Phylogeny and temporal divergence of the seagrass family Zosteraceae using one nuclear and three chloroplast loci

Seagrasses are among the most productive habitats in the marine realm, performing several crucial physical and biological ecosystem services. One group of seagrasses is the family Zosteraceae, which includes three to four genera and >20 species inhabiting temperate waters of both the northern and southern hemisphere. Species delineation depends on the type of data used, ranging from morphological to molecular. The main goal of this study was to better understand the evolution and divergence within the family, using a broad taxon sampling (>90 individuals) representing all species across the entire biogeographical range in both hemispheres and a four-locus approach (ITS1, matK, rbcL, psbA-trnH). The concatenated four-locus analysis supported earlier studies showing four genera in the family: Phyllospadix, Zostera, Nanozostera and Heterozostera. Four species were resolved within the genus Zostera, four within Nanozostera and two within Heterozostera. No distinction was revealed between H. nigracaulis (Australia) and H. chiliensis (Chile), suggesting a very recent introduction to Chile. A time-calibrated phylogeny using the rbcL gene revealed an early divergence of Zostera-Nanozostera/Heterozostera at 14.4 Ma, followed by a late Miocene radiation of Nanozostera-Heterozostera at 6.4 Ma, and the H. polychalymas-H. nigracaulis/tasmanica/chiliensis split at 2.3 Ma. Zostera asiatica diverged from other species of Zostera at 4.6 Ma. Phylogenetic analyses indicated that matK was the most informative single locus, whereas psbA-trnH (a widely used barcoding locus) was unable to resolve any entities within the Zosteraceae. A commonly used barcoding combination for plants, rbcL/matK, distinguished all genera, but was unable to resolve several species.