Hybridity of mainly asexually propagating duckweeds in genus Lemna - dead end or breakthrough?

Hybridity of mainly asexually propagating duckweeds in genus Lemna - dead end or breakthrough? | 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 Hybridity of mainly asexually propagating duckweeds in genus Lemna - dead end or breakthrough? View ORCID Profile Yuri Lee , View ORCID Profile Luca Braglia , View ORCID Profile Anton Stepanenko , View ORCID Profile Jörg Fuchs , View ORCID Profile Veit Schubert , View ORCID Profile Silvia Gianì , View ORCID Profile Leone Ermes Romano , View ORCID Profile Giovanna Aronne , View ORCID Profile Chiara Forti , View ORCID Profile Ingo Schubert , View ORCID Profile Laura Morello doi: https://doi.org/10.1101/2025.08.11.667838 Yuri Lee 1 Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR) , Via Alfonso Corti 12, 20133 Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yuri Lee Luca Braglia 1 Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR) , Via Alfonso Corti 12, 20133 Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Luca Braglia Anton Stepanenko 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) , 06466 Gatersleben, Stadt Seeland, Germany 3 Department of Molecular Genetics, Institute of Cell Biology and Genetic Engineering , NASU, 03143 Kyiv, Ukraine Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anton Stepanenko Jörg Fuchs 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) , 06466 Gatersleben, Stadt Seeland, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jörg Fuchs Veit Schubert 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) , 06466 Gatersleben, Stadt Seeland, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Veit Schubert Silvia Gianì 1 Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR) , Via Alfonso Corti 12, 20133 Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Silvia Gianì Leone Ermes Romano 4 Department of Agricultural Sciences, University of Naples “Federico II” Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Leone Ermes Romano Giovanna Aronne 4 Department of Agricultural Sciences, University of Naples “Federico II” Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giovanna Aronne Chiara Forti 1 Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR) , Via Alfonso Corti 12, 20133 Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chiara Forti Ingo Schubert 2 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) , 06466 Gatersleben, Stadt Seeland, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ingo Schubert For correspondence: lauraemmamaria.morello{at}cnr.it schubert{at}ipk-gatersleben.de Laura Morello 1 Institute of Agricultural Biology and Biotechnology, National Research Council (IBBA-CNR) , Via Alfonso Corti 12, 20133 Milan, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laura Morello For correspondence: lauraemmamaria.morello{at}cnr.it schubert{at}ipk-gatersleben.de Abstract Full Text Info/History Metrics Preview PDF Summary The cosmopolitan, mainly vegetatively propagating, organ-reduced monocotyledonous aquatic duckweeds are the smallest and fastest growing angiosperms, distributed world-wide and flower rarely in nature. Recently, we reported intra- and interspecific hybrids and ploidy variants in the genus Lemna . Thus, contrary to the expectation, sexual propagation may occasionally occur within and between Lemna species. Our main goal was to uncover whether the ecologically successful hybrids are evolutionary dead ends, or initiate further speciation and novel sexual recombination. We investigated flower development, pollen viability, seed set and seed germination in hybrids and their parental species and characterized genome size and genetic markers in the progenies. Intraspecific crosses yielded fertile progeny, but all dihaploid and triploid interspecific hybrids were male sterile. Only an established allotetraploid hybrid reproduced sexually, while colchicine-induced allotetraploids from dihaploids did not re-gain sexual competence so far. We concluded that only established allotetraploid hybrids represent an evolutionary break-through in duckweeds. Our results regarding sexual traits within the duckweed genus Lemna , and the sexual competence of diverse hybrids i) pave the way for further investigation in this understudied field, ii) provide fundamental data regarding the evolutionary potential of duckweed hybrids and iii) are important for future breeding efforts on this emerging crop. Introduction Hybridization, resulting from sexual crosses between genetically distinct populations or taxa and yielding fertile progeny ( Mallet, 2007 ; Abbott et al., 2013 ; as cited in Vallejo-Marin and Hiscock, 2016 ), has long been considered as a major mechanism of speciation in the plant kingdom ( Stebbins, 1959 ; Abbott, 1992 ; Arnold, 1997 ; Rieseberg & Willis, 2007 ; Soltis & Soltis, 2009 ; Abott & Loren, 2012; Depotter et al., 2016 ). Indeed, it has been estimated that at least 25% of vascular plants hybridize naturally ( Mallet, 2005 ; Vallejo-Marin & Hiscock, 2016 ). The ability to reproduce sexually is a prerequisite for hybridization which in turn is often related to polyploidization. Therefore, understanding the mechanism of sexual reproduction in a given plant can elucidate its evolutionary origin and potential. Although most plant species reproduce sexually and some can generate hybrids in overlapping geographic areas (hybrid zones), other plants (for example some aquatics) reproduce extensively without sex ( Kliber & Eckert, 2005 ; Y.Y. Zhang et al., 2010 ), making it more difficult to understand their speciation and evolutionary potential. The cosmopolitan, monocotyledonous aquatic duckweeds, the family Lemnaceae Martinov, emerged as model plants in basic research as well as for diverse commercial exploitation ( Zhang et al., 2010 ; Tonon et al., 2017 ; Sońta et al., 2019 ; Acosta et al., 2021 ; Romano et al., 2024 ; Song et al., 2025 ), due to their small size and rapid growth. Some species of the duckweed genus Lemna L., despite their mainly asexual propagation, revealed an unexpected ability to form interspecific hybrids, as recently reported ( Braglia et al., 2021a , b , 2024 ; Ernst et al., 2025 ; Stepanenko et al., 2025 ; Michael et al., 2025 , preprint). The genus Lemna , the largest in the duckweed family, has evolved around 2.7–41.7 Ma [crown age] ago from a common ancestor in North America ( Tippery & Les, 2020 ). It includes four sections ( Alatae Hegelm., Biformes Landolt, Lemna L. and Uninerves Hegelm.; Bog et al., 2020 ) and, so far, 14 taxa including three nothospecies: Lemna aoukikusa Beppu et Murata, L . × mediterranea Braglia et Morello, and L. × japonica Landolt ( Ernst et al., 2025 ). Plants of the Lemna genus are simply structured. They consist of just a leaf-like organ of a few millimetres in diameter, called frond, and one vestigial root. During vegetative propagation, new daughter fronds arise from two meristematic pockets in the mother frond and form colonies of 2 to 50 ( L. trisulca L.) cohering individuals. They double their biomass in two or three days under optimal conditions ( Rusoff et al., 1980 ; Landolt & Kandeler, 1987 ; Ziegler et al., 2015 , 2023 ). The occasionally appearing flower is surrounded by a calathiform spathe with one pistil and two stamens, different from the related genera Wolffia Horkel ex Schleid. and Wolffiella Hegelm. which have only one pistil and one stamen per flower ( Landolt, 1986 ). Although all Lemna species have been reported to flower in nature, this seems to occur on an annual basis only for some species, at some places, e.g. L . aequinoctialis Welw., while being a rare event for others, e.g. L. valdiviana Phil. and L. obscura (Austin) Daubs. The natural flowering rate of the majority of species is estimated as 1.5 to 14%, mainly based on herbarium specimens, although some species, such as those of section Alatae , can reach up to 60% flowering fronds in nature (see p.167–169 in Landolt, 1986 ). Thus, the occurrence of natural interspecific hybrids in the two Lemna sections, Lemna and Alatae , was rather unexpected. Lemna × japonica (unidirectional cross L . minor L. ♀ × L . turionifera Landolt ♂, see Braglia et al., 2021a , b ; Ernst et al., 2025 ) and L . × mediterranea ( L . gibba L. ♀ × L . minor ♂ and reciprocal, see Braglia et al., 2024 ) are common in sect. Lemna . Both hybrids include dihaploid (homoploid) and more commonly triploid cytotypes, with reciprocal subgenome compositions in L . × japonica ( Braglia et al., 2024 ; Ernst et al., 2025 ; Michael et al., 2025 , preprint). In the sect. Alatae , the L . aequinoctialis complex includes hybrids between L . perpusilla