Models have always been central to inferring molecular evolution and to reconstructing phylogenetic trees. Their use typically involves the development of a mechanistic framework reflecting our understanding of the underlying biological processes, such as nucleotide substitu- tions, and the estimation of model parameters by maximum likelihood or Bayesian inference. However, deriving and optimizing the likelihood of the data is not always possible under complex evolutionary scenarios or even tractable for large datasets, often leading to unrealistic simplifying assumptions in the fitted models. To overcome this issue, we coupled stochastic simulations of genome evolution with a new supervised deep learning model to infer key parameters of molecular evolution. Our model is designed to directly analyze multiple sequence alignments and estimate per-site evolutionary rates and divergence, without requiring a known phylogenetic tree. The accuracy of our predictions matched that of likelihood-based phylogenetic inference, when rate heterogeneity followed a simple gamma distribution, but it strongly exceeded it under more complex patterns of rate variation, such as codon models. Our approach is highly scalable and can be efficiently applied to genomic data, as we showed on a dataset of 26 million nucleotides from the clownfish clade. Our simulations also showed that the integration of per-site rates obtained by deep learning within a Bayesian framework led to significantly more accu- rate phylogenetic inference, particularly with respect to the estimated branch lengths. We thus propose that future advancements in phylogenetic analysis will benefit from a semi-supervised learning approach that combines deep-learning estimation of substitution rates, which allows for more flexible models of rate variation, and probabilistic inference of the phylogenetic tree, which guarantees interpretability and a rigorous assessment of statistical support.

BACKGROUND: Independent origins of carnivory in multiple angiosperm families are fabulous examples of convergent evolution using a diverse array of life forms and habitats. Previous studies have indicated that carnivorous plants have distinct evolutionary trajectories of plastid genome (plastome) compared to their non-carnivorous relatives, yet the extent and general characteristics remain elusive.
RESULTS: We compared plastomes from 9 out of 13 carnivorous families and their non-carnivorous relatives to assess carnivory-associated evolutionary patterns. We identified inversions in all sampled Droseraceae species and four species of Utricularia, Pinguicula, Darlingtonia and Triphyophyllum. A few carnivores showed distinct shifts in inverted repeat boundaries and the overall repeat contents. Many ndh genes, along with some other genes, were independently lost in several carnivorous lineages. We detected significant substitution rate variations in most sampled carnivorous lineages. A significant overall substitution rate acceleration characterizes the two largest carnivorous lineages of Droseraceae and Lentibulariaceae. We also observe moderate substitution rates acceleration in many genes of Cephalotus follicularis, Roridula gorgonias, and Drosophyllum lusitanicum. However, only a few genes exhibit significant relaxed selection.
CONCLUSIONS: Our results indicate that the carnivory of plants have different effects on plastome evolution across carnivorous lineages. The complex mechanism under carnivorous habitats may have resulted in distinctive plastome evolution with conserved plastome in the Brocchinia hechtioides to strongly reconfigured plastomes structures in Droseraceae. Organic carbon obtained from prey and the efficiency of utilizing prey-derived nutrients might constitute possible explanation.

Coding sequence evolution is influenced by both natural selection and neutral evolutionary forces. In many species, the effects of mutation bias, codon usage, and GC-biased gene conversion (gBGC) on gene sequence evolution have not been detailed. Quantification of how these forces shape substitution patterns is therefore necessary to understand the strength and direction of natural selection. Here, we used comparative genomics to investigate the association between base composition and codon usage bias on gene sequence evolution in butterflies and moths (Lepidoptera), including an in-depth analysis of underlying patterns and processes in one species, Leptidea sinapis. The data revealed significant G/C to A/T substitution bias at third codon position with some variation in the strength among different butterfly lineages. However, the substitution bias was lower than expected from previously estimated mutation rate ratios, partly due to the influence of gBGC. We found that A/T-ending codons were overrepresented in most species, but there was a positive association between the magnitude of codon usage bias and GC-content in third codon positions. In addition, the tRNA-gene population in L. sinapis showed higher GC-content at third codon positions compared to coding sequences in general and less overrepresentation of A/T-ending codons. There was an inverse relationship between synonymous substitutions and codon usage bias indicating selection on synonymous sites. We conclude that the evolutionary rate in Lepidoptera is affected by a complex interaction between underlying G/C -> A/T mutation bias and partly counteracting fixation biases, predominantly conferred by overall purifying selection, gBGC, and selection on codon usage.

