The endomicrobiome and weed invasiveness in Mediterranean ecosystems worldwide

Domain: chemistry
Published: 2026-02-23
Editorial outcome: minor_revision

Abstract
Mediterranean ecosystems, one of the most significant global hotspots of biodiversity, are threatened by invasive weeds. Although endomicrobiomes – the vast array of microbes colonising living plant tissues – are known to affect plant fitness, their contribution to weed invasiveness remains virtually unknown. Here, we experimentally assess the role of the endomicrobiome in the invasiveness of Taraxacum officinale (common dandelion), a widespread weed in Mediterranean ecosystems worldwide. In a culling experiment across five generations, we compare the fitness of T. officinale from these ecosystems on five continents grown with intact or reduced native seed-borne endomicrobiomes. Additionally, we report a competition experiment with F1 and F5 individuals assessing their impacts on native local Asteraceae species. We found that T. officinale individuals harboring intact endomicrobiomes show faster and more favorable trait development compared with individuals with reduced endomicrobiomes. Enhanced competitiveness of endomicrobiome-colonised T. officinale plants with local Asteraceae species is apparently caused by increased synthesis of allelochemicals in shoots and rhizosphere soil, with gene expression analyses also showing the endomicrobiome to affect the expression by T. officinale of stress response and RNA-directed DNA methylation genes. Our findings provide insights into the mechanisms underlying weed invasiveness in Mediterranean ecosystems.

Introduction
Mediterranean ecosystems cover only 5% of the Earth’s land surface and yet support 20% of vascular plant species, and are thus considered to be global biodiversity hotspots1. Currently, these ecosystems face multiple and severe threats, including invasion by alien weed species, which presents a significant risk to their rich native biodiversity and the well-being of their ~500 million human inhabitants2. Invasive alien plant species cause huge economic losses, with plant invasions incurring costs totalling ~$4 billion per annum worldwide3, and almost $10 billion over three decades in the Mediterranean Basin alone4. Invasive species can disrupt ecosystem services, such as water availability and nutrient cycling, and also increase the frequency and intensity of wildfires, compounding their ecological and economic impacts5,6. Hence, understanding the mechanisms underlying the success of alien plant species remains a fundamental goal in invasion ecology7. This knowledge is key to curtailing the impacts of alien species and to developing effective programs to manage invasive weeds in Mediterranean ecosystems8,9.

Non-native plant species colonising new habitats often undergo adaptive evolution, explaining – at least in part – the success of the invasion process10–12. Following arrival in a new community, a lag phase occurs until a plant species increases in abundance and transitions to becoming invasive13. This lag phase is primarily due to initially low population size and genetic variability, which require time to increase and diversify in response to local selection pressures8. For instance, invasive plant species demonstrate remarkable climate adaptability in their invading ranges14, manifested as differentiation among populations in key functional traits enhancing fitness and mechanisms of environmental tolerance15,16. However, although research has focused on genetic differentiation or local adaptation as mechanisms underlying the rapid adaptation rates of invasive species17, the potential role of the endomicrobiome – i.e., the microbes (predominantly fungi and bacteria) colonising mature and healthy plant tissues18 – has garnered little attention as a factor explaining the invasiveness of weed species (but see refs. 19, 20).

The endomicrobiome is known to confer ecological advantages and physiological benefits to plants, including enhanced nutrient uptake, competitive ability, and tolerance to biotic and abiotic stresses21–23. However, its role in plant invasiveness in new habitats remains unknown7,24,25. In one of the few studies to have empirically measured feedbacks between the host genotype and the endomicrobiome, repeated exposures to a herbicide led to herbicide-resistant populations of the invasive weed Echinochloa crus-galli26, which stemmed from co-adaptation between host plants and their vertically transmitted seed microbiome27,28. After five generations, the selected microbiome showed enrichment in a bacterial taxon (Pantoea deleyi SH-355) capable of inducing the expression of genes associated with the metabolism of xenobiotics, such as those encoding for cytochrome P450 monooxygenases and glutathione transferase27. Similarly, an initially strong genotypic effect of Solanum lycopersicum on its endomicrobiome declined over successive generations, resulting in a convergent yet robust microbiome resistant to external invasions29. Although these studies suggest a significant role of the microbiome in plant invasiveness, empirical evidence linking endomicrobiomes to plant competitive ability is still absent from the literature.

Accordingly, we conducted a series of laboratory selection (i.e., culling) experiments using Taraxacum officinale F.H. Wigg. (Asteraceae, common dandelion), a species originating in Eurasia that has become one of the most successful and widespread invasive weeds worldwide30. For this, we collected seeds of T. officinale from Mediterranean ecosystems on five continents and conducted experiments to investigate changes in the invasiveness traits of plants grown across five generations with intact or reduced local seed-borne endomicrobiomes. We selected and reproduced genotypes with the highest performance in each generation, measured by an integrated performance index based on plant fitness proxies. By calculating Haldanes31, we compared the micro-evolutionary rates of the index across generations. Furthermore, we evaluated the competitive ability of T. officinale grown with its intact seed-borne endomicrobiome in the first (F1) and fifth (F5) generations against a phylogenetically closely-related plant species native to each of the five Mediterranean ecosystems studied. We explored potential mechanisms involved in this competitive interplay by measuring soil and shoot concentrations of allelochemical phenolic compounds32. Finally, the expression by T. officinale of genes associated with different aspects of invasiveness, including fitness, environmental tolerance and epigenetic variation, was measured in the F1 and F5 generations. Whilst our experiments could not differentiate between changes in plant phenotype caused by the seed-borne endomicrobiome and genetic drift or maternal effects and other factors33 – and thus did not explicitly test for microbiome-associated adaptive evolution – this approach enabled us to determine whether the endomicrobiome influences the development of fitness traits associated with the invasiveness of a globally important weed.

Results
Our study provides evidence that the vertically transmitted seed-borne endomicrobiome enhances the development across several generations of traits associated with the invasiveness of T. officinale. It indicates that endomicrobiome-associated traits, such as the enhanced expression of stress tolerance genes and production of allelochemicals, confer competitive ability on the weed against native plant species. Although our experiments did not track genetic lineage, measure genome-wide variation, artificially select microbial communities or involve reciprocal transplants of host and microbiome34, they nevertheless detected consistent, multigenerational shifts in fitness-related traits of T. officinale colonised by seed-borne endomicrobiomes. Whilst other factors, notably maternal effects33, could in part explain the plant phenotypic changes reported here, the observations support responses directly mediated by the seed-borne endomicrobiome.

