DNA Barcoding Assisting Identification Of Rhodophyta In The Intertidal Zone Of Shandong Peninsula

Traditional taxonomy of Rhodophyta mainly depends on morphologicalcharacter of algae, reproductive way and life history type. However, the red algaehave a high degree of variability in intraspecific and interspecific morphology, whichoften change a lot when algae are suffered from different environmental impact, andin their different life periods. It leads to lots of controversies about the classificationof many species for a long time. Therefore, only a few experts can accuratelydistinguish from similar species. This research used the morphology method andDNA barcode technology to identify the red algae which collected from intertidalzone in Shandong Peninsula.Total600samples of the algae (Rhodophyta) were collected from October2011to December2012in intertidal zone around Shandong, China. Firstly, we identifiedby morphological method based on some special characters and microscopic structure.Referring illustrated handbooks and literatures, the samples were identified as7orders,16families,30genera and35species, but there were still some samples thatcould not been accurately identified, and only could be confirmed belong to20orders,families or genera.DNA barcoding was used in identification of these samples assistingmorphological method. Three DNA markers were chosen, LSU (partial28S rRNAgene), UPA (partial23S rRNA gene, universal plastid amplicon) and COI (partialcytochrome c oxidase I gene).527sequences were obtained for LSU,515sequencesfor UPA and332sequences for COI, the amplification efficiency of LSU and UPAmarkers was greater than COI. Among the three markers, LSU was the mostconservative, COI showed higher discrimination ability than LSU and UPA. LSU hadno intraspecific divergence and interspecific divergence was0.002-0.160; For UPA,intraspecific divergence was0-0.003, interspecific divergence was0.011-0.123; COI had largest divergences with0.002-0.008, intraspecific and0.144-0.238interspecificdivergences.DNA barcoding using the three markers powerfully assisted taxonomicidentification. In NJ trees of the three markers,600samples were divided into56species, belonging to7orders,20families,40genera. Except LSU, all sequencesexhibited appropriate divergence among Rhodophyta species on the UPA and COIphylogenetic trees. Three specimen (representative as QDHQW20111124-R23), dueto lack of their reproductive structure characteristics, which hard identified onmorphologically could be classified to Heterosiphonia crispella by all three NJ trees,but we found that there is no distribution in China, therefore, this species maybe acryptic or invasive species in investigated region. Heterosiphonia japonicai wasindicated that more closely related to Dasya than to Heterosiphonia.The result showed using UPA and COI can distinguish red algae speciessuccessfully, LSU was not suitable to discriminate some species for its mostconservative, but it can be used at family or genus level. The COI has the largestbarcoding gap, but due to its hardest in amplification and sequencing, appropriateprimers of this gene should be explored. UPA and COI were recommended to beapplied as complementary markers. In generally, morphological and molecularmethods would better to be used together.