# Insight on the heterocyte patterning and the proheterocyte division in the toxic cyanobacterium Kaarinaea lacus gen. nov., sp. nov., and its genomic potential for natural products

Aniket Saraf, Eddy Blondet, Anne Boullié, Alexis Criscuolo, Muriel Gugger

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## To cite this version:

Aniket Saraf, Eddy Blondet, Anne Boullié, Alexis Criscuolo, Muriel Gugger. Insight on the heterocyte patterning and the proheterocyte division in the toxic cyanobacterium Kaarinaea lacus gen. nov., sp. nov., and its genomic potential for natural products. Harmful Algae, 2025, 142, pp.102792. 10.1016/j.hal.2024.102792. pasteur-05113070

HAL Id: pasteur-05113070

https://pasteur.hal.science/pasteur-05113070v1

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# Insight on the heterocyte patterning and the proheterocyte division in the toxic cyanobacterium *Kaarinaea lacus* gen. nov., sp. nov., and its genomic potential for natural products

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Aniket Saraf<sup>a</sup>, Eddy Blondet<sup>a</sup>, Anne Boullié<sup>a</sup>, Alexis Criscuolo<sup>b,#</sup>, Muriel Gugger<sup>a,#,*</sup>

## ARTICLE INFO

### Keywords:

- Cyanobacteria

- Proheterocyte division

- Heterocyte glycolipid

- Pattern formation

- Nostocales

## ABSTRACT

Nostoc sp. 152 (= PCC 9237<sup>T</sup>), a toxic cyanobacterium, isolated from mixed cyanobacterial bloom in a Finnish freshwater lake was reassessed using phylogenetic, morphological and genomic analyses. Multilocus and 16S rRNA gene phylogenetic analyses confirmed that this strain represents a novel Nostocalean genus for which we propose the name *Kaarinaea lacus* gen. nov., sp. nov., in accordance with the International Code of Nomenclature of Prokaryotes. The most intriguing morphological feature exhibited by PCC 9237<sup>T</sup> is the occasional division of proheterocytes. Based on our observations, we propose a hypothesis for the sequence of events taking place leading to this phenomenon. The other interesting feature includes the unusual heterocyte patterning resulting in the development of heterocytes in series in the old culture followed by its fragmentation at this site. Among the genes involved in heterocyte differentiation and patterning, *patC* and three homologs of *hetP* were not found from the genome of PCC 9237<sup>T</sup>. Furthermore, genomic investigations revealed a variant of heterocyte glycolipid gene cluster with a reduced *hglB*, but having additional gene coding for a protein with TubC-N terminal docking domain. Finally, the presence of a *pks2*-like gene cluster, whose product may interfere with the cellular differentiation, and an anachelin-like gene cluster demands further investigations.

## 1. Introduction

Cyanobacteria are one of the most successful inhabitants of the Earth and are reported from diverse habitats such as freshwater, brackish water, oceans, soils or alkaline marshes. In addition, they can also be found in extreme habitats such as cold and arid deserts, or thermal springs (Gaysina et al., 2019). Apart from being omnipresent, cyanobacteria play an important role in the global carbon and nitrogen cycles, making them ecologically significant (Sanchez-Baracaldo et al., 2022). However, they are also well-known to produce toxic compounds (cyanotoxins) that have adverse impacts on humans and other animals (Sivonen and Jones, 1999). Among the cyanotoxins, microcystins (MCs) are the most commonly occurring and widely investigated (Merel et al., 2013), and multiple variants of MCs have been reported to be produced by various genera of cyanobacteria (Christiansen et al., 2003; Fiore et al., 2009; Sivonen et al., 1992). In 1990, O-acetylated MC variants were described from a freshwater *Nostoc* sp. 152 isolated from mixed

cyanobacterial bloom in a Finnish lake (Sivonen et al., 1990). Later, another toxic cyclic peptide, nostophycin, was discovered from the same strain (Fuji et al., 1999).

This toxic strain was originally assigned to the genus *Nostoc* because of its overall morphological resemblance to its members. This first taxonomic assignment was supported a decade later by a phylogenetic analysis based on 16S rRNA gene sequences, where *Nostoc* sp. 152 clustered with *Nostoc punctiforme* PCC 73102 and *Nostoc* sp. PCC 9709 (Lyra et al., 2001; Sivonen et al., 1990). However, using a similar approach with an increased taxon sampling, *Nostoc* sp. 152 was located distant from the other *Nostoc* strains (Oksanen et al., 2004). Further, when the *Nostoc sensu stricto* clade was established, *Nostoc* sp. 152 clustered distantly from this clade (Lukešová et al., 2009; Reháková et al., 2007). The establishment of *Nostoc sensu stricto* opened the way for naming the *Nostoc*-like taxa. To date, 15 *Nostoc*-like genera have been validly described using a polyphasic approach (Alvarenga et al., 2021; Bagchi et al., 2017; Cai and Li, 2020; Cai et al., 2019, 2020; Genuario

<footnote><sup>a</sup> Institut Pasteur, Université Paris Cité, Collection of Cyanobacteria, Paris F-75015, France</footnote>

<footnote><sup>b</sup> Institut Pasteur, Université Paris Cité, GIPhy - Genome Informatics and Phylogenetics, Biological Resource Center of Institut Pasteur, Paris F-75015, France</footnote>

<footnote>* Corresponding author.
E-mail address: muriel.gugger@pasteur.fr (M. Gugger).</footnote>

<footnote># These authors share last authorship</footnote>
et al., 2015; Hentschke et al., 2017; Hrouzek et al., 2013; Lee et al., 2021; Pal et al., 2024; Reháková et al., 2007; Saraf et al., 2019; Soares et al., 2020), but none of these studies reevaluated *Nostoc* sp. 152. Since *Nostoc* sp. 152 is a toxic cyanobacterium, it is crucial to assign its true taxonomic identity; this will indeed increase our understanding regarding the diversity of toxin-producing heterocytous cyanobacteria which are currently reported from a few genera, including the recently described *Heteroscytonema* (Sendall and McGregor, 2018) and *Iningai-nema* (McGregor and Sendall, 2017).

