 786 MW; population: 700);

point P4: 50° 13′ 48.90″ N; 19° 13′ 24.45″ E (suburbs of Jaworzno (Śląskie Voivodeship) in the vicinity of a 1,345 MW power plant; number of inhabitants: 95 500);

K1 point: 50° 10′ 11.36″ N; 18° 40′ 34.35″ E (Czerwionka—Leszczyny in the Śląskie Voivodeship, in the vicinity of a small coking plant; number of inhabitants: 27 300);

K2 point: 50° 3′ 19.76″ N; 18° 30′ 21.69″ E (Popielów—a suburban district of Rybnik in the Śląskie Voivodeship, surrounded by a small working coking plant; population:3 300);

K3 point: 50° 21′ 24.08″ N; 19° 21′ 37.46″ E (Łęka—Dąbrowa Górnicza district, in the Śląskie Voivodeship, surrounded by a large coking plant; number of inhabitants: 700);

K4 point: 50° 21′ 0.47″ N; 18° 53′ 15.44″ E (Bytom—a city in the Śląskie Voivodeship, a small coking plant located on the outskirts of the city; population: 174 700).

The state of air pollution with particulate matter in the area investigated in the study is affected by various local sources of pollution emissions. At the measurement sites P1, P2, P3 and P4, the emissions are mainly from power plant chimneys, but also from auxiliary processes, i.e. coal storage and its transport. In addition, the recorded emissions are also influenced by other industrial plants operating in the vicinity of the measurement sites, domestic and municipal sector and the impact of automotive industry. The measurement sites K1, K2, K3 and K4 involve primarily the emissions accompanying the processes of coal coking as well as auxiliary processes, i.e. coal deposition, its transmission, management of products and post-production wastes. Additionally, they are affected by the emissions from industrial plants and low emission sources operating in this area, as well as the emission from the combustion of solid fuels for domestic or municipal purposes, as well as by the automotive industry.Sampling processThe samples of suspended dust (PM10), respirable fraction (PM2.5) and submicron particulate matter (PM1) were collected using the Dekati PM10 cascade impactor serial No. 6648 by Dekati (Finland) with the air flow rate of (1.8 {mathrm{m}}^{3}/mathrm{h}). The impactor Dekati PM10 guarantees the collection of dust samples for three cutpoint diameters: 10 μm, 2.5 μm and 1 μm. For the sampling at the first, second and third stages of the impactor, polycarbonate filters were used (Nuclepore 800 203, with the diameter of 25 mm, by Whatman International Ltd., Maidstone, UK). At the fourth stage, the dust was collected on a Teflon filter for particles ≤ 1 μm in diameter (Pall Teflo R2PJ047, 47 mm in diameter, by Pall International Ltd., New York, NY, USA). The average volume of air passing through the filters was approximately 300 m3. The impactor’s capture efficiency was characterized by the uncertainty below 2.8%. The mass of dust collected at the individual stages of the impactor was determined by the gravimetric method, and it was referenced to the volume of passed air (left(mathrm{mu g}/{mathrm{m}}^{3}right)) according to the PN-EN1234122. All impactor samples were analysed by inductively coupled plasma mass spectrometry (ICP-MS).The samples were collected at a height of 1.5 m from the ground, i.e. in the breathing zone for people. The respective dust fractions were collected in 7-day cycles from 28 May to 24 September 2014 (16 weeks) in the surroundings of four working coal-fired power plants and from 4 May to 28 August 2015 (16 weeks) in the surroundings of four working coking plants. The measurement campaign comprised four measurement sessions separately for each sampling site. One session comprised dust sampling at each stage of the Dekati PM10 cascade impactor and filters used for reference. The filters were taken back after study period and labeled during the collection process in the field and stored in the plastic containers for safe transportation and storage in laboratory for further analysis.In each measurement session, blind filters were stored at the sampling site, but they were not subjected to exposure. The sample data were corrected from these blanks. The length of the measurement cycles was conditioned by the need to collect an appropriate amount of research material (with the aerodynamic diameter of the dust grains  10 μm). Analogous (7-day) periods of dust sampling were used in the studies by4,23.Polycarbonate and Teflon filters were conditioned before and after dust collection at a temperature of 20 ± 1 °C (relative humidity 50%(pm ) 5%) for 48 h, and then weighed on a microbalance with an accuracy of 1 (mathrm{mu g}) (MXA5/1, by RADWAG, Poland).Taking into account the measurement sessions at four sites in the surroundings of the power plant (P1 (div) P4) and at four sites in the surroundings of the coking plant (K1 (div) K4), the aggregate number of samples exceeded 450.Chemical analysisThe qualitative and quantitative analysis of the obtained solutions was performed by inductively coupled plasma mass spectrometry using an ICP-MS instrument (NexION 300D, PerkinElmer, Inc., Waltham, MA, USA). For all elements determined simultaneously, the same parameters of the instrument were used, which are presented in the publications20,21,24.As standards for the determination of 75As, 111Cd, 59Co, 53Cr, 200Hg, 55Mn, 60Ni, 206Pb, 121Sb and 82Se, we applied the 1000 (mathrm{mu g}/{mathrm{cm}}^{3}) CertPUR ICP multi-element standard solution VI for ICP-MS by Merck, Germany. Ten repetitions were performed for all samples. The determined limits of detection (LOD) were based on 10 independent measurements for blank test. For the results obtained in that way, the mean value and the value of the standard deviation SD were calculated. The values of LOD for individual elements were determined on the basis of the dependence (1):$$mathrm{LOD}= {mathrm{x}}_{mathrm{sr}}+ 3mathrm{SD}$$
(1)

