Diversity of Frankia Populations in Root Nodules of Geographically Isolated Arizona Alder Trees in Central Arizona (United States)ᰔ† Allana K

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Diversity of Frankia Populations in Root Nodules of Geographically Isolated Arizona Alder Trees in Central Arizona (United States)ᰔ† Allana K. Welsh,1 Jeffrey O. Dawson,2 Gerald J. Gottfried,3 and Dittmar Hahn1* Texas State University, Department of Biology, 601 University Drive, San Marcos, Texas 786661; University of Illinois at Urbana-Champaign, Department of Natural Resources and Environmental Sciences, 1201 South Dorner Drive, Urbana, Illinois 618012; and USDA Forest Service, Rocky Mountain Research Station, Tonto National Forest, 2324 E. McDowell Road, Phoenix, Arizona 850063

Received 13 May 2009/Accepted 26 August 2009

The diversity of uncultured Frankia populations in root nodules of Alnus oblongifolia trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of nifH gene fragments. Sequences were retrieved from Frankia populations in nodules of four trees from each of three and their levels of diversity compared using spatial genetic clustering methods and (162 ؍ mountaintops (n single-nucleotide or 1, 3, or 5% sequence divergence thresholds. With the single-nucleotide threshold level, 45 different sequences with signiﬁcant differences between the mountaintops were retrieved, with the southern site partitioning in a separate population from the two other sites. Some of these sequences were identical in nodules from different mountaintops and to those of strains isolated from around the world. A high level of diversity that resulted in the assignment of 14 clusters of sequences was also found on the 1% divergence level. Single-nucleotide and 1% divergence levels thus demonstrate microdiversity of frankiae in root nodules of A. oblongifolia trees and suggest a partitioning of diversity by site. At the 3 and 5% divergence levels, however, diversity was reduced to three clusters or one cluster, respectively, with no differentiation by mountaintop. Only at the 5% threshold level do all Frankia strains previously assigned to one genomic group cluster together.

Frankia spp. are nitrogen-fixing actinomycetes that form tops may provide an opportunity to get new insights into the root nodules in symbiosis with more than 200 species of non- diversity and biogeography of these Frankia populations. leguminous woody plants in 24 genera of angiosperms (5, 24, Specific factors that drive Frankia diversification are pres- 43). These actinorhizal plants have an almost worldwide dis- ently unclear, even though there are preferences among tribution and can live in soils with low nitrogen availability and Frankia strains for specific host plants, separating strains into thus exploit habitats not favorable for growth of many other host infection groups and subgroups (15, 22, 28). Frankia plant species (12). Alnus oblongifolia Torr. (Arizona alder) is strains infecting Casuarina plants have been shown to have an actinorhizal plant that can be found in mountainous regions coevolved with their host plant, illustrating the importance of in northern Mexico and the southwestern United States. the host plant in shaping the diversity and evolution of these Within the southwestern United States, isolated populations of strains (44). However, for most Frankia strains, no simple Arizona alder trees are frequently found along streams drain- pattern of coevolution is present (3). While phylogenetic anal- ing the southern edge of the Colorado Plateau and the scat- yses reveal three clades for each of the partners in this symbi- tered mountain ranges found throughout central Arizona. The osis, Frankia populations within one clade may form root nod- alder sites are in mountains that are surrounded by deserts, ules with plants in more than one clade (4). This lack of grasslands, brush or woodland types, and forests and as such correlating phylogenies is likely due to Frankia populations are home to many endemic species that have developed as a occupying two distinct ecological niches, root nodules and soil, consequence of geographic isolation (47). where symbiotic or saprophytic growth conditions may drive Alnus oblongifolia grows in unique moist environments in diversification of Frankia populations differently (3). Thus, the this desert region, specifically along perennial streams of can- complex divergence patterns in Frankia phylogeny may best be yons, primarily at elevations between 1,400 and 2,300 m, and explained in a geographic mosaic theory of coevolution in has been shown to form effective root nodules in nature (13). which multiple confounding factors, like geographic isolation, Because mountainous sites inhabited by A. oblongifolia are geographically isolated, analyses of Frankia populations in plant host preferences, and environmental factors, converge to nodules of A. oblongifolia trees growing on different mountain- shape the evolutionary patterns of Frankia (3, 46). One aspect of the geographic mosaic theory of coevolution is allopatric speciation, the divergent evolution of geographically isolated populations, which may be a potential driver * Corresponding author. Mailing address: Department of Biol- of Frankia diversification (38, 50). Comparative analysis of ogy, Texas State University, 601 University Drive, San Marcos, TX Frankia populations on isolated mountainous habitats may be 78666. Phone: (512) 245-3372. Fax: (512) 245-8713. E-mail: dh49 a unique opportunity to test if geographic divergence is indeed @txstate.edu. driving Frankia evolution. The isolation of Frankia populations † Supplemental material for this article may be found at http://aem .asm.org/. in root nodules of trees growing on different mountaintops ᰔ Published ahead of print on 4 September 2009. may permit differentiation, as the effects of neutral drift, pop-