The first and only published microphotography of *Nostoc* sp. 152 (Sivonen et al., 1990) indicated that this strain probably produces heterocytes solitary and/or in series of two to three, a rarely reported morphological trait in the genus *Nostoc* (Komárek, 2013). Most of the studies on heterocyte refer to the pattern formation in both *Anabaena cylindrica* and *A. catenula* (Wilcox et al., 1973a), as well as the genetic mechanisms involved in heterocyte differentiation and pattern formation in *Trichormus* sp. PCC 7120 (formerly named *Nostoc* and *Anabaena*) (Zeng and Zhang, 2022). Moreover, the cellular functions and the exchanges between the different types of cells were thoroughly investigated (Flores and Herrero, 2010; Herrero et al., 2016). In addition, few studies examined the proheterocyte regression (Wilcox et al., 1973b) and germination of heterocyte in *Anabaena cylindrica*, *A. hallensis*, *A. steloides*, *Brachytrichia bulani*, *Calothrix weberi*, *Gloeotrichia raciborskii*, *Nostoc commune*, *N. ellipsosporum*, *N. linckia*, *N. microscopicum*, *Rivularia mangini*, *Tolypothrix elenkinii* and *T. lanata* (Canabaeus, 1929; Desikachary, 1945; Geitler, 1921; Hollerbach, 1928; Iyengar and Desikachary, 1944; Steinecke, 1931; Wolk, 1965).

In the present study, we conducted a thorough taxonomic reassessment of *Nostoc* sp. 152 (= PCC 9237T; T indicates PCC 9237 to be type strain) using a combination of morphological, phylogenetic and genomic analyses. Our results confirmed that PCC 9237T represents a novel Nostocalean genus for which the name *Kaarinaea lacus* gen. nov., sp. nov. is proposed under the provisions of the International Code of Nomenclature of Prokaryotes. Morphologically, *Kaarinaea lacus* PCC 9237T exhibits interesting attributes, notably the division of proheterocytes. Furthermore, an in-depth genomic investigation of the strain revealed novelty related to the heterocyte patterning, heterocyte glycolipid gene cluster and uncharacterized natural products.

# 2. Materials and methods

## 2.1. Culturing of the strain

The axenic strain *Nostoc* sp. 152, cultured on Z8₀ liquid medium (Kotai, 1972; recipe at https://norcca.scrol.net/node/3963), was deposited by Kaarina Sivonen in 1992 in the collection Pasteur Cultures of Cyanobacteria (PCC; Gugger et al., 2023). This culture was spread on agar (0.8%) plates with Z8₀ media for cyanobacteria, and one trichome of this strain was used to establish the culture of the strain PCC 9237T. The details of the purification process can be found in Haande et al. (2017). Since then, the monoclonal strain has been maintained axenically by regular transfer every 12 weeks on Z8₀ + 1 mM of NaHCO₃ agar slants in a culture room illuminated with 5 µmol photons m⁻² s⁻¹ of light with a photoperiod of 13/11 h of light/dark cycle at 22 °C. For the morphological examination, the strain was transferred in Z8₀ and BG11₀ (Rippka et al., 1979) liquid medium and similar agar plates in a culture room illuminated with 5–8 µmol photons m⁻² s⁻¹ of continuous light at 25 °C. We further tested the capacity of this strain to grow actively in high cellular density photobioreactors of 10 mL (CellDeg, Germany) with 3 % CO₂, 200 rpm orbital agitation (Unimax 2010, Heidolph) in Z8₀, in Z8₀ (2×) and in BG11 (2×) for 96 h under LED illumination mimicking daylight with an intensity increasing continuously from 30 to 130 µmol photons m⁻² s⁻¹.

## 2.2. Morphological analysis

Morphological evaluation of PCC 9237T was conducted using a Zeiss Axioskop2 microscope (Oberkochen, Germany) fitted with a Leica MC170 HD (Wetzlar, Germany) camera for the microphotographs. Phenotypic characteristics such as the overall appearance of the filament, the size and shape of vegetative cells and heterocytes, the patterning of heterocytes, the appearance of the sheath, and the mode of reproduction were carefully documented. The samples for microscopic observation were prepared using 10 % glycerol in water as the mounting medium to prevent the cells from undergoing death by bursting under a long term exposure to the light. The steps involve placing 5–10 µL of the sample (depending on the density) on the glass slide, and mixing it with an equal amount of mounting medium before covering with the glass cover slip.

## 2.3. Genomic DNA extraction, whole genome sequencing, assembly and annotation

A volume of 40 mL liquid cultures in late exponential to linear growth phase was centrifuged at 12,000 g for 10 min at 20 °C. After washing twice with sterile distilled water, the pellet was immediately frozen in liquid nitrogen prior to being lyophilized. DNA of the lyophilized pellet was extracted using Genomic DNA Isolation - NucleoBond® AXG columns (Macherey-Nagel, Hoerdt, France) according to manufacturer's instructions for bacterial DNA using columns NucleoBond® AXG 20.

For long read sequencing, libraries were prepared using the native barcoding kit 96 V14 (Oxford Nanopore Technologies, ONT) following the manufacturer's protocol, except no shearing was performed. The whole genome sequencing of the strain PCC 9237T was carried out on a PromethION flow cell (FLO-PRO114M) and sequenced for 72 h on a P2 solo sequencer (ONT). Base calling was carried out using MinKNOW v23.11.3 with Dorado v7.2.12, yielding 552,000 long reads (1080,852,290 sequenced bases, 142× average sequencing depth). The whole genome sequencing of strain PCC 9237T was also carried out twice on a NextSeq500 apparatus (2 × 150 bps paired-end protocol), yielding 2782,569 reads pairs (=1535,287 + 1247,282; 112× sequencing depth and 311 bps insert size, on average).

Whole genome assembly was performed from long reads using Flye v2.9.3 (Kolmogorov et al., 2019). To correct putative assembly errors, short reads were next aligned against the assembled contigs using bwa-mem2 v2.2.1 (Vasimuddin et al., 2019), and a final consensus of the aligned short reads was derived using SAMtools v1.18 (Danecek et al., 2021). To assess genome sequence authenticity, 16S rRNA segments were extracted using Barnapp v0.9 (https://github.com/tseemann/barrnap), and next aligned using blastn against the 16S rRNA segment derived from Sanger sequencing (GenBank accession: AJ133161). Completeness and contamination indices of the whole genome assembly were estimated using CheckM v1.1.3 (Parks et al., 2015).

