The nematode Caenorhabditis elegans can benefit from its coexisting fungal microbiome member Barnettozyma californica

The nematode Caenorhabditis elegans can benefit from its coexisting fungal microbiome member Barnettozyma californica | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The nematode Caenorhabditis elegans can benefit from its coexisting fungal microbiome member Barnettozyma californica View ORCID Profile Carola Petersen , Hanne Griem-Krey , Christina Martínez Christophersen , View ORCID Profile Hinrich Schulenburg , View ORCID Profile Michael Habig doi: https://doi.org/10.1101/2025.02.17.638710 Carola Petersen 1 Evolutionary Ecology and Genetics, Christian-Albrechts University of Kiel , Germany , Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Carola Petersen Hanne Griem-Krey 1 Evolutionary Ecology and Genetics, Christian-Albrechts University of Kiel , Germany , 2 Fungal Evolutionary Genetics, Christian-Albrechts University of Kiel , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christina Martínez Christophersen 1 Evolutionary Ecology and Genetics, Christian-Albrechts University of Kiel , Germany , Find this author on Google Scholar Find this author on PubMed Search for this author on this site Hinrich Schulenburg 1 Evolutionary Ecology and Genetics, Christian-Albrechts University of Kiel , Germany , 3 Max-Planck Institute for Evolutionary Biology , Ploen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hinrich Schulenburg Michael Habig 2 Fungal Evolutionary Genetics, Christian-Albrechts University of Kiel , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Habig For correspondence: mhabig{at}bot.uni-kiel.de Abstract Full Text Info/History Metrics Preview PDF Abstract The nematode Caenorhabditis elegans is known to feed on and interact with bacteria in its environment and has become a model organism for microbiome studies. However, whether and how C. elegans interacts with co-occurring fungi remains largely unknown, despite the presence of many fungal species in its natural habitat. Here, we isolate the yeast Barnettozyma californica from a mesocosm experiment with C. elegans and characterize its genome and interaction with the nematode. We find that B. californica is ingested by C. elegans and can serve as a sole, albeit poor, food source. Yet, when present together with Escherichia coli OP50, the fungus can lead to higher population growth and altered foraging behavior, suggesting that this fungi-bacteria mixture provides a better food source than bacteria alone. This effect varied between different natural C. elegans strains, suggesting a genomic basis for the nematode’s interaction with B. californica . The fully assembled and annotated genome of the isolated B. californica strain does not indicate any obvious candidate genes for its interaction with C. elegans and/or E. coli OP50. Overall, our results provide an intriguing example of the complexity and multi-level relationship between naturally interacting fungi, bacteria, and animals. Graphical Abstract Barnettozyma californica is a member of the fungal microbiome of the model nematode Caenorhabditis elegans and can serve as food for worms. When combined with E. coli , this fungus can increase population growth and reduce foraging behaviour dependent on C. elegans genotype, revealing a genetic influence on host-fungus-bacterium interactions. Download figure Open in new tab Introduction Caenorhabditis elegans inhabits a diverse microbial community in its natural environment, including bacteria and fungi. These microbes play various roles in the life cycle of C. elegans – serving as food, competitors, commensals, or pathogens – and can also be part of its microbiome. The interaction of C. elegans with pathogens such as the bacteria Bacillus thuringiensis , Pseudomonas aeruginosa , microsporidia of the genus Nematocida , the oomycete Myzocytiopsis humicola , or the Orsay virus have been studied over the last decades ( Tan, Mahajan-Miklos and Ausubel, 1999 ; Troemel et al ., 2008 ; Schulte et al ., 2010 ; Félix et al ., 2011 ; Osman et al ., 2018 ; Tecle and Troemel, 2022 ; Tran and Luallen, 2024 ; Zárate-Potes, Schulenburg and Dierking, 2024 ). More recently, interest has expanded to include the worm’s bacterial microbiome with the first studies published in 2016 ( Berg et al ., 2016 ; Dirksen et al ., 2016 ; Samuel et al ., 2016 ; Schulenburg and Félix, 2017 ). These and subsequent studies have shown that the bacterial microbiome of C. elegans is distinct from its surrounding microbial environment under both natural and experimental conditions ( Berg et al ., 2016 ; Dirksen et al ., 2016 , 2020 ; Samuel et al ., 2016 ; Petersen et al ., 2023 ; Zimmermann et al ., 2024 ; Johnke et al ., 2025 ). In C. elegans , unclassified Enterobacteriaceae are the most common bacteria, along with members of the genera Pseudomonas , Stenotrophomonas , Ochrobactrum , and Sphingomonas ( Zhang et al ., 2017 ). The composition, assembly, stability, and variation of the microbiome are influenced by the host’s developmental stage and genotype ( Dirksen et al ., 2016 ; F. Zhang et al ., 2021 ; Zimmermann et al ., 2024 ). The majority of bacterial strains isolated from either the worm’s habitat or directly from its intestine support robust C. elegans growth. However, some can be pathogenic ( Samuel et al ., 2016 ; Griem-Krey et al ., 2023 ; Gonzalez and Irazoqui, 2024 ). To better understand the influence of microbial communities on the host, a simplified synthetic 12-member microbiota community, known as the CeMbio resource, was developed ( Dirksen et al ., 2020 ). All CeMbio bacteria can colonize the C. elegans intestine, either as a community or individually, to varying degrees. Most strains accelerate the worm’s development compared to those grown on standard lab food, Escherichia coli OP50 ( Dirksen et al ., 2020 ). However, some CeMbio bacteria shorten the lifespan of immunodeficient animals ( Gonzalez and Irazoqui, 2024 ). Beneficial effects, such as the protective role of Pseudomonas lurida MYb11 against the pathogen B. thuringiensis Bt679, can be disrupted by p38 MAPK signaling inhibition. This suggests that microbiota effects on C. elegans can be context-dependent ( Kissoyan et al ., 2019 , 2022 ; Griem-Krey et al ., 2023 ; Pees et al ., 2024 ). Like bacteria, many fungi are found in the natural habitat of C. elegans ( Dirksen et al ., 2016 ). In contrast to its interactions with naturally co-occurring bacteria, very little is known about its interactions with co-occurring fungi. The focus has primarily been on pathogenic interactions. Spores of Drechmeria coniospora and other natural fungal endoparasites of C. elegans , such as Hirsutella