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Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli.

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.

Blotting, Southern

Comparative genomics and phylogenetic analysis of three Malvaceae species on the basis of chloroplast genomes.

INTRODUCTION: The Malvaceae family shows rich species diversity and has substantial economic and medicinal value. However, the frequent interspecific hybridization among members of this family has resulted in confused phylogenetic relationships among the groups, limiting the usefulness of traditional classification methods. METHODS: This study aimed to investigate the phylogenetic relationships among selected taxa of Malvaceae by evaluating 23 chloroplast (CP) genomes, including three newly assembled CP genomes. Among these three genomes, the CP genome of Hibiscus schizopetalus L. was reported for the first time, while the CP genomes of Alcea rosea L. and Hibiscus grewiifolius L., which have been deposited in NCBI, were re-analyzed here alongside newly generated data for comparative purposes. In addition, 20 downloaded CP genomes encompassing 13 genera were analyzed using SNPs in whole CP genomes data. RESULTS: The results showed that the genomes ranged from 160,403 to 161,978 base pairs in length and consisted of small single copies (SSCs) and large single copies (LSCs) separated by two inverted repeat sequences (IRs), forming a typical quadripartite circular structure. The entire genome sequence showed relative conservation across species in terms of structure, GC content, codon usage, and gene composition. The mutation sites were mainly located in the LSC and SSC regions, and the variability in the non-coding regions was higher than that in the coding regions. The nucleotide polymorphism (Pi) analysis identified the non-coding regions such as ndhF-rpl32 and psbZ-trnG as high variable hotspots. A maximum likelihood phylogenetic tree was constructed based on SNPs in whole CP genomes data. The phylogenetic analysis divided these 23 species into five highly supported clades. It also revealed a close sister-group relationship between Abelmoschus and Hibiscus species, suggesting that Hibiscus may have a separate lineage from okra species. DISCUSSION: In conclusion, the increasing availability of CP genome resources will enhance our understanding of the classification and evolutionary patterns of the Malvaceae family. The development of molecular markers will provide important molecular evidence for precise identification and classification revision of plants in this family.

Malvaceae

A phylogenetic analysis of the mycoplasmas: basis for their classification.

Small-subunit rRNA sequences were determined for almost 50 species of mycoplasmas and their walled relatives, providing the basis for a phylogenetic systematic analysis of these organisms. Five groups of mycoplasmas per se were recognized (provisional names are given): the hominis group (which included species such as Mycoplasma hominis, Mycoplasma lipophilum, Mycoplasma pulmonis, and Mycoplasma neurolyticum), the pneumoniae group (which included species such as Mycoplasma pneumoniae and Mycoplasma muris), the spiroplasma group (which included species such as Mycoplasma mycoides, Spiroplasma citri, and Spiroplasma apis), the anaeroplasma group (which encompassed the anaeroplasmas and acholeplasmas), and a group known to contain only the isolated species Asteroleplasma anaerobium. In addition to these five mycoplasma groups, a sixth group of variously named gram-positive, walled organisms (which included lactobacilli, clostridia, and other organisms) was also included in the overall phylogenetic unit. In each of these six primary groups, subgroups were readily recognized and defined. Although the phylogenetic units identified by rRNA comparisons are difficult to recognize on the basis of mutually exclusive phenotypic characters alone, phenotypic justification can be given a posteriori for a number of them.

Base Sequence

Insights Into the Structural Features, Codon Usage Patterns, and Phylogenetic Analysis in Neoniphon argenteus (Teleostei: Holocentriformes) Based on Complete Mitochondrial Genome.