After five generations of experimental selection based on a performance index calculated from integrated measurements of maximum photosynthetic rate, flower head and viable seed production (see “Methods”), distinct developmental trajectories were observed between T. officinale individuals grown with their intact or reduced seed-borne endomicrobiomes (hereafter referred to as E+ and E- plants, respectively). E- plants, which were generated using a fungicide and a bactericide with no effects on plant growth (see “Methods”), consistently exhibited slower and less positive trait development than E+ plants, which was evident in the performance index for the T. officinale populations from each continent and the global mean (Fig. 1) and the three individual traits from which the index was calculated (Supplementary Figs. 1–3). The highly significant endomicrobiome × time (generation) factor in all linear mixed models (Supplementary Data 1–4), along with the significant differences in fitted slopes between E- and E+ plants (Supplementary Data 5), supported these findings. Microevolutionary rate, expressed in Haldanes (the average change in the performance index, in units of standard deviations and corrected for the number of generations) was significantly faster in E+ plants than in E- plants, again for populations from each continent and for the global mean (Fig. 1, and Table 1, Supplementary Fig. 4). Similarly, supplementary experiments confirmed that the endomicrobiome enhanced the performance of T. officinale, with E- plants inoculated with seed-borne microbes displaying the same maximum photosynthetic rate, flower production, biomass and survival as E+ plants (Supplementary Fig. 5).

These findings highlight the central role of the seed-borne endomicrobiome in enhancing the fitness of the invasive weed species T. officinale. The enhanced photosynthetic rate, flower head production and viable seed output observed in E+ plants across generations demonstrate how the endomicrobiome boosts traits linked to competitive capacity, specifically the ability to convert available resources (i.e., photosynthetic rates) into reproductive output (i.e., flowers and viable seeds). Nevertheless, populations from each continent varied in their responses to seed-borne endomicrobiomes, with clear inter-generational reductions in the performance and micro-evolutionary rates of E- T. officinale plants from Europe, South America and Africa, but null and positive effects on those of E- T. officinale from North America and Oceania over five generations, respectively (Fig. 1). This variation was reflected in the global trend, which, despite showing significant differences between E- and E+ plants in both the individual traits and the overall performance index, showed far more variability within the two groups relative to those observed in each Mediterranean ecosystem (Fig. 1; and Supplementary Fig. 1–3).

Having established that the endomicrobiome positively influences the fitness of T. officinale from Mediterranean ecosystems, we sought to further evaluate the ecological implications of this effect for the invasiveness of the species. To this end, we assessed competition, measured by the relative competition intensity index35, between E+ T. officinale and a local native Asteraceae species from each continent, and compared values of the index between the F1 and F5 generations. Although this experimental design did not include a direct comparison between F5 plants grown with intact or reduced endomicrobiomes, it measured the cumulative impacts of selection over five generations on the competitiveness of T. officinale grown with its intact seed-borne endomicrobiome. After five generations, E+ T. officinale plants exhibited increased competitiveness, as indicated by the relative biomass gains among F5 plants compared with F1 individuals, and the detrimental effect on local native plant species (Fig. 2). Although relative biomass gains among E+ T. officinale F5 plants were not observed in all populations, significant biomass losses were evident across all Mediterranean ecosystems among native Asteraceae species, mainly when grown with E+ T. officinale F5 individuals compared with F1 plants (relative to monoculture, Fig. 2; Supplementary Data 6). Interestingly, despite the absence of inter-generational changes in relative biomass gain among T. officinale plants from California and Chile, these populations nevertheless exerted negative effects on their respective local native species in the F5 compared with the F1 generation (Fig. 2).

Although we cannot exclude the possibility of maternal effects influencing the outcome of this experiment33, these findings suggest that T. officinale benefits from seed-borne endomicrobiomes that enhance its competitive advantage over Asteraceae species native to each continent (see “Methods”). They partially support the evolution of increased competitive ability hypothesis36, and indicate that the endomicrobiome, and not a lack of specialised predators or enemies, could explain the enhanced performance of T. officinale in its invaded range. They also suggest that, if microbiomes had been exchanged between T. officinale and each Asteraceae species (see “Methods”), then those associated with E+ seeds did not benefit the native plant species. In addition to the increased competitive ability hypothesis, a further mechanism, the novel weapons hypothesis37,38, has been proposed to explain the role of microbes in improving plant invasiveness7. This hypothesis states that the competitive advantage of invasive plants derives from their ability to release allelopathic chemicals that inhibit the growth of native plants lacking protective mechanisms against these compounds. We accordingly measured phenol concentrations in the soil and shoots of T. officinale and its local native Asteraceae species when grown in interspecific competition or in monoculture. The experiment revealed that when subjected to competition, E+ T. officinale plants increase the production of phenolics in surrounding soil and in shoots, with a more pronounced effect when grown in interspecific competition compared with in monoculture, and with this pattern being consistent across all Mediterranean ecosystems (Figs. 3–4). In contrast, no changes to phenolic concentrations in soils and shoot tissues were observed between F1 and F5 native Asteraceae plants grown in monoculture (Figs. 3–4).

While the role of host-microbe interactions in shaping the macroevolutionary trajectories of plants is well-recognised39, our findings suggest that the endomicrobiome significantly influences plant competitiveness over timescales of just a few generations. They build upon previous research demonstrating that microbes can enhance plant fitness by inducing physiological changes such as increased stress tolerance and the production of defence compounds40–42. Mechanisms involved include the secretion into the rhizosphere of chemicals mimicking plant hormones, the modulation of plant gene expression to trigger physiological changes enhancing plant stress tolerance, and the synthesis of plant growth-promoting hormones40. Collectively, our competition experiments demonstrate that microbial symbionts may significantly enhance the invasive potential of T. officinale, primarily through the increased synthesis of phenolic compounds, which have well-established roles in plant defence against herbivory and competitive interactions with other plants43. These findings align with recent research showing that inoculation with fungal endophytes from extreme environments augments plant fitness and stress tolerance by promoting the production of allelopathic phenolic compounds in the rhizosphere44.

We compared the relative expression of genes in E- and E+ T. officinale plants in order to explore the role of putative molecular mechanisms in the observed inter-generational changes. Independent t-tests showed that, from a global perspective, intact endomicrobiomes typically increased the expression by T. officinale of CHS, COR47, HSP and LEC genes across generations (Fig. 5). These genes are associated with the synthesis of secondary metabolites such as phenols, tolerance to drought and heat and increased seed production, respectively, supporting the findings described above. In addition, increased expression of CHROMOMETHYLASE3 (CMT3), a gene associated with DNA methylation maintenance, was observed in E+ plants (Supplementary Fig. 6), indicating that an increase in DNA methylation activity in E+ plants on specific (although yet to be discovered) genes may enhance their expression levels45. Similarly, in the medicinal plant species Salvia miltiorrizhiza, increased methylation levels of genes associated with the biosynthesis of phenolic acids and tanshinones is concomitant with both increased gene expression and contents of these compounds45. Furthermore, in the plant species Artemisia annua, the endophytic symbiont Pseudonocardia sp. upregulates genes associated with the synthesis of artemisinin, a potent drug for malaria treatment46,47, which inhibits the germination and vegetative growth of several other plant species48. The only gene that was under-expressed by E+ T. officinale plants was IAA12 (Fig. 5), which is associated with auxin repression, perhaps accounting for the increased flower head production of plants colonised by the endomicrobiome (Supplementary Fig. 2). As for the phenotypic results, gene expression profiles varied to some extent between populations, with no inter-generational effects on the expression of the CHS and HSP genes in the Spanish population, the COR47 gene in Australian and American populations and the LEC gene in the South African population (Fig. 5). These observations suggest that endomicrobiomes from different geographical locations may differ in their abilities to influence plant gene transcription across generations. However, collectively, our findings suggest that the endomicrobiome affects gene expression in T. officinale (including RNA-dependent DNA methylation mechanisms), likely underpinning the invasiveness of the weed in Mediterranean ecosystems.

