HIV Subtypes and Drug-resistance-associated Mutations in US Blood Donors, 2015-2020.

Background: Monitoring genotypes of HIV infections in blood donors may provide insights into infection trends in the general population. Methods: HIV RNA was extracted from plasma samples of blood donors confirmed as HIV positive by blood screening nucleic acid and antibody tests. HIV genome target regions were amplified using nested real time-polymerase chain reaction followed by next-generation sequencing. Sequences were compared to those in the Los Alamos National Laboratory (LANL) database. Sequences were also assessed for drug resistance mutations (DRM) using the Stanford HIV DRM Database. Results: From available HIV-positive donations collected between 1 September 2015 and 31 December 2020, 563 of 743 (75.8%) were successfully sequenced; 4 were subtype A, 543 subtype B, 5 subtype C, 1 subtype G, 5 circulating recombinant forms (CRF), and 2 were subtype B and D recombinants. Overall, no significant differences between blood donor and available LANL genotypes were found, and the genotypes of newly acquired versus prevalent HIV infections in donors were similar. The proportion of non-B subtypes and CRF remained a small fraction, with no other subtype or CRF representing more than 1% of the total. DRM were identified in 122 (21.6%) samples with protease inhibitor, nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor DRMs identified in 4.9%, 4.6% and 14.0% of samples, respectively. Conclusions: HIV genetic diversity and DRM in blood donors appear representative of circulating HIV infections in the US general population and may provide more information on infection diversity than sequences reported to LANL, particularly for recently transmitted infections.

Embedding of Markov matrices for d ⩽ 4.

The embedding problem of Markov matrices in Markov semigroups is a classic problem that regained a lot of impetus and activities through recent needs in phylogeny and population genetics. Here, we give an account for dimensions d ⩽ 4 , including a complete and simplified treatment of the case d = 3 , and derive the results in a systematic fashion, with an eye on the potential applications. Further, we reconsider the setup of the corresponding problem for time-inhomogeneous Markov chains, which is needed for real-world applications because transition rates need not be constant over time. Additional cases of this more general embedding occur for any d ⩾ 3 . We review the known case of d = 3 and describe the setting for future work on d = 4 .

Domestication and evolutionary histories of specialized gut symbionts across cephalotine ants.

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes.

Detecting diversifying selection for a trait from within and between-species genotypes and phenotypes.

To quantify selection acting on a trait, methods have been developed using either within or between-species variation. However, methods using within-species variation do not integrate the changes at the macroevolutionary scale. Conversely, current methods using between-species variation usually discard within-species variation, thus not accounting for processes at the micro-evolutionary scale. The main goal of this study is to define a neutrality index for a quantitative trait, by combining withinand between-species variation. This neutrality index integrates nucleotide polymorphism and divergence for normalizing trait variation. As such, it does not require estimation of population size nor of time of speciation for normalization. Our index can be used to seek deviation from the null model of neutral evolution, and test for diversifying selection. Applied to brain mass and body mass at the mammalian scale, we show that brain mass is under diversifying selection. Finally, we show that our test is not sensitive to the assumption that population sizes, mutation rates and generation time are constant across the phylogeny, and automatically adjust for it.

An updated LSU database and pipeline for environmental DNA identification of arbuscular mycorrhizal fungi.

Recent work established a backbone reference tree and phylogenetic placement pipeline for identification of arbuscular mycorrhizal fungal (AMF) large subunit (LSU) rDNA environmental sequences. Our previously published pipeline allowed any environmental sequence to be identified as putative AMF or within one of the major families. Despite this contribution, difficulties in implementation of the pipeline remain. Here, we present an updated database and pipeline with (1) an expanded backbone tree to include four newly described genera and (2) several changes to improve ease and consistency of implementation. In particular, packages required for the pipeline are now installed as a single folder (conda environment) and the pipeline has been tested across three university computing clusters. This updated backbone tree and pipeline will enable broadened adoption by the community, advancing our understanding of these ubiquitous and ecologically important fungi.

Phylogenetic Survival Analysis.

Going back in time through a phylogenetic tree makes it possible to evaluate ancestral genomes and assess their potential to acquire key polymorphisms of interest over evolutionary time. Knowledge of this kind may allow for the emergence of key traits to be predicted and pre-empted from currently circulating strains in the future. Here, we present a novel genome-wide survival analysis and use the emergence of drug resistance in Mycobacterium tuberculosis as an example to demonstrate the potential and utility of the technique.

Epidemiological, virological and clinical characterization of a Dengue/Zika outbreak in the Caribbean region of Costa Rica 2017-2018.

