Tracking karyotype dynamics by flow cytometry reveals de novo chromosome duplications in laboratory cultures of Macrostomum lignano

Tracking karyotype dynamics by flow cytometry reveals de novo chromosome duplications in laboratory cultures of Macrostomum lignano | 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 Tracking karyotype dynamics by flow cytometry reveals de novo chromosome duplications in laboratory cultures of Macrostomum lignano View ORCID Profile Stijn Mouton , Lisa Glazenburg , View ORCID Profile Eugene Berezikov doi: https://doi.org/10.1101/2025.10.30.685567 Stijn Mouton 1 European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen , Groningen 9700AD, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stijn Mouton For correspondence: s.m.mouton{at}umcg.nl e.berezikov{at}umcg.nl Lisa Glazenburg 1 European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen , Groningen 9700AD, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Eugene Berezikov 1 European Research Institute for the Biology of Ageing, University Medical Center Groningen, University of Groningen , Groningen 9700AD, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Eugene Berezikov For correspondence: s.m.mouton{at}umcg.nl e.berezikov{at}umcg.nl Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT The flatworm Macrostomum lignano is a versatile invertebrate model organism with a growing molecular toolbox. Genome assembly and detailed karyotyping revealed that M. lignano is a hidden polyploid species with a recent whole-genome duplication. Its karyotype consists of six small and two large chromosomes ( 2n = 8 ), with the large chromosomes originating from the fusion of duplicated ancestral chromosomes. However, 2n = 9 and 2n = 10 karyotypes with duplicated large chromosomes were also observed in animals from both laboratory cultures and field samples, prompting us to further investigate this phenomenon. To this end, we optimized a flow cytometric approach that enables easy and rapid studies of tens or even hundreds of animals simultaneously to gain insight into the karyotype polymorphisms present in a culture, and consistently tracked karyotype dynamics in multiple cultures over a period of 26 months. We demonstrate that de novo duplications of the large chromosome in M. lignano can spontaneously appear under laboratory conditions and can become dominant in laboratory cultures. Since uncontrolled chromosomal duplications can complicate genetic studies in laboratory model organisms, we propose an approach to easily control the karyotype of experimental cultures by regular karyotyping M. lignano subcultures using flow cytometry and replacing cultures with de novo chromosome duplications as needed. SUMMARY STATEMENT We present a flow cytometric approach to identify karyotypic polymorphisms in Macrostomum lignano cultures, demonstrate de novo chromosome duplications under laboratory conditions, and propose a solution for controlling culture karyotypes. INTRODUCTION Macrostomum lignano is a free-living marine flatworm that is approximately 1 mm long ( Fig. 1A ). While this transparent worm might seem unremarkable at first, it is gaining traction as a model organism to study diverse biological questions ( Wudarski et al., 2020 ), including regeneration ( Hall et al., 2024 ; Kibet et al., 2025 ), bio-adhesion ( Wunderer et al., 2019 ), and male fertility ( Weber et al., 2020 ). A key factor in M. lignano’s appeal as a model system is its ease of culturing under laboratory conditions. Large cultures can be easily generated and maintained by keeping worms in Petri dishes with artificial sea water (ASW) and diatoms as a food source. M. lignano is a non-self-fertilizing hermaphrodite that reproduces exclusively in a sexual manner ( Schärer and Ladurner, 2003 ). The worms lay abundant single-cell fertilized eggs, and the egg-to-egg generation time is 2-3 weeks ( Morris et al., 2004 ; Wudarski et al., 2019 ) Notably, the eggs can be microinjected, which has enabled the development of robust transgenesis methods ( Wudarski et al., 2017 ), and M. lignano remains the only flatworm species in which transgenesis is currently possible. To support functional studies, transcriptome and genome assemblies have been created and are publicly available at the Flatworms and Acoels Genome Browser. Download figure Open in new tab Figure 1. Karyotype evolution in laboratory cultures of Macrostomum lignano . (A) Brightfield image of an adult worm. Scalebar: 100 µm. (B) Karyotype polymorphisms with an increasing number of large chromosomes that can be found in M. lignano . Scalebars: 10 µm. (C) Dynamics of karyotype polymorphisms present in 12 experimental subcultures over a period of 26 months. All subcultures started with only the 2n = 8 karyotype. The bars indicate the percentage of subcultures that also contain the 2n = 9 and 2n = 10 karyotypes as a function of time. (D-F) Flow cytometric histograms based on Propidium Iodide Area, which visualize karyotype polymorphisms present