A peer-reviewed open-access journal @) NeoBiota Advancing research on alien species and biological invasions NeoBiota 99: 93-108 (2025) DOI: 10.3897/neobiota.99.144628 Research Article Unraveling the microbiome dynamics of the invasive Acacia longifolia: a closer look at seeds and nodules Joana G. Jesus'!”®, Andreia Anjos'®, Cristina Maguas'®, Helena Trindade’”© 1 Centre for Ecology, Evolution and Environmental Change (CE3C), Faculty of Sciences of the University of Lisbon (FCUL) & Global Change and Sustainability Institute (CHANGE), Lisbon, Portugal 2 Faculty of Sciences of the University of Lisbon (FCUL), Lisbon, Portugal Corresponding author: Joana G. Jesus (jgjesus@fc.ul.pt) OPEN Qaceess Academic editor: Elizabeth Wandrag Received: 16 December 2024 Accepted: 7 April 2025 Published: 5 June 2025 Citation: Jesus JG, Anjos A, Maguas C, Trindade H (2025) Unraveling the microbiome dynamics of the invasive Acacia longifolia: a closer look at seeds and nodules. NeoBiota 99: 93-108. https://doi.org/10.3897/ neobiota.99.144628 Copyright: © Joana G. Jesus et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Acacia longifolia, a species native to Australia, is an aggressive invasive in Mediterranean-type eco- systems worldwide. Its success in diverse habitats, expanding from coastal dunes to forests, is often attributed to its ability to establish interactions with a variety of microbes, including bacteria and fungi. This study investigates the seed and root-nodule microbiomes of A. /ongifolia to understand the roles these microbial communities play in its adaptation and invasive behaviour. Using high-throughput se- quencing, we characterized the bacteriome and mycobiome associated with the plant, considering nod- ules and seeds, and the surrounding soil in three different locations in Portugal with different climate conditions (North, Center and South), and a comparison between two different habitats (Dune versus Forest). Our results reveal a dynamic interaction between A. longifolia and its microbial partners, sup- porting the importance of these plant-microbe interactions in nutrient acquisition and stress tolerance for A. longifolia, ultimately leading to their impact in an invaded ecosystem. The seed microbiome of A. longifolia was less diverse than for nodules but with more functions assigned, while nodules showed a broader diversity, assigned to more specific functions. Here we provide evidence for the role of seed mi- crobiota in germination and seed-to-seedling transition along with the beneficial role of nodulation in development and seedling-to-sapling switch. We also propose a local signature for microbial communi- ties as we found a dissimilarity in microbial partners when considering habitat, with dune communities showing a functional plasticity, aiding A. /ongifolia to cope in such nutrient-limiting environment. For forests, functions more related with plant and microbe associations are evidenced, possibly facilitating interspecific competition. These findings contribute to an understanding of the plant-microbe interac- tions and dynamics that underpin A. longifolia ecological success as an invasive plant. Key words: Dissimilarity, functional prediction, habitat adaptation, microbial communities Introduction Invasive plant species are a major driver of biodiversity loss and ecosystem deg- radation worldwide, being responsible for disrupting native plant communities and altering soil properties (Millennium Ecosystem Assessment 2005; Pysek et al. 2012; Dostal 2024). Among such species, Acacia longifolia Andrews (Willd.), a leguminous shrub native to Australia, has become highly invasive in Mediterra- nean-type ecosystems, mainly in coastal dunes where it was introduced to prevent erosion (Peperkorn et al. 2005). In Portugal, A. /ongifolia is no longer restricted to 93 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia coastal dune ecosystems, extending to native and non-native forests from the north to the south of Portugal (Colago et al. 2023). This spread raises the question of what adaptive traits this species possesses that allow its success. Several factors are responsible for the spread of Acacia spp., including the pro- duction of a high number of seeds, high seed longevity, and increased seed germi- nation after fire events, just to mention a few (e.g., Richardson and Kluge 2008; Palmer 2016; Riveiro et al. 2020). Being an invasive species, a key factor that could trigger its success is the microbiome (Lau and Suwa 2016). Following an intro- duction, particularly in Fabaceae-belonging species, studies raised the hypothesis of these plants’ inadaptation, considering the absence of compatible rhizobia (i.e., nitrogen-fixing bacteria) (Simonsen et al. 2017). However, in the case of Acacia spp., their promiscuity can