where: xśr—mean concentration value of the element, (mathrm{g}/{mathrm{dm}}^{3}), SD—standard deviation.The determination correctness of the content of the elements was verified with the use of certified reference materials: European Reference Material ERM-CZ120 and Standard Reference Material SRM 1648a (National Institute of Standards and Technology, USA). The recovery with the use of the said certified reference materials was respectively as follows: As (111% for ERM-CZ120 and 96% for SRM 1648a), Cd (97% and 105%), Co (108% and 97%), Cr (103% and 94%), Mn (106% and 100%), Ni (107% and 102%), Pb (107% and 105%) and Sb (99% and 91%). The certified reference materials did not contain Hg or Se. More

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Despite only focusing on one female Chinese pangolin, LF28, our study, to our knowledge, is the first to provide highly detailed records on the nursing behavior of this poorly studied but critically endangered species. During the entire tracking period, the body weight of LF28 increased from 2 kg at the age of 1 year to 3 kg at the age of 2 years, and LF28 reached her maximum body weight of 4 kg at the age of 3 years. Based on the uninterrupted monitoring between Dec 2014 and June 2016, LF28 gave birth to her first offspring when she was 3 years old and another one at the age of 4 years (Fig. 1). Both infants were born in early December, which were in accordance with the peak birth season of the species10. Our observations confirmed that the Chinese pangolin is a seasonal breeder in the wild, and they give birth once a year10,11. Also, they can give birth in consecutive years with a litter size of one17.Other studies (n = 4) have found that the lightest weight, or youngest age, a female Chinese pangolin can give birth at the age of 2 years or weight of 3 kg11,17,18, which indicated that they can conceive at an age of 1–2 years. Therefore, the first birth of LF28, which took place when she was 3 years old, might suggest a delay in pregnancy or sex maturation. However, information concerning the average primipara age for this species is not available to date, more research, especially in the wild, is necessary.Our results indicate that female Chinese pangolins will carry their offspring frequently from one nursing burrow to another during the entire nursing period. In the case of LF28, nursing burrows were only some of the resting burrows utilized and were predominantly located within the core area (MCP75) of her home range (Fig. 4), despite the close proximity to human settlements. This suggests that familiarity of the environment or food resource availability should be important considerations in nursing burrow selection.Nursing burrows were normally used only once during the same nursing period, with durations varying from 1 day to more than 1 month (Fig. 5). This frequent relocation behavior should be important to avoid predation of the newborn. Our monitoring showed that small carnivores, such as ferret-badgers or crab-eating mongoose, will enter the nursing burrow, which may suggest they are searching for prey. Therefore, this could reflect a potential threat to the infant pangolin, especially when the mother is absent for foraging8.Burrows where LF28 gave birth were not only used for the longest duration after birth, they were also used before parturition. Similar to our findings, a previous study reported that both males and females will collect and pull hay into the resting burrow in the wintertime3. Therefore, in addition to providing insulation, the hay could also serve as necessary bedding for the delivery and nursing of offspring. Other functions of hay that have been proposed include false barriers that can act as predator deterrent structures19.Our records revealed at least two different adult male pangolins approaching and entering the nursing burrows multiple times throughout the nursing period. Most of these visits lasted only minutes, whereas a few lasted longer. During one long visit, in March, mating behavior was observed, therefore the occurrence of post-partum estrus, or even ovulation, may be likely for this species. In captivity, mating behavior was also observed between