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Tan, H.W., Heenan, P.B., De Meyer, S.E., Willems, A. and Andrews, M. (2015) Diverse novel mesorhizobia nodulate New Zealand native species. Systematic and Applied Microbiology, 38 (2). pp. 91-98.

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Accepted Manuscript

Title: Diverse novel mesorhizobia nodulate New Zealand native Sophora species

Author: Heng Wee Tan Peter B. Heenan Sofie E. De Meyer Anne Willems Mitchell Andrews

PII: S0723-2020(14)00175-1 DOI: http://dx.doi.org/doi:10.1016/j.syapm.2014.11.003 Reference: SYAPM 25664

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Received date: 25-10-2014 Revised date: 12-11-2014 Accepted date: 14-11-2014

Please cite this article as: H.W. Tan, P.B. Heenan, S.E. De Meyer, A. Willems, M. Andrews, Diverse novel mesorhizobia nodulate New Zealand native Sophora species, Systematic and Applied Microbiology (2014), http://dx.doi.org/10.1016/j.syapm.2014.11.003

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1 Diverse novel mesorhizobia nodulate New Zealand native Sophora 2 species 3 4 Heng Wee Tana, Peter B. Heenanb, Sofie E. De Meyerc, Anne Willemsd, Mitchell 5 Andrewsa* 6 7 aFaculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, NZ 8 bAllan Herbarium, Landcare Research, Lincoln 7640, NZ 9 cCentre for Rhizobium Studies, Murdoch University, Murdoch, Western Australia 6150, Australia 10 dDepartment of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 11 Ghent, Belgium 12 13 A B S T R A C T 14 15 Forty eight rhizobial isolates from New Zealand (NZ) native Sophora spp. growing in natural 16 ecosystems were characterised. Thirty eight isolates across five groups showed greatest similarity to 17 ciceri LMG 14989T with respect to their 16S rRNA and concatenated recA, glnll and 18 rpoB sequences. Seven isolates had a 16S rRNA sequence identical to M. amorphae ATCC 19665T 19 but showed greatest similarity to M. septentrionale LMG 23930T on their concatenated recA, glnll and 20 rpoB sequences. All isolates grouped closely together for their nifH, nodA and nodC sequences, 21 clearly separate from all other in the Genbank database. None of the type strains closest to 22 the Sophora isolates based on 16S rRNA sequence similarity nodulated Sophora microphylla but they 23 all nodulated their original host. Twenty one Sophora isolates selected from the different 16S rRNA

24 groupings produced N2-fixing nodules on three Sophora spp. but none nodulated any host of the type 25 strains for the related species. DNA hybridisations indicated that these isolates belong to novel 26 Mesorhizobium spp. that nodulate NZ native Sophora species. 27 28 Keywords: 29 Mesorhizobium 30 Sophora 31 Phylogeny 32 Symbiosis genes 33 34 Introduction 35 36 Most legumes ( family ) have the capacity to fix atmospheric nitrogen (N2) via 37 symbiotic (generally termed ‘rhizobia’) in root nodules which gives them an advantage under 38 low soil nitrogen (N) conditions if other factors are favourable for growth [1,2,21]. There are four 39 genera of nativeAccepted legume of the sub-family Papilionoideae Manuscript on the main New Zealand (NZ) islands. 40 These genera are the closely related (23 endemic species), (2 endemic 41 species) and Montigena (1 endemic species) in the ‘Carmichaelinae’ clade, tribe , and 42 Sophora (8 endemic species) in the tribe Sophoreae [11,12,13,27]. All four genera are capable of 43 forming nodules [21], but genotypic data on the rhizobia which induce nodules on the Sophora spp. 44 are limited.

* Corresponding author. Tel.: +64 3 4230692 E‐mail address: [email protected]

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45 Previously, sequences were obtained for the small subunit ribosomal RNA (16S rRNA) gene of five 46 isolates of Sophora spp. growing in natural ecosystems [34]. Four isolates were most closely 47 grouped to the Mesorhizobium ciceri and M. loti type strains while the fifth isolate, aligned closely with 48 Rhizobium leguminosarum strains. This study did not assess the ability of the isolated strains to

49 nodulate NZ native legumes or sequence any of their N2-fixation (nif) or nodulation (nod) genes but 50 subsequent work showed that although the R. leguminosarum strain (ICMP 14642) produced nodules

51 on Sophora microphylla, these nodules did not fix N2 [33, http://scd.landcareresearch.co.nz]. 52 Tan et al. [25] examined ten bacterial strains isolated from NZ Carmichaelinae growing in natural 53 ecosystems which grouped close to the M. huakuii type strain in relation to their 16S rRNA and 54 nitrogenase iron protein gene (nifH) sequences. These strains showed novel DNA recombinase A 55 gene (recA), glutamine synthetase ll gene (glnll), N-acyltransferase nodulation protein A gene (nodA) 56 and N-acetylglucosaminyltransferase nodulation protein C gene (nodC) sequences. Seven strains 57 selected produced functional nodules on Carmichaelia spp. and Clianthus puniceus but did not 58 nodulate two Sophora species indicating that within NZ native legumes, at least some bacterial strains 59 are specific to Carmichaelinae species. Tan et al. [24] tested the ability of eleven isolates, two from 60 Montigena and three each from Carmichaelia spp., Clianthus puniceus and Sophora spp. to nodulate 61 Montigena. Two of the Sophora strains were taken from a previous study [34] and showed 16S rRNA 62 similar to M. loti/ M. ciceri but the third had a 16S rRNA sequence similar to M. huakuii. All three 63 isolates from Sophora spp. were shown to nodulate their original host. Only isolates from 64 Carmichaelia spp. and Clianthus puniceus that produced functional nodules on their original host and 65 strains from Montigena produced functional nodules on Montigena. Strains that nodulated Montigena, 66 Carmichaelia spp. and Clianthus puniceus had variable 16S rRNA, recA and glnll gene sequences, 67 but specific nifH, nodA and nodC gene sequences different from those of the Sophora isolates. 68 These results indicate that Montigena, Carmichaelia spp. and Clianthus spp. share at least some 69 rhizobia with each other but not with Sophora spp. and that the ability of different rhizobial strains to 70 produce functional nodules on the different NZ native legume genera is associated with specific 71 symbiosis genes. Here we focus on bacterial isolates from Sophora spp. growing in natural 72 ecosystems and characterise them with respect to their 16S rRNA, recA, glnll, rpoB, nifH, nodA and 73 nodC gene sequences, ability to nodulate a range of legume species and DNA hybridisation tests with 74 their most closely related rhizobial type strains on the basis of gene sequence similarity. 75 76 Materials and methods 77 78 Bacterial isolatesAccepted Manuscript 79 80 Forty five bacterial isolates from Sophora spp. sampled in April 2011 or April 2012 under natural 81 conditions in the current study plus the three Sophora isolates previously shown to nodulate their host 82 [24] were studied. Here, bacteria were isolated from sampled at six sites in the South Island: 83 alluvial limestone river terrace, Waima/Ure River, Marlborough (41° 52´S 174° 0´E, 147m; Field site 84 1); alluvial outwash river fan, Pororari River, Westland (42° 6S´ 171° 20´E, 1m; Field site 2); margin of 85 estuary, Saltwater Creek, Greymouth, Westland (42° 30´S 171° 9´E, 2m; Field site 3); margin of 86 Greywacke rock outcrop, Kowai River, Springfield, Canterbury (43° 19´S 171° 46´E, 612m; Field site

