Morphology and Molecular Phylogeny of the Genus Stigeoclonium (Chaetophorales, Chlorophyta) from China, Including Descriptions of the Pseudostigeoclonium gen. nov.

Stigeoclonium is a genus of green algae that is widely distributed in freshwater habitats around the world. The genus comprises species with variously developed prostrates and erect systems of uniseriate branched filaments and grows attached to a wide range of different surfaces. It holds significant promise for applications in water quality indicators, sewage treatment, and the development of high-value-added products. Nevertheless, our comprehension of Stigeoclonium remains unclear and perplexing, particularly regarding its fundamental systematic taxonomy. Recent molecular analyses have revealed that the morphologically well-defined genus Stigeoclonium is polyphyletic and requires taxonomic revision. Phylogenetic analysis based on a single molecular marker and limited samples is insufficient to address the polyphyletic nature of Stigeoclonium. In the present study, 34 out of 45 strains of Stigeoclonium were newly acquired from China. Alongside the morphological data, a concatenated dataset of three markers (18S rDNA + ITS2 + tufA) was utilized to determine their molecular phylogeny. The phylogenetic analysis successfully resolved the broadly defined Stigeoclonium into three robustly supported clades (Stigeoclonim tenue clade, S. farctum clade, and S. helveticum clade). The morphological characteristics assessment results showed that the cell type of the main axis-producing branch, considered a crucial morphological characteristic of the Stigeoclonium taxonomy, did not accurately reflect the real phylogeny of the genus. A new taxonomical classification of the genus Stigeoclonium was proposed based on zoospores’ germination types, which aligned well with the phylogenetic topologies. Species where zoospores showed erect germination (S. helveticum clade) formed a distinct monophyletic clade, clearly separated from the other two clades, with zoospores showing prostrate germination or pseudo-erect germination. Consequently, a new genus, Pseudostigeoclonium gen. nov., is suggested to include all species in the broadly defined Stigeoclonium with zoospores with erect germination. The taxonomic diversity is supported by distinctive morphological differences and phylogenetic divergence within the broadly defined Stigeoclonium identified in this study. Further evaluation of the genus Stigeoclonium is necessary, especially via examining additional specimens and re-evaluating morphological characters under precisely defined laboratory conditions.


Introduction
Stigeoclonium is an important primary producer that is widely distributed in freshwater ecosystems around the world.They often attach to inorganic substrates or other large algae and higher aquatic plant surfaces.Some species of Stigeoclonium have high application prospects in terms of water quality indication, wastewater treatment [1][2][3], and lipid production [4].
The genus Stigeoclonium, comprising approximately 80 described species, was initially established by Kützing [5] and belongs to the order Chaetophorales (Chlorophyceae, Chlorophyta) [6].Stigeoclonium was originally defined based on the type species Stigeoclonium tenue, encompassing algae forming vibrant green tufts with uniseriate branched heterotrichous filaments [5].Further delimitation of the genus occurred through the examination of various vegetative traits, such as cellular dimensions, branching degree, the presence/absence of hairs, thallus color, and habitat, resulting in the description of 29 species [7].Subsequent taxonomic revisions by different phycologists led to varied concepts and proposed species.Islam [8] critically reviewed the genus based on 73 specimens worldwide, recognizing 28 species, with only 10 being considered valid.Printz [9] reported 42 species, but 11 remained not fully understood.These phycologists primarily delimited Stigeoclonium species based on branching types and the shape and size of cells in the erect system.
Stigeoclonium is a heterotrichous alga, which is composed of an erect system and a prostrate system.Cox and Bold [10] introduced a species concept, focusing on the morphological features of the prostrate system and considering it more stable than the erect system.This approach identified seven species from 81 Stigeoclonium-like specimens collected in Texas, which belonged to three groups.Francke and Simons [11] refined these methods, confirming four groups (S. helveticum group, S. aestivale group, S. tenue group, and S. farctum group) from 150 strains collected in the Netherlands.Simons et al. [12] further merged groups based on detailed studies on the morphological development of the prostrate system of Stigeoclonium, with particular emphasis on the types of zoospore germination, resulting in three groups: S. helveticum, S. tenue, and S. farctum, each with a single species.
Despite these efforts, universally recognized taxonomic criteria for Stigeoclonium remain elusive.Various authors have proposed species and variants based on different concepts.John et al. [13] focused on the morphology of the erect system, describing nine species.Branco et al. [14] identified six species in southeastern Brazil by considering the characteristics of both prostrate and erect systems.Skinner and Entwisle [15] described three species in Australia following the species concept of Simons et al. [12].
The genus Stigeoclonium has undergone comprehensive study for nearly two centuries, resulting in numerous species descriptions based on morphology.However, the traditional taxonomic framework established by Kützing [5] has persisted without significant changes.The genus Stigeoclonium was considered a well-defined monophyletic genus for a long time until Caisová et al. [16] first used SSU rDNA to study the phylogeny of Stigeoclonium, which contained nine molecular sequences of six species of Stigeoclonium, confirming that Stigeoclonium was polyphyletic, challenging the previous notion.Notably, species sharing the same name, including the type species S. tenue, did not form cohesive clusters, adding complexity to the phylogenetic understanding of Stigeoclonium [17,18].
While molecular phylogenetic studies have primarily concentrated on the order Chaetophorales as a whole, investigations specific to Stigeoclonium have been limited.To address these challenges, the present study generated new sequences for 18S rDNA, ITS2, and tuf A. A concatenated dataset, combined with morphological data, was employed to (1) re-evaluate morphological characteristics and (2) reassess the broadly defined genus Stigeoclonium, leading to the proposal of a new genus.Locality: Heilongjiang province, China; on a rock in a stream; freshwater.

