Rhinolophus is highly speciose. Over the last 15 years, the number of its recognized species has grown from 77 to 106, but knowledge of their interrelationships has not kept pace. Species limits and phylogenetic relationships of this morphologically conservative group remain problematic due both to poor sampling across the Afrotropics and to repeated instances of mitochondrial-nuclear discordance. Recent intensive surveys in East Africa and neighboring regions, coupled with parallel studies by others in West Africa and in Southern Africa, offer a new basis for understanding its evolutionary history.The Old World insectivorous bat genus b (1140 bp) and four independent and informative nuclear introns (2611 bp) for 213 individuals and incorporated sequence data from 210 additional individuals on GenBank that together represent 24 of the 33 currently recognized Afrotropical Rhinolophus species. We addressed the widespread occurrence of mito-nuclear discordance in Rhinolophus by inferring concatenated and species tree phylogenies using only the nuclear data. Well resolved mitochondrial, concatenated nuclear, and species trees revealed phylogenetic relationships and population structure of the Afrotropical species and species groups.We investigated phylogenetic relationships and intraspecific genetic variation in the Afro-Palearctic clade of Rhinolophidae using broad sampling. We sequenced mitochondrial cytochrome-Rhinolophus species groups analyzed, suggesting as many as 12 undescribed cryptic species; these include several instances of sympatry among close relatives. Coalescent lineage delimitation offered support for new undescribed lineages in four of the six African groups in this study. On the other hand, two to five currently recognized species may be invalid based on combined mitochondrial and/or nuclear phylogenetic analyses. Validation of these cryptic lineages as species and formal relegation of current names to synonymy will require integrative taxonomic assessments involving morphology, ecology, acoustics, distribution, and behavior. The resulting phylogenetic framework offers a powerful basis for addressing questions regarding their ecology and evolution.Multiple well-supported and deeply divergent lineages were resolved in each of the six African The online version of this article (10.1186/s12862-019-1485-1) contains supplementary material, which is available to authorized users. We remain in an era of biological discovery , even foRhinolophus offers an instructive example. The sole living genus of the Paleotropical  family Rhinolophidae, Rhinolophus is the second-most speciose genus of bat (after Myotis). Over the last 15 years, the number of its recognized species has grown from 77  to 106 ropical; , 12, an Rhinolophus is restricted to Africa and the Palearctic; it includes 7 species groups whose names represent the nomenclatural framework for this study: (1) R. landeri group ; (2) R. euryale group ; (3) R. capensis group ; (4) R. adami group ; (5) R. ferrumequinum group ; (6) R. maclaudi group ; (7) R. fumigatus group . Of the 33 currently recognized Afrotropical Rhinolophus species ) from eastern Eurasia is strongly supported as sister to four endemic Afrotropical R. clivosus clades and two R. ferrumequinum + R. clivosus clades whose distributions include North Africa, Europe, and the Middle East. Second, eastern Eurasian R. xinanzhongguoensis , and in this study as R. simulator 2. This simulator clade is distributed in Tanzania, Malawi, Zambia, Mozambique, and South Africa. Rhinolophus rhodesiae is likewise nested within R. simulator clade 2 that includes sequences from DRC, Botswana, Zambia, Malawi, Mozambique, Zimbabwe, and South Africa. There is no indication of mitochondrial introgression involving either R. gorongosae or R. rhodesiae. Rhinolophus cf. denti/simulator is a deeply diverged, monophyletic clade from southeast Africa with uncertain relationships to R. denti (southern Africa) and sympatric R. simulator 1 + 2. The membership of R. deckenii in the ferrumequinum group is challenged by its well supported sister relationship to the fumigatus group, rather than to R. clivosus + R. ferrumequinum  is uncertain, although R. ruwenzorii + R. willardi + R. deckenii are strongly supported as sister to members of the fumigatus group.The remaining clades supported as monophyletic in the cyt-num Fig. . The pos6 generations. We discarded the first 20% of each run, resulting in 8,000 trees in the posterior distributions. ESS values for all posterior parameters were greater than 300 in the combined species tree analysis of 32,000 trees. The species tree  strongly supports the sister relationship of the R. landeri group to the remaining African groups in the tree; c) strongly supports R. landeri as sister to R. alcyone + R. lobatus + R. cf. landeri; and d) strongly supports all the species group assignments made by Csorba et al. 2003, with the exception of R. deckenii (which had been uncertainly placed in ferrumequinum group) and R. ruwenzorii and R. willardi (maclaudi group), but here recovered in the fumigatus group. In contrast to the concatenated analysis, R. alcyone is poorly supported as sister to R. lobatus + R. cf. landeri clade. R. gorongosae and R. rhodesiae are respectively members of well supported monophyletic clades that also include specimens assigned to simulator 1 and simulator 2 in the cyt-b tree . Although we did not have access to tissue samples from R. smithersi, R. cohenae, and R. mabuensis, and so lack intron data, cyt-b sequences from GenBank indicate that these recently described taxa are minimally divergent from R. hildebrandtii (< 1% in cyt-b), and their recognition would render it paraphyletic.The phylogenetic relationships of Afrotropical l et al. , based oRhinolophus names long regarded as synonyms: R. lobatus  from R. landeri and R. rhodesiae  from R. swinnyi. They described the new species R. gorongosae on the basis of integrative data that included a suite of morphological variables, but their molecular phylogenetic analyses relied solely on cytochrome-b. Their putative R. gorongosae  had anomalously long branches , instead of with alcyone and lobatus , led us to compare this sequence to GenBank accessions using BLASTn. The BLASTn query showed 100% identity of their R. landeri  with Bos taurus. When tissue from this voucher specimen and another (DM12622), also from Liberia, was extracted and new sequence data generated, those individuals were found to nest deeply within the R. blasii clade 1 . However, they did not test this hypothesis with genetic data. In this study, mitochondrial, concatenated nuclear loci, and the species tree  all strongly infer the very close relationship of R. rhodesiae to R. simulator clade 1. Although the cyt-b tree recovers both gorongosae and rhodesiae as paraphyletic, the concatenated nuclear phylogeny recovers them in two separate monophyletic clades  among populations of clivosus ; R. sinio et al. , 25, andin China . Potentiussed in . Taylor ussed in  suggestecapensis group, a clade from Cameroon (ROM 68963), Gabon (FMNH 167770), and western DRC (FMNH 219476) is strongly supported in the cyt-b gene tree  and western (R. denti knorri) Africa. The specimens from DRC and Gabon were included in the concatenated nuclear intron tree and found to nest well within R. simulator, which is sister to R. denti. Rhinolophus alcyone alticolus Sanborn, 1936  and five specimens newly sequenced for cyt-b in this study (indicated by blue font). DM14815 is included twice in the tree (both the GenBank sequence and a newly generated sequence from this study). Nodal support is indicated above branches. Museum acronyms are defined in Additional file Maximum likelihood gene tree inferred for cyt-Additional file 6:Maximum likelihood gene trees inferred for nuclear introns using IQ-TREE (A–D) and Bayesian gene trees inferred for nuclear introns using MRBAYES (E–H). Nodal support is indicated above branches. Museum acronyms are defined in Additional file


Mycobacterium avium  was the organism most commonly isolated, followed by Mycobacterium intracellulare . Postoperative pneumothorax and atelectasis developed in four (8%) patients and one (2%) patient, respectively. Five patients received postoperative antibiotic therapy. Over a median follow-up period of 18.0 months, one patient with residual lesions after surgery started macrolide-based therapy due to aggravated symptoms. Conclusions: Most NTM granulomas can be treated completely by surgical resection without antibiotic therapy, and microbiological examination of surgical specimens is important for optimal management.Background: Limited data are available regarding the detailed characteristics and outcomes of surgically resected nontuberculous mycobacterial (NTM) granulomas. Methods: We evaluated the characteristics of 49 NTM granulomas presenting as solitary pulmonary nodules (SPNs) between January 2007 and December 2016. Results: Twenty-five patients (51%) were male and 27 (55%) were never-smokers. Seven (14%) patients had a history of tuberculosis. More than half (51%) of patients were asymptomatic. On chest computed tomography, the median SPN diameter was 18 mm, and approximately half of all SPNs (49%) were located in the upper lobes on chest computed tomography. NTM strain were preoperatively isolated from sputum , bronchial wash fluid , and needle biopsy specimens .  