# Harboring Starships: The Accumulation of Large Horizontal Gene Transfers in Domesticated and Pathogenic Fungi

Samuel O'donnell, Gabriela Rezende, Jean-Philippe Vernadet, Alodie Snirc, Jeanne Ropars

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To cite this version:

Samuel O'donnell, Gabriela Rezende, Jean-Philippe Vernadet, Alodie Snirc, Jeanne Ropars. Harboring Starships: The Accumulation of Large Horizontal Gene Transfers in Domesticated and Pathogenic Fungi. Genome Biology and Evolution, 2025, 17 (7), 10.1093/gbe/evaf125. http://dx.doi.org/10.1093/gbe/evaf125

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# Harbouring *Starships*: The accumulation of large Horizontal Gene Transfers in Domesticated and Pathogenic Fungi

Samuel O'Donnell¹*, Gabriela Rezende¹, Jean-Philippe Vernadet¹, Alodie Snirc¹, Jeanne Ropars¹*

¹Universite Paris-Saclay, CNRS, AgroParisTech, Ecologie Société et Evolution, 91190, Gif-sur-Yvette, France

*Corresponding authors: [samuel.a.odonnell@gmail.com](mailto:samuel.a.odonnell@gmail.com), [jeanne ropars@cnrs.fr](mailto:jeanne.ropars@cnrs.fr)

**S. O'Donnell**: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualisation, Writing-original draft, Writing-review and editing. **G. Rezende**: Investigation. **J.-P. Vernadet**: Data Curation, Investigation, Software. **A. Snirc**: Investigation. **J. Ropars**: Conceptualization, Data Curation, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing-original draft, Writing-review and editing.

ORCIDs

Samuel O'Donnell: https://orcid.org/0000-0003-2447-1993

Jeanne Ropars: https://orcid.org/0000-0002-3740-9673
22 **Abstract**

Human-related environments, including food and clinical settings, present microorganisms with atypical and challenging conditions that necessitate adaptation. Several cases of novel horizontally acquired genetic material associated with adaptive traits have been recently described, contained within giant transposons named *Starships*. While a handful of *Starships* have been identified in domesticated species, their abundance has not yet been systematically explored in human-associated fungi. Here, we investigated whether *Starships* have shaped the genomes of two major genera of fungi occurring in food and clinical environments, *Aspergillus* and *Penicillium*, providing a unique opportunity to study several independent events of adaptation to similar niches. We found in all cases that the domesticated strains or species exhibited significantly greater *Starship* content compared with close relatives from non-human-related environments, containing an enrichment in genes involved in adaptation to food. We found a similar pattern in clinical contexts. Our findings have clear implications for agriculture, human health and the food industry as we implicate *Starships* as a widely recurrent mechanism of gene transfer aiding the rapid adaptation of fungi to novel environments.

37 **Key words**

adaptation; transposable elements; moulds; filamentous fungi; fermented food; clinical;
*Penicillium*; *Aspergillus*; human-made environments; accurate phylogeny; HGT

40 **Significance**

Recently, giant transposons carrying genes likely involved in adaptation, named *Starships*, have been identified in some pathogenic and domesticated fungi that move by horizontal transfer
between phylogenetically distant species. However, whether *Starships* are widespread and have
shaped the genomes of human-related species has yet to be explored. We focused our study on
the two fungal genera *Aspergillus* and *Penicillium*, which contain several phylogenetically distant
species adapted to human-related environments, either pathogenic or domesticated for
fermenting food products worldwide. They provide a unique opportunity to study independent
events of adaptation to similar niches. We found in all cases that the domesticated and pathogenic
strains or species exhibited significantly greater *Starship* content compared with close relatives
from non-human-related environments, carrying genes enriched in functions relevant for
adaptation. Our study thus showed that *Starships* are a widely recurrent mechanism aiding the
rapid adaptation of fungi to novel environments through horizontal gene transfer.

53

Introduction

A central question in evolutionary biology is to understand how species adapt to their
environment. Indeed, identifying genomic footprints of adaptation is of interest for predicting how
species can face global changes. Horizontal gene transfer (HGT) and transposable elements
(TEs) are key contributors to adaptive evolution. First recognized as a major mechanism of
adaptation in bacteria, it is now accepted that HGT also occurs in eukaryotes (Keeling 2024),
facilitating, for example, the acquisition of both virulence genes in fungal plant pathogens (Friesen
et al. 2006) and carotenoid production in aphids (Moran and Jarvik 2010). TEs are mobile genetic
elements present in all domains of life, capable of moving within and between genomes through
horizontal transfer events (Zhang et al. 2020). This movement generates genetic variability and
genome plasticity, fostering the emergence of novel adaptive variants, particularly in species
coping with new environments (Schrader and Schmitz 2019).

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66 Fungi are models for studying adaptation in eukaryotes given their relatively small genomes and their experimental assets. Additionally, many have adapted to human-related environments.
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68 Some *Aspergillus* species included problematic human pathogens (e.g. *A. fumigatus*, *A. udagawae* and *A. felis*). Other species have been domesticated to ferment food (e.g. beer, bread, sake, tamari, cheese) or for industrial purposes (enzyme and protein synthesis) and to produce antibiotics (penicillin). In particular, several species from the genera *Aspergillus* and *Penicillium* play roles in the production of cheese (e.g. *Penicillium camemberti* for the production of soft cheeses and *P. roqueforti* for blue cheeses), cured sausages (e.g. *P. nalgiovense* and *P. salamii*) and both soy and rice based products such as soy sauce, miso and alcohols (e.g. *Aspergillus oryzae*, *A. sojae* and *A. luchuensis*). Considering domestication is the result of strong and recent selection by humans on some phenotypic traits, these domesticated fungi are prime models for looking at genomic footprints of adaptation as they should be still visible in the genomes.
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75 Horizontal gene transfers have been identified as a recurrent genomic mechanism involved in adaptation in domesticated fungi, such as the budding yeast *Saccharomyces cerevisiae* (Novo et al. 2009; Legras et al. 2018), the dry-cured meat *Penicillium* species *P. nalgiovense* and *P. salamii* (Lo et al. 2023) as well as in the cheese species *P. camemberti* and *P. roqueforti* (Cheeseman et al. 2014; Ropars et al. 2015). The very same HGTs have been identified in a variety of distantly related *Penicillium* fungi used for both cheese and meat production giving us cases of convergent adaptation (Cheeseman et al. 2014; Ropars et al. 2015; Lo et al. 2023).
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87 Recently, these HGTs found in domesticated *Penicillium* fungi have been formally identified as *Starships*, a superfamily of giant transposons (Gluck-Thaler et al. 2022; Lo et al. 2023). These large TEs, ranging from tens to hundreds of kilobases, contain a structurally conserved tyrosine recombinase with a DUF3435 domain, also called the Captain, and can house an extensive and diverse array of cargo genes not involved in transposition (Gluck-Thaler et al. 2022; Urquhart et al. 2023). Notably, these cargo genes have already been linked to, sometimes retroactively,
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92 resistance to formaldehyde (Urquhart et al. 2024), heavy metals (Urquhart et al. 2022), adaptation
93 to food production (Cheeseman et al. 2014; Ropars et al. 2015; Lo et al. 2023) and plant
94 pathogenicity (Bucknell et al. 2024; Peck et al. 2024), suggesting that *Starships* and their
95 associated cargo genes may facilitate rapid adaptation to challenging environments. This may be
96 particularly relevant to both newly emerging and treatment resistant pathogens within the
97 *Aspergillus* genus.

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99 Due to both their size and the frequent presence of repetitive material, *Starships* are often
100 fragmented in short-read assembled genomes and the analysis of their structure and impact can
101 be hindered (Cheeseman et al. 2014; Gluck-Thaler et al. 2022). Fortunately, long-read assembled
102 genomes have enabled the discovery and description of complete *Starship* elements along with
103 their surrounding genomic context (Vogan et al. 2021; Gluck-Thaler et al. 2022; Bucknell and
104 McDonald 2023). Here, we focused on the fungal genera *Penicillium* and *Aspergillus*, each
105 comprising various species of considerable significance to humans, to test whether human-
106 related strains, compared to environmental, have accumulated a greater amount of accessory
107 material from *Starships* insertions.

108 To do so, we first compiled a genome database containing more than 1,600 public and newly-assembled genomes, manually curated information on the isolation origins of each strain and generated phylogenies using >3000 single copy orthologs to ensure accurate species identification. We then used the tool *starfish* to identify tyrosine recombinase/Captain genes within all these genomes, using their abundance as a proxy for the number of *Starships* present.
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113 All strains or species associated with food production or clinical contexts exhibited significantly greater *Starship* content compared with closely related strains or species from environmental sources. Second, we developed a genome-graph based pipeline to extract both complete *Starships* and large *Starship*-like regions from long-read genomes. We found that *Starships* were mostly unique, suggesting independent acquisition events, but that same cargo genes were found
118 in different *Starships*. Importantly, specific gene functions were enriched in *Starships* of
119 domesticated fungi, relevant with adaptation to food. This includes several functionally relevant
120 cargo clusters, present across multiple *Starships*, thus greatly enhancing the list of adaptive
121 convergence cases between distinct lineages. Additionally, we unveiled a novel element present
122 in a subset of *Starships*, a MYB/SANT gene, often located in *Starships* at the opposite edge to
123 the Captain gene, therefore likely denoting the downstream border to these *Starships*. In
124 summary, our findings corroborate the building evidence that *Starships* play a distinguished role
125 in rapid adaptation of fungi to novel environments, including those with human significance.

126

Results

127

**Accumulation of Starships in human-associated fungal**
**isolates**

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129 Using 1516 (*368 Penicillium* and 1148 *Aspergillus*) publicly assembled genomes and 157 newly
130 assembled *Penicillium* genomes, alongside manually curated isolation origins, we tested whether
131 food and clinical environments have any discernible relationship to *Starship* content (table S1).
132 We grouped strains according to six isolation origins, namely environment-clinical, i.e. strains
133 isolated from clinical environment but not associated with disease; clinical for strains associated
134 with disease; environment-food, i.e. strains isolated from the close environment of food, such as
135 cheese ripening caves; food-production for strains isolated from food; environment-saline for
136 strains isolated from saline environment other than food; and environment-other which
137 corresponds to strains isolated from all other environments different from those cited here. Due
138 to the limitations in using short-read generated assemblies, which make up the vast majority of
139 publicly available genomes, we use solely the number of identified DUF3435-domain containing
140 genes (hereafter named Captains) as a proxy for the number of *Starship* insertions considering
141 their conserved and unique position in *Starships* (Gluck-Thaler et al. 2022; Gluck-Thaler and
142 Vogan 2024).

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144 To ensure the accuracy of the species identification for all genomes used in this study, we also
145 constructed BUSCO-based phylogenetic species trees for each genus (Fig. 1; fig. S3; fig. S4;
146 fig. S5; fig. S6; table S1; File S1; File S2). Using these genus wide datasets, we see greater
147 *Starship* content in strains isolated from food-production in both *Aspergillus* and *Penicillium*
148 compared to environment-other and environment-clinical (p <= 0.0002 Fig. 1B; Fig. 1D). In
149 *Aspergillus*, two additional isolation categories were identified, namely clinical and environment-
150 saline; environment-saline strains had significantly greater *Starship* content than all other isolation
151 categories (p <= 0.0002), whereas clinical contained more Captains than both environment-other
152 and environment-clinical (p <=0.01; **Fig. 1B; Fig. 1D**).

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154 Importantly, in order to confirm that we are indeed detecting, in some cases, horizontally
155 transferred Captains located within a *Starship*, we looked at the discrepancies between the
156 pairwise identity of Captain proteins and the average amino acid identity (AAI) of their respective
157 genomes estimated using 50 BUSCO proteins. We only analysed Captains with a pairwise identity
158 >=95% and generated a non-redundant dataset, accounting for species, identity and Captain
159 length, comprising 9,821 Captain proteins (fig. S7). We then visualised alignments between
160 genomes with large differences between their AAI and Captain identity to show that Captains are
161 associated with regions with similarity unexplained by vertical inheritance (fig. S7).

