Applying early divergent characters in higher rank taxonomy of Melampsorineae (Basidiomycota, Pucciniales)

ABSTRACT Rust fungi in the order Pucciniales represent one of the largest groups of phytopathogens, which occur on mosses, ferns to advanced monocots and dicots. Seven suborders and 18 families have been reported so far, however recent phylogenetic studies have revealed para- or polyphyly of several morphologically defined suborders and families, particularly in Melampsorineae. In this study, a comprehensive phylogenetic framework was constructed based on a molecular phylogeny inferred from rDNA sequences of 160 species belonging to 16 genera in Melampsorineae (i.e. Chrysomyxa, Cerospora, Coleopuccinia, Coleosporium, Cronartium, Hylospora, Melampsora, Melampsorella, Melampsoridium, Milesina, Naohidemyces, Pucciniastrum, Quasipucciniastrum, Rossmanomyces, Thekopsora, Uredinopsis). Our phylogenetic inference indicated that 13 genera are monophyletic with strong supports, while Pucciniastrum is apparently polyphyletic. A new genus, Nothopucciniastrum was therefore established and segregated from Pucciniastrum, with ten new combinations proposed. At the family level, this study further demonstrates the importance of applying morphologies of spore-producing structures (basidia, spermogonia, aecia, uredinia and telia) in higher rank taxonomy, while those traditionally applied spore morphologies (basidiospores, spermatia, aeciospores, urediniospores and teliospores) represent later diverged characters that are more suitable for the taxonomy at generic and species levels. Three new families, Hyalopsoraceae, Nothopucciniastraceae and Thekopsoraceae were proposed based on phylogenetic and morphological distinctions, towards a further revision of Pucciniales in line with the phylogenetic relationships.


Introduction
Plant parasitic rusts, taxonomically as members of the order Pucciniales, are one of the most diverse groups of fungal pathogens, with over 7 800 species recognised worldwide (Arthur 1934;Hiratsuka et al. 1992;Zhao et al. 2021;Aime et al. 2006;Webster and Weber 2007). Rusts are obligate parasites with up to five different spore types, including basidiospores, spermatia, aeciospores, urediniospores, teliospores, as well as different lifestyles (micro-, hemi-, demi-, or macrocyclic) that occur on a single (autoecious), or alternate between two unrelated host plants (heteroecious) (Cummins and Hiratsuka 1983). Many species are wreaking havoc on agricultural and forest crop plants, resulting in significant economic losses (Cummins and Hiratsuka 2003). Due to the serious threats posed to these crops, Puccinia spp. on wheat and Melampsora lini on flax were listed in the "top 10 most important fungal pathogens" in a recent global survey of important plant pathogens (Dean et al. 2012). Furthermore, many rust species, such as Austropuccinia psidii (myrtle rust), Cronartium ribicola (white pine blister rust), Hemileia vastatrix (coffee rust), Phakopsora pachyrhizi (Asian soybean rust) etc., are known to be among the most important and threatening species to agriculture and forestry (Ono et al. 1992;Beenken 2014. Despite the importance of rust fungi, their taxonomy is still debated due to the lack of information on host alternation, and morphological characteristics of their various spore stages (Kuprevich and Tranzschel 1957;Savile 1976;Aime 2006).
