Recent Advances in the Ecology of Bloom-Forming Raphidiopsis (Cylindrospermopsis) raciborskii: Expansion in China, Intraspecific Heterogeneity and Critical Factors for Invasion

Water blooms caused by the invasive cyanobacterium Raphidiopsis raciborskii occur in many reservoirs in the tropical and subtropical regions of China. In recent decades, this species has spread rapidly to temperate regions. Phenotypic plasticity and climate warming are thought to promote the worldwide dispersion of R. raciborskii. However, investigations into the genetic and phenotypic diversities of this species have revealed significant intraspecific heterogeneity. In particular, competition between R. raciborskii and Microcystis aeruginosa was highly strain dependent. Although the concept of an ecotype was proposed to explain the heterogeneity of R. raciborskii strains with different geographic origins, microevolution is more reasonable for understanding the coexistence of different phenotypes and genotypes in the same environment. It has been suggested that intraspecific heterogeneity derived from microevolution is a strong driving force for the expansion of R. raciborskii. Additionally, temperature, nutrient fluctuations, and grazer disturbance are critical environmental factors that affect the population establishment of R. raciborskii in new environments. The present review provides new insights into the ecological mechanisms underlying the invasion of R. raciborskii in Chinese freshwater ecosystems.


Introduction
Cyanobacterial blooms are severe environmental problems in eutrophic freshwater ecosystems [1,2]. Raphidiopsis raciborskii (previously known as Cylindrospermopsis raciborskii) is a filamentous bloom-forming cyanobacterium belonging to Nostocales with heterocysts and akinetes ( Figure 1A). Upon the occurrence of an R. raciborskii bloom, a significant amount of cell filaments usually distributes evenly in the water column ( Figure 1B). As a major producer of hepatotoxic cylindrospermopsin (CYN) and its analogs ( Figure 1C) [3][4][5][6], R. raciborskii often proliferates in lakes or reservoirs in tropical and subtropical zones [7,8], posing a significant threat to ecological safety. In the past decades, the occurrence frequency of this species has been significantly increased over the world, including both subtropical and temperate zones of the globe [7,9,10]. Therefore, R. raciborskii was suggested to be an invasive cyanobacterium, and much attention has been paid to its dispersion routes and adaptation mechanisms [9,11,12]. Based on the phylogenetic relationship of strains isolated worldwide, three hypotheses have been proposed for the biogeographic origin and global dispersion of R. raciborskii [7]. First, R. raciborskii was suggested to be derived from tropical regions in Africa due to its temperature dependence and the high diversity of African strains [13]. After invasion and colonization in Australia, R. raciborskii was dispersed throughout Eurasia and America [13]. Similarly, a recent study on the biogeography of R. raciborskii supports central Africa as the original dispersion center for nontoxic strains of this species [14]. The ability to produce CYN and paralytic shellfish poison (PSP) is thought to be acquired after dispersion to new settlements in tropical Africa and the South American continent [14]. Second, it was thought that R. raciborskii was extinct in most areas of the world during the Pleistocene Ice Age, except for the warm refuges of each continent at low latitudes [15]. The surviving R. raciborskii strains were dispersed to high-latitude areas when the global climate became warm. Third, R. raciborskii was thought to be derived from a tropical region in America and dispersed to other continents [11]. Considering that phylogeographic analysis was strongly affected by the genetic variation of the strains used in different studies [16], sufficient strains from different continents and environments should be investigated to clarify the dispersion route of R. raciborskii.
