New host associations and a novel species for the gall-inducing acacia rust genus Ravenelia in South Africa.

Trees in the genus Vachellia (previously Acacia) are commonly infected by the gall-inducing rusts Raveneliamacowaniana and R.evansii. Rust galls bearing aecial infections and relating uredinial and telial infections on the leaves of nine Vachellia species not previously recorded to be infected by Ravenelia spp. have recently been collected in South Africa. The rust fungi causing these infections were characterised using molecular phylogenetic analyses of DNA sequence data of the LSU and ITS rDNA regions as well as morphological examinations. The host range of R.macowaniana and R.evansii was thus re-assessed and extended from four to nine species and from one to three species, respectively. Application of Principal Component Analyses (PCA) of telial morphological characters provided evidence of an effect of the host species on the teliospore morphology in R.evansii, but only minor effects in R.macowaniana. A novel gall-inducing Ravenelia sp. closely related to R.macowaniana, was found on Vachelliaxanthophloea and it is described here as R.xanthophloeae.


Introduction
Trees in the genus Vachellia (formerly Acacia subg. Acacia) and referred to here as acacias make up one of the most prominent floral elements of the Southern African es having aecial galls were collected and subsequently dried between paper sheets in a plant press. In total, 49 specimens were studied based on morphology and 31 of these could be used for phylogenetic analyses based on DNA-sequence data. Ten of the 49 specimens were either type or voucher specimens collected in the late 19 th and early 20 th century and used by Doidge for her studies of the southern African Ravenelia spp. (Doidge 1927(Doidge , 1939. These herbarium specimens were used only for morphological comparisons with the newly collected material (Table 1). Three specimens were deposited in KR, all others in PREM.

DNA extraction and PCR
Spores from individual sori were collected separately using sterile insect needles. Genomic DNA extractions were made using the INNUPrep Plant DNA Kit (Analytik Jena, Germany) following the manufacturer's protocols with the following modifications: Spores were crushed using a Retsch mixer mill MM2000 (Retsch, Haan, Germany) by shaking them together with 2 steel beads of 2.5 mm diameter in a 2.0 ml Eppendorf tube. This process was repeated in three consecutive cycles. In the first step, the closed tubes were cooled in liquid nitrogen and immediately shaken for 2 min at 100 Hz. Thereafter 10-40 μl of lysis buffer was added to the tube to loosen spore remnants from the inner side of the Eppendorf tube lid using a vortex mixer followed by a centrifugation step. Samples were again cooled in liquid nitrogen and shaken for an additional 2 min at 100 Hz followed by centrifugation for 1 min at 6000 rcf. The last two steps were repeated once.
For PCR of the ribosomal nrITS and LSU rDNA gene regions, the Taq-DNA-Polymerase Mix (PeqLab, Erlangen, Germany) was used with the primers ITS1F (Gardes and Bruns 1993) and RustITS1F (Toome and Aime 2014), respectively and ITS4BR (Vialle et al. 2009). PCR of the LSU rDNA region was performed using the primer pairs LR0R and LR6 (Vilgalys and Heester 1990). Two additional primers (5.8SrustF: 5'-CGA TGA AGA ACA CAG TGA AAT GTG; D1D2RustR: 5'-CTY TGC TAT CCT GAG GGA) were designed with improved specificity for Ravenelia and that reduced amplification of ascomycetous non-target organisms. The thermal cycling conditions for primers ITS1F/ITS4BR and RustITS1F/ITS4BR were as follows: 2 min at 96 °C followed by 40 cycles of 20 sec at 96 °C, 40 sec at 50 °C and 50 sec at 72 °C, final extension was for 5 min at 72 °C; for primers LR0R/LR6: 3 min at 96 °C followed by 40 cycles of 30 sec at 95 °C, 40 sec at 49 °C and 1 min at 72 °C, final extension was for 7 min at 72 °C; for primers 5.8SrustF/D1D2rustR: 3 min at 96 °C followed by 40 cycles of 30 sec at 96 °C, 45 sec at 54 °C and 1 min 20 sec at 72 °C, final extension was for 7 min at 72 °C.
PCR products were purified using Sephadex G-50 columns (Sigma-Aldrich, Steinheim, Germany). Where PCR products showed only weak bands on agarose gels, purification was undertaken using the Zymo Research DNA Clean & Concentrator Freiburg,Germany) following the manufacturer's pro- Table 1. List of specimens included in the present study, including host information, collection data and GenBank accession numbers of rDNA sequences.   tocol. DNA sequencing was carried out in both directions using the same primers as those used for PCR on a 3130XL Genetic Analyzer (Applied Biosystems) at the sequencing service of the Faculty of Chemistry and Biochemistry of the Ruhr University Bochum, Germany.