February and July10,20. Although there is no direct evidence yet, these adult male visits suggest that at least some of them were for mate-searching. It has been proposed that while mammalian females spend more energy on parental care, males often invest more energy towards seeking mates21. For solitary and fossorial species such as the pangolin, a male’s mate-finding tactics can be critical for mating success, especially due to the low population density22,23. Male pangolins most likely depend on olfactory cues to locate females in heat. In mammals, female chemical signals have important roles in sexual attraction and facilitating sexual receptivity24,25,26,27. Female Chinese pangolins tend to defecate close to the burrow during the nursing period (N.C.M. Sun unpubl. data), therefore, despite the frequent relocation behavior expressed by the mother, it was likely to generate sufficient olfactory information for male pangolins.It is also possible that female pangolins will mate more than once, even with different males, during the same nursing period. Sun et al.17 have reported that certain female Chinese pangolins exhibited a lack of mate fidelity based on microsatellite marker assessments. Our observation provides additional support for this phenomenon. Multiple mating with the same or different males has been observed in several solitary carnivores28,29,30,31. For males, frequent pre-copulatory encounters with females may offer advantages that increase opportunities for mating compared to males that are less familiar with females32,33. Hypotheses concerning the advantages of females exhibiting promiscuity have also been widely proposed, including direct benefits (e.g., stimulation of reproduction, fertilization assurance, mate retention etc.) and genetic benefits (e.g., choice of paternity, sperm competition, inbreeding avoidance etc.)34,35.Interestingly, during two separate visitations adult males exhibited excavation behavior, and both events took place shortly after parturition. This excavation behavior at a parturition burrow has never been reported before for male pangolins, therefore, further research is needed to better understand the role male pangolins play in parental care.The fetus of LF28’s second offspring detected in the ultrasonographic image in Aug. 15 provided additional information on the gestation length of the species. Following the fetal and extra-fetal structure development of small-sized (3–8 kg) dogs described in Luvoni and Grioni36 and Kim and Son37, we estimated the gestation period of this fetus may have lasted 30–40 days or less. The implantation of the blastocyst, therefore, most likely occurred in early July. This infant pangolin was born on Dec. 8 later that year, and the gestation length was estimated to be around 150 days, which was shorter than previous reports4,9,10. This was the first estimation of gestation length of the Chinese pangolin based on physiological evidence under natural conditions.Our findings of the gestation period, which took place later in the year (July–December), coupled together with the occurrence of post-partum estrus and mating earlier in the year (December–May), suggests that delayed implantation likely takes place in this species, as proposed by Chin et al.11. This also explains why there was such an extensive variation in the gestation length, from 180 to more than 372 days, determined based on the observation of mating behavior and parturition in captivity10,11,18. More studies on the reproductive physiology for this species are necessary.Lastly, the present study also demonstrated that the difficulties associated with researching the life history and behaviors of the elusive pangolin could be alleviated with the use of technologies (e.g., camera trapping, radio tracking, etc.). This is especially true for non-migratory fossorial species if one has an appropriate knowledge of their home range or residential environment. There are more and more new technologies and devices that have been developed and applied to wildlife research in the field, which should greatly improve our understanding and promote conservation efforts of endangered species such as the pangolin. More

