Comparing Complete Mitochondrion Genome of Bloom-forming Macroalgae from the Southern Yellow Sea, China

. The green tide in the Southern Yellow Sea which has been erupting continuously for 14 years. Dominant species of the free-floating Ulva in the early stage of macroalgae bloom were Ulva compressa , Ulva flexuosa , Ulva prolifera , and Ulva linza along the coast of Jiangsu Province. In the present study, we carried out comparative studies on complete mitochondrion genomes of four kinds of bloom-forming green algae, and provided standard morphological characteristic pictures of these Ulva species. The maximum likelihood phylogenetic analysis showed that U. linza is the closest sister species of U. prolifera . This study will be helpful in studying the genetic diversity and identification of Ulva species.


Introduction
Green tides, which occur widely in many coastal areas, are caused primarily by flotation, accumulation, and excessive proliferation of green macroalgae, especially the members of the genus Ulva [1][2][3]. China has the high frequency outbreak of the green tide [4][5][6][7][8][9][10]. Especially, the world's largest green tide events, have occurred annually from 2007 to 2020 along the coast of the Yellow Sea, China, seriously affecting marine environments and ecological services functions [11][12][13][14][15]. An interesting phenomenon called Ulva species succession were found at the early stage of the blooms in the Southern Yellow Sea. The initial floating species was identified as Ulva compressa; Ulva flexuosa and Ulva linza were the second floating species; and Ulva prolifera was the last [16][17][18]. However, during the drifting process of the blooms, U. prolifera became the dominant species and other species disappeared gradually [19]. Thus, it was meaningful to carry out comparative studies on organelle genomes of these bloom-forming green algae.

The specimen and data preparation
In our previous studies, mitochondrion genome of U. flexuosa (NC035809; KX455878) [20], U. linza (NC029701; KU189740) [21], U. compressa (KX595276), and U. prolifera (KU161104) were conducted [22]. We downloaded these sequences from US National Center for Biotechnology Information (NCBI: www.ncbi.nlm.nih.gov/) ( Table 1). A total of 6 complete mitochondrion genome of Ulva and 1 outgroup called Pseudendoclonium akinetum (AY359242) were used during this study. Also, specimens were stored in the herbarium of Shanghai Ocean University Museum (SHOUM). Based on these four kinds of Ulva specimens, we took clear morphological photographs by using the camera model GE-X2600.

Result and discussion
A Maximum-likelihood (ML) phylogenetic tree with 6 complete mitochondrion genome of Ulva and 1 outgroup called P. akinetum (AY359242) was constructed by using the MEGA 7 software (Figure 2) [21][22], which showed U. prolifera is closely related to U. linza. In addition, the standard morphological characteristics of these Ulva species were presented in Figure 2.
The formation of the green tide in the Southern Yellow Sea of China is complicated. Many researches demonstrated that the significant amount of attached Ulva species on the Pyropia aquaculture rafts were considered as the main source of the blooms in the Southern Yellow Sea [23][24][25]. The attached Ulva species consisted of four species which were U. compressa, U. linza, U. flexuosa and U. prolifera. During the harvest season of Pyropia aquaculture, as a kind of fouling macroalgae, the attached Ulva were removed artificially, resulting in the initial formation of the blooms.
These four kinds of Ulva species could be distinct from the complete mitochondrion genomes (Figure 2). Also, the morphological characteristics of these bloomforming species are different during the initial formation of the bloom process (Figure 2), and in conbine with these methods above, we can distinguish the Ulva species.
The largest green tide bloom areas in the Southern Yellow Sea of China from 2007 to 2019 was shown in Figure 3. China has the high frequency outbreak of the green tide, in 2009, the cover area of green tide outbreaks in the Southern Yellow Sea of China has reached 2,100 km 2 (Figure 3) [26][27][28]. Maybe through the application of biological prevention, strengthening breeding management and other methods, we can fundamentally solve the problem of green tides [29][30].

Conclusion
In this thesis, we analyzed complete mitochondrion genomes of the free-floating Ulva species during the early stage of macroalgae bloom in the Southern Yellow Sea and provided standard morphological pictures of these Ulva species, which will be useful for studying the genetic diversity and phylogenetic history of Ulva species in the Southern Yellow Sea.