Mycobacterium avium complex (including M. avium and M. intracellulare), M. abscessus (including M. abscessus subsp. abscessus and M. abscessus subsp. massiliense), and M. kansasii [Non-tuberculous mycobacteria-pulmonary disease (NTM-PD) is a chronic progressive infectious disease, and its burden is rapidly increasing worldwide ,2. Althokansasii . NTM-PD kansasii ,5.NTM-PD can also present as solitary pulmonary nodules (SPNs) without cavitary lesions or bronchiectasis ,6. StudiM. avium complex [M. avium complex. There is lack of evidence supporting this approach and limited data on outcomes of NTM SPNs. Therefore, in this study, we evaluated the yield of non-invasive diagnostic modalities for detecting NTM SPNs, and clinico-radiological features and outcomes of surgically resected NTM SPNs.Current guidelines state that, in the absence of other radiographic evidence of NTM-related disease, surgical resection of the SPN alone may be curative and antibiotic therapy is unnecessary, especially for  complex ,9. Howev complex , and it Patients who were diagnosed with a solitary NTM granuloma between January 2007 and December 2016 were retrospectively identified among patients who underwent thoracic surgery for benign lung disease at Samsung Medical Center  using medical records. A solitary NTM granuloma was diagnosed as follows: (1) solitary lung nodule defined as a round or oval lesion, on chest CT with no nodular clusters in the same lobe except for lesions adjacent to the main nodule; (2) positive NTM culture from a respiratory specimen such sputum, bronchial washing fluid, or lung biopsy; and (3) histopathologic features of an inflammatory granuloma. Patients who had been treated with chemotherapy before surgery were excluded given the potential effects on pathologic features. Postoperative chemotherapy was prescribed at the discretion of the attending physician. Forty-nine patients who underwent surgical resection of an NTM granuloma were included in the final analysis. The Institutional Review Board of Samsung Medical Center approved the study protocol. Informed consent was waived as this was a retrospective study.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Institutional Review Board of Samsung Medical Center is the local ethics committee that reviewed and approved the protocol. Informed consent was obtained from all individual participants included in the study.rpoB gene. Beginning in June 2014, species identification was conducted via nested multiplex polymerase chain reaction and a reverse-hybridization assay of the internal transcribed spacer region .Sputum, bronchial washing fluid, or tissue was obtained for microbiological evaluation. Acid-fast bacilli smears and cultures were conducted using standard methods. NTM species were identified using polymerase chain reaction-restriction fragment length polymorphism analysis or reverse-blot hybridization of the max) were measured [All patients underwent chest CT within two months prior to surgery. Two of the authors (Y.C. and B.W.J.) reviewed the CT images. The maximal long-axis diameter of the nodule was measured, and CT findings including the presence of calcification, satellite lesion, and location were analyzed. All data with inter-observer disagreement were re-evaluated until consensus was reached. Positron emission tomography with fluorodeoxyglucose (FDG-PET) images were available for 41 patients and maximum standardized uptake values (SUVmeasured .Data are presented as medians (interquartile range (IQR)) for continuous variables or numbers (percentage) for categorical variables. Statistical analysis was performed using SPSS 25.0 .2 (IQR 21.2–25.1 kg/m2) and 27 (55%) patients were never-smokers. Seven (14%) patients had a history of tuberculosis and 13 (27%) patients had a history of solid tumors. More than half (51%) of all patients were asymptomatic and their SPNs were detected at regular checkups or during the course of follow-up for other diseases. The remaining patients presented cough (37%), sputum (29%), or blood tinged sputum/hemoptysis (8%). No patients had a previous history of NTM disease.Characteristics of the 49 study patients are presented in max was 4.9 (IQR 2.5–8.7), and 76% (31/41) of them had relatively high uptake (SUVmax > 2.5) (Radiologic characteristics of NTM nodules are summarized in x > 2.5) B.Microbiological data of 49 NTM nodules according to diagnostic modality are presented in M. avium  was the most commonly isolated, followed by M. intracellulare . M. kansasii, M. gordonae, and M. szulgai were identified in one  patient each. Interestingly, a mixed infection with M. avium and M. kansasii was identified in one  patient.Species identification was performed in 31 patient specimens . Treatment outcomes are described in M. avium complex (2 M. avium and 1 M. intracellulare), and one had M. kansasii disease. The remaining one patient had unidentified NTM but treated with macrolide-containing regimen. During a median follow-up period of 18.0 months (IQR 5.0–57.5 months), most  of patients who did not receive postoperative antibiotic therapy were clinically stable without complication or recurrence of NTM disease. Only one patient with few residual lesions was started macrolide-based therapy due to aggravated respiratory symptoms for four months after surgery (M. intracellulare).Five patients received postoperative antibiotic therapy with a median treatment duration of 6.2 months (IQR 4.6–17.6 months); of them, three patients had n = 9) and those who did not (n = 19) [M. avium complex with other granulomas showed that no patients developed new nodules or disseminated disease during follow-up [Our data showed that NTM granulomas presenting as SPNs have favorable outcomes. Most cases can be treated completely by surgical resection without perioperative antibiotic therapy. Ninety-eight percent of 44 patients who did not receive postoperative antibiotics were stable without complications or recurrence of NTM disease. Postoperative antibiotic therapy was prescribed in only five of our patients, but baseline characteristics between those who received antibiotics after surgery and those who did not were significantly different (data not shown). These data support the current guidelines , which i(n = 19) . A studyollow-up . HoweverM. avium complex was cultured from bronchial washing fluid in only one patient [In our study, the rate of NTM species isolation from sputum or bronchial washing fluid was very low, about 50%. These findings suggest that non-invasive testing using respiratory specimens may frequently be non-diagnostic or cannot exclude NTM granulomas, and thus microbiological examinations using tissue specimens are also important for the diagnosis of suspected NTM granuloma. Consistent with our data, other studies of NTM granulomas revealed that preoperative bronchoscopy did not play a significant role in the microbiological diagnosis of NTM SPNs. For example, in a 1981 study that evaluated 20 granulomas presenting as SPNs, bronchoscopy was not diagnostic, but  patient . In a Ja patient . In othe patient ,16,17, amax uptake on FDG-PET/CT scan, which suggests that FDG-PET/CT is likely not useful for differentiating NTM SPNs from malignant nodules. NTM SPN is a unique phenotype of NTM-PD that is frequently difficult to radiologically distinguish from malignancy. However, FDG-PET can be used to evaluate the glucose metabolism of SPNs and thereby identify an active lesion regardless of whether the lesion is benign or malignant. Granulomatous nodules such as tuberculomas or NTM SPNs can have positive results as shown in our previous study that compared characteristics of tuberculomas and NTM SPNs [In our study, more than three-fourths 76%) of NTM SPNs showed high SUV6% of NTMNTM SPNs ,19.M. kansasii, M. szulgai, and M. gordonae, but the most common causative organism was M. avium. The reason for this phenomenon is not known, but most previous studies reported similar results. In 1981, Gribetz et al. reported that M. avium complex was the most common causative pathogen (60%) in 20 NTM SPNs [M. avium as a causative organism [M. avium was the causative strain in 82% (9/11) and 58% (7/12) of patients with NTM SPNs, respectively [M. avium [M. avium complex among NTM SPN is due to specific bacterial tropism or some other phenomenon. Additionally, given that NTM SPNs caused by rare strains such as M. kansasii, M. gordonae, M. fortuitum, or M. haemophilum have been reported [Interestingly, we identified rare organisms including NTM SPNs . In a prorganism . Recent ectively ,17. It ireported ,22,23,24There were several limitations in this study. First, this was a single center, retrospective study. However, we included the largest number of patients with surgically resected NTM SPNs as possible. Second, more than 30% of identified nodules did not undergo NTM identification testing, which is why we were unable to evaluate prognosis according to organism. Third, the post-operative follow-up period may not have been long enough to evaluate the long-term prognosis of NTM SPNs.In conclusion, the present study suggests that most NTM granulomas can be treated safely by surgical resection without postoperative antibiotic therapy, and microbiological examinations including mycobacterial culture of surgical specimens is important for optimal management of these patients.


Pyropia yezoensis, one of the most economically important marine algae, suffers from the biotic stress of the oomycete necrotrophic pathogen Pythium porphyrae. However, little is known about the molecular defensive mechanisms employed by Pyr. yezoensis during the infection process. In the present study, we defined three stages of red rot disease based on histopathological features and photosynthetic physiology. Transcriptomic analysis was carried out at different stages of infection to identify the genes related to the innate immune system in Pyr. yezoensis. In total, 2139 up-regulated genes and 1672 down-regulated genes were identified from all the infected groups. Pathogen receptor genes, including three lectin genes (pattern recognition receptors (PRRs)) and five genes encoding typical plant R protein domains (leucine rich repeat (LRR), nucleotide binding site (NBS), or Toll/interleukin-1 receptor (TIR)), were found to be up-regulated after infection. Several defense mechanisms that were typically regarded as PAMP-triggered immunity (PTI) in plants were induced during the infection. These included defensive and protective enzymes, heat shock proteins, secondary metabolites, cellulase, and protease inhibitors. As a part of the effector-triggered immunity (ETI), the expression of genes related to the ubiquitin-proteasome system (UPS) and hypersensitive cell death response (HR) increased significantly during the infection. The current study suggests that, similar to plants, Pyr. yezoensis possesses a conserved innate immune system that counters the invasion of necrotrophic pathogen Pyt. porphyrae. However, the innate immunity genes of Pyr. yezoensis appear to be more ancient in origin compared to those in higher plants.  