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Article

Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order

by
Guilherme Scotta Hentschke
1,*,
Zakaria Mohamed
2,
Alexandre Campos
1 and
Vitor M. Vasconcelos
1,3
1
Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Terminal de Cruzeiros de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
2
Department of Botany and Microbiology, Faculty of Science, Sohag University, Sohag 82524, Egypt
3
Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4069-007 Porto, Portugal
*
Author to whom correspondence should be addressed.
Toxins 2024, 16(10), 451; https://doi.org/10.3390/toxins16100451
Submission received: 19 September 2024 / Revised: 17 October 2024 / Accepted: 18 October 2024 / Published: 21 October 2024
(This article belongs to the Special Issue New Reports of Toxigenic Cyanobacteria and Cyanotoxins in Understudied Regions)
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Abstract

:
In this paper, we examine the filamentous cyanobacterial strain NILCB16 and describe it as a new species within the genus Pegethrix. The original population was sampled from a mat growing in an irrigation canal in the Nile River, Egypt. Initially classified under Plectonema or Planktolyngbya, the strain is a potential producer of the toxins microcystin and β-N-Methylamino-L-Alanine (BMAA). Additionally, we reviewed the taxonomic relationships between the Oculatellales genera. To describe the new species, we conducted a polyphasic study, encompassing 16S rRNA gene phylogenetic analyses performed using both Maximum Likelihood and Bayesian methods, sequence identity (p-distance) analysis, 16S-23S ITS secondary structures, and morphological and habitat comparisons. The phylogenetic analysis revealed that strain NILCB16 clustered within the Pegethrix clade with strong phylogenetic support, but in a distinct position from other species in the genus. The strain shared a maximum 16S rRNA gene identity of 97.3% with P. qiandaoensis and 96.1% with the type species, P. bostrychoides. Morphologically, NILCB16 can be differentiated from other species in the genus by its lack of false branching. Our phylogenetic analyses also show that Pegethrix, Cartusia, Elainella, and Maricoleus are clustered with strong phylogenetic support. They exhibit high 16S rRNA gene identity and are morphologically indistinguishable, suggesting they could potentially be merged into a single genus in the future.
Keywords:
biodiversity; new species; 16S rRNA gene; microcystin; BMAA
Key Contribution: This paper describes a new species of the cyanobacterial genus Pegethrix and accesses the taxonomic status of Oculatellales genera.

1. Introduction

Cyanobacteria are photosynthetic and prokaryotic microorganisms that initially evolved about 3500 million years ago [1]. They are found in a wide range of habitats, including in terrestrial, marine, and freshwater environments, as well as in harsh environments like hot springs [2,3,4]. Cyanobacteria play a vital role in the global ecosystem by producing oxygen and fixing carbon and nitrogen [5]. Cyanobacteria present a complex and challenging taxonomy, with numerous cryptic taxa being frequently misidentified [6].
Classification of cyanobacteria based on their morphological characteristics has led to taxonomic confusion, because of their simple structure and the similar characteristics among different taxa [7]. However, the emergence of the polyphasic taxonomic approach, which combines morphological, eco-physiological, biochemical, and molecular traits, resulted in substantial revisions of cyanobacterial classification and the establishment of new orders, families, genera, and species [8,9,10].
An example of this can be observed in the order Oculatellales, where strains previously assigned to Leptolyngbya by morphological analysis have been reclassified using a polyphasic approach into numerous clades and new genera such as Drouetiella [11], Thermoleptolyngbya [3], Timaviella [11], Trichotorquatus [12], and Pegethrix [10].
Among the recently described genera of this order, Pegethrix has seven characterized species including Pegethrix botrychoides, P. olivacea, P. convoluta, P. indistincta [11], P. atlantica [13], P. sichuanica [14], and P. qiandaoensis [10]. Nevertheless, the majority of Pegethrix species are geographically confined to the USA [11,13], with two species reported from China [10,14] and one from the Azores Islands, Portugal [13]. Nevertheless, strains isolated from various regions of the world that had morphological traits like Pegethrix, but were mistakenly assigned to Lyngbya/Phormidium/Plectonema and Leptolyngbya [9,11], should be re-evaluated using the polyphasic approach in order to ascertain their exact phylogenetic position.
In Egypt, the irrigation canals split from the Nile River, especially those located in a subtropical region, have been influenced by high temperature, humidity, intense sunlight exposure, and increasing anthropogenic activities [15]. These conditions promoted the proliferation of microalgae and resulted in frequent harmful cyanobacterial blooms in these water bodies during warm months every year [16]. The cyanobacterial communities in some irrigation canals in Upper Egypt are dominated by a Leptolyngbya-related morphotype, and based on morphological characteristics, this cyanobacterium was previously reported under Plectonema [17] or Planktolyngbya [18]. Interestingly, ELISA and HPLC analyses linked this cyanobacterium to the production of microcystin –YR and –LR [17]. Furthermore, the toxin β-N-Methylamino-L-Alanine (BMAA) was also reported in mats where this cyanobacterium was collected [17,18].
Therefore, the main aim of this study is to apply a polyphasic approach to clarify the phylogenetic position of a Leptolyngbya-related morphotype, which was isolated (strain NILCB16) from a Nile River irrigation canal. The present study determined the position of this cyanobacterium within the genus Pegethrix, as the new species Pegethrix niliensis sp. nov. Additionally, this paper reviews the close phylogenetic relationship among Pegethrix, Cartusia, Elainella, Maricoleus, and Drouetiella.