## 2.4. Genomic investigation

Overall gene prediction and annotation were performed using Bakta v1.9.3 (Schwengers et al., 2021). COG categories were determined using COGNiz v1.1 (https://gitlab.pasteur.fr/GIPhy/COGNiz) with the 2020 update of the COG database (Galperin et al., 2021). Gene clusters involved in the synthesis of natural products were inferred using antiSMASH v7.1.0.1 (Blin et al., 2023). Genes involved in heterocyte differentiation and patterning were first searched using tblastn with *Trichormus* sp. PCC 7120 protein sequences as queries, and the corresponding predicted coding sequences (CDS) were next gathered; syntheses of the neighbouring CDS were checked in a few cases using the synteny tool on the MicroScope platform (Vallenet et al., 2020). Of note, one CDS (corresponding to the gene *patS*) was not predicted by Bakta,
but subsequently detected using our tblastn search analyses, and therefore manually added to the set of predicted/annotated CDS (locus tag: NOSPCC9237_16672).

## 2.5. Phylogenetic analyses

A multilocus phylogenetic tree was inferred using the 120 loci suggested by Parks et al. (2017). For each of these 120 loci, the associated specific scoring matrix (PSSM; available at https://giphy.pasteur.fr/PhyloM/bacteria/) was used as a query to perform a tblastn search against each considered genome assembly, and every first tblastn hit was translated into an amino acid sequence. For each locus, gathered amino acid sequences were aligned using MAFFT v7.505 (Katoh and Standley, 2013, 2016) and the aligned characters were next processed using BMGE v2.0 (Criscuolo and Gribaldo, 2010) to select those suited for phylogenetic analysis. Maximum Likelihood (ML) phylogenetic inference from the concatenation of the resulting 120 multiple sequence alignments (43,887 aligned characters) was carried out using IQ-TREE v2.2.2.2 (Minh et al., 2020) with evolutionary model LG+F+I+R6, derived by minimizing the Bayesian information criterion.

The 16S rRNA gene sequences of the representatives of the *Nostoc sensu stricto* and other *Nostoc*-like genera were gathered from the Gen-Bank repository (Supplementary Table S1). The sequences were initially aligned using MAFFT v7 (Katoh et al., 2019) and manually curated to ensure that the folded secondary structure of the 16S rRNA gene was maintained. A total of 1435 aligned nucleotide characters were obtained after discarding those containing >50 % undefined character states. A ML phylogenetic tree was inferred using IQ-TREE v2.2.2.2 with evolutionary model GTR+F+I+R4, derived by minimizing the Bayesian information criterion.

For each aligned sequence dataset (i.e. 16S rRNA and multilocus), 500 bootstrap-based replicates were generated (Felsenstein, 1985). Bootstrap-based phylogenetic trees were inferred from these replicates by optimizing the ML, Minimum Evolution (ME) and Maximum Parsimony (MP) criteria using IQ-TREE, FastME v2.1.6.4 (Lefort et al., 2015) and TNT v1.5 (Goloboff and Catalano, 2016), respectively. Branch supports of the two ML phylogenetic trees (see above) were then assessed using bootstrap proportions (Felsenstein, 1985) derived from both ME and MP bootstrap-based trees, as well as transfer bootstrap expectation (TBE) indices (Lemoine et al., 2018) derived from ML bootstrap-based trees. Finally, the aligned 16S rRNA gene sequences were also analyzed using PhyloBayes v4.1 (Lartillot et al., 2009) with evolutionary model CAT-GTR (two chains of 210,000 cycles each, with 10,000 burn-in ones), in order to also assess posterior probability supports for the internal branches of the 16S rRNA gene ML phylogenetic tree. The trees were visualized and annotated in iTOL (Letunic and Bork, 2021) and edited in Adobe Illustrator.

## 2.6. 16S-23S ITS rRNA region analysis

The ITS rRNA region was recovered from the genome of PCC 9237T and the folded secondary structures corresponding to the D1-D1', BoxB, V2 and V3 helices were determined using Mfold (Zuker, 2003) on the UNAFold web server (http://www.unafold.org/).

## 2.7. Secondary structure prediction

The structure of the N-terminal part of the TubC-N gene was investigated using AlphaFold Protein structure database with the TubC-N domain containing protein of *Nostoc* sp. NIES-4103 (https://alphafold.ebi.ac.uk/entry/A0A1Z4S4V9) and others. The alignments of the N-terminal part of the predicted protein coded by TubC-N gene compared to the N-terminal beta-hairpin docking domain of AerJ halogenase from *Microcystis* sp. NIES-98 (PDB accession: 8AQR; found in the RCSB protein data bank, https://www.rcsb.org) were performed using Clustal O from SnapGene® vers. 5.1.7 (https://www.snapgene.com).

Conserved positions and hydrophobicity were compared to the experimental structure of A8QR, and further drawn and named as identified in Richter et al. (2008).

# 3. Results and discussion

This study aimed to reassess the taxonomic identity of a toxic Nostocalean strain, *Nostoc* sp. 152, isolated from a Finnish freshwater bloom sample by conducting detailed morphological, phylogenetic and genomic analyses. At the time of sample collection, the water bloom was dominated by *Aphanizomenon flos-aquae*, and the strain isolated into the culture was assigned to the genus *Nostoc* based on morphological resemblance in terms of the cells' shape and the filament's overall appearance (Sivonen et al., 1990). However, the distant phylogenetic positioning of PCC 9237T from *Nostoc sensu stricto* clade (Lukesová et al., 2009; Řeháková et al., 2007) indicated it to be different from true *Nostoc*. It was therefore necessary to ascertain the true taxonomic identity of PCC 9237T.

## 3.1. Genome of the strain PCC 9237T

We obtained a genome assembly of the strain PCC 9237T which is 7572,127 bps in length on 16 contigs with a G+C content of 40.7 mol% (assembly/contig accessions: GCA_964211855/CAXOSP010000000). Among the 16 assembled contigs, five were assessed as circular, including the longest one (6610,623 bps) that can be considered as the complete chromosome (accession: CAXOSP010000001). Genome sequence authenticity was assessed by comparing the 16S rRNA gene sequence derived from Sanger sequencing (GenBank accession AJ133161) against the three assembled 16S rRNA segments identified within the assembled contigs (locus tags: NOSPCC9237_00010, 15960 and 20975), each leading to > 99.79 % pairwise sequence similarity. Completeness and contamination indices of the genome assembly are 99.56 % and 0.22 %, respectively. The genome assembly contains 97 tRNA, 9 rRNA and 6489 coding sequences. Among the cluster of orthologous groups (COG) predicted, a high abundance of genes was assigned to signal transduction mechanisms [T], followed by energy production and conversion [C], cell wall/membrane/envelope biogenesis [M] and coenzyme transport and metabolism [H] (Supplementary Fig. S1).

