Molecular Identification and Toxin Analysis of Alexandrium spp. in the Beibu Gulf: First Report of Toxic A. tamiyavanichii in Chinese Coastal Waters

The frequency of harmful algal blooms (HABs) has increased in China in recent years. Information about harmful dinoflagellates and paralytic shellfish toxins (PSTs) is still limited in China, especially in the Beibu Gulf, where PSTs in shellfish have exceeded food safety guidelines on multiple occasions. To explore the nature of the threat from PSTs in the region, eight Alexandrium strains were isolated from waters of the Beibu Gulf and examined using phylogenetic analyses of large subunit (LSU) rDNA, small subunit (SSU) rDNA, and internal transcribed spacer (ITS) sequences. Their toxin composition profiles were also determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). All eight strains clustered in the phylogenetic tree with A. pseudogonyaulax, A. affine, and A. tamiyavanichii from other locations, forming three well-resolved groups. The intraspecific genetic distances of the three Alexandrium species were significantly smaller than interspecific genetic distances for Alexandrium species. Beibu Gulf isolates were therefore classified as A. pseudogonyaulax, A. affine, and A. tamiyavanichii. No PSTs were identified in A. pseudogonyaulax, but low levels of gonyautoxins (GTXs) 1 to 5, and saxitoxin (STX) were detected in A. tamiyavanichii (a total of 4.60 fmol/cell). The extremely low level of toxicity is inconsistent with PST detection above regulatory levels on multiple occasions within the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. Understanding the toxicity and phylogeny of Alexandrium species provides foundational data for the protection of public health in the Beibu Gulf region and the mitigation of HAB events.


Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among

Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and other organisms through the food web. Humans who consume shellfish containing PSTs can become poisoned; PSTs thus represents a major health hazard [1,5]. Alexandrium is a common species in the coastal waters of China. Over the past 15 years, 24 HABs due to Alexandrium spp. have occurred in the Bohai, Yellow, and South China Seas [5], with disastrous consequences for local marine fisheries, ecosystems, and coastal landscapes.
The morphological identification of Alexandrium is based primarily on the fine structure of the apical pore complex, the first apical and sixth precingular plates, the presence/absence of a ventral pore and its location, the shape and size of sulcal plates, and whether they form chains [6][7][8]. However, morphological differences between the many species of Alexandrium are small. Some characteristics are plastic, such as the presence or absence of a ventral pore and the position of the anterior connecting pore. The identification of Alexandrium is thus inadequate when only morphological standards are applied. One newer method of identifying marine microalgae is DNA molecular identification. Applying this method to Alexandrium classification along with phylogenetic studies can help to identify species in this genus [8,9]. Studies on the molecular identification of Alexandrium have mostly focused on its large (LSU rDNA) and small (SSU rDNA) subunit, and non-coding internal transcribed spacer (ITS) ribosomal DNA sequences. For example, the Alexandrium tamarense species complex can be classified into A. catenella (Group Ⅰ , conspecific as A. fundyense), A. mediterraneum (Group Ⅱ), A. tamarense (Group Ⅲ), A. pacificum (Group Ⅳ), and A. australiense (Group Ⅴ) based on rDNA sequences [6,[10][11][12]. New species A. diversaporum, A. fragae, and A. pohangense have recently been identified based on their cell morphology and 18S rDNA, 28S rDNA, and ITS ribosomal gene sequences [8,13,14]. Gu et al. [15,16] also isolated and germinated multiple strains of Alexandrium from cysts in sediments along the coast of China, and discovered the newly recorded species, A. andersonii, A. fraterculum, A. leei, A. ostenfeldii, A. pseudogonyaulax, A. tamutum through LSU rDNA and ITS sequence analysis.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.

Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and other organisms through the food web. Humans who consume shellfish containing PSTs can become poisoned; PSTs thus represents a major health hazard [1,5]. Alexandrium is a common species in the coastal waters of China. Over the past 15 years, 24 HABs due to Alexandrium spp. have occurred in the Bohai, Yellow, and South China Seas [5], with disastrous consequences for local marine fisheries, ecosystems, and coastal landscapes.
The morphological identification of Alexandrium is based primarily on the fine structure of the apical pore complex, the first apical and sixth precingular plates, the presence/absence of a ventral pore and its location, the shape and size of sulcal plates, and whether they form chains [6][7][8]. However, morphological differences between the many species of Alexandrium are small. Some characteristics are plastic, such as the presence or absence of a ventral pore and the position of the anterior connecting pore. The identification of Alexandrium is thus inadequate when only morphological standards are applied. One newer method of identifying marine microalgae is DNA molecular identification. Applying this method to Alexandrium classification along with phylogenetic studies can help to identify species in this genus [8,9]. Studies on the molecular identification of Alexandrium have mostly focused on its large (LSU rDNA) and small (SSU rDNA) subunit, and non-coding internal transcribed spacer (ITS) ribosomal DNA sequences. For example, the Alexandrium tamarense species complex can be classified into A. catenella (Group Ⅰ , conspecific as A. fundyense), A. mediterraneum (Group Ⅱ), A. tamarense (Group Ⅲ), A. pacificum (Group Ⅳ), and A. australiense (Group Ⅴ) based on rDNA sequences [6,[10][11][12]. New species A. diversaporum, A. fragae, and A. pohangense have recently been identified based on their cell morphology and 18S rDNA, 28S rDNA, and ITS ribosomal gene sequences [8,13,14]. Gu et al. [15,16] also isolated and germinated multiple strains of Alexandrium from cysts in sediments along the coast of China, and discovered the newly recorded species, A. andersonii, A. fraterculum, A. leei, A. ostenfeldii, A. pseudogonyaulax, A. tamutum through LSU rDNA and ITS sequence analysis.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.

Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and other organisms through the food web. Humans who consume shellfish containing PSTs can become poisoned; PSTs thus represents a major health hazard [1,5]. Alexandrium is a common species in the coastal waters of China. Over the past 15 years, 24 HABs due to Alexandrium spp. have occurred in the Bohai, Yellow, and South China Seas [5], with disastrous consequences for local marine fisheries, ecosystems, and coastal landscapes.
The morphological identification of Alexandrium is based primarily on the fine structure of the apical pore complex, the first apical and sixth precingular plates, the presence/absence of a ventral pore and its location, the shape and size of sulcal plates, and whether they form chains [6][7][8]. However, morphological differences between the many species of Alexandrium are small. Some characteristics are plastic, such as the presence or absence of a ventral pore and the position of the anterior connecting pore. The identification of Alexandrium is thus inadequate when only morphological standards are applied. One newer method of identifying marine microalgae is DNA molecular identification. Applying this method to Alexandrium classification along with phylogenetic studies can help to identify species in this genus [8,9]. Studies on the molecular identification of Alexandrium have mostly focused on its large (LSU rDNA) and small (SSU rDNA) subunit, and non-coding internal transcribed spacer (ITS) ribosomal DNA sequences. For example, the Alexandrium tamarense species complex can be classified into A. catenella (Group Ⅰ , conspecific as A. fundyense), A. mediterraneum (Group Ⅱ), A. tamarense (Group Ⅲ), A. pacificum (Group Ⅳ), and A. australiense (Group Ⅴ) based on rDNA sequences [6,[10][11][12]. New species A. diversaporum, A. fragae, and A. pohangense have recently been identified based on their cell morphology and 18S rDNA, 28S rDNA, and ITS ribosomal gene sequences [8,13,14]. Gu et al. [15,16] also isolated and germinated multiple strains of Alexandrium from cysts in sediments along the coast of China, and discovered the newly recorded species, A. andersonii, A. fraterculum, A. leei, A. ostenfeldii, A. pseudogonyaulax, A. tamutum through LSU rDNA and ITS sequence analysis.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.

Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and other organisms through the food web. Humans who consume shellfish containing PSTs can become poisoned; PSTs thus represents a major health hazard [1,5]. Alexandrium is a common species in the coastal waters of China. Over the past 15 years, 24 HABs due to Alexandrium spp. have occurred in the Bohai, Yellow, and South China Seas [5], with disastrous consequences for local marine fisheries, ecosystems, and coastal landscapes.
The morphological identification of Alexandrium is based primarily on the fine structure of the apical pore complex, the first apical and sixth precingular plates, the presence/absence of a ventral pore and its location, the shape and size of sulcal plates, and whether they form chains [6][7][8]. However, morphological differences between the many species of Alexandrium are small. Some characteristics are plastic, such as the presence or absence of a ventral pore and the position of the anterior connecting pore. The identification of Alexandrium is thus inadequate when only morphological standards are applied. One newer method of identifying marine microalgae is DNA molecular identification. Applying this method to Alexandrium classification along with phylogenetic studies can help to identify species in this genus [8,9]. Studies on the molecular identification of Alexandrium have mostly focused on its large (LSU rDNA) and small (SSU rDNA) subunit, and non-coding internal transcribed spacer (ITS) ribosomal DNA sequences. For example, the Alexandrium tamarense species complex can be classified into A. catenella (Group Ⅰ , conspecific as A. fundyense), A. mediterraneum (Group Ⅱ), A. tamarense (Group Ⅲ), A. pacificum (Group Ⅳ), and A. australiense (Group Ⅴ) based on rDNA sequences [6,[10][11][12]. New species A. diversaporum, A. fragae, and A. pohangense have recently been identified based on their cell morphology and 18S rDNA, 28S rDNA, and ITS ribosomal gene sequences [8,13,14]. Gu et al. [15,16] also isolated and germinated multiple strains of Alexandrium from cysts in sediments along the coast of China, and discovered the newly recorded species, A. andersonii, A. fraterculum, A. leei, A. ostenfeldii, A. pseudogonyaulax, A. tamutum through LSU rDNA and ITS sequence analysis.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.