162 Accumulation of *Starships* in moulds domesticated for food-production

163 We have previously shown that three *Penicillium* species have been domesticated for
164 cheesemaking, namely *P. roqueforti* used for blue cheeses worldwide such as Roquefort or Stilton
(Dumas et al. 2020) (Fig. 1A), *P. camemberti* used for soft cheeses such as Camembert (Ropars et al. 2020) and *P. biforme* present on many natural cheese rinds and also found on some dried sausages (Ropars et al. 2020; Lo et al. 2023), with *P. biforme* and *P. camemberti* being sister clades (Fig. 1A). Our analysis shows that *P. biforme* and the two varieties of *P. camemberti*; i.e. var. *camemberti* and var. *caseifulvum*, contain significantly more *Starships* than their wild sister species *P. fuscoglaucum* and *P. palitans* (p <= 0.026; Fig. 2A; fig. S8). *Penicillium roqueforti* has been domesticated independently at least twice (Dumas et al. 2020), with one population containing strains of Roquefort PDO cheeses (named Roquefort population) and another population containing strains from all blue cheeses produced worldwide (named Non-Roquefort population). A new cheese population has been recently described (Crequer et al. 2023), with strains isolated from an uninoculated blue cheese from the village Termignon in the French Alps (named the Termignon population). The two HGTs identified previously in the non-Roquefort population (*cheesyTer* (Ropars et al. 2015) and *Wallaby* (Cheeseman et al. 2014), retroactively identified as *Starships* (Gluck-Thaler et al. 2022; Urquhart et al. 2022)), have also been identified in the Termignon population. We find that strains from the wood/lumber and silage populations have significantly fewer *Starships* than cheese-related strains from both the Non-Roquefort and Termignon populations (p <= 0.027; Fig. 2B; fig. S9). However, we also see that the Roquefort population has significantly fewer *Starships* than other cheese strains (p <= 0.01). We newly defined a 'Contaminants' population, comprising strains isolated from contaminated food products, with a *Starship* count similar to the Non-Roquefort and Termignon clades. *Penicillium psychrosexualis* is the sister species of *P. roqueforti* and is not associated with the cheese environment, as well as *P. carneum*, the sister species of the *P. roqueforti* - *P. psychrosexualis* clade. Although not significant because of too few strains in *P. psychrosexualis* and *P. carneum*, we found less *Starships* in these two latter species than in *P. roqueforti*. Non-Roquefort and Termignon cheese populations.

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Penicillium

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biforme

camemberti

caseifolvum

roqueforti

nalgiovense

salamii

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Aspergillus

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flavus

sojae

luchuensis

fumigatus

<figure><img src="image_5.png" /></figure>
Figure 1. Greater *Starship* content in clinical, food-related and environment-saline strains with phylogenetic evidence of species identification. Captain count per strain and isolation category along the phylogeny of *Penicillium* and *Aspergillus* using 525 and 1148 genome assemblies respectively. (A) 3154 single copy BUSCO genes were extracted from each assembly and used to generate a Maximum-Likelihood tree with SH-like support. Tree was rooted using *Aspergillus* genomes which have been removed. (B) Boxplots and dotplots of the number of Captains per assembly split by isolation category for *Penicillium* genus. (C) 3258 single copy BUSCO genes were extracted from each assembly and used to generate a Maximum-Likelihood tree with SH-like support. Tree was rooted using *Penicillium* genomes which have been removed. (D) Boxplots and dotplots of the number of Captains per assembly split by isolation category for *Aspergillus* genus. (A and C) Trees are labelled by species specific nodes and scale bars represent 0.01 substitutions per site for branch lengths. Red outlines encompass seven examples of species independently domesticated for food production. Each assembly forms a row that contains a bar on the right with the number of Captain genes detected, coloured by isolation category (clinical = orange; environment-other = green; environment-saline = purple; environment-clinical = yellow; environment-food = light-blue; food-production=dark-blue; unknown=grey; NA=black). (B and D) P-values for the differences between groups were calculated using Wilcoxon rank-sum tests with Bonferroni adjusted p-values for multiple tests (* = p <= 0.05; ** = p <= 0.01; *** = p <= 0.001; **** = p <= 0.0001). Non-significant differences are not labelled.

In dry-cured meats, two *Penicillium* species are isolated in most cases, namely *P. nalgiovense* and *P. salamii* (Iacumin et al. 2009; Perrone et al. 2015; Lo et al. 2023). Other *Penicillium* species have also been isolated from fermented meat products, including *P. biforme* (also found in cheese) and *P. nordicum*. On the contrary to *Penicillium* cheese species, dry-cured meat strains of *P. salamii* and *P. nalgiovense* are not genetically distinct from their wild counterparts when considering SNPs, but very few non-dried sausage strains were analysed (Lo et al. 2023). However, dry-cured meat strains carry HGTs, which are likely *Starships*, that are shared between all three species and absent from non-dry-cured meat strains (Lo et al. 2023). Our analysis found that *P. salamii* contains significantly more *Starships* than its wild sister species *P. olsonii* (p < 0.0001; **Fig. 2C; fig. S10**). Furthermore, within the *P. salamii* species, strains isolated from dry-cured meat exhibit significantly more *Starships* compared to environment-other isolated strains (p = 0.00158). For *P. nalgiovense* we show that it contains significantly more *Starships* than both sister species *P. chrysogenum* and *P. rubens* (p < 0.0001; **Fig. 2D; fig. S11**). As in *P. salamii*, *P*.
nalgiovense strains isolated from dry-cured meats appear to have more *Starships* compared to their environmentally isolated counterparts (environment-other group).

Two distantly related species of *Aspergillus*, namely *A. sojae* and *A. oryzae*, are used for fermenting soybean- and rice-based products such as miso and sake (Gibbons et al. 2012; Acevedo et al. 2025). We show that *A. sojae* contains a significantly larger number of *Starships* compared to highly genetically similar *A. parasiticus* strains isolated from environmental sources (p = 0.00083; **Fig. 3A; fig. S12**). Similarly, *A. oryzae* displays a significant increase in *Starship* count when compared to strains of the closely related species *A. flavus* and *A. minisclerotigenes* (p < 0.0001; **Fig. 3B; fig. S13**). Moreover, within *A. oryzae*, strains with food-production origins contain significantly more *Starships* than environment-other isolated strains (p < 0.0001). Of particular note, the few clinical isolates of *A. oryzae* contain, on average, a similar number of *Starships* as the food-production isolates and were phylogenetically distinct (**fig. S13**). The last food-related *Aspergillus* species, *A. luchuensis*, primarily used in alcohol production, contains significantly more *Starships* than its closely related and primarily environmental sister species *A. tubingensis* (p = 0.00018; **Fig. 3C; fig. S14**).

In *A. niger*, although not significant due to few publicly available genomes, the number of Captains in the food-production, environment-food and clinical isolates is higher than the average number of Captains in the environment-other isolates (**fig. S15; fig. S16**). *Aspergillus niger* is widely used in industry for the production of enzymes, proteins and secondary metabolites, yet no industrial strains are publicly available.
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Domesticated *Penicillium* species compared by Captain count to their close relatives and split by isolation origins. Each row contains a specific subset of species shown in the phylogeny on the far left (maximum likelihood tree). The species specific nodes display a triangle with corners extending to both the maximum and minimum of the branch lengths. Scales of branch lengths are displayed above each phylogenetic tree on the left panel. Boxplots and dotplots show the Captain count</figcaption></figure>
distribution with the dots coloured by isolation origins. Dotted squares on the far right of rows B, C and D, are zooms of dotted squares on the middle panels, and display boxplots and dotplots showing the Captain count distribution according to isolation origin within each domesticated species. For row B, *P. roqueforti* is divided into several genetic clusters, and Captain count distribution is shown for each genetic cluster. P-values for the differences between groups were calculated using Wilcoxon rank-sum tests with Bonferroni adjusted p-values for multiple tests (* = p <= 0.05; ** = p <= 0.01; *** = p <= 0.001; **** = p <= 0.0001). Non-significant differences are not labelled.

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<figure><img src="image_9.png" /></figure>

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**Figure 3.** Domesticated and pathogenic *Aspergillus* species compared by Captain count to their close relatives and split by isolation origins. Each row contains a specific subset of species shown in the maximum likelihood phylogenetic tree on the far left. Scales of branch lengths are displayed above each phylogenetic tree on the left panel. The species-specific nodes display a triangle with corners extending to both the maximum and minimum of the branch lengths. Boxplots and dotplots show the Captain count distribution with the dots coloured by isolation origins. Dotted squares on the far right of rows B and D, display a subset of information similarly highlighted to their left. P-values for the differences between groups were calculated using Wilcoxon rank-sum tests with Bonferroni adjusted p-values for multiple tests (* = p <= 0.05; ** = p <= 0.01; *** = p <= 0.001; **** = p <= 0.0001). Non-significant differences are not labelled.

266 Accumulation of *Starships* in clinical isolates

267 Aspergillosis encompasses a spectrum of diseases caused by species within the genus 268 *Aspergillus* and represents a significant health concern globally. Although almost 20 species have 269 been implicated as causative agents (Pfaller et al. 2006), the majority of infections are caused by 270 four species, namely *A. fumigatus*, *A. flavus*, *A. niger* and *A. terreus*. *Aspergillus fumigatus* is 271 currently the major air-borne fungal pathogen, with a relatively high incidence and mortality, 272 comparable to *Candida* and *Cryptococcus* species (Bongomin et al. 2017; Denning 2024). We 273 found that clinical strains of *A. fumigatus* contain on average more *Starships* than environmental 274 strains (tagged as environment-other; p = 0.02; **Fig. 3D**). Notably, the clinical strains are 275 phylogenetically distinct and widely distributed (**fig. S17**). *Aspergillus udagawae* and *A. felis* are 276 two species considered emerging causes of invasive aspergillosis (Sugui et al. 2010; Barrs et al. 277 278 279 280

281 Looking at *Starship* content across the entire *Aspergillus* genus, the species *A. montevidensis/amstelodami* stands out with an average of 46 Captains (range: 44-48) across nine 282 283 284
of 11 (Fig. 1; fig. S19). This species is both considered pathogenic (Fernandez-Pittol et al. 2022) and commonly found in fermented foods such as teas, meju and cocoa beans. Additionally, A. montevidensis is closely related to A. cristatus, another species domesticated for tea fermentation and with high salt tolerance (Xie et al. 2024), with an average of 30 captains.

## Accumulation of specific genes functionally relevant for food adaptation in *Starships*

Using long-read genomes we aimed to, first, confirm our earlier results and secondly, explore both the cargo content and structure of *Starships*. To do so, we employed a robust genome-graph based pipeline, using pggb and odgi (Guarracino et al. 2022; Garrison et al. 2024), to conduct clade-specific all-vs-all whole genome alignments, exclusively using contiguous long-read assembled genomes. This new approach allowed us to extract large candidate *Starship*-like regions (SLRs) from all genomes simultaneously and without reference bias. Initially, all regions larger than 30kb and showing any presence/absence variation in the graph were extracted. These regions were then classified as a SLR if at least one *Starship*-related gene was present (see Methods section 'Starship-like region detection'). We performed alignments of these SLRs and further confirmed that 1) these SLRs are completely absent in some close relatives and 2) inserted at non-homologous loci in the different genomes, further supporting the horizontal acquisition of these regions (examples provided in fig. S20-28). We did not aim to further characterise these *Starship*-like regions, for example by identifying more precisely the *Starship* borders, nor require the presence of a Captain, considering fragmented assemblies, structural variation and Repeat Induced Point-mutation (RIP) can disrupt the detection of full length elements and/or *Starship*-related genes (Gluck-Thaler et al. 2022).
309 Using 76 long read assemblies from 36 species, we constructed 11 clade-specific genome-graphs
310 and detected a total of 857 *Starship*-like regions with a combined total of 202Mb and 963 Captains
311 (table S2). Of the 857 regions, 79% contained at least one Captain. With this data, we were able
312 to recapitulate what we previously discovered, i.e., that food and clinical strains consistently have
313 a larger number of *Starships* compared to their environmental relatives, and additionally that this
314 equates to a larger sum total of *Starship*-related material (Fig. 4; fig. S29). The importance of the
315 ability to detect all regions associated with *Starship*-like genes, considering the variation in
316 *Starship* size, is highlighted by certain cases where although the number of Captains were similar,
317 the amount of *Starship*-like material could differ largely. For example, the *A. oryzae* strain KBP3,
318 with a similar number of Captains, contains over twice as much *Starship*-like material compared
319 to other strains (Fig. 4A). Additionally, we were able to assess which regions were shared
320 between different genomes within each genome graph constructed. This highlighted that some
321 very closely related strains have a large number of recently acquired, unique *Starships* (Fig. 4A;
322 fig. S29). A clear example is between the two varieties of *P. camemberti*, var. *caseifulvum* and
323 var. *camemberti*, which share 99.9% DNA identity and yet contain 12/26 and 12/21 unique
324 *Starship*-like regions respectively, suggesting *Starships* have accumulated independently after
325 the divergence of the two varieties. This result was verified by manually inspecting alignments of
326 all *Starship*-like regions.