## 3.2. Proposition of a new genus within the Nodulariaceae

The multilocus phylogenetic tree of the Nostocales order reconstructed in this study (Fig. 1, Supplementary Fig. S2) placed PCC 9237T within the family Nodulariaceae as delineated by Strunecký et al. (2023), therefore confirming its separation from *Nostoc sensu stricto* and other *Nostoc*-like genera of the family Nostocaceae as delineated by Strunecký et al. (2023) such as *Mojavia* (Reháková et al., 2007), *Komarekiella* (Hentschke et al., 2017), and *Desmonostoc* (Hrouzek et al., 2013). Further, within the family Nodulariaceae, PCC 9237T was clearly separated at the root of the clade containing *Aliinostoc* (Bagchi et al., 2017), *Desikacharya* (Saraf et al., 2019), *Purpureonostoc* (Cai and Li, 2020), *Pseudoaliinostoc* (Lee et al., 2021) and even from *Trichormus* (Ralf ex Bornet et Flahault) *Komárek and Anagnostidis* (1989) (the latter genus comprising the well-known laboratory strains PCC 7120 and ATCC 29413). Moreover, PCC 9237T was more distantly related to other *Nostoc*-like genera within Nodulariaceae such as *Ahomia* (Pal et al., 2024), *Atlanticothrix*, *Amazonocrinis*, *Dendronialum* (Alvarenga et al., 2021), *Halotia* (Genuario et al., 2015), *Anabaenopsis* (Miller, 1923), *Chrysoporum* (Zapomělová et al., 2012), *Nodularia* (Bornet and Flahault, 1886) and *Umezakia* (Watanabe, 1987).

Since two *Nostoc*-like genera, *Compactonostoc* (Cai et al., 2019) and *Parakomarekiella* (Soares et al., 2020), did not have genome representatives, we reconstructed a 16S rRNA gene phylogenetic tree with the representatives of *Nostoc* and all the other *Nostoc*-like genera
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Multilocus phylogenetic tree showing the position of *Kaarinaea lacus* PCC 9237T within the order Nostocales. This maximum likelihood (ML) phylogenetic tree includes 98 taxa belonging to the order Nostocales; three *Chroococcidiopsis* taxa were used as an outgroup. Branch supports were assessed using ML-based transfer bootstrap expectations/Minimum Evolution (ME) bootstrap proportions/Maximum Parsimony (MP) bootstrap proportions. ‘-’ indicates branch support values lower than 50 %. Bar, 0.1 amino acid substitutions per site.</figcaption></figure>

(Supplementary Fig. S3). In this phylogenetic tree, PCC 9237T and its equivalent *Nostoc* sp. 152 (AJ133161) formed a well-supported and distinct clade with the Norwegian cyanobiont *Nostoc* sp. SKS8 and another Nordic freshwater *Nostoc* sp. ARC 64, sharing >99 % nucleotide similarity with both strains (*Kaarinaea* clade in blue in Supplementary Fig. S3). In coherence with the multilocus phylogenetic tree (Fig. 1), the clade *Kaarinaea* was distantly related to *Nostoc sensu stricto* and other *Nostoc*-like genera clades. Every nucleotide similarity of PCC 9237T 16S rRNA gene against the ones of *Nostoc* and other *Nostoc*-like genera was in the range 93.57–97.42 % (Supplementary Fig. S3). Although this range does not completely satisfy the threshold proposed by Yarza et al. (2014) for delineation of prokaryotic genera, it is now well-established that this threshold does not always hold true in the order Nostocales (Bohunická et al., 2015; Kaštovský et al., 2014; Pal et al., 2024). From the multilocus and 16S rRNA gene phylogenetic analyses carried out in this study, we gather enough evidence indicating that PCC 9237T (along with two other strains) represents a novel evolutionary lineage. Therefore, we propose the new genus *Kaarinaea* with the type species *Kaarinaea lacus*

PCC 9237T.

### 3.3. *The morphology of Kaarinaea lacus PCC 9237T*

We further investigated the morphological attributes of strain PCC 9237T in different media and under several modes of cultivation (on solid or in liquid media, low versus high cellular density). The culture is green and dense in solid Z80, but light green in liquid Z80, as if the liquid version of this medium would not be enough to promote a vigorous growth of this cyanobacterium (Supplementary Fig. S4). On the contrary, the culture looks dark green and dense in both solid and liquid BG110 and liquid Z8. Also, the strain is capable of high cellular density cultivation (up to OD750 6, Supplementary Fig. S4) in 2-fold concentrated Z80 liquid medium supplemented with 3 % CO₂ bubbling. The trichomes of PCC 9237T were either long (~300 cells) or short (~20 cells) under all the conditions examined, except in Z8 (one-month-old culture) in which the length ranged from 20 to 80 cells. However, the overall morphological characteristics of the strain remained unchanged
under all the tested conditions. The trichomes were uniserial, unbranched, untapered and delicate (evident from the bursting of vegetative cells under a long term exposure to light while performing microscopic examination). The trichomes were constricted at the crosswalls and enveloped by a thin mucilaginous layer without prominent sheaths. The vegetative cells were granulated, barrel-shaped to cylindrical, and (3)4.4–5.8(7) µm long and 3.3–4 µm wide (Fig. 2a). The heterocytes were primarily developed at the intercalary position and less frequently at the terminal position, and were either oval, spherical or cylindrical-shaped (Fig. 2a-b,e). The intercalary heterocytes were (4) 5.4–6.8(8) µm long and (4)4.5–5(6) µm wide, whereas the terminal heterocytes were smaller, i.e. (3)4.4–5.5(6) µm long and (3)3.9–4.5(5) µm wide. Reháková et al. (2007) reported terminal heterocyte to be absent in PCC 9237T (Nostoc sp. Fin 152 in their study); however, this assertion remains doubtful as this strain was not investigated by the authors in their study, nor associated with any citation. In the fresh medium (Z8₀ and BG11₀) and in high density cultivation, a large number of dividing vegetative cells were observed in the trichomes, and single heterocytes developed at the intercalary position. In older cultures (Z8₀ and BG11₀) of five weeks or more, the trichomes presented fewer dividing vegetative cells, and at least two contiguous heterocytes developed at the intercalary position (Fig. 2c-d; **Supplementary Fig. S5b-c**). Furthermore, the development of multiple contiguous heterocytes was never observed at the terminal position. PCC 9237T also displayed morphological plasticity when transferred from Z8₀ agar slant to BG11₀ liquid media. Notably, in certain trichomes, vegetative cells dividing perpendicularly to the axis of the trichome were observed

(Supplementary Fig. S5a). However, this character was lost, approximately three weeks after the transfer.