in a culture. The different peaks (1-3) represent diploid cells with different karyotypes and thus different amounts of DNA. Arrows represent cells in the G2 and mitotic cell cycle phases that do not impact karyotype interpretation. (D) Example histogram representing the 2n = 8 karyotype at the start of the experiment. (E) The first appearance of a second peak (2) arising due to the duplication of a large chromosome, thus representing a 2n = 9 karyotype. (F) The first appearance of the 2n = 10 karyotype (peak 3) at 22 months. Assembling the genome ( Wudarski et al., 2017 ) and detailed karyotyping ( Zadesenets et al., 2016 ; Zadesenets et al., 2017a ; Zadesenets et al., 2017b ) revealed that M. lignano is a hidden polyploid species. The modern genome of this species was formed through a recent whole-genome duplication, followed by re-diploidization, during which all ancestral chromosomes fused into one large chromosome ( Zadesenets et al., 2020 ; Zadesenets et al., 2023 ). This resulted in a 2n=8 karyotype with one pair of large and three pairs of small metacentric chromosomes ( Fig. 1B ) ( Egger and Ishida, 2005 ). However, this karyotype appears unstable. Substantial karyotype polymorphisms were observed between individual M. lignano worms, both in laboratory cultures and in field-collected specimens. The main variation is aneuploidy of the large chromosome, resulting in 2n = 9 and 2n = 10 karyotypes ( Fig. 1B ) ( Zadesenets et al., 2016 ). A gain or loss of small chromosomes has only been observed in some rare cases ( Zadesenets et al., 2017b ). Interestingly, tri- and tetrasomy of the large chromosome do not adversely affect worm morphology and fertility. On the contrary, aneuploid karyotypes are not only tolerated but are increasingly prevalent after long-term culturing under laboratory conditions ( Zadesenets et al., 2016 ; Zadesenets et al., 2020 ). To date, it remains unclear whether this increasing number of aneuploid worms is due to the fitness advantages of a few aneuploid worms present when starting laboratory cultures from field samples or if de novo duplications of the large chromosome can occur in the laboratory ( Zadesenets et al., 2016 ; Zadesenets et al., 2020 ). By consistently tracking the karyotype dynamics of laboratory cultures for 26 months, we unambiguously demonstrated that de novo chromosome duplications occur under laboratory conditions. This experiment was performed by combining simple culturing techniques with flow cytometry of large numbers of Propidium Iodide (PI)-labelled nuclei. The fluorescence intensity of PI represents the amount of DNA in the nucleus. Karyotype polymorphisms with varying numbers of large chromosomes are therefore visualized as separate peaks in PI-based histograms ( Fig. 1F , Fig. 3C ). This makes flow cytometry an easy and fast method for studying karyotype dynamics of M. lignano . RESULTS Creating a wild-type M. lignano culture without karyotype polymorphisms To study whether chromosome duplications can occur de novo in laboratory cultures, we first took several steps to obtain a culture without any karyotype polymorphisms, called NL12S. For this purpose, we made subcultures of the NL10 M. lignano culture by randomly selecting 30 juvenile worms and making 15 isolated pairs, of which 14 gave rise to starting subcultures. NL10 is a wild-type laboratory culture that was established from worms collected near Lignano, Italy, in 2014 (Wudarski, 2017) and was propagated in the laboratory for 3 years before we started this study. Flow cytometry-based karyotyping of these subcultures revealed that only 6 of them were characterized by a single 2n=8 karyotype (Suppl. Fig. 1A). Those 6 subcultures were further grown to include hundreds of worms, and were analyzed again 6 weeks later. Only four of them showed a clear single peak and were thus characterized by a single 2n=8 karyotype (Suppl. Fig. 1B). These subcultures were combined, renamed NL12S (‘S’ for ‘single’), and further grown as a novel laboratory culture without chromosomal duplications. Chromosome duplications occur de novo in laboratory cultures of M. lignano Starting with pairs of worms, 12 experimental subcultures of the NL12S culture were established and examined every two months for a period of 26 months. Flow cytometry-based karyotyping performed after two and four months confirmed that all subcultures initially contained only the 2n=8 karyotype ( Fig. 1D ; Suppl. Fig. 2 - 13). Already after 6 months, the first chromosome duplications, leading to a 2n=9 karyotype, were observed in subcultures 2 and 7 ( Fig. 1C, E ). During the first year of the experiment, these remained the only two subcultures, representing 17% of all studied cultures, in which chromosome duplication occurred (Suppl. Fig. 2 - 13). From 14 months onwards, chromosome duplications were also observed in other subcultures ( Fig. 1C ; Suppl. Fig. 2 - 13). By the end of the experiment at 26 months, seven subcultures (58%) included the 2n=9 karyotype ( Fig. 1C , Fig. 