Methods
Seed collection and production of plant material
Seeds of Taraxacum officinale (Asteraceae) were collected from Mediterranean ecosystems on five continents (Fig. 1). Three populations, geographically distanced by ca. 40–60 km, were selected on each continent (North America: 37°17´N–121°44W´, 36°55´N–121°31´W and 36°37´N–121°48´W; South America: 34°08´S–70°44´W, 34°37´S–70°52´W and 34°57´N–71°12´W; Europe: 37°44´N–00°54´E, 38°06´N–00°40´E and 37°32´N–01°23´E; Africa: 33°45´S–18°30´E, 34°04´S–18°50´E and 34°20´S–19°12´E; Oceania: 34°43´S–138°39´E, 35°07´S–138°02´E and 35°20´S–139°02´E). In each population, ~five seeds per individual were collected from ca. 50 widely spaced maternal plants in ruderal areas. All seeds were stored in paper bags under dry conditions until their use in the manipulative experiments. Seeds from each of the 15 populations were pooled and germinated under glasshouse conditions (air temperature 29±4°C and solar radiation 1723±365 μmol m-2 s-1 PAR during daylight hours). The F0 generation of T. officinale was germinated from this seed pool and was grown in a glasshouse at the University of Talca, Chile. F0 plants were placed in 750-ml plastic pots filled with autoclaved field soil and sand (2:1), and irrigated every three days with 75 ml of tap water for five months until they produced seeds. These seeds were used to obtain F1 experimental plants. Plants from the F1 generation were germinated at 30±2°C on wet filter paper in plastic boxes, and the seedlings were planted in 1 L capacity plastic pots filled with autoclaved potting soil (field soil and sand, 2:1 v/v).

One week after the appearance of the first true leaf, half of the plants were irrigated with 50 µg mL−1 of rifampicin, a potent and broad-spectrum systemic antibiotic, and the commercial broad-spectrum systemic fungicide Benlate, to obtain individuals with a reduced complement of local seed-borne endomicrobiomes (hereafter referred to as E- plants). The remainder were irrigated with sterile tap water in order to retain their local endomicrobiomes, i.e., the vertically transmitted seed-borne endomicrobiome acquired from each of the five continents (hereafter E+ plants). By propagating from the seeds of E- or E+ plants over five generations, we were able to maintain plants with reduced or intact endomicrobiomes, respectively (Supplementary Fig. 7). In agreement with previous studies42,56, treatment with rifampicin or Benlate did not result in any visible symptoms or detectable differences in the growth or development of E- plants. The presence of the endomicrobiome was confirmed by observing aniline blue-stained fungal hyphae in leaf, stem and root cross-sections in ca. 2% of plants (Supplementary Fig. 7). In addition, surface-sterilised leaf, stem and root fragments from the same T. officinale individuals were pressed into potato dextrose agar (PDA, Difco, USA) in 90 mm diameter Petri dishes and incubated for 15 days at 27±2°C. These analyses confirmed that E- plants were apparently free of seed-borne endomicrobiomes. Only plants in which fungal hyphae were observed, or from which fungi or bacteria were grown, were considered to be colonised by endomicrobiomes and were used in the experiments described below.

Finally, two seeds from each individual plant from each population were randomly selected to assess for the presence of the seed endomicrobiome. First, one seed was cut in half transversely with a razor blade and was stained with aniline blue prior to observation at 400× magnification (Motic BA310) to record stained fungal hyphae. Second, the remaining seed from each plant was pressed into potato dextrose agar (PDA, Difco) in 90 mm diameter Petri dishes prior to being incubated at 18°C for 2 weeks. Maternal plants were considered to be endomicrobiome-reduced when no fungal hyphae were visually detected in seeds and no hyphal or bacterial growth was observed in the culture medium. Conversely, plants were considered to have intact endomicrobiomes when stained hyphae were visible in the seeds and when either fungal hyphae or bacteria grew from the seeds onto the medium. Although we did not confirm the absence of bacterial cells from E- seeds using microscopy, it is unlikely that unculturable bacteria survived in rifampicin-treated seeds, since this antibiotic has a high affinity for bacterial DNA-dependent RNA polymerase57. Consequently, irrespective of culturability, it inhibits protein synthesis, halting the growth of, and ultimately killing, bacterial cells.

Culling experiment
To measure the performance of E- and E+ T. officinale plants, we calculated an integrated index of seed output, flower head production and photosynthetic rate in a five generation laboratory culling experiment58. Five E- and five E+ plants from each of the 15 populations were grown for five generations at 33±3°C in the same sterile soil and sand mixture as described above. The use of a single sterilised substrate allowed the effect of the vertically transmitted seed-borne endomicrobiome on T. officinale to be isolated, eliminating variability between populations caused by the influence of local soil microbiota on seed-borne endomicrobiomes51. The mixture had a water potential of ca. −36±8 kPa, mimicking frequent summer conditions in Mediterranean ecosystems. Capitula from E− and E+ individuals from each of the 15 populations were enclosed in nylon mesh bags and were allowed to self-pollinate. Although T. officinale is capable of cross-pollination in sexually reproducing diploid individuals, apomixis is common in polyploids, leading to offspring genetically identical to the mother plants59. Therefore, any potential variation in the mating and or reproductive traits reflects the characteristics of the experimental populations. From each population in each generation, the three individuals with the highest seed output (or, as tiebreakers, flower head production or photosynthetic rate) were selected to produce the next generation. Seeds from these selected individuals were used to generate five new plants for the next generation, always maintaining 15 individuals per condition (E- and E+) and generation in each of the three populations from each continent.

Cumulative flower head production (i.e., the number of floral heads) was recorded every three days on each T. officinale individual. Each flower head was labelled in order to avoid duplicate counts. Once a flower head was closed, indicating the start of seed development, it was carefully enclosed in a nylon mesh bag to protect it against seed dispersal. These bagged flower heads were then collected, and the viable seed output was determined by calculating the proportion of filled seeds to the total number of florets per flower head, accounting for both filled, aborted and predated seeds. Finally, maximum photosynthetic rate (Amax) was recorded on healthy leaves with an Infra-Red Gas Analyzer (CIRAS-2, PP-Systems Haverhill, Amesbury, MA, USA). During the measurements, the leaf chamber was set at a CO2 concentration of 400 ppm, temperature of 30°C and irradiance of 1500 μmol m−2 s−1 PAR.