Neoniphon argenteus, a widely distributed nocturnal coral reef fish in the family Holocentridae, plays an important role in maintaining coral reef ecosystem health, yet its phylogenetic position remains poorly resolved. To bridge this gap, we sequenced and analyzed the complete mitochondrial genome of a specimen from the South China Sea to characterize its structural features, codon usage patterns, and phylogenetic relationships. The 16,569 bp mitogenome (GenBank: PP190474.1) encodes 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and two non-coding regions, exhibiting a distinct A + T bias. All tRNAs fold into typical cloverleaf secondary structures except tRNA-Ser (AGN), which lacks the dihydrouridine (DHU) arm. The control region contains palindromic motifs (TACAT/ATGTA) capable of forming hairpin structures and five conserved sequence blocks, whereas the OL region harbors a conserved 5'-GCCGG-3' motif. RSCU analysis revealed 31 frequently used codons (RSCU > 1) with a pronounced preference for A/C-ending codons. The ΔRSCU method identified 10 candidate optimal codons (GCA, CAA, GAA, GGA, AUU, CUA, CCA, CGA, ACA, and GUC). Selection pressure analysis using EasyCodeML and site-specific models indicated that all PCGs are predominantly under purifying selection, with no significant evidence of pervasive positive selection. ND6 exhibited elevated pairwise Ka/Ks ratios (mean = 1.209 ± 0.047), consistent with reduced selective constraint rather than adaptive evolution. Phylogenetic analysis of 19 Holocentriformes species using maximum likelihood and Bayesian inference with partitioned models based on 13 PCGs and two rRNA genes (12S and 16S) assigned all taxa to two well-supported subfamilies (Holocentrinae and Myripristinae). Within Holocentrinae, Neoniphon species form a monophyletic clade nested within a paraphyletic Sargocentron, suggesting that the genus Sargocentron as currently defined is not monophyletic. This study provides useful baseline molecular data for further exploration of the evolutionary history of N. argenteus and other members of Holocentriformes.

Holocentridae

Epidemiological and phylogenetic analysis of anthrax in Kazakhstan in 2024.

BACKGROUND: Anthrax remains an important zoonotic disease in Kazakhstan due to the persistence of environmental reservoirs and long-standing endemic foci. Despite ongoing surveillance, the epidemiological characteristics and genetic diversity of circulating Bacillus anthracis strains in the country remain incompletely understood. METHODS: A retrospective epidemiological and phylogenetic investigation of anthrax outbreaks reported in Kazakhstan during 2024 was conducted. Epidemiological data were collected for all laboratory-confirmed human cases and associated outbreak foci. Confirmation of infection was performed by PCR, and B. anthracis isolates were obtained from clinical, environmental and animal-associated samples. Whole-genome sequencing and core-genome single nucleotide polymorphism (cgSNP) analysis were used to characterize the genetic relationships among isolates and to determine their phylogenetic placement. RESULTS: Nine anthrax outbreaks were identified across four regions of Kazakhstan (Almaty, Zhambyl, Atyrau, and West Kazakhstan), resulting in 20 confirmed human cases. All patients were male, with the highest proportion occurring among individuals aged 36-55 years (45%). The mean patient age was 43.9 years (range: 16-64 years). Most infections were associated with slaughtering infected livestock (65%), followed by handling contaminated meat (15%). PCR confirmed infection in all 20 human cases. Culture yielded 17 human-derived B. anthracis isolates from 14 patients and 17 environmental/animal-derived isolates, resulting in 34 isolates in total. Of these, 22 representative isolates underwent whole-genome sequencing. Phylogenetic analysis revealed the circulation of two major lineages. Isolates from Atyrau and West Kazakhstan clustered within the Trans-Eurasian (TEA/STI) lineage. Atyrau isolates formed a tight cluster differing by only 21-32 cgSNPs, consistent with a shared epidemiolocal source, whereas the West Kazakhstan isolate was highly divergent. Zhambyl and Almaty region belonged to the A.Br.Ames lineage but diverged into two distinct sublineages. Zhambyl region isolates demonstrated minimal divergence from the global reference genome Ames Ancestor, differing by only 16-31 SNPs. Almaty region isolates formed an endemic subclone, separated from the reference group by approximately 114 SNPs. Comparison with the Ames Ancestor and Sterne reference strains demonstrated substantial genetic divergence. CONCLUSION: Anthrax outbreaks in Kazakhstan during 2024 were primarily associated with livestock exposure and occurred within established endemic regions. Whole-genome sequencing revealed the coexistence of distinct TEA and Ames lineages, including evidence of persistent local transmission and long-term evolutionary stability of endemic B. anthracis populations. These findings enhance understanding of anthrax epidemiology in Central Asia and support the integration of genomic surveillance into national outbreak investigation programs.

Anthrax

Dominance behavior: a phylogenetic analysis in the mouse.

Tube-dominance behavior differed significantly in C57BL/6By and BALB/cBy mice. Evaluation of these two inbred strains, and their recently developed recombinant inbred strains permitted the derivation of an hypothesis regarding possible genetic control of this behavior in mice. The results were analyzed by means of patristic matrices with accompanying phylogenetic tree analysis that marks the first occasion that such analysis has been used for a behavioral trait. Based on the data obtained and the statistical analyses and groupings of the various strains, the most satisfactory hypothesis that can be proposed at this time is one which assumes that dominance behavior, as measured in this experiment, is influenced genetically by at least 3 loci. Attempts to link these loci were not successful.

