Raveneliapiepenbringiae and Raveneliahernandezii, two new rust species on Senegalia (Fabaceae, Mimosoideae) from Panama and Costa Rica

Abstract Two new rust species, Raveneliapiepenbringiae and R.hernandezii (Pucciniales) on Senegalia spp. (Fabaceae) are described from the Neotropics (Panama, Costa Rica). A key to the species on neotropical Senegalia spp. is provided. Molecular phylogenetic analyses based on 28S rDNA sequence data suggest that the representatives of Senegalia rusts distributed in the neotropics evolved independently from species known from South Africa. This is further supported by the teliospore morphology, which is characterised by uniseriate cysts in the neotropical Senegalia rusts and contrasting multiseriate cysts in the paleotropic Ravenelia species that infect this host genus.


Introduction
With more than 200 described species, the genus Ravenelia is amongst the most speciose genera within the rust fungi (Pucciniales) (Cummins and Hiratsuka 2003). In the tropics and subtropics, members of this genus parasitise a diverse range of hosts of the legume family (Fabaceae), including Caesalpinioideae, Faboideae and Mimosoideae. Numerous species of Ravenelia are known from the neotropics, mostly from Mexico (Cummins 1978), Brazil (Dianese et al. 1993, Rezende andDianese 2001;Hennen et al. 2005) and Argentina (Hernández and Hennen 2002).
However, in the neotropics, occurrence of Ravenelia species is poorly known in other countries such as Panama and Costa Rica. Preliminary checklists of abundant fungi in Central America report only a single species of Ravenelia in Panama (R. entadae) (Piepenbring 2006) and 18 species of Ravenelia in Costa Rica, respectively (Berndt 2004).
Specimens of a rust fungus on Senegalia hayesii (Benth.) Britton and Rose were collected in Panama in 2013. Another species of Ravenelia was discovered through the analysis of herbarium specimens of the U.S. National Fungal Collections (BPI) on Senegalia tenuifolia (L.) Britton and Rose. On the basis of morphological and molecular data, these two specimens were herein analysed and described respectively as Ravenelia piepenbringiae and R. hernandezii.

Light-and electron microscopic investigations
Spores representing different spore stages were scraped from the leaf surfaces of dried herbarium specimens and stained in lactophenol solution on microscope slides. For the analysis of soral structures, hand sections were prepared under a stereomicroscope. Samples were microscopically studied with a Zeiss Axioplan Light Microscope and Zeiss Axi-oCam. Cellular structures were measured using ZEN 2 (Blue Edition) Software. Infected leaflets of the herbarium specimens were mounted on double-sided sticky carbon tape on metal stubs and coated with gold in a Sputtercoater BAL-TEC SCD OSO (Capovani Brothers Inc, USA). Superficial ornamentation of spores was investigated using a ZEISS Sigma VP scanning electron microscope at the Ruhr-University Bochum, Germany.

DNA extraction and PCR
Genomic DNA extractions were carried out using the INNUPrep Plant DNA Kit (Analytic Jena, Germany) according to the manufacturer's protocol. Spores were milled in a Retsch Schwingmühle MM2000 (F. Kurt Retsch GmbH &Co KG, Haan, Germany), using two steel beads and liquid nitrogen in three consecutive cycles. An amount of 40 ml of lysis buffer was added to loosen spore remnants by vortexing from the Eppendorf tube lid, followed by centrifuging in a final cycle. Polymerase chain reaction (PCR) of 28S rDNA was conducted using the Taq-DNA-Polymerase Mix (PeqLab, Erlangen, Germany). To compensate for small amounts of spores applied for DNA extractions up to 5ml of genomic DNA extraction were used as the template in 25 ml reactions. Primer pair LR0R (Moncalvo et al. 1995) and LR6 (Vilgalys and Heester 1990) were used to obtain sequences of the 28S rDNA, with thermal cycling conditions set at 96 °C (3 min) followed by 40 cycles of 30 sec at 95 °C, 40 sec at 49 °C and 1 min at 72 °C, with a final extension for 7 min at 72 °C. PCR products, which showed only weak bands on agarose gels, were purified with Zymo Research DNA Clean & Concentrator-5 Kit (ZymoResearch Corp., Irvine, USA), according to the manufacturer's protocol. The remaining PCR products were purified using Sephadex G-50 columns (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Sequencing was carried out in both directions using the same primers as in PCR at the sequencing service of the Faculty of Chemistry and Biochemistry of the Ruhr-University Bochum, Germany and by GATC (GATC Biotech, Konstanz, Germany)

Phylogenetic analyses
Sequences were screened against the NCBI Genbank using the BLAST algorithm to check for erroneously amplified contaminations and were afterwards edited manually using Sequencher 5.0 software (Gene Codes Corp., Michigan, USA). In total, 26 sequences were included (  Rodriguez et al. 1990) and 1000 generations. Four representative species of Endoraecium (KJ862335, KJ862298, KJ862337, KJ862344) were set as multiple outgroups. Maximum Parsimony (MP) analyses were carried out using MEGA6 (Tamura et al. 2013) using the heuristic search option with tree bisection-reconnection (TBR) branch swapping algorithm with 10 initial trees using random step-wise addition. The reliability of topology was tested using the bootstrap method with 1000 replicates.