help in finding suitable partners within soil microor- ganisms in the novel habitat (Birnbaum et al. 2012; Klock et al. 2015; Jesus et al. 2020). Some studies already show evidence for a co-introduction of rhizobial partners (Rodriguez-Echeverria 2010; Crisdstomo et al. 2013) or the possibility of novel symbiotic relations (e.g., Ndlovu et al. 2013; Jesus et al. 2020, 2023). The ability of A. longifolia to fix nitrogen, alongside with its rapid growth and disper- sal, enables it to outcompete native vegetation. This causes significant changes in ecosystem structure and function, with Acacia spp. playing a role as ecosystem-en- gineers (Stock et al. 1995; Marchante et al. 2004, 2008, 2009; Yelenik et al. 2007). This includes high water consumption (Marchante et al. 2003), nutrient cycles’ alterations (Ulm et al. 2017; Meira-Neto et al. 2018) and increased biomass pro- duction (Ulm et al. 2019, 2022). Soil is considered essential to ecosystem functioning hosting a high biodiversity (Delgado-Baquerizo et al. 2016; Schuldt et al. 2018; Liu et al. 2023) impacting soil vitality, which is mostly dependent on microbial communities’ activity and interactions (Johns 2017). This is critical in invaded areas, where invasive plants significantly alter soil communities to establish and spread further, while simulta- neously causing profound changes that constrain native plant diversity (e.g., Lau and Suwa 2016). Plant-associated microorganisms play crucial roles in plant devel- opment, growth and plasticity (Bulgarelli et al. 2013; Imam et al. 2016; Jain et al. 2024) and, particularly, seed-associated microbes are crucial for germination and early-establishment (Fukami 2015; Bergmann and Leveau 2022). Seed microbiota can include endophytes and epiphytes. While endophytes reside inside seed tissues and are vertical- or horizontally transmitted, epiphytes are colonizers of seed sur- face and may not become part of the seed microbiome (Barret et al. 2015). As it was proposed by several studies (e.g., Malfanova et al. 2013; Truyens et al. 2015), seed microbiota can be a hotspot of beneficial bacteria and fungi, making this mi- crobiota an important player on seed-to-seedling transition (Fenner and Thomp- son 2005; Leck et al. 2008). Also, previous studies on A. longifolia showed that in its invasive range, this species has a combination of endophytes that include plant growth promoting bacteria, providing extra capabilities that helps growth and ex- pansion (Condessa et al. 2024). Furthermore, root-nodules house several growth promoters and provide a direct source of nitrogen, crucial in the seedling-to-sap- ling transition (Desbrosses and Stougaard 2011; Pucciariello et al. 2019). From soil-to-seed and from soil-to-nodule, studies have been raised hypothesizing a dy- namism that could determine plant adaptation, and these transitions can be local- and individual-dependent, respectively (Nelson 2018; Wang and Zhang 2023). The diversity and dynamics of microbial communities associated with seeds and NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 94 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia root-nodules require further investigation in this aggressive invasive plant, as they may hypothetically trigger new dispersal events and serve as a lever for successful adaptation, facilitating drastic environmental changes. Accordingly, this study aims to (i) explore how seed and root-nodule microbi- omes vary across different habitats and edaphoclimatic conditions, (ii) identify key species within the bacteriome and mycobiome of seeds and root-nodules at a local scale and (iii) determine the potential functional roles played by these microbial communities. These findings could elucidate how microbial communities support the invasiveness of A. /ongifolia, ultimately contributing to mitigate its impacts on native biodiversity and ecosystem functioning. Material and methods Field sampling Sampling was performed in spring 2023 (ie., during March) in three locations with distinct climate conditions in Portugal’s mainland: Mira (hereafter referred as “North” and described as “Very Humid”), Fonte da Telha (“Center” and “Hu- mid”) and Vila Nova de Mil Fontes (“South” and “Semi-Humid”), respectively. Classification of these locations was performed according to De Martonne aridity index (De Martonne 1925). At each location, two habitats invaded by Acacia lon- gifolia were selected, a Dune and a Forest, in which ten A. longifolia young saplings were selected (ca. 60 + 13 cm in height), with saplings separated by at least 10 m from each other (to cover microsite variability). Each sapling was carefully dug up to collect roots with attached nodules, which were stored in silica gel to dehydrate until further processing. Ten soil samples (10—20 cm in depth) were