1. Introduction
There is an open debate in the microbial biogeography literature regarding whether or not microorganisms are biogeographically structured [
1,
2,
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, bacteria, fungi and protists, which points to the existence of microbial biogeographic structure [
7,
8,
9,
10,
11,
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 Alphaproteobacteria, 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 that they are incapable of nodulating each other’s hosts [
16,
17,
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,
21,
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,
25,
26,
27,
28,
29]. These factors also affect general plant 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,
33,
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) plants 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,
45,
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 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
Podalyria 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 Fynbos 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.
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 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 of nodulation from these soils would indicate that the restricted distribution of
I. superba is due to rhizobia specificity.