rhossiliensis , Haptoglossa dickii , and Catenaria anguillulae , infect nematodes and complete their vegetative life cycle within infected hosts. Upon infection, D. coniospora produces enterotoxins in the epidermis of C. elegans which manipulate the host’s immune responses (X. Zhang et al ., 2021 ). Additionally, C. elegans has been used as a model organism to study hosts immune responses towards fungal human pathogens ( Madende et al ., 2020 ). Innate immune responses of C. elegans towards fungal pathogens involve antimicrobial peptides, lectins, lysozymes, the p38 and ERK mitogen-activated protein kinase pathways, as well as the DAF-2 and TGF-ß/DBL-1 signaling pathways ( Zugasti and Ewbank, 2009 ; Zugasti et al ., 2016 ). Moreover, C. elegans produces chemical compounds with antifungal activity, making it a valuable infection model ( Breger et al ., 2007 ; Madende et al ., 2020 ). A completely different type of interaction between fungi and nematodes involves nematode-trapping fungi, carnivorous microorganisms that capture and digest nematodes using specialized trapping structures ( Jiang, Xiang and Liu, 2017 ). These include Arthrobotrys spp. and Drechslerella stenobrocha , which ensnare nematodes with constricting ring traps or adhesive traps ( Yang et al ., 2007 ; Nordbring-Hertz, Jansson and Tunlid, 2011 ; Liu et al ., 2012 ), highlighting the diverse range of possible interactions between fungi and nematodes. Apart from these few well-characterized interactions between fungi and nematodes, and despite the fact that fungi have been isolated directly from natural C. elegans strains ( Dirksen et al ., 2016 ), little is known about fungi as members of the nematode’s microbiome. In a recent study examining C. elegans in laboratory compost mesocosms, several fungal species were identified, with Barnettozyma californica strains being particularly abundant in the worms ( Petersen et al ., 2023 ). Interestingly, worm populations that lived for multiple generations in a microbial community that included Barnettozyma showed a particularly high fitness. This raises the question of whether the observed fitness advantage was directly caused by Barnettozyma fungi and whether this fungal taxon plays an important role for C. elegans – either as a food source or as a member of its microbiota. Barnettozyma is a genus in the order Phaffomycetales, which was established in 2023 based on a phylogenetic analysis of 290 BUSCO genes ( Groenewald et al ., 2023 ). The genus Barnettozyma was originally described in 2008 based on a multigene phylogenetic analysis, which included B. californica (previously known as Williopsis californica ) ( Kurtzman, Robnett and Basehoar-Powers, 2008 ). B. californica has a worldwide distribution ( Kurtzman, 2011 ), with its type strain CBS 252 first isolated from soil in California in 1931 ( Lodder, 1932 ). Although relatively few genomic sequences are available for B. californica , the first draft genomes – produced using short-read sequencing technologies – were obtained from two B. californica isolates from Ireland ( Mullen et al ., 2018 ). To date, no chromosome-level genome assemblies exist for this species. B. californica has demonstrated the ability to grow on xylose and can utilize a broader range of carbon sources than other tested members of the Barnettozyma genus. It also exhibits ammonium reduction capabilities and can utilize both nitrite and nitrate ( Falih and Wainwright, 1995 ; Kurtzman, 2011 ; Shen et al ., 2018 ). Additionally, B. californica strain K1, isolated from surface sediments of the Pearl River, has been shown to perform heterotrophic nitrification-aerobic denitrification ( Fang et al., 2021 ). Recently, another member of the Barnettozyma genus, B. botsteinii , was discovered in the intestinal tract of the termite Macrotermes bellicosus . Like B. californica , B. botsteinii can grow on xylose, and its ability to degrade plant-derived polymers suggests a potential role in assisting termite metabolism ( Arrey et al ., 2021 ). In summary, despite its wide distribution and presence in various environments, many aspects of B. californica ’s life history and genomic characteristics remain to be explored. Additionally, how B. californica interacts with C. elegans and whether and how it directly affects the nematode’s fitness is unknown. Here, we characterize the interaction between B. californica and C. elegans and show that B. californica can serve as a food source and influence the behavior of C. elegans . When provided as the sole food source, it is less beneficial than bacterial food but can still support the complete life cycle of C. elegans . In contrast, when B. californica is offered as food in combination with bacteria – presumably more representative of C. elegans ’ natural environment – it can increase C. elegans fitness, though this effect depends on the nematode’s genotype. Thus, we demonstrate that a fungus co-occurring with C. elegans can influence the worm’s fitness. Results We first isolated B. californica MYf642 from a mesocosm experiment and characterized its interaction with C. elegans . We then analyzed the genome of B. californica MYf642 to determine whether it reflects its interaction with C. elegans . C. elegans ingests B. californica MYf642 Worms exposed to the microbiome of a specific compost mesocosm consistently showed a higher relative abundance of ASVs from the fungal genus Barnettozyma ( Petersen et al ., 2023 ). We isolated B. californica MYf642 from this mesocosm and tested whether C. elegans ingests it. To visualize B. californica MYf642, we used calcofluor white staining, which specifically binds to chitin. We also used the C. elegans strain dkIs37[ act-5p::GFP::pgp-1 ], in which the intestinal basal membrane is fluorescently labeled to aid localization. Our results show that C. elegans ingests B. californica MYf642, with fungal cells observed throughout the whole intestine, posterior to the grinder ( Fig. 1 , Fig. S1 ). However, it is not entirely clear whether intact B. californica cells can pass through the grinder unaltered. Additionally, we observed and documented feeding behavior of C. elegans including ingestion, passage through the worm intestine and excretion of B. californica MYf642 (Movies S1-2). Control worms fed solely on E. coli OP50 did not show any stained cells in their intestine because E. coli does not contain chitin ( Fig. S1 ). Additionally, previous studies have shown that intact E. coli cells are not found posterior to the grinder in C. elegans . Download figure Open in new tab Figure 1: C. elegans ingest Barnettozyma californica MYf642 (A–F) C. elegans dkIs37[ act-5p::GFP::pgp-1 ] can ingest B. californica MYf642, with