collected close to each of the saplings (i.e., one sample per sapling) and stored in a freezer (-80 °C) for microbial assessment. Mature pods were collected directly from the branches of ten adult A. longifolia trees, with each tree again selected based on proximity to the collected saplings. Seeds were then separated from pods and stored in paper bags at room temperature pending further analysis. All the material used for sample col- lection was disinfected with ethanol among saplings in each habitat and location. An overview of each location and their climatic conditions including temperature and precipitation in the six months before sampling date are presented in Suppl. material 1: fig. S1. Also, soil physicochemical parameters were assessed to character- ize each location, and this data is presented in Suppl. material 1: table S1. Soil anal- ysis were performed in Laboratorio de Plantas e Solos da Universidade de Tras-os- Montes e Alto Douro (UTAD), Portugal. A vegetation survey was conducted using a 60 x 60 cm quadrat nearby each sapling to understand other plant species presence and cover. Most of the species were herbaceous whose influence is mostly negligible. Sample processing The three biological samples were prepared for high-throughput sequencing: root-nod- ules, hereafter named nodules, seeds and soils in composite samples, ending up with 18 samples to represent each biological sample from each habitat in each location. Next-generation sequencing (NGS) through Oxford Nanopore © was per- formed to infer microbial communities of nodules, seeds and soil from each habitat in each location. Both nodules and seeds were surface disinfected before NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 95 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia processing. For pooled seeds (n = 3 from each adult tree) and pooled nodules (n = 5 from each sapling) samples, total DNA was extracted using the modified CTAB protocol (Yuan 2019). Regarding soil, total DNA was extracted from 10 g of each soil using the DNeasy PowerMax Soil Kit from Qiagen, following the manufacturer's instructions. DNA purity was assessed by absorbance at 260 nm using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA). The extracted DNA was stored at -20 °C pending further processing. For all samples, 12.5 pL of eluted DNA obtained in the extraction protocol was used for library preparation. Sequencing runs were performed using R10.4.1 flow cells on a Pro- methION sequencing platform and sequencing data were acquired in real time using MinKNOW 18.08.2 software. Sequencing data was stored in fastq files, with each file representing a batch of 4000 reads. Sequencing data was derived from 16S or 25S - 28S amplicons (for Bacteria and Fungi, respectively), with the exclusion of low-quality reads. The remaining reads underwent size selection, retaining those with lengths between 1200 bps and 1700 bps, accomplished through prinseq-lite (Schmieder and Edwards 2011). Taxonomic classification employed a Lowest Common Ancestor approach, uti- lizing an index based on k-mers that map to the lowest common ancestor of all genomes known to encompass a specific k-mer (Wood et al. 2019). Statistical analysis The composition of bacterial and fungal communities was used to generate heat- maps based on the abundance data of the top 10 genera identified in each sam- ple. Abundance data were transformed using z-score normalization to enhance the comparability across samples. Samples (rows) and Operational Taxonomic Units (OTUs, columns) were clustered hierarchically based on relative abundance pro- files, using the Bray-Curtis distance and the Ward’s method as clustering algorithm. All OTUs, for both bacteriome and mycobiome, were used to calculate Shan- non-Wiener (Shannon 1948) and Chao (richness) (Chao 1984) indexes consider- ing biological sample x habitat x location (i.e., 18 composite samples tested). To explore differences in microbial communities among samples, a Principal Coordi- nates Analysis (PCoA) was performed using the Bray-Curtis dissimilarity index to construct the distance matrix. The first two principal coordinates, which explain most of the variance in the data, were visualized to highlight clustering patterns among samples, which statistical significance was tested using PERMANOVA (Permutational Multivariate Analysis of Variance). Functional predictions for bacterial and fungal communities were performed using all classified taxa from each biological sample, applying FAPROTAX v1.2.6 (Louca et al. 2016) for bacteria and FungalTraits (Polme et al. 2020) for fungi. Presence and absence matrixes were built according to these databases and a cu- mulative relative abundance for each identified function was calculated. Ternary plots were generated to visually