2. Results

  • Description of the new taxon
  • Order Oculatellales
  • Family Oculatellaceae
  • Pegethrix niliensis G. S. Hentschke sp. nov.
  • Figure 1
In liquid medium, growing cespitose from the bottom of the flask, forming erect fascicles, or with filaments attached to the flask walls. Filaments long, straight, flexuous or wavy, entangled or forming fascicles. Sheaths firm, colorless, tightly embracing the trichome (up to 1 μm) or widened (up to 3 μm), containing only one trichome. Trichomes facultatively constricted, isopolar, cylindrical. Apical cells rounded. Cells isodiametric, longer or shorter than wide. Cell content homogenous or granulose. Cell measurements: 1.8–3.5 μm long and 2.3–2.7 μm wide.
Etymology: niliensis is derived from the Latin word “Nilus”, which refers to the Nile River. The suffix “-ensis” indicates origin or place.
Holotype: A metabolically inactive (lyophilized) biomass of the strain NILCB16 was deposited at the microorganism’s herbarium of the Faculty of Science, Sohag University, under the number SOU00197.
Type strain: NILCB16 (PQ314731).
Habitat: Mat at the margin of a freshwater irrigation canal from Nile River, Egypt.
Toxins 16 00451 g001
Figure 1. Pegethrix niliensis sp. nov. (a) Thalli growing in liquid medium. (b,c) Filaments with entangled arrangement and forming fascicles. (d) Details of trichomes. (e) Constricted filament with necridium. (f) Filament with widened sheath. (g) Details of trichomes. Arrows indicate necridia.
Figure 1. Pegethrix niliensis sp. nov. (a) Thalli growing in liquid medium. (b,c) Filaments with entangled arrangement and forming fascicles. (d) Details of trichomes. (e) Constricted filament with necridium. (f) Filament with widened sheath. (g) Details of trichomes. Arrows indicate necridia.
Toxins 16 00451 g001
The first-round phylogeny (Figure 2) demonstrated that the order Oculatellales was monophyletic (ML = 100), encompassing the genera Oculatella, Tildeniella, Calenema, Shahulinema, Thermoleptolyngbya, Drouetiella, Pegethrix, Cartusia, Maricoleus, Elainella, Albertania, Egbenema, Trichotorquatus, Komarkovaea, Timaviella, and Shackletoniella. Among these, the genera Pegethrix, Elainella, Cartusia, and Maricoleus formed a highly supported cluster (ML = 100), indicating a close phylogenetic relationship among them. Drouetiella was positioned as a sister group to this cluster (ML = 0.8).
This phylogeny was also able to separate the Pegethrix species in distinct clades. Our isolate P. niliensis sp. nov. (labeled as SH) was placed at the base of the Pegethrix clade, along with “Leptolyngbya” WR9, with robust phylogenetic support (ML = 90), suggesting a close relationship with this genus. However, P. niliensis sp. nov. was phylogenetically distinct from the clades of the already established species within the genus, suggesting that it is a new taxon worthy to be described. The genus Elainella was found to be polyphyletic, as E. chongqingensis CCNU0012 was phylogenetically separated from the type species strain E. saxicola UPOC E1. Between these two Elainella species, the Maricoleus and Cartusia clades were placed.
The second-round ML and BI phylogenies confirmed the polyphyletic status of Elainella (Figure 3). As in the first-round analysis, the second-round analysis showed the genera Pegethrix, Elainella, Cartusia, Maricoleus, and Drouetiella forming a strongly supported cluster (ML = 91, BI = 1). The Pegethrix clade was robustly defined with strong statistical support (ML = 96, BI = 1). In these analyses, as in the first-round tree, P. niliensis sp. nov. was phylogenetically separated from the other species within the genus, suggesting that it represents a new taxon worthy of description.
The 16S rRNA gene identity analysis confirmed a close relationship among the genera Pegethrix, Elainella, Cartusia, Maricoleus, and Drouetiella. Comparisons between their respective clades revealed that their 16S rRNA identity values were frequently above 95% (Table S1). When comparing only the reference/type strains, Pegethrix bostrychoides GSE-PSE-MK47-15B shared 16S rRNA gene identity values of 95.8% with Elainella saxicola UPOC E1, 95.7% with Cartusia fontana Kovacik 1999/1-LC, 94.9% with Maricoleus vaginatus WZU 0102, and 94% with Drouetiella lurida LUKESOVA1986/6.
P. niliensis sp. nov. also exhibited high 16S rRNA gene identity with all these genera, with values ranging from 94% to 97.6% with Pegethrix, 96.8% to 97.6% with Cartusia, 96.3% to 97.6% with Elainella, 95.1% to 95.9% with Drouetiella and 96.8% with Maricoleus.
The 16S-23S ITS secondary structure analysis aligned with the previous analyses, indicating close relationship among all these genera (Figure 4, Figure 5, Figure 6 and Figure 7). All of them presented the sequence 5′-CAUCCCA-3′ in the basal lateral bulge of the D1-D1′ helix, with the only exception of P. altantica, which showed a single substitution resulting in the sequence 5′-CAUCUCA-3′ (Figure 4). No diagnostic characters in the secondary structures were found to separate these genera. In contrary, the sequence 5′-CAUCCCA-3′ at the lateral bulge can be considered a constant character for them. As for all the compared genera, the D1-D1′ helix of P. niliensis sp. nov. also featured the sequence 5′-CAUCCCA-3′ at the lateral bulge. However, the remaining regions of this helix, as well as the V2, Box B, and V3 helices, differed from the others in sequence, length, and structure.
The V2, Box B, and V3 helices showed differences in sequence, length, and structure across the compared genera (Figure 5, Figure 6 and Figure 7). This variability was also evident among the Pegethrix species, which displayed significant differences in sequence, length, and structure across all ITS helices.
Morphologically, Pegethrix, Cartusia, Elainella, and Drouetiella are quite similar and cannot be distinguished. They all exhibit entangled or fascicled filaments. The trichomes contain necridia, may be facultatively constricted, and exhibit apical cells rounded, not tapered. Their sheaths are firm and colorless. Their cells are isodiametric, longer or shorter than wide, with granulose content. All these genera inhabit humid (freshwater) terrestrial environments. Maricoleus is also morphologically similar but is a marine genus (Table 1). P. niliensis sp. nov. is from freshwater and fits into the description of any of these genera, presenting the same morphological characters. At the species level, we compared P. niliensis sp. nov. with all Pegethrix species, as shown in Table 2. It was evident that, morphologically, all Pegethrix species are similar, but present some differences, as detailed in the Discussion section.
All of our results considering 16S rRNA gene phylogenetic analysis, identity, 16S-23S ITS secondary structures, and morphological and habitat analyses indicate that Pegethrix, Cartusia, Maricoleus, Elainella, and Douetiella are very closely related genera. However, our isolate was phylogenetically clustered to Pegethrix and fit into the circumscription of this genus, and because of that, we herein describe the new species Pegethrix niliensis sp. nov.