Torr. × L . aequinoctialis , previously described in Japan as L . aoukikusa ( Beppu et al., 1985 ), its triploid and tetraploid backcrosses to L . aequinoctialis as well as more recent reciprocal hybrids of L . perpusilla × L . aequinoctialis ( Stepanenko et al., 2025 ). The origin of these hybrids is shown schematically in Fig. 1 . The genetic diversity and the wide geographic distribution of the hybrid accessions ( Braglia et al., 2021b , 2024 ; Volkova et al., 2023 ; Schmid et al., 2024 ; Romano et al., 2025 ; Stepanenko et al., 2025 ; Michael et al., 2025 , preprint) document their successful recurrent interspecific hybridization. However, information is missing which factors favour hybridization and what is the impact of hybridization on the evolution of the prevailingly asexually propagating Lemna accessions. Moreover, information on sexual traits of Lemna species is scarce and hampered by the uncertainty of morphology-based species determination as well as the occurrence of cryptic hybrids ( Braglia et al., 2021b , 2024 ; Stepanenko et al., 2025 ). Download figure Open in new tab Figure 1. The origin of the investigated hybrids of section Lemna (A) and sect. Alatae (B) of genus Lemna . Only gamete types involved in hybridization are shown. Each coloured ellipse indicates one chromosome set of presumed female or male gametes. Lpe = L . perpusilla , Lae = L . aequinoctialis ; Capital letters indicate involved genomes. U indicates the L. × aoukikusa chromosomes in backcrosses. The dashed circle surrounding the haploid L . × aoukikusa gamete indicates the lack of a dihaploid L . × aoukikusa accession in our collection. Considering hybridity, possible misclassifications and occasional mislabelling of clones over time, some previous species assignments might be unreliable. This makes some data questionable and calls for a more systematic revision. The reproductive success of duckweed hybrids is almost unknown and it is urgent to answer the question whether Lemna hybrids are an evolutionarily dead-end or a breakthrough for prospective speciation. Knowledge about reproductive traits is also instrumental for ongoing exploitation of duckweed as new crops for feed and food production as well as for future breeding efforts. In this study, we focused on the possible role of interspecific hybridization in duckweed evolution, and how different mating systems can shape the direction of the genetic flow. We report a systematic, comparative analysis of the flower organ morphology and reproductive traits of natural and artificially induced inter- and intraspecific hybrids in sections Lemna and Alatae of the genus Lemna . Upon flower induction, male and female organ development, pollen viability, seed set, seed viability, and genome size of seeds and progenies were investigated in genetically and cytogenetically characterized clones of hybrid accessions and their parental species. Dihaploid L . × mediterranea and L . × japonica accessions were treated with colchicine to investigate potential restoration of fertility in the resulting allotetraploid progeny. Our results show how different hybrids have different evolutionary prospects. Materials and Methods Plant material and clone maintenance We selected representative clonal accessions of interspecific Lemna hybrids and their parental species, based on previous genetic and cytogenetic studies ( Braglia et al., 2024 ; Ernst et al., 2025 ; Stepanenko et al., 2025 ). Clones were from the original Landolt’s collection, from other collections, or collected by authors, as indicated in Table 1 ; All accessions, are now available at the duckweed collection of the Institute of Agricultural Biology and Biotechnology (IBBA) in Milan (Italy; Morello et al., 2025 ). All clones were cultured under axenic conditions in SH or Hoagland medium ( Hoagland & Arnon, 1950 ; Schenk & Hildebrandt, 1969 ) supplemented with 0.1 or 0.2% sucrose. The pH was adjusted to 5.0. View this table: View inline View popup Table 1. Duckweed accessions used in this study Genome duplication via colchicine treatment To investigate whether dihaploid hybrids can recover fertility when becoming allotetraploid, we treated 20 healthy fronds of the dihaploid clones L . × mediterranea 6861 and L . × japonica 8434 for 24 h with 0.05% colchicine, washed twice in sterile water, and subsequently propagated individual fronds in at least two rounds up to 5–10 fronds, until the clonal populations were stably tetraploid. After each round of clonal propagation genome size was measured. Genomic in situ hybridization (GISH) Fronds grown in SH medium with 0.5% sugar were treated in 0.02 M 8-hydroxyquinoline at 37□ for 1 h and then fixed in fresh 3:1 (absolute ethanol:acetic acid) for 24 h, subsequently washed twice in 10 mM Na-citrate buffer pH 4.6 for 10 min each, before and after softening in 2 ml PC enzyme mixture [2% pectinase and 2% cellulase in Na-citrate buffer] for 60 min at 37□, before maceration and squashing in 60% acetic acid on glass slides. After freezing in liquid nitrogen, the slides were treated with pepsin, (50 µg pepsin/ml in 0.01N HCl, 5 min at 37□), post-fixed in 4% formaldehyde in 2 × SSC for 10 min, rinsed twice in 2 × SSC, 5 min each, dehydrated in an ethanol series (70, 90 and 96%, 2 min each) and air-dried. The genomic DNA probes of diploid L . minor and L . gibba , respectively, were prepared following the procedure described by Hoang & Schubert (2017) . First, genomic DNA isolated from L . minor (8703) and L . gibba (9577) was sonicated to obtain fragments of 500□1000 bp which served as template for labelling with specific fluorescent dyes. The DNA was subjected to nick-translation using the Atto488 and Alexa594 NT Labeling Kits (Jena Bioscience, Jena, Germany), respectively. After precipitation with 96% ethanol, probe pellets were dissolved in 100 μl hybridization buffer (50% (v/v) formamide, 20% (w/v) dextran sulfate in 2 × SSC, pH 7). Labelled genomic DNA of L . minor and L . gibba was applied to well-spread chromosome preparations of the species of probe origin and those of presumed hybrids. Probes were denatured at 95□ for 5 min and chilled on ice for 10 min before adding 10 µl of each probe per slide. Then, the chromosome preparations were denatured together with the probes on a heating plate at 80□ for 3 min, followed by incubation in a moist chamber at 37□ for at least 16 h. Post-hybridization washing was done as described ( Lysak et al., 2006 ) with minor modifications. Microscopic preparations were analyzed via spatial structured illumination microscopy (3D-SIM) as described ( Weisshart et al., 2016 ; Stepanenko et al., 2025 ). We used an Elyra 7 microscope system equipped with a 63 × 1.4 □1 Plan-Apochromat objective and the ZENBlack software (Carl Zeiss GmbH, Jena, Germany). Image stacks were captured separately for each fluorochrome using 405 nm (DAPI), 488 nm (Atto488) and 561 nm (Alexa594) laser lines for excitation and appropriate emission filters ( Weisshart et al., 2016 ; Kubalova et al., 2021 ). Flower induction and sexual organ observation Flower induction (FI) was carried out based on the methods of Lee et al. (2024) and Romano et al. (2025) . Asexually propagated fronds were cultured in plastic boxes (10 × 10 cm) with 100 ml of modified, NH + free 20 -1 Hutner liquid medium ( Hutner, 1953 ). The medium basically included 1% sucrose and the pH was adjusted to 6.2. We added 30 µM Salicylic Acid (SA) to clones of sect. Lemna , or 10 µM Benzoic Acid (BA) to sect. Alatae , and kept them under a 16 h light/8 h dark photoperiod (white light, 11,750 lux-150 μmol m □2 s □1 ) at 25□ for 10–14 days. To monitor flower development, the fronds showing flower primordia were transferred to 90 × 20 mm petri dishes, in the same FI medium made semisolid by including 0.3% agar. To facilitate cross-pollination, the jars containing flowering duckweeds in liquid medium were briefly gently shaken every morning for at least two weeks. Pollen viability test by Alexander staining, DAPI staining and germination test For pollen observation, fronds immobilized on the semi-solid FI medium were observed daily to select flowers having freshly opened, mature anthers. In case a mature anther did not spontaneously open, hypodermic needles were used to dissect the pollen sac. The middle part of filament was carefully cut by micro-scissors and moved onto a glass slide into a drop of 5 μl Alexander staining solution ( Alexander, 1969 ) under a stereomicroscope. Pollen release was supported by hypodermic needles before the slide was covered with a coverslip and checked after 30–60 minutes’ incubation using an optical microscope. For each clone, 10 anthers (from 10 flowers) were individually