The invasion success of R. raciborskii was mainly ascribed to its phenotypic plasticity and growth optimization under different environments [9,[17][18][19][20]. R. raciborskii is tolerant to low and high light intensity and a wide range of temperatures [19,[21][22][23][24][25]. This species has flexible strategies for utilizing various forms of nitrogen (N), such as accumulating nitrogen as cyanophycin [26] and switching to N2 fixation under N depletion [27][28][29]. With a strong affinity for phosphate, R. raciborskii is capable of absorbing and storing a lot of phosphorus (P) [18,30]. After depletion of inorganic P, dissolved organic phosphorus can be used by R. raciborskii through the secretion of alkaline phosphatase (ALP) [31,32]. Thus, R. raciborskii can be dominant in the phytoplankton community at both very low and high N:P ratios [33]. In addition, it has been suggested that this species has a competitive advantage over other bloom-forming cyanobacteria under N-or P-limited conditions [30,34,35]. For example, toxic R. raciborskii is favored by the allelopathic effects of CYN on the surrounding phytoplankton [36][37][38][39].
Recent investigations have revealed high genetic and phenotypic diversity within the population of R. raciborskii [40][41][42]. The adaptation mechanisms of different strains were divergent and the competitive advantage of R. raciborskii against Microcystis aeruginosa Based on the phylogenetic relationship of strains isolated worldwide, three hypotheses have been proposed for the biogeographic origin and global dispersion of R. raciborskii [7]. First, R. raciborskii was suggested to be derived from tropical regions in Africa due to its temperature dependence and the high diversity of African strains [13]. After invasion and colonization in Australia, R. raciborskii was dispersed throughout Eurasia and America [13]. Similarly, a recent study on the biogeography of R. raciborskii supports central Africa as the original dispersion center for nontoxic strains of this species [14]. The ability to produce CYN and paralytic shellfish poison (PSP) is thought to be acquired after dispersion to new settlements in tropical Africa and the South American continent [14]. Second, it was thought that R. raciborskii was extinct in most areas of the world during the Pleistocene Ice Age, except for the warm refuges of each continent at low latitudes [15]. The surviving R. raciborskii strains were dispersed to high-latitude areas when the global climate became warm. Third, R. raciborskii was thought to be derived from a tropical region in America and dispersed to other continents [11]. Considering that phylogeographic analysis was strongly affected by the genetic variation of the strains used in different studies [16], sufficient strains from different continents and environments should be investigated to clarify the dispersion route of R. raciborskii.
The invasion success of R. raciborskii was mainly ascribed to its phenotypic plasticity and growth optimization under different environments [9,[17][18][19][20]. R. raciborskii is tolerant to low and high light intensity and a wide range of temperatures [19,[21][22][23][24][25]. This species has flexible strategies for utilizing various forms of nitrogen (N), such as accumulating nitrogen as cyanophycin [26] and switching to N 2 fixation under N depletion [27][28][29]. With a strong affinity for phosphate, R. raciborskii is capable of absorbing and storing a lot of phosphorus (P) [18,30]. After depletion of inorganic P, dissolved organic phosphorus can be used by R. raciborskii through the secretion of alkaline phosphatase (ALP) [31,32]. Thus, R. raciborskii can be dominant in the phytoplankton community at both very low and high N:P ratios [33]. In addition, it has been suggested that this species has a competitive advantage over other bloom-forming cyanobacteria under N-or P-limited conditions [30,34,35]. For example, toxic R. raciborskii is favored by the allelopathic effects of CYN on the surrounding phytoplankton [36][37][38][39].
Recent investigations have revealed high genetic and phenotypic diversity within the population of R. raciborskii [40][41][42]. The adaptation mechanisms of different strains were divergent and the competitive advantage of R. raciborskii against Microcystis aeruginosa was strain dependent [43][44][45][46]. These findings indicated apparent intraspecific heterogeneity in R. raciborskii. A comprehensive investigation of R. raciborskii diversity and dispersion in local ecosystems can provide new insights into its invasion success. R. raciborskii has been detected in many lakes and reservoirs in China [8,35,[47][48][49][50][51][52]. The recent expansion of R. raciborskii in Chinese freshwater bodies, its intraspecific heterogeneity, and critical factors for its invasion are the focus of this review.