3. Discussion

Although the primary objective of this paper is to describe a new species of Pegethrix, our analysis yielded two other significant findings: (1) Pegethrix, Cartusia, Elainella, and Maricoleus are clustered together, present high 16S rRNA gene identity values, and are morphologically indistinguishable from each other and (2) Elainella is currently polyphyletic.
To address these issues, two potential solutions are possible: (1) split Elainella into two genera or (2) merge the above-cited genera into a single monophyletic genus. We advocate in favor that in the future Pegethrix, Cartusia, Maricoleus, and Elainella should be merged into a single genus for the reasons discussed below. The case of Drouetiella is more complicated as it shows lower 16S rRNA gene identity values when compared to the other genera (Table S1) and probably warrants classification as a separate genus.
We conducted a polyphasic analysis and found that Pegethrix, Cartusia, Maricoleus, and Elainella form a monophyletic cluster (Figure 2 and Figure 3). Within this cluster, the 16S rRNA gene identity values were high, with all genera sharing more than 95% identity. According to the phylogenetic results, the morphological comparisons, the monophyletic concept of cyanobacterial genera [19], and the 16S rRNA gene threshold of Yarza et al. [20], which suggests that strains with more than 94.5% 16S rRNA gene identity should belong to the same genus, there is no justification for the separation of these genera. Moreover, their habitats are similarly characterized by humid rocky walls or freshwater environments, except for Maricoleus, which is marine. Another piece of evidence is that the 16S-23S ITS secondary structures of the D1-D1′ helix of all the genera featured a conserved region, 5′-CAUCCCA-3′, in the lateral bulge. Furthermore, no diagnostic region in these structures is present to distinguish these genera (e.g., there is no unique region among the Pegethrix helices which can distinguish it from the other genera). These results align with the original publications of these genera and their species [11,21,22], which also demonstrated that this group is monophyletic, with high 16S rRNA gene identity and very similar morphological and ecological traits. However, they were treated as different taxa.
In their original description, Pegethrix, Cartusia, and Drouetiella were distinguished among them and from Elainella solely based on 16S-23S ITS secondary structures and the length of certain 16S-23S ITS domains [11]. However, we believe this criterion is not robust, as our analysis revealed that these genera share significant similarities in their D1-D1′ helices, particularly in the conserved region of the lateral bulge. Furthermore, if the 16S-23S ITS regions are used as a diagnostic criterium to separate these genera, then logically, all Pegethrix species would also need to be classified as different genera, given the significant variation among their helices (Figure 4, Figure 5, Figure 6 and Figure 7). Additionally, the 16S rRNA gene identities among the Pegethrix species are sometimes lower than those observed between strains from different genera. For example, the Pegethrix strains Pegethrix FACHB3566 and P. olivacea GSEPSEMK4615A share 96.37% of 16S rRNA gene identity, while strains from different genera, such as Cartusia fontana KOVACIK1999/1 and P. indistincta GSETBCMK07GA, share 96.47% of 16S rRNA gene identity. In our analyses, we could not include Cartusia and P. qiandoaensis 16S-23S ITS secondary structures, because their sequences are not available in public databases.
We also highlight that interpreting 16S-23S ITS secondary structures can be highly subjective, with some researchers identifying differences, while others find similarities among them, as seen in this case. The problem of interpretation of secondary structures is discussed by Oliveira et al. [23], who demonstrated that genera from different orders can also exhibit similar 16S-23S ITS secondary structures. Additionally, some studies [24,25,26] showed that different operons within the same strain can have varying lengths in their conserved domains, complicating comparisons even more. According to Jusko et al. [27], “analyses of the 16S–23S ITS region are highly subject to the effects of multiple ribosomal operons in a genome and evidence is mounting that widespread issues may have arisen due to the lack of consideration of this phenomenon” [25]. For the reasons presented in our article, we believe that Pegethrix, Cartusia, Elainella, and Maricoleus should be merged in the future. In this case, the name adopted should be Elainella, which is the oldest genus [28]. Drouetiella, however, exhibits lower 16S rRNA gene identity values with these genera and possibly warrants classification as a separate genus.
