Effect of Calcium Ion Supplementation on Oral Microbial Composition and Biofilm Formation In Vitro.

The oral cavity contains a variety of ecological niches with very different environmental conditions that shape biofilm structure and composition. The space between the periodontal tissue and the tooth surface supports a unique anaerobic microenvironment that is bathed in the nutrient-rich gingival crevicular fluid (GCF). During the development of periodontitis, this environment changes and clinical findings reported a sustained level of calcium ion concentration in the GCF collected from the periodontal pockets of periodontitis patients. Here, we report the effect of calcium ion supplementation on human oral microbial biofilm formation and community composition employing an established SHI medium-based in vitro model system. Saliva-derived human microbial biofilms cultured in calcium-supplemented SHI medium (SHICa) exhibited a significant dose-dependent increase in biomass and metabolic activity. The effect of SHICa medium on the microbial community composition was evaluated by 16S rRNA gene sequencing using saliva-derived microbial biofilms from healthy donors and periodontitis subjects. In this study, intracellular microbial genomic DNA (iDNA) and extracellular DNA (eDNA) were analyzed separately at the genus level. Calcium supplementation of SHI medium had a differential impact on iDNA and eDNA in the biofilms derived from healthy individuals compared to those from periodontitis subjects. In particular, the genus-level composition of the eDNA portion was distinct between the different biofilms. This study demonstrated the effect of calcium in a unique microenvironment on oral microbial complex supporting the dynamic transformation and biofilm formation.

Oral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin K in mice.

Periodontitis is a highly prevalent disease leading to uncontrolled osteoclastic jawbone resorption and ultimately edentulism; however, the disease onset mechanism has not been fully elucidated. Here we propose a mechanism for initial pathology based on results obtained using a recently developed Osteoadsorptive Fluogenic Sentinel (OFS) probe that emits a fluorescent signal triggered by cathepsin K (Ctsk) activity. In a ligature-induced mouse model of periodontitis, a strong OFS signal is observed before the establishment of chronic inflammation and bone resorption. Single cell RNA sequencing shows gingival fibroblasts to be the primary cellular source of early Ctsk. The in vivo OFS signal is activated when Toll-Like Receptor 9 (TLR9) ligand or oral biofilm extracellular DNA (eDNA) is topically applied to the mouse palatal gingiva. This previously unrecognized interaction between oral microbial eDNA and Ctsk of gingival fibroblasts provides a pathological mechanism for disease initiation and a strategic basis for early diagnosis and treatment of periodontitis.

A Denture Use Model Associated with Candida spp. in Immunocompetent Male and Female Rats.

Denture stomatitis (DS) is a common infection in denture wearers, especially women. This study evaluated the induction of DS using acrylic devices attached to the palate of rats combined with inoculation of Candida spp. Immunocompetent male and female rats received a carbohydrate-rich diet. Impressions were taken from the rats' palate to individually fabricate acrylic devices. Mono- and multispecies biofilms of C. albicans, C. glabrata, and C. tropicalis were grown on the devices, which were then cemented on posterior teeth and kept in the rats' palate for four weeks. Microbial samples from the palate and the device were quantified. Oral microbiome of rats inoculated with C. albicans was analyzed by 16S rRNA gene sequencing. Log10(CFU/mL) were analyzed by mixed or two-way MANOVA (α = 0.05). Candida spp. and acrylic device did not induce palatal inflammation macroscopically nor microscopically. Although there was an increase (p < 0.001) of the total microbiota and female rats demonstrated higher (p = 0.007) recovery of Candida spp. from the palate, the gender differences were not biologically relevant. The microbiome results indicate an increase in inflammatory microbiota and reduction in health-associated micro-organisms. Although Candida spp. and acrylic device did not induce DS in immunocompetent rats, the shift in microbiota may precede manifestation of inflammation.

Oral Microbiome: Streptococcus mutans/Caries Concordant-Discordant Children.