Phylogenetic analyses
Sequences were screened against the NCBI GenBank using the BLASTn algorithm (Altschul et al. 1990) to exclude erroneously amplified contaminants from further processing. Forward and reverse strands of the rust sequences were assembled using Sequencher 5.0 software (Gene Codes Corp., Ann Arbor, MI, USA) and, where necessary, manually edited. In total, 32 sequences were used to construct an alignment of the nrITS and LSU rDNA sequence data, respectively, using MAFFT v6.832b (Katoh and Standley 2014) (Lanave et al. 1984) with gamma distributed substitution rates. The analyses were run with a rapid bootstrap analysis using 1000 bootstrap replicates. The analyses were first conducted for each dataset separately and topological congruence was checked visually. As no conflict of supported phylogenetic groupings was observed, the final phylogeny was inferred by combining both datasets of the nrITS and LSU rDNA sequences applying the same methodology as for individual datasets.
Parsimony network analyses were performed using TCS v1.21 (Clement et al. 2000) and the same sequence alignments that were used for the phylogenetic analyses. Gaps were deleted from calculations and the default connection limit of 95% was used.

Light-and electron microscopic investigations
The spores of the dried herbarium specimens (Table 1) were scraped from leaf surfaces and mounted in lactophenol on microscope slides. A minimum of ten teliospores and 30 urediniospores per specimen were examined. Minimum, maximum and mean values are provided in Table 2 PREM10698 (R. glabra) and PREM20727 (R. glabra) were examined at the facilities of the ARC-Plant Protection Institute (ARC-PPRI), Roodeplaat, South Africa using a Leica Dialux 22 EB microscope and a ColorView III CCD colour camera. Measurements were made using analySIS LS software (LS Research Software GmbH, Germany). The remaining specimens were studied at the Ruhr University Bochum, Germany, using a Zeiss Axioplan light microscope. Morphological characteristics were measured using Cell^D v. 3.1 imaging software (Olympus Soft Imaging Solutions GmbH, Germany) and Zen2 lite (Blue Edition) V. 2.0.0.0 (Carl Zeiss Microscopy, 2011, Jena, Germany). Photographs were obtained using a Color View microscope camera (Olympus Soft Imaging System, Germany). For detailed investigations of the spore-surface structures, scanning electron microscopy (SEM) was used. For this purpose, infected leaflets from the herbarium specimens were mounted on double-sided adhesive carbon tape on metal stubs and coated with gold in a sputter coater BAL-TEC SCD OSO (Capovani Brothers Inc, USA). Subsequently, the samples were examined using a ZEISS Sigma VP scanning electron microscope.

