Application Of Molecular Markers To The Researches On Pelagic Copepods In The Chinese Coastal Regions

In terms of their size, diversity and abundance, copepods can be regarded as the insects of the seas. Marine copepods are the most abundant and conspicuous component of a plankton sample. They are by far the most important primary consumers in the marine ecosystem and play a vital role by linking primary production to higher trophic levels. Furthermore, copepods are abundant and thus represent an ecologically important source of carbon flux. However, researches on copepods are usually hampered by the difficulties in species identification based on morphology in isolate owning to the subtle differences between species. The conservative trends of morphological evolution in copepods also confuse the phylogenetic researches in copepods. Molecular markers can offer us alternative tools for phylogenetic analysis at broad range of levels from population to inter-order relationships. Here we investigate copepods at different phylogenetic levels via separate molecular markers including COX1, 18S rRNA and complete mitochondrial genome. The thesis paper was divided into 4 independent issues:1. The mitochondrial genome of C. sinicus with multiple long noncoding regions and reshuffled gene order, useful molecular markers for phylogenetic analysis.With greatest diversity and richest abundance, copepods exhibit extensive ecological radiation in the marine ecosystem. Calanus sinicus dominates continental shelf waters in the Northwest Pacific Ocean and plays an important role in local ecosystem by linking primary production to higher trophic levels. Lacking effective molecular markers, several phylogenetic issues on copepods are still elusive from population to higher levels such as their phylogenetic position within pancrustacea. Being genome-level informative, mitochondrial DNA sequences are used as markers not only for population genetic studies but also for phylogenetic studies. Concurrence of multiple noncoding regions and reshuffled gene arrangement makes the mitochondrial genome of C. sinicus remarkably distinctive from other arthropods, including copepods. Furthermore, there are another suit of particularities found in mitogenome of C. sinicus, such as low AT content and symmetrical nucleotide composition between strands, abbreviated stop codons for some PCGs, as well as extended length of ATP6 and ATP8 relative to other copepods. Monophyly of copepods and basal split between Calanoida and Podoplea were well resolved while Maxillopoda were resolved polyphyletic. The closest affinity between Cyclopoida and Poecilostomatoida supports Boxshall in reassigning the latter subordinate to the former. The occurrence of mosaic sub-genomic fragments (mosaic cycler sequence) during laboratory work calls attention to the application of LA PCR to mitogenome sequencing in copepods, and may give an additional evidence of mitochondrial recombination. The accelerated mutational rates within copepods as well as interaction of non-coding regions with recombination in C. sinicus mitogenome give rise to present highly rearranged mitochondrial genome.2. Molecular phylogeny and divergence estimation of calanoid copepods based on 18S rRNA and mtCOX1 genes.Calanoid copepods, which are major component in all plankton samples, exhibit extensive radiation in the marine ecosystem. They are by far the most successful colonizers of pelagic realm. Their successful colonization is closely, if not completely, associated with the evolutionary history they have encountered. Here we tested the hypothesis regarding phylogenetic relationships of Calanoid copepods with molecular evidences.Secondary structures for 18S rRNA of calanoid copepods were constructed based on their consensus sequences. Owing to functional constraints, the structures are almost unchangeable among calanoid copepods with some exceptions in the genus of Acartia. Acartia species expanded loop regions in the helix 43, which was refolded into new helixes, being Helix E43₁ and Helix E43₂. The structure has been proved useful to assist in aligning 18S rRNAs and in giving more reasonalbe evolutionary models. Our phylogenetic analysis based on 18S rRNAs confirmed monophyly of Calanoida, Misophrioida and Siphonostomatoida. Cyclopoida and Poecilostomatoida which were resolved reciprocal paraphyletic coalescent into a monophyletic clade, supporting Boxhall in subordinating Poecilostomatoida to Cyclopoida. Misophrioida penetrated into Harpacticoida and destroyed the monophyly of the latter. In accordance with the results from 18S rRNA dataset, monophyly of Eucalanoidea, Clausocalanoidea, Diaptomoidea and Arietelloidea were well resolved by the combined dataset of both 18S rRNA and mtCOX1 gene. Though