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87 4); alluvial Greywacke river terrace, upper Rakaia River, Canterbury (43° 26´S 171° 34´E, 357m; Field 88 Site 5) and among Haast Schist rock outcrop, Waitaki River, Otago (44° 53´S 170° 48´E, 126m; Field 89 site 6). Field sites were selected to represent low rainfall in the eastern South Island (< 1000 mm per 90 year; sites 1, 4-6) and high rainfall in the western South Island (> 2500 mm per year, sites 2, 3) [16]. 91 All isolates are deposited in the International Collection of Microorganisms from Plants (ICMP), 92 Landcare Research, Auckland, NZ. The ICMP number, Sophora species host and field site sampled 93 are given for the isolates on the phylogenetic trees. The type strains of M. amorphae (ICMP 15022 = 94 ACCC 19665T), M. ciceri (ICMP 13641 = LMG 14989T), M. huakuii (ICMP 11069 = IAM 14158T) and 95 M. loti (ICMP 4682 = LMG 6125T) were obtained from the ICMP collection directly. 96 For bacteria isolated in this study, root nodules were surface sterilised, crushed in sterile water and 97 the bacterial suspension was streaked onto yeast mannitol agar (YMA) [26] and incubated at 25°C in 98 the dark for 5 days as described previously [25]. A purified culture was obtained by sub-culture from 99 each plate. Each culture was inoculated into a suspension of yeast mannitol broth (YMB) [26] and 100 used for preparation of subcultures for DNA extraction or inoculum. 101 102 DNA sequencing 103 104 DNA was extracted from the bacterial cultures grown in YMB using the standard Qiagen-Gentra 105 PUREGENE DNA Purification Kit for gram-negative bacteria. The 16S rRNA, recA, glnll, nifH, nodA 106 and nodC genes were amplified with appropriate primer sets and PCR conditions as described 107 previously [25] except that for most strains, the nodC primers NodCfor540 (TGA-TYG-AYA-TGG- 108 ART-AYT-GGC-T) and NodCrev1160 (CGY-GAC-ARC-CAR-TCG-CTR-TTG) [20] were used. The 109 rpoB83F (CCT- SAT-CGA-GGT-TCA-CAG-AAG-GC) and rpoB1061R (AGC-GTG-TTG-CGG-ATA- 110 TAG-GCG) primers [17] were used for rpoB amplification. The PCR products were separated by 111 electrophoresis in 1% (w/v) agarose gels, stained with ethidium bromide (0.5 μg mL-1) and viewed 112 under UV light. PCR products of the expected size were sequenced by the Bio-Protection Research 113 Centre Sequencing Facility, Lincoln University. DNA sequence data were viewed via Sequence 114 Scanner v 1.0 software (©Applied Biosystems) and edited and assembled using DNAMAN Version 6 115 (©Lynnon Biosoft Corporation). 116 117 Phylogenetic analyses 118 119 DNA sequences of individual genes and concatenated recA, glnll and rpoB for the Sophora isolates 120 were aligned andAccepted Maximum Likelihood (ML) trees constructed Manuscript using the partial deletion method with 121 an 80% cut off in MEGA5 software [23]. Bootstrap support for each node was evaluated with 500 122 replicates. The most closely related Mesorhizobium type strains on the Genbank sequence database 123 [www.ncbi.nlm.nih.gov/genbank] were used for all trees. In addition, sequences of closely related

124 non-type strains were included in the nifH and nodC trees and those of strains shown to produce N2 125 fixing nodules on NZ Carmichaelinae species in the nodA and nodC trees. Only bootstrap values 126 ≥50% are shown for each tree. MEGA5 model test was performed to select a model of nucleotide 127 substitution and the ‘best’ model (lowest Bayesian Information Criterion (BIC) score) used for each 128 gene. GenBank accession numbers for all genes are shown in the figures.

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129 130 Nodulation and N2 fixation studies 131 132 All bacterial strains isolated in the current study were inoculated onto their original host legume 133 species. In addition, twenty one of the isolates of variable 16S rRNA, recA, glnll and rpoB gene 134 sequence groups, but which all nodulated their host Sophora sp. were inoculated onto Sophora 135 microphylla, Sophora prostrata, Sophora longicarinata, Carmichaelia australis and Clianthus 136 puniceus. Mesorhizobium amorphae ACCC 19665T, M. ciceri LMG 14989T, M. huakuii IAM 14158T 137 and M. loti LMG 6125T were inoculated onto the three Sophora species and the Sophora isolates 138 were also inoculated onto Amorpha fruticosa, Cicer arietinum, Astragalus sinicus and Lotus 139 corniculatus the legume species from which respectively, the M. amorphae [29], M. ciceri [18], M. 140 huakuii [5] and M. loti [14] type strains were originally isolated. The type strains were inoculated onto 141 their original host as a positive control. Seeds were obtained from different sources: Carmichaelia 142 australis from Proseed, North Canterbury, NZ; Clianthus puniceus and all Sophora spp. from New 143 Zealand Tree Seeds, Rangiora, NZ; Amorpha fruticosa from the Agroforestry Research Trust, Devon, 144 UK; Astragalus sinicus and Lotus corniculatus from the Margot Forde Germplasm Centre, Palmerston 145 North, NZ and Cicer arietinum from the Binn Inn, Christchurch, NZ. 146 All plant procedures were carried out under gnotobiotic conditions. All seeds, except those of 147 Cicer arietinum were, in sequence, soaked in concentrated sulphuric acid for 30-60 minutes, rinsed 148 with sterile water, soaked in hot (∼ 60°C) sterile water which was left at room temperature overnight 149 then transferred to 1.5% water agar plates and kept in the dark to germinate. After germination, each 150 seedling was transferred to a polyethylene terephthalate jar containing vermiculite and supplied with a 151 complete nutrient solution [25]. Plants were grown in a chamber with a 16 hour photoperiod (400 152 μmol photons m-2 s-1) at a constant 22°C. Seeds of Cicer arietinum were soaked in 20% commercial 153 bleach (0.5 g L-1 sodium hypochlorite) for 20 min, rinsed with 96% ethanol, air dried then soaked in 154 hot sterile water and left to cool overnight. Cicer arietinum was germinated and grown in 0.75 L pots 155 (four seedlings per pot) containing autoclaved N-free potting mix under natural daylight in a 156 glasshouse. The potting mix base was 80% composted bark and 20% pumice (1-4 mm) to which was 157 added 1 g L-1 agricultural lime (primarily calcium carbonate), 0.3 g L-1 superphosphate (9P-11S-20Ca; 158 Ravensdown, NZ), and 0.3 g L-1 Osmocote (6 months, ON-OP-37K), 0.3 g L-1 Micromax trace 159 elements and 1 g L-1 Hydraflo, all three obtained from Everris International, Geldermalsen, The 160 Netherlands. The pH of the medium was 5.8. 161 At 5-10 days after sowing, seedlings were inoculated with 10 ml of the appropriate bacterial strain 162 grown to log phase.Accepted Uninoculated plants supplied with ManuscriptYMB only were used as controls. There were 163 3 replicates per treatment. All plants were supplied with sterile water as required. Plants were 164 inspected at three weekly intervals for nodulation and at 70-90 days after inoculation were tested for 165 nitrogenase activity using the acetylene reduction assay (ARA) [6]. All values taken as negative were 166 similar to controls and in the range 2.1 to 4.5 μL ethylene L-1. All values taken as positive were one to 167 two orders of magnitude greater than this (ranging from 198.9 to 2107.3 μL ethylene L-1). After the 168 ARA, rhizobial strains were isolated from three to six nodules per treatment and their 16S rRNA and

