Comparing the Usefulness of Distance, Monophyly and Character-Based DNA Barcoding Methods in Species Identification: A Case Study of Neogastropoda

DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species

Comparative Transcriptome Analysis of Toxic and Non-Toxic Nassarius Communities and Identification of Genes Involved in TTX-Adaptation

Nassarius has caused serious people poisoning and death incident as a popular food due to tetrodotoxin (TTX) accumulation in their body. Understanding the genetic basis of tetrodotoxin (TTX) transformation and resistance in animals could lead to significant insights into adaptive evolution to toxins and toxin poisoning cures in medicine. Here we performed comparative transcriptome analysis for toxic and non-toxic communities in Nassarius succinctus and Nassarius variciferus to reveal their genetic expression and mutation patterns. For both species, the cellular and metabolic process, and binding and catalytic activity accounted for the top classification categories, and the toxic communities generally produced more up-regulated genes than non-toxic communities. Most unigenes and different expression genes were related to disease, e.g., heat shock protein and tissue factor pathway inhibitors, which involve detoxification and coagulation. In mutation levels, the sodium channel gene of N. succinctus had one amino acid mutation “L”, which is different from that of other animals. In conclusion, the comparative transcriptome analysis of different species and populations provided an important genetic basis for adaptive evolution to toxins, health and toxin poisoning cure research for TTX in marine gastropoda mollusk. Future studies will focus on the action mechanism of the important functional gene for TTX accumulation and resistance

Sequences of the primers used in the PCRs.

<p>Sequences of the primers used in the PCRs.</p

Bayesian tree of the 16S rDNA locus.

<p>Posterior probabilities and bootstrap values were included. The number of individuals included in each species was shown in brackets by the species name. Species showing cryptic diversity were marked in red. Species that could be identified as synonyms were marked in blue.</p

Bayesian tree of the COI locus.

<p>Posterior probabilities and bootstrap values were included. The number of individuals included in each species was shown in brackets by the species name. Species showing cryptic diversity were marked in red. Species that could be identified as synonyms were marked in blue. Representative shells of species available were illustrated.</p

Frequency distribution of COI intraspecific and interspecific (congeneric) K2P distances in <i>Nassarius</i>.

<p>Frequency distribution of COI intraspecific and interspecific (congeneric) K2P distances in <i>Nassarius</i>.</p

Sampling sites in this study.

<p>The letter codes correspond to geographic locations listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047276#pone.0047276.s001" target="_blank">table S1</a>.</p

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta)

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella–like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential “specific barcode” for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes.Waller Creek Working Grou