ArticlePDF Available

Cryptic diversity and significant cophylogenetic signal detected by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae‐Juncaceae

March 2021
Journal of Systematics and Evolution 59(4)

March 2021
59(4)

Authors:

Université de Montréal

Plants play important roles as habitat and food for a tremendous diversity of specialist animals and fungi. The disappearance of any plant species can lead to extinction cascades of its associated biota. In consequence, documenting the diversity and specificity of plant‐associated organisms is of high practical relevance in biodiversity conservation. Here we present the first large‐scale molecular investigation into the diversity, host‐specificity, and cophylogenetic congruence of an especially rich plant‐fungal association, the rust fungi (Pucciniaceae) of Cyperaceae and Juncaceae. Using the largest rust fungi DNA barcoding dataset published to date (252 sequences, 82 taxa), we reject the presence of a global ITS2‐28S barcode gap, but find a local gap in Cyperaceae‐Juncaceae rusts, and suggest the existence of many cryptic species in North America, with some broadly‐circumscribed species possibly corresponding to >10 cryptic species. We test previous hypotheses of correlations between the phylogenies of rust fungi and their Cyperaceae‐Juncaceae hosts using a combination of global‐fit and event‐based cophylogenetic methods. Significant cophylogenetic signal is detected between rusts and their hosts, but the small number of cospeciations argues for preferential host jumps as the driving process behind these correlations. In addition, temporal congruence between the origin of major Carex clades and their rusts suggests that host diversification may have promoted parasite diversification. Finally, we discuss the relevance of rust infection patterns to the systematics of Cyperaceae, highlight some taxonomic problems uncovered by the analyses, and call attention to the promise of DNA barcoding for bridging knowledge gaps in poorly studied plant‐associated microorganisms. This article is protected by copyright. All rights reserved.

Barcode gap in the ITS2-28S dataset. Histogram of within-OTU (blue) and between-OTU (orange, hatched) k-mer distances between samples, showing important overlap and suggesting the absence of a global barcode gap. Inset shows violin and dot plots of the ratio between minimum between-OTU and maximum within-OTU k-mer distances (log scale). A local barcode gap is present in OTUs with a ratio above 2.

…

Rarefaction curves of the eleven North American rust species and aggregates included in rarefaction and richness analyses. The four aggregates where >10 individuals were sequenced are identified by name.

…

Tanglegrams showing associations between rust fungi and their monocot (A) or dicot (B) hosts. Links colored in dark blue are highly congruent, those in dark red are highly discordant, and those in light grey are purely random. The amount of cophylogenetic signal is also expressed at branch nodes using the same color codes. A pure cospeciation scenario would be visualized as tanglegrams without any overlapping lines, and dark blue colors throughout. These tanglegrams show uneven cophylogenetic signal (both dark blue and dark red are present).

…

Sequence statistics for the ITS2-28S dataset.

…

Cophylogenetic signal and estimated coevolutionary events between rust fungi and their monocot and dicot hosts. An asterisk (*) indicates a statistically significant value at alpha=0.05.

…

Figures - uploaded by Étienne Léveillé-Bourret

Content may be subject to copyright.

Content uploaded by Étienne Léveillé-Bourret

Content may be subject to copyright.

This article has been accepted for publication and undergone full peer review but has

not been through the copyediting, typesetting, pagination and proofreading process,

which may lead to differences between this version and the Version of Record. Please

cite this article as doi: 10.1111/jse.12740.

Accepted Article

Étienne Léveillé-Bourret ORCID iD: 0000-0002-0069-0430

Cryptic diversity and significant cophylogenetic signal detected

by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae-

Juncaceae

Étienne Léveillé-Bourret1*, Quinn Eggertson2, Sarah Hambleton2, Julian R.

Starr3

1Institut de Recherche en Biologie Végétale and Département de Sciences

Biologiques, Université de Montréal, 4101 Sherbrooke E, Montréal, Québec, H1X

2B2, Canada.

2Biodiversity and Bioresources, Agriculture and Agri-Food Canada, 960 Carling

Avenue, Ottawa, Ontario, K1A 0C6, Canada.

3Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario,

K1N 6N5, Canada.