The mitochondrial genomes of Bilateria are relatively conserved in their protein-coding, rRNA, and tRNA gene complement, but the order of these genes can range from very conserved to very variable depending on the taxon. The supposedly conserved gene order of Annelida has been used to support the placement of some taxa within Annelida. Recently, authors have cast doubts on the conserved nature of the annelid gene order. Various factors may influence gene order variability including, among others, increased substitution rates, base composition differences, structure of noncoding regions, parasitism, living in extreme habitats, short generation times, and biomineralization. However, these analyses were neither done systematically nor based on well-established reference trees. Several focused on only a few of these factors and biological factors were usually explored ad-hoc without rigorous testing or correlation analyses. Herein, we investigated the variability and evolution of the annelid gene order and the factors that potentially influenced its evolution, using a comprehensive and systematic approach. The analyses were based on 170 genomes, including 33 previously unrepresented species. Our analyses included 706 different molecular properties, 20 life-history and ecological traits, and a reference tree corresponding to recent improvements concerning the annelid tree. The results showed that the gene order with and without tRNAs is generally conserved. However, individual taxa exhibit higher degrees of variability. None of the analyzed life-history and ecological traits explained the observed variability across mitochondrial gene orders. In contrast, the combination and interaction of the best-predicting factors for substitution rate and base composition explained up to 30% of the observed variability. Accordingly, correlation analyses of different molecular properties of the mitochondrial genomes showed an intricate network of direct and indirect correlations between the different molecular factors. Hence, gene order evolution seems to be driven by molecular evolutionary aspects rather than by life history or ecology. On the other hand, variability of the gene order does not predict if a taxon is difficult to place in molecular phylogenetic reconstructions using sequence data or not. We also discuss the molecular properties of annelid mitochondrial genomes considering canonical views on gene evolution and potential reasons why the canonical views do not always fit to the observed patterns without making some adjustments. [Annelida; compositional biases; ecology; gene order; life history; macroevolution; mitochondrial genomes; substitution rates.].

BACKGROUND: Plastomes of heterotrophic plants have been greatly altered in structure and gene content, owing to the relaxation of selection on photosynthesis-related genes. The orchid tribe Gastrodieae is the largest and probably the oldest mycoheterotrophic clade of the extant family Orchidaceae. To characterize plastome evolution across members of this key important mycoheterotrophic lineage, we sequenced and analyzed the plastomes of eleven Gastrodieae members, including representative species of two genera, as well as members of the sister group Nervilieae.
RESULTS: The plastomes of Gastrodieae members contain 20 protein-coding, four rRNA and five tRNA genes. Evolutionary analysis indicated that all rrn genes were transferred laterally and together, forming an rrn block in the plastomes of Gastrodieae. The plastome GC content of Gastrodia species ranged from 23.10% (G. flexistyla) to 25.79% (G. javanica). The plastome of Didymoplexis pallens contains two copies each of ycf1 and ycf2. The synonymous and nonsynonymous substitution rates were very high in the plastomes of Gastrodieae among mycoheterotrophic species in Orchidaceae and varied between genes.
CONCLUSIONS: The plastomes of Gastrodieae are greatly reduced and characterized by low GC content, rrn block formation, lineage-specific reconfiguration and gene content, which might be positively selected. Overall, the plastomes of Gastrodieae not only serve as an excellent model for illustrating the evolution of plastomes but also provide new insights into plastome evolution in parasitic plants.

Considering the phylogenetic differences in the taxonomic framework of the Chaetophorales as determined by the use of nuclear molecular markers or chloroplast genes, the current study was the first to use phylotranscriptomic analyses comparing the transcriptomes of 12 Chaetophorales algal species. The results showed that a total of 240,133 gene families and 143 single-copy orthogroups were identified. Based on the single-copy orthogroups, supergene analysis and the coalescent-based approach were adopted to perform phylotranscriptomic analysis of the Chaetophorales. The phylogenetic relationships of most species were consistent with those of phylogenetic analyses based on the chloroplast genome data rather than nuclear molecular markers. The Schizomeriaceae and the Aphanochaetaceae clustered into a well-resolved basal clade in the Chaetophorales by either strategy. Evolutionary analyses of divergence time and substitution rate also revealed that the closest relationships existed between the Schizomeriaceae and Aphanochaetaceae. All species in the Chaetophorales exhibited a large number of expanded and contracted gene families, in particular the common ancestor of the Schizomeriaceae and Aphanochaetaceae. The only terrestrial alga, Fritschiella tuberosa, had the greatest number of expanded gene families, which were associated with increased fatty acid biosynthesis. Phylotranscriptomic and evolutionary analyses all robustly identified the unique taxonomic relationship of Chaetophorales consistent with chloroplast genome data, proving the advantages of high-throughput data in phylogeny.

