G C A T T A C G G C A T genes

Article Differential Preference of Burkholderia and to pH and Soil Types in the Core Cape Subregion, South Africa

Meshack Nkosinathi Dludlu * ID , Samson B. M. Chimphango, Charles H. Stirton ID and A. Muthama Muasya

Department of Biological Sciences, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa; [email protected] (S.B.M.C.); [email protected] (C.H.S.); [email protected] (A.M.M.) * Correspondence: [email protected]; Tel.: +27-21-650-3684

Received: 31 October 2017; Accepted: 13 December 2017; Published: 22 December 2017

Abstract: Over 760 legume species occur in the ecologically-heterogeneous Core Cape Subregion (CCR) of South Africa. This study tested whether the main symbionts of CCR legumes (Burkholderia and Mesorhizobium) are phylogenetically structured by altitude, pH and soil types. Rhizobial strains were isolated from field nodules of diverse CCR legumes and sequenced for 16S ribosomic RNA (rRNA), recombinase A (recA) and N-acyltransferase (nodA). Phylogenetic analyses were performed using Bayesian and maximum likelihood techniques. Phylogenetic signals were determined using the D statistic for soil types and Pagel’s λ for altitude and pH. Phylogenetic relationships between symbionts of the narrowly-distributed Indigofera superba and those of some widespread CCR legumes were also determined. Results showed that Burkholderia is restricted to acidic soils, while Mesorhizobium occurs in both acidic and alkaline soils. Both genera showed significant phylogenetic clustering for pH and most soil types, but not for altitude. Therefore, pH and soil types influence the distribution of Burkholderia and Mesorhizobium in the CCR. All strains of Indigofera superba were identified as Burkholderia, and they were nested within various clades containing strains from outside its distribution range. It is, therefore, hypothesized that I. superba does not exhibit rhizobial specificity at the intragenic level. Implications for CCR legume distributions are discussed.

Keywords: ; Burkholderia; Mesorhizobium; Core Cape Subregion; Southern Africa; Cape Peninsula

1. Introduction There is an open debate in the microbial biogeography literature regarding whether or not microorganisms are biogeographically structured [1–3], thanks to the Baas Becking hypothesis that “everything is everywhere, but the environment selects” [4]. The premise of the hypothesis is that since microorganisms are small, they reproduce rapidly, they have dormancy stages and they have high dispersal potential; it follows that they should not be limited by geographical barriers and distances [5,6]. However, there is a growing body of evidence from studies on archaea, , fungi and protists, which points to the existence of microbial biogeographic structure [7–12]. Like the other microorganisms alluded to above, the various rhizobial genera exhibit some notable biogeographic structuring at local, regional, continental and global scales [13]. For example, while Burkholderia is the predominant symbiont of mimosoid legumes in the Brazilian Cerrado and Caatinga Biomes [14], the Mimosoid legumes occurring in Mexico are predominantly nodulated by , particularly the genera Rhizobium and Ensifer [15]. Genome level studies have also shown that the Burkholderia species that nodulate Mimosoid legumes in South America are genetically distinct from those that nodulate papilionoid legumes in the CCR of South Africa, such

Genes 2018, 9, 2; doi:10.3390/genes9010002 www.mdpi.com/journal/genes Genes 2018, 9, 2 2 of 23 that they are incapable of nodulating each other’s hosts [16–18]. Furthermore, a recent study of the symbionts of legumes found in the sub-Himalayan region of India showed that they are nodulated by distinct Bradyrhizobium strains that represent new species to science [19]. Likewise, legumes of the Core Cape Subregion (CCR) of Southern Africa are predominantly nodulated by unique Burkholderia and Mesorhizobium strains [20–22], whereas those from the Grassland and Savannah biomes of the region are largely nodulated by unique strains of Bradyrhizobium [23]. Therefore, the distribution of rhizobia is as prone to biogeographic limitations as other living organisms. Some of the factors that influence the growth and distribution of rhizobia species include pH, temperature, salinity and the distribution of suitable hosts [24–29]. These factors also affect general growth and nodule development [30,31]; hence, they can influence levels of nitrogen fixation. Notably, rhizobia species differ in their sensitivity to these factors. For example, species of the genus Burkholderia can tolerate acidic soil conditions, whereas they are replaced by alpha-rhizobia in alkaline habitats [32–34]. This could explain the predominance of Burkholderia in the acidic soils of the Cerrado, Caatinga biomes and other parts of South America [14,35], and in South Africa’s CCR, where it associates with diverse legume tribes including the Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae [20,21]. However, unlike in South America, Burkholderia is not the only dominant rhizobial symbiont in the CCR. Mesorhizobium is also an abundant symbiont, associated with a wide range of legumes in the tribes Crotalarieae, Galegeae, Genisteae and Psoralea [21,22], and the reasons for its dominance are yet to be determined. Contrary to Burkholderia’s genus-wide predilection for acidic soils [34], Mesorhizobium species exhibit differential tolerance to environmental stress, including heavy metals, pH, salinity and temperature [27,36,37]. In terms of pH, Mesorhizobium species can tolerate a wide range of pH conditions (3–10), despite an optimal range of pH 6–8 [36,38]. For example, Mesorhizobium was found to be the dominant symbiont of Cicer arietinum L. (chickpea) growing on alkaline soils in China [39]. On the other hand, a study of Mesorhizobium strains nodulating chickpea plants in Portuguese soils showed that some strains were able to tolerate acidic conditions down to a minimum of pH 3 [36]. This suggests that the predominance of Mesorhizobium in the CCR (in addition to Burkholderia) might be linked to its wide-ranging tolerance to different pH conditions. Notably, while acidic soil conditions are more prevalent in the CCR, particularly in the sandstone-derived soils, patches of near neutral and alkaline soils (e.g., granite, limestone and shale) also exist [40,41]. Based on the discussion above, it appears that Burkholderia is more sensitive to pH, and hence, soil type, than Mesorhizobium. Therefore, in the case of the CCR, it is hypothesized that the distribution of Burkholderia species is structured by soil type and pH; while Mesorhizobium should be more dispersed. Moreover, Burkholderia should only dominate in the acidic soils, being replaced by Mesorhizobium species in neutral and alkaline soils. Apart from the effects of edaphic factors on the growth and distribution of rhizobia, some studies have found correlations between turnover in the diversity of rhizobia and altitude. For example, Bontemps and co-workers [14] observed that discrete Burkholderia species complexes were restricted to specific altitudes in the Brazilian Caatinga and Cerrado biomes. Likewise, turnover in Sinorhizobium community assemblages along elevation gradients were observed in Northern China [42]. Since differences in altitude are directly related to changes in humidity and temperature [43], the correlations between altitude and rhizobial diversity suggest that rhizobial lineages vary in their sensitivity and tolerance to these attributes. The evident influence of altitudinal gradients on microbial diversity is not unique to rhizobia as similar patterns have been reported for other microorganisms, e.g., non-rhizobial bacteria and fungi [10,44–46]. Considering that altitude is highly variable in the CCR and that it is one of the major drivers of the diversification of the CCR flora [47], it is hypothesized that altitude influences rhizobial diversity and turnover in CCR landscapes. Considering that the soils of the CCR are generally oligotrophic [48] and the observation that legumes have a high nitrogen-demanding lifestyle [49,50], nitrogen fixation must be a key strategy for their success in the region. Since the distribution of rhizobia is constrained by environmental factors (as previously discussed), legumes might fail to establish in habitats where their rhizobial Genes 2018, 9, 2 3 of 23 symbionts are lacking [51,52]. Therefore, legumes that are highly specific in the kinds of rhizobia that they associate with might be restricted to habitats where their specific symbionts are present. A study by Lemaire and co-workers [21] showed that CCR legumes of the tribe Podalyrieae are exclusively nodulated by Burkholderia species. A subsequent study, which sampled multiple disjunct populations of the widespread calyptrata Willd., found high levels of genetic diversity between the Burkholderia strains that nodulate the species [53]. This indicates that while P. calyptrata exhibits symbiotic specificity towards the genus Burkholderia, it associates with diverse lineages within Burkholderia, and this could explain its widespread distribution. Studies on the diversity of symbionts that nodulate geographically-restricted taxa are lacking for the CCR, yet such studies could shed light on the potential influence of rhizobia specificity on legume distributions. For the CCR, one such taxon is Indigofera superba C.H. Stirt., a rare legume species that is restricted to the Kleinrivier Mountains within the biome of the CCR [54]. It occurs on sandstone-derived soils, at altitudes of 100–300 m [55]. It occurs in sympatry with some widespread legume species, such as Aspalathus carnosa Eckl. & Zeyh., Indigofera filifolia Thunb. and Psoralea pullata C.H. Stirt. Its rhizobial symbionts are presently unknown, and it is hypothesized that rhizobia specificity contributes to its limited distribution. The main objectives of the present study were to determine if the ecological parameters; altitude, pH and soil type influence the distribution of rhizobial symbionts that nodulate various legumes of the Cape Peninsula as a microcosm of the CCR and to determine the diversity and phylogenetic position of rhizobia that associate with the narrowly-distributed I. superba in the CCR. The first objective was pursued through molecular characterization of rhizobial strains isolated from nodules of legume species collected in the field across the Cape Peninsula. These were analyzed together with the data from a previous study [21] that sampled broadly within the CCR. It was postulated that if an ecological parameter limits the distribution of symbionts within the landscape, then each habitat type should predominantly harbor symbionts that are suitably adapted to the local conditions. Such symbionts would likely be genetically similar. Therefore, a significant phylogenetic signal would be expected for that parameter, i.e., closely-related species would occupy similar habitats [56]. Thus, tests for phylogenetic signals for the three ecological parameters were conducted based on phylogenies of housekeeping and nodulation genes of the rhizobial strains. For the study of rhizobial symbionts of the rare I. superba, field nodules were sampled from multiple populations across its distribution range, and a phylogeny of its symbionts was reconstructed in a matrix that included symbionts of diverse legumes from diverse habitats within the CCR.

