What lurks beneath the surface? The hidden Frankia biodiversity in Casuarina nodules across continents

What lurks beneath the surface? 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The hidden Frankia biodiversity in Casuarina nodules across continents Nadia Binte Obaid, András Patyi, Fede Berckx, Maru Bernal-Gómez, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8701053/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background and Aims Actinorhizal root nodule symbioses are formed between a diverse group of mostly woody dicotyledonous plants and nitrogen-fixing soil Actinomycetota of the genus Frankia . One of the most ecologically relevant actinorhizal plants are (Allo-)Casuarina species, used widely in shelter belts and phytoremediation due to their high tolerance to abiotic stresses and ability to thrive on marginal soils. All sequenced Frankia strains isolated from (Allo-)Casuarina nodules via traditional techniques show high sequence identity and belong to a single species, Frankia casuarinae . This lack of diversity in nodules is unusual in actinorhizal symbioses. We hypothesised that (Allo-)Casuarina nodules are colonized by Frankia strains that cannot be cultivated and exhibit genome erosion. Methods To test this, we directly sequenced nodule metagenomes from four countries, followed by reconstruction of metagenome-assembled genomes (MAGs). Results Our findings show that the dominant Frankia strains in field samples were far more diverse than the isolated strains and included MAGs with substantial genome reduction – one exhibiting over 25% reduction compared to F. casuarinae . Notably, we observed erosion of two types of [NiFe] hydrogenases, a phenomenon linked to evolution toward obligate symbiosis in other Frankia groups. Conclusion These results suggest that potentially obligate symbionts may dominate nodules in nature but had gone undetected by conventional approaches. For applications such as reforestation or tsunami shelter belts, crushed, nodule-derived strains may offer superior ecological compatibility. We speculate that Frankia strains followed two different evolutionary trajectories; one, towards obligate symbiosis accompanied by strong genome erosion, and two, towards rhizosphere colonization involving limited genome erosion. root nodules symbiotic nitrogen fixation obligate symbiont Frankia Casuarina evolution [NiFe] hydrogenase genome erosion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Actinorhizal symbioses are root nodule symbioses between nitrogen-fixing soil Actinomycetota of the genus Frankia and a diverse group of mostly woody Angiosperm species from eight different families belonging to three different orders (Pawlowski and Demchenko, 2012 ). Amongst the host plants, (Allo-)Casuarina species are of particular importance; due to their high tolerance against abiotic stresses like high salinity, drought, flooding and heavy metal pollution (Batista-Santos et al., 2015 ; Diagne et al., 2013 ; Jeddi et al., 2021 ), they are often used in phytoremediation, coastal shelter belts and as windbreaks in agriculture. Apart from that, they are also used to provide fuel wood and charcoal, contributing to local economies. Therefore, they have been introduced in many countries in warmer climatic regions (Maity and Pawlowski, 2021 ). Phylogenetically, Frankia strains can be grouped into four clades called clusters, three of which (clusters 1–3) encompass symbiotic strains, while cluster-4 strains are non-symbiotic (Pawlowski and Demchenko, 2012 ). The clusters are loosely linked to host specificity – meaning that specific species of Frankia only infect specific actinorhizal hosts. For example, while the basal genus of the family Casuarinaceae – Gymnostoma – can be nodulated by cluster-3 strains (Savouré and Lim, 1991 ; Steane et al., 2003 ; Kates et al., 2024 ), species of two other genera in the same family, Casuarina and Allocasuarina , are nodulated by cluster-1c strains (Pawlowski and Demchenko, 2012 ). Thus far, genome sequences have been published for twelve cluster-1c Frankia strains isolated from (Allo-)Casuarina trees growing on five different continents (Australia, Asia, Africa, North America, South America). These genomes have at least 98% average nucleotide identify (ANI) with each other ( Supplementary Figure S1 ), i.e., they belong to the same species, Frankia casuarinae (Nouioui et al., 2016 ). While the members of F. casuarinae sequenced thus far show some differences regarding salt tolerance (Oshone et al., 2017 ), there is a surprising lack of overall diversity. In 1996, Rouvier et al. , published an attempt to assess the diversity of Casuarinaceae-nodulating strains in their native distribution range by isolating DNA directly from nodules collected in Australia and performing Restriction Fragment Length Polymorphism (RFLP). They found at least seven different groups. Why is this diversity ostensibly not represented by the genomes of F. casuarinae strains isolated around the world via traditional culturing methods? The easiest explanation is because isolation of a species is based on the capability of saprotrophic growth. This restriction to the analysis of isolated strains might have introduced a bias: since no species except for F. casuarinae were isolated from nodules of (Allo-)Casuarina , then these must be the only species growing in the host nodules. However, it is entirely possible that other species of Frankia do grow in the nodules of (Allo-)Casuarina , but lack the ability to be grown in culture, and were therefore never isolated. This has been seen previously; one example is the isolation of non-symbiotic cluster-4 Frankia strains from surface-sterilized nodules of members of Cucurbitales infected by Frankia strains that could never be cultivated (Mirza et al., 1994 ; Carlos-Shandley et al. , 2021; Gueddou et al., 2022 ). Given the thick periderm of mature actinorhizal nodule lobes, fully effective surface sterilization is difficult, i.e., in the absence of a vast majority of cultivable microsymbionts, the occasional isolation of epiphytic Frankia strains from surface-sterilized nodules is not surprising. However, the lack of cultivable microsymbionts is not assumed, as in the cases of the isolation of all known members of the cluster-3 species Frankia irregularis from surface-sterilized nodules of Casuarina spp. on three different continents (strain R43 from Casuarina cunninghamiana in the USA, Pujic et al., 2015 ; strain CcI49 from C. cunninghamiana in Egypt, Mansour et al., 2017 ; and strain G2 T from C. equisetifolia in Guadeloupe, Nouioui et al., 2018 ). Like the cluster-4 strains, these strains cannot nodulate the hosts from whose nodules they were isolated, and careful analysis has shown that they are not present in the infected cells of nodules of Casuarina spp. but rather seem to live epiphytically on the nodule surface (Vemulapally et al., 2019 ). F. irregularis strains can nodulate Frankia cluster-3 host plants from the Elaeagnaceae family (Pujic et al., 2015 ; Mansour et al., 2017 ; Nouioui et al., 2018 ), but so far, no representative of this species has ever been isolated from Elaeagnaceae nodules. In summary, non-endophytic Frankia strains have been ostensibly isolated from surface-sterilized actinorhizal nodules several times, mostly from nodules induced by as-of-yet-uncultivated strains, but three times from nodules of Casuarina spp. The fact that the isolated strains did not represent the nodule endophytes was only recognized because these strains were unable to nodulate their Casuarina “hosts”. Inocula of hosts of the members of Frankia cluster-2 collected in the field represent strain assemblages, some members of which can represent inefficient symbionts that cannot fix nitrogen in nodules (Nguyen et al., 2019 ) and/or symbionts of other host plants (Berckx et al., 2022 ). What if F. casuarinae strains were commonly part of the inocula of Casuarinaceae hosts, and because they had a significantly higher saprotrophic potential than the other strains in the inocula, were always the strains that ended up being isolated? There are also examples of cluster-1 Frankia strains that could never be grown in culture despite several attempts, namely Alnus spp.-infective strains that form spores in nodules (Pozzi et al., 2020 ; Herrera-Belaroussi et al., 2020 ). They show strong genome reduction and, interestingly, the loss of one of the [NiFe] hydrogenases (Pawlowski et al., 2024 ). [NiFe] hydrogenases are required to recycle H 2 which is lost during nitrogenase reaction (Islam et al., 2020 ). Based on Søndergaard et al., ( 2016 ), [NiFe] hydrogenases can be grouped into 29 classes based on the amino acid sequences of their large subunits. Three distinct forms of the 500 + amino acid large subunit of [NiFe] uptake hydrogenase, HupL, were found in symbiotic Frankia strains. Genomes of cluster-1 and cluster-3 strains contain the genes for a [NiFe] hydrogenase of group 1h (synton 1; Søndergaard et al., 2016 ; Pawlowski et al., 2024 ). Genomes of cluster-1 strains and genomes of cluster-2 strains from the continental lineage (Berckx et al., 2022 ) additionally contain the genes for a [NiFe] hydrogenase of group 2a (synton 2), while genomes of cluster-3 strains and genomes of cluster-2 strains from the island lineage contain the genes for a [NiFe] hydrogenase of type 1f (synton-3; Pawlowski et al., 2024 ). Expression analyses showed that while the genes encoding type 1h [NiFe] hydrogenase were expressed at higher levels during saprotrophic growth than in planta , the genes encoding type 2a or 1f [NiFe] hydrogenase were expressed at higher levels in planta than during saprotrophic growth (Pawlowski et al., 2024 ). This can be interpreted to mean that type 2a or type 1f [NiFe] hydrogenase is required for symbiotic nitrogen fixation while type 1h [NiFe] hydrogenase is required for saprophytic growth in culture. This hypothesis is supported by the fact that (a) Søndergaard et al., ( 2016 ) assigned a function in retrieval of protons for nitrogenase to types 2a and 1f, but not 1h, (b) cluster-2 Frankia strains, which with two exceptions could never be grown in culture, indicating a reduced saprotrophic potential, only contain genes for a [NiFe] hydrogenase of type 2a (continental lineage) or type 1f (island lineage) and (c) the erosion of the type 1h [NiFe] hydrogenase synton in strains that form spores in nodules of Alnus spp. (Herrera-Belaroussi et al., 2020 ; Pawlowski et al., 2024 ). Thus, the loss of type 1h [NiFe] hydrogenase in Frankia so far has been found to be correlated with reduced saprotrophic potential and genome reduction. Some Frankia strains additionally contain a type 3b cytosolic [NiFe] hydrogenase (Søndergaard et al., 2016 ; Pawlowski et al., 2024 ). The corresponding synton was found in some representatives of cluster-3 and cluster-1a. Interestingly, it is present – and intact – in all genomes available from F. casuarinae strains published by 2024, suggesting that this enzyme is required for saprotrophic and/or symbiotic growth of F. casuarinae. This begs the questions: (a) do Frankia strains fixing nitrogen in the nodules of (Allo-)Casuarina lack the ability to be grown in culture, and were thus never isolated, and (b) is the loss of type 1h and/or 3b [NiFe] hydrogenases in Frankia generally correlated with reduced saprotrophic potential and genome reduction? We directly sequenced the metagenomes of nodules from different Casuarina spp. trees collected in the field within the original distribution area of the genus, i.e., in Australia, Papua New Guinea, Singapore and French Polynesia (Fig. 1 ) using a method that had been established for nodules of cluster-2 (Nguyen et al., 2019 ; Berckx et al., 2022 , 2024 ) and cluster-1a Frankia strains that could never be cultivated (Herrera-Belaroussi et al., 2020 ). Analysis of the metagenome-assembled genomes showed that most nodules were inhabited by Frankia strains showing higher diversity than the cultivable Casuarina -infective strains sequenced thus far, as well as significant genome reduction and erosion of type 1h and often also of type 3b [NiFe] hydrogenase. We also determined that several of these strains belong to a species other than F. casuarinae. Materials and Methods Plant material Nodules and photosynthetic branchlets were harvested from Casuarina spp. trees in different locations in New South Wales (NSW, Australia), Papua New Guinea, Singapore and French Polynesia. Nodules were kept in either 70% EtOH or RNAlater (Malmstrom, 2015) for transport, while photosynthetic branchlets were air-dried. Australian sampling locations are described in Supplementary Figure S2 . Sampling locations and host species are listed in Table 1 ; Herbarium voucher numbers are listed in Supplementary Table S1 ; a map of all sampling locations is shown in Fig. 1 . The names of the MAGs begin with the abbreviation of the host species (e.g., Cg, Casuarina glauca ) followed by an abbreviation of the sampling site (e.g., TB, Trial Bay) and a number (Table 1 ). The names of the MAGs from Tahiti and surrounding islands are called CeTa-number-full name of sampling place; the MAG from Rurutu is called CeRuru1. DNA isolation For isolation of total nodule DNA, the storage fluid was first removed by centrifuging nodule samples in Eppendorf tubes for 5 min at maximum speed and then pipetting off the liquid. Nodule metagenomes were isolated as described by Nguyen et al., ( 2019 ) and Berckx et al., ( 2022 ) or using the E.Z.N.A. HP Plant DNA kit (OMEGA BIO-TEK, VWR, Sweden). In either case, Polyclar AT (Serva, Germany) was added with a ratio of 1:2 Polyclar AT to sample weight during grinding in liquid nitrogen, and one additional ultrasonic homogenization step was performed using the Bandelin Sonopuls UW 2070 ultrasonic homogenizer with the MS 73 probe attached (Bandelin Electronic, Germany) after adding the lysis buffer. Host plant species determination The distribution of Casuarina and Allocasuarina species in Australia, and a description of the morphology of their photosynthetic branchlets, is available at PlantNET and in Castle ( 2008 ). These data were used to identify the putative host plants for the samples from New South Wales (Australia; Supplementary Table S2 ). Based on the branchlet anatomy, the samples from inland sites came from Casuarina cunninghamiana , and the samples collected close to the sea from C. glauca. For the samples from Papua New Guinea, Singapore and French Polynesia, we relied on published data regarding the distribution of (Allo-)Casuarina species. In case of Papua New Guinea, only two Casuarina species are documented, C. equisetifolia at the coast and C. oligodon in the mountains (Nuberg et al., 2015 ); the nodule samples were collected in the mountains (Fig. 1 ). The two trees used for sampling in Coney Island Park (Singapore) were identified as C. equisetifolia on the park web site ( https://www.nparks.gov.sg/florafaunaweb/flora/2/7/2793 ). Only one Casuarina species is documented in French Polynesia, namely C. equisetifolia (Dotte-Sarout and Kahn, 2017 ). Nodule metagenome sequencing and assembly Sequencing of the DNA from nodules was performed as previously described (Berckx et al., 2024 ). The raw reads were subjected to binning to remove plant DNA sequences and sequences of unrelated bacterial strains and then assembled into Frankia metagenome-assembled genomes (MAGs) as described before (Berckx et al., 2022 ). MAG analysis For phylogenetic analyses based on core genome, and calculations of Average Nucleotide Identity (ANI) and Average Amino-acid Identity (AAI), we used the EDGAR 3.0 platform (Blom et al., 2009 , 2016 ; Dieckmann et al., 2021 ), which provides robust tools for comparative genomics. Specifically, ANI values were determined by computing fastANI scores, which assess the nucleotide similarity between two genomes using consecutive 1,020 nucleotide fragments across the core genome and averaging the results. AAI scores were calculated based on alignments of core genome-encoded proteins. This combined approach enabled a comprehensive and high-resolution comparison of the genomes studied and will help identify potential species and genus boundaries. A core genome tree and a 16S tree were also constructed for the MAGs. The core genome tree was inferred using the EDGAR3.0 platform. For construction of the 16S rRNA phylogenetic tree, 16S gene sequences corresponding to the dominant Frankia strain were first identified within the datasets. This was achieved through two complementary strategies: (i) by aligning the entire metagenome assembly to reference 16S sequences from established Frankia strains – using, for example, 16S of Frankia casuarinae CcI3 T as a query sequence for targeted retrieval by means of BLASTn (Altschul et al., 1990 ), and (ii) by generating additional metagenome assemblies using SPAdes v3.15.4 (Bankevich et al., 2012 ) with reduced subsets of the dataset and then again comparing to 16S sequences from established Frankia strains by applying BLASTn. The latter approach was employed to mitigate potential oversampling and assembly biases that can arise from high-abundance sequences. Identified 16S rRNA gene sequences were then aligned and used for phylogenetic reconstruction to assess the taxonomic placement of the dominant Frankia strain within the sample. The sequences were then used to construct a phylogenetic tree on MEGA (version 12.0.4; Kumar et al., 2024 ) using the Maximum Likelihood method. 