*Corresponding author: etienne.leveille-bourret@umontreal.ca

Minister of Agriculture and Agri-Food Canada

Accepted Article

Abstract

Plants play important roles as habitat and food for a tremendous diversity of

specialist animals and fungi. The disappearance of any plant species can lead to

extinction cascades of its associated biota. In consequence, documenting the

diversity and specificity of plant-associated organisms is of high practical relevance in

biodiversity conservation. Here we present the first large-scale molecular

investigation into the diversity, host-specificity, and cophylogenetic congruence of an

especially rich plant-fungal association, the rust fungi (Pucciniaceae) of Cyperaceae

and Juncaceae. Using the largest rust fungi DNA barcoding dataset published to date

(252 sequences, 82 taxa), we reject the presence of a global ITS2-28S barcode gap,

but find a local gap in Cyperaceae-Juncaceae rusts, and suggest the existence of

many cryptic species in North America, with some broadly-circumscribed species

possibly corresponding to >10 cryptic species. We test previous hypotheses of

correlations between the phylogenies of rust fungi and their Cyperaceae-Juncaceae

hosts using a combination of global-fit and event-based cophylogenetic methods.

Significant cophylogenetic signal is detected between rusts and their hosts, but the

small number of cospeciations argues for preferential host jumps as the driving

process behind these correlations. In addition, temporal congruence between the

origin of major Carex clades and their rusts suggests that host diversification may

have promoted parasite diversification. Finally, we discuss the relevance of rust

infection patterns to the systematics of Cyperaceae, highlight some taxonomic

problems uncovered by the analyses, and call attention to the promise of DNA

barcoding for bridging knowledge gaps in poorly studied plant-associated

microorganisms.

Accepted Article

Brief abstract

Phylogenies of Cyperaceae, Juncaceae and their rusts (Pucciniaceae) are congruent

despite few cospeciation events, suggesting that preferential host jumps generated the

cophylogenetic signal. Host diversification may have promoted parasite

diversification given the temporal congruence between the origin of major Carex

clades and their rusts. This large DNA barcoding effort (252 sequences, 82 taxa) also

uncovered many potential cryptic species.

KEYWORDS: Coevolution, host-parasite, molecular phylogenetics,

phytopathology, Pucciniomycotina, rushes, sedges, Uredinales.

1 Introduction

An important motivation for targeting vascular plants in conservation efforts is the

crucial role they play as food and habitat for a tremendous diversity of specialist

animals and fungi (Caro, 2010). Specialized associations are common in

phytophagous arthropods and plant-associated fungi, and it is estimated that there

could be three to six arthropods and two to five specialist fungi for every species of

Accepted Article

vascular plant (Ødegaard, 2000; Zhou & Hyde, 2001; Schmit & Mueller, 2007;

Blackwell, 2011; Blackwell & Vega, 2018). The extinction of any plant species can

thus trigger extinction cascades of its associated biota, and of species that depend in

turn on this biota (Koh, 2004; Brodie et al., 2014). Documenting diversity of plant-

associated organisms and their degree of host specificity is thus of high practical

relevance for conservation, especially considering that most plant-associated

arthropods and microfungi remain undescribed (Hawksworth and Rossman, 1997;

Larsen et al., 2017). In this study, we present the first large-scale molecular

investigation into the diversity, host-specificity, and cophylogenetic congruence of a

particularly rich plant-fungal association, the rust fungi of Cyperaceae (sedges) and

Juncaceae (rushes).

Cyperaceae (ca. 5500 species) and Juncaceae (ca. 440 species) are sister

families of mostly wind-pollinated, grass-like plants that are particularly abundant in

wetlands, temperate forest understories, and arctic-alpine habitats (Goetghebeur,

1998; Kirschner, 2002; Govaerts et al., 2007). As a consequence of their diversity

and ecological prominence, Cyperaceae and Juncaceae host highly diverse fungal

assemblages, being amongst the most heavily used families by two extremely

diverse lineages of plant parasites, the smut (Ustilaginales) and rust (Pucciniales)

fungi (Arthur, 1934; Cummins & Hiratsuka, 2003; Henderson, 2004; Savile, 1979;

Vánky, 2012). The high diversity and specificity of fungal parasites of Cyperaceae

and Juncaceae fostered early interest into using them as an aid for plant

classification, with the underlying expectation that parasite phylogeny would mirror

the phylogeny of their hosts, a principle known as “Fahrenholz’s rule” (Eichler, 1948;

Fahrenholz, 1913; Klassen, 1992). Rust and smut fungi provided some of the earliest

evidence supporting the close relationship between Cyperaceae and Juncaceae

Accepted Article

(Piepenbring et al., 1999; Savile, 1990; Riess et al., 2019), and they formed the basis

for one of the first phylogenetic hypotheses for Carex L. (Savile & Calder, 1953), a

genus comprising more than a third of all Cyperaceae species and ranking amongst

the largest in the world (ca. 2000 species; Frodin, 2004; Global Carex Group, 2015).