represent the relative abundances of key functional groups assigned to the different OTUs identified in both microbial communities (see Suppl. material 1: tables S2, S3). Data analyses were performed using the packages betapart (Baselga et al. 2023), ggtern (Hamilton and Ferry 2018), microeco (Liu et al. 2021), pheatmap (Kol- de 2019), rstatix (Kassambara 2023), stats and vegan (Oksanen et al. 2022) in R (v.4.2.2) (R Core Team 2023). NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 96 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia Cluster II Cluster I Cluster ITI Results Regarding the top 10 genera of bacterial communities, they were grouped in two main clusters, one including mostly soils (Cluster I) and the other comprising seeds and nodules’ communities (Cluster II) (Fig. 1). The cluster that contained most of the seed samples (Cluster III) included genera that were not the most abundant in soil, mostly including the phylum Cyanobacteriota, and the genera Synechococcus, Oscillatoria, Nostoc and Stanieria. For nodules, assigned to Cluster IV, Bradyrhizobium was a representative genus along with Bacillus, Paenibacillus and Stenotrophomonas. In soils, Paraburkholderia, Conexibacter, Mucilaginibacter, Paludibaculum and Candidatus Koribacter were more dominant among samples (Cluster V). There was no clear clustering based on location, however, dispersed clusters were formed formed considering habitat, especially for soil, in Cluster I, with Dune samples presented together as well as Forest samples. Regarding the top 10 genera for fungal communities, three main clusters grouped biological samples: Cluster I for nodules, Cluster II for seeds and Cluster II for soil (Fig. 2). Considering the communities found, there were two main clusters, one that included the representative genera (i.e., more abundant) in soils (Cluster IV) and an- other for nodules and seeds (Cluster V). Amanita, Russula, Saitozyma, Fusarium and Acremonium were among the more abundant genera for Cluster IV; included in seed mycobiome, Alternaria, Aureobasidium, Entoloma, Cladosporium and Curvularia were identified, while for nodules, Solicoccozyma, Umbelopsis, Serendipita and Tuber were among the most abundant. In this case, there was a more evident distinction when considering habitat, especially for seeds (i.e., higher z-score value of 2 to 4). Cluster IV Cluster V aieeae con OSS cn Center_Dune_Seed Center_Dune_Nodule 3 2 1 North_Dune_Nodule 0 Center_Forest_Nodule Synechococcus | _ Oscillatoria Stanieria 1 South_Dune_Nodule 2 3 North_Dune_Soil North_Forest_Nodule Planktothrix Geminocystis South_Forest_Nodule North_Forest_Seed North_Dune_Seed South_Dune_Seed Crinalium | | Center_Forest_Seed North_Forest_Seed South_Dune_Soil Center_Dune_Soil North_Forest_Soil Center_Forest_Soil South_Forest_Soil a «8 *® & »&» S&S SB & &® BH BH BB = b «@ fk & oe “ES "a — = . SESE SS SERS EE PPERPS Ess sees sss acshteePeerestess SERPES SES 8 sere s os = © = = = os Ss 8 = is. 4 o S$ ss © & & & 8 £ € Ss 2 f= 2 $8 & = 6 = 8® 6 6 8S a . 3 £55 €¢ & 6 & 5 #8 G 8 SS HS St R S § | “2 & te 8B 2 © 8 s & & § 2 3 = Ps 2 oS = = = ¢ = 3 & Ses ess 8 st SEs § SE = « Ss BA & = & = & = 8 8 8 £ S&S BF & & EF &® 8 = = = 2 = & s $ S$ = = S mM 68 g E EPs sessvsrsssessssezrse = = = & aa = a a oe a 3 mq = i) 5 < s 5 5 = MY Ss Ry 3 3 a) = = = S 1s) Figure 1. Heatmap based on bacterial communities at genus level (top 10) considering biological samples (nodule, seed and soil), habitat (dune and forest) and location (North, Center and South). Samples (rows) and Operational Taxonomic Units - OTUs (columns) were clustered hierarchically based on relative abundance profiles. These values were standardized in z-scores (varying between -3 and 3, against each mean value). Sample names stand accordingly: Location_Habitat_Biological sample. NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 97 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia Cluster IV Cluster V 4 North_Dune_Soil Center_Forest_Soil 2 South_Forest_Soil North_Forest_Soil Center_Dune_Soil z South_Dune_Soil sail 4 Center_Dune Seed Cluster III North_Dune_Seed South Dune Seed Center_Forest_Seed Cluster II South_Forest_Seed Center_Forest_Nodule North_Dune_Nodule South Dune_Nodule North_Forest_Nodule Cluster I North_Forest_Seed Center_Dune_Nodule South_Forest_Nodule & © 8 & gS ose 268 & Fe es SS ee & Ses sek s&s BS 8 & 28 8 S&S £ 8 &§ & & & 8 es & & aet eee Pete Esa RES EE ERS aE ERE SEP ES EEC SC SPEC ER ERP EE PEER TES ET SES Bos a 328585 2 €&€ SS 2s @€ g§@ & &€ 3 8 = § = 3 8 5 8 & 3 6 5 3 S$ #3 BEE & 6 a 3 2 E SA SSESRP ERS RES ERPESEERERP ES ES SSCS SER REZ ESE RES ES PTR ZS ETE ETERS EC ES 6 5 @ & 8 = 2 5 S$ § &§ & & ¢ ££ E£ = EBEeseSeteaBS SE SES F&F ESERBEEEF BEPE SE SESE FE = 2 S68 Ss 2 8 5 Bf ES 2 2 8 SSO BRR a sESEtESsGPeREVPtr,g