327
328 We then investigated whether *Starship*-cargo was functionally enriched for certain COG
329 categories compared to non-cargo genes. We used our four newly assembled and annotated
330 long-read assemblies from three cheese-related strains (*P. camemberti* var. *caseifulvum*
331 ESE00019, *P. camemberti* var. *camemberti* LCP06093 and *P. biforme* LCP05531) and a closely
332 related environmental strain (*P. fuscoglaucum* ESE00090). Using all COGs, and identifying genes
333 without any COGs, we saw a very strong enrichment in genes without a COG annotation in all
334 three genomes (fig. S30). In our cheese related strains, we also see the depletion of several other
335 COG categories. Only the Z (Cytoskeleton) category was enriched in P. biforme. We then filtered
336 out COGs that were absent, unknown (S), or associated with TEs (L and K) and found, only in
337 cheese strains, several COGs significantly enriched in cargo genes compared to the non-cargo
338 gene dataset (fig. S31). Combining the data from all three genomes showed the same COG
339 differences (Fig. 4B). The enriched COGs are E (Amino acid transport and metabolism), I (Lipid
340 transport and metabolism), M (Cell wall/membrane/envelope biogenesis), P (Inorganic ion
341 transport and metabolism) and Z. On the contrary, there is a depletion of COGs A (RNA
342 processing and modification), C (Energy production and conversion), H (Coenzyme transport and
343 metabolism), J (Translation, ribosomal structure and biogenesis), U (Intracellular trafficking,
344 secretion, and vesicular transport) and Y (Nuclear structure).
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346
<figure><img src="image_12.png" /><figcaption>**Figure 4.** Genome graphs based *Starship* statistics and functional enrichment showed a greater number of *Starship*-like material and gene COG annotations relevant for the food environment. (A) Each set of genomes represents a genome-graph constructed with the assemblies for each strain present. The darker, black outlined column represents unique *Starship*-like material and Captains within them. (B and C) *Starship* cargo genes were analysed for functional enrichment, comparing the total proportion of each COG within the entire dataset. COG categories: A - RNA processing and modification; B - Chromatin structure and dynamics; C - Energy production and conversion; D - Cell cycle control, cell division, chromosome partitioning; E - Amino acid transport and metabolism; F - Nucleotide transport and metabolism; G - Carbohydrate transport and metabolism; H - Coenzyme transport and metabolism; I - Lipid transport and metabolism; J - Translation, ribosomal structure and biogenesis; K - Transcription; L - Replication, recombination and repair; M - Cell wall/membrane/envelope biogenesis; N - Cell motility; O - Posttranslational modification, protein turnover, chaperones; P - Inorganic ion transport and metabolism; Q - Secondary metabolites biosynthesis, transport and catabolism; R - General function prediction only; S - Function unknown; T - Signal transduction mechanisms; U - Intracellular trafficking, secretion, and vesicular transport; V - Defense mechanisms; W - Extracellular structures; Y - Nuclear structure; Z - Cytoskeleton. All p-values</figcaption></figure>
360 were calculated from hypergeometric tests and adjusted for multiple comparisons using Benjamini-Hochberg (· = p <= 0.1; * = p <= 0.05; ** = p <= 0.01; *** = p <= 0.001). (B) Cargo and non-cargo gene COG annotations for three cheese related strains (*P. camemberti var. caseiformum* ESE00019, *P. camemberti var. camemberti* LCP6093 and *P. bifforme* LCP05531). (C) Cargo gene COG annotations were grouped by strain isolation origins and compared for functional enrichment.

364
365 We also tested cargo COGs depending on the isolation origins of the strain by using all annotated
366 long-read assemblies. We compared all cargo from strains classified as 'food-production', 'clinical'
367 and 'environment-other' (Fig 4C). We found no significant differences between clinical and
368 environment-other cargo. Interestingly, several differences were significant when comparing
369 either clinical and environment-other cargo to food-production cargo. Cargo from food-related
370 isolates are enriched for several categories (Fig 4C).

371 *Starships* contain cargo-clusters for rapid adaptation to human-related environments

373
374 Using the *Starship*-like regions, we found not only genes of general interest such as those related
375 to drug resistance (CDR1, metallo-beta-lactamase, FCR1, PDR16, ergosterol biosynthesis
376 genes, YOR1, ...), pathogenicity (RBT1, SEF1, AGS1, CAT1, ...) and metal ion homeostasis
377 (VCX1, ZRT2, SMF3, ...) (table S3.6), but also gene clusters likely involved in lactose metabolism,
378 dityrosine biosynthesis, salt tolerance, arsenic resistance and ethanol utilisation (Fig. 5; fig. S32;
379 fig. S33; fig. S34; table S3). In all cases, the rapid loss of synteny at the edges of each cluster
380 suggests that they have all been acquired as cargo-clusters several times by different *Starships*.

381
382 Lactose metabolism and dityrosine synthesis clusters in cheese and cured-meat Penicillium species acquired independently several times in different Starships
We previously discovered *CheesyTer*, a retroactively identified *Starship* (Ropars et al. 2015; Gluck-Thaler et al. 2022; Urquhart et al. 2023), shared between species primarily used for cheese production, and containing two genes, a beta-galactosidase and a lactose permease, likely important for lactose consumption (Ropars et al. 2015). Here, we were able to identify that the beta-galactosidase (LAC4) and the lactose permease are actually contained within a larger lactose related gene cluster. The other genes within this cluster are two C6 zinc-fingers, a glycoside hydrolase of the family 71 (GH71) and a D-lactate dehydrogenase (DLD1) (Fig. 5B), likely play a direct role in lactose metabolism (table S3.1), and all of which were present in *CheesyTer* (Ropars et al. 2015). Although our current sampling of this cluster suggests all genes are core, there is an apparent lower identity and truncated reading frames in DLD1 and one of the two C6-zinc fingers. In addition to the previously described species *P. roqueforti*, *P. camemberti*, *P. biforme* and *P. nalgiovense*, we also found this gene cluster in genomes from *P. solitum*, *P. salamii*, *P. nordicum* and *P. crustosum*, which are all found in food environments, although *P. crustosum* is often considered purely a food contaminant (Pitt and Hocking 2009). These genomes represent multiple, independent events of adaptation to food, for both cheese and cured-meat. Additionally, *P. biforme* (LCP05531) contained two copies of the cluster in different *Starship*-like regions (Fig. 5B-C).

We also identified another biosynthesis cluster, namely the dityrosine cluster, containing two genes, DIT1 and DIT2 that cluster tail-to-tail as already described across a variety of ascomycetes (Fig. 5A), particularly the Saccharomycetes (Nickles et al. 2023). These genes have been shown in *Saccharomyces cerevisiae* to be involved in the biosynthesis of dityrosine within the spore cell wall and enhance their tolerance to stresses (Briza et al. 1994). In *Candida albicans*, with the same cluster, a dit2 mutant was found to be both more susceptible to the antifungal drug caspofungin and to exhibit hyphal growth in minimal media (Melo et al. 2008).

410
<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 5.** Cargo-clusters in *Starship*-like regions. Genes within a subset of *Starship*-like regions were aligned and centred upon genes within the respective gene cluster, A) a dityrosine synthesis or B) lactose metabolism clusters. Genes conserved within the cluster are highlighted in orange. Captains are coloured in red. Colour of links between genes indicate pairwise identity as indicated in the scale. The presence/absence of each cluster within all genomes in a heatmap aligned against the BUSCO based phylogeny for C) *Penicillium* and D) *Aspergillus*. The colour for each block in the heatmap indicates presence and the isolation origins classification</figcaption></figure>
417 (clinical = orange; environment-other = green; environment-saline = purple; environment-clinical = yellow; environment-food = light-blue; food-production=dark-blue; unknown=grey; NA=black).

419
420 We describe for the first time a DIT1/DIT2 cluster in two distinct *Starship-like* regions (**Fig. 5A**; **table S3.2**). In addition to DIT1 and DIT2, this cluster contains DTR1, a multi-drug resistance
421 protein and putative dityrosine transporter of the major facilitator superfamily (Felder et al. 2002),
422 and ATG22, a vacuolar effluxer for amino acids after autophagy that primarily targets tyrosine
423 (Yang et al. 2006). Notably, this set of four genes has been previously described as a co-regulated
424 dityrosine cluster in *Pichia stipitis* (Jeffries and Van Vleet 2009). This cluster has been
425 independently acquired in different *Starships*, in the cheese species *P. camemberti* var.
426 *camemberti* (LCP06093), *P. camemberti* var. *caseifulvum* (ESE00019) and *P. biforme*
427 (LCP05531). Of note, there are five copies of the cluster within *P. camemberti* var. *caseifulvum*
428 (ESE00019), involving four different *Starships* of which one, ESE00019:HTR34, contains a
429 duplicate copy without DTR1 and an apparent splitting of DIT2 into two ORFs (**Fig. 5A,C**). Similar
430 in respects to the lactose cluster, the dityrosine cluster is found in cheese and cured-meat related
431 strains.
432

433
434 **Expanding upon the Arsenic resistance cluster of Hephaestus**

435 Although arsenic is a naturally occurring toxic compound present ubiquitously in the environment,
436 elevated concentrations are found in certain areas due to human-activities such as mining and
437 agriculture (Patel et al. 2023). Arsenic resistance has evolved many times (William and
438 Magpantay 2024), including the ACR cluster in *S. cerevisiae* (Stefanini et al. 2022) and there is
439 also evidence for the horizontal transmission of arsenic resistance genes by large ICE elements
440 in bacteria (Arai et al. 2019). An arsenic cluster found within the *Starship Hephaestus* contains
441 five genes (arsH, arsC, arsB, Pho80 and arsM) and confers arsenic resistance in the
442 environmental fungus *Paecilomyces variotii* (Urquhart et al. 2022). With a larger sampling of all
arsenic clusters in *Starships*, we describe here the cluster's variable content, made up of only three core-cluster genes, namely, *arsH*, *arsC* and *arsB*, and additional genes that may be frequent but not required (fig. S32; table S3.3). Among these cluster-accessory genes, several were frequently present, specifically two genes with no functional description available and a basic region leucine zipper (bZIP), potentially playing a similar role to the bZIP transcription factor ARR1/YAP8, which is both required for the transcription of ARR2 and ARR3 in *S. cerevisiae* (Wysocki et al. 2004) and activated by arsenite binding (Navarro et al. 2022). Additionally, two other cluster-accessory genes were found close to the cluster in several different *Starship*-like regions; TRX1, a thioredoxin both inhibited by arsenic and shown to confer resistance (Park 2020) and a heavy metal associated p-type ATPase, similar to *pcaA*, shown to confer resistance to cadmium and lead in *Hephaestus*, possibly involved in arsenic/arsenite transport (Antonucci et al. 2017). Several genomes had multiple copies of the cargo-cluster genes, whether from several unique *Starships* and/or duplications within *Starships*. Aside from the previously described species (*P. variotii*, *A. fumigatus*, *A. sydowii*, *A. primulinus* and *A. varians* (Urquhart et al. 2022)), we detected the cluster in six *Penicillium* species (*P. chrysogenum/rubens*, *P. brevicompactum*, *P. freii*, *P. crustosum*, *P. nordicum* and *P. biforme*) and a further one in *A. niger* (Fig. 5C-D). Notably, this list contains more environmental species, deviating from the other clusters that contain primarily food-related isolates, and a combination of species from both genera.

## A highly variable salt tolerance gene cluster in food-related species

We also found more extensive evidence for a cluster briefly described in Urquhart et al. 2022 within *A. aureolatus* and containing genes involved in salt tolerance (NHA1, ENA1/2 and SAT4) (fig. S33; table S3.4). This cluster contains a more varied array of accessory genes and their structure. However, focussing on ENA2 as a central component, involved in Na+ efflux to allow salt tolerance (Ariño et al. 2010), we see other genes commonly cluster within its vicinity. The two
468 other core genes include the previously described SAT4/HAL4, involved in salt tolerance (Mulet
469 et al. 1999; Urquhart et al. 2022), and a TRK2-like gene involved in potassium transport (Mulet et
470 al. 1999). In the majority of cases, both genes are found clustered with ENA2, however, their
471 variable orientation and position relative to ENA2 exemplify this cluster's diverse arrangement
472 (fig. S33). Additionally, this cluster contains multiple instances of accessory genes such as TRK1
473 (Potassium transporter, activated by SAT4 and required for SAT4 salt tolerance (Mulet et al.
474 1999)), pacC/RIM101 (TF involved in pH signal transduction; confers salt tolerance through the
475 regulation of ENA2-like genes (Caracuel et al. 2003)), ERG20 (involved in sterol biosynthesis
476 shown to impact salt-tolerance (Kodedová and Sychrová 2015)), and several others (table S3.4).
477 This gene cluster repertoire clearly highlights its functional role centred around ion homeostasis
478 and salt tolerance.

479
480 Further cementing this clusters' functional relevance is that it was found in multiple configurations
481 and unique *Starships* in several strains all related to food-production. For example, three clusters
482 were found each in *P. salamii* (PN007), *P. solitum* (strain12) and *P. camemberti* var. *caseifulvum*
483 (ESE00019), that is a cured-meat and two cheese-related strains respectively. These three
484 strains, belonging to distant species, all shared the same *Starship* with one of these cluster
485 configurations.

