Population Genetic Structure of Sargassum horneri , the Dominant Species of Golden Tide in the Yellow Sea

: Sargassum horneri golden tides are increasingly becoming a marine ecological problem in the Yellow Sea (YS) and East China Sea. To understand the genetic relationship between the attached S. horneri along the China coast and the floating biomass in the YS, we used partial rbcL , ITS2 , cox1 , cox3 , and cob-cox2 to analyze the population genetic evolution of 165 Sargassum samples. The results showed that all samples were a single species of S. horneri . Partial sequences of each gene had major haplotypes, and other haplotypes evolved from the occurrence of base mutations. The cob-cox2 gene haplotype research showed only the attached samples from ZJ, LN, and KR contained Hap3, and the distribution proportions of Hap2 and Hap4 in SS and the YS were closest to the distribution of the attached samples from SD. These novel findings provided information about the genetic evolutionary relationship between attached S. horneri along the coast of China and floating S. horneri in the YS, as well as new ideas for tracing the source of floating S. horneri in the YS from a molecular biological perspective.


Introduction
The genus Sargassum is a large group of 354 species of brown algae commonly found along tropical and temperate coasts.It is one of the most abundant genera in the Order Fucales, which is widely distributed throughout the world [1][2][3][4].Eutrophication is caused by the enrichment of nitrogen and phosphorus nutrients in water [5,6] and can cause macroalgal blooms to develop rapidly in the sea [7,8].Some species in the genus Sargassum have air sacs that allow the algae to float for a long time at the sea surface, where they can grow rapidly via vegetative propagation and subsequently form large floating biomass.These blooms form "Sargasso seaweed beds" or "golden tides" [9].
As an invasive species of macroalgae, Sargassum has invaded European shores and the Mediterranean, and even drifting individuals have been observed in the sea area near the Canary Islands, becoming a potential species responsible for the "Sargassum golden tide".Golden seaweed tides are a global environmental and social problem, that have been occurring along Ireland's eastern coastline since the 1990s [10].Recently, significant accumulations of drifting Sargassum have been observed on the sea surface in specific regions such as the Gulf of Mexico in the Atlantic Ocean [6,11], the East China Sea and the Yellow Sea.In the Atlantic region, two species namely S. natans and S. fluitans dominate these accumulations [6], while in the East China and Yellow Seas only S. horneri is found.The proliferation of this invasive genus can lead to substantial modifications in the original

Sample Collection
Of the 165 samples collected, the attached samples were collected from coastal areas of Liaoning (LN), Shandong (SD), Zhejiang (ZJ), Fujian (FJ), Guangdong (GD) Provinces of China and Jeju Island (KRJJ) from South Korea, and floating samples were collected from Subei Shoal (SS) and the YS (Table 1).The algal thalli from each site were sampled randomly and cleaned with distilled seawater to remove attached organisms and debris.The samples then were preserved at −20 °C for DNA extraction.

Sample Collection
Of the 165 samples collected, the attached samples were collected from coastal areas of Liaoning (LN), Shandong (SD), Zhejiang (ZJ), Fujian (FJ), Guangdong (GD) Provinces of China and Jeju Island (KRJJ) from South Korea, and floating samples were collected from Subei Shoal (SS) and the YS (Table 1).The algal thalli from each site were sampled randomly and cleaned with distilled seawater to remove attached organisms and debris.The samples then were preserved at −20 • C for DNA extraction.

Satellite Remote Sensing
Satellite remote sensing is one of the most efficient tools for natural hazard monitoring and assessment.The Haiyang-1C (HY-1C) satellite, which was launched in September 2018, has a spatial resolution of 50 m and revisiting frequency of 3 days.In this study, images from the HY-1C were used qualitatively to provide a time-series view of the Sargassum distribution pattern.The HY-1C satellite images were provided by the East China Sea Environmental Monitoring Center of State Oceanic Administration (Shanghai) to monitor drifting seaweeds in 2021.

