Molecular Phylogenetics Of Acipenseriformes

Acipenseriformes(Sturgeons and paddlefishes)is the only extant order under the Chondrostei(Osteichthyes,Actinopterygii),and occupies a very important position along the evolution of vertebrates.This group,known as “living fossils”,contains 2families,6 genera and 27 extant species,which are widely distributed in the northern hemisphere.There were fossils of the Acipenseriformes about 200 million years ago(Mya)in the Jurassic period,but their morphological changes were very small.Along with unique benthic life characteristics,both special life history and near extinction or critically endangered status,Acipenseriformes has become a hot research topic in phylogeny,ecology and conservation biology.There were many studies making great progress in phylogenetics of Acipenseriformes,but those studies were mainly focus on a few mitochondrial genes.In recent years,some mitochondrial genes and even the whole mitochondrial genomes were sequenced in Acipenseriformes,which makes it possible to reconstruct the phylogenetic relationship of the group from the perspective of mitochondrial molecular markers.However,due to the characteristics of maternal inheritance and rapid evolution rate of mitochondrial DNA,nuclear molecular markers become the first choice for the phylogenetic study of ancient groups.In particular,the nuclear protein-coding(NPC)markers are the best choice to study the deep-level phylogenetic relationships because of its high conservatism in exons.In addition,many species of Acipenseriformes are polyploidy because of the widespread genome duplication,make the nuclear molecular markers are extremely deficient.With the development of sequencing technologies and the analyzing methods,especially,basedon transcriptome & reference genome and multi-genome alignment development nuclear markers have already reported,which provide new ideas for development nuclear molecular markers for Acipenseriformes.In order to making a more comprehensive analysis of the phylogenetics of Acipenseriformes,we used the existing mitochondrial genome data and self-developed nuclear markers to reconstruct the phylogeny and to estimate divergence time of Acipenseriformes.The main results are as follows:(1)Phylogenetics and divergence time analyses of Acipenseriformes based on mitochondrial genome dataCurrently,the study of phylogenetic relationships of Acipenseriformes were mainly focus on mitochondrial genes,which is rarely involved in mitochondrial genome.Because compare with mitochondrial genes,mitochondrial genome is more difficult to developed.With the progress of sequencing technique and related the development of universal primers for Acipenseriformes,make it easier to sequenced the whole mitogenome.Mitogenome provides more phylogenetic information,compared with mitochondrial genes,it is a better choice for phylogeny.There are 21 species of Acipenseriformes mitogenomes in the database,including 2 families and 5 genera.In order to comprehensively analysis of the phylogenetic relationships of Acipenseriformes,we choose cytb and ND5 for phylogenetic analysis,which currently contain 26 species including 2 families and 6 genera.We reconstructed phylogenetic trees by using maximum likelihood(ML)and bayesian inference method(BI)based on mitogenomes and concatenated dataset.The results of all trees are highly consistent showed that(i)Acipenseridae can be divided into three clades(clade 1,clade 2,clade 3);(ii)the genus Scaphirhynchus located in base position of Atlantic clade;(iii)the genus Huso is not a monophyletic group,H.dauricus and H.huso disperse and nested in genus Acipenser;(iv)A.sturio and A.oxyrinchus are sister species,located in base position of Acipenseridae.The results of concatenated dataset suggested that genus Pseudoscaphirhynchus cluster with A.stellatus as a sister group,which consistent with previous research based on mitochondrial data.We use the concatenated dataset for divergence time calculations and results showed that genus Pseudoscaphirhynchus divergence with A.stellatus at 24.1 Mya,and the time of origin genus Scaphirhynchus in the late cretaceous(99.7 Mya).In order to calculate the divergence time more accurately,we suggested that the fossil record and the data size should be increased.(2)Phylogenetics and divergence time analyses of Acipenseriformes based on nuclear molecular markersNuclear genes have better analytical ability to deep-level phylogenetic relationships.However,Acipenseriformes species have no published reference genome sequences data until now that leads to phylogenetic relationships focus on mitochondrial genes,nuclear markers extremely deficient.In this study,four transcriptomes of sturgeon and Lepisosteus oculatus genome as a database,this analyzed with bioinformatics approach and screened the candidate single-copy genes for developing NPC markers.In order to ensure the universal primers,we screening 193 nuclear single-copy genes,which selected from 1850 candidate genes and at least one exon is larger than 700 bp,then we choose 43 nuclear single-copy genes for primer design,most of them just have one exon.Finally,we successfully develop 30 newly NPC markers for 16 species of Acipenseriformes,ranging from 467 bp to 993 bp with an average length of 698 bp.The success rate is 100% in 27 NPC markers,93.75% in 2NPC markers,87.5% in 1 NPC markers.This data set consists of 30 nuclear markers in16 species of Acipenseriformes.Total alignment length is 20946 bp,which is a large data set of nuclear protein-coding genes for phylogenetic study of Acipenseriformes till now.The supermatrix was separately analysed with both maximum-likelihood(ML)and Bayesian inference(BI)methods under best scheme calculated by PartitionFinder 2.Analyses of individual gene showed that no single gene was able to recover all nodes identified with the concatenated data.The construction of the species tree was completed by ASTRAL.The results showed that Both ML tree and BI tree were produced highly similar topologies,moreover,most nodes have strongly support(BP?75%,PP ?0.95).The species tree produced similar topologies with gene trees(ML tree and BI tree)in most lineages,all of their support(i)divided Acipenseridae into three clades(clade 1,clade 2,clade 3);(ii)the genus Huso is not a monophyletic group;(iii)P.kaufmanni also located in base position of Atlantic clade.We also calculated the divergence times between the 16 species of Acipenseriformes,and the results showed that the nuclear gene data set was narrower and more accurate in the credibility intervals.The divergence time of the genus Pseudoscaphirhynchus is 103.3 Mya,far more than all previous studies,but due to incomplete species,the actual divergence time may be a little younger than the result.The successful development and application of NPC markers not only provides a strategy for the development of nuclear markers for lackreference genome sequences data taxa,but also provides a new perspective and insight for phylogenetic relationships of Acipenseriformes.