2. Materials and Methods

2.1. Study Site, Nodule Sampling and Rhizobia Isolation The primary study area was the Cape Peninsula, which is located on the south westernmost tip of the Core Cape Subregion of South Africa. Details of its climatic, edaphic, physiographic and vegetation characteristics and the selection of sampling sites are as described by Dludlu and co-workers [57]. Root nodules of legume species occurring at each site were collected and transported to the laboratory, where they were kept at 4 ◦C before the isolation of rhizobia, which took place within 2–5 days of sampling. Rhizobia were isolated and cultured using standard protocols [58] on yeast extract mannitol agar (YEMA), with the exception that for the surface sterilization of the nodules, a 4% solution of sodium hypochlorite (NaOCl) was used instead of acidified mercuric chloride. Rhizobial isolates were incubated at 28 ◦C for three to ten days depending on their growth rates, and pure cultures were obtained by sub-culturing on fresh YEMA plates. Purified cultures were suspended in 20% (v/v) glycerol solution and stored in a −80 ◦C freezer for long-term storage. This method of obtaining rhizobial cultures was chosen over the direct sequencing of DNA from the nodules because it allows to produce a pure culture that can be authenticated for nitrogen fixing properties. Furthermore, previous studies from our laboratory have shown that each nodule is occupied by a single dominant rhizobial strain [18], but nodules may be colonized by non-rhizobial bacteria. Genes 2018, 9, 2 4 of 23

2.2. DNA Extraction, Amplification and Sequencing DNA was extracted using a modified version [59] of the cetyl trimethylammonium bromide (CTAB) DNA Extraction protocol [60]. Polymerase chain reactions (PCR) were conducted to amplify 16S ribosomic RNA (rRNA), recombinase A (recA) and N-acyltransferase (nodA) using an Applied Biosystems GeneAmp 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA). Primer pairs used were 16S-f27 and 16S-r1485 [61,62] for 16S rRNA; recA-63F and recA-504R [63] for recA; and nodA-1F and nodA-2R [64] for nodA. Each PCR reaction had a total volume of 25 µL: comprising 19.92 µL of water, 2 µL of 10× buffer (Buffer A) that contained 1.5 mM Mg2+, 0.4 µL of 10 mM dNTP, 0.8 µL each of forward and reverse primers (10 µM), 0.08 µL of Taq polymerase (Kapa Biosystems, Cape Town, South Africa) and 1 µL of template DNA. All DNA regions were amplified according to the reaction conditions described by the authors of the primers, i.e. Weisburg and co-workers [61] for 16S rRNA, Gaunt and co-workers [63] for recA and Haukka and co-workers [64] for nodA. PCR products were loaded onto ethidium bromide agarose gels (1%) and subjected to electrophoresis using 0.5× Tris Borat EDTA (TBE). The gels were observed under UV light (Wavelength = 365 nm) to identify successfully amplified samples. Amplified products were enzymatically purified using the Exo/SAP protocol [65] and sent to Macrogen (Macrogen, Amsterdam, The Netherlands) for sequencing with the same primers used for PCR amplification. Newly generated sequences were deposited in the GenBank database, and the accession numbers for 16S rRNA range from MG593870–MG593941, MG704159-MG704225 for recA and MG704226-MG704280 for nodA.

2.3. Contig Assembly and Phylogenetic Analyses The forward and reverse DNA sequence contigs were assembled using the Staden package Version 2.0.0 [66] and aligned using the online version of MAFFT [67]. Identification of the isolated strains was achieved by comparing individual sequences with publically available sequences on GenBank, using the Basic Local Alignment Search Tool (BLAST) of Altschul and co-workers [68]. The highest matching (% similarity) GenBank sequences for the various strains are provided as part of the supplementary materials (Table S1). The newly-generated sequences were combined with those from the study by Lemaire and co-workers [21], which sampled various legume species throughout the CCR to allow for a broader representation. The alignments were viewed in Bioedit Version 7.1.9 [69], and equivocally aligned fragments were adjusted manually. Phylogenetic analyses of the aligned matrices were performed on the Cyberinfrastructure for Phylogenetic Research (CIPRES) web portal (https://www.phylo.org), through a maximum likelihood (ML) approach, using RaxML Version 8.2.10 [70] and Bayesian inference (BI), as implemented in MrBayes Version 3.2.6 [71]. The ML analyses employed the General Time Reversible with categorized rates (GTRCAT) substitution model, and statistical support on nodes was evaluated using the non-parametric rapid bootstrapping technique [72], with 1000 replicates. For the BI analysis, the best model of nucleotide substitution was determined using jModelTest2 Version 2.1.6 [73], employing the Bayesian Information Criterion (BIC). The BI analyses were run for as many generations as necessary to achieve chain convergence (5–10 million generations). A conservative burn-in of 25% was applied to all BI analyses, and convergence of the chains was assessed using Tracer Version 1.6 [74]. To determine the combinability of the different DNA data partitions, the approach used by Pirie and co-workers [75,76] was employed. The DNA sequence data for the different genes were first analyzed separately by ML techniques as described above, and the resulting tree topologies were examined for conflicting nodes with ≥70% bootstrap support. Nodes that had <70% bootstrap support were considered unsupported, and thus, when no supported conflict was observed, the partitions were considered combinable. This approach was chosen over the widely used incongruence length difference (ILD) test [77] because the ILD only tests for overall incongruence between partitions without detecting local conflict that is due to specific taxa or clades [76]. There was no conflict observed between 16S rRNA and recA, and therefore, these partitions were combined in subsequent analyses. Genes 2018, 9, 2 5 of 23

However, the nodA partition had significantly supported conflict with both chromosomal markers, and therefore, it was analyzed separately. For the study of the diversity of rhizobia associated with I. superba, root nodules were sampled from six populations of the species across its distribution range in Vogelgat Private Nature Reserve (Hermanus, Western Cape, South Africa), sampling multiple (at least five) individuals per population to capture any potential genetic variation within and between populations. Root nodules from other legumes (i.e., Aspalathus carnosa Eckl. & Zeyh., Indigofera candolleana Meisn., P. pullata C.H. Stirt. and Psoralea restioides Eckl. & Zeyh.) that occur in the same locality as I. superba were also sampled to determine phylogenetic relationships between their symbionts. One chromosomal gene (recA) and one nodulation gene (N-acetylglucosaminyltransferase (nodC)) were sequenced for this study. Additional sequences from previous studies [53,78] on CCR legumes were incorporated into the dataset to determine the phylogenetic position of I. superba strains relative to strains nodulating other legumes in the CCR. Some sequences for reference strains, downloaded from GenBank were also included (Table S1).

