Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors. Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that primarily colonizes the subgingival tissues in the oral cavity. This asaccharolytic anaerobe can adapt to and survive in the low oxygen tension conditions characteristic of periodontal pockets [P. gingivalis can tolerate microenvironments with low oxygen [P. gingivalis also metabolizes amino acids (AAs) and peptides for energy and as a supply of carbon [ pockets . Howeverw oxygen . Hemin ow oxygen ,4. Howevf carbon .P. gingivalis with various other bacterial species and evaluate its effects on the microbial community within the biofilm environment [Over 700 bacterial species of diverse microbial flora are estimated to inhabit the oral cavity . New culironment ,12.P. gingivalis during the colonization of periodontal tissues and the pathogenesis of periodontitis [The new culture-dependent techniques employ a variety of media prior to analysis using sensitive mass spectrometric and sequencing techniques, such as matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) and 16S ribosomal RNA (rRNA) sequencing, to identify bacterial species ,14. Futudontitis .P. gingivalis is considered to be a late colonizer, often co-aggregating at the top layer with initial and secondary colonizers [P. gingivalis, are recognized as opportunistic or keystone pathogens [The subgingival biofilm, within periodontal pockets, comprises over 300 different species . In thislonizers ,18. Whilathogens .P. gingivalis acts as a dynamic interface between the oral pathogen and its local environment. Successful acquisition of nutrients facilitates growth, while effective tissue colonization ensures bacterial survival. These are highly dependent on virulence factors expressed or released by P. gingivalis  [Gram-negative bacteria, including es (CPS) . Bacteries (CPS) . Carboxyes (CPS) . The capes (CPS) . Additioes (CPS) .P. gingivalis pathogenicity by enabling the evasion of detection by the immune cells and increasing the resistance to phagocytosis [P. gingivalis lacking a capsule, increased interleukin (IL)-1β, IL-6, and IL-8 cytokine production [P. gingivalis are also more invasive, although there are some conflicting reports about this capsular effect. The highest degree of bacterial invasion is observed when human coronary artery endothelial cells are incubated with wild-type P. gingivalis compared to other P. gingivalis strains [P. gingivalis strains were more efficient at invading gingival fibroblasts [Neisseria meningitides, associated with septicemia and meningitis, are known to downregulate capsule synthesis and assembly during adhesion to target cells [P. gingivalis infection of subgingival tissue, reducing the capsule production to increase adhesion. Consistent with this, in contrast to P. gingivalis cultured from healthy sites, P. gingivalis cultured from diseased periodontal sites are highly invasive to KB cells (a subline of HeLa cells) [P. gingivalis strains are associated with increased virulence, implying that capsule expression contributes to bacterial survival and its ability to impact host immune responses.Encapsulation contributes to ocytosis . Furtheroduction . The preoduction . In addi strains . Howeverroblasts . In thiset cells . A simila cells) . OverallP. gingivalis expresses two distinct types of fimbriae that vary in length, classified as major and minor. The long fimbrial polymer is primarily composed of the repeating fimbrillin (Fim) A subunits [P. gingivalis strains produce different overall FimA monomer sizes with some amino-terminus sequence heterogeneity [fimA gene, encoding the FimA protein, has six recognized gene variants: type I, Ib, II, III, IV, and V [fim genes: fimX, pgmA, and fimABCDE [fimB leads to the production of extended fimbriae in P. gingivalis strains ATCC33277 and 381 [P. gingivalis mutants to extracellular matrix (ECM) protein fibronectin or to type I collagen [Characteristic of gram-negative bacterial cells, fimbriae are thin protruding appendages attached to the outer membrane. subunits , which asubunits . Variousogeneity . The fimV, and V , and belfimABCDE . FimB re and 381 . FimC, F and 381 ,62. DefiP. gingivalis short fimbriae [mfa1 gene is part of a four-gene cluster operon comprising mfa1-4, while the mfa5 gene appears to be independently transcribed [Enterococcus faecalis and Streptococcus agalactiae, the vWF type A domain containing pilus proteins are known to mediate bacterial cell adhesion to host tissue [Minor fimbrial antigen 1 (Mfa1) is the main structural protein of fimbriae . The mfanscribed . Formatinscribed . In addinscribed . Mfa4 conscribed ,68. For t tissue ,70.P. gingivalis fimbriae serve dual functions during the pathogenesis of periodontitis: bacterial adhesion and invasion. The long and short fimbriae support bacterial colonization of gingival tissue and ensure its survival. First, fimbriae enable bacterial adhesion to host cells. Purified fimbriae from P. gingivalis cells adhere to human gingival cells in a concentration and time-dependent manner [P. gingivalis strains deficient in long fimbriae [fimA and mfa1 genes [P. gingivalis invasiveness is dependent on fimbriae. A FimA deficient strain has diminished potential for bacterial invasion of gingival epithelial and fibroblast cells [t manner . Impairefimbriae . Similara1 genes . Second,st cells ,73. The st cells ,74.P. gingivalis-induced periodontitis. Gnotobiotic rats exhibit significant bone loss after infection with fimbriae expressing wild-type P. gingivalis [P. gingivalis or (b) infected with a fimA knockout strain [fimA and mfa1 [P. gingivalis pathogenicity.Alveolar bone destruction is a hallmark feature of ngivalis . Howevert strain ,76. Consand mfa1 . Moreoveand mfa1 . Such chand mfa1 . In contP. gingivalis is asaccharolytic, other types of biomolecules are used for its energy and carbon supply needs. Gingipains cleave host proteins to generate peptides and amino acids, which in turn supply some of these requirements. Proteolysis of host proteins also facilitates bacterial invasion and colonization of subgingival tissues. Furthermore, gingipains can undermine the host immune responses allowing P. gingivalis to evade neutrophil-mediated bacterial clearance. Overall, gingipains play a pivotal role in exerting bacterial pathogenicity and catalyzing the degradation of a broad spectrum of host proteins to support the proliferation and invasion of the periodontium.Gingipains are surface-expressed or secreted cysteine proteases that can cleave a variety of host proteins in plasma, ECM, and in association with immune cells ,79,80,81P. gingivalis expresses two distinct forms of gingipains, arginine- and lysine-specific, that hydrolyze peptide bonds at carboxy termini of arginine or lysine residues, respectively [rpgA, rgpB, and kgp genes, respectively [P. gingivalis and Prevotella intermedia, suggesting a contributing role in periodontal biofilm formation [P. gingivalis gingipains are expressed in three main forms: membrane-bound monomeric proteins, membrane-bound multimeric complexes, and secreted soluble proteins. Purified RgpA, RgpB, and Kgp are synthesized as single-chain glycoprotein enzymes and expressed along the surface of P. gingivalis [ectively . This cyectively . The ginectively . Hemagglectively . Hgp15 aectively ,86. Hgp1ormation . Gingipaormation . P. gingngivalis ,90,91. Rngivalis ,93. Outengivalis .P. gingivalis virulence during infection of the oral cavity. These cysteine proteases mediate tissue colonization to establish a niche in the subgingival regions and degrade host proteins to provide essential nutrients for bacterial growth. Furthermore, gingipain-mediated subversion of the host immune responses promotes the proinflammatory environment that contributes to pathogenic persistence and dysbiosis of the oral microbiota.Some gingipain expression is central in the development of P. gingivalis adheres to human oral epithelial cells, while strains deficient in arginine gingipain activity have reduced attachment to these cells [P. gingivalis was demonstrated toward type I collagen, fibronectin, and laminin [P. gingivalis with inactivated rgpA gene. Adherence to immobilized collagen is also diminished in the rgpA knockout [During the initial stages of infection, gingipains assist bacterial adhesion to subgingival tissues. Wild-type se cells ,95. ECM se cells . Similar laminin . Reducedknockout .P. gingivalis in the subgingival sulcus. The binding of adhesin proteins to epithelial cells can be detected even if wild-type P. gingivalis is treated with a protease inhibitor, implying that protease functions of gingipains are not involved [P. gingivalis cells [Gingipain adhesin domains mediate tissue colonization of involved . Howeveris cells . Moreoveis cells ,99,100.P. gingivalis expressed fimbriae are heavily implicated as primary virulence factors initiating bacterial adhesion to host cells. However, gingipain proteolytic activity plays a role in effective fimbrial function. Post-translational processing of precursor fimbrillin proteins is crucial for proper fimbrial assembly along the outer membrane [rgpA and rgpB [membrane . Decreasand rgpB . Additioand rgpB . In thisand rgpB . This efand rgpB . Thus, gP. gingivalis bacterial cells within oral epithelial cells [P. gingivalis localization within gingival epithelial cells [P. gingivalis [P. gingivalis infiltrates the entirety of a human oral mucosal tissue model, including internalization within gingival epithelial cells, as well as penetration into the basement membrane and lamina propria [rgpA, rgpB, kgp) gingipain knockout strain, however, is primarily localized to the epithelial surface. Gingival tissue invasion involves cleavage of CAMs and cytoskeletal remodeling. Increased fibronectin and its fragments are observed in gingival crevicular fluid (GCF) from patients with periodontitis but not from healthy subjects [Following the attachment to cell surfaces, gingipains facilitate the invasion of gingival tissues. Transmission electron micrographs from invasion assays illustrate internalized al cells . Similaral cells . In contngivalis . Wild-ty propria . The trisubjects . Similarsubjects . Moreovesubjects .P. gingivalis can cause connective tissue proteolysis by upregulating activation and downregulating inhibition of matrix metalloproteases (MMPs) [Purified RgpA and Kgp can digest laminin directly . Howevers (MMPs) ,113,114.s (MMPs) . These ms (MMPs) .P. gingivalis-mediated infection of gingival epithelial cells triggers significant depolymerization of actin and disassembly of microtubules in host cells [rgpA, rgpB, kgp) knockout is incubated with human gingival epithelial cells [rgpA, rgpB) knockout, implying that Kgp is the effective enzyme. This is confirmed by dose and time-dependent degradation of isolated actin by purified Kgp [Gingipains can alter the host cell's cytoskeletal responses to facilitate bacterial invasion. st cells . Such acal cells . Howeverfied Kgp . In contP. gingivalis is known to break down the intercellular junctional complexes, thus disrupting this important epithelial barrier function [P. gingivalis with Madin-Darby