Introduction
The genus Alexandrium is an important toxic and harmful algal blooms (HABs) dinoflagellate distributed in coastal waters worldwide [1]. This genus was first described by Halim [2] and it has since become one of the most studied dinoflagellate genera. Among the over 30 species of Alexandrium reported to date, at least half can cause paralytic shellfish poisoning (PSP) [3,4]. Paralytic shellfish toxins (PSTs) accumulate in shellfish, fish, and other organisms through the food web. Humans who consume shellfish containing PSTs can become poisoned; PSTs thus represents a major health hazard [1,5]. Alexandrium is a common species in the coastal waters of China. Over the past 15 years, 24 HABs due to Alexandrium spp. have occurred in the Bohai, Yellow, and South China Seas [5], with disastrous consequences for local marine fisheries, ecosystems, and coastal landscapes.
The morphological identification of Alexandrium is based primarily on the fine structure of the apical pore complex, the first apical and sixth precingular plates, the presence/absence of a ventral pore and its location, the shape and size of sulcal plates, and whether they form chains [6][7][8]. However, morphological differences between the many species of Alexandrium are small. Some characteristics are plastic, such as the presence or absence of a ventral pore and the position of the anterior connecting pore. The identification of Alexandrium is thus inadequate when only morphological standards are applied. One newer method of identifying marine microalgae is DNA molecular identification. Applying this method to Alexandrium classification along with phylogenetic studies can help to identify species in this genus [8,9]. Studies on the molecular identification of Alexandrium have mostly focused on its large (LSU rDNA) and small (SSU rDNA) subunit, and non-coding internal transcribed spacer (ITS) ribosomal DNA sequences. For example, the Alexandrium tamarense species complex can be classified into A. catenella (Group Ⅰ , conspecific as A. fundyense), A. mediterraneum (Group Ⅱ), A. tamarense (Group Ⅲ), A. pacificum (Group Ⅳ), and A. australiense (Group Ⅴ) based on rDNA sequences [6,[10][11][12]. New species A. diversaporum, A. fragae, and A. pohangense have recently been identified based on their cell morphology and 18S rDNA, 28S rDNA, and ITS ribosomal gene sequences [8,13,14]. Gu et al. [15,16] also isolated and germinated multiple strains of Alexandrium from cysts in sediments along the coast of China, and discovered the newly recorded species, A. andersonii, A. fraterculum, A. leei, A. ostenfeldii, A. pseudogonyaulax, A. tamutum through LSU rDNA and ITS sequence analysis.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.
Beibu Gulf is a natural, semi-enclosed, shallow gulf located in the northwestern continental shelf of the South China Sea. It is bordered on the west, north, and east by Vietnam, Guangxi, and Hainan Island, respectively. Beibu Gulf has long been regarded as the last clean sea zone in Chinese coastal waters. However, the number, scale, and areas of algal blooms in the region have significantly increased recently, and the number of species causing HABs has also increased [17,18]. Accurate identification of causative species is the biological basis for HAB prevention and monitoring. For Alexandrium spp. in the Beibu Gulf, a detailed investigation is needed. Alexandrium cysts in sediments from the Beibu Gulf near Fangchenggang have been germinated and then identified by optical microscopy, scanning electron microscopy, and LSU rDNA and ITS sequence analyses as A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) [6,10,16]. Cysts in samples from Fangchenggang and Beihai have been identified by optical microscopy as A. minutum and A. pacificum (as A. tamarense) [6,10,19,20]. Alexandrium pacificum (as A. tamarense and A. catenella) [6,10,11,21,22] is so far the only species that have been recorded from the waters in Beibu Gulf. Although Alexandrium cysts serve as seed banks in algal blooms, the species composition and toxicity of Alexandrium in the waters are inconsistent with these cysts [23,24]. Therefore, the species composition of the natural waters of Beibu Gulf needs further study.
Alexandrium catenella, A. minutum, A. ostenfeldii, and A. pacificum (as A. tamarense) species complex from the Bohai, Yellow, East China Sea, and South China Seas contain PSTs [6,10,16,25,26]. Wang et al. [27] also detected C1, C2, gonyautoxins (GTX)3, and 5, and NEO in the ATGX02 strain of A. pacificum (as A. tamarense) germinated near Weizhou Island in the Beibu Gulf, among which, C2 and GTX5 accounted for the highest proportion (>90%). The toxin content was 30.67-40.97 fmol/cell, but the PSP toxin was undetectable in the AAGX01 strain of A. affine. Zou et al. [26] tested the same ATGX02 strain of A. pacificum and found a toxin that included GTX1, 2, 3, and 4, dcSTX, and STX, which differed from Toxins 2021, 13, 161 3 of 16 that found by Wang et al. [27]. The toxin content was 4.03 fmol/cell, and the toxicity was 0.69 pg STXeq/cell [26], classifying it as a low-toxicity algal strain. In the study of Gu et al. [16], a total of 18 strains of A. affine, A. andersonii, A. leei, A. pseudogonyaulax, and A. pacificum (as Atama complex, Group IV) were germinated from cysts in the Beibu Gulf near Fangchenggang, but these strains' PSTs were not analyzed. Currently, data about toxin production by Alexandrium strains in the natural waters of the Beibu Gulf remain scant, although PST contamination of shellfish was reported in this area [28][29][30], and occasionally exceeds food safety thresholds [28,29]. The source of those PSTs remains unknown.
Here we classify the species and define the toxicity of Alexandrium isolates from Beibu Gulf waters and characterize the toxin-producing algae that can cause PST contamination of shellfish. We isolated eight Alexandrium strains from Weizhou Island in the Beibu Gulf, an area with a high incidence of HABs [17] and analyzed their characteristic LSU rDNA, SSU rDNA, and ITS sequences to achieve their species identification. We also applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze toxin composition and content. Here, we describe the molecular phylogeny of three Alexandrium species in the Beibu Gulf, their relationships with Alexandrium in other marine areas, and differences in toxin production.

