Differences In Branch Lengths Research Articles

Rapid identification and subsequent contextualization of an outbreak of methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit using nanopore sequencing.

Outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) are well described in the neonatal intensive care unit (NICU) setting. Genomics has revolutionized the investigation of such outbreaks; however, to date, this has largely been completed retrospectively and has typically relied on short-read platforms. In 2022, our laboratory established a prospective genomic surveillance system using Oxford Nanopore Technologies sequencing for rapid outbreak detection. Herein, using this system, we describe the detection and control of an outbreak of sequence-type (ST)97 MRSA in our NICU. The outbreak was identified 13 days after the first MRSA-positive culture and at a point where there were only two known cases. Ward screening rapidly defined the extent of the outbreak, with six other infants found to be colonized. There was minimal transmission once the outbreak had been detected and appropriate infection control measures had been instituted; only two further ST97 cases were detected, along with three unrelated non-ST97 MRSA cases. To contextualize the outbreak, core-genome single-nucleotide variants were identified for phylogenetic analysis after de novo assembly of nanopore data. Comparisons with global (n=45) and national surveillance (n=35) ST97 genomes revealed the stepwise evolution of methicillin resistance within this ST97 subset. A distinct cluster comprising nine of the ten ST97-IVa genomes from the NICU was identified, with strains from 2020 to 2022 national surveillance serving as outgroups to this cluster. One ST97-IVa genome presumed to be part of the outbreak formed an outgroup and was retrospectively excluded. A second phylogeny was created using Illumina sequencing, which considerably reduced the branch lengths of the NICU isolates on the phylogenetic tree. However, the overall tree topology and conclusions were unchanged, with the exception of the NICU outbreak cluster, where differences in branch lengths were observed. This analysis demonstrated the ability of a nanopore-only prospective genomic surveillance system to rapidly identify and contextualize an outbreak of MRSA in a NICU.

Phylogenomics of the Major Tropical Plant Family Annonaceae Using Targeted Enrichment of Nuclear Genes.

Targeted enrichment and sequencing of hundreds of nuclear loci for phylogenetic reconstruction is becoming an important tool for plant systematics and evolution. Annonaceae is a major pantropical plant family with 110 genera and ca. 2,450 species, occurring across all major and minor tropical forests of the world. Baits were designed by sequencing the transcriptomes of five species from two of the largest Annonaceae subfamilies. Orthologous loci were identified. The resulting baiting kit was used to reconstruct phylogenetic relationships at two different levels using concatenated and gene tree approaches: a family wide Annonaceae analysis sampling 65 genera and a species level analysis of tribe Piptostigmateae sampling 29 species with multiple individuals per species. DNA extraction was undertaken mainly on silicagel dried leaves, with two samples from herbarium dried leaves. Our kit targets 469 exons (364,653 bp of sequence data), successfully capturing sequences from across Annonaceae. Silicagel dried and herbarium DNA worked equally well. We present for the first time a nuclear gene-based phylogenetic tree at the generic level based on 317 supercontigs. Results mainly confirm previous chloroplast based studies. However, several new relationships are found and discussed. We show significant differences in branch lengths between the two large subfamilies Annonoideae and Malmeoideae. A new tribe, Annickieae, is erected containing a single African genus Annickia. We also reconstructed a well-resolved species-level phylogenetic tree of the Piptostigmteae tribe. Our baiting kit is useful for reconstructing well-supported phylogenetic relationships within Annonaceae at different taxonomic levels. The nuclear genome is mainly concordant with plastome information with a few exceptions. Moreover, we find that substitution rate heterogeneity between the two subfamilies is also found within the nuclear compartment, and not just plastomes and ribosomal DNA as previously shown. Our results have implications for understanding the biogeography, molecular dating and evolution of Annonaceae.

The Y-chromosome tree bursts into leaf: 13,000 high-confidence SNPs covering the majority of known clades.

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes.