Regarding the species P. niliensis sp. nov., it is phylogenetically clustered to Pegethrix with strong statistical support, and because of that, we assigned it to this genus. After this assignment at the genus level, we also found that P. niliensis sp. nov. was phylogenetically distinct from previously described species of Pegethrix. The 16S rRNA gene identity matrix (Table S1) indicated that the identity values shared between P. niliensis sp. nov. and other Pegethrix species were always below 98.7%, the threshold for species delimitation as defined by Yarza et al. [20]. According to these authors, strains with 16S rRNA gene identity values below this threshold are considered to represent different species. The highest 16S rRNA gene identity value was found with Pegethrix FACHB3566, which was only 97.6% identical to P. niliensis sp. nov.
Morphologically, P. niliensis sp. nov. differs from P. bostrychoides, P. olivacea, P. qiandaoensis, and P. atlantica in that it lacks false branching. P. indistincta is unique among these species in that it can present more than one trichome within a single sheath. While P. niliensis sp. nov. is morphologically more similar to P. sichuanica, it can be distinguished from this species through 16S rRNA gene phylogenetic and identity analyses. The occurrence of Pegethrix niliensis sp. nov. as a constituent of cyanobacterial blooms/mats in Egyptian irrigation canals fits ecologically with P. atlantica and P. qiandaoensis inhabiting freshwater lakes in the Azores Islands and China, respectively. However, the freshwater habitat of this new species distinguishes it from the remaining five described species, which are primarily found in hot springs, rocks, and soil crusts (Table 2).
Another important remark of our work is that if hypothetically Pegethrix, Cartusia, Elainella, Maricoleus, and Drouetiella were merged, the polyphyletic status of Elainella would also be addressed. Our three phylogenetic analyses reveal that E. chongqingensis formed a distinct clade separate from the type species E. saxicola. In fact, E. chongqingensis was more closely related to Maricoleus and Cartusia than to E. saxicola. Although among these taxa, Maricoleus was the most recently described, the article that described this genus [22] did not show a phylogeny that encompassed E. chongqingensis, preventing a direct comparison with our findings. However, this phylogeny also presented Pegethrix, Elainella, Maricoleus, Cartusia, and Drouetiella as a monophyletic cluster with 16S rRNA gene identity values higher than 95%, in agreement with our results. According to that, we believe that the polyphyletic nature of Elainella further supports the need to review and merge these genera into a single, cohesive genus.
In conclusion, our paper represents a contribution to biodiversity research. It introduces a new species from the Nile River (Egypt), a region that is largely unexplored in terms of cyanobacterial diversity. This type strain was isolated from an irrigation canal, where toxic Leptolyngbya-like populations commonly form blooms and produce microcystin and BMAA [17,18]. Thus, this identification is of utmost importance for understanding bloom development, toxicity risk assessment in the aquatic environment, and water management in the region. Additionally, our study provides insights into the taxonomy of the Oculatellales, highlighting its complexity and the close relation among its genera. Notwithstanding, considering that the studied strain was sampled from an environment with common toxic blooms, further studies are needed, namely on the toxic potential of the species (toxin profiling). This information will enable an assessment of the risks of its occurrence in the environment and any eventual implications to human health.