The taxonomic ranks of plant-parasitic rusts vary at the generic and suprageneric levels. Rust fungi have been classified into different classes (Basidiomycetes, Pucciniomycetes or Urediniomycetes) or orders (Pucciniales or Uredinales) at various time periods (Plowright 1889;Dietel 1928;Cummins 1971;Hiratsuka et al. 1992). Rust fungi were recently found to be monophyletic, and the order Pucciniales was proposed to accommodate these plant parasitic rusts (Aime et al. 2006;Hibbett et al. 2007;Zhao et al. 2016). At the family level, the traditional taxonomy of rust fungi changed dramatically over time, and they were divided into 2-14 families based on criteria applied at the time. At early 20 th century, all rusts were initially divided into 2-4 families on the basis of the presence of teliospore pedicels and basidial formation (Dietel 1928;Arthur 1934;Hiratsuka 1955). Such a taxonomic treatment has been widely debated, as many apparently unrelated genera have been placed in the same family simply because of morphological similarities in their teliospores (Wilson and Henderson 1966;Leppik 1972;Hiratsuka and Sato 1982). Later, spermogonial morphology was introduced as criterion and the taxonomic importance of this criterion at the family level was further evaluated (Hiratsuka and Cummins 1963;Savile 1976). Hiratsuka and Cummins summarised 12 morphological types of spermogonia in rust fungi and categorised them into six major groups based on the structure of spermogonia (Cummins and Hiratsuka 1983). Thus, a taxonomic scheme with 14 families was proposed and uredinologists universally accepted this taxonomic classification (Hiratsuka and Cummins 1983;Hiratsuka et al. 1992;Cummins and Hirastuka 2003). However, several defined families, such as Chaconiaceae, Pucciniaceae, Pucciniastraceae, Pucciniosiraceae and Uropyxidaceae, have been revealed to be poly-or paraphyletic in molecular phylogenetic studies (Maier et al. 2003;Wingfield et al. 2004;Aime 2006). Aime (2006) has roughly divided the order Pucciniales into three suborders based on molecular phylogeny: Melampsorineae, Mikronegeriineae and Uredinineae, but the polyphyly of several morphologically defined families remains unresolved (Maier et al. 2003;Wingfield et al. 2004;Beenken et al. 2012;Beenken and Wood 2015;Qi et al. 2019). Thereafter, Aime and McTaggart (2021) presented a high-rank classification of the Pucciniales, in which the order was divided into seven suborders and 18 families. Among these families, all rusts with sessile teliospores were classified in the suborder Melampsorineae, with 16 genera included in four families, Coleosporiaceae, Melampsoraceae, Milesinaceae and Pucciniastraceae based on their aecial similarities. However, these families showed significant morphological differences in the structures of spermogonia and telia, which have long been employed as the main criteria for family classification (Cummins andHiratsuka 1983, 2003). As a result, the taxonomic placement of these 16 genera remains a point of contention, and it is necessary to reassign those genera in the proper family in the suborder Melampsorineae.
In this study, we conducted molecular phylogenetic analyses and morphological reappraisal of Melampsorineae. The objectives of this study were: (1) to evaluate the monophyly of traditional morphologically defined genera and determine their familial placements and generic boundaries in Melampsorineae; (2) to propose a taxonomic amendment towards establishing monophyletic families in Melampsorineae.

Molecular phylogeny and supergeneric-level delimitation
We have included sequence data from our previous taxonomic studies on genera in Melampsorineae (Zhao et al. 2014(Zhao et al. , 2015(Zhao et al. , 2016(Zhao et al. , 2017(Zhao et al. , 2020(Zhao et al. , 2021Qi et al. 2019) as well as some newly generated sequence data from our one unpublished paper (under review), and detailed information of specimens, host species and GenBank accession numbers has been listed in Table 1. In addition, rDNA sequence data from previous taxonomic studies on Pucciniales, particularly those in Melampsorineae, were included in the final alignment (Table 1). Those sequences were acquired from taxonomic references and retrieved from NCBI (https:// www.ncbi.nlm.nih.gov/) based on the accession number. To determine the phylogenetic relationships of genera in the suborder Melampsorineae, rDNA ITS and LSU sequences of representative taxa from the genera Chrysomyxa, Coleopuccinia, Coleosporium, Cronartium, Hylospora, Melampsora, Melampsorella, Melampsoridium, Milesina, Naohidemyces, Pucciniastrum, Quasipucciniastrum, Rossmanomyces, Thekopsora, and Uredinopsis were chosen for phylogenetic studies (Table 1). Two Gymnosporangium species were selected as outgroups. The majority of the sequence data came from samples that had detailed morphological information. For phylogenetic analyses, raw sequence data were aligned by BioEdit v. 7.0.9 (Hall 1999), and multiple alignments were performed with MAFFT v. 7.394 (Katoh et al. 2017). Ambiguous alignment positions were manually adjusted before the final analyses. Topologies were constructed based on maximum likelihood (ML) analyses using RAxML v. 0.95 (Stamatakis 2006). Bayesian Markov Chain Monte Carlo (MCMC) analyses were performed using MrBayes v. 3.1.2 (Huelsenbeck and Ronquist 2001), and Bayesian posterior probabilities (Bpp) were calculated. In ML and Bayesian analyses, the best-fit substitution model was estimated using Modeltest v. 3.7 (Posada and Crandall 1998).