2.4. Determination of Phylogenetic Signals Analyses of phylogenetic signals for the various ecological parameters were conducted in R [79] using the phylogenetic trees constructed above as input and the corresponding parameters’ data as described below. Data for soil types of the sampling sites were extracted from a geological map of the CCR (shapefiles were kindly provided by the Geology Department, University of Cape Town, Western Cape, South Africa) using the site Global Positioning System (GPS) information collected during fieldwork. Soil type was coded as a binary character for each of the four soil types from which the legumes had been sampled (granite, limestone, sandstone and shale), as follows: 1, when the site belonged to a particular soil type, and 0, if it did not (Table S2). Phylogenetic structuring of rhizobial strains by soil type was tested using the D statistic, which measures phylogenetic signal for a discrete binary trait [80]. This was implemented using the ‘phylo.d’ function of the ‘Caper’ package, which calculates the value of D and tests for its significant departure from a random association and the clumping expected under a Brownian motion model [81]. The statistic D = 0 denotes a phylogenetically-conserved trait under a Brownian model, while D = 1 indicates a random distribution of traits on the tips of the phylogeny, and D < 0 indicates a strong phylogenetic signal, while D > 1 points toward phylogenetic overdispersion [80]. Significance testing was conducted using 10,000 permutations. Altitude data for the sampled sites were recorded during field surveys using a GPS, and the soil pH was determined using the methods described by Dludlu and co-workers [57]. The raw data for altitude and pH are provided as part of the supplementary materials (Table S3). Pagel’s λ [82] was used to test for the presence of phylogenetic signal for these two continuously varying parameters. This metric ranges from 0–1, where 0 indicates that the trait evolves independently of the phylogeny and 1 indicates that the trait evolves according to the shared evolutionary history of the phylogeny’s tips, i.e., presence of phylogenetic signal [83]. The metric has proven to be robust to incomplete phylogenetic information and the presence of polytomies in the phylogenetic tree [84,85], making it suitable for the present study. The analyses were conducted using the ‘phylosig’ function of the ‘phytools’ package [86], employing 10,000 simulations for significance testing.

3. Results

3.1. Strain Identification and Phylogenetic Analyses All strains isolated as part of this study were identified to the genus level, based on BLAST [68] search results of individual sequences, as belonging to either Burkholderia or Mesorhizobium. All Burkholderia strains had at least 97% similarity to known South African strains, while the Mesorhizobium strains were similar to rhizobial strains from various parts of the world. Strains isolated Genes 2018, 9, 2 6 of 23 from the legume genera Aspalathus L. (except for Aspalathus callosa, Aspalathus capensis and A. carnosa), Argyrolobium Eckl. & Zeyh., Otholobium C.H. Stirt. and Psoralea L. were identified as Mesorhizobium. Burkholderia strains were from Amphithalea Eckl. & Zeyh., Aspalathus L., Bolusafra Kuntze., Dipogon Liebm., Indigofera L., Lebeckia Thunb., Podalyria Willd., Rafnia Thunb. and Virgilia Poir. Phylogenetic analyses were conducted separately for each of the two genera to allow for independent analyses of phylogenetic signals within each genus. The aligned 16S rRNA matrix of Burkholderia consisted of 67 strains and 1540 characters (total aligned length), while that of Mesorhizobium had 73 strains and 1520 characters. The recA matrix for Burkholderia had 67 strains and 951 characters, while that of Mesorhizobium had 67 strains and 886 characters. The Bayesian and ML analyses of the individual chromosomal gene regions produced trees of similar topologies, and in all cases, the recA tree was better resolved than that of the 16S rRNA. The trees from the concatenated matrices were better resolved and more strongly supported (Figures1 and2) than the individual gene trees. The aligned nodA matrix for Burkholderia had 74 rhizobial strains and 734 characters, while that of Mesorhizobium had 41 strains and 674 characters. The Bayesian and ML trees had similar topologies and were well supported (Figures3 and4). However, for both Burkholderia and Mesorhizobium, the nodA topologies were incongruent to those of the chromosomal gene trees, suggesting disparate evolutionary histories between the chromosomal and nodulation genes. Genes 2018, 9, 2 7 of 23 Genes 2018, 9, 2 7 of 22

FigureFigure 1. PhylogeneticPhylogenetic relationships relationships of of BurkholderiaBurkholderia strainsstrains based based on on16S 16S ribosomic ribosomic RNA RNA (rRNA) (rRNA) and andrecombinase recombinase A (recA A (recA) data.) data. Names Names of ofthe the legume legume hosts hosts and and the the rhizobial rhizobial strain strain numbers numbers are inin parentheses.parentheses. StrainStrain numbersnumbers with with the the prefixes prefixes OD- OD (i.e.,- (i.e., collector collector name: name: Oscar Oscar Dlodlo) Dlodlo) and MM-and MM- (i.e., collector(i.e., collector name: name: Muthama Muthama Muasya) Muasya) are are from from the the study study by by Lemaire Lemaire and and co-workers co-workers [21 [21].]. AllAll otherother strainsstrains werewere newlynewly generatedgenerated inin thisthis study.study. MaximumMaximum likelihoodlikelihood (ML)(ML) bootstrapbootstrap (%)(%) andand BayesianBayesian InferenceInference (BI)(BI) posteriorposterior probabilitiesprobabilities areare shownshown aboveabove andand belowbelow nodes,nodes, respectively.respectively. ColoredColored circlescircles indicateindicate thethe soil soil types types of of the the sites sites where where the legumesthe legumes and theirand symbiontstheir symbionts were collected, were collected, blue: granite, blue: pink:granite, sandstone, pink: sandst yellow:one, shale.yellow: shale.

Genes 2018, 9, 2 8 of 23 Genes 2018, 9, 2 8 of 22

Figure 2. Phylogenetic relationships of Mesorhizobium strains based on 16S rRNA and recA data. Figure 2. Phylogenetic relationships of Mesorhizobium strains based on 16S rRNA and recA data. Names ofNames the legume of the legume hosts and hosts rhizobial and rhizobial strain numbers strain numb are iners parentheses. are in parentheses. Strain numbers Strain numbers with the with prefixes the OD-prefixes and OD- MM- and are MM- from theare studyfrom the by Lemairestudy by and Lema co-workersire and co-workers [21]. The rest[21]. were The newlyrest were generated newly ingenerated this study. in this ML study. bootstrap ML bootstrap (%) and BI(%) posterior and BI posterior probabilities probabilities are shown are aboveshown and above below and nodes,below respectively.nodes, respectively. Colored Colored circles indicate circles indicate the soil typesthe soil of thetypes sites of wherethe sites the where legumes the and legumes their symbionts and their weresymbionts collected, were blue: collected, granite; blue: green: granite; limestone; green: pink:limestone; sandstone, pink: sandstone, yellow: shale. yellow: shale.

Genes 2018, 9, 2 9 of 23 Genes 2018, 9, 2 9 of 22

Figure 3. Phylogenetic relationships of Burkholderia strains based on N-acyltransferase (nodA) data. Figure 3. Phylogenetic relationships of Burkholderia strains based on N-acyltransferase (nodA) data. Names of the legume hosts and rhizobial strain numbers are in parentheses. Strain numbers with the Names of the legume hosts and rhizobial strain numbers are in parentheses. Strain numbers with prefixes OD- and MM- are from the study by Lemaire and co-workers [21]. The rest were newly the prefixes OD- and MM- are from the study by Lemaire and co-workers [21]. The rest were newly generated in this study. ML bootstrap (%) and BI posterior probabilities are shown above and below generated in this study. ML bootstrap (%) and BI posterior probabilities are shown above and below nodes, respectively. Colored circles indicate the soil types of the sites where the legumes and their nodes, respectively. Colored circles indicate the soil types of the sites where the legumes and their symbionts were collected, blue: granite; pink: sandstone, yellow: shale. symbionts were collected, blue: granite; pink: sandstone, yellow: shale.

Genes 2018, 9, 2 10 of 23 Genes 2018, 9, 2 10 of 22

Figure 4. Phylogenetic relationships of Mesorhizobium strains based on nodA data. Names of the Figure 4. Phylogenetic relationships of Mesorhizobium strains based on nodA data. Names of the legume hosts and rhizobial strain numbers are in parentheses. Strain numbers with the prefixes OD- legume hosts and rhizobial strain numbers are in parentheses. Strain numbers with the prefixes OD- and MM- are from the study by Lemaire and co-workers [21]. The rest were newly generated in this and MM- are from the study by Lemaire and co-workers [21]. The rest were newly generated in this study. ML bootstrap (%) and BI posterior probabilities are shown above and below nodes, study. ML bootstrap (%) and BI posterior probabilities are shown above and below nodes, respectively. respectively. Colored circles indicate the soil types of the sites where the legumes and their Colored circles indicate the soil types of the sites where the legumes and their symbionts were collected, symbionts were collected, blue: granite; green: limestone; pink: sandstone, yellow: shale. blue: granite; green: limestone; pink: sandstone, yellow: shale.