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169 nodC genes sequenced. In all cases, the 16S rRNA and nodC sequences for the strain recovered 170 from nodules from the inoculation assays were identical to those of the strain used as inoculant. 171 172 DNA-DNA hybridisation 173 174 For DNA-DNA hybridisation, high-molecular weight DNA was prepared [19]. DNA-DNA hybridisations 175 were performed using a microplate method and biotinylated probe DNA [7]. The hybridisation 176 temperature was 48 °C ± 1 °C. Reciprocal reactions (A × B and B × A) were performed in 177 quadruplicate for each DNA pair and their variation was within the limits of this method [10]. 178 179 Results and discussion 180

181 In the current study, forty eight bacterial isolates from Sophora spp. were shown to produce N2-fixing 182 nodules on their host species. These isolates separated into three major groupings on the basis of 183 their 16S rRNA sequences (Fig. 1). Group one, comprising thirty eight isolates from four different 184 Sophora species across eight different field sites separated into five sub-groups with high similarity 185 (99.60-100% similarity, 1260 bp) to M. ciceri LMG 14989T isolated from Cicer arietinum in Spain [18]. 186 The second group, comprising seven isolates of Sophora longicarinata from Field site 1, showed 16S 187 rRNA sequences (1260 bp) identical (100% similarity) to that of the M. amorphae type strain which 188 was isolated from Amorpha fruticosa growing in Beijing, China, although there is evidence that its 189 origin was the native range of Amorpha fruticosa which is South Eastern and Mid-Western USA 190 [28,29]. The third group contains three isolates (ICMP 19550, ICMP 19559 and ICMP 19567) aligning 191 closely, based on 16S rRNA gene analysis, with M. huakuii IAM 14158T which was isolated from 192 Astragalus sinicus nodules sampled in Nanjing province, China [5]. These data indicate that Sophora 193 spp. and Carmichaelinae species in NZ [24,25] can be nodulated by Mesorhizobium spp. with 16S 194 rRNA gene sequences similar to M. ciceri, M. huakuii and M. amorphae. 195 The forty eight mesorhizobial isolates from Sophora spp. separated into eight groups and three 196 individual isolates (ICMP 11719, ICMP 19513 and ICMP 19550) on the basis of their concatenated 197 recA, glnll and rpoB sequences, separate from all Mesorhizobium type strains. Generally, isolates 198 from the same field site grouped together. This apparent link between recA, glnll and rpoB 199 sequences and field site may be at least in part, due to adaptation of the bacteria to local conditions 200 outside the host plant and this warrants further study. Seven of the groups and the three individual 201 isolates (40 isolates) clustered around M. ciceri LMG 14989T (84.5-86.5 % similarity, 1800 bp), while 202 one group of eight isolates, all from Sophora longicarinata sampled at field site 1, was closest to Accepted ManuscriptT 203 (87.9% similarity, 1800 bp) M. septentrionale LMG 23930 isolated from Astragalus adsurgens 204 growing in Northern China [8]. This second group contained all seven isolates with a 16S rRNA 205 sequence identical to M. amorphae ACCC 19665T and ICMP 19567 which had a 16S rRNA sequence 206 closest to M. huakuii IAM 14158. 207 The nifH gene sequences for all forty eight isolates clustered together (97.4-100% similarity, 280 208 bp) closest to but separate from (92.5-95.6% similarity, 280 bp) M. alhagi CCNWXJ12-2T isolated 209 from Alhagi sparsifolia in Xinjiang province, China [3]; Mesorhizobium strain CCNWSX672 isolated 210 from Coronilla varia in Shaanxi province, China [35]; and M. camelthorni CCNWX340-4T isolated from

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211 Alhagi sparsifolia in Xinjiang Province, China [4] (Fig. 3). The nifH sequences of the forty eight 212 Sophora isolates differed (92.5-94.7% similarity, 280 bp) from the nifH sequences of the seven strains

213 previously shown to induce N2 fixing nodules on Carmichaelinae but not Sophora species which were 214 identical (six strains) or very similar to that of the M. huakuii type strain [24,25]. 215 The nodA sequences for the Sophora isolates showed 97.4-100% similarity to each other (420 bp) 216 and clearly separated from those of all Mesorhizobium type strains and rhizobial symbionts of NZ 217 Carmichaelinae species (Fig. 4A). The strains aligned closest to (86.5-88.4% similarity, 420 bp) the 218 M. albiziae type strain isolated from Albizia kalkora in Suchian province, Southern China [30,31]. The 219 nodC sequences for the Sophora isolates showed 98.9-100% similarity to each other (650 bp) and as 220 for nodA sequences were clearly separated from all Mesorhizobium type strains and rhizobial 221 symbionts of NZ Carmichaelinae species (Fig. 4B). Here, the isolates aligned closest to M. albizeae 222 CCBAU 61158T (91.5-91.7% similarity, 650 bp) and Mesorhizobium strain CBAU03074 (93.5-94.2% 223 similarity, 650 bp) isolated from Astragalus scaberrimus in temperate China which had a 16S rRNA 224 sequence identical to M. septentrionale [37]. Overall, the data obtained in the current and previous 225 studies provide strong evidence that mesorhizobia are the major, if not exclusive, rhizobial partners of 226 Sophora spp. in NZ. These mesorhizobia had variable 16S rRNA, recA, glnll and rpoB but specific 227 nifH, nodA and nodC gene sequences. A similar generalisation was made for rhizobia that nodulate 228 NZ Carmichaelinae species [25] but the symbiosis genes of the two groups are very different from 229 each other. Considering work on Sophora outside NZ, 16S rRNA and recA gene sequences of 75 230 rhizobial isolates indicated that Mesorhizobium sp., Agrobacterium sp., Ensifer sp., Phyllobacterium 231 sp. and Rhizobium sp. could effectively nodulate Sophora alopecuroides grown in different regions of 232 the Loess Plateau in China [36]. These isolates had diverse nifH and nodA genes similar to those of 233 rhizobial isolates from a range of legume genera in the same region indicating that these legumes 234 may be able to share these rhizobia. However, as for rhizobia from NZ Sophora, there was a 235 correlation between genotype and geographical origin for rhizobia isolated from Sophora 236 alopecuroides.

237 Previously, rhizobia shown to produce N2 fixing nodules on Carmichaelinae species did not 238 nodulate NZ Sophora spp. [24,25]. Here, twenty one Sophora isolates from the different 16S rRNA/ 239 housekeeping gene groupings were inoculated onto Sophora microphylla, Sophora prostrata, 240 Sophora longicarinata, Clianthus puniceus and Carmichaelia australis (Table 1). All Sophora rhizobial 241 isolates tested, regardless of their 16S rRNA or concatenated recA, glnll and rpoB gene grouping,

242 produced N2 fixing nodules on the three Sophora species but none induced N2 fixing nodules on

243 Carmichaelia australisAccepted. However, eighteen of the twenty Manuscript one isolates produced N2 fixing nodules on 244 Clianthus puniceus. Clianthus puniceus, therefore, can share some rhizobia with Sophora spp. and 245 others with Carmichaelia spp. (Table 1) and Montigena [24]. Host range in rhizobia is at least in part 246 determined by the structure of the lipo-chitin oligosaccharide ‘Nod factors’ synthesised by the 247 products of the nodulation genes such as nodA and nodC [6,15]. Therefore, the substantial

248 differences in the nodA and nodC gene sequences between isolates that produce N2 fixing nodules 249 on NZ Sophora spp. or Carmichaelia spp. and Montigena are likely to be important in determining the 250 specificity of the different groups of strains. It is thus unexpected that rhizobial symbionts of Sophora