2 ). Download figure Open in new tab Figure 2. Karyotype polymorphisms present after 26 months. Overview of the Propidium Iodide Area (PI-A)-based histograms of the 12 experimental subcultures at the end of the study after 26 months. The number of karyotype polymorphisms present and the ratio of nuclei between them varies considerably. Download figure Open in new tab Figure 3. Flow cytometric approach to identify karyotype polymorphisms in Macrostomum lignano cultures (A) Gating strategy to identify karyotype polymorphisms present in a culture. ‘FSC-A’ represents the size, ‘PI-A’ represents the total Propidium Iodide (PI) fluorescence, ‘PI-W’ represents the time a nuclei spends while passing through the laser, and ‘Count’ represents the number of visualized nuclei. The first plot shows all the measured particles (black). Gate A represents the selection of all particles with PI labelling and thus represents the nuclei. The second plot shows only the nuclei selected in gate A (blue). Gate B (red) selects the single nuclei and discriminates the clusters. The third plot is a histogram showing only the single nuclei. The large peak at the 50-value represents all diploid cells with a 2n = 8 karyotype. The small peak at the 100-value represents nuclei in the G2 and M phases of the cell cycle with double the amount of DNA. (B) A plot visualizing the size of particles (FSC-A) as a function of their internal complexity (SSC-A). All events are shown in black and include the nuclei and cellular debris. Single nuclei are shown in red. Due to the small size of nuclei in M. lignano , there is considerable overlap between debris and intact nuclei, making this plot type less valuable for gating. (C) Propidium Iodide Area (PI-A)-based histogram representing a culture with three karyotype polymorphisms (1-3) present. The presence of one or two additional large chromosomes increases the amount of DNA in diploid nuclei, causing a clear shift in total PI fluorescence, resulting in this peak pattern. The 2n = 10 karyotype was first observed in subculture 2 at 22 months ( Fig. 1C, F ). By the 26 months timepoint, three subcultures (25%) included this karyotype polymorphism ( Fig. 1C ). At this time, the ratio of karyotypes present in each subculture clearly varied, as shown in Figure 2 . It is worth noting that it is impossible to distinguish worms with different karyotypes by microscopy, suggesting that the karyotype has no clear impact on the morphology or motility of the worms. Interestingly, once a novel karyotype polymorphism with additional large chromosomes appeared, the number of worms in the subculture with this polymorphism always increased over time during our studies, as judged by the increased number of nuclei with a higher DNA content (Suppl. Fig. 2 – 13). Moreover, the karyotype dynamics of experimental subcultures appeared to be unidirectional. Additional chromosomes can be gained but are never lost. These trends are illustrated by subculture 2 in Figure 1 (D-F) . Taken together, this further suggests that aneuploid worms could have fitness advantages and slowly outnumber worms with the original 2n=8 karyotype. As a consequence, the 2n=10 karyotype will become dominant, or even the only one, in laboratory cultures of M. lignano in the long run (Suppl. Fig. 14) Controlling culture karyotypes After the experiment, we maintained the subcultures with the single 2n = 8 karyotype. Six years after the start of the experiment, only subculture 10 showed no additional karyotype polymorphisms. For experiments, we aim to maintain a large wild-type culture without chromosome polymorphisms (NL12S) at all times. To achieve this, we perform annual flow cytometry of the NL12S culture. When chromosome duplications were observed, the culture was discarded, and a new NL12S culture was grown from subculture 10. To keep track of the origin of the NL12S culture, the year in which it was made was added to the name. Currently, we are working with NL12S23 and provide this culture to the community. If needed, novel subcultures can be established from NL12S23 to replace the culture. DISCUSSION In this study, we characterized karyotype dynamics of M. lignano over a period of 26 months using a methodology based on flow cytometry of Propidium Iodide (PI)-labeled nuclei. The essence of this technique is that the intensity of PI fluorescence represents the amount of DNA in the nuclei, which increases with the duplication of large chromosomes, and therefore reflects the karyotype polymorphisms present in a culture. This flow cytometric approach has been previously used to estimate the genome size of multiple eukaryotic species by comparing samples of species with unknown genome sizes to control samples of species with known genome sizes ( Hare and Johnston, 2011 ). During the genome sizing of M. lignano ( Wudarski et al., 2017 ), we recognized the potential of this method for identifying karyotype variations in this species. Traditional metaphase chromosome preparations are essential