4. Materials and Methods

4.1. Sampling, Isolation of Strains, and Morphological Analysis

The strain NILCB16 was isolated from a benthic mat collected from an irrigation canal in Sohag Province, Egypt (26°62′ N and 31°64′ E), in September 2021. This irrigation canal is a permanent branch of the Nile River and serves about 155,000 acres. This water source was classified as oligotrophic–eutrophic water body dominated by cyanobacteria (1.1 × 105 cells L−1), with physico-chemical parameters characterized by high water temperature (29 °C), slightly alkaline pH (7.8), and high nutrient concentrations (NO3: 1.8 mg L−1; PO4: 0.07 mg L−1) during summer months [18]. The isolation was performed by inoculating a single filament from the natural population into a culture flask with Z8 liquid medium [29]. The strain is currently maintained at the Algal Culture Collection at Sohag University, in Z8 liquid medium under the following conditions: 25 °C, 16꞉8 h in a light–dark cycle, with a light intensity of 40 μmol m−2 s−1) under fluorescent light.
For morphological analysis, the strain was microphotographed and analyzed by the LEICA LAS version 4.12.0 image analysis software (Leica Microsystems Limited and CMS GmbH, Heerbrugg, Switzerland). The measurements were performed for each characteristic of the strains (20 to 30 measurements) and were carried out at various positions of the slide preparation.