486
487 An ethanol utilisation cluster in food spoilage species

488 The model system of ethanol utilisation in *Aspergillus nidulans* requires both alcA and aldA,
489 encoding for alcohol and aldehyde dehydrogenases that convert alcohol into acetate via
490 acetaldehyde, and the transcription factor alcR, which strongly induces the expression of alcA
491 and aldA (Felenbok et al. 2001). Alongside alcR and alcA, three other genes make up an alc gene
492 cluster and are regulated by alcR. One of these three genes, alcS, is likely an acetate transporter
493 (Robellet et al. 2008). We found a *Starship* gene cluster containing alcR, alcS, alcA (ADH1) and
494 aldA (ALD5) (**fig. S34; table S3.5**). Additionally, ERT1/AcuK was found in several clusters,
495 another ethanol regulated transcription factor (Gasmi et al. 2014). This cluster was found in strains
496 of *P. nordicum*, *P. canescens*, *P. antarcticum*, *P. bialowiezense*, *P. digitatum*, *P. verrucosum* and
497 *P. expansum*, of which the latter four are all related to food spoilage, primarily in fruit.

498

499 **Cargo-clusters within the whole genera**

500 Having leveraged long-read assemblies in order to describe complete cargo-clusters, we then
501 took a broader look at their distribution within all genomes of *Penicillium* and *Aspergillus* used in
502 this study (**Fig. 5C-D; fig. S35; table S3.7**). We found in most cases the presence of each cluster
503 within the same species, however there were three main exceptions; The lactose cluster was
504 found in 10/39 genomes of *Aspergillus sydowii*, the arsenic cluster was found in most strains of
505 *P. roqueforti* (deviating from the other clusters found only in food-related clades) and the salt
506 cluster was found in 2/8 *A. tamarii* strains. Looking at the isolation origins of all strains with each
507 cluster, more environment-other strains are found containing the ethanol and the arsenic cluster,
508 whilst the other three clusters are found primarily in strains from food, most notably the dityrosine
509 cluster (**fig. S35**).

510 **A new gene identified at the opposite border to Starship**
511 **Captains**

512
513 During preliminary pairwise comparisons and manual inspection of *Starship*-like regions, we
514 identified genes with a particular MYB/SANT protein domain that were frequently present and
515 *Starship*-like region specific (**fig. S36; Table 1**). Additionally we noticed that these genes were
not only frequently close to the edge of *Starship*-like regions but both in the opposite sense and at the opposite edge to the Captain, if present. Using our newly generated list of *Starship*-like regions, we generated a non-redundant dataset to look at this association. In total, nearly 1/4 (23%) of the non-redundant elements contained both a MYB/SANT and a Captain gene, whilst an additional 9% contained only the MYB/SANT gene. We also compared the distance of each *Starship*-related gene, including the MYB/SANT, to the closest edge. This showed that DUF3435 and MYB/SANT genes are on average significantly closer to an edge than all other *Starship*-related genes (p <= 0.0001; **Fig. 6A**). We also looked at the relative distance of each *Starship*-related gene from each Captain, within the same *Starship*-like region, and showed that MYB/SANT genes are significantly further away than other *Starship*-related genes (p <= 0.0008; **Fig. 6B**). These results affirm the idea that MYB/SANT genes, like Captains (DUF3435), appear to have a structurally conserved position at the edge of *Starships* opposite to the Captain.

Additionally, we have many examples of *Starships* found within contigs, therefore more likely to represent complete *Starship* elements, and/or have been transferred between distant genomes that are clearly bordered by a Captain at one edge and the MYB/SANT gene at the other (**Fig. 6C; fig. S23; fig. S25; fig. S26; fig. S33; fig. S36; fig. S37**). The six given examples all contained Terminal Inverted Repeats flanking Captain and MYB elements. This configuration was also detected in four of the previously identified complete *Starships* from Gluck-Thaler et al. 2022, in genera as distant as *Fusarium* and *Macrophomina* (**fig. S37**). Notably, we also found genes that contained both the DUF3435 and MYB/SANT protein domains, including in the previously annotated *Starship* (*Starship-Aory1*) (Gluck-Thaler et al. 2022) (**fig. S37**). Using the set of non-redundant *Starship*-like regions, we also compared their size separated by whether they contained both Captain and MYB/SANT genes together, either one of the genes without the other (e.g. contains a Captain but no MYB/SANT gene), or neither. We see that *Starship*-like regions containing both genes were significantly larger than those containing only the Captain or neither
542 (p <= 0.00012; Fig. 6D). No significant difference in size was observed with *Starship*-like regions
543 containing only the MYB/SANT gene although the size is on average smaller. Considering both
544 Captain- and MYB/SANT-only regions would be subject to the same impact of assembly breaks
545 and structural variations, this further suggests that there is an association between larger
546 *Starships* and the presence of MYB/SANT genes.

547
548 Lastly, we constructed a MYB/SANT database using all genes identified as MYB/SANT *Starship-*
549 related genes and used this to replace the Captain database in *starfish*. *Starfish* was then run on
550 the same long and short-read genome datasets for *Penicillium* and *Aspergillus*. The MYB/SANT
551 count recapitulated much of the same differences when comparing clades and we see a strong
552 correlation between MYB/SANT count and Captain count, where the number of Captains is on
553 average slightly more than MYB/SANT genes (Fig. 6E).

<figure><img src="image_15.png" /><figcaption>**Figure 6.** The structural association of MYB/SANT genes with *Starship*-like regions. (A) *Starship*-related genes and their relative distance to the closest edge of their associated Starship-like region. (B) The relative distances of *Starship*-related genes to Captains within the same *Starship*-like region. (C) Six examples of *Starship*-like regions indicated by grey rectangles highlighting the position</figcaption></figure>
of Captains (red rectangles), MYB/SANT genes (blue) and other *Starship-like* regions (grey). (D) The size of *Starship*-like regions that contain both a Captain and MYB/SANT gene, only a Captain, only a MYB/SANT gene or neither. (E) The linear relationship between the number of Captains and MYB/SANT genes per assembly. Pearson coefficients were calculated for *Aspergillus* and *Penicillium* species together and separately. The dotted red line is a straight line indicating a one to one ratio. (A, B, D) P-values for the differences between groups were calculated using Wilcoxon rank-sum tests with Bonferroni adjusted p-values for multiple tests (* = p <= 0.05; ** = p <= 0.01; *** = p <= 0.001; **** = p <= 0.0001). Non-significant differences are not labelled.

567

Discussion

There is now growing evidence that *Starships* are widespread in a subphylum of filamentous fungi, namely the Pezizomycotina (Bucknell and McDonald 2023; Gluck-Thaler and Vogan 2024; Urquhart et al. 2024), horizontally transferring between individuals. The Captain (DUF3435), a gene encoding a tyrosine site-specific recombinase, has been experimentally shown to be the only gene required for the excision and reintegration of the entire *Starships* (Urquhart et al. 2023); it has also been shown that *Starships* are not duplicated in genomes (cut and paste model) and that genes present in *Starships* are very rarely duplicated in genomes (Gluck-Thaler et al. 2022). In our dataset, we also confirmed these previous findings, i.e. no multiple copies of same *Starships* but we found duplicated cargo genes in some cheese strains within different *Starships*. When analysing some specific *Starships* with long-read assemblies, we found that i) the same *Starships* found in different species were located at non-homologous loci (fig. S20; fig. S22; fig. S23; fig. S24), ii) the presence of *Starships* was not following the phylogeny and iii) all strains of a given species did not carry the same *Starships*, confirming *Starships* are horizontally transferred. Much of what we have found and discussed here further elevates the idea of the recently proposed *Starship* compartment (Urquhart et al. 2024) as a pervasive region within genomes with distinct evolutionary trajectories that in turn influence the evolution of their hosts.

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# Identifying the other edge of large *Starships*

585

The only structurally conserved gene within *Starships* described so far is the putative tyrosine recombinase, also referred as Captain/DUF3435, positioned close to one edge of *Starships* (Gluck-Thaler et al. 2022; Urquhart et al. 2023). Here we show that a MYB/SANT transcription factor is present in a subset of *Starships* across the Pezizomycotina and structurally positioned at the opposite edge to the Captain. This combination of genes, a transposase and MYB/SANT gene, can also be found in Harbinger transposons, both of which are required for transposition (Sinzelle et al. 2008); Harbinger DNA transposons have been identified in diverse phyla of life including protists, plants, insects and vertebrates. Considering this similarity, we could hypothesise that the MYB/SANT gene in *Starships* plays a similar role, aiding in both the nuclear import and TIR recruitment of the transposase. However, MYB/SANT do not appear required for *Starships*, as they are often absent from seemingly complete *Starships* and are found at a lower frequency than Captains. Therefore, MYB/SANT presence may be only beneficial for transposition, particularly in larger *Starships*, as our data suggests. Alternatively, the MYB/SANT TF may impact the movement of other *Starships* also present within the same genome. Much like previous HGT regions retroactively described as *Starships*, we found MYB/SANT genes in previously described *Starships* confirming their presence regardless of the pipeline used to discover the region. Furthermore, a recent analysis in the *Paecilomyces* genus using the *Starship* detection tool starfish found two horizontally transferred *Starships*, *Hades* and *Demeter*, flanked both by Captains and MYB/SANT genes (Urquhart et al. 2025).
Starships, a convergent mechanism for food and clinical
filamentous fungi to rapidly adapt to their environments

Using both inter- and intra-species comparisons, of varying phylogenetic distances with seven independent events of domestication for food, our findings reveal that all species/populations linked to food production in both the *Aspergillus* and *Penicillium* genera exhibit a distinct increase in the number of *Starships* compared to their environmentally isolated counterparts (Fig. 1; Fig. 2; Fig. 3; Fig. 4; table S1) within seven independent instances of species domesticated for food production, including cheeses, dry-cured meats, and various soy and rice-based foods. Furthermore, many genes within *Starships* have putative functions relevant for adaptation to food environments, such as genes involved in lactose metabolism and salt tolerance. We found, building upon previous observations (Cheeseman et al. 2014; Ropars et al. 2015; Lo et al. 2023), that *Starships* and their food relevant cargo are commonly shared among food-related species and in turn highlighting *Starships* as a generalisable mechanism for these filamentous fungi to rapidly adapt to these new niches.

Fermented food environments are rich in terms of both nutrients and microbes (number and species diversity), including bacteria, yeasts and filamentous fungi, making them ideal media for genetic exchange (Wolfe and Dutton 2015). For example, several *Penicillium* and *Aspergillus* species have been described to co-occur in cheeses alongside many other fungi (Martin and Cotter 2023). These exchanges between fungi co-occuring in the cheese mimics what has been described in cheese-related bacteria, as HGT events appear to be common, contain genes with functional relevance for cheese adaptation and in some cases facilitated by ICEs (Bonham et al. 2017). Similar to ICEs, due to a *Starship's* relatively large size, these transfer events are certainly
629 costly and should be maintained under strong selection. This is particularly relevant in some cases
630 such as in the cheese-related species *P. camemberti* var. *caseifulvum*, where we identified that
631 greater than 15% of the ESE00019 genome is made up of *Starship*-related material. Furthermore,
632 considering that *Starships* have been transferred between species from the same food-related
633 environments (Cheeseman et al. 2014; Ropars et al. 2015; Lo et al. 2023), that they can be very
634 numerous in a single host genome and that most *Starships* identified are unique, even if at times
635 containing similar cargo, this establishes their crucial role in adaptation and their ease of transfer;
636 extrapolated, additional cargo may yet be acquired in domesticated strains. However, we still
637 don't know how often *Starships* move or whether they can be lost as easily as they are acquired.
638 Importantly, domesticated fungi are typically clonally propagated and the absence of regular
639 sexual reproduction may promote the accumulation of *Starships* in these domesticated lineages.
640
641 Although food environments could encourage the likelihood of cross species *Starship* transfers
642 (assuming they only require physical contact (Urquhart et al. 2025)), this may only be part of the
643 story, particularly due to elevated *Starship* content in clinical strains. A recent and thorough
644 investigation into *Starships* in *A. fumigatus* showed that they not only promote strain
645 heterogeneity, counterbalancing the lack of sexual reproduction, but also that certain *Starships*
646 are enriched in clinical strains (Gluck-Thaler et al. 2024). The current prevailing theory is that
647 clinical strains of *Aspergillus*, with pathogenic adaptations, have acquired these prior to
648 colonisation (Rhodes et al. 2022). The primary example in *A. fumigatus*, azole resistance, is said
649 to develop in agricultural settings due to the use of azole based antifungal treatments. This would
650 therefore likely be the same pressure driving the increase in *Starship* content and, considering
651 that most environmental strains of *A. fumigatus* (classified as environment-other) came from
652 agricultural settings, this may explain why there is a smaller difference between clinical and
653 environmental strains within *A. fumigatus*, compared to differences in other *Aspergillus* species
654 with food-related strains such as *A. oryzae* and *A. sojae*.
655 **Starships**, a more general mechanism to rapidly adapt and
656 **survive to extreme environments?**

657
658 Beyond food and clinical isolates, we were also able to identify a relative increase in *Starships* in
659 several phylogenetically distant fungi occurring in saline environments, unrelated to food.
660 However, food environments linked to *Starships* are almost all salted, i.e. cheese, cured meat,
661 soy sauce, miso, suggesting salt could play a part in *Starship* movement. A variety of stresses,
662 including salt, have already been identified as a means of inducing the transfer of plasmids and
663 TE transposition (Beuls et al. 2012). In this scheme, certain conditions such as high salinity might
664 not only select for *Starships* that confer resistance but may also increase general *Starship* mobility
665 and subsequent accumulation. However, we also identified potentially elevated *Starships* in
666 strains isolated from heavy metal contaminated soil (fig. S38) aligning with the previously
667 described *Starship* acquired heavy metal resistance (Urquhart et al. 2022; Urquhart et al. 2024).
668 Furthermore, the ethanol utilisation cluster was found primarily in species known to cause post-
669 harvest disease in fruits. Ethanol resistance and utilisation may be in response to early colonising
670 yeast that can rapidly produce volatile ethanol in order to deter competition (Freimoser et al.
671 2019).