Phylogenetic Analyses
Sequence data from the nrITS and LSU rDNA gene regions were obtained for all 31 newly collected specimens. The alignment of the nrITS sequence dataset had a total length of 764 bp with 133 variable sites of which 131 positions were parsimony informative. The aligned sequences of the LSU rDNA dataset had a length of 922 bases and comprised 31 sequences with 45 variable sites and 40 parsimony informative positions. The combination of the nrITS and LSU rDNA datasets resulted in an alignment with a total length of 1686 nucleotides comprising 32 sequences. The sequence alignment and phylogenetic tree of the combined rDNA sequence data set was deposited at TreeBASE (http://purl.org/phylo; submission IDS22307). Maximum likelihood analysis of the combined dataset resulted in a phylogenetic tree that consisted of three highly supported groups representing R. evansii, R. macowaniana and a novel Ravenelia species described below (Fig. 1A).
A second group included the sequences of eleven specimens, five of which originated from V. karroo and were identified as R. macowaniana (KR-M-43657, PREM61222, PREM61221, PREM61210, KR-M-43406). Five specimens were collected from V. natalitia (PREM61214, PREM61862, PREM61218, PREM61219, PREM61216) and one originating from V. permixta (PREM61875) also clustered in this group. The latter two hosts are newly reported for R. macowaniana.
A distinct clade, nested within the R. macowaniana group, was represented by three Ravenelia specimens that were isolated from V. xanthophloea (PREM61215, PREM61213, PREM61000) suggesting that it represents a novel taxon. For PREM61213, two identical sequences were obtained, one derived from aeciospores and one from teliospores.
The parsimony network analysis, based on the combined set of nrITS and LSU rDNA sequence data, separated three distinct groups each comprising the same specimens representing R. evansii, R. macowaniana and the novel Ravenelia species in our phylogenetic analysis, respectively (Fig. 1B). Network analysis relying on LSU alone could not separate R. macowaniana from the novel taxon, while separation of these two groups was observed based on nrITS alone (not shown). The R. evansii group consisted of six haplotypes of 17 sequences that differed by a maximum of two substitutions from the inferred ancestral sequences (PREM61005 and PREM61227). In R. macowaniana, sequence divergence was more pronounced and consisted of nine haplotypes in a total of eleven sequences. The highest rate of six substitutions was observed for specimen PREM61221 relative to the inferred ancestral sequence (KR-M-43657). Specimens collected on V. xanthophloea had only one substitution.

Ravenelia evansii
The teliospore morphology of R. evansii specimens showed a considerable overall variability in all six investigated teliospore characteristics (Suppl. material 1: Fig. S1, Table 2). The voucher specimens PREM61869 (on V. borleae), PREM61876 and PREM61868 (both on V. exuvialis) had significantly smaller teliospores compared to the remaining specimens, but variation in this trait could also be observed within single host associations, e.g. within those from V. davyi and V. robusta (Suppl. material 1: Fig. S1).
The principal component analysis (PCA) of teliospore characteristics clustered several individuals derived from specific hosts into distinct groups (Fig. 2C, D). Individuals that originated from V. borleae and V. exuvialis clustered more closely and could be separated from those individuals that were collected from V. davyi (Fig. 2D). The separation of these individuals was supported by PC1 which could explain 37.6% of the overall variability. The traits 'cells in diameter' and 'teliospore diameter' were characteristics that corresponded best with this axis (Fig. 2C). These results indicated, that the latter two characters were the most variable traits to separate these individuals with different host associations. However, individuals from V. luederitzii var. retinens, V. robusta var. robusta, V. sieberiana var. woodii and V. swazica showed only weak separation, i.e. less clear patterns of morphological separation.

Ravenelia macowaniana
Specimens representing R. macowaniana were morphologically more homogeneous compared to R. evansii. Here, only spore characteristics such as 'probasidial cell width' and 'epispore thickness' were often significantly different between investigated specimens (Suppl. material 2: Fig. S2). There was little variation for the characters 'teliospore diameter', 'probasidial cell length' and the number of 'cells in diameter'. These characters varied distinctly only between single specimens, e.g. PREM61226 originating from V. natalitia had significantly larger teliospores in comparison to specimen PREM61888 that was also collected from this host (Suppl. material 2: Fig. S2).
For the specimens of R. macowaniana, PC1 and PC2 could explain 36.2% and 22.4% of the similarity, respectively. However, unlike in R. evansii, single teliospore characteristics did not differ significantly in terms of host association ( Fig. 2A).