Molecular studies have since confirmed that phylogenies of Carex and their smut

fungi are correlated (Escudero, 2015; Hendrichs et al., 2005). Likewise, the close

relationship between Carex and Trichophorum Pers., first proposed by Kukkonen &

Timonen (1979) based on the presence of Anthracoidea Bref. smuts on both genera,

was recently confirmed by molecular phylogenetic studies (Léveillé-Bourret et al.,

2014, 2018b).

In his seminal paper on the use of fungi as aids in plant classification, Savile

(1979) proposed that young lineages of rust fungi infect young plant genera, whereas

older rust lineages remain confined to their initial hosts, thereby providing a basis for

the phylogenetic classification of plants. This work influenced many classical studies

on higher-level plant classification (Takhtajan, 1980; Dahlgren, 1983; Thorne, 2000),

and it was cited as evidence for several infrafamilial classifications, including the

most recent complete treatments of Cyperaceae tribes and genera (Bruhl, 1995;

Goetghebeur, 1998). However, no study has yet re-examined Savile’s hypothesis of

a correlation between the phylogenies of Cyperaceae genera and their rust parasites.

Rust fungi (>8400 species; Pucciniales, Basidiomycota) take their name from their

usually bright-orange replicative spores (Aime et al., 2014). They are responsible for

several economically devastating crop diseases such as crown rust of oats, stem rust

of wheat, and white pine blister rust (Saari & Prescott, 1985; Geils et al., 2010; Liu &

Hambleton, 2012, 2013), but they are also ecologically important as mediators of

plant competition in natural environments (Barnes et al., 2005; Price et al., 1988;

Accepted Article

Rice & Westoby, 1982), and as food for many fungal and arthropod hyper-parasites

(Lutz et al., 2004a, 2004b; Nischwitz et al., 2005; Nelsen, 2010; Henk et al., 2011;

Trakunyingcharoen et al., 2014).

Rusts are infamously known for possessing some of the most complex life cycles

found in nature, often involving as many as five morphologically and functionally

distinct spore stages (O basidiospores, I spermatia, II aeciospores, III urediniospores,

IV teliospores) on two distantly related plant species (Jackson, 1931; Ono, 2002).

These two plant species are called “telial” and “aecial” hosts, which refers to the main

specialised spore type produced on each host. The monocotyledonous species of

Cyperaceae and Juncaceae are telial hosts used for clonal population growth

through the spread of urediniospores (stage III), and for the production of teliospores

(IV), thick-walled spores used for overwintering. In the spring, these teliospores

germinate into basidia, producing basidiospores (O) that infect a new dicotyledonous

aecial host. Spermatia (I) and receptive hyphae are produced on the aecial host, and

their fusion eventually results in the production of aeciospores (II) that complete the

cycle by infecting another telial host. From such complex host-alternating

(heteroecious) life-cycles, a great variety of reduced life cycles involving fewer spore

types (microcyclic) or the infection of a single host (autoecious) have evolved

(Jackson, 1931; Petersen, 1974; Ono, 2002).

Cyperaceae and Juncaceae are common hosts for rusts in one of the two main

clades of the giant genus Puccinia Chevall. (ca. 3000 species), including species of

the nested polyphyletic genera Aecidium Pers. p.p. and Uromyces (Link) Unger

(Aime, 2006; Maier et al., 2007, 2003; van der Merwe et al., 2007). These rusts

alternate between Cyperaceae-Juncaceae and at least 15 families of dicotyledons,

such as Asteraceae, Grossulariceae or Urticaceae, but each rust species normally

Accepted Article

infects a single or a few closely-related plant species for both their telial and aecial

hosts (Arthur, 1934; Savile, 1972). Host fidelity should be especially important in the

aecial hosts because the infection of different hosts would make outcrossing more

difficult. This hypothesis would explain why cophylogenetic signal is higher and host

jumps less frequent in aecial hosts than in telial hosts, at higher taxonomic levels

(Aime et al., 2018). However, no study has yet examined the pattern at lower

taxonomic levels. We here hypothesize that a similar pattern will be found in the rusts

of Cyperaceae-Juncaceae, with a higher cophylogenetic in aecial dicotyledon hosts

than in telial Cyperaceae-Juncaceae hosts.

Using one of the largest rust fungi DNA barcoding dataset produced to date and

recent developments in cophylogenetic analysis of host-parasite associations, we

aim to answer the following questions: (1) how many species of rust fungi are there

on Cyperaceae-Juncaceae hosts?; (2) is the phylogeny of rust fungi correlated with

the phylogeny of their Cyperaceae-Juncaceae hosts?; (3) if it is, what are the

processes responsible for such cophylogenetic congruence?; and (4) are host jumps

more rare in the aecial dicotyledonous hosts (sexual stage), than in the telial

Cyperaceae-Juncaceae hosts?