One of the major modes of reproduction in PCC 9237T was by the fragmentation of the trichome at the site of heterocytes in series. In these series, a single or multiple heterocyte(s) start(s) to degenerate, leading to the fragmentation of the trichomes into two parts (Fig. 2e). The daughter trichomes may have one or more heterocyte(s) at the terminal end based on the fragmentation. In such a condition, based on our long-term observation of the strain, it can be concluded that, except for the heterocyte attached to the vegetative cell, the remaining heterocytes degenerate and are eventually shed off from the trichome (Fig. 2 g-h). Later, the daughter trichomes grow into longer trichomes and develop heterocytes solitarily rather than in pairs or series. This mode of reproduction has been reported from *Purpureonostoc tibetica* CHAB 5880T (Cai et al., 2019), *Amazonocerinis malviyae* 19C-PS<sup>T</sup> (Kumar et al., 2022) and *Ahomia kamrupensis* ASM11-PS<sup>T</sup> (Pal et al., 2024); however, the distant phylogenetic clustering in the 16S rRNA gene phylogenetic tree differentiates PCC 9237<sup>T</sup> from these three species (Supplementary Fig. S2, Supplementary Table S1). Among the well-characterized *Nostoc* species included in *Komárek* (2013), the formation of heterocytes in series was reported from *N. kihlmanii*. Interestingly, *N. kihlmanii* has also been reported from Finnish waters (Tikkanen, 1986, as reported in Sivonen et al., 1990); however, the absence of periderm in PCC 9237<sup>T</sup> differentiates it from *N. kihlmanii*. The other mode of reproduction is by the formation of hormogonia, which can be morphologically differentiated from the mature trichome (Fig. 2f-g). The hormogonium cells were smaller than the vegetative cells of the mature trichome. We also

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Morphological attributes of *Kaarinaea lacus* PCC 9237T: (a) trichomes with barrel-shaped and cylindrical granulated vegetative cells, spherical and cylindrical heterocytes; (b) trichome with terminal heterocyte; (c) trichomes with heterocytes in series of three or more and degenerating heterocyte within the series; (d) trichome with three mature heterocytes (H) and one proheterocyte (PH); (e) trichome with oval shaped heterocyte and fragmentation of the trichome at the site of heterocytes in series; (f-g) hormogonia; (h) young trichomes with solitary intercalary heterocytes and terminal heterocytes at one or both ends; (i) degenerated heterocytes attached to the functional heterocytes after fragmentation; (j-k) filament primordium. Scale bar, 10 µm.</figcaption></figure>
observed filament primordium (Fig. 2j-k; as described by Mollenhauer et al., 1994), indicating that the hormogonium is transformed into the filament primordium stage before the formation of the mature filament. In our growing conditions, we never observed the presence of akinetes, despite the report by Reháková et al. (2007). Among the *Kaarinaea* clade (in blue in **Supplementary Fig. S3**), only strain SKS8 was available for comparison and its overall filament morphology was similar to PCC 9237T, except for the presence of pointed terminal heterocytes. Also, the frequency of terminal heterocytes in SKS8 was higher than in PCC 9237T. Furthermore, we occasionally observed the heterocytes in series of two or three in SKS8, but not more in 5-weeks-old culture (data not shown). Based on the available evidence (morphological and genetic), the species level identity of *Kaarinaea* sp. SKS8 remains uncertain and needs to be confirmed either by 16S-23S ITS rRNA region (the folded secondary structures for PCC 9237T are provided in **Supplementary Fig. S6**) and/or whole genome analyses.

### 3.4. Heterocyte patterning in *Kaarinaea lacus* PCC 9237T and genes involved in pattern formation

Heterocyte patterning in cyanobacteria is a well-studied phenomenon, and our current understanding of the pattern formation is largely based on the extensive studies conducted on the model organism *Trichormus* sp. PCC 7120. Under nitrogen depletion condition, PCC 7120 develops heterocytes at an interval of ~10 cells following a semi-regular pattern (see the review of Zeng and Zhang, 2022). However, some individual mutants of PCC 7120 were shown to develop multiple contiguous heterocytes (Callahan and Buikema, 2001; Corrales-Guerrero et al., 2014; Yoon and Golden, 1998, 2001; Zhang et al., 2007). Notably, for PCC 9237T, without any mutational experiments, we observed solitary heterocytes in the young culture and multiple contiguous heterocytes in culture older than five weeks or more. Therefore, we performed a comparative genomic analysis between PCC 7120 and PCC 9237T to identify the genes previously identified as responsible for heterocyte differentiation and pattern formation.