1,000 bootstraps were used for the tree calculation. Statistical analysis of genomes and MAGs Genome size and GC content of genomes were derived from contig information on GenDB (Meyer et al., 2003 ), a genome annotation system for prokaryotic genomes, to visualize the relationship between genome size and GC content. To determine whether the genome sizes significantly differed among the known cultivable strains and the strains in this study, an ANOVA test followed by Tukey pairwise comparison was carried out, and the results were illustrated in a merged dotplot and violin plot. Data analysis and preparation of plots were done on RStudio (Posit Team, 2024 ). For digital DNA:DNA hybridization (dDDH) studies, the genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available under https://tygs.dsmz.de , for a whole genome-based taxonomic analysis (Meier-Kolthoff and Göker, 2019 ). The analysis also made use of recently introduced methodological updates and features (Meier-Kolthoff et al., 2022 ). Information on nomenclature, synonymy, and associated taxonomic literature was provided by TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de ) (Meier-Kolthoff et al., 2022 ). The results were provided by the TYGS on 2025-09-05. For phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5 (Meier-Kolthoff et al., 2013 ). 100 distance replicates were calculated each. Digital DDH values and confidence intervals were calculated using the recommended settings of the GGDC 4.0 (Meier-Kolthoff et al., 2013 , 2022 ). Analysis of [NiFe] hydrogenase operons [NiFe] hydrogenase genes of all Frankia strains in this study were identified via BlastP searches on GenDB (Meyer et al., 2003 ), using Frankia casuarinae CcI3 T genes as queries. Contig information were used to create plots of the hydrogenase syntons on RStudio (Posit Team, 2024 ), using the package gggenes (Wilkins, 2023). Protein modelling Protein structure analysis was done via Alphafold (Jumper et al., 2021 ; Abramson et al., 2024 ). Results Identification of the host species through morphology MAGs were obtained from a total of 21 nodule samples. Based on the sampling locations in NSW, the host tree species could be narrowed down to two Casuarina species ( C. cunninghamiana, C. glauca ). The morphology of the photosynthetic branchlets showed that all inland samples from NSW were from C. cunninghamiana while all samples collected at or close to a beach came from C. glauca (Fig. 1 , Table 1 , Supplementary Figure S2, S3, Supplementary Table S1, S2) . In summary, MAGs were obtained from Casuarina equisetifolia in Singapore (Coney Island, two samples) and French Polynesia (Tahiti, Moorea and Rurutu, four samples); from Casuarina oligodon in Papua New Guinea (Local Level Government Bulolo-Wau, Morobe Province, three samples); and from Casuarina cunninghamiana and Casuarina glauca in New South Wales (NSW, Australia, 12 samples). Details of the MAGs are given in Table 1 ; the accession numbers of the MAGs are given in Supplementary Table S3. Table 1 Details of Frankia MAGs from Casuarina spp. 1 trees from self-sown material of unknown origin. Country Location MAG host Genome size [bp] % [GC] BUSCO N50 coverage contigs Australia Casuarina Sands CcCS1 C. cunninghamiana 4,072,291 70.45 88.50% 12,484 bp 258x 429 Casuarina Sands CcCS3 C. cunninghamiana 4,183,436 70.43 91.20% 15,747 bp 156x 367 Wombeyan Caves CcWB2 C. cunninghamiana 4,309,795 70.41 83.80% 19,335 bp 356x 336 Wombeyan Caves CcWB3 C. cunninghamiana 4,284,996 70.4 90.50% 14,875 bp 74x 403 Molonglo CcMO1 C. cunninghamiana 5,271,872 70.17 87.80% 18,394 bp 50x 405 Arakoon CgARK3 C. glauca 4,169,588 70.26 81.20% 14,809 bp 36x 376 Jervis Bay CgJB1 C. glauca 4,238,692 70.3 89.90% 20,036 bp 71x 292 Jervis Bay CgJB3 C. glauca 4,238,692 70.3 89.90% 20,036 bp 71x 292 Kioloa CgKIO3 C. glauca 4,249,750 70.32 93.20% 20,463 bp 110x 291 Roseville CgROS2 C. glauca 4,066,875 70.22 87.80% 12,945 bp 110x 448 Trial Bay CgTRI1 C. glauca 4,089,891 70.24 87.20% 13,537 bp 20x 409 Trial Bay CgTRI2 C. glauca 4,185,287 70.28 91.30% 15,352 bp 45x 372 Singapore Coney Island 1 CeCAU1 C. equisetifolia 5,145,236 70.12 91.90% 31,920 bp 160x 263 Coney Island 1 CeCAU2 C. equisetifolia 4,965,976 70.11 91.20% 25,973 bp 90x 302 Papua New Guinea Morobe-Bulolo CoBUL1 C. oligodon 3,786,542 70.46 91.90% 14,424 bp 208x 368 Morobe-Wau CoWAU3 C. oligodon 3,786,542 70.39 87.20% 16,007 bp 350x 342 Morobe-Wau CoWAU4 C. oligodon 3,702,336 70.37 79.80% 14,865 bp 450x 363 French Polynesia Rurutu CeRuru1 C. equisetifolia 5,221,813 70.23 96,7% 12,788 bp 24x 544 Tahiti, Mahina CeTa2Mahina C. equisetifolia 3,875,375 70.18 83,6% 6,032 bp 17x 745 Tahiti, Moorea CeTa3Moorea C. equisetifolia 5.462.711 70.39 98,6% 31,927 bp 87x 288 Tahiti, Puna'auia CeTa4Puna'auia C. equisetifolia 4,166,592 69.78 77,7% 6,142 bp 20x 798 Do these MAGs represent a new species? A core genome tree was inferred using all MAGs obtained in this study combined with the genome sequences of isolated Frankia casuarinae strains available at NCBI by 2024 ( Supplementary Table S4 ), along with type strains of other Frankia species. The tree was rooted with Cryptosporangium arvum DSM44712 (NCBI RefSeq accession: GCF_000585375.1) and Jatrophihabitans endophyticus DSM45627 (NCBI RefSeq accession: GCF_900129455.1) (Fig. 2 ). The tree shows that the strains from Singapore and Papua New Guinea, and one of the strains from French Polynesia (CeTa2Mahina), clustered with the known isolated Frankia casuarinae strains, while all strains from Australia and the other three strains from French Polynesia (CeRuru1, CeTa3Moorea and CeTa4Puna’auia) mapped to a different branch, suggesting that they were likely to represent a novel species. Furthermore, among the strains collected from Australia, strains coming from nodules that were collected close from the beach and strains coming from nodules collected in-land also displayed a clear separation, indicating that they formed separate subclades within the species. Similarly, the genomes of isolated F. casuarinae strains and the genomes of strains coming from Singapore, Papua New Guinea and CeTa2Mahina from French Polynesia also showed a clear separation in the core genome tree, i.e., they formed separate subclades of F. casuarinae. Are we dealing with two different species, or with two subspecies of the same species? Jain et al. ( 2018 ) propose 95% ANI as cutoff value for demarcating prokaryotic species, while Ciufo et al. ( 2018 ) propose 96%. fastANI analysis (Dieckmann et al., 2021 ) of the 21 new strains studied shows that the two strains from Singapore (CeCAU1 and CeCAU2), the three strains from Papua New Guinea (CoBUL1, CoWAU3 and CoWAU4), and one strain from French Polynesia (CeTa2Mahina) had ≥ 97% ANI among them (Fig. 3 ). Two of the remaining three strains from French Polynesia (CeRuru1 and CeTa3Moorea) and all strains from Australia had < 96% identity with the cultivable F. casuarinae strains but had ≥ 97% ANI with each other (Fig. 3 ). Overall, fastANI analysis of the genomes of Casuarina -infective Frankia strains shows a border of 95–96% between the two branches (Fig. 3 ). Interestingly, the MAG CeTa4Puna’auia takes a slightly ambiguous position between both groups with less than 96% ANI with most strains of both; when Average Amino-acid Identity is analysed it shares more than 96% AAI with the F. casuarinae strains but more than 97% AAI with the MAGs of the potential novel species (AAI; Supplementary Figure S4 ). In short, core genome tree and the ANI analysis suggest that the strains from Australia and three of the strains from French Polynesia represent a novel Frankia species or subspecies. Genome reduction in most MAGs from Casuarina nodules Most of the MAGs sequenced in this study were smaller than 5 MB, i.e., below the range found for the genomes of isolated F. casuarinae strains (Fig. 4 A). CcMO1, one of the MAGs from NSW, was an exception among the NSW strains; it was isolated from nodules collected from a fallen tree with its roots in the air ( Supplementary Figure S2 ). Two of the MAGs from French Polynesia, CeRuru1 and CeTa3Moorea, based on genome size also did not exhibit genome reduction compared to the cultivable strains, while CeTa2Mahina showed some reduction and the last strain, had a much smaller genome (Table 1 ). In contrast, the two F. casuarinae strains from Singapore showed no genome reduction, whereas all three F. casuarinae strains from Papua New Guinea, had significantly smaller genomes. One-way ANOVA revealed that there was a statistically significant difference in mean genome size between at least two groups [F(4, 28) = 20.89, p = 4.52e-08]. Tukey’s HSD Test for multiple comparisons found that the mean value of genome size was significantly different between the published F. casuarinae strains and the MAGs from Papua New Guinea [p = 0. 0.0000006, 95% CI = (-2.20, -0.94)], between the published F. casuarinae strains and the MAGs from Australia [p = 0. 0.0000005, 95% CI = (-1.40, -0.61)], and between the published F. casuarinae strains and the MAGs from French Polynesia [p = 0.0271632, 95% CI = (-1.18, -0.05)]. There was no statistically significant difference in size between the genomes of isolated F. casuarinae strains and the MAGs from Singapore. These results are shown in Fig. 4 B. Overall, 16 out of 21 MAGs sequenced in this study were markedly smaller (3.7–4.3 MB) than the genomes of cultivable F. casuarinae strains published thus far. Genome erosion: which genes were lost in the new Frankia MAGs? Genome reduction in cluster-1 Frankia has also been documented for cluster-1a strains that form spores in the nodules of alder species and could never be cultured despite numerous attempts (Herrera-Belaroussi et al., 2020 ; Pozzi et al., 2020 ; Pawlowski et al., 2024 ). In the Pawlowski et al., ( 2024 ) study, Frankia cluster-1 strains were shown to contain two or three different types of [NiFe] hydrogenases, encoded by synton-1, synton-2 and, in case of F. casuarinae , synton-4. Genome erosion in cluster-1a was first visible for synton-1 (type 1h [NiFe] hydrogenase; Søndergaard et al., 2016 ). Therefore, syntons 1, 2 and 4 were analysed for the new MAGS. With the exception of CeTa3Moorea and CeRuru1, all MAGs showed erosion of synton-1 (type 1h [NiFe] hydrogenase; Fig. 5 A) while synton-2 (type 2a [NiFe] hydrogenase) remained intact (data not shown). The functions of the proteins encoded by synton-1 are given in Supplementary Table S5 . All MAGs from nodules collected from C. glauca trees growing at the beach in NSW (CgJB1, CgJB3, CgTRI1, CgTRI2, ACgARK3, CgKIO3, CgROS2) do not contain the maturation protease hupD1 and contain at least one stop codon in the ORF of hupL1 , precluding enzyme function. The strains collected from C. cunninghamiana further inland (CcCS1, CcCS3, CcWB2, CcWB3, CcMO1) had no intact hypB1, hupL1 and hypF1 genes. In CgTRI2 and CgROS2, the 3’-parts of the synton ( hypF1 – hypC1 – hypD1 – hypE1 ) were separated from the 5’-parts. Similarly, in CcWB3, CcCS1 and CcMO1, the 3’-parts of the operon ( hupL1 – hypD1 – hypC1 – hypE1 – hypF1 ) were separated from the 5’-parts. Among the MAGs from French Polynesia, CeRuru1 contained all genes of synton-1, but the 5’ and 3’ parts of the syntons were in different parts of the genome. CeTa3Moorea was the only MAG containing an intact synton-1 not divided in parts by transposition. CeTa4Puna’auia contained no functional copy of hupL1, hypF1 and hypD1 ; moreover, synton-1 was divided in four parts scattered throughout the genome. CeTa2Mahina had lost most genes of synton-1 completely, while hypF1, hypD1 and hypE1 were truncated. The MAGs from Papua New Guinea – CoBUL1, CoWAU3 and CoWAU4 – had lost the entire synton-1 encoding type 1h [NiFe] hydrogenase. The MAGs from Singapore, CeCAU1 and CeCAU2, while ostensibly not showing genome reduction, had no intact copies of hupS1, hupL1 , and hupD1 genes, i.e., the strains had lost the structural proteins of type 1h [NiFe] hydrogenase. Erosion of synton-4 encoding type 3b cytosolic [NiFe] hydrogenase was also found (Fig. 5 B). The complete operon of F. casuarina CcI3 T as well as the other cultivable strains consists of six genes, hyhB – cNMP-DB– hyhG – hyhS – hyhL – M52 . Gene names were taken from Søndergaard et al., ( 2016 ) except for cNMP-BD and the maturation protease M52. Supplementary Table S5 shows the function of each protein. Among the MAGs from nodules collected from C. glauca growing at the beach in NSW, only CgKIO3 and CgTRI1 showed erosion of the type 3b [NiFe] hydrogenase synton; in both cases, hyhG was truncated. In CgKIO3, hyhB was interrupted by a stop codon, while in CgTRI1, hyhL was broken in two; one of the parts was also truncated. Among the MAGs collected from inland C. cunninghamiana plants, hyhL and M52-4 were fused in CcMO1, CcCS1 and CcCS3, while hyhB was truncated in CcWB2 and CcWB3. HyhL contained a stop codon in CcWB2, while it was truncated in CcWB3. CcCS1 also contained a stop codon in hyhS. Among the MAGs from C. equisetifolia growing in French Polynesia, we found that synton-4 was divided into two (CeRuRu1, CeTa4Puna’auia) or three (CeTa2Mahina) parts in different areas of the genome, indicating transposase activity. In CeRuRu1, three genes were truncated ( hyhB, hyhS and hyhL ); furthermore, the ORFs of hyhS and hyhL were fused. In CeTa3Moorea, hyhB, hyhS and hyhL were truncated and the ORFs of hyhS and hyhL were fused. In CeTa4Puna’auia, hyhS was divided in two truncated parts, hyhG was lost entirely and cNMP-BD and M52-4 were truncated. In CeTa2Mahina, hyhG was truncated while hyhL was divided into two parts in different areas of the genome; one of the parts was truncated. The MAGs from C. equisetifolia growing in Singapore contained the full-size synton-4 like F. casuarinae CcI3 T . In all MAGs from nodules of C. oligodon growing in Papua New Guinea, the ORF of hyhB was interrupted by a stop codon. In CoWAU3, the M52-4 gene was truncated, while in CoWAU4, hyhG was truncated and hyhL was interrupted by a stop codon. In summary, we found erosion of synton-1 encoding type 1h [NiFe] hydrogenase in all MAGs sequenced in this study except for CeTa3Moorea and presumably CeRuru1 (we do not know whether a promoter was affected by the transposition). Erosion of synton-4 encoding type 3b cytosolic [NiFe] hydrogenase was observed for all MAGs except for the ones from Singapore and most of the MAGs from nodules of C. glauca trees growing at the beach in NSW. This is particularly interesting since some of the MAGs that showed erosion of type 1h and/or type 3b hydrogenase (CcMO1, CeCAU1, CeCAU2, CeRuru1 and CeTa3Moorea) did not show an obvious reduction in overall size compared to the genomes of the isolated strains (5.0–5.5 compared to 5.0–5.6 MB in the cultivable strains; Fig. 4 A). Which genes distinguish F. casuarinae and the potential new species? This question is difficult to answer since while several genomes of cultivable F. casuarinae strains are available, the [NiFe] hydrogenase data show that all MAGs sequenced in this study show a higher degree of genome erosion than the cultivable ones. We compared the genomes of the isolated F. casuarinae strains with the MAGs of CeTa3Moorea