Investigation On Biological Characteristics Of The Different Ploidy Crucian Carps (Carassius Auratus L.) In The Dongting Water System And Their Evolutionary Relationship

The importance of polyploidization is found enlarging in the evolution process and has gained more and more public attention. In fish species, polyploidization occurred more commonly than in other kinds of vertebrates. The crucian carps are regarded as ancient tetraploids. It has been one of the most important freshwater species for Chinese aquaculture and is especially abundant in the Dong-ting water system of Hunan province. Crucian carp used to be considered as all diploid forms (2n=100). However, triploid forms (3n=150+) have been reported in China since 1980s.In this study, the wild population of crucian carp living in the Dong-ting water system in Hunan province were sampled. After a large scale of detection was set up to monitor the ploidy diversity of the population, totally three different ploidy of crucian carps were found co-existent in the Dong-ting water system. The biological characteristics of the population were comparatively studied, leading to further consideration of the possible origin and probable evolutionary pathway of polyploid. The primary coverage includes:1. Since 2005, the ploidy diversity of the wild population of crucian carps in Dong-ting water system was systematically monitored by DNA content measurement and further by chromosome count on kidney tissue. Accordingly, besides the diploid forms (2n=100), there were a great amount of triploid forms (3n=150+) and a few of tetraploid forms (4n=200) found in the population. The triploids made up the majority of the population with an increasing proportion every year. Only a few tetraploids were detected, all of which were identified as all female. Most importantly, this is the first reports on the discovery of tetraploid forms in the wild crucian carp population. The discovery of coexistence of three different ploidy in the wild crucian carp population of Dong-ting water system was of great significance in discussing the genetic diversity of crucian carps at chromosome level, providing excellent material for study on the origin of polyploidization and fish evolution.2. Morphological analyses on the three different ploids of wild crucian carp population in Dong-ting water system and comparative investigation of their erythrocytes were employed. Accordingly, there was no obvious variety in their countable and measurable traits. The three different ploids with similar appearance could not be directly distinguished by their configuration. The erythrocyte nuclear volumes increased among diploids, triploids and tetraploids with the increasing ploidy. Erythrocytes with two nuclei resulting from amitosis were observed in triploids and tetraploids but not found in diploids.3. The gonads of the three ploids of common crucian carps in Dong-ting water system were systemically compared by tissue section, scanning electron microscope (SEM) and transmission electron microscope (TEM). The tissue section of the ovaries showed that the ovary structures of the three forms were normal with isochronous development of common crucian carps and could produce functional eggs. Under SEM observation, the mature spermatozoa of diploids and triploids had the normal external form, comprising by a head, a connecting piece, and a tail. With the increase of ploidy level, the mean head size of spermatozoa increased. Under TEM observation, all the mature spermatozoa in the testis of diploids and triploids had normal ultra-structures. However, in the testis of 3nCC there were certain a few of vacuoles in the head of the spermatozoa. According to these results, the three different forms in the wild crucian carp population possessed normally developed gonads which could produce functional spermatozoa and eggs, forcefully supporting the fertility of triploids at cytology level.4. Series of crossings between the females of three different ploidy of wild crucian carps and the males of some differently related species were set up to compare the reproductive patterns of the wild crucian carps. The results came back that the three different forms possessed specific reproductive patterns. The diploids were indentified as normal bisexual fertile. The all-female tetraploids could produce progenies through natural gynogenesis. The triploids possessed a complex pattern that they could be bisexual fertile when their eggs met homogenous spermatozoa of the male triploids or they reproduced all-female progenies through natural gynogenesis when their eggs were activated by related spermatozoa of other kinds.5. The complete mitochondrial genomes of the three different forms of common crucian carp were sequenced. Comparative analyses on the sequences showed that the relationship among the three different ploidy were very close, and the genetic distance within the population of common crucian carps was smaller than that between the common crucian carps and red crucian carps. Novel Sox-HMG markers were employed to compare the nuclear genomes of the three different ploidy forms at molecular level. Allogenetic DNA fragments were found in triploids and tetraploids. Moreover, the 1900-bp fragments of Sox4 gene which could be amplified only in species of the genus of C. were also obtained in common crucian carps. The phylogenetic tree that was structured by the sequences of these 1900-bp fragments were divided into two clades:one including the diploid common crucian carp and red crucian carp; and the other including the triploid and tetraploid forms of common crucian carp. The molecular evidences that were mentioned above directly proved that the different ploidy forms in the wild population of common crucian carp shared the original maternal parent. The triploid and tetraploid genomes probably not resulted from direct duplication of the diploid genome but from hybridization between diploid individuals and some other related species. Consequently, the possible origin and evolutionary pathway of polyploid common crucian carps were investigated at the molecular level. Considering the appearance time of the polyploid forms, human economic activity was further presumed as the main reason of genome duplication of the common crucian carps. The conclusion provides valuable information to study how hybridization contributes to animal evolution.