Plants suffer from a variety of diseases that are caused by fungi ,2,3, oomEctocarpus siliculosus and Saccharina japonica. E. siliculosus-Eurychasma dicksonii has been used as a model of Phaeophyta-oomycete interaction [E. siliculosus responds to infection by strengthening the cell wall, producing protease inhibitors, reactive oxygen species (ROS), and halogen metabolism [Laminaria digitate, a model organism in a macroalgae immune study, activates ROS [S. japonica, could get infected by several alginic acid-decomposing bacterial strains [S. japonica generates ROS after the successful recognition of bacterial PAMPs flg22, to fight the pathogen [S. japonica post-infection [Similar to plants, algae can potentially get infected by a variety of pathogens ,16,17. Seraction ,19. E. stabolism ,21. Lamiates ROS ,23, releates ROS , and empates ROS  in respo strains . S. japopathogen . Furthernfection .Pyropia yezoensis, one of the economically important red algae that is massively cultivated in East Asia, is susceptible to Alternaria [Olpidiopsis porphyrae [Phoma sp. [Pythium porphyrae [Pyr. yezoensis. The symptoms first appear as red needle-sized lesions on the surface of the thallus, followed by expansion to the whole thallus in two–three days, thus leading to the large-scale necrosis of Pyr. yezoensis [Pyt. porphyrae hyphae could be observed in the lesion [Pyr. yezoensis does possess a defense mechanism to resist the infection. Recently, research discovered that the red rot disease is distributed widely in Western Pacific coastal regions, including China [Pyr. yezonesis during the infection of red rot disease. It is reported that Pyropia tenera could produce reactive oxygen species (ROS), inducing heat shock proteins (HSPs) and cell wall-associated hydrolases to prevent the spread of the oomycete pathogen [Pyr. yezoensis, proteins related to photosynthesis, energy, and carbohydrate metabolism is shown to be suppressed in response to Pyt. porphyrae infection [Pyr. yezoensis might possess an innate immune system to defend against infection. To validate the hypothesis, differentially expressed genes in Pyr. yezoensis during Pyt. porphyrae infection is obtained by transcriptomic analysis in the current study. Immunity mechanisms in Pyr. yezoensis were described and summarized in the current study.ternaria , Olpidioorphyrae , and Phohoma sp. . Red rotorphyrae , is lethezoensis . Interese lesion , which ing China , South Kng China , Japan [ng China , Philippng China , and Newng China . Howeverpathogen . In Pyr.nfection . We hypoIn total, 2139 up-regulated genes and 1672 down-regulated genes were identified in the comparison of the slightly infected stage vs. the control group and the severely infected stage vs. the slightly infected stage. As shown in http://pfam.xfam.org/). In Pyr. yezoensis, lectin-containing genes and LRR-containing genes were seen, whereas EGF  or lysine motif-containing genes were unable to be found. Among these genes, only lectin-containing genes were predicted as transmembrane proteins by transmembrane region analysis. Transcriptome analysis also showed that the expression of three lectin-containing genes was significantly increased during infection. Compared with the healthy group, these up-regulated genes were specifically expressed in different infected stages: C-type lectin-containing gene py02002.t1 was only up-regulated in the slightly infected stage by 1.72-fold; two L-type lectin-containing genes py02275.t1 and py02843.t1 were only up-regulated in the severely infected stage by 2.36 and 2.44-fold, respectively.PRRs, located on membranes, are the initial pathogen receptors in the innate immune response . Genes cPyr. yezoensis transcripts containing typical R-protein domains such as LRR, NBS, and TIR are summarized in Twenty-three Pyr. yezoensis, NADPH-oxidase (nicotinamide adenine dinucleotide phosphate oxidase) gene py00308.t1 up-regulated constantly during the process of infection. The expression of py00308.t1 increased by 1.68 and 2.36-folds in the slightly and severely infected stages, respectively, compared to that in the healthy group. The up-regulation of antioxidizes, such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), were related to scavenging extra ROS. The expression of POD in Pyr. yezoensis increased by 1.6-fold and 2.63-fold during the infection. Two CAT genes were found to be upregulated. CAT-1 was strictly up-regulated in the severely infected stage (FC = 1.87) and CAT-2 was up-regulated in both the slightly (FC = 1.70) and severely infected stages (FC = 1.76). One gene of SOD was up-regulated in both the slightly (FC = 1.60) and severely infected stages (FC = 1.65).The expression pattern of the respiratory burst and cellular anti-oxidation related genes, which includes NADPH-oxidase and antioxidant enzymes, is represented in Pyr. yezoensis, we identified a cellulase enzyme that could degrade the cellulose, which is the major component of the Pyt. porphyrae cell wall. Compared with the healthy Pyr. yezoensis, the expression of py05706.t1 increased by 3.84-fold and 3.57-fold in the slightly and severely infected stages  using their protein sequences [py03343.t1, could interact with the catalytic center  of the metalloproteinase. We speculated that Pyr. yezoensis might have secreted py03343.t1 as an extracellular inhibitor to combine with metalloproteinase in order to restrict the damage from the pathogen.The expression of transcript hy group . This prequences . As showPyr. yezoensis could synthesize and accumulate secondary metabolites in response to infection.F520, a marker for estimating plant secondary metabolite concentration in multi-colour fluorescence imaging (MCFI) was measured during infection. Compared to the control group, F520 intensity of the infected group increased with a significant difference from four-days post-infection when the lesion rate was only 3.7% and increased continuously with the spread of the lesion A. As shopy07137.t1, isoquercetin synthesis gene py00829.t1, and 2-coumaric acid (precursor of coumarin) synthesis gene py05984.t1. Only two genes were up-regulated in the severely infected stage—caffeoyl-CoA O-methyltransferase py04307.t1 and neohesperidin synthesis gene py00132.t1. However, another neohesperidin synthesis gene py01289.t1 was up-regulated in both the slightly and severely infected stages.The results were further confirmed by transcriptome data. Transcriptome analysis showed that eight genes related to phenolic biosynthesis were up-regulated significantly during infection (FC > 1.5) . The exppy04518.t1 and py08669.t1, which were up-regulated to ~200-fold post-infection.The transcriptome data also showed that twelve heat shock protein genes  were up-regulated (FC > 1.5) after infection . CompareIn plants, hypersensitivity reaction (HR), a form of programmed cell death, is the key disease-resistance mechanism in ETI . MetacasPyr. yezoensis, all processes related to the ubiquitination system were up-regulated post-Pyt. porphyrae infection  [Pyr. yezoensis secreted the metalloproteinase inhibitor as a “bait” to occupy the metalloproteinase catalytic center and to protect the host protein degradation by pathogen metalloproteinase.Protease inhibitors (PIs), a part of the plant ETI, protect the host by inhibiting pathogen proteases and regulating the activity of host protease . It has ibition) ,55,56. Iibition) ). It appPyr. tenera, several cell-wall associated hydrolases were up-regulated after Pyt. porphyrae invasion [Pyr. yezoensis cellulase belongs to the glycosyl hydrolase family 5 (cellulase) and was continuously up-regulated during the whole infection process. It might be possible that Pyr. yezoensis secreted cellulase to degrade cellulose, thereby inhibiting the formation and expansion of Pyt. porphyrae hyphae.Plants can inhibit the fungal pathogens by secreting chitinase and β-1, 3-glucanase that degrade fungal cell wall during infection ,58. In Pinvasion . In the Porphyra umbilicalis [Pyr. tenera, the expression of putative R-proteins showed no significant change during the infection of Pyt. Porphyrae [Pyr. tenera, six up-regulated putative R-proteins could be observed in Pyr. yezoensis. The increase in expression indicated the plausibility of these genes being involved in the Pyr. yezoensis innate immunity. However, owing to their simple protein structures, the function of these genes might be limited in the interaction of Pyr. yezoensis and Pyt. porphyrae.R proteins are essential for the plant to activate ETI and create specificity in disease resistance. R proteins with a conserved protein structure of LRR-NBS are extensively present in higher plants. Genes encoding LRR or NBS could also be found in the genome of ilicalis . In Pyr.orphyrae .Unlike PPyr. yezoensis innate immune system.Several findings demonstrated that large HSPs, such as HSP70s and HSP90s, could recognize pathogen effectors specifically and activate the defense mechanisms against pathogens . ResultsPseudomonas syringae infection [S. japonica, depending on the cell ultrastructural changes during infection [Pyrenophora teres and Magnaporthe grisea [Pyt. porphyrae could feed on the dead cells or tissues. Nevertheless, PCD related genes that were up-regulated included metacaspase, endonuclease G, and cytochrome C, and these could still be identified in Pyr. yezoensis transcripts during infection. These results indicated that PCD was involved in the immune response of Pyr. yezoensis against Pyt. porphyrae.Hypersensitive response (HR), a form of programmed cell death, was triggered after recognition of the R protein and the effectors at the site of infection to prevent the spread of the pathogen . It has nfection . Howevernfection . It is we grisea ,66. As aPyr. yezoensis, genes related to UPS were up-regulated during infection. Especially in the severely infected stage, all parts of the UPS were up-regulated significantly, indicating that UPS might play a crucial role in Pyr. yezoensis to defend against the invasion of Pyt. porphyrae hyphae in the severely infected stage.In plants, ubiquitin-proteasome system (UPS) was associated with the innate immunity mechanism in a variety of ways, such as PTI modulation, programmed cell death, and signal transduction . E3 was Pyr. yezoensis. Compared with the innate immune system related genes in plants and algae, we reasonably deduced the overall view of the Pyr. yezoensis innate immunity  as a chaperone [Pyr. yezoensis lectin to activate the downstream defense genes.PRRs are transmembrane proteins with functional ectodomains binding to PAMPs . Based o lectins . It has haperone . TherefoChromochloris zofingiensis and Botryococcus braunii [Pyr. yezoensis. As shown in Porphyra umbilicalis, such as NBS+TPR and NBS+WD40. The fusion event of NBS and LRR was unable to be found in Por. Umbilicalis [Arabidopsis R proteins. Due to the lack of LRR, TIR-NBS and