![]() This maze of steps, algorithms, and file formats can be daunting and time-consuming for many scientists. Finally, these programs often use very different input and output file formats. for phylogenetic tree reconstruction, commonly used programs are IQ-TREE, RAxML, MrBayes, etc. For example, for sequence alignment, there are MAFFT, MUSCLE, PRANK, and etc. For each of these steps, there are commonly different software programs available to choose from. Furthermore, conducting multilocus phylogenetic analyses comprises a large number of steps: selecting and downloading sequences, filtrating sequences, preparing gene sequences (extracting genes from a multilocus dataset, such as organelle genomes or identifying orthologous loci from genomic data), sequence alignment, alignment trimming (optional), alignment concatenation, selecting optimal partitioning schemes, selecting optimal substitution models, phylogenetic tree inference (often using several different algorithms to assess the topological stability), and finally visualization and annotation of the phylogenetic tree. A large number of molecular phylogenetics-related algorithms and software programs have been developed to address these developments, but they are often complicated and confusing for beginners, experimental biologists, and in general insufficiently computer-savvy researchers. For example, the application of multilocus phylogeny (inferring phylogenetic trees using multiple loci or genes) has provided new insights into many historically controversial relationships as well as contributed to our understanding of the evolutionary history of life on earth. Aside from the traditional objective of inferring evolutionary relationships among different lineages, the rapid generation of genomic data in the last two decades has facilitated the application of molecular phylogenetics to various aspects of biological sciences, such as population changes, migration patterns, the adaptive evolution of species associated with specific environments, and etc. Molecular phylogenetics aims to reconstruct the evolutionary history of life using genetic markers, such as nucleotide and amino acid sequences. This protocol will help researchers quick-start their way through the multilocus phylogenetic analysis, especially those interested in conducting organelle-based analyses. 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). 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-to-noise calculation, saturation analysis, spurious species identification, and etc. In this protocol, we aim to explain how to conduct each step of the phylogenetic pipeline and tree-based analyses in PhyloSuite. 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. 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. ![]() Phylogenetic analysis has entered the genomics (multilocus) era. ![]()
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