Effects of the endomicrobiome on plant competitive ability
The influence of the endomicrobiome on the ability of T. officinale to compete with Asteraceae species native to each continent was tested by calculating the relative competition intensity index (RCI; 35), using final biomass as the response variable. For each of the five Mediterranean ecosystems, 36 pots (1-L capacity) containing the same mixture of sterile soil and sand as described above were prepared, for a total of 180 pots. Nine pots were each planted with two T. officinale individuals grown from E+ seeds, and another nine pots were each planted with two individuals of native Asteraceae species occupying similar ecological niches (Leontodon taraxacoides from Europe, Taraxacum californicum from North America, Hypochaeris scorzonerae from South America, Ursinia nana from Africa and Actites megalocarpus from Oceania). All native individuals used in the manipulative experiments were obtained from seeds germinated under the same conditions as those described above. The remaining 18 pots were each planted with two individuals of T. officinale (nine pots) and two individuals of the native Asteraceae species (nine pots). This assay was conducted with 360 T. officinale individuals from F1 and F5 (nine individuals × four treatments × two generations × five ecosystems = 360 plants). The 72 pots containing plants from each continent were divided equally between four automated growth chambers set to the same environmental conditions as those described above. The pots were randomised and rotated among chambers to avoid chamber effects. Note that microbiomes may have been exchanged between T. officinale and each of the native Asteraceae species (and vice versa) in these experiments. After 75–90 days, the plants were harvested and the relative competitive impact of T. officinale (invasive) on its native counterpart (native) was calculated as RCI native=([biomass native:native – biomass native:invasive]/biomass native:native), and the resistance of the native counterpart to invasion by T. officinale was calculated as RCI invasive=([biomass invasive:invasive – biomass invasive:native]/biomass invasive:invasive), where invasive and native represent the biomasses of T. officinale and its native counterpart, respectively42.

To assess the potential effect of plant-to-plant competition mediated through the exudation of allelochemicals, we measured the total content of phenolic compounds in fresh biomass and in the soil in which the plant had been grown using the Folin-Ciocalteu assay (with gallic acid as the standard60;). Briefly, the phenols in each sample were extracted with methanol/H2O (4:1, v/v) and homogenised by sonication. The extracts were then stirred for 1.5 h at room temperature and centrifuged at 4,200 g before the supernatant was recovered. Supernatant (100 μl) was oxidised with 200 μl 10% (v/v) Folin-Ciocalteu reagent, neutralised with 800 μl sodium carbonate, and incubated for 2 h at room temperature. The absorbance of the resulting solution was measured at 765 nm, using an Epoch microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). Finally, phenol concentration was calculated using a standard curve of gallic acid60.

Effects of the endomicrobiome on T. officinale gene expression levels
To assess whether the seed-borne endomicrobiome exerts a positive effect on the ecophysiological performance of T. officinale through genetic control, we recorded the differential expression of five genes linked to traits related to invasiveness. The gene expression included in our assessment corresponded to those associated with floral development [IAA12], drought stress tolerance [COR47], synthesis of phenolic compounds [CHS–chalcone isomerase], heat tolerance [HSP], seed production [LEC], and DNA Methylation [CMT3]. Gene expression was assessed in E- and E+ T. officinale plants in generations F1 and F5. To select these genes, a reference transcriptome assembly of T. officinale was obtained from publicly available raw Illumina reads (NCBI accession PRJNA316842; SRA libraries SRR5342876, SRR5342878, SRR5342880, SRR5342882, SRR5342884, SRR5342886). These reads were assembled de novo locally using Trinity version 2.15.1 into 102,882 contigs. The transcriptional abundance was then estimated by aligning raw reads to this transcriptome using BOWTIE, and by counting mapped reads with RSEM61. This transcriptome was also annotated with Mercator4 V5.0 and the Diamond BlastX alignment tool using the NCBI NR protein database as a reference. From this annotated set, five protein-encoding contigs, involved in different plant molecular mechanisms and showing in silico transcriptional evidence (number of mapped reads > 0), were selected for downstream expression analysis using qPCR. Selected contigs encode for: Chalcone-Flavanone Isomerase type III (CHS, “Chalcone”), Dehydrin-COR47 (COR47), Heat Shock Protein 70 (HSP70), Indole-3-acetic acid inducible 12 (IAA12), LEAFY COTYLEDON 1-LIKE (LEC1-LIKE) and CHROMOMETHYLASE3 (CMT3). Using the contigs’ sequences as templates, specific qPCR primers were designed using NCBI Primer Blast. The primers used are listed in Supplementary Data 14.

Statistical analyses
Linear mixed models (LMMs) were employed to examine inter-generational (temporal) variations in Amax, flower head and viable seed production, and the integrated performance index. These analyses were done separately for E- and E+ T. officinale plants. The continent of origin was integrated into the error structure of the LMMs as a random factor, and its significance was assessed using marginal and conditional R2 coefficients. In the context of mixed models, these coefficients quantify the variance explained solely by the fixed factors or the overall model (i.e., fixed and random factors), respectively62. Hence, within each LMM, the difference between the coefficients equals the role of the random factor in the model’s performance, i.e., to the local inter-population variability present in each region. In addition, to explore the overall trend, a global analysis was also conducted including data from all 15 populations (three from each continent) in the same model.

For each trait, as well as for the integrated performance index, deviations of fitted slopes from zero were tested with LMMs. Moreover, the impacts of symbiotic status on these trends were determined by the statistical significance of the endomicrobiome × generation interaction in LMMs, indicating differences in regression slopes between plants with different endomicrobiome statuses. In addition, we calculated the micro-evolutionary rate for each trait and the integrated performance index in Haldanes31. The Haldane units scale the change in a trait across generations (i.e., the model slope) by the magnitude of trait variation. Specifically, we computed Haldanes as the difference between the mean trait values in the F1 and F5 generations standardised by five times the pooled standard deviation of the population63.

For the competition experiments, we examined differences in the relative competition index (RCI) for soil and shoot phenol concentrations for E+ F1 and F5 T. officinale plants, and their corresponding local native Asteraceae species, after being grown together (native – T. officinale) or in monoculture (native – native or T. officinale – T. officinale). Since these variables were derived from competition experiments, the results represent both the effect of invasive T. officinale on the performance of the local native Asteraceae species (the “native” experimental group) and the effect on T. officinale of competing against the local native species (the “invasive” experimental group). One-sample t-tests were used to determine whether RCI averaged for each experimental group was statistically different from zero (where 0 = no effect of competition) and independent two-sample t-tests were used to compare the RCI averaged between generations (F1 vs. F5) within each experimental origin, with significant differences indicating changes to the invasion potential of T. officinale.