and CeRuru1. The MAGs of CeTa3Moorea and CeRuru1 contain a gene that is not present in any other Frankia genome sequenced to date, encoding a bifunctional enzyme encompassing isocitrate lyase (N-terminal) and malate synthase (C-terminal), thus combining the two functions required for the glyoxylate shunt of the tricarboxylic acid (TCA) cycle (Woo et al., 2024 ; Fig. 6 ; Supplementary Table S6 ). Such bifunctional enzymes have so far only been found in Euglena gracilis (Nakazawa et al., 2011 ) and Caenorhabditis elegans (Liu et al., 1995 ). F. casuarinae strains, like cluster-2 Frankia strains (Berckx et al., 2025 ), lack the glyoxylate shunt since they do not contain a gene for isocitrate lyase, although such a gene is present in most cluster-1 genomes other than those of F. casuarinae (Berckx et al., 2025 ), even in those of cluster-1a Frankia strains that form spores in nodules of Alnus glutinosa and have a reduced genome (Herrera-Belaroussi et al., 2020 ; Pozzi et al., 2020 ). Most cultivable F. casuarinae strains except for CcI156 also lost malate synthase (NCBI accession WP_076804337.1). Interestingly, CeRuru1 contains a complete malate synthase gene and CeTa3Moorea a truncated one, but not a complete isocitrate lyase gene ( Supplementary Table S6 ). Since the gene of the bifunctional enzyme cannot be found in any MAG analyzed in this study except for those of CeTa3Moorea and CeRuru1, nor in the genomes of cultivable F. casuarinae strains, it must be concluded that it – and thus, the glyoxylate shunt – got lost early during genome erosion. Thus, genome erosion led to loss of the glyoxylate shunt of the TCA cycle in all genomes of (Allo-)Casuarina -infective Frankia strains known to date. On the other hand, we found some genes that are only present in F. casuarinae , not in any MAG of the novel species. The cultivable F. casuarinae strains contain four genes encoding polyhydroxybutyrate depolymerase (NCBI accessions OAA30795, OAA26147, OAA26975, OAA25310 in F. casuarinae CcI3 T ), one of them (OAA30795) encoding a member of the secretome (Mastronuncio et al. , 2008). No homologs of the secreted PBH depolymerase, which had been suggested to be involved in the degradation of suberin, and of OAA25310, were found in any MAG of the potential new species. Discussion The sequencing of 21 MAGs from nodules of Casuarina spp. in their original distribution area yielded 16 MAGs showing distinct genome erosion (3.7–4.3 compared to 5.0–5.6 MB in the cultured strains of Frankia casuarinae ), while all MAGs showed erosion of the genes encoding [NiFe] hydrogenase type 1h and/or type 3b. Thus, the loss of these two [NiFe] hydrogenases seems to have occurred very early during genome erosion in cluster-1c Frankia. The plasma membrane type 1h [NiFe] hydrogenase (synton-1) has been linked to hydrogenotrophic respiration using O 2 as a terminal electron acceptor; the enzyme is supposed to scavenge electrons from atmospheric H 2 to fuel the respiratory chain during carbon-deficiency (Søndergaard et al., 2016 ). In Frankia , this enzyme was lost in species that show reduced saprotrophic potential and genome erosion (cluster-2 strains, cluster-1 strains that form spores in nodules and could never be cultured despite multiple attempts), suggesting that it is not essential for the symbiotic lifestyle (Pawlowski et al., 2024 ). The results of this study are consistent with this hypothesis; furthermore, the fact that erosion of synton-1 was found in all genomes except for that of CeTa2Moorea suggests that even in genomes that have sizes comparable to those of cultured Frankia casuarinae strains (CcMO1, CeCAU1, CeCAU2, CeRuru1; 5.0–5.3 MB), genome erosion had begun. Thus, except for CeRuRu1 and presumably CeTa3Moorea, all MAGs sequenced in this study showed erosion of [NiFe] type 1h hydrogenase (synton-1), a phenomenon linked to reduced saprotrophic potential in cluster-1a strains. This could explain why no strains of their respective phylogenetic groups (Fig. 2 ) have been isolated from (Allo-)Casuarina nodules in the past using traditional methods. To date, no Frankia strain with a genome size below 5 MB has been successfully cultivated. The cytosolic bidirectional group 3b [NiFe] hydrogenase encoded by synton-4 can couple the oxidation of NADP to the fermentative evolution of H 2 (Søndergaard et al., 2016 ). Its pattern of occurrence is interesting: it is present in only a few genomes from cluster-1a and − 3 strains but it is present in all genomes of isolated F. casuarinae strains (Pawlowski et al., 2024 ). There is no clear grouping of MAGs where it is ostensibly not eroded – CeCAU1, CeCAU2, CgARK1, CgJB1, CgJB3, CgTRI2. Two of them come from Coney Island in Singapore from Casuarina equisetifolia trees with unclear pedigree, the other four from NSW are from nodules of Casuarina glauca trees growing close to the sea; in short, the data available are not suited to build a hypothesis about the function of synton-4. We can conclude, however, that its erosion does not seem to be linked to host specificity. It might be linked to salt stress as all genomes from nodules of inland strains, including CcMO1 with its genome size of 5.27 MB, did show erosion of synton-4. At any rate, the fact that it was found to be eroded or even completely lost in strains showing genome erosion, as is typical for the evolutionary trajectory towards obligate symbiosis, indicates that it is more relevant for the saprotrophic than for the symbiotic lifestyle. Do the Australian MAGs and the three French Polynesian MAGs represent a new species, separate from F. casuarinae ? The percentage of species separation is not unambiguously defined (Ciufo et al., 2018 ; Jain et al., 2018 ). An Average Nucleotide Identity (ANI) matrix (Fig. 3 ) and an Average Amino-acid Identity (AAI) matrix ( Supplementary Figure S4) are somewhat ambiguous regarding CeTa4Puna’auia and of CcMO1. A 16S phylogenetic tree rooted with the 16S of the type strain of a cluster-1a Frankia species, Frankia alni ACN14a T provides a clear separation between the MAGs from NSW (Australia), CeTa3Moorea and CeTa4Puna’auia versus F. casuarinae CcI3 T , the MAGs from Papua New Guinea and Singapore and CeTa2Mahina (Fig. 7 ). However, on the 16S level we do not see the separation between the MAGs from C. glauca nodules and C. cunninghamiana nodules that we see in the core genome tree (Fig. 2 ), calling the usefulness of 16S phylogeny for Frankia cluster-1c species separation into question (see also Pozzi et al., 2018 ). Therefore, we used the option for pairwise calculation of digital DNA:DNA hybridization (dDDH) values calculations) provided by the TYGS web server (Meier-Kolthoff and Göker 2019 ), where the species border is defined at 70%. Figure 8 shows the results. There is a clear separation of F. casuarinae and the novel species, with CeTa4Puna’auia taking a slightly ambiguous position, displaying maximally 69.8% dDDH with any genome of F. casuarinae and and minimally 70.2% dDDH with any genome of the novel species. In summary, both genomic and phylogenetic data (ANI, 16S, core genome tree, dDDH) grouped together the MAGs from NSW, CeRuru1, CeTa3Moorea and CeTa4Puna’auia in the same species, proposed as Candidatus Frankia pacificiensis. This species can be found in root nodules of C. equisetifolia and C. glauca at the coasts of the Society Islands and NSW, and in root nodules of C. cunninghamiana in inland NSW. The strongest support for a long-term separation of F. casuarinae and the novel species is the existence of the unique gene for a bifunctional enzyme combining isocitrate lyase and malate synthase in the MAGs CeTa3Moorea and CeRuru1. However, the fact that both MAGs also contain a malate synthase gene (although truncated in CeTa3Moorea) might be taken to suggest that the gene for the bifunctional enzyme was acquired by horizontal gene transfer. The closest homologs of both parts of the bifunctional enzyme (isocitrate lyase and malate synthase) are enzymes from cluster-3 Frankia strains (data not shown); however, no gene for a bifunctional enzyme was detected in cluster-3 Frankia . Does the core genome tree (Fig. 2 ) tell us something about the evolution of Frankia casuarinae ? In F. casuarinae we see a clear separation between the cultured strains and the MAGs from Papua New Guinea and Singapore (from C. equisetifolia and C. oligodon ) that is not related to genome erosion which is minimal in the MAGs from Singapore (see Fig. 5 A regarding the [NiFe] hydrogenase type 1h synton). In contrast, in Ca. F. pacificiensis we see a clear separation between MAGs from C. glauca growing close to the sea – i.e., under conditions of salt stress – and from C. cunninghamiana growing inland. The three MAGs from C. equisetifolia growing close to the sea in French Polynesia occupy the root position (Fig. 2 ). Given the lower similarity among the four MAGs from French Polynesia, one of which represents F. casuarinae , compared to the much more conserved MAGs from NSW, and the fact that one representative of F. casuarinae was found in French Polynesia (CeTa2Mahina), we conclude that F. casuarinae and the novel species diverged in French Polynesia and that CeTa4Puna’auia, with its slightly ambiguous position between the two species, is closest to this point of separation. These data could be further interpreted to mean that the new species evolved in coastal areas of French Polynesia and adapted to inland areas after reaching NSW. However, they could also be interpreted to mean that the new species is divided in groups of strains with different host specificity. Phylogenetic analysis on the plant side has shown that C. equisetifolia is the earliest divergent species in the genus Casuarina , followed by C. cunninghamiana (Kates et al., 2024 ). The grouping of the MAGs would be consistent with co-evolution between host and microsymbiont. To return to the original research question whether Frankia strains fixing nitrogen in the nodules of (Allo-)Casuarina lack the ability to be grown in culture, and were therefore never isolated, and whether the loss of type 1h and/or 3b [NiFe] hydrogenases in Frankia is generally correlated with reduced saprotrophic potential and genome erosion, the results are affirmative. In most cases of field-collected nodules in the original distribution area of the host plants, we find in 16 out of 21 cases strong genome erosion and in the remaining five cases beginning genome erosion; in all cases type 1h and/or 3b [NiFe] hydrogenases had lost functionality. This resembles the situation in alder-infective cluster-1 strains that sporulate in nodules (Pozzi et al. , 2015; Herrera-Belaroussi et al., 2020 ; Pozzi et al., 2020 ; Pawlowski et al., 2024 ). Why did we not find some nodules without detectable genome erosion although the cultivable strains are definitely able to nodulate (Allo-)Casuarina spp.? In case of in planta sporulating strains, i.e., in cluster-1a strains with genome erosion on an evolutionary trajectory towards obligate symbiosis, infectivity and rhizospheric competitiveness were increased compared to cultivable strains (Cotin-Galvan et al., 2016 ). Thus, they were likely to outcompete any saprotrophs in the inoculum. Our results suggest that the same was the case for ( Allo-)Casuarina -infective strains with genome erosion. Altogether, evolution towards reduced saprotrophic capacity and stronger symbiotic competitiveness seems to have happened in several lineages of Frankia cluster-1a and cluster-1c, as well as in cluster-2 (Persson et al., 2015 ), except that in the latter, only two cultivable strains were identified (Gtari et al., 2015 ; Gueddou et al. , 2018). Did it also happen in cluster-3 which encompasses symbiotic Frankia strains with the largest genomes (Ghodhbane-Gtari et al., 2020 )? Direct sequencing of total nodule DNA and MAG assembly will show whether also here, some strains evolved towards obligate symbiosis. Conclusion Frankia strains present in nodules of Casuarina spp. in its original distribution area show greater diversity than strains isolated from Casuarina spp. nodules and show genome erosion compared to those cultivable strains (in 76% of the cases, strong genome erosion). Thus, a focus on Frankia strains isolated from nodules has rendered a misleading picture of Frankia diversity, and, presumably, its adaptation to ecological conditions and specific host species. Abbreviations AAI average amino acid identity ANI average nucleotide identity MAG Metagenome-assembled genome NSW New South Wales Declarations Statements and declarations Open access funding provided by Stockholm University. This project was supported by the Swedish Research Council Vetenskapsradet (2019–05540 to K.P.) and the Carl Tryggers foundation (CTS22: 2145 to K.P.). The bioinformatics support of the BMBF-funded project ‘Bielefeld-Giessen Center for Microbial Bioinformatics’ (BiGi) and the BMBF grant FKZ 031A533 within the German Network forBioinformatics Infrastructure (de.NBI) are gratefully acknowledged. The bioinformatics support of the ‘Bielefeld-Giessen Center for Microbial Bioinformatics’ (BiGi) within the German Network for Bioinformatics Infrastructure BMFTR grants: W-de.NBI-004, W-de.NBI-010, BMBF grant FKZ 031A533) are gratefully acknowledged. The authors have no relevant financial or non-financial interests to disclose. Author contributions Nadia Binte Obaid : formal analysis, supervision, writing – original draft. András Patyi : investigation, formal analysis, writing – original draft. Fede Berckx – supervision, writing – review & editing. Maru Bernal-Gomez : formal analysis, writing – review & editing. Andrea Lavello : investigation, writing – review & editing. Andreas Brachmann : investigation, writing – review & editing. Daniel Wibberg : formal analysis, writing – review & editing. Jochen Blom : data curation, writing – review & editing. Jörn Kalinowski : writing – review & editing. Sara Mehrabi : investigation, formal analysis, writing – review & editing. Ivan R. Kennedy : investigation, writing – review & editing. Philippe Normand : investigation, formal analysis, writing – review & editing. Ulrike Mathesius : investigation, writing – review & editing. Katharina Pawlowski : conceptualization, funding acquisition, project administration, supervision, investigation, formal analysis, writing – original draft. 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Biomass Bioenergy 78:126–139. https://doi.org/10.1016/j.biombioe.2015.04.010 Oshone R, Ngom M, Abebe-Akele F, Simpson S, Morris K, Sy MO, Champion A, Thomas WK, Tisa LS (2016) Permanent Draft Genome sequence of Frankia sp. strain Allo2, a salt-tolerant nitrogen-fixing Actinobacterium isolated from the root nodules of Allocasuarina . Genome Announcements 4(3):e00388–e00316. https://doi.org/10.1128/genomeA.00388-16 Oshone R, Ngom M, Chu F, Mansour S, Sy MO, Champion A, Tisa LS (2017) Genomic, transcriptomic, and proteomic approaches towards understanding the molecular mechanisms of salt tolerance in Frankia strains isolated from Casuarina trees. BMC Genomics 18(1):633. https://doi.org/10.1186/s12864-017-4056-0 Pawlowski K, Demchenko KN (2012) The diversity of actinorhizal symbiosis. 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Mol Syst Biol 20(3):170–186. https://doi.org/10.1038/s44320-024-00017-w Supplementary Files SupplementaryTableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 11 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor invited by journal 06 Feb, 2026 Editor assigned by journal 05 Feb, 2026 First submitted to journal 01 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8701053","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588843907,"identity":"c3e64244-7a60-4897-b4cf-99756d7b1116","order_by":0,"name":"Nadia Binte Obaid","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"Binte","lastName":"Obaid","suffix":""},{"id":588843908,"identity":"ccde1df6-8ca1-449d-b7a5-9f96c6b949f0","order_by":1,"name":"András 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14:06:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8701053/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8701053/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102754692,"identity":"13866704-217f-48cc-b742-552f51bd1daa","added_by":"auto","created_at":"2026-02-16 09:39:00","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195288,"visible":true,"origin":"","legend":"\u003cp\u003eHarvesting locations. Nodules were collected from \u003cem\u003eCasuarina equisetifolia\u003c/em\u003e in Singapore (orange, Coney Island), from\u003cem\u003e Casuarina glauca\u003c/em\u003eand \u003cem\u003eCasuarina cunninghamiana \u003c/em\u003ein Australia (blue, New South Wales), from \u003cem\u003eCasuarina oligodon\u003c/em\u003e in Papua New Guinea (yellow), and from\u003cem\u003e Casuarina equisetifolia\u003c/em\u003ein French Polynesia (green).