# O short'branch Microsporidia, where art thou?
## Identifying diversity hotspots for future sampling

Megan Gross, Lubomír Rajter, Frédéric Mahé, David Bass, Cédric Berney,
Nicolas Henry, Colomban de Vargas, Micah Dunthorn

► To cite this version:

Megan Gross, Lubomír Rajter, Frédéric Mahé, David Bass, Cédric Berney, et al.. O short'branch Microsporidia, where art thou? Identifying diversity hotspots for future sampling. European Journal of Protistology, 2024, 96, pp.126119. 10.1016/j.ejop.2024.126119 . hal-04908383

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Research Paper

# O short-branch Microsporidia, where art thou? Identifying diversity hotspots for future sampling

<figure><img src="image_3.png" /></figure>

Megan Gross<sup>a,b,*</sup>, Ľubomír Rajter<sup>c</sup>, Frédéric Mahé<sup>d,e</sup>, David Bass<sup>f,g,h</sup>, Cédric Berney<sup>i,j</sup>, Nicolas Henry<sup>i,k</sup>, Colomban de Vargas<sup>i,j</sup>, Micah Dunthorn<sup>a</sup>

## ARTICLE INFO

Keywords:

Metabarcoding

Parasites

Protists

SSU rRNA

V4 region

## ABSTRACT

Short-branch Microsporidia were previously shown to form a basal grade within the expanded Microsporidia clade and to branch near the classical, long-branch Microsporidia. Although they share simpler versions of some morphological characteristics, they do not show accelerated evolutionary rates, making them ideal candidates to study the evolutionary trajectories that have led to long-branch microsporidian unique characteristics. However, most sequences assigned to the short-branch Microsporidia are undescribed, novel environmental lineages for which the identification requires knowledge of where they can be found. To direct future isolation, we used the EukBank database of the global UniEuk initiative that contains the majority of the publicly available environmental V4 SSU rRNA gene sequences of protists. The curated OTU table and corresponding metadata were used to evaluate the occurrence of short-branch Microsporidia across freshwater, hypersaline, marine benthic, marine pelagic, and terrestrial environments. Presence-absence analyses infer that short-branch Microsporidia are most abundant in freshwater and terrestrial environments, and alpha- and beta-diversity measures indicate that focusing our sampling effort on these two environments would cover a large part of their overall diversity. These results can be used to coordinate future isolation and sampling campaigns to better understand the enigmatic evolution of microsporidians' unique characteristics.

## 1. Introduction

Parasites comprise a large fraction of Earth's biodiversity (Dobson et al., 2008; Loker and Hofkin, 2022; Mahé et al., 2017) and their transition from free-living forms to a parasitic lifestyle occurred in numerous independent events. This convergent evolution among phylogenetically unrelated taxa resulted in similar strategies of host-invasion, transmission between, and survival within their hosts (Poulin, 2011; Poulin and Randhawa, 2015). One clade of obligate intracellular parasites is the Microsporidia (Keeling and Fast, 2002;

Vávra and Lukeš, 2013; Weiss and Becnel, 2014). While the diversity of Microsporidia and their infection mechanisms are somewhat understood, it is still not clear how their unique characteristics, and thus how this clade itself, evolved (Keeling and Fast, 2002).

Microsporidians infect a wide range of animals and some protists (Adl et al., 2019; Becnel and Andreadis, 1999; Foissner and Foissner, 1995; Vávra and Lukeš, 2013; Vossbrinck and Debrunner-Vossbrinck, 2005). Thus far, more than 1300 species are described (Franzen, 2008; Weiss and Becnel, 2014), many of them being harmful and emergent pathogens of socio-economic importance (Fries, 1993; Keeling

<footnote><sup>a</sup> Natural History Museum, University of Oslo, 0562 Oslo, Norway</footnote>

<footnote><sup>b</sup> Department of Ecology, University of Kaiserslautern-Landau RPTU, 67663 Kaiserslautern, Germany</footnote>

<footnote><sup>c</sup> Institute for Zoology, University of Cologne, 50923 Cologne, Germany</footnote>

<footnote><sup>d</sup> CIRAD, UMR PHIM, 34398 Montpellier, France</footnote>

<footnote><sup>e</sup> PHIM, Univ Montpellier, CIRAD, INRAE, Institut Agro, IRD, 34398 Montpellier, France</footnote>