fungal cells observed throughout the intestine (A, C, D, F) (white or light blue). The apical intestinal membrane of C. elegans dkIs37[ act-5p::GFP::pgp-1 ] expresses GFP and is shown in white (B, E) or yellow (C, F). Calcofluor white (CFW) stained B. californica MYf642 is depicted in white (A, D) or light blue (C, F). Worms were visualized using confocal laser scanning microscopy. Scale bars represent 50 µm. Images were false-colored using ImageJ. B. californica MYf642 is a poor food source for C. elegans alone, but can significantly increase the fitness when presented alongside bacterial food Next, we tested whether B. californica can serve as a sole food source or, in combination with the standard laboratory food source E. coli OP50, support the entire life cycle and influence the fitness of C. elegans . To account for the genetic variability of C. elegans and avoid restricting our analysis to the canonical laboratory strain N2, we included eleven additional strains from the C. elegans mapping population provided by the Caenorhabditis Natural Diversity Resource (CaeNDR) ( Cook et al ., 2017 ; Crombie et al ., 2024 ). In a population growth assay, three L4 larvae of each C. elegans strain were added to microbial lawns consisting of either B. californica MYf642, E. coli OP50, or a combination of both (which would, by containing both a fungal and a bacterial component, presumably be more akin to the microbial composition in the natural habitat of C. elegans ). The number of offspring was determined after five days ( Fig. 2A ). For all C. elegans strains tested, B. californica MYf642 as the sole food source resulted in significantly lower population growth compared to the E. coli OP50 control. On average, the number of offspring on B. californica ranged from 0.4% to 5.1% of the respective offspring count on E. coli OP50, with a maximum of an average of 79 offspring per original worm in strain CB4856 and a minimum of 20 on average in strain JU775. Although more than three adult worms were observed after five days – indicating that B. californica MYf642 can support the complete life cycle of C. elegans – the low number of offspring per worm compared to on E. coli suggests that B. californica is a poor food source on its own. Download figure Open in new tab Figure 2: B. californica MYf642 in combination with E. coli OP50 as food results in a worm strain-dependent effect on the number of C. elegans offspring and behavioral response A) Outline of the population growth assay: Three synchronized L4 larvae are transferred onto a microbial lawn and the population size is evaluated after 5 days. B) Outline of the microbial lawn leaving assay: Approximately 30 synchronized L4 larvae are put onto a central microbial lawn and after 2 h and 24 h the fraction of nematodes outside the microbial lawn is determined. C-D) The figure shows the effect on population growth (C) and the behavioral response (D) on E. coli OP50 (gray), E. coli OP50 combined with B. californica MYf642 (dark green), and B. californica MYf642 alone (light green). B. californica MYf642 as the sole food source results in significantly lower number of offspring per worm and a higher number of worms outside the microbial lawn after 24 h across all worm strains (compared to E. coli OP50 as sole food source). When combined with E. coli OP50, B. californica MYf642 leads to a higher number of offspring in C. elegans strains EG4725 and JU258, while significantly lowering the number of offspring in strains CB4856, DL238, and LKC34. In JU258 and CB4856, this is associated with fewer or more worms leaving the microbial lawn, respectively. Significant differences (determined by the Wilcoxon rank sum test with Holm correction for multiple testing) compared to E. coli OP50 as the sole food source are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), ns = non-significant. n = 8 The poor population growth of C. elegans on B. californica MYf642 alone contrasts with the results observed when the fungus was combined with bacterial food ( Fig. 2A ). On the combined microbial lawn, for eight C. elegans strains, population growth was not significantly different from that on the E. coli OP50 control. For three other strains (CB4856, DL238, and LKC34), the number of offspring was significantly lower on the mixed microbial lawn, reaching 59%, 19%, and 53% of the respective E. coli OP50 control levels. Most interestingly, for C. elegans strains EG4725 and JU258, the number of offspring was significantly higher on the mixture of E. coli OP50 and B. californica MYf642. Here, the mean number of offspring per worm was 150% and 252% of the respective E. coli OP50 control, indicating a strong positive effect on population growth. Using population growth as a proxy for fitness, we conclude that B. californica MYf642 can influence the fitness of C. elegans when present alongside a bacterial food source. This effect varies from positive to negative, depending on the C. elegans genotype. B. californica MYf642 affects the behavior of C. elegans in a genotype-dependent manner Next, we hypothesized that B. californica might influence the exploratory behavior of the nematode, similar to how different bacterial species affect dietary choice behavior in C. elegans ( Shtonda and Avery, 2006 ; Meisel and Kim, 2014 ). To test this, we placed approximately 30 synchronized L4 worms onto a microbial lawn consisting of either B. californica MYf642, E. coli OP50, or a mix of these two. The microbial lawn-leaving behavior of twelve C. elegans strains was evaluated by measuring the fraction of worms found outside the microbial lawn after 2 h and 24 h ( Fig. 2B for 24 h data and Fig. S2 for 2 h data). When placed on a B. californica MYf642 lawn, a significantly larger fraction of worms was found outside the microbial lawn after 24 h compared to on the E. coli OP50 control. This suggests that the nematodes increase their foraging behavior due to B. californica being a poor food source. This contrasts with the situation when the microbial lawn contained both the fungi and the bacteria. Here, we observed C. elegans genotype-dependent variation in the response. After 24 h, a significantly higher fraction of worms left the mixed microbial lawn in strain CB4856 – one of the strains that also exhibited lower population growth on these mixed microbial lawns. Interestingly, a lower fraction of worms left the mixed microbial lawn for the C. elegans strains JU258 (which displayed higher population growth on these mixed microbial lawns) and JU775. This suggests that, for these C. elegans strains, the mixed microbial lawn may provide a more favorable environment. Although there appears to be a correlation between the