Ravenelia sp. nov.
Due to similar teliospore characteristics, R. macowaniana was compared using PCA to individuals of the undescribed Ravenelia species collected on V. xanthophloea in order to characterise and, if possible, to contrast both morphologies. The PCA separated two groups that corresponded well with R. macowaniana and the novel Ravenelia species and showed very little overlap in morphological characteristics (Fig. 2B). Separation between both species groups was mostly seen in PC1 that could explain 42.5% of the overall variability with the 'ornamentation length' corresponding best to this axis (Fig. 2B). Although less distinct in the PCA, mean values of teliospore characters also revealed that the characters 'epispore thickness', 'probasidial cell length' and especially the 'teliospore diameter' and 'cells in diameter' are valuable characters for the discrimination of both species (Fig. 3). All spore measurements, derived from the six defined spore characteristics that were used for PCA, are available as an excel-file in the Suppl. materials 3, 4: tables S1, S2: 'PCA-   Figure 4C). Peridia and peridial cells could not be described because only disintegrated aecia were present in the dried herbarium material.
Notes. In South Africa, R. macowaniana, R. glabra Kalchbr. & Cooke and R. deformans (Maublanc) Dietel are the only known species that exhibit two-layered probasidial cells and smooth teliospores. While the first character is shared by R. xanthophloeae, the teliospore surface bears small and irregularly arranged small warts clearly visible in SEM (Fig. 4F). However, these can easily be overlooked in light microscopy ( Fig. 4H) and could potentially lead to misidentification. Specifically, R. macowaniana differs from R. xanthophloeae in the overall size of its teliospores (Table 2; Figure 4K) and the urediniospores have four equatorial germ pores whereas those of R. xanthophloeae have five to six equatorial germ pores. The teliospores of the microcyclic R. glabra Kalchbr. & Cooke are about twice the size (120-160 μm) of those of R. xanthophloeae and its oblong urediniospores are significantly larger (32-48 × 14-21 μm). This rust has also been reported only from Calpurnia sylvatica (Burch.) E. Mey (Fabaceae) (Doidge 1927). The demicyclic R. deformans (Maublanc) Dietel was synonymised with the neotropical R. hieronymi Speg. based on nearly identical morphology and congruent life cycle characteristics (Hernandez and Hennen 2003;Hennen et al. 2005) but conspecificity of these two rust fungi is doubtful as they infect distinct host species and occur each on different continents. However, both species produce aecia that induce malformations in young branches, which is a characteristic similar to the newly described R. xanthophloeae. With a size of 60-120 μm and 75-120 μm, respectively, the teliospores of R. deformans and R. hieronymi are, however, on average significantly larger and develop intermingled with the aecia (Doidge 1927), while R. xanthophloeae is macrocyclic and aecia, uredinia and telia are produced in spatially separated sori.
The teliospores of R. xanthophloeae may also be confused with those of R. natalensis Syd., P. Syd & Pole-Evans, but they are significantly smaller in size (30-50 μm diam.) and possess extraordinarily long and persistent pedicels (up to 110 μm; Sydow 1912, Fig. 4I). In R. natalensis, the aparaphysate uredinia and telia are confluent and cover large areas on the branches of the host (Sydow and Sydow 1912; own observations), while the specimens of R. xanthophloeae examined in this study have minute uredinia with numerous paraphyses and telia not exceeding 200 μm in diameter.
New host records. Morphological and molecular phylogenetic analyses based on nrITS and nrLSU data confirmed new host records for R. macowaniana and R. evansii that will be reported in the following section. An emended species decription for R. evansii is also provided.  Emended description. Telia subepidermally erumpent, dark brown to blackish, scattered or in loose groups on the abaxial side of leaflets, sori on the comparatively large leaflets of V. robusta ssp. robusta often forming concentric rings of 2.2-3.3 mm in diameter, single sori (120)230-500(710) μm in diameter with the largest telia appearing to develop in concentric arranged groups, subcircular to elongated; paraphyses lacking; teliospores circular to subcircular from topview, topside convex to almost hemispherical from lateral view, chestnut brown, (47)74-103(124) μm in diameter with (3)5-7(8) probasidial cells in a cross-section; single probasidial cells mostly single-layered, sometimes central cells and in rare events single cells two-layered, (16)23-30(39) μm from lateral view and (11)18-25(34) μm from top view; each probasidial cell with 3-5(8) spines; cysts hyaline and smooth, uniseriate and each cyst appears to be divided by a faint constriction, of the same number as peripheral probasidial cells, swelling in water but only slightly in lactophenol, pedicels compound, not persisting. Specimens examined. All specimens examined for the emended species description of R. evansii representing new host associations are given in Table 1.