Molecular Identification of Sargassum Samples
Total DNA was extracted using a Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions.The regions selected for polymerase chain reaction (PCR) amplification and automated sequencing were as follows: (1) partial rbcL in the chloroplast genome, (2) partial ITS2 in ribosomal DNA, and (3) partial cox1, cox3, and cob-cox2 in the mitochondrial genome.Table 2 shows information about the primers used.The PCR cycling procedures for ITS2 and cob-cox2 were described previously [33,34].Amplification was initiated with denaturing at 94 • C for 4 min, followed by 38 cycles of 94 • C for 45 s, annealing at 50 • C for rbcL, cox1, and cox3 for 45 s, and 72 • C for 90 s, and then a final extension at 72 • C for 10 min.Neighbor-joining analyses of the aligned sequences were performed with MEGA 5.05 [35] and the reliability of each branch was tested with 1000 bootstrap replications.All samples were identified as S. horneri.

Haplotype Statistics and Analysis of cob-cox2 and cox3 Partial Sequences
We used the Alignment Editor of BioEdit7.0sequence software to optimize the gene sequences of the samples that we collected from eight collection sites [37].Of the available sequences from the eight collection sites (Table 1), we counted and analyzed haplotypes using DNASP5.0software [38].The network diagrams of cob-cox2 and cox3 partial sequences and pedigrees obtained in this study were constructed using PopART1.7 software [39].

Blooming Process of Drifting Sargassum Species in 2021
The evolution of the golden tides was depicted by a long time-series based on satellite remote sensing images in the YS and ECS in 2021 (Figure 1h).The Sargassum rafts generally drifted southwards and expanded from March to April, and then they moved northwards and gradually decayed in June.On 5 April, drifting algae were distributed from the ECS to the south YS.Sargassum bloom patches began to congregate to form a much larger bloom from 5 April to 26 April, with a cover of up to 104 km 2 .The maximum distribution area of 69,285 km 2 occurred on 9 April (Figure 1g).A large biomass of floating Sargassum was stranded on P. yezoensis cultivation rafts located on Subei Shoal during a coastal survey in May (Figure 1e).These patches then moved northwards.Between 30 April and 6 June, algal cover gradually decreased, fragmented into several small patches, and then disappeared in the YS.

