Determining Mitochondrial Genome Sequence Form Formalin Fixed Sample And Reassessing The Phylogenetic Interrelationships Among The Acipenseriformes With Mitochondrial Genomes And Thousands Of Nuclear Coding Sequences

The Acipenseriformes, commonly known as the sturgeons, consists of two families,the Polyodontidae and the Acipenseridae. The Acipenseriform is the only existing order under the Chondrostei with first fossil appeared in the Jurassic, approximately 200 million years before present. The Acipenseriformes take a special place in vertebrate evolution mainly because of their old age and their relatively slow evolutionary rate.The Acipenseriformes contains 27 species, which are widely distributed. Due to overfishing for meat and caviar, damming of rivers that hold their spawning ground, and water pollution, many acipenseriform species have been extirpated or highy endangered in recent years. Therefore, reassessing the evolutionary interrelationships among the acipenseriforms became urgent, so that it could help in solving taxonomic controversies in the Acipenseriformes and in conserving the endangered sturgeon species.This research contains three chapters:In Chapter one, I have extracted DNA from samples of Polyodon spathula that treated with formalin for different length of time, as well as three specimens of Psephurus gladius that have been preserved in formalin for more than 40 years using an ancient DNA method. I made biotinylated baits using mitochondrial genome of fresh paddlefish(P. spathula) DNA as template, to capture the mitochondrial genome of formalin-fixed samples. Then I sequenced the enriched libraries on Illumina next-generation sequencing(NGS) platforms to obtain the mitochondrial genomes. The genome sequences I obtained for P. spathula were 16,524 bp in length. Comparing to reference sequence generated with traditional Sanger sequencing method, the coverageof the sequenced genome was 100%, the ratio of on-target reads was above 45% and the error rate was zero. I also studied the relationship between DNA fragmentation and time used for formalin treatment. The results showed that when the length of the target amplicon was 650 bp, it could be amplified in 1-h fixed sample only. When the length of the amplicons was 41 bp, 129 bp or 305 bp, a trace of product could be amplified for samples treated with formalin for up to 150 days. According to Q-ratio scores, we found the longer of formalin-fixed time, the lower Q-ratio score became. In other word, the longer of formalin-fixed time, the greater degree of DNA fragmentation occurred. These results augment the base knowledge for using DNA extraction from formalin-fixed samples in molecular studies.In Chapter two, I have obtained sequences of the mitochondrial genomes and 1,202single-coping nuclear genes from nine acipenseriforms species using the gene-capture and NGS method. I have reconstructed the phylogenetic interrelationships among the acipenseriforms using both the mitochondrial genome and the nuclear loci and have compared the results.The length of mitochondrial genome of the nine sturgeon species varied between16,443 bp to 16,785 bp. They all consisted of 13 protein-coding genes, 2 r RNA genes,22 t RNA genes and a D-loop sequence. When building the phylogenetic tree using mitochondrial genome data or protein-coding gene exclude third-coding loci, 2 r RNA gene, and 22 t RNA gene, I have obtained the same tree topology. Using paddlefish as the outgroup, the other eight species could be divided into three clades: Scaphirhynchus platorynchus as a clade; Acipenser sinensis, A. dabryanus, A. schrenckii and Huso dauricus as the second clade; A. gueldenstaedtii, A. ruthenus and H. huso as the third clade. Huso is not monophyletic genus, because the two species of Huso were found embedded separately within the genus Acipenser. Acipenser sinensis and A. dabryanus were confirmed as sister species.In Chapter three, using RNA baits made on 14,054 single-copy nuclear coding sequences of Lepisosteus oculatus,I have captured and sequenced 1,202 target genesfrom nine acipenseriform species. The concatenated length of all genes totaled 330,060 bp. Using L. oculatus as outgroup, the 8 acipenseriform species could be divided into three clades: P. spathula as the first clade; A. sinensis, A. dabryanus, A. schrenckii and H. dauricus as the second clade; A. gueldenstaedtii, A. ruthenus and H. huso as the third branch. Similar to the mitochondrial genome analysis, Huso was not a monophyletic taxonomic unit, because its two species were found embedded separately within the genus Acipenser; A. sinensis and A. dabryanus were confirmed as closely related. The difference between the nuclear gene tree and the mitochondrial tree was that H.dauricus was grouped with A. schrenckii instead of being sister to a clade containing A.sinensis, A. dabryanus and A. schrenckii.