Taxonomy and Phylogeny of Rust Fungi on Hamamelidaceae

Hamamelidaceae is composed of woody plant taxa of important economic value; however, reports on diseases affecting these plants are rare. Three kinds of rusts were studied, of which the first one is characterized by catenulate spores in peridiate columnar sori on Sycopsis sinensis, the second one produces two-celled pedicellate teliospores in pulvinate sori on S. sinensis and Corylopsis spp., and the last one produces aeciospores in cup-shaped aecia on Hamamelis spp. Phylogenetic analyses indicated that the three species belong to the same genus in Pucciniaceae. The first fungus is distinct in teliospore morphology, producing one-celled catenulate spores in peridiate columnar sori and molecular phylogeny from species of other genera. Thus, it is described herein as a new genus and species Novopuccinia sycopsis-sinensis in Pucciniaceae. The latter two species were reported as Puccinia corylopsidis and Aecidium hamamelidis, respectively. However, phylogenetic analysis using ITS and 28S genes has revealed that these are closely related to the new genus and species. By combining host, distribution, and evolutionary hypothesis of rust fungi with endocyclic life cycle, these are reclassified as N. corylopsidis and N. hamamelidis. Taxonomic descriptions, illustrations, and a key to rust fungal species occurring in Hamamelidaceae in Asia are provided.


INTRODUCTION
Several Hamamelidaceae plant species are of significant ecological value in the subtropical humid evergreen broad-leaved forest community (Xu, 2001). Some species such as Sycopsis sinensis also have special habitat requirements and therefore show scattered distribution in the whole range of the subtropical humid evergreen broad-leaved forests. S. sinensis has been listed as a class of protected species in several provinces (Zang and Bian, 2003;Xu et al., 2007).
Rust fungi, Pucciniales, are obligate plant parasites and comprise one of the largest groups in Basidiomycota. Approximately one half of the nearly 8,000 described rust species belong to the genus Puccinia Pers., which is mainly characterized by two-celled teliospores (Index Fungorum) 1 . Several molecular phylogenetic studies have shown that Puccinia spp. is a polyphyletic group and some species are misclassified under this genus name (Aime, 2006;Minnis et al., 2012;Beenken and Wood, 2015). These recent studies have revealed that the taxonomy of the species within the huge genus Puccinia is worth exploring.
In this study, three distinct symptoms with different sporulation of rust fungi were observed on Hamamelidaceae trees. Of these, two fungi produce telia; i.e., one produces twocelled pedicellate spores in pulvinate sori on S. sinensis and Corylopsis sp., while another generates one-celled catenulate spores in peridiate columnar sori on S. sinensis. Long intercalary cells are prominent in this fungus. The other produces spermogonia and cup-shaped aecia on Hamamelis spp. This study aimed to determine the taxonomic status and relationship of three Hamamelidaceae fungi by morphological, molecular phylogenetic, host, and evolutionary analyses of these endocyclic rust fungi. A new genus, Novopuccinia, and three species, namely, N. sycopsis-sinensis sp. nov., N. corylopsidis comb. nov., and N. hamamelidis comb. nov., are described and presented here in detail. All of the rust species of Hamamelidaceae are listed, and their hosts and distribution are provided.

Study Site and Specimens
Four rust specimens were collected from diseased S. sinensis on Mt. Sanqingshan, northeast Jiangxi Province, China (Figure 1), in 2017. Rust specimens on Hamamelis sp. were collected from Ibaraki, Japan, by Y. Ono (IBAR). The host tree was identified by referring to the Flora of China 2 . These specimens were deposited at the Museum of the Beijing Forestry University (BJFC), Beijing, China. Individual specimens are listed under each species in the Taxonomy section. Photomicrographs of type specimens of P. corylopsidis were provided by the U. S. National Fungus Collections (BPI).

Morphology
Rust sori from fresh collections were free-hand sectioned using a double-edged blade under a binocular microscope. Spores were scraped from sori on the dried specimens. Thin sections and spores were mounted in a drop of lactophenol solution on a microscope slide and observed, measured, and photographed using a Leica DM3000 upright microscope (Leica, Germany). For scanning electron microscopy (SEM), fragments of dried leaves with sori and spores were mounted on SEM stubs with double-sided tape. The samples were coated with gold using a Hitachi SCD-005 sputter coater and examined with a Hitachi S-3400 scanning electron microscope (Hitachi, Tokyo, Japan) operated at 5 kV.