examined. Only pollen grains whose whole cytoplasm looked deeply stained were considered as viable. As we usually could see naturally dropped pollen grain germination on the FI medium during flower development observation ( Fig. 2A , inset), we used the same media without any SA or BA addition for in vitro pollen germination test in petri dishes. The medium was equilibrated to room temperature for 1–3 h before use. Just-opened anthers were individually moved onto the germination plate and pollen grains were carefully spread on the agar surface with hypodermic needles at 25 ± 1□. Petri dishes were then sealed with parafilm, and kept under the same light and temperature condition as for FI, for 24 h. Pollen germinability was calculated as percentage of germinated pollen per anther. Download figure Open in new tab Figure 2. Flowers and pollen of Lemna minor , L . turionifera and L . gibba. (A□C) L. minor 5500. (A) Flowering plant with mature anthers while pistil is already wilted and (inset) germinating pollen; (B) Alexander-stained pollen; and (C) DAPI-stained trinucleate pollen with one vegetative nucleus (red arrow) and two sperm nuclei (white arrows). (D□F) L . turionifera 9434. (D) Flowering plant with mature pistil and not yet developed stamen (arrow head); (E) Alexander-stained pollen grains and (F) germinating pollen. (G□I) L . gibba 7742a. (G) bearing a fruit having typically winged margin and pigment on surface (arrow head); (inset) dehiscing anther (an) and self-pollinated maturing stigma (st); (H) Alexander-stained pollen; and (I) germinating pollen, white arrows indicate elongating pollen tubes. Based on preliminary Alexander staining or pollen germination test, the pollen maturation stage was determined by fluorescent nuclei after staining with 0.1% (w/v) DAPI (4’,6-diamidino-2-phenylindole) solution in deionized water. We put 10 not naturally-opening anthers (from 10 flowers) on a glass slide into 8 μl of DAPI solution and opened the pollen sac with a needle to spread pollen grains. After overnight-incubation in the dark, pollen was observed and photographed under a fluorescence microscope (AXIO Observer 7, Zeiss, Oberkochen, Germany). Artificial cross-pollination Two closely related, diploid L . aequinoctialis clones, 9661 and NGY122 ( Stepanenko et al., 2025 ), were cultured under FI condition for two weeks. As soon as stigma was carrying a droplet of exudate, six flowering fronds of 9661, chosen as maternal parent, were transferred to the semisolid medium. We carefully cut the anther filament of NGY122 flowers whose anthers had just opened, with micro-scissors, and directly touched the stigma of the maternal plant. All cross-pollination procedures were conducted under a binocular microscope (Wild Heerbrugg, Switzerland) in the sterile bench hood. Seed germination Mature seeds of BOG0007, 9661 × NGY122, NGY142 and 6746YL, 7742a, were collected and each seed was independently moved to 24-well-plates with 2 ml of Hoagland medium (including 0.2% sucrose) under 16 h of light (11,750 lux-150 μmol m□² s□¹) at 25□. Seeds were cultured for 45 days, and their germination was checked every morning by microscopy. DNA extraction, TBP amplification, capillary electrophoresis and data analysis Total DNA of the three F1 hybrid progenies of L . aequinoctialis 9661 × NGY122 was extracted from 100 mg of fresh fronds, after squeezing at 30 Hz for 80 sec using a TissueLyser II (Qiagen, Aarhus, Denmark) in 2-ml Eppendorf tube with 5□8 mm stainless steel beads. DNA was then isolated according to the standard protocol of the DNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark), the DNA quality and amount were evaluated by UV absorbance with the Nanodrop 2000C Spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, United States), and DNA was stored at −20□ until use. Tubulin-Based Polymorphism (TBP) profiling via 1st and 2nd intron amplifications was conducted using 30 ng of genomic DNA, following Braglia et al. (2021a) . The PCR amplicons were checked on 2% agarose gels, before running Capillary electrophoresis (CE) separation. The data collection was independently performed for the two beta-tubulin intron amplifications, using Gene Mapper Software v.5.0 tools (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to Braglia et al. (2021a) . The peak size (base pairs) and height (in relative fluorescence units = RFUs) composing the CE-TBP profiles of the L. aequinoctialis hybrid progeny were collected in Microsoft Office Excel files, and compared with those of the two parental accessions. Flow cytometric ploidy measurements To find out whether sexual progeny is of homogeneous and stable ploidy, we measured the genome size of the seed-derived progeny of the tetraploid L . × aoukikusa accession NGY142 which frequently self-pollinates in vitro ( Lee et al., 2024 ). Asexually propagated fresh fronds from 20 out of 50 seeds, after incubation in Hoagland medium (0.2% sucrose) under 16 h of light per day (11,750 lux-150 μmol m□² s□¹) at 25□ for 50 days, were chopped with a razor blade together with an appropriate amount of fronds of the parental clone NGY142 and applying the ‘CyStain PI Absolute P’ nuclei extraction and staining kit (Partec-Sysmex). After filtering the suspension of nuclei through a 50 µm mesh (CellTrics, Sysmex-Partec), samples were analyzed on a CyFlow Space flow cytometer (Partec-Sysmex, Görlitz, Germany). The remaining 30 seeds were immediately collected and kept in a refrigerator at 4□ for two months in the dark. Each seed was independently chopped using the ‘CyStain UV Ploidy’ nuclei extraction and staining kit (Partec-Sysmex) in isolation buffer according to manufacturer’s instructions. The nuclear suspension was filtered and measured on a BD Influx cell sorter (BD Biosciences). The peak indicating 2C nuclear DNA content of embryo cells was adjusted based on 2C nuclei of maternal fronds. Results While flowering has been reported for all species in the Lemna section Lemna , detailed descriptions of sexual organ development, fruiting and mature seed formation are rather scarce ( Landolt 1986 ; Sarin et al., 2023 ). Accessions belonging to five different species of the Lemna genus and their natural hybrids of different ploidy, a natural tetraploid clone of L . gibba , as well as an artificial intraspecific L. aequinoctialis hybrid were investigated ( Table S1 ). View this table: View inline View popup Supporting Table 1. Genome size, ploidy level, and flowering traits of accessions used in this study. Sexual traits differ within sect. Lemna Flowers usually appeared 12□14 days after treatment with the appropriate phytohormones. The genus Lemna predominantly showed flowers whose pistil develops first and stamens grow asynchronously. However, maturation time of female and male organs typically differed among species. Lemna minor and L . turionifera have protogynous flowers and are self-sterile These two sister species showed a very similar flower development, but a distinctive trait was identified: the spathe colour, transparent in L . minor , and reddish in L. turionifera , reflected different frond pigmentation ( Fig. S1 ). Flowers were mostly strictly protogynous ( Fig. 2A, D ), the pistil came out from the frond pouch and matured first, as deduced from the droplet of stigmatic fluid which has been traditionally considered as a signal of pistil maturity ( Landolt, 1986 ). The two stamens started growing, maturing, and shedding viable pollen grains asynchronously ( Fig. 2 , Table 2 ), when the pistil started wilting. Pollen appeared regularly shaped and, after DAPI staining, was normally trinucleate ( Fig. 2C ) and viable, as observed by frequent pollen grain germination on the semisolid nutrient medium ( Fig. 2A , inset). As pistil and stamens did not mature simultaneously, self-pollination was prevented. In addition, no fruit or seed has ever been obtained in liquid medium even after gently shaking the flasks, suggesting also cross-pollination between flowers from different fronds of the same clone is impaired. Therefore, L . minor and L . turionifera can be considered self-incompatible. Download figure Open in new tab Supporting Figure 1. Excised Flowers of sect. Lemna . Flower organs are surrounded by a spathe (arrows), transparent in Lemna minor and L . gibba (white arrows). Deeply purplish or with few purple spots in L. turionifera and L . × japonica , respectively (red arrows). The spathe of L. gibba has been detached from the flower. (Scale bars = 200 μm). A detailed explanation of flower structure is in Fig. 7C . View this table: View inline View popup Table 2. Seed germination rate of Lemna gibba , L . aequinoctialis , L . perpusilla and L . × aoukikusa