672 **Cargo-clusters drive the functional convergence of Starships**

673
674 Our data on *Starship*-related gene clusters highlights that ‘cargo-clusters’ frequently move among
675 different *Starships* as single elements. This is supported by the recently published results of
676 Urquhart et al. 2023, where they describe the same cargo-cluster, responsible for resistance to
677 formaldehyde, in several different *Starships*, referred to as cargo-swapping (Urquhart et al. 2024).
678 Within the formaldehyde cluster, it was experimentally shown that ssfB granted much greater 679 formaldehyde resistance compared to the six other genes and that it was the only gene 680 consistently maintained within the cluster, whilst the other genes' presence varied (Urquhart et al. 681 2024). The arsenic cluster in the *Starship Hephaestus* has similarly been shown to have been 682 acquired as a single element including a truncated flanking gene (Urquhart et al. 2022). Here, we 683 showed several cargo-clusters, unlike those containing biosynthetic pathways, that contain 684 variable gene content with similarly functional roles. For example, the arsenic cluster, previously 685 described in the *Starship Hephaestus*, contains a set of three core genes (ArsC, ARR3 and ArsH) 686 alongside seven accessory cargo-cluster genes (fig. S32). This variable arrangement is similar 687 to that of arsenic gene islands in prokaryotes (Ben Fekih et al. 2018). It is therefore possible that 688 cargo-clusters may only orient themselves around certain more functionally important genes, 689 providing a more malleable adaptive structure whilst conserving the clusters' function. This is 690 particularly likely considering that cargo is in itself accessory material. 691

692 In conclusion, *Starships* thus appear to be a way of rapidly adapting and surviving to novel 693 conditions, depending on the cargo genes acquired, having been employed heavily within human- 694 related environments. By focusing on recent evolutionary events, domestication and 695 pathogenesis, we were able to clearly detect more *Starships* in food and clinical isolates 696 compared to their environmental counterparts. Furthermore, we have described numerous cargo 697 genes involved in resistance to salt, sulfites and even drugs. Many of these are antimicrobial 698 compounds employed by humans for food preservation or against pathogens of both humans and 699 plants. This may become important as fungal pathogens are becoming increasingly resistant to 700 antifungal treatments (Yin et al. 2023) and their ability to rapidly acquire and transfer resistance 701 would certainly worsen treatment outcomes. Our findings have thus clear implications for the 702 future of agriculture, human health and the food industry as we provided a comprehensive 703 overview on a widely recurrent mechanism of gene transfer between filamentous fungi by focusing
704 on two genera. This also indicates a need for caution regarding the use of bioengineered fungal
705 strains in our agriculture or food systems, as already proposed in the fermented food species
706 Aspergillus oryzae, which was successfully genetically modified using the CRISPR-Cas9 system
707 (Maini Rekdal et al. 2024). We cannot predict how these genes will be spread and whether they
708 can be future threats.

709 # Methods

710 **Sampling**

711 We isolated 68 new Penicillium strains from cheese rinds. To do so, we collected different kinds
712 of cheese rinds (e.g. crottin de chavignol France, Tommes France, Cuesta blue Portugal,
713 Caerphilly UK), often obtained by donors. After at least 24 hours at -80°C to get rid of acarians,
714 we performed two 1:100 successive dilutions in sterile water of pieces of rinds to obtain isolated
715 spores on solid malt extract agar. We checked the species identity using the beta-tubulin locus
716 using bt2a and bt2a primers (seq primers). **Table S1** recapitulates strain details, including species
717 identification, origin and country of origin.

718

719 **DNA extraction, genome sequencing**

720 We used the Nucleospin Soil Kit (Macherey-Nagel, Düren, Germany) for DNA extraction of the
721 68 newly isolated strains grown for five days on malt extract agar. DNA was sequenced using
722 Illumina technologies (paired-end, 100bp and 150bp) (table S1).

723

724 Illumina genome assembly

725 We assembled the genomes of the 68 newly isolated strains and of the 49 already published
726 strains (Ropars et al 2020; **table S1**). Illumina reads were checked with FastQC (v0.11.9)
727 (Andrews 2020). Leading or trailing low quality or N bases below a quality score of three were
728 removed with Trimmomatic (v0.36) (Bolger et al. 2014). For each read, only parts that had an
729 average quality score higher than 20 on a four base window were kept, and only reads with at
730 least a length of 36 bp were kept. Cleaned reads were assembled with SPAdes (v3.15.3)
731 (Prjibelski et al. 2020) not using unpaired reads using the --careful parameter.
732

733 Long-read genome assembly

734 We newly assembled 3 genomes (ESE00019, ESE00090 and LCP05531) with Oxford Nanopore
735 Technology (ONT), newly assembled 1 genomes (LCP05904) with Pacbio and re-assembled 3
736 genomes (LCP06093, PN007, PN016) with publicly available Pacbio data. We assembled each
737 dataset with a variety of assemblers and with different preprocessed read datasets and settled on
738 two assemblies depending on the technology. Additionally, illumina data was available for all ONT
739 sequenced strains, and one re-assembled strain, PN016. For ONT sequenced strains, data was
740 basecalled using Guppy (v5; model r941_min_fast_g507) and assembled using Canu (v2.2)
741 (Koren et al. 2017) with all raw reads and Flye (v2.9-b1768) (Kolmogorov et al. 2019) after using
742 Filtlong (v0.2.1) (github.com/rrwick/Filtlong) to remove all reads less than 5kb. These assemblies
743 were merged using Ragtag's (v2.1.0) (Alonge et al. 2022) patch function with the Canu assembly
744 as the 'target' and the flye assembly as the 'query'. A second round of merging was done replacing
745 the flye assembly with the merged assembly from the first round. This merged assembly was then
746 polished with Medaka (v1.6.0) (github.com/nanoporetech/medaka) using all the ONT reads. The
747 assembly was then polished using the illumina reads with Hapo-G (v1.3.2) (Aury and Istace 2021).
748 For Pacbio based genomes, both the Canu and Flye assembly was assembled using the Filtlong
749 based reads, removing all reads shorter than 5kb and merging was performed using Quickmerge
750 (v0.3) (Chakraborty et al. 2016) with the flye assembly as the 'reference' and the Canu assembly
751 as the 'query'. One round of Hapo-G polishing was performed for PN017. All assemblies were
752 then treated to remove redundant contigs. Contig alignment was performed by BLAST (Camacho
753 et al. 2009) and contigs were removed if both 99% or more was covered by another larger contig
754 with 99% or more identity. For the contig naming; contigs were organised by largest to smallest
755 then numbered and mitochondrial contigs were identified by BLASTing all contigs against a
756 manually identified complete mitochondrial genome from ESE00019.

757
758 We assessed genome completeness using the BUSCO (v5.3.1) eurotiales_odb10 database
759 (Manni et al. 2021) and Merqury (-k 18) (Rhie et al. 2020) for those with illumina data (table S4).
760 We also compared genomes using assembly statistics (table S4). Additionally, we compared our
761 long read assemblies to short-read assemblies if available. This shows that long-read assembly
762 and reassembling of these genomes resulted in significant improvements in contiguity, BUSCO
763 and Merqury completeness compared to previous assemblies both long and short.

764
765 Long-read genome annotation
766 All genomes excluding PN016 and PN007 were annotated. Genomes were first analysed by
767 RepeatModeler (v2.0.2) (Flynn et al. 2020) using the *BuildDatabase* and *RepeatModeler* functions
768 (-engine ncbi). Repeat families were further clustered using cd-hit-est (v4.8.1) (-d 0 -aS 0.8 -c 0.8
769 -G 0 -g 1 -b 500) (Li and Godzik 2006). This repeat library was then fed to RepeatMasker (v4.1.2-
770 p1) (http://www.repeatmasker.org) to identify and mask all repeats (-xsmall -s). The masked
771 assembly was then annotated by Braker2 (v2.1.6) (--fungus --gff3) (Brůna et al. 2021) using a set
772 of all BUSCOs in fungal taxonomic groups (Manni et al. 2021). Braker was then rerun using the
773 previous run's augustus protein hints for 4 rounds. The resulting protein dataset was then
774 analysed using the initial BUSCO database, showing 99.9% of all proteins were annotated for
775 ONT based assemblies and 94.7-94.6% for Pacbio based assemblies. The resulting proteins
776 were annotated using Funannotate (v1.8.11) (Palmer and Stajich 2020). Firstly a funannotate
777 database was generated using the *setup* command (-b fungi). We then used the funannotate's
778 *iprscan* wrapper (Jones et al. 2014) and emapper (v2.1.8) (Cantalapiedra et al. 2021) to annotate
779 protein domains and Eggnog functions respectively. The masked genome, the braker gff3, the
780 funannotate fungi database, the iprscan output and the eggnog annotations were then combined
781 using funannotate's *annotate* function.

782

783 Phylogenomics

784 We placed all genomes used in our study within a phylogenomic framework, separated into the
785 *Penicillium* and *Aspergillus* genera, using several genomes of the other genus for rooting (**table
786 S1**). This served several purposes, such as verifying species identification for public assemblies
787 (which were frequently incorrect and have been tagged in **table S1**), confirming species
788 relationships including their distances and highlighting complex species naming systems such as
789 in *Aspergillus* species complexes. This genome dataset excluded any NCBI genomes labelled as
790 abnormal or from metagenomic data. All genomes were analysed by BUSCO using the
791 eurotiales_odb10 database (Manni et al. 2021). Any genome with less than 80% complete single
792 copy BUSCOs was removed from further analysis. We then established a protein dataset
793 consisting of only BUSCOs contained within 99% of all genomes (3154/4190 and 3258/4190 for
794 *Penicillium* and *Aspergillus* respectively) which were then used to build each tree. Using BUSCOs
795 allowed us to heavily limit any impact of horizontally transferred genes on the topography. The
796 BUSCO datasets were aligned by mafft (v7.310) (Katoh and Standley 2013) and trimmed by trimal
797 (v1.4.rev15) (Capella-Gutiérrez et al. 2009). All protein alignments were concatenated using
798 *catfasta2phyml/catfasta2phyml.pl* (https://github.com/nylander/catfasta2phyml) (-c -f) to produce
799 a single fasta file. We then used this to generate a species tree with VeryFastTree (-double-
800 precision) (v4.0.3) with the default SH-like node support calculations (**Supp. files 1 and 2**) (Piñeiro
801 et al. 2020). The resulting treefile was analysed in R (R Core Team 2024). Each tree was rooted
using the outgroup genomes with treeio::MRCA (v1.22) (Wang et al. 2020), ape::root.phylo (v5.7.1) (Paradis and Schliep 2019), TreeTools::Preorder (v1.10.0) (Smith 2019) and treeio::droptip and visualised using the ggtree suite (Yu et al. 2017). Renaming of species was performed, based on the phylogeny as noted in **table S1**, with most being supported by other publications, also noted in **table S1**. It should be kept in mind that renaming is specific to the genome, regardless of the strain name assigned in NCBI. Renaming efforts were mainly targeted towards species with numerous genomes and we do not want to enter the debate surrounding the naming of *A. terreus*/*A. pseudoterreus*. For phylogenies with collapsed clades (e.g **Fig. 2** and **Fig. 3**) we used *scaleClade* (scale=0.1) then *collapse* (mode='mixed') from the ggtree suite. Therefore these species or clade specific nodes display a triangle a tenth the size of the actual branches and with corners extending to both the maximum and minimum of these scaled branch lengths. The phylogenetics pipeline can be found at https://github.com/SAMtoBAM/publicgenomes-to-buscophylogeny.