FFE SsBERSE ES B 6 & S & EE = 2 8B & § © ¢ & = 2 =: § = E 3 gets. Ss Fe SS PREG Ps FseaRETE SE FRE SE a bras se 5 £ = Ss § 3 a = - = S € Ss = ses: 3 Go § Saez ses 5 ; P E = a es 2% POs = E es ber as gece Bes 5 foe i 3 = = GS = % 4 | 5 2 4 = F Q x 3 < Figure 2. Heatmap based on fungal communities at genus level (top 10) considering biological samples (nodule, seed and soil), habitat (dune and forest) and location (North, Center and South). Samples (rows) and Operational Taxonomic Units - OTUs (columns) were clustered hierarchically based on relative abundance profiles. The values were standardized in z-scores (varying between -4 and 4, against each mean value). Sample names stand accordingly: Location_Habitat_Biological sample. Por both bacterial and fungal communities, dissimilarity analysis highlighted the clustering by biological sample, regardless of both habitat and location (Fig. 3, Suppl. material 1: fig. S2). Within the bacterial communities, there was greater dissimilarity between soils and nodules or seeds, resulting in an overlap between the latter two. Overall, bacterial communities were more dissimilar within nod- ules than when compared with seeds. In fungal communities, there was a greater similarity among different samples within each biological sample, and a higher dis- similarity between the three biological samples, regardless of habitat and location. Soil samples had the highest number of OTUs with a greater abundance of bac- teria compared to fungi. In contrast, seeds and nodules showed a higher prevalence Bacterial communities Fungal communities 050 : : ey x q 0251 Oe : : -@° : ab eo ie & 0.25 (7 0.25 0.00 PCo 1 (44.6 %) PCo 1 (50.5 %) @ Nodule A Seed ® Soil Figure 3. Principal component analysis (PCoA) based on dissimilarity matrixes with Bray-Curtis distance method, using classified Oper- ational Taxonomic Units (OTUs) for bacterial and fungal communities, considering the three biological samples (nodules, seeds and soil), each habitat (dune and forest) and the three locations (North, Center and South). Colors and shapes were assigned to each biological sample: red circles for nodules, cream triangles for seeds and light brown squares for soil. Ellipses were used to highlight the clustering of each bio- logical sample [(for bacterial communities: pseudo-F = 10.05, p = 0.001, 999 permutations), with biological sample explaining 57.3% of the variance in community composition (R’ = 0.573); for fungal communities: pseudo-F = 15.21, p = 0.001, 999 permutations), with biological sample explaining 66.9% of the variance in community composition (R’ = 0.669)]. Note that y-axis has different scales in each community. NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 98 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia of fungi than bacteria, with seeds displaying the lowest overall OTU counts for bacteria (Suppl. material 1: table S4). In both microbial communities, while biological sample and habitat had an in- fluence on diversity and richness, location did not influence significantly. Also, soil exhibited the highest diversity and richness comparing with nodules and seeds. For bacterial communities, nodules were more diverse and showed richer communities than seeds. Overall, dune samples had higher diversity and richness than forest samples (Fig. 4a). In contrast, for fungal communities, seeds had higher values of diversity and richness than nodules and showed greater variability among samples. A similar pattern of higher dispersion of diversity was observed across habitats, with dune samples again displaying higher values (Fig. 4b). For functional characterization, considering bacterial communities, the most common function in nodules was assigned to Nitrogen metabolism, mostly nitro- gen fixation, higher in samples from dunes (88%) compared to forests (ca. 44%) (Fig. 5a, Suppl. material 1: table $5). General metabolism related functions were also present in nodules, accounting for 60% for methylotrophy in dunes, and only 25% in forest samples. Also, when considering Defense, including aromatic a) Bacterial communities Biological sample Habitat 40004 | | | © ia al | 3000 4 Oo | [=i F ial ial é al pe = - 2000+ i ~ me of? ,0 | of .e Ne te Biological sample y Yon »A @ Nodule ‘ > ‘ i > 3 4 A Seed Diversity ® Soil b) Fungal communities Biological sample Habitat Habitat © Dune @ Forest 2000 7 a BI :- : rf & 1600) A A 3 @ e ® 2 oame eave 12004 © e A A 2 3 4 2 3 } Diversity Figure 4. Diversity (Shannon-Wiener index) and Richness (Chao index) considering a bacterial and b fungal communities, for biological samples (nodule, seed and soil), habitat (dune and forest), regardless of location (North, Center and South). Note that y-axis has different scales in each community. NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 