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/75ec478f9995734542bf370e.jpg"},{"id":102754678,"identity":"ba061dd9-7f78-4cf7-9067-bc35455c6fdc","added_by":"auto","created_at":"2026-02-16 09:38:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":164102,"visible":true,"origin":"","legend":"\u003cp\u003eCore genome tree of genomes of \u003cem\u003eCasuarina\u003c/em\u003e-infective strains including the type strain \u003cem\u003eFrankia casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e, rooted with \u003cem\u003eCryptosporangium arvum\u003c/em\u003e DSM44712 and \u003cem\u003eJatrophihabitans endophyticus\u003c/em\u003e DSM45627. Tree for 60 genomes, build out of a core of 262 genes per genome, 15,720 in total. The core has 114,822 amino acid residues per bp per genome, 6,889,320 in total. Phylogeny was inferred using the neighbour-joining algorithm as implemented in the PHYLIP package. There is a clear separation between the published genomes of cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains (bold) and the MAGs sequenced directly from nodules mapping with \u003cem\u003eF. casuarinae\u003c/em\u003e, and a deeper separation between the branch including the genomes of isolated \u003cem\u003eFrankia casuarinae\u003c/em\u003e strains, the MAGs from Singapore and Papua New Guinea and CeTa2Mahina on one side (red lines), and the MAGs from NSW, CeRuRu1, CeTa3Moorea and CeTa4Puna’auia on the other side (blue lines). A red circle is used to mark separation between \u003cem\u003eF. casuarinae\u003c/em\u003e and the potential new species. The scale bar represents 0.2 nucleotide substitutions per site. Asterisks indicate genome erosion.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/fbc0b693bab4855860474b32.jpg"},{"id":102754628,"identity":"cb36b69a-35fc-4726-87a7-cf0f3c516f0a","added_by":"auto","created_at":"2026-02-16 09:38:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":689938,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAverage nucleotide identity (fastANI) matrix for the MAGs sequenced in this study, compared to the genomes of isolated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFrankia casuarinae \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003estrains (labelled by bold print)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e The matrix suggests that the MAGs from NSW (Australia) and French Polynesia, except for CeTa2Mahina, either represent a novel \u003cem\u003eFrankia\u003c/em\u003e species different from\u003cem\u003e F. casuarinae\u003c/em\u003e (ANI \u0026lt; 96), or a new subspecies of \u003cem\u003eF. casuarinae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/bb56b3777da0ce57bd9968d5.jpg"},{"id":102754669,"identity":"2a11da13-43ef-412c-bde5-b1c51b0e0bfc","added_by":"auto","created_at":"2026-02-16 09:38:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":196377,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Scatterplot of genome size vs GC content for the MAGs from Australia, French Polynesia, Singapore and Papua New Guinea, with the genomes of cultivated \u003cem\u003eFrankia casuarinae\u003c/em\u003estrains for comparison. (B) Violin plot demonstrating the differences in genome size among the MAGs sequenced in this study and genomes of isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains in NCBI. One-way ANOVA showed that there was a statistically significant difference in mean genome size between at least two groups (p = 4.52e-08). A Tukey HSD test showed the groups between which the genome sizes were significantly different: the \u003cem\u003epublished Frankia casuarinae\u003c/em\u003e strains had a significantly larger mean genome size than the Australian MAGs, the MAGs from Papua New Guinea and the MAGs from French Polynesia, but not significantly larger than the MAGs from Singapore.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/fa8ce7e7d3de76bf3c162316.jpg"},{"id":102754695,"identity":"e0e4e576-6b1a-4d62-9305-80c100a3bdfa","added_by":"auto","created_at":"2026-02-16 09:39:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1289385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSyntons 1 encoding type 1h [NiFe] hydrogenase (A) and syntons 4 encoding [NiFe] hydrogenase type 3b (B) of the MAGs from NSW, Singapore, French Polynesia and Papua New Guinea.\u003c/strong\u003e Syntons-1 and 4 of the \u003cem\u003eF. casuarinae \u003c/em\u003etype strain CcI3\u003csup\u003eT\u003c/sup\u003e have been added for comparison. The scale bar represents 2000 bp. Letters a-c over genes denote fragmentation of a gene into multiple ORFs with stop codons between them; trN denotes truncation at the N terminus, trC denotes truncation at the C terminus; trM denotes that a gene is missing a segment in the middle; fs denotes two ORFs fused together.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/3d14d22c3939d98c530ae1b6.jpg"},{"id":102754634,"identity":"4c2522cf-274f-47aa-83e8-6810bbfafe7b","added_by":"auto","created_at":"2026-02-16 09:38:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":423909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBifunctional enzymes of CeTa3Moorea and CeRuru1 combining isocitrate lyase and malate synthase. (A) Amino acid of the CeTa3Moorea enzyme.\u003c/strong\u003e The isocitrate sequence (blue) and the malate synthase sequence (yellow) are connected by a short linker. The malate synthase sequence is followed by a 111 aa C-terminal domain (red) that does not show any homology with database sequences. \u003cstrong\u003e(B-D) \u003c/strong\u003eData obtained with Alphafold (Jumper \u003cem\u003eet al., \u003c/em\u003e2021; Abramson \u003cem\u003eet al., \u003c/em\u003e2024). \u003cstrong\u003e(B) shows the Alphafold structure prediction of the bifunctional protein seen from different angles\u003c/strong\u003e, colours as described in (A). \u003cstrong\u003e(C) shows the confidence level of the prediction\u003c/strong\u003e for the different parts of the protein. \u003cstrong\u003e(D)\u003c/strong\u003e \u003cstrong\u003eshows the alignment of the two domains\u003c/strong\u003e with the Predicted Aligned Error (PAE) plot, allowing users to assess predicted domain packing confidence by AlphaFold.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/3ef1fb415fd8101b1d0609b7.jpg"},{"id":102754589,"identity":"6711fc8f-1890-46ab-93bc-d2f672a2b67e","added_by":"auto","created_at":"2026-02-16 09:38:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":122776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e16S maximum likelihood phylogenetic tree of strains of the novel species and of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFrankia casuarinae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e CcI3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e (bold) \u003c/strong\u003eas calculated by MEGA (version 12.0.4; Kumar \u003cem\u003eet al., \u003c/em\u003e2024). Bootstrap values (1,000 replications) are presented. A red circle is used to mark the point of separation of the two species. The scale bar represents 0.002 nucleotide substitutions per site.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/e1d64dfffeb21d05a8ed95bc.jpg"},{"id":102754860,"identity":"00a57afe-2dfd-42be-a9ee-20e898b22f46","added_by":"auto","created_at":"2026-02-16 09:40:22","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":402368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDigital DNA:DNA hybridization (Meier-Kolthoff and Göker, 2019) between the genomes of the cultivable \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. casuarinae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strains and the MAGS sequenced in this study. \u003c/strong\u003eGenomes of the same species should show \u0026gt;70% dDDH.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/89b1b6d585f535fcd56e68c7.jpg"},{"id":102755200,"identity":"3019bacc-f3c4-4e21-bf79-a4f495901894","added_by":"auto","created_at":"2026-02-16 09:42:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5314839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/8f916288-1160-40b0-841c-d2f4d9acf487.pdf"},{"id":102754621,"identity":"5130fab2-c5ce-44b6-b5da-af856bb4bbf9","added_by":"auto","created_at":"2026-02-16 09:38:34","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2934388,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8701053/v1/a051544a96d13dec8e6ef078.docx"}],"financialInterests":"","formattedTitle":"What lurks beneath the surface? The hidden Frankia biodiversity in Casuarina nodules across continents","fulltext":[{"header":"Introduction","content":"\u003cp\u003eActinorhizal symbioses are root nodule symbioses between nitrogen-fixing soil Actinomycetota of the genus \u003cem\u003eFrankia\u003c/em\u003e and a diverse group of mostly woody Angiosperm species from eight different families belonging to three different orders (Pawlowski and Demchenko, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Amongst the host plants, \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e species are of particular importance; due to their high tolerance against abiotic stresses like high salinity, drought, flooding and heavy metal pollution (Batista-Santos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Diagne et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Jeddi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), they are often used in phytoremediation, coastal shelter belts and as windbreaks in agriculture. Apart from that, they are also used to provide fuel wood and charcoal, contributing to local economies. Therefore, they have been introduced in many countries in warmer climatic regions (Maity and Pawlowski, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhylogenetically, \u003cem\u003eFrankia\u003c/em\u003e strains can be grouped into four clades called clusters, three of which (clusters 1\u0026ndash;3) encompass symbiotic strains, while cluster-4 strains are non-symbiotic (Pawlowski and Demchenko, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The clusters are loosely linked to host specificity \u0026ndash; meaning that specific species of \u003cem\u003eFrankia\u003c/em\u003e only infect specific actinorhizal hosts. For example, while the basal genus of the family Casuarinaceae \u0026ndash; \u003cem\u003eGymnostoma \u0026ndash;\u003c/em\u003e can be nodulated by cluster-3 strains (Savour\u0026eacute; and Lim, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Steane et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Kates et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), species of two other genera in the same family, \u003cem\u003eCasuarina\u003c/em\u003e and \u003cem\u003eAllocasuarina\u003c/em\u003e, are nodulated by cluster-1c strains (Pawlowski and Demchenko, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thus far, genome sequences have been published for twelve cluster-1c \u003cem\u003eFrankia\u003c/em\u003e strains isolated from \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e trees growing on five different continents (Australia, Asia, Africa, North America, South America). These genomes have at least 98% average nucleotide identify (ANI) with each other (\u003cb\u003eSupplementary Figure S1\u003c/b\u003e), i.e., they belong to the same species, \u003cem\u003eFrankia casuarinae\u003c/em\u003e (Nouioui et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While the members of \u003cem\u003eF. casuarinae\u003c/em\u003e sequenced thus far show some differences regarding salt tolerance (Oshone et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), there is a surprising lack of overall diversity. In 1996, Rouvier \u003cem\u003eet al.\u003c/em\u003e, published an attempt to assess the diversity of Casuarinaceae-nodulating strains in their native distribution range by isolating DNA directly from nodules collected in Australia and performing Restriction Fragment Length Polymorphism (RFLP). They found at least seven different groups. Why is this diversity ostensibly not represented by the genomes of \u003cem\u003eF. casuarinae\u003c/em\u003e strains isolated around the world via traditional culturing methods?\u003c/p\u003e \u003cp\u003eThe easiest explanation is because isolation of a species is based on the capability of saprotrophic growth. This restriction to the analysis of isolated strains might have introduced a bias: since no species except for \u003cem\u003eF. casuarinae\u003c/em\u003e were isolated from nodules of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e, then these must be the only species growing in the host nodules. However, it is entirely possible that other species of \u003cem\u003eFrankia\u003c/em\u003e do grow in the nodules of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e, but lack the ability to be grown in culture, and were therefore never isolated. This has been seen previously; one example is the isolation of non-symbiotic cluster-4 \u003cem\u003eFrankia\u003c/em\u003e strains from surface-sterilized nodules of members of Cucurbitales infected by \u003cem\u003eFrankia\u003c/em\u003e strains that could never be cultivated (Mirza et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Carlos-Shandley \u003cem\u003eet al.\u003c/em\u003e, 2021; Gueddou et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the thick periderm of mature actinorhizal nodule lobes, fully effective surface sterilization is difficult, i.e., in the absence of a vast majority of cultivable microsymbionts, the occasional isolation of epiphytic \u003cem\u003eFrankia\u003c/em\u003e strains from surface-sterilized nodules is not surprising.\u003c/p\u003e \u003cp\u003eHowever, the lack of cultivable microsymbionts is not assumed, as in the cases of the isolation of all known members of the cluster-3 species \u003cem\u003eFrankia irregularis\u003c/em\u003e from surface-sterilized nodules of \u003cem\u003eCasuarina\u003c/em\u003e spp. on three different continents (strain R43 from \u003cem\u003eCasuarina cunninghamiana\u003c/em\u003e in the USA, Pujic et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; strain CcI49 from \u003cem\u003eC. cunninghamiana\u003c/em\u003e in Egypt, Mansour et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; and strain G2\u003csup\u003eT\u003c/sup\u003e from \u003cem\u003eC. equisetifolia\u003c/em\u003e in Guadeloupe, Nouioui et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Like the cluster-4 strains, these strains cannot nodulate the hosts from whose nodules they were isolated, and careful analysis has shown that they are not present in the infected cells of nodules of \u003cem\u003eCasuarina\u003c/em\u003e spp. but rather seem to live epiphytically on the nodule surface (Vemulapally et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eF. irregularis\u003c/em\u003e strains can nodulate \u003cem\u003eFrankia\u003c/em\u003e cluster-3 host plants from the Elaeagnaceae family (Pujic et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Mansour et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nouioui et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), but so far, no representative of this species has ever been isolated from Elaeagnaceae nodules. In summary, non-endophytic \u003cem\u003eFrankia\u003c/em\u003e strains have been ostensibly isolated from surface-sterilized actinorhizal nodules several times, mostly from nodules induced by as-of-yet-uncultivated strains, but three times from nodules of \u003cem\u003eCasuarina\u003c/em\u003e spp. The fact that the isolated strains did not represent the nodule endophytes was only recognized because these strains were unable to nodulate their \u003cem\u003eCasuarina\u003c/em\u003e \u0026ldquo;hosts\u0026rdquo;.