Genes 2018, 9, 2 11 of 23

3.2. Analyses of Phylogenetic Signals For Burkholderia, 72% of the strains occurred on sandstone, 24% on granite, 4% on shale and none were on limestone-derived soils, whereas for Mesorhizobium, 54% of the strains were on sandstone, 20% on granite, 17% on shale and 9% on limestone-derived soils (Table S2). From the chromosomal gene tree, a comparison of the phylogenetic D statistic with the random shuffling of parameter values along the tips of the phylogeny showed a significant phylogenetic signal for sandstone (D = 0.133; p = 0.00) and a strong phylogenetic signal for granite (D = −0.22; p = 0.00) for Burkholderia. Mesorhizobium had significant phylogenetic signals for sandstone (D = 0.433; p = 0.0009) and granite (D = 0.252; p = 0.0006) and a strong phylogenetic signal for limestone-derived (D = −0.359; p = 0.0006) soils (Table1). On the other hand, when the D statistic was compared to the Brownian threshold model, all but the Mesorhizobium on shale-derived soils were as clumped on the phylogeny as expected under a Brownian motion model (Table1).

Table 1. Results of the tests of phylogenetic signals on soil types using the D statistic on the combined chromosomal gene (16S ribosomic RNA (rRNA) and recombinase A (recA)), and the N-acyltransferase (nodA) trees of Burkholderia and Mesorhizobium.

p-Value p-Value DNA Region Genus Soil Type D Random Shuffle Brownian Motion Granite −0.220 0.00 0.717 Burkholderia Sandstone 0.133 0.00 0.384 16S rRNA Shale 0.956 0.375 0.144 and recA Granite 0.252 0.0006 0.235 Limestone −0.359 0.0006 0.744 Mesorhizobium Sandstone 0.133 0.0009 0.056 Shale 0.975 0.419 0.001 Granite −0.208 0.00 0.715 Burkholderia Sandstone 0.082 0.00 0.424 Shale 0.617 0.160 0.291 nodA Granite 1.752 0.833 0.032 Limestone −0.871 0.0005 0.914 Mesorhizobium Sandstone 0.162 0.002 0.423 shale 0.492 0.118 0.218

For the nodA phylogeny, Burkholderia showed similar patterns of phylogenetic signal as observed for the chromosomal genes (Table1). Despite having similar patterns to those of the chromosomal genes for sandstone and limestone-derived soils, nodA results for Mesorhizobium showed evidence of random dispersion of parameter values for granite-derived soils (D = 1.752; p = 0.833), with a significant departure (p = 0.032) from a Brownian threshold model (Table1), indicating a lack of phylogenetic structure for this parameter. Analyses based on the chromosomal gene trees showed significant phylogenetic signals for pH for both Burkholderia (Pagel’s λ = 0.642; p = 0.019) and Mesorhizobium (Pagel’s λ = 0.508; p = 0.027). On the other hand, altitude showed no significant phylogenetic signal on either Burkholderia (Pagel’s λ = 0.402; p = 0.081) or Mesorhizobium (Pagel’s λ = 0.217; p = 0.097). Similar patterns were observed for analyses based on the nodA trees of both rhizobial genera (Table2). Genes 2018, 9, 2 12 of 23

Table 2. Results of the tests of phylogenetic signals for altitude and pH using Pagel’s λ on the chromosomal (16S rRNA and recA) and nodA trees of Burkholderia and Mesorhizobium.

Genes 2018, 9, 2 12 of 22 Genus Gene Type Variable Pagel’s λ p-Values Table 2. Results of the tests of phylogeneticAltitude signals for altitude0.402 and pH using Pagel’s0.081 λ on the Chromosomal chromosomal (16S rRNA and recA) and nodA pHtrees of Burkholderia and0.643 Mesorhizobium. 0.019 Burkholderia Genus Gene TypeAltitude Variable0.093 Pagel’s λ 0.389p-Values nodA Altitude 0.402 0.081 Chromosomal pH 0.840 0.0008 pH 0.643 0.019 Burkholderia Altitude 0.217 0.097 Chromosomal Altitude 0.093 0.389 nodA pH 0.508 0.027 Mesorhizobium pH 0.840 0.0008 Altitude Altitude 0.767 0.217 0.9990.097 nodA Chromosomal pH pH 0.912 0.508 0.0160.027 Mesorhizobium Altitude 0.767 0.999 nodA 3.3. Diversity of Rhizobial Symbionts of Indigofera superba pH 0.912 0.016

In total,3.3. 87 andDiversity 86 strains of Rhizobial were Symbionts isolated of and Indigofera successfully superba sequenced for recA and nodC, respectively, from I. superba andIn itstotal, sympatric 87 and legumes86 strains inwere Vogelgat isolated Private and successfully Nature Reserve sequenced (South for Africa). recA and All nodC strains, that were isolatedrespectively, from from the rootI. superba nodules and its of sympatricI. superba legumeswere identified in Vogelgat (based Private on Nature BLASTn Reserve searches (South on GenBank) asAfrica).Burkholderia All strains, and that the were highest isolated matches from (the≥ root95% nodules similarity) of I. weresuperba known were identifiedBurkholderia (basedspecies on from South Africa,BLASTn i.e.,searchesBurkholderia on GenBank) dilworthii as Burkholderia, Burkholderia, and the kirstenboschensis highest matches, (Burkholderia ≥ 95% similarity) rhynchosiae were , Burkholderia sprentiaeknown Burkholderiaand Burkholderia species tuberum from . TheSouth highest Africa, matching i.e., Burkholderia (% similarity) dilworthii GenBank, Burkholderia sequences kirstenboschensis, Burkholderia rhynchosiae, Burkholderia sprentiae and Burkholderia tuberum. The highest for the various strains are provided as part of the supplementary materials (Table S1). On the other matching (% similarity) GenBank sequences for the various strains are provided as part of the hand, all strainssupplementary isolated from materialsP. pullata (Table, which S1). On occurs the other in sympatryhand, all stra withins I.isolated superba from, were P. pullataMesorhizobium, which . The BI and MLoccurs analyses in sympatry of the withrecA I. superbaand nodC, werematrices Mesorhizobium produced. The BI some and ML fairly analyses resolved of the andrecA and supported nodC topologies (Figuresmatrices5 producedand6). Indigofera some fairly superba resolvedsymbionts and supported were parttopologies of multiple (Figures distinct 5 and clades,6). Indigofera most of which includedsuperba strains symbionts isolated were from part of other multiple legume distinct species clades, that most occur of which outside included its distributionstrains isolated range from in the CCR in bothother thelegumerecA speciesand the thatnodC occurtrees outside (Figures its distribution5 and6). range in the CCR in both the recA and the nodC trees (Figures 5 and 6).

A)

Figure 5. Cont.

