Quasipucciniastrum agrimoniae, gen. et sp. nov. on Agrimonia (Rosaceae) from China

ABSTRACT A new rust genus, Quasipucciniastrum, typified by Q. agrimoniae sp. nov., is proposed based on distinct morphological characters and phylogenetic placement. This genus is characterised by its uredinial ostiolar peridial cells with rough surface and sessile, multicellular teliospores with apparently thickened apical wall. Molecular phylogenetic analyses using internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) and the large subunit (LSU) rDNA revealed that this genus is sister to the genus Cronartium (Cronartiaceae), but morphologically it is distinct from Cronartium in the sessile teliospores that are divided by vertical septa. Morphologically, Quasipucciniastrum should be compared to Pucciniastrum (Pucciniastraceae) in its multicellular teliospores, but they were phylogenetically distant.


Introduction
Agrimonia Ldb. species, known as "common agrimony", are perennial herbaceous flowering plants widely distributed in the temperate regions of the Northern Hemisphere, and they have been commonly planted for ornamentation and medicinal use (Lu 2001). Common agrimony is economically and horticulturally important, but their growths have been frequently threatened by rust diseases. Hitherto, seven rust species have been recorded on Agrimonia species (Farr and Rossman 2018), among which, Pucciniastrum agrimoniae (Dietel) Tranzschel (Pucciniastraceae) and its synonymies, P. agrimoniaeeupatoriae (DC.) Lagerh.
During our study on rust fungi in China, a morphologically distinct species was found on Agrimonia pilosa. It produces Milesia-type uredinia and ostiolar peridial cells with rough surface, and have subglobose teliospores divided by vertical septa. These characters are to some extent, similar to Pucciniastrum spp. (Pucciniastraceae), but the rDNA ITS and LSU sequences showed its close relationship to genus Cronartium Fr. (Cronartiaceae) rather than Pucciniastrum. Our critical morphological and molecular comparisons of this fungus with Cronartium, Pucciniastrum and other related genera suggested that this rust fungus represents a new genus herein described as Quasipucciniastrum agrimoniae gen. et sp. nov.

Morphological examinations
Morphological characteristics of all specimens were observed using a dissecting microscope (DM), the light microscope (LM) and the scanning electron microscope (SEM). The methods for morphological analyses as outlined by Zhao et al. (2013), Zhao et al. (2014), 2017) were followed. Fifty measurements of sori and spores from each specimen were recorded, and morphological characteristics in uredinial and telial stages, i.e. the position of uredinia and telia, the ornamentation and dimension of urediniospores, the dimension and shape of ostiolar cells, the position and shape of peridial cells, the dimension of teliospores, the position and shape of teliospores were examined.

DNA extraction, amplification and sequencing
For the fungal specimens, several sori from each specimen were excised and DNA were extracted from all studied herbarium specimens using Gentra Puregene Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. From the crude extracts, 1-3 μl DNA templates were directly used for the polymerase chain reaction (PCR) amplification of the internal transcribed spacer regions and intervening 5.8S nrRNA gene (ITS) and the large subunit (LSU) rDNA. rDNA ITS regions were amplified using the primer pairs ITS1F (Gardes and Bruns 1993)/ITS4 (White et al. 1990), Rust2inv (Aime 2006)/ITS4BR (Feau et al. 2009), and fragment of LSU was amplified using the primer pairs ITS4BRf/LR6 (Vilgalys and Hester 1990), LR1R/LR3 and LR3R/LR6 (Vilgalys and Hester 1990). PCR was performed under the following conditions: denaturation at 95°C for 5 min; followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 55°C for 1 min and elongation at 72°C for 1 min, finally with an extension step at 72°C for 10 min. Purification and sequencing of PCR amplicons were carried out at the Tianyi Huiyuan Company, Beijing.

Molecular phylogeny
Full-length ITS and partial LSU regions were amplified from 23 specimens, and we included 16 specimens from genus Cronartium and Peridermium for comparable studies because they have high similarity to rust fungus on Agrimonia in rDNA sequences. Their herbarium numbers, host plants, geographical origins and GenBank accession numbers are listed in Table 1. Besides, a total of 148 sequences from closely related species were retrieved from GenBank for phylogenetic comparison (Table 2). Pileolaria terebinthi (DC.) Castagne was used as outgroup. In the final dataset, ITS and LSU were concatenated and the final alignment includes 124 specimens with a length of 1836 characters (743 for ITS, 1093 for 28S).
Sequences were manually aligned by using BioEdit 7.0.9 (Hall 1999), and multiple alignments were performed using MAFFT 7 (Katoh and Standley 2013). Gaps were treated as missing data for all analyses. The Akaike Information Criteria (AIC) in Modeltest 3.7 (Posada and Crandall 1998) was used to estimate the best-fit substitution models, and GTR+I + G was selected as the best evolutionary model. Maximum Likelihood (ML) analyses were performed using RAxML 8.0.0 (Stamatakis and Alachiotis 2010), and Bayesian Markov chain Monte Carlo (MCMC) analyses were performed by MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001). Supported values of ML and Bayesian posterior probability (Bpp) were indicated in the phylogenetic tree.