<footnote><sup>f</sup> Cefas, International Centre for Aquatic Animal Health, Weymouth, Dorset DT4 8UB, United Kingdom</footnote>

<footnote><sup>g</sup> Sustainable Aquaculture Futures, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, United Kingdom</footnote>

<footnote><sup>h</sup> Department of Life Sciences, The Natural History Museum, London SW7 5BD, United Kingdom</footnote>

<footnote><sup>i</sup> CNRS, Sorbonne Université, FR2424, ABiMS, Station Biologique de Roscoff, 29680 Roscoff, France</footnote>

<footnote><sup>j</sup> Sorbonne Université, CNRS, Station Biologique de Roscoff, UMR7144, ECOMAP, 29680 Roscoff, France</footnote>

<footnote><sup>k</sup> Research Federation for the Study of Global Ocean Systems Ecology and Evolution, FR2022/Tara GOSEE, 75016 Paris, France</footnote>

<footnote>* Corresponding author at: Department of Ecology, University of Kaiserslautern-Landau RPTU, 67663 Kaiserslautern, Germany. E-mail address: gross.megan@rptu.de (M. Gross).</footnote>
and Fast, 2002; Kent et al., 1989; Stentiford et al., 2016; Weber et al., 1994). While microsporidians display a wide complexity in their infection pathways using a unique polar filament for host invasion, they otherwise have had a rather reductive evolution in their cellular organization (Dean et al., 2018; Katinka et al., 2001; Keeling and Corradi, 2011). The loss of many DNA repair enzymes (Gill and Fast, 2007) and the presence of many fast-evolving genes (Thomarat et al., 2004), may have enhanced the process of genome reduction, thereby playing a major role in the evolution of Microsporidia (Cuomo et al., 2012).

Accelerated evolutionary rates caused long-branch attraction artefacts and initial phylogenetic inferences placed the microsporidians as an early branching eukaryote clade (Keeling and McFadden, 1998; Vossbrinck et al., 1987). Subsequent phylogenetic analyses that took into account variable base substitutions and included numerous loci changed our understanding of their placement within the eukaryotic tree of life (Corradi and Keeling, 2009; Park and Poulin, 2021). It is now widely accepted that microsporidians are closely related to the Fungi (Brown and Doolittle, 1999; Corradi and Keeling, 2009; Gill and Fast, 2006; Hirt et al., 1999; Keeling, 2014, 2003; Strassert and Monaghan, 2022; Voigt et al., 2021). However, although many studies made important contributions to unravel their diversity and phylogeny, as well as shedding light on the unique characteristics that make the microsporidians such pivotal parasites, it still remains unclear how these characteristics evolved.

Bass et al. (2018) expanded our understanding of the relationship between microsporidians and many different microsporidian-like protists that were previously assumed to group together with rozzellids (parasites of Chytridiomycetes, Blastocladiomycetes, and Oomycetes) within the ‘cryptomycotan’ clade. They used small subunit rRNA (SSU rRNA) from sequenced isolates and environmental metabarcoding data to infer that these microsporidian-like lineages form a basal grade of parasites that group with metchnikovellids and classical Microsporidia, thereby demonstrating that the phylogenetic scope of the microsporidians is greater than originally assumed. This expanded microsporidian clade includes all classical Microsporidia, Metchnikovellida, and Chytridiopsida, which they collectively named ‘long-branch Microsporidia’ (referring to the relatively long branches within phylogenetic trees), and the basal grade with less divergent SSU rRNA gene sequences, which they named ‘short-branch Microsporidia’ (Bass et al., 2018; Corsaro et al., 2019).