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FIG. 1. Locations of the mountaintops in central and southern Arizona sampled for uncultured Frankia populations from the root nodules of Alnus oblongifolia trees growing near perennial streams on these mountains. The scale bar indicates 100 km. Site 1 is Oak Creek in the Coconino National Forest (35°00.6ЈN, 111°44.3ЈW), site 2 is the Workman Creek watershed in the Sierra Ancha Wilderness of Tonto National Park (33°49.1ЈN, 110°55.8ЈW), and site 3 is the Sabino Canyon watershed in the Coronado National Forest (32°26.1ЈN, 110° 45.5ЈW).

ulation bottlenecks and adaptation to even slight environmen- primers nifHf1 and nifHr (34, 49) and sequenced as described tal differences cause the accumulation of mutations which may previously (49). Initially, nifH gene sequences were obtained lead to allopatry (38), as indicated for other bacteria (37, 51). from 24 nodules from one tree from each mountaintop to However, Frankia populations are capable of forming spores determine the level of sampling required to capture the diver- that allow them to survive transport from one hospitable hab- sity present. A rarefaction curve was generated using DOTUR itat to another (26). Additionally, Frankia strains, particularly (41) with a threshold level of divergence set to 3%, which was those of the Alnus host infection group, seem to have a cos- found to group Frankia strains into appropriate genomic mopolitan distribution (4) because strains from the same spe- groups in a previous study (34). Based on rarefaction analyses, cies or genomic group have been isolated from all over the 10 nodules were sampled from the remaining three trees from world (see references 1 and 19) and have been found in soils each mountaintop, for a total of 54 nodules from each moun- with no extant actinorhizal plants (9, 25, 30, 39). tain. Sequences of amplified nifH gene fragments of uncul- The aim of this study was to determine if uncultured Frankia tured Frankia populations from 54 A. oblongifolia nodules from populations in root nodules of A. oblongifolia trees isolated each of three mountaintops (GenBank accession numbers on mountaintops within different geologic regions of Arizona FJ977167 to FJ977328) were aligned with those of the three showed signs of endemism in a functional gene, nifH, and whether pure cultures of Frankia populations (GenBank accession that unique diversity could be correlated with differences in numbers FJ977329 to FJ977331) and sequences of 46 other Frankia populations from root nodules among mountaintops. strains or uncultured populations analyzed in previous studies Nodules were collected in June of 2008 from four trees at each of (34, 49) or retrieved from public databases and analyzed using three mountaintop sampling sites, each separated from the near- maximum likelihood, maximum parsimony, neighbor-joining, est by 150 km, proceeding from north to south, within 1° longitude and Bayesian analyses as described previously (49). of each other along a 300-km latitudinal gradient in southern Phylogenetic analyses of this data set of 211 sequences pro- Arizona (Fig. 1). Site 1 (Oak Creek in the Coconino National duced similar topologies independent of the methodology used Forest, 35°00.6ЈN, 111°44.3ЈW) was a sandy alluvial soil located (data not shown) and assigned all sequences in nodules of A. near Oak Creek at an elevation of 1,703 m, site 2 (Workman oblongifolia to frankiae of the Alnus host infection group (see Creek watershed in the Sierra Ancha Experimental Forest of Fig. S1 in the supplemental material). The analysis retrieved 45 Tonto National Forest, 33°49.1ЈN, 110°55.8ЈW) a streamside different sequences in these nodules from A. oblongifolia, dif- soil high in organic matter at an elevation of 2,073 m, and site fering from each other by at least one nucleotide, with most of 3 (Sabino Canyon in Coronado National Forest, 32°26.1ЈN, the changes being synonymous. For presentation purposes, to 110°45.5ЈW) a sandy loam soil adjacent to Sabino Creek at an show the relationship of frankiae from root nodules of A. elevation of 2,310 m. Each site was in a different geologic oblongifolia to available pure cultures, the complete data set province: the Colorado Plateau Province was in the north, the was reduced to 51 representative sequences, including 21 se- Central Highlands Province in a transition zone, and the Basin quences from frankiae in root nodules of A. oblongifolia, and and Range Province within the Madrean Archipelago in the was reanalyzed using the above-mentioned parameters (Fig. south (8, 32). Nodules were stored in cold 95% ethanol until 2). Several of the sequences obtained from root nodules were analyzed. identical or nearly identical (i.e., single-nucleotide differences) DNA was extracted from individual lobes of different root to those of strains or uncultured Frankia populations from nodules, and a 606-bp fragment of nifH, the structural gene for other parts of the world (Fig. 2). For example, sequence AO3- nitrogenase reductase, was amplified using Frankia-specific 14nodF was identical to nifH gene fragment sequences from VOL. 75, 2009 FRANKIA POPULATIONS IN A. OBLONGIFOLIA ROOT NODULES 6915