Discussion
The new rust species, R. xanthophloeae, was found only on the fever tree Vachellia xanthophloea. In South Africa, this tree species is naturally confined to habitats with shallow watertables in low-altitude areas of the northeastern KwaZulu-Natal, Mpumalanga and Limpopo Provinces (Coates Palgrave 2005, Smit 2008). However, it is frequently planted as ornamental at higher altitudes throughout Southern Africa, where infections by R. macowaniana on V. karroo are common. Yet, despite extensive sampling efforts, no rust has been reported from V. xanthophloea in these regions, suggesting that R. xanthophloeae might currently be restricted to the native range of its host tree. Furthermore, V. xanthophloea is apparently resistant to infection by the frequently cooccurring and closely related R. macowaniana. This observation lends additional support to the separation of R. macowaniana and R. xanthophloeae as distinct species. Sequence divergence was smaller amongst the specimens of R. evansii than within R. macowaniana (Fig. 1). This is in contrast to teliospore morphology, where the six examined teliospore traits showed considerable variability in R. evansii, but very little variation in R. macowaniana. Specifically, an effect of the host association on teliospore morphology could be demonstrated and this was most pronounced in specimens of R. evansii collected from V. borleae, V. davyi and V. exuvialis. It has been demonstrated in other fungal and oomycetous plant pathogens that infraspecific variation of spore traits might correlate with host species (Savile 1976, Lutz et al 2005, Runge and Thines 2011. However, mechanisms leading to such host-associated differences in rust fungi remain obscure. Savile (1976) hypothesised that differences in host compatibility of rust fungi potentially lead to differences in nutrient supply and could consequently influence morphological features. It was also speculated that host anatomy such as the thickness of the cuticle and epidermis might influence spore morphology (Scholler et al. 2011). Clearly, experimental studies that focus on the differential effects of the host and environment on morphological character expression in the rust fungi are needed to resolve this question.
In the present study, the host ranges of R. macowaniana and R. evansii were expanded from one to three and from four to nine hosts, respectively. Thus, these two rust species have a broader host range then previously reported and parasitise several co-occurring acacia species in the South African savannah biome. This is in contrast to recent findings in the genus Endoraecium that infect Australian wattles (Acacia s. str., formerly Acacia subg. Phyllodineae). Based on morphological and molecular phylogenetic studies, species previously thought to have a broad host range were split into several species infecting mostly a single tree species (Berndt 2011, McTaggart et al. 2015. In this case, a general pattern of co-speciation of the parasites with their hosts was suggested (McTaggart et al. 2015). In the South African acacia rusts however, the shared distribution ranges of their hosts may prevent the rusts from speciation by recurrent gene flow between metapopulations. In contrast, Ravenelia xanthophloeae appears to infect only V. xanthophloea. In the phylogenetic analyses, this rust was closely related to R. macowaniana, which suggests a more recent speciation of both species. The host of R. xanthophloeae is eco-geographically clearly separated from the hosts of R. macowaniana and the different niches of the host most likely contributed considerably to the divergence of the parasite species.

Figure S1
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