The Yellow Sea green tide has occurred in successive years since 2007. The floating U. prolifera consists of a genetically unique ecotype, different from other attached populations located in the intertidal zones around the Yellow Sea. Although these two ecotypes could hybridize under laboratory conditions, the floating ecotype of U. prolifera has remained its genetically distinct for years, strongly suggesting a special reproductive strategy that might be related to its blooming.
U. prolifera has a complex life cycle and a variety of reproductive methods. The sexual life cycle of U. prolifera is diplohaplontic and isomorphic, consisting of alternating generations of dioecious, and haploid gametophytes and diploid sporophytes. Because these two generations have identically shaped thalli, it is hard to discriminate them based on morphological characteristics, especially during their vegetative growth stages, which makes it very difficult to study life history of U. prolifera. It is necessary to set up a method which can identify the generation and sex of algae quickly and accurately, and replace the time-consuming and laborious traditional method to obtain the survey data with real ecological significance. In this study, we first developed and validated the sex-specific molecular markers of U. prolifera, and further studied the genetic rule of the new markers in various reproductive modes of U. prolifera. Based on the above results, the marker was detected the green tide floating U. prolifera adults from 2016 to 2020, the germination of micro-propagules collected during the green tide, and the offspring algae induced by laboratory reproduction. The results are as follows:
1. Successfully developed specific molecular markers for different mating types of U. prolifera.
A large number of samples were collected from different populations of U. prolifera from China and Japan, together with four individuals from four other Ulva species were selected for the development of MT-related markers. Based on the sex-related region reported in U. partita, two pairs of specific molecular markers for different mating types were developed by homologous cloning PRA1 gene from U. prolifera. The marker was tested in the sexual life history of U. partita which was constructed in the laboratory, and the results confirmed that the amplified product was an allele and related to mating type. Compared with existing methods for identifying generation or sex, these MT-related molecular markers have obvious advantages: 1) it can distinguish the generation and sex of the sample at the same time; 2) it can be detected by one-step PCR, and the operation is simple; 3) it is suitable for the high-throughput synchronous detection of the sample.
2. The genetic rule of the new marker in various reproduction modes of U. prolifera was clarified, and the special reproduction mode of in situ somatic cell regeneration was described for the first time at subcellular level.
In addition to sexual reproduction, three atypical reproductive methods, apomeiosis, parthenogenesis, and in situ somatic embryogenesis were constructed, the genetic rule of the new marker in the total four propagation modes was elucidated and verified by flow cytometry. The results showed that only in sexual reproduction, there were segregation and aggregation of markers, and in the three atypical reproduction modes, the Ploidy or sex of the progenies was the same as that of the female parent. In addition, it is the first time to describe the specific characteristics of in situ somatic cell regeneration at the subcellular level, which is essentially a vegetative propagation, it was confirmed that this type of reproduction can occur in sporophytes, gametophytes and other species of Ulva.
3. The distribution of generation and sex of floating U. prolifera for 5 years and its inter-annual variation were revealed.
A large number of floating ecotype of U. prolifera were detected from 2016 to 2020 by using the new markers. The results showed that: 1) The sporophytes and gametophytes were mixed in 2016, the sporophytes were absolutely dominant in 2017 and 2018, and the gametophytes were absolutely dominant in 2019 and 2020; 2) At different stages of the green tide outbreak, the generation and sex distribution of the algae were basically consistent, suggesting that the increase of green tide biomass was mainly dependent on growth and vegetative propagation; 3) The characteristics of generation and sex distribution of algal often show obvious inter-annual changes. Combined with the detection results of germination of micro-propagules, it is confirmed that there is an active sexual reproduction process; 4) No matter what the generation composition of green tide floating algae is, the micro-propagules came from various reproductive modes, and the generation distribution of germinating algal were sporophytes : gametophytes = 1 : 1; 5) The generation distribution was highly synchronized in four consecutive years (2017-2020) , which does not accord with the typical characteristics of alternating generations, suggesting that the marine environment has strong selectivity to the dominant generation.
4. A life-history strategy for the floating ecotype of U. prolifera is proposed
At the outbreak of green tide each year, the floating ecotypes of U. prolifera produced two types of micro-propagules (sporophytes : gametophytes = 1 : 1) by various reproduction methods. It is inferred that the germination of the two types of micro-propagules was strongly selected by environmental factors, thus affecting the generation distribution of the green tide floating algae, at the same time, the same biomass of the two generations in the micro-propagules ensures that the floating ecotypes of U. prolifera can effectively cope with the changing marine environment to achieve a stable outbreak for many years. In addition, the floating ecotypes of U. prolifera can not only have sexual reproduction, but also maintain their genetic specificity, suggesting that it have special mechanisms to achieve de facto reproductive isolation from attached populations.
The above research can provide important theoretical support for revealing the biological causes of the green tide of U. prolifera in the Yellow Sea.