Molecular phylogeny
The combined ITS and LSU dataset included 106 sequences of ITS and 123 sequence of LSU from 125 rust samples. The dataset comprised aligned length of 1836 characters, of which, 1162 characters are constant, and 640 are variable with 533 parsimony informative sites. Both ML and Bayesian inference resulted in a highly concordant topology ( Figure 1)

Notes
The new genus Quasipucciniastrum is characterised by its Milesia-type uredinia with well-developed ostiolar cells, well-developed peridial cells, hypophyllous telia producing subglobose teliospores which is divided by vertical and oblique septa under host epidermis. This genus resembles Pucciniastrum (Pucciniastraceae, Pucciniales) but differs in producing hypophyllous telia, subglobose teliospores with apparently thickened apical wall. Within family Pucciniastraceae, other genera clearly differed from Quasipucciniastrum in uredinial ostiole and teliospores. Genera Calyptospora, Hyalopsora, Milesina and Uredinopsis differ from this new genus in the type of ostiole, position of telia and type of teliospores, while Melampsorella and Melampsoridium differ from Quasipucciniastrum mainly in their unicellular teliospores without septa (Cummins and Hiratsuka 2003). In addition, Melampsorella has Milesia-type uredinia with discrete ostiole, which also clearly differs from Quasipucciniastrum.

Etymology
Named after the host plant of the type specimen.

Discussion
In this study, we recognised a new genus Quasipucciniastrum on Agrimonia pilosa, and described a new species Q. agrimoniae based on morphological and molecular evidences. Hitherto, rust species in genera Puccinia Pers., Pucciniastrum, Thekopsora, Uredo Pers. and Uropyxis J. Schröt. have been recorded on Agrimonia species, but Quasipucciniastrum clearly differs from above-mentioned genera by its hypophyllous telia, subepidermal teliospores with subglobose shape, and multicellular teliospores with thickened apical wall. rDNA based phylogenies further supported the independence of Quasipucciniastrum from these genera, especially Pucciniastrum and other genera in Pucciniastraceae, which have similar uredinial and telial morphologies. Here we confirmed the close relationship of Quasipucciniastrum and Cronartium (Figure 1). Quasipucciniastrum is currently best placed in Cronartiaceae, together with Cronartium, although this should be better confirmed after the examination of the morphological characters in spermogonial and aecial stages of Q. agrimoniae. Morphologically, genus Cronartium owned Milesia-type uredinia that is similar to Quasipucciniastrum, but its columnar telia and unicellular teliopsores are embedded in a common matrix (Cummins and Hiratsuka 2003). Hitherto, we are not successful to obtain the spermogonial and aecial stages of Q. agrimoniae. Further investigation on its life cycles and detailed morphological examination of spermogonia and aecia are necessary.
The rust fungus on Agrimonia pilosa was previously frequently recognised as Pucciniastrum agrimoniae due to its ostiolar cells and subepidermal teliospores divided by vertical septa (Tai 1979;Guo 1989;Zhang et al. 1997;Cao and Li 1999;Zhuang and Wei 1999;Cao et al. 2000;Zhuang 2001Zhuang , 2005. P. agrimoniae was first described on A. pilosa from Western Siberia Borus Mountains, Altai in Russia by Tranzschel (1895). Based on the original description of P. agrimoniae from its type specimens and other Russian materials (Tranzschel 1895;Sydow and Sydow 1915;Kuprevich and Tranzschel 1957), our novel species Quasipucciniastrum agrimoniae on A. pilosa resembles P. agrimoniae in several aspects but still clearly differs in its 2 to 6 celled and subglobose teliospores with apparently thickened apical wall. Similarly P. agrimoniae-like rust on A. eupatoria was once reported to bear phylogenetic affinities to Cronartium flaccidum and C. ribicola (Cronartiaceae) Maier et al. (2003). Those specimens used by Maier et al. (2003) have been shown to be conspecific to Q. agrimoniae in our study. As for P. agrimoniae, currently there is no type-derived sequence to confirm its phylogenetic affinities. Since the type specimen of P. agrimoniae was not obtained from all possibly deposited herbaria, it might have been lost. Neotypification is thus needed using a new and suitable specimen from the original host and locality.
Hitherto, several genera in Pucciniastraceae have been delimitated based on morphological characters in teliospores (Cummins and Hiratsuka 2003), even at generic level, the position of telia and morphology of teliospores have long been used as important taxonomic criteria (Hiratsuka 1958;Cummins and Hiratsuka 2003;Liang et al. 2005;Yang 2015). Although there have been much debates concerning the generic classification based on telial morphologies, recent phylogenetic studies and our study supported the monophyly of all sampled genera in Pucciniastraceae, thus, supported the effectiveness of telial morphologies as taxonomic criteria at generic level. In addition, our morphological and molecular studies further emphasised the importance of the uredinial morphology at generic level because Quasipucciniastrum and Cronartium with Milesia-type uredinia show a much closer relationship than those with Caeoma-type uredinia (e.g. Cronartium, Chrysomyxa). These overlooked morphological characters appeared to be very useful in delimiting taxa at generic and suprageneric level. Further comprehensive studies need to be conducted to evaluate the effectiveness of these morphological characters in rust taxonomy.