Aggression

Phylogenetic analysis of Alloglossidium Simer, 1929 (Digenea: Plagiorchiiformes: Macroderoididae) with discussion of the origin of truncated life cycle patterns in the genus.

Alloglossidium comprises 9 species of North American plagiorchiiform digeneans using ictalurid catfish, freshwater crustacea, and hirudinid leeches as definitive hosts. Two hypotheses about the evolution of this array of definitive hosts were examined using phylogenetic systematic analysis. Two most parsimonious trees, based on 15 homologous series derived from morphological data, each indicated the 2 species utilizing ictalurid catfish definitive hosts are basal members of the group, whereas the 2 species using freshwater crayfish definitive hosts and the 5 utilizing leech definitive hosts each comprise relatively derived monophyletic sister groups. The results suggest that species using crustaceans as definitive hosts are derived by life cycle truncation, whereas those using leeches as definitive hosts appear to be derived through a switch from crustaceans to leeches.

Animals

The first two complete mitochondrial genomes for the genus Neotrichoporoides (Hymenoptera, Eulophidae) and their phylogenetic analysis.

Neotrichoporoides belongs to the family Eulophidae (Hymenoptera: Chalcidoidea). As a group of parasitic wasps, it plays an indispensable role in the biological control of agricultural and forest pests and in maintaining ecosystem balance. To date, only nine complete mitochondrial genomes of Eulophidae have been sequenced worldwide, including the two newly sequenced species in this study. To enrich our understanding of the mitochondrial genomic diversity of Eulophidae and to provide preliminary insights into its phylogenetic relationships, we sequenced and comparatively analyzed the mitochondrial genomes of two Neotrichoporoides species. The mitogenomes of N. nyemitawus (GenBank: PZ188956; 15,164 bp) and N. viridimaculatus (GenBank: PX794932; 15,297 bp) contain 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and one control region (CR), and exhibit a strong AT bias, with AT contents of 85.5% and 85.0%, respectively. We further analyzed mitochondrial gene rearrangements across 17 species from Encyrtidae, Eulophidae and Pteromalidae and summarized family-specific rearrangement characteristics. tRNA rearrangements were detected in all three families. Eulophidae harbors conserved PCGs, while the inverse transposition of trnA and transposition of trnV are likely reported for the first time within this family. The two Neotrichoporoides species differ only in the arrangement of several tRNAs. Comparative analysis of PCGs revealed differences in molecular evolutionary rates among genes, with ATP8, ND2 and ND4 evolving faster than the others. Phylogenetic analysis based on mitochondrial genome sequences showed that species from two subfamilies formed a monophyletic group, and congeneric species clustered into a single clade. This study contributes to resolving phylogenetic relationships within Eulophidae and further deepens our understanding of this family.

Eulophidae

Limitations of the evolutionary parsimony method of phylogenetic analysis.

Lake's evolutionary parsimony (EP) method of constructing a phylogenetic tree is primarily applied to four DNA sequences. In this method, three quantities--X, Y, and Z--that correspond to three possible unrooted trees are computed, and an invariance property of these quantities is used for choosing the best tree. However, Lake's method depends on a number of unrealistic assumptions. We therefore examined the theoretical basis of his method and reached the following conclusions: (1) When the rates of two transversional changes from a nucleotide are unequal, his invariance property breaks down. (2) Even if the rates of two transversional changes are equal, the invariance property requires some additional conditions. (3) When Kimura's two-parameter model of nucleotide substitution applies and the rate of nucleotide substitution varies greatly with branch, the EP method is generally better than the standard maximum-parsimony (MP) method in recovering the correct tree but is inferior to the neighbor-joining (NJ) and a few other distance matrix methods. (4) When the rate of nucleotide substitution is the same or nearly the same for all branches, the EP method is inferior to the MP method even if the proportion of transitional changes is high. (5) When Lake's assumptions fail, his chi2 test may identify an erroneous tree as the correct tree. This happens because the test is not for comparing different trees. (6) As long as a proper distance measure is used, the NJ method is better than the EP and MP methods whether there is a transition/transversion bias or whether there is variation in substitution rate among different nucleotide sites.

Base Sequence

Mitochondrial genomic characteristics and phylogenetic analysis of Cunninghamella elegans (Mucorales: Cunninghamellaceae).