The short-branch Microsporidia include the partially-characterized lineages *Mitosporidium*, *Morellospora*, *Nucleophaga*, and *Paramicrosporidium*, which are variously known to be parasites of *Daphnia* (Haag et al., 2014) and different amoebae (Corsaro et al., 2014a, 2014b; Michel et al., 2000, 2009a, 2009b). Some of them share morphological traits with the long-branch Microsporidia, such as simpler versions of the polar filament (with a similar function) but differ greatly in other characteristics (Bass et al., 2018; Corsaro et al., 2014a, 2014b; Haag et al., 2014). For example, they do not show rapid rates of evolution and have less reduced genomes that are more similar to those of *Rozella* and canonical Fungi (Haag et al., 2014; Quandt et al., 2017). The short-branch Microsporidia also include many uncharacterized environmental lineages that may be parasitic as well (Doliwa et al., 2021), such as LMK11. By further investigating the short-branch Microsporidia, we can better understand the intriguing evolution that occurred among the classical long-branch Microsporidia, which ultimately resulted in trait reductions and increased complexity of their extrusion apparatus. However, as the short-branch Microsporidia are undersampled and understudied, we do not yet know where to focus attempts in order to isolate more of them, to further investigate the partially characterized lineages, and to newly characterize the environmental lineages.

In this study, we used data from Berney et al. (2023) that collected and analyzed all available environmental metabarcoding data of the protistan hypervariable V4 region of the nuclear SSU rRNA gene. After extracting short-branch Microsporidia operational taxonomic units (OTUs), we asked in which environments short-branch Microsporidia

are present and if there are differences in their abundance and diversity throughout these environments. Our results pave the road for future studies in which we aim to better understand the evolution of the Microsporidia, by directing where we should go to isolate and sequence more short-branch Microsporidia.

## 2. Material and methods

### 2.1. Dataset

Microsporidian sequences and their corresponding metadata used in this study came from a beta version of the EukBank database (Berney et al., 2023) as part of the global UniEuk initiative (Berney et al., 2017). Briefly, to develop the EukBank database, metadata from each bio-project was downloaded from public repositories such as the European Nucleotide Archive (EMBL/EBI-ENA) and the Sequence Read Archive (NCBI). Environmental V4 SSU rRNA raw sequences were downloaded from the EMBL/EBI-ENA EukBank umbrella project, clustered with Swarm ver. 3 using default parameters with the fastidious option on (Mahé et al., 2022), checked for chimeras using VSEARCH ver. 2.21.0 (Rognes et al., 2016), merged with MUMU ver. 1.0.1 (Mahé, 2021), and taxonomically assigned using the stampa pipeline (https://github.com/frederic-mahe/stampa/) and EukRibo ver. 1 reference database (Berney et al., 2023). From this, an OTU (operational taxonomic unit) occurrence table was generated. OTU, taxonomy, and metadata tables can be found in the supplements (Suppl. Files 1–3). All further steps were conducted using R ver. 4.3.1 (R Development Core Team, 2012). The codes are available in HTML format (Suppl. File 4).

### 2.2. Modification of OTU table

Modifications of the dataset were performed using the R package tidyverse ver. 1.3.1 (Wickham et al., 2019). The OTU, taxonomy, and metadata tables were matched against each other to make sure that OTUs and samples were identical. To verify that the dataset only contained OTUs annotated to the short-branch Microsporidia, the taxonomy table was searched for the keywords ‘canonical’ and ‘classical’ Microsporidia. All OTUs to which this applied were removed. To correctly infer the environment in which short-branch Microsporidia can be found, every sample was assigned to either of the five different environments, i.e., ‘freshwater’, ‘hypersaline’, ‘marine benthic’, ‘marine pelagic’ or ‘terrestrial’ using the information provided in the metadata table. However, for some samples, there was not enough information provided in the metadata table itself to correctly assign it to one of the environments. To address this knowledge gap, we searched inside the EMBL-EBI Ontology Code for equivalent information and added it to the metadata table.

### 2.3. Statistical analysis

Analyses and visualizations were performed with the R packages tidyverse ver. 2.0.0 (Wickham et al., 2019), ggpubr ver. 0.6.0 (Kassambara, 2020), rstatix ver. 0.7.2 (Kassambara, 2021), maps ver. 3.4.2 (Brownrigg, 2021), phyloseq ver. 1.46.0 (McMurdie and Holmes, 2013), metagMisc ver. 0.5.0 (Mikryukov, 2021), and venn ver. 1.12 (Dusa, 2021). All analyses were performed on the unrarefied dataset to ensure that samples were not lost due to poor sample coverage. However, as the sequencing depth varies between different studies due to different sampling efforts and procedures, all analyses were tested on the rarefied dataset as well, to make sure that this did not influence the outcome. The quality of the taxonomic annotation was examined based on the number of OTUs per similarity value to the reference database and was visualized as bar charts. Sampling maps, based on latitude/longitude information, were created to compare the total amount of samples against samples for which short-branch microsporidian OTUs were found. Samples were color-coded based on the five different
environments. To compare the number of unique and shared OTUs present in the different environments, Venn diagrams were created. Additionally, OTUs occurring in all investigated environments were aligned with MUSCLE ver. 3.8.425 (Edgar, 2004) to check for sequence similarity. For community analysis, OTU, metadata, and taxonomy tables were transformed and then merged into a phyloseq object. Observed and estimated richness (Chao1) was compared between the