4.2. DNA Extraction, PCR Amplification, and Sequencing

The cyanobacterial filaments were harvested from the cultures, and the total genomic DNA (gDNA) of the strains were extracted using the PureLink Genomic DNA kit (Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions provided for Gram-negative bacteria. For the amplification (PCR) of the 16S rRNA gene, the primers 27SF [30] and 23SR [31] were used. The PCR reaction was performed in a Veriti Thermal Cycler (Veriti 9902, Applied Biosystems, Thermo Fisher Scientific, Singapore). The final reaction volume was 20 μL, consisting of 5.9 μL of molecular biology-grade water, 4 μL of Green GoTaq Flexi Buffer, 2 μL of MgCl2, 2 μL of each forward and reverse primer, 1.5 μL of deoxynucleotide triphosphate (dNTPs), 0.5 μL of bovine serum albumin (BSA), 0.1 μL of GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA), and 2 μL of genomic DNA [32]. The 16S rRNA gene sequence was obtained upon PCR amplification in the following conditions: initial denaturation at 94 °C for 5 min, followed by 10 cycles of denaturation at 94 °C for 45 s, annealing at 57 °C for 45 s, and extension at 72 °C for 2 min. This was followed by an additional 25 cycles of denaturation at 92 °C for 45 s, annealing at 54 °C for 45 s, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 7 min. The PCR product was separated using 1% (w/v) agarose gel stained with SYBR Safe DNA gel stain (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), and DNA fragment of the expected size were excised from the gel and purified using the NZYGelpure kit (Nzytech, Lisbon, Portugal), following the manufacturer’s instructions. Finally, the purified fragments were sent for sequencing (separately) with primers 359F and 781R [33], 1494R and 27SF [30], 23SR [31,34], and 1114F. The sequencing was performed by sanger dideoxy sequencing at GATC Biotech (Ebersberg, Germany), and the nucleotide sequences obtained were manually inspected for quality and assembled using the Geneious Prime 2023.2.1 software (Biomatters Ltd., Auckland, New Zealand). Before phylogenetic analysis, the sequence was checked for possible chimera formation using the DECIPHER software 2.27.2 [35]. To assess the presence and quality of the DNA obtained from extraction and PCR, we performed electrophoresis on a 1% (w/v) and 1.5% (w/v) agarose gel stained with SYBR Safe DNA gel stain (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA), respectively. The confirmation of high-molecular-weight DNA was based on the presence of clear bands observed in the gel. The sequence was deposited in GenBank (National Center for Biotechnology Information, NCBI) under the code PQ314731.

4.3. Phylogenetic Analysis

The phylogenetic analyses were conducted in two rounds. In the first round, we aimed to position our strains among the cyanobacterial genera. For that, we aligned the 16S rRNA gene sequences of our isolate with sequences of cyanobacterial reference strains and additional sequences retrieved from GenBank (NCBI) by BLAST. The final alignment comprised 419 sequences and 813 nucleotide informative sites. Phylogenetic reconstruction was performed using the FastTree method [36], with the bootstrap value set to the default of 1000 as per the manual. The command used to run the phylogeny was “FastTree -gtr -nt alignment_file> tree_file”. The resulting tree was edited using iTOL [37].
In the second round of analysis, to confirm that the strain NILCB16 represents a new species, we selected the cyanobacterial sequences most closely related to them. This included genera from the order Oculatellales and additional genera from related orders such as Nodosilineales and Leptolyngbyales. This selection resulted in a total of 112 sequences and 894 nucleotide positions analyzed. Then, the phylogenetic trees were built using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses. GTR + G + I evolutionary model was selected by MEGA11: Molecular Evolutionary Genetics Analysis version 11 [38]. The robustness of ML tree was estimated by bootstrap percentages, using 1000 replications using IQ-Tree online version v1.6.12 [39]. The Bayesian tree was constructed in two independent runs, with four chains each, for 5 × 106 generations. Burnin fraction was set to 0.25 and sample frequency was 1000, using MrBayes [40] in Cipres Gateway [41]. The processing and visualization of these trees were conducted using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 1 August 2024).
For all analyses, the sequences were aligned using MAFFT [42] and the outgroup used was Gloeobacter violaceus PCC 8105 (AF132791). A similarity (p-distance) matrix was generated using MEGA11, and the 16S-23S ITS secondary structures of D1-D1′, V2, Box B, and V3 helices were folded using MFold [43], according to [44].

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/toxins16100451/s1, Table S1. 16S rRNA gene identity (p-distance) matrix comparing P. niliensis sp. nov. with its most related phylogenetic taxa [38,45].

Author Contributions

Conceptualization, G.S.H.; writing—original draft preparation, G.S.H.; writing—review and editing, Z.M. and A.C.; supervision, V.M.V.; funding acquisition, A.C. and V.M.V. All authors have read and agreed to the published version of the manuscript.

Funding

This work received funding from the European Union’s Horizon 2020 research and innovation program, from the Portuguese Foundation for Science and Technology (FCT) Projects UIDB/04423/2020 and UIDP/04423/2020, and the WP9—Portuguese Blue Biobank under the Blue Economy Pact—Project No. C644915664-00000026—co-funded by Programa de Recuperação e Resiliência PRR, The Portuguese Republic, and the European Union.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The 16S rRNA gene and 16S-23S ITS sequences are available in GenBank (NCBI) under the ID PQ314731.

Conflicts of Interest

The authors declare no conflicts of interest.