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251 spp., and Carmichaelia spp. and Montigena can induce functional nodules on Clianthus puniceus. 252 This indicates that Clianthus puniceus is a more promiscuous rhizobial host than Carmichaelia spp., 253 Montigena or Sophora spp. Previously, M huakuii IAM 14158T was shown to nodulate Carmichaelia 254 spp. and Clianthus puniceus [25]. Here, none of the type strains closest to the Sophora isolates on 255 16S rRNA sequences (M. ciceri, M. amorphae or M. huakuii depending on strain) nor the M. loti type 256 strain, isolated from nodules of Lotus corniculatus in Wanganui, NZ [14], produced nodules on

257 Sophora microphylla, Sophora prostrata or Sophora longicarinata (Table 1) but they all induced N2 258 fixing nodules on their original host (data not shown). Also, none of the 21 Sophora isolates 259 nodulated any of the type strain hosts (data not shown) indicating a specific symbiosis with Sophora 260 spp. 261 It is generally accepted that strains within a species should exhibit more than 70% DNA 262 hybridisation [9,22,32]. In the current study, DNA-DNA hybridisation values ranged from 30.1-52.1% 263 between the five Sophora isolates selected from the different 16S rRNA/ housekeeping gene groups 264 indicating that these groups are quite different from each other (Table 2). Additionally, these strains 265 showed only 26.1-36.3% similarity to M. ciceri LMG 14989T. Also, isolate ICMP 19515 which was 266 identical to M. ciceri LMG 14989T on 16S rRNA sequence and closest to M. ciceri on concatenated 267 housekeeping gene sequences showed only 28.9% similarity to M. ciceri LMG 14989T. Similarly, 268 strain ICMP 19557 which had a 16S rRNA sequence identical to M. amorphae ACCC 19665T and 269 showed greatest similarity to M. septentrionale LMG 23930 on concatenated recA, glnll and rpoB 270 gene sequences showed 28.8% similarity to M. septentrionale LMG 23930T and 32.3% similarity to 271 ICMP 19515. Thus, DNA-DNA hybridisations indicate that the isolates belong to novel 272 Mesorhizobium spp. 273 In this study, DNA sequencing, DNA hybridisations and inoculation experiments have revealed that 274 a significant diversity of novel Mesorhizobium spp. nodulate NZ native Sophora spp. The ‘drivers’ for 275 this diversity are unknown and require further study. Abiotic characteristics of the sampling sites may 276 be important drivers as the field sites represent a variety of South Island habitats, including parent 277 rock type (e.g. schist, greywacke and limestone), substrate (alluvium and rock outcrop), and rainfall (> 278 2500 mm in western South Island, < 1000 mm in eastern South Island). 279 280 Acknowledgements 281 282 This work was supported by the Brian Mason Scientific and Technical Trust and a Lincoln University 283 Doctoral Scholarship (HWT). We thank Liesbeth Lebbe for excellent technical assistance. 284 285 References Accepted Manuscript 286 287 [1] Andrews, M., James, E.K., Sprent, J.I., Boddey, R.M., Gross, E., dos Reis, F.B. (2011) 288 Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained 289 using 15N natural abundance. Plant Ecol. Divers. 4, 131-140. 290 [2] Andrews, M., Raven, J.A., Lea, P.J. (2013) Do plants need nitrate? The mechanisms by 291 which nitrogen form affects plants. Ann. Appl. Biol. 163, 174-199. 292 [3] Chen, W-M., Zhu, W-F., Bontemps, C., Young, J.P.W., Wei, G.H. (2010) Mesorhizobium 293 alhagi sp. nov., isolated from wild Alhagi sparsifolia in north-western China. Int. J. Syst. Evol. 294 Microbiol. 60, 958-962.

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295 [4] Chen, W-M., Zhu, W-F., Bontemps, C., Young, J.P.W., Wei, G-H. (2011) Mesorhizobium 296 camelthorni sp. nov., isolated from Alhagi sparsifolia. Int. J. Syst. Evol. Microbiol. 61, 574- 297 579. 298 [5] Chen, W.X., Li, G.S., Qi, Y.L., Wang, E.T., Yuan, H.L., Li, J.L. (1991) Rhizobium huakuii sp. 299 nov. isolated from the root nodules of Astragalus sinicus. Int. J. Syst. Bacteriol. 41, 275-280. 300 [6] Cummings, S.P., Gyaneshwar, P., Vinuesa, P., Farruggia, F.T., Andrews, M., Humphry, D., 301 Elliot, G.N., Nelson, A., Orr, C., Pettitt, D., Shah, G.R., Santos, S.R., Krishnan, H.B., Odee, 302 D., Moreira, F.M.S, Sprent, J.I., Young, J.P.W., James, E.K. (2009) Nodulation of Sesbania 303 species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia. Environ. Microbiol. 304 11, 2510-2525. 305 [7] Ezaki, T., Hashimoto, Y., Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid- 306 deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter 307 hybridization in which radioisotopes are used to determine genetic relatedness among 308 bacterial strains. Int. J. Syst. Bacteriol. 39, 224-229. 309 [8] Gao, J-L., Turner, S.L., Kan, F.L., Wang, E.T., Tan, Z.Y., Qiu, Y.H., Gu, J., Terefework, Z., 310 Young, J.P.W., Lindström, K., Chen, W.X. (2004) Mesorhizobium septentrionale sp. nov. and 311 Mesorhizobium temperatum sp. nov., isolated from Astragalus adsurgens growing in the 312 northern regions of China. Int. J. Syst. Evol. Microbiol. 54, 2003-2012. 313 [9] Goris, J., Konstantinidis, K.T., Klappenbach, J.A., Coenye, T., Vandamme, P., Tiedje, J.M. 314 (2007) DNA-DNA hybridization values and their relationship to whole-genome sequence 315 similarities. Int. J. Syst. Evol. Microbiol. 57, 81-91. 316 [10] Goris, J., Suzuki, K., De Vos, P., Nakase, T., Kersters, K. (1998) Evaluation of a microplate 317 DNA-DNA hybridization method compared with the initial renaturation method. Can. J. 318 Microbiol. 44, 1148-1153. 319 [11] Heenan, P.B. (2000) Clianthus (Fabaceae), in New Zealand: a reappraisal of Colenso’s 320 . New Zeal. J. Bot. 38, 361-371. 321 [12] Heenan, P.B., Dawson, M.I., Wagstaff, S.J. (2004) The relationship of Sophora sect. 322 Edwardsia (Fabaceae) to Sophora tomentosa, the type species of the Sophora, 323 observed from DNA sequence data and morphological characters. Bot. J. Linn. Soc. 146, 324 439-446. 325 [13] Heenan, P.B., de Lange, P.J., Wilton, A.D. (2001) Sophora (Fabaceae) in New Zealand: 326 taxonomy, distribution, and biogeography. New Zeal. J. Bot. 39, 17-53. 327 [14] Jarvis, B.D.W., Pankhurst, C.E., Patel, J.J. (1982) Rhizobium loti, a new species of legume 328 root nodule bacteria. Int. J. Syst. Bacteriol. 32, 378-380. 329 [15] Kobayashi, H., Broughton, W.J. (2008) Fine-tuning of symbiotic genes in rhizobia: flavonoid 330 signal transduction cascade. In: Dilworth, M.J., James, E.K., Sprent, J.I., Newton, W.E., 331 (Eds.), Nitrogen-fixing legume symbioses, Springer, Dordrecht, pp. 117-152. 332 [16] Leathwick, J.R., Wilson, G., Stephens, R.T.T. (2002) Climate surfaces for New Zealand. 333 Landcare Research Contract Report LC9798/126. 334 [17] Martens M, Dawyndt P, Coopman R, Gillis M, De vos P, Willems A. (2008) Advantages of 335 multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping 336 genes in the genus Ensifer (including former Sinorhizobium). Int. J. Syst. Evol. Microbiol. 58, 337 200-214. 338 [18] Nour, S.M., Fernandez, M.P., Normand, P., Cleyet-Marel, J-C. (1994) Rhizobium ciceri sp. 339 nov., consisting of strains that nodulate chickpeas (Cicer arietinum L.). Int. J. Syst. Evol. 340 Microbiol. 44, 511-522. 341 [19] Pitcher, D.G., Saunders, N.A., Owen, R.J. (1989) Rapid extraction of bacterial genomic DNA 342 with guanidium thiocyanate. Lett. Appl. Microbiol. 8, 151-156. 343 [20] Sarita,Accepted S., Sharma P.K., Priefer, U.B., Prell, J. Manuscript(2005). Direct amplification of rhizobial nodC 344 sequences from soil total DNA and comparison to nodC diversity of root nodule isolates. 345 FEMS Microbiol. Ecol. 54, 1-11. 346 [21] Sprent, J.I. (2009) Legume nodulation: a global perspective. New Delhi, Wiley-Blackwell. 347 [22] Stackebrandt, E., Goebel, B.M. (1994) Taxonomic note: a place for DNA-DNA reassociation 348 and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. 349 Syst. Bacteriol. 44, 846-849. 350 [23] Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011) MEGA5: 351 Molecular evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 352 2731-2739.