Genes 2018, 9, 2 13 of 23 Genes 2018, 9, 2 13 of 22

Burkholderia sp. (Indigofera superba 328N2) 82 Burkholderia sp. (Indigofera superba 328N6) Burkholderia sp. (Indigofera superba 328N8) 63 0.97 KM188317.1 Burkholderia sp. BL27 ind4 R3 0.72 KM188320.1 Burkholderia sp. BL29 ind6 R2 KM188297.1 Burkholderia sp BL21 ind5 R3 0.72 98 Burkholderia sp. (Aspalathus carnosa 334N8) B) Burkholderia sp. (Aspalathus carnosa 334N9) 1.00 Burkholderia sp. (Indigofera superba 326N16) Burkholderia sp. (Indigofera superba 325N1) Burkholderia sp. (Indigofera superba 325N2) Burkholderia sp. (Indigofera superba 325N3) 0.59 Burkholderia sp. (Indigofera superba 325N4) Burkholderia sp. (Indigofera superba 325N5) Burkholderia sp. (Indigofera superba 325N6) Burkholderia sp. (Indigofera superba 325N7) 0.55 KM188266.1 Burkholderia sp. BL11 ind3 R1 0.80 KM188280.1 Burkholderia sp. BL17 R1 61 Burkholderia sp. (Indigofera superba 326N17) 0.83 KM188301.1 Burkholderia sp. BL25 ind1 R1 0.70 0.84 KM188288.1 Burkholderia sp. BL18 ind8 R3 64 KM188324.1 Burkholderia sp. MM6665C I2 R1 99 Burkholderia sp. (Indigofera candolleana 330N1) 0.90 Burkholderia sp. (Indigofera candolleana 330N8) 1.00 Burkholderia sp. (Indigofera candolleana 330N9) 100 KM188260.1 Burkholderia sp. BL9 ind2 R2 1.00 62 KM188307.1 Burkholderia sp. BL25 ind3 R6 0.85 50 KM188283.1 Burkholderia sp. BL18 ind3 R2 81 0.90 KM188286.1 Burkholderia sp. BL18 ind8 R1 83 0.69 HQ849152.1 Burkholderia phytofirmans LMG 22487 1.00 1.00 JX009159.2 Burkholderia dipogonis DL7 94 100 KM188274.1 Burkholderia sp. BL15 R3 1.00 KM188278.1 Burkholderia sp. BL16 ind3 1.00 KM188298.1 Burkholderia sp. BL23 ind1 R1 0.74 AY619664.1 Burkholderia fungorum LMG 16225 Burkholderia sp. (Indigofera superba 328N1) Burkholderia sp. (Indigofera superba 328N4) Burkholderia sp. (Indigofera superba 328N7) 91 Burkholderia sp. (Indigofera superba 333N12) 0.77 Burkholderia sp. (Indigofera superba 333N20) Burkholderia sp. (Indigofera superba 333N23) 0.72 Burkholderia sp. (Indigofera superba 333N9) KM188313.1 Burkholderia sp. BL27 ind3 R6 0.65 Burkholderia sp. (Indigofera superba 333N13) 55 0.76 Burkholderia sp. (Indigofera superba 333N16) Burkholderia sp. (Indigofera superba 328N3) 100 0.78 96 Burkholderia sp. (Indigofera superba 333N10) Burkholderia sp. (Indigofera superba 333N14) 1.00 0.81 Burkholderia sp. (Indigofera superba 333N25) 53 91 Burkholderia sp. (Indigofera superba 333N8) 77 AY619669.1 Burkholderia caledonica LMG 19076 0.89 1.00 HF544404.1 Burkholderia kirstenboschensis 65 0.98 HE994060.1 Burkholderia dilworthii WSM3556 HE994063.1 Burkholderia rhynchosiae WSM3930 73 0.99 HQ398589.1 Burkholderia phenoliruptrix AC1100 Burkholderia sp. (Indigofera superba 325NX) 1.00 Burkholderia sp. (Indigofera superba 326N10) Burkholderia sp. (Indigofera superba 326N13) Burkholderia sp. (Indigofera superba 326N2) Burkholderia sp. (Indigofera superba 326N3) Burkholderia sp. (Indigofera superba 326N4) 97 Burkholderia sp. (Indigofera superba 326N7) Burkholderia sp. (Indigofera superba 326N9) 1.00 Burkholderia sp. (Indigofera superba 333N15) Burkholderia sp. (Indigofera superba 333N22) Burkholderia sp. (Indigofera superba 333N24) Burkholderia sp. (Indigofera superba 333N4) Burkholderia sp. (Indigofera superba 333N5) Burkholderia sp. (Indigofera superba 333N6) KM188299.1 Burkholderia sp. BL23 ind2 R2 55 KM188261.1 Burkholderia sp. BL10 ind2 R1 0.66 0.62 KM188293.1 Burkholderia sp. BL21 ind4 R1 0.99 HQ849164.1 Burkholderia xenovorans LMG 21463 HQ398579.1 Burkholderia ginsengisoli LMG 24044 KM188306.1 Burkholderia sp. BL25 ind3 R1 Burkholderia sp. (Indigofera candolleana 330N6) Burkholderia sp. (Indigofera candolleana 330N7) Burkholderia sp. (Indigofera superba 326N1) 88 Burkholderia sp. (Indigofera superba 326N11) Burkholderia sp. (Indigofera superba 326N12) 1.00 Burkholderia sp. (Indigofera superba 326N5) Burkholderia sp. (Indigofera superba 326N6) 74 Burkholderia sp. (Indigofera superba 326N8) Burkholderia sp. (Aspalathus carnosa 334N7) 1.00 Burkholderia sp. (Indigofera candolleana 330N4) Burkholderia sp. (Indigofera candolleana 330N5) 61 Burkholderia sp. (Indigofera superba 326N14) Burkholderia sp. (Indigofera superba 326N15) 0.71 Burkholderia sp. (Indigofera superba 326N18) Burkholderia sp. (Indigofera superba 333N1) Burkholderia sp. (Indigofera superba 333N11) Burkholderia sp. (Indigofera superba 333N18) Burkholderia sp. (Indigofera superba 333N2) 57 Burkholderia sp. (Indigofera superba 333N3) 88 Burkholderia sp. (Indigofera candolleana 330N2) 0.97 Burkholderia sp. (Indigofera superba 333N19) 0.70 Burkholderia sp. (Indigofera superba 333N27) KF791820.1 Burkholderia sp. MM5819 68 89 AY619674.1 Burkholderia tuberum LMG 21444 Burkholderia sp. (Aspalathus carnosa 334N6) 0.87 1.00 Burkholderia sp. (Indigofera superba 333N26) Burkholderia sp. (Aspalathus carnosa 334N1) 64 Burkholderia sp. (Aspalathus carnosa 334N2) Burkholderia sp. (Aspalathus carnosa 334N4) 0.99 Burkholderia sp. (Aspalathus carnosa 334N5) KM188279.1 Burkholderia sp. BL16 ind4 R2 Burkholderia sp. (Aspalathus carnosa 334N3) 1.00 Burkholderia sp. (Indigofera candolleana 330N3) 85 85 Burkholderia sp. (Indigofera superba 328N5) Burkholderia sp. (Indigofera superba 333N17) 1.00 88 0.55 Burkholderia sp. (Indigofera superba 333N21) Burkholderia sp. (Indigofera superba 333N7) 1.00 KM188270.1 Burkholderia sp. BL13 R2 100 HE994077.1 Burkholderia sprentiae WSM5005 69 AY619667.1 Burkholderia phymatum STM815 1.00 99 0.94 EU294397.1 Burkholderia sp. Br3407 AY619662.1 Burkholderia caribensis LMG 18531 99 1.00 FN543898.1 Burkholderia diazotrophica FN543850.1 Burkholderia sp. JPY345 95 1.00 95 DQ514539.1 Burkholderia unamae MTI 641 1.00 EU294398.1 Burkholderia sp. Br3437 1.00 HM598380.1 Burkholderia cepacia ATCC 25416 HE687279.1 Cupriavidus taiwanensis LMG19425 KF802753.1 Mesorhizobium sp. MM5397 100 KF802758.1 Mesorhizobium sp. OD13 0.98 KF802766.1 Mesorhizobium sp. MM5440 KF802783.1 Mesorhizobium sp. OD15 0.78 99 KF802749.1 Mesorhizobium sp. OD14 0.98 KF802752.1 Mesorhizobium sp. OD18 0.71 100 KF802782.1 Mesorhizobium sp. MM5675 1.00 100 KF802787.1 Mesorhizobium sp. OD119 57 KF802754.1 Mesorhizobium sp. MM5618 0.70 1.00 KF802776.1 Mesorhizobium sp. MM5334 0.71 KF802765.1 Mesorhizobium sp. MM5398 0.65 98 Mesorhizobium sp. (Psoralea pullata 327N11) 73 KF802756.1 Mesorhizobium sp. MM5361 50 1.00 Mesorhizobium australicum WSM2073 0.97 0.92 73 KF802755.1 Mesorhizobium sp. OD108 KF802759.1 Mesorhizobium sp. OD31 95 0.62 KF802775.1 Mesorhizobium sp. OD32 1.00 76 KF802778.1 Mesorhizobium sp. MM5369 100 0.69 KF802781.1 Mesorhizobium sp. MM5357 KF802791.1 Mesorhizobium sp. MM5343 1.00 85 Mesorhizobium sp. (Psoralea pullata 327N10) Mesorhizobium sp. (Psoralea pullata 327N4) 0.98 Mesorhizobium sp. (Psoralea pullata 327N7) 92 Mesorhizobium sp. (Psoralea pullata 327N8) Mesorhizobium sp. (Psoralea pullata 327N3) 1.00 Mesorhizobium sp. (Psoralea pullata 327N5) 0.68 Mesorhizobium sp. (Psoralea pullata 327N6) Mesorhizobium shangrilense CCBAU 65327 64 KR154529.1_Mesorhizobium_sp._MM5382 0.97 Mesorhizobium erdmanii USDA 3471 0.59 Mesorhizobium opportunistum WSM2075 0.84 76 Mesorhizobium ciceri CCANP20 0.77 0.91 52 Mesorhizobium loti CCBAU 01461 Mesorhizobium abyssinicae AC98c 0.98 51 0.74 Mesorhizobium tamadayense CCANP122 99 KF802785.1 Mesorhizobium sp. MM5462 0.99 KF802750.1 Mesorhizobium sp. OD48 1.00 KF802751.1 Mesorhizobium sp. OD47 61 Mesorhizobium mediterraneum USDA 3392 0.74 Mesorhizobium robiniae CCNWYC 115 1.00 54 Mesorhizobium alhagi MQ15 100 0.76 89 Mesorhizobium huakuii CCBAU 65318 1.00 KF802774.1 Mesorhizobium sp. OD42 0.99 Mesorhizobium albiziae CCBAU 61158 KF802763.1_Rhizobium.sp. OD49 FJ652641.1 Pseudomonas aeruginosa PAK 2.0 Figure 5. (A) Phylogenetic relationships of rhizobial strains based on recA data, showing the Figure 5. (Aphylogenetic) Phylogenetic position relationships of strains isolated of rhizobial from Indigofera strains superba based (red on nodes)recA indata, relation showing to rhizobial the phylogenetic positionstrains of other of strains legumes isolated in the CCR, from blue:Indigofera Mesorhizobium superba strains;(red black: nodes) Burkholderia in relation, green: to outgroup. rhizobial strains of other(B legumes) Detailed inversion the CCR, of Figure blue: 5A),Mesorhizobium showing the strains;tip labels black: and MLBurkholderia Bootstrap support, green: values outgroup. (%) (B) Detailed versionabove the of nodes Figure and5A), Bayesian showing posterio the tipr probabilities, labels and below ML Bootstrap the branches. support values (%) above the nodes and Bayesian posterior probabilities, below the branches.

