DNA Taxonomy In Marine Bivalves: DNA Barcoding In Mytilidae And Ostreidae, And Cryptic Diversity In The Pen Shell Atrina Pectinata

Accurate species delimitation and identification is a challenging problem for biologists. Adozen operational methods, mostly based on DNA sequences, have been proposed, but it isstill problematic when delimiting species. DNA barcoding has become a well-funded, globalenterprise since its proposition as a rapid and practical molecular method to identify anddiscovery species, although the latter utility has been repeatedly criticized. On the contrary, anintegrative approach using multiple lines of evidence to delimit species is increasinglypopularized. As a big, diverse group in marine realm, bivalve biodiversity has probably beenunderestimated because of a lack of scrutiny of most taxa as well as the absence of powerfultools. In the present disertation, we assessed the utility of DNA barcoding techniques in theidentification of specimens from Mytilidae and Ostreidae, and we also explored the crypticdiversity in the pen shell Atrina pectinata in combination of different sources of data tomethodologically aid studies of taxonomy, biodiversity conservation and evolution of marinetaxa.1. A test of the utility of DNA barcoding in the bivalve family MytilidaeThis study firstly tested whether the standard DNA barcoding method is suitable for speciesidentification of the bivalve family Mytilidae. Here,52samples belonging to eleven speciesof Mytilidae were analyzed using mitochondrial COI (mtCOI) gene as well as mitochondrial16S rDNA. Neighbor-joining trees of mtCOI and16S rDNA sequences using Kimura’s2-parameter (K2P) substitution model showed that most of the species were reciprocallymonophyletic with no exceptions. Pairwise K2P genetic distances among all species andwithin species were calculated in MEGA. And the results indicated that the genetic distancesbetween species showed up to>20%, much greater than distances within species when exceptional species and individuals were excluded. Mitochondrial DNA heteroplasmy wasdetected in Mytilus galloprovincialis, Musculus senhousia and Hormomya mutalilis. Ourresults suggested that DNA barcoding is a promising tool for species identification ofMytilidae, but the interpretation should be cautious in light of the doubly uniparentalinheritance of mtDNA (DUI) and mtDNA heteroplasmy.2. Identifying the true oysters (Ostreidae) with mitochondrial phylogeny anddistance-based DNA barcodingOysters (family Ostreidae), with high levels of phenotypic plasticity and wide geographicdistribution, are a challenging group for taxonomists and phylogenetics. Based on theGenBank database, the present study used two mitochondrial fragments, mtCOI and16SrDNA, to assess whether oyster species could be identified by phylogeny and distance-basedDNA barcoding techniques. Relationships among species were estimated by the phylogeneticanalyses of both genes, and then pairwise K2P inter-and intraspecific genetic divergenceswere assessed. Species forming well differentiated clades in the molecular phylogenies wereidentical for both genes even when the closely related species were included. Intraspecificvariability of16S rDNA overlapped with interspecific divergence. However, average intra-and interspecific genetic divergences for mtCOI were0–1.4%(maximum2.2%) and2.6–32.2%(minimum2.2%), respectively, indicating the existence of a barcoding gap. Theseresults confirm the efficacy of species identification in oysters via DNA barcodes andphylogenetic analysis.3. Cryptic diversity and hybridization revealed by multiple sources of data in AtrinapectinataCryptic species have been increasingly revealed in the marine realm through an analyticalapproach incorporating multiple lines of evidence (e.g., mtDNA, nuclear genes andmorphology). Illustrations of cryptic taxa improve our understanding of species diversity andevolutionary histories within marine animals. The pen shell Atrina pectinata is known toexhibit extensive morphological variations that may harbour cryptic diversity. In this study,we investigated16A. pectinata populations along the coast of China and one from Japan to explore possible cryptic diversity and hybridization using a combination of mitochondrial(mtCOI) and nuclear (ribosomal internal transcribed spacer, nrITS) genes as well asmorphology. Phylogenetic analyses of mtCOI ‘DNA barcoding gene’ sequences resolved sixdivergent lineages with intralineage divergences between0.4%and0.8%. Interlineagesequence differences ranged from4.3%to22.0%, suggesting that six candidate crypticspecies are present. The nrITS gene revealed five deep lineages with Kimura2-parameterdistances of3.7–30.3%. The five nuclear lineages generally corresponded to mtCOI lineages1–4and (5+6), suggestive of five distinct evolutionary lineages. Multiple nrITS sequences ofsignificant variance were found within an individual, clearly implying recent hybridizationevents between among the evolutionary lineages, which contributed to cytonucleardiscordance. Morphologically, five morphotypes matched the five genetic lineages, althoughthe intermediates may well blur the boundaries of different morphotypes. This studydemonstrates the importance of combining multiple lines of evidence to explore speciescryptic diversity and past evolutionary histories.