DNA Extraction and PCR Amplification
Genomic DNA was extracted from the sori by a method described by Tian et al. (2004). The primer pairs NL1 and NL4 for nuc rDNA 28S (28S) (O'Donnell, 1993) and Rust2inv and ITS4rust for Nuc rDNA internal transcribed spacer region (5.8S-ITS2) (Aime, 2006;Beenken et al., 2012) were used in this study. The PCR conditions were fully described by Liu et al. (2018). The PCR amplification products were estimated visually by electrophoresis in 1.5% agarose gels. DNA sequencing was performed using an ABI PRISM R 3730xl DNA Analyzer with BigDye R Terminater Kit v. 3.1 (Invitrogen) at the Shanghai Invitrogen Biological Technology Company Limited (Beijing, China).

Phylogenetic Analyses
The sequences of the 28S and 5.8S-ITS2 genes from this study and reference sequences obtained from GenBank (Supplementary Table 1) were aligned and edited manually using online MAFFT 3 (Katoh and Standley, 2013). The alignments were concatenated for phylogenetic analyses. The alignments were concatenated for phylogenetic analyses. Maximum parsimony (MP) analyses were conducted with PAUP v. 4.0b10 (Swofford, 2003), using 1,000 heuristic search replicates with random additions of sequences with the tree bisection and reconnection (TBR) branch swapping algorithm. All molecular characters were unordered and given equal weight; analyses were performed with gaps treated as missing data. All equally parsimonious trees found were saved in the MP analyses. Other calculated parsimony scores were tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency (RC). MP bootstrap analyses with 1,000 replicates were performed in the same way, with 10 rounds of replicates of heuristic search with random addition of sequences and subsequent TBR branch swapping during each bootstrap replicate. ML analysis was performed with a GTR + I + G substitution model selected by MrModeltest v.2.3 (Posada and Crandall, 1998). Branch support was evaluated with a bootstrapping (BS) method with 1,000 replicates. For Bayesian Inference (BI), the best-fit model of substitution among those implementable in MrBayes was estimated separately for each gene using jModeltest (Darriba et al., 2012) on the CIPRES portal, based on the Bayesian Information Criterion (BIC). According to the results of MrModeltest, Bayesian Inference (BI) of the 28S was performed using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003), implementing the GTR + I + G model. BI was performed by a Markov chain Monte Carlo (MCMC) algorithm (Rannala and Yang, 1996). The MCMC chains were run for 1 million generations and saved every 1,000 generations. Sequence alignments were deposited at TreeBASE 4 under the accession number 27,477.

RESULTS
To confirm the phylogenetic placement of the fungi in Hamamelidaceae and their relationship, a phylogenetic tree was established based on 28S (Figure 2). The phylogenetic results were mostly congruent with recent studies of rust fungi (Aime and Mctaggart, 2020). However, the support value was lower because only the 28S gene was used in the analysis. In this tree, the new genus was clustered with the Pucciniaceae, which was represented by 14 genera and separated from every genus. In addition, the 28S and 5.8S-ITS2 genes of 46 rust taxa from 13 genera of Pucciniaceae were analyzed, using Dasyspora guianensis as outgroup taxa (Figure 3). The MP analyses of the combined 28S and ITS contained 899 characters. Of these, 537 characters were constant, 105 variable characters were parsimony uninformative, and 257 were parsimony informative. There were 11 equally most parsimonious trees, with the first tree (TL = 1,183, CI = 0.496, RI = 0.630, RC = 0.314). The phylogenetic trees obtained from ML and BI analyses with the MCMC algorithm coincided with the MP tree shown in Figure 3. The rust fungi on Hamamelidaceae form three separate clades on one big branch and represent three different species, namely, Novopuccinia corylopsidis, N. hamamelidis, and N. sycopsissinensis (Figure 3). The type species of Puccina, namely, P. graminis, was placed within the Puccinia/Uromyces subclade. Novopuccinia was distantly related to the clade represented by type species P. graminis and closely related to Stereostratum, with lower support value (Figure 3). Descriptions: Spermogonia subepidermal and mostly globose. Aecia subepidermal in origin, cupulate with well-developed peridium (Aecidium-type). Uredinia subepidermal in origin and becoming erumpent (Uredo-type). Telia subepidermal in origin, becoming erumpent, either pulvinate or columnar; teliospores one-celled and catenulate with a long intercalary cell or twocelled and pedicellate.

