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AnimalsThe experimental yaks were divided into four groups: 1-day old, 30-days-old, 180-days-old and adult. Three yaks were selected for each group, regardless of sex, and purchased from a local herdsmen in Haiyan County of Qinghai Province. All of the yaks showed a good nutritional status, and appeared healthy with no apparent diseases or conditions. The yaks were sacrificed by exsanguination in a slaughterhouse. The lungs were obtained immediately after the yak had died, and tissue samples were immediately collected from the diaphragmatic lobe of right lungs (to ensure that obvious blood vessels and the trachea were not gathered). The tissue samples were divided into three parts. One part was cut into 1 cm3 sections and fixed with 4% paraformaldehyde (PFA). The other two parts were cut into 1 mm3 pieces; one part was fixed with 2.5% glutaraldehyde, and the other was put into a freezing tube and placed into liquid nitrogen.Ethics statementThis study was approved by the Institutional Animal Care and Use Committee of Qinghai University (Xining, China). All methods were carried out in accordance with the ARRIVE guidelines and the Animal Ethics Procedures and Guidelines of the People’s Republic of China. No local regulations or laws were overlooked. All yaks used in this study were purchased from local farmers.Haematoxylin and eosin stainingLung tissue samples (1 cm3) were fixed in 4% PFA, dehydrated in 30%, 50%, 75%, 95% and 100% ethanol and then treated with xylene before embedding in paraffin. Paraffin-embedded lung tissues were cut into 4 µm sections. The sections were deparaffinized in xylene, and sections were stained either with haematoxylin and eosin (HE) (Y&K Bio, Xi’an, China) or Masson’s trichrome stain, to examine general morphology.ImmunohistochemistryThe unstained, deparaffinized sections were rinsed with Phosphate Buffered Saline with Twen-20 (PBST) 3 times for 5 min each time. Then, endogenous peroxidase was quenched using 3% peroxide-methanol at room temperature in the dark for 25 min, and then the samples were placed on a decolorizing shaking table 3 times, for 5 min each. The slides were then incubated with 3% foetal bovine serum (Sangon Biotech, Shanghai, China) at room temperature for 25 min. The serum was discarded, and rabbit anti-cattle CD34 and rabbit anti-CD34 polyclonal antibodies (Proteintech group, Wuhan, China) diluted in phosphate buffer saline (PBS) were added. CD34 is a transmembrane glycoprotein known as an angiogenesis marker. The sections were incubated in the primary antibodies overnight at 4 °C. Then, the sections were rinsed in Phosphate Buffered Saline with Twen-20 (PBST) (3 × 5 min), goat anti-rabbit IgG was added, and the sections were incubated for 30 min at 37 °C. 3,3-Diaminobenzidin (DAB) was added to the sections to visualise antibody binding, and the sections were washed 3 times in PBST. Haematoxylin was used to counterstain the nucleus prior to the samples being dehydrated and mounted.An Olympus BX51 microscope was used to take photomicrographs of the microstructures, images depict 1000× magnification. Transmission electron microscopy.The TEM lung tissue samples were processed using previously published methods16. Fresh lung samples (1 mm3) were fixed with glutaraldehyde (2.5%, 24 h) and postfixed with osmium tetroxide (1%, 2 h). The samples were dehydrated in a series of increasing concentrations of ethanol and embedded in Epon812. After preparing semithin sections, ultrathin sections were double stained with uranyl acetate and lead citrate. A 10,000× magnification was used to observe and photograph the sections with a JEM 1230 electron microscope (JEOL, Tokyo, Japan) set at 120 kV.Quantitative real-time PCR (qPCR)The gene expression levels in lung tissues from the yaks in the four age groups were analysed using qPCR. Total RNA was isolated with TRIzol® reagent (Invitrogen, CA, USA). cDNA was obtained by reverse transcription of total RNA using the SYBR PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time; Takara, Dalian, China). The forward and reverse primers sequences for the qPCR are shown in Table 1. The genes expression levels were detected using TB Green™ Premix Ex Taq™ II (TIi RNaseH Plus; Takara, Dalian, China) according to the manufacturer’s instructions. The 2−ΔΔCT method was used to analyse the relative expression of target genes, and the housekeeping gene β-actin was used for normalization.Table 1 Primer sequences.Full size tableWestern blot analysisEqual amounts of proteins of yak lung tissue in different development stages were harvested. These proteins were separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Sangon Biotech, Shanghai, China). PVDF membranes were blocked in 10% non-fat (skimmed) milk for 3 h and then incubated in rabbit anti-VEGFA polyclonal antibody (OriGene, Maryland, USA) at 4 °C overnight. The membranes were then incubated with a goat anti-rabbit IgG antibody (Abcam, Cambridge, UK) for 2 h being washed 3 times (10 min / time) with Tris-buffered saline with Twen-20 (TBST; containing 0.1% Twen-20). All antibodies were diluted according to the manufacturer’s instructions. Immunoblots were analysed by autograph using a Gel Doc™ XR + Gel documentation system (BIO-RAD, California, USA).Statistical analysisThe experimental data are showed as the mean ± standard deviation (SD). The differences between the four groups were compared using one-way ANOVA. P values at less than 0.05 were considered significantly different. 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

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