for identifying different karyotype polymorphisms in a species. However, once these are known, the flow cytometric approach provides several advantages over the traditional method because it is easier, faster, less biased as it is less dependent on personal skills, and it provides information on a large number of nuclei, reflecting the karyotype polymorphisms of numerous worms in a laboratory culture. Using this approach, we unambiguously demonstrated that de novo duplications of large chromosomes occur in laboratory cultures of M. lignano . Moreover, these duplications are not rare, as they occurred in seven of the 12 studied subcultures (58%) within 26 months. Within one year, two subcultures obtained a 2n=9 karyotype; therefore, we recommend that the Macrostomum community evaluate the karyotype of important laboratory cultures at least once a year. The combination of flow cytometry-based karyotyping and the creation of subcultures starting from worm pairs provides a way to study karyotype dynamics and control culture karyotypes. Subcultures can be made at any time, but maintaining a small number of subcultures that are evaluated yearly makes it possible to quickly grow a large experimental culture with only the 2n=8 karyotype when required. Importantly, these tools are not only convenient for maintaining the 2n=8 karyotype, but could also be used to accelerate the generation of cultures with increasing numbers of worms with karyotype polymorphisms and to obtain cultures with only 2n=9 or 2n=10 karyotypes. This is facilitated by the unidirectional nature of karyotype changes in laboratory cultures. As previously reported ( Zadesenets et al., 2016 ), the number of aneuploid worms appears to increase over time in laboratory conditions. Moreover, although chromosomes can be gained, the loss of additional chromosomes has not yet been observed. Taken together, these results suggest that aneuploidy is tolerated and can even result in fitness advantages in M. lignano . Tolerance to aneuploidy is not unique to M. lignano and has been previously described in other representatives of the flatworm genus Macrostomum and species from different taxa, including plants, fungi, and protozoa ( Zadesenets and Rubtsov, 2025 ). Interestingly, polyploid organisms are more tolerant to aneuploidy, particularly those that have recently undergone whole-genome duplication ( Zadesenets and Rubtsov, 2025 ). M. lignano falls within this category as it is a hidden polyploid with the large chromosome being formed by a fusion of all ancestral small chromosomes ( Zadesenets et al., 2023 ). This unusual genomic composition of M. lignano makes it a special case where the concepts of aneuploidy and polyploidy merge because the duplication of the large chromosome also means another duplication of the ancestral whole genome. Whole-genome duplications have been reported in different invertebrate lineages, including rotifers, snails, nematodes, and arthropods ( Au et al., 2025 ). However, despite invertebrates comprising more than 95% of all described animal species, the study of WGD events in invertebrate lineages is still in its infancy compared to that in plants, fungi, and vertebrates. M. lignano represents an interesting and convenient invertebrate model to perform research on the mechanisms of karyotype and genome evolution after a recent whole genome duplication event. MATERIAL AND METHODS Macrostomum lignano cultures Worms were kept in Petri dishes with Guillard’s f/2 medium ( Anderson et al., 2005 ), a nutrient-enriched artificial sea water medium, at a salinity of 32‰. The dishes also contained pre-grown diatoms of the unicellular species Nitzschia curvilineata (SAG, Göttingen, Germany) as a food source. Petri dishes with worms and diatoms were maintained with a 14h/10h day/night rhythm and a temperature of 20°C or 25°C. Increasing the temperature from 20°C to 25°C increases the fertility of worms and shortens the total generation time without inducing stress ( Wudarski et al., 2019 ), which enables growing larger cultures in less time. Worms were transferred weekly or biweekly to new Petri dishes containing fresh medium and diatoms. Creating and maintaining experimental subcultures Experimental subcultures of M. lignano were created by placing pairs of juvenile worms in separate wells of 6-well plates containing f/2 medium and diatoms. Once they reached adulthood, the couples began reproducing, resulting in subcultures. All worms within a well were transferred weekly to new wells containing fresh media and food. When a well contained multiple adult worms, they were transferred to a Petri dish for further subculture growth. Most subcultures contained hundreds of worms within 2 months, and all worms were transferred weekly to new dishes with fresh medium and diatoms to provide ad libitum food. Because all worms were transferred, the subcultures included a mix of different generations of worms. Multi-well plates and Petri dishes with worms were maintained at 25°C with a 