canine kidney (MDCK) cells results in reduced labeling intensity of occludin and E-cadherin [Epithelial cells in gingival tissues form tight and adherent junctional complexes via specialized protein–protein interactions . These cfunction . Incubatcadherin . Westerncadherin .P. gingivalis with access to underlying connective tissue. This creates an environment conducive to bacterial colonization of the periodontium, an important step in the pathogenesis of periodontitis. The compromising of the structural integrity of gingival epithelium may lead to further tissue destruction, a hallmark feature of P. gingivalis-induced periodontitis [P. gingivalis, inhibits abscess formation and mortality [P. gingivalis and protects mice against tissue invasion. Similarly, bacterial preincubation with cysteine proteinase inhibitors prevents the development of necrotic lesions along the mouse abdomen and protects the mice from death [rgpA, rgpB) strain [kgp gene knockout strain also improves survivability in mice. Taken together, either (a) gingipain gene inactivation or (b) pretreatment of P. gingivalis culture with gingipain inhibitors attenuates bacterial virulence resulting in diminished protein degradation and reduced tissue invasion.Gingipain-mediated degradation of epithelial CAMs, as well as hydrolysis of actin and junctional proteins, provide dontitis . Immunizortality . This imom death . Additio) strain . Even atP. gingivalis is a porphyrin auxotroph lacking the enzymatic machinery for porphyrin biosynthesis [Iron is an essential metal required for a wide range of physiological processes, including DNA replication/repair, mitochondrial function, myelin synthesis, and red blood cell (RBC) mediated oxygen transport ,129,130.ynthesis . As a reynthesis .P. gingivalis. Gingipains play a central role in iron acquisition, starting with gingipain-mediated hemagglutination and hemolysis. RgpA and Kgp contain four hemagglutinin/adhesin domains, which mediate P. gingivalis interactions with RBCs [rgpA, rgpB, kgp) knockout strain is typically characterized by inhibited RBCs hemagglutination potential, while single RgpA or Kgp knockout strains support reduced RBC binding [P. gingivalis is suppressed [rgpA, rgpB, kgp) knockout is used, suggesting that gingipains may not be the primary sources of the hemolytic function [RBCs represent a major iron reserve in the host, which can serve as an accessible iron source for the nutritional requirements of ith RBCs . Purifieith RBCs . The tri binding . Agglutippressed . Furtherppressed . Howeverfunction .P. gingivalis [Hemoglobin is a globular heterotetrameric protein comprising two α and two β polypeptide chains, each of which noncovalently binds to its own single heme prosthetic group . Gingipangivalis ,142,144.P. gingivalis relies on host proteins to supply its metabolic energy and nutrient needs. Peptide fragments appear to be the preferred nutrient for bacterial growth. Minimal metabolism of free AAs is observed in P. gingivalis culture media, with none becoming fully depleted [P. gingivalis bacterial cells incubated with peptides, and free AA, preferentially hydrolyze peptides containing aspartate, glutamate, leucine, and valine [P. gingivalis culture medium, in addition to human serum albumin, inhibits bacterial growth and partially reduces the ability to degrade the protein [rgpA or kgp), double gene , or triple gene  knockout strains are unable to grow in human serum supplemented with hemin [As an asaccharolytic pathogen, depleted . This lidepleted . Washed d valine . In contd valine . Gingipa protein . Furtherth hemin . Howeverth hemin .P. gingivalis to successfully colonize the periodontium and proliferate within the subgingival region. To this end, the bacteria (a) evade the host immune response and (b) subvert the immune cell-mediated bacterial clearance. Avoiding detection by the host immune system is pivotal to bacterial survival within the oral microenvironment. However, P. gingivalis can also manipulate the host's inflammatory response. Initial infection in the oral cavity induces the production of a multitude of chemokines and cytokines. Increased IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α are released following incubation of wild-type P. gingivalis with cultured human gingival fibroblast cells, oral epithelial cells, or periodontal ligament cells from healthy subjects [P. gingivalis have significantly higher serum levels of IL-1β, IL-6, IL-8, and TNF-α, compared to controls or compared to mice infected with a mutant strain deficient in functional fimbriae [Neutralizing host immune defenses is necessary for subjects ,149,150.fimbriae . Elevatefimbriae ,154,155.Inflammation is a physiologic host response to pathogenic infection. Infected cells secrete proinflammatory mediators that serve as chemotactic signals to recruit immune effector cells, which mediate bacterial clearance. IL-1β is a proinflammatory cytokine commonly secreted by monocytes or macrophages . It is aP. gingivalis-induced IL-6 expression increases bone resorption by downregulating osteoprotegerin (OPG) [IL-6 is a proinflammatory cytokine secreted by gingival and periodontal ligament fibroblasts ,162. P. in (OPG) . OPG is in (OPG) . RANKL bin (OPG) . Gingipain (OPG) ,166. Thein (OPG) .IL-8 is a chemokine secreted by gingival epithelial cells, gingival fibroblasts, and periodontal ligament fibroblasts ,169. It P. gingivalis lipopolysaccharide (LPS) [P. gingivalis-infected cathepsin K knockout mice [TNF-α is a pleiotropic proinflammatory cytokine primarily secreted by activated macrophages . It workde (LPS) . In the de (LPS) . Furtherout mice .P. gingivalis infection progresses deeper into the periodontium, the release of chemokines and cytokines may be altered. Membrane-expressed gingipains can exploit the host inflammatory responses by degrading proinflammatory cytokines. Human gingival epithelial cells treated with wild-type P. gingivalis elicit the release of IL-1β, IL-6, and IL-8 [rgpA, rgpB, kgp) knockout strain, but not with the double  knockout, abolishes cytokine degradation. Similarly, transient increases in IL-1β, IL-6, or IL-8 levels are observed upon incubation of P. gingivalis with human gingival fibroblasts [P. gingivalis or with gingipain-deficient strains lead to significantly higher IL-6 and IL-8 secretion from fibroblasts.As and IL-8 . Howeverroblasts . HoweverP. gingivalis [P. gingivalis. Kgp is implicated as the primary gingipain involved in disrupting the cytokine signaling network since minimal effects are observed when epithelial cells are treated with a Kgp-null strain [P. gingivalis gingipains promote a persistent proinflammatory environment within the subgingival region to extract nutrients from host proteins and evade host immune cells.Additional inflammatory gingipain targets, including chemokine (C-C motif) ligand (CCL) 2, CCL5, chemokine (C-X-C motif) ligand (CXCL) 1, and CXCL10, are suppressed following a challenge of human gingival fibroblasts with ngivalis . In contl strain . Taken tSuccessful colonization of the periodontium is crucial to establishing a subgingival niche and fostering bacterial proliferation. Subsequently, long-term bacterial survival requires evasion of the host immune responses to avoid bacterial killing. Impaired immune responses enable a proinflammatory environment that favors persistent infection, characteristic of chronic periodontitis.P. gingivalis viability in human serum is not significantly affected by incubation with functionally competent complement factors [rgpA, rgpB, or kgp) or double  gingipain gene inactivation significantly reduces P. gingivalis survival in human serum, suggesting a contributing role for these cysteine proteases in disruption of the complement bactericidal functions. Gingipains cleave and degrade complement components C3, C4, and C5 [P. gingivalis strain [P. gingivalis. Uptake of the RgpA deficient strain by polymorphonuclear leukocytes (PMNs) is also significantly improved in the presence of complement factors [The complement system, integral to the host immune defenses, comprises a group of plasma proteins that generally contribute to pathogen identification and elimination. However, wild-type  factors . Single , and C5 . Purifies strain . Further factors . HoweverP. gingivalis improves the survival of subsequently added Escherichia coli [E. coli viability in a concentration-dependent manner. The reduction of the bactericidal effect of human serum is most and least efficiently achieved by RgpA and Kgp, respectively [C3a is a proinflammatory anaphylatoxin mediating the degranulation of eosinophils and chemotaxis of mast cells ,185. Thehia coli . Similarectively .C4b-binding protein (C4BP) is a circulating inhibitor of the complement system . C4BP inP. gingavlis occurs following bacterial incubation with human serum deficient in C4BP. Conversely, exogenously added C4BP reduces C9 surface accumulation on P. gingivalis to levels seen with normal human serum. The binding of Haemophilus influenzae to C4BP does not affect the cofactor function of the complement system inhibitor [P. gingivalis with an evasion mechanism that attenuates host immune defenses by downregulating C3b-mediated opsonization and phagocytosis.C4BP can interact with the hemagglutinin/adhesin domains of RgpA gingipain . In addinhibitor . Rather,nhibitor . SimilarP. gingivalis can impair bacterial clearance by degrading proinflammatory mediators involved with neutrophil recruitment. Reduced levels of secreted IL-8 are observed in human gingival epithelial cells following incubation with P. gingivalis [P. gingivalis, the expected IL-8-mediated neutrophil transmigration through the epithelial layer becomes attenuated [The release of chemokines and cytokines at an infection site leads to the recruitment of neutrophils, which serve as the host’s first line of defense against pathogenic bacteria . P. gingngivalis . In additenuated .P. gingivalis virulence by disrupting neutrophil recruitment and neutrophil functions at the infection site. The release of TNF-α and several interleukins contributes toward neutrophil recruitment. PMN responses are reduced in mice deficient in TNF receptors following wild-type P. gingivalis infection [Surface-expressed gingipains can mediate nfection . Moreovenfection . Similarnfection .P. gingivalis in mice elicits a proinflammatory response with the release of several cytokines, including IL-17 [P. gingivalis or sham infected [P. gingivalis have significant neutrophil infiltration into the gingival epithelium and adjacent connective tissues compared to sham-infected mice [P. gingivalis tends to suppress the IL-17-mediated signaling and the subsequent neutrophil infiltration.Oral inoculation of ng IL-17 . Howeverng IL-17 ,201. Furinfected . Similarted mice . This imP. gingivalis to exploit host inflammatory and neutrophil responses. The bacterial infection triggers neutrophil recruitment via chemokine/cytokine release. P. gingivalis expressed gingipains degrade inflammatory mediators to attenuate neutrophil functions and promote a chronic proinflammatory environment. In turn, the inflammation-mediated breakdown of gingival tissue