results of the microbial lawn-leaving assay at 24 h and the population growth assay ( Fig. S3 ), this correlation is not statistically significant. In addition to the 24-h time point, we also tested lawn-leaving behavior after 2 h. Interestingly, at this earlier time point, four C. elegans strains – including CB4856, which at 24 h showed a much larger fraction outside the mixed microbial lawn – remained more on the mixed microbial lawn than on the bacterial lawn. In summary, B. californica MYf642 affects the foraging behavior of C. elegans when mixed with E. coli OP50 in a genotype-dependent manner. The genome of B. californica MYf642 comprises seven chromosomes and one mitochondrial genome Having established that B. californica MYf642 affects the fitness and behavior of C. elegans , we next sequenced and assembled its genome as a resource for future experimental analyses and also to identify patterns that might help us understand its interaction with C. elegans . Using PacBio HiFi CLR reads we compared three different assembly methods (see Methods and Materials). The best assembly contained seven nuclear contigs and one mitochondrial genome, resulting in a total genome size of 12.1 Mb ( Fig. 3A , Table S1). A full genomic phylogeny based on orthologous proteins identifies the isolate MYf642 to fall within the species B. californica ( Fig. S4A ). We compared the eight nuclear contigs with four publicly available, more fragmented assemblies of B. californica and found a high degree of synteny between orthologous genes ( Fig. 3B , Fig. S4B, C ). With one exception, no contig from the fragmented assemblies spans more than one contig of the B. californica MYf642 assembly. The exception is the fragmented assembly of B. californica strain PB4207, where one contig shows synteny with both TIG09 and TIG15 of the assembly. However, this synteny discrepancy is also present compared to the other three fragmented assemblies of B. californica , suggesting that it is either due to a structural variation or an assembly error in PB4207. This conclusion is supported by the presence of telomeric repeats at both ends of five of the seven contigs assembled here by us ( Fig. 3A ). Although we did not detect the canonical 5’-TTAGGG-3’ telomeric repeat, we identified a novel telomeric repeat sequence, 5’-GTGTGG-3’, which differs from the wide variety of telomeric repeats previously described in ascomycetous yeasts, especially species within the subphylum Saccharomycotina ( Červenák et al ., 2021 ). The telomeric repeat sequence we identified is similar to but shorter than those described for other members of the Phaffomycetaceae ( Červenák et al ., 2021 ). The presence of telomeric repeats at both ends of five contigs, along with the strong synteny across all published B. californica genomes, supports our conclusion that B. californica MYf642 has seven nuclear chromosomes. Download figure Open in new tab Figure 3: Chromosome-level assembly of B. californica MYf642 A) Tapestry report on contig sizes and the presence of telomere repeats in the seven contigs of the B. californica MYf642 assembly. Five of the seven contigs show telomeres at both ends. B) Synteny plot based on orthologous genes between B. californica MYf642 and four publicly available fragmented assemblies of B. californica , showing high levels of synteny across all B. californica MYf642 contigs. The absence of syntenic regions spanning more than one contig or scaffold suggests that the B. californica MYf642 contigs represent individual chromosomes (see also Fig. S7). C) Synteny plot between B. californica MYf642 and other species within the Barnettozyma genus reveals high structural variation, which increases with phylogenetic distance. The genome of B. californica MYf642 is densely populated with genes and contains a low proportion of repetitive and transposable elements We annotated TEs, genes, tRNAs, and functionally characterized putative secreted proteins, effectors, proteases, and Carbohydrate-Active Enzymes (CAZymes), along with gene-wise relative synonymous codon usage (Tables S1 and S2). The genome of B. californica MYf642 contains 5,893 genes, comparable to the 5,802–5,803 genes reported in the fragmented assemblies of B. californica strains UCD09 and UCD5803 ( Mullen et al ., 2018 ). The genome is gene-dense, with genes covering 70.5% of the genome, while TEs account for a relatively low 2.1%. Of the gene transcripts, 312 contain a secretion signal, and among these, a relatively small number (78) are putative effectors – small secreted proteins that are thought to interact with potential hosts ( Jones and Dangl, 2006 ) or other microorganisms ( Snelders et al ., 2020 ). Additionally, we identified 191 putative proteases, 143 putative CAZymes, and a total of 428 TEs and repeats (Table S2). Among the TEs, hAT transposons, a widespread class II DNA transposon ( Atkinson, 2015 ), are the most frequent in the B. californica MYf642 genome (Table S3). Furthermore, we functionally annotated all transcripts using Blast2GO (Table S4). The genome of Barnettozyma californica MYf642 is similar to other members of the Barnettozyma genus We next compared the synteny of genes from our chromosome-level assembly with high-continuity genome assemblies of other Barnettozyma species to assess whether the high synteny observed within B. californica also extends to other species in the genus. Our results show that this is not the case. Instead, we find a low level of synteny between different Barnettozyma species. The closely related B. populi shows a relatively high level of synteny, with few inversions and translocations. In contrast, more distantly related species show much lower levels of synteny ( Fig. 3C and Fig. S4B ). We, therefore, conclude that synteny across species within the Barnettozyma genus is generally low. We also compared the genome functional composition of 74 additional species within the Phaffomycetales order, including Komagataella pastoris as an outgroup to discern patterns of evolution and adaptation in the family. Notably, Komagataella was previously classified within the Phaffomycetaceae ( Kurtzman, 2011 ), but genome-scale analyses now place it under the Pichiales , making it a suitable outgroup for this study ( Shen et al ., 2018 ; Groenewald et al ., 2023 ). To correlate phylogenetic relationships with genome functional composition, we reconstructed a maximum likelihood phylogenetic tree based on concatenated protein sequences of single-copy orthologous genes (a total of 657 genes) ( Fig. 4 , right). To ensure comparability between the species, we functionally reannotated each of the assemblies using the same bioinformatics pipeline employed for the annotation