Annonaceae substitution rates: a codon model perspective

The Annonaceae includes cultivated species of economic interest and represents an important source of information for better understanding the evolution of tropical rainforests. In phylogenetic analyses of DNA sequence data that are used to address evolutionary questions, it is imperative to use appropriate statistical models. Annonaceae are cases in point: Two sister clades, the subfamilies Annonoideae and Malmeoideae, contain the majority of Annonaceae species diversity. The Annonoideae generally show a greater degree of sequence divergence compared to the Malmeoideae, resulting in stark differences in branch lengths in phylogenetic trees. Uncertainty in how to interpret and analyse these differences has led to inconsistent results when estimating the ages of clades in Annonaceae using molecular dating techniques. We ask whether these differences may be attributed to inappropriate modelling assumptions in the phylogenetic analyses. Specifically, we test for (clade-specific) differences in rates of non-synonymous and synonymous substitutions. A high ratio of nonsynonymous to synonymous substitutions may lead to similarity of DNA sequences due to convergence instead of common ancestry, and as a result confound phylogenetic analyses. We use a dataset of three chloroplast genes (rbcL, matK, ndhF) for 129 species representative of the family. We find that differences in branch lengths between major clades are not attributable to different rates of non-synonymous and synonymous substitutions. The differences in evolutionary rate between the major clades of Annonaceae pose a challenge for current molecular dating techniques that should be seen as a warning for the interpretation of such results in other organisms.

Cardinal characters on a slippery slope – A re-evaluation of phylogeny, character evolution, and evolutionary rates in the jelly lichens (Collemataceae s. str)

Recent molecular systematic studies have indicated that the traits currently used for generic delimitation in the jelly lichens (Collemataceae s. str.), may not characterize monophyletic groups. Here we reconstruct the phylogeny of Collemataceae using Bayesian and maximum likelihood analyses based on mitochondrial (mtSSU rDNA) and nuclear (nuLSU rDNA, Beta-tubulin and MCM7) markers of 70 Collemataceae species. We studied the evolution of four morphological and ecological characters traditionally used to delimit genera and infra-generic groups. Finally, we tested if differences in branch-lengths between clades are due to differences in rates of molecular evolution. Eleven strongly supported groups were recovered in the resulting well-resolved and well-supported phylogeny. The presence/absence of a eucortex, which is currently used as the cardinal character to define genera in the group, does not characterize monophyletic groups corresponding to the genera as currently circumscribed. Ancestral state reconstruction indicates that the most recent common ancestor of the jelly lichens most likely was saxicolous/terricolous, lacked a tomentum, and had transversally septate ascospores. Although the cortex state could not be reconstructed for the ancestor of the family, our observations indicate that a lack of cortex may have an evolutionary advantage in saxicolous/terricolous species in semi-arid environments, as non-corticate species tends to be larger and occur in higher frequency and abundance in such regions, compared to corticate species. A significant evidence for faster evolutionary rates was found in a lineage mainly including taxa that occur in the wet tropics and humid temperate regions, compared to other lineages. We suggest that this can explain the greater diversity of Collemataceae in tropical and humid areas.

Eumalacostracan phylogeny and total evidence: limitations of the usual suspects.

BackgroundThe phylogeny of Eumalacostraca (Crustacea) remains elusive, despite over a century of interest. Recent morphological and molecular phylogenies appear highly incongruent, but this has not been assessed quantitatively. Moreover, 18S rRNA trees show striking branch length differences between species, accompanied by a conspicuous clustering of taxa with similar branch lengths. Surprisingly, previous research found no rate heterogeneity. Hitherto, no phylogenetic analysis of all major eumalacostracan taxa (orders) has either combined evidence from multiple loci, or combined molecular and morphological evidence.ResultsWe combined evidence from four nuclear ribosomal and mitochondrial loci (18S rRNA, 28S rRNA, 16S rRNA, and cytochrome c oxidase subunit I) with a newly synthesized morphological dataset. We tested the homogeneity of data partitions, both in terms of character congruence and the topological congruence of inferred trees. We also performed Bayesian and parsimony analyses on separate and combined partitions, and tested the contribution of each partition. We tested for potential long-branch attraction (LBA) using taxon deletion experiments, and with relative rate tests. Additionally we searched for molecular polytomies (spurious clades). Lastly, we investigated the phylogenetic stability of taxa, and assessed their impact on inferred relationships over the whole tree. We detected significant conflict between data partitions, especially between morphology and molecules. We found significant rate heterogeneity between species for both the 18S rRNA and combined datasets, introducing the possibility of LBA. As a test case, we showed that LBA probably affected the position of Spelaeogriphacea in the combined molecular evidence analysis. We also demonstrated that several clades, including the previously reported and surprising clade of Amphipoda plus Spelaeogriphacea, are 'supported' by zero length branches. Furthermore we showed that different sets of taxa have the greatest impact upon the relationships within molecular versus morphological trees.ConclusionRate heterogeneity and conflict between data partitions mean that existing molecular and morphological evidence is unable to resolve a well-supported eumalacostracan phylogeny. We believe that it will be necessary to look beyond the most commonly utilized sources of data (nuclear ribosomal and mitochondrial sequences) to obtain a robust tree in the future.