The increase in incidence and geographical expansion of viruses transmitted by the Aedes mosquitoes, such as dengue (DENV) and zika (ZIKV) in the Americas, represents a burden for healthcare systems in tropical and subtropical regions. These and other under-detected arboviruses co-circulate in Costa Rica, adding additional complexity to their management due to their shared epidemiological behavior and similarity of symptoms in early stages. Since diagnostics of febrile illness is mostly based on clinical symptoms alone, we gathered acute-phase serum and urine from 399 samples of acute dengue-like cases from two healthcare facilities of Costa Rica, during an outbreak of arboviruses from July 2017 to May 2018, and tested them using molecular and serological methods. The analyses showed that of the clinically presumptive arbovirus cases that were reported, only 39.4% (n=153) of the samples were confirmed positive by RT-PCR to be DENV (DENV (10.3%), CHIKV (0.2%), ZIKV (27.3%), or mixed infections (1.5%). RT-PCR for other alphaviruses and flaviviruses, and PCR for Leptospira sp were negative. Furthermore, to assess flavivirus positivity in post-acute patients, the negative sera were tested against Dengue-IgM. 20% of sera were found positive, confounding even more the definitive number of cases, and emphasizing the need of several distinct diagnostic tools for accurate diagnostics. Molecular characterization of the prM and E genes from isolated viruses revealed that the American/Asian genotype of DENV-2 and the Asian lineage of ZIKV were circulating during this outbreak. Two different clades of DENV-2 American/Asian genotype were identified to co-circulate in the same region and a difference in the platelet and leukocyte count was noted between people infected with each clade, suggesting a putative distinct virulence. Our study sheds light on the necessity for healthcare strategies in managing arbovirus outbreaks, emphasizing the importance of comprehensive molecular and serological diagnostic approaches, as well as molecular characterization. This approach aids in enhancing our understanding of the clinical and epidemiological aspects of arboviral diseases during outbreaks. Our research highlights the need to strengthen training programs for health professionals and the need to increase research-based on laboratory evidence for diagnostic accuracy, guidance, development and implementation of public health interventions and epidemiological surveillance.

Genetic diversity of porcine circoviruses 2 and 3 circulating among wild boars in the Moscow Region of Russia.

Porcine circoviruses (PCVs) are widely distributed in swine herds. PCV2, the significant swine pathogen, causes infections characterized by growth and development disorders, skin lesions, and respiratory distress. PCV3 has been circulating worldwide and can be associated with various clinical signs and disease developments. Wild boars are the main reservoir of these pathogens in wildlife and can create an alarming threat to pig farming. In Russia, three PCV2 genotypes (PCV2a, PCV2b, and PCV2d) were identified in pig farms. Additionally, PCV3 was observed in pig herds during the monitoring studies in the country. However, data considering the circulation of PCVs in herds of wild boars in Russia is scant. For this purpose, we performed PCR assays of the samples from 30 wild boars hunted in the Moscow Region of Russia in 2021-2023. The ratios of wild boars positive for PCV2, PCV3, or coinfected were 50, 10, and 13.3%, respectively. Additionally, we sequenced 15 PCV2 and four PCV3 complete genomes and conducted phylogenetic analysis, which divided PCV2 isolates into two groups: PCV2d and PCV2b. The study showed a high infection rate of PCV2 among wild boars, with PCV2d dominance. Simultaneously, PCV3 also circulates among wild boars. The obtained results can provide a basis for the development of preventive measures to support infection transmission risks between farm and wild animals.

Utility of various genome lengths in diversity and evolution analyses of Hepatitis E virus.

The aim of this study was to investigate to what extent fragments of the HEV genome could be used for accurate diagnostics and inference of viral population-scale processes. For this, we selected all the published whole genome sequences from the NCBI GenBank and trimmed them to various fragment lengths (ORF1,2,3, ORF1, ORF2, ORF3, 493 nt in ORF2 and 148 nt in ORF2). Each of the fragment lengths was used to infer the richness and diversity of the viral sequence types, typing accuracy, and potential use in phylodynamics. The results obtained from the different fragments were compared. We observed that, generally, the longer the nucleic acid fragment used in typing, the better the accuracy in predicting the viral subtype. However, the dominant HEV subtypes circulating in Europe were relatively well classified even by the 493 nt fragment, with false negative rates as low as 8 in 1000 typed sequences. Most fragments also give comparable results in analyses of population size, albeit with shorter fragments showing a broader 95 % highest posterior density interval and less obvious increase of the viral effective population size. The reconstructed phylogenies of a heterochronous subset indicated a good concordance between all the fragments, with the major clades following similar branching patterns. Furthermore, we have used the HEV sequence data from the Netherlands available in the HEVnet database as a case study for reconstruction of population size changes in the past decades. This data showed that molecular and epidemiological results are concordant and point to an increase in the viral effective population size underlying the observed increase in incidence of acute HEV infection cases. In the absence of whole genome sequencing data, the 493 bp fragment can be used for analyzing HEV strains currently circulating in Europe, as it is informative for describing short term population-scale processes.