Sequence Analysis
The average lengths of the LSU rDNA, SSU rDNA, and ITS sequences obtained from the eight strains of Alexandrium from the Beibu Gulf were 1382, 1712, and 586 bp, respectively. The average base composition of the LSU rDNA sequences was 27.0% A, 30.8% T, 25.5% G, and 16.7% C, respectively, and the A+T content was greater than the G+C content (57.8% vs. 42.2%). The average base composition of the SSU rDNA sequences was 27.6% A, 29.5% T, 25.1% G, and 17.8% C, and the A+T content was greater than G+C content (57.1% vs. 42.9%). The average base composition of the ITS sequences was 24.4% A, 33.4% T, 23.9% G, and 18.3% C, and the A+T content was greater than G+C content (57.8% vs. 42.2%). These sequence composition characteristics were similar to those of the Alexandrium sequences that were used to construct the phylogenetic tree (Table 1). Among the three sequences, the SSU rDNA sequence had the highest and lowest proportions of conserved (82.6%) and variable (15.5%) sites, respectively, confirming that the SSU rDNA sequence is more conserved than the LSU rDNA and the ITS sequences (Table 1).

Phylogenetic Tree Analysis
Phylogenetic trees of the LSU rDNA, SSU rDNA, and ITS sequences were constructed using Goniodoma polyedricum, Lingulodinium polyedra, and Scrippsiella acuminata as outgroups (Figures 1-3). The topological structures of the trees using the maximum likelihood and Bayesian inference were generally consistent; thus, only the maximum likelihood trees are shown. The three phylogenetic trees all showed that five of the Alexandrium strains from the Beibu Gulf (APBG01, APBG02, APBG03, APBG04, APBG05) and A. pseudogonyaulax of 99/1.00, 98/1.00, and 99/1.00, respectively (Figures 1-3). A. tamarense, A. fundyense, and A. catenella clustered together in a large branch, with bootstrap values/Bayesian posterior probabilities of 99/1.00, 84/1.00, and 100/1.00, respectively, and the remaining Alexandrium species also formed clear branches with strong bootstrap values and posterior probabilities (Figures 1-3).     The ATBG01 strain of A. tamiyavanichii from Beibu Gulf was most closely related to the Japanese AT0112T06 (LSU) and TAMI22012 (SSU) strains and the Malaysian AcSM01 (ITS) strain, with genetic distances of 0.000, 0.001, and 0.000, respectively, whereas the genetic distances of LSU rDNA, SSU rDNA, and ITS with other A. tamiyavanichii strains worldwide were 0.000-0.013, 0.001-0.002, and 0.000-0.060, respectively. The intraspecific genetic distances of the three A. tamiyavanichii sequences were 0.000-0.013, 0.000-0.002, The Jukes-Cantor genetic distance matrix calculated based on LSU rDNA, SSU rDNA, and ITS sequences showed genetic distances among the A. pseudogonyaulax APBG01, APBG02, APBG03, APBG04, APBG05 strains of 0.000-0.014, 0.000-0.004, and 0.002-0.028, respectively. The A. pseudogonyaulax strains with the highest homology were the APFC01 and APFC02 strains from the South China Sea (LSU), the OF2AUG09 strain from Norway (SSU), the APFC01 and APFC02 strains from the South China Sea, the APCH01 strain from the Yellow Sea, and the CAWD54 strain from New Zealand (ITS), with genetic distances of 0.000-0.002, 0.000-0.004, and 0.000, respectively, whereas the genetic distances of LSU rDNA, SSU rDNA, and ITS from other A. pseudogonyaulax strains worldwide were 0.000-0.021, 0.000-0.009, and 0.000-0.037, respectively. The intraspecific genetic distances of the three sequences of A. pseudogonyaulax were 0.000-0.021, 0.000-0.009, and 0.000-0.037, respectively, and generally smaller than the interspecific genetic distances of 0.009-0.881, 0.000-0.111, and 0.206-1.368 for Alexandrium species.