different environments and results were visualized as boxplots. Wilcoxon rank sum tests were used to assess the pairwise differences in richness among the environments (we recognized the dataset was not normally distributed because many samples were low abundant, but we retained all samples to get a complete view of the distributions across the environments). To investigate similarity patterns between the five different environments, the OTU table was transformed into presence/

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Map of all sample locations (upper panel) and map of samples that include short-branch Microsporidia (lower panel). Colors represent the environment in which the samples were collected.</figcaption></figure>
absence data, and a non-metric multidimensional scaling (NMDS), using
the Jaccard metric, was performed.

**3. Results**

**3.1. Sampling and sequencing**

Of the 13,045 samples taken from across the globe, the majority
came from marine pelagic environments (62.89 %), followed by
terrestrial (19.36 %), marine benthos (13.58 %), freshwater (3.92 %),
and hypersaline (0.25 %) environments (Fig. 1). The majority of the
terrestrial samples were taken in areas in the northern hemisphere,
especially within Europe. There were also many countries for which no
samples targeting the V4 region were available including the whole
African continent, Australia, and also parts of South America such as
Brazil. From the initial dataset, 1,403,019,176 environmental V4
sequencing reads clustered into 460,147 OTUs that were taxonomically
assigned to the protists in general. After filtering out all non-short-
branch microsporidian sequences, 6,796,304 (0.48 %) reads remained
that were clustered into 1,741 short-branch microsporidian OTUs. From
these short-branch Microsporidia data, 94.9 % of the reads and 69.8 % of
the OTUs were more than 90 % similar to references in the EukRibo
(Berney et al., 2022) reference database (Fig. 2).

**3.2. Distributions across environments**

Of the 13,045 samples, we found short-branch Microsporidia OTUs within 3,063 (23.27 %) of them (Fig. 1). A majority of these samples (1,794) and OTUs (1,279) were found in terrestrial environments (Fig. 3). Although there were more samples from marine environments, freshwater samples accounted for 879 OTUs compared to 578 for marine pelagic and 563 OTUs for marine benthos. A similar picture was found when searching for unique and shared OTUs across the different environments. Terrestrial environments had the highest number of unique OTUs (604) followed by freshwater (192). There were 155 OTUs shared between freshwater and terrestrial environments, 216 that occurred within every environment with the exception of hypersaline, and two OTUs that occurred in all five environments. These two OTUs found in all five environments had a high similarity to accessions in the taxonomic reference database (>98 %). Both had *Paramicrosporidium* clone LKM-46 (van Hannen et al. 1999) as their closest reference and they were 98.26 % similar to each other.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Similarity of short-branch Microsporidia to the taxonomic reference database for number of reads (A) and number of OTUs (B).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Venn-diagram showing the number of unique and shared OTUs across the five investigated environments. Sections in which no number appears indicate zero OTUs.</figcaption></figure>

**3.3. Community richness and heterogeneity**

The observed richness of short-branch Microsporidia OTUs showed that the average number of OTUs per sample was highest for terrestrial, followed by freshwater environments (Fig. 4). Estimated richness using Chao1 predicted slightly higher diversity values for the different environments. Terrestrial environments had the highest diversity (Chao1: 15 ± 18.82 SE) followed by freshwater (Chao1: 11 ± 44.13 SE). Marine benthos, marine pelagic, and hypersaline environments showed very low average diversity estimates (respectively 3 ± 30.39 SE, 2 ± 15.54 SE, and 1 ± 1.50 SE) and differed significantly from freshwater and terrestrial environments (Suppl. Table 1). Although most samples showed an observed richness of <50 OTUs per sample, 11 freshwater and six marine benthos samples were found with >100 OTUs per sample. Among these, three out of 11 freshwater sample sites were from lake Pollevann in Norway (unpublished study), seven came from lake Sana-bria in Spain (unpublished study), and one sample originated from lake Augstsee in Austria (unpublished study). All of the six marine benthos samples were from Norwegian fjords (unpublished study). In contrast to the differences in observed and estimated OTU richnesses, there were no clear differences in the communities of short-branch Microsporidia across the environments (Fig. 5). Non-metric multidimensional scaling showed that the majority of samples clustered together with the exception of some marine benthic, marine pelagic, and terrestrial samples.