815

## Strain isolation origins and classification

For each genome assembly we attempted to manually gather information on the strains' isolation origins. Classifying these origins into simple categories was then also done manually. For environmental origins we used several environmental categories, i.e. environment-other, environment-clinical, environment-food and environment-saline. Environment-saline was used for *Aspergillus* strains where the environment would be generally considered as extreme in its saline content such as sea-water or saline soils. Environment-food was used primarily for contamination of processed food-products such as unwanted growth on dairy products. Environment-clinical strains mainly come from a single study which isolated fungi from healthy human faecal samples. Other strains in this latter category were isolated from hospital and/or human samples but unassociated with disease. **Table S1** contains the detailed and simplified isolation origins.
827

828 **Starfish identification on all (short- and long-read) genomes**

829 In order to estimate the *Starship* count within and between species, we used *starfish* to identify
830 DUF3435/Captain genes in both long and short-read assemblies. The Captain count was then
831 used as a proxy for the number of *Starships*. We ran *starfish annotate* with default settings and
832 the default Captain database (YRsuperfamRefs.faa). The number of captains and the isolation
833 origins per genome was then plotted against the phylogeny in R. To reorder plots of Captain
834 counts against phylogenetic trees, bar graphs were made by ggplot (Wickham 2016) then
835 combined with ggtree (v3.6.2) (Yu et al. 2017) phylogenies using the R library aplot (Yu 2023).
836 To identify changes in *Starship* content, species known to have been domesticated for food
837 production or cause disease were compared against either wild sister species, or within species
838 split by isolation origins. In all cases, to test for differences in Captain counts between species
839 and/or isolation environments, we performed Wilcoxon rank-sum tests with Bonferroni adjusted
840 p-values for multiple comparisons.
841
842 We tested the accuracy of Captain count as a proxy by looking for positive correlations between
843 Captain count and genome size within species/species-complexes in which a large number of
844 genomes were available. To do so, we used the Pearson correlation coefficient in R. In all cases
845 we detected strong and significant positive relationships (**fig. S1; fig. S2**) establishing that we are
846 likely correct in equating Captain count with *Starship* insertions. Note that in Fig. 1, two strains of
847 *P. janthinellum* were removed due to an extremely high Captain count which was most likely
848 erroneous due to no corresponding increase in genome size.
849
850 In *A. fumigatus*, in order to remove any bias in how genomes were assembled across projects, we
851 restricted the *Starship* isolation comparison to a large subset of 252/336 genomes, containing
852 both environment-other (202/252) and clinical (50/252) isolates, that all originated from two
853 bioprojects/studies (PRJNA697844 and PRJNA595552) from the same group and therefore were
854 all assembled and treated with the same pipeline. In doing so we still see, as shown with all
855 genomes, that clinical strains contain more *Starships* (p=0.034; fig. S39).

856
857 **Pairwise captain identity and Average Amino Acid Identity**
858 To also test the assumption that captain occurrence is associated with HGT we tested the
859 similarity between captains across all species. We concatenated all captain proteins identified by
860 **starfish** and constructed a Diamond database (Buchfink et al. 2021). We then searched this
861 database using the same entire set of captain proteins, all vs all, using **diamond blastp**, keeping
862 only matches where both the reference and query protein were covered >=95% and an amino
863 acid identity >=95% (--very-sensitive --id 95 --query-cover 95 --subject-cover 95 --no-self-hits).
864 This gave us a set of highly similar captain proteins based on length and composition.

865
866 For all pairs of genomes identified by captain similarity we calculated the average amino acid
867 identity (AAI) using 50 random BUSCOs, all of which were used to build both the Penicillium and
868 Aspergillus phylogenies. We analysed the BUSCO dataset in the same fashion as the Captains,
869 using Diamond, but with only a filter for 80% reference and query coverage (--very-sensitive --
870 query-cover 80 --subject-cover 80 --no-self-hits).

871
872 Due to some species having many assemblies, and in order to focus on identifying likely examples
873 of HGT, we reduced the dataset to a non-redundant set. That is, we filtered for unique pairs of
874 captains in terms of both the captain length and identity, the AAI and the two species involved.
875 Following this we examined examples of high discrepancy between pairwise Captain identity and
876 their respective genomes AAI, i.e. where Captains from genomes were much more similar than
877 the BUSCOs. To examine these cases we aligned the entire genomes using nucmer, extracting
878 all regions that aligned >7.5kb, taking 50kb up and downstream of these alignments and
879 visualising the loci alongside the captain position using gggenomes (fig. S20-28).
880

881 **Genome graph**

882 Using NCBI (Sayers et al. 2022) we identified publicly available, contiguous (relatively high contig
883 N50), long-read technology assemblies within species of interest and combined them with our
884 newly assembled genomes (table S2). We separated them into groups based on their relatedness
885 and generated genome graphs for each using pggb (v0.5.0-hdfd78af) (-p 75 -s 30000 -m -S -k
886 19-G 7919,8069 -Y_) (Garrison et al. 2024). We also set the option -n as the number of genomes
887 used per grouping. The maximum distance (-p) was only reduced to 50% (-p 50) for the
888 Brevicompacta graph to help with the larger distances between P. brevicompactum and P.
889 salamii. The script for generating a genome-graph and detecting Starship-like regions can be
890 found here https://github.com/SAMtoBAM/pggb_starship_pipe line.
891

891

892 **<u>Starship-like region detection</u>**

893 To extract presence and absence data we used the optimized dynamic genome/graph
894 implementation (odgi v0.8.1-1-ge91b1cd) (Guarracino et al. 2022) *paths function*, extracting both
895 the paths (contigs) and their associated genomes from the genome-graph. We then fed this into
896 odgi *pav* (*pav* for presence/absence variants) alongside 1kb sliding windows for each genome
897 (generated using bedtools (v2.26.0) (Quinlan and Hall 2010) *makewindows*). This calculated the
898 number of genomes covering each 1kb window for each contig for all genomes. We then extracted
899 all regions absent from at least one genome, merged these windows within 1bp using bedtools
900 *merge*, removed all remaining regions smaller than 10kb then remerged regions with a max gap
901 of 20 kb. The final resulting regions were then filtered for a minimum size of 30kb. This dataset
902 was then annotated with braker, emapper and funannotate, as with the whole genomes, except
903 using only a single round of braker annotation.

904
905 Following this the resulting annotation files were searched for protein domains and Eggnogs
906 indicative of *Starship*-related genes. Table 1 below shows the list of genes and their respective
907 tags used to find them. These *Starship*-related genes are made up of previously identified
908 components of starships from Gluck-Thaler et al. 2022 (DUF3435/Captain, DUF3723, PLPs)
909 alongside newly added conidiophore related genes (CRGs) and the MYB/SANT. These new
910 genes alongside the previously identified genes were shown to be highly starship specific in all
911 genomes analysed.

912
913 Table 1
914 *Starship*-related genes used for identifying *Starship*-like regions and their identifying features. Identifying features come from
915 annotation methods and are a combination of InterPro protein domains, InterPro protein families and EggNog categories.

<table><thead><tr><th>Starship-related gene</th><th>Annotation features</th></tr></thead><tbody><tr><td>DUF3435/Captain</td><td>IPR021842 (with no other IPRs)<br>IPR021842 + IPR013087 + IPRXXXXXX<br>IPR021842 + IPR011010 (with no other IPRs)<br>IPR021842 + IPR011010 + IPR013762 (with no other IPRs)<br>IPR021842 + IPR013087 + IPR011010 + IPR013762 (with no other IPRs)</td></tr><tr><td>MYB/SANT</td><td>IPR001005 (with no other IPRs)<br>IPR001005 + IPR009057 (with no other IPRs)<br>IPR001005 + IPR009057 + IPR013087 (with no other IPRs)</td></tr><tr><td>DUF3723</td><td>IPR022198 (with no other IPRs)</td></tr><tr><td>PLP</td><td>ENOG503NY7U</td></tr><tr><td>CRG</td><td>ENOG503P5Q6</td></tr></tbody></table>
<table><thead><tr><td>Starship-related gene</td><td>Annotation features</td></tr></thead><tbody><tr><td>DUF3435/MYB hybrid</td><td>IPR021842 + IPR001005 (with no other IPRs)</td></tr></tbody></table>

The *Starship*-related gene annotation and identification pipeline was initially tested on the *Starships* released in Gluck-Thaler et al. 2022 showing we were able to effectively identify the *Captains* in 33/39 (85%) of these *Starships* and additionally MYB/SANT genes at the distal end of 4/39 (fig. S27).

Additionally *starfish* (Gluck-Thaler and Vogan 2024) was used on each dataset similarly in order to detect DUF3435/Captain genes using the default database (YRsuperfamRefs.faa) and default settings. Additional Captains, that were not already identified, were added to the set of *Starship*-related genes within the defined candidate regions. Notably this added an additional 338/986 Captains (34%). Any region containing a *Starship*-related gene was then considered a *Starship*-like region (Supp. file 3). By comparing the number of bases associated with all presence-absence variant (PAV) regions and *Starship*-like regions (SLRs), we show there is no relationship between the two, therefore confirming that we are measuring distinct changes in SLR content and not an overall increase in accessory material (fig. S40).

*Starship*-like regions were reduced to a non-redundant set using a typical repeat clustering method of 85% identity and 85% coverage using BLAST, taking the largest SLR in each cluster. The SLRs in each cluster were then aligned against one another and a genome identified as missing the SLR using nucmer from MUMMER4 (Marçais et al. 2018). The aligned regions were then visualised using R and gggenomes.

## Functional enrichment
For functional enrichment we used COG gene annotations all from the same annotation pipeline.

Genes were classified by their COG category or given 'Absent' in the absence of annotation. Our
first dataset contained our four newly assembled and annotated long-read assemblies. This
contains three strains from cheese-related species (ESE00019, LCP06093, LCP05531) and one
strain from an environmental species (ESE00090). For each genome we separated genes into
those present or absent from the *Starship*-like regions detected by the genome-graph. For each
genome, all genes were annotated simultaneously, therefore we limit bias in annotation pipelines
or runs that could explain differences in cargo and non-cargo genes. Hypergeometric tests for
enrichment and depletion were performed for each COG category and the p-value was adjusted
for multiple comparisons (Benjamini-Hochberg) using hyper and pzeqjust respectively (R Core
Team 2024). We also performed an analysis on the combined cargo and non-cargo genes from
all three cheese-related strains, which showed the same differences. Second, we performed the
same tests on the COG categories of cargo in *Starship*-like regions, split into groups based on
the strains isolation category, either food, clinical or environment. The above tests were all
performed with and without the COG categories 'S', 'L', 'K' and 'Absent'. S and Absent are
unknown categories. L and K are commonly associated with Transposable elements and our
Starship-like regions were not masked prior to annotation.

956

957 Clinker

A gff3 file for each *Starship-like* region, containing a gene cluster, was converted to an EMBL file
using EMBLmyGFF3 (v2.3) (Norling et al. 2018). The EMBL file was then converted to a genbank
file using biopython (SeqIO.convert(sys.stdin, 'embl', sys.stdout, 'genbank')) (Cock et al. 2009).
This two step process avoided any formatting issues given by other tools that converted gff3 files
directly to gbk. We then ran Clinker (v0.0.28) (-i 0.5), with a minimum of 50% sequence alignment
identity, on all the genbank files simultaneously for each gene cluster. The html output was
964 manually modified in order to highlight the clusters and shorten the length of regions. The
965 Saccharomyces Genome Database (Engel et al. 2022), Candida genome database (Skrzypek et
966 al. 2017), fungidb (Basenko et al. 2018) and NCBI (Sayers et al. 2022) were indispensable in
967 manually investigating the function and relevance of these genes.

968
969 **Cargo-clusters in all assemblies**
970 All assemblies used in this study (Table S1) were combined within a single BLAST database and
971 queried using the cluster-core genes for each cargo-cluster, with a minimum query coverage and
972 identity of 95%. A cargo-cluster was considered present if all core genes within cargo-clusters
973 contained a match. The presence/absence plot (Fig 6) was generated using ggplot and geom_tile
974 (Wickham 2016) then combined with ggtree (v3.6.2) (Yu et al. 2017) phylogenies using the R
975 library aplot (Yu 2023).
976

976
977 **Acknowledgements**

978 We would like to thank Emile Gluck-Thaler for his insights and suggestions on the manuscript.

979 **Funding**
980 This work was funded by the Artifice ANR-19-CE20-0006-01 ANR grant to J.R. and in the
981 framework of a research program co-funded by the CNIEL led by J.R. (INTERFUNIVERSE).
982 **Data availability**

983 Genomic raw data and assemblies are available at NCBI under the bioproject PRJNA1241262.

984 All scripts are available on github and links provided in their respective methods section.

985 **References**

986 Acevedo KL, Eaton E, Leite J, Zhao S, Chacon-Vargas K, McCarthy CM, Choi D, O'Donnell S, 987 Gluck-Thaler E, Yu J-H, et al. 2025. Population Genomics of *Aspergillus sojae* is Shaped 988 by the Food Environment. Genome Biol. Evol. 17:evaf067.

989 Alonge M, Lebeigle L, Kirsche M, Jenike K, Ou S, Aganezov S, Wang X, Lippman ZB, Schatz 990 MC, Soyk S. 2022. Automated assembly scaffolding using RagTag elevates a new tomato 991 system for high-throughput genome editing. Genome Biol. 23:258.

992 Andrews S. 2020. FastQC: a quality control tool for high throughput sequence data. Available 993 online at: http://www.bioinformatics.babraham.ac.uk/projects/fastqc.