Diploid Lemna gibba has homogamous flowers and is self-fertile So far, L . gibba ( Fig. 2G , inset and Table S1 ) is the only species of sect. Lemna , having homogamous flowers. In the two diploid strains, 7742a and LER010, flowering started from the 12th day of flower induction. Even though the pistil appeared first, the first stamen immediately started growing and matured before the pistil withered. Also the second stamen usually matured while the pistil was still viable. Regularly shaped, viable, trinucleate pollen grains frequently dropped onto the mature stigma ( Fig. 2g, H, I ). Self-pollination yielded a fruit with red pigment on the surface and winged margins ( Fig. 2G ), usually carrying one to three seeds (frequently only one fully developed and one or two aborted). Seeds were obtained easily from both L . gibba clones, and over 50% of seeds germinated in the nutrition medium after one day ( Table 2 ). Tetraploid Lemna gibba has homogamous, but aberrant and self-sterile flowers Natural tetraploid L . gibba (7245) started becoming slightly gibbous from the seventh day after flower induction. The first flower primordia were detected after one month. Usually, the first stamen and the pistil appeared together. During pistil growth, the yellowish first anther started withering. The second stamen developed soon after. However, stigma sometimes did not mature until stamens wilted. Hence, flowers of 7245 are not strictly homogamous. Shape and size of pollen grains were irregular, and the number of nuclei was variable ( Fig. 3D ), indicating at least partial male sterility. Naturally dropped pollen grains on the immature stigma and its germination on solid medium were sometimes observed, but fruit or seeds were never found. The ovary usually contained one or two apparently normal ovules, sometimes enlarging while a droplet was still present on the stigma, suggesting a parthenocarpic development of fruit without fertilization. Irregular pistils (double-pistils from one ovary, cracked stigma and style, and winged style, as if spathe and style were combined), were observed in 7 out of 40 flowers ( Fig. 3A□C ). Download figure Open in new tab Figure 3. Sexual organ abnormalities of the natural tetraploid Lemna gibba 7245. (A) Double-pistil-(pi)-occurrence on the same ovary (ov), the first anther (an1) is already wilted, the second anther (an2) is releasing pollen grains; (B) cracked, abnormal stigma (st) with two dehiscing anthers (an), fr = frond; (C) winged style (stl) and one dehiscing anther; (D) DAPI-stained pollen grains. The number of nuclei varies from zero to three (normal pollen), one vegetative (red arrow) and two sperm nuclei (white arrows). Lemna × japonica hybrids have protogynous flowers and are male-sterile Representative clones of two different cytotypes of L . × japonica [dihaploid (MT): 9421, triploid (MMT): 8627] showed strictly protogynous flowers as do the parental species. The spathe color in both cytotypes was pale red or purple spotted, intermediate between parents ( Fig. S1 ). As the pistil developed, the stigma emitted the exudate normally ( Fig. 4A ). Both anther filaments started growing after the pistil wilted, but anthers never dehisced ( Fig. 4A , inset). The pollen grains were usually irregularly shaped and not roundish. Alexander staining was weak, and none, or occasionally one or two nuclei were detectable by DAPI-staining ( Fig. 4B, C ). Fruits or seeds never occurred even in liquid medium, indicating male sterility of dihaploid and triploid hybrids. Download figure Open in new tab Figure 4. Sexual organs of Lemna × japonica . (A) Flowering plants of dihaploid 9421; (inset) a non-dehiscent anther pushed out by a nascent daughter frond; (B) Alexander-stained, and (C) DAPI-stained pollen grains. White arrow head indicates the droplet on the stigma. Dihaploid and triploid Lemna × mediterranea hybrids are male-sterile Both cytotypes of the natural interspecific hybrid L . × mediterranea : 6861 [dihaploid (MG)] and LER021, 7763, 9248 [triploid (GGM)] always developed pistil first ( Fig. 5A ), as the parental species L . minor . Stamens of dihaploid L . × mediterranea 6861 started arising when the stigma had no exudation anymore. Anthers were already whitish when stamens started growing and never dehisced until withering ( Fig. 5B ). On the other hand, triploid L . × mediterranea accessions (LER021, 7763, 9248), with two subgenomes from the female parent L . gibba, and one from L. minor , the pollen donor, sometimes showed apparently normally developed stamens with yellowish anthers, dehiscing when the stigma was still covered by the droplet ( Fig. 5C ). Despite such a tendency to homogamy, no seed was ever produced due to pollen sterility. Pollen grains were irregularly shaped and sized, never fully stained by Alexander staining solution ( Fig. 5D ). Most pollen seemed to be empty; only rarely one or two nuclei were visible by DAPI staining ( Fig. 5E ). Download figure Open in new tab Figure 5. Sexual organs of Lemna × mediterranea . (A) The whole flowering plant. (B) Whitish, indehiscent anthers (an1 - an2) of dihaploid 6861. (C) Bursting anthers of triploid LER021, next to the pistil (pi) with mature stigma with droplet; (D) Alexander-stained and (E) DAPI-stained pollen grains of clone 6861. (F-G) Unfertilized, enlarged ovaries (red arrows) of dihaploid 6861 and triploid 9248. Occasionally we found enlarged ovaries (parthenocarpy) from both L . × mediterranea cytotypes (ca. 10%), after their flowers completely withered ( Fig. 5F, G ). The enlarged ovaries harboured 1□2 brownish immature seed(s) which never completed development. The colchicine-induced allotetraploids Lemna × mediterranea and L . × japonica remained sterile Somatic whole-genome doubling (WGD) in the dihaploid hybrids L . × mediterranea 6861 and L . × japonica 8434 was induced by colchicine treatment to test for restoration of fertility, because meiotic bivalent formation should be possible upon chromosome duplication. The allotetraploid clone 6861b displayed larger fronds ( Fig. 6A ) and duplicated genome size (477 vs. 973 Mb/1C, Table S1 ). GISH with L . gibba and L . minor differently labelled fluorescent DNA probes showed duplication of each parental chromosome set in 6861b ( Fig. 6B ), compared to the original clone 6861 ( Fig. 6C ), while in the triploid clone 7763, only the chromosomes of the maternal parent L . gibba were duplicated ( Fig. 6D ). Download figure Open in new tab Figure 6. (A) Fronds of the natural dihaploid clone 6861 (left) are smaller than those of the artificially induced tetraploid descendant 6861b progeny (right). (B□D) Fluorescence in situ hybridization with genomic DNA (GISH) of Lemna minor (red) and L . gibba (green) on mitotic chromosomes of L . × mediterranea clones. (B) colchicine-induced tetraploid 6861b. (C) dihaploid 6861, (D) triploid 7763. The flower development in the artificial tetraploid was similar to that of the original diploid 6861. Anthers were yellowish and seemed vigorous at first, but the color turned gray while the filament became longer, and never dehisced. Most pollen was irregularly shaped and empty, only occasionally harboring one or two but never three nuclei. Thus, fertility could not be restored in the artificial induction of allotetraploidy in L . × mediterranea clone 6861b. Clone 6861b also showed aberrant flowers, with two fused carpels, enclosed by one spathe ( Fig. 7A□C ) together with one pair of stamens. Like in the dihaploid and triploid cytotypes, enlarged ovaries (fruit) were detected, and even double flowers, where the coalescent ovaries sometimes swelled together ( Fig. 7D, E ). Doubled ovaries contained none or one whitish, immature seed, which later became aborted. Some of the parthenocarpic fruits showed fissures in the central part and cracked during growth. Download figure Open in new tab Figure 7. Sexual organs of the induced allotetraploid hybrids. (A□E) Lemna × mediterranea 6861b (A) Fronds with emerging pistils (white arrow heads). (inset) Pistils (pi) are made up by one carpel each. (B) Two carpels and a pair of stamens surrounded by a transparent spathe. (C) Schematic presentation of (B) anther (an), ovary (ov), pistil (pi) and spathe (sp). (D) Fronds bearing parthenocarpic fruits (red arrows). (E) Different developmental stages of excised flower organs: (a□b) Ovaries bearing two and one ovules, respectively (dotted-lined circles indicate ovule position); (c) parthenocarpic fruit after the stigma completely withered. (F□H) L . × japonica 8434a. (F) Protogynous flowering plants with mature pistil (white arrow head), and dehiscent anthers (inset and yellow arrow head) while pistil is already