of B. californica MYf642. Our results confirm the monophyletic origins of the genera within the Phaffomycetaceae , specifically Barnettozyma , Cyberlindnera , Phaffomyces , and Starmera . However, the Wickerhamomycetaceae , which contains the genus Wickerhamomyces , does not appear monophyletic and is interspersed among species of the Phaffomycetaceae . Although outside the main focus of this study, it is important to note that the non-monophyletic characteristics of the Wickerhamomyces genus, which we here inferred from 657 orthologous genes, contrast with earlier analyses based on internal transcribed spacer (ITS) and large ribosomal subunit (LSU) sequences ( Arrey et al ., 2021 ; Nundaeng et al ., 2021 ), or elongation factor-1α (EF-1α) and small ribosomal subunit (SSU) sequences ( Kobayashi, Kanti and Kawasaki, 2017 ). The functional genome composition of B. californica MYf642 is similar to that of other species within the genus Barnettozyma and other species in the Phaffomycetales . Despite a high variation in certain genomic features, such as transposable element content (ranging from 0.2% to 7.4%), secreted proteins (239–482), effector candidates (58–248), proteases (175–238), and CAZymes (112–172), none of these variations appear to correlate with phylogenetic relationships, nor does B. californica MYf642 stand out as an extreme in any category. Additionally, we observed high variation in codon usage bias, as predicted by gene-wise relative synonymous codon usage (RSCU), and the predicted numbers of tRNAs. However, neither codon usage bias nor tRNA numbers appeared to be correlated or follow any phylogenetic pattern, and again nor does B. californica MYf642 stand out as an extreme. In conclusion, the functional genome composition of B. californica MYf642 is broadly similar to other related species, despite the observed variations in certain genomic traits. Download figure Open in new tab Figure 4: Phylogeny and summary of functional annotation of Phaffomycetales Left: Maximum Likelihood (ML) phylogenetic tree of single-copy orthologous proteins from the genera Starmera , Phaffomyces , Cyberlindnera , Barnettozyma , and Wickerhamomyces , with Komagataella pastoris used as an outgroup. The genus Wickerhamomyces (pink), part of the family Wickerhamomycetaceae , is polyphyletic and interspersed among other genera from the family Phaffomycetaceae . Right: High variation is observed in the presence of transposable elements (TEs), secreted proteins, putative effectors, putative CAZymes, and proteases. The number of TEs and codon usage patterns vary greatly between species and do not correlate with phylogenetic relationships. B. californica MYf642 contains unique genes associated with terpenoid metabolism and transport We next compared the gene composition of B. californica MYf642 with representative members of other genera in the Phaffomycetales for which high-continuity genome assemblies are available. A total of 4,258 orthogroups were shared across all six species, while 28 orthogroups were unique to B. californica MYf642 ( Fig. 5A, B ). Based on these 28 orthogroups and functional gene annotations (Table S4), we conducted a GO-term enrichment analysis. This analysis identified several significantly over-represented GO terms, including “terpenoid metabolic processes,” “positive regulation of (R)-carnitine transmembrane transport,” and “polyamine transmembrane transport.” Interestingly, terpenoids and terpenes, form the largest group of secondary metabolites in fungi ( González-Hernández et al ., 2023 ). Additionally, we tested whether genes involved in nitrification and denitrification, previously reported in the B. californica K1 strain – specifically amoA, nirK, nosZ ( Fang et al ., 2021 ) – were present in B. californica MYf642. However, none of these genes were detected by BLAST searches using the sequences described in ( Fang et al ., 2021 ). Therefore, we conclude that B. californica MYf642 does not possess the genetic capacity for heterotrophic nitrification-aerobic denitrification. Download figure Open in new tab Figure 5: Identification of proteins restricted to B. californica MYf642 and associated GO terms A) Venn diagram of orthologous proteins among representative species of the Phaffomycetales compared to the B. californica MYf642 isolate, identifying 28 clusters with a total of 83 proteins that are restricted to B. californica MYf642 and do not have orthologs in the other representative species. B) Results of the GO term enrichment analysis for the 83 proteins restricted to B. californica MYf642 compared to the reference set (all proteins encoded by the B. californica MYf642 genome), with significance thresholds of p = 0.05 and FDR = 0.1. Discussion Here, we show that the fungus B. californica , which is found in diverse environments and was isolated as a putatively beneficial microbiome member of C. elegans from a mesocosm experiment, is ingested and used as food by C. elegans . On its own, it appears to be a poor food source, leading to increased C. elegans foraging behavior. However, when combined with a bacterial food source, it can have a positive fitness effect and reduce foraging activity. Furthermore, we report the chromosome-level assembly of the B. californica MYf642 genome, which includes a novel type of telomeric repeat. The genome is highly compact and does not exhibit any distinct features that set it apart from other Phaffomycetales yeasts and that could thus explain its interaction with C. elegans and its bacterial food. The interaction between C. elegans and fungi is poorly understood, and the extent to which fungi contribute to the C. elegans diet remains unknown. Our study is one of the few that demonstrate that fungi can serve as a food source for C. elegans . Similarly, the basidiomycete yeasts Cryptococcus kuetzingii and C. laurentii have been used as C. elegans food sources in the lab ( Mylonakis et al ., 2002 ). However, for neither Cryptococcus species was a direct association with C. elegans shown, nor were they isolated from a C. elegans environment. Notably, in our experiments, the positive effect on C. elegans population growth was observed only when B. californica MYf642 was present together with E. coli OP50. This suggests that, in addition to directly affecting C. elegans biology, B. californica MYf642 may influence it indirectly via interactions with E. coli OP50. In general, fungi and bacteria that share habitats can interact through the production of antibiotics, signalling molecules, and other compounds, such as siderophores that manipulate nutrient availability ( Deveau et al ., 2018 ; Scherlach and Hertweck, 2020 ; Pierce et al ., 2021 ). Currently, the