Dental caries remains the most common chronic disease in children, and the respective etiology is not fully understood. Though Streptococcus mutans is an important factor in the initiation and progression of caries, its presence is not always associated with the disease. The existence of caries discordant populations, in which S. mutans counts do not correlate with caries experience, poses a challenging problem. This study explored the possible correlation of S. mutans and other microorganism levels on caries-associated ecology of caries-concordant and discordant populations. A total of forty-seven children were analyzed in this study and stratified into four clinical groups based on their S. mutans levels in saliva (HS/LS: High/low S. mutans) and caries experience. Streptococcus mutans levels were determined by culture-based selective plating. The salivary microbiome of caries concordant and discordant populations was investigated by 16S rRNA gene sequencing and downstream bioinformatics analysis. The salivary microbial communities significantly clustered based on S. mutans levels and independent of their caries experience. In addition to S. mutans levels, significant differences in the abundance of other species were observed between HS and LS groups. Interestingly, disease-associated species such as Veillonella dispar, Streptococcus spp., and Prevotella spp. were significantly increased in HS groups and may contribute, in combination with S. mutans, to the caries progression. Furthermore, health-associated species exhibited higher abundance in the LS groups, such as Veillonella rogosae, Haemophilus sp., and Alloprevotella spp. but their possible contribution to the caries process remains to be elucidated. This study provides evidence that S. mutans may play a role in shaping the salivary microbial community. Our results highlight that future caries research should consider additional species as health/disease microbial markers in conjunction with S. mutans to improve diagnosis and caries management of the caries-discordant population.

The impact of fixed orthodontic appliances and clear aligners on the oral microbiome and the association with clinical parameters: A longitudinal comparative study.

INTRODUCTION: Orthodontic treatment interferes with oral hygiene and promotes plaque retention, which leads to gingival inflammation and enamel demineralization. Although removable clear aligners (CAs) are designed to improve oral hygiene compared with fixed appliances (FAs), comprehensive studies comparing their respective effects on the oral microbiome are limited. This longitudinal study investigated the microbial changes during orthodontic treatment with FA and CA in correlation with clinical parameters. METHODS: Clinical parameters and supragingival plaque were collected from 12 study participants for the FA or CA treatment groups at baseline and at least twice at the 1, 3, 6, and 12-month follow-up appointments. The plaque was also harvested from the aligner tray for the CA group. Microbiome composition was determined via 16S rRNA gene sequencing, compared between groups, and correlated with clinical parameters. RESULTS: Plaque (PI) and gingival indexes (GI) increased significantly in the FA but not the CA group. Beta but not alpha diversities of the microbial communities were distinct between the 2 treatment groups, even though genus-level differences were not significant except for Leptotrichia. The CA tray harbors a unique plaque community. Elevated PI and GI in the FA group correlated with a higher abundance of disease-related genera. CONCLUSIONS: Orthodontic treatments trigger appliance-related plaque community shifts from baseline, and the CA tray environment attracts distinct microbial communities. In comparison with FA, the use of CA resulted in better oral health index outcomes, which is reflected by the corresponding PI and GI-associated oral microbial communities.

Omics and interspecies interaction.

Interspecies interactions are key determinants in biofilm behavior, ecology, and architecture. The cellular responses of microorganisms to each other at transcriptional, proteomic, and metabolomic levels ultimately determine the characteristics of biofilm and the corresponding implications for health and disease. Advances in omics technologies have revolutionized our understanding of microbial community composition and their activities as a whole. Large-scale analyses of the complex interaction between the many microbial species residing within a biofilm, however, are currently still hampered by technical and bioinformatics challenges. Thus, studies of interspecies interactions have largely focused on the transcriptional and proteomic changes that occur during the contact of a few prominent species, such as Porphyromonas gingivalis, Streptococcus mutans, Candida albicans, and a few others, with selected partner species. Expansion of available tools is necessary to grow the revealing, albeit limited, insight these studies have provided into a profound understanding of the nature of individual microbial responses to the presence of others. This will allow us to answer important questions including: Which intermicrobial interactions orchestrate the myriad of cooperative, synergistic, antagonistic, manipulative, and other types of relationships and activities in the complex biofilm environment, and what are the implications for oral health and disease?

Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation.