994 Antonucci I, Gallo G, Limauro D, Contursi P, Ribeiro AL, Blesa A, Berenguer J, Bartolucci S, 995 Fiorentino G. 2017. An ArsR/SmtB family member regulates arsenic resistance genes 996 unusually arranged in *Thermus thermophilus* HB27. Microb. Biotechnol. 10:1690-1701.

997 Arai N, Sekizuka T, Tamamura Y, Kusumoto M, Hinenoya A, Yamasaki S, Iwata T, Watanabe-998 Yanai A, Kuroda M, Akiba M. 2019. Salmonella Genomic Island 3 Is an Integrative and 999 Conjugative Element and Contributes to Copper and Arsenic Tolerance of *Salmonella* 1000 *enterica*. Antimicrob. Agents Chemother. 63:10.1128/aac.00429-19.

1001 Ariño J, Ramos J, Sychrová H. 2010. Alkali Metal Cation Transport and Homeostasis in Yeasts. 1002 Microbiol. Mol. Biol. Rev. 74:95-120.

1003 Aury J-M, Istace B. 2021. Hapo-G, haplotype-aware polishing of genome assemblies with 1004 accurate reads. NAR Genomics Bioinforma. 3:1qab034.

1005 Barrs VR, van Doorn TM, Houbraken J, Kidd SE, Martin P, Pinheiro MD, Richardson M, Varga J, 1006 Samson RA. 2013. Aspergillus felis sp. nov., an Emerging Agent of Invasive Aspergillosis 1007 in Humans, Cats, and Dogs. PLoS ONE 8:e64871.

1008 Basenko EY, Pulman JA, Shanmugasundram A, Harb OS, Crouch K, Starns D, Warrenfeltz S, 1009 Aurrecoechea C, Stoeckert CJ, Kissinger JC, et al. 2018. FungiDB: An Integrated 1010 Bioinformatic Resource for Fungi and Oomycetes. J. Fungi 4:39.

1011 Ben Fekih I, Zhang C, Li YP, Zhao Y, Alwathnani HA, Saquib Q, Rensing C, Cervantes C. 2018. 1012 Distribution of Arsenic Resistance Genes in Prokaryotes. Front. Microbiol. 9:2473.

1013 Beuls E, Modrie P, Deserranno C, Mahillon J. 2012. High-Salt Stress Conditions Increase the 1014 pAW63 Transfer Frequency in *Bacillus thuringiensis*. Appl. Environ. Microbiol. 78:7128- 1015 7131.

1016 Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. 1017 Bioinformatics 30:2114-2120.
1018 Bongomin F, Gago S, Oladele RO, Denning DW. 2017. Global and Multi-National Prevalence of
1019 Fungal Diseases—Estimate Precision. J. Fungi 3:57.

1020 Bonham KS, Wolfe BE, Dutton RJ. 2017. Extensive horizontal gene transfer in cheese-associated
1021 bacteria. Handley K, editor. eLife 6:e22144.

1022 Briza P, Eckerstorfer M, Breitenbach M. 1994. The sporulation-specific enzymes encoded by the
1023 DIT1 and DIT2 genes catalyze a two-step reaction leading to a soluble LL-dityrosine-
1024 containing precursor of the yeast spore wall. Proc. Natl. Acad. Sci. U. S. A. 91:4524–4528.

1025 Brůna T, Hoff KJ, Lomsadze A, Stanke M, Borodovsky M. 2021. BRAKER2: automatic eukaryotic
1026 genome annotation with GeneMark-EP+ and AUGUSTUS supported by a protein database.
1027 NAR Genomics Bioinforma. 3:1qaa108.

1028 Buchfink B, Reuter K, Drost H-G. 2021. Sensitive protein alignments at tree-of-life scale using
1029 DIAMOND. Nat. Methods 18:366–368.

1030 Bucknell A, Wilson HM, Santos KCG do, Simpfendorfer S, Milgate A, Germain H, Solomon PS,
1031 Bentham A, McDonald MC. 2024. Sanctuary: A *Starship* transposon facilitating the
1032 movement of the virulence factor ToxA in fungal wheat pathogens.
1033 bioRxiv:2024.03.04.583430.

1034 Bucknell AH, McDonald MC. 2023. That's no moon, it's a Starship: Giant transposons driving
1035 fungal horizontal gene transfer. Mol. Microbiol.

1036 Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. 2009.
1037 BLAST+: architecture and applications. BMC Bioinformatics 10:421.

1038 Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J. 2021. eggNOG-
1039 mapper v2: Functional Annotation, Orthology Assignments, and Domain Prediction at the
1040 Metagenomic Scale. Mol. Biol. Evol. 38:5825–5829.

1041 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. 2009. trimAl: a tool for automated alignment
1042 trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972–1973.

1043 Caracuel Z, Casanova C, Roncero MIG, Di Pietro A, Ramos J. 2003. pH Response Transcription
1044 Factor PacC Controls Salt Stress Tolerance and Expression of the P-Type Na+-ATPase
1045 Ena1 in Fusarium oxysporum. Eukaryot. Cell 2:1246–1252.

1046 Chakraborty M, Baldwin-Brown JG, Long AD, Emerson JJ. 2016. Contiguous and accurate de
1047 novo assembly of metazoan genomes with modest long read coverage. Nucleic Acids Res.
1048 44:e147.

1049 Cheeseman K, Ropars J, Renault P, Dupont J, Gouzy J, Branca A, Abraham A-L, Ceppi M,
1050 Conseiller E, Debuchy R, et al. 2014. Multiple recent horizontal transfers of a large genomic
1051 region in cheese making fungi. Nat. Commun. 5:2876.

1052 Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, Friedberg I, Hamelryck T, Kauff
1053 F, Wilczynski B, et al. 2009. Biopython: freely available Python tools for computational
1054 molecular biology and bioinformatics. Bioinformatics 25:1422–1423.

1055 Crequer E, Ropars J, Jany J-L, Caron T, Coton M, Snirc A, Vernadet J-P, Branca A, Giraud T,
1056 Coton E. 2023. A new cheese population in *Penicillium roqueforti* and adaptation of the five
1057 populations to their ecological niche. Evol. Appl. 16:1438–1457.

1058 Denning DW. 2024. Global incidence and mortality of severe fungal disease. Lancet Infect. Dis.
1059 7:e428-e438

1060 Dumas E, Feurtey A, Rodríguez de la Vega RC, Le Prieur S, Snirc A, Coton M, Thierry A, Coton
1061 E, Le Piver M, Roueyre D, et al. 2020. Independent domestication events in the blue-cheese fungus Penicillium roqueforti. Mol. Ecol. 29:2639-2660.

1063 Engel SR, Wong ED, Nash RS, Aleksander S, Alexander M, Douglass E, Karra K, Miyasato SR,
1064 Simison M, Skrzypek MS, et al. 2022. New data and collaborations at the Saccharomyces
1065 Genome Database: updated reference genome, alleles, and the Alliance of Genome
1066 Resources. Genetics 220:iyab224.

1067 Felder T, Bogengruber E, Tenreiro S, Ellinger A, Sá-Correia I, Briza P. 2002. Dtr1p, a Multidrug
1068 Resistance Transporter of the Major Facilitator Superfamily, Plays an Essential Role in
1069 Spore Wall Maturation in Saccharomyces cerevisiae. Eukaryot. Cell 1:799-810.

1070 Felenbok B, Flipphi M, Nikolaev I. 2001. Ethanol catabolism in *Aspergillus nidulans*: A model
1071 system for studying gene regulation. In: Progress in Nucleic Acid Research and Molecular
1072 Biology. Vol. 69. Academic Press. p. 149-204.

1073 Fernandez-Pittol M, Alejo-Cancho I, Rubio-García E, Cardozo C, Puerta-Alcalde P, Moreno-García E, Garcia-Pouton N, Garrido M, Villanueva M, Alastruey-Izquierdo A, et al. 2022. Aspergillosis by cryptic *Aspergillus* species: A case series and review of the literature. Rev. Iberoam. Micol. 39:44-49.

1077 Flynn JM, Hubley R, Goubert C, Rosen J, Clark AG, Feschotte C, Smit AF. 2020. RepeatModeler2
1078 for automated genomic discovery of transposable element families. Proc. Natl. Acad. Sci.
1079 117:9451-9457.

1080 Freimoser FM, Rueda-Mejia MP, Tilocca B, Migheli Q. 2019. Biocontrol yeasts: mechanisms and applications. World J. Microbiol. Biotechnol. 35:154.

1082 Friesen TL, Stukenbrock EH, Liu Z, Meinhardt S, Ling H, Faris JD, Rasmussen JB, Solomon PS,
1083 McDonald BA, Oliver RP. 2006. Emergence of a new disease as a result of interspecific
1084 virulence gene transfer. Nat. Genet. 38:953-956.

1085 Garrison E, Guarracino A, Heumos S, Villani F, Bao Z, Tattini L, Hagmann J, Vorbrugg S, Marco-Sola S, Kubica C, et al. 2024. Building pangenome graphs. Nat. Methods 21:2008-2012.

1087 Gasmi N, Jacques P-E, Klimova N, Guo X, Ricciardi A, Robert F, Turcotte B. 2014. The Switch
1088 from Fermentation to Respiration in *Saccharomyces cerevisiae* Is Regulated by the Ert1
1089 Transcriptional Activator/Repressor. Genetics 198:547-560.

1090 Gibbons JG, Salichos L, Slot JC, Rinker DC, McGary KL, King JG, Klich MA, Tabb DL, McDonald
1091 WH, Rokas A. 2012. The Evolutionary Imprint of Domestication on Genome Variation and
1092 Function of the Filamentous Fungus *Aspergillus oryzae*. Curr. Biol. 22:1403-1409.

1093 Gluck-Thaler E, Forsythe A, Puerner C, Stajich JE, Croll D, Cramer RA, Vogan AA. 2024. Giant
1094 transposons promote strain heterogeneity in a major fungal pathogen.
1095 bioRxiv:2024.06.28.601215.

1096 Gluck-Thaler E, Ralston T, Konkel Z, Ocampos CG, Ganeshan VD, Dorrance AE, Niblack TL,
1097 Wood CW, Slot JC, Lopez-Nicora HD, et al. 2022. Giant Starship Elements Mobilize
1098 Accessory Genes in Fungal Genomes. Mol. Biol. Evol. 39:msac109.

1099 Gluck-Thaler E, Vogan AA. 2024. Systematic identification of cargo-mobilizing genetic elements
1100 reveals new dimensions of eukaryotic diversity. Nucleic Acids Res.:gkae327.

1101 Guarracino A, Heumos S, Nahnsen S, Prins P, Garrison E. 2022. ODGI: understanding
1102 pangenome graphs. Bioinformatics 38:3319-3326.

1103 Iacumin L, Chiesa L, Boscolo D, Manzano M, Cantoni C, Orlic S, Comi G. 2009. Moulds and ochratoxin A on surfaces of artisanal and industrial dry sausages. Food Microbiol. 26:65-
1105 70.

1106 Jeffries TW, Van Vleet JRH. 2009. Pichia stipitis genomics, transcriptomics, and gene clusters.

1107 FEMS Yeast Res. 9:793–807.

1108 Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, McWilliam H, Maslen J, Mitchell A,

1109 Nuka G, et al. 2014. InterProScan 5: genome-scale protein function classification.

1110 Bioinformatics 30:1236–1240.

1111 Katoh K, Standley DM. 2013. MAFFT Multiple Sequence Alignment Software Version 7:

1112 Improvements in Performance and Usability. Mol. Biol. Evol. 30:772–780.

1113 Keeling PJ. 2024. Horizontal gene transfer in eukaryotes: aligning theory with data. Nat. Rev.

1114 Genet.:1–15.

1115 Kodedová M, Sychrová H. 2015. Changes in the Sterol Composition of the Plasma Membrane

1116 Affect Membrane Potential, Salt Tolerance and the Activity of Multidrug Resistance Pumps

1117 in *Saccharomyces cerevisiae*. PLoS ONE 10:e0139306.

1118 Kolmogorov M, Yuan J, Lin Y, Pevzner PA. 2019. Assembly of long, error-prone reads using

1119 repeat graphs. Nat. Biotechnol. 37:540–546.

1120 Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. 2017. Canu: scalable and

1121 accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome

1122 Res. 27:722–736.

1123 Legras J-L, Galeote V, Bigey F, Camarasa C, Marsit S, Nidelet T, Sanchez I, Couloux A, Guy J,

1124 Franco-Duarte R, et al. 2018. Adaptation of S. cerevisiae to Fermented Food Environments

1125 Reveals Remarkable Genome Plasticity and the Footprints of Domestication. Mol. Biol.

1126 Evol. 35:1712–1727.

1127 Li W, Godzik A. 2006. Cd-hit: a fast program for clustering and comparing large sets of protein or

1128 nucleotide sequences. Bioinformatics 22:1658–1659.