\u003c/p\u003e \u003cp\u003eInocula of hosts of the members of \u003cem\u003eFrankia\u003c/em\u003e cluster-2 collected in the field represent strain assemblages, some members of which can represent inefficient symbionts that cannot fix nitrogen in nodules (Nguyen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and/or symbionts of other host plants (Berckx et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). What if \u003cem\u003eF. casuarinae\u003c/em\u003e strains were commonly part of the inocula of Casuarinaceae hosts, and because they had a significantly higher saprotrophic potential than the other strains in the inocula, were always the strains that ended up being isolated? There are also examples of cluster-1 \u003cem\u003eFrankia\u003c/em\u003e strains that could never be grown in culture despite several attempts, namely \u003cem\u003eAlnus\u003c/em\u003e spp.-infective strains that form spores in nodules (Pozzi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They show strong genome reduction and, interestingly, the loss of one of the [NiFe] hydrogenases (Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). [NiFe] hydrogenases are required to recycle H\u003csub\u003e2\u003c/sub\u003e which is lost during nitrogenase reaction (Islam et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Based on S\u0026oslash;ndergaard et al., (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), [NiFe] hydrogenases can be grouped into 29 classes based on the amino acid sequences of their large subunits. Three distinct forms of the 500\u0026thinsp;+\u0026thinsp;amino acid large subunit of [NiFe] uptake hydrogenase, HupL, were found in symbiotic \u003cem\u003eFrankia\u003c/em\u003e strains. Genomes of cluster-1 and cluster-3 strains contain the genes for a [NiFe] hydrogenase of group 1h (synton 1; S\u0026oslash;ndergaard et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Genomes of cluster-1 strains and genomes of cluster-2 strains from the continental lineage (Berckx et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) additionally contain the genes for a [NiFe] hydrogenase of group 2a (synton 2), while genomes of cluster-3 strains and genomes of cluster-2 strains from the island lineage contain the genes for a [NiFe] hydrogenase of type 1f (synton-3; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExpression analyses showed that while the genes encoding type 1h [NiFe] hydrogenase were expressed at higher levels during saprotrophic growth than \u003cem\u003ein planta\u003c/em\u003e, the genes encoding type 2a or 1f [NiFe] hydrogenase were expressed at higher levels \u003cem\u003ein planta\u003c/em\u003e than during saprotrophic growth (Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This can be interpreted to mean that type 2a or type 1f [NiFe] hydrogenase is required for symbiotic nitrogen fixation while type 1h [NiFe] hydrogenase is required for saprophytic growth in culture. This hypothesis is supported by the fact that (a) S\u0026oslash;ndergaard et al., (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) assigned a function in retrieval of protons for nitrogenase to types 2a and 1f, but not 1h, (b) cluster-2 \u003cem\u003eFrankia\u003c/em\u003e strains, which with two exceptions could never be grown in culture, indicating a reduced saprotrophic potential, only contain genes for a [NiFe] hydrogenase of type 2a (continental lineage) or type 1f (island lineage) and (c) the erosion of the type 1h [NiFe] hydrogenase synton in strains that form spores in nodules of \u003cem\u003eAlnus\u003c/em\u003e spp. (Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, the loss of type 1h [NiFe] hydrogenase in \u003cem\u003eFrankia\u003c/em\u003e so far has been found to be correlated with reduced saprotrophic potential and genome reduction.\u003c/p\u003e \u003cp\u003eSome \u003cem\u003eFrankia\u003c/em\u003e strains additionally contain a type 3b cytosolic [NiFe] hydrogenase (S\u0026oslash;ndergaard et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The corresponding synton was found in some representatives of cluster-3 and cluster-1a. Interestingly, it is present \u0026ndash; and intact \u0026ndash; in all genomes available from \u003cem\u003eF. casuarinae\u003c/em\u003e strains published by 2024, suggesting that this enzyme is required for saprotrophic and/or symbiotic growth of \u003cem\u003eF. casuarinae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThis begs the questions: (a) do \u003cem\u003eFrankia\u003c/em\u003e strains fixing nitrogen in the nodules of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e lack the ability to be grown in culture, and were thus never isolated, and (b) is the loss of type 1h and/or 3b [NiFe] hydrogenases in \u003cem\u003eFrankia\u003c/em\u003e generally correlated with reduced saprotrophic potential and genome reduction? We directly sequenced the metagenomes of nodules from different \u003cem\u003eCasuarina\u003c/em\u003e spp. trees collected in the field within the original distribution area of the genus, i.e., in Australia, Papua New Guinea, Singapore and French Polynesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using a method that had been established for nodules of cluster-2 (Nguyen et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Berckx et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and cluster-1a \u003cem\u003eFrankia\u003c/em\u003e strains that could never be cultivated (Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Analysis of the metagenome-assembled genomes showed that most nodules were inhabited by \u003cem\u003eFrankia\u003c/em\u003e strains showing higher diversity than the cultivable \u003cem\u003eCasuarina\u003c/em\u003e-infective strains sequenced thus far, as well as significant genome reduction and erosion of type 1h and often also of type 3b [NiFe] hydrogenase. We also determined that several of these strains belong to a species other than \u003cem\u003eF. casuarinae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eNodules and photosynthetic branchlets were harvested from \u003cem\u003eCasuarina\u003c/em\u003e spp. trees in different locations in New South Wales (NSW, Australia), Papua New Guinea, Singapore and French Polynesia. Nodules were kept in either 70% EtOH or RNAlater (Malmstrom, 2015) for transport, while photosynthetic branchlets were air-dried. Australian sampling locations are described in \u003cb\u003eSupplementary Figure S2\u003c/b\u003e. Sampling locations and host species are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Herbarium voucher numbers are listed in \u003cb\u003eSupplementary Table S1\u003c/b\u003e; a map of all sampling locations is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The names of the MAGs begin with the abbreviation of the host species (e.g., Cg, \u003cem\u003eCasuarina glauca\u003c/em\u003e) followed by an abbreviation of the sampling site (e.g., TB, Trial Bay) and a number (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The names of the MAGs from Tahiti and surrounding islands are called CeTa-number-full name of sampling place; the MAG from Rurutu is called CeRuru1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA isolation\u003c/h3\u003e\n\u003cp\u003eFor isolation of total nodule DNA, the storage fluid was first removed by centrifuging nodule samples in Eppendorf tubes for 5 min at maximum speed and then pipetting off the liquid. Nodule metagenomes were isolated as described by Nguyen et al., (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and Berckx et al., (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) or using the E.Z.N.A. HP Plant DNA kit (OMEGA BIO-TEK, VWR, Sweden). In either case, Polyclar AT (Serva, Germany) was added with a ratio of 1:2 Polyclar AT to sample weight during grinding in liquid nitrogen, and one additional ultrasonic homogenization step was performed using the Bandelin Sonopuls UW 2070 ultrasonic homogenizer with the MS 73 probe attached (Bandelin Electronic, Germany) after adding the lysis buffer.\u003c/p\u003e\n\u003ch3\u003eHost plant species determination\u003c/h3\u003e\n\u003cp\u003eThe distribution of \u003cem\u003eCasuarina\u003c/em\u003e and \u003cem\u003eAllocasuarina\u003c/em\u003e species in Australia, and a description of the morphology of their photosynthetic branchlets, is available at PlantNET and in Castle (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These data were used to identify the putative host plants for the samples from New South Wales (Australia; \u003cb\u003eSupplementary Table S2\u003c/b\u003e). Based on the branchlet anatomy, the samples from inland sites came from \u003cem\u003eCasuarina cunninghamiana\u003c/em\u003e, and the samples collected close to the sea from \u003cem\u003eC. glauca.\u003c/em\u003e For the samples from Papua New Guinea, Singapore and French Polynesia, we relied on published data regarding the distribution of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e species. In case of Papua New Guinea, only two \u003cem\u003eCasuarina\u003c/em\u003e species are documented, \u003cem\u003eC. equisetifolia\u003c/em\u003e at the coast and \u003cem\u003eC. oligodon\u003c/em\u003e in the mountains (Nuberg et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); the nodule samples were collected in the mountains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The two trees used for sampling in Coney Island Park (Singapore) were identified as \u003cem\u003eC. equisetifolia\u003c/em\u003e on the park web site (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.nparks.gov.sg/florafaunaweb/flora/2/7/2793\u003c/span\u003e\u003cspan address=\"https://www.nparks.gov.sg/florafaunaweb/flora/2/7/2793\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Only one \u003cem\u003eCasuarina\u003c/em\u003e species is documented in French Polynesia, namely \u003cem\u003eC. equisetifolia\u003c/em\u003e (Dotte-Sarout and Kahn, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eNodule metagenome sequencing and assembly\u003c/h3\u003e\n\u003cp\u003eSequencing of the DNA from nodules was performed as previously described (Berckx et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The raw reads were subjected to binning to remove plant DNA sequences and sequences of unrelated bacterial strains and then assembled into \u003cem\u003eFrankia\u003c/em\u003e metagenome-assembled genomes (MAGs) as described before (Berckx et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMAG analysis\u003c/h3\u003e\n\u003cp\u003eFor phylogenetic analyses based on core genome, and calculations of Average Nucleotide Identity (ANI) and Average Amino-acid Identity (AAI), we used the EDGAR 3.0 platform (Blom et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Dieckmann et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which provides robust tools for comparative genomics. Specifically, ANI values were determined by computing fastANI scores, which assess the nucleotide similarity between two genomes using consecutive 1,020 nucleotide fragments across the core genome and averaging the results. AAI scores were calculated based on alignments of core genome-encoded proteins. This combined approach enabled a comprehensive and high-resolution comparison of the genomes studied and will help identify potential species and genus boundaries.\u003c/p\u003e \u003cp\u003eA core genome tree and a 16S tree were also constructed for the MAGs. The core genome tree was inferred using the EDGAR3.0 platform. For construction of the 16S rRNA phylogenetic tree, 16S gene sequences corresponding to the dominant \u003cem\u003eFrankia\u003c/em\u003e strain were first identified within the datasets. This was achieved through two complementary strategies: (i) by aligning the entire metagenome assembly to reference 16S sequences from established \u003cem\u003eFrankia\u003c/em\u003e strains \u0026ndash; using, for example, 16S of \u003cem\u003eFrankia casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e as a query sequence for targeted retrieval by means of BLASTn (Altschul et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), and (ii) by generating additional metagenome assemblies using SPAdes v3.15.4 (Bankevich et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with reduced subsets of the dataset and then again comparing to 16S sequences from established \u003cem\u003eFrankia\u003c/em\u003e strains by applying BLASTn. The latter approach was employed to mitigate potential oversampling and assembly biases that can arise from high-abundance sequences. Identified 16S rRNA gene sequences were then aligned and used for phylogenetic reconstruction to assess the taxonomic placement of the dominant \u003cem\u003eFrankia\u003c/em\u003e strain within the sample. The sequences were then used to construct a phylogenetic tree on MEGA (version 12.0.4; Kumar et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) using the Maximum Likelihood method. 1,000 bootstraps were used for the tree calculation.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis of genomes and MAGs\u003c/h2\u003e \u003cp\u003eGenome size and GC content of genomes were derived from contig information on GenDB (Meyer et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), a genome annotation system for prokaryotic genomes, to visualize the relationship between genome size and GC content. To determine whether the genome sizes significantly differed among the known cultivable strains and the strains in this study, an ANOVA test followed by Tukey pairwise comparison was carried out, and the results were illustrated in a merged dotplot and violin plot. Data analysis and preparation of plots were done on RStudio (Posit Team, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor digital DNA:DNA hybridization (dDDH) studies, the genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available under \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, for a whole genome-based taxonomic analysis (Meier-Kolthoff and G\u0026ouml;ker, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The analysis also made use of recently introduced methodological updates and features (Meier-Kolthoff et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Information on nomenclature, synonymy, and associated taxonomic literature was provided by TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://lpsn.dsmz.de\u003c/span\u003e\u003cspan address=\"https://lpsn.dsmz.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Meier-Kolthoff et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The results were provided by the TYGS on 2025-09-05.\u003c/p\u003e \u003cp\u003eFor phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5 (Meier-Kolthoff et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). 100 distance replicates were calculated each. Digital DDH values and confidence intervals were calculated using the recommended settings of the GGDC 4.0 (Meier-Kolthoff et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of [NiFe] hydrogenase operons\u003c/h3\u003e\n\u003cp\u003e[NiFe] hydrogenase genes of all \u003cem\u003eFrankia\u003c/em\u003e strains in this study were identified via BlastP searches on GenDB (Meyer et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), using \u003cem\u003eFrankia casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e genes as queries. Contig information were used to create plots of the hydrogenase syntons on RStudio (Posit Team, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), using the package gggenes (Wilkins, 2023).\u003c/p\u003e\n\u003ch3\u003eProtein modelling\u003c/h3\u003e\n\u003cp\u003eProtein structure analysis was done via Alphafold (Jumper et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Abramson et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the host species through morphology\u003c/h2\u003e \u003cp\u003eMAGs were obtained from a total of 21 nodule samples. Based on the sampling locations in NSW, the host tree species could be narrowed down to two \u003cem\u003eCasuarina\u003c/em\u003e species (\u003cem\u003eC. cunninghamiana, C. glauca\u003c/em\u003e). The morphology of the photosynthetic branchlets showed that all inland samples from NSW were from \u003cem\u003eC. cunninghamiana\u003c/em\u003e while all samples collected at or close to a beach came from \u003cem\u003eC. glauca\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSupplementary Figure S2, S3, Supplementary Table S1, S2)\u003c/b\u003e. In summary, MAGs were obtained from \u003cem\u003eCasuarina equisetifolia\u003c/em\u003e in Singapore (Coney Island, two samples) and French Polynesia (Tahiti, Moorea and Rurutu, four samples); from \u003cem\u003eCasuarina oligodon\u003c/em\u003e in Papua New Guinea (Local Level Government Bulolo-Wau, Morobe Province, three samples); and from \u003cem\u003eCasuarina cunninghamiana\u003c/em\u003e and \u003cem\u003eCasuarina glauca\u003c/em\u003e in New South Wales (NSW, Australia, 12 samples). Details of the MAGs are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; the accession numbers of the MAGs are given in \u003cb\u003eSupplementary Table S3.