mechanism underlying the positive effects of B. californica MYf642 in the presence of E. coli OP50 remains unclear. One possibility is that the fungus produces compounds that are limited in a pure bacterial culture. Many fungi are known to produce secondary metabolites with great structural diversity ( Macheleidt et al ., 2016 ), which may be beneficial to C. elegans . Alternatively, and not mutually exclusively, B. californica may induce changes in E. coli that are beneficial to C. elegans . In any case, based on our results, this effect also appears to be dependent on the genotype of the nematode. We also found that the foraging behavior of C. elegans was affected by B. californica MYf642 when present in combination with E. coli OP50, and this effect was again dependent on the C. elegans genotype. It is known that genetically different C. elegans strains vary in their lawn-leaving behavior in response to food bacteria and pathogens ( Reddy et al ., 2009 ; Bendesky et al ., 2011 ; Nakad et al ., 2016 ). Leaving a microbial lawn can be an avoidance behavior toward certain pathogens, such as Serratia marcescens ( Pradel et al ., 2007 ) or B. thuringiensis ( Schulenburg and Müller, 2004 ; Hasshoff et al ., 2007 ; Nakad et al ., 2016 ). However, C. elegans can also distinguish between high- and low-quality food bacteria. Worms tend to search for high-quality food that supports growth, while they avoid or abandon low-quality food ( Shtonda and Avery, 2006 ). Thus, the increased foraging behavior in the presence of B. californica MYf642 indicates that it is a poor food source, while the reduced foraging behavior of some C. elegans strains when present with the mixture of fungi and bacteria indicates that this is a good food source. In microbial lawn-leaving assays, nematodes often return to the food multiple times within minutes to hours, which might explain the differences between our 2-h and 24-h data in the lawn-leaving assay. The genomes of fungi are shaped by interactions with other organisms that share the same habitat, including other microorganisms. The discovery of penicillin by Alexander Fleming in 1929 was the first indication that fungi manipulate their microbial environment through what we now recognize as secondary metabolites. Fungi also produce small secreted proteins, so-called effectors. Until recently, the role of effectors in plant-associated fungi was largely thought to be limited to interactions with the host and the suppression of the host immune system ( Jones and Dangl, 2006 ). More recently, effectors have been shown to influence the bacterial and fungal microbiome of plants, suggesting that this may have been their ancestral function ( Snelders et al ., 2018 , 2020 , 2021 ). Although we were unable to identify any unique secondary metabolite clusters or effectors in the genome of B. californica MYf642 that might explain the fungus’s positive impact – both in this study and in the mesocosm study ( Petersen et al ., 2023 ) – this does not rule out the existence of yet-undiscovered genetic components involved in these interactions. Additionally, it is puzzling that we did not find substantial genetic overlap with the termite-associated B. botsteinii ( Arrey et al ., 2021 ). Thus, the fungal genetic components underlying the interaction with C. elegans and E. coli OP50 remain unknown. In conclusion, both the microbial lawn-leaving assay and the population growth assay demonstrated that B. californica MYf642 affects C. elegans behavior when presented jointly with bacteria, in a genotype-dependent manner. The use of genetically diverse mapping strains from the CaeNDR has allowed us to identify these patterns, which are not apparent in the standard laboratory N2 strain. The interaction between C. elegans , B. californica MYf642, and E. coli OP50 that we present here is an intriguing example of the complexity and multi-level nature of interactions between fungi, bacteria, and animals – for which C. elegans is one of the ideal model organisms. Supplementary Figures Download figure Open in new tab Figure S1: Stained Barnettozyma californica MYf642 but no stained E. coli OP50 in the C. elegans intestine B. californica MYf642 can be found in various regions of the C. elegans intestine (A, C) (white or light blue) while E. coli OP50 cells do not contain chitin and hardly pass the worm grinder and are therefore not stained by the calcofluor white staining (D, F). The apical intestinal membrane of C. elegans dkIs37[ act-5p::GFP::pgp-1 ] expresses GFP and is shown in white (B, E) or yellow (C, F). Calcofluor white (CFW) stained B. californica MYf642 is shown in white (A, D) or light blue (C, F). Worms were visualized using confocal laser scanning microscopy. The scale bars have a length of 100 µm. Images were false-colored using ImageJ. Download figure Open in new tab Figure S2: B. californica MYf642 in combination with E. coli OP50 as food results in a strain-dependent effect on the behavioral response after 2 h The figure shows the behavioral response of the indicated C. elegans strains on E. coli OP50 (gray), E. coli OP50 combined with B. californica MYf642 (dark green), and B. californica MYf642 alone (light green). B. californica MYf642 as the sole food source results in a higher number of worms outside the microbial lawn after 2 h for all strains (compared to E. coli OP50 as sole food source), with the exception of CX11314. When combined with E. coli OP50, B. californica MYf642 leads to genotype dependent variation in the fraction of worms outside the microbial lawn. Significant differences (determined by the Wilcoxon rank sum test with Holm correction for multiple testing) compared to E. coli OP50 as the sole food source are indicated as follows: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), ns = non-significant. n = 8 Download figure Open in new tab Figure S3: Correlation between lawn-leaving behavior and population growth rate The relative number of offspring per worm for each C. elegans strain on the mixed microbial lawn (normalized to the E. coli OP50 control) is plotted against the relative fraction of worms outside the microbial lawn for each C. elegans strain (normalized to the E. coli OP50 control). A linear regression line is indicated by the dashed line. Download figure Open in new tab Figure S4: Network phylogeny of species within the genus Barnettozyma and details on synteny within B. californica support chromosome-level assemblies and extensive structural variation within the Barnettozyma genus A) NeigborNet phylogeny of 2710 single copy orthologous proteins of species within the genus Barnettozyma and with E. anomalus as an outgroup. The isolate B. californica MYf642 falls within