: RadD, a major adhesin of oral fusobacteria, is part of a four-gene operon encoding the small lipoprotein FAD-I and two currently uncharacterized small proteins encoded by the rapA and rapB genes. Previously, we described a role for FAD-I in the induction of human B-defensin 2 (hBD2) upon contact with oral epithelial cells. Here, we investigated potential roles for fad-I, rapA, and rapB in interspecies interaction and biofilm formation. Gene inactivation mutants were generated for each of these genes in the nucleatum and polymorphum subspecies of Fusobacterium nucleatum and characterized for their adherence to partner species, biofilm formation, and operon transcription. Binding to Streptococcus gordonii was increased in all mutant strains with Δfad-I having the most significant effect. This increased adherence was directly proportional to elevated radD transcript levels and resulted in significantly different architecture and height of the biofilms formed by Δfad-I and S. gordonii compared to the wild-type parent. In conclusion, FAD-I is important for fusobacterial interspecies interaction as its lack leads to increased production of the RadD adhesin suggesting a role of FAD-I in its regulation. This regulatory effect does not require the presence of functional RadD.

Correction: Shokeen, B., et al. Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation. Microorganisms 2020, 8, 70.

The authors wish to make the following corrections to this paper [...].

Streptococcus mutans SpaP binds to RadD of Fusobacterium nucleatum ssp. polymorphum.

Adhesin-mediated bacterial interspecies interactions are important elements in oral biofilm formation. They often occur on a species-specific level, which could determine health or disease association of a biofilm community. Among the key players involved in these processes are the ubiquitous fusobacteria that have been recognized for their ability to interact with numerous different binding partners. Fusobacterial interactions with Streptococcus mutans, an important oral cariogenic pathogen, have previously been described but most studies focused on binding to non-mutans streptococci and specific cognate adhesin pairs remain to be identified. Here, we demonstrated differential binding of oral fusobacteria to S. mutans. Screening of existing mutant derivatives indicated SpaP as the major S. mutans adhesin specific for binding to Fusobacterium nucleatum ssp. polymorphum but none of the other oral fusobacteria tested. We inactivated RadD, a known adhesin of F. nucleatum ssp. nucleatum for interaction with a number of gram-positive species, in F. nucleatum ssp. polymorphum and used a Lactococcus lactis heterologous SpaP expression system to demonstrate SpaP interaction with RadD of F. nucleatum ssp. polymorphum. This is a novel function for SpaP, which has mainly been characterized as an adhesin for binding to host proteins including salivary glycoproteins. In conclusion, we describe an additional role for SpaP as adhesin in interspecies adherence with RadD-SpaP as the interacting adhesin pair for binding between S. mutans and F. nucleatum ssp. polymorphum. Furthermore, S. mutans attachment to oral fusobacteria appears to involve species- and subspecies-dependent adhesin interactions.

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6.

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.

Differential regulation of type III secretion and virulence genes in Bordetella pertussis and Bordetella bronchiseptica by a secreted anti-σ factor.

The BvgAS phosphorelay regulates ∼10% of the annotated genomes of Bordetella pertussis and Bordetella bronchiseptica and controls their infectious cycles. The hierarchical organization of the regulatory network allows the integration of contextual signals to control all or specific subsets of BvgAS-regulated genes. Here, we characterize a regulatory node involving a type III secretion system (T3SS)-exported protein, BtrA, and demonstrate its role in determining fundamental differences in T3SS phenotypes among Bordetella species. We show that BtrA binds and antagonizes BtrS, a BvgAS-regulated extracytoplasmic function (ECF) sigma factor, to couple the secretory activity of the T3SS apparatus to gene expression. In B. bronchiseptica, a remarkable spectrum of expression states can be resolved by manipulating btrA, encompassing over 80 BtrA-activated loci that include genes encoding toxins, adhesins, and other cell surface proteins, and over 200 BtrA-repressed genes that encode T3SS apparatus components, secretion substrates, the BteA effector, and numerous additional factors. In B. pertussis, BtrA retains activity as a BtrS antagonist and exerts tight negative control over T3SS genes. Most importantly, deletion of btrA in B. pertussis revealed T3SS-mediated, BteA-dependent cytotoxicity, which had previously eluded detection. This effect was observed in laboratory strains and in clinical isolates from a recent California pertussis epidemic. We propose that the BtrA-BtrS regulatory node determines subspecies-specific differences in T3SS expression among Bordetella species and that B. pertussis is capable of expressing a full range of T3SS-dependent phenotypes in the presence of appropriate contextual cues.

Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus.