99 Joana G. Jesus et al.: Unraveling the micr a) obiome dynamics of the invasive Acacia longifolia Bacterial communities Dune Forest Seed Seed ~100 ~100 ¥ 2 \ 2 “80 , -80 / Ls rd & / 6 \ / \ ov \ a & \ y = -60 WS y oe 60 \g 7 Z e « \ ys a \ Functional group f/f [A 7 \ Y / a \ “ @* \ MA 5 Ez 40 \ = Defense S = General metabolism b & £ = Nitrogen Metabolism . > 2 e é° " || -20 = Sulphur Metabolism 2 & = Interactions 2 « @ Nodule 8 S Ss S Ss Soil a, ; Pai ' | | . I y ¥ Ss " S S Nodule S > € > iS Soil Soil Soil Functional group 48 wm Li ] b) Fungal communities ee == Interaction Dune Forest = Decomposition == Growth Seed Seed fv \ ee, 83 \ Habitat 2, = r iia ¢ m vs \go @ Dune re | \ / / \ % i Forest s/ B t Nua o B/ = : Y ee “ & J 7 Ne Ne Pi ,, a) - a aol % e 7) C \ \ we f - ae a das 40 \ ae Se : b/ e % e LY Y & ® Ps 20 2/ th ga ap J ey Ag PA é og val =] \ a \ 2 W.Va TAT CAT SRP ke 2,/ ms Nodule s > © = SS Soil Nodule S = SS = = Soil ~ —— s Soil Soil z Figure 5. Ternary plots presenting functional predictions for a) bacterial and b) fungal communities using FAPROTAX v1.2.6 (Louca et al. 2016) and Fungal Traits (Pélme et al. 2020), respectively. Each function abundance (mean values considering the three locations, North, Center and South) is presented for each biological sample (nodules, seeds and soil) and for each habitat (dune and forest). Colors are assigned to functional groups identified and shapes to habitat. NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 compounds degradation, ca. 36% were found in forest samples compared to 22% for dunes. In dunes, Sulfur metabolism related functions were also registered, ac- counting for 24% of sulfur compound degradation. Seeds gathered a greater variety of functions, and all functional groups identified were included in dune habitat. A greater abundance of different General metab- olism related functions (e.g., chitinolysis, ligninolysis and manganese oxidation), Nitrogen-related metabolism (mainly nitrogen respiration), Sulfur metabolism (i.e., dark oxidation of sulfur compounds) and Defense, as aromatic compound degradation was found. Considering forests, the potential functional pool was mostly present in soils. For fungal communities, there was a more evident distinction between habitats, having Interaction as the main functional group associated with forest, and dune relied more on Growth and Decomposition (Fig. 5b, Suppl. material 1: table S6). Nodules from forest samples had functional machinery more abundant in Eco- logical Strategies (i.e., Lifestyle) and Interaction-related functions, including my- corrhiza and endophytes, particularly ectomycorrhizal (ca. 29%) and endophytic capacity via root and leaves (ca. 47%, each), respectively. Dunes had the same functional groups but more abundantly, highlighting the plant pathogens (64%) and soil saprotrophs (60%) alongside endophytic capacity via roots (62%). 100 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia Discussion Acacia longifolia nodules and seeds showed distinct microbial compositions sug- gesting potential functions tailored to their specific roles in plant's lifecycle and ecological strategies. Indeed, we observed for the first time A. longifolia seed mi- crobiome that seems to be the determinant for plant establishment despite its low diversity. On the other hand, nodules seem to be important for plant develop- ment, reflecting a more targeted functional machinery, especially in nutrient-poor environments as found in dune ecosystems. Altogether, this study points out the adaptative capacity of A. Jongifolia to interact with soil communities and cope with surrounding conditions, and its limitations. Among bacterial communities, the phyla Pseudomonadota, Actinomycetota, Bacillota and Bacteroidota have been more described for both seeds and nodules microbiomes, where they are considered important, rendering its high abundance in soils (Fierer et al. 2012; Barret et al. 2015; Etesami 2022 and references within). In our study, alongside with these phyla, we identified a considerable presence of Cyanobacteriota inside both structures. A more similar microbiome was found among seed samples slightly varying in abundance. Previous studies suggest a vertical transference from mother-plant (Johnston-Monje and Raizada 2011; Links et al. 2014), corroborating the diver- gence on seed compared to soil communities. ‘This is evidenced by the lower abun- dance of the Cyanobacteriota in soils that gain representation in seed bacteriome. However, this more homogeneous microbiome provides a diverse set of functions that could be essential for germination and early establishment, such as variable metabolic pathways or defense strategies. The higher abundance of genera from Cyanobacteriota could be explained through their