DNA Barcoding And Molecular Phylogeny Of The Pteriina And Arcoida

DNA barcoding is attracting the wide attention as a new method to indentify species. It canpropose a solution to the limitations of the traditional morphological identification andpromote the development of the biodiversity studies. DNA barcoding has been usedsuccessfully in many animal groups since it was proposed. But the practice of barcoding hasalso received much criticism. DNA sequences enable not only the identification of specieseffectively, but also the inference of phylogenetic relationships, i.e., molecular systematics.The development of molecular systematics can help us understand the evolutionaryrelationships among species profoundly. The Pteriina is the commercially important group inbivalve molluscs and many species of the Arcoida have been cultured and exploited, but fewstudies focus on the biodiversity and phylogenetics of the two groups. This study assesses theutility of DNA barcoding, then applies the DNA barcoding to the Pteriina and Arcoida species,and finally presents the phylogenetic relationships of the two groups using multiple molecularmarkers. The main results are listed as follows:1. DNA barcoding distinguishes the species of the family PectinidaeWe have sequenced partial sequences of mitochondrial COI and16S rRNA genes from63specimens of8species of Pectinidae. Sequences from homologous regions of four otherspecies of this family were gathered from GenBank. Comparisons of within and betweenspecies levels of sequence divergence showed that the genetic distances, within and betweenspecies of COI sequences, ranged from0.000to0.020(average0.0048) and from0.133to0.344(average0.284), respectively, and that of16S sequences varied from0.000to0.008(average0.001) and from0.053to0.309(average0.231), respectively. These indicate thegenetic variation between species exceeds variation within species either based on COI or16Sgene. The so-called ‘‘barcoding gap’’ exist. The neighbour-joining trees based on COI and16S genes showed that all species fell into reciprocally monophyletic clades with high bootstrap values. All the results above evidence that these scallop species can be efficientlyidentified by DNA barcoding. Evolutionary relationships of Pectinidae were also examinedusing Bayesian inference approach. The results are almost consistent with Waller’sclassification, though differences emerged both at the subfamily and the genus level, whichwas proposed on the basis of shell microstructure and the morphological characteristics ofjuveniles.2. DNA barcoding of the PteriinaWe have sequenced the COI gene and16S rRNA gene from75individuals of the Pteriina.The K2P genetic distances between sequences were calculated and the phylogenetic treeswere constructed. The results showed that the individuals identified as Pinctada sp.1andPinctada sp.2should be the species Pinctada chemnitzi, the two individuals named asIsognomon sp. and the one individual named as Plicatula sp. should be the species Isognomonperna and Plicatula simplex respectively, and four individual identified as Spondylus sp. didnot form monophyletic clades. The75individuals of the Pteriina in this study should beassigned to17species. The genetic distances between species for the COI and16S rRNAgene exceeded the distances within species and reached the10×threshold. The phylogenetictrees showed that all species appeared as a monophyletic group. These indicate DNAbarcoding can indentify species of the Pteriina effectively. In addition, by comparing COIgene and16S rRNA gene, the COI gene should be the more suitable DNA barcoding standardgene for the Pteriina.3. Molecular phylogeny of the PteriinaIn our study, we use three nuclear genes (28S,18S, H3) and one mitochondrial (16S) geneto reconstruct the phylogenetic relationships of the Pteriina. The new sequences together withthe sequences from Temkin (2010) were analysed. The results showed that except Mytiloideaand Arcoidea, all other superfamilies formed two major clades with high support values. Thefirst clade was composed of Pterioidea, Ostreoidea and Pinnoidea. Pterioidea was the sistergroup of Ostreoidea, and Pinnoidea formed a clade with Pterioidea+Ostreoidea. The secondclade included Pectinoidea and Anomioidea. The Plicatulidae within Pectinoidea formed a clade with Anomioidea, which make Pectinoidea appear as paraphyletic. Except Pectinoidea,all other superfamilies were supported monophyletic. The Pteriina defined by Newell (1969)was not monophyletic. The phylogenetic relationships within Pterioidea are consistent withthe results of Temkin (2010). The Pectinidae and Spondylidae within Pectinoidea formedsister group, indicating the relationship between them is very close. The Plicatulidae did notgroup together with Ostreoidea, which indicates the Plicatulidae is raised to the superfamilyrank and assigned to Ostreina with Ostreoidea and Dimyoidea by Waller (1978) isinappropriate. The phylogenetic results of our study powerfully challenge to the currentclassification systems based on morphology.4. DNA barcoding of the ArcoidaWe apply here DNA barcoding based on mitochondrial COI gene to arcoid133individuals.19homologous sequences were obtained from GenBank. Molecular diversity indices such asvariable sites, number of haplotypes and the K2P distances were calculated and thephylogenetic trees were constructed. Combining morphology with molecular data indicatesthe133specimens of Arcoida could be assigned to24species. The biggest intraspecificdistance was0.155observed between Wenchang population and other populations withinTegillarca granosa. The smallest interspecific distance was0.064observed betweenScapharca globosa and Scapharca sp. Because of the deep genetic divergence within T.granosa, there was an overlap between genetic variation within species and variation betweenspecies. Nevertheless, NJ and Bayesian trees showed that all species fell into reciprocallymonophyletic clades with high bootstrap values. Our results evidence that the DNA barcodingbased on COI gene can efficiently identify species, reveal genetic differentiation amongpopulations within species and correct mistakes caused by morphological identification.5. Molecular Phylogeny of the ArcoidaWe present the phylogenetic relationships of the Arcoida based on Bayesian inference andMaximum Likelihood analyses using three nuclear (28S,18S, H3) and two mitochondrial(COI,12S) genes in our study. The resulting topologies were discussed with traditional family,subfamily and genus arrangements and previous molecular studies. We confirmed monophyly of the order Arcoida, the family Noetiidae, the subfamily Anadarinae, Striarcinae andsupported the inclusion of the Glycymerididae in the Arcoidea. The Arcinae and the generaArca, Barbatia, Scapharca, Anadara and Glycymeris were not supported monophyletic,suggesting many taxonomic issues exist. The Noetiidae, Cucullaeidae, and Glycymerididaedid not appear as sister groups to the Arcidae, rather as groups within the Arcidae. This studypowerfully suggests the current taxonomy of the Arcoida, especially the Arcidae, is veryconfused and requires lots of revision based on morphological, paleontological and moleculardata.6. The complete mitochondrial genome of Cucullaea labiataThe complete mitochondrial genome of C. labiata is determined using long-PCRamplification, primer walking and shotgun sequencing approaches. The entire mitochondrialgenome sequence of C. labiata was25845bp in length, and contained36genes including12protein-coding genes (atp8is absent),2ribosomal RNAs, and22tRNAs. The COI gene of C.labiata was very special, within which a651bp non-coding region was inserted and devidedthe COI gene into two parts of1182bp and414bp. The role of this non-coding region andhow it affects the function of COI gene need to be further studied. In C. labiata, a lot ofnon-coding regions were observed, among which the largest was6057bp and containedtandem repeat sequences. Compared with other Pteriomorphia species, it bore a novel geneorder. C. labiata was viewed as basal in the phylogenetic trees based on amino acid sequencesof12protein-coding genes.