Finally, we also used one and two-sample, two-tailed t-tests, to evaluate whether the endomicrobiome influences the relative gene expression of the five stress response genes evaluated in T. officinale across generations. We first obtained a relative ratio between the expression levels of genes in E- and E+ T. officinale plants averaged among individuals from the F1 and F5 generations. We then estimated the significance of the endomicrobiome in determining the relative expression levels of each gene in each generation by determining how different these ratio values were from 1 using one-sample, two-tailed t-test (where 1 = no effect of the endomicrobiome). To determine the occurrence of a given inter-generational change, we searched for significant differences in the expression ratios between F1 and F5 plants using two-sample, two-tailed t-tests, with values > 1 indicating positive effects of the endomicrobiome on gene expression and those < 1 indicating significant genetic repression. Consequently, inter-generational changes (F1 vs. F5) in relative gene expression levels were obtained for T. officinale plants from each Mediterranean ecosystem. To control for multiple comparisons across all pairwise t-tests (both one-sample and two-sample), we applied Benjamini–Hochberg false discovery rate correction64 at q=0.05 within appropriate test families, ensuring conservative and statistically robust data interpretation. All data recorded were incorporated into the analyses. All datasets were tested for normality using the Shapiro-Wilks test, analyses were performed in the R language and statistical environment v.4.2.265 using the “nlme” package (v.3.1-163) for fitting LMMs66, the “emmeans” package (v. 1.11.1) for slopes estimation and paired comparisons67 and the “ggplot2” package (v. 4.0.0) for data visualisation68.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The publication is interesting and is of high interest.\nI have just some minor comments:\n263-274 it is not a proof that the plants have no endophytes. You may just have kill the culturable fraction of microorganisms\nbut not the whole community, especially bacteria. you just show absecne of hyphae in E- but not bacteria. So you may need\nto rephrase part of your paragraph\nThere is the need to provide more details on how seed E- were propagated and how plants look like after E-. Was there\nseveral generations of plants derived from the seeds that have been studied or only one?\nYou might need to further proof your hypothesis by applying microbes deriving from E+ on new generation of E- plants and\nwait new seed offspring to show that the endophytic part of the microbiome is really involved in the conclusion you said.", "reviewer_2": "In general, this is a very interesting and well written manuscript. The research is a very clear demonstration of seed borne\nmicroevolution and how this constitutes a robust strategy for weed invasiveness. Below are a set of comments on specific\naspects of the manuscript.\nSince the endo-microbiome was the focus of this study, autoclaved field soil and sand was used to rule out the possibility of\ncontaminants and focus on those microbes within the seeds. As a food for thought – would it be possible that the authors\nwould get near similar results when the plants were grown in the soils of the different continents studied.\nLines 270 to 273 – Somewhere between 90 and 99 % of the microbes associated with plants cannot be grown in culture.\nWhile it is most likely that the root pieces the authors tested would have had at least some microbes in them that would grow\nin the agar growth medium utilized if they were contaminated with a representative segment of the microbiome micro-flora,\nthere is a small chance that unculturable microbes were present. The authors should probably acknowledge this.\nIn line 284 the authors acknowledge the presence of apomixis which allows for self-pollinated seed production. However, for\nTaraxacum officinale the apomixis can go beyond simple self pollination; this species is also capable of undergoing\npollination but then producing embryos from vegetative cells and so that the resulting embryo is genetically the same as the\nmaternal plant. The authors should acknowledge this and provide a few sentences of discussion.\nIn lines 307 to 308 the authors indicate that Taraxacum officinale plants were grown in pots with specific Asteraceae plants.\nIs there some likelihood of exchange of microbiome species between the two plant species? I realize that this is\nunavoidable, and feel that the experiments were very valuable in any case, but it still might be useful to at least comment on\nthis.\nFinally, while the quality of the language in this manuscript was very high, a copy of the manuscript is attached an this has a\nfew very minor suggestions on it.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "REVIEWER COMMENTS\nReviewer #1\nOverall Comment: The publication is interesting and is of high interest. I have just some minor\ncomments:\nResponse: Thank you very much for your positive feedback on our manuscript. We aimed to\naddress all your minor concerns below.\nComment 1: 263-274 it is not a proof that the plants have no endophytes. You may just have kill\nthe culturable fraction of microorganisms but not the whole community, especially bacteria. you\njust show absence of hyphae in E- but not bacteria. So you may need to rephrase part of your\nparagraph. There is the need to provide more details on how seed E- were propagated and how\nplants look like after E-. Was there several generations of plants derived from the seeds that have\nbeen studied or only one?\nResponse: Thank you for pointing out that unculturable bacteria may have been present in E- seeds.\nAlthough we did not confirm the absence of bacterial cells from E- seeds using microscopy, we are\nconfident that their exposure to rifampicin (see L382) would have removed almost all bacteria,\nregardless of their culturability, from the seeds. Rifampicin, a potent and broad-spectrum antibiotic,\nhas a high affinity for bacterial DNA-dependent RNA polymerase. Irrespective of their\nculturability, it hence inhibits protein synthesis and consequently kills bacteria (see Campbell et al.\n2001, https://doi.org/10.1016/S0092-8674(01)00286-0). We have added text to L404–407 in the\nMethods section to clarify this (‘Although we did not confirm the absence of bacterial cells from\nE- seeds using microscopy, it is unlikely that unculturable bacteria survived in rifampicin-treated\nseeds, since this antibiotic has a high affinity for bacterial DNA-dependent RNA polymerase (53),\nand hence, irrespective of culturability, inhibits protein synthesis, halts growth and ultimately kills\nbacterial cells.’) and have added Campbell et al. (2001) to the citations list. In response to the\nreviewer’s other points above, we have added two sentences to L385–389 (‘By propagating from\nthe seeds of E- or E+ plants over five generations, we were able to maintain endomicrobiome-free\nand endomicrobiome-colonised plants, respectively (Fig. S7). In agreement with previous studies\n(40, 52), treatment with rifampicin or Benlate did not result in any visible symptoms or detectable\ndifferences in the growth or development of E- plants.’).\nComment 2: You might need to further proof your hypothesis by applying microbes deriving from\nE+ on new generation of E- plants and wait new seed offspring to show that the endophytic part of\nthe microbiome is really involved in the conclusion you said.\nResponse: In order to address this comment, we conducted a new experiment demonstrating that\nthe E+ phenotype can be restored by inoculating previously disinfected E- individuals with micro-\norganisms isolated from E+ seeds. In this experiment, we reintroduced the endomicrobiome from\nE+ seeds into E- plants and monitored subsequent changes in phenotypic traits. The results showed\nthat the traits of re-inoculated plants were the same as those of E+ plants, with marked\nimprovements in survival rate, photosynthetic capacity, biomass and flower production relative to\nE- plants. We have added a new Figure showing these data (Fig. S5) and have added text to L134–\n137 to describe the findings (Supplementary experiments similarly confirmed that the\nendomicrobiome enhanced the performance of T. officinale, with E- plants inoculated with seed-\nborne microbes displaying the same maximum photosynthetic rate, flower production, biomass and\nsurvival as E+ plants (Fig. S5)). All of this evidence underscores the crucial role of the\nendomicrobiome in enhancing the performance, and hence the invasive potential, of T. officinale.