recently described mixotrophy (Mufioz-Marin et al. 2020), which renders resilience to the host plant. Host selec- tion can ensure the success of establishment, which is also corroborated by the di- verse functions assigned to carbon, nitrogen and sulfur cycles, especially in dunes, making seed microbiota highly plastic and efficient regardless of lower diversity. Additionally, nodules also have a higher abundance of Cyanobacteriota cor- roborating previous results from our group (Jesus et al. 2023). It can be suggested that these bacteria are available via seeds, considering their low abundance in soils, highlighting a selection from host plant for stable partners within plant microbi- ome. Bradyrhizobium was the main genus associated with these structures, along with other Rhizobia as extensively described (e.g., Rodriguez-Echeverria 2010; Crisdstomo et al. 2013; Barret et al. 2015; Jesus et al. 2020), intimately asso- ciated with nitrogen fixation in both habitats. However, considering the habitat conditions, differences were observed, that balanced nutrient pools with energy demand, acknowledging nodulation as a costly process. On the other hand, in forests, it is less abundant, and these soils show a higher diversity of functions associated with the identified microbiome. This suggests that A. longifolia can be able to recruit a set of microorganisms more capable of triggering interactions, es- pecially regarding fungal communities. This environmental shift allows this species to be more competitive and highlight nodulation as a process that enhances plant development. Indeed, while seedlings are growing to saplings, there is an essential carbon and nitrogen dynamics required (Zheng 2009), which can be facilitated by a more specialized microbiome. Furthermore, methylotrophy is more abundant in dunes; this process is essential in carbon cycling and plant growth, particularly NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 101 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia in nutrient-poor environments, where single-carbon compounds are more readily available than multi-carbon ones (as revised by Elbasiouny et al. 2022). Apart from these more specific communities, Pseudomonas and Stenotrophomas were consistently present across all samples, regardless of habitat and location, be- ing present in soil, seeds, and nodules. This corroborates previous studies that highlight the contribution of beneficial surrounding microbial communities to plant microbiome, as well as, if a microorganism is dominant in soil, it increases the probability of becoming dominant inside the plant (e.g., Bulgarelli et al. 2013; Lebeis et al. 2015; Flores-Duarte et al. 2022; Kumar et al. 2023). Environmental reservoir, niche specialization and priority effects determine this, which explains the differences in abundance, accounting for nodules and seed inherent filtering/ selection processes. It is known that microbial lifestyle, i-e., being mutualistic or pathogenic, is clearly expressed in specific contexts. The specificity in which this (dis)advantage is explored considers both plant and microbe when in association, as suggested by Nelson (2018). This is relevant given the higher abundance of potential plant pathogens in seeds. Given that microbial functional redundancy is highly present in soils, the presence of a microorganism does not necessarily equate to it performing a specific function. On the contrary, we found less segre- gation between seed and nodules communities, but a higher divergence in poten- tial functions. So, the promiscuity associated with a great adaptative behavior of A, longifolia makes it a highly plastic species. Regarding mycobiome and the acquisition of fungal partners to the seeds, local assembly has been proposed as more relevant than host selection (Klaedtke et al. 2016); the main important phyla are Ascomycota followed by Basidiomycota (Links et al. 2014). In this study, which includes genera from both phyla, the community found seems to behave as endophytic, given its dissimilarity from soil communities, suggesting its heritage from mother plant. This includes the genera Entoloma, Cladosporium and Curvularia found in seeds. In nodules, So- licoccozyma was found, being also described as an endophytic fungus (Sarabia et al. 2018), having a role in nutrient cycling and being described as tolerant to a range of environmental conditions. Regarding dunes, Umbelopsis, Serendipita and T7uber were among the main genera and their roles in decomposition (Janicki et al. 2022), as beneficial root-associated fungus (Mahdi et al. 2022) and ecto- mycorrhizal (Monaco et al. 2022), respectively, could potentiate survival under more nutrient-limiting conditions. The simultaneous presence of these genera in seeds and root-nodules