Molecular Analysis Based on Marker Genes in Ribosomes, Mitochondria, and Chloroplasts
Comparison of the partial sequences of ITS2, rbcL, cox1, cob-cox2, and cox3 revealed genetic variation sites in all five genes (Figure 2).We identified 10 variant sites in the ITS2 partial sequences (two insertion mutations), as well as seven haplotypes and 10 single nucleotide polymorphisms (SNPs).We found three variant sites in the rbcL partial sequences (one mutation type with insertion and one with deletion), four haplotypes, and three SNPs.Seven variant sites were present in the cox1 partial sequences (one deletion mutation), with four haplotypes and seven SNPs.We identified 12 variant sites in the cob-cox2 partial sequences (including one deletion mutation), with 12 haplotypes and 12 SNPs present.Finally, we found 16 variant sites (no insertion or deletion mutations), 10 haplotypes, and 16 SNPs in the cox3 partial sequences.These results showed that the gene sequence polymorphism was higher on mitochondria than on ribosomes or chloroplasts.Among the five genes analyzed, the partial sequences of the mitochondrial cob-cox2 and cox3 had high polymorphisms with more variation sites, so they are more suitable for further population diversity analysis.
three SNPs.Seven variant sites were present in the cox1 partial sequences (one deletion mutation), with four haplotypes and seven SNPs.We identified 12 variant sites in the cob-cox2 partial sequences (including one deletion mutation), with 12 haplotypes and 12 SNPs present.Finally, we found 16 variant sites (no insertion or deletion mutations), 10 haplotypes, and 16 SNPs in the cox3 partial sequences.These results showed that the gene sequence polymorphism was higher on mitochondria than on ribosomes or chloroplasts.Among the five genes analyzed, the partial sequences of the mitochondrial cob-cox2 and cox3 had high polymorphisms with more variation sites, so they are more suitable for further population diversity analysis.An analysis of the percentage of haplotypes composition of the partial sequences of the five genes revealed the presence of major haplotypes (high percentages) in all of them (Figure 3).In the partial sequence of the ITS2 gene, the major haplotype was Hap1 and the minor haplotype was Hap2, and the total proportion of both was 94.48%.Among the partial rbcL sequences, Hap1 accounted for 97.93% of the cases.Hap1, Hap3, Hap4, and Hap21 shared some proportion of the distribution among the partial cob-cox2 sequences; except for 35.92% of Hap2, the analysis of partial cob-cox2 showed high richness and diversity.Of the cox1 and cox3 partial sequences, the major haplotypes were Hap1 (86.90%) and Hap3 (75.86%), respectively.In addition, Hap2 (11.72%) was a minor haplotype in cox1, and Hap9 and Hap23 (9.66% for both) were minor haplotypes in cox3.The genetic evolutionary relationship between major and minor haplotypes requires further analysis.
except for 35.92% of Hap2, the analysis of partial cob-cox2 showed high richness and diversity.Of the cox1 and cox3 partial sequences, the major haplotypes were Hap1 (86.90%) and Hap3 (75.86%), respectively.In addition, Hap2 (11.72%) was a minor haplotype in cox1, and Hap9 and Hap23 (9.66% for both) were minor haplotypes in cox3.The genetic evolutionary relationship between major and minor haplotypes requires further analysis.The relationships among the major haplotypes and other haplotypes were further analyzed by generating a NJ phylogenetic evolution tree for the partial sequences of the ITS2, rbcL, cox1, cob-cox2, and cox3 genes (Figure 4).The ITS2 partial sequence tree showed that the major haplotype, Hap1, was at the start of the main clade of the evolutionary tree, the minor haplotype Hap2 was on a branch of this main clade, and both floating and attached samples were clustered in Hap1 and Hap2 (Figure 4).Notably, in the evolutionary trees for the rbcL, cox1, cob-cox2, and cox3 partial sequences, both floating and attached samples were clustered simultaneously only in the main haplotypes ( ), and all were at the position where the evolutionary tree branching began.In addition, many of the haplotypes that accounted for a relatively small proportion of the total were derived from the major haplotypes, and there was a relatively close genetic evolution relationship with the major haplotypes.The relationships among the major haplotypes and other haplotypes were further analyzed by generating a NJ phylogenetic evolution tree for the partial sequences of the ITS2, rbcL, cox1, cob-cox2, and cox3 genes (Figure 4).The ITS2 partial sequence tree showed that the major haplotype, Hap1, was at the start of the main clade of the evolutionary tree, the minor haplotype Hap2 was on a branch of this main clade, and both floating and attached samples were clustered in Hap1 and Hap2 (Figure 4).Notably, in the evolutionary trees for the rbcL, cox1, cob-cox2, and cox3 partial sequences, both floating and attached samples were clustered simultaneously only in the main haplotypes (

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples ), and all were at the position where the evolutionary tree branching began.In addition, many of the haplotypes that accounted for a relatively small proportion of the total were derived from the major haplotypes, and there was a relatively close genetic evolution relationship with the major haplotypes.

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant (Figure 5b).
Analysis of the population diversity and genetic evolutionary relationships of the 2021 samples based on partial sequences of cob-cox2 (Figure 5c) and cox3 (Figure 5d) revealed the same regularity as those of previous years' samples, with more haplotypes and more abundant population diversity in the attached samples from the ZJ coast.

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant (Figure 5b).
Analysis of the population diversity and genetic evolutionary relationships of the 2021 samples based on partial sequences of cob-cox2 (Figure 5c) and cox3 (Figure 5d) revealed the same regularity as those of previous years' samples, with more haplotypes and more abundant population diversity in the attached samples from the ZJ coast.

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant (Figure 5b).
Analysis of the population diversity and genetic evolutionary relationships of the 2021 samples based on partial sequences of cob-cox2 (Figure 5c) and cox3 (Figure 5d) revealed the same regularity as those of previous years' samples, with more haplotypes and more abundant population diversity in the attached samples from the ZJ coast.

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant (Figure 5b).
Analysis of the population diversity and genetic evolutionary relationships of the 2021 samples based on partial sequences of cob-cox2 (Figure 5c) and cox3 (Figure 5d) revealed the same regularity as those of previous years' samples, with more haplotypes and more abundant population diversity in the attached samples from the ZJ coast. ).