14h/10h day/night rhythm. Tracking karyotype evolution of M. lignano laboratory cultures To study the de novo appearance of karyotype polymorphisms under laboratory conditions, 12 experimental subcultures of wild-type worms were created. These subcultures were maintained at 25°C and studied every 2 months for a total period of 26 months. The karyotype polymorphisms were analysed using flow cytometry of PI-labelled single-nuclei suspensions of at least 100 worms. The first timepoint (2 months) deviates as it lacks for subcultures 3, 6, and 9, and the analysis of subcultures 1, 10, and 11 was performed with less than 100 worms. This is due to differences in the initial growth of the subcultures. Generating single-nuclei suspensions of Macrostomum lignano To prepare a suspension of PI-labelled single nuclei, at least 100 worms were selected and starved for 24 h to eliminate residual diatoms present in the gut. The worms were then collected in an Eppendorf tube, and the f/2 medium was removed and replaced with 200 µl 1x Accutase (Sigma, A6964). After 15 min of incubation at room temperature, the medium containing worms was pipetted up and down to break the worms into smaller fragments. After 15 min of incubation, the fragments were dissociated into a single-cell suspension by pipetting. Then, 800 µL f/2 medium was added, and the single-cell suspension was transferred to a 15 ml Falcon tube. The cells were pelleted by centrifugation at 1000 rpm for 5 min. at 4°C using a centrifuge with a swing-bucket rotor. The supernatant was aspirated, and the cell pellet was resuspended in 1 ml nuclei isolation buffer (100 mM Tris-HCl pH 7.4, 154 mM NaCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.02% BSA, 0.1% NP-40 in MilliQ water) with RNAse A (10 µg/ml) and PI (10 µg/ml). The nuclei suspension was passed through a 45 µm filter into the tubes for flow cytometry. The samples were then incubated in the dark for 15 min on ice. Flow cytometry-based karyotyping The nuclei suspensions were examined using a BD FacsCanto II Cell Analyzer. Flow cytometry enables the rapid investigation of large numbers of nuclei and, thus, many worms in a culture. As PI intercalates into the major groove of DNA, its fluorescence intensity reflects the amount of DNA present in the nuclei. Therefore, duplications of the large chromosome of M. lignano lead to clear shifts in PI intensity, visualizing the karyotype polymorphisms present in the studied culture ( Fig. 3C ). The shift in PI intensity is probably not enough to identity a change in the number of small chromosomes, but these changes are rare ( Zadesenets et al., 2017b ) and therefore not the focus of this study. The analysis was performed using a straightforward gating strategy in the Kaluza Analysis Software ( Fig. 3 A). Initially, PI-labelled nuclei were selected to distinguish intact nuclei from the cellular debris. Flatworm cell and nuclei suspensions contain large amounts of cellular debris, and it can be di?cult to distinguish debris fragments from nuclei based on size (FSC) and internal complexity (SSC) because of the small size of M. lignano cells and nuclei ( Fig. 3B ). We then discriminated the nuclei aggregates and doublets based on the PI Area and Width characteristics. Finally, a histogram of the PI Area was used to visualize the fluorescence intensity of the PI labelling of the DNA, which reflects the karyotypes present in the culture. Karyotyping with metaphase chromosome preparations Chromosome slides were prepared as previously described ( Wudarski et al., 2017 ; Zadesenets et al., 2016 ). First, the bodies of worms were amputated, and the head fragments were left to regenerate for 48 h to increase the number of mitotic cells. The regenerating heads were collected and treated with 0.2% colchicine (Sigma, C9754-100mg) in f/2 medium for 4 h at room temperature (RT). This step arrests mitosis at the metaphase. The head fragments were then treated with hypotonic 0.2% KCl solution for 1 h at RT. Next, the fragments were placed on clean glass slides in a mixture of 4:3:3 distilled water:ethanol:glacial acetic acid and macerated into small pieces using pulled glass pipettes. A mixture of 1:1 ethanol:glacial acetic acid was added dropwise to the cells, followed by pure glacial acetic acid. The slide with the fixed material was dried at 60°C and stained using Vectashield with DAPI (Vectrolabs, H-1200). Images were captured using a Zeiss Axio Scope A1 microscope with an MRc5 digital camera. AUTHOR CONTRIBUTIONS Conceptualization: S.M., E.B.; Funding Acquisition: E.B.; Experiments: S.M., L.G.; writing: S.M., E.B. COMPETING INTERESTS The authors declare no competing or financial interests. FUNDING This work was supported by UMCG core funding to EB. DATA AND RECOURCE AVAILABILITY All relevant data and details of the resources can be found in this article and supplementary figures. FIGURE LEGENDS Supplementary Figure 1 . Selection of NL10 subcultures to create NL12S . (A) Propidium Iodide Area (PI-A)-based histograms