the species B. californica . B) Details of orthologous proteins based synteny for TIG09 and TIG15 (left) and TIG19 (right) show that the apparent structural variation is unique to the B. californica strain PB4207, where it is located at the end of a contig. All other previously published assemblies show no structural variation compared to B. californica MYf642. Hence this apparent structural variation in B. californica strain PB4207 could be due to a misassembly. C) Details of synteny for TIG01 (left) and TIG10 (right) demonstrate extensive structural variation, which increases with phylogenetic distance. Panels B) and C) show details from Figure 3 panel B) and C), respectively. Supplementary Tables Table S1: Overview of B. californica MYf642 genome assembly and characteristics Table S2: Comparison of the functional annotations of the genomes used for the phylogenetic analysis Table S3: Overview of the transposable elements (TE) and repeat elements (RE) in the B. californica MYf642 assembly Table S4: Functional annotation of proteins encoded by B. californica MYf642 Table S5: Genome assembly accessions included in this study Supplementary Movies Captions Movie S1: Feeding behavior of C. elegans including ingestion, passage through the worm intestine and excretion of B. californica MYf642 Movie S2: Feeding behavior of C. elegans including ingestion, passage through the worm intestine and excretion of B. californica MYf642 Methods and Materials Microbial and C. elegans strains E. coli OP50 and B. californica MYf642 were cultured overnight in a shaking incubator (180 rpm) in LB medium at 37 °C or 28 °C, respectively. We used the 12 C. elegans strains CB4856, CX11314, DL238, LKC34, ED3017, EG4725, JT11398, JU258, JU775, MY16, MY23, and N2 from a mapping population provided by the Caenorhabditis Natural Diversity Resource (CaeNDR) ( Cook et al ., 2017 ; Crombie et al ., 2024 ). Additionally, the C. elegans strain dkIs37[ act-5p::GFP::pgp-1 ] was used to locate stained cells of B. californica MYf642 in the C. elegans intestine. All C. elegans strains were maintained on nematode growth medium (NGM) on E. coli OP50. Isolation of Barnettozyma californica MYf642 We isolated B. californica MYf642 from a 100-day-old laboratory compost mesocosm, originating from a long-term evolution experiment with C. elegans and its associated microbes. Worms from this 100-day-old laboratory compost showed improved fitness and enrichment of Barnettozyma in a follow-up common garden experiment when exposed to the microbial community from day 100 ( Petersen et al ., 2023 ). To isolated Barnettozyma californica MYf642, a random compost sample was mixed with 25 ml M9 with 0.025% Triton X-100 (M9-T). A 300 µl aliquot of this mixture was homogenized with 10 zirconia beads (1 mm) in a Bead Ruptor 96 (OMNI International, Kennesaw, GA, USA) at 30 Hz for 3 min, then frozen in 10% dimethyl sulfoxide (DMSO) at −80 °C. Thawed material was serially diluted (10⁻¹ to 10⁻⁴) in sterile phosphate buffered saline (PBS), and 50 µl of each dilution was plated onto potato dextrose agar (39 g/l, Carl Roth, Karlsruhe, Germany). Plates were incubated at room temperature for 10 days. Morphologically distinct colonies were picked upon appearance and purified by re-culturing for at least three times. Pure isolates were grown in lysogeny broth (LB) on a circular shaker for 2 days at 25 °C and preserved in 35% glycerol at −80 °C. Crude DNA was extracted by adding 5 µl liquid culture to 19.5 µl 5x PCR buffer with 0.5 µl proteinase K (20 mg/ml), followed by freezing at −80 °C for 30 min, digestion at 50 °C for 1 h and heat inactivation at 95 °C for 15 min. For identifying yeasts and fungi, 1 µl crude DNA served as template in a 50 µl PCR reaction using DreamTaq DNA polymerase (Thermo Fisher Scientific, Darmstadt, Germany) to amplify the internal transcribed spacer (ITS) region with the primers ITS1f (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS4r (5’-TCCTCCGCTTATTGATATGC-3’) ( White et al ., 1990 ; Gardes and Bruns, 1993 ). A one-day culture of the B. californica MYf642 isolate in LB medium, grown at 28 °C and 200 rpm, was used for DNA isolation following a modified version of the cetyltrimethylammonium bromide (CTAB) DNA extraction protocol ( Plissonneau, Stürchler and Croll, 2016 ). Briefly, the cells were harvested by centrifugation, washed twice, and 400 µl of the cell pellet was crushed using a mortar and pestle with liquid nitrogen. Following extraction with CTAB, DNA was isolated twice using phenol:chloroform:isoamylalcohol and precipitated with isopropanol. The DNA was washed three times with ethanol before being resuspended in 1x TE buffer. Sequencing was performed using the PacBio Revio platform at BGI Techsolutions (Hong Kong), resulting in 308,037 CCS reads with a mean length of 17,689 bp. Calcofluor white staining Calcofluor white stain is a fluorochrome that binds to cellulose and chitin in cell walls of fungi. L4 larvae of the C. elegans strain dkIs37[act-5 p :: GFP ::pgp-1] expressing PGP-1::GFP in the apical intestinal membrane ( Sato et al ., 2007 ) were transferred to nematode growth medium (NGM) plates inoculated with B. californica MYf642. After 24 h, adult worms were washed from the plates with M9-buffer containing 0.3% Tween20 (M9-T20). 500 µl of the worm-containing M9-T20 was added to a 2-ml tube, and 500 µl of 10 mM tetramisole was added to stop the ingestion and excretion of microbes. After about 2 minutes, the pellet of immobilized worms was transferred to a new tube. The worms were fixed and stained by adding 250 µl of calcofluor white staining solution consisting of equal parts calcofluor white stain and ethanol. After 3 h, the staining solution was removed, the worms were washed three to four times with M9-T20 and then examined for stained B. californica MYf642 in their intestine using confocal laser scanning microscopy (ZEISS LSM 880). Population growth assay The population growth rate is a measure for worm fitness as it combines information on the developmental time, reproductive rate and survival. To start the assay, three C. elegans larvae in the fourth larval stage (L4) were picked to a 1-ml lawn of either B. californica MYf642, E. coli OP50 or a 1:1 mixture of both at OD 600 10 on 9 cm peptone-free medium plates. After 5 days at 20 °C, the worms were washed from the plates to 15 ml Falcon tubes using 5 ml M9-T. The liquid in the Falcon tubes was weighed for later calculations and then frozen at −20 °C until the worms were counted. Three subsamples were counted per sample, from which a mean value was calculated to account for variations in worm numbers. The total number of offspring over five days was calculated by dividing