Genes 2018, 9, 2 14 of 23 Genes 2018, 9, 2 14 of 22

A)

Figure 6. Cont.

Genes 2018, 9, 2 15 of 23 Genes 2018, 9, 2 15 of 22

Burkholderia sp. (Indigofera superba 333N12) 100 Burkholderia sp. (Indigofera superba 333N8) 1.00 Burkholderia sp. (Indigofera superba 333N9) KR154722.1 Burkholderia sp. BL27 I3R6 94 Burkholderia sp. (Indigofera superba 333N10) B) Burkholderia sp. (Indigofera superba 333N14) 100 0.96 Burkholderia sp. (Indigofera superba 333N25) 1.00 89 Burkholderia sp. (Indigofera_superba_333N13) 0.97 Burkholderia sp. (Indigofera superba 333N23) Burkholderia sp. (Indigofera superba 333N16) 73 Burkholderia sp. (Indigofera superba 333N20) 99 KT390810.1 Burkholderia unamae BRUESC684 0.96 0.79 KT390835.1 Burkholderia nodosa BRUESC913 100 1.00 99 1.00 KT390830.1 Burkholderia tropica BRUESC901 100 1.00 EU386155.1 Burkholderia mimosarum PAS44 100 KT390820.1 Burkholderia sartisoli BRUESC853 1.00 100 DQ888213.1 Burkholderia phymatum STM815 1.00 KT390815.1 Burkholderia diazotrophica BRUESC845 1.00 KT390825.1 Burkholderia sabiae BRUESC861 0.74 97 KR154716.1 Burkholderia sp BL21 I5R3 60 0.96 KR154721.1 Burkholderia sp BL25 I3R6 0.71 KR154726 1 Burkholderia sp BL9 I2R2 KR154724.1 Burkholderia sp. BL29 I6R2 100 Burkholderia sp. (Indigofera superba 326N13) 1.00 Burkholderia sp. (Indigofera superba 326N2) Burkholderia sp. (Indigofera superba 326N9) Burkholderia sp. (Aspalathus carnosa 334N8) Burkholderia sp. (Aspalathus carnosa 334N9) Burkholderia sp. (Indigofera superba 325N3) Burkholderia sp. (Indigofera superba 325N4) 0.92 82 Burkholderia sp. (Indigofera superba 325N5) 0.95 Burkholderia sp. (Indigofera superba 325N6) Burkholderia sp. (Indigofera superba 325N7) Burkholderia sp. (Indigofera superba 326N16) 91 Burkholderia sp. (Indigofera superba 326N17) Burkholderia sp. (Indigofera superba 326N7) 1.00 Burkholderia sp. (Indigofera superba 326N8) Burkholderia sp. (Indigofera superba 325NX) 63 KR154706.1 Burkholderia sp. BL11 I3R1 KR154712.1 Burkholderia sp. BL18 I3R2 0.94 91 KR154714.1 Burkholderia sp. BL18 I8R3 93 0.96 KR154719.1 Burkholderia sp. BL25 I1R1 86 1.00 KR154725.1 Burkholderia sp. BL6665C I2R2 1.00 KR154708.1 Burkholderia sp. BL15 R3 KR154720.1 Burkholderia sp. BL25 I3R1 Burkholderia sp. (Aspalathus carnosa 334N1) 82 Burkholderia sp. (Aspalathus carnosa 334N5) 0.94 Burkholderia sp. (Aspalathus carnosa 334N6) Burkholderia sp. (Indigofera superba 333N26) Burkholderia sp. (Aspalathus carnosa 334N2) Burkholderia sp. (Aspalathus carnosa 334N3) Burkholderia sp. (Aspalathus carnosa 334N4) Burkholderia sp. (Aspalathus carnosa 334N7) EF566977.1 Burkholderia tuberum DUS833 Burkholderia sp. (Indigofera superba 326N1) Burkholderia sp. (Indigofera superba 326N11) Burkholderia sp. (Indigofera superba 326N12) Burkholderia sp. (Indigofera superba 326N14) Burkholderia sp. (Indigofera superba 326N15) Burkholderia sp. (Indigofera superba 326N18) 100 Burkholderia sp. (Indigofera superba 326N3) 1.00 Burkholderia sp. (Indigofera superba 326N5) Burkholderia sp. (Indigofera superba 326N6) Burkholderia sp. (Indigofera superba 333N1) Burkholderia sp. (Indigofera superba 333N11) Burkholderia sp. (Indigofera superba 333N17) 76 Burkholderia sp. (Indigofera superba 333N18) Burkholderia sp. (Indigofera superba 333N19) 0.99 Burkholderia sp. (Indigofera superba 333N2) 0.92 Burkholderia sp. (Indigofera superba 333N21) Burkholderia sp. (Indigofera superba 333N27) Burkholderia sp. (Indigofera superba 333N3) Burkholderia sp. (Indigofera superba 333N7) KR154710.1 Burkholderia sp. BL16 I4R2 HG934336.1 Burkholderia dilworthii WSM4206 84 KR154713.1 Burkholderia sp. BL18 I8R1 93 0.98 KR154723.1 Burkholderia sp. BL27 I4R3 KR154709.1_Burkholderia sp. BL16 R3 0.61 86 0.93 KR154715.1 Burkholderia sp. BL21 4R1 1.00 KR154717.1 Burkholderia sp. BL23 I1R1 100 JX009155.2 Burkholderia dipogonis DL12 1001.00 KR154711.1 Burkholderia sp. BL17 R1 99 HG425183.1 Burkholderia rhynchosiae WSM3930 99 1.00 HG425199.1 Burkholderia sprentiae WSM5005 1.00 KR154707.1 Burkholderia sp. BL13 R2 1.00 Burkholderia sp. (Indigofera superba 326N4) KR154705.1 Burkholderia sp. BL10 I2R1 Burkholderia sp. (Indigofera superba 326N10) Burkholderia sp. (Indigofera superba 333N22) 61 Burkholderia sp. (Indigofera superba 333N24) 100 0.86 Indigofera_superba_333N4 Indigofera_superba_333N5 1.00 KR154718.1 Burkholderia sp. BL23 I2R2 93 Burkholderia sp. (Indigofera superba 333N15) 62 Mesorhizobium sp. (Psoralea pullata 327N10) 1.00 Mesorhizobium sp. (Psoralea pullata 327N11) 0.96 KR154636.1 Mesorhizobium sp. OD119 79 Mesorhizobium