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Toxins 16 00451 g002
Figure 2. First-round phylogeny. FastTree phylogeny with cyanobacterial reference strains. The genera of Oculatellales are colored. The upper image is a section of the circular tree.
Figure 2. First-round phylogeny. FastTree phylogeny with cyanobacterial reference strains. The genera of Oculatellales are colored. The upper image is a section of the circular tree.
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Toxins 16 00451 g003
Figure 3. Second-round phylogeny based on Maximum Likelihood. The bootstrap values and the Bayesian posterior probabilities are indicated at the nodes.
Figure 3. Second-round phylogeny based on Maximum Likelihood. The bootstrap values and the Bayesian posterior probabilities are indicated at the nodes.
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Toxins 16 00451 g004
Figure 4. D1-D1′ helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
Figure 4. D1-D1′ helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
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Toxins 16 00451 g005
Figure 5. V2 helix of the 16S-23S ITS region of P. niliensis sp. nov and its closest related taxa.
Figure 5. V2 helix of the 16S-23S ITS region of P. niliensis sp. nov and its closest related taxa.
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Toxins 16 00451 g006
Figure 6. Box B helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
Figure 6. Box B helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
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Toxins 16 00451 g007
Figure 7. V3 helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
Figure 7. V3 helix of the 16S-23S ITS region of P. niliensis sp. nov. and its closest related taxa.
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Table 1. Morphological and habitat comparisons between P. niliensis sp. nov. and other phylogenetically and morphologically related genera.
Table 1. Morphological and habitat comparisons between P. niliensis sp. nov. and other phylogenetically and morphologically related genera.
P. niliensis sp. nov.PegethrixCartusiaMaricoleusElainellaDrouetiella
Trichome/filaments mode of growthErect fascicles from the bottom of the culture flasks (liquid medium)Radially spreadingNot describedBenthic massesFascicles or tuftsNot described
FilamentsLong, wavy, intensely entangled or forming fasciclesEntangled or in fascicles, with nodule formation. Straight, flexuous, sometimes coiled, with nodules.Sometimes in fascicles. Straight or flexuous, sometimes with more than one trichome in a sheathEntangled, rarely with more than one trichome within a sheathForming fasciclesSolitary or in fascicles. Straight, flexuous, or coiled
TrichomesNot tapered. Apical cells roundedNot tapered. Apical cells roundedNot tapered. Apical cells roundedNot taperedNot tapered.Not tapered. Apical cells
False branchingNot presentRare, single or doubleNot presentFacultativeYes, single and doubleRare, single
Constrictions at cell wallsFacultativeFacultativeFacultativeFacultativeFacultativeFacultative
SheathsFirm, colorless, attached to the trichomeFirm to soft and thin or widened, colorlessFirm, thin or widened, colorlessFacultative, layered, colorless, thin or widenedFirm, thin, colorless Firm, thin, colorless
MotilityNoYesNot describedNot describedNoNot described
Cell shapeIsodiametric, longer or shorter than wideIsodiametric, longer or shorter than wideIsodiametric or shorter than wideIsodiametricIsodiametric or longer than wideIsodiametric, longer or shorter than wide
Cell contentHomogenous or with granuleHomogenous or with granuleHomogenous or with granuleGranularOften with granulesHomogenous or with granule
NecridiaYesYesYesNot describedYesFacultative
Cell dimensions (μm)2.1–3.1 long × 1.9–2.8 wide1–3 long × 1.3–3.3 wide1.3–2 long × 1.8–3.5 wide1.3–5.4 long × 1.4–4.21.7–2.6 long × 1.3–3.8 wide2.1–5.4 long × 1.7–2.1 wide
HabitatBenthic mat. Irrigation canal from Nile River, EgyptTerrestrial. Seep wallsTerrestrialMarineOn rock in a lakeTerrestrial
Table 2. Morphological and habitat comparisons between P. niliensis sp. nov. and other Pegethrix species.
Table 2. Morphological and habitat comparisons between P. niliensis sp. nov. and other Pegethrix species.
P. niliensis sp. nov. P. bostrychoidesP. olivaceaP. atlanticaP. indistinctaP. sichuanicaP. convolutaP. qiandaoensis
Trichome/filaments mode of growthErect fascicles from the bottom of the culture flasks (liquid medium)Radial fasciculation, penetrating the agarSpreading radially, flat and mucilaginous or moundedNot describedNot describedRadially spreading, with loose fasciculation or clusteredRadially spreading, growing into the agarNot described
