DNA Barcoding And Phylogeny Of Neogastropoda

The order Neogastropoda (Gastropoda: Caenogastropoda) represents a species rich (approx.16,000living species) marine invertebrate gastropod group and has adapted to almost every marineenvironment. It contains many well-known, diverse, and ecologically significant families (such asMuricidae, Nassariidae, Turridae and Littorinidae) and has a well-established morphologicaltaxonomic system. However, the identification of neogastropod taxa is often difficult since themorphological characters (shell characters and the anatomy of the digestive system) that speciesidentification bases on are not only varied within groups but also easily to be impacted byenvironment.DNA barcoding has been proposed as a method that will make species identification faster andmore accessible using a small fragment of DNA sequence, particularly in species with complexaccessible morphology, and has been successfully used in animals, plants and microorganism. Thisstudy employs DNA barcoding and some molecular markers to study the species identification andphylogeny of Neogastropoda, especially for the economic species of Muricidae and Nassarius.1. DNA barcoding and phylogeny of MuricidaeThis study employs a DNA barcoding method to identify17known and easily confused muricidspecies (120individuals) from the whole China coast based on mitochondrial cytochrome c oxidasesubunit I (COI) and16S rDNA sequences, and nuclear ITS-1and28S rRNA sequences. Thephylogeny of muricid subfamilies is also analysed based on all mitochondrial and nuclear sequences.The universal COI and16S rDNA primers did not work broadly across the study group, necessitatingthe redesign of muricid specific COI and16S rDNA primers. This study demonstrates that COI geneis a suitable marker for barcoding muricids, which can distinguish all muricid species studied.Phylogenetic analysis of16S rDNA, ITS-1and28S rRNA data also provide good support for thespecies resolution observed in COI data. The relationships of muricid subfamilies are resolved based on the separate and combined gene data that showed the monophyly of each the subfamiliesErgalataxinae, Rapaninae, Ocenebrinae and Muricinae, especially that Ergalataxinae did not fallwithin Rapaninae.2. Cryptic diversity in NassariusThis study reported a comprehensive barcoding analysis of22Nassarius species. Themitochondrial and nuclear sequences and the morphological characters are integrated to determine13Nassarius species studied and reveal four cryptic species and one pair synonyms. Distance,monophyly, and character–based barcoding methods were employed. Such successful identificationand unexpected cryptic discovery is significant for Nassarius in food safety and species conversationand remind us to pay more attention to the hidden cryptic biodiversity ignored in marine life. Distance,monophyly, and character–based barcoding methods are all very helpful in identification but thecharacter-based method shows some advantages.3. Molecular phylogeny of NeogastropodaThis study reports the most extensive molecular study of the group published to date with increasedneogastropod taxa, multiple representatives of caenogastropod outgroups, and additional genesequences. Data were collected from the entire18S rRNA, histone H3, and three partialmitochondrial genes. Maximum parsimony, maximum likelihood and Bayesian analyses wereconducted. In the caenogastropod phylogenetic framework, Hypsogastropoda was contradictedowing to the inclusion of Cerithioidea. Contrary to previous molecular studies, all the resultsrecovered Neogastropoda as a monophyletic group, which confirms the monophyly ofNeogastropoda and the validity of morphological synapomorphies that usually define Neogastropodaas monophyletic. Tonnoidea was shown to be paraphyletic with respect to Ficidae, and together thisgroup formed a monophyletic clade as the sister group to Neogastropoda, which supported the ‘‘highmesogastropod’’ hypothesis of the origin of Neogastropoda. All neogastropod families were strongly supported except Buccinidae, Turridae and Cancellariidae. This study shed light on the status ofNeogastropoda, a controversial group, within Caenogastropoda.4. Comparing the usefulness of DNA barcoding methodsThis study analyzed108COI and10216S rDNA sequences of40species of Neogastropoda froma wide phylogenetic range to assess the performance of distance, monophyly and character-basedmethods of DNA barcoding. The distance-based method for both COI and16S rDNA genesperformed poorly in terms of species identification. Obvious overlap between intraspecific andinterspecific divergences for both genes was found. The ‘‘10×rule’’ threshold resulted in lumpingabout half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI coulddistinguish all species studied. However, the16S rDNA tree could not distinguish some closelyrelated species. In contrast, the character-based barcode method for both genes successfully identified100%of the neogastropod species included, and performed well in discriminating neogastropodgenera. This present study demonstrates the effectiveness of the character-based barcoding method forspecies identification in different taxonomic levels, especially for discriminating the closely relatedspecies. While distance-and monophyly-based methods commonly use COI as the ideal gene forbarcoding, the character-based approach can perform well for species identification using relativelyconserved gene markers (e.g.,16S rDNA in this study). Nevertheless, distance and monophyly-basedmethods, especially the monophyly-based method, can still be used to flag species.