Raveneliopsis, a new genus of ravenelioid rust fungi on Cenostigma (Caesalpinioideae) from the Brazilian Cerrado and Caatinga

ABSTRACT The multicellular discoid convex teliospore heads represent a prominent generic feature of the genus Ravenelia. However, recent molecular phylogenetic work has shown that this is a convergent trait, and that this genus does not represent a natural group. In 2000, a rust fungus infecting the Caesalpinioid species Cenostigma macrophyllum (= C. gardnerianum) was described as Ravenelia cenostigmatis. This species shows some rare features, such as an extra layer of sterile cells between the cysts and the fertile teliospores, spirally ornamented urediniospores, as well as strongly incurved paraphyses giving the telia and uredinia a basket-like appearance. Using freshly collected specimens of Rav. cenostigmatis and Rav. spiralis on C. macrophyllum, our phylogenetic analyses based on the nuc 28S, nuc 18S, and mt CO3 (cytochrome c oxidase subunit 3) gene sequences demonstrated that these two rust fungi belong in a lineage within the Raveneliineae that is distinct from Ravenelia s. str. Besides proposing their recombination into the new genus Raveneliopsis (type species R. cenostigmatis) and briefly discussing their potentially close phylogenetic affiliations, we suggest that five other Ravenelia species that are morphologically and ecologically close to the type species of Raveneliopsis, i.e., Rav. corbula, Rav. corbuloides, Rav. parahybana, Rav. pileolarioides, and Rav. Striatiformis, may be recombined pending new collections and confirmation through molecular phylogenetic analyses.


INTRODUCTION
Within the rust fungi (Pucciniales), the suborder Raveneliinae consists of four families: the Ochropsoraceae, Tranzscheliaceae, Phakopsoraceae, and Raveneliaceae. Whereas the first three families appear to represent natural groups, the monophyly of the Raveneliaceae remains dubious to this day, due to the unresolved deeper branching patterns in phylogenic reconstructions of this suborder (Aime and McTaggart 2021). The repeated evolution of convergent morphologies further complicates a better understanding of their evolutionary history and became a challenge to taxonomists. Morphologically, the genus Ravenelia (Pucciniales; Raveneliineae), parasitic on members of the Fabaceae, is known for its multihyphal pedicellate discoid to hemispherical multicellular teliospore "heads." This complex structure typically consists of one or two layers of probasidial cells subtended by hygroscopic and sterile cyst cells. Similar ravenelioid spore heads are also found in Kernkampella, parasitic on Phyllanthaceae not on Fabaceae. They are distinguished from Ravenelia by its uniquely papillate teliospore germ pores and patelliform stratum of sterile cells in between the teliospore layer and the cyst cells around the terminal end of the pedicel (Berndt and Freire 2000;Rajendren 1970).
Morphological as well as ecological characters in Ravenelia are diverse (Dietel 1906) and were used in the early literature to subdivide the genus (Dietel 1906;Long 1903;Sydow 1921;Sydow and Sydow 1915). In addition, divergence in teliospore ontogeny among Ravenelia species further reveals the heterogeneity of this genus (Berndt 1995;Hiermath and Pavgi 1976), but its significance for segregation has been disputed (Hiermath and Pavgi 1979). However, the polyphyletic origin of Ravenelia has only recently been proven by molecular phylogenetic studies (Aime and McTaggart 2021). Berndt and Freire (2000) described a Ravenelia species on Cenostigma macrophyllum (as C. gardnerianum) that showed some peculiarities. Like Kernkampella, this rust fungus showed an intercalary layer of supposedly sterile cells between the probasidial cells and the cysts, as well as distinctive protruding ornamentations originating from specialized cells at the edge of the discoid spore heads. Also, the urediniospores showed spirally arranged striate ornamentations, which were similarly observed in R. corbula (Baxter 1966), R. corbuloides (Hennen and Cummins 1990), R. pileolarioides (Sydow and Sydow 2016), R. spiralis (Hennen and Cummins 1990), and to a lesser extent in R. striatispora (Cummins and Baxter 1975).
Recently, several species of Poincianella and Caesalpinia were transferred to Cenostigma with 14 species today recognized throughout the Neotropics, with most species known from the Brazilian Cerrado ) and a few from the Caatinga ecoregion (Gagnon et al. 2016). In field trips organized by the Mycological Collection of Herbarium UB (Universidade de Brasília), conducted in the Brazilian Cerrado, we collected rust fungi on Cenostigma macrophyllum that resembled Ravenelia cenostigmatis and R. spiralis. Therefore, the aim of the present study was to verify their identities and to evaluate their phylogenetic relationships with other Ravenelia species, as revealed by phylogenetic analyses based on sequences of the nuc 28S rDNA (28S), nuc 18S rDNA (18S), and the mitochondrial cytochrome c oxidase subunit 3 (CO3) gene.