BACKGROUND AND AIMS: Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus. METHODS: For simple sequence repeat (SSR) isolation, a genomic library enriched for (GA)(n) repeats was constructed from C. roseus 'Nirmal' (CrN1). In addition, GTMs were also designed from 12 genes of the TIA (terpenoid indole alkaloid) pathway - the medicinally most significant pathway in C. roseus. An F(2) mapping population was also generated by crossing two diverse accessions of C. roseus CrN1 (Nirmal)×CrN82 (Kew). KEY RESULTS: A new set of 314 STMS markers and 64 GTMs were developed in this study. A segregating F(2) mapping population consisting of 111 F(2) individuals was generated. For generating the linkage map, a set of 423 co-dominant markers (378 newly developed and 45 published earlier) were screened for polymorphism between the parental genotypes, of which 134 were identified to be polymorphic. A total of 114 markers were mapped on eight linkage groups that spanned a 632·7 cM region of the genome with an average marker distance of 5·55 cM. Further, the mechanism of hypervariability at the gene-targeted loci was investigated at the sequence level. CONCLUSIONS: For the first time, a large array of STMS markers and GTMs was generated in the model medicinal plant C. roseus. Moreover, the first microsatellite marker-based linkage map was described in this study. Together, these will serve as a foundation for future genomics studies related to quantitative trait loci analysis and molecular breeding in C. roseus.

Advancing the STMS genomic resources for defining new locations on the intraspecific genetic linkage map of chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea (Cicer arietinum L.) is an economically important cool season grain legume crop that is valued for its nutritive seeds having high protein content. However, several biotic and abiotic stresses and the low genetic variability in the chickpea genome have continuously hindered the chickpea molecular breeding programs. STMS (Sequence Tagged Microsatellite Sites) markers which are preferred for the construction of saturated linkage maps in several crop species, have also emerged as the most efficient and reliable source for detecting allelic diversity in chickpea. However, the number of STMS markers reported in chickpea is still limited and moreover exhibit low rates of both inter and intraspecific polymorphism, thereby limiting the positions of the SSR markers especially on the intraspecific linkage maps of chickpea. Hence, this study was undertaken with the aim of developing additional STMS markers and utilizing them for advancing the genetic linkage map of chickpea which would have applications in QTL identification, MAS and for de novo assembly of high throughput whole genome sequence data. RESULTS: A microsatellite enriched library of chickpea (enriched for (GT/CA)n and (GA/CT)n repeats) was constructed from which 387 putative microsatellite containing clones were identified. From these, 254 STMS primers were designed of which 181 were developed as functional markers. An intraspecific mapping population of chickpea, [ICCV-2 (single podded) × JG-62 (double podded)] and comprising of 126 RILs, was genotyped for mapping. Of the 522 chickpea STMS markers (including the double-podding trait, screened for parental polymorphism, 226 (43.3%) were polymorphic in the parents and were used to genotype the RILs. At a LOD score of 3.5, eight linkage groups defining the position of 138 markers were obtained that spanned 630.9 cM with an average marker density of 4.57 cM. Further, based on the common loci present between the current map and the previously published chickpea intraspecific map, integration of maps was performed which revealed improvement of marker density and saturation of the region in the vicinity of sfl (double-podding) gene thereby bringing about an advancement of the current map. CONCLUSION: An arsenal of 181 new chickpea STMS markers was reported. The developed intraspecific linkage map defined map positions of 138 markers which included 101 new locations.Map integration with a previously published map was carried out which revealed an advanced map with improved density. This study is a major contribution towards providing advanced genomic resources which will facilitate chickpea geneticists and molecular breeders in developing superior genotypes with improved traits.

Isolation of microsatellites from Catharanthus roseus (L.) G. Don using enriched libraries.

Catharanthus roseus is an indispensable source of the anticancerous alkaloids-vincristine and vinblastine, even though they are produced in trace amounts in vivo. In order to increase the yield of alkaloids, in vitro tissue culture studies are carried out which result in a large number of lines/cultures. For identification and characterization of the in vitro cultures, microsatellites in the form of STMS (Sequenced Tagged Microsatellite Sites) markers are used for identification of genetic polymorphism. STMS markers are also used for assessment of genetic diversity within natural populations as well as for construction of genetic linkage maps. Isolation of microsatellites and development of STMS markers typically involves library construction and screening, DNA sequencing, polymerase chain reaction (PCR) primer design, and PCR optimization. This chapter details two approaches for the isolation of microsatellite loci. The first approach is based on PCR using microsatellite containing primers which also have degenerate bases at the 5 cent-end that act as anchors preventing the primers from slippage to the 3 cent-end and the subsequent loss of polymorphism. The multi-locus PCR amplified product is cloned and sequenced. Though this method generates a large number of microsatellites, the major drawback is the high redundancy observed in this method. The second approach described in this chapter is based on the construction of a microsatellite enriched library which involves preferential cloning of the microsatellite enriched fraction of genomic DNA. This method therefore necessitates the isolation of microsatellites through hybridization with biotin labeled oligoprobe followed by their capture with streptavidin-coated magnetic beads. In comparison to the first approach, this approach yields less redundant clones with high microsatellite enrichment. Moreover enriched libraries are 40-60 times more efficient than the conventional small insert genomic libraries.