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353 [24] Tan, H.W., Heenan, P.B., Ridgway, H.J., Andrews, M. (2013) The New Zealand alpine 354 endemic Montigena novae-zelandiae (Fabaceae) shares rhizobial symbionts with 355 Carmichaelia and Clianthus. New Zeal. J. Bot. 356 [25] Tan, H.W., Weir, B.S., Carter, N., Heenan, P.B., Ridgway, H.J., James, E.K., Sprent, J.I., 357 Young, J.P.Y., Andrews, M. (2012) Rhizobia with 16S rRNA and nifH similar to 358 Mesorhizobium huakuii but novel recA, glnll, nodA and nodC genes are symbionts of New 359 Zealand Carmichaelinae. PLoS ONE 7(10): e47677. 360 [26] Vincent, J.M. (1970) A manual for the practical study of root-nodule bacteria. IBP Handbook 361 15. Blackwell, Oxford, United Kingdom. 362 [27] Wagstaff, S.J., Heenan, P.B., Sanderson, M.J. (1999) Classification, origins and patterns of 363 diversification in New Zealand Carmichaelinae (Fabaceae). Am. J. Bot. 86, 1346-1356. 364 [28] Wang, E.T., Rogel, M.A., Sui, X.H., Chen, W.X., Martínez-Romero, E., van Berkum, P. (2002) 365 Mesorhizobium amorphae, a rhizobial species that nodulates Amorpha fruticosa, is native to 366 American soils. Arch. Microbiol. 178, 301-305. 367 [29] Wang, E.T., van Berkum, P., Sui, X.H., Beyene, D., Chen, W.X., Martínez-Romero, E. (1999). 368 Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and 369 description of Mesorhizobium amorphae sp. nov. Int. J. Syst. Bacteriol. 49, 51-65. 370 [30] Wang, F.Q., Wang, E.T., Liu, J., Chen, Q., Sui, X.H., Chen, W.F., Chen, W.X. (2007) 371 Mesorhizobium albiziae sp. nov., a novel bacterium that nodulates Albizia kalkora in a 372 subtropical region of China. Int. J. Syst. Evol. Microbiol. 57, 1192-1199. 373 [31] Wang, F.Q., Wang, E.T., Zhang, Y.F., Chen, W.X. (2006) Characterization of rhizobia isolated 374 from Albizia spp. in comparison with microsymbionts of Acacia spp. and Leucaena 375 leucocephala grown in China. Syst. Appl. Microbiol. 29, 502-517. 376 [32] Wayne, L., Brenner, D., Colwell, R., Grimont, P., Kandler, O., Krichevsky, M., Moore, L., 377 Moore, W., Murray, R., Stackebrandt, E. (1987) Report of the ad hoc committee on 378 reconciliation of approaches to bacterial systematics. Int. J. Syst. Bact. 37, 463-464. 379 [33] Weir, B.S. (2006) Systematics, specificity and ecology of New Zealand rhizobia. PhD thesis. 380 University of Auckland. 381 [34] Weir, B.S., Turner, S.J., Silvester, W.B., Park, D-C., Young, J.M. (2004) Unexpectedly diverse 382 Mesorhizobium strains and Rhizobium leguminosarum nodulate native legume genera of New 383 Zealand, while introduced legume weeds are nodulated by Bradyrhizobium species. Appl. 384 Environ. Microbiol. 70, 5980-5987. 385 [35] Yang, W., Kong, Z., Chen, W., Wei, G. (2013) Genetic diversity and symbiotic evolution of 386 rhizobia from root nodules of Coronilla varia. Syst. Appl. Microbiol. 36, 49-55. 387 [36] Zhao, L., Deng, Z., Yang, W., Cao, Y., Wang, E., Wei, G. (2010) Diverse rhizobia associated 388 with Sophora alopecuroides grown in different regions of Loess Plateau in China. Syst. Appl. 389 Microbiol. 33, 468-477. 390 [37] Zhao, C.T., Wang, E.T., Chen, W.F., Chen, W.X. (2008) Diverse genomic species and 391 evidences of symbiotic gene lateral transfer detected among the rhizobia associated with 392 Astragalus species grown in the temperate regions of China. FEMS Microbiol. Lett. 286, 263- 393 273. 394 395 Accepted Manuscript

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395

396 Legends to Figures 397 398 Fig. 1. 16S rRNA gene maximum likelihood (ML) tree (ca. 1260 bp) of bacterial strains isolated from 399 New Zealand native Sophora spp. and selected Mesorhizobium type strains. Sl = Sophora 400 longicarinata; Sm = Sophora microphylla; Sp = Sophora prostrata; St = Sophora tetraptera. The 401 Kimura 2‐parameter + Gamma distribution (K2 + G) model was used to construct the tree. Numbers 402 on branches are bootstrap % from 500 replicates (shown only when ≥ 50%). The tree was rooted 403 with Bradyrhizobium elkani USDA 76T. Scale bar = 2% sequence divergence (2 substitutions per 100 404 nucleotides). *Isolated in previous study [34]. FS = field site. 405 406 Fig. 2. Concatenated recA, glnII and rpoB gene maximum likelihood (ML) tree (ca. 1800 bp) of 407 bacterial strains isolated from New Zealand native Sophora spp. and selected Mesorhizobium type 408 strains. Sl = Sophora longicarinata; Sm = Sophora microphylla; Sp = Sophora prostrata; St = Sophora 409 tetraptera. The Tamura‐Nei with gamma distribution (TN93 + G) model was used to construct the 410 tree. Numbers on branches are bootstrap % from 500 replicates (shown only when ≥ 50%). The 411 trees were rooted with Bradyrhizobium elkani USDA 76T. Scale bar = 5% sequence divergence (5 412 substitutions per 100 nucleotides). *Isolated in previous study [34]. FS = field site. 413 414 Fig. 3. nifH gene maximum likelihood (ML) tree (ca. 280 bp) of bacterial strains isolated from New 415 Zealand native Sophora spp. and selected Mesorhizobium type and non‐type strains. Sl = Sophora 416 longicarinata; Sm = Sophora microphylla; Sp = Sophora prostrata; St = Sophora tetraptera. The 417 Tamura 3‐parameter with gamma distribution (T92 + G) model was used to construct the tree. 418 Numbers on branches are bootstrap % from 500 replicates (shown only when ≥ 50%). The trees 419 were rooted with Azorhizobium caulinodans ORS571T. Scale bar = 5% sequence divergence (5 420 substitutions per 100 nucleotides). *Isolated in previous study [34]. FS = field site. 421 422 Fig. 4. nodA (A) and nodC (B) gene maximum likelihood (ML) trees (ca. 420 bp and 650 bp 423 respectively) of bacterial strains isolated from New Zealand native Sophora spp., selected 424 Mesorhizobium type and non‐type strains and strains shown to produce functional nodules on NZ 425 Carmichaelinae species (superscript C). Sl = Sophora longicarinata; Sm = Sophora microphylla; Sp = 426 Sophora prostrata; St = Sophora tetraptera. The Tamura 3‐parameter with gamma distribution and 427 invariant sites (T92 + G + I) model was used to construct the trees. Numbers on branches are 428 bootstrap % from 500 replicates (shown only when ≥ 50%). The trees were rooted with 429 Azorhizobium caulinodans ORS571T. Scale bars = 1% sequence divergence (1 substitution per 100 430 nucleotides). *Isolated in previous study [34]. FS = field site. 431 432 Accepted Manuscript