Distribution and Dispersion in China
The first reported R. raciborskii bloom in Chinese water bodies occurred in 2002 at 21 • 59 N-22 • 25 N and 113 • 05 E-113 • 25 E [53]. To date, the presence of this species has been observed from 20 • 09 N to 39 • 59 N and between 100 • 03 E and 121 • 41 E, that is, from the tropical to temperate regions of China (Table S1, Figure 2). R. raciborskii was distributed in the basins of Zhujiang River, Yangtze River, Huaihe River, Yellow River, Haihe River, Yongjiang River, Qiantang River, and in the southeastern rivers in Fujian and Taiwan ( Figure 2). Dominance or high biomass of this species was observed in most of the basins, except in the Haihe and Qiantang River basins. The Haihe River basin is the northernmost line with the presence of R. raciborskii in China [50]; this species may have dispersed to this basin recently and has not been fully adapted to the local environment. Given that the Qiantang River basin is located in a subtropical zone with favorable temperature conditions for the proliferation of R. raciborskii, its distribution in this basin needs further investigation. Although historical data on R. raciborskii distribution are lacking, it should be noted that bloom events of this species have increased in recent decades. In 2006, only a low concentration of R. raciborskii filaments was observed in Liangzi lake (30 • 10 N, 114 • 36 N) [48]. However, its biomass reached 52.03 mg L −1 , with an obvious water bloom between 2013 and 2017 [35]. This finding indicates that R. raciborskii has evolved from an occasional species to a dominant species in Liangzi lake, which is likely a typical succession process after the invasion of new water bodies.

Distribution and Dispersion in China
The first reported R. raciborskii bloom in Chinese water bodies occurred 21°59′ N-22°25′ N and 113°05′ E-113°25′ E [53]. To date, the presence of this s been observed from 20°09′ N to 39°59′ N and between 100°03′ E and 121°41′ E, th the tropical to temperate regions of China (Table S1, Figure 2). R. raciborskii w uted in the basins of Zhujiang River, Yangtze River, Huaihe River, Yellow Ri River, Yongjiang River, Qiantang River, and in the southeastern rivers in Fujia wan ( Figure 2). Dominance or high biomass of this species was observed in m basins, except in the Haihe and Qiantang River basins. The Haihe River basin is ernmost line with the presence of R. raciborskii in China [50]; this species may persed to this basin recently and has not been fully adapted to the local env Given that the Qiantang River basin is located in a subtropical zone with favo perature conditions for the proliferation of R. raciborskii, its distribution in this b further investigation. Although historical data on R. raciborskii distribution are should be noted that bloom events of this species have increased in recent d 2006, only a low concentration of R. raciborskii filaments was observed in Li (30°10′ N, 114°36′ N) [48]. However, its biomass reached 52.03 mg L -1 , with a water bloom between 2013 and 2017 [35]. This finding indicates that R. raci evolved from an occasional species to a dominant species in Liangzi lake, whi a typical succession process after the invasion of new water bodies. The wide distribution of R. raciborskii in the same basin ( Figure 2) indicate species might be dispersed from upstream lakes to downstream lakes throug ( Figure 3). On the other hand, lakes located separately in floodplain could be large floods, creating opportunities for R. raciborskii to invade new environme The wide distribution of R. raciborskii in the same basin ( Figure 2) indicated that this species might be dispersed from upstream lakes to downstream lakes through the river (Figure 3). On the other hand, lakes located separately in floodplain could be linked by large floods, creating opportunities for R. raciborskii to invade new environments. Water diversion projects (WDPs) also provide an important dispersion route for aquatic organisms. For example, fish invasion has occurred through the south-to-north WDP in China [54]. This WDP is designed to divert water from humid subtropical areas to arid or semi-arid temperate areas and thus is favorable for the dispersion of R. raciborskii. Five lakes in the Huaihe River basin were linked with three reservoirs and one pond in the Yellow and Haihe River basins by the east route of the south-to-north WDP (Table S1), and abundant R. raciborskii were observed in all of these water bodies. In addition, the filaments of this species can be conveyed between lakes via natural water used for the transport of aquatic products and ballast water of commercial ships [7]. Owing to their high survivability, the akinetes of R. raciborskii are likely to be transported by migratory birds via feet or gut carryover [55].
Int. J. Environ. Res. Public Health 2023, 20, x FOR PEER REVIEW 4 of diversion projects (WDPs) also provide an important dispersion route for aquatic orga isms. For example, fish invasion has occurred through the south-to-north WDP in Chin [54]. This WDP is designed to divert water from humid subtropical areas to arid or sem arid temperate areas and thus is favorable for the dispersion of R. raciborskii. Five lakes the Huaihe River basin were linked with three reservoirs and one pond in the Yellow an Haihe River basins by the east route of the south-to-north WDP (Table S1), and abunda R. raciborskii were observed in all of these water bodies. In addition, the filaments of th species can be conveyed between lakes via natural water used for the transport of aquat products and ballast water of commercial ships [7]. Owing to their high survivability, th akinetes of R. raciborskii are likely to be transported by migratory birds via feet or g carryover [55]. CYN is synthesized by a series of enzymes encoded by cyr genes [56]. Characterizin the distribution and variation of cyr genes can provide insights into the dispersion CYN-producing R. raciborskii strains. A systematic investigation into the sequences of cy and cyrJ genes in Chinese water bodies divided them into four and three main sequen types, respectively (Table 1) [48]. It was shown that low-latitude reservoirs contain mo sequence types. Given that Itype4a f and Itype4a r are Itype1 variants with transpositio elements, and that Jtype2b is a Jtype2a variant with a deletion of six nucleotides, the Ti gang reservoir presumably contains all the ancestral cyrI and cyrJ sequence types. Th finding indicated that low-latitude lakes in Southeast China are potential dispersion ce ters of toxic R. raciborskii. Similarly, the tropical regions in Southeast China were al thought to be a glacial refuge for higher plant species [57]. These findings provide furth evidence for the refuge hypothesis about R. raciborskii dispersion [15]. PSP-producing raciborskii strains were also isolated and identified from Chinese water bodies [58,59]. Ph logenetic analysis revealed that these strains were closely related to strains from the Nor and South American continents [58]. This finding may imply the dispersion of PSP-pr ducing strains from America to China because the ability of R. raciborskii to produce PS was proposed to originate from America [14]. CYN is synthesized by a series of enzymes encoded by cyr genes [56]. Characterizing the distribution and variation of cyr genes can provide insights into the dispersion of CYN-producing R. raciborskii strains. A systematic investigation into the sequences of cyrI and cyrJ genes in Chinese water bodies divided them into four and three main sequence types, respectively (Table 1) [48]. It was shown that low-latitude reservoirs contain more sequence types. Given that Itype4a f and Itype4a r are Itype1 variants with transposition elements, and that Jtype2b is a Jtype2a variant with a deletion of six nucleotides, the Tiegang reservoir presumably contains all the ancestral cyrI and cyrJ sequence types. This finding indicated that low-latitude lakes in Southeast China are potential dispersion centers of toxic R. raciborskii. Similarly, the tropical regions in Southeast China were also thought to be a glacial refuge for higher plant species [57]. These findings provide further evidence for the refuge hypothesis about R. raciborskii dispersion [15]. PSP-producing R. raciborskii strains were also isolated and identified from Chinese water bodies [58,59]. Phylogenetic analysis revealed that these strains were closely related to strains from the North and South American continents [58]. This finding may imply the dispersion of PSP-producing strains from America to China because the ability of R. raciborskii to produce PSP was proposed to originate from America [14].