Maximum Likelihood Estimation for Unrooted 3-Leaf Trees: An Analytic Solution for the CFN Model.

Maximum likelihood estimation is among the most widely-used methods for inferring phylogenetic trees from sequence data. This paper solves the problem of computing solutions to the maximum likelihood problem for 3-leaf trees under the 2-state symmetric mutation model (CFN model). Our main result is a closed-form solution to the maximum likelihood problem for unrooted 3-leaf trees, given generic data; this result characterizes all of the ways that a maximum likelihood estimate can fail to exist for generic data and provides theoretical validation for predictions made in Parks and Goldman (Syst Biol 63(5):798-811, 2014). Our proof makes use of both classical tools for studying group-based phylogenetic models such as Hadamard conjugation and reparameterization in terms of Fourier coordinates, as well as more recent results concerning the semi-algebraic constraints of the CFN model. To be able to put these into practice, we also give a complete characterization to test genericity.

Mitonuclear compatibility is maintained despite relaxed selection on male mitochondrial DNA in bivalves with doubly uniparental inheritance.

Mitonuclear coevolution is common in eukaryotes, but bivalve lineages that have doubly uniparental inheritance (DUI) of mitochondria may be an interesting example. In this system, females transmit mtDNA (F mtDNA) to all offspring, while males transmit a different mtDNA (M mtDNA) solely to their sons. Molecular evolution and functional data suggest oxidative phosphorylation (OXPHOS) genes encoded in M mtDNA evolve under relaxed selection due to their function being limited to sperm only (vs. all other tissues for F mtDNA). This has led to the hypothesis that mitonuclear coevolution is less important for M mtDNA. Here, we use comparative phylogenetics, transcriptomics, and proteomics to understand mitonuclear interactions in DUI bivalves. We found nuclear OXPHOS proteins coevolve and maintain compatibility similarly with both F and M mtDNA OXPHOS proteins. Mitochondrial recombination did not influence mitonuclear compatibility and nuclear-encoded OXPHOS genes were not upregulated in tissues with M mtDNA to offset dysfunction. Our results support that selection maintains mitonuclear compatibility with F and M mtDNA despite relaxed selection on M mtDNA. Strict sperm transmission, lower effective population size, and higher mutation rates may explain the evolution of M mtDNA. Our study highlights that mitonuclear coevolution and compatibility may be broad features of eukaryotes.

Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

Objective: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. Methods: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Results: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-ß-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Conclusion: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against ß-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

Repetitive Sequences, Codon Usage Bias and Phylogenetic Analysis of the Plastome of Miliusa glochidioides.

Annonaceae is the largest family in Magnoliales, exhibiting the greatest diversity among and within genera. In this study, we conducted an analysis of repetitive sequences and codon usage bias in the previously acquired plastome of Miliusa glochidioides. Using a concatenated dataset of shared genes, we constructed the phylogenetic relationships among 27 Annonaceae species. The results showed that the size of the plastomes in the Annonaceae ranged from 159 to 202 kb, with the size of the inverted repeat region ranging from 40 to 65 kb. Within the plastome of M. glochidioides, we identified 42 SSRs, 36 tandem repeats, and 9 dispersed repeats. These SSRs consist of three nucleotide types and eight motif types, with a preference for A/T bases, primarily located in the large single-copy regions and intergenic spacers. Tandem and dispersed repeat sequences were predominantly detected in the IR region. Through codon usage bias analysis, we identified 30 high-frequency codons and 11 optimal codons. The plastome of M. glochidioides demonstrated relatively weak codon usage bias, favoring codons with A/T endings, primarily influenced by natural selection. Phylogenetic analysis revealed that all four subfamilies formed monophyletic groups, with Cananga odorata (Ambavioideae) and Anaxagorea javanica (Anaxagoreoideae) successively nested outside Annonoideae + Malmeoideae. These findings improve our understanding of the plastome of M. glochidioides and provide additional insights for studying plastome evolution in Annonaceae.

Sources of Human Immunodeficiency Virus Infections Among Men Who Have Sex With Men With a Migration Background: A Viral Phylogenetic Case Study in Amsterdam, The Netherlands.