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After 90 years of taxonomic uncertainties, using phenotypic, phylogenetic, and population genetics analyses, the two uncultivated fungi causing skin disease in humans and dolphins, long known as Lacazia loboi8, are now placed as separate species within the genus Paracoccidioides. Early studies using phenotypic or phylogenetic data alone erroneously placed these two fungal pathogens in different genera and species3,4,5,6,7,8,12,13,15,16,17,24,25. This trend persisted for years2,13,16,17,25. For instance, recent studies using several partial DNA sequences recovered from Brazilian humans with skin disease in phylogenetic analyses concluded that the genus Lacazia, the accepted name at that time, was an independent taxon from Paracoccidioides species16,24,25. Their phylogenetic data was correct, but their analyses missed the inclusion of DNA from the uncultivated pathogen causing skin disease in dolphins. This was an understandable mistake, since the collection and processing specimens from infected dolphins is highly regulated and the fact that the etiology of dolphins’ disease was long believed to be the same as that in humans, as shown in Fig. 1 and Table 1. Although P. cetii has numerous phenotypic differences with Paracoccidioides species (Table 1, Fig. 1), in the pass used to group them in separated clusters2,3,7,8, our data showed they share several phylogenetic features in common (Figs. 4, 5 and 6). With the addition of P. cetii DNA sequences, the phylogenetic support of closely related Paracoccidioides species dramatically changed. For example, P. loboi clustered in a monophyletic group sister to P. lutzii, even with the inclusion of homologous dimorphic Onygenales DNA sequences as outgroup (Figs. 4b, 5), whereas the support of monophyletic species within the genus weakened (Figs. 4, 5 and 6). More dolphin DNA sequences from different geographical locations must be sequenced to understand P. cetii´s true evolutionary traits.Several studies reported geographical cryptic speciation among Paracoccidioides species14,24,26,27,28. In those analyses the presence of at least five species within the genus, including P. lutzii, was found14,15,24,27,29,30. Recent genome sequencing in phylogenetic analysis tend to validate these findings26,28,29. Although the DNA sequences of P. loboi were used in some of the analyses, the human skin pathogen was always placed as an independent genus from that in Paracoccidioides species16,24,25. The placement of P. cetii sister to P. americana DNA sequences in this study, indicates the use of phenotypic or phylogenetic characteristics without the inclusion of anomalous species, can lead to inaccuracies in the taxonomic and phylogenetic classification of these type of microbes. For instance, our data, using several statistical tools, consistently showed the presence of different clusters within Paracoccidioides species. In our analyses, P. americana, P. cetii, P. lutzii, and P. loboi were placed in monophyletic groups sister to the remaining Paracoccidioides species (Figs. 2, 3, 4, 5 and 6). Therefore, the addition of P. cetii to the genus Paracoccidioides not only confirmed that the genus has indeed a high level of speciation but, indicates that the concept of species delimitation in this genus must be revisited12,31.Recently, Vilela et al.16, using phylogenetic analysis of five different genes, showed P. loboi shared the same ancestor with Paracoccidioides species. The results in our study support their proposal. The main obstacle of this hypothesis at that time was the phenotypic features of P. loboi (Fig. 1). However, if P. loboi and P. cetii (both uncultivated and subcutaneous pathogens) share the same ancestor with other Paracoccidioides species (cultivated and causing systemic infections), the likelihood that the ancestor of Paracoccidioides species could growth in culture, as previously suggested, is a strong possibility16. If this concept is correct, when in the evolutionary history of P. cetii and P. loboi they lost the capacity to grow in culture? What evolutionary pressure triggered such a change? Sadly, as is common in neglected pathogens such as P. cetii and P. loboi key questions such as these, remain without an answer. Interestingly, the uncultivated feature found in these two neglected fungi was also reported in a strain of Histoplasma capsulatum infecting monkeys, suggesting that an uncultivated ancestral trait in the Onygenales dimorphic fungi may be at work32. However, the evolutionary pressures that triggered such ancestral feature remains an enigma.The report of new human cases of paracoccidioidomycosis loboi acquired by traveling to endemic areas2,3,4,5,33,34,35,36, suggests P. loboi may has a similar phenotype (hyphae with conidia) to the one displayed by Paracoccidioides species in nature and in culture. Thus, it may be present in specific ecological niches in the endemic areas (around the Amazon basin and other Latin American big rivers)2,14,15,25. Therefore, it is possible P. cetii and P. loboi may have a phenotype in nature similar to that of Paracoccidioides species (hyphae with conidia). Under this scenario, both uncultivated pathogens display a mycelia form with conidia and the classic life cycle style of dimorphic fungi in nature25. As is the case in other dimorphic fungi, these