Phenotypic and Genetic Diversity
In previous studies on the environmental adaptability of R. raciborskii, it has often been regarded as a homogenous population because of the highly similar 16S rRNA genes of different strains. For example, most present studies suggest that R. raciborskii is a mesophile with a tolerance to low temperatures [7,19,60,61]. However, laboratory experimentation has found that several strains maintain a high growth rate at 15 • C, while the growth of some strains is completely inhibited under low-temperature conditions [17,47]. These findings indicate that low-temperature tolerance is not an intrinsic characteristic of R. raciborskii and that significant intraspecific heterogeneity exists for the temperature adaptability of this species. Similarly, the intraspecific heterogeneity of R. raciborskii has also been found in its response to light intensity [9,62,63] and conductivity [64], as well as its strategies for the utilization of N [26,27] and P [41]. In addition, varied growth rates, morphologies, and toxicities have been observed in genetically similar isolates of R. raciborskii [65,66].
In contrast to the 16S rRNA gene, the RNA polymerase C1 (rpoC1) gene and ITS-L sequence, which is the larger fragment of the inter-transcribed sequence of rRNA, are useful molecular markers for discriminating different R. raciborskii strains [58,67]. The strains were classified into nontoxic, CYN-producing, and PSP-producing clusters based on the phylogenetic analysis of rpoC1 and ITS-L [58]. This result is supported by the genomic variations between R. raciborskii strains [16,42,68]. The relationship between genetic diversity and phenotypic variation remains to be clarified and requires further systematic investigation.
The presence of cyr genes is the main genetic difference between CYN-producing and non-CYN-producing strains [48]. However, genomic variations were also found in genes associated with stress and adaptation, which are probably related to the physiological role of CYN [42]. In fact, CYN has been shown to contribute to the successful invasion and survival of R. raciborskii [4,5]. For example, CYN-producing strains have a competitive advantage under nutrient-replete conditions [69]. In comparison to nontoxic R. raciborskii, toxic strains have a more efficient response to inorganic phosphorus and could become dominant in the community [70]. A recent study also found that toxic strains were more competitive under Fe-starved conditions [71].
Previous investigations have revealed that the cell quotas of CYN varied significantly between R. raciborskii strains [65,66,72]. However, the genetic basis of this finding remains unknown. As aforementioned, sequence variations were observed for cyrI and cyrJ genes, which encodes a hydroxylase and a sulfotransferase, respectively [48,[73][74][75]. These two enzymes catalyze tailoring reactions in the last two steps of CYN biosynthesis [56]. An inactivated mutation of cyrI inhibits the synthesis of CYN, leading to an accumulation in the intermediate product 7-deoxy-CYN [76]. Therefore, variations in cyr genes may change the activity of the enzymes encoded by them and further affect CYN synthesis efficiency.