1129 Lo Y-C, Bruxaux J, Rodríguez de la Vega RC, O'Donnell S, Snirc A, Coton M, Le Piver M, Le

1130 Prieur S, Roueyre D, Dupont J, et al. 2023. Domestication in dry-cured meat *Penicillium*

1131 fungi: Convergent specific phenotypes and horizontal gene transfers without strong genetic

1132 subdivision. Evol. Appl. 16:1637–1660.

1133 Maini Rekdal V, van der Luijt CRB, Chen Y, Kakumanu R, Baidoo EEK, Petzold CJ, Cruz-Morales

1134 P, Keasling JD. 2024. Edible mycelium bioengineered for enhanced nutritional value and

1135 sensory appeal using a modular synthetic biology toolkit. Nat. Commun. 15:2099.

1136 Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. 2021. BUSCO Update: Novel and

1137 Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring

1138 of Eukaryotic, Prokaryotic, and Viral Genomes. Mol. Biol. Evol. 38:4647–4654.

1139 Marçais G, Delcher AL, Phillippy AM, Coston R, Salzberg SL, Zimin A. 2018. MUMmer4: A fast

1140 and versatile genome alignment system. PLoS Comput. Biol. 14:e1005944.

1141 Martin JGP, Cotter PD. 2023. Filamentous fungi in artisanal cheeses: A problem to be avoided or

1142 a market opportunity? Heliyon 9:e15110.

1143 Melo NR, Moran GP, Warrilow AGS, Dudley E, Smith SN, Sullivan DJ, Lamb DC, Kelly DE,

1144 Coleman DC, Kelly SL. 2008. CYP56 (Dit2p) in Candida albicans: Characterization and

1145 Investigation of Its Role in Growth and Antifungal Drug Susceptibility. Antimicrob. Agents

1146 Chemother. 52:3718–3724.

1147 Moran NA, Jarvik T. 2010. Lateral transfer of genes from fungi underlies carotenoid production in
1148 aphids. Science 328:624-627.

1149 Mulet JM, Leube MP, Kron SJ, Rios G, Fink GR, Serrano R. 1999. A Novel Mechanism of Ion
1150 Homeostasis and Salt Tolerance in Yeast: the Hal4 and Hal5 Protein Kinases Modulate the
1151 Trk1-Trk2 Potassium Transporter. Mol. Cell. Biol. 19:3328-3337.

1152 Navarro C, Navarro MA, Leyva A. 2022. Arsenic perception and signaling: The yet unexplored
1153 world. Front. Plant Sci. 13:993484.

1154 Nickles GR, Oestereicher B, Keller NP, Drott MT. 2023. Mining for a new class of fungal natural
1155 products: the evolution, diversity, and distribution of isocyanide synthase biosynthetic gene
1156 clusters. Nucleic Acids Res. 51:7220-7235.

1157 Norling M, Jareborg N, Dainat J. 2018. EMBLmyGFF3: a converter facilitating genome annotation
1158 submission to European Nucleotide Archive. BMC Res. Notes 11:584.

1159 Novo M, Bigey F, Beyne E, Galeote V, Gavory F, Mallet S, Cambon B, Legras J-L, Wincker P,
1160 Casaregola S, et al. 2009. Eukaryote-to-eukaryote gene transfer events revealed by the
1161 genome sequence of the wine yeast *Saccharomyces cerevisiae* EC1118. Proc. Natl. Acad.
1162 Sci. 106:16333-16338.

1163 Palmer JM, Stajich J. 2020. Funannotate v1.8.1: Eukaryotic genome annotation.

1164 Paradis E, Schliep K. 2019. ape 5.0: an environment for modern phylogenetics and evolutionary
1165 analyses in R. Bioinformatics 35:526-528.

1166 Park WH. 2020. Upregulation of thioredoxin and its reductase attenuates arsenic trioxide-induced
growth suppression in human pulmonary artery smooth muscle cells by reducing oxidative
1167 stress. Oncol. Rep. 43:358-367.
1168

1169 Patel KS, Pandey PK, Martín-Ramos P, Corns WT, Varol S, Bhattacharya P, Zhu Y. 2023. A
1170 review on arsenic in the environment: contamination, mobility, sources, and exposure. RSC
1171 Adv. 13:8803-8821.

1172 Peck LD, Llewellyn T, Bennetot B, O'Donnell S, Nowell RW, Ryan MJ, Flood J, Vega RCR de la,
1173 Ropars J, Giraud T, et al. 2024. Horizontal transfers between fungal *Fusarium* species
1174 contributed to successive outbreaks of coffee wilt disease. PLOS Biol. 22:e3002480.

1175 Perrone G, Samson RA, Frisvad JC, Susca A, Gunde-Cimerman N, Epifani F, Houbraken J. 2015.
1176 Penicillium salamii, a new species occurring during seasoning of dry-cured meat. Int. J.
1177 Food Microbiol. 193:91-98.

1178 Pfaller MA, Pappas PG, Wingard JR. 2006. Invasive Fungal Pathogens: Current Epidemiological
1179 Trends. Clin. Infect. Dis. 43:S3-S14.

1180 Piñeiro C, Abuín JM, Pichel JC. 2020. Very Fast Tree: speeding up the estimation of phylogenies
1181 for large alignments through parallelization and vectorization strategies. Bioinformatics
1182 36:4658-4659.

1183 Pitt JI, Hocking AD. 2009. Fungi and Food Spoilage. Springer Science & Business Media

1184 Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. 2020. Using SPAdes De Novo
1185 Assembler. Curr. Protoc. Bioinforma. 70:e102.

1186 Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features.
1187 Bioinformatics 26:841-842.

1188 R Core Team. 2024. R: A language and environment for statistical computing. R Found. Stat.
1189 Comput. Vienna Austria [Internet]. Available from: https://www.R-project.org/
1190 Rhie A, Walenz BP, Koren S, Phillippy AM. 2020. Merqury: reference-free quality, completeness, and phasing assessment for genome assemblies. Genome Biol. 21:245.

1192 Rhodes J, Abdolrasouli A, Dunne K, Sewell TR, Zhang Y, Ballard E, Brackin AP, van Rhijn N, 1193 Chown H, Tsitsopoulou A, et al. 2022. Population genomics confirms acquisition of drug-1194 resistant *Aspergillus fumigatus* infection by humans from the environment. Nat. Microbiol. 7:663-674.

1196 Robellet X, Flipphi M, Pégot S, MacCabe AP, Vélot C. 2008. AcpA, a member of the 1197 GPR1/FUN34/YaaH membrane protein family, is essential for acetate permease activity in 1198 the hyphal fungus *Aspergillus nidulans*. Biochem. J. 412:485-493.

1199 Ropars J, Didiot E, Rodríguez de la Vega RC, Bennetot B, Coton M, Poirier E, Coton E, Snirc A, 1200 Le Prieur S, Giraud T. 2020. Domestication of the Emblematic White Cheese-Making 1201 Fungus *Penicillium camemberti* and Its Diversification into Two Varieties. Curr. Biol. CB 1202 30:4441-4453.e4.

1203 Ropars J, Rodríguez de la Vega RC, López-Villavicencio M, Gouzy J, Sallet E, Dumas É, Lacoste 1204 S, Debuchy R, Dupont J, Branca A, et al. 2015. Adaptive Horizontal Gene Transfers 1205 between Multiple Cheese-Associated Fungi. Curr. Biol. 25:2562-2569.

1206 Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, Connor R, Funk K, Kelly C, 1207 Kim S, et al. 2022. Database resources of the national center for biotechnology information. 1208 Nucleic Acids Res. 50:D20-D26.

1209 Schrader L, Schmitz J. 2019. The impact of transposable elements in adaptive evolution. Mol. 1210 Ecol. 28:1537-1549.

1211 Sinzelle L, Kapitonov VV, Grzela DP, Jursch T, Jurka J, Izsvák Z, Ivics Z. 2008. Transposition of 1212 a reconstructed Harbinger element in human cells and functional homology with two 1213 transposon-derived cellular genes. Proc. Natl. Acad. Sci. 105:4715-4720.

1214 Skrzypek MS, Binkley J, Binkley G, Miyasato SR, Simison M, Sherlock G. 2017. The Candida 1215 Genome Database (CGD): incorporation of Assembly 22, systematic identifiers and 1216 visualization of high throughput sequencing data. Nucleic Acids Res. 45:D592-D596.

1217 Smith MR. 2019. TreeTools: create, modify and analyse phylogenetic trees. Comprehensive R 1218 Archive Network

1219 Stefanini I, Di Paola M, Liti G, Marranci A, Sebastiani F, Casalone E, Cavalieri D. 2022. 1220 Resistance to Arsenite and Arsenate in *Saccharomyces cerevisiae* Arises through the 1221 Subtelomeric Expansion of a Cluster of Yeast Genes. Int. J. Environ. Res. Public. Health 1222 19:8119.

1223 Sugui JA, Vinh DC, Nardone G, Shea YR, Chang YC, Zelazny AM, Marr KA, Holland SM, Kwon- 1224 Chung KJ. 2010. *Neosartorya udagawae* (*Aspergillus udagawae*), an Emerging Agent of 1225 Aspergillosis: How Different Is It from *Aspergillus fumigatus*? J. Clin. Microbiol. 48:220-228.

1226 Urquhart A, Vogan AA, Gluck-Thaler E. 2024. Starships: a new frontier for fungal biology. Trends 1227 Genet. 40:1060-1073.

1228 Urquhart AS, Chong NF, Yang Y, Idnurm A. 2022. A large transposable element mediates metal 1229 resistance in the fungus *Paecilomyces variotii*. Curr. Biol. CB 32:937-950.e5.

1230 Urquhart AS, Gluck-Thaler E, Vogan AA. 2024. Gene acquisition by giant transposons primes 1231 eukaryotes for rapid evolution via horizontal gene transfer. Sci. Adv. 10:eadp8738.

1232 Urquhart AS, O'Donnell S, Gluck-Thaler E, Vogan AA. 2025. A natural mechanism of eukaryotic 1233 horizontal gene transfer. bioRxiv:2025.02.28.640899.
1234 Urquhart AS, Vogan AA, Gardiner DM, Idnurm A. 2023. *Starships* are active eukaryotic
1235 transposable elements mobilized by a new family of tyrosine recombinases. Proc. Natl.
1236 Acad. Sci. 120:e2214521120.

1237 Vogan AA, Ament-Velásquez SL, Bastiaans E, Wallerman O, Saupe SJ, Suh A, Johannesson H.
1238 2021. The Enterprise, a massive transposon carrying Spok meiotic drive genes. Genome
1239 Res. 31:789-798.

1240 Wang L-G, Lam TT-Y, Xu S, Dai Z, Zhou L, Feng T, Guo P, Dunn CW, Jones BR, Bradley T, et al. 2020. Treeio: An R Package for Phylogenetic Tree Input and Output with Richly Annotated and Associated Data. Mol. Biol. Evol. 37:599-603.

1243 Wickham H. 2016. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York
1244 Available from: https://ggplot2.tidyverse.org

1245 William VU, Magpantay HD. 2024. Arsenic and Microorganisms: Genes, Molecular Mechanisms, and Recent Advances in Microbial Arsenic Bioremediation. Microorganisms 12:74.

1247 Wolfe BE, Dutton RJ. 2015. Fermented Foods as Experimentally Tractable Microbial Ecosystems. Cell 161:49-55.

1249 Wysocki R, Fortier P-K, Maciaszczyk E, Thorsen M, Leduc A, Odhagen Å, Owsianik G,
1250 Ulaszewski S, Ramotar D, Tamás MJ. 2004. Transcriptional Activation of Metalloid
1251 Tolerance Genes in Saccharomyces cerevisiae Requires the AP-1-like Proteins Yap1p and
1252 Yap8p. Mol. Biol. Cell 15:2049-2060.

1253 Xie L, Zhou L, Zhang R, Zhou H, Yang Y. 2024. Material Composition Characteristics of Aspergillus cristatus under High Salt Stress through LC-MS Metabolomics. Molecules 29:2513.

1256 Yang Z, Huang J, Geng J, Nair U, Klionsky DJ. 2006. Atg22 Recycles Amino Acids to Link the Degradative and Recycling Functions of Autophagy. Mol. Biol. Cell 17:5094-5104.

1258 Yin Y, Miao J, Shao W, Liu X, Zhao Y, Ma Z. 2023. Fungicide Resistance: Progress in Understanding Mechanism, Monitoring, and Management. Phytopathology® 113:707-718.

1260 Yu G. 2023. aplot: Decorate a "ggplot" with Associated Information. Available from: https://github.com/YuLab-SMU/aplot

1262 Yu G, Smith DK, Zhu H, Guan Y, Lam TT-Y. 2017. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol. Evol. 8:28-36.

1265 Zhang H-H, Peccoud J, Xu M-R-X, Zhang X-G, Gilbert C. 2020. Horizontal transfer and evolution of transposable elements in vertebrates. Nat. Commun. 11:1362.

1267