BACKGROUND: Men and women with a migration background comprise an increasing proportion of incident human immunodeficiency virus (HIV) cases across Western Europe. METHODS: To characterize sources of transmission in local transmission chains, we used partial HIV consensus sequences with linked demographic and clinical data from the opt-out AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort of people with HIV in the Netherlands and identified phylogenetically and epidemiologically possible HIV transmission pairs in Amsterdam. We interpreted these in the context of estimated infection dates, and quantified population-level sources of transmission to foreign-born and Dutch-born Amsterdam men who have sex with men (MSM) within Amsterdam transmission chains. RESULTS: We estimate that Dutch-born MSM were the predominant sources of infections among all Amsterdam MSM who acquired their infection locally in 2010-2021, and among almost all foreign-born Amsterdam MSM subpopulations. Stratifying by 2-year intervals indicated time trends in transmission dynamics, with a majority of infections originating from foreign-born MSM since 2016, although uncertainty ranges remained wide. CONCLUSIONS: Native-born MSM have predominantly driven HIV transmissions in Amsterdam in 2010-2021. However, in the context of rapidly declining incidence in Amsterdam, the contribution from foreign-born MSM living in Amsterdam is increasing, with some evidence that most local transmissions have been from foreign-born Amsterdam MSM since 2016.

Cladistics of Rhipicephalus microplus and laboratory assessment of acaricidal efficacy of different Cassia fistula extracts against R. microplus larvae.

The present study aimed to analyze the cladistics and population structure analysis of Rhipicephalus microplus ticks infesting buffaloes in Haryana, India, as well as the assessment of the anti-tick efficacy of the ethanolic extracts of Cassia fistula (bark, pod pulp, and flowers) against R. microplus larvae. The molecular characterization and population structure analysis were performed by targeting the amplification of the partial mitochondrial cytochrome C oxidase subunit 1 (cox1) gene, whereas anti-tick efficacy was evaluated using a larval packet test. The sequences generated herein revealed a homology of 98.26-100% to the GenBank-archived R. microplus sequences. In population structure analysis, high haplotype (0.500 ± 0.265) and low nucleotide (0.002 ± 0.001) diversities were recorded for the sequences generated in this study. Significantly negative neutrality indices were recorded for the overall dataset. The extracts were found to significantly affect mortality rates in a dose-dependent manner, and the ethanolic extracts of the bark, pod pulp, and flowers of C. fistula exhibited median lethal concentration (LC50) values of 27.989, 40.457, and 49.43 mg/mL, respectively. The LC50 value recorded for the combination of the ethanolic extracts of the bark, flower, and pod pulp of C. fistula was 19.724 mg/mL, whereas the synthetic acaricide ivermectin had an LC50 value of 351.56 mg/mL. In conclusion, R. microplus populations infesting cattle and buffalo hosts in India exhibited negligible genetic differentiation and high gene flow between them. Additionally, the combination of all C. fistula extracts could serve as a potential substitute for the synthetic acaricide.

Allometry and ecology shape eye size evolution in spiders.

Eye size affects many aspects of visual function, but eyes are costly to grow and maintain. The allometry of eyes can provide insight into this trade-off, but this has mainly been explored in species that have two eyes of equal size. By contrast, animals possessing larger visual systems can exhibit variable eye sizes within individuals. Spiders have up to four pairs of eyes whose sizes vary dramatically, but their ontogenetic, static, and evolutionary allometry has not yet been studied in a comparative context. We report variable dynamics in eye size across 1,098 individuals in 39 species and 8 families, indicating selective pressures and constraints driving the evolution of different eye pairs and lineages. Supplementing our sampling with a recently published phylogenetically comprehensive dataset, we confirmed these findings across more than 400 species; found that ecological factors such as visual hunting, web building, and circadian activity correlate with eye diameter; and identified significant allometric shifts across spider phylogeny using an unbiased approach, many of which coincide with visual hunting strategies. The modular nature of the spider visual system provides additional degrees of freedom and is apparent in the strong correlations between maximum/minimum investment and interocular variance and three key ecological factors. Our analyses suggest an antagonistic relationship between the anterior and posterior eye pairs. These findings shed light on the relationship between spider visual systems and their diverse ecologies and how spiders exploit their modular visual systems to balance selective pressures and optical and energetic constraints.

The fundamental role of character coding in Bayesian morphological phylogenetics.