Among the 60 PCC 7120 genes, 56 homolog genes were found in the genome of PCC 9237T (**Supplementary Table S2**), including the twelve genes *devH*, *hetC*, *hetF*, *hetF'*, *hetL*, *hetZ*, *patA*, *patB*, *patD*, *patL*, *patN* and *patU3*, whose product's network has been suggested to play a crucial role in heterocyte development and patterning (Xu et al., 2022). In addition, a small RNA Yfr1, shown to be involved in heterocyte differentiation (Brenes-Alvarez et al., 2016), was also found. In strain PCC 7120, the gene *patS* was shown to participate in establishing the initial pattern by the diffusion of a pentapeptide RGSGR, whereas *hetN* (whose product also contains the RGSGR pentapeptide) was reported to maintain this pattern; both these genes are present in PCC 9237T, together with *patX*, which contains the pentapeptide RG(S/T)GR and was shown to play a role in heterocyte patterning (Elhai and Khudyakov, 2018; Khudyakov et al., 2020). Furthermore, we found the gene *hetP* (regulator of heterocyte commitment) in the genome of PCC 9237T, but none of its three homologs (*asl1930*, *alr2902* and *alr3234*) that play distinct and opposite regulatory roles to *hetP* (Videau et al., 2016). In addition, the gene *patC*, suggested to be involved in selecting the sites of heterocyte differentiation (Corrales-Guerrero et al., 2014), was absent from the genome of PCC 9237T. The amino acid similarity of the heterocyte differentiation and patterning genes against their homologous sequences in *Trichormus* sp. PCC 7120 were >50 %, with the exception of *hetL* (38.64 %) and *hepS* (35.42 %, see **Supplementary Table S2**). The absence of the three *hetP* homologs and of the *patC* gene in PCC 9237T does not completely explain why solitary heterocytes can be observed in the early stages and series of heterocytes in the older cultures of PCC 9237T. Among the 38.5 % of the 6489 CDS of the PCC 9237T genome which are without known function, other gene(s) may be involved in the patterning.

Since the genomes of *Purpureonostoc tibetica* CHAB 5880T, *Amazonocrinis malviyae* 19C-PST and *Ahomia kamrupensis* ASM11-PST were

unavailable (as well as those of the closely related strains of *Kaarinea lacus* PCC 9237T reported to form heterocytes in series), a comparative genomic analysis could not be conducted within the Nostocales clade. A targeted effort in genome sequencing of these species will provide valuable information to design studies on heterocyte differentiation and patterning.

### 3.5. Division of proheterocyte observed in *Kaarinaea lacus* PCC 9237

While growing the strain PCC 9237T, we occasionally observed a single or several cell(s) undergoing division adjoined by degenerated heterocytes on either side (Fig. 3a). These cells were of width similar to the one of heterocyte, but appeared to perform cellular division. In the first step, a two-celled stage is formed, followed by three- and four-celled stages that were always enclosed within an common outer membrane (Fig. 3a-e). However, in some cases, the two-celled stage was released by rupturing of the common outer membrane (Fig. 3c). Also, the three- and four-celled stages were rarely observed compared to the two-celled stage, whereas we never observed higher celled stages within the common outer membrane as previously discussed (Desikachary, 1945). The presence of the outer membrane around the dividing cells revealed that the heterocyte differentiation was initiated before the division occurred. Moreover, the dividing cells were pale yellow or light green, as opposed to the dark green of the vegetative cells. The dividing stages exhibited in PCC 9237T resembled heterocyte germination stages reported previously in *Nostoc linckia* with a germinating heterocyte surrounded by degenerated heterocytes (Geitler, 1921). This study also reported the rare occurrence of heterocyte germination from *Anabaena hallensis*, *Nostoc commune*, *N. ellipsosporum* and *Tolypothrix lanata*, which exhibited the division stages comparable to the ones we observed. However, unlike in case of PCC 9237T where the cells were surrounded by degenerated heterocytes, the dividing cells in the above taxa were adjacent to either vegetative cells or mature heterocytes.

Decades later, Wilcox et al. (1973b), in their study on *Anabaena catenula*, provided evidence indicating that it was proheterocytes (and not heterocytes) that have the ability to regress and undergo division. Based on our current knowledge of heterocyte differentiation, the morphological and physiological changes that are occurring in mature heterocytes (Komárek, 2013; Muro-Pastor and Hess, 2012), and the results obtained in this study, we are more prone to name the cells in PCC 9237T, which undergo division within a common outer membrane as proheterocytes. This phenomenon was observed in both Z80 and BG110 media, and our long-term inspection suggests that in PCC 9237T the proheterocyte possibly went beyond the commitment stage, and the differentiation process reversed during morphogenesis and maturation stages. More recently, heterocyte germination was also reported from *Dendronalium phyllosphericum* CENA369 and *Atlanticothrix silvestris* CENA357 (Alvarenga et al., 2021), but the revaluation of both strains at our request revealed that they were not heterocyte (personal communication with Dr. Danillo Oliveria Alvarenga).

As we observed fragmentation of trichome at the site of heterocytes in chains, several dividing proheterocytes within chains of heterocytes, and the absence of some of the heterocyte patterning genes, the two following hypotheses can be proposed for the sequence of events that took place in PCC 9237T.

(i) The fragmentation of the trichomes.

The heterocytes are developed consecutively in series followed by the degeneration of the oldest heterocyte(s). This results in the fragmentation of the trichome as discussed in Section 3.3 (Fig. 2e, **Supplementary Fig. S7**).

(ii) Proheterocyte division within a chain of heterocytes.

Instead of heterocytes developing consecutively, two heterocytes
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Stages of proheterocyte division in *Kaarinaea lacus* PCC 9237T: (a) three proheterocytes in different stages of division leading to a two-celled stage; (b) two-celled stage with intact common outer membrane; (c) release of two celled stage from the common outer membrane; (d) three-celled stage with degenerated heterocytes attached on either end; (e) four-celled stage within a common outer membrane. Scale bar, 10 µm.</figcaption></figure>

may be developed a few cells apart from each other (Fig. 4, stage 1). Further, it elicits a gradual development of proheterocytes between them, but the central proheterocytes would remain at the early stage of development while the other proheterocytes would progress up to the mature heterocyte stage (Fig. 4, stage 2–3). This would lead to a condition where the central proheterocyte/s is/are in between the heterocytes that continue their maturation until degeneration, resulting in the

loss of direct communication between the central proheterocyte(s) and the rest of the trichome (Fig. 3a,d, Fig. 4, stage 4–6).

The latter hypothesis has a consequence as it was previously shown that the loss of communication between the developing proheterocyte and the trichome results in the regression and the subsequent division of the proheterocyte (Wilcox et al., 1973a). Of the multiple fragments that were generated in this study, the regression frequency was highest in the

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Schematic representation of the hypothetical stages leading to proheterocyte division in *Kaarinaea lacus* PCC 9237T.</figcaption></figure>
‘P fragment’. In this fragment, the proheterocyte was completely isolated and not attached to vegetative cells on either side. Moreover, the isolated proheterocytes were shown to regress, even at the advanced level of development (Wilcox et al., 1973b). In PCC 9237T, dividing proheterocytes were observed when they were attached to degenerated heterocytes in either side, or when they were completely detached from the trichome, mimicking the condition of the ‘P fragment’ of Wilcox et al. (1973b). Despite attempts to obtain more systematic divisions of proheterocytes as previously described through a cold thermic shock (Desikachary, 1945), we were unable to force the development of these structures. The laboratory conditions which can elevate the proheterocyte division, the molecular mechanisms involved, and the stage at which the differentiation process of proheterocytes is reverted, all need to be further investigated.