\nReviewer #2\nOverall Comment: In general, this is a very interesting and well written manuscript. The research\nis a very clear demonstration of seed borne microevolution and how this constitutes a robust\nstrategy for weed invasiveness. Below are a set of comments on specific aspects of the manuscript.\nResponse: Thank you for the positive evaluation of our work and for highlighting some of the key\nfindings of our study.\nComment 1: Since the endo-microbiome was the focus of this study, autoclaved field soil and sand\nwas used to rule out the possibility of contaminants and focus on those microbes within the seeds.\nAs a food for thought – would it be possible that the authors would get near similar results when\nthe plants were grown in the soils of the different continents studied.\nResponse: Thank you for this insightful observation. We agree that growing T. officinale plants in\nnative soils from each continent could have yielded additional insights. However, we used a single\nsterile soil-sand mixture in order to reduce variability between the responses of T. officinale\npopulations from different continents to the seedborne microbiome. Although the effect of the\nseedborne microbiome on the invasiveness of T. officinale might differ between soils, the success\nof the weed in all 15 sites from which it was sampled, as well as its invasiveness worldwide, suggest\nthat the seed-borne endomicrobiome has consistent beneficial effects on the weed species across\ndiverse edaphic conditions. In order to address this point, we have adjusted the text at L247–248\nto ‘Despite all T. officinale plants being grown in the same sterile soil and sand mixture (see\nMethods), variation was observed here in the phenotypic and genotypic responses of different\npopulations of the species.’, at L254–280 to ‘…Interactions with native soil microbiomes may\nintroduce further variation into the response of T. officinale to the seed-borne endomicrobiome\n(49). However, the successful establishment of the weed species in all five Mediterranean-type\necosystems studied here, as well as its global success (30), suggests that it benefits from positive\neffects of the seed-borne endomicrobiome across diverse edaphic conditions.’ and at L382–384 to\n‘The use of a single sterilized substrate allowed us to isolate the effect of the vertically transmitted\nendomicrobiome on T. officinale and eliminated variability between populations caused by the\ninfluence of local soil microbiota on seed-borne endomicrobiomes (49)’.\nComment 2: Lines 270 to 273 – Somewhere between 90 and 99 % of the microbes associated with\nplants cannot be grown in culture. While it is most likely that the root pieces the authors tested\nwould have had at least some microbes in them that would grow in the agar growth medium utilized\nif they were contaminated with a representative segment of the microbiome micro-flora,\nthere is a small chance that unculturable microbes were present. The authors should probably acknowledge\nthis.\nResponse: Please see the response above to reviewer 1’s first comment.\nComment 3: In line 284 the authors acknowledge the presence of apomixis which allows for self-\npollinated seed production. However, for Taraxacum officinale the apomixis can go beyond simple\nself pollination; this species is also capable of undergoing\npollination but then producing embryos from vegetative cells and so that the resulting embryo is genetically the same as the\nmaternal plant. The authors should acknowledge this and provide a few sentences of discussion.\nResponse: Thank you for your observation. As suggested, we have expanded this section by adding\nfurther text at L387–389. We state that the exact nature of the mating system or reproductive traits\nin the individuals from each population is unknown, but that our results remain valid in\ndemonstrating how the endophytic microbiome enhances the microevolution of T. officinale\n(‘Although T. officinale is capable of cross-pollination in sexually reproducing diploid individuals,\napomixis is common in polyploids, leading to offspring genetically identical to the mother plants\n(55). Therefore, any potential variation in the mating and or reproductive traits reflects the\ncharacteristics of the experimental populations.’).\nComment 4: In lines 307 to 308 the authors indicate that Taraxacum officinale plants were grown\nin pots with specific Asteraceae plants. Is there some likelihood of exchange of microbiome species\nbetween the two plant species? I realize that this is\nunavoidable, and feel that the experiments were very valuable in any case, but it still might be useful to at least comment on\nthis.\nResponse: We appreciate this observation, as it raises an intriguing question regarding a potential\nexchange of microbiomes between plants. To date, there is very limited evidence supporting the\ninterspecific exchange of endophytic microbiomes between plant species. As the reviewer points\nout, the exchange of seedborne microbiomes between E+ T. officinale and the five native\nAsteraceae plant species (or between the five Asteraceae species and T. officinale) cannot be ruled\nout in our study. We have added brief text to L174–176 to discuss this point (‘… They also suggest\nthat, if exchange of microbiomes between T. officinale and each Asteraceae species had occurred\n(see Methods), then those associated with E+ seeds did not benefit the native plant species.’) and\nalso alert the reader to the possibility of the exchange of microbiomes between plants at L428–429\n(‘Note that seed-borne microbiomes may have been exchanged between T. officinale and each of\nthe native Asteraceae species (and vice versa) in these experiments’).\nComment 5: Finally, while the quality of the language in this manuscript was very high, a copy of\nthe manuscript is attached and this has a few very minor suggestions on it.\nResponse: Thank you very much. The grammatical suggestions have been incorporated into the\nrevised version of the manuscript.\nReviewer #3\nOverall Comment: I co-reviewed this very good manuscript with one of the reviewers who\nprovided the listed reports. This is part of the Nature Communications initiative to facilitate\ntraining in peer review and to provide appropriate recognition for Early Career Researchers who\nco-review manuscripts.\nResponse: The initiative promoted by Nature Communications is an excellent way to enhance the\neditorial experience of early-career researchers. Undoubtedly, a “double” review not only helps to\nidentify any unclear aspects of the study that may need clarification, but also facilitates a deep\ndiscussion between both reviewers about the study’s overall contribution to advancing the field.\nThank you for the collaborative work.\nWe appreciate the opportunity to address the comments of the reviewer #4, which have significantly improved our\nmanuscript. We provide detailed responses below (in blue text) to their points, based in the revised version with\ntrack of changes.\nReviewer #4 (Dr. Maren L. Friesen)\nMajor Comments\nComment 1. There's likely to be a huge amount of genetic drift happening in this experiment, and it seems\nimplausible that genetic changes are occurring in the host plant given that they are selecting a single best plant for\nthe next generation out of a population of 5 plants. Thus, the phenotypic changes observed could be due to (i)\nshifts in the microbiome that influence traits and/or (ii) maternal effects. I suggest that the discussion of possible\nselection on plant genotypes be revised (around line 236) and these possibilities discussed. Work by Mueller should\nbe cited and discussed regarding shifts in the microbiome driving changes in plant traits, e.g., Mueller UG, Juenger\nTE, Kardish MR, Carlson AL, Burns KM, Edwards JA, Smith CC, Fang CC, Des Marais DL. Artificial selection\non microbiomes to breed microbiomes that confer salt tolerance to plants. MSystems. 2021 Dec 21; (6):e01125-\n21. Maternal effects could arise in this experiment since plants seem to benefit from the presence of their\nunperturbed microbiomes; offspring of these plants are likely to be more fit and have different trait values simply\nbecause their mothers were more fit and not necessarily because of genetic changes in the plant (highly\nunlikely with this selection design) or the composition of the microbiome. My suspicion is that some combination of\nmaternal effects and microbiome shifts are occurring in these experiments, given how consistent the effects are for\nlines from diverse geographic origins.