Reconsidering the generation time hypothesis based on nuclear ribosomal ITS sequence comparisons in annual and perennial angiosperms

BackgroundDifferences in plant annual/perennial habit are hypothesized to cause a generation time effect on divergence rates. Previous studies that compared rates of divergence for internal transcribed spacer (ITS1 and ITS2) sequences of nuclear ribosomal DNA (nrDNA) in angiosperms have reached contradictory conclusions about whether differences in generation times (or other life history features) are associated with divergence rate heterogeneity. We compared annual/perennial ITS divergence rates using published sequence data, employing sampling criteria to control for possible artifacts that might obscure any actual rate variation caused by annual/perennial differences.ResultsRelative rate tests employing ITS sequences from 16 phylogenetically-independent annual/perennial species pairs rejected rate homogeneity in only a few comparisons, with annuals more frequently exhibiting faster substitution rates. Treating branch length differences categorically (annual faster or perennial faster regardless of magnitude) with a sign test often indicated an excess of annuals with faster substitution rates. Annuals showed an approximately 1.6-fold rate acceleration in nucleotide substitution models for ITS. Relative rates of three nuclear loci and two chloroplast regions for the annual Arabidopsis thaliana compared with two closely related Arabidopsis perennials indicated that divergence was faster for the annual. In contrast, A. thaliana ITS divergence rates were sometimes faster and sometimes slower than the perennial. In simulations, divergence rate differences of at least 3.5-fold were required to reject rate constancy in > 80 % of replicates using a nucleotide substitution model observed for the combination of ITS1 and ITS2. Simulations also showed that categorical treatment of branch length differences detected rate heterogeneity > 80% of the time with a 1.5-fold or greater rate difference.ConclusionAlthough rate homogeneity was not rejected in many comparisons, in cases of significant rate heterogeneity annuals frequently exhibited faster substitution rates. Our results suggest that annual taxa may exhibit a less than 2-fold rate acceleration at ITS. Since the rate difference is small and ITS lacks statistical power to reject rate homogeneity, further studies with greater power will be required to adequately test the hypothesis that annual and perennial plants have heterogeneous substitution rates. Arabidopsis sequence data suggest that relative rate tests based on multiple loci may be able to distinguish a weak acceleration in annual plants. The failure to detect rate heterogeneity with ITS in past studies may be largely a product of low statistical power.

Ecdysozoan Clade Rejected by Genome-Wide Analysis of Rare Amino Acid Replacements

As the number of sequenced genomes from diverse walks of life rapidly increases, phylogenetic analysis is entering a new era: reconstruction of the evolutionary history of organisms on the basis of full-scale comparison of their genomes. In addition to brute force, genome-wide analysis of alignments, rare genomic changes (RGCs) that are thought to comprise derived shared characters of individual clades are increasingly used in genome-wide phylogenetic studies. We propose a new type of RGCs designated RGC_CAMs (after Conserved Amino acids-Multiple substitutions), which are inferred using a genome-scale analysis of protein and underlying nucleotide sequence alignments. The RGC_CAM approach utilizes amino acid residues conserved in major eukaryotic lineages, with the exception of a few species comprising a putative clade, and selects for phylogenetic inference only those amino acid replacements that require 2 or 3 nucleotide substitutions, in order to reduce homoplasy. The RGC_CAM analysis was combined with a procedure for rigorous statistical testing of competing phylogenetic hypotheses. The RGC_CAM method is shown to be robust to branch length differences and taxon sampling. When applied to animal phylogeny, the RGC_CAM approach strongly supports the coelomate clade that unites chordates with arthropods as opposed to the ecdysozoan (molting animals) clade. This conclusion runs against the view of animal evolution that is currently prevailing in the evo-devo community. The final solution to the coelomate-ecdysozoa controversy will require a much larger set of complete genome sequences representing diverse animal taxa. It is expected that RGC_CAM and other RGC-based methods will be crucial for these future, definitive phylogenetic studies.