of 14 NL10 subcultures. Several subcultures have additional peaks representing karyotype polymorphisms. As these measurements are performed on a small number of worms (<100), different peaks are less distinct from each other than in other experiments. (B) The subcultures which showed a single peak, were analysed again 6 weeks later. Only four subcultures (3; 6; 9; and 12) still showed a single peak and were merged into a new NL12S culture with a single 2n = 8 karyotype. Supplementary Figure 2 . Karyotype dynamics of NL12S subculture 1 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, and 2 represent the 2n = 9 karyotype. Supplementary Figure 3 . Karyotype dynamics of NL12S subculture 2 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, 2 represent the 2n = 9 karyotype, 3 represent the 2n = 10 karyotype. Note that the data for months 2, 6, and 22 are also represented in main Figure 1 . Supplementary Figure 4 . Karyotype dynamics of NL12S subculture 3 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. This culture did not obtain chromosome duplications, and is characterized by a 2n = 8 karyotype for the complete period. Supplementary Figure 5 . Karyotype dynamics of NL12S subculture 4 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, and 2 represent the 2n = 9 karyotype. Supplementary Figure 6 . Karyotype dynamics of NL12S subculture 5 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. This culture did not obtain chromosome duplications, and is characterized by a 2n = 8 karyotype for the complete period. Supplementary Figure 7 . Karyotype dynamics of NL12S subculture 6 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, and 2 represent the 2n = 9 karyotype. Supplementary Figure 8 . Karyotype dynamics of NL12S subculture 7 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, 2 represent the 2n = 9 karyotype, 3 represent the 2n = 10 karyotype. Note that the data for the sixth month is also represented in main Figure 1 . Supplementary Figure 9 . Karyotype dynamics of NL12S subculture 8 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, and 2 represent the 2n = 9 karyotype. Supplementary Figure 10 . Karyotype dynamics of NL12S subculture 9 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. This culture did not obtain chromosome duplications, and is characterized by a 2n = 8 karyotype for the complete period. Supplementary Figure 11 . Karyotype dynamics of NL12S subculture 10 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. This culture did not obtain chromosome duplications, and is characterized by a 2n = 8 karyotype for the complete period. Supplementary Figure 12 . Karyotype dynamics of NL12S subculture 11 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. This culture did not obtain chromosome duplications, and is characterized by a 2n = 8 karyotype for the complete period. Supplementary Figure 13 . Karyotype dynamics of NL12S subculture 12 for a period of two years . The subculture was analysed every 2 months and data are visualised as (PI-A)-based histograms. The number of peaks is indicated: 1 represent the 2n = 8 karyotype, and 2 represent the 2n = 9 karyotype. Supplementary Figure 14 . Long-term karyotype dynamics . The NL12S23 culture as a reference of a culture with only the 2n = 8 karyotype represented as a single peak (1), located at the 50 value on the X-axis. The NL10 culture after 11 years of culturing without interfering with the karyotype. The 2n = 8 , and corresponding peak (1) completely disappeared. There is a small peak (2) representing the 2n = 9 karyotype, and a large peak (3) representing the 2n = 10 karyotype. This suggests that, in the long run, the worms with the 2n = 10 karyotype outcompete the others. ACKNOWLEDGEMENTS We thank Kirill Ustyantsev for providing feedback on the manuscript and Frank Beltman for helping with metaphase chromosome preparations. REFERENCES ↵ Anderson , R. A. , Berges , R. A. , Harrison , P. 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Share Tracking karyotype dynamics by flow cytometry reveals de novo chromosome duplications in laboratory cultures of Macrostomum lignano Stijn Mouton , Lisa Glazenburg , Eugene Berezikov bioRxiv 2025.10.30.685567; doi: https://doi.org/10.1101/2025.10.30.685567 Share This Article: Copy Citation Tools Tracking karyotype dynamics by flow cytometry reveals de novo chromosome duplications in laboratory cultures of Macrostomum lignano Stijn Mouton , Lisa Glazenburg , Eugene Berezikov bioRxiv 2025.10.30.685567; doi: https://doi.org/10.1101/2025.10.30.685567 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 Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41911) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13371) Ecology (19887) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22482) Immunology (17728) Microbiology (40363) Molecular Biology (17163) Neuroscience (88536) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15129) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9817) Zoology (2269)