## 3.6. Biosynthetic gene clusters in *Kaarinaea lacus* PCC 9237T

The biosynthetic gene cluster (BGC) prediction for the synthesis of natural products into the genome of PCC 9237T enabled the identification of 15 regions on the chromosome and one region on one of the other (plasmids) contigs. The BGCs of PCC 9237T are predicted to code for one polyketide synthase (PKS) cluster, six non ribosomal peptide synthetase (NRPS)/PKS hybrid clusters, and nine gene clusters of ribosomally synthesized and post-translationally modified peptides (RiPPs) belonging to lanthipeptides, proteusin, terpenes and lassopeptides. Three BGCs were already associated with known natural products. The first is a 57kb-long NRPS/PKS gene cluster involved in the synthesis of MC with a *mcyL* gene to perform the O-acetylation of ADDA, shown to synthesize the O-acetylated MC variant (Fewer et al., 2013). The second is also a NRPS/PKS cluster involved in the synthesis of nostophycins, and split into four genes spanning on 45 kb, instead of the three adjacent genes of 41.5 kb found previously using PCR and sequencing from a fosmid library (Fewer et al., 2011). The third BGC with known function

was a PKS cluster encoding heterocyte glycolipid with some variation described below (see *subSection 3.6.1*). The remaining predicted clusters had a too low similarity to a known cluster associated with a natural product. However, two NRPS/PKS hybrid clusters were of interest and detailed hereafter (see *subSection 3.6.12* and 3.6.3).

### 3.6.1. Heterocyte glycolipid gene cluster

Only one BGC was dedicated to the heterocyte glycolipids (HG) in PCC 9237T, but it showed a significant difference from the well-studied HG BGC model of the strain PCC 7120 (Fan et al., 2005) as well as from homologous ones (named cluster family CF1) in numerous heterocytous cyanobacteria (Calteau et al., 2014) (Fig. 5). This variant BGC in PCC 9237T was highly similar (89 %) to a BGC in 'Nostoc sp.' NIES-4103, a strain that is phylogenetically unrelated to the *Nostoc sensu-stricto* clade (Hirose et al., 2021), as it belongs to the *Amazonocerinis/Dendronalium/Ahomia* clade in our multilocus phylogenetic tree (**Supplementary Fig. S2**). As this variant HG BGC was also found in two recently described species (*Dendronalium phyllosphericum* CENA369 reported to form heterocytes in pairs and *Atlancothrix silvestris* CENA357) we further investigated their potentials. These variant HG BGCs were highly similar to each other (83 %) and only 66 % homologous to the CF1 HG BGCs from PCC 7120 and PCC 73102 (Calteau et al., 2014), which share 63 % homology among themselves and synteny (Fig. 5). Furthermore, the variant HG BGCs were < 60 % homologous to CF2, a second HG BGC present in PCC 73102 and 10 other heterocytous cyanobacteria (Calteau et al., 2014), for which the production of glycolipid was however never demonstrated.

The *hglB* gene, essential for the glycolipid layer of heterocytes (Garg and Maldener, 2021), is reduced to the acyl carrier protein (ACP) module, whereas the C-terminal thioesterase reductase (TER) module is missing in these CF1 variant HG BGCs. Instead, the HG BGC ended with another gene that codes for a protein with N-terminal domain related to the docking domains of TubC (Richter et al., 2008). In addition, this

<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Heterocyte glycolipid biosynthetic gene cluster in *Kaarinaea lacus* PCC 9237T and relatives. *Nostoc punctiforme* PCC 73102 and *Trichomus* sp. PCC 7120 (up) were used as references, while ‘*Nostoc* sp.’ NIES-4103, *Dendronalium phyllosphericum* CENA369 and *Atlancothrix silvestris* CENA357 (bottom) showed the same variation at the end of the cluster. Every coding sequence was compared against each other using blastp (E-values ≤ 1e-20, identity ≥ 50 %). Amino acid similarity is indicated by light grey (61 - 70 %), grey (71 - 80 %) and black (81–90 %). Coded enzymes: KS, β-ketoacyl synthase; AT, acyltransferase; KR, ketoacyl reductase; ACP, acyl carrier protein; DH, dehydratase; ER, enoyl reductase; TER, thioester reductase.</figcaption></figure>
predicted N-terminal domain was highly similar to the recently available structure of the N-terminal beta-hairpin docking domain of the aeruginosin halogenase of *Microcystis* sp. NIES-98 (PDB: 8AQR, **Supplementary Fig. S8**). The TubC-N terminal docking domain of the last genes of the HG BGC in *Kaarinaea lacus* PCC 9237T, 'Nostoc sp.' NIES-4103, *Dendronalium phyllosphericum* CENA369 and *Atlancothrix silvestris* CENA357 contains residues, likely ready to form an αββαα topology of this family of β-hairpin docking elements (**Supplementary Fig. S8**).

### 3.6.2. *pks2*-like gene cluster

The other most promising PKS/NRPS cluster in PCC 9237T is a large 44kb-long gene cluster (Fig. 6) that encodes 10 PKS modules, with a truncated KS and one NRPS module predicted to incorporate a cysteine, as well as several accessory proteins and transporters. The closest relative cluster (94.9 % homology) is the *pks2* cluster found in *Nostoc punctiforme* PCC 73102 (= ATCC 29133), which possesses nine PKS modules and similar NRPS and accessory or transporter genes (Liaimer et al., 2011). The two clusters are alike and may encode highly similar compounds (Fig. 6). To our knowledge, the family of compounds encoded by *Nostoc punctiforme* PCC 73102 remains to be discovered despite its already known activity to interfere with cellular differentiation of the host. Having another producer of a related compound could ease the discovery of this family of active molecules.