Competition between R. raciborskii and M. aeruginosa
M. aeruginosa is the dominant species in most eutrophic water bodies, both in China and worldwide [51,77]. Competition against M. aeruginosa is inevitable for R. raciborskii during invasion. However, recent culture experiments have produced inconsistent results regarding the competition between R. raciborskii and M. aeruginosa (Table 2). Under light-or P-limited conditions, both R. raciborskii and M. aeruginosa are likely to dominate the mixed culture of these two species with equal starting biovolumes [45]. R. raciborskii, with Nfixation capability, was a more successful invader than M. aeruginosa when N was depleted in batch culture [35]. Likewise, the model prediction of species competition outcomes is strongly affected by the growth variability of strains [78]. These findings demonstrate the intraspecific heterogeneity for R. raciborskii and M. aeruginosa [44].

Ecotype and Microevolution
In several studies, R. raciborskii was assumed to have evolved into different ecotypes with special physiological characteristics in its original habitats, and the geographic dispersion of this species is a dynamic selection process for existing ecotypes [60,61,72]. The concept of an ecotype is reasonable for understanding the intraspecific heterogeneity of R. raciborskii in different environments but not for explaining the coexistence of different phenotypes and genotypes in the same environment [16,65]. Heterogeneity between coexisting strains is better defined as microevolution, the concept of which refers to minor variations within species [80]. Microevolution is the source of adaptive variation for organisms, and the accumulation of microevolution may further lead to new speciation. The ecotypes of R. raciborskii were presumably established by the natural selection of heterogeneous strains generated during microevolution.

Temperature and Effective Accumulated Temperature (EAT)
Temperature is an important ecological factor that drives the global distribution of R. raciborskii [10,13,81]. Climate warming is expected to increase the biomass of this species and promote its dispersion in new habitats (Table 3). In addition to its proliferation in warm areas, the dominance and bloom of this species were observed in cool water between 10 • C and 15 • C [22,47]. This finding indicates that R. raciborskii is likely to disperse and survive in most freshwater bodies in China. Under nutrient-replete conditions, the potential biomass that R. raciborskii can reach is probably determined by the Effective Accumulated Temperature (EAT) affecting the heat that organisms can obtain during multiplication seasons. To date, there are no available data on the effects of EAT on the growth of R. raciborskii. In China, the EAT varies with latitude from south to north and altitude from east to west. It is necessary to investigate the relationship between EAT and the distribution of R. raciborskii to assess the bloom risk this species presents.

