Phylogenetic analysis has entered the genomics (multilocus) era. For less experienced researchers, conquering the large number of software programs required for a multilocus-based phylogenetic reconstruction can be somewhat daunting and time-consuming. PhyloSuite, a software with a user-friendly GUI, was designed to make this process more accessible by integrating multiple software programs needed for multilocus and single-gene phylogenies and further streamlining the whole process. In this protocol, we aim to explain how to conduct each step of the phylogenetic pipeline and tree-based analyses in PhyloSuite. We also present a new version of PhyloSuite (v1.2.3), wherein we fixed some bugs, made some optimizations, and introduced some new functions, including a number of tree-based analyses, such as signal-tonoise calculation, saturation analysis, spurious species identification, and etc. The step-by-step protocol includes background information (i.e., what the step does), reasons (i.e., why do the step), and operations (i.e., how to do it). This protocol will help researchers quick-start their way through the multilocus phylogenetic analysis, especially those interested in conducting organellebased analyses.

The long-prevailing paradigm of the adaptive evolution of organism complexity has been challenged in the last two decades by a competing theory that nonadaptive processes are the major driver of evolutionary patterns (Lynch, 2007;Lynch et al., 2006;Pouyet et al., 2017). Mitochondrial genomes (mitogenomes) were not spared this proposed paradigm shift, as their evolution in animals is suggested

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BackgroundSeveral studies have shown that the phylogeny of Annelida is pronouncedly data- and methodology-influenced. Inversions of the origin of replication (ORI) in mitochondrial genomes may interfere with phylogenetic reconstruction, as well as other evolutionary studies in Arthropoda. ORI events and their impacts on evolutionary analyses remain very poorly understood in other metazoan lineages. This also includes the Annelida, in which a handful of studies merely reported inverted base composition skews in isolated lineages.ResultsWe observed that some of the inverted-skew species exhibited long branches and ‘rogue’ behaviour in phylogenetic analyses. This made us hypothesise that ORI events may be a major factor interfering with phylogenetic reconstruction in Annelida. We first inferred the evolutionary history of ORI events in Annelida on a dataset comprising almost all available annelid mitogenomes (174). We identified twelve new ORI species and six ORI events in total. Most ORI events occurred at lower taxonomic levels (species, genus, family), but one was mapped to a common ancestor of sister-families Sabellidae & Serpulidae, and one to the entire suborder Hirudiniformes. In comparison to non-ORI lineages, ORI lineages exhibited a higher: mutational saturation, number of nonsynonymous substitutions, dN/dS ratios, hydropathicity of the translated gene products, and compositional heterogeneity; but a lower codon bias index. They also exhibited relaxed purifying selection pressures. We found several putative examples of ORI events causing artefactual clustering of lineages with homoplastic base composition biases in phylogenetic analyses. We attempted several different strategies aimed at stabilizing the topology, such as the removal of ORI species and third codon sites, but none of these managed to fully stabilize the topology.ConclusionsWe inferred the evolutionary history of skews in annelids and explored the underlying mechanisms by which ORI events may cause artefactual clustering. We conclude that ORI events may be one of the causes for the chaotic relationships of Annelida. In the context of currently available strategies for phylogenetic reconstruction, mitogenomes are not suitable for studying the phylum-level and class-level phylogeny of Annelida. Given the strong evidence that ORIs may interfere with evolutionary studies in annelids, researchers should pay close attention to indications of skew inversions.

Background Within the class Enoplea, the earliest-branching lineages in the phylum Nematoda, the relatively highly conserved ancestral mitochondrial architecture of Trichinellida is in stark contrast to the rapidly evolving architecture of Dorylaimida and Mermithida. To better understand the evolution of mitogenomic architecture in this lineage, we sequenced the mitogenome of a fish parasite Pseudocapillaria tomentosa (Trichinellida: Capillariidae) and compared it to all available enoplean mitogenomes. Results P. tomentosa exhibited highly reduced noncoding regions (the largest was 98 bp), and a unique base composition among the Enoplea. We attributed the latter to the inverted GC skew (0.08) in comparison to the ancestral skew in Trichinellidae (-0.43 to -0.37). Capillariidae, Trichuridae and Longidoridae (Dorylaimida) generally exhibited low negative or low positive skews (-0.1 to 0.1), whereas Mermithidae exhibited fully inverted low skews (0 to 0.05). This is indicative of inversions in the strand replication order or otherwise disrupted replication mechanism in the lineages with reduced/inverted skews. Among the Trichinellida, Trichinellidae and Trichuridae have almost perfectly conserved architecture, whereas Capillariidae exhibit multiple rearrangements of tRNA genes. In contrast, Mermithidae (Mermithida) and Longidoridae (Dorylaimida) exhibit almost no similarity to the ancestral architecture. Conclusions Longidoridae exhibited more rearranged mitogenomic architecture than the hypervariable Mermithidae. Similar to the Chromadorea, the evolution of mitochondrial architecture in enoplean nematodes exhibits a strong discontinuity: lineages possessing a mostly conserved architecture over tens of millions of years are interspersed with lineages exhibiting architectural hypervariability. As Longidoridae also have some of the smallest metazoan mitochondrial genomes, they contradict the prediction that compact mitogenomes should be structurally stable. Lineages exhibiting inverted skews appear to represent the intermediate phase between the Trichinellidae (ancestral) and fully derived skews in Chromadorean mitogenomes (GC skews = 0.18 to 0.64). Multiple lines of evidence (CAT-GTR analysis in our study, a majority of previous mitogenomic results, and skew disruption scenarios) support the Dorylaimia split into two sister-clades: Dorylaimida + Mermithida and Trichinellida. However, skew inversions produce strong base composition biases, which can hamper phylogenetic and other evolutionary studies, so enoplean mitogenomes have to be used with utmost care in evolutionary studies.