An accurate timescale of evolutionary history is essential to testing hypotheses about the influence of historical events and processes, and the timescale for evolution is increasingly derived from analysis of DNA sequences. But variation in the rate of molecular evolution complicates the inference of time from DNA. Evidence is growing for numerous factors, such as life history and habitat, that are linked both to the molecular processes of mutation and fixation and to rates of macroevolutionary diversification. However, the most widely used methods rely on idealised models of rate variation, such as the uncorrelated and autocorrelated clocks, and molecular dating methods are rarely tested against complex models of rate change. One relationship that is not accounted for in molecular dating is the potential for interaction between molecular substitution rates and speciation, a relationship that has been supported by empirical studies in a growing number of taxa. If these relationships are as widespread as current evidence suggests, they may have a significant influence on molecular dates.
We simulate phylogenies and molecular sequences under three different realistic rate variation models-one in which speciation rates and substitution rates both vary but are unlinked, one in which they covary continuously and one punctuated model in which molecular change is concentrated in speciation events, using empirical case studies to parameterise realistic simulations. We test three commonly used \"relaxed clock\" molecular dating methods against these realistic simulations to explore the degree of error in molecular dates under each model. We find average divergence time inference errors ranging from 12% of node age for the unlinked model when reconstructed under an uncorrelated rate prior using BEAST 2, to up to 91% when sequences evolved under the punctuated model are reconstructed under an autocorrelated prior using PAML.
We demonstrate the potential for substantial errors in molecular dates when both speciation rates and substitution rates vary between lineages. This study highlights the need for tests of molecular dating methods against realistic models of rate variation generated from empirical parameters and known relationships.

With some 7300 species of small nonvascular spore-producing plants, liverworts represent one of the major lineages of land plants. Although multi-locus molecular phylogenetic studies have elucidated relationships of liverworts at different taxonomic categories, the backbone phylogeny of liverworts is still to be fully resolved, especially for the placement of Ptilidiales and the relationships within Jungermanniales and Marchantiales. Here, we provided phylogenomic inferences of liverworts based on 42 newly sequenced and 24 published liverwort plastid genomes representing all but two orders of liverworts, and characterized the evolution of the plastome in liverworts. The structure of the plastid genome is overall conserved across the phylogeny of liverworts, with only two structural variants detected from simple thalloids, besides 18 out of 43 liverwort genera showing intron variations in their plastomes. Complex thalloid liverworts maintain the most plastid genes, and seem to undergo fewer gene deletions and pseudogenization events than other liverworts. Plastid phylogenetic inferences yielded mostly robustly supported relationships, and consistently resolved Ptilidiales as the sister to Porellales. The relative ratio of silent substitutions across the three genetic compartments (i.e., 1:15:10, for mitochondrial:plastid:nuclear) suggests that liverwort plastid genes have the potential to evolve faster than their nuclear counterparts, unlike in any other major land plant lineages where the mutation rate of nuclear genes overwhelm those of their plastid and mitochondrial counterparts.

We assembled the plastome of the temperate, Southern Hemisphere liana Muehlenbeckia australis from high throughput sequencing data (paired-end Illumina reads) generated from total genomic DNA sequencing libraries. M. australis\' chloroplast genome sequence (GenBank: MG604297) is 163,484 bp in length and composed of long single copy (LSC; 88,166 bp) and short single copy (SSC; 13,486 bp) regions flanked by inverted repeats (IR; 30,916 bp each) typical for angiosperms. The plastome includes 131 genes comprising 83 protein-coding genes, 37 transfer RNA genes, eight ribosomal RNA genes, two possible pseudogenes, psbL and rpl23 with internal stop codons, and truncated repeats of ndhF and rps19 at IR boundaries.

Cyanophora is the glaucophyte model taxon. Following the sequencing of the nuclear genome of C. paradoxa, studies based on single organelle and nuclear molecular markers revealed previously unrecognized species diversity within this glaucophyte genus. Here, we present the complete plastid (ptDNA) and mitochondrial (mtDNA) genomes of C. kugrensii, C. sudae, and C. biloba. The respective sizes and coding capacities of both ptDNAs and mtDNAs are conserved among Cyanophora species with only minor differences due to specific gene duplications. Organelle phylogenomic analyses consistently recover the species C. kugrensii and C. paradoxa as a clade and C. sudae and C. biloba as a separate group. The phylogenetic affiliations of the four Cyanophora species are consistent with architectural similarities shared at the organelle genomic level. Genetic distance estimations from both organelle sequences are also consistent with phylogenetic and architecture evidence. Comparative analyses confirm that the Cyanophora mitochondrial genes accumulate substitutions at 3-fold higher rates than plastid counterparts, suggesting that mtDNA markers are more appropriate to investigate glaucophyte diversity and evolutionary events that occur at a population level. The study of complete organelle genomes is becoming the standard for species delimitation and is particularly relevant to study cryptic diversity in microbial groups.