Haplotype Network Analysis Based on the Partial cob-cox2 and cox3 Sequences
The haplotype network map of cob-cox2 showed that the attached samples from the Zhejiang (ZJ) coast had the most haplotypes with more abundant population diversity compared to the other sampling locations.Of concern were samples from ZJ that shared Hap circles with samples from all regions and that shared common haplotypes.The attached samples from ZJ, Liaoning (LN), and Jeju Island from South Korea (KRJJ) shared Hap3 circles, which were also the only Hap circles present in the South Korea (KR) samples.Except for the attached samples from LN and KRJJ, other regional samples were present in the two main haplotypes Hap2 and Hap4 (Figure 5a).
The cox3 gene haplotype network map also showed that the attached samples from the ZJ coast had more haplotypes that were not found in samples collected in other regions.Some attached samples from LN were genetically distant from other regional samples.In addition, the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant (Figure 5b).
Analysis of the population diversity and genetic evolutionary relationships of the 2021 samples based on partial sequences of cob-cox2 (Figure 5c) and cox3 (Figure 5d) revealed the same regularity as those of previous years' samples, with more haplotypes and more abundant population diversity in the attached samples from the ZJ coast.

Analysis of Different Haplotype Occupancy in Different Geographic Regions Based on the Partial cob-cox2 and cox3 Sequences
In the regional percentage distribution of partial cob-cox2 sequences (Figure 5e), the attached samples from ZJ had more haplotypes, with the most diverse sites of genetic variation.The attached samples from LN did not share two haplotypes (Hap2 and Hap4) that were characteristic of samples from the China coast, and the proportion of Hap2 showed a decreasing trend from north to south.In addition, only the attached samples from ZJ, LN, and KR contained Hap3.The distribution proportions of Hap2 and Hap4 in SS and the YS were closest to the distribution of the attached samples from SD.In the regional percentage distribution of partial cox3 sequences (Figure 5f), Hap3 was predominant in all regional haplotype compositions along the coast of China.The results of the analysis of the attached samples from ZJ were the same as those for cob-cox2, with more haplotypes and the most diverse sites of gene variation.Hap9 was only present

Analysis of Different Haplotype Occupancy in Different Geographic Regions Based on the Partial cob-cox2 and cox3 Sequences
In the regional percentage distribution of partial cob-cox2 sequences (Figure 5e), the attached samples from ZJ had more haplotypes, with the most diverse sites of genetic variation.The attached samples from LN did not share two haplotypes (Hap2 and Hap4) that were characteristic of samples from the China coast, and the proportion of Hap2 showed a decreasing trend from north to south.In addition, only the attached samples from ZJ, LN, and KR contained Hap3.The distribution proportions of Hap2 and Hap4 in SS and the YS were closest to the distribution of the attached samples from SD.In the regional percentage distribution of partial cox3 sequences (Figure 5f), Hap3 was predominant in all regional haplotype compositions along the coast of China.The results of the analysis of the attached samples from ZJ were the same as those for cob-cox2, with more haplotypes and the most diverse sites of gene variation.Hap9 was only present in in the attached samples from the ZJ and SD regions, and this haplotype was absent in other regions.In addition, four regional samples (LN, Guangdong (GD), YS, and KRJJ) had region-specific haplotypes.

Discussion
Floating S. horneri has been present in the western Pacific Ocean for many years, but it was not a research focus because of its small biomass and range in early years [14][15][16].However, a large outbreak of floating S. horneri biomass in the YS and ECS in 2017 resulted in stranded algae in the raft area of P. yezoensis aquaculture in Jiangsu Province, which caused direct economic losses as high as $78 million [29].
S. horneri is widely distributed all over the world [1-3], especially in the western Pacific [20].According to the incomplete statistics from https://www.gbif.org(accessed on 17 January 2022), S. horneri was observed 145 times in Japan, 14 times in South Korea, and 16 times in China between January 2000 and September 2021 along the coast of China, Korea, and Japan.We reinvestigated the distribution of S. horneri along the coast of China and found both attached and floating S. horneri in Dalian, Shandong, Zhejiang, Fujian, and Guangdong Provinces.Our survey added 34 distribution sites along the coast of China to the list on the GBIF site (Figure 6a).S. horneri mainly grows on rocks or stone marshes in the intertidal and subtidal zones under natural conditions [19].However, large biomass of S. horneri have a strong relationship with aquaculture along the coast of China, and materials such as cables and drift in raft areas contribute greatly to the attached growth of S. horneri.Studies have found that the body length and biomass of intertidal S. horneri are much lower than those of S. horneri on rafts in aquaculture areas [24,40].As the number and density of air sacs increases, the buoyancy of air sacs may exceed the attachment ability of their holdfasts.With the combined action of wind, waves, and currents, the ultra-long thalli in aquaculture areas are easily detached [10].Large amounts of S. horneri in aquaculture areas are not welcomed by farmers because algal growth and surface coverage in raft areas affect the growth of cultured species, and fishers cut them off and discard them at will.Large scale floating accumulation of S. horneri on the sea surface is caused by the combined effects of the alga itself, natural conditions, and human factors [24]. in the attached samples from the ZJ and SD regions, and this haplotype was absent in other regions.In addition, four regional samples (LN, Guangdong (GD), YS, and KRJJ) had region-specific haplotypes.