wilted; (G) DAPI-stained pollen grains. Red and white arrows indicate one vegetative and two sperm nuclei, respectively in trinucleate pollen grains; (H) Pollen grains do not germinate. Genome-duplicated allotetraploid L . × japonica 8434a (471 vs. 940 Mb/1C, Table S1 ) showed protogynous flowers ( Fig. 7F ). However, different from the original dihaploid clone 8434 which showed indehiscent anthers, 8434a naturally opened anthers at maturity, spreading pollen grains ( Fig. 7F , inset). Few pollen grains were trinucleate but no pollen germinated in vitro ( Fig. 7G, H ). Artificial pollination was not successful. Fruiting was never detected. Sexual reproduction in sect. Alatae Section Alatae includes the two sister species: i) Lemna perpusilla , distributed in North Eastern America, and ii) L . aequinoctialis , ubiquitous across tropical, subtropical and temperate regions, worldwide ( Landolt, 1986 ; Bog et al., 2020 ; Stepanenko et al., 2025 ). A third presumed species, was described by Beppu et al. (1985) as L. aoukikusa , but considered by Landolt (1986) and Borisjuk et al. (2015) as a geographic variant of L. aequinoctialis . Recent evidence based on plastid markers, flower development ( Lee et al., 2024 ), genetic and cytogenetic evidence ( Stepanenko et al., 2025 ), suggested that the presumed L. aoukikusa accessions represent a fertile allotetraploid hybrid between L . aequinoctialis and L . perpusilla originated relatively far in the past, because plastid and nuclear sequences already diverged from those of the parental species ( Stepanenko et al., 2025 ). Genetic evidence also showed further gene flow between L . × aoukikusa and L . aequinoctialis as well as the existence of other, more recent hybrids between L . aequinoctialis and L . perpusilla in America ( Stepanenko et al., 2025 ). Given the complexity of the group, investigation of reproductive processes and sexual traits in fully characterized accessions, and in comparison with the parental species, is fundamental to identify distinctive traits and to understand the evolutionary potential of these hybrids. Lemna aequinoctialis displays protogynous, self-sterile flowers but inter-clonal fertility Two diploid L . aequinoctialis accessions 9661, NGY122 showed mostly strictly protogynous flowers ( Fig. 8A ). The anthers appeared after the pistil had already matured. While the first stamen matured, the droplet on stigma disappeared and pistil wilted. Fruits or seeds were never obtained even after facilitating cross-pollination through gently shaking of the liquid medium. This suggests self-incompatibility of this species. Fruit and seed traits have been considered the most relevant for species determination in section Alatae , in particular the development of indehiscent fruits in L . perpusilla in comparison with free-threshing seeds of L . aequinoctialis (Kandler & Hügel, 1974). Albeit L . aequinoctialis is known as self-sterile ( Beppu et al., 1985 ; Lee et al., 2024 ; this work), artificial cross-pollination between the two clones was successfully conducted and yielded seed. This was also useful to compare seed traits between parental species and hybrids. Download figure Open in new tab Figure 8. Diagram of flower development and seed formation in the genus Lemna sect. Alatae. (A) Two different kinds of protogynous flower: (A1) when the anther release pollen grains, the pistil wilts already from the middle part of the style, but inter-clonal pollination is possible in BOG0007; (A2) After pistil wilts, anthers wilt without dehiscence (B) Two different kinds of homogamous flowers: (B1) the pistil matures first but stamen(s) grow while the pistil is still receptive (stigma has droplet), thus pollen can reach the stigma; (B2) when the first stamen grows, the pistil is still immature (short style, no receptive droplet at stigma) but along with dehiscence of the first anther, the pistil matures and pollen grains may reach the stigma for self-pollination. Numbers in the diagram indicate the developing order of organs. SI: self-incompatible, MS: male sterile, SC: self-compatible. Out of six reciprocal artificial cross-fertilization trials between L . aequinoctialis accessions 9661 and NGY122, three yielded fruit and seed. When the three yellowish-green fruits matured, the pericarp became very thin, transparent, and finally the pale brown-colored, mature seed naturally dropped from the fruit. All three seeds (named P07, P12, P14) germinated 14□19 days after transfer to nutrient medium without any cold treatment ( Fig. 9A, B ), and fronds started propagating clonally. TBP profiling showed different segregation of parental alleles in the three F1 hybrids, proving that the seeds were not derived from self-pollination ( Fig. S2 ). The three progenies have similar genome size of 488□491Mb/1C, indicating they are diploid, as their parental clones ( Table S1 ). Flowering induced in the F1 lineages of P07, P12, and P14 showed protogynous, self-sterile flowers identical to those of the parental clones. Download figure Open in new tab Supporting Figure 2. Capillary electrophoresis TBP profile comparison (1st and 2nd intron regions) of three Lemna aequinoctialis artificial intraspecific hybrids P07, P12 and P14 (9661 × NGY122) and their parents proves inter-clonal fertility. The red arrows indicate typical alleles from parental clones. Download figure Open in new tab Figure 9. Seeds and their germination in Lemna sect. Alatae . (A) Naturally dropped, ripe seeds of Lemna aequinoctialis (9661 × NGY122). (B) Cotyledon of L . aequinoctialis is emerging from seed; operculum (op) and hypocotyl (hy) are visible. (C) Seeds of L . perpusilla BOG0007 kept in the fruit attached to the dying mother frond. The seed is germinating from an indehiscent fruit (yellow arrow). Each scale bar indicates 500 μm. Lemna perpusilla displays variable flower development and differs regarding self-compatibility The pistil always developed first in L . perpusilla , but two types of flower development were observed among three diploid North American accessions analysed (BOG0007, 8539 and 8479). BOG0007 displayed incomplete protogyny with occasional overlap of pistil and stamen maturation. Anthers naturally dehisced and spread pollen. Fruits were not produced by colonies fixed on semisolid medium, but by gently shaking in the liquid medium, fronds were easily fruiting. Hence, even if direct self-pollination is impeded by protogyny, BOG0007 ( Fig. 8A 1) can be pollinated by other genetically identical individuals. Only four out of 29 fruits naturally shed ripe seed, sinking in the liquid medium; the other 25 fruits kept their seed inside, still attached to the senescing frond, as it is reported to occur in L . perpusilla . Nineteen out of these 25 seeds germinated without any cold treatment at the next day after transfer to nutrient medium ( Fig. 9C ). In clones 8473 and 8539, the first stamen immediately developed following the pistil and matured while the stigma was still covered with exudation ( Fig. 8B 1). Indeed, we could frequently see the pollen grains on the same flower’s stigma in solid medium culture. However, despite pollen of 8539 was regularly shaped and sized, and stably trinucleate, we never found fruits or seeds, neither on solid nor in liquid media ( Table S2 ). Therefore, these two homogamous strains are self-incompatible, in contrast to BOG0007. View this table: View inline View popup Download powerpoint Supporting Table 2. Pollen viability (Alexander staining) and germination rate of Lemna species and hybrids. (-) not tested.not tested. Ancestral tetraploid Lemna × aoukikusa hybrids between L . perpusilla and L . aequinoctialis are self-compatible The flower development and self-compatibility of L . × aoukikusa were described in detail previously ( Beppu et al., 1985 ; Lee et al., 2024 ) and are confirmed here for clones NGY140, NGY142 and 6746YL: while the first two were already demonstrated to belong to the L . × aoukikusa tetraploid group ( Stepanenko et al., 2025 ), the assignment of clone 6746YL to L . × aoukikusa was confirmed by nuclear markers ( Fig. S3 ) and genome size measurement ( Table S1 ) in this work. As already shown by Lee et al. (2024) , the 6746YL clone displayed the typical homogamous flower development of L . aoukikusa ; the pistil appeared after the first stamen matured, followed by the second stamen ( Fig. 8B 2). We could spontaneously get seeds after flowers withered on the semisolid medium, confirming self-compatibility. Seeds of NGY142 and 6746YL naturally dropped when fruit ripened, and 30% (NGY142) to 53% (6746YL) of seeds started germinating from day 11 and 4, respectively ( Table 2 ). Although BA-induced flowering