DNA barcode assessment of Ceramiales(Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales （Rhodophyta） were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene （LSU）, universal plastid amplicon （UPA）, and partial mitochondrial cytochrome c oxidase subunit I gene （COl）. No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Syrnphyocladia latiuscula, and Neosiphoniajaponica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD （Barcode of Life Data） Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might he a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H.japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

Identifying the ‘unidentified’ fungi: a global-scale long-read third-generation sequencing approach

Molecular identification methods,in particular high-throughput sequencing tools,have greatly improved our knowledge about fungal diversity and biogeography,but many of the recovered taxa from natural environments cannot be identified to species or even higher taxonomic levels.This study addresses the phylogenetic placement of previously unrecognized fungal groups by using two complementary approaches:(i)third-generation amplicon sequencing analysis of DNA from global soil samples,screening out ITS reads of<90%similarity to other available Sanger sequences,and(ii)analysis of common fungal taxa that were previously indicated to be enigmatic in terms of taxonomic placement based on the ITS sequences alone(so-called top50 sequences).For the global soil samples,we chose to amplify the full rRNA gene operon using four partly overlapping amplicons and multiple newly developed primers or primer combinations that cover nearly all fungi and a vast majority of non-fungal eukaryotes.We extracted the rRNA 18S(SSU)and 28S(LSU)genes and performed phylogenetic analyses against carefully selected reference material.Both SSU and LSU analyses placed most soil sequences and top50 sequences to known orders and classes,but tens of monophyletic groups and single sequences remained outside described taxa.Furthermore,the LSU analyses recovered a few small groups of sequences that may potentially represent novel phyla.We conclude that rRNA genes-based phylogenetic analyses are efficient tools for determining phylogenetic relationships of fungal taxa that cannot be placed to any order or class using ITS sequences alone.However,in many instances,longer rRNA gene sequences and availability of both SSU and LSU reads are needed to improve taxonomic resolution.By leveraging third-generation sequencing from global soil samples,we successfully provided phylogenetic placement for many previously unidentified sequences and broadened our view on the fungal tree of life,with 10-20%new order-level taxa.In addition,the PacBio sequence data greatly extends fungal class-level information in reference databases.