Phylogenetic Analysis of Sea Lamprey Hox Genes

Phylogenetic analysis was performed on Hox paralog groups with 4 or more members in sea lamprey: groups 4, 8, 9, 11 and 13. For each paralog group, predicted Sea lamprey Hox protein sequences were aligned against homologs from other vertebrate species and amphioxus, retrieved from Genbank. Our approach was informed by the experiences detailed by Kuraku et al^{89 (link)}, Qiu et al^{90 (link)}, Mehta et al^{17 (link)} and Manousaki et al⁹¹ . In selecting jawed vertebrate taxa for these analyses, we avoided teleost fish and Xenopus laevis as these lineages have undergone additional genome duplication events, which can lead to their co-orthologous genes/proteins being more derived than those from non-duplicated lineages. Thus, we opted for Elephant shark (C. milii) and coelacanth (L. menadoensis) as Chondrichthian and ‘basal’ Sarcopterygian representatives respectively, both of which having slowly evolving protein-coding genes and well characterized Hox gene complements^{92 (link),93 (link)}. Urochordates are the sister group of vertebrates but the divergent nature of their Hox genes led us to favor the cephalochordate amphioxus as a source for outgroup sequences in our analyses. We chose to perform protein alignments rather than DNA alignments due to the high coding GC content in lamprey, which can result in artifactual clustering of lamprey genes in DNA trees. Nevertheless, the unique pattern of amino-acid composition in lamprey proteins is an unavoidable complicating factor that impinges on their phylogenetic analysis and can lead to artifactual clustering of lamprey proteins, as described in Qiu et al^{90 (link)}. The MEGA7^{41 (link)} software suite was used for sequence alignment, best-fit substitution model evaluation and phylogeny reconstruction. Protein alignments were performed with full available length protein sequences using MUSCLE^{41 (link)}. Best-fit substitution models were evaluated and chosen for each alignment. Maximum likelihood, neighbor joining and maximum parsimony approaches were used for phylogenetic analysis, with 100 bootstrap replicates generated for node support. For each method, all positions in the alignment containing gaps and missing data were eliminated.