Taxonomy
Habitat and distribution: Currently only known on Hamamelidaceae in Asia.
Another telium/teliospore type is similar to those of Puccinia in two-celled with pedicellate (Figure 4), but it can be distinguished from almost all other species by thick-walled teliospores and Hamamelidaceae host. Our phylogenetic results also supported the independence of this type of rust on Hamamelidaceae from the large genus Puccinia (Figures 2, 3).
Novopuccinia sycopsis-sinensis Y. Liu & Y. M. Liang, sp. nov. Etymology: Named after the host name of the type species.
Habitat and distribution: On Hamamelis japonica (spermogonial/aecial stages) and Sasamorpha spp. (uredinial/ telial stages) in Japan. Notes: Dietel (1899) described this fungus as Aecidium hamamelidis based on the Aecidium-type Aecia on Hamamelis plants. It is also related to the bamboo rust P. mitriformis S. Ito reported by Hiratsuka and Sato (1970). We did not observe the type specimen of A. hamamelidis, but specimens that were collected from the same region and host with type collection were researched. For further study, here we designate BJFC-R3798 as the epitype specimen. Our molecular phylogenetic analysis recovered A. hamamelidis within the new genus, with strong support (Figures 2, 3), hence the new combination in Novopuccinia.

DISCUSSION
In this study, three rust taxa obtained from Hamamelidaceae were identified based on morphologic characters and phylogeny based on the 28S and 5.8S-ITS2 sequences. As a result, Novopuccinia typified with N. sycopsis-sinensis is proposed as a new genus in Pucciniaceae. The two known species, P. corylopsidis and Aecidium hamamelidis, were combined to the new genus (N. corylopsidis and N. hamamelidis) by the same host plant, the molecular data, and endocyclic life cycle.
Novopuccinia includes two different teliospore types. The first type is represented by type species N. sycopsis-sinensis with one-celled teliospores and belongs to the kind of taxa, whose telia are cupulate, usually with peridia, and teliospores are verrucose and catenulate with intercalary cells. These taxa are considered to have been derived from a Puccinia-Uromyces complex by reduction of the life cycle through the endophylloid pathway; their occurrence on the same host plants may nonetheless provide clues on the relationship between microcyclic and long-cycled autoecious rust fungi (Jackson, 1931;Buriticá and Hennen, 1980). Members of this complex are closely related to the environment and occur mainly in tropical and other warm regions (Berndt, 2018). The second type is similar to Puccinia-like teliospores and represented by P. corylopsidis and Aecidium hamamelidis (synonym: Puccinia mitriformis), but phylogenetically closely related to Novopuccinia sycopsis-sinensis (Figures 2, 3). While teliospore characters are deemed important to distinguishing genera and families within Pucciniales (Cummins and Hiratsuka, 2003), molecular phylogenies show that their morphological characteristics may not always define higher taxa (Aime, 2006;Beenken and Wood, 2015). Consequently, several genera have diverged from the genus Puccinia sensu lato, despite their similarity in two-celled teliospore morphology such as Allodus (Minnis et al., 2012), Austropuccinia (Beenken, 2017), and Puccorchidium (Beenken and Wood, 2015). However, genera that have been separated from Puccinia based on differences in their morphology appear within Puccinia, such as Uromyces, Diorchidium, Cumminsiella, and Miyagia (Figures 2, 3; Aime, 2006;Van Der Merwe et al., 2008;Beenken and Wood, 2015). New combined species, Novopuccinia corylopsidis and N. hamamelidis, were defined by combining their distant relationship to type species of Puccinia clade, their close relationship with type species N. sycopsis-sinensis (Figures 2, 3), common hosts, and evolution hypothesis of the endocyclic life cycle.