was not successful for clone NGY140, fronds, kept in the dark for one month, naturally bore seeds after flower wilting. A few seeds already germinated on semisolid medium in the petri dish. Thus, all tested L . × aoukikusa accessions are self-compatible. Download figure Open in new tab Supporting Figure 3. TBP tree of sect. Alatae clones used in this study. Left and right side of coloured bar indicates maternal and paternal species, respectively. The tetraploid hybrid Lemna × aoukikusa, accession NGY142 generates tetraploid progeny by self-pollination Genome size measurement of 20 progenies of the self-pollinating tetraploid hybrid L . × aoukikusa NGY142 showed stable and homogenous ploidy, with a 2C DNA amount, representing the cell population in G1 or G0 phase of the cell cycle, identical to the fronds of the parental accession, in all samples ( Fig. 10A ). Download figure Open in new tab Figure 10. Ploidy of the seed progeny of Lemna × aoukikusa NGY142 after self-pollination. (A) Nuclei isolated from mother plants; (B) single seed showing 2C and 4C peaks (the latter possibly representing cells in G2 phase); (C) single seed with an additional 3C peak (apparently endosperm remnants). Out of 20 seeds immediately incubated in nutritional medium, 15 showed only 2C and 4C peaks (G0/G1 and G2 cells of the embryo) ( Fig. 10B ), while five showed an additional 3C peak, as expected for the triploid endosperm ( Fig. 10C ). However, none of 30 seeds which were kept at 4□ before testing displayed a 3C peak ( Fig. 10B ), suggesting endosperm degradation. Of the more recent natural hybrids of Lemna aequinoctialis and L . perpusilla the tetraploid is fertile, but the triploid is not Another tetraploid natural hybrid BOG0001 ( L . aequinoctialis × L . perpusilla ), showed homogamous flowers similar to the more ancient tetraploid hybrids ( L . × aoukikusa ; Fig. 8B 2). The pistil normally developed with exudation, two stamens dehisced and naturally dispersed pollen grains. Pollen grains, looked vital after Alexander staining (92.4 ± 8.9%), displayed three nuclei after DAPI staining and germinated. Fruits and seeds were often detected on solid medium (38 seeds from 85 flowers). However, different from L . × aoukikusa , ripe seed usually did not drop from the fruit, as in its paternal species, L . perpusilla . We also analysed clone 7006, a natural triploid hybrid ( L . aequinoctialis × L . perpusilla , Fig. S3 ). Different from L. × aoukikusa , the triploid hybrid developed the pistil first, and sometimes stamens developed when the pistil had not yet withered ( Fig. 8A 1). Although stamen(s) sometimes dehisced, the released pollen was irregularly sized and shaped, and not viable, like in the triploid hybrids of section Lemna . Fruits were never found. Natural backcross progenies of Lemna × aoukikusa to L . aequinoctialis seem to be sterile Both triploid backcross hybrid clones 0098 and 9669 had protogynous flower, the prolonged filament appeared one day after pistil completely wilted and the whitish anthers never dehisced. Flower induction with BA did not function regularly for clone NGY128, which is the only known tetraploid backcross hybrid in sect. Alatae . We got flowers only once but were unable to induce flowering again under the same conditions, thus we could observe only few flowers. The flowers were protogynous, the pistil matured with a normal droplet and anthers naturally dehisced. However, pollen grain size and shape were irregular and little pollen appeared viable after Alexander staining. DAPI staining showed trinucleate as well as empty pollen (not shown). Fruit set was never detected. Apparently, male and female sexual processes are often disturbed, making sexual reproduction a rare or even impossible event. Discussion Sexual reproduction and its role in the evolutionary path of mainly clonally propagating duckweed is an underestimated issue. Large intraspecific genetic diversity, polyploidy and the recently discovered interspecific hybrids, however, highlighted the role of sexual reproduction in duckweeds, albeit occurring only occasionally in many species. Despite the loss of hundreds of genes in the course of evolution ( Michael et al., 2021 ; Ware et al., 2023 ), duckweed reduced but did not completely abandon sexuality as apparently did some other clonal plant species ( Eckert, 2001 ). So far, most studies on duckweed flowering focused on its hormonal or light regulation rather than on reproductive morphology and physiology ( Hillman 1957 , 1961 , 1963 ; Maheshwari & Chauhan, 1963 ; Maheshwari & Gupta, 1967 ; Cleland & Tanaka, 1979 ; Tanaka & Cleland, 1980 ; Cleland & Ben-Tal, 1982 ; Khurana & Maheshwari, 1983 ; for review see Kandeler, 1984 ). Furthermore, in older publications, the identification of the plant species investigated, was often uncertain, due to organ reduction, overlapping morphological traits, frequent changes in taxonomic treatment and overlooked hybridity. Only recently optimal conditions for in vitro flower induction ( Fu et al., 2017 ; Fourounjian et al., 2021 ) and genomics and transcriptomics of flowering have been addressed for diverse Lemnaceae ( Fu et al., 2020 ; Yoshida et al., 2021 ; Fu et al., 2024 ), albeit neither nuclear markers nor genome size measurement to confirm species identity were included. Our study, conducted under the same inducing conditions on 29 Lemna accessions, representing different interspecific hybrids of the genus Lemna and their parental species, revealed variation in reproductive pathways between and within species and their hybrids. Different reproductive behaviour in section Lemna and the likely reasons for hybrid sterility Lemna turionifera and L. minor revealed the same mating system, with protogynous flowers as most of the Araceae Juss. ( Mayo et al., 1997 ), in accordance with previous findings ( Landolt, 1986 p.179; Lee et al., 2023 ). In spite of mature, vital pollen, they produce no seeds, suggesting that self-incompatibility (or genetic deficiencies), in addition to dichogamy, largely avoid self-fertilization within clonal populations in these species. Moreover, such mechanisms favour outbreeding within and between the two species as shown by the various L . × japonica hybrids ( Braglia et al., 2021b ; Schmid et al., 2024 ; Ernst et al. 2025 ; Michael et al., 2025 , preprint). As L. minor is always the maternal parent, it can be hypothesized that either pollen-stigma incompatibility exists between L. minor and L. turionifera , or that L. turionifera has a reduced female fertility, due to the absence of RanGAP genes, as suggested ( Ernst et al, 2025 ). The two dihaploid and one triploid L . × japonica clones showed normal flower development, but anthers remained indehiscent, full of unviable, irregularly shaped pollen grains ( Table S2 ). Male meiosis seems to be affected in both subgenome combinations, consistently with what observed in another triploid cytotype having two L. turionifera subgenomes (clone 7182, MTT), which displayed the same features ( Lee, 2023 ). Similar male-sterile flowers and unviable pollen, were reported for L . minor clones ( Fourounjian et al., 2021 ) which were identified only by plastid markers, suggesting they could have been L . × japonica instead. Conversely, homogamy and self-compatibility were confirmed by our observation for L . gibba on both diploid clones investigated: 7742a, derived from the widely investigated clone G3 ( Kandeler, 1955 ), and LER010, recently collected in nature ( Romano et al., 2025 ). Both clones produce abundantly seeds of high germination rate in liquid and on semi-solid medium, as reported by Landolt for L. gibba 7741 (G2; Kandeler, 1955 ), and later confirmed ( Fu et al., 2017 ; Fourounjian et al., 2021 ). Abundant seed production in L . gibba also occurs in nature ( Landolt, 1957 , p.372; 1986; Giuga, 1973 ). However, the natural autotetraploid L . gibba 7245, while showing normal vegetative growth and propagation, displays dichogamous flower and frequent flower abnormalities (winged style, enlarged ovary, two pistils from one ovary) under our conditions ( Fig. 3 ). It is not yet clear whether at least a few normal male and female gametes are produced by 7245, but no seed was ever produced from 40 flowers observed. Fertility in neo-polyploids, in particular autopolyploids and odd polyploids, is often reduced because of meiotic chromosome mis-segregation, but also by aberrant anatomy such as lacking pollen tube formation in Arabidopsis ( Westermann et al., 2024 ). A spontaneous somatic whole genome duplication in diploid L . gibba under long-term stress in vitro has been described, but floral traits were not investigated ( Sarin et al., 2023 ). If female fertile tetraploid L. gibba clones spontaneously occur, they could have contributed to generate the natural triploids of L . × mediterranea with two L. gibba subgenomes. Aberrant flowers were also observed in dihaploid L. × mediterranea , showing double flowers and parthenocarpic fruits (see below). Hybridization and polyploidy are both known for their genetic and genomic impact, involving genome rearrangements and transposon activation, whose extension may vary in nature and intensity across species or between different lines of a species, impair transcriptome regulation and potentially cause phenotypic alterations ( Soltis et al., 2016 ). Several rounds of selfing may be required in order to stabilize genomes after hybridization and/or polyploidization ( Mason & Wendel, 2020 ). However, the predominantly clonal reproduction in Lemna tolerates mutations affecting sexual reproduction. Aberrant flower and fruit development occur in sterile Lemna × mediterranea Lemna is known for having bisexual flowers with a monocarpellate gynoecium. Several abnormal flowers, as we observed in tetraploid L. gibba , have been reported for the genus in the past (e.g. basal part of two carpels which are independently surrounded by spathe fused to each other) ( Witztum, 1966 cited in Landolt, 1986 ; p.91), but polycarpellate flowers and parthenocapic fruits found in the hybrid L . × mediterranea have never been reported before. Abnormal flowers mediated by gene mutations have been well studied in Arabidopsis thaliana ( Bowman et al., 1989 ; Lenhard et al., 2001 ; Yumul et al., 2013 ). Possibly, formation of aberrant flowers in L . × mediterranea could be the consequence of asynchronous or conflicting expression of flower development genes, which differ in expression time between the parents and disrupt normal flower development in the hybrids. Such flower defects were not observed in L . × japonica, whose parental species display similar flower development. L . × mediterranea , similar as L . × japonica, is male-sterile, although in some cases anthers dehisce and shed pollen over the mature stigma, as observed in the early description of the hybrid under the name L . symmeter Giuga ( Giuga, 1973 ). Transcriptome studies have been performed to dissect flower development in L. gibba ( Fu et al., 2020 ). Abnormal flowers of L . gibba hybrids and polyploids may represent future targets for identifying genes responsible for either homogamy or protogyny in duckweed. A second unusual trait of dihaploid, triploid and induced tetraploid male-sterile L . × mediterranea clones, including those which have indehiscent anthers, is the initiation of fruit development without pollination and fertilization, called parthenocarpy. This phenomenon is frequently found in tomato, grape, eggplant and strawberry ( Rotino et al., 1997 ; Acciarri et al., 2000 ; Donzella et al., 2000 ; Mezzetti et al., 2004 ; Costantini et al., 2007 ) as a selected trait to obtain seedless fruits. The frequent occurrence of parthenocarpy among polyploid and hybrid species suggests wide hybridization as possible force driving parthenocarpy ( Picarella & Mazzuccato, 2019 ). This is in line with the observed flower abnormalities in L . gibba hybrid and tetraploid clones, and could also be due to disrupted events of reproductive development. Parthenocarpy could be mediated by genetic factors or environmental stress, eventually altering hormonal regulation. Phytohormones (i.e. auxin, gibberellin, salicylic acid) activate molecular mechanisms that provoke development of parthenocarpy in some plants ( Wang et al., 2005 ; Goetz et al., 2006 ; Zhang et al., 2021 ). Although we cannot exclude that SA, used to induce flowering, also affected fruit development in L . × mediterranea, the abnormal polycarpellate flowers and parthenocarpic fruits were not observed in L . × japonica or in Lemna section Alatae after SA application. From these data, and the lack of natural fertile tetraploid hybrids, despite quite a large number of collection clones investigated ( Braglia et al., 2021b , 2024 ; Michael et al., 2025 , preprint), we conclude that all dihaploid and triploid hybrids of L. minor are sexually deficient and represent dead-ends regarding further evolution via sexual recombination. This is not surprising considering disturbed meiotic chromosome segregation, as long as not a (rare) mechanism of regular segregation of entire chromosome complements is active, as e.g., in pentaploid dogroses ( Herklotz et al., 2025 ). In the dogroses, homologous chromosome complements form bivalents during meiosis I and are inherited biparentally, while unique chromosome sets are inherited uniparentally via female gametes only. Fertile natural allotetraploids could theoretically overcome this problem via regular bivalent formation, but such hybrids are not known among a large number of collected clones for section Lemna ( Braglia et al., 2021b , 2024 ; Michael et al., 2025 , preprint). Colchicine-induced tetraploidy in the dihaploid L . × mediterranea 6861 and L. × japonica 8434 so far failed to restore hybrid fertility, which occurred for instance in Brassica ( Katche & Mason, 2023 ). The induced polyploids produced no fertile male gametes and no seeds, similar to the original dihaploid ancestors. In nature, polyploidy of interspecific hybrids occurs either via early somatic genome duplication of dihaploid hybrid genomes, or via fusion of unreduced parental gametes ( Olsen et al., 2006 ). However, the degree of immediate fertility of allotetraploids is related to the degree of divergence between the genomes of the parental taxa, i.e. the degree of homologous vs. homoeologous meiotic chromosome pairing ( Olsen et al., 2006 ; Contreras & Ranney, 2007 ). Even in successful cases, the initial fertility of allopolyploids is usually very low and gets stabilized only after selection during several selfing generations ( Katche & Mason, 2023 ). In fact, natural, normally developing, fertile allotetraploids occur in hybrids of the section Alatae . Mating systems vary within section Alatae The section Alatae , traditionally considered as including only two species, L . aequinoctialis and L . perpusilla , also includes their hybrid L . × aoukikusa ( Beppu et al., 1985 ; Lee et al., 2024 ), ecologically separated, but able to backcross to L. aequinoctialis , thus forming the L . aequinoctialis species complex ( Stepanenko et al., 2025 ). Different from the sterile F1 hybrids of section Lemna , L . × aoukikusa seems to be an established lineage in which somatic exchange between homeologous chromosomes took place, as deduced from GISH experiments ( Stepanenko et al., 2025 ). Whether L . × aoukikusa originated from unreduced female and male gametes or by somatic doubling of a dihaploid hybrid is not known, and can only be estimated by a systematic search for occurrence of unreduced gametes in the parental species. Our study made clear that diploid L . aequinoctialis is protogynous and self-sterile ( Table S1 ), but manual inter-clonal crossing yields fertile diploid progeny. This holds at least for Asian accessions, in agreement with the description of Beppu et al. (1985) for L . aequinoctialis sensu stricto , and by Lee et al. (2024) , but is in contrast with previous data reporting this species as homogamous and self-compatible ( Landolt, 1986 ). However, most older studies, including Landolt’s description, were based on the famous accession 6746 ( Hillman, 1958 ; Oda, 1962 ), then identified as a tetraploid hybrid ( Stepanenko et al., 2025 ; this work). However, it has to be noted that under the same ID 6746 diverse taxa apparently occur in different collections. Already in 1965 two different 6746 lineages from different laboratories, showed different photoperiodic responses ( Umemura and Oota, 1965 ). We also find a similar occurrence in this study, working on clones from two different collections. In fact, clone 6746YL, corresponds to L . × aoukikusa according to Lee et al. (2024) , as confirmed by TBP in this study. Conversely, clone 6746 coming from the original Landolt’s collection and described by Stepanenko et al. (2025) is also a tetraploid hybrid, with L . perpusilla as maternal parent, but it belongs to the recent hybrid cluster by TBP genotyping ( Fig. S3 ). In this clone 6746 ( Stepanenko et al., 2025 ), flowers could not be induced so far. It has been considered that seeds of L . perpusilla do not germinate without cold treatment ( Kandeler & Hügel, 1974 ). However, we detected that ca. 80% seeds of L . perpusilla (BOG0007) can germinate without cold treatment ( Table 2 ). While we used only one clone for seed germination, but several flower developmental