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433 Table 1. Host specificity of rhizobial strains used in this study. 434 435 16S rRNA Strain Host Sophora Clianthus Carmichaelia grouping microphylla prostrata longicarinata puniceus australis M. ciceri ICMP 11719 S. tetraptera Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ M. ciceri ICMP 14330 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ M. ciceri ICMP 19513 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ M. ciceri ICMP 19514 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19515 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19519 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19520 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ M. ciceri ICMP 19560 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19561 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19545 S. prostrata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19546 S. prostrata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19523 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19528 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. ciceri ICMP 19535 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. amorphae ICMP 19557 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. amorphae ICMP 19558 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. amorphae ICMP 19568 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. amorphae ICMP 19569 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod‐ M. huakuii ICMP 19559 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ Nod+Fix‐ M. huakuii ICMP 19567 S. longicarinata Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ Nod+Fix‐ M. huakuii ICMP 19550 S. microphylla Nod+Fix+ Nod+Fix+ Nod+Fix+ Nod+Fix‐ Nod+Fix‐ M. ciceriT ICMP 13641 Cicer arietinum Nod‐ Nod‐ Nod‐ N.D. N.D. T Accepted Manuscript M. amorphae ICMP 15022 Amorpha fruticosa Nod‐ Nod‐ Nod‐ N.D. N.D. M. huakuiiT ICMP 11069 Astragalus sinicus Nod‐ Nod‐ Nod‐ N.D. N.D. M. lotiT ICMP 4682 Lotus corniculatus Nod‐ Nod‐ Nod‐ N.D. N.D. 436

437 Nod+ = all plants nodulated; Nod‐ = no plants nodulated; Fix+ = N2 fixing nodules; Fix‐ = nodules not fixing N2

438 N.D. Not determined

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439 Table 2. % DNA‐DNA hybridisation between pairs of Sophora isolates and Sophora isolates and their closest Mesorhizobium type strain. 440 Strains ICMP 19512 ICMP 19535 ICMP 19523 ICMP 19545 ICMP 19560 LMG ICMP ICMP LMG 14989 19515 19557 23930

ICMP 19512 100 ICMP 19535 43.6 100 ICMP 19523 52.1 38.2 100 ICMP 19545 41.1 32.4 32.8 100 ICMP 19560 36.1 30.1 36.6 38.4 100 M. ciceri LMG14989T 29.2 31.1 27.0 26.1 36.3 100 ICMP 19515 N.D. N.D. N.D. N.D. N.D. 28.9 100 ICMP 19557 N.D. N.D. N.D. N.D. N.D. 29.2 31.8 100 M. septentrionale LMG 23930T N.D. N.D. N.D. N.D. N.D. N.D. 32.3 28.8 100 441 442 N.D. Not determined

443

Accepted Manuscript

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ICMP 19526 (Sm) (KC237416) FS6 Fig. 1 ICMP 19540 (Sm) (KC237429) FS2 16S rRNA ICMP 19539 (Sm) (KC237428) FS2 ICMP 19538 (Sm) (KC237427) FS2 ICMP 19537 (Sm) (KC237426) FS2 ICMP 19536 (Sm) (KC237425) FS2 ICMP 19535 (Sm) (KC237424) FS2 ICMP 19532 (Sm) (KC237422) FS3 ICMP 19531 (Sm) (KC237421) FS3 ICMP 19530 (Sm) (KC237420) FS3 ICMP 19529 (Sm) (KC237419) FS3 ICMP 19528 (Sm) (KC237418) FS3 ICMP 19527 (Sm) (KC237417) FS6 ICMP 19525 (Sm) (KC237415) FS6 ICMP 19524 (Sm) (KC237414) FS6 ICMP 19523 (Sm) (KC237413) FS6 ICMP 19521 (Sm) (KC237404) FS4 ICMP 19520 (Sm) (KC237403) FS4 ICMP 14330 (Sm) (AY491067)* ICMP 11719 (St) (AY491065)* ICMP 19533 (Sm) (KC237423) FS3 90 ICMP 19522 (Sm) (KC237405) FS4 ICMP 19560 (Sl) (KC237406) FS1 ICMP 19561 (Sl) (KC237407) FS1 ICMP 19562 (Sl) (KC237408) FS1 ICMP 19563 (Sl) (KC237409) FS1 ICMP 19513 (Sm) (KC237395) FS5 93 ICMP 19519 (Sm) (KC237402) FS4 ICMP 19512 (Sm) (KC237394) FS5 ICMP 19545 (Sp) (KC237410) FS1 ICMP 19546 (Sp) (KC237411) FS1 52 ICMP 19547 (Sp) (KC237412) FS1 71 M. ciceri UPM-Ca7 (U07934)T ICMP 19518 (Sm) (KC237401) FS5 ICMP 19551 (Sm) (KC237400) FS5 58 ICMP 19517 (Sm) (KC237399) FS5 ICMP 19516 (Sm) (KC237398) FS5 ICMP 19515 (Sm) (KC237397) FS5 ICMP 19514 (Sm) (KC237396) FS5 68 T M. shangrilense CCBAU 65327 (EU074203) M. australicum WSM2073 (NR102452)T M. qingshengii CCBAU 33460 (NR109565)T 57 M. loti LMG 6125 (X67229)T M. albiziae CCBAU 61158 (NR043549)T ICMP 19567 (Sl) (KC237385) FS1 ICMP 19550 (Sm) (KC237386) FS3 Accepted ICMP 19559 (Sl)Manuscript (KC237384) FS1 M. huakuii IAM 14158 (D12797)T ICMP 19557 (Sl) (KC237387) FS1 ICMP 19564 (Sl) (KC237389) FS1 ICMP 19565 (Sl) (KC237390) FS1 ICMP 19566 (Sl) (KC237391) FS1 ICMP 19568 (Sl) (KC237392) FS1 59 ICMP 19558 (Sl) (KC237388) FS1 M. amorphae ACCC 19665 (AF041442)T ICMP 19569 (Sl) (KC237393) FS1 M. septentrionale SDW 014 (AF508207)T B. elkanii USDA 76 (U35000)T