Morphological Observations
Species of Stigeoclonium were highly variable in morphology, especially in the erect system.For instance, Stigeoclonium farctum specimens displayed different morphologies when growing epiphytically on a plastic cup in their natural habitat compared with those cultured in medium.Filaments cultured on solid medium were abundantly branched, whereas those in liquid medium had extremely long, upright filaments with rare branching (Figure 1).
In this study, three types of zoospore germination were clearly observed (Figure 2).The genus Stigeoclonium was grouped into three clades based on zoospore germination types: The Stigeoclonim helveticum clade (Pseudostigeoclonium gen.nov.):The Stigeoclonim helveticum clade exclusively included the species Stigeoclonium, primarily including Stigeoclonium helveticum and Stigeoclonium sp.This clade was characterized by the strictly erect germination of zoospores (Figure 2A) and a prostrate system consisting of one Ulothrix-like holdfast cell or some very short prostrate filaments and/or slender rhizoids growing out from the basal cell of an erect axis [19,20].
The Stigeoclonim tenue clade: The Stigeoclonim tenue clade included some species of Stigeoclonium and the genus Caespitella.This clade was characterized by an approximately prostrate germination of zoospores.The original zoospore cell unipolar germination usually gave rise to a lateral outgrowth at one side to first form the prostrate part, and later, a pointed erect filament began to develop (Figure 2D).We placed all species with an approximately prostrate germination of zoospores and an irregular and mostly extensive open prostrate system, with or without rhizoids, into this clade.
The Stigeoclonim farctum clade: The Stigeoclonim farctum clade comprised some species of Stigeoclonium and the genus Fritschiella.This clade was characterized by an approximately prostrate or pseudo-erect germination of zoospores (Figure 2B,C,E).What was different from the Stigeoclonim tenue clade was that the original zoospore cell bipolar germination gave rise to a lateral outgrowth on two sides to form the prostrate part, with the exception of the Fritschiella tuberosa (approximately gregarious on moist soil or silt, including drying rainwater puddles), which showed the original unipolar germination of zoospore cells, and S. aestivale, which showed the original unipolar germination of zoospore cells, to first form the erect part.We placed all species with an approximately prostrate and pseudo-erect germination of zoospores, a regular compact development of the prostrate system that results in a closed, pseudoparenchymatous disk, an open star-like form, or intermediate forms and a poorly developed erect system, into this clade.In addition, three cell types of the main axis-producing branch were observed (Figure 3).Species with a main axis and primary branches were similar, and usually, no specialized or modified cells were present on the main axis producing the branches (Figure 3A).Species showed transitions in which some differentiation began between the cells of the main axis.Some cells gradually became long and some short, with the latter commonly producing branches (Figure 3B).Species with a main axis usually consisted of two types of cells.The long cells usually did not produce branches, while the small and short cells usually produced lateral primary branches (Figure 3C).In addition, three cell types of the main axis-producing branch were observed (Figure 3).Species with a main axis and primary branches were similar, and usually, no specialized or modified cells were present on the main axis producing the branches (Figure 3A).Species showed transitions in which some differentiation began between the cells of the main axis.Some cells gradually became long and some short, with the latter commonly producing branches (Figure 3B).Species with a main axis usually consisted of two types of cells.The long cells usually did not produce branches, while the small and short cells usually produced lateral primary branches (Figure 3C).