Results
For ML and Bayesian analyses, a dataset containing selected species from 16 genera in Melampsorineae was used. Phylogenetic trees using the combined dataset yielded higher confident values for the generic level than that of the single locus tree, with 570 bp nucleotide positions for ITS and 800 bp for LSU. An overview of the inferred topology is given in Figure 1. Melampsorineae is divided into 16 well-supported clades. The monophylies of several genera in Melampsorineae, i.e. Ceropsora, Chrysomyxa, Coleopuccinia, Cronartium, Coleosporium, Hylospora, Melampsorella, Melampsoridium, Naohidemyces, Quasipucciniastrum, and Thekopsora, were confirmed, in agreement with previous studies (Aime et al. 2018;Qi et al. 2019;Zhao et al. 2020;Aime and McTaggart 2021). Pucciniastrum, the type genus of the Pucciniastraceae, was split into two different clades. The type species of Pucciniastrum, P. epilobii clustered with other nine species (i.e. P. circaeae, P. guttatum, P. lanpingensis, P. minimum, P. myosotidii, P. nipponicum, P. pustulatum, P. rubiae, and P. verruculosum) in one clade, whilst 10 other Pucciniastrum species (i.e. P. actinidiae, P. boehmeriae, P. corni, P. fagi, P. hikosanense, P. kusanoi, P. miyabeanum, P. styracinum, P. tiliae and P. yoshinagai) were in another distinct clade. Species of Milesina and Uredinopsis were found in a same clade, with the later merged into the Milesina species, forming a sister clade to M. vogesiaca.
Morphological comparison of each clade is shown in Figure 1. Morphologies in spore-producing structures (i.e. spermogonia, aecia, uredinia, telia and basidia), exhibited the strongest association with the phylogeny. Based on our morphological comparisons and phylogenetic analyses of 16 genera in Melampsorineae, ten families, including three new families, Hyalopsoraceae, Nothopucciniastraceae, Thekopsoraceae, and one new genus, Nothopucciniastrum, were proposed. These families differ from their phylogenetically allied families in their spore-producing structures (basidia, spermogonia, aecia, uredinia and telia), as shown in Figure 2. Previous studies have demonstrated that sporeproducing structures were phylogenetically informative at the family level (Zhao et al. 2020(Zhao et al. , 2021Aime and McTaggart 2021), and we have further confirmed that these characters throughout the life cycle are of great importance to facilitate natural familial delimitation, especially in the suborder Melampsorineae (Figure 3).
Notes -Chrysomyxa was previously placed in Chrysomyxaceae, Coleosporiaceae, or Melampsoraceae by different taxonomists based on various morphological criteria in teliospores (Sydow and Sydow 1915;Dietel 1928;Leppik 1972;Savile 1976). Cummins and Hiratsuka (1983) placed Chrysomyxa in Coleosporiaceae together with Coleosporium by emphasising the importance of spermogonial and teliospore's morphologies, however, telial differences between the two genera have been clearly demonstrated (Sydow and Sydow 1915;Crane et al. 2005). Chrysomyxa was then placed in Coleosporiaceae by Aime and McTaggart (2021), along with Coleosporium, Cronartium, Quasipucciniastrum, Rossmanomyces and Thekopsora, but these genera in this broadly defined family have high morphological variations in the structures of spermogonia and telia, which had long been used as important criteria at the familial level (Cummins and Hiratsuka 2003).
The polyphyly of the family Coleosporiaceae defined by Aime and McTaggart (2021) has been revealed by our previous molecular phylogenetic analyses (Zhao et al. 2020(Zhao et al. , 2021. Our current findings further demonstrated the phylogenetic distinction of Chrysomyxa from Coleosporium, and two genera have different spermogonial, uredinial and telial morphologies ( Figure 1). Furthermore, the divergence time of two genera and their median ages were in a general range of divergent time of families in Basidiomycota (Aime et al. 2018;He et al. 2019). All these findings supported the taxonomic treatments of Dietel and Neger (1900) and Leppik (1972), who placed the genus Chrysomyxa in the Chrysomyxaceae.