provides essential nutrients for bacterial growth.Taken together, gingipains help The glycosylated transmembrane glycoprotein CD99 is expressed on the surface of gingival epithelial, gingival fibroblast, and endothelial cells ,204. CD9Complement C5 is proteolytically activated to form C5a and C5b fragments. Gingipains are able to cleave C5, releasing C5a, which can serve as an anaphylatoxin and contribute to neutrophil chemotaxis . This prP. gingivalis subverts neutrophil antimicrobial functions by manipulating the complement system and the TLR signaling pathway. Cleavage of C5 by P. gingivalis gingipains produces endogenous C5a, which interacts with the C5a receptor (C5aR), to inhibit toll-like receptor (TLR) 2 mediated induction of IL-12 [P. gingivalis strain, exogenously added C5a promotes bacterial survival by reducing neutrophil-mediated phagocytosis [P. gingivalis-infected wild-type mice are treated with a C5aR antagonist or an anti-TLR2 mAb [P. gingivalis. Similarly, bacterial killing is increased when isolated neutrophils are challenged with P. gingivalis in the presence of a C5aR antagonist or with an anti-TLR2 mAb [P. gingivalis [P. gingivalis survival.of IL-12 . IL-12 iof IL-12 . Howeverocytosis . ConversTLR2 mAb . In addiTLR2 mAb . Furtherngivalis . ClearlyP. gingivalis virulence, attenuating neutrophil function and enhancing bacterial survival. Suppression of the neutrophil antimicrobial function may further lead to the overt growth of other common bacterial species in oral biofilm. Consequently, this can remodel the host commensal microbiota into a dysbiotic state favoring proinflammatory conditions and further aggravating periodontitis's pathogenesis.Gingipain-mediated C5aR cleavage can be observed when isolated human neutrophils are incubated with purified Kgp . Prior pP. gingivalis evasion of neutrophils. On the contrary, P. gingivalis viability is higher in MyD88 knockout mice [P. gingivalis via phagocytosis is MyD88 dependent [P. gingivalis [P. gingivalis-mediated digestion of MyD88 is reduced after treatment with a C5aR antagonist or with an anti-TLR2 mAb [P. gingivalis survival involves the exploitation of the C5aR-TLR2 receptor crosstalk and inactivation of the MyD88 protein.Myeloid differentiation primary response 88 (MyD88) is a downstream adaptor protein that mediates TLR2 signaling for the induction of proinflammatory cytokines and for the host inflammatory response . This suout mice . In addiependent . Howeverngivalis . MoreoveTLR2 mAb . This imPeriodontitis is a proinflammatory state mediated by a pathogenic infection within subgingival tissues. The pathophysiology of periodontitis includes a chronic inflammatory environment that may ultimately progress to a breakdown of gingival tissues, including periodontal ligament and destruction of supporting structures for teeth . GradualP. gingivalis.Removal of the oral biofilm is key to reducing the tissue destruction associated with periodontitis. Scaling and root planing (SRP) remains the gold standard for non-surgical therapy in patients . HoweverContributing factors to the formation of oral biofilm include increased opportunistic bacterial colonization, periodontitis, dental prosthetics, poor oral hygiene, and smoking ,220,221.Actinomyces (A. oris), Streptococcus (S. gordonii and S. mutans), or Veillonella (V. denticariosi and V. parvula) [Actinomyces sp. and Streptococcus sp., based on meta-transcriptome analyses of human supragingival dental biofilm, was 3–12% and 12–19%, respectively [P. gingivalis. The surface-expressed polypeptides of S. gordonii, Streptococcal surface protein A (SspA), and SspB interact with Mfa1, a protein component of the P. gingivalis fimbriae [Biofilm formation comprises cell-to-cell interactions among multiple bacterial species. Early colonizers of the tooth surface include gram-positive anaerobic bacteria such as parvula) ,230. Theectively . Followifimbriae ,233. Mfafimbriae .Prevotella intermedia, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum, can interact with early colonizers [F. nucleatum and P. gingivalis are observed in coaggregation assays [P. gingivalis demonstrated reduced integration into biofilms formed by mutant F. nucleatum strains deficient in the outer membrane proteins, fibroblast activation protein 2 (Fap2), and arginine (R)-inhibitable adhesin (RadD) [Secondary colonizers, such as lonizers . Subsequn assays . P. gingn (RadD) .P. gingivalis and F. nucleatum involve a galactoside moiety and a lectin-like adhesin (FomA), respectively [P. gingivalis PK 1924 (serotype K5) can bind to F. nucleatum [Interactions between ectively ,238,239.ucleatum . Consequucleatum .P. gingivalis, transform the biofilm microbiota into a dysbiotic community, which undermines the host immune response and exploits the inflammatory responses to infection. Biofilm buildup propagates persistent chemokine and cytokine production, which is associated with bacterially induced-inflammation of gingival tissues [Together, dental biofilm encompasses a diverse bacterial community of over 300 species . Keyston tissues . UltimatP. gingivalis, as a late colonizer that adheres to earlier colonizers, is identified as a keystone pathogen implicated in the pathogenesis and progression of periodontitis [P. gingivalis, disrupt this homeostatic balance and shift the commensal microbial community to a pathogenic state [P. gingivalis mediates a reinforcing cycle of periodontal dysbiosis leading to enhanced bacterial pathogenicity [P. gingivalis. Inflammation and gingipain-mediated degradation of gingival tissue proteins provide peptides, iron, and other nutrients crucial for bacterial growth and further progression of infection.During biofilm formation, dontitis ,243. Thedontitis . Under pdontitis . Ordinardontitis . Howevergenicity . This opgenicity . ChronicP. gingivalis contributes to the growth of such a microbial community. However, the expression and release of such proteases in the periodontal pocket also appear to be coordinated via signaling from other community members [P. gingivalis with the host as well as other microbial surfaces, are further aided by the adhesive properties of fimbriae [Dysbiotic polymicrobial communities characteristically develop increased reliance on the nutrients from the serum-like transudate produced during periodontal inflammation . The ado members . This mi members . Moreovefimbriae . ConsequIn turn, the host tissues respond by upregulating the expression of various genes, including ferric ion binding protein, several proto-oncogenes, an ankryn repeat, and a β-enolase . The hosP. gingivalis can potentially extend beyond the oral cavity. Tissue damage from routine oral hygiene practices or dental procedures may facilitate the entry of the periodontopathogen into the systemic circulation [P. gingivalis can trigger the activation of prothrombotic mediators, including platelets, increasing the risk for thrombosis [P. gingivalis expressed gingipains are cysteine proteases, which can activate plasma serine protease coagulation factors [cat/Km) of FIX activation is comparable to that observed for a physiological activator, activated factor VII (FVIIa)-tissue factor (TF) complex [Notechis scutulus scutulus venom but lower compared to venom from Oxyuranus scutellatus [The pathogenicity of culation . P. gingrombosis . Concentrombosis . P. ging factors . Purifie factors ,38,39. I factors ,38,39. T complex ,38. Seve complex ,258. In  complex . In addiP. gingivalis interaction with platelets can induce platelet activation and subsequent aggregation [P. gingivalis, isolated platelets undergo intracellular calcium mobilization [rgpA and rgpB) gingipain knockout mutant. A single (kgp) gingipain mutant did elicit changes in intracellular calcium levels, however, this was significantly lower compared to platelet exposure to the wild-type strain. Similarly, platelet aggregation is observed following the incubation of P. gingivalis with isolated platelets. However, platelet aggregation depends on the ratio of platelet/bacteria, consistent with the possibility of either a threshold phenomenon or multiple competing platelet interactions. In whole blood, platelet expression of CD62P, an adhesion molecule expressed on surfaces of activated platelets, increases following preincubation of high P. gingivalis colony-forming units (CFU) with or without subsequent ADP stimulation [P. gingivalis CFU, particularly as preincubation time is extended. This suggests a dose and a time dependence for the impact of P. gingivalis preincubation on platelet surface CD62P expression.Bacterial infection is often transient for individuals with a robust immune system. However, serious thrombotic complications can develop from persistent infection, including infective endocarditis and sepsis-associated disseminated intravascular coagulation . A distiregation . Intraceregation . Consistlization . Howevermulation . ConversP. gingivalis may promote an excitable state in platelets that results in rapid activation following subsequent interaction with physiologic agonists. At lower P. gingivalis levels, platelet responses may be triggered with prolonged preincubation times. In this context, whole blood from generalized aggressive periodontitis and periodontitis patients is associated with higher platelet activation [P. gingivalis with whole blood from patients with the peripheral arterial disease (PAD) [P. gingivalis infusion into rats [P. gingivalis with whole blood also impacts platelet plug formation under shear conditions [P. gingivalis preincubation times past 7.5 min significantly reduces the time for platelet plug-mediated aperture occlusion in the Platelet Function Analyzer (PFA-100). Thus, platelet plug formation time in whole blood is affected both by the P. gingivalis concentration and by the duration of bacterial preincubation.High levels of tivation ,265. Morse (PAD) . Similarnto rats . Preincunditions . ExtendiP. gingivalis levels below those needed for the occlusion time shortening [P. gingivalis. However, the activated platelets may be insufficient to trigger full platelet aggregation. Consequently, the spent activated platelets become refractory to platelet plug formation, leading to a prolonged occlusion time. Alternatively, if P. gingivalis is capable of interacting with multiple platelet activation pathways with characteristic interaction affinities, then multiple platelet functions could be triggered in a concentration-dependent manner. As a result, P. gingivalis in whole blood may trigger a variety of time-dependent processes, some of which are possibly functionally opposing [Interestingly, a prolongation of the occlusion time can be observed at certain ortening . This isopposing .P. gingivalis in a preincubation time-dependent manner. Moreover, bacterial exposure to whole blood can trigger the neutrophil release of nuclear DNA, also known as neutrophil extracellular traps (NETs). Such release of NETs in response to P. gingivalis is known to be at least in part dependent on an interaction between activated platelets and neutrophils [P. gingivalis with the various blood cells does not only potentially alter their cell-specific functions