Fig. 4

Timetree analysis of advanced members of the colonial volvocine algae including the Thai strains of Volvox africanus. Note that the divergence time between heterothallic Volvox reticuliferus and homothallic V. africanus and the divergence between Thai and Japanese strains of V. africanus are “11.44 (8.48–15.43) MYA” and “5.04 (2.84–8.95) MYA”, respectively. Species names in orange or black indicate homothallic or heterothallic sexuality, respectively. Tree topology was inferred by maximum likelihood (ML) analyses of 6021 base pairs of five chloroplast genes from 47 operational taxonomic units [TreeBASE (Vos et al. 2010, 2012) ID 28191] with a model selected by MEGA X (Kumar et al. 2018). Asterisks on branches indicate 80% or more bootstrap values (based on 1000 replicates) by the ML analyses. A timetree was inferred by applying the RelTime method (Tamura et al. 2012, 2018) to the ML phylogenetic tree whose branch lengths were calculated using the ML method and the General Time Reversible substitution model (Nei and Kumar 2000). The timetree was computed using two calibration constraints [C1 (65–90 MYA) and C2 (50–90 MYA)] based on TimeTree: the Time Scale of Life <http://www.timetree.org/> (Liss et al. 1997; Herron et al. 2009). Outgroup was designated as in the previous study (Yamamoto et al. 2021). Evolutionary analyses were conducted in MEGA X (Kumar et al. 2018)