\nReply: We appreciate the reviewer’s insightful critique and agree that caution is essential when interpreting\nmultigenerational phenotypic changes as evidence of evolutionary divergence. In response, we have revised the\ntext to acknowledge these important caveats more explicitly and to reposition our findings within the appropriate\nconceptual framework. Specifically, we added a new paragraph to the Results and Discussion section at L380–390\nto clarify that our experimental approach did not involve tracking lineages or assessing genomic variation –\nmethodologies previously used in microbial or host–microbiome evolutionary studies – and to also recognise that\nmaternal environmental effects may have contributed to the observed patterns (‘Whilst our experiments did not\nin’t\ninvolve tracking genetic lineage or assessing genome-wide variation, they were designed to detect consistent,\nmultigenerational shifts in fitness related traits in a controlled environment. In this context, the endomicrobiome\napparently acted as a selective filter, shaping phenotypic distribution in a direction consistent with\nmicroevolutionary divergence. This form of quasi-natural selection aligns with conceptual frameworks in plant\nmicroevolution under global change (52). We acknowledge that our approach did not include reciprocal transplants\nof host and microbiome or artificial selection on microbial communities, as implemented in other microbiome\nselection studies (53). Furthermore, we cannot entirely rule out the contribution of maternal environmental effects\n(34) to the observed patterns, particularly in the interspecific competition experiment. Nevertheless, the repeatable\nand directional nature of trait changes across multiple generations, combined with the enhanced performance of T.\nofficinale grown with its native microbiome, strongly support a microevolutionary response mediated by the seed-\nborne endomicrobiome.’). As suggested by the reviewer, we have included a citation here to the article by Mueller\net al. (2021), and have included a citation to Gianoli & Molina-Montenegro (2021), which reports that the\nendomicrobiome acts as a selective filter, driving trait distributions under global change in a manner consistent\nwith quasi-natural selection, and hence supports the observations from our study. We have also added a citation to\nVan Allen et al. (2021), which discusses the influence of maternal effects in interspecific competition experiments.\nWe believe that these additions improve the clarity and balance of our interpretation, and we thank the reviewer\nfor prompting this important revision.\nComment 2: There is no way that their experiment is actually axenic and I believe it is disingenuous to claim\notherwise. It is hard to keep plants axenic even in closed containers, and impossible that plants grown in a\ngreenhouse are completely free from microbes. The authors see strong effects of microbial transfer as well as\nstrong differences between E+ and E- plants, so they are clearly influencing the microbiome. But as all hospitals\nknow, antibiotics cannot completely kill all bacteria and antibiotic resistance evolves all too readily. For accuracy,\nI suggest describing E- as “endomicrobiome reduced” rather than claiming they are completely free of microbes.\nMuch more rigorous methodology and testing would be needed to support that claim.\nReply: We agree with this comment, and in the revised version have modified the text throughout to clarify that\nE- plants are those with reduced microbiomes, including in the Abstract at L39 (‘… T. officinale… grown with\nintact and reduced native seed-borne endomicrobiomes), in the Introduction at L99 and 102 (‘… to investigate the\nmicroevolution of the species grown… with intact or reduced local seed-borne endomicrobiomes…’) and at the\nstart of the Results and Discussion at L120 (‘… distinct micro-evolutionary trajectories were observed between T.\nofficinale individuals grown with their intact or reduced seed-borne endomicrobiomes...’). At L121–122, we have\nalso inserted text to direct the reader to the methods describing how plants with reduced microbiomes were\nproduced (‘E- plants, which were generated using a fungicide and a bactericide with no effects on plant growth\n(see Methods)’).\nComment 3. The competition experiment, while interesting, is not designed to test whether or not the change in\ncompetitiveness is due to the endomicrobiome. Rather it is comparing the effect of four generations in the\ngreenhouse (F5 vs F1). The authors would have needed to compare E+ to E- plants in terms of their competitive\nability. Some discussion and revision of conclusions from this experiment are needed.\nReply: The purpose of this experiment was to evaluate whether selection in the presence of the endomicrobiome\naffected plant competitive ability. The initial culling experiment allowed us to assess the response of individual\nplants grown with intact or reduced endomicrobiomes to a selection process lasting for several generations (Figure\n1). Subsequently, we aimed to test whether this selection process, beyond shaping individual phenotypes (Table\n1), also resulted in greater plant competitive ability. To this end, we designed an experiment to assess whether F\n5\nplants with intact endomicrobiomes exhibited greater competitive ability than their ancestral lineages (F ) and a\n1\nphylogenetically close Asteraceae species on each continent (Figure 2). We acknowledge that comparing F plants\n5\nwith intact or reduced endomicrobiomes would have provided additional insights into the extent to which\ncompetitive ability can be attributed to the endomicrobiome. However, while this would indeed be an interesting\napproach, it was beyond the scope of our study. Following the reviewer’s valuable suggestion, we have\nincorporated text in the Results and Discussion section at L196–199 acknowledging this limitation (‘Although this\nexperimental design did not include a direct comparison between F plants grown with intact or reduced\n5\nendomicrobiomes, it measured the cumulative impacts of selection over five generations on the competitiveness\nof T. officinale grown with its intact seed-borne endomicrobiome.’). In order to further tone down the revised\narticle, the title of the sub-section has been changed from ‘The endomicrobiome boosts the capacity of invasive\nplants to outcompete native species’ to ‘The role of the endomicrobiome in interspecific competition’, and text has\nbeen added to L207–208 (‘Although we cannot exclude the possibility of maternal effects influencing the outcome\nof this experiment (34)…’). Similarly, at L225, we have altered ‘provide the first empirical evidence’ to ‘suggest’,\nso that the text now reads ‘our results suggest that the endomicrobiome significantly influences plant\ncompetitiveness on microevolutionary timescales’. We have also inserted ‘may’ into the text at L231, so that L230–\n233 now read ‘Collectively, our competition experiments demonstrate that microbial symbionts may significantly\nenhance the invasive potential of T. officinale…’.\nComment 4: Conceptually, although it is certainly interesting to see such large microbiome effects on plant traits,\nI’m not quite sure how this translates to controlling invasive plants. Killing the microbiomes of entire plant\ncommunities seems both challenging and problematic--the spread of antibiotic resistance genes is a major concern\nand as a society we need to be reducing our use of these powerful compounds. eg. Naghavi M, Vollset SE, Ikuta\nKS, Swetschinski LR, Gray AP, Wool EE, Aguilar GR, Mestrovic T, Smith G, Han C, Hsu RL. Global burden of\nbacterial antimicrobial resistance 1990–2021: a systematic analysis with forecasts to 2050. The Lancet. 2024 Sep\n28;404(10459):1199-226.\nReply: We agree that controlling the microbiomes of invasive plants as a biological control strategy would be\nlogistically challenging. We have accordingly shortened the final sentence of the first paragraph in the Conclusions\nand outlook section (now L292–294) to ‘These findings hold promise for the management of this, and other,\ninvasive plant species in Mediterranean ecosystems through microbiome manipulation (48) rather than through\nthe application of glyphosate, a herbicide with potential adverse effects on human health and the environment\n(49)’.\nMinor Comments\nComment 5: line 153: in nature all plants are E+, do you mean to compare Europe/Asia as “native” E+? It seems\nimportant to give information on where T. officinale is native to at some point in the Introduction.\nReply: The text at L96–97 has been reworded to ‘… using Taraxacum officinale… a species originating in Eurasia\nthat has become one of the most successful and widespread invasive weeds worldwide…’. In addition, in the\nrevised version we have clarified that the native species used in the competition experiment are all Asteraceae\nspecies native to each continent (see L209).