Among the ca. 120 to 160 genera of rust fungi (Cummins and Hiratsuka 2003; Kirk et al. 2008), Puccinia is readily recognized by the two-celled teliospores and the shape of the spermogonia (Cummins and Hiratsuka 2003). Uromyces with one-celled teliospores is typically differentiated from Puccinia, although some species of Puccinia have both one-celled (mesospores) and two-celled teliospores, e.g., P. lagenophorae. Teliospore morphology is homoplasious, and Puccinia and Uromyces were polyphyletic in systematic studies based on the LSU and SSU regions of nuclear ribosomal DNA (Maier et al. 2007; Aime 2006), and the two nuclear genes: elongation factor and β- tubulin (Van der Merwe et al. 2007). Some rust fungi have teliospores morphologically similar to Puccinia, but are not closely related or have an uncertain systematic position. For example, Allodus podophylli has two-celled teliospores convergent with Puccinia. A systematic analysis based on the nLSU and nSSU regions of rDNA determined Allodus and Puccinia were unrelated (Minnis et al. 2012). Puccinia psidii, which spread from South America to much of the Pacific region and South Africa, now infects 30 genera of Myrtaceae out of its natural host range (Pegg et al. 2013). It has two-celled teliospores, but its placement within the Pucciniales is unknown. Phylogenetic analyses of the nLSU and nSSU (Pegg et al. 2013) and the protein-coding gene beta-tubulin (Van Der Merwe et al. 2008) indicated that P. psidii was sister to the Pucciniaceae. Several families and genera of rust fungi are polyphyletic, namely the Raveneliaceae, Phakopsoraceae, and Pucciniaceae. These polyphyletic families and genera await resolution by molecular phylogenetic analyses.

Species identification and numbers

Rust fungi are usually considered host-specific (Cummins and Hiratsuka 2003), although some, e.g., Puccinia psidii and P. lagenophorae, infect multiple host genera (McTaggart et al. 2014; Pegg et al. 2013). Some species of rust fungi are heteroecious, requiring two hosts in different families to complete their life cycle, e.g., P. graminis on Triticum (Poaceae) and Berberis (Berberidaceae).

Rust fungi have a complicated life cycle with up to five spore states (Cummins and Hiratsuka 2003). Consequently, up to three names have been proposed for the same taxon based on different life cycle stages. To add to the confusion, there are two systems of terminology that describe these spore states, one based on morphology (Laundon 1967), and the other on ontogeny (Arthur and Kern 1926; Cummins and Hiratsuka 2003; Hiratsuka 1973). These systems of terminology were summarised by Hennen and Hennen (2000).

Species of rust fungi are often identified on the basis of their host specificity, and monographs were organized by plant family (Sydow and Sydow 1904; McAlpine 1906; Cummins 1971, 1978). Morphological characters of the teliospores and urediniospores, such as size, apex shape, and wall thickness, ornamentation, and germ pore position and number, are useful for species identification.

Molecular diagnostic tools have been developed for some species of Puccinia based on the ITS region of rDNA, e.g., P. coronata (Beirn et al. 2011; Pfunder et al. 2001), P. kuehnii (Glynn et al. 2010) and P. psidii (Langrell et al. 2008). The ITS region has successfully distinguished phylogenetic species in Uromyces (Barilli et al. 2011) and it was used in combination with TEF to resolve the taxonomy of P. melampodii (Seier et al. 2009). However, the ITS region was polymorphic in Puccinia lagenophorae (Littlefield et al. 2005; Scholler et al. 2011), and Morin et al. (2009) discovered a paralogous copy of the ITS region, which may have resulted from a hybridization event. A paralogous copy of the ITS region was also reported in P. kuehnii in the study by Virtudazo et al. (2001). Polymorphisms and paralogous copies are caveats for studies based on the ITS region in rust fungi.

Molecular phylogeny

Large-scale systematic studies of rust fungi have focused mainly on the SSU and LSU regions of rDNA  (Aime 2006; Beenken et  al.  2012;  Dixon  et  al.  2010;  Maier et al. 2003, 2007; Minnis et al. 2012; Wingfield et  al. 2004; Yun et al. 2011) (Table). Protein coding genes such as beta-tubulin (Morin et al. 2009;  Van  der  Merwe  et al. 2007, 2008) and elongation factor (TEF) (Seier et al. 2009; Van der Merwe et al. 2007) were successfully used  at the family, genus and species level in rust fungi, although beta-tubulin required cloning rather than direct sequencing of PCR product. Liu et al. (2013)  included ITS, beta-tubulin, ribosomal polymerase subunit 2 (RPB2) and cytochrome c oxidase subunit 1 (COI) in a systematic study to resolve the P. coronata species complex. They discussed the difficulty of PCR amplification of older herbarium specimens, and that DNA repair was successful in some cases. Vialle et al. (2009) compared mitochondrial genes to rDNA markers in two genera of rusts, Chrysomyxa and Melampsora. They found rDNA had better species resolution than mitochondrial genes. Mitochondrial genes were since used in studies of the genera Chrysomyxa (Feau et al. 2011) and Dasyspora (Beenken et al. 2012), but have not yet been used for Puccinia.

Recommended genetic markers

• The large subunit of nrDNA (LSU)–is useful for genus and species-level identification of all rust fungi
• The internal transcribed spacer (ITS)–is useful for species-level identification, but may contain polymorphic sites and paralogous copies. Rust specific primers are recommended.