FIG. 2. Maximum parsimony tree of a subset of uncultured Frankia populations from root nodules of Alnus oblongifolia and other pure cultures and uncultured Frankia populations created using 522 bp of the nifH gene. Numbers outside parentheses reﬂect bootstrap support values, and numbers in parentheses reﬂect bootstrap support or posterior probabilities from neighbor-joining, maximum likelihood, and Bayesian analyses. Representatives from clusters A and B are indicated. Strains in boldface belong to Frankia genospecies 1 (see reference 20 for a summary). Strain EAN1pec was used as the outgroup.

four Frankia strains isolated from around the world (CpI1 in ever, were not significant (P ϭ 0.165). Significant differences in Massachusetts [7], ArI3 in Oregon [6], AvsI4 in Washington the levels of diversity of Frankia populations among mountain- [2], and Ai14a in Finland [48]). Identical sequence does not tops were also recovered, accounting for 14.6% of the variation mean that these are identical strains but does suggest that in diversity, suggesting differences by site in the diversity of certain genotypes may have a ubiquitous distribution (34). Frankia populations recovered at the single-nucleotide level of Sequences retrieved from Frankia populations in root nod- differences. ules of A. oblongifolia were organized into populations by the To explore this geographic component in more detail, spa- tree that they were isolated from and by mountaintop (18) for tial genetic clustering methods were used in GENELAND analysis of molecular variance (AMOVA) using Arlequin ver- version 3.1.4, which utilizes a Bayesian algorithm to make sion 3.01 (17). The AMOVA settings included 16,000 permu- population assignments and weights by using geographic coor- tations and a more conservative proportion of differences for dinates (20). The analyses of 54 variant characters among the matrix criteria. AMOVA includes both differences in se- 162 root nodule sequences proceeded in two stages, similar to quences at the single-nucleotide level and differences in abun- what was observed by Coulton et al. in 2006, except that the dance of sequences present (40) and indicated significant dif- number of populations in the data set initially fluctuated be- ferences in sequence diversity within trees (P Ͻ 0.001), with tween 1 and 24 and 1 million generations were run with a most of the variation in diversity (83.6%) found within popu- burn-in of 100,000 (10). Spatial and nonspatial settings were lations of Frankia from each A. oblongifolia tree. Differences in used, and uncertainty in coordinates was tested at3mand sequence diversity among trees on each mountaintop, how- 10 m; however, all analyses yielded two populations in the data 6916 WELSH ET AL. APPL.ENVIRON.MICROBIOL.