TIR-X proteins could interact with NBS-LRR [Nearly all the identified R proteins typically shared commonly conversed domains of TNL (TIR-NBS-LRR) or nTNL (NBS-LRR) . Recent  braunii . In the ilicalis . The pheilicalis . Similar NBS-LRR . This suPyr. yezoensis, which included cellulase, metalloproteinase inhibitor and NADH-oxidase, were clustered into a clade of Rhodophyta, which was separate from the clade of plants and Chlorophyta. Therefore, our findings indicated that Pyr. yezoensis defense genes preserved the original character in the evolutionary process of the plant innate immune system.As shown in Pyr. yezoensis might have had an ancient origin.Endosymbiosis drives the evolution and diversification of eukaryotic algae. Plastids originated from cyanobacteria and created three “primary algae”: Glaucophyta, Rhodophyta, and Chlorophyta. Chlorophyta further evolved to land plants . It has Pyr. yezoensis. Therefore, we speculated that, similar to the early defense responses of plants, these early-response genes might potentially be genes related to red rot disease resistance in Pyr. yezoensis.The innate immune system is considered to be the source of disease resistance in plants, such as non-host resistance and R protein relate resistance ,78,79. IA. thaliana, the hypermethylated mutant was shown to be susceptible to Hyaloperonospora arabidopsidis and Fusarium oxysporum, while the mutant that DNA methylation establishment impaired was more resistant to both pathogens [A. thaliana was more susceptible to H. arabidopsidis after the mutation of histone lysine methyltransferase ATXR7 [Pyr. yezoensis, a variety of enzymes related to DNA and histone modification were shown to be differentially expressed during red rot disease. We speculated that epigenetic modification was a defense regulation mechanism and might be related to disease resistance development in Pyr. yezoensis.Epigenetic modification plays an important role in plant resistance to fungal and oomycete pathogens. In athogens ,85. A. tse ATXR7 . Moreovese ATXR7 ,88. In PPyr. yezoensis was used as the host in the infection test. Gametophytes were cultured under 40 μmol photons m−2 s−1 at 10 °C and a photoperiod of light: dark = 12:12 h to obtain thalli. Boiled seawater, supplemented with a Provasoli’s enrichment solution medium (PES) [A lab-cultured pure line RZ of um (PES) , was usePyt. porphyrae strain of NBRC33253 was used as a pathogen in the infection test. The hyphae of NBRC33253 were cultured at 24 °C in a medium of 50% seawater glucose–glutamate medium (SGG) [The um (SGG) . Zoosporum (SGG) .0) was obtained after dark adaption for 15 min. Maximal fluorescence in the dark-adapted state (FM) was measured after illuminating it with a saturating pulse (2000μmol photons m−2 s−1). Maximum PSII quantum yield was calculated using the formula FV/FM = (FM − F0)/FM. FV/FM of a different area in the infected thallus was analyzed by FluorCam software7 version 1.2.5.7 on a manual mode. Three biological replicates were performed in each group.The value of the maximum PSII quantum yield was measured by FluorCam 800MF . Minimal fluorescence (F5 zoospores mL−1) in a flask with PES incubated under 40 μmol photons m−2 s−1 at 15 °C and a photoperiod of light: dark = 12:12 h for 10 days. Healthy Pyr. yezoensis thalli were used as the control group. Three biological replicates were performed in each group.Healthy RZ thalli (0.4 g fresh weight) (1.2 ± 0.2 cm in width and 9.3 ± 1.1 cm in length) were exposed to 200 mL zoospore suspension  were ground in liquid nitrogen and a total RNA was extracted using the Plant RNA Kit , following the manufacturer’s instructions. DNase I (Omega) was used to digest the DNA RNA extraction. The quantity and integrity/quality of the total RNA extracted was examined on a Nanodrop and Agilent 2100 bioanalyzer .1μg of total RNA was used to construct a cDNA library using VAHTS™ mRNA-seq V3 Library Prep Kit for Illumina , following the manufacturer’s protocol. High-throughput sequencing was performed on the Illumina HiSeq 2000 platform in a paired-end 150 bp run.https://dataview.ncbi.nlm.nih.gov/object/PRJNA560692?reviewer = 99tc7nravboeun8gna76hrjorp).The datasets generated are available in the Sequence Read Archive (SRA) repository accessible through NCBI BioProject ID PRJNA560692 (Pyr. yezoensis was was used as a reference genome (DDBJ/ENA/GenBank: WMLA00000000) to filter the reads from infected groups. The genome index was built using Bowtie2 and clean reads were aligned with the genome of Pyr. yezoensis using Tophat software. Gene expression was quantified by cuffquant and normalized by cuffnorm to obtain the value of the number of fragments per kilobase per million (FPKM) for each group. Cuffdiff was used to identify differentially expressed genes and the data were manually screened to identify the significantly differentially expressed genes with thresholds of FPKM  >  3 and >1.5-fold (or < 0.67 fold) change. ALL protein sequences used in current study was listed in The genome of www.omicshare.com/tools). A KEGG pathway analysis was performed using OmicShare tools (www.omicshare.com/tools).GO enrichment analysis was performed using OmicShare tools, which is a free online platform for data analysis  to identify proteins containing a signal peptide [(1) All differentially expressed protein sequences were submitted to SignalP 4.1 online  was used to analyze whether transmembrane domains were present in the proteins obtained from SignalP 4.1. Thus, transmembrane proteins were separated from secretory proteins.(2) TMHMM v2.0 (http://www.cbs.dtu.dk/services/TargetP/). Proteins localized in mitochondria, chloroplasts, and other subcellular locations were identified and distinguished. Extracellular secretory proteins were obtained for further analysis.(3) Subcellular localization was analyzed by Target P 1.1 Server , LRR(PF00560), and TIR(PF01582) were generated on the PFAM website (http://pfam.xfam.org) in the Stockholm format. HMMbuild, from HMMER package 3.1b2, was used to create Hidden Markov model (HMM) of the three domains. The profile of HMM was used to search for transcripts with a cut-off E-value of 10−5.HMMsearch was used to predict proteins that had LRR, NBS, and TIR domains in The MCFI of healthy and infected thalli was performed using Multi-color FluorCam . The thallus was tiled with a little boiled seawater in Petri dishes and then kept on the sample table. The excitation wavelength used was 355 nm. The fluorescence spectra of blue (F440), green (F520), red (F690), and far red (F740) were collected. A fluorescence image was displayed with a false-color scale by the FluorCam software7 version 1.2.5.7 . Further, F520 of a different area in the infected thallus was analyzed by FluorCam software7 version 1.2.5.7 on manual mode. Three biological replicates were performed in each group.Protein sequences of genes related to lectin, R proteins, cellulase, NADPH-oxidaseRBOH, and metalloproteinase inhibitors were collected from GenBank. The GenBank accession number of all the sequences are listed in Pyr. yezoensis based on the transcriptomic data during the infection of red rot disease. The conserved innate immune system seen in higher plants were also revealed in the red algae. Pyr. yezoensis lectins might act as PRRs to activate PTI after the recognition of carbohydrate PAMPs. PTI mechanisms included the up-regulation of ROS, secondary metabolism, metalloproteinase inhibitor, cellulose, and small HSPs in response to infection. PCD and UPS, like ETI, might be activated as a defense mechanism to infection after effector recognition by putative R proteins and HSP70s. Nevertheless, compared to the homologs in higher plants, Pyr. yezoensis defense genes appeared to have ancient characteristics. The protein structure of pathogen receptors, which included PRRs and R proteins, were simple with a single functional domain. Therefore, our study provides valuable clues not only for understanding the innate immunity mechanisms of red algae, but also for tracing the origin of the innate immune system in plants. Furthermore, our results provided molecular resources for the breeding of red rot disease resistance strains in Pyr. yezoensis, which will continue the sustainable development of nori industries.In the current study, we outlined the defense response of red alga


Lactobacillus differs from the animal model used (MRL/lpr mice or NZB/W F1 mice). This may indicate that interactions between gut microbes and the host, rather than the enrichment of certain gut microbes, are especially significant in terms of SLE development. Enterococcus gallinarum and Lactobacillus reuteri, both of which are possible gut pathobionts, become translocated into systemic tissue if the gut epithelial barrier is impaired. The microbes then interact with the host immune systems, activating the type I IFN pathway and inducing autoantibody production. In addition, molecular mimicry may critically link the gut microbiome to SLE. Gut commensals of SLE patients share protein epitopes with the Ro60 autoantigen. Ruminococcus gnavus strain cross-reacted with native DNA, triggering an anti-double-stranded DNA antibody response. Expansion of R. gnavus in SLE patients paralleled an increase in disease activity and lupus nephritis. Such insights into the link between the gut microbiota and SLE enhance our understanding of SLE pathogenesis and will identify biomarkers predicting active disease.Systemic lupus erythematosus (SLE) is an autoimmune disease featuring enhanced expression of type I interferon (IFN) and autoantibody production triggering inflammation of, and damage to, multiple organs. Continuing research efforts focus on how gut microbes trigger systemic autoimmunity and SLE. The gut microbial communities of mice and humans with lupus have been investigated via high-throughput sequencing. The Firmicutes-to-Bacteroidetes ratio is consistently reduced in SLE patients, regardless of ethnicity. The relative abundance of  SLE is characterized by autoantibody production and immune complex deposition reflecting dysregulation of both the innate and adaptive immune systems. Loss of tolerance to various self-antigens is a critical feature of SLE. Nuclear antigens released during apoptosis are normally cleared without activating the immune system. However, in SLE, external stimuli such as ultraviolet (UV) light, an infection, or a toxin may increase the apoptotic cell load . Increasoduction . Thus, aoduction ,11.The etiology of SLE is multifactorial. Environmental and genetic factors interact to trigger SLE development. Differences in SLE development between monozygotic twins suggest that environmental triggers are important in terms of lupus development in genetically susceptible individuals . UV lighRecently, growing evidence suggests that the microbiota plays a role in SLE ,22. The 11 cells/g in the ascending colon, 107–8 cells/g in the distal ileum, and 102–3 cells/g in the proximal ileum and jejunum; over 2000 species of bacteria have been described [Humans host diverse microorganisms in the gut lumen. The number of microorganisms varies along the gut, being 10escribed . Metagenescribed . It is nescribed . The gutescribed . The gutescribed . Importaescribed ,31. The escribed . Howeverescribed ,34. WhenSeveral diseases are associated with gut dysbiosis. Early studies found that obesity correlated with a shift in the gut microbial community ,36. Tranlpr mutation in the gene encoding Fas protein can spontaneously develop many lupus autoantibodies and lupus manifestations [Lactobacillus constitutes the microbiota of human gastrointestinal and genitourinary tracts. Some Lactobacillus species are used as probiotics since they are known to have anti-inflammatory properties [Lactobacillus spp. was most prominent before (not after) disease onset [Lactobacillus might play a preventive role in terms of lupus pathogenesis. Indeed, Lactobacillus addition reduced proteinuria and levels of lupus autoantibodies and improved the renal pathology scores in/of MRL/lpr mice [Spontaneous and induced animal models of lupus have been employed to determine the effect of the gut microbiota on lupus. MRL/lpr mice with the stations . The gutstations . Lactobaoperties . Lachnosoperties . Lachonooperties . The sevoperties . The relse onset . Thus, Llpr mice .Lactobacillus played an opposite role in studies using different lupus mouse models. NZB/W F1 mice, another spontaneous lupus model, developed lupus slowly [Lactobacillus increased dramatically during lupus development; this rise was reversed by dexamethasone [Lactobacillus species tended to correlate positively with poorer renal function and higher-level systemic autoimmunity [However, s slowly . Luo et s slowly . The guts slowly . The relethasone . The relimmunity .Lactobacillus reuteri alone exacerbated lupus [L. reuteri, the genus Desulfovibrio, and the family Rikennellaceae were enriched. However, only Lactobacillus spp. (L. reuteri and L. johnsonni) were translocated to internal organs, and only L. reuteri (thus not L. johnsonni) induced the IFN gene signature and systemic autoimmunity. Resistant starch (fermented to short-chain fatty acids) suppressed L. reuteri growth both in vitro and in vivo. This reduced gut epithelial permeability in vivo, and decreased type I IFN expression and improved lupus nephritis.In TLR7-dependent mouse models, the lupus-prone TLR7.1 Tg mice and imiquimod (a TLR7 agonist)-induced mice, ed lupus . In fecaLactobacillus and the changes mediated by Lactobacillus differ among the various mouse models of lupus. The mechanisms underlying lupus development are not identical among the mouse strains; the gut microbiota compositions and associated host interactions vary. IFN-α plays the predominant role in TLR7-dependent mouse models, whereas IFN-γ is more important in MRL/lpr mice [Prevotella copri triggered arthritis, whereas inoculation with Prevotella histicola suppressed arthritis [The effects of relative abundance of lpr mice . In addirthritis . In addirthritis . Work onBacteroidetes is increased, showing the decreased ratio of Firmicutes to Bacteroidetes compared to healthy controls [The human gut microbiome consists principally of the phyla Firmicutes and Bacteroidetes ,65. The controls ,68,69,70controls . Rather,Lactobacillus spp. was increased in the gut microbiota of SLE patients compared to healthy controls, consistent with the rise in the relative abundance of L. reuteri in lupus-prone mice [Lactobacillus spp. spectrum was not clarified in human fecal samples.The relative abundance of one mice . HoweverRuminococcus gnavus (of the family Lachnospiraceae) in the gut microbiota of 61 female SLE patients varying in terms of disease activity [R. gnavus reflected the extent of disease activity and were prominent in patients with lupus nephritis [R. gnavus was also abundant in fecal samples from spondyloarthritis patients; the levels reflected disease activity [R. gnavus levels have been observed in the gut microbiome of patients with IBD; the R. gnavus strains of IBD patients and healthy controls differed [Azzouz et al. were the first to describe the overabundance of activity . Gut expephritis . Interesephritis . Patientephritis . Thus, tactivity . Moreovediffered . Works o+ T cells are activated, and inflammatory cytokines such as IL-6 are released. IL-6 plays a pivotal role in lupus progression in MRL/lpr mice by inducing autoantibody production by B cells and suppressing the activities of Treg cells [Intestinal epithelial integrity is maintained in healthy subjects. Normally, gut-resident bacteria do not transfer to internal organs. However, if the gut barrier is disrupted, gut commensals are exposed to the host immune systems of various organs. A “leaky gut” initiates systemic autoimmunity. The role of an impaired gut barrier in lupus pathogenesis has been well-studied in mice ,59,63. Teg cells ,75.Enterococcus gallinarum in gut leakage and bacterial translocation inducing the development of lupus autoantibodies [E. gallinarum, a gut commensal, was detected in the mesenteric veins, MLNs, and liver of (NZW × BXSB) F1 mice both via tissue cultures and in situ assays; C57BL/6 mice did not exhibit systemic bacterial growth [E. gallinarum increased the frequencies of plasmacytoid dendritic cells (DCs), the source of the SLE IFN signature [E. gallinarum-induced autoantigens stimulated the aryl hydrocarbon receptor (AhR)-CYP1A1 pathway, in turn triggering Th17 cell activation and anti-dsDNA antibody production [E. gallinarum alone, but not after colonization by Enterococcus faecalis or Bacteroides thetaiotaomicron [E. gallinarum limited bacterial translocation and the lupus manifestations. Thus, E. gallinarum per se may be a gut pathobiont in the SLE context. Notably, E. gallinarum has been found in liver biopsy samples of both SLE patients and autoimmune hepatitis patients [Antibiotic-treated or germ-free lupus-prone mice exhibit enhanced gut integrity and lower-level systemic autoimmunity ,63. Thuspatients .E. gallinarum was present in the small intestine, the bacterium was not detected in the feces of lupus-prone mice via 16S rRNA sequencing [Although quencing . This maAutoantibodies to the 60 kDa Ro protein are common in SLE patients. It was found early that the lupus autoantigen Ro60 cross-reacted with the Ebstein-Barr virus nuclear antigen-1 (EBNA-1), suggesting that lupus humoral autoimmunity was initiated via molecular mimicry of EBNA-1 and Ro60 antigens .Bacteroides thetaiotaomicron, which expressed human anti-Ro60 antibodies in the blood [Recently, commensal bacteria have been shown to trigger autoreactive T and B cell responses in susceptible individuals. Peptides derived from the oral, gut, skin, and vaginal microbiotas activated Ro60-reactive T cells . Greilinhe blood . In summR. gnavus correlated with lupus disease activity and lupus nephritis [R. gnavus overgrowth was associated with the development of serum antibodies against strain RG2. Patients with high titers of IgG anti-RG2 antibodies exhibited active lupus nephritis. Of the various strains of R. gnavus, extracts of RG2, but not RG1, cross-reacted with lupus anti-dsDNA antibodies, suggesting that cross-reactivity was strain-restricted. Cell wall lipoglycans of the RG2 strain contained the antigenic properties for anti-dsDNA antibody responses. Molecular mimicry between RG cell wall moieties and native DNA molecules may trigger or exacerbate SLE and lupus nephritis.Intestinal expansion of ephritis . R. gnavSLE is more common in females than in males. Both female and male lupus-prone mice spontaneously develop lupus. However, female mice develop a more severe disease than male mice. A previous study from Zhang et al. investigated the gut microbiota of female and male MRL/lpr mice; the patterns differed markedly . Female The sex difference in the gut microbiota emerges in adulthood, suggesting that the gut microbiota is affected by sex hormones . CastratLactobacillus treatment reversed proteinuria and improved the renal pathology scores in female MRL/lpr mice [Lactobacillus, both mock-castrated and castrated mice exhibited increased gut colonization of Lactobacillales. However, only castrated mice evidenced reduced proteinuria and improved renal pathology scores; mock-castrated mice did not. In summary, Lactobacillus was therapeutic in both female lupus-prone mice and castrated male lupus-prone mice, but not in intact male mice. Thus, androgenic hormones may suppress the therapeutic effect of the gut microbiota.Sex hormones may influence the effects of the gut microbiota in lupus-prone mice. lpr mice . In malePotential mechanisms of the gut microbiota triggering the autoimmunity of SLE are summarized in E. gallinarum and L. reuteri cross the gut epithelium to induce type I IFN expression and anti-dsDNA antibody production. Molecular mimicry is critical in terms of autoimmunity induction by gut microbes. Gut commensals from humans (including SLE patients) contain proteins with epitopes homologous to those of the Ro60 autoantigen. Such proteins bind to B and T cells to activate (inappropriate) immune responses. In addition, overexpression of specific gut microbes indicates increased disease activity and predicts lupus nephritis. The gut microbiome aids our understanding of SLE pathogenesis and can serve as a biomarker predicting active disease.Exploration of the gut microbiome in murine and human lupus has afforded new insights into the role played by the microbiota in SLE. Several taxa are either enriched or depleted in the lupus gut, as revealed via high-throughput sequencing. The ratio of Firmicutes to Bacteroidetes is reduced in SLE patients, regardless of ethnicity. Most studies were performed in female mice or patients since sex hormones can affect the gut microbiota. Gut pathobionts interact with hosts by translocating to systemic tissues and, thereby, activating the immune system.