Discussion
Alexandrium species are distributed worldwide, and many of them are toxic [1]. Determining the species composition, toxicity, and biogeographic distribution of Alexandrium spp. in a marine area is a prerequisite for HAB control and PSP mitigation. Alexandrium margalefii and A. tamiyavanichii are recently discovered species in China using optical and fluorescence microscopy [31,32]. Ten species of Alexandrium, A. affine, A. andersonii, A. catenella, A. fraterculum, A. leei, A. minutum, A. ostenfeldii, A. pseudogonyaulax, A. pacificum (as A. tamarense), and A. tamutum, have been identified in the East China, South China, Yellow, and Bohai Seas using optical, fluorescence and scanning electron microscopy, and rDNA molecular identification techniques [6,10,16,33]. While A. acatenella, A. catenella (as A. fundyense), A. hiranoi, A. minutum, A. ostenfeldii, A. pseudogonyaulax, A. pacificum, and A. tamiyavanichii were identified in Hong Kong waters morphologically [6,34], but molecular sequences are not available to confirm their identities. Considering the identification of Alexandrium is inadequate when only morphological standards are used, the real number of Alexandrium species in Chinese waters still remains to be determined.
Eight Beibu Gulf Alexandrium strains were identified based on LSU rDNA, SSU rDNA, and ITS sequences as A. affine, A. pseudogonyaulax, and A. tamiyavanichii. These results are unsurprising, as A. pseudogonyaulax and A. affine are globally distributed and have been reported in the sediments of Beibu Gulf previously [16], whereas A. tamiyavanichii is a warm-water species distributed in temperate to tropical regions such as Japan, Thailand, Malaysia, Brazil, and Mexico [35][36][37][38][39]. Both A. affine and A. pseudogonyaulax have been identified in the East China, South China, and Yellow Seas [16], while A. tamiyavanichii has been found only in the East China Sea [31] and in waters near Hong Kong [34] based on morphology. This is the first report of A. tamiyavanichii from the Beibu Gulf confirmed with molecular sequences. This is also the first report on the toxicity of A. tamiyavanichii from Chinese coastal waters.
Except for the A. tamarense species complex, the three phylogenetic trees generally formed clear branches of Alexandrium species with good bootstrap values. The positions of the three Beibu Gulf species on the trees were consistent with those of other published studies [13,14], indicating that Alexandrium species can be distinguished using LSU rDNA, SSU rDNA, and ITS sequences. However, the phylogenetic tree constructed using SSU rDNA has occasional exceptions, because A. hiranoi, A. pseudogonyaulax, and strains APBG01-05 clustered together (Figure 2). Alexandrium hiranoi and A. pseudogonyaulax are difficult to identify using SSU rDNA gene sequences, which have been noted previously [13,40].
Generally, algal strains of the same species from the same or similar geographic locations have small base differences and are closely related; the present findings on A. tamiyavanichii are consistent with this. A. tamiyavanichii from the Beibu Gulf is most closely related to the Japanese strains AT0112T06 and TAMI22012, and the Malaysian strain AcSM01, and relatively distantly related to the Brazilian PSII strain. A relationship between the different strains of A. affine and A. pseudogonyaulax in terms of genetic distance and geographical was not obvious, however. For example, in the ITS tree, Beibu Gulf A. affine had close homology with strains of this species from the geographically distant waters of Spain, Australia, and Gibraltar, and less homology with algal strains from the geographically close waters of China and Japan. The LSU rDNA and ITS trees of A. pseudogonyaulax also showed that strains with different geographical origins clustered on the same large branch with no obvious small-branch topology. Others have also noted similar phenomena [14,16], which might be associated with the physiological and ecological features of Alexandrium species [1,4,41].