in response to this pathogen but can also impact their physiologic cell-cell interactions.Platelets are involved in a variety of ways with leukocyte functions, including those of neutrophils. Platelet-neutrophil interactions are believed to be mediated by an interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) . Such introphils . This imP. gingivalis expressed gingipains, however, can also cleave and activate PAR-1 and PAR-4 [Protease-activated receptors (PARs), members of the GPCR family, are characterized by a unique activation mechanism. The amino terminus of PARs is cleaved to expose an auto-activating tethered ligand that triggers intracellular signal transduction via an internal salt bridge formation . Human pnd PAR-4 . RgpA isnd PAR-4 . Thrombind PAR-4 . The parnd PAR-4 . Furthernd PAR-4 . Similarnd PAR-4 . In thisP. gingivalis, platelet aggregation is observed in platelet-rich plasma (PRP) treated individually or in combination with inhibitors for Rgp or Kgp [P. gingivalis strain deficient in adhesin domains only or with a strain deficient in Rgp, Kgp, and adhesin domains does not induce platelet aggregation [P. gingivalis in the presence of anti-FcγRIIa mAb inhibits platelet aggregation [P. gingivalis and an anti-glycoprotein (GP) Ibα mAb. However, aggregation of washed platelets, treated with gingipain deficient strains, is restored if anti-P. gingivalis immunoglobulin G (IgG) is added [P. gingivalis can induce platelet aggregation independent of gingipains via pathways that involve contributing roles from FcγRIIa, IgG, and GPIbα.However, the proteolytic functions of gingipains are not solely responsible for mediating platelet aggregation. In the presence of p or Kgp . This sup or Kgp . Incubatregation . Howeverregation . Similaris added . Taken tP. gingivalis in the pathogenesis of oral inflammatory processes. In addition, the potential impact on the functioning of certain blood cells, such as platelets and neutrophils, is also considered, particularly regarding their subsequent roles in increased prothrombotic risks. However, this review does not represent an encyclopedic compilation of everything currently known about this pathogen. Similarly, it does not detail the impact of this pathogen on every cell type that it may come in contact with and possibly infect. Of particular interest might be the effects of P. gingivalis on the cells and tissues of blood vessel walls. That would be a worthy topic for a review in its own right.This review represents the current understanding from a basic science perspective of the role of P. gingivalis is an opportunistic pathogen that infects the subgingival tissues of the oral cavity. Virulence factors, most notably arginine- and lysine-specific gingipains, play a central role in mediating P. gingivalis pathogenicity. Gingipains are involved with all aspects of P. gingivalis-induced infection, including nutrient acquisition essential for bacterial growth, tissue breakdown to facilitate bacterial invasion, and degradation of cytokines to disrupt the host inflammatory responses. Gingipains also contribute to P. gingivalis-mediated subversion of host immune responses by cleaving complement proteins and exploiting the TLR signaling pathway to attenuate neutrophil functions. Ineffective bacterial clearance reinforces persistent P. gingivalis infection and propagates chronic inflammation, driving the periodontitis pathophysiology. A growing body of evidence further suggests a potential circulatory prothrombotic role of P. gingivalis. Platelet activation and aggregation are observed in the presence of this periodontopathogen. Future studies should include further characterization of the thrombotic mechanisms triggered by P. gingivalis and its expressed virulence factors.


Streptomyces malaysiensis F913, was identified as a SARP family regulator. Disruption of nigR abolished nigericin biosynthesis, while complementation of nigR restored nigericin production, suggesting that NigR is an essential positive regulator for nigericin biosynthesis. Overexpression of nigR in Streptomyces malaysiensis led to significant increase in nigericin production compared to the wild-type strain. Nigericin production in the overexpression strain was found to reach 0.56 g/L, which may be the highest nigericin titer reported to date. Transcriptional analysis suggested that nigR is required for the transcription of structural genes in the nig gene cluster; quantitative RT-PCR analysis revealed that the expression of structural genes was upregulated in the nigR overexpression strain. Our study suggested that NigR acts in a positive manner to modulate nigericin production by activating transcription of structural genes and provides an effective strategy for scaling up nigericin production.Nigericin is a polyether antibiotic with potent antibacterial, antifungal, antimalarial and anticancer activity. NigR, the only regulator in the nigericin biosynthetic gene cluster in  Streptomyces are well known for their extraordinary capacity to produce bioactive secondary metabolites in industrial, agricultural and medical applications. Secondary metabolism in Streptomyces is regulated in a complex fashion in response to environmental manipulations. The regulation of secondary metabolite biosynthesis is governed by various regulators, including cluster-situated regulators (CSRs), pleiotropic regulators and global regulators [Streptomyces antibiotic regulatory protein (SARP) family transcriptional regulators constitute the most frequently occurring transcriptional regulators, which serve mainly as activators in antibiotic biosynthesis [Streptomyces coelicolor [actIV-orfA operon and actIV-orf1 operon activates their transcription [S. coelicolor [Streptomyces ansochromgenes (nikkomycin cluster), CcaR in Streptomyces clavuligerus (cephamycin-clavulanic acid supercluster) and VlmI in Streptomyces viridifaciens  [Streptomyces cacaoi subsp. asoensis [polR, the activator of key structural genes such as polC and polB [gulators . Global gulators . CSRs usynthesis . ActII-Oelicolor . Bindingcription ; the expelicolor . SARP facluster) ,8,9. Apaasoensis , which cand polB . Though and polB . + and K+ concentration [Paecilomyces variotii and Candida albicans [Streptomyces sp. DSM4137; the biosynthetic pathway of nigericin was proposed based on analyses of intermediates accumulated in disruption mutants of several structural genes [Streptomyces malaysiensis F913 is a soil-borne actinomycete with high algicidal activity [S. malaysiensis F913 has a nigericin biosynthetic gene cluster (nig gene cluster) which showed high synteny with the reported homologous gene cluster in Streptomyces sp. DSM413. In this paper, we determined the role of the only regulatory gene, nigR, in the nigericin biosynthesis gene cluster in S. malaysiensis F913. The results suggest that NigR positively regulated nigericin biosynthesis by activating the transcription of structural genes. Overexpression of nigR significantly improved nigericin production. Polyether antibiotics are a unique class of ionophores with metal ion-binding ability and lipophilic characteristics . One majntration ,17. Nigealbicans . In vivoalbicans ,20. The al genes . Howeveractivity . Whole gS. malaysiensis F913 genome yielded 25 contigs, assembled into 6 scaffolds (GenBank accession number: LJIW00000000). The fine genome map of S. malaysiensis F913 was 11,608,319 bp in length without any plasmid, the chromosome encoded 9497 predicated genes, and the total length of the genes was 10,011,963 bp. The nig gene cluster was identified by searching the genome sequence of S. malaysiensis F913. As the nigericin biosynthesis gene cluster in S. malaysiensis F913 showed high synteny with the reported cluster in Streptomyces sp. DSM413, we used the same gene name as proposed for the original discovered cluster  and SMART (http://smart.embl-heidelberg.de (accessed on 25 May 2022). The results suggested that NigR is a SARP family regulator, with an N-terminal trans-Reg-C domain (amino acids 19–96) (Pfam no. PF00486) which may play a role in DNA binding, and a bacterial transcriptional activator domain (amino acids 103–248) (Pfam no. PF03704) located in the C-terminal  was tested for nigericin production. Unlike the S. malaysiensis F913 wild-type strain (WT), nigericin production was abolished in the nigR disruption mutant , was used as internal control. The results suggest that NigR is essential for the expression of structural genes; the promoters of structural genes are potential targets of NigR. A consensus sequence of “CGWWWCCG” was identified in the promoter of orf9, intergenic region of nigD-C and nigAI-nigE  tests were performed to detect the transcription of the gene in the  by NigR . A primegAI-nigE .nigR overexpression in S. malaysiensis F913, we constructed nigR overexpression vectors pSET152::PhrdBR and pSET152::PkasOR. pSET152, pSET152::PhrdBR and pSET152::PkasOR were introduced into S. malaysiensis F913 to generate F913-pSET152, F913-hrdBR and F913-kasOR, respectively. Nigericin production was increased in both F913-hrdBR and F913-kasOR strains compared to F913 and F913-pSET152 controls. Compared to the F913 strain, nigericin production rose by 68% in F913-hrdBR and 54% in F913-kasOR at day five  assays to detect the interaction of NigR with promoters of target genes [Nigericin has a broad spectrum of biological activities. Recent research has focused on its anticancer activities ,27. Prevet genes . Streptomyces species. Secondary metabolite titers can be influenced by global regulatory networks and pathway-specific regulation, which makes these regulators perfect targets for the rational engineering of overproduction strains. Overexpression of positive regulatory genes is a well-established strategy for strain improvement. By multiplying copy numbers of sanG, nikkomycin production was increased significantly [Streptomyces fradiae, insertion of a second copy of the positive regulator tylR under control of the strong constitutive ermE* promoter caused a 50% improvement in tylosin production [ermE* promoter from Saccharopolyspora erythraeus is the most widely used strong constitutive promoter; other optional tools, such as SF14p, are also available [kasO* promoter and hrdB promoter, have been developed, and have shown better performance in some Streptomyces species [kasO* promoter and hrdB promoter as cis regulatory elements to drive nigR expression. The results suggest that both promoters can upregulate nigericin expression and the F913-hrdBR strain showed higher nigericin production. The mean production of nigericin in the F913-hrdBR strain was 0.56 g/L. To our knowledge, this is the highest nigericin titer reported to date, representing a new step for scaling-up the production of nigericin. Promoter strength varied in different Streptomyces strains, implying that more promoters need to be screened to choose the most suitable one for strain improvement in S. malaysiensis F913. Secondary metabolites are synthesized from precursors through multistep biosynthetic pathways with the cells. Medium and fermentation process optimization are also required to further increase the