MATERIAL AND METHODS
Collection and preservation of material. -The holotype of R. cenostigmatis was originally described using material collected at the center of the Brazilian semiarid Caatinga Biome "at the road PI 140 between São Raimundo Nonato and Canto do Buriti, ca. 30 km from São Raimundo Nonato City" (Berndt and Freire 2000) Morphological characterization. -For the macromorphological study of the fungarium specimens, a Leica 205C stereomicroscope (Leica Microsystems, Nassloch, Germany) was used. Photography and measurements were performed with a Leica DM 2500 light microscope equipped with Nomarski optics, coupled to a Leica DFC 490 digital camera. Measurements for all structural components, image capture, and editing were processed with Leica Qwin 3 software. For scanning electron microscopy, fresh samples were attached to the surface of cylindrical metal stubs, fixated using Karnovisky reagent, post-fixed on osmium tetroxide, and contrasted with uranyl acetate. After dehydration on an acetone gradual series (30%-100%) for approximately 20 min on each stage, leaf pieces were criticalpoint dried and metal coated with gold using a Leica SCD050 sputter coater. Dry telia and uredinia were directly coated using the same device. In both situations, analyses were conducted on a Jeol JSM 7001 F scanning electron microscope (Tokyo, Japan).
Taxon and sequence sampling.-Taxon sampling in this study was chosen to cover major lineages and genera within the families Raveneliaceae, Phakopsoraceae, Tranzscheliaceae, and Ochropsoraceae of the suborder Raveneliineae (Aime and McTaggart 2021). Newly obtained sequences are derived from Herbaria KR (Natural History Museum, Karlsruhe, Germany) and UB (University of Brasilia, Brazil).