Phylogenetic Analysis of the Stigeoclonium
A total of 135 new sequences, including 18S rDNA, ITS2, and tufA, were generated in this study.The best models were selected for the BI analysis: TrNef + I + G for 18S rDNA,

Phylogenetic Analysis of the Stigeoclonium
A total of 135 new sequences, including 18S rDNA, ITS2, and tuf A, were generated in this study.The best models were selected for the BI analysis: TrNef + I + G for 18S rDNA, GTR + G for ITS2 rDNA, GTR + G for the first codon position matrix of tuf A, and GTR + I + G for the second and third codon positions matrix.The GTRGAMMA model was used for maximum likelihood analysis.
A saturation index analysis (Iss = 0.386 < Iss.c = 0.802) showed that the sequence matrix was not saturated and could be used for phylogenetic analysis.The 71-taxa alignment consisted of 2687 positions (18S rDNA + ITS2 + tuf A) (see Appendix A).A total of 743 sites in these nucleotides were variable, of which 573 sites were parsimoniously informative and 170 sites were singleton sites.The average amount of A, T, C, and G was 24.69%, 26.04%, 20.89%, and 28.38%, respectively, of which A + T (50.73%) was greater than G + C (49.27%).The transition/transversion ratio was 1.61.

Discussion
Based on concatenated datasets and additional samples, we still had to accept the fact that the Stigeoclonium was paraphyletic.Clearly, the morphologically defined genus Stigeoclonium was unnatural and needed re-evaluation.This study obtained a well-resolved molecular phylogeny with moderate to high support values for Stigeoclonium.All species of Stigeoclonium were divided into three clades with robust support, located in either the family Fritschiellaceae or the family Chaetophoraceae.This provided a basis for further discussion on the phylogenetic relationships within the paraphyletic genus Stigeoclonium.Fritschiellaceae diverged into three well-supported sister lineages.The family Chaetophoraceae included the genera Chaetophora, Draparnaldia, and the Stigeoclonium helveticum clade.The S. helveticum clade showed basal divergence as a sister of the Chaetophora and Draparnaldia, while the family Fritschiellaceae comprised the genus Chaetophoropsis, S. tenue clade, and S. farctum clade.The S. tenue clade showed basal divergence as a sister of the Chaetophoropsis and S. farctum clade.The S. tenue clade and S. farctum clade, with a similar morphology, were well separated from each other.The three clades of Stigeoclonium were further split into seven molecular groups.All Stigeoclonium species were dispersed over these two families and were recovered as independent monophyletic clades (Stigeoclonium helveticum clade, S. tenue clade, and S. farctum clade) with moderate to robust support values (BP/PP, 80/0.85 to 100/1.00).Species of Stigeoclonium with zoospore germination of an erect type (Stigeoclonium helveticum clade) formed a monophyletic clade in the Chaetophoraceae, with a robust support value (BP/PP, 100/1.00),which obviously branched independently of the S. tenue clade and S. farctum clade (within the Fritschiellaceae) with zoospore germination prostrate or the pseudo-erect type in our phylogenetic trees.In addition, multiple sequences with the same species name were phylogenetically distantly related, including the type species of the genus Stigeoclonium (S. tenue in bold; Figure 1) and Caespitella pascheri (type species of Caespitella).Two sequences with the same name, S. tenue, were dispersed over the Fritschiellaceae and the Chaetophoraceae instead of clustering together.