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDetails of\u003c/b\u003e \u003cb\u003eFrankia\u003c/b\u003e \u003cb\u003eMAGs from\u003c/b\u003e \u003cb\u003eCasuarina\u003c/b\u003e \u003cb\u003espp.\u003c/b\u003e \u003csup\u003e1\u003c/sup\u003etrees from self-sown material of unknown origin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCountry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMAG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ehost\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenome size [bp]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e% [GC]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBUSCO\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN50\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ecoverage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003econtigs\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003eAustralia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasuarina Sands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcCS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. cunninghamiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,072,291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e88.50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12,484 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e258x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e429\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCasuarina Sands\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcCS3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. cunninghamiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,183,436\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15,747 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e156x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e367\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWombeyan Caves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcWB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. cunninghamiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,309,795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19,335 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e356x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e336\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWombeyan Caves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcWB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. cunninghamiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,284,996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e90.50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14,875 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e74x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e403\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolonglo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCcMO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. cunninghamiana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,271,872\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18,394 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e50x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e405\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArakoon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgARK3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,169,588\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e81.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14,809 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e36x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e376\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJervis Bay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgJB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,238,692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20,036 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e71x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJervis Bay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgJB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,238,692\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e89.90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20,036 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e71x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKioloa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgKIO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,249,750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e93.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e20,463 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e110x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e291\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRoseville\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgROS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,066,875\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12,945 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e110x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e448\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrial Bay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgTRI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,089,891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13,537 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e409\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrial Bay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCgTRI2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. glauca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,185,287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15,352 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e45x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e372\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSingapore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConey Island\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeCAU1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,145,236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31,920 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e160x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e263\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConey Island\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeCAU2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,965,976\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e25,973 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e90x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e302\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePapua New Guinea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorobe-Bulolo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoBUL1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. oligodon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,786,542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.90%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14,424 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e208x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e368\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorobe-Wau\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoWAU3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. oligodon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,786,542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.20%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16,007 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e350x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e342\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMorobe-Wau\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoWAU4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. oligodon\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,702,336\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e79.80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14,865 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e450x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e363\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFrench Polynesia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRurutu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeRuru1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5,221,813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e96,7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e12,788 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e544\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTahiti, Mahina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeTa2Mahina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3,875,375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e83,6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6,032 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e17x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e745\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTahiti, Moorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeTa3Moorea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.462.711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e70.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e98,6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e31,927 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e87x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTahiti, Puna'auia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCeTa4Puna'auia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. equisetifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,166,592\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e69.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e77,7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6,142 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e20x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e798\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDo these MAGs represent a new species?\u003c/h2\u003e \u003cp\u003eA core genome tree was inferred using all MAGs obtained in this study combined with the genome sequences of isolated \u003cem\u003eFrankia casuarinae\u003c/em\u003e strains available at NCBI by 2024 (\u003cb\u003eSupplementary Table S4\u003c/b\u003e), along with type strains of other \u003cem\u003eFrankia\u003c/em\u003e species. The tree was rooted with \u003cem\u003eCryptosporangium arvum\u003c/em\u003e DSM44712 (NCBI RefSeq accession: GCF_000585375.1) and \u003cem\u003eJatrophihabitans endophyticus\u003c/em\u003e DSM45627 (NCBI RefSeq accession: GCF_900129455.1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The tree shows that the strains from Singapore and Papua New Guinea, and one of the strains from French Polynesia (CeTa2Mahina), clustered with the known isolated \u003cem\u003eFrankia casuarinae\u003c/em\u003e strains, while all strains from Australia and the other three strains from French Polynesia (CeRuru1, CeTa3Moorea and CeTa4Puna\u0026rsquo;auia) mapped to a different branch, suggesting that they were likely to represent a novel species. Furthermore, among the strains collected from Australia, strains coming from nodules that were collected close from the beach and strains coming from nodules collected in-land also displayed a clear separation, indicating that they formed separate subclades within the species. Similarly, the genomes of isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains and the genomes of strains coming from Singapore, Papua New Guinea and CeTa2Mahina from French Polynesia also showed a clear separation in the core genome tree, i.e., they formed separate subclades of \u003cem\u003eF. casuarinae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAre we dealing with two different species, or with two subspecies of the same species? Jain et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) propose 95% ANI as cutoff value for demarcating prokaryotic species, while Ciufo et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) propose 96%. fastANI analysis (Dieckmann et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) of the 21 new strains studied shows that the two strains from Singapore (CeCAU1 and CeCAU2), the three strains from Papua New Guinea (CoBUL1, CoWAU3 and CoWAU4), and one strain from French Polynesia (CeTa2Mahina) had\u0026thinsp;\u0026ge;\u0026thinsp;97% ANI among them (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Two of the remaining three strains from French Polynesia (CeRuru1 and CeTa3Moorea) and all strains from Australia had\u0026thinsp;\u0026lt;\u0026thinsp;96% identity with the cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains but had\u0026thinsp;\u0026ge;\u0026thinsp;97% ANI with each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Overall, fastANI analysis of the genomes of \u003cem\u003eCasuarina\u003c/em\u003e-infective \u003cem\u003eFrankia\u003c/em\u003e strains shows a border of 95\u0026ndash;96% between the two branches (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Interestingly, the MAG CeTa4Puna\u0026rsquo;auia takes a slightly ambiguous position between both groups with less than 96% ANI with most strains of both; when Average Amino-acid Identity is analysed it shares more than 96% AAI with the \u003cem\u003eF. casuarinae\u003c/em\u003e strains but more than 97% AAI with the MAGs of the potential novel species (AAI; \u003cb\u003eSupplementary Figure S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn short, core genome tree and the ANI analysis suggest that the strains from Australia and three of the strains from French Polynesia represent a novel \u003cem\u003eFrankia\u003c/em\u003e species or subspecies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome reduction in most MAGs from\u003c/b\u003e \u003cb\u003eCasuarina\u003c/b\u003e \u003cb\u003enodules\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMost of the MAGs sequenced in this study were smaller than 5 MB, i.e., below the range found for the genomes of isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). CcMO1, one of the MAGs from NSW, was an exception among the NSW strains; it was isolated from nodules collected from a fallen tree with its roots in the air (\u003cb\u003eSupplementary Figure S2\u003c/b\u003e). Two of the MAGs from French Polynesia, CeRuru1 and CeTa3Moorea, based on genome size also did not exhibit genome reduction compared to the cultivable strains, while CeTa2Mahina showed some reduction and the last strain, had a much smaller genome (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the two \u003cem\u003eF. casuarinae\u003c/em\u003e strains from Singapore showed no genome reduction, whereas all three \u003cem\u003eF. casuarinae\u003c/em\u003e strains from Papua New Guinea, had significantly smaller genomes. One-way ANOVA revealed that there was a statistically significant difference in mean genome size between at least two groups [F(4, 28)\u0026thinsp;=\u0026thinsp;20.89, p\u0026thinsp;=\u0026thinsp;4.52e-08]. Tukey\u0026rsquo;s HSD Test for multiple comparisons found that the mean value of genome size was significantly different between the published \u003cem\u003eF. casuarinae\u003c/em\u003e strains and the MAGs from Papua New Guinea [p\u0026thinsp;=\u0026thinsp;0. 0.0000006, 95% CI = (-2.20, -0.94)], between the published \u003cem\u003eF. casuarinae\u003c/em\u003e strains and the MAGs from Australia [p\u0026thinsp;=\u0026thinsp;0. 0.0000005, 95% CI = (-1.40, -0.61)], and between the published \u003cem\u003eF. casuarinae\u003c/em\u003e strains and the MAGs from French Polynesia [p\u0026thinsp;=\u0026thinsp;0.0271632, 95% CI = (-1.18, -0.05)]. There was no statistically significant difference in size between the genomes of isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains and the MAGs from Singapore. These results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, 16 out of 21 MAGs sequenced in this study were markedly smaller (3.7\u0026ndash;4.3 MB) than the genomes of cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains published thus far.