suggest a seed-to-nodule transmission (or vice-versa), and this intimate integration could trigger new specific interactions as proposed by Nelson (2018). Future studies should be focused on in-depth transcriptomics aiming to fully understand these communities and understanding in detail the transformative role of A. /ongifolia as an invasive plant in soils. In this study, we found differences in microbial composition between the nodules and seeds per se, but further changes in the potential functional “status” can increase those differences, as suggested previously by Degens et al. (2000). The importance of metabolic plasticity, i.e., variety of functions, for some of the microorganisms is potentially raised here, hypothesizing two distinct roles for nodule and seed microbial communities. Nodule communities depend less on carbon acquisition, since the host plant provides the carbohydrates need- ed in exchange for fixed nitrogen; while for seeds, this plasticity can be im- portant given the already known role of seed microbiome in germination and NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 102 Joana G. Jesus et al.: Unraveling the microbiome dynamics of the invasive Acacia longifolia early establishment (Fukami 2015; Bergmann and Leveau 2022; Condessa et al. 2024). So, here we highlight this intimate interaction with microbial com- munities: an adjustment on seed microbiome is found to ease germination and seed-to-seedling transition allowing establishment, while nodule microbiome is more selected to increase development and seedling-to-sapling switch. Indeed, the increased knowledge of invasive plant-microbiome interactions will contrib- ute to a broader understanding of plant local adaptation in novel ecosystems. Conclusions Our study suggests that Acacia longifolia adapts the microbiome according to its needs, depending on the surrounding conditions. The microbiome of this invasive plant species plays a pivotal role in the plant’s success across environments, aiding invasive success. Our findings suggest a trade-off between microbial diversity and functional specialization in seed microbiome, proposing that even with lower di- versity, their functionality is maintained or even increased to support the plant’s initial establishment. On the other hand, the nodule microbiome reflects a greater selection of microbes, each contributing to specific physiological processes nec- essary for growth. In nodules, apart from Bradyrhizobium and nitrogen fixation contribution, a diverse and richer microbial community is harbored, performing specific functions (such as defense and metabolism-related) to sustain plant devel- opment in varying and challenging habitats. ‘The interaction between A. longifolia and its microbiome highlights the role of microbial partners in invasive plants’ ability to adapt to different ecosystems. This is particularly relevant in Mediterra- nean-type ecosystems under climate change. The plants’ success in invasive range can be partly attributed to these efficient and specialized microbial associations that enhance both nutrient uptake and resistance to edaphic fluctuations. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding JGJ is funded by the PhD research fellowship 2021.08482.BD from Fundagao para a Ciéncia e Tecnologia (FCT). This work was funded by FCT through CE3C - Centre for Ecology, Evolution and Environmental Changes unit funding (UIDB/00329/2025) and by the Project INVASIVES “Biological invasions across terrestrial and marine habitats: histories, common traits, and ecological impacts” funded by FCiéncias.ID through FCUL budget. Author contributions JGJ and AA led the data collection and curation. JGJ led the formal analysis, visualization and writing — original draft. CM and HT were responsible for supervision, validation and writing — review & editing. AA was involved in validation and writing — review & editing. HT and CM was involved in funding acquisition. All authors were responsible for conceptualization and gave final approval for publication. NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 103 Joana G. 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Plant Signaling & Behavior 4: 584-591. https://doi.org/10.4161/psb.4.7.8540 Supplementary material 1 Unraveling the microbiome dynamics of the invasive Acacia longifolia: a closer look on seeds and nodules Authors: Joana G. Jesus, Andreia Anjos, Cristina Maguas, Helena Trindade Data type: docx Explanation note: Complementary tables to the information that is included in the manuscript. Copyright notice: This dataset is made available under the Open Database License (http://opendata- commons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/neobiota.99.144628.suppl1 NeoBiota 99: 93-108 (2025), DOI: 10.3897/neobiota.99.144628 108