propagules could then contact susceptible hosts (human, dolphins) switching from hyphae → yeast thus, causing subcutaneous infections. Perhaps due to abnormalities on the molecular mechanisms of yeast → hyphae conversion (mutations?), once the hyphae → yeast conversion occurs, it cannot longer switch back from yeast to hyphal phase. However, the yeast phase of both pathogens can infect other hosts, as had been demonstrated in accidental and experimental infection with yeast-like cells from infected humans and dolphins2,37,38,39,40,41,42. Despite attempts made by the Broad Institute (https://www.broadinstitute.org/fungal-genome-initiative/lacazia-loboi-sequencing), only fragmented genomic information is available for P. loboi, and the genome of P. cetii is yet to be sequence. We hypothesize that the genomes of both uncultivated pathogens may hide important genomic clues that could answer this and other evolutionary questions.Several P. cetii DNA sequences recovered from dolphins captured in Brazil, Cuba, Japan, and the USA are currently available in the database (Table S1)19,20,21,22,23. The complete ITS DNA sequences from Brazilian and Cuban dolphins with paracoccidioidomycosis ceti, showed high percentage of identify with the DNA sequences in this study (ITS = 100%) whereas the partial Gp43 DNA sequences from a Japanese dolphin (471 bp) had 98.62% identity with P. cetii DNA sequences from dolphins captured in the Americas. During Gp43 DNA alignment of Japanese and USA dolphins, a five nucleotides gap was consistently present in the DNA sequences of USA dolphins. Moreover, two additional 266 bp GP43 DNA sequences extracted from a Japanese dolphin (Lagenorhynhus obliquidens) with paracoccidioidomycosis ceti showing, 99.62% identity with P. brasiliensis (sensu lato). In our analyses, these two sequences (only 110 bp could be used) clustered also with P. brasiliensis (Fig. 4, red rectangle). However, the same DNA sequences clustered close to P. cetii in haplotype analysis indicating a fragile relationship (Fig. 3). If P. cetii DNA sequences from Japanese dolphins are accurate, the differences in the genetic makeup of these two populations of uncultivated pathogens is intriguing and deserve further analysis. Our data suggest P. cetii strains causing paracoccidioidomycosis ceti in Japanese and USA dolphins, likely are evolving into two different populations.According to Teixeira et al.24, the estimated time for genetic divergence in Paracoccidioides species was calculated around 33 million years. Although, others have questioned this result31, Carruthers et al.43, cautioned that the use of linage-specific data usually demonstrate approximate divergence time regardless of the number of loci interrogated. Nonetheless, according to these reports, Paracoccidioides species probably diverged from their ancestor from a fraction of a million of years (P. restrepiensis and P. venezuelensis) to 10–30 million of years (P. lutzii and P. brasiliensis, sensu lato)24,31. Conversely, dolphins evolved into aquatic mammals ~ 50 to 30 million years ago, around late Paleocene period (Eocene, Oligocene epochs)44. According to fossil records, South America at this time had a large body of water crossing from the north Atlantic Ocean to what is today Bolivia, Brazil, Ecuador, Colombia, Peru and Venezuela45, all endemic areas of these species3,4,5,24,26,29, that lasted for millions of years. A similar situation occurred in what is today the estuary of the Amazon River. The current location of Paracoccidioides species (including P. loboi), coincide with the locations of such geological periods, and then it is quite possible that during the time following these geological events, an ancestor of P. cetii first encountered dolphins entering these areas. Since humans came to South Americas only ~ 15,000-year ago46, likely the ancestor of Paracoccidioides species infected dolphin first and later humans. Whether this event had a role on the pathogenic capabilities of the genus to infect mammals is difficult to determine, nonetheless it is an intriguing possibility.Working with uncultivated pathogens infecting the skin of mammals is challenging. Not only because collecting specimens from these species (dolphins are protected species and human cases are located in poor remote rural areas) is extremely difficult, but because open lesions usually harbor numerous environmental contaminants, which in the past had led to erroneous conclusions on the classifications of these two anomalous pathogens2,8,15,16,25,47. Furthermore, these unusual fungi are not in the list of neglected pathogens, thus discouraging investigators to submit proposals to funding organizations. Previous studies using P. loboi in phenotypic or phylogenetic analyses placed this anomalous pathogen away from the genus Paracoccidioides2,4,15,16,25. This study found that the use of phenotypic or phylogenetic approaches without the inclusion of DNA from infected dolphins, likely led previous studies to flawed data15,16,25. Thus, the failure of including organisms sharing a common ancestor, based in phenotypic or phylogenetic traits alone, could result in incomplete or incorrect assessment of the investigated populations. This study showed that the interpretation of taxonomic and/or phylogenetic data could be affected by missing neighboring anomalous taxa. More