**4. Discussion**

Our data show that short-branch Microsporidia are ubiquitous pro-
tists that occur in all of the investigated environments with many of
them not closely related to already sequenced isolates (Figs. 1 and 2).
Although widespread, we nevertheless found differences in their abun-
dance across environments, with terrestrial accounting for the highest
number of samples with short-branch Microsporidia, followed by
freshwater (Figs. 1 and 4). However, since there were no clear com-
munity differences between environments (Fig. 5), we can infer that
sampling in terrestrial and freshwater environments would cover a large
fraction of the overall diversity of the short-branch Microsporidia. These
<figure><img src="image_7.png" /><figcaption>Fig. 4. Alpha-diversity per environment for the observed number of OTUs and estimated number of OTUs using Chao1.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>Fig. 5. Non-metric multidimensional scaling plot of all samples across the five different environments using the Jaccard index as the dissimilarity measure.</figcaption></figure>

findings can be used to direct future isolation of short-branch
Microsporidia.

Using environmental sequencing data targeting the SSU rRNA gene has revealed an incredible diversity of unicellular eukaryotes, including microsporidians (Logares et al., 2020; Massana et al., 2015; Santoferrara et al., 2020; de Vargas et al., 2015). Many of these studies led to novel findings and highlighted that Microsporidia are widespread parasites with a large part of their diversity that remains undescribed (Ardila-Garcia et al., 2013; Dubuffet et al., 2021; Murareanu et al., 2021; Williams et al., 2018). However, since short-branch Microsporidia are generally referred to as 'microsporidian-like', and because they are frequently considered to group indistinguishably from the rozzellids, published studies often did not include them when investigating the diversity and distribution of long-branch Microsporidia (Bass et al., 2018). Additionally, it was not until Bass et al. (2018) that the

relationship between long-branch and short-branch Microsporidia was solidly known. Here, with our dataset of 3,063 samples and only selecting short-branch Microsporidia sequences, our results show that this high diversity not only applies to the long-branch Microsporidia, but also applies to the short-branch Microsporidia (Fig. 1). We also found that around 30 % of the OTUs analyzed here had no close reference (<90 % similarity) within the taxonomic reference database (Fig. 2), and may therefore represent further undescribed lineages, underpinning the need to find and sequence more short-branch Microsporidia.

Beyond the standard problems of using an environmental meta-
barcoding approach to evaluate protistan environmental diversity
(Santoferrara et al., 2020), the uneven sampling in our study could have
led to an underestimation of short-branch Microsporidia in some parts of
Earth. In particular, large parts of Africa, Australia as well as of North
and South America are underrepresented or not represented at all in this
study, and there was uneven sampling in the different layers of the
oceans. Additionally, this study analyzed just V4 data derived from
general eukaryotic primers. General eukaryotic primers are known to
amplify many of the short-branch Microsporidia (Bass et al., 2018),
while Microsporidia-specific primers tend just to amplify the long-
branch Microsporidia (Doliwa et al., 2023). Even with these limita-
tions in mind, our results still show that terrestrial and freshwater en-
vironments harbor a large diversity of short-branch Microsporidia that
warrant further exploration.