DNA extraction, PCR amplification, and DNA
sequencing.-Spores were isolated from infected leaves using a sterile insect needle under a stereomicroscope. Spores were placed in 1.5-mL Eppendorf tubes and subsequently crushed in three consecutive freeze-thaw cycles as described in . The subsequent DNA extraction was done using the Qiagen DNeasy Blood & Tissue Kit (Valencia, California) following the manufacturer's protocol. Polymerase chain reactions (PCRs) to amplify fragments of the 28S, 18S, and CO3 gene regions were performed using primer pairs LR0R/LR6 (Moncalvo et al. 1995;Vilgalys and Hester 1990), NS1/Rust18SR (Aime 2006;White et al. 1990), and CO3-R1/CO3-F1 (Vialle et al. 2009), respectively, following conditions described in Ebinghaus et al. (2020), Ebinghaus et al. (2022)). PCR products were sequenced at Macrogen (Seoul, South Korea) using the PCR primers. Forward and reverse sequence reads were assembled and edited using Sequencher 5.0 (Gene Codes, Ann Arbor, Michigan) and checked against the National Center for Biotechnology Information (NCBI) GenBank database using BLASTn (Altschul et al. 1990). All sequences obtained in this study were deposited in GenBank and listed in TABLE 1.    Sequence analyses.-Two data sets were constructed in this study. An overview data set, A, was built to confirm or refute the generic identity as currently recognized for Ravenelia cenostigmatis and R. spiralis. This was achieved by covering major lineages and genera within the families Raveneliaceae, Phakopsoraceae, Tranzscheliaceae, and Ochropsoraceae of the suborder Raveneliineae (Aime and McTaggart 2021). Based on this initial analysis, a second data set, B, was constructed in order to narrow down taxon sampling and to potentially improve lineage resolution. Data set A comprised alignments of the 28S and CO3 gene regions, whereas data set B additionally comprised the 18S gene region. Alignments were constructed by using the MAFFT algorithm as implemented in the GUIDANCE2 Web interface and applying the L-INS-i strategy for the 28S and the 6mer approach for the 18S and CO3 sequence data sets, respectively (Landan and Graur 2008;Larget and Simon 1999;Penn et al. 2010;Katoh and Standley 2014). To improve accuracy of the alignments, 100 bootstraps repeats and a maximum of 100 reiterates were chosen. In the resulting 28S and 18S alignments, unreliable columns below an automatically generated confidence score were filtered. Due to the codon structure of the CO3 gene sequences, the unfiltered alignment was used. All alignments were subsequently checked and manually edited in MEGA 7.0.26 (Kumar et al. 2016). The 28S alignment was furthermore used to construct an indelcoded binary data set according to Simmons and Ochotena (2000) using FastGap 1.2 (Borchsenius 2009).
The subsequent maximum likelihood (ML) analyses were conducted in IQ-TREE 2.1.3 (Minh et al. 2020) as partitioned data sets with 10 000 ultrafast bootstraps (UFboots; Hoang et al. 2018). UFboots were tested by 10 000 replicates of a Shimodaira-Hasegawa approximate likelihood-ratio test (SH-aLRT; Guindon et al. 2010). We included FreeRate heterogeneity models to find best-fit models for the respective data sets using ModelFinder (Kalyaanamoorthy et al. 2017) and applied a codon model for the gene-encoding CO3 sequences. The alignments and tree files are available as SUPPLEMENTARY DATA 1-9. (SUPPLEMENTARY FIG. 1), the Phakopsoraceae, Tranzscheliaceae, and Ochropsoraceae were resolved as highly significantly supported monophyletic groups within the Raveneliineae. In data set B (FIG. 1), only the Ochropsoraceae were moderately supported. Due to a lack of resolution in deeper nodes, the Raveneliaceae appeared polyphyletic in all analyses, as previously shown by Aime and McTaggart (2021). Correspondingly, relationships between recognized families and genera remain obscure (FIG. 1; SUPPLEMENTARY FIG. 1).

Phylogenetic analyses.-In all analyses using data set A
Ravenelia cenostigmatis and R. spiralis were resolved as sister species with highly significant support in all analyses and always grouped distantly from Ravenelia s. str. Instead, they showed affiliation to Kernkampella breyniae, Newinia heterophragmitis, and Tegillum scitulum, albeit this relationship was not found to be supported in any of the analyses (FIG. 1; SUPPLEMENTARY FIG. 1).

TAXONOMY
Our phylogenetic analyses had shown that the ravenelioid rust fungi infesting the genus Cenostigma and having striate and spirally arranged urediniospore ornamentation as well as strongly incurved paraphyses represent a distinct lineage within the Raveneliineae and are herein proposed as members of the new genus Raveneliopsis.
The new genus proposed here includes R. cenostigmatis (Berndt and Freire 2000) (type species) and R. spiralis (Hennen and Cummins 1990), as follows.
Diagnosis: Differs from Raveneliopsis cenostigmatis by lack of appendages on the verrucose teliospore heads but showing identically ornamented urediniospores.
Description and illustrations: See Hennen and Cummins (1990)  Notes: There are four other Ravenelia species on Cenostigma that may also belong to this new genus, as they have the same characteristics of striate urediniospores, incurved paraphyses, and basket-like sori. These rust fungi can be distinguished from the Raveneliopsis species by details in their teliospore morphologies and their reported host associations: (i) R. corbula exhibits uredinioid paraphysate aecia and produces verrucose teliospore heads with all peripheral spores ornamented by a single 13-µm-high, conical or capitate appendage. This species was described on Cenostigma eriostachys (originally named Caesalpinia eriostachys) from Sinaloa, Mexico (Baxter 1966). (ii) Ravenelia corbuloides shows uredinioid paraphysate aecia and smooth teliospore heads with uniseriate cysts that were described as appressed or semipendant and was only reported on Cenostigma bracteosum (originally Caesalpinia bracteosa) in Bahia, Brazil (Hennen and Cummins 1990). (iii) No aecia are known for R. pileolarioides, with mostly verrucose teliospores resembling those of R. corbuloides but additionally showing some intermixed tuberculate spores (Sydow and Sydow 1916). The host of R. pileolarioides was originally stated as Pithecellobium sp., but according to Hennen et al. (2005) and Hennen and Cummins (1990), it was misidentified and represents Cenostigma pyramidale (as Caesalpinia pyramidalis). This rust is known from Ceará in northwestern Brazil. (iv) Ravenelia parahybana (Viégas 1945) was treated as a synonym to R. pileolarioides by Hennen et al. (2005) based on the verrucose teliospores and was collected in Alagoa Grande, Paraíba, in the Brazilian Northeast.
Beside those four species on Cenostigma, another rust showing ravenelioid teliospores and striate spirally arranged urediniospore ornamentations was described on the Mimosoideae host Havardia mexicana (= Pithecellobium mexicanum) from Sinaloa, Mexico, as Ravenelia striatispora (Cummins and Baxter 1975). Despite it partially sharing characteristics of the urediniospores, we consider close relationship of R. striatispora with the former species doubtful due to the distinctly deviant host association, the aparaphysate sori, and the interconnected striations of the urediniospores.