Quercus) comprise more than 400 species worldwide and centres of diversity for most sections lie in the Americas and East/Southeast Asia. The only exception is the Eurasian sect. Cerris that comprises about 15 species, most of which are confined to western Eurasia. This section has not been comprehensively studied using molecular tools. Here, we assess species diversity and provide a first comprehensive taxonomic and phylogeographic scheme of western Eurasian members of sect. Cerris using plastid (trnH-psbA) and nuclear (5S-IGS) DNA variation with a dense intra-specific and geographic sampling. Chloroplast haplotypes primarily reflected phylogeographic patterns originating from interspecific cytoplasmic gene flow within sect. Cerris and its sister section Ilex. We identified two widespread and ancestral haplotypes, and locally restricted derived variants. Signatures shared with Mediterranean species of sect. Ilex, but not with the East Asian Cerris oaks, suggest that the western Eurasian lineage came into contact with Ilex only after the first (early Oligocene) members of sect. Cerris in Northeast Asia had begun to radiate and move westwards. Nuclear 5S-IGS diversification patterns were more useful for establishing a molecular-taxonomic framework and to reveal hybridization and reticulation. Four main evolutionary lineages were identified. The first lineage is comprised of Q. libani, Q. trojana and Q. afares and appears to be closest to the root of sect. Cerris. These taxa are morphologically most similar to the East Asian species of Cerris, and to both Oligocene and Miocene fossils of East Asia and Miocene fossils of western Eurasia. The second lineage is mainly composed of the widespread Q. cerris and the narrow endemic species Q. castaneifolia, Q. look, and Q. euboica. The third lineage comprises three Near East species , well adapted to continental climates with cold winters. The forth lineage appears to be the most derived and comprises Q. suber and Q. crenata. Q. cerris and Q.  trojana displayed high levels of variation; Q. macrolepis and Q. euboica, previously treated as subspecies of Q. ithaburensis and Q. trojana, likely deserve independent species status. A trend towards inter-specific crosses was detected in several taxa; however, we found no clear evidence of a hybrid origin of Q. afares and Q. crenata, as currently assumed.Oaks ( Studies on the genetic diversity of forest species across their distributional ranges are relevant for genetic resource inventories and devising conservation strategies . ComparaQuercus L.) are an ideal model for comparative phylogeographic studies. They are common, often (co-)dominant vegetation elements and include several widely distributed and ecologically diverse species (http://www.ipni.org). At the same time, taxonomic ambiguity and reticulate evolution during formation of species make sampling and phylogenetic tree inference difficult.Oaks , Protobalanus (western North America), Ponticae , Virentes (southeastern US into Mesoamerica), and Quercus (with most species in North America and about 30 species in Eurasia). The Palearctic-Indomalayan subgenus Cerris includes sections Cyclobalanopsis (East Asia), Ilex and Cerris (across Eurasia). For some of these groups, detailed infragroup phylogenies and assessment of main biogeographic patterns have recently been published . The understudied sect. Cerris (Cerris oaks) currently includes 13 species and a few unresolved taxa (BSk) and warm temperate or snow climates with different precipitation regimes, including more arid summer-dry and (more) mesic per-humid and winter-dry regimes , and sect. Ilex appears to have been present in middle Eocene strata of southern China  of disputed or unresolved status. The ranges of Cerris oaks vary substantially in size and in the degree of contact with other species of the same section and the sister section Ilex. The Qinghai-Tibet Plateau and the Himalayan front-hills, home of several species of sect. Ilex, separate the East Asian and the western Eurasian taxa. The central and eastern Mediterranean region comprise most of the group’s diversity , which decreases westward  and eastward  and three are geographically extremely limited . A hybrid origin has been postulated for Q. crenata  and Q. afares  were studied using plastid microsatellite variation (Q. suber) and Pleistocene (Q. cerris), respectively. Local investigations focussing on conservation were conducted on Q. trojana in Italy and Q.  libani in Iran using nuclear microsatellites  ‘Euro-Med’, comprising the most distinct haplotypes dominating in the western Mediterranean, a plastid lineage that diverged before the radiation of plastid pools in Subgenus Cerris, (2) ‘WAHEA’ , distributed from Anatolia/Levant to East Asia, and sister to (3) ‘Cerris-Ilex’, centred on the Aegean Sea and shared with co-occurring members of sect. Cerris. Haplotype variation of the trnH-psbA intergenic spacer was determinant for the phylogeographic inferences; this marker also showed the highest variation rate among several plastid regions in 35 Chinese oak species  to assess species coherence and delimitation, (ii) to infer inter-species relationships, (iii) to gain insight into the origin and diversification of the group. We further hypothesize that signals from both markers may combine to reveal potential zones of secondary genetic contact between already established species and to gain insight into the putative hybrid status of Q. crenata and Q. afares. All data will likely contribute to identify biogeographic hotspots for further in-depth studies on species diversity, isolation, introgression and natural hybridisation in oaks.Clearly, full comprehension of the drivers of speciation of sect. Quercus sect. Cerris in western Eurasia ]). The positive clones, selected on LB/Ampicillin plates, were identified by colony PCR using the amplification primers. Five to ten recombinant clones per individual were sequenced with the vector-specific universal primers (SP6/T7) at LGC Genomics . The GenBank trnH-psbA sequences of several East Asian members of sect. Ilex , East Asian species of sect. Cerris  and further sequence accessions of the investigated group were included in the analyses. In addition, all 5S IGS sequences of Quercus sect. Cerris available on GenBank were included in the final dataset, and the sequences of Q. baloot and Q. floribunda were used as outgroups, based on We combined previously studied  and new  Eurasia ; some intrnH-psbA region were inferred with Network 4.6.1.1 (http://www.fluxus-engineering.com/), treating gaps as fifth state. The MJ algorithm was invoked with default parameters , in order to handle large datasets and multistate characters.Eye-checked electropherograms were aligned in MEGA7 . Highly g2cef (available at http://www.goeker.org/mg/distance/) was used to transform the primary character matrix  into a character consensus matrix of the individuals (‘hosts’) using an association file defining the list of clone sequences belonging to the same individual. The uncorrected pairwise distances of the primary character matrix  was calculated and used as input to the program pbc  was then used to infer a phylogenetic network using the Neighbour-Net (NN) algorithm  tree with RAxML v8.2  using thgram pbc . This prlgorithm  implemenitsTree4 .Cerris were analysed (trnH-psbA) and 856 nuclear (5S-IGS) sequence accessions. The nuclear data  included 651 newly sequenced clones and 205 accessions from Q. baloot/Q. floribunda sequences, used here as outgroups , while the marker variation in the remaining taxa converged to zero.After removing the 34-bp inversion, the nd 0.008 . The high = 0.515). Haplotype H1 hit 100% sequence identity with three non-representative individuals assigned to East Asian species of sect. Ilex in Genbank . Haplotypes H2, H5–H7 and H11 showed 100% sequence identity with Mediterranean members of sect. Ilex  samples from Turkey, the Balkans and Italy; H5–H7 were found in Q. brantii, Q. cerris and Q. macrolepis from Turkey, Iran, and Israel. They were all shared with Q. coccifera and Q. ilex of the Aegean ‘Cerris-Ilex’ lineage. Haplotype H11 found in Iberian samples of Q. suber was shared with Q. ilex of the ‘Euro-Med’ lineage. Rare haplotypes restricted to a single species were H7 (one accession of Q. cerris), H8  and H11/H12 ; all other haplotypes were shared by more than one species of sect. Cerris. Quercus cerris, the most widespread and ecologically diverse species of sect. Cerris, showed the highest number of haplotypes (eight), followed by Q. brantii (five) and Q. macrolepis (four). In the latter species, haplotype H6, exclusively found in Q. brantii, was also found in a suspected hybrid Q. macrolepis × Q. brantii (sample ml27). All samples of Q. ithaburensis exhibited a single haplotype (H9). The geographically (more) restricted taxa Q. afares, Q. castaneifolia, Q. crenata and Q. trojana subsp. euboica (henceforth: Q. euboica) showed only the most frequent and widespread haplotype (H1).The total matrix was 503-bp characters long, including several indels (1–8 bp) and six polymorphic sites resulting in twelve haplotypes (labelled H1–H12) with a medium overall diversity (ct. Ilex . H2 is tCerris (Q. acutissima and Q. variabilis) displayed a higher variation at the trnH-psbA locus, although mostly due to indels. A higher number of haplotypes was found in these species; none of them was shared with any species of sect. Ilex available in gene banks , and only one haplotype was shared between the two species.In comparison , the twoQ. suber from Iberian Peninsula, North Morocco). One further PIC separated H9, including all individuals of Q. ithaburensis, two Q. look, two Israelian and one Italian Q.  cerris individuals. A single PIC also defined H4, including three co-occurring Q. trojana and Q. macrolepis accessions from the same locality in western Turkey, and another PIC was limited to two Q. cerris and Q. libani accessions from southern Turkey, corresponding to H3. Q. suber and Q. libani. The haplotypes of Q. suber and Q. look displayed the highest mean divergence from all other species. Quercus variabilis appeared more similar to its western Eurasian counterparts, while Q. acutissima was highly distinct.No shared parsimony informative characters (PICs) were found in the East Asian samples. In contrast, three PICs were exclusively shared by haplotypes H11 and H12 ; this latter represents a unique, early diverged plastid lineage, most frequent in the western Mediterranean populations of sect. Ilex (Ilex (Q. phylliraeoides), the plastid sister lineage of ‘Cerris-Ilex’  are derivates of the ‘Cerris-Ilex’ and ‘Euro-Med’ main types , shared by both sections in the Aegean and the western Mediterranean regions. As shown in The haplotype network  shows thct. Ilex . Based ois-Ilex’ ; (L2) a trnH-psbA, 5S-IGS sequence variation appeared generally correlated with the taxonomy of the studied individuals, and allowed inferences on potential reticulation and inter-species relationships within the western Eurasian members of sect. Cerris. The 5S-IGS clones varied greatly in sequence features and length . For instance, all Q. brantii clones displayed an intra-specific (ATTT)1−7 simple sequence repeat (SSR) variation. In all the other species, this motif was either absent (replaced by a 5–12 bp long poly-T) or consisting of one to two repeats, with the exception of two clones of the suspected hybrid Q. macrolepis × Q. brantii  that showed four to five repetitions. Two clones of Q. brantii  