production of nigericin in engineered overexpression strains. Combinational mutation and culture condition optimization have been carried out to improve nigericin production; however, titer improvement of nigericin production by overexpressing regulators has not been reported ,30. Afteficantly . In Streoduction . The ermvailable . In rece species ,34. In tStreptomyces malaysiensis F913, a natural nigericin producer, was isolated from the soil of Southwest University; the accession number of its genome is LJIW00000000. Streptomyces malaysiensis F913 and their derivatives were grown on MS agar  at 28 °C. Liquid Gause’s medium  was used for nigericin production. All antibiotics were purchased from Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. The bacterial strains and plasmids used in this study are listed in nigR disruption vectors, a 2329 bps PCR product from primer NigRdm-upF/R .For nigericin production, spore suspensions were inoculated into liquid Gause’s medium and incubated for 168 h at 28 °C before sampling. For analysis of nigericin, culture supernatants were extracted twice with an equal volume of ethyl acetate, and mycelia were extracted with 50 mL of methanol. Combined extracts were dried by removing the solvent under reduced pressure at 50 °C. The dried pellet was then dissolved in 1 mL methanol. High-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1260 HPLC system and ZORBAX SB-C18 column  . HPLC conditions were as follows: a linear gradient of MeOH/HS. malaysiensis F913 and DM01R strains after inoculation for 96 h and used as templates for analysis of gene expression. For qRT-PCR analysis, total RNAs were isolated from mycelium of F913, F913-hrdBR and F913-kasOR strains grown in Gause’s medium at various fermentation times . The qRT-PCR was performed by a LightCycle 96 instrument . All experiments were carried out in triplicate. The relative expression levels of all samples were calculated using the 2−ΔΔCT method [hrdB (a housekeeping sigma factor in Streptomyces). Significance tests were calculated by means of a Student’s t-test (GraphPad Prism 6).The primers used in RT-PCR and qRT-PCR are listed in T method . All qRTS. malaysiensis F913 was extracted, followed by RNase treatment. The draft genome sequence of S. malaysiensis F913 was achieved using Illumina Hiseq2000 sequencing system at NovoGene Bioinformatics Institute in Beijing, China. The paired-end reads generated by the Illumina sequencer were assembled by using SOAPdenovo 1.05 [The genomic DNA of ovo 1.05 . Functioovo 1.05 . The putovo 1.05 .nig gene clusters determined in this study have been submitted to the GenBank database under accession number ON664936.The nucleotide sequences of S. malaysiensis F913, was identified as a SARP family regulator in this study. Disruption of nigR resulted in loss of nigericin production, while complementation of nigR restored nigericin production. The targets of NgrR might be structural genes in the nig gene cluster, given the knockout of ngrR abolishes the transcription of all biosynthesis genes. Nigericin overproducing strains were constructed through overexpression of nigR. The maximum production of nigericin was achieved in the F913-hrdBR strain at about 0.56 g/L, which was about 1.5-fold higher than S. malaysiensis F913. These results extended our understanding about the regulatory mechanism of nigericin biosynthesis, and have also provided a promising strategy for scaling up the production of nigericin.NigR, the only regulator in the nigericin biosynthetic gene cluster in


We used DNA-based fingerprinting and statistical analyses of leaf shapes, ecological niches, and distribution ranges for eight predefined morphotaxa to judge their ranks as species or subspecies and propose a taxonomical treatment for the surveyed group with six species (two of them with two subspecies). Having clarified the taxonomic structure of the ancestral diploid (the ‘warps and wefts’) and the subsequent tetraploid layer (the ‘picks of the fabric’), we will be able to provide a taxonomy for the remainder of this well-known plant group and study its reticulate evolutionary history.The delimitation of species as the most important rank in biological classification is an essential contribution of taxonomy to biodiversity research, with all of its evolutionary, ecological, political, and legislative ramifications. Species delimitation is extremely tricky in plant groups evolving by polyploidisation (multiplication of chromosome sets) because the rapid formation of new, reproductively isolated lineages (species) is often not paralleled by conspicuous genetic, morphological, physiological, and/or ecological differentiation. Having clarified the taxonomy of diploid , the present study aims at the elaboration of a specific and subspecific taxonomic treatment of the tetraploid members of the genus. Following an integrative taxonomic approach, species-level decisions on eight predefined morphotaxon hypotheses were based on genetic/genealogical, morphological, ecological, and geographical differentiation patterns. ddRADseq fingerprinting and SNP-based clustering revealed genetic integrity for six of the eight morphotaxa, with no clear differentiation patterns observed between the widespread L. ircutianum subsp. ircutianum and the N Spanish (Cordillera Cantábrica) L. cantabricum and the S French L. delarbrei subsp. delabrei  and L. meridionale . The inclusion of differentiation patterns in morphological (leaf dissection and shape), ecological , and geographical respects  together with the application of a procedural protocol for species-rank decisions (the ‘Wettstein tesseract’) led to the proposal of an acknowledgement of the eight predefined morphotaxon hypotheses as six species (two of them with two subspecies). Nomenclatural consequences following from these results are drawn and lead to the following new combinations: Leucanthemum delarbrei subsp. meridionale (Legrand) Oberpr., T.Ott & Vogt, comb. nov. and Leucanthemum ruscinonense (Jeanb. & Timb.-Lagr.) Oberpr., T.Ott & Vogt, comb. et stat. nov.Based on the results of a preceding species-delimitation analysis for the diploid representatives of the genus  The delimitation of species as the most paramount rank in biological classification is an essential contribution of taxonomy to biodiversity research, with all of its evolutionary, ecological, political, and legislative ramifications and corollaries. Following Stuessy  and ZachGeneland [iBPP [Delineate [The ‘unified species concept’ proposed by De Queiroz  was a gaGeneland  for the Geneland ,10 for mnd [iBPP  for genend [iBPP  for genend [iBPP ,14,15,16elineate ).Species formation by polyploidy—a speciation mode realised in significant numbers of pteridophyte and angiosperm groups—poses considerable problems for species delimitation : while pLeucanthemum Mill.  comprises 39 [x) to dodecaploid (12x), and one species [L. lacustre (Brot.) Samp.) from Portugal even showing a chromosome number of 2n = 22x = 198 (docosaploid level). Leucanthemum is distributed over the whole European continent and extends into Northern Asia (the tetraploid L. ircutianum DC. is found in Siberia), while some species were also introduced into temperate regions of the Northern and Southern Hemispheres [The genus rises 39  to 42 sprises 39 , with plispheres . The curispheres  and, morLeucanthemum representatives based on multicopy nuclear markers (nrDNA ETSs), AFLP fingerprinting, and single-copy nuclear markers [L. cacuminis, L. eliasii, and L. pyrenaicum).Previous studies addressed the species delimitation and phylogeny of the diploid  markers ,25,26,27 markers . This fiL. ageratifolium group, despite the frequent presence of hybrid individuals in this group. Finally, Wagner et al. [Leucanthemum species was dated to the last 1.9 (1.1–2.9) Ma, arguing for the strong influence of Pleistocene oscillations on species formation in this genus. Most recently, Ott et al. [Leucanthemum representatives.Species delimitation in a morphologically close-knit group of taxa in the clade with the apomorphic nrDNA ETS ribotypes was subsequently carried out by Wagner et al. , who user et al.  presentet et al.  combinedLeucanthemum species have been the subject of previous studies. Oberprieler et al. [L. ircutianum subsp. ircutianum based on AFLP markers. One year later, Greiner et al. [L. pseudosylvaticum is able to form fertile offspring when crossed with its diploid relative L. pluriflorum, suggesting a relatively recent diversification. Another study of the L. pluriflorum group by Greiner et al. [L. pseudosylvaticum and L. corunnense, respectively, and raised L. pseudosylvaticum, which was formerly a subspecies of L. ircutianum, to species rank. Finally, the most recent contribution to the taxonomy of the tetraploid Leucanthemum representatives by Oberprieler et al. [L. ircutianum subsp. ircutianum and its amphi-Adriatic counterpart L. ircutianum subsp. leucolepis based on AFLP fingerprinting and sequence variations in nuclear and plastid DNA.In contrast to the extensive investigation of species delimitation and the phylogenetic relationships of diploids, the evolutionary histories of only a few tetraploid r et al.  found inr et al.  showed tr et al.  producedr et al.  suggesteLeucanthemum taxa. Applying the conceptual framework for species-rank decisions proposed by Oberprieler [L. corunnenese Lago, for which an autotetraploid origin based on the diploid L. pluriflorum has been shown in a previous study [The present contribution aims at a comprehensive and integrative assessment of all tetraploid rprieler , morpholus study .L. ircutianum subsp. ircutianum, the following seven geographically more restricted tetraploid taxa were considered as entities, for which an integrative taxonomic treatment was envisioned in the present contribution: these comprise the three species endemic to the Iberian Peninsula, L. cantabricum Sennen, L. crassifolium (Lange) Lange, and L. pseudosylvaticum (Vogt) Vogt & Oberpr., along with the NE Spanish and SW French L. delarbrei Timb.-Lagr. subp. delarbrei, subsp. ruscinonense (Jeanb. & Timb.