\nComment 6: line 168: cut traits, it's redundant.\nReply: Done.\nComment 7: line 202: not necessarily “directly”, there could be many mechanisms.\nReply: This word has been removed from the revised version.\nComment 8: line 218: broad spectrum antibiotics/antifungals are hardly safer!\nReply: The text here referring to the use of biocides for controlling the seed-borne endomicrobiome has been\ndeleted. Please see the response to the reviewer’s fourth comment.\nComment 9: line 321: do you mean 15 individuals it says five.\nReply: Thank you for this observation. In the revised version, we have clarified that in each generation, the three\nindividuals with the highest fitness values were selected, and from each of these, five new individuals were\ngenerated for the next generation. This process ensured a constant population size of 15 individuals per generation.\nThis clarification now makes it clear why there are 15 individuals in each generation, rather than 5, as might have\nbeen inferred from the previous version of the manuscript. Please see L481.\nComment 10: line 367: I would replace this with gene expression.\nReply: Done.\nComment 11: line 383: please give primer sequences.\nReply: In the revised version of our manuscript, we have added a new supplementary table (Table S14) with the\nprimer sequences used in this study. See L552–553.\nComment 12: Table 1: statistics need to be corrected for multiple testing.\nReply: We thank the reviewer for her valuable observation regarding the statistical treatment of multiple\ncomparisons. As she correctly notes, the interpretation of t-tests across multiple datasets may lead to inflated Type\nI error rates if not adjusted for multiple testing. To address this concern, we have revisited all pairwise t-tests\nperformed in the manuscript and applied Benjamini–Hochberg False Discovery Rate (FDR) correction to control\nfor false positives while maintaining adequate statistical power. This approach ensures that all p-values associated\nwith univariate comparisons are now adjusted for multiplicity, providing a more conservative interpretation of the\ndata.\nImportantly, among the 212 t-tests performed, just two – the Haldane rate for flower production in Oceania (E+),\nand the F –F soil-phenol comparison for invasive plants in Chile – changed their significance status after\n1 5\ncorrection, demonstrating the overall robustness of the patterns and trends originally reported. The\nmicroevolutionary rate for the former response is now reported as non-significant in both Table 1 and\nSupplementary Table 13, and all corresponding descriptions in the Results and Discussion have been updated\naccordingly. We have added text to the Statistical Analyses section to indicate that all t-tests were adjusted for\nmultiple comparisons (‘To control for multiple comparisons across all pairwise t-tests (both one-sample and two-\nsample), we applied Benjamini–Hochberg false discovery rate correction (63) at q = 0.05 within appropriate test\nfamilies, ensuring conservative and statistically robust data interpretation.’), and have added a citation to\nBenjamini & Hochberg (1995) to the reference list.\nPlease note that Supplementary Tables S6–S13 have also been updated to incorporate the results of the new tests\nto correct for FDR. Each table now includes an additional column (q_bh) indicating the Benjamini–Hochberg\nFDR–adjusted q-values for every t-test performed, together with a clear statement in the table legends specifying\nthat statistical significance corresponds to the corrected values.\nPlease note that other small adjustments have been made to the text, which do not alter its sense. Finally, we have\nacknowledged Dr Friesen’s helpful input at L405.\nWe hope that these edits to the manuscript are satisfactory and look forward to hearing from you again.\nThank you for your time,\nProfessor Marco Molina-Montenegro\npp. Ian Acuña-Rodríguez, Cristian Atala, Gabriel Ballesteros, Fernando Carrasco-Urra, Eduardo Castro-Nallar,\nManuel Delgado-Baquerizo, Víctor Escobedo, Fernando Fuentes-Peñailillo, Leidy García, Sergio Guajardo-Leiva,\nPedro Gundel, Rasme Hereme, Raúl Ochoa-Hueso, Luis Pertierra, David Richardson, Cristian Torres-Díaz,\nAndrea Ueno, Ariel Villalón, Amy Zanne and Kevin Newsham"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Accepted. Good work!", "reviewer_2": "The authors have successfully addressed my comments, providing clarifications and additional information. I think that this\nmanuscript is now suitable for acceptance as a publication.", "reviewer_4": "MAJOR COMMENTS:\n1. There's likely to be a huge amount of genetic drift happening in this experiment, and it seems implausible that genetic\nchanges are occurring in the host plant given that they are selecting a single best plant for the next generation out of a\npopulation of 5 plants. Thus, the phenotypic changes observed could be due to (i) shifts in the microbiome that influence\ntraits and/or (ii) maternal effects. I suggest that the discussion of possible selection on plant genotypes be revised (around\nline 236) and these possibilities discussed. Work by Mueller should be cited and discussed regarding shifts in the\nmicrobiome driving changes in plant traits, e.g., Mueller UG, Juenger TE, Kardish MR, Carlson AL, Burns KM, Edwards JA,\nSmith CC, Fang CC, Des Marais DL. Artificial selection on microbiomes to breed microbiomes that confer salt tolerance to\nplants. MSystems. 2021 Dec 21;6(6):e01125-21. Maternal effects could arise in this experiment since plants seem to benefit\nfrom the presence of their unperturbed microbiomes; offspring of these plants are likely to be more fit and have different trait\nvalues simply because their mothers were more fit and not necessarily because of genetic changes in the plant (highly\nunlikely with this selection design) or the composition of the microbiome. My suspicion is that some combination of\nmaternal effects and microbiome shifts are occurring in these experiments, given how consistent the effects are for lines from diverse\ngeographic origins.\n2. There is no way that their experiment is actually axenic and I believe it is disingenuous to claim otherwise. It is hard to\nkeep plants axenic even in closed containers, and impossible that plants grown in a greenhouse are completely free from\nmicrobes. The authors see strong effects of microbial transfer as well as strong differences between E+ and E- plants, so\nthey are clearly influencing the microbiome. But as all hospitals know, antibiotics cannot completely kill all bacteria and\nantibiotic resistance evolves all too readily. For accuracy, I suggest describing E- as “endomicrobiome reduced” rather than\nclaiming they are completely free of microbes. Much more rigorous methodology and testing would be needed to support\nthat claim.\n3. The competition experiment, while interesting, is not designed to test whether or not the change in competitiveness is due\nto the endomicrobiome. Rather it is comparing the effect of four generations in the greenhouse (F5 vs F1). The authors\nwould have needed to compare E+ to E- plants in terms of their competitive ability. Some discussion and revision of\nconclusions from this experiment are needed.\n4. Conceptually, although it is certainly interesting to see such large microbiome effects on plant traits, I’m not quite sure how\nthis translates to controlling invasive plants. Killing the microbiomes of entire plant communities seems both challenging and\nproblematic--the spread of antibiotic resistance genes is a major concern and as a society we need to be reducing our use of\nthese powerful compounds. eg. Naghavi M, Vollset SE, Ikuta KS, Swetschinski LR, Gray AP, Wool EE, Aguilar GR,\nMestrovic T, Smith G, Han C, Hsu RL. Global burden of bacterial antimicrobial resistance 1990–2021: a systematic analysis\nwith forecasts to 2050. The Lancet. 2024 Sep 28;404(10459):1199-226.\nMinor comments:\nline 153: in nature all plants are E+, do you mean to compare Europe/Asia as “native” E+? It seems important to give\ninformation on where T. officinale is native to at some point in the Introduction\nline 168: cut traits, it's redundant\nline 202: not necessarily “directly”, there could be many mechanisms\nline 218: broad spectrum antibiotics/antifungals are hardly safer!\nline 321: do you mean 15 individuals it says five\nline 367: I would replace this with gene expression\nline 383: please give primer sequences and qpcr conditions\nTable 1: statistics need to be corrected for multiple testing\nBest wishes,\nMaren L. Friesen"}, "author_response": ""}]

Decision letter