FilamentsLong, wavy, intensely entangled or forming fasciclesLong or short,
sometimes forming nodules or loosely to tightly spirally coiled		Long or short, frequently irregularly bent due to uneven cell division along filament. Sometimes loosely coiled to form irregular nodulesLong, fasciculate, straight or loosely
coiled		Long, with variation in width between young
and mature trichomes. Rarely with more than one
trichome sharing a common sheath		Straight or slightly bentFasciculate, long,
straight or slightly bent, frequently forming
loose to compact nodules		Long. Not forming nodules
TrichomesNot tapered. Apical cells roundedNot tapperedCell division along trichomes often irregular, producing cells with variable shape
and width		Not taperedNot tapered. Apical cells roundedApical cells roundedNot taperedNot tapered. Apical cells rounded
False branchingNot presentRare, singleYesRare, singleRare, single or doubleNot describedSometimes singly or doubly false branchedSingle or double
Constrictions at cell wallsFacultativeMore or less constricted at the distinctly visible cross-wallsConstricted at indistinctly visible cross-wallsNot or
slightly constricted at the visible cross-walls		Not or slightly constricted at distinctly visible cross-wallsNot or slightly constricted at the cross-wallsNot or slightly constricted at distinctly visible cross-walls,Not or slightly constricted at visible cross-walls
SheathsFirm, colorless, tightly embracing the trichome (up to 1 μm) or widened (up to 3 μm)Firm, colorless, usually attached
to trichome, occasionally softer, widened, sometimes irregular and stratified		Sheath firm,
colorless, usually attached to trichome, occasionally widened		Firm,
colorless, attached to the trichome		Firm, usually attached to trichome, occasionally
widened, rarely irregular and stratified, absent in immature filaments		Firm, colorless, thin,
usually attached to the trichome, occasionally distinct,
clear, but occasionally widened		Firm, colorless, usually attached to trichome, occasionally widened, rarely irregular and stratifiedFirm, attached to the trichome, occasionally widened
Cell shapeIsodiametric, longer or shorter than wideSlightly shorter than wide to longer than wideCells occasionally isodiametric, shorter than
wide in meristematic regions		Mostly
shorter than wide		Isodiametric, often shorter than wide especially in
meristematic zones, slightly longer than wide in young trichomes		Isodiametric
or slightly shorter than wide		Slightly shorter than wide to longer than wideIsodiametric, longer or shorter than wide
Cell content (light microscopy)Homogenous or with granuleRarely with a single central granuleLarge central granuleSometimes with a unique central granuleNot describedPolyphosphate body commonly
visible in nucleoid region		Sometimes with a single central granuleNot described
NecridiaYesYesYesYesYesYesYesNot described
Cell dimensions (μm)2.1–3.1 long × 1.9–2.8 wide1.0–3.0 long × 1.5–2.5–(3.0) wide1.7–2.6 long × 1.9–3.5 wide2.4 long × 1.8–3.0 wide(1.3)–1.7–2.7 long × 1.9–3.3 wide 1.0–2.5–(3.7) long × 1.3–2.5 (3.2) wide(1.3)1.7–2.7 long × 2.3–4.0 wide
HabitatBenthic mat. Irrigation canal from Nile River, EgyptTerrestrial. Sandstone seep wall, UT, USATerrestrial. Sandstone seep wall, UT, USARocky substrate over lakes, Azores, PortugalTerrestrial. Seep wall and waterfall in Navajo
Sandstone, UT, USA		Terrestrial. Brick wall alongside mountain, Sichuan, ChinaTerrestrial. Large seep wall and waterfall in Navajo
Sandstone		Planktonic. Freshwater
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Hentschke, G.S.; Mohamed, Z.; Campos, A.; Vasconcelos, V.M. Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order. Toxins 2024, 16, 451. https://doi.org/10.3390/toxins16100451

AMA Style

Hentschke GS, Mohamed Z, Campos A, Vasconcelos VM. Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order. Toxins. 2024; 16(10):451. https://doi.org/10.3390/toxins16100451

Chicago/Turabian Style

Hentschke, Guilherme Scotta, Zakaria Mohamed, Alexandre Campos, and Vitor M. Vasconcelos. 2024. "Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order" Toxins 16, no. 10: 451. https://doi.org/10.3390/toxins16100451

APA Style

Hentschke, G. S., Mohamed, Z., Campos, A., & Vasconcelos, V. M. (2024). Description of Pegethrix niliensis sp. nov., a Novel Cyanobacterium from the Nile River Basin, Egypt: A Polyphasic Analysis and Comparative Study of Related Genera in the Oculatellales Order. Toxins, 16(10), 451. https://doi.org/10.3390/toxins16100451

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