The type species of Novopuccinia, namely, N. sycopsis-sinensis, is microcyclic in the life cycle and produces catenulate, roughsurfaced teliospores with intercalary cells between them. The similar life cycle pattern and morphological characteristics and close phylogenetic relationship to N. corylopsidis and N. hamamelidis (Figures 2, 3) indicate that N. sycopsissinensis was derived from a hypothetical puccinioid ancestor by reduction of the life cycle through the endophylloid pathway (Jackson, 1931;Buriticá and Hennen, 1980). In this pathway, morphologically Aecidium-type telia of a derived microcyclic species are produced on a spermogonial/aecial host plant of a putative ancestral macrocyclic heteroecious species. Similarly, N. corylopsidis might have been derived from the same hypothetical ancestor by reduction of the life cycle through the telioid pathway (Jackson, 1931;Buriticá and Hennen, 1980). The latter evolutionary pathway is wellknown and is accounted for by Tranzschel's Law (Shattock and Preece, 2000). These two evolutionary phenomena and resulting correlations between macrocyclic heteroecious species and microcyclic species have been corroborated to a slight extent by a molecular phylogenetic method. Early successful studies include graminicolous puccinias (Zambino and Szabo, 1993), macrocyclic heteroecious Cronarium and endocyclic Peridermium (Vogler and Bruns, 1998), the P. monoica species complex (parasitizing on Brassicaceae and Poaceae) (Roy et al., 1998), and the P. hemerocallidis species complex (parasitizing Caprifoliaceae and Xanthorrhoeaceae) (Chatasiri et al., 2006).
Sycopsis sinensis (Hamamelidaceae) has been listed as a protected species in China based on its ecological value and rarity (Zang and Bian, 2003;Xu et al., 2007). No disease has been reported in this species to date. Two distinct rust fungi on two adjacent S. sinensis trees, N. sycopsissinensis and N. corylopsidis, were found in the present study. N. corylopsidis has earlier been reported on Corylopsis sp. (Cummins, 1951), and now we observe host expansion and parasitism involving S. sinensis. At the same time, we suspect that the species had mutated to adapt to the environment, thus forming a new rust N. sycopsis-sinensis that also causes disease.
In addition to these three species, two more species have been reported on Hamamelidaceae in Japan, namely, P. sasicola (Hara) Hino & Katumoto on C. glabrescens (spermogonial/aecial stages) and Sasamorpha borealis (telial stage), P. sakamotoi Hirats. f. & Yoshin on Distylium spp. (Hiratsuka, 1942;Hino and Katumoto, 1955). The former is heteroecious and demicyclic; its teliospores are thick-walled, similar to those of N. corylopsidis and N. hamamelidis (Hino and Katumoto, 1955;Hiratsuka et al., 1992). However, it differs from the latter two species by its demicyclic life cycle and hosts during telia stage. The latter is demicyclic and autoecious on Distylium, cylindrical telia are similar to N. sycopsis-sinensis (Figure 4), and two-celled teliospores with thick walls are similar to N. corylopsidis and N. hamamelidis (Figure 5; Hiratsuka et al., 1992). However, teliospores of the latter are one-celled with elongated pedicellike intercalary cells, while P. sakamotoi produces two-celled teliospores without intercalary cells (Hiratsuka, 1942). An autoecious and demicyclic life cycle can also distinguish it from similar species. No molecular data have been used to analyze the relationship between these two species and Novopuccinia on Hamamelidaceae, although we may assume that they are closely related to N. corylopsidis and N. hamamelidis based on the observation of similar teliospores, hosts of spermogonial/aecial stages, and distribution. Further investigations will confirm this conjecture.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www. ncbi.nlm.nih.gov/genbank/ MN943642, MN943643, MW136693, MW136697, MW386782, MW386783, MW394517.

AUTHOR CONTRIBUTIONS
YLa and CT conceived and designed the experiments. YLu, BC, and WL performed the experiments. YLu and YO analyzed the data. YLu wrote the manuscript. YO and YLa revised and approved the final version of the manuscript. All authors contributed extensively to the work presented in this manuscript.

FUNDING
The National Natural Science Foundation Program of China (No. 31870628) supported this study.