Cunninghamella, a filamentous fungal genus with important biomedical and biochemical value, lacks any fully annotated mitochondrial genome to date. Herein, we presented the first complete mitogenome of Cunninghamella elegans, a circular 41,552 bp molecule (GC 27.86%) encoding 14 conserved protein-coding genes, 2 rRNA genes, 24 tRNA genes, and 6 non-conserved ORFs. Structural comparison with related species (Absidia glauca and Gongronella sp. w5) revealed dynamic evolution in intron and repeat elements. Phylogenetics places C. elegans within Cunninghamellaceae, with Gongronella as its closest relative. This reference mitogenome will underpin future evolutionary and taxonomic investigations of this industrially and medically significant lineage.

Cunninghamella elegans

The complete chloroplast genome of Cynanchum hemsleyanum and its phylogenetic analysis.

C. hemsleyanum chloroplast genome is 157,356 bp with a quadripartite structure, 37.99% GC, and 132 genes (87 protein-coding, 37 tRNA, 8 rRNA). Phylogenomic analysis places it as sister to C. thesioides with 100% bootstrap support. This resource aids molecular identification, genetic diversity, and evolutionary studies in Apocynaceae.

Cynanchum hemsleyanum

Mitochondrial genome characteristics and phylogenetic analysis of Ramaria longispora.

This study, for the first time, assembled and annotated the complete mitochondrial genome of R. longispora using high-throughput sequencing technology. The genome is a circular molecule with a total length of 157,712 bp and a GC content of 31.55%. It encodes 71 genes, including 15 core protein-coding genes (PCGs), 25 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, 5 free-stranding open reading frames (ORFs), and 24 intronic ORFs. Among these, most free-stranding ORFs have unknown functions but include a DNA polymerase gene, while the intronic ORFs primarily encode LAGLIDADG and GIY-YIG endonucleases. The mitochondrial genome contains 39 introns. Phylogenetic analyses based on 15 core PCGs using Bayesian inference (BI) and maximum likelihood (ML) methods revealed that this R. longispora is most closely related to Ramaria flavescens and Ramaria ichnusensis. This study provides foundational data for mitochondrial genome research in the Ramaria genus and offers important references for taxonomic and evolutionary studies of this group.

Mitochondrial genome

Complete genome characterization, phylogenetic analysis, and capsid P2 variation of a goose astrovirus genotype 2 isolate from Guizhou, China.

Goose astrovirus genotype 2 (GAstV-2) is associated with gout and renal disease in goslings, but its occurrence in Guizhou Province remains poorly documented. We isolated a GAstV-2 strain, designated GZJP2024, from goslings with visceral gout on a farm in Jinping County, Guizhou Province, China. PCR detected GAstV-2 but not goose parvovirus, goose reovirus, Tembusu virus, fowl adenovirus, or goose astrovirus genotype 1. Serial passage in goose embryos produced mortality and hemorrhagic lesions during the third passage. Whole-genome sequencing yielded a 7,251-nt genome containing three overlapping open reading frames (ORF1a, ORF1b, and ORF2). Sequence identity and phylogenetic analyses assigned GZJP2024 to the GAstV-2 lineage. ORF1b was the most conserved coding region, whereas ORF2 was more variable. Comparison with consensus sequences from representative GAstV-2 strains identified five amino acid substitutions in ORF1a and ten in ORF2. Four ORF2 substitutions (E456D, L540Q, S608T, and A614T) occurred in the capsid P2 domain and overlapped or neighbored predicted B-cell epitope-rich regions. Template-based mapping placed E456D, S608T, and A614T on exposed regions of a spike-like capsid structure. These findings document a GAstV-2 isolate from a gout-affected goose farm in Guizhou and provide sequence data for future regional surveillance.

Capsid P2

Structural features in TAR RNA of human and simian immunodeficiency viruses: a phylogenetic analysis.

A comparative analysis of TAR RNA structures in human and simian immunodeficiency viruses reveals the conservation of certain structural features despite the divergence in sequence. Both the TAR elements of HIV-1 and SIV-chimpanzee can be folded into relatively simple one-stem hairpin structures. Chemical and RNAase probes were used to analyze the more complex structure of HIV-2 TAR RNA, which folds into a branched hairpin structure. A surprisingly similar RNA conformation can be proposed for SIV-mandrill, despite considerable divergence in nucleotide sequence. A third structural presentation of TAR sequences is seen for SIV-african green monkey. These results are generally consistent with the classification of HIV-SIV viruses in four subgroups based on sequence analyses (both nucleotide- and amino acid-sequences). However, some conserved TAR structures were detected for members of different virus subgroups. It is therefore proposed that RNA structure analysis might provide an additional tool for determining phylogenetic relationships among the HIV-SIV viruses.