Previous tests of A. pseudogonyaulax did not reveal any PSTs, but did find the toxin goniodomin A (GDA) [40,42,43]. We did not detect PSTs in five strains of Beibu Gulf A. pseudogonyaulax either; whether they can produce GDA awaits further investigation. Two strains of Beibu Gulf A. affine were lost during culture maintenance, so PSTs could not be analyzed. Current literature suggests that A. affine is largely nontoxic [16,44] or only potentially toxic [45]. However, A. affine strains with low PST toxin content (<1.0-2.28 fmol/cell), including NeoSTX, STX, and GTX1-4, have been isolated from Ha Long Bay, Vietnam, near the Beibu Gulf [46]. A. tamiyavanichii strains from various marine areas worldwide, including the Beibu Gulf strain described herein, can produce PST toxins, but toxin composition and content differ significantly (Table 3). We detected only GTX1-5 and STX toxins in the Beibu Gulf strain, which is similar to the PSTs composition of the Chula 6 strains from Thailand and the KOSKP01, KOSKP02, and KOSKP03 strains from Malaysia [38,47,57], but different from other A. tamiyavanichii strains from these countries as well as strains from Japan, Brazil, and South Africa ( Table 3). The range in PST content of A. tamiyavanichii reported in the literature is 1.17 fmol/cell-3.07 × 10 6 fmol/cell. The PST content of one A. tamiyavanichii strain isolated from the recent cases of PSP outbreak at Kuantan Port, Malaysia [38] reached an extreme maximum of 3.07 × 10 6 fmol/cell, which is 100,000 times higher than strain ATBG01 in this study. The Beibu Gulf A. tamiyavanichii strain is a low-toxicity algal strain, but more strains need to be tested in the future, and measurements from bloom populations are needed as well.
Based on this and previous studies, Beibu Gulf Alexandrium species appear to be either non-toxic or weakly toxic [16,26,27]. This is, however, inconsistent with the high level of PST detection rate in Beibu Gulf shellfish, which have exceeded food safety standards on multiple occasions (Table 4).
Possible explanations for this inconsistency include the following: (1) Low-toxicity algal strains are widely distributed in the Beibu Gulf and survive for long periods. PSTs can thus accumulate in shellfish through time to levels sufficient to exceed regulatory standards. Anderson et al. [62] noted a similar phenomenon in Daya Bay, China where A. pacificum has very low toxicity of 7.2-12.7 fmol/cell, yet PSP poisoning still occasionally occurs there.
(2) Based on reports of A. tamiyavanichii toxin content varying by more than 5 orders of magnitude among isolates from the same locations in Malaysia and by factors of 857 in Thailand and 651 in Japan (Table 3), it seems very likely that other highly toxic A. tamiyavanichii strains occur in the Beibu Gulf that were not isolated. Lowabundance Alexandrium spp. in seawater can be missed due to limitations of small numbers of culture isolations, and thus highly toxic strains could easily be overlooked. Furthermore, because isolates were only established from Weizhou Island in the Beibu Gulf, and not from other highly productive areas of the Gulf, such as Qinzhou Bay and Tieshan Harbor, the presence of highly toxic species in these regions cannot be ruled out. In this regard, it is of note that the PST composition of shellfish from the Beibu Gulf differs significantly from the A. tamiyavanichii isolates analyzed here (Tables 3 and 4). This could reflect biotransformation (see below) and/or different toxic Alexandrium species or strains from those analyzed here. (3) Alexandrium in field populations can grow faster and produce more toxins than laboratory cultures, as has been observed by others through variations in temperature, salinity, light, and nutrients [63][64][65][66]. Brosnahan et al. [67] recently reported in situ growth rates for A. catenella that were more than twice those observed in laboratory cultures under similar growth conditions. (4) Metabolism and bioconversion processes in different shellfish greatly affect the accumulation and potency of PSTs [68,69]. For example, the PSP toxicity of Paphia undulata is over 1000 times higher than that of Meretrix lusoria in the Beibu Gulf [29]. Thus, further study of biotransformation of PSTs in different shellfish from the Beibu Gulf is needed. (5) Although Alexandrium spp.are the principal causative organisms of PSP in many regions, the presence of other PST-producing microalgae, such as Gymnodinium catenatum and Pyrodinium bahamense [70,71], and atypical toxin-producing organisms such as brackish cyanobacteria, as well as calcareous red macroalgae [72], cannot be ruled out.