Discussion
Based on concatenated datasets and additional samples, we still had to accept the fact that the Stigeoclonium was paraphyletic.Clearly, the morphologically defined genus Stigeoclonium was unnatural and needed re-evaluation.This study obtained a well-resolved molecular phylogeny with moderate to high support values for Stigeoclonium.All species of Stigeoclonium were divided into three clades with robust support, located in either the family Fritschiellaceae or the family Chaetophoraceae.This provided a basis for further discussion on the phylogenetic relationships within the paraphyletic genus Stigeoclonium.
In cases where paraphyly or polyphyly is demonstrated within a genus, a common approach is to designate one clade as "genus sensu stricto" and analyze the morphological characteristics used in the original diagnosis [16].Before that, it is necessary for us to reexamine its type species.However, re-examination of the type species revealed two stains of S. tenue (S. tenue CCAC 3492B HF920647 and CCAP 477/11A FN824374), which are clearly unrelated and located in Fritschiellaceae and Chaetophoraceae, respectively.Based on a comprehensive sample observation and literature analysis, the zoospore germination type of the type species (S. tenue) was consistently identified as prostrate [10][11][12].On the other hand, recent research has provided evidence indicating that taxa within the family Fritschiellaceae exhibit zoospore germination of the prostrate or pseudo-erect type and, in contrast, taxa belonging to Chaetophoraceae display zoospore germination of the erect type [19].Consequently, the inference that was drawn was that S. tenue CCAC 3492B (HF920647), situated in the Fritschiellaceae, was more likely to represent Stigeoclonium sensu stricto.This conclusion aligned with the understanding that the germination characteristics of zoospores played a crucial role in delineating the taxonomy of Stigeoclonium.
After identifying Stigeoclonium sensu stricto, we next tried to find some effective morphological characteristics to revise the genus Stigeoclonium.The characteristic cell type of the main axis-producing branch was considered the most important morphological characteristic in the traditional taxonomy of Stigeoclonium by Islam [8], although it did not reflect the real phylogeny of the genus Stigeoclonium in this study.Instead, the characteristics of zoospore germination types correlated well with the phylogenetic results.Drawing upon the morphological features of the prostrate system and the germination type of zoospores, various phycologists have categorized the genus Stigeoclonium into distinct groups [10][11][12].Despite the fact that none of these grouping schemes matched the phylogenetic results well, the important studies mentioned above [10][11][12] provide a valuable indication that the germination type of zoospores might serve as a dependable morphological characteris-tic [20].This observation is consistent with findings from prior studies [19], reinforcing the significance of zoospore germination type in understanding the taxonomy of Stigeoclonium.
The broadly defined genus Stigeoclonium diverged into three well-supported clades in our phylogenetic analyses using a concatenated dataset, which was initially unexpected.By observing the zoospore germination type of representative species and combining the phylogeny results, the genus Stigeoclonium could be divided into at least six groups-the S. helveticum group, S. aestivale group, S. variabile group, S. farctum group, S. tenue group and S. pascheri group-and three clades-the S. tenue clade, S. farctum clade, and S. helveticum clade.The life history and reproductive development characteristics of S. helveticum (S. helveticum group) have been well studied, providing us with many taxonomic insights [23].All species with erect zoospore germination types (S. helveticum group) of the broadly defined Stigeoclonium formed a separate, monophyletic clade that was clearly separate from the S. tenue clade (Stigeoclonium sensu stricto) and the S. farctum clade with zoospore germination of the prostrate and pseudo-erect types.Thus, in light of the morphological and molecular differences, we erected a new genus, Pseudostigeoclonium gen.nov., and amended the Stigeoclonium.The genus Pseudostigeoclonium was newly erected due to its unique morphological characters, as follows: the erect filaments are unbranched in the actively growing stage or sparsely branched in older stages; the prostrate systems are underdeveloped, attached to the substrate by a sparse, rhizoid-like basal system developed from the lower or middle cells; zoospore germination is of the erect type.This clearly distinguishes Pseudostigeoclonium from Stigeoclonium sensu stricto.
Usually, samples with typical characteristics of Stigeoclonium are confidently classified within the genus.However, some species of Stigeoclonium (such as S. terrestre and S. pascheri) spun off to create the new genera [24,25].Vischer [25] discovered and isolated a new Stigeoclonium-like taxon with cell division that was entirely apical, distinguishing it from the Stigeoclonium with intercalary cell division and, rather than describing it as a new species of Stigeoclonium, created the new genus Caespitella with the type species C. pascheri.There is a lack of consensus among phycologists regarding this taxonomic approach.Printz [9] and Bourrelly [26] recognized this genus based on the morphological characteristics of the erect system.However, Cox and Bold [10] reincorporated the genus into Stigeoclonium based on detailed life history observations, which was accepted by Shyam and Sarma [27].Caisová et al. [16] endorsed the establishment of the genus Caespitella based on the limited samples and molecular data of Stigeoclonium.In this study, two stains of Caespitella pascheri (S. pascheri HB201611 OP236750 and SAG 410-1 FN824387) (type species of Caespitella) were not independent of the genus Stigeoclonium; instead, they were dispersed within the S. pascheri group.Moreover, the key morphological characteristics (zoospore germination types and prostrate system) were found to be more reliable and were not significantly different between Caespitella pascheri and other Stigeoclonium.Thus, proposing Caespitella pascheri as a separate genus raised questions.Vischer [25] was, at the time, intensively studying both S. helveticum and C. pascheri.These two species exhibited significant differences in growth pattern, both in liquid and solid agar, which might have influenced Vischer's belief that they belonged to different genera.In fact, apical cell division and intercalary cell division have been observed in the same species [10].Apical cell division is common in prostrate filaments, while intercalary cell division is common in erect filaments [10].The authentic strains of C. pascheri and S. helveticum could also be well distinguished on the phylogenetic tree.Considering the crucial evidence presented by Cox and Bold [10] and the molecular phylogeny in this study, it is suggested that C. pascheri should be considered a member of Stigeoclonium.
It is noteworthy that the sequences with the same species name were not clustered into one clade in the genus Stigeoclonium (e.g., S. tenue, S. pascheri), which may be for the following reasons: (1) the presence of cryptic species within the genus Stigeoclonium, where species share similar morphological characteristics but exhibit significant differences in molecular data; (2) insufficient knowledge among phycologists regarding the genus Stigeoclonium, leading to the use of inadequate morphological characteristics for classification Each gene set was initially aligned with MAFFT 7.2 [36] and manually refined using Seaview v. 4.32 [37].jModelTest v.2.1.4[38] was used to select the best-fitting evolutionary models for each marker according to the Bayesian information criterion calculations.All genes were then concatenated using Phyutility 2.2 [39] (see Appendix A).Mutational saturation was evaluated by looking at variable positions of the alignments using DAMBE 5.6 [40].
Phylogenetic reconstructions were performed using RAxML 8.0 [41] and MrBayes3.1.2[42].Bootstrap analyses with 1000 replicates of the ML dataset were performed to estimate the statistical reliability.For Bayesian analyses, Markov chain Monte Carlo analyses were run with four Markov chains (three heated; one cold) for 4 × 10 6 generations, with trees sampled every 1000 generations.It was assumed that a stationary distribution was reached when the average standard deviation of the split frequencies between two runs was lower than 0.01.The first 25% of the calculated trees were discarded as burn in, and the remaining samples were used to construct a Bayesian consensus tree and to infer posterior probabilities.The bootstrap values and posterior probabilities are presented at the nodes.The resulting phylogenetic trees were edited using Figtree 1.4.2(http://tree.bio.ed.ac.uk/software/ figtree/ (accessed on 19 January 2024).