DISCUSSION
The Cenostigma rusts examined in this study were originally identified as Ravenelia cenostigmatis Bernd & Freire and R. spiralis Hennen & Cummins by morphological comparisons. However, our molecular phylogenetic analyses refuted their affiliation with the genus Ravenelia s. str. but revealed their distinct position within the Raveneliinae. Consequently, we have recombined these two species into the new genus Raveneliopsis.
The Raveneliopsis species show some unique features within Raveneliaceae but also differ from other rust fungi that have ravenelioid multicellular teliospore heads. Berndt and Freire (2000) commented on and illustrated the unusual structural characteristics of the teliospore head of R. cenostigmatis when compared with other Ravenelia species. They specifically emphasized the teliospore heads with one or two rows of small, appressed sterile cells located between the teliospores and the subjacent hygroscopic cysts.
According to Berndt and Freire (2000), R. cenostigmatis would be closely related to Kernkampella, which represents the only known genus with ravenelioid teliospore heads that also shows an additional layer of compressed ("patelliform") sterile cells between the teliospores and the cyst cells. However, the decision to segregate the two genera was supported by diverse host associations (Caesalpinioideae for R. cenostigmatis and Phyllanthaceae for Kernkampella) and distinct papillate germ pores of the teliospores in the case of Kernkampella species, reinforced by our phylogenetic analyses.
Noteworthily, additional phylogenetic associations with species of the former Uropyxidaceae, especially Tegillum, Newinia, and Prospodium were found in our analyses, albeit these relationships were not statistically supported and the proximity to Prospodium was found only once ( SUPPLEMENTARY FIG. 1). These rust fungi are characterized by telial morphologies that appear distinctly different from those of Raveneliopsis.
Tegillum scitulum produces unicellular teliospores, but which are also formed in basket-like sori (Sydow 1937). Newinia, on the other hand, forms multicellular transversely septate teliospores, whereas the teliospores of Prospodium are puccinioid and are often formed with appendages basally on the pedicels. Common to the latter three genera, however, are the frequently formed type 7 spermogonia as well as paraphysate uredinioid aecidia. This discussion highlights the problems that need to be addressed in further taxonomic studies within the Raveneliineae. They are mainly due to the lack of appropriate data both in terms of molecular markers and inclusion of key species that can reveal the evolution of convergent morphologies and determine taxonomically relevant traits. Specifically, the inclusion of taxa such as Tegillum fimbricatum and Newinia kigeliae and N. thaiana, as well as genera such as Mimema and Phragmopyxis, which have potential key features such as strongly curved paraphyses and type 7 spermogonia (Cummins and Hiratsuka 2003), may provide new insights into the evolution of these rust fungi.

ACKNOWLEDGMENTS
The authors are grateful to Dr. Christiane Ceriani Aparecido from Instituto Biológico de São Paulo/Brazil for the access to the IBI isotypes of Ravenelia cenostigmatis and R. spiralis, and to Dr. Dagmar Triebel from the Botanische Staatssammlung München for the access to the holotype of R. cenostigmatis. The authors also thank Dr. David Rabern Simmons, herbarium curator at The Arthur Fungarium (Purdue University), for helping with remote access to type materials, illustrations, and photos.

DISCLOSURE STATEMENT
No potential conflict of interest was reported by the author(s).