patterns have been identified within L . perpusilla ( Fig. 8 ), it is possible that in future clone-specific requirements for seed germination will be uncovered. In contrast to L . aequinoctialis , L . perpusilla seems to have different mating types ( Fig. 8 ): i) protogynous flowers unable to self-pollinate but compatible with pollen from other fronds of the same clone ( Fig. 8A 1), and ii) homogamous but self-incompatible flowers, never producing seeds with pollen from the same clone ( Fig. 8B 1). Thus, within the section Alatae we can observe all combinations of mating systems: homogamy, dichogamy, self-compatibility and self-incompatibility, meaning the two mechanisms of controlling outcrossing are independent and can regulate outcrossing frequency in a population. The allotetraploid Lemna × aoukikusa can generate stable sexual progeny Some apomictic plants can form endosperm autonomously, i.e. without central cell fertilization, and result in a nuclear DNA content represented by 2C:4C instead of a 2C:3C embryo:endosperm ratio ( Šarhanová et al., 2024 ). Because 90% of 50 L . × aoukikusa NGY142 seeds obtained after self-pollination displayed a 2C:4C ratio, apomictic reproduction appeared as a possible option of their origin. However, we did not observe any fruit development from 20 emasculated flowers, thus apomixis can be excluded. A 3C endosperm peak was only detected in five of the 20 seeds kept in the nutritional medium. Possibly, a small endosperm is rapidly resorbed during embryo maturation or endosperm is not regularly formed in duckweeds, as in some families where endosperm nuclei degenerate rapidly as in the genus Janusia A. Juss. (Malpighiaceae) ( Souto & Oliveira, 2014 ). Alternatively, some duckweeds do not develop primary endosperm nuclei (exalbuminous seeds) as in Podostemaceae. Considering that complete breakdown of 3C endosperm only happens during seed germination ( Boesewinkel & Bouman, 1995 ), but even seeds collected immediately from fruits displayed no 3C peak, the majority of seeds were most likely spontaneously deficient of 3C endosperm cells. Because a 4C peak was detected in seeds but never in progeny fronds, possibly part of embryonic cells is arrested in G2. The ploidy of 20 tested seed progenies was the same as that of the original maternal NGY142 plants, indicating that unreduced gametes were not involved. Usually, newly synthesized allopolyploids need several generations of sexual reproduction to become meiotically stable, i.e. forming exclusively bivalents between homologs instead of irregular chromosomal configurations ( Mason & Wendel, 2020 ). Section Alatae includes the most regularly flowering species among the mainly vegetatively propagating genus Lemna ( Landolt, 1986 ). This facilitates the origin of interspecific hybrids such as L . × aoukikusa, if parental species are sympatric. Evolutionary prospects differ between interspecific Lemna hybrids Multiple, interspecific hybridization in Lemna section Lemna yielded dihaploid and triploid cytotypes, ( Braglia et al., 2024 ; Michael et al., 2025 , preprint). Both kind of hybrids are quite uncommon in wild plants, due to frequent sterility, but if at least partially fertile, they may serve as bridges to tetraploidy as demonstrated for some species ( Ramsey & Schemske, 1998 ; Husband, 2004 ; Bartolić et al., 2025 ) or eventually directly lead to dihaploid speciation ( Abbott et al., 2010 ; Yakimowski & Rieseberg 2014 ). The prominence of triploids in duckweed has been imputed to the absence of the argonaute gene AGO6, which mutated in Arabidopsis leads to loss of the ‘triploid block’, an embryo/endosperm genomic unbalance impairing triploid seed germination ( Ernst et al., 2025 ). Flower induction in four L . × mediterranea and four L . × japonica clones of different ploidy invariably showed hybrid sterility and production of abnormal, unviable pollen. Natural tetraploid L . × japonica and L . × mediterranea accession were never found so far. This suggests rare fusion of two unreduced gametes (or infrequent early genome doubling in dihaploids). Colchicine-induced allotetraploid L . × mediterranea and L . × japonica derived from male-sterile natural dihaploid hybrids, could not restore fertility. Therefore, both L. minor hybrids seem to represent highly successful asexual lineages, but evolutionary dead-ends. Conversely, self-fertility of allotetraploid hybrids between the two sister species, L. perpusilla and L. aequinoctialis , demonstrates that allotetraploidization is a possible speciation mechanism in the Alatae section, allowed by the reproductive isolation from the parental species, as underlined by sterility of the triploid and tetraploid backcross of L. × aoukikusa to L. aequinoctialis (0098, 9669, NGY128). Whole Genome Sequencing of the parental species and their hybrid L . aoukikusa will help dating the origin of this mainly vegetatively propagating duckweed nothospecies, and understanding how and at what pace its speciation proceeds. Search for further natural hybrids and generation of artificial ones will uncover the limits and opportunities of natural hybridization as well as of artificial interspecific crossing in breeding efforts for duckweeds as emerging crops. Author contribution YL, IS and LM planned and conceptualized the research. YL, LB, AS, JF, VS, SG, LER and CF performed experiments. IS, AG and LM supervised. YL wrote the original manuscript. LB, IS, GA and LM reviewed and edited the manuscript. LM administrated project & funding. Publication ethics The authors declare no competing interests. Acknowledgements We appreciate our colleagues in both IBBA and IPK for their kind support, Prof. Klaus J. Appenroth (Friedrich Schiller University Jena, DE) and Dr. Manuela Bog (University of Greifswald, DE) for sharing their duckweed collection. This study was carried out within project AGRITECH National Research Center funded by EU Next-Generation EU PNRR Missione 4 Componente 2, Investimento 1.4 – (D.D. 1032 17/06/2022, CN00000022) to IBBA-CNR, and Project IR0000032 – ITINERIS - Italian Integrated Environmental Research Infrastructures System. We acknowledge IBISBA-IT CNR-IBBA node for the access provided to the infrastructure (microscope). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them. Anton Stepanenko was supported by a Ukraine Distinguished Fellowship of the German National Academy of Sciences Leopoldina. Funder Information Declared AGRITECH National Research Center funded by EU Next-Generation EU PNRR Missione 4 Componente 2, Investimento 1.4 , D.D. 1032 17/06/2022, CN00000022 IR0000032 – ITINERIS – Italian Integrated Environmental Research Infrastructures System Ukraine Distinguished Fellowship of the German National Academy of Sciences Leopoldina References ↵ Abbott RJ . 1992 . Plant invasions, interspecific hybridization and the evolution of new plant taxa . Trends in ecology & evolution 7 : 401 – 405 . OpenUrl PubMed Abbott RJ , Loren HR . 2021 . Hybrid speciation . eLS 2 : 1 – 9 . OpenUrl ↵ Abbott RJ , Albach D , Ansell S , Arntzen JW , Baird SJE , Bierne N , Boughman JW , Brelsford A , Buerkle CA , Buggs R et al. 2013 . Hybridization and speciation . Journal of Evolutionary Biology 26 : 229 – 246 . OpenUrl CrossRef PubMed ↵ Abbott RJ , Hegarty MJ , Hiscock SJ , Brennan AC . 2010 . Homoploid hybrid speciation in action . Taxon 59 : 1375 – 1386 . 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Yuri Lee , Luca Braglia , Anton Stepanenko , Jörg Fuchs , Veit Schubert , Silvia Gianì , Leone Ermes Romano , Giovanna Aronne , Chiara Forti , Ingo Schubert , Laura Morello bioRxiv 2025.08.11.667838; doi: https://doi.org/10.1101/2025.08.11.667838 Share This Article: Copy Citation Tools Hybridity of mainly asexually propagating duckweeds in genus Lemna - dead end or breakthrough? Yuri Lee , Luca Braglia , Anton Stepanenko , Jörg Fuchs , Veit Schubert , Silvia Gianì , Leone Ermes Romano , Giovanna Aronne , Chiara Forti , Ingo Schubert , Laura Morello bioRxiv 2025.08.11.667838; doi: https://doi.org/10.1101/2025.08.11.667838 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 (7635) Biochemistry (17691) Bioengineering (13892) Bioinformatics (41936) Biophysics (21452) Cancer Biology (18588) Cell Biology (25504) Clinical Trials (138) Developmental Biology (13378) Ecology (19899) Epidemiology (2067) Evolutionary Biology (24320) Genetics (15609) Genomics (22506) Immunology (17736) Microbiology (40394) Molecular Biology (17181) Neuroscience (88605) Paleontology (666) Pathology (2832) Pharmacology and Toxicology (4824) Physiology (7641) Plant Biology (15153) Scientific Communication and Education (2045) Synthetic Biology (4294) Systems Biology (9825) Zoology (2271)