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGenome erosion: which genes were lost in the new\u003c/b\u003e \u003cb\u003eFrankia\u003c/b\u003e \u003cb\u003eMAGs?\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenome reduction in cluster-1 \u003cem\u003eFrankia\u003c/em\u003e has also been documented for cluster-1a strains that form spores in the nodules of alder species and could never be cultured despite numerous attempts (Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pozzi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the Pawlowski et al., (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) study, \u003cem\u003eFrankia\u003c/em\u003e cluster-1 strains were shown to contain two or three different types of [NiFe] hydrogenases, encoded by synton-1, synton-2 and, in case of \u003cem\u003eF. casuarinae\u003c/em\u003e, synton-4. Genome erosion in cluster-1a was first visible for synton-1 (type 1h [NiFe] hydrogenase; S\u0026oslash;ndergaard et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, syntons 1, 2 and 4 were analysed for the new MAGS. With the exception of CeTa3Moorea and CeRuru1, all MAGs showed erosion of synton-1 (type 1h [NiFe] hydrogenase; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) while synton-2 (type 2a [NiFe] hydrogenase) remained intact (data not shown). The functions of the proteins encoded by synton-1 are given in \u003cb\u003eSupplementary Table S5\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll MAGs from nodules collected from \u003cem\u003eC. glauca\u003c/em\u003e trees growing at the beach in NSW (CgJB1, CgJB3, CgTRI1, CgTRI2, ACgARK3, CgKIO3, CgROS2) do not contain the maturation protease \u003cem\u003ehupD1\u003c/em\u003e and contain at least one stop codon in the ORF of \u003cem\u003ehupL1\u003c/em\u003e, precluding enzyme function. The strains collected from \u003cem\u003eC. cunninghamiana\u003c/em\u003e further inland (CcCS1, CcCS3, CcWB2, CcWB3, CcMO1) had no intact \u003cem\u003ehypB1, hupL1\u003c/em\u003e and \u003cem\u003ehypF1\u003c/em\u003e genes. In CgTRI2 and CgROS2, the 3\u0026rsquo;-parts of the synton (\u003cem\u003ehypF1 \u0026ndash; hypC1 \u0026ndash; hypD1 \u0026ndash; hypE1\u003c/em\u003e) were separated from the 5\u0026rsquo;-parts. Similarly, in CcWB3, CcCS1 and CcMO1, the 3\u0026rsquo;-parts of the operon (\u003cem\u003ehupL1 \u0026ndash; hypD1 \u0026ndash; hypC1 \u0026ndash; hypE1 \u0026ndash; hypF1\u003c/em\u003e) were separated from the 5\u0026rsquo;-parts.\u003c/p\u003e \u003cp\u003eAmong the MAGs from French Polynesia, CeRuru1 contained all genes of synton-1, but the 5\u0026rsquo; and 3\u0026rsquo; parts of the syntons were in different parts of the genome. CeTa3Moorea was the only MAG containing an intact synton-1 not divided in parts by transposition. CeTa4Puna\u0026rsquo;auia contained no functional copy of \u003cem\u003ehupL1, hypF1\u003c/em\u003e and \u003cem\u003ehypD1\u003c/em\u003e; moreover, synton-1 was divided in four parts scattered throughout the genome. CeTa2Mahina had lost most genes of synton-1 completely, while \u003cem\u003ehypF1, hypD1\u003c/em\u003e and \u003cem\u003ehypE1\u003c/em\u003e were truncated. The MAGs from Papua New Guinea \u0026ndash; CoBUL1, CoWAU3 and CoWAU4 \u0026ndash; had lost the entire synton-1 encoding type 1h [NiFe] hydrogenase. The MAGs from Singapore, CeCAU1 and CeCAU2, while ostensibly not showing genome reduction, had no intact copies of \u003cem\u003ehupS1, hupL1\u003c/em\u003e, and \u003cem\u003ehupD1\u003c/em\u003e genes, i.e., the strains had lost the structural proteins of type 1h [NiFe] hydrogenase.\u003c/p\u003e \u003cp\u003eErosion of synton-4 encoding type 3b cytosolic [NiFe] hydrogenase was also found (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). The complete operon of \u003cem\u003eF. casuarina\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e as well as the other cultivable strains consists of six genes, \u003cem\u003ehyhB \u0026ndash; cNMP-DB\u0026ndash; hyhG \u0026ndash; hyhS \u0026ndash; hyhL \u0026ndash; M52\u003c/em\u003e. Gene names were taken from S\u0026oslash;ndergaard et al., (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) except for \u003cem\u003ecNMP-BD\u003c/em\u003e and the maturation protease M52. \u003cb\u003eSupplementary Table S5\u003c/b\u003e shows the function of each protein. Among the MAGs from nodules collected from \u003cem\u003eC. glauca\u003c/em\u003e growing at the beach in NSW, only CgKIO3 and CgTRI1 showed erosion of the type 3b [NiFe] hydrogenase synton; in both cases, \u003cem\u003ehyhG\u003c/em\u003e was truncated. In CgKIO3, \u003cem\u003ehyhB\u003c/em\u003e was interrupted by a stop codon, while in CgTRI1, \u003cem\u003ehyhL\u003c/em\u003e was broken in two; one of the parts was also truncated. Among the MAGs collected from inland \u003cem\u003eC. cunninghamiana\u003c/em\u003e plants, \u003cem\u003ehyhL\u003c/em\u003e and \u003cem\u003eM52-4\u003c/em\u003e were fused in CcMO1, CcCS1 and CcCS3, while \u003cem\u003ehyhB\u003c/em\u003e was truncated in CcWB2 and CcWB3. \u003cem\u003eHyhL\u003c/em\u003e contained a stop codon in CcWB2, while it was truncated in CcWB3. CcCS1 also contained a stop codon in \u003cem\u003ehyhS.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAmong the MAGs from \u003cem\u003eC. equisetifolia\u003c/em\u003e growing in French Polynesia, we found that synton-4 was divided into two (CeRuRu1, CeTa4Puna\u0026rsquo;auia) or three (CeTa2Mahina) parts in different areas of the genome, indicating transposase activity. In CeRuRu1, three genes were truncated (\u003cem\u003ehyhB, hyhS\u003c/em\u003e and \u003cem\u003ehyhL\u003c/em\u003e); furthermore, the ORFs of \u003cem\u003ehyhS\u003c/em\u003e and \u003cem\u003ehyhL\u003c/em\u003e were fused. In CeTa3Moorea, \u003cem\u003ehyhB, hyhS and hyhL\u003c/em\u003e were truncated and the ORFs of \u003cem\u003ehyhS\u003c/em\u003e and \u003cem\u003ehyhL\u003c/em\u003e were fused. In CeTa4Puna\u0026rsquo;auia, \u003cem\u003ehyhS\u003c/em\u003e was divided in two truncated parts, \u003cem\u003ehyhG\u003c/em\u003e was lost entirely and \u003cem\u003ecNMP-BD\u003c/em\u003e and \u003cem\u003eM52-4\u003c/em\u003e were truncated. In CeTa2Mahina, \u003cem\u003ehyhG\u003c/em\u003e was truncated while \u003cem\u003ehyhL\u003c/em\u003e was divided into two parts in different areas of the genome; one of the parts was truncated. The MAGs from \u003cem\u003eC. equisetifolia\u003c/em\u003e growing in Singapore contained the full-size synton-4 like \u003cem\u003eF. casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e. In all MAGs from nodules of \u003cem\u003eC. oligodon\u003c/em\u003e growing in Papua New Guinea, the ORF of \u003cem\u003ehyhB\u003c/em\u003e was interrupted by a stop codon. In CoWAU3, the \u003cem\u003eM52-4\u003c/em\u003e gene was truncated, while in CoWAU4, \u003cem\u003ehyhG\u003c/em\u003e was truncated and \u003cem\u003ehyhL\u003c/em\u003e was interrupted by a stop codon.\u003c/p\u003e \u003cp\u003eIn summary, we found erosion of synton-1 encoding type 1h [NiFe] hydrogenase in all MAGs sequenced in this study except for CeTa3Moorea and presumably CeRuru1 (we do not know whether a promoter was affected by the transposition). Erosion of synton-4 encoding type 3b cytosolic [NiFe] hydrogenase was observed for all MAGs except for the ones from Singapore and most of the MAGs from nodules of \u003cem\u003eC. glauca\u003c/em\u003e trees growing at the beach in NSW. This is particularly interesting since some of the MAGs that showed erosion of type 1h and/or type 3b hydrogenase (CcMO1, CeCAU1, CeCAU2, CeRuru1 and CeTa3Moorea) did not show an obvious reduction in overall size compared to the genomes of the isolated strains (5.0\u0026ndash;5.5 compared to 5.0\u0026ndash;5.6 MB in the cultivable strains; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWhich genes distinguish\u003c/b\u003e \u003cb\u003eF. casuarinae\u003c/b\u003e \u003cb\u003eand the potential new species?\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis question is difficult to answer since while several genomes of cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains are available, the [NiFe] hydrogenase data show that all MAGs sequenced in this study show a higher degree of genome erosion than the cultivable ones. We compared the genomes of the isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains with the MAGs of CeTa3Moorea and CeRuru1.\u003c/p\u003e \u003cp\u003eThe MAGs of CeTa3Moorea and CeRuru1 contain a gene that is not present in any other \u003cem\u003eFrankia\u003c/em\u003e genome sequenced to date, encoding a bifunctional enzyme encompassing isocitrate lyase (N-terminal) and malate synthase (C-terminal), thus combining the two functions required for the glyoxylate shunt of the tricarboxylic acid (TCA) cycle (Woo et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; \u003cb\u003eSupplementary Table S6\u003c/b\u003e). Such bifunctional enzymes have so far only been found in \u003cem\u003eEuglena gracilis\u003c/em\u003e (Nakazawa et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and \u003cem\u003eCaenorhabditis elegans\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). \u003cem\u003eF. casuarinae\u003c/em\u003e strains, like cluster-2 \u003cem\u003eFrankia\u003c/em\u003e strains (Berckx et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), lack the glyoxylate shunt since they do not contain a gene for isocitrate lyase, although such a gene is present in most cluster-1 genomes other than those of \u003cem\u003eF. casuarinae\u003c/em\u003e (Berckx et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), even in those of cluster-1a \u003cem\u003eFrankia\u003c/em\u003e strains that form spores in nodules of \u003cem\u003eAlnus glutinosa\u003c/em\u003e and have a reduced genome (Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pozzi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains except for CcI156 also lost malate synthase (NCBI accession WP_076804337.1). Interestingly, CeRuru1 contains a complete malate synthase gene and CeTa3Moorea a truncated one, but not a complete isocitrate lyase gene (\u003cb\u003eSupplementary Table S6\u003c/b\u003e). Since the gene of the bifunctional enzyme cannot be found in any MAG analyzed in this study except for those of CeTa3Moorea and CeRuru1, nor in the genomes of cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains, it must be concluded that it \u0026ndash; and thus, the glyoxylate shunt \u0026ndash; got lost early during genome erosion. Thus, genome erosion led to loss of the glyoxylate shunt of the TCA cycle in all genomes of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e-infective \u003cem\u003eFrankia\u003c/em\u003e strains known to date.\u003c/p\u003e \u003cp\u003eOn the other hand, we found some genes that are only present in \u003cem\u003eF. casuarinae\u003c/em\u003e, not in any MAG of the novel species. The cultivable \u003cem\u003eF. casuarinae\u003c/em\u003e strains contain four genes encoding polyhydroxybutyrate depolymerase (NCBI accessions OAA30795, OAA26147, OAA26975, OAA25310 in \u003cem\u003eF. casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e), one of them (OAA30795) encoding a member of the secretome (Mastronuncio \u003cem\u003eet al.\u003c/em\u003e, 2008). No homologs of the secreted PBH depolymerase, which had been suggested to be involved in the degradation of suberin, and of OAA25310, were found in any MAG of the potential new species.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe sequencing of 21 MAGs from nodules of \u003cem\u003eCasuarina\u003c/em\u003e spp. in their original distribution area yielded 16 MAGs showing distinct genome erosion (3.7\u0026ndash;4.3 compared to 5.0\u0026ndash;5.6 MB in the cultured strains of \u003cem\u003eFrankia casuarinae\u003c/em\u003e), while all MAGs showed erosion of the genes encoding [NiFe] hydrogenase type 1h and/or type 3b. Thus, the loss of these two [NiFe] hydrogenases seems to have occurred very early during genome erosion in cluster-1c \u003cem\u003eFrankia.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe plasma membrane type 1h [NiFe] hydrogenase (synton-1) has been linked to hydrogenotrophic respiration using O\u003csub\u003e2\u003c/sub\u003e as a terminal electron acceptor; the enzyme is supposed to scavenge electrons from atmospheric H\u003csub\u003e2\u003c/sub\u003e to fuel the respiratory chain during carbon-deficiency (S\u0026oslash;ndergaard et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In \u003cem\u003eFrankia\u003c/em\u003e, this enzyme was lost in species that show reduced saprotrophic potential and genome erosion (cluster-2 strains, cluster-1 strains that form spores in nodules and could never be cultured despite multiple attempts), suggesting that it is not essential for the symbiotic lifestyle (Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The results of this study are consistent with this hypothesis; furthermore, the fact that erosion of synton-1 was found in all genomes except for that of CeTa2Moorea suggests that even in genomes that have sizes comparable to those of cultured \u003cem\u003eFrankia casuarinae\u003c/em\u003e strains (CcMO1, CeCAU1, CeCAU2, CeRuru1; 5.0\u0026ndash;5.3 MB), genome erosion had begun.\u003c/p\u003e \u003cp\u003eThus, except for CeRuRu1 and presumably CeTa3Moorea, all MAGs sequenced in this study showed erosion of [NiFe] type 1h hydrogenase (synton-1), a phenomenon linked to reduced saprotrophic potential in cluster-1a strains. This could explain why no strains of their respective phylogenetic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) have been isolated from \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e nodules in the past using traditional methods. To date, no \u003cem\u003eFrankia\u003c/em\u003e strain with a genome size below 5 MB has been successfully cultivated.\u003c/p\u003e \u003cp\u003eThe cytosolic bidirectional group 3b [NiFe] hydrogenase encoded by synton-4 can couple the oxidation of NADP to the fermentative evolution of H\u003csub\u003e2\u003c/sub\u003e (S\u0026oslash;ndergaard et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Its pattern of occurrence is interesting: it is present in only a few genomes from cluster-1a and \u0026minus;\u0026thinsp;3 strains but it is present in all genomes of isolated \u003cem\u003eF. casuarinae\u003c/em\u003e strains (Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There is no clear grouping of MAGs where it is ostensibly not eroded \u0026ndash; CeCAU1, CeCAU2, CgARK1, CgJB1, CgJB3, CgTRI2. Two of them come from Coney Island in Singapore from \u003cem\u003eCasuarina equisetifolia\u003c/em\u003e trees with unclear pedigree, the other four from NSW are from nodules of \u003cem\u003eCasuarina glauca\u003c/em\u003e trees growing close to the sea; in short, the data available are not suited to build a hypothesis about the function of synton-4. We can conclude, however, that its erosion does not seem to be linked to host specificity. It might be linked to salt stress as all genomes from nodules of inland strains, including CcMO1 with its genome size of 5.27 MB, did show erosion of synton-4. At any rate, the fact that it was found to be eroded or even completely lost in strains showing genome erosion, as is typical for the evolutionary trajectory towards obligate symbiosis, indicates that it is more relevant for the saprotrophic than for the symbiotic lifestyle.