Conclusions
The Beibu Gulf region is experiencing rapid economic growth, frequent HABs, and the increasingly serious problem of PST contamination of shellfish. To explore this issue, potential PST toxin-producing algae in the Alexandrium genus were isolated from Weizhou Island, a HAB hotspot in the Beibu Gulf and characterized phylogenetically and toxicologically. For the first time, A. affine, A. pseudogonyaulax, and A. tamiyavanichii were definitively identified in plankton samples from the Beibu Gulf, among which, A. tamiyavanichii is new to this body of water. This species showed small base differences from Asian strains of the same species from nearby locations. There was no observed relationship between genetic distance and the geographical distribution of various A. affine and A. pseudogonyaulax strains. A. pseudogonyaulax strains did not produce PSTs, whereas A. tamiyavanichii produced 4.6 fmol/cell of toxin, indicating that it is a low-toxicity strain in comparison to global isolates. This finding of low toxicity is inconsistent with the detection of relatively high levels of PSTs in shellfish from the Beibu Gulf, suggesting that higher toxicity strains may occur in those waters, but were unsampled. Other explanations including biotransformation of PSTs in shellfish and the presence of other PST-producing algae are also suggested. This is the first report of toxic A. tamiyavanichii in the coastal waters in China. In future studies, potential PST-producing microalgae and toxic shellfish should be collected from the major localities in the Beibu Gulf to further elucidate species diversity, biogeographical distribution, toxin-producing characteristics, and relationships with PST vectors in the region. This would provide much-needed insights into PST sources and aid in the protection of public health in the Beibu Gulf.

Algal Source and Culture Conditions
Water samples containing phytoplankton collected from the waters of Weizhou Island in the Beibu Gulf four times between the summer 2018 and summer 2019 (Figure 4), were transported and stored as described in the "Specification for Marine Monitoring" (GB/T17378-2007). The samples were visually assessed using a TS100 inverted microscope (Nikon Corp., Tokyo, Japan). Algal cells of interest were isolated from water samples using capillary tubes, washed 3 or 4 times with sterilized seawater, then cultured, resulting in eight Alexandrium strains ( Table 5). The culture conditions were f/2 medium, 22 • C, light intensity 150 µE/m 2 /s, and a 12:12 h light-dark cycle. The algal strains ATBG01 and APBG0 were cultured to the exponential growth phase, then toxin was extracted from 6 × 10 6 algal cells. with PST vectors in the region. This would provide much-needed insights into PST sources and aid in the protection of public health in the Beibu Gulf.

Algal Source and Culture Conditions
Water samples containing phytoplankton collected from the waters of Weizhou Island in the Beibu Gulf four times between the summer 2018 and summer 2019 (Figure 4), were transported and stored as described in the "Specification for Marine Monitoring" (GB/T17378-2007). The samples were visually assessed using a TS100 inverted microscope (Nikon Corp., Tokyo, Japan). Algal cells of interest were isolated from water samples using capillary tubes, washed 3 or 4 times with sterilized seawater, then cultured, resulting in eight Alexandrium strains ( Table 5). The culture conditions were f/2 medium, 22 °C, light intensity 150 μE/m 2 /s, and a 12:12 h light-dark cycle. The algal strains ATBG01 and APBG0 were cultured to the exponential growth phase, then toxin was extracted from 6 × 10 6 algal cells.