-Lagr.) Vogt et al. and L. meridionale Legrand and the amphi-Adriatic L. ircutianum subsp. leucolepis (Briq. & Cavill.) Vogt & Greuter ) according to Ott et al. [Differences in leaf shape, an important delimitation criterion for he genus  and werehe genus ) and calt et al. . Subsequhttps://github.com/hbldh/pyefd (accessed on 31 January 2023)). For the EFA, we used 20 harmonics, normalised the descriptors, and applied principal component analysis (PCA) for decorrelation and feature extraction to the non-constant descriptors .Using the binary masks, we conducted EFA and calculated LDIs using the Python packages scikit-image , numpy , scikit-To find differences in general leaf shape, we used the first 15 principal components (PCs), explaining 50% of the total variance with two different testing strategies: (1) the permutation-based test for differences in Euclidean distance in PC space as applied by ; and (2)Leucanthemum taxa were reconstructed using ecological niche modelling (ENM) and compared using permutation-based statistical tests. For this purpose, we retrieved the collection locations of 1470 individuals from several herbaria ). In addition to the climate rasters for presence, we retrieved paleoclimate rasters for the last glacial maximum  and the last interglacial  from Worldclim Bioclim and subjected them to the same preprocessing, but with additional recoding of temperature, since LGM and LIG datasets used Bioclim 1 temperature encoding. For the edaphic variables, we averaged the raster values of the three mentioned depth levels to obtain a single raster for each soil variable. Since it was computationally not tractable to work with all 29 rasters, we applied principal component analysis (PCA) with standardisation implemented in the R package ‘ENMtools’ v1.0.6 [Ecoclimatological and edaphic niches of the tetraploid herbaria  and obtaherbaria  from Wor’ v1.0.6  for featL. meridionale using MaxEnt v3.4.4 [MaxEnt as species distribution, 200 replicates, a species range of 50 km, and 1000 background points with a range of 20 km. Leucanthemum meridionale was excluded from these comparisons because this species is endemic to a very limited area, causing all collection points to fall into the same raster cell and thus rendering ENM impossible. Potential niches at LGM and LIG were also reconstructed using MaxEnt with the same parameters as for the PC rasters, but this time with LGM and LIG as projection rasters and including collection data for the diploid species provided by Ott et al. [To compare the ecological niches of the different taxa, we reconstructed potential distribution ranges for all tetraploid taxa except t v3.4.4 , with 50t et al. .L. meridionale again due to its restricted sampling area as a point endemic, was assessed by evaluating the overlap of spatial distributions using the same data as for the ENM analyses. Unfortunately, there are no comprehensive distribution rasters available for the tetraploid Leucanthemum representatives, which is why we had to resort to approximating the geographic distribution from the available sampling points. We applied the method proposed by Ott et al. [MaxEnt v3.4.4; [p-values were corrected for multiple testing using Bonferroni´s method.The geographic co-distribution of the tetraploid taxa, excluding t et al. , which a v3.4.4; ) and sub v3.4.4;  with 400Leucanthemum species . ddRAD Illumina sequencing  using the restriction enzymes Pst1 and ApeK1, including demultiplexing and adapter clipping, was conducted by LGC Genomics .Double-digest RADseq  was con20 taxa; ) and 35 iPyrad v0.9.81 [Demultiplexed and adapter clipped reads from the diploid and tetraploid samples were assembled using  v0.9.81  against  v0.9.81 ; the min v0.9.81 ,46).https://github.com/simjoly/pofad (accessed on 31 January 2023)) were calculated using SplitsTree4 v4.15.1 [iPyrad using a custom Python and C tool (https://github.com/TankredO/nei_vcf (accessed on 31 January 2023)). With SNP-based Nei distances, differences in variant (SNP) frequencies are directly included in distance calculation, rendering this kind of distance metric particularly useful for comparing diploid and polyploid samples.Neighbor-nets based on SNP-based Nei distances  (k)k-means (WKM) clustering [Leucanthemum representatives. WKM clustering is a semi-supervised consensus (ensemble) clustering technique, allowing for a priori-defined fuzzy pairwise must-link and must-not-link constraints. Roughly, WKM clustering fits a number of k-means clusterings , each for a random subset of features (‘variables’) and data points (‘samples’), and with a random number of clusters (k). For each k-means run, clustering-level and cluster-level consistencies are calculated, which measure deviations from the fuzzy constraints concerning the whole clustering and single clusters, respectively. In addition, the mean silhouette index is calculated as an internal clustering-quality metric. The three values are non-linearly combined to obtain a clustering weight . The clustering co-association matrix, i.e., the matrix determining which samples were clustered together , is multiplied by the clustering weight. All weighted co-association matrices are summed  to obtain a consensus (co-association) matrix. Finally, a consensus clustering is calculated using this consensus matrix via hierarchical or spectral clustering.Weighted ensemble of random ustering  was applLeucanthemum representatives, the previously calculated SNP-based Nei distances  were subjected to a principal coordinate analysis (PCoA) with Lingoes negative eigenvalue correction, and the first principal coordinates (PCos) explaining at least 50% of the variance were selected. Based on the PCo scores, WKM clustering was applied with must-link constraints for the individuals of each of the taxa L. cantabricum, L. crassifolium, L. delarbrei subsp. delarbrei, L. delarbrei subsp. ruscinonense, L. ircutianum subsp. leucolepis, and L. meridionale and must-not-link constraints among individuals from L. delarbrei subsp. delarbrei and L. meridionale. The fraction of random features and samples was set to 0.60 and 0.80, respectively. The number of k-means runs was set to 5000, and k was allowed to take values from 2 to 14, including the lower and upper bounds. Finally, the consensus matrix was subjected to an average-linkage hierarchical clustering for each of the k-values, and the clustering scoring that was best according to the Bayesian information criterion (BIC) and the Calinski–Harabasz criterion (CH) was selected. All analyses were performed using pyckmeans v0.9.4 (https://github.com/TankredO/pyckmeans (accessed on 31 January 2023)).For clustering of the tetraploid SNiPloid [SNiPloid algorithm, our script compares two parent individuals  with one child individual  on a per-SNP basis, distinguishing and counting the frequencies of five different SNP categories: categories 1 and 2 count so-called inter-specific SNPs, where exactly one parental SNP is identical to the child SNP and the other not. Categories 3 and 4 are so-called derived SNPs, meaning that an SNP is unique to the child individual, while the parents are homozygous for the same variant; patterns 3 and 4 cannot be distinguished based on unphased SNP data. Category 5 SNPs are homeo-SNPs, where the child is heterozygous (polymorphic), combining both parents’ homologous and monomorphic alleles. For all categories, only SNPs where both parents were homozygous (monomorphic) were considered.To find the potential diploid parental species of the tetraploid taxa under study, we applied a custom version of SNiPloid , as propSNiPloid . Very siSNiPloid approach to look for signs of allopolyploid speciation and expected recently formed allotetraploids to express a high number of category 5 SNPs, while longer established allotetraploid species should express a higher frequency of derived SNPs . Finally, category 1 and 2 SNPs may indicate gene flow to the putative parents or alternatively be an additional signature of recent polyploidisation. For each tetraploid taxon and each possible pair of diploid parent species, therefore, we applied our custom Python implementation of the SNiPloid algorithm to the concatenated SNP output returned by iPyrad (.snps ouput). For each triplet, we tested all individuals and calculated the arithmetic means of SNP patterns 1 through 5.We applied the p-value < 0.01 for all but three taxon pairs  and L. cantabricum (N Spain)–L. delarbrei subsp. ruscinonense  were significant at an alpha level of 0.01 . Maps depicting predicted potential distribution ranges of the diploids and tetraploids based on recent, LGM, and LIG bioclimatic variables are provided in the supporting information  and L. delarbrei subsp. ruscinonense –L. ircutianum subsp. ircutianum (large parts of Europe) were significant, suggesting a deviation from sympatric distributions for most of the tetraploid taxa.In the tests on geographical range overlap , all taxOf 216,551,342 raw reads, 81,517,011 demultiplexed, adapter-clipped, and restriction-enzyme-filtered reads were mapped against the reference of Ott et al. . The resL. vulgare group , and a single individual (437-01) was ungrouped with any cluster. For L. delarbrei subsp. ruscinonense, two clusters with two (accessions 139-01 and 355-03) and three  individuals were observed in quite distant positions in the network. When subjecting the tetraploid accessions alone to Neighbor-net clustering , Corsica (437-01), Germany , Italy (87-01), and Montenegro (177-01), while the second one, closer to L. cantabricum, contains the two accessions from SW France (106-01 and 343-01). Additionally, all accessions of L. delarbrei subsp. ruscinonense now cluster together, and the network reconstruction indicates that there are closer genetic similarities between (a) L. delarbrei subsp. delarbrei and L. meridionale (both S France) and (b) L. crassifolium (N Spain), L. pseudosylvaticum (NW Spain), and L. delarbrei subsp. ruscinonense , while accessions of L. ircutianum subsp. leucolepis  form a cluster relatively isolated from these.The network reconstruction based on SNP-based Nei distances for the complete dataset  placed all tetraploid taxa into the so-called re group A. While ustering B, samplek = 2, but also had a local optimum at six clusters. The consensus clustering with the co-association matrix at k = 6  and L. ircutianum subsp. ircutianum and L. cantabricum (yellow cluster), while the remaining assignments of accessions to clusters followed their taxonomic classifications.The optimal number of clusters according to the Bayesian information criterion  was found to be six B, while at k = 6 A merged SNiPloid approach to infer parental diploids are provided in the supporting information  included as a parental species.Plots of the category proportions for all taxon triplets surveyed in the ormation . We founLeucanthemum taxa based on the same sources of evidence for species delimitation: genetic diversification, morphological discontinuities, ecological differentiation, and information on the geographical distribution of taxa. In addition to the methodological approach taken in the diploid case, we additionally tried to incorporate genealogical aspects into this integrative taxonomy of Leucanthemum tetraploids: by trying to infer the parentage of tetraploid taxa from RADseq-based single-nucleotide polymorphism (SNP) data, we hoped for additional arguments for a classification scheme that incorporates the evolutionary history of the study group. This follows the rationale that, by elucidating the combination of diploid genomes in the polyploids, the disentangling of auto- and alloploid formations of these taxa and the knowledge of their independent vs. non-independent evolutionary trajectories could be used in taxonomic decisions.Having provided an integrative classification scheme for the diploid representatives of the genus , the preLeucanthemum taxa under study. This disappointing result is somewhat unexpected in consideration of the huge amount of genomic information received from the GBS fingerprinting, with over 150,000 SNPs from over 7000 loci. However, our present analyses simultaneously point towards the probable explanation for the unresolved evolutionary patterns in this plant group: all eight surveyed tetraploid taxa appear to be closely related to the close-knit species group of diploids around L. vulgare (Leucanthemum was dated to the last 1.9 (1.1–2.9) Ma [L. vulgare group is presumably not older than 400 ka, polyploid species formation from these diploid ancestors has to be assumed to have occurred not earlier than the Middle  or Late Pleistocene (0.13–0.01 Ma) with its glaciation cycles of the Mindel, Elster, Riss, and Würm eras.Unfortunately, the present analyses provide us only with a quite limited notion about the evolutionary origins of the tetraploid  vulgare A, for wh–2.9) Ma  and the SNiPloid-based strategy proposed by Wagner et al. [Salix polyploids revealed no patterns in Leucanthemum. We think that the observed failure of the mentioned strategy, in contrast to its successful application in Salix tetraploids, is not due to a lack of sufficient loci and sampled SNPs  but due to the fact that the putative parental diploid species in Salix are much older than in Leucanthemum. He et al. [Salix subg. Chamaetia/Vetrix clade, in which the two diploids S. purpurea L. and S. repens L.