Discussion
Floating S. horneri has been present in the western Pacific Ocean for many years, but it was not a research focus because of its small biomass and range in early years [14][15][16].However, a large outbreak of floating S. horneri biomass in the YS and ECS in 2017 resulted in stranded algae in the raft area of P. yezoensis aquaculture in Jiangsu Province, which caused direct economic losses as high as $78 million [29].
S. horneri is widely distributed all over the world [1][2][3], especially in the western Pacific [20].According to the incomplete statistics from https://www.gbif.org(accessed on 17 January 2022), S. horneri was observed 145 times in Japan, 14 times in South Korea, and 16 times in China between January 2000 and September 2021 along the coast of China, Korea, and Japan.We reinvestigated the distribution of S. horneri along the coast of China and found both attached and floating S. horneri in Dalian, Shandong, Zhejiang, Fujian, and Guangdong Provinces.Our survey added 34 distribution sites along the coast of China to the list on the GBIF site (Figure 6a).S. horneri mainly grows on rocks or stone marshes in the intertidal and subtidal zones under natural conditions [19].However, large biomass of S. horneri have a strong relationship with aquaculture along the coast of China, and materials such as cables and drift in raft areas contribute greatly to the attached growth of S. horneri.Studies have found that the body length and biomass of intertidal S. horneri are much lower than those of S. horneri on rafts in aquaculture areas [24,40].As the number and density of air sacs increases, the buoyancy of air sacs may exceed the attachment ability of their holdfasts.With the combined action of wind, waves, and currents, the ultra-long thalli in aquaculture areas are easily detached [10].Large amounts of S. horneri in aquaculture areas are not welcomed by farmers because algal growth and surface coverage in raft areas affect the growth of cultured species, and fishers cut them off and discard them at will.Large scale floating accumulation of S. horneri on the sea surface is caused by the combined effects of the alga itself, natural conditions, and human factors [24]. in the attached samples from the ZJ and SD regions, and this haplotype was absent in other regions.In addition, four regional samples (LN, Guangdong (GD), YS, and KRJJ) had region-specific haplotypes.