### 3.6.3. Anachelin-like gene cluster

Finally, the genome of PCC 9237T contain also a NRPS/PKS cluster (bordered by CRISPR-associated genes) that is highly similar (95 %) to the anachelins BGC of *Anabaena cylindrica* NIES 19, which produces catecholate siderophores (Itou et al., 2001), as well as the non-functional version of this BGC of the co-identical strain *Anabaena cylindrica* PCC 7122 (Calteau et al., 2014). It remains to investigate which type of siderophores is produced by the enzymes coded by this BGC.

## 4. Taxonomic treatment

### Description of *Kaarinaea* gen. nov.

*Kaarinaea* (Kaa.ri.nae'a. N.L. fem. n. *Kaarinaea*, named in honor of Prof. Kaarina Sivonen for her immense contribution to the field of cyanobacterial research).

Filaments long or short, uniseriate, unbranched and of the same width throughout the entire length. Trichomes constricted at the crosswalls, without prominent sheath, may be enveloped by mucilaginous layer. Vegetative cells barrel-shaped to cylindrical and granulated. Heterocytes intercalary and terminal; spherical, oval or cylindrical-shaped; solitary, geminate or in series. Modes of reproduction include fragmentation of the trichome and formation of hormogonia.

Type species: *Kaarinaea lacus*

Description of *Kaarinaea lacus* sp. nov.

*lacus* (la'cus. L. gen. n. *lacus*, of a lake)

Filaments long or short, uniseriate, unbranched and of the same

width throughout the entire length. Trichomes constricted at crosswalls in all the stages of the life cycle, enveloped by thin mucilaginous layer and without prominent sheath. Vegetative cells granulated, barrel-shaped to cylindrical, (3)4.4-5.8(7) µm long and 3.3-4 µm wide. Heterocytes usually intercalary rarely terminal; solitary, geminate or in series of three or more; oval, spherical or cylindrical-shaped. Terminal heterocytes (3)4.4-5.5(6) µm long and (3)3.9-4.5(5) µm wide. Intercalary heterocytes (4)5.4-6.8(8) µm long and (4)4.5-5(6) µm wide. Occasionally, dividing proheterocytes were observed. Modes of reproduction include fragmentation of the trichome by degeneration of one or more heterocytes at the site of heterocytes developed in series and formation of hormogonia. Akinetes not observed.

The type strain PCC 9237T (= UHCC 0152T) was isolated from the water bloom sample from Lake Sääksjärvi in Finland. The genomic DNA G + C content is 40.67 mol % (based on the genome sequence). The 16S rRNA gene and the whole genome sequences are available under the GenBank/EMBL/DDBJ accession numbers AJ133161 and CAX-OSP010000000, respectively.

## 5. Conclusions

The present work highlights the importance of genomic analysis in the taxonomic studies of cyanobacteria. The multilocus and 16S rRNA gene phylogenetic analyses conducted in this study support the creation of the genus *Kaarinaea* with the type species *Karrinaea lacus* PCC 9237T. Morphologically, the division of proheterocyte (under our laboratory culture conditions) is the most intriguing feature observed in PCC 9237T. Moreover, the occurrence of solitary intercalary heterocytes in the young culture, but the development of chains of intercalary heterocytes in the old culture is the other striking feature of this strain. Furthermore, the genomic investigation in the context of heterocyte patterning indicated the role of currently unknown gene(s). The other noteworthy features of PCC 9237T include the presence of a variant of a functional heterocyte glycolipid biosynthetic gene cluster, as well as gene clusters closely related to *pks2*-like and anachelin ones. These observations indicate *Kaarinaea lacus* PCC 9237T as a potential candidate in elucidating further insights into the heterocyte differentiation and patterning within the Nostocales order.

### CRediT authorship contribution statement

**Aniket Saraf:** Writing - review & editing, Writing - original draft, Visualization, Validation, Software, Methodology, Formal analysis, Data curation, Conceptualization. **Eddy Blondet:** Validation, Methodology, Investigation. **Anne Boullié:** Validation, Investigation. **Alexis Criscuolo:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Muriel Gugger:** Writing - review & editing, Writing - original draft, Visualization, Validation, Supervision, Software, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** *pks2*-like biosynthetic gene cluster (BGC) predicted from *Kaarinaea lacus* PCC 9237T compared to the *pks2* BGC of *Nostoc punctiforme* PCC 73102. The PKS and NRPS modules are in green and blue, respectively. Amino acid similarities > 90 % are indicated in grey. Coded enzymes: KS, β-ketoacyl synthase; AT, acyltransferase; KR, ketoacyl reductase; ACP, acyl carrier protein; DH, dehydratase; ER, enoyl reductase; Cy, cyclisation; A, adenylation; T, thiolation; Cys, cysteine.</figcaption></figure>
Declaration of competing interest

The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper.

Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments

This work was supported by a Pasteur-Roux-Cantarini fellowship of Institut Pasteur. Institut Pasteur is thanked for funding the PCC collection. The Centre de Ressources Biologiques de l'Institut Pasteur (CRBIP), the Biomics Platform and the Mutualized Platform for Microbiology (P2M) of Institut Pasteur are thanked for the sequencing of the genome of PCC 9237T. Thomas Cokelaer, Rania Ouazahrou, Chloé Baum, Elodie Turc from Biomics Platform, C2RT, Institut Pasteur, Paris, France, were supported by France Génomique (ANR-10-INBS-09) and IBISA. This work used the computational and storage services provided by the IT department at Institut Pasteur. The LABGeM (CEA/Genoscope & CNRS UMR8030), the France Génomique and French Bioinformatics Institute national infrastructures (funded as part of Investissement d'Avenir and France 2030 programs managed by Agence Nationale pour la Recherche, contracts ANR-10-INBS-09, ANR-11-INBS-0013 and ANR-21-ESRE-0048) are acknowledged for support within the MicroScope annotation platform. Dr. Anton Liaimer of the UiT-Arctic University of Norway (Tromsø, Norway) is deeply thanked for providing the strain SK8S included in this study. We are grateful to Dr. Ahmed Haouz from Crystallography Platform (Institut Pasteur) for discussion on the TubC structure. We also thank Prof. Aharon Oren for his help with the Latin etymology of the scientific name. We are thankful for the comments of the anonymous reviewers.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.hal.2024.102792.

Data availability

It is described in the methods where the data came from including a link to the data.

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