—the two putative parents of the tetraploid S. caesia Vill. analysed by [SNiPloid—are dated as having diverged from each other in the Early Miocene, around 20 Ma ago. It appears obvious that in a constellation like this, with the long evolutionary independence of diploid precursor lineages and a relatively recent formation (Pleistocene) of a tetraploid taxon, the SNiPloid-based strategy proposed by Wagner et al. [Leucanthemum case, with its comparatively young diploid lineages.It is comprehensible, therefore, that our present efforts to trace the parenthood of diploid species for the formation of tetraploid taxa by application of the r et al.  for Salie et al.  provide lysed by  using SNr et al.  will revSNiPloid approach is strongly influenced by the filtering of RADseq reads gained from polyploids. This is due to the fact that, when parameters (cluster thresholds) are optimised in iPyrad to avoid under- and oversplitting of loci and to minimise paralogy, paralogy caused by whole-genome duplication  will be also reduced and this could diminish potential additive allelic signals of allopolyploidy. However, we circumvented this problem by applying a reference-based assembly of reads from tetraploids  insteadSNiPloid-based strategy, some relationships among taxa at the two ploidy levels were revealed by the SNP-based network reconstruction  at the vertex of a parallelogram at their base in the NeighborNet network of L. pseudosylvaticum sharing one edge (genome) with L. pluriflorum/L. eliasii, one may feel justified in hypothesising the sharing of the other edge (genome) with one of the diploid species making up the left side of the network. Owing to the fact that all other members of this subgroup are presently not found in the Iberian Peninsula, L. gracilicaule—a diploid endemic to the region around Valencia in SE Spain—could then be the most probable candidate for the other parental taxon of L. pseudosylvaticum, if an extinct diploid may not have acted as such. The latter scenario receives plausibility from studies on other polyploid complexes that have encountered so-called ‘ghost (sub)genomes’ of extinct diploids in polyploid taxa .L. pseudosylvaticum, hypotheses about the formation of the other seven tetraploid taxa included in the present study remain even more obscure due to the nearly star-like structure in the right part of the NeighborNet network of L. gaudinii and L. vulgare (with its two subspecies L. vulgare subsp. vulgare and subsp. pyrenaicum) amongst accessions of the tetraploid taxa L. ircutianum (with its two subspecies L. ircutianum subsp. ircutianum and subsp. leucolepis) and L. cantabricum may point towards the participation of either of these two diploids (or a joint ancestor of both) in the formation of the latter. In the case of L. ircutianum subsp. ircutianum, it has been shown by Oberprieler et al. [L. vulgare subsp. vulgare may have acted as the paternal partner in this allopolyploidisation and L. virgatum as the maternal partner [L. virgatum quite distantly from all members of the L. vulgare cluster. Strangely enough, however, the cpDNA-based analysis of Greiner et al. [L. virgatum (or haplotypes closely related to these) could not only be found in accessions of L. ircutianum subsp. ircutianum but also in some or even all representatives of L. ircutianum subsp. leucolepis, L. cantabricum, and L. crassifolium included in the mentioned study. Finally, chloroplast haplotypes characterised for L. delarbrei subsp. delarbrei and subsp. ruscinonense (sub L. monspeliense) were found to be closely related to L. halleri and L. vulgare, respectively [Leucanthemum.In contrast to the evolutionary history of r et al.  that L.  partner . At leasr et al.  demonstrectively . In summ(k)k-means (WKM) clustering [L. cantabricum and L. ircutianum subsp. ircutianum and for L. meridionale and L. delarbrei subsp. delarbrei, respectively. In both cases, this was found to correspond to the close positions of accessions from these taxon pairs in the NeighborNet network for the tetraploids (Leucanthemum and [Rhodanthemum). The lack of an MSC-based model adapted for polyploids hampers coalescent-based species delimitation here. However, since we have demonstrated in Leucanthemum diploids that pattern-based methods, such as consensus k-means  clusterns , while the latter is endemic to Puy de Wolf, a serpentine mountain c. 65 km SW of the Monts du Cantal. The situation is different for the other taxon pair, for which genetic differentiation was found to be not significant according to BIC and CH scores. In this case, L. cantabricum, with four accessions from two populations in NW Spain , is also distinct but nested in a highly diverse cluster representing L. ircutianum subsp. ircutianum, with eight accessions from eight populations sampled throughout the European range of the taxon.It is obvious from both the NeighborNet network  and the L. cantabricum in the present analysis are the westernmost ones in the distributional range of the species that is mainly distributed in the W Pyrenean mountains and the Cordillera Cantábrica in N Spain [locus classicus of the taxon being around 250 km to the east of the sampled populations. (b) In his revision of the genus for the Iberian Peninsula, Vogt [L. cantabricum (sub L. ircutianum subsp. cantabricum) as being the main difference between this taxon and L. ircutianum subsp. ircutianum. The author, however, reports also that the morphological variation observed in the former taxon is considerable and that morphologically intermediate individuals with respect to the latter are observed frequently. Sampling of accessions from the centre of the distributional range of L. cantabricum  may reveal more pronounced genetic differences to L. ircutianum subsp. ircutianum, and the two populations sampled for the present study may turn out to be intermediate forms rather than pure representatives of L. cantabricum. Nevertheless, the undeniable closeness of the two taxa and their connectedness via intermediates will remain an unequivocal genealogical pattern.Two circumstances, however, are considered noteworthy here in evaluating the genetic/genealogical relationship of the two taxa: (a) The two populations sampled for  N Spain , the locla, Vogt  considerLeucanthemum. As demonstrated by many taxonomic treatments of the genus in floras of European countries , other When compared across all possible pair-wise test combinations, leaf dissection (as measured by LDI) discriminates among the eight morphotaxa more profoundly than leaf outline : while only three (11%) of the 28 possible pair-wise comparisons of LDIs revealed no significant differences between taxa, 14 (50%) and 12 (43%) of the tests addressing leaf-shape differences showed non-significant results. We think that these contrasting results do not only have a biological reason, but also a methodological one: in its present and here-applied formulation (comparison of the length of leaf outline with leaf area), LDI does not only measure the intensity of leaf incision, but also the deviation of the leaf shape from a perfect circle. That means that it also captures leaf shape. In contrast, elliptic Fourier analysis (EFA) not only exclusively captures the outline of leaves, but also (with higher harmonics) the subdivision of the leaf lamina. However, since parameters gained with the latter harmonics of the EFA are down-weighted against parameters from earlier ones in a principal component analysis (PCA), it may be justified to consider both measures applied here  as overlapping with respect to what they capture in terms of leaf morphology, but with a tendency towards assessment of leaf dissection with the former and leaf outline with the latter.L. meridionale was included in the pair-wise comparisons: while six of the seven comparisons concerning LDIs revealed significance, the majority of EFA-based comparisons resulted in non-significant results. The former is quite reasonable when the habit of the species is studied both in its natural habitat and in a herbarium: compared with all other tetraploid taxa of Leucanthemum, L. meridionale, with its reduced scape size and its filigree leaves, resembles more the diploid representatives of the genus than the tetraploid ones. This led Tison and de Foucault [L. vulgare and L. graminifolium. The somewhat reduced habit of L. meridionale may be a consequence of the exceptional habitat preference of the species for serpentine soils; the lack of a significant difference in LDIs with respect to L. delarbrei subsp. delarbrei, which is found on volcanic soils further north and is genetically closely related with it, however, may also point towards a close phylogenetic relationship between these two tetraploid Leucanthemum taxa.Discrepancies between LDIs and EFA descriptors of leaf morphology were found to be especially pronounced when Foucault  among otL. ircutianum subsp. ircutianum and subsp. leucolepis, for which all three tests remained non-significant (L. ircutianum subsp. asperulum as a synonym) are allopatrically distributed in the Apennine Peninsula but sympatrically distributed in the Balkan Peninsula and form morphologically intermediate forms through hybridisation in overlapping regions of their distribution ranges. The main difference between the two taxa is in the colouration of the margins of involucral bracts, which are black to dark-brown in subsp. ircutianum and hyaline in subsp. leucolepis. In genetic respects, the two taxa represent two significantly distinct ; it rather led to the detection of patterns of bimodality in the spatial and ecoclimatological distributions of the species pairs compared. Therefore, the overwhelming number of significant test results (even after Bonferroni correction for multiple testing) for both geographical (2 of 21 tests) or ecological  differences between species pairs may just represent tendencies in spatial or ecoclimatological differentiation and not complete discontinuities. Nevertheless, when looking at the distribution