3.7 Phylogenetic analysis and gene family evolution
The phylogenetic position of C. fluminea and other representative species was estimated based on single-copy orthologs. Three time points for the most recent common ancestor (MRCA) were estimated by TimeTree. The differentiation time of C. gigas and C. virginica was 72.9 (63.2 - 82.7) million years ago (Mya) (Plazzi & Passamonti, 2010); that of B. platifrons and M. coruscus was 387 (308 - 481) Mya (Peterson, Cotton, Gehling, & Pisani, 2008); that of C. fluminea and six marine bivalves was 492 (472 - 516) Mya (Stöger, et al., 2013; Huang, et al., 2018). We utilized these time of MRCA to calibrate the phylogenetic tree, resulting in the phylogenetic tree constructed by seven bivalves and four other marine species (Figure 4C). As shown, all marine bivalves were clustered together, especially those belong to the same family. The phylogenetic tree showed that C. fluminea and the closest genetic relatives, the ancestors of marine bivalves (family Mytilidae represented by B. platifronsand M. coruscus ; family Ostreidae represented by C. gigasand C. virginica; family Pteriidae represented by P. imbricata; family Pectinidae represented by M. yessoensis ) diverged ~ 492.00 million years ago. This evidence suggests that C. fluminea is a kind of bivalve, which is obviously different from marine bivalves. It is suggested that bivalves were divided into two groups, and they survive in freshwater and seawater respectively for a long time (million years). Some references showed that Heterodonta living in the freshwaters originated in the Paleozoic (Moore & Raymond, 1969; Cope & Veliger, 1995) and Veneroida diversified during the Mesozoic and Cenozoic eras (Stanley, 1968). The ancestors of C. fluminea may have invaded and migrated to freshwater from the ocean since millions of years ago, and they have evolved to fill various freshwater habitat.