shared a four-bp insertion with several clones of sympatric Q. macrolepis individuals (ml20–ml22). Three Q. libani individuals  displayed a long indel (ca. 100 bp) in (nearly) all clones  and Q. trojana ; the latter, however, is another suspected hybrid (Q. trojana x Q. libani).In contrast to libani variant’. One (22 bp) was shared by single clones of two Q. cerris individuals , four clones of a Q. look individual  and two clones of Q. trojana . The second (∼100 bp), largely overlapping with the deletion of the ‘short libani variant’, but beginning a few basepairs downstream, was shared by one clone of Q. cerris  and one clone of Q. macrolepis . An 8-bp deletion occurred exclusively in Q. suber and Q. crenata, with the exception of single clones of samples su07, su09 (N.E. and S. Spain), su37 (Croatia), su53 , cr02 , two clones of sample cr04 (Slovenia) and cr06 , and three clones of sample cr05 (Croatia). The same deletion also occurred in two clones of sample tj08, a Q. suber × Q. trojana cultivation hybrid. Further deletions (1–60 bp) were scattered along the alignment and found only in single individuals . Finally, an 18-bp highly variable region was exclusively found in some clones of four co-occurring Q. trojana samples .Two other deletions were detected in the same region of the ‘short Q. brantii, Q. cerris, Q. trojana, Q. look; see also Q. afares, Q. castaneifolia, Q. libani, Q. ithaburensis, Q. macrolepis and Q. euboica showed high species coherence. Quercus suber and Q. macrolepis showed the highest number of intra-individual and intra-specifically shared clones, whereas Q. cerris, Q.  trojana and Q. ithaburensis displayed the highest levels of unique variants. No variants were shared between Q. trojana and Q. euboica; Q. suber and Q. crenata (but not Q. cerris) shared 69 identical sequences and are the genetically most similar taxon pair. The pairwise uncorrected p-distance range of the total dataset was much higher than for the plastid marker (0–0.209), with highest values scored by Q. cerris and Q. trojana. The mean intra-specific molecular diversity estimated within sequence pairs , treated as subspecies of Q. ithaburensis in current regional floras, was similar to values detected between these taxa and the other species . Likewise, the divergence recorded between the putative conspecific Q. trojana and Q. euboica (0.0266) was comparable to the estimates calculated between these and other taxa . The putative hybrid taxon Q. crenata displayed the lowest divergence (0.0197) with Q. suber, one of the assumed parental species, and a slightly higher estimate  with Q. cerris (0.0266), the other putative parental species.The main diversity values of the investigated dataset are reported in ce pairs  was loweQ. baloot and Q. floribunda (West-Asian members of sect. Ilex) showed four main topological features (grades/clades) generally coherent with taxonomy , (2) Q. brantii, Q. ithaburensis and Q. macrolepis (the most highly supported clade: BSML = 84), (3) Q. trojana (a large heterogeneous grade), and (4) Q. cerris . Quercus libani clones  were present in all clades/grades except grade 1. A moderately supported clade (BS = 63) including all Q. afares clones was placed as sister to the main clade including clades/grades 2–4; Q. castaneifolia clones were placed within grade 3. Clones of Q. ithaburensis also occurred in grade 3, Q. brantii and Q. crenata in clade 4, Q. look and Q. euboica in grade 3 and 4. A few clones of Q. cerris and Q. trojana occurred scattered across the tree .The clone-based ML tree rooted on taxonomy . These gQ. macrolepis × brantii hybrid, one was identical to another Q. macrolepis clone  and the other two clustered together with Q. brantii. Likewise, three and two of the five clones sequenced in sample tj08, a Q. trojana × Q. suber hybrid, clustered within the respective parental subtrees; the same applies to the five clones of the sample tj33, a supposed Q. libani × Q. trojana hybrid. Conversely, all the three clones sequenced in sample tj02, another tree determined as possible Q. libani × Q. trojana hybrid, clustered with Q.  trojana.Of the three clones sequenced from individual ml27, a suspected Cerris, is the least coherent cluster and equivalent to a grade in a corresponding outgroup-rooted  tree. This lineage included, in the AVG network, Q. afares, four out of five Q. libani individuals and about half of the Q. trojana individuals . The reason for this is that the PBC transformation has a higher chance to capture evolutionary signals  oaks, Q. brantii, Q. ithaburensis and Q.  macrolepis, with two Q. brantii individuals , one Q. crenata individual , and one Q. ithaburensis individual . The placement of one Q. libani individual , with normal-long variants in the clone sample, and one Q. cerris , Q. trojana , Q. suber , Q. brantii (br06), Q. ithaburensis , and Q. macrolepis (ml10). Some of these samples had unique deletions or highly divergent regions in their clones . The networks produced with individuals represented by ≥2, ≥3 (and ≥5) clones did not change this structure (Quercus castaneifolia (represented by two clones) and one sample of Q. crenata  formed part of the ‘occidental’ lineage, cluster 2; one sample of Q. suber  was included in cluster 1, the ‘oriental’ lineage of Sect. Cerris . The same holds even more for the ‘Vallonea’ oaks (Cluster 4), which appear to have split before the remainder of western Eurasian Cerris (but long-branch/-edge attraction with the extreme long-edged outgroup needs to be considered). A clear signal in the PBC network  individuals of Q. cerris and Q. libani  Q. libani  and its ecological adaptability. Likewise, Q. cerris also displayed a high nuclear (5S-IGS) diversity  and it is the only species of sect. Cerris naturalized on the British Islands (Cfb) and cultivated all over continental Europe . Indeed, establishment of a large range across the geologically and ecologically dynamic West Eurasian region might have provided many opportunities for diversification, isolation, drift, conservation of variants and eventual reticulation with sibling species.The strikingly high haplotype richness of ange cf. , mirrorsiversity , Fig. 3.Quercus suber, instead, displayed the lowest number of unique 5S-IGS variants (like Q. macrolepis) with a low diversity  or reflect ancient reticulation and retention of ancestral signatures .iversity  and 5, wQ. libani, the high haplotype diversity coincides with a moderate diversity at the 5S-IGS locus, characterized by interesting variation among the cloned sequences. Two individuals  show potential introgression . Together with Q. brantii, Q. libani is the easternmost oak among the western Eurasian Cerris, and it is extremely variable at the morphological and ecological level (occurring in climates ranging from Csa to Dsb). For instance, Q. libani, and introgression phenomena with other co-occurring oaks have been postulated , but individuals of Q. trojana can be found in two different clusters in the 5S-IGS network. Reflecting its more limited distribution and climatic niche , the haplotype diversity is substantially lower in Q. trojana s.l. (Q. trojana + Q. euboica) than in Q. cerris  ranges. Accordingly, the two endemic taxa Q. afares and Q.  castaneifolia appeared the least diverse . In addition, Q. euboica is characterized by special edaphic conditions, growing on serpentine rocks. All these data indicate that the Euboean oak should be better considered as an independent species requiring special protection. Another (hairy) variant of Q. trojana has been locally described at the south-eastern margin of the species’ range, in South-central Turkey , the two previous assessments of this oak as synonym of Q. ithaburensis or Q. ithaburensis ×  Q. libani hybrid . Interestingly, Q. brantii appeared as the most diverse of the three taxa , confirmed the hybrid identity of a few samples included in our dataset . The haplotypes of these samples , also allowed identification of the maternal species. This finding confirms that these oaks can occasionally hybridize in sympatry, and evidence for such hybridization is found in the nuclear genome  spatial proximity, introgressions could be suggested for samples Q. brantii  and Q. macrolepis  sharing sequence features, and South Anatolian Q. libani (li01) and Q. trojana  sharing sequence features and trnH-psbA haplotypes. Outside Anatolia, evidence of reticulation (shared variants) can be traced between Israelian Q. cerris and Q. look  and between Balkan Q. cerris and Q. suber . Introgression and past hybridization events between all these species or their precursors is a possible explanation. At the same time, retention of ancestral traits cannot be discarded, as Q. cerris  and Q. trojana (5S-IGS) cover most variability found in sect. Cerris and are highly variable, especially in Anatolia.Further instances of hybridization and/or introgression events could be inferred from common sequence features, inter-specifically shared variants  and the large extent of variants shared with Q. suber can be interpreted as evidence of co-existing both (1) Q. cerris × Q. suber F1 hybrids and (2) introgressive forms into either Q. cerris  or Q. suber , in partial agreement with Q. suber and Q. cerris is traditionally used as a diagnostic character of Q. crenata), which seems inconsistent with their presumable different genome composition. Clearly, the hybrid/introgressed phenotypes can be affected by several phenomena such as segregation, epistasis, heterosis, and maternal origin . Also, Q. cerris shares plastid and nuclear signatures with other species of sect. Cerris, including geographically isolated, morphologically distinct taxa such as Q. afares, Q. castaneifolia, Q. euboica , and Q. look , which are part of the ‘occidental’ lineage (see ‘Results’). Besides occasional hybridizations Q. cerris  lineage, representing a geographic-evolutionary gradient . Quercus crenata may then just represent the remainder of the ancestral form from which Q. suber evolved rather than being the product of secondary contact between Q. cerris and Q. suber.In this context, the unresolved taxonomic status of  results –5. The sl origin . At the Q. suber . Being cQ. afares  as suggested by Cerris types and should be detectable unless the F1 hybrids, with Q. suber as a maternal parent, only backcrossed with the local Q. suber but not Q. canariensis. However, genetic exchange with local Q. suber can be excluded, since no Q. suber-typical 5S-IGS variants, or obvious phylogenetic linkage with the Q. crenata-suber lineage, were found in Q. afares. Ongoing next-generation target sequencing of the 5S-IGS region  showed, so far, no evidence for a clone-sampling artefact in the studied individuals of Q. afares. The possibility of incomprehensive clone-sampling can thus be discarded. Analoguous to Q. crenata, the ancestry level of Q. afares may explain earlier findings interpreted towards a hybrid origin: being much closer to the common ancestor of sect. Cerris than Q. suber, this species may have retained (some) genetic imprints today found in members outside its section. This would explain also the association of Q. afares with two other, geographically very distant, individuals of the ‘oriental’ lineage .We also found no evidence to support the hybrid origin of Cerris, allowing occasional crosses. Indeed, our results demonstrate that Q. trojana and Q. libani, Q. brantii and Q. macrolepis, Q. cerris and Q. suber, and Q. trojana and Q. suber are interfertile and hybridize in the