ranges of the eight tetraploid morphotaxa under study  or by small-scale habitat differences—especially in edaphic factors—that have not been captured by the geography-based ecoclimatological niche modelling underlying the test-setup, L. meridionale being adapted to serpentine soils and L. crassifolium to saliferous coastal habitats.In geographical respects, the distribution ranges of the eight morphotaxa clearly follow an allopatric pattern, with exceptions being the observed non-significant differentiation between  regions ,23 and tSpecies delimitation in plants—especially in hybridising plant groups (syngameons) and polyploid complexes—is a problematic matter . The appLeucanthemum—the ”warps and wefts” of this polyploid complex [Here, we follow a procedural protocol for species-rank decisions designed by Oberprieler  that is  complex .Leucanthemum polyploidy complex, the present study is extremely helpful in terms of providing a pattern-based species delimitation at the tetraploid level and its integrative taxonomical conceptualisation. Aiming at a reproducible, objective classification of eight morphotaxon hypotheses in terms of delimitation and ranking, we have analysed genetic, ecological, and morphological variation together with information from taxon distributions, summarised in L. meridionale, which is a point endemic of a serpentine mountain in S France , this taxon has been omitted from pair-wise comparisons that are dependent on modelling procedures based on spatial distribution data comprising more than a single raster cell . Due to its described endemicity, L. meridionale  is allopatrically distributed with all other tetraploid Leucanthemum taxa except L. ircutianum subsp. ircutianum, which is found in parapatry on non-serpentine soils surrounding Puy de Wolf (and sampled with accession 343-01). Its ecological niche is uniquely determined by edaphic factors connected to serpentine soils  rather than climatic ones.Despite the somewhat pessimistic outlook on the phylogenetics of the Leucanthemum tetraploids in seeking the most reasonable ranking for closely related species hypotheses (morphotaxa) leads to the following reasoning:Application of the ‘Wettstein tesseract’ tool to the present study group of Lecanthemum crassifolium (N Spain), L. delarbrei subsp. ruscinonense (E Pyrenees and northeastern foothills), and L. pseudosylvaticum (W Iberian Peninsula) are best considered as being genetically closely related but independent lineages , L. eliasii , L. graminifolium (S France), L. pluriflorum subsp. cantabricum .(a) lineages  that merlineages  and leaflineages  that haviflorum; ,30), theiflorum; , L. pluriense in ) shares iulae in ), L. buriasii in ), L. gauricum in ), and boLeucanthemum delarbrei subsp. delarbrei  and L. meridionale  are allopatrically distributed sister groups  r groups  that lacr groups  and showr groups  but exhir et al.  was founL. cantabricum and the two subpecies of L. ircutianum also show close genetic relationships in the NeighborNet network of L. ircutianum subsp. ircutianum shows a closer genetic similarity with L. cantabricum than with L. ircutianum subsp. leucolepis. When following the conceptual framework of species-rank decision making with the ‘Wettstein tesseract’, the significant differentiations between L. ircutianum subsp. ircutianum and subsp. leucolepis in genetic, geographical, and ecoclimatological aspects would argue for an acknowledgement of the two taxa at species level . The main argument for treating the two entities as independent species, following the logic of von Wettstein [(c) Finally, ettstein , is thatettstein  have shoettstein , we propL. ircutianum, on the other hand, is less equivocal for L. cantabricum (Cordillera Cantábrica in N Spain) following the here-presented results: the two taxa are allopatrically distributed and show ecoclimatological and morphological differences (more strongly dissected leaves in L. cantabrica) but lack genetic differentiation  Lange in Willk. & Lange, Prodr. Fl. Hispan. 2: 96. 1865 ≡Leucanthemum pallens var. crassifolium Lange in Vidensk. Meddel. Dansk Naturhist. Foren. Kjøbenhavn, ser. 2, 3: 77. 1861 ≡Leucanthemum ircutianum subsp. crassifolium (Lange) Vogt in Ruizia 10: 127. 1991—Lectotype : In rupibus maritimis ad Portugalete, Cantabria, Oct. 1851, Herb. Joh. Lange (C! (C10007128)).(1) Notes.—Endemic to the Cantabrian coast between Asturias and the Basque region (NE Spain and SW France). Habitats are coastal rocks and salt-influenced coastal slopes from sea level to 20 m. Leucanthemum crassifolium is characterised by succulent leaves  in Mém. Acad. Sci. Clermont-Ferrand 20: 508. 1878.—Lectotype: Not yet designated. =Chrysanthemum leucanthemum var. pinnatifidum Lecoq. & Lamotte, Cat. Pl. Plateau Central: 227. 1847 ≡Leucanthemum vulgare var. pinnatifidum (Lecoq. & Lamotte) Briq. & Cavill. in Burnat, Fl. Alpes Marit. 6: 91. 1916 ≡Leucanthemum ircutianum var. pinnatifidum (Lecoq. & Lamotte) D. Löve & J.-P. Bernard in Svensk. Bot. Tidskr. 53: 444. 1959.—Ind. loc.: “AR.- Mont Dore; pâturages et pentes herbeuses de Chaudefour, bords du chemin de Sancy à Vassivière. Bozat! AR.”—Lectotype (designated here by Vogt & Oberprieler): Mont Dore, Sancy, 15 (..) 1844, leg. H. Lecoq & M. Lamotte (P! (P00729975)).(2a) Notes.—Endemic to the siliceous rocks of the old volcano cones of the central Massif Central in France . Its habitats are siliceous rocks and meadows between 1550 m and 1750 m. Leucanthemum delarbrei subsp. delarbrei is characterised by strongly dissected leaves, from pinnatifid to bipinnatisect  Oberpr., T. Ott & Vogt, comb. nov. ≡Leucanthemum meridionale Legrand in Bull. Soc. Bot. France 28: 56. 1881 (basionym) ≡Leucanthemum vulgare var. (‘χ’) meridionale (Legrand) Rouy, Fl. France 8: 274. 1903 ≡Chrysanthemum leucanthemum f. (‘g’) meridionale (Legrand) Fiori in Fiori & Béguinot, Fl. Italia 3: 239. 1903 ≡Leucanthemum vulgare subsp. meridionale (Legrand) Nyman, Consp. Fl. Eur., Suppl. 2: 169. 1889.—Ind loc.: “Habite dans les interstices des rochers serpentineuses du puy de Wolf, près de Firmy (Aveyron); fleurit de fin mai à juillet. Je reçus cette plante en 1879 ... leg. F(rère) Saltel .”—Lectotype (designated here by Vogt & Oberprieler): Puy de Wolf, pr. Firmy; mai, juin 1879 (Aveyron).—Gallia merid., Saltel.—Comm. Le Grand (P! (P00729957)).(2b) Notes.—Endemic to the Puy de Wolf , a serpentine mountain in the western Massif Central. It is found in dry and open, south-facing grassland on serpentine soil between 400 m and 600 m. Leucanthemum meridionale is characterised by its lanky habitus resembling the diploid L. vulgare or a member of the genus Leucanthemopsis with quite narrow leaves and cuneate leaf bases (af bases .Leucanthemum ircutianum DC. (ircutianumsubsp. ), Prodr. 6: 47. 1838.—Lectotype : In pratis, 1828, Turczaninoff a Irkoutsk, Turcz.: 1830 (G-DC! (G00451151)).(3a) Notes.—Besides the diploid L. vulgare Lam., this taxon is the most widely distributed species of the genus [Leucanthemum ircutianum subsp. ircutianum is morphologically very similar to L. ircutianum subsp. leucolepis (see he genus . It is phe genus  but has epis see  but can Leucanthemum ircutianum subsp. cantabricum (Sennen) Vogt in Ruizia 10: 121. 1991 ≡Leucanthemum cantabricum Sennen, Diagn. Nouv.: 50. 1936 ≡Leucanthemum vulgare subsp. cantabricum Sennen, Diagn. Nouv. 50. 1936 .—Lectotype : Santander: La Calda de Besaya, rochers silliceux humides, 5.6.1927, E. Leroy (BC-Sennen!).(3b) Notes.—Distributed between the western foothills of the Pyrenees (Spain and France), along the northern slopes of the Cantabrian Mountains to Asturias and Galicia in the west. Habitats include road and meadow margins, meadows, and pastures; the elevational distribution ranges from sea level to 800 m. Leucanthemum cantabricum is characterised by dissected (pinnatisect to pinnatipartite), non-succulent leaves (L. ircutianum by Vogt [t leaves  and dark by Vogt  before b by Vogt .Leucanthemum ircutianum subsp. leucolepis (Briq. & Cavill.) Vogt & Greuter in Willdenowia 33: 41. 2003 ≡Leucanthemum leucolepis (Briq. & Cavill.) Gajić in Josifović, Fl. SR Srbije 9: 185. 1977 ≡Leucanthemum vulgare subsp. leucolepis Briq. & Cavill. in Burnat, Fl. Alpes Marit. 6: 93. 1916 ≡Chrysanthemum leucanthemum subsp. leucolepis (Briq. & Cavill.) Schinz & Thell., Fl. Schweiz, ed. 4, 1: 685. 1923 ≡Leucanthemum leucolepis (Briq. & Cavill.) Horvatić in Acta Bot. Croat. 22: 214. 1963, nom. inval.  ≡Leucanthemum pallens subsp. leucolepis (Briq. & Cavill.) Faverger in Anales Inst. Bot. Cavanilles 32: 1236. 1975.—Lectotype : Flora Italica Exsiccata, curantibus Adr. Fiori, A. Béguinot, R. Pampanini, number 175—Etruria, Prov. di Firenze, Vallombrosa, in pratis, alt. 900–1000 m., solo pingui, siliceo, 19.6.1904, A. Fiori (G-BU! (G00848032)).(3c) Notes.—A taxon with an amphi-Adriatic distribution, with populations throughout Italy and along the Adriatic coast of the Balkan Peninsula. The taxon was described as a subspecies of the diploid L. vulgare but was subsequently considered a subspecies of the hexaploid L. pallens due to its white hyaline margins of involucral bracts or seen as an independent species. The observation of an allopatric distribution with L. ircutianum subsp. ircutianum as more Mediterranean facies of the species, together with the observation of hybrid individuals and hybrid swarms in areas of joint occurrence of the two tetraploid taxa in the Apennine and Balkan Peninsulas by Oberprieler et al. [L. ircutianum. In contrast to L. ircutianum subsp. ircutianum, it has paler hyaline margins of the involucral bracts.r et al.  argued fLeucanthemum pseudosylvaticum (Vogt) Vogt & Oberpr. in Ann. Bot. (Oxford), n.s., 111: 1121. 2013 ≡Leucanthemum ircutianum subsp. pseudosylvaticum Vogt in Ruizia 10: 134. 1991.—Holotype: Portugal, Distrito Porto, Serra do Marão, Mesão Frio-Amarante, feuchter Hang südlich der Paßhöhe, ca. 800 m, 20.07.1986, R. Vogt 4711 & E. Bayón (M! (M-0030173)).(4) Notes.—Distributed throughout the western part of the Iberian Peninsula . Its habitats comprise road margins and slopes, margins of creeks, and ditches, at an elevational range from 100 m to 1600 m. Leucanthemum pseudosylvaticum is characterised by leaves with proximally (sub)entire margins  Oberpr., T.Ott & Vogt, comb. et stat. nov. ≡Leucanthemum palmatum var. ruscinonense Jeanb. & Timb.-Lagr. in Mém. Acad. Sci. Toulouse, ser 8, 1(2): 192. 1879 (basionym) ≡Leucanthemum monspeliense var. ruscinonense (Jeanb. & Timb.-Lagr.) O. Bolòs & Vigo in Collect. Bot. (Barcelona) 17: 91. 1988 ≡Leucanthemum cebennense var. ruscinonense (Jeanb. & Timb.-Lagr.) Gaut., Catal. Rais. Fl. Pyr. Orient.: 232. 1898 ≡Leucanthemum delarbrei subsp. ruscinonense (Jeanb. & Timb.-Lagr.) Vogt, Florian Wagner & Oberpr., Fl. Iber. 16(3): 1866. 2019—Ind loc.: “... les Alberes ...”.—Holotype: ... la tour de la Massane dans la altura, Pyr. Orient., 21.5.1877, E. Timbal-Lagrave .(5) Notes.—Endemic to the eastern Pyrenees (Spain and France) and the SW parts of the Massif Central . Habitats comprise road margins, creeks, and stony slopes, between 250 m and 1900 m. It is characterised by strongly dissected leaves, pinnatifid to bipinnatisect , which