Development of chickpea EST-SSR markers and analysis of allelic variation across related species.

Despite chickpea being the third important grain legume, there is a limited availability of genomic resources, especially of the expressed sequence tag (EST)-based markers. In this study, we generated 822 chickpea ESTs from immature seeds as well as exploited 1,309 ESTs from the chickpea database, thus utilizing a total of 2,131 EST sequences for development of functional EST-SSR markers. Two hundred and forty-six simple sequence repeat (SSR) motifs were identified from which 183 primer pairs were designed and 60 validated as functional markers. Genetic diversity analysis across 30 chickpea accessions revealed ten markers to be polymorphic producing a total of 29 alleles and an observed heterozygosity average of 0.16 thereby exhibiting low levels of intra-specific polymorphism. However, the markers exhibited high cross-species transferability ranging from 68.3 to 96.6% across the six annual Cicer species and from 29.4 to 61.7% across the seven legume genera. Sequence analysis of size variant amplicons from various species revealed that size polymorphism was due to multiple events such as copy number variation, point mutations and insertions/deletions in the microsatellite repeat as well as in the flanking regions. Interestingly, a wide prevalence of crossability-group-specific sequence variations were observed among Cicer species that were phylogenetically informative. The neighbor joining dendrogram clearly separated the chickpea cultivars from the wild Cicer and validated the proximity of C. judaicum with C. pinnatifidum. Hence, this study for the first time provides an insight into the distribution of SSRs in the chickpea transcribed regions and also demonstrates the development and utilization of genic-SSRs. In addition to proving their suitability for genetic diversity analysis, their high rates of transferability also proved their potential for comparative genomic studies and for following gene introgressions and evolution in wild species, which constitute the valuable secondary genepool in chickpea.

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.).

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT)n and (CT/GA)n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08-0.91) and 0.74 (0.23-0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.

Identification of microsatellite markers from Cicer reticulatum: molecular variation and phylogenetic analysis.

Microsatellite sequences were cloned and sequenced from Cicer reticulatum, the wild annual progenitor of chickpea (C. arietinum L.). Based on the flanking sequences of the microsatellite motifs, 11 sequence-tagged microsatellite site (STMS) markers were developed. These markers were used for phylogenetic analysis of 29 accessions representing all the nine annual Cicer species. The 11 primer pairs amplified distinct fragments in all the annual species demonstrating high levels of sequence conservation at these loci. Efficient marker transferability (97%) of the C. reticulatum STMS markers across other species of the genus was observed as compared to microsatellite markers from the cultivated species. Variability in the size and number of alleles was obtained with an average of 5.8 alleles per locus. Sequence analysis at three homologous microsatellite loci revealed that the microsatellite allele variation was mainly due to differences in the copy number of the tandem repeats. However, other factors such as (1) point mutations, (2) insertion/deletion events in the flanking region, (3) expansion of closely spaced microsatellites and (4) repeat conversion in the amplified microsatellite loci were also responsible for allelic variation. An unweighted pairgroup method with arithmetic averages (UPGMA)-based dendrogram was obtained, which clearly distinguished all the accessions (except two C. judaicum accessions) from one another and revealed intra- as well as inter-species variability in the genus. An annual Cicer phylogeny was depicted which established the higher similarity between C. arietinum and C. reticulatum. The placement of C. pinnatifidum in the second crossability group and its closeness to C. bijugum was supported. Two species, C. yamashitae and C. chorassanicum, were grouped distinctly and seemed to be genetically diverse from members of the first crossability group. Our data support the distinct placement of C. cuneatum as well as a revised classification regarding its placement.