The family name Chrysomyxaceae is therefore resurrected here. Aime and McTaggart (2021) established a new genus Rossmanomyces, which is phylogenetic allied to Chrysomyxa but differs in forming a systemic sporothallus in telia. Here we placed genera Chrysomyxa and Rossmanomyces in the family Chrysomyxaceae.  Dietel, in Engler and Prantl, Nat. Pflanzenfam., Teil. I (Leipzig)  Notes -Coleosporiaceae has undergone multiple taxonomic reassignments over the years based on various morphological criteria in teliospores and spermogonia (Wilson and Henderson 1966;Hiratsuka et al. 1992;Cummins and Hiratsuka 2003). Recently, Aime and Taggart (2021) included seven genera in this family, despite the fact that the spore-producing structures (i.e.  Thekopsora was classified in the new family Thekopsoraceae. Thus, Coleosporiaceae is emended with a narrower concept based on the type genus Coleosporium, which is distinctive from other families in telia with internal basidia and unicellular teliospores in 1-layered crust, wall thick and gelatinising above (Figure 1). Dietel, in Engler and Prantl, Nat. Pflanzenfam., Teil. I (Leipzig)  Notes -Cronartium was originally placed in the family Pucciniaceae (Dietel 1897), but then classified as a member of Cronartiaceae, Coleosporiaceae, Melampsoraceae or Pucciniastraceae in different time periods based on various morphological criteria in teliospores or spermogonia (Dietel 1928;Cummins andHiratsuka 1983, 2003;Aime and Taggart 2021). Recent molecular phylogenetic studies confirmed the placement of Cronartium in the suborder Melampsorineae (Aime 2006;Aime et al. 2018), but its relationship with several other genera, including Chrysomyxa, Coleosporium, Diaphanopellis, Quasipucciniastrum, Rossmanomyces and Thekopsora, was still uncertain (Aime and Taggart 2021). Our current results and previous phylogenetic studies of the order Pucciniales (Zhao et al. 2020(Zhao et al. , 2021 clearly revealed the phylogenetic distinction between Cronartium and the above-mentioned genera (Figure 1), and Cronartium clade differs from other families in the spore-producing structures, especially in Group II spermonogia, Milesia-type Spermogonia Group I (type 1), subepidermal, with concave hymenia, bounding structures lacking. Aecia Peridermium-type, subepidermal, erumpent, with peridia, aeciospores catenulate, verrucose. Uredinia Milesiatype, subepidermal, urediniospores mostly echinulate, borne singly. Telia intraepidermal, consisting of spores in the epidermal cells, teliospores aseptate or multiseptate, with oblique septa. Basidia external.

Family: Cronartiaceae
Notes -Milesina, Naohidemyces and Uredinopsis were classified in Pucciniastraceae based on their teliospores that are embedded in the host tissue and the Peridermium-type aecia (Cummins and Hiratsuka 1984;Cummins and Hiratsuka 2003). Phylogenetically, Milesia, Naohidemyces and Uredinopsis clustered in one phylogenetic group, and separated from Hyalopsora, Melampsorella, Melampsoridium, Pucciniastrum and Thekopsora (Figure 1). In morphology, Milesia, Naohidemyces and Uredinopsis have similar morphological features in all spore-producing structures, but clearly differentiate themselves from other families in Milesiatype uredinia without ostiolar cells and wall colourless ( Figure 1). Thus, Aime and McTaggart (2021) proposed Milesinaceae to accommodate these three genera. Our results is in agreement of Aime and McTaggart (2021 Notes -The Thekopsora clade, including the type of the genus, Thekopsora areolata, was phylogenetically close to Cronartium but distinct from Pucciniastrum species (Figure 1), in agreement with Aime et al. (2018). In morphology, it resembles Coleopuccinia, Hylospora, Melampsoridium, and Pucciniastrum, but differs from these genera in the aecia, uredinia and telia (Figure 1; Yang 2015). It also differs from the phylogenetically allied family Cronartiaceae in the structures of spermogonia, uredinia and telia. Thus, a new family Thekopsoraceae is proposed to accommodate the genus Thekopsora.