Discussion
Floating S. horneri has been present in the western Pacific Ocean for many years, but it was not a research focus because of its small biomass and range in early years [14][15][16].However, a large outbreak of floating S. horneri biomass in the YS and ECS in 2017 resulted in stranded algae in the raft area of P. yezoensis aquaculture in Jiangsu Province, which caused direct economic losses as high as $78 million [29].
S. horneri is widely distributed all over the world [1][2][3], especially in the western Pacific [20].According to the incomplete statistics from https://www.gbif.org(accessed on 17 January 2022), S. horneri was observed 145 times in Japan, 14 times in South Korea, and 16 times in China between January 2000 and September 2021 along the coast of China, Korea, and Japan.We reinvestigated the distribution of S. horneri along the coast of China and found both attached and floating S. horneri in Dalian, Shandong, Zhejiang, Fujian, and Guangdong Provinces.Our survey added 34 distribution sites along the coast of China to the list on the GBIF site (Figure 6a).S. horneri mainly grows on rocks or stone marshes in the intertidal and subtidal zones under natural conditions [19].However, large biomass of S. horneri have a strong relationship with aquaculture along the coast of China, and materials such as cables and drift in raft areas contribute greatly to the attached growth of S. horneri.Studies have found that the body length and biomass of intertidal S. horneri are much lower than those of S. horneri on rafts in aquaculture areas [24,40].As the number and density of air sacs increases, the buoyancy of air sacs may exceed the attachment ability of their holdfasts.With the combined action of wind, waves, and currents, the ultra-long thalli in aquaculture areas are easily detached [10].Large amounts of S. horneri in aquaculture areas are not welcomed by farmers because algal growth and surface coverage in raft areas affect the growth of cultured species, and fishers cut them off and discard them at will.Large scale floating accumulation of S. horneri on the sea surface is caused by the combined effects of the alga itself, natural conditions, and human factors [24].S. horneri mainly grows on rocks or stone marshes in the intertidal and subtidal zones under natural conditions [19].However, large biomass of S. horneri have a strong relationship with aquaculture along the coast of China, and materials such as cables and drift in raft areas contribute greatly to the attached growth of S. horneri.Studies have found that the body length and biomass of intertidal S. horneri are much lower than those of S. horneri on rafts in aquaculture areas [24,40].As the number and density of air sacs increases, the buoyancy of air sacs may exceed the attachment ability of their holdfasts.With the combined action of wind, waves, and currents, the ultra-long thalli in aquaculture areas are easily detached [10].Large amounts of S. horneri in aquaculture areas are not welcomed by farmers because algal growth and surface coverage in raft areas affect the growth of cultured species, and fishers cut them off and discard them at will.Large scale floating accumulation of S. horneri on the sea surface is caused by the combined effects of the alga itself, natural conditions, and human factors [24].
In recent years, the area of aquaculture along the coast of China has gradually increased.The water in the culture area is highly eutrophic and suitable for the growth and reproduction of S. horneri.S. horneri absorbs nutrients from water, causing a large-scale outbreak, which inhibits the nutrient absorption of aquatic products and indirectly affects the local economic development.It will also destroy the local ecological environment and have a negative impact on the local economy, tourism and transportation.The mussel cultivation industry in China is mainly concentrated around Gouqi Island in Zhejiang Province, and the local cultivation area has shown a steadily increasing trend (remained above 1000 ha after 2015) from year to year (Figure 6b).Since 2005, there has been a dramatic increase in the area of cultivation of the brown alga Saccharina japonica in Rongcheng City of Shandong Province, which reached 4000 ha by 2016 (Figure 6c).Some studies have shown that the source of golden tides is located along the coast of Shandong and Zhejiang Provinces [13,18,41].Specifically, the fast-growing aquaculture industry provides more sessile substrate for the attached growth of S. horneri, which in turn provides more biomass for floating golden tide outbreaks and may be the key to such outbreaks in the YS and ECS in recent years.In addition, the aquaculture areas along the coast of southern Korea and Japan are showing an increasing trend and may also supply considerable biomass to the floating golden tides in the YS and ECS.
Studies of the origin of golden tides in the YS and ECS have mainly focused on morphology, physical oceanography, satellite remote sensing, and molecular identification.Floating S. horneri can be transported into the ECS and even coastal Japan by the Kuroshio current [42] and further into the YS [17].The YS floating S. horneri mainly originates from the coast of Zhejiang Province, but we cannot ignore the possibility of multiple sources along the coast in Shandong Province of China and southern Korea [18,29].Xing et al. (2017) predicted the source region of floating S. horneri from October 2016 to January 2017 to be Rongcheng City of Shandong Province.In summary, there may be dual sources of floating golden tides in the YS and ECS, and their origin could be season dependent.
Population diversity of floating S. horneri in the YS was low [30].We evaluated several partial sequences of ITS2, rbcL, cox1, cob-cox2, and cox3, which revealed a higher genetic diversity in attached S. horneri populations along the coast of China than in floating samples from the YS.
Although floating golden tides have serious impacts on the aquaculture industry along the coast of the western Pacific, the role of S. horneri in ecological remediation, resourcebased utilization, and marine biodiversity conservation cannot be ignored [43,44].From a dialectical perspective, we first need to rationally protect and support the unique ecosystem of the floating Sargasso seaweed bed and then we need to actively develop golden tide warning systems in order to prepare for the inshore drift of large amounts of biomass.