\u003c/p\u003e \u003cp\u003eDo the Australian MAGs and the three French Polynesian MAGs represent a new species, separate from \u003cem\u003eF. casuarinae\u003c/em\u003e? The percentage of species separation is not unambiguously defined (Ciufo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Jain et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). An Average Nucleotide Identity (ANI) matrix (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and an Average Amino-acid Identity (AAI) matrix (\u003cb\u003eSupplementary Figure S4)\u003c/b\u003e are somewhat ambiguous regarding CeTa4Puna\u0026rsquo;auia and of CcMO1. A 16S phylogenetic tree rooted with the 16S of the type strain of a cluster-1a \u003cem\u003eFrankia\u003c/em\u003e species, \u003cem\u003eFrankia alni\u003c/em\u003e ACN14a\u003csup\u003eT\u003c/sup\u003e provides a clear separation between the MAGs from NSW (Australia), CeTa3Moorea and CeTa4Puna\u0026rsquo;auia versus \u003cem\u003eF. casuarinae\u003c/em\u003e CcI3\u003csup\u003eT\u003c/sup\u003e, the MAGs from Papua New Guinea and Singapore and CeTa2Mahina (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, on the 16S level we do not see the separation between the MAGs from \u003cem\u003eC. glauca\u003c/em\u003e nodules and \u003cem\u003eC. cunninghamiana\u003c/em\u003e nodules that we see in the core genome tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), calling the usefulness of 16S phylogeny for \u003cem\u003eFrankia\u003c/em\u003e cluster-1c species separation into question (see also Pozzi et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, we used the option for pairwise calculation of digital DNA:DNA hybridization (dDDH) values calculations) provided by the TYGS web server (Meier-Kolthoff and G\u0026ouml;ker \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where the species border is defined at 70%. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the results. There is a clear separation of \u003cem\u003eF. casuarinae\u003c/em\u003e and the novel species, with CeTa4Puna\u0026rsquo;auia taking a slightly ambiguous position, displaying maximally 69.8% dDDH with any genome of \u003cem\u003eF. casuarinae\u003c/em\u003e and and minimally 70.2% dDDH with any genome of the novel species.\u003c/p\u003e \u003cp\u003eIn summary, both genomic and phylogenetic data (ANI, 16S, core genome tree, dDDH) grouped together the MAGs from NSW, CeRuru1, CeTa3Moorea and CeTa4Puna\u0026rsquo;auia in the same species, proposed as \u003cem\u003eCandidatus\u003c/em\u003e Frankia pacificiensis. This species can be found in root nodules of \u003cem\u003eC. equisetifolia and C. glauca\u003c/em\u003e at the coasts of the Society Islands and NSW, and in root nodules of \u003cem\u003eC. cunninghamiana\u003c/em\u003e in inland NSW.\u003c/p\u003e \u003cp\u003eThe strongest support for a long-term separation of \u003cem\u003eF. casuarinae\u003c/em\u003e and the novel species is the existence of the unique gene for a bifunctional enzyme combining isocitrate lyase and malate synthase in the MAGs CeTa3Moorea and CeRuru1. However, the fact that both MAGs also contain a malate synthase gene (although truncated in CeTa3Moorea) might be taken to suggest that the gene for the bifunctional enzyme was acquired by horizontal gene transfer. The closest homologs of both parts of the bifunctional enzyme (isocitrate lyase and malate synthase) are enzymes from cluster-3 \u003cem\u003eFrankia\u003c/em\u003e strains (data not shown); however, no gene for a bifunctional enzyme was detected in cluster-3 \u003cem\u003eFrankia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDoes the core genome tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) tell us something about the evolution of \u003cem\u003eFrankia casuarinae\u003c/em\u003e? In \u003cem\u003eF. casuarinae\u003c/em\u003e we see a clear separation between the cultured strains and the MAGs from Papua New Guinea and Singapore (from \u003cem\u003eC. equisetifolia\u003c/em\u003e and C. \u003cem\u003eoligodon\u003c/em\u003e) that is not related to genome erosion which is minimal in the MAGs from Singapore (see Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA regarding the [NiFe] hydrogenase type 1h synton). In contrast, in \u003cem\u003eCa.\u003c/em\u003e F. pacificiensis we see a clear separation between MAGs from \u003cem\u003eC. glauca\u003c/em\u003e growing close to the sea \u0026ndash; i.e., under conditions of salt stress \u0026ndash; and from \u003cem\u003eC. cunninghamiana\u003c/em\u003e growing inland. The three MAGs from \u003cem\u003eC. equisetifolia\u003c/em\u003e growing close to the sea in French Polynesia occupy the root position (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Given the lower similarity among the four MAGs from French Polynesia, one of which represents \u003cem\u003eF. casuarinae\u003c/em\u003e, compared to the much more conserved MAGs from NSW, and the fact that one representative of \u003cem\u003eF. casuarinae\u003c/em\u003e was found in French Polynesia (CeTa2Mahina), we conclude that \u003cem\u003eF. casuarinae\u003c/em\u003e and the novel species diverged in French Polynesia and that CeTa4Puna\u0026rsquo;auia, with its slightly ambiguous position between the two species, is closest to this point of separation.\u003c/p\u003e \u003cp\u003eThese data could be further interpreted to mean that the new species evolved in coastal areas of French Polynesia and adapted to inland areas after reaching NSW. However, they could also be interpreted to mean that the new species is divided in groups of strains with different host specificity. Phylogenetic analysis on the plant side has shown that \u003cem\u003eC. equisetifolia\u003c/em\u003e is the earliest divergent species in the genus \u003cem\u003eCasuarina\u003c/em\u003e, followed by \u003cem\u003eC. cunninghamiana\u003c/em\u003e (Kates et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The grouping of the MAGs would be consistent with co-evolution between host and microsymbiont.\u003c/p\u003e \u003cp\u003eTo return to the original research question whether \u003cem\u003eFrankia\u003c/em\u003e strains fixing nitrogen in the nodules of \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e lack the ability to be grown in culture, and were therefore never isolated, and whether the loss of type 1h and/or 3b [NiFe] hydrogenases in \u003cem\u003eFrankia\u003c/em\u003e is generally correlated with reduced saprotrophic potential and genome erosion, the results are affirmative. In most cases of field-collected nodules in the original distribution area of the host plants, we find in 16 out of 21 cases strong genome erosion and in the remaining five cases beginning genome erosion; in all cases type 1h and/or 3b [NiFe] hydrogenases had lost functionality. This resembles the situation in alder-infective cluster-1 strains that sporulate in nodules (Pozzi \u003cem\u003eet al.\u003c/em\u003e, 2015; Herrera-Belaroussi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pozzi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pawlowski et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhy did we not find some nodules without detectable genome erosion although the cultivable strains are definitely able to nodulate \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e spp.? In case of \u003cem\u003ein planta\u003c/em\u003e sporulating strains, i.e., in cluster-1a strains with genome erosion on an evolutionary trajectory towards obligate symbiosis, infectivity and rhizospheric competitiveness were increased compared to cultivable strains (Cotin-Galvan et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, they were likely to outcompete any saprotrophs in the inoculum. Our results suggest that the same was the case for (\u003cem\u003eAllo-)Casuarina\u003c/em\u003e-infective strains with genome erosion. Altogether, evolution towards reduced saprotrophic capacity and stronger symbiotic competitiveness seems to have happened in several lineages of \u003cem\u003eFrankia\u003c/em\u003e cluster-1a and cluster-1c, as well as in cluster-2 (Persson et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), except that in the latter, only two cultivable strains were identified (Gtari et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gueddou \u003cem\u003eet al.\u003c/em\u003e, 2018). Did it also happen in cluster-3 which encompasses symbiotic \u003cem\u003eFrankia\u003c/em\u003e strains with the largest genomes (Ghodhbane-Gtari et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)? Direct sequencing of total nodule DNA and MAG assembly will show whether also here, some strains evolved towards obligate symbiosis.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cem\u003eFrankia\u003c/em\u003e strains present in nodules of \u003cem\u003eCasuarina\u003c/em\u003e spp. in its original distribution area show greater diversity than strains isolated from \u003cem\u003eCasuarina\u003c/em\u003e spp. nodules and show genome erosion compared to those cultivable strains (in 76% of the cases, strong genome erosion). Thus, a focus on \u003cem\u003eFrankia\u003c/em\u003e strains isolated from nodules has rendered a misleading picture of \u003cem\u003eFrankia\u003c/em\u003e diversity, and, presumably, its adaptation to ecological conditions and specific host species.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAAI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eaverage amino acid identity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eaverage nucleotide identity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMetagenome-assembled genome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSW\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNew South Wales\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eStatements and declarations\u003c/h2\u003e\n\u003cp\u003eOpen access funding provided by Stockholm University. This project was supported by the Swedish Research Council Vetenskapsradet (2019\u0026ndash;05540 to K.P.) and the Carl Tryggers foundation (CTS22: 2145 to K.P.). The bioinformatics support of the BMBF-funded project \u0026lsquo;Bielefeld-Giessen Center for Microbial Bioinformatics\u0026rsquo; (BiGi) and the BMBF grant FKZ 031A533 within the German Network forBioinformatics Infrastructure (de.NBI) are gratefully acknowledged. The bioinformatics support of the \u0026lsquo;Bielefeld-Giessen Center for Microbial Bioinformatics\u0026rsquo; (BiGi) within the German Network for Bioinformatics Infrastructure BMFTR grants: W-de.NBI-004, W-de.NBI-010, BMBF grant FKZ 031A533) are gratefully acknowledged.\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eNadia Binte Obaid\u003c/strong\u003e: formal analysis, supervision, writing \u0026ndash; original draft. \u003cstrong\u003eAndr\u0026aacute;s Patyi\u003c/strong\u003e: investigation, formal analysis, writing \u0026ndash; original draft. \u003cstrong\u003eFede Berckx\u003c/strong\u003e \u0026ndash; supervision, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eMaru Bernal-Gomez\u003c/strong\u003e: formal analysis, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eAndrea Lavello\u003c/strong\u003e: investigation, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eAndreas Brachmann\u003c/strong\u003e: investigation, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eDaniel Wibberg\u003c/strong\u003e: formal analysis, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJochen Blom\u003c/strong\u003e: data curation, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJ\u0026ouml;rn Kalinowski\u003c/strong\u003e: writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eSara Mehrabi\u003c/strong\u003e: investigation, formal analysis, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eIvan R. Kennedy\u003c/strong\u003e: investigation, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003ePhilippe Normand\u003c/strong\u003e: investigation, formal analysis, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eUlrike Mathesius\u003c/strong\u003e: investigation, writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eKatharina Pawlowski\u003c/strong\u003e: conceptualization, funding acquisition, project administration, supervision, investigation, formal analysis, writing \u0026ndash; original draft.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank Tiberius Jimbo (Papua New Guinea Forest Research Institute) for collecting the nodules from Papua New Guinea, and Jarkko Salosj\u0026auml;rvi and Jia Jun Ngiam (Nanyang Technological University Singapore) for collecting the nodules from Singapore.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eGenome sequences were submitted to NCBI\u0026rsquo;s GenBank; accession numbers are provided (\u003cstrong\u003eTable\u0026nbsp;1; Supplementary Table S3\u003c/strong\u003e).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaumgardt K, Charoenpanich P, McIntosh M, Schikora A, Stein E, Thalmann S, Kogel K-H, Klug G, Becker A, Evguenieva-Hackenberg E (2014) RNase E affects the expression of the acyl-homoserine lactone synthase gene \u003cem\u003esinI\u003c/em\u003e in \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e. 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Mol Syst Biol 20(3):170\u0026ndash;186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s44320-024-00017-w\u003c/span\u003e\u003cspan address=\"10.1038/s44320-024-00017-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"root nodules, symbiotic nitrogen fixation, obligate symbiont, Frankia, Casuarina, evolution, [NiFe] hydrogenase, genome erosion","lastPublishedDoi":"10.21203/rs.3.rs-8701053/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8701053/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground and Aims\u003c/b\u003e\u003c/p\u003e \u003cp\u003eActinorhizal root nodule symbioses are formed between a diverse group of mostly woody dicotyledonous plants and nitrogen-fixing soil Actinomycetota of the genus \u003cem\u003eFrankia\u003c/em\u003e. One of the most ecologically relevant actinorhizal plants are \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e species, used widely in shelter belts and phytoremediation due to their high tolerance to abiotic stresses and ability to thrive on marginal soils. All sequenced \u003cem\u003eFrankia\u003c/em\u003e strains isolated from \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e nodules via traditional techniques show high sequence identity and belong to a single species, \u003cem\u003eFrankia casuarinae\u003c/em\u003e. This lack of diversity in nodules is unusual in actinorhizal symbioses. We hypothesised that \u003cem\u003e(Allo-)Casuarina\u003c/em\u003e nodules are colonized by \u003cem\u003eFrankia\u003c/em\u003e strains that cannot be cultivated and exhibit genome erosion.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test this, we directly sequenced nodule metagenomes from four countries, followed by reconstruction of metagenome-assembled genomes (MAGs).\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur findings show that the dominant \u003cem\u003eFrankia\u003c/em\u003e strains in field samples were far more diverse than the isolated strains and included MAGs with substantial genome reduction \u0026ndash; one exhibiting over 25% reduction compared to \u003cem\u003eF. casuarinae\u003c/em\u003e. Notably, we observed erosion of two types of [NiFe] hydrogenases, a phenomenon linked to evolution toward obligate symbiosis in other \u003cem\u003eFrankia\u003c/em\u003e groups.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThese results suggest that potentially obligate symbionts may dominate nodules in nature but had gone undetected by conventional approaches. For applications such as reforestation or tsunami shelter belts, crushed, nodule-derived strains may offer superior ecological compatibility. We speculate that \u003cem\u003eFrankia\u003c/em\u003e strains followed two different evolutionary trajectories; one, towards obligate symbiosis accompanied by strong genome erosion, and two, towards rhizosphere colonization involving limited genome erosion.\u003c/p\u003e","manuscriptTitle":"What lurks beneath the surface? The hidden Frankia biodiversity in Casuarina nodules across continents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 09:27:30","doi":"10.21203/rs.3.rs-8701053/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2026-02-11T21:40:17+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T09:23:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-02-06T21:39:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T02:58:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-02-01T16:15:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c076a263-54fb-442d-bc96-0bc9e859553d","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-17T00:22:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 09:27:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8701053","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8701053","identity":"rs-8701053","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}