ambiguously resolved with low node confidence support, monophyly of the superfamily Calanoidea was confirmed by the best likelihood score for the Calanoidea-monophyletic constraint tree during the hypothesis testing. Based on 18S rRNA, divergence time for the order Calanoida was estimated at 400 Mya while the pelagic forms originated 398 Mya, which is in congruent with Bradford's hypothesis. The high turbidity during early Devonian may trigger the explosion of pelagic animals, as supported by fossil evidence from ostracods. About 343 million years before, the clade comprising of Calanoidea-Clausocalanoidea and Diaptomoidea diversified. Extinction event at late Denovian should have accelerated this divergence which gave rise to the lineage that fits the pelagic realms better. Divergence dating of advanced groups (calanoids from species in Clanoidea-Clausoclanoidea with Myelin-like sheath covered nerve fibers) was estimated at 246 Mya, when the turbidity decrease to a minimum level and the abundance of predator retained a high level. The extinction event at the boundary of Permian and Triassic should have driven the divergence process.3. DNA barcoding the calanoid copepods in the Chinese coastal regionsCopepods play an important role in the marine ecosystem. Accurate copepod species identification is thus necessary for understanding the conditions of local marine ecosystem. However, even the most skillful experts may get confused since the copepods resemble each other with subtle morphological differences. Even fewer clues are available for larval identifications. DNA barcodes provide an alternative approach for species identification.222 sequences covering 90 zooplanktons were identified in our study, AT content of which is 61.1%. Notable substitution saturation is apparent in all codons and particularly in the wobble ones, which preclude their utility in higher-level phylogenetic studies. Intraspecific variation of 224 calanoid copepods ranged from 0 to 26.8% with the means of 1.9% while congeneric intraspecific variation ranged from 0.3% to 36.1% with the means of 23.62%. Significant barcode gap was observed (t=18.574, p<<0.01). Most individuals belonging to the lineages whose intra- and inter-specific variation overlapped have been proved to encounter cryptic speciations. The majority of morphological species were clustered together in a monophyletic clade. These findings suggest that mtCOX1 is an appropriate barcoding tool for calanoid copepods and the diversity of calanoid copepods have been overlooked based on morphology. Using this molecular approach, several marine copepods in China were revised here.4. Population genetics of Calanus sinicus in the Chinese coastal regions Calanus sinicus dominates continental shelf waters in the Northwest Pacific Ocean and plays an important role in local ecosystem by linking primary production to higher trophic levels. Lacking effective molecular markers, phylogenetic relationships among populations of the species are still elusive.For the preliminary study, COX1 genes of 1060 bp in length and complete ribosomal internal transcriptional spacers (ITS1-5.8S-ITS2) were adopted for population genetic analysis in 50 individuals of C. sinicus sampled from Yalujiang estuary, Changjiang estuary, the South China sea and the Cold Water Mass in the Yellow Sea. ITS sequences were revealed highly conservative with no variable sites in all sequences analyzed. Though more variable, only 15 variable sites were detected in COX1 genes, representing 14 haplotypes. Specific haplotypes can be found in separate sampling stations, indicating that self-recruitment may exist in local areas. However, H1 haplotype was shared by all stations with high frequencies, which illustrate the homological status between populations. As was confirmed by AMOVA analysis, no genetic structure could be resolved in C. sinicus with available markers, suggesting that high dispersal potential may lead to a strong gene flow among different regions. However, consensus is far away from being reached. Since more variable markers are needed.11 nearly complete C. sinicus mitochondrial genomes from Yalujiang estuary, Changjiang estuary, Cold Water Mass and Korea nearshore were screened for hyper-variable markers for population studies. Within the 16,670 bp alignment, there are a total of 401 variable sites. Indel variations all present in non-coding regions and transitions dominate the SNPs. Three"hot-spots", especially the hyper-variable microsatellite locus in LNRs provide rich polymorphism for population studies. By the means of 3 hyper-variable regions, all individuals could be assigned to a unique haplotype. Compared to the COX1 gene, more information could be obtained from mitochondrial genome for the population studies.