0.02 Page 13 of 16 ICMP 19531 (Sm) FS3 ICMP 19532 (Sm) FS3 Fig. 2 98 ICMP 19530 (Sm) FS3 recA + glnII + rpoB ICMP 19529 (Sm) FS3 ICMP 19528 (Sm) FS3 99 ICMP 19533 (Sm) FS3 ICMP 19535 (Sm) FS2 ICMP 19536 (Sm) FS2 ICMP 19537 (Sm) FS2 99 99 ICMP 19538 (Sm) FS2 ICMP 19539 (Sm) FS2 ICMP 19540 (Sm) FS2 88 ICMP 11719 (St)* ** ICMP 14330 (Sm)*** ICMP 19524 (Sm) FS6 ICMP 19526 (Sm) FS6 89 100 ICMP 19523 (Sm) FS6 ICMP 19525 (Sm) FS6 ICMP 19527 (Sm) FS6 ICMP 19512 (Sm) FS5 ICMP 19519 (Sm) FS4 ICMP 19520 (Sm) FS4 100 ICMP 19521 (Sm) FS4 ICMP 19522 (Sm) FS4 ICMP 19559 (Sl) FS1 ICMP 19563 (Sl) FS1 ICMP 19560 (Sl) FS1 100 ICMP 19561 (Sl) FS1 ICMP 19562 (Sl) FS1 ICMP 19513 (Sm) FS5 ICMP 19546 (Sp) FS1 100 ICMP 19547 (Sp) FS1 ICMP 19545 (Sp) FS1 M. ciceri T 68 ICMP 19516 (Sm) FS5 ICMP 19515 (Sm) FS5 98 ICMP 19517 (Sm) FS5 100 ICMP 19514 (Sm) FS5 ICMP 19551 (Sm) FS5 ICMP 19518 (Sm) FS5 ICMP 19550 (Sm) FS3 T 65 M. plurifarium M. huakuii T M. loti T T 94 M. amorphae 100 M. caraganae T M. septentrionale T 73 ICMP 19567 (Sl) FS1 ICMP 19557 (Sl) FS1 97 Accepted ICMP 19558 (Sl)Manuscript FS1 ICMP 19564 (Sl) FS1 99 ICMP 19565 (Sl) FS1 ICMP 19568 (Sl) FS1 ICMP 19566 (Sl) FS1 ICMP 19569 (Sl) FS1 M. mediterraneum T M. gobiense T 98 M. tianshanense T 69 50 M. tarimense T M. albiziae T B. elkanii T

0.05

Page 14 of 16 ICMP 19531 (Sm) (KC237485) FS3 ICMP 19532 (Sm) (KC237486) FS3 Fig. 3 ICMP 19530 (Sm) (KC237484) FS3 ICMP 19529 (Sm) (KC237483) FS3 nifH ICMP 19528 (Sm) (KC237482) FS3 ICMP 19563 (Sl) (KC237473) FS1 ICMP 19562 (Sl) (KC237472) FS1 ICMP 19561 (Sl) (KC237471) FS1 ICMP 19560 (Sl) (KC237470) FS1 ICMP 19518 (Sm) (KC237465) FS5 ICMP 19551 (Sm) (KC237464) FS5 ICMP 19517 (Sm) (KC237463) FS5 ICMP 19516 (Sm) (KC237462) FS5 ICMP 19515 (Sm) (KC237461) FS5 ICMP 19514 (Sm) (KC237460) FS5 ICMP 19566 (Sl) (KC237452) FS1 ICMP 19565 (Sl) (KC237451) FS1 ICMP 19564 (Sl) (KC237450) FS1 ICMP 19550 (Sm) (KC237438) FS3 ICMP 19559 (Sl) (KC237436) FS1 74 ICMP 19558 (Sl) (KC237449) FS1 ICMP 19524 (Sm) (KC237478) FS6 ICMP 19525 (Sm) (KC237479) FS6 89 ICMP 19523 (Sm) (KC237477) FS6 ICMP 19526 (Sm) (KC237480) FS6 ICMP 19527 (Sm) (KC237481) FS6 ICMP 19535 (Sm) (KC237488) FS2 ICMP 19536 (Sm) (KC237489) FS2 ICMP 19537 (Sm) (KC237490) FS2 61 ICMP 19538 (Sm) (KC237491) FS2 ICMP 19539 (Sm) (KC237492) FS2 ICMP 19540 (Sm) (KC237493) FS2 ICMP 19545 (Sp) (KC237474) FS1 ICMP 19546 (Sp) (KC237475) FS1 97 95 ICMP 19547 (Sp) (KC237476) FS1 ICMP 19533 (Sm) (KC237487) FS3 ICMP 14330 (Sm) (KC237456)* ICMP 19568 (Sl) (KC237453) FS1 ICMP 19567 (Sl) (KC237437) FS1 ICMP 19569 (Sl) (KC237454) FS1 ICMP 11719 (St) (KC237455)* ICMP 19512 (Sm) (KC237458) FS5 61 ICMP 19513 (Sm) (KC237459) FS5 ICMP 19519 (Sm) (KC237466) FS4 ICMP 19520 (Sm) (KC237467) FS4 ICMP 19521 (Sm) (KC237468) FS4 ICMP 19522 (Sm) (KC237469) FS4 ICMP 19557 (Sl) (KC237448) FS1 T M. amorphae ACCC 19665 (EU267714) T M. huakuii ICMP 11069 (JQ963082) T M. plurifarium LMG 11892 (EU267717) T 87 M. robiniae CCNWYC 115 (EU849570) T M. albiziae CCBAU 61158 (DQ311093) T M. septentrionale SDW014 (EU130411) T 100 M. ciceri UPM-Ca7 (DQ450928) Accepted ManuscriptT M. mediterraneum UPM-Ca36 (DQ450930) T 52 M. shangrilense CCBAU 65327 (EU877532) T M. sangaii SCAU7 (EU514544) T M. qingshengii CCBAU 33460 (JQ339911) T M. tianshanense A-1BS (DQ450934) T M. temperatum SDW018 (EU130410) T M. gobiense CCBAU 83330 (EU252606) T M. caraganae CCBAU 11299 (EU130422) Mesorhizobium sp. CCNWSX672 (JF273843) T M. camelthorni CCNWXJ40-4 (GU220806) 51 T M. alhagi CCNWXJ12-2 (FJ481884) T M. loti NZP 2213 (DQ450929) T 59 M. tarimense CCBAU 83306 (EU252607) T A. caulinodans ORS571 (AP009384)