Phylogenetic trees establish a historical context for the study of organismal form and function. Most phylogenetic trees are estimated using a model of evolution. For molecular data, modeling evolution is often based on biochemical observations about changes between character states. For example, there are four nucleotides, and we can make assumptions about the probability of transitions between them. By contrast, for morphological characters, we may not know a priori how many characters states there are per character, as both extant sampling and the fossil record may be highly incomplete, which leads to an observer bias. For a given character, the state space may be larger than what has been observed in the sample of taxa collected by the researcher. In this case, how many evolutionary rates are needed to even describe transitions between morphological character states may not be clear, potentially leading to model misspecification. To explore the impact of this model misspecification, we simulated character data with varying numbers of character states per character. We then used the data to estimate phylogenetic trees using models of evolution with the correct number of character states and an incorrect number of character states. The results of this study indicate that this observer bias may lead to phylogenetic error, particularly in the branch lengths of trees. If the state space is wrongly assumed to be too large, then we underestimate the branch lengths, and the opposite occurs when the state space is wrongly assumed to be too small.

Characterization of the complete chloroplast genome of Rhodiola sachalinensis and comparative analysis with its congeneric plants.

Rhodiola, belonging to the Crassulaceae family, is a perennial herbaceous plant genus. There are about 90 Rhodiola species worldwide, some of which have been reported to have medicinal properties. Rhodiola sachalinensis is a perennial medicinal herb within this genus and, in the present study, its chloroplast genome was sequenced, assembled, annotated and compared with 24 other Rhodiola species. The results obtained show that the chloroplast genome of R. sachalinensis is 151 595 bp long and has a CG content of 37.7%. The inverted repeats (IR) region of the Rhodiola chloroplast genome is the most conserved region, with the main differences being observed in the ycf1 and ndhF genes at the IRb-small single copy boundary, and rps19 and trnH genes at the IRa-large single copy boundary. Phylogenetic analysis showed that Rhodiola species form two major clades, and species with recorded medicinal properties, clustered together in one branch except for R. dumulosa. Within the genus, R. sachalinensis is most closely related to Rhodiola rosea, although comparative analyses showed that only R. sachalinensis and Rhodiola subopposita contained the psbZ gene, which encodes a highly conserved protein subunit of the Photosystem II core complex. Overall, the present study contributes to the understanding of the chloroplast genome of Rhodiola species, and provides a theoretical basis for the study of their genetic diversity and possible use as medicinal plants.

Characterization of the family-level Borreliaceae pan-genome and development of an episomal typing protocol.

Background: The Borreliaceae family includes many obligate parasitic bacterial species which are etiologically associated with a myriad of zoonotic borrelioses including Lyme disease and vector-borne relapsing fevers. Infections by the Borreliaceae are difficult to detect by both direct and indirect methods, often leading to delayed and missed diagnoses. Efforts to improve diagnoses center around the development of molecular diagnostics (MDx), but due to deep tissue sequestration of the causative spirochaetes and the lack of persistent bacteremias, even MDx assays suffer from a lack of sensitivity. Additionally, the highly extensive genomic heterogeneity among isolates, even within the same species, contributes to the lack of assay sensitivity as single target assays cannot provide universal coverage. This within-species heterogeneity is partly due to differences in replicon repertoires and genomic structures that have likely arisen to support the complex Borreliaceae lifecycle in which these parasites have to survive in multiple hosts each with unique immune responses. Results: We constructed a Borreliaceae family-level pangenome and characterized the phylogenetic relationships among the constituent taxa which supports the recent taxonomy of splitting the family into at least two genera. Gene content pro les were created for the majority of the Borreliaceae replicons, providing for the first time their unambiguous molecular typing. Conclusion: Our characterization of the Borreliaceae pan-genome supports the splitting of the former Borrelia genus into two genera and provides for the phylogenetic placement of several non-species designated isolates. Mining this family-level pangenome will enable precision diagnostics corresponding to gene content-driven clinical outcomes while also providing targets for interventions.

The polyphyletic Caucasus-centred Campanula subg. Scapiflorae (Campanulaceae) revisited with a newly circumscribed C. sect. Tridentatae for its core clade.

Campanula L. is among the genera with the highest number of endemics in the Caucasus ecoregion. A group of attractive alpine and subalpine perennial rosette plants with short single-flowered stems centred in the Caucasus has been treated as Campanulasubg.Scapiflorae or at other ranks, with considerably varying circumscription and classification. Molecular phylogenetic analysis of three plastid DNA regions (trnK/matK, petD, rpl16) of a strongly extended sampling, comprising 23 of the 27 commonly accepted taxa (85%) with 330 accessions built on and guided by the results of our previous study of the group, confirmed the polyphyly of C.subg.Scapiflorae in any of its circumscriptions. The core clade of the group comprises exclusively endemics and near-endemics of the Caucasus and is treated here as C.sect.Tridentatae in a revised circumscription. The phylogenetic relationships of the disparate other elements of the Scapiflorae group are outlined.