We found that 216 of the short-branch Microsporidia OTUs were
shared between freshwater, marine benthos, marine pelagic, and
terrestrial environments, and 155 OTUs were shared between fresh-
water and terrestrial environments, thus increasing our level of confi-
dence in the reality of this diversity (Fig. 3). Although early
classifications divided Microsporidia into the ‘Aquasporidia’, ‘Mar-
inosporidia’ and ‘Terresporidia’ (Vossbrinck and Debrunner-Vossbrinck,
2005), more recent studies have shown that many species are associated
with more than one environment (Murareanu et al., 2021). This het-
erogeneity may be the result of their hosts being widespread and
occurring in different environments themselves (Park et al., 2020). For
example, Murareanu et al. (2021) found that 27.4 % of microsporidian
hosts were classified to occur in more than one environment.
Furthermore, some microsporidians have been classified as generalists and can infect more than one host species in different environments (Stentiford et al., 2016). Both of these scenarios could apply to many lineages of short-branch Microsporidia, especially for those two OTUs that were associated with all environments. Despite the many shared OTUs, we also found a considerable number of unique OTUs within freshwater and terrestrial environments (Fig. 3; 192 and 604 OTUs, respectively) that may allow to resolve some of the uncharacterized diversity. In addition, although the large diversity of environmental lineages of short-branch Microsporidia was first shown in neotropical rainforest soils by Bass et al. (2018), we found more diversity within 17 samples taken from freshwater and marine benthos environments of the northern hemisphere. These samples came from the freshwater lake Pollevann and fjords close to Oslo, Norway as well as from lake Sana-bria, Spain.

The greater diversity and abundance of short-branch Microsporidia in freshwater lakes is also in agreement with the assumption that evolutionary transitions to long-branch Microsporidia have occurred in freshwater environments due to the accessibility to free-living amoebae as transfer hosts (Corsaro et al., 2019). However, our results also showed that many short-branch Microsporidia OTUs were found within samples from Norwegian fjords. One possible explanation may be that many of the free-living amoebae that have been identified as hosts have also been described from marine environments (Page, 1977), or related amoebae in marine and non-marine environments are similarly infected by short-branch Microsporidia. This could also apply to the two OTUs that were shared across all five investigated environments and which had *Parame microsporidium* as the closest genus. These references were originally found in freshwater environments. The original sequence assigned to *Paramicrosporidium* clone LKM-46 was isolated from cultures containing water from lake Ketelmeer, Netherlands, and various sources of detritus (van Hannen et al., 1999). However, it was not clear in the original study that the sequences were derived from parasitic species and what their potential hosts were.

The findings of this study also further our understanding of what is known about the partially characterized lineages so far. For example, *Mitosporidium daphniae* infects the water flea *Daphnia*, which occurs in freshwater habitats, and *Nucleophaga terricolae* infects free-living amoebae that were isolated from the bark of trees (Corsaro et al., 2016; Michel et al., 2012). The same applies to other partially-characterized lineages that were also isolated from hosts derived from freshwater or terrestrial samples (Corsaro et al., 2016, 2020; Michel et al., 2000, 2009a). A recent study investigating host-parasite interactions in a freshwater lake found that phytoplankton and microzooplankton (e.g., ciliates) are potential hosts of Microsporidia and suggested that they could be of greater importance for the functioning of lake ecosystems than previously known (Chauvet et al., 2022). The same could apply to the short-branch Microsporidia since many of their identified hosts are protists and microscopic animals.

## 5. Conclusion

Using environmental metabarcoding data from samples taken across the world, our study shows that there is a tremendous diversity of short-branch Microsporidia, especially in freshwater and terrestrial environments. In specific, some samples originating from Norway and Spain were particularly rich in short-branch Microsporidia. These data can be used to direct future sampling campaigns, with the goal of isolating and further characterizing partially described as well as novel environmental lineages. Furthering our knowledge about these lineages of short-branch Microsporidia may allow us to better understand the evolution that occurred among the long-branch Microsporidia.

### CRediT authorship contribution statement

Megan Gross: Writing – original draft, Visualization, Investigation,

Funding acquisition, Formal analysis. Ľubomír Rajter: Writing – review & editing, Writing – original draft, Supervision, Formal analysis. Frédéric Mahé: Writing – review & editing, Writing – original draft, Supervision, Formal analysis, Data curation, Conceptualization. David Bass: Writing – review & editing, Writing – original draft, Conceptualization. Cédric Berney: Writing – review & editing, Data curation. Nicolas Henry: Writing – review & editing, Data curation. Colomban de Vargas: Writing – review & editing. Micah Dunthorn: Writing – review & editing, Writing – original draft, Supervision, Investigation, Conceptualization.

### Declaration of competing interest

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

### Data availability

Data will be made available on request.

### Acknowledgements

This work was funded by an Erasmus + grant to M.G. EukBank was made possible by funds from the Gordon and Betty Moore Foundation.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejop.2024.126119.

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