Reconciling Extreme Branch Length Differences: Decoupling Time and Rate through the Evolutionary History of Filmy Ferns

The rate of molecular evolution is not constant across the Tree of Life. Characterizing rate discrepancies and evaluating the relative roles of time and rate along branches through the past are both critical to a full understanding of evolutionary history. In this study, we explore the interactions of time and rate in filmy ferns (Hymenophyllaceae), a lineage with extreme branch length differences between the two major clades. We test for the presence of significant rate discrepancies within and between these clades, and we separate time and rate across the filmy fern phylogeny to simultaneously yield an evolutionary time scale of filmy fern diversification and reconstructions of ancestral rates of molecular evolution. Our results indicate that the branch length disparity observed between the major lineages of filmy ferns is indeed due to a significant difference in molecular evolutionary rate. The estimation of divergence times reveals that the timing of crown group diversification was not concurrent for the two lineages, and the reconstruction of ancestral rates of molecular evolution points to a substantial rate deceleration in one of the clades. Further analysis suggests that this may be due to a genome-wide deceleration in the rate of nucleotide substitution.

Implications from a 28S rRNA gene fragment for the phylogenetic relationships of halichondrid sponges (Porifera: Demospongiae)

Halichondrida is a pivotal demosponge order of which the classification underwent major changes in the recent history. The monophyly of this order and its intra-ordinal phylogeny cannot be reliably determined on the basis of morphology. Here we present a 28Sr RNA gene tree of selected halichondrids, which supports the hypothesis of halichondrid non-monophyly and elucidates further inter-ordinal relationships. We enlarged the analysis by previously published sequences, discuss how previous analyses suffer from taxon bias and analyse the resulting phylogenetic implications. Most halichondrid families (in particular Axinellidae und Dictyonellidae) cluster polyphyletic and the molecular classification of several genera does not agree with the current (morphological) system. Zusammenfassung Die Systematik der Ordnung Halichondrida ist von Bedeutung fur die Phylogenie aller Demospongien und hat in den letzten Jahren grose Veranderungen vollzogen. Dennoch konnte die Monophylie dieser Ordnung und ihrer Familien noch nicht mit morphologischen Merkmalen deutlich gemacht werden. Hier prasentieren und diskutieren wir eine 28SrDNA-Phylogenie reprasentativer Halichondrien. Teils wurde diese Stammbaumrekonstruktion durch Sequenzen aus anderen Arbeiten erweitert und der Einfluss des Taxonsets auf das Resultat analysiert. Die Monophylie der Ordnung wird durch diese Fragmentanalyse nicht gestutzt, und mehrere Familien (insbesondere Axinellidae und Dictyonellidae) gruppieren polyphyletisch. Die Position mehrerer Gattungen, insbesondere Axinyssa und Stylissa, ist nicht im Einklang mit dem aktuellen (morphologischen) System.

Bayesian and maximum likelihood phylogenetic analyses of protein sequence data under relative branch-length differences and model violation

BackgroundBayesian phylogenetic inference holds promise as an alternative to maximum likelihood, particularly for large molecular-sequence data sets. We have investigated the performance of Bayesian inference with empirical and simulated protein-sequence data under conditions of relative branch-length differences and model violation.ResultsWith empirical protein-sequence data, Bayesian posterior probabilities provide more-generous estimates of subtree reliability than does the nonparametric bootstrap combined with maximum likelihood inference, reaching 100% posterior probability at bootstrap proportions around 80%. With simulated 7-taxon protein-sequence datasets, Bayesian posterior probabilities are somewhat more generous than bootstrap proportions, but do not saturate. Compared with likelihood, Bayesian phylogenetic inference can be as or more robust to relative branch-length differences for datasets of this size, particularly when among-sites rate variation is modeled using a gamma distribution. When the (known) correct model was used to infer trees, Bayesian inference recovered the (known) correct tree in 100% of instances in which one or two branches were up to 20-fold longer than the others. At ratios more extreme than 20-fold, topological accuracy of reconstruction degraded only slowly when only one branch was of relatively greater length, but more rapidly when there were two such branches. Under an incorrect model of sequence change, inaccurate trees were sometimes observed at less extreme branch-length ratios, and (particularly for trees with single long branches) such trees tended to be more inaccurate. The effect of model violation on accuracy of reconstruction for trees with two long branches was more variable, but gamma-corrected Bayesian inference nonetheless yielded more-accurate trees than did either maximum likelihood or uncorrected Bayesian inference across the range of conditions we examined. Assuming an exponential Bayesian prior on branch lengths did not improve, and under certain extreme conditions significantly diminished, performance. The two topology-comparison metrics we employed, edit distance and Robinson-Foulds symmetric distance, yielded different but highly complementary measures of performance.ConclusionsOur results demonstrate that Bayesian inference can be relatively robust against biologically reasonable levels of relative branch-length differences and model violation, and thus may provide a promising alternative to maximum likelihood for inference of phylogenetic trees from protein-sequence data.

