MORPHOLOGY AND MOLECULAR PHYLOGENETIC ANALYSIS OF FRESHWATER HILDENBRANDIA IN CHINA

This study explored morphological observation and molecular phylogenetic analysis of two freshwater Hildenbrandia specimens from Yuxi, Yunnan Province (encoded as YZYUNNAN) and Jiaozuo, Henan Province (encoded as YZHENAN) in China. Morphological observation and molecular phylogenetic analysis were conducted on these specimens. Phylogenetic relationship of genus Hildenbrandia was constructed based on the obtained gene sequence and related molecular data previously reported in China. Ancestral geographical origin of freshwater Hildenbrandia was inferred based on reliable phylogenetic relationship and modern distribution pattern of each sample. The results are consistent using Bayesian Inference, Maximum Likelihood and Neighbor-joining based on rbcL, psbA and UPA sequences. The genetic distances of three molecular sequences between specimens collected and H. jigongshanensis were smaller compared with another freshwater species. The uncorrected p-distances of rbcL sequences between specimens and H. jigongshanensis were within intraspecific divergence of freshwater Hildenbrandia species. Phylogenetic trees showed that specimens YZYUNNAN and YZHENAN formed an independent branch with H. jigongshanensis, and obtained high supporting values. Specimens YZYUNNAN and YZHENAN shared close relationship with H. jigongshanensis based on molecular phylogeny. The morphological measurements of collected specimens were in consistent with another freshwater Hildenbrandia species H. rivularis. It has been acknowledged that the widely reported freshwater species H. angolensis and H. rivularis are distinguished by morphometric measurements, with H. angolensis characterized by smaller mean cell and filament dimensions (cells 4.0×4.4 µm, filaments 46.5 µm) and H. rivularis by larger parameters (cells 5.8×6.6 µm, filaments 55.3 µm). H. jigongshanensis is separated from other freshwater members by significantly larger cell dimensions (9.8—19.6)×(9.9—10.4) µm and filament height (364—409 µm). The morphological measurements of H. japananense, the cell size (8.4×10.5 μm) and filament height (308—491 μm) were overlapping with H. jigongshanensis whereas identified as a new species based on molecular sequences. with (5.0—8.0)×(4.0—6.7) μm and (4.8—8.0)×(4.8—6.4) μm in cellular diameter and 42—50 μm in thallus height, respectively. There exits contradiction in species identification based on traditional morphological taxonomy and modern molecular methods. By referring the criteria of species identification in other freshwater red algal taxa, the specimens YZYUNNAN and YZHENAN were determined to be H. jigongshanensis based on molecular evidences. The molecular phylogenetic study indicated H. jigongshanensis was firstly described from specimens collected from Jigongshan Mountain, Henan Province, China and follow reported in Niangziguan, Shanxi Province, China. It was found from Jiaozuo, Henan Province and Yunnan Province for the first time. This study expands the geographical distribution and genetic diversity of genus Hildenbrandia in China. Additionally, the traditional morphological characters including thallus height and cellular diameter were found variable under different environments and thus not reliable taxonomic criteria. Molecular-assisted species identification and phylogenetic analysis were necessary for genus Hildenbrandia due to its simple morphological structures. There are 4 widely accepted freshwater Hildenbrandia species up to now and H. rivularis proved monophyletic whereas H. angolensis was paraphyletic. Molecular phylogenetics in this study showed the monophyly of H. jigongshanensis and its close relationship with another freshwater species H. japananense. All freshwater Hildenbrandia species formed an independent clade from the marine species, suggesting that the freshwater Hildenbrandia was derived from the marine populations. The 4 freshwater species of Hildenbrandia were special in geographical distribution, with populations distributed in Europe were mainly H. rivularis, populations distributed in North America were mainly H. angolensis, and H. jigongshanensis and H. japananense were only reported from China and Japan respectively. Ancestral geographical origin reconstruction showed the freshwater Hildenbrandia was derived in North America and dispersed to Eastern Caribbean, Latin America and Eurasia. Sexual reproduction has not been documented for freshwater forms of Hildenbrandia and they reproduced only by asexual modes including fragmentation or gemmae, resulting in rare genetic exchange among species. It was speculated that the special species-related geographical pattern of genus Hildenbrandia was formed after dispersal events and independent evolution in each geographical area. The freshwater Hildenbrandia in North America derived ancestrally and had a long evolutionary history and rich genetic diversity, consistent with the paraphyly of the species H. angolensis distributed in this area.