sp. (Psoralea pullata 327N3) 0.93 Mesorhizobium sp. (Psoralea pullata 327N5) 0.95 KR154637.1 Mesorhizobium sp. MM5343 0.97 KR154631.1 Mesorhizobium sp. MM5382 0.87 KR154632.1 Mesorhizobium sp. MM5357 KR154633.1 Mesorhizobium sp. MM5675 100 KR154623.1 Mesorhizobium sp. OD31 0.78 1.00 KR154629.1 Mesorhizobium sp. OD32 Mesorhizobium sp. (Psoralea restioides 329bN2) Mesorhizobium sp. (Psoralea pullata 327N6) 88 Mesorhizobium sp. (Psoralea restioides 329bN3) 0.93 Mesorhizobium sp. (Psoralea restioides 329bN4) 58 Mesorhizobium sp. (Psoralea restioides 329bN5) 0.94 Mesorhizobium sp. (Psoralea restioides 329bN6) 77 Mesorhizobium sp. (Psoralea pullata 327N16) 0.88 0.98 Mesorhizobium sp. (Psoralea pullata 327N17) Mesorhizobium sp. (Psoralea pullata 327N18) Mesorhizobium sp. (Psoralea restioides 329bN1) Mesorhizobium sp. (Psoralea pullata 327N2) Mesorhizobium sp. (Psoralea pullata 327N23) 75 Mesorhizobium sp. (Psoralea pullata 327N25) 0.86 Mesorhizobium sp. (Psoralea pullata 327N7) 56 Mesorhizobium sp. (Psoralea pullata 327N8) Mesorhizobium sp. (Psoralea pullata 327N1) 62 99 0.51 Mesorhizobium sp. (Psoralea pullata 327N19) 0.75 1.00 Mesorhizobium sp. (Psoralea pullata 327N22) 98 Mesorhizobium sp. (Psoralea pullata 327N21) 98 1.00 Mesorhizobium sp. (Psoralea pullata 327N24) 100 Mesorhizobium sp. (Psoralea pullata 327N20) 1.00 1.00 Mesorhizobium sp. (Psoralea pullata 327N4) 93 0.56 KR154635.1 Mesorhizobium sp. MM5462 100 1.00 Mesorhizobium sp. (Psoralea pullata 327N26) KR154630.1 Mesorhizobium sp. MM5334 1.00 KR154628.1 Mesorhizobium sp. OD42 55 KR154624.1 Rhizobium sp. OD49 72 KR154618.1 Mesorhizobium sp. MM5397 75 0.71 KR154619.1 Mesorhizobium sp. MM5618 100 0.99 KR154615.1 Mesorhizobium sp. MM5369 0.99 72 1.00 90 KR154626.1 Mesorhizobium sp. MM5440 0.96 0.80 KR154634.1 Mesorhizobium sp. OD15 100 74 KR154613.1 Mesorhizobium sp. OD14 1.00 80 KR154622.1 Mesorhizobium sp. OD13 0.98 KR154625.1 Mesorhizobium sp. MM5398 100 77 0.99 0.99 KR154617.1 Mesorhizobium sp. OD18 1.00 92 KR154620.1 Mesorhizobium sp. OD108 0.52 KR154614.1 Mesorhizobium sp. OD48 60 1.00 KR154616.1 Mesorhizobium sp. OD47 0.87 95 KR154621.1 Mesorhizobium sp. MM5361 1.00 KR154627.1 Mesorhizobium sp. MM5378 100 GQ847995.1 Mesorhizobium shonense AC39a 1.00 GQ848002.1 Mesorhizobium abyssinicae AC98c 0.68 KR514316.1 Mesorhizobium acaciae RITF1220 0.88 100 EU849563.1 Mesorhizobium robiniae CCNWYC 115 781.00 JF907686.1 Mesorhizobium amorphae CCNWGS0123 92 EF050784.1 Mesorhizobium gobiense CCBAU 83330 0.83 KP251787.1 Mesorhizobium septentrionale SDW014 53 1.00 78 0.80 KP251768.1 Mesorhizobium temperatum CCBAU 01821 100 1.00 EU130405.2 Mesorhizobium caraganae CCBAU 11299 1.00 JN129450.1 Mesorhizobium sangaii SCAU27 100 EU722487.1 Mesorhizobium alhagi CCNWXJ052 100 JX885708.1 Mesorhizobium ciceri CKr16 98 0.57 KU894874.1 Mesorhizobium opportunistum SAM96 0.68 1.00 99 91 1.00 KU894877.1 Mesorhizobium tamadayense SAM106 1.00 68 0.95 JQ339886.1 Mesorhizobium qingshengii CCBAU 33455 0.98 JX298870.1 Mesorhizobium camelthorni A24 GQ848005.1 Mesorhizobium hawassense AC99b KC237564.1 Mesorhizobium waitakense ICMP 14330 63 KC237575.1 Mesorhizobium kowhaii ICMP 19520 0.98 KC237580.1 Mesorhizobium calcicola ICMP 19562 100 KC237584.1 Mesorhizobium newzealandense ICMP 19547 100 1.00 KC237601.1 Mesorhizobium sophorae ICMP 19540 100 1.00 KC237573.1 Mesorhizobium cantuariense ICMP 19518 1.00 KC237562.1 Mesorhizobium waimense ICMP 19569 DQ311092.1 Mesorhizobium albiziae CCBAU 61158 100 EF549514.1 Mesorhizobium tarimense CCBAU 83321 1.00 KM192344.1 Mesorhizobium erdmanii USDA 3471 LN681559.1 Mesorhizobium olivaresii CGS22 2.0 Figure 6. (A) Phylogenetic relationships of rhizobial strains based on nodC data, showing the Figure 6. (A) Phylogenetic relationships of rhizobial strains based on nodC data, showing the phylogenetic position of strains isolated from I. superba (red nodes) in relation to rhizobial strains of phylogenetic position of strains isolated from I. superba (red nodes) in relation to rhizobial strains other legumes in the CCR, blue: Mesorhizobium strains; and black: Burkholderia strains. (B) Detailed of other legumes in the CCR, blue: Mesorhizobium strains; and black: Burkholderia strains. (B) Detailed version of Figure 6A), showing the tip labels and ML and Bootstrap support values (%) above version of Figure6A), showing the tip labels and ML and Bootstrap support values (%) above branches branches and Bayesian posterior probabilities below branches. and Bayesian posterior probabilities below branches.

