We examined the phylogeny and biogeography of the glassperch family Ambassidae (Teleostei), which is widely distributed in the freshwater, brackish and marine coastal habitats across the Indo-West Pacific region. We first built a comprehensive time-calibrated phylogeny of Ambassidae using five genes. We then used this tree to reconstruct the evolution of the salinity preference and ancestral areas. Our results indicate that the two largest genera of Ambassidae, Ambassis and Parambassis, are each not monophyletic. The most recent common ancestor of Ambassidae was freshwater adapted and lived in Australia about 56 million years ago. Three independent freshwater-to-marine transitions are inferred, but no marine-to-freshwater ones. To explain the distribution of ambassids, we hypothesise two long-distance marine dispersal events from Australia. A first event was towards Southeast Asia during the early Cenozoic, followed by a second one towards Africa during mid-Cenozoic. The phylogenetic signal associated with the salinity adaptation of these events was not detected, possibly because of the selective extinction of intermediate marine lineages. The Ambassidae shares two characteristics with other freshwater fish groups distributed in continental regions surrounding the Indian Ocean: They are too young to support the hypothesis that their distribution is the result of the fragmentation of Gondwana, but they did not retain the phylogenetic signal of their marine dispersal.
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