Extraction of DNA, PCR Amplification, and Sequencing
Fresh algae (30 mL) in the exponential growth phase were collected and sedimented by centrifugation at 7000× g for 1 min, then the supernatants were discarded. Total DNA was obtained using BioFastSpin Plant Genomic DNA Extraction Kits (Bioer, Hangzhou, China), and characteristic LSU rDNA, SSU rDNA, and ITS fragments were amplified by PCR. The amplification primers (5′3′) were listed in Table 6. The PCR reaction volume was 40 μL, and included 20 μL of 2 × Es Taq Master Mix (CoWin Biosciences, Cambridge, MA, USA), 1 μL of DNA template, 17 μL of ddH2O, and 1 μL each of forward and reverse primer. The PCR program comprised pre-denaturation at 94 °C for 5 min, 35 cycles of

Extraction of DNA, PCR Amplification, and Sequencing
Fresh algae (30 mL) in the exponential growth phase were collected and sedimented by centrifugation at 7000× g for 1 min, then the supernatants were discarded. Total DNA was obtained using BioFastSpin Plant Genomic DNA Extraction Kits (Bioer, Hangzhou, China), and characteristic LSU rDNA, SSU rDNA, and ITS fragments were amplified by PCR. The amplification primers (5 →3 ) were listed in Table 6. The PCR reaction volume was 40 µL, and included 20 µL of 2 × Es Taq Master Mix (CoWin Biosciences, Cambridge, MA, USA), 1 µL of DNA template, 17 µL of ddH 2 O, and 1 µL each of forward and reverse primer. The PCR program comprised pre-denaturation at 94 • C for 5 min, 35 cycles of 94 • C for 30 s, 56 • C for 30 s, and 72 • C for 30 s, and elongation at 72 • C for 5 min. The PCR products were sequenced at a commercial laboratory (TsingKe Biological Technology Company (Beijing, China).

Sequence Analyses
We searched the LSU rDNA, SSU rDNA, and ITS sequences of genes from the eight Alexandrium strains in the NCBI database using BLAST, and downloaded 43 LSU rDNA, 34 SSU rDNA, and 44 ITS sequences with high species similarity and coverage. We used the online BioEdit v7.2.5 software (https://bioedit.software.informer.com/, accessed on 8 January 2021), for multiple sequence alignment of the obtained sequences. We calculated and analyzed genetic distances within and between species and calculated base compositions, conserved, variable, parsimony informative, and singleton sites, and transition/transversion ratios using online MEGA-X v10.1.5 software (https://www. megasoftware.net/, accessed on 10 January 2021). We confirmed that GIR+I+G was the best alternative model using the AIC function in downloadable Modeltest3.7 software (https://sourceforge.net/projects/modeltest/, accessed on 10 January 2021). We constructed a maximum likelihood phylogenetic tree for the sequences obtained using MEGA-X v10.1.5 software, selected GIR+I+G as the best alternative model, and tested the confidence of the branches by running 1000 bootstrap replications. We constructed a Bayesian phylogenetic tree using Bayesian inference (BI) in MrBayes v3.1.2 software ( http://nbisweden.github.io/MrBayes/, accessed on 13 January 2021), again with GIR+I+G as the best replacement model, and 4 Markov chain operations were run for 1 × 10 6 generations for Bayesian analysis, with sampling every 500 generations. The phylogenetic tree was viewed and downloaded using FigTree v1.4.0 software, and edited using Adobe Acrobat DC v2020.013.20074.

Toxin Extraction
Algal suspensions (6 L) in the exponential growth phase were centrifuged at 4000 rpm for 8 min (15 • C), the supernatant was decanted, and the sedimented microalgal cells were stored at −80 • C. After three freeze-thaw cycles, stored algal cells were suspended in 10 mL of 0.1 mol/L acetic acid, sonicated on ice for 20 min, and centrifuged at 8000 rpm for 10 min at 4 • C. The supernatant was collected, the pellet was extracted again, the supernatants were combined and adjusted to a volume of 20 mL with 0.1 mol/L acetic acid, and 1.0 mL was passed through 0.22 µm mobile phase filter membranes (Shanghai ANPEL Laboratory Technologies Inc., Shanghai, China) for LC-MS/MS analysis.