Conclusions
Compared with previous studies, the investigation and sampling area of S. horneri in this study was the widest, and it was the first comprehensive investigation and community genetic structure analysis of S. horneri along the coast of China.The analysis of cox3 showed the attached samples from along the coast of ZJ and Shandong (SD) shared Hap9 and were connected to Hap11, which was unique to the attached samples from KRJJ.The genetic evolution between the samples from KR and the attached samples from other regions of China (CN) was relatively distant.The source of floating S. horneri golden tides in the YS was traced, which suggested the possibility of two sources for S. horneri golden tides, and the huge attached biomass in local aquaculture of Zhejiang and Shandong Provinces of China may provide a source for golden tides in the YS.In addition, researchers should think from a dialectical perspective about the prevention and management of S. horneri.

Figure 1 .
Figure 1.General situation of Sargassum blooms: (a-d) are, in order, the S. horneri biomass grown in coastal aquaculture areas of Shandong, Zhejiang, Fujian, and Guangdong Provinces; (e) S. horneri biomass stranded on P. yezoensis cultivation rafts located on Subei Shoal, and (f) drifting S. horneri biomass in the YS; (g) Long time-series trend of golden tides based on satellite remote sensing images in the Yellow Sea and the East China Sea in 2021; (h) The distribution and coverage areas of the golden tide that evolved over time in the YS and ECS in 2021.

Figure 1 .
Figure 1.General situation of Sargassum blooms: (a-d) are, in order, the S. horneri biomass grown in coastal aquaculture areas of Shandong, Zhejiang, Fujian, and Guangdong Provinces; (e) S. horneri biomass stranded on P. yezoensis cultivation rafts located on Subei Shoal, and (f) drifting S. horneri biomass in the YS; (g) Long time-series trend of golden tides based on satellite remote sensing images in the Yellow Sea and the East China Sea in 2021; (h) The distribution and coverage areas of the golden tide that evolved over time in the YS and ECS in 2021.

Figure 5 .
Figure 5. Haplotype network and composition percentage (%) of the S. horneri samples collected in the large-scale spate-temporal sampling along the coast of China.Samples from the Yellow Sea and Subei Shoal were floating samples, all others were attached samples.(a) cob-cox2 and (b) cox3 in 2017-2021; (c) cob-cox2 and (d) cox3 in 2021; (e) cob-cox2 and (f) cox3 in 2017-2021.

Figure 5 .
Figure 5. Haplotype network and composition percentage (%) of the S. horneri samples collected in the large-scale spate-temporal sampling along the coast of China.Samples from the Yellow Sea and Subei Shoal were floating samples, all others were attached samples.(a) cob-cox2 and (b) cox3 in 2017-2021; (c) cob-cox2 and (d) cox3 in 2021; (e) cob-cox2 and (f) cox3 in 2017-2021.

Figure 6 .
Figure 6.Distribution of S. horneri along the coast of China, South Korea, and Japan from January 2000 to present ( : floating samples found during reinvestigation; : attached samples found during reinvestigation) (a) and an overview of culture area and total production of mussels and S. japonica along the coast of Zhejiang (b) and Shandong Provinces (c) over successive years.

Figure 6 .
Figure 6.Distribution of S. horneri along the coast of China, South Korea, and Japan from January 2000 to present (

Figure 6 .
Figure 6.Distribution of S. horneri along the coast of China, South Korea, and Japan from January 2000 to present ( : floating samples found during reinvestigation; : attached samples found during reinvestigation) (a) and an overview of culture area and total production of mussels and S. japonica along the coast of Zhejiang (b) and Shandong Provinces (c) over successive years.

Figure 6 .
Figure 6.Distribution of S. horneri along the coast of China, South Korea, and Japan from January 2000 to present ( : floating samples found during reinvestigation; : attached samples found during reinvestigation) (a) and an overview of culture area and total production of mussels and S. japonica along the coast of Zhejiang (b) and Shandong Provinces (c) over successive years.

:
attached samples found during reinvestigation) (a) and an overview of culture area and total production of mussels and S. japonica along the coast of Zhejiang (b) and Shandong Provinces (c) over successive years.

Table 1 .
Information about the S. horneri samples collected from the coast of China, Subei Shoal, and the YS.

Table 1 .
Information about the S. horneri samples collected from the coast of China, Subei Shoal, and the YS.

Table 2 .
Information about the five marker genes in chloroplasts (rbcL), ribosomes (ITS2), and mitochondria (cox) used in this study.