sequences. Seven sequence clusters were found only in nodules of trees from one mountaintop, five were present in nodules from trees of two mountaintops, and two were detected on all three mountaintops (identified as A and B in Fig. 3). Cluster A was dominant overall (n ϭ 73; 44% of all nodules recovered) and was found on all three mountaintops at various frequen- cies (Fig. 3). Cluster B was also found on all three mountaintops (n ϭ 23; 14% of all nodules recovered) but was dominant on one site (site 3, Sabino Canyon) and barely detected on the FIG. 3. Graphical representation of output from SONS (42) for clusters from uncultured Frankia populations from root nodules of other mountaintops, supporting the geographic uniqueness of Alnus oblongifolia trees (n ϭ 54) isolated on three different mountain- this site (Fig. 3). The number of clusters decreased to three tops in central and southern Arizona (sites 1 to 3). The inner circle when a 3% divergence threshold was used. All three clusters represents 14 clusters recovered using a 1% diversity threshold, the were present on all three mountaintops, but in various fre- middle circle represents 3 clusters recovered using a 3% diversity threshold, and the outer circle represents 1 cluster recovered using a quencies (Fig. 3). At this threshold, however, pure cultures 5% diversity threshold. A and B designate the only clusters at the 1% belonging to the same genomic group still did not cluster in the diversity threshold, representing ϳ5-nucleotide differences, for uncul- same group. Only when the threshold was set to 5% did all the tured frankiae recovered from all three mountaintops. pure cultures from Frankia genomic group 1 (see reference 21 for a summary) cluster together. At a threshold of 5%, all Frankia populations in nodules of A. oblongifolia were placed set and assigned individuals in the same way. Sequences of 108 in one group, suggesting no differentiation by mountaintop and root nodule Frankia strains were unambiguously assigned to a limited overall level of diversity, with one cluster present, one population, corresponding to those from sites 1 and 2, and compared to the potential presence of at least six clusters of 54 sequences of root nodule Frankia strains were unambigu- frankiae within the Alnus host infection group described in ously assigned to the other population, corresponding to site 3. previous studies (34, 49). Low overall diversity has also been Regions encompassing both site 1 and site 2 have some geo- described to occur in other studies of natural Frankia diversity graphic connectivity by forest along the Mongollon Rim and in root nodules of various alder species (11, 23, 27, 29). have been placed in the Central Highlands Floristic Subdivi- Differences in nitrogenase activity and nodulation capacity sion and thus might have been expected to be more similar (8, have been reported for Frankia strains of the same genomic 31). However, site 3 is isolated from the other two sites on group on the same alder species in the same soils (14, 15). account of being surrounded by desert on four sides and was These differences in effectiveness and infectivity of strains in placed in the Southeastern Arizona Floristic Subdivision, sup- the same species group have been suggested to be evidence of porting the comparative uniqueness of site 3 revealed by our the effects of plant host shaping symbiotic Frankia diversity spatial clustering analysis (8, 31). under different environmental conditions (3). Thus, the varia- Three additional threshold levels, i.e., the 1, 3, and 5% tions in diversity and abundance seen at the single-nucleotide divergence levels, were subsequently used to compare the lev- or 1% diversity level may reflect preferences by A. oblongifolia els of diversity of nodule populations among mountaintops. for one strain over another in the different environmental While the 1% level, corresponding to ϳ5-nucleotide differ- conditions on each mountaintop and microscale differences ences, was arbitrarily chosen, the 3 and 5% levels represented among trees on the same mountaintop. Genetic differences thresholds previously used to assign Frankia strains of the same among Arizona alder populations are unknown, as is the extent species or genomic group (34) and uncultured root nodule to which seed and pollen dispersals occur among these isolated frankiae of the Alnus or Elaeagnus host infection groups (34, populations. Nonetheless, there were no morphological differ- 49) into the same cluster on the basis of comparative sequence ences among trees in populations sampled, and the trees were analyses of nifH gene fragments. To formulate the assignment all the same species. In contrast, Frankia diversity in root of clusters at these three levels of differentiation, the complete nodules has been shown to be affected by different edaphic data set was reduced by removing all sequences but those conditions, like soil type and pH (35, 45), or environmental representing frankiae in nodules of A. oblongifolia. This data effects, like elevation (27, 29), which are different on each set of 162 nifH gene fragments was executed using PAUP* mountaintop. However, these variations in diversity may also 4.05b, where an uncorrected distance matrix was created and reflect random chance and small sample size (16), because analyzed using DOTUR to assign taxa to clusters at various rarefaction analysis at the 1% diversity level did not indicate thresholds of diversity and then in SONS to compare mem- saturation of sampling for any mountaintop (data not shown). berships in these clusters by mountaintop (42). A similar Determination of reasons for selective nodulation by specific DOTUR/SONS analysis was completed on the entire data set of strains of Frankia becomes highly speculative. Some evidence 211 sequences to describe the groupings of uncultured nodule suggests that active Frankia populations in the soil may be populations with pure cultures representing various genomic preferentially selected by the host for nodulation (33, 36). groups. Pie charts displaying the clusters of frankiae in root Previous research in our laboratory has confirmed the impor- nodules of A. oblongifolia at 1%, 3%, and 5% differences were tance of the plant host in selecting Frankia strains for symbiosis generated (Fig. 3). when the same soil was inoculated into six different actinorhi- At the 1% threshold level, analyses using the SONS program zal plant species and resulted in six different diversity profiles demonstrated the presence of 14 clusters of sequences (Fig. 3). (34). Additionally, we have shown that the same actinorhizal Half of these clusters were represented by three or fewer plant species inoculated with soils from five different conti- VOL. 75, 2009 FRANKIA POPULATIONS IN A. OBLONGIFOLIA ROOT NODULES 6917 nents resulted in five different diversity profiles, demonstrating Pawlowski and W. E. Newton (ed.), Nitrogen-fixing actinorhizal symbioses. the effects of soil type and history on root nodule Frankia Springer-Verlag, Berlin, Germany. 22. Ho¨nerlage, W., D. Hahn, K. Zepp, and J. Zeyer. 1994. A hypervariable 23S diversity (49). rRNA region provides a discriminating target for specific characterization of Frankia microdiversity in root nodules of A. oblongifolia uncultured and cultured Frankia. Syst. Appl. Microbiol. 17:433–443. 23. Huguet, V., J. McCray Batzli, J. F. Zimpfer, P. Normand, J. Dawson, and recovered in this study shows a clear geographic pattern, but M. P. Fernandez. 2001. Diversity and specificity of Frankia strains in nodules the reasons for these patterns are unclear. The limited Frankia of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis deter- diversity in A. oblongifolia root nodules is likely due to a com- mined by rrs gene polymorphism. Appl. Environ. Microbiol. 67:2116–2122. 24. Huss-Danell, K. 1997. 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