0.05 Page 15 of 16 ICMP 19538 (Sm) (KC237540) FS2 ICMP 19530 (Sm) (KC237592) FS3 Fig. 4A ICMP 19539 (Sm) (KC237541) FS2 Fig. 4B ICMP 19531 (Sm) (KC237593) FS3 ICMP 19537 (Sm) (KC237539) FS2 ICMP 19529 (Sm) (KC237591) FS3 nodA nodC ICMP 19536 (Sm) (KC237538) FS2 ICMP 19528 (Sm) (KC237590) FS3 ICMP 19535 (Sm) (KC237537) FS2 ICMP 19527 (Sm) (KC237589) FS6 ICMP 19527 (Sm) (KC237530) FS6 ICMP 19526 (Sm) (KC237588) FS6 ICMP 19526 (Sm) (KC237529) FS6 ICMP 19525 (Sm) (KC237587) FS6 ICMP 19525 (Sm) (KC237528) FS6 ICMP 19524 (Sm) (KC237586) FS6 ICMP 19524 (Sm) (KC237527) FS6 ICMP 19523 (Sm) (KC237585) FS6 ICMP 19523 (Sm) (KC237526) FS6 ICMP 19547 (Sp) (KC237584) FS1 ICMP 19547 (Sp) (KC237525) FS1 ICMP 19546 (Sp) (KC237583) FS1 ICMP 19545 (Sp) (KC237582) FS1 54 ICMP 19546 (Sp) (KC237524) FS1 ICMP 19563 (Sl) (KC237581) FS1 ICMP 19545 (Sp) (KC237523) FS1 ICMP 19562 (Sl) (KC237580) FS1 ICMP 19522 (Sm) (KC237518) FS4 ICMP 19561 (Sl) (KC237579) FS1 ICMP 19521 (Sm) (KC237517) FS4 61 ICMP 19560 (Sl) (KC237578) FS1 ICMP 19520 (Sm) (KC237516) FS4 ICMP 19522 (Sm) (KC237577) FS4 ICMP 19519 (Sm) (KC237515) FS4 ICMP 19521 (Sm) (KC237576) FS4 ICMP 19512 (Sm) (KC237507) FS5 ICMP 19520 (Sm) (KC237575) FS4 ICMP 11719 (St) (DQ100400)* ICMP 19519 (Sm) (KC237574) FS4 ICMP 14330 (Sm) (DQ100401)* ICMP 19512 (Sm) (KC237566) FS5 ICMP 19540 (Sm) (KC237542) FS2 ICMP 14330 (Sm) (KC237564)* ICMP 19559 (Sl) (KC237496) FS1 ICMP 11719 (St) (KC237563)* 97 ICMP 19562 (Sl) (KC237521) FS1 ICMP 19532 (Sm) (KC237594) FS3 ICMP 19563 (Sl) (KC237522) FS1 60 ICMP 19535 (Sm) (KC237596) FS2 ICMP 19560 (Sl) (KC237519) FS1 ICMP 19536 (Sm) (KC237597) FS2 ICMP 19561 (Sl) (KC237520) FS1 ICMP 19537 (Sm) (KC237598) FS2 ICMP 19513 (Sm) (KC237508) FS5 97 ICMP 19538 (Sm) (KC237599) FS2 ICMP 19550 (Sm) (KC237498) FS3 ICMP 19539 (Sm) (KC237600) FS2 ICMP 19514 (Sm) (KC237509) FS5 ICMP 19559 (Sl) (KC237544) FS1 ICMP 19515 (Sm) (KC237510) FS5 ICMP 19540 (Sm) (KC237601) FS2 ICMP 19516 (Sm) (KC237511) FS5 ICMP 19533 (Sm) (KC237595) FS3 ICMP 19517 (Sm) (KC237512) FS5 ICMP 19550 (Sm) (KC237546) FS3 ICMP 19551 (Sm) (KC237513) FS5 ICMP 19513 (Sm) (KC237567) FS5 100 ICMP 19518 (Sm) (KC237514) FS5 ICMP 19515 (Sm) (KC237569) FS5 56 ICMP 19528 (Sm) (KC237531) FS3 99 ICMP 19516 (Sm) (KC237570) FS5 ICMP 19529 (Sm) (KC237532) FS3 ICMP 19517 (Sm) (KC237571) FS5 ICMP 19530 (Sm) (KC237533) FS3 ICMP 19514 (Sm) (KC237568) FS5 ICMP 19531 (Sm) (KC237534) FS3 ICMP 19518 (Sm) (KC237573) FS5 ICMP 19532 (Sm) (KC237535) FS3 ICMP 19551 (Sm) (KC237572) FS5 ICMP 19533 (Sm) (KC237536) FS3 ICMP 19567 (Sl) (KC237545) FS1 ICMP 19557 (Sl) (KC237556) FS1 93 ICMP 19568 (Sl) (KC237505) FS1 97 ICMP 19569 (Sl) (KC237506) FS1 ICMP 19558 (Sl) (KC237557) FS1 ICMP 19567 (Sl) (KC237497) FS1 ICMP 19564 (Sl) (KC237558) FS1 ICMP 19557 (Sl) (KC237500) FS1 77 ICMP 19565 (Sl) (KC237559) FS1 63 ICMP 19558 (Sl) (KC237501) FS1 99 ICMP 19566 (Sl) (KC237560) FS1 ICMP 19564 (Sl) (KC237502) FS1 ICMP 19568 (Sl) (KC237561) FS1 ICMP 19569 (Sl) (KC237562) FS1 ICMP 19565 (Sl) (KC237503) FS1 Mesorhizobium sp. (EU753421) ICMP 19566 (Sl) (KC237504) FS1 80 M. albiziae CCBAU 61158 (DQ311092)TTT M. albiziae CCBAU 61158 (GQ167236)TTT M. camelthorni CCNWXJ40-4 (EU722491)TTT M. loti NZD 2213 (L06241)TTT 99 TTT TTT M. alhagi CCNWXJ12-2 (EU722486) 100 M. australicum WSM2073 (AY601528) TTT M. caraganae CCBAU 11299 (EU130405) M. opportunistum WSM2075 (AY601530)TTT 98 TTT M. sangaii SCAU7 (JN129438) M. ciceri UPM-Ca7 (AJ300247)TTT 100 TTT M. septentrionale SDW014 (GQ167237) TTT 97 M. mediterraneum UPM-Ca36 (AJ300248) 63 TTT M. temperatum SWD018 (GQ167238) ICMP 12690 (Mn) (DQ100404) TTT 94 65 M. gobiense CCBAU 83330 (EF050784) 100 ICMP 11541 (Clp) (DQ100406) TTT 75 M. tianshanense USDA 3592 (DQ407291) TTT 99 M. huakuii CCBAU 2609 (GQ167249) TTT M. plurifarium ORS 1032 (FJ745283) TTT M. caraganae CCBAU 11299 (GQ167250) TTT M. robiniae CCNWYC 115 (EU849563) TTT 67 100 M. tianshanense USDA3592 (AJ250142) TTT 69 M. amorphae ACCC19665 (AF217261) TTT 85 M. temperatum SDW018 (AJ579887) Ccc ICMP 11541 (Clp) (JQ963100) TTT M. camelthorni CCNWXJ40-4 (EU722478) Ccc ICMP 18942 (Mn) (JQ963091) TTT M. alhagi CCNWXJ12-2 (EU722474) Ccc 100 ICMP 12690 (Mn) (JQ963094) TTT 99 Ccc 58 M. amorphae ICMP15022 (GQ375855) ICMP 19041 (Ca) (JQ963095) TTT Ccc M. robiniae CCNWYC 115 (EU849558) 72 ICMP 13190 (Ca) (JQ963096) TTT 72 Accepted M. plurifarium ICMP 13640 (DQ100407) Manuscript Ccc ICMP 19418 (Ca) (JQ963097) TTT Ccc M. septentrionale SDW014 (AJ579893) ICMP 19042 (Cm) (JQ963098) Ccc TTT ICMP 18942 (Mn) (JQ963065) M. qingshengii CCBAU 33460 (JQ339881) Ccc TTT ICMP 19041 (Ca) (JQ963068) M. mediterraneum UPM-Ca36 (DQ407293) 80 Ccc TTT ICMP 13190 (Ca) (DQ100386) M. ciceri UPM-Ca7 (DQ407292) 100 99 Ccc TTT ICMP 19418 (Ca) (JQ963069) 98 M. huakuii ICMP 11069 (KC854806) Ccc TTT ICMP 19042 (Cm) (JQ963070) M. tarimense CCBAU 83306 (EF050786) TTT TTT A. caulinodans ORS571 (S70039) 99 M. loti NZP 2213 (DQ450939) TTT A. caulinodans ORS 571 (AP009384) 0.1 0.1

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