the mean worm count of the three subsamples by the number of counted microliters multiplied by the total weight of the sample in gram. The offspring number was divided by the three original worms to obtain the offspring per worm. Microbial lawn leaving assay C. elegans can perceive the quality of its food as well as the presence of pathogens and adapt its behavior accordingly ( Shtonda and Avery, 2006 ; Meisel and Kim, 2014 ). To determine the behavior of C. elegans towards B. californica MYf642, we performed a microbial lawn leaving assay. Approximately 30 synchronized L4 worms were pipetted to a 60 µl microbial spot of B. californica MYf642, E. coli OP50 or a 1:1 mixture of both adjusted to OD 600 10 on 6 cm peptone-free medium plates. The number of worms on and outside the spot was counted after 2 h and 24 h to analyze the early and late lawn leaving behavior. The escape rate was then calculated by dividing the number of worms outside the microbial spot by the total number of counted worms. Bioinformatics Analysis Genome assembly The code used for all subsequent bioinformatics analyses has been deposited here: https://github.com/michaelH-git/BarnettozymaCalifornica_genomeAnalysis.git . Briefly, assemblies were generated using Canu (2.2) ( Koren et al ., 2017 ), HiFiasm (0.19.9-r616) (Cheng et al., 2021), and Flye (2.9.3-b1797) (Kolmogorov et al., 2019). The assembly quality was checked using QUAST (v5.2.0) ( Mikheenko et al ., 2018 ). After visual inspection, the Canu-based assembly was selected for all subsequent analyses. The quality of this assembly was further evaluated using Tapestry (1.0.1) ( Davey et al ., 2020 ), and contigs that showed no coverage by the original reads were discarded (40 contigs, totaling 1.3 Mb). Of the remaining nine contigs, the smallest one (TIG20) showed synteny to mitochondrial genomes. This sequence was further polished and annotated using MitoHiFi (3.2.1) ( Uliano-Silva et al ., 2023 ), resulting in a 48,643 bp circular contig representing the mitogenome. For the remaining eight contigs representing the nuclear genome, telomere sequences were identified using TelFinder ( Q et al ., 2023 ), and the quality of the genome was rechecked using Tapestry. Functional annotation of genomes The list and accessions of the genome assemblies included in this study is given in Table S5. Genomes were functionally annotated using the following tools. Transposable and repetitive elements were identified using EDTA (2.2.0) ( Ou et al ., 2019 ). In cases where no transposable elements (TEs) were detected, repetitive sequences were called using EarlGrey (4.4.4) ( Baril, Galbraith and Hayward, 2024 ). These annotations were used to softmask the genomes for gene annotation. Genes were annotated using BRAKER3 (3.0.8) ( L et al ., 2024 ) with protein evidence from OrthoDB11 restricted to fungi. Secondary metabolite clusters were annotated using antiSMASH (7.1.0) ( Blin et al ., 2021 ), and functional annotation of predicted proteins was performed using eggNOG-mapper (2.1.12) ( Cantalapiedra et al ., 2021 ) and InterProScan ( Blum et al ., 2021 ). Genome completeness was assessed with BUSCO (5.7.1) ( Simão et al ., 2015 ), while tRNAs were predicted using tRNAscan-SE (2.0.12) ( Chan et al ., 2021 ). Codon usage was estimated by determining the gene-wise relative synonymous codon usage (gRSCU) with BioKit ( Steenwyk and Buida, 2022 ). Putatively secreted proteins were identified using SignalP (5.0b) ( Almagro Armenteros et al ., 2019 ), and putative effectors were predicted using EffectorP3 (3.0) ( Sperschneider and Dodds, 2022 ). Putative CAZymes (carbohydrate-active enzymes) were identified using dbCAN ( Zhang et al ., 2018 ). All these annotations were combined using Funannotate ( Funannotate , 2017 ). For B. californica MYf642 the functional annotation was further improved using Blast2GO ( Conesa and Götz, 2008 ). Fisher’s Exact test for GO-term enrichment was conducted using this improved annotation in Blast2GO. Phylogenetic analysis Identification of single copy orthologs for all genomes of the phylogenetic analysis was conducted using Orthofinder ( Emms and Kelly, 2019 ) restricted to one transcript for each gene. All single copy orthologous proteins shared among all species (657) were concatenated and aligned using MAFFT and the phylogenetic tree determined by IQTree using the LG+I+G model and generating a consensus tree of 1000 bootstrapings ( Minh et al ., 2020 ). Determination of orthologous proteins between B. californica MYf642, B. botsteinii (GCA_020280145.1), C. sargentensis (GCA_020995425.1), S. stellimalicola (GCA_030580055.1), W. anomalus (GCA_019321675.1) and [Candida] orba (GCA_003708145.3) to represent all genera within the phylogenetic analysis was performed using OrthoVenn3 ( Sun et al ., 2023 ). GO term enrichment analysis was performed on those genes unique to B. californica MYf642. Synteny analysis was conducted using GENESPACE (v1.3.1) ( Lovell et al ., 2022 ). Data availability Sequencing reads, genome assemblies and annotations for B. californica MYf642 are deposited using Bioproject PRJNA1224554. Acknowledgements CP and HS are thankful for support by the CRC1182 projects A1.1 and A4.3 funded by the German Science Foundation. The graphical abstract and Fig2 were created in BioRender. Habig, M. (2025) https://BioRender.com/y15j225 https://BioRender.com/d81s260 References ↵ Almagro Armenteros , J.J. et al. 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Share The nematode Caenorhabditis elegans can benefit from its coexisting fungal microbiome member Barnettozyma californica Carola Petersen , Hanne Griem-Krey , Christina Martínez Christophersen , Hinrich Schulenburg , Michael Habig bioRxiv 2025.02.17.638710; doi: https://doi.org/10.1101/2025.02.17.638710 Share This Article: Copy Citation Tools The nematode Caenorhabditis elegans can benefit from its coexisting fungal microbiome member Barnettozyma californica Carola Petersen , Hanne Griem-Krey , Christina Martínez Christophersen , Hinrich Schulenburg , Michael Habig bioRxiv 2025.02.17.638710; doi: https://doi.org/10.1101/2025.02.17.638710 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Ecology Subject Areas All Articles Animal Behavior and Cognition (7624) Biochemistry (17651) Bioengineering (13871) Bioinformatics (41884) Biophysics (21424) Cancer Biology (18566) Cell Biology (25463) Clinical Trials (138) Developmental Biology (13365) Ecology (19867) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22477) Immunology (17714) Microbiology (40331) Molecular Biology (17148) Neuroscience (88487) Paleontology (666) Pathology (2828) Pharmacology and Toxicology (4817) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)