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants13050748/s1,Table S1: The newly obtained voucher number, taxon, collection information, and their GenBank accession numbers of Chaetophorales used for 18S rDNA, ITS2, and tuf A sequence analyses.

Figure 4 .
Figure 4. ML and Bayesian phylogenetic tree constructed using a concatenated dataset (18S rDNA + ITS2 + tufA) of the Chaetophorales.The numbers on the nodes represent the bootstrap support values (BP)/posterior probabilities (PP) above 50/0.50.Two sequences of S. tenue from our study are shaded in bold.

Figure 4 .
Figure 4. ML and Bayesian phylogenetic tree constructed using a concatenated dataset (18S rDNA + ITS2 + tuf A) of the Chaetophorales.The numbers on the nodes represent the bootstrap support values (BP)/posterior probabilities (PP) above 50/0.50.Two sequences of S. tenue from our study are shaded in bold.Stigeoclonium species were dispersed over the family Fritschiellaceae or family Chaetophoraceae and intermixed with the other genera.The family Chaetophoraceae and family

Author
Contributions: B.L. and G.L. planned and designed the research and wrote the manuscript; Q.L. performed the sampling and data analysis; Q.D. edited the manuscript; H.Z. edited the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This work was supported by the Science & Technology Fundamental Resources Investigation Program (Grant No. 2022FY100400) and the National Natural Science Foundation of China (Grant No. 32000168).
ImplicationsPseudostigeoclonium B. Wen Liu, Q. Mei Lan, Q. Yu Dai, Huan Zhu et G. Xiang Liu gen.nov.Filaments consist of erect and prostrate systems.The erect filaments are unbranched in the actively growing stage or sparsely branched in the older stage.The branches usually alternate, are rarely approximate or opposite, and branches are long, bluntly attenuating, or ending in a colorless hair.Cells of the main filament and branches are cylindrical and either slightly constricted or not.Chloroplasts are single and parietal; pyrenoid 1-3; typically two per cell.The prostrate systems are underdeveloped, attached to the substrate by a sparse, rhizoid-like basal system developed from the lower or middle cells.Sexual reproduction produces biflagellate isogametes, and asexual reproduction produces four-flagellated zoospores.