wild. Further investigations  are needed to distinguish between ancient hybridization with subsequent incomplete lineage sorting and retention of ancestral traits, to clarify the status of several other samples that may represent both phenomena based on shared 5S-IGS variants, the occurrence of unique sequence features, length of terminal branches and odd-placing in the phylogenetic reconstructions. Adequately addressing these issues would be of great relevance to identify relict populations and/or past contact/hybrid zones, to assess the hybridization ability of species growing in sympatry, and further define the evolutionary history of the Cerris oaks.Aside from putative hybrid species and swarms , our data demonstrates a general permeability of species boundaries in members of Sect. trnH-psbA differentiation patterns, and with respect to the plastid tree provided in From the clusters identified by the 5S-IGS network based on the average inter-individual clone  and PBC-Q. suber and Q. crenata. Sample cr05 likely represents a F1 hybrid with Q. cerris. The same holds for tj08, a Q. trojana x Q. suber hybrid. A second lineage, tentatively termed the ‘occidentalis’ lineage includes the widespread Q. cerris and the geographically restricted Q. castaneifolia, Q. euboica and Q. look. The third lineage collects the Eastern Mediterranean ‘Vallonea’ oaks Q. brantii, Q. ithaburensis, and Q. macrolepis, with three potential outliers: br02, br03 and it03 (possible recent or ancient hybrids). The last, least coherent group, the ‘oriental’ lineage, includes Q. afares, Q. libani and Q. trojana. Aside from supposed hybrids and introgressed individuals (see section above), these groups almost perfectly match previous taxonomic observations  sublineages occur in Italy and might correspond to the two main nuclear gene pools identified by Cerris: the corkish oaks (Q. crenata + Q. suber) and the ‘Vallonea’ (or Aegilops) oaks . Quercus euboica possibly originated by geographical isolation from a proto-trojana still close to the proto-cerris . It is impossible to provide absolute dates for the final isolation events. Comparison with intra- and inter-species 5S-IGS divergence in the other two main lineages of western Eurasian oaks , and younger than the main split within the ‘ilicoid’ oaks, i.e., of Q. ilex from Q. aucheri (+Q. coccifera).Overall, the complex genetic differentiation patterns can only be explained by longer ongoing free genetic exchange or more recent common origin of the ‘oriental’ and ‘occidental’ lineages than in case of the other two lineages within western Eurasian o-cerris . Interes clade 4 , hence, Ilex  fossil record of Cerris in (North-)East Asia (starting from early Oligocene) predates earliest records in western Eurasia (Oligocene/Miocene boundary) by ca. 10 Ma, thus rejecting the hypothesis that sect. Cerris evolved from the western stock of sect. Ilex populations with ‘WAHEA’ haplotypes (Cerris must have been (very) large in contrast to their East Asian siblings. The East Asian species of sect. Cerris are more heterogenous (Q. phylliraeoides (the Japanese Ilex oak), but also from the ‘WAHEA’ haplotypes of sect. Ilex and Q. baroni  and derived (L2) haplotype groups of ‘Cerris-Ilex’ lineage, and two haplotypes representing the ‘Euro-Med’ lineage characteristic for western populations of sect. Ilex , describncestral ; Fig. 1.ncestral  and in araeoides , Fig. 1,plotypes . The effrogenous , and difIlex, Q. coccifera in particular (Ilex is the lack of plastid structuring (and diversity) in the central and western Mediterranean region, indicating a rather recent, singular colonization by the master population, clearly not affected by Oligocene micro-plate tectonics as suggested for Q. suber by Once established in the Mediterranean region H1, H2; , local brticular . A notabCsa, Csb, Dsb) can be considered as trigger for increased genetic drift, possibly linked to speciation. The Aegilops oaks, Q. brantii, Q. ithaburensis, and Q. macrolepis, a well-circumscribed group based on 5S-IGS differentiation (The derived L2 ‘Cerris-Ilex’ haplogroup H5–H10) starts in Anatolia and extends further east (Iran) and south (Levant). In addition to isolation during range establishment, specialization to drier climates  and the very limited, amphi-Adriatic distribution of haplotype H2 in Q. macrolepis  and the systematic relationships of Q. look are clarified. Although we observed the occurrence of occasional F1 hybrids, possible intrograded individuals and several potential outlier individuals across the studied range, we could not confirm the hybrid origin of Q. afares and Q. crenata. The fossil record corroborates major inferences about the origin and diversification of the section.The present study is the first to include all putative species of Cerris and Ilex. A future focus should be on all Hindukush to western Himalayan species and the Japanese Q.  phylliraeoides, the north-easternmost member of sect. Ilex, which has plastid signatures very similar to the western Eurasian members of sect. Cerris but not to the geographically closer East Asian species of sect. Cerris. The entire fossil record of sections Cerris and Ilex should then be recruited to infer age estimates, following the recent example of the genus Fagus  outgroup when inferring a tree  is reported.The tree was rooted on Click here for additional data file.10.7717/peerj.5793/supp-5File S5Individuals represented by more than 2, 3 and 5 clones are included (cut-offs specimens are indicated).Click here for additional data file.10.7717/peerj.5793/supp-6Supplemental Information 1This Online Supporting Archive includes data and analysis files of Simeone et al. ”Comparative systematics and phylogeography of Quercus Section Cerris in western Eurasia: inferences from plastid and nuclear DNA variation” in several subfolders.Click here for additional data file.


Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel’s tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis. North American forest trees have been exposed to a number of unprecedented health challenges that are likely to be exacerbated by warming temperatures . These cPinus albicaulis Engelm.  is a widely distributed western North American tree species of subalpine and treeline elevations ranging from about 37° to 55° N latitude. It functions as an ecological keystone and foundation species in these high elevation communities . Ov. Ov11]. ght DBH; .GenAlEx 6.5 , [, [P. albfamilies . The lowfamilies . The sucsibirica . While tP. albicaulis previously studied using allozyme analysis [P. albicaulis—including allozymes [Our secondary objective was to provide a case example of the utility of these microsatellites for population genetic research by comparing genetic diversity in two populations of analysis . Despiteanalysis . Comparellozymes  and SNPsllozymes —the micrllozymes . The surP. albicaulis, or a presence/absence polymorphism in some chromosomal lineages. In these instances, null alleles fail to amplify, leading to decreased observed heterozygosity and apparent reaction failure in null homozygous individuals [IS values, which are large and positive for both populations, leading to potentially erroneous inferences of moderate inbreeding. Six loci—RPS119, RPS127, P5, P45, P63, and PisATG0012—were estimated to have high null allele frequencies in both populations. Loci that diverge from HWE and show evidence of null alleles should be used with caution as they inflate homozygosity and thus may not be applicable for all analyses  and are most likely the result of allelic dropout . All loci that violate HWE are transferred loci, which may indicate imperfect conservation of primer binding sites in ividuals . The potanalyses . Lookingm mating . For stuMantel's test revealed low genetic differentiation among populations and high gene flow, with no correlation between geographic distance and genetic relatedness among populations. Within populations, Mantel’s correlation shows no spatial autocorrelation at Henderson Mountain, with individuals no more or less closely related genetically to those nearby geographically than expected at random. There is weak evidence for isolation by distance in the Mt. Washburn population, with a positive Mantel correlation indicating that genetic distance between individuals increases with increasing physical distances. However, the AMOVA, which revealed that 9% of genetic differentiation occurred between populations, indicates greater population differentiation compared to SNP findings . A largeP. albicaulis reveal that locus transferability is a viable option for developing microsatellites in five-needle pines, with the caveat that null alleles may show high frequencies in some interspecific comparisons. Other efforts in cross-species amplification in Pinus, however, have had mixed results, with some cases appearing successful [P. albicaulis genome .Our results with ccessful , 83, 84,ccessful , 85. It P. albicaulis genetic diversity, population structure, and parentage. Employing high-level PCR multiplexing [P. albicaulis into five “seed zones” in the Inland West [P. albicaulis, and for the exploration of climate change mitigation strategies . These s e.g., , 88). Si. SiP. als , ca, caP. alS1 FigA) difference in mean number of alleles (-0.74) and 95% confidence interval of the difference (-2.29 to 0.81) and B) difference in mean expected heterozygosity (-0.015) and 95% confidence interval of the difference (-0.178 to 0.148) of 23 microsatellite loci for two populations of Pinus albicaulis Engelm. (Pinaceae): Henderson Mountain, Custer Gallatin National Forest, MT, and Mount Washburn, Yellowstone National Park, WY. Plotted points are the difference in means, Washburn minus Henderson, and the lines are the 95% confidence intervals around those points.(TIF)Click here for additional data file.S2 Figxy versus the observed Rxy (red line) for 9999 permutations, from Mantel’s test on two Pinus albicaulis Engelm. (Pinaceae) populations . Rxy is the correlation between geographic and genetic distance between individuals. A positive value indicates individuals are more closely related genetically to those nearby geographically than random, and a negative value indicating individuals are less closely related to those nearby.Frequency distribution of random R(TIF)Click here for additional data file.S3 FigPinus albicaulis Engelm. (Pinaceae) populations. A) Henderson Mountain, Custer Gallatin National Forest, MT showing no correlation between genetic and geographic distances  and B) Mount Washburn, Yellowstone National Park, WY showing a positive correlation .Correlation between geographic distance and genetic distance, from Mantel’s test on two (TIF)Click here for additional data file.S1 TablePinus albicaulis Engelm. (Pinaceae). Bold entries successfully transferred.Locus name, source species of primer development, and allele size range for 49 primers screened for transferability to (DOCX)Click here for additional data file.S2 TablePinus albicaulis Engelm. (Pinaceae) sampled at two populations .Minimum, maximum, and mean diameter at breast height (DBH) in centimeters compared between (DOCX)Click here for additional data file.S3 TablePinus albicaulis Engelm. (Pinaceae) sampled at two populations . Rxy and P(rxy-rand > = rxy-data) from Mantel’s correlation, showing isolation by distance and spatial autocorrelation, testing for each population and for all individuals combined.Minimum, maximum, and mean distance between (DOCX)Click here for additional data file.