Nutrient Fluctuations
The coexistence of multiple species is frequently observed during cyanobacterial blooms in eutrophic water bodies [51]. This finding implies that competitive exclusion does not occur, which may be attributed to nutrient repletion in the environment. For example, R. raciborskii may invade into an M. aeruginosa-dominated culture and survive with relatively low biomass [35]. The coexistence of R. raciborskii and M. aeruginosa has been found in many subtropical water bodies located in the Yangtze River basin [35]. Sometimes, R. raciborskii becomes the dominant species in the phytoplankton community. Considering intraspecific heterogeneity, it cannot be predicted that R. raciborskii will eventually compete against M. aeruginosa.
Niche and fitness differences are thought to determine invasion success in bacterial communities [82]. Field investigations have demonstrated that Raphidiopsis, rather than Microcystis, benefit from low P concentrations [83]. This finding was supported by laboratory experiments indicating that R. raciborskii could be advantageous under P-limited conditions [34,84]. This advantage may be enhanced using the allelopathic effects of CYN, which can induce extracellular ALP production in other phytoplankton species [39,85]. In addition, stoichiometric analysis revealed a lower metabolic cost of synthesizing CYN than that of producing ALP [39]. Moreover, N limitation may favor the growth of R. raciborskii strains with N-fixation capability. However, previous studies have claimed that this highenergy-consuming function cannot support the proliferation of R. raciborskii [40]. Nutrient fluctuations frequently occur in natural water bodies [84,86,87]. Intermittent P and N limitations may create opportunities for R. raciborskii to become dominant in the phytoplankton community [87] and proliferate when nutrients are repleted (Table 3).

Grazer Disturbance
Local grazers have a significant impact on the structure of phytoplankton communities [88]. Daphnia feeding on R. raciborskii can hamper the population establishment of this species [88,89], whereas copepods, preferentially grazing on non-Raphidiopsis species, can promote the persistence of R. raciborskii [90,91] (Table 3). Therefore, a low grazing pressure or loss of top-down control is a critical factor for the invasion success of R. raciborskii [88]. In addition, CYN-producing strains may be more advantageous because of the toxicity of CYN on zooplankton [5].

Conclusions
R. raciborskii has spread widely in Chinese freshwater bodies in recent decades. In addition to its adaptability to various environments, the intraspecific heterogeneity of R. raciborskii presumably has a significant contribution to the species' rapid expansion. Temperature, nutrient fluctuations, and grazer disturbance are important factors that affect the invasion success of R. raciborskii.

Hypothesis for the Invasion Success of R. raciborskii
Field populations of R. raciborskii are mixtures of heterogeneous strains. When the mixtures were dispersed in new environments, strains with strong adaptability could survive under natural selection and evolve into ecotypes with specific genetic and phenotypic characteristics. Some ecotypes of R. raciborskii are likely to proliferate and form blooms under appropriate conditions. In summary, the invasion success of R. raciborskii may be ascribed to sufficient intraspecific heterogeneity, rather than the adaptability of individual strains.

Future Outlook
Given that R. raciborskii has a growth advantage under nutrient-limited conditions, the expansion of this species may benefit from a reduction in P and N during the remediation of lake eutrophication [92]. To enhance our understanding of the bloom-forming mechanisms of R. raciborskii, knowledge of the intraspecific heterogeneity of R. raciborskii should be strengthened in the future. To accurately discriminate between strain differences, quantitative traits should be defined and investigated systematically. The growth rate, photosynthetic rate, electron transfer rate, affinity for inorganic carbon, pigment content, extracellular ALP activity, toxin content, and critical requirements for nutrients, temperature, and light intensity, are candidate quantitative traits for R. raciborskii. Moreover, highly variable genetic markers, such as ITS-L, can be used to discriminate strain differences at the molecular level. The consistency between molecular markers and quantitative traits should be carefully evaluated. Moreover, it is crucial to identify genes controlling quantitative traits to provide more reliable genetic evidence for the heterogeneity of R. raciborskii.
When a specific field population of R. raciborskii is investigated, many strains should be isolated to fully capture their variability. Considering the effects of in-culture evolution on laboratory experiments [93], strains should be maintained under conditions similar to their original habitats, and their quantitative traits should be characterized after isolation as soon as possible. To develop a worldwide database describing the intraspecific heterogeneity of R. raciborskii, unified and standard experimental methods and procedures are required. Furthermore, attention should be paid to the effects of EAT on the distribution and heterogeneity of R. raciborskii for assessing its bloom risk.