Mitochondrial Genes and Mammalian Phylogenies: Increasing the Reliability of Branch Length Estimation

Since branch lengths provide important information about the timing and the extent of evolutionary divergence among taxa, accurate resolution of evolutionary history depends as much on branch length estimates as on recovery of the correct topology. However, the empirical relationship between the choice of genes to sequence and the quality of branch length estimation remains ill defined. To address this issue, we evaluated the accuracy of branch lengths estimated from subsets of the mitochondrial genome for a mammalian phylogeny with known subordinal relationships. Using maximum-likelihood methods, we estimated branch lengths from an 11-kb sequence of all 13 protein-coding genes and compared them with estimates from single genes (0.2-1.8 kb) and from 7 different combinations of genes (2-3.5 kb). For each sequence, we separated the component of the log-likelihood deviation due to branch length differences associated with alternative topologies from that due to those that are independent of the topology. Even among the sequences that recovered the same tree topology, some produced significantly better branch length estimates than others did. The combination of correct topology and significantly better branch length estimation suggests that these gene combinations may prove useful in estimating phylogenetic relationships for mammalian divergences below the ordinal level. Thus, the proper choice of genes to sequence is a critical factor for reliable estimation of evolutionary history from molecular data.

Chloroplast microsatellites reveal population genetic diversity in red pine, Pinus resinosa Ait.

Variation in paternally inherited chloroplast microsatellite (cpSSR) DNA was used to study population genetic structure in red pine (Pinus resinosa Ait.), a species characterized by morphological uniformity, no allozyme variation, and limited RAPD variation. Using nine cpSSR loci, a total of 23 chloroplast haplotypes and 25 cpSSR alleles were found among 159 individuals surveyed in seven widely separated populations. The total genetic diversity, HT, was 0.618, but haplotype differentiation among populations was low (GST = 0.121). All populations were distinguished from each other by their haplotype compositions, and only one haplotype was common among all populations. Based on average squared composite cpSSR length differences (stepwise haplotypes), within-population diversity was relatively high for only one population (D 2 SH = 0.443). Frequency distributions of pairwise SSR differences among individuals within different populations, as well as branch length differences in neighbour-joining dendrograms, indicated recovery from one or more population bottlenecks, and may be explained by metapopulation dynamics.

Testing the generation time hypothesis using DNA/DNA hybridization between artiodactyls

AbstractHeterogeneous rates of molecular change between some mammalian lineages are commonly explained by contrasts in generation time length. Here the generation time hypothesis is tested by comparing the relative rates of molecular change in related artiodactyl taxa differing by their generation time. A demographic model based on allometric relations with the adult body weight is used to estimate the cohort generation time in Bovidae and Cervidae families (Artiodactyla, Mammalia). Two pairs of closely related taxa (two cervids, two bovids) were selected, each showing clear ratios (1.5 to 3.5 times) in their generation time. Rates of genetic change in non‐repeated nuclear DNA were estimated by DNA/DNA hybridization experiments performed among these ruminants and a camelid outgroup. Relative rate tests were applied to the two pairs of ingroup taxa differing by their generation time, in order to test if shorter generation time would correspond to higher rate of molecular change. Contradictory statistical results did not show a greater accumulation of nucleotide changes in the lineage leading to the short generation time species. The recorded differences in branch lengths of sister taxa were either conflicting or too small (relative to the contrasted generation times) to reveal a generation time effect. Alternative hypotheses are suggested to explain these preliminary results.