Phylogenetic reappraisal of rust families and genera
To date, more than 7 800 rust species have been described in 289 genera in Pucciniales (Laundon 1965;Kirk et al. 2008). Cummins and Hiratsuka (2003) in their monograph "Illustrated Genera of Rust Fungi" included 120 holomorphic genera and 13 asexual typified genera, in which most genera are traditionally morphologically defined. At the family level, since the first system proposed by Dietel (1897), different mycologists have classified 289 recognised genera into 2-14 families mainly based on morphological characters in spermogonia and teliospores (Arthur 1907(Arthur , 1934Kuprevich and Tranzschel 1957;Wilson and Henderson 1966;Azbukina 1972;Cummins and Hiratsuka 1984;Buriticá 1991;Hiratsuka et al. 1992;Cummins and Hiratsuka 2003). Although urediniologists have generally accepted Cummins and Hiratsuka (2003)'s taxonomy system, subsequent studies have found numerous inconsistencies between this system and the molecular phylogeny (Wingfield et al. 2004;Aime 2006;Aime et al. 2018;Zhao et al. 2020). A definite family level resolution has not been obtained due to the lack of a comprehensive molecular phylogenetic study within the order Pucciniales. In our previous studies on the whole order, we proposed four new families based on extensive phylogenetic and morphological comparisons, and also recognised inconsistencies between morphologically-defined families in Melampsorineae suborder and their molecular phylogenetic relationships (Zhao et al. 2020(Zhao et al. , 2021. Thus, we conducted phylogenetic studies of 16 genera in the Melampsorineae suborder, and confirmed boundaries at the familial and generic level. Traditional morphology-defined families such as Coleosporiaceae, Chrysomyxaceae, and Melampsoraceae have been shown to be monophyletic with the core of species around the type (Figure 1). Pucciniastraceae has been found to be polyphyletic and its traditional members scattered in numerous discrete lineages. This family has been redefined by emphasising the importance of traditional taxonomic criteria used by Cummins and Hiratsuka (2003) and Aime and McTaggart (2021). In this study, we further emphasised several newly recognised criteria for familial delineation, i.e. the structures of uredinia and telia, including the colour of urediniospores, the existence of peridia in uredinia and telia, the existence of ostiolar cells in uredinia and their ornamentations. Our phylogenetic studies further emphasised that morphologies throughout their life cycles, especially the uredinial and aecial morphologies, are of great diagnostic value in delineating at the familial and generic level.

Importance of applying early diverged characters in higher rank taxonomy
Hitherto, classification of the rust fungi at species, generic and family levels relies on morphological features of different spores and spore-producing structures in different stages throughout the life cycles. Spore morphologies, such as basidiospores, spermatia, aeciospores, urediniospores and teliospores, as well as spore-producing structures like basidia, spermogonia, aecia, uredinia and telia, were hitherto not seriously investigated to see if they are phylogenetically significant at particular taxonomic levels.
Our previous and current studies on the phylogeny of Pucciniales, particularly those in the Melampsorineae, have revealed that morphological characters, especially spore-producing structures throughout the whole life cycle, such as basidia spermogonia, aecia, uredinia and telia, were phylogenetically more informative at higher taxonomic ranks (family level). By contrast, spore morphologies such as basidiospores, spermatia, aeciospores, urediniospores and teliospores, were phylogenetically more informative at lower taxonomic rank (species level) (Tian et al. 2004;Crane et al. 2005;Feau et al. 2009, Beenken 2014Zhao et al. 2015Zhao et al. , 2017. These spore-producing structures represent early diverged morphological characters, while spore morphologies have already been proved to be recently diverged characters by our continuous investigations (Figure 1; Zhao et al. 2020Zhao et al. , 2021. Our findings were further supported by the successive evolutionary process of rust fungi, because the structures of basidia, spermogonia, aecia, uredinia and telia might be directly influenced at early adaptation stages of rust fungi when they shifted from ferns to conifers and angiosperms (Leppik 1965). Based on these findings, taxonomic significance of morphological features in different spore stages throughout the whole life cycle in the rust fungi is proposed as shown in Figure 4. It is quite obvious that the polyphylies of many traditionally defined genera and families were resulted from the inappropriate use of recently diverged characters (particularly morphology of teliospores) at higher level taxonomy, and the use of the early diverged characters at lower-level taxonomy. Apparently these recently diverged characters have evolved more than once in different lineages during the evolutionary process.
Among the early diverged characters, the structures of spermogonia and telia have long been employed for classification at the family level, but the structures of aecia and uredinia have long been overlooked at higher taxonomic ranks (Hiratsuka and Cummins 1963;Hiratsuka and Hiratsuka 1980;Cummins andHiratsuka 1983, 2003). Our studies indicated the importance of aecia and uredinia morphology for higher level classification, especially differences in spore ontogeny, hymenium shape, position in host tissues, presence of intercalary cells and paraphyses. Until now, fourteen different morphological types in aecial and uredinial structure have been recognised (Kenny 1970;Sato and Sato 1985). These morphological variations appear to be very useful criteria at family and generic level taxonomy in rust fungi.