Genes 2018, 9, 2 16 of 23

4. Discussion The main objective of this study was to determine if the three ecological parameters, altitude, pH and soil type, show phylogenetic structuring for the two predominant rhizobial genera, Burkholderia and Mesorhizobium [21], in the Core Cape Subregion of South Africa. For both genera, the results showed significant phylogenetic signals for soil type and pH, but not for altitude. Soil type can be viewed as an indicator of the nutrient status of the habitats based on the literature [87,88] and on the results of Dludlu and co-workers [57], which showed that sandstone habitats are the most nutrient-impoverished relative to the granite and shale substrates. Limestone soils are generally more fertile than the granite, sandstone and shale substrates [48,89]. Soil type is also related to pH, with the following general ranges, sandstone pH: 3–4.5, granite pH: 4.5–5.5, shale pH: 5.5–6.5 and limestone pH: > 6.5 [48,88,90]. Therefore, it is unsurprising that the results show similar patterns for pH and soil type. Consistent with previous studies [14,34], Burkholderia strains showed a preference for acidic soils, as indicated by the large proportion (72%) of its strains that were collected from the highly acidic sandstone habitats and its complete absence in the limestone habitats, which have alkaline conditions (Table S2). The findings of significant phylogenetic signals on the acidic sandstone and granite habitats indicate that in addition to the genus-wide preference for acidic conditions, Burkholderia strains are not randomly distributed within these soil types, but closely-related strains tend to occupy similar habitats with respect to soil type and pH. Thus, these ecological parameters have a significant influence on Burkholderia’s distribution within the CCR landscape. On the other hand, the results indicated that Mesorhizobium tolerates a wider range of soil types and pH conditions because it had nearly equal proportions of its strains isolated from the highly acidic and infertile sandstones and the higher pH and nutrient rich substrates (Table S2). The finding of significant phylogenetic signals for granite, limestone and sandstone and for pH indicates that despite the wider tolerance range of Mesorhizobium as a genus, the distribution of various strains is phylogenetically structured. Thus, for each of the different soil types and pH conditions of the CCR, there are particular strains of Mesorhizobium that are adapted to them. This is consistent with observations from other biomes showing that Mesorhizobium species exhibit high diversity in their tolerance to various pH conditions [27,37,91]. This could explain the predominance of Mesorhizobium (in addition to Burkholderia) in the CCR [21]. Overall, the results suggest that Mesorhizobium has a wider soil type and pH tolerance range than Burkholderia, and strains of both genera exhibit phylogenetic clustering within their distribution ranges. The finding of a significant phylogenetic signal for granite-derived soils (for Mesorhizobium) based on the chromosomal gene tree, versus a lack of phylogenetic signal for the same parameter on the nodA tree suggests that the chromosomal and nodulation genes have different evolutionary histories, possibly due to horizontal inheritance of the nodulation genes. This would be unsurprising as studies [20,78] show that horizontal gene transfer (HGT) is a common phenomenon among CCR rhizobia, leading to conflicting phylogenetic signals between chromosomal and nodulation genes. The observed variation in the biogeographical structuring of the different rhizobia with respect to soil type and pH has implications for the biogeography of legumes in the CCR. This is particularly the case considering that distinct edaphic habitats are characterized by discrete legume assemblages in the Cape Peninsula [57], which points to an important role of edaphic factors in driving legume biogeography. Considering that soil nutrients are a limiting factor to plants in the CCR [48], the ecological advantage that nitrogen fixation confers on legumes must be key to their success in such an environment. Therefore, if edaphic factors also limit the distribution of rhizobia, legumes that exhibit high rhizobial specificity, e.g., species of the tribe Podalyrieae, which are only nodulated by Burkholderia [21], might fail to establish in habitats that are unsuitable for their rhizobial symbionts [51,52]. In such a case, the biogeography of such legumes would also be driven by the distribution of their specific symbionts. This could explain the sparse representation of the tribe Podalyrieae in the limestone habitats (three out of 104 species), whereas most of its species occur in sandstone habitats (i.e., where Burkholderia are the predominant symbionts) in the CCR [92]. On the Genes 2018, 9, 2 17 of 23 contrary, promiscuous legume lineages, e.g., Aspalathus and Indigofera, or those that are nodulated by Mesorhizobium, e.g., Psoralea and Otholobium [21], are widespread in diverse soil types of the CCR [92]. These patterns suggest that rhizobia play a significant role in the distribution of legumes in the CCR. Furthermore, in a glasshouse experiment where legumes from the Fynbos and Grassland biomes were grown in soils from both biomes, Fynbos legumes were only able to nodulate in Fynbos soil (Lemaire and co-workers, unpublished [93]). This indicates a potential role of rhizobia specificity in driving the distribution of legumes in the various biomes of Southern Africa. This, therefore, opens up avenues for further research. For example, can rhizobia specificity explain why some Cape clades that occur outside the Fynbos are restricted to sandstone habitats? Furthermore, why are genistoid legumes that occur in the CCR nodulated by Mesorhizobium [21], whereas those of the Great Escarpment are nodulated by Bradyrhizobium?[23]. Although altitude is highly heterogeneous and it has been found to play a significant role in driving plant diversification in the CCR [47], the results of the present study showed no evidence of phylogenetic structuring of the two predominant rhizobial genera, for this ecological parameter. Similar results were obtained in the study conducted by Lemaire and co-workers [21] for both the chromosomal (16S rRNA) and nodA genes of Mesorhizobium. Likewise, the nodA genetic diversity in Burkholderia was not significantly correlated with altitude [21]. The only disparity is that they [21] found a positive correlation between altitude and genetic diversity of 16S rRNA for Burkholderia. The disparity is likely because the current study considered overall phylogenetic signals based on a combination of both 16S rRNA and recA, whereas the previous study only considered genetic distances of a single chromosomal marker: 16S rRNA. These findings are contrary to the observed biogeographic structuring of Burkholderia communities along altitudinal gradients in Brazil [14]. Such conflicting results have also been observed in other rhizobial genera, e.g., Sinorhizobium in Northern China, where the diversity of nodule isolates from different sites was correlated with altitude [42], versus central China, where they found no correlation between altitude and the genetic variation of rhizobial strains cultured from soils collected from sites of different altitudes [94]. Although the latter study was conducted under glasshouse conditions while the former was based on field nodules, the soils used for the trapping experiments were from sites of different altitudes [94], which validates comparing the two studies. Considering that the glasshouse trapping experiments were conducted under the same conditions for all the different soils, changes in rhizobial diversity (if any) as a result of the glasshouse conditions would have to be homogeneous across the samples. However, the sampling of the present study and that of Lemaire and co-workers [21] spanned an altitude range of 10–1000 m above sea level, whereas the highest altitude in the CCR is 2249 m [47]. Thus, the current data may not be sufficient to allow for conclusive inferences on the role of altitude in rhizobial biogeography for the region. Hence, future studies, sampling higher altitude areas could allow for further investigation of the effect of altitude on rhizobia diversity and turnover in the CCR. The finding that all strains isolated from the root nodules of the rare I. superba belong to the genus Burkholderia (despite the availability of Mesorhizobium, which was isolated from its sympatric species, P. pullata) suggests a potential symbiotic specificity at the generic level. However, the dispersion of the different strains in several distinct clades points towards association with multiple divergent lineages within the genus Burkholderia. If these strains that cluster with divergent lineages are capable of nodulating I. superba, it would be a similar scenario to that of the widespread P. calyptrata, which is nodulated by strains from several distinct lineages within Burkholderia [53], i.e., no symbiotic specificity at the intragenic level. The results also suggest that the strains isolated from I. superba are not genetically distinct since they were part of various clades that included strains isolated from legumes that occur outside its distribution range. Overall, these results lead to the hypothesis that I. superba does not exhibit rhizobia specificity at the intragenic level. More studies are required to test this hypothesis, and this could involve testing if the various strains are able to induce nodulation on I. superba and determining if I. superba is able to form nodules in soils from outside its distribution range. A lack Genes 2018, 9, 2 18 of 23 of nodulation from these soils would indicate that the restricted distribution of I. superba is due to rhizobia specificity.

5. Conclusions The study of the legume-rhizobia relationship in Southern Africa is still at its infancy, and although the CCR has received more attention relative to the rest of the sub-continent, only a small proportion of its legume diversity has been studied. Nevertheless, the patterns that are emerging from these few studies suggest that these below ground mutualists of the legumes might be significant drivers of the distribution of legumes in the CCR. The findings of the present study suggest that while Burkholderia has an affinity for the acidic and nutrient-poor soils of the CCR, Mesorhizobium has a wider soil type and pH tolerance range, allowing various strains to thrive in habitats of varying edaphic stress. The presence of such ecologically diverse symbionts, coupled with the edaphic heterogeneity of the CCR landscape provide opportunities for the legumes to diversify, and this might explain the high species richness of the family. With the finding that rhizobia contribute towards the structuring of legume assemblages in the CCR, it is plausible that rhizobia also have a strong influence on the structuring of legume assemblages within and across the different biomes of Southern Africa, and this provides a potential direction for future research.

Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/9/1/2/s1. Table S1: BLASTn search results for the various rhizobial strains isolated for this study, Table S2: List of rhizobial strains with binary scoring of their soil types, Table S3: List of rhizobial strains used with the altitude and pH data of their sites. Acknowledgments: The authors would like to thank Benny Lemaire and Edward Chirwa for assistance with field and laboratory experiments. The Table Mountain National Parks (Cape Research Centre, Tokai, Cape Town, Western Cape, South Africa and the Vogelgat Private Nature Reserve (Hermanus, Western Cape, South Africa) are acknowledged for providing permits to conduct the research within their reserves. Funding for this research was provided by the National Research Foundation of South Africa (Grant Number 81818: Biology of Cape Legumes). Two anonymous reviewers, who commented on an earlier version of the manuscript, are gratefully acknowledged. Author Contributions: All authors conceived of and designed the experiments. M.N.D. performed the experiments and analyzed the data. A.M.M and S.B.M.C. contributed reagents and materials. C.H.S. identified the legume specimens. M.N.D wrote the paper with contributions from all authors. All authors read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; nor in the decision to publish the results.

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