Base Composition

A phylogenetic analysis of the genus Leuconostoc based on reverse transcriptase sequencing of 16 S rRNA.

The phylogenetic interrelationships of members of the genus Leuconostoc and some heterofermentative lactobacilli, which phenotypically resemble leuconostocs, were investigated by comparative analysis of their 16 S rRNA sequences. The six species, Leuconostoc mesenteroides, Leu. carnosum, Leu. citreum, Leu. gelidum, Leu. lactis and Leu. pseudomesenteroides exhibited a high degree of sequence similarity with each other and formed a phylogenetically coherent group, quite separate from all other lactic acid bacteria investigated. The species Leu. paramesenteroides was found to be phylogenetically distinct from the Leu. mesenteroides group of species and formed a natural grouping with the heterofermentative lactobacilli, Lb. confusus, Lb. kandleri, Lb. minor and Lb. viridescens. The rRNA sequence of the acidophilic species, Leu. oenos, displayed exceptionally low levels of homology with all of the other taxa examined. The 16 S sequence of Leu. oenos showed major nucleotide differences in relatively highly conserved positions of the molecule indicating this species is phylogenetically distinct and warrants a separate genus.

Base Sequence

Phylogenetic analysis of the genus Listeria based on reverse transcriptase sequencing of 16S rRNA.

The phylogenetic interrelationships of members of the genus Listeria were investigated by using reverse transcriptase sequencing of 16S rRNA. The sequence data indicate that at the intrageneric level the genus Listeria consists of the following two closely related but distinct lines of descent: (i) the Listeria monocytogenes group of species (including Listeria innocua, Listeria ivanovii, Listeria seeligeri, and Listeria welshimeri) and (ii) the species Listeria grayi and Listeria murrayi. At the intergeneric level a specific phylogenetic relationship between the genera Listeria and Brochothrix was evident. The sequence data clearly demonstrated that the genus Listeria is phylogenetically remote from the genus Lactobacillus and should not be included in an extended family Lactobacillaceae.

Base Sequence

Rhinebothrium devaneyi n. sp. (Eucestoda: Tetraphyllidea) and Echinocephalus overstreeti Deardorff and Ko, 1983 (Nematoda: Gnathostomatidae) in a thorny back ray, Urogymnus asperrimus, from Enewetak Atoll, with phylogenetic analysis of both species groups.

The new species is a member of an apparently monophyletic group within the genus that includes R. flexile, R. walga, R. himanturi, R. burgeri, R. euzeti, R. hawaiiensis, R. urobatidium, R. paratrygoni, R. ditesticulum, R. tetralobatum, R. margaritense, R. biorchidum, and R. spinicephalum. All of these species have bothridia with medial longitudinal septa, a constriction at mid-bothridium, and, primitively, at least 42 loculi per bothridium and 17-22 testes per proglottid. Of the above, the new species is apparently most closely related to R. burgeri, with which it shares an increased number of testes (30-43) per proglottid, a V-shaped ovary, and a muscular genital pore. The new species is distinct by virtue of possessing 94-152 loculi per bothridium--no other known species has more than 78. This is the second report of Echinocephalus overstreeti from a stingray. It represents a new host, U. asperrimus, and a new location, Enewetak Atoll. Phylogenetic and biogeographic analysis of each species group suggests an ancient Tethys Sea-circum-Pacific origin and evolution. This supports the hypothesis of ancient Pacific origins for potamotrygonid stingrays.

Animals

Phylogenetic analysis and identification of different serovars of Mycobacterium intracellulare at the molecular level.

Comparative 16S rRNA sequencing was used to infer the phylogenetic relationship among different serovars of the Mycobacterium avium-M. intracellulare complex as well as to define signature nucleotides characteristic for different serovars. In general, the groups defined by rRNA sequencing reflect the classification obtained with sensitin tests and pathogenicity examinations in chickens. Unique 16S rRNA sequence patterns could be defined for (1) M. avium, (2) M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11, (3) M. intracellulare serovars 12, 13, 14, 15, 17, 19 and 20, (4) M. intracellulare serovar 7 and (5) M. intracellulare serovar 18. Phylogenetically, groups 1 and 2 on one hand and groups 3, 4 and 5 on the other hand each share a common ancestor. M. paratuberculosis was indistinguishable from M. intracellulare serovars 4, 5, 6, 8, 9, 10 and 11 by this kind of analysis.

Base Sequence