2 Material and Methods

2.1 Taxonomic sampling

A total of 254 rust-infected herbarium specimens from the Canadian National

Mycological Herbarium (DAOM) at Agriculture and Agri-Food Canada (AAFC) in

Ottawa were selected for DNA extraction and sequencing. Included were

representatives of 53 rust taxa (species, subspecies or varieties) in the genera

Aecidium (1), Puccinia (43) and Uromyces (9) and two unidentified specimens. In

addition, 94 sequences previously generated for DAOM specimens using the same

Accepted Article

methods were also included in the analyses, representing an additional 29 Puccinia

and four Uromyces taxa. The DAOM specimens successfully sequenced and

included in the analyses for these 86 species are listed in Data S1 with their

GenBank species names and accessions, collection metadata, and AAFC DNA

numbers.

Of the total 348 specimens included in the study (254 newly sampled + 94

previously-sequenced), 50 were collected before 1930, 260 from 1930 to 1990, 37

since 1990, and one had no date recorded. The majority of rusts sampled were

collected on host plants in Cyperaceae (165) and Juncaceae (21) or alternate hosts

in Asteraceae (96) and Grossularicaeae (21), but other represented families are

Araliaceae, Celastraceae, Elaeagnaceae, Iridaceae, Lamiaceae, Lythraceae,

Onagraceae, Orobanchaceae, Primulaceae, and Urticaeae. Specimens were

primarily from Canada (184), USA (71), and countries in the European Union (51),

with the rest from Africa (5), Asia (8), New Zealand or Australia (12), South America

(11), Bermuda (1), Mexico (4), and Dominican Republic (1). Fourty four rust

sequences representing 17 additional species were acquired from GenBank when

they had a Cyperaceae or Juncaceae as host, were related to Cyperaceae-

Juncaceae rusts as evidenced from BLAST searches and preliminary phylogenetic

analyses, or to serve as outgroups (Austropuccinia Beenken and Dasyspora Berk. &

M.A. Curtis, Puccinia spp. on Poaceae; Data S1).

2.2 DNA extraction and sequencing

The amount of infected leaf tissue sampled per specimen depended on the sizes

of lesions and degree of infection. Infected leaf tissue was excised with scalpels, or

with 1 mm, 1.5 mm, 2 mm or 3 mm biopsy punches. Most often, two 2 mm biopsy

punches were used to excise infected regions of plant tissue. Samples were similarly

Accepted Article

excised from uninfected areas, from the same leaf as the rust whenever possible, for

host-only extractions. Methods for genomic DNA extraction were as described in

Hambleton et al. (2019) using a Macherey-Nagel NucleoMag® 96 Trace kit

(Macherey Nagel GmbH & Co. KG, Düren, Germany) and a KingFisher Flex

magnetic particle processor (Thermo Fisher Scientific Oy, Yantaa, Finland). The

protocol was modified as follows: prior to extraction, samples were homogenized

using liquid nitrogen and sterile disposable micro-centrifuge tube pestles (PES-15-B-

SI Axygen, Corning, NY USA), before suspending the DNA in 70 µl of elution buffer.

Methods for PCR amplification and sequencing of the nuclear rDNA ITS2 and

partial 28S region (ITS2-28S) for the rusts, and a portion of the ribulose-1,5-

bisphosphate carboxylase/oxygenase large subunit (rbcL) gene for the hosts, were

as described in Demers et al. (2017), with some modifications. For the rusts, PCR

fragments were typically amplified and sequenced using primers Rust2inv (Aime,

2006) and ITS4Ru1 (Rioux et al., 2015), which target ITS2 plus ca. 300 bp of the 28S

gene, or Rust2inv and ITS4 (White et al., 1990), which only target the ITS2 region,

when the amplification of longer fragments failed. For select samples, the reverse

primers LR5 or LR6 were used to target longer 28S fragments (Vilgalys & Hester,

1990). A few putatively misidentified host species were also barcoded for a fragment

of the external transcribed spacer region of rDNA (ETS-1f; Starr et al., 2003) using

primers ETS-1f and 18S-R or for the whole internal transcribed spacer region (ITS;

Cheng et al., 2016) using primers ITS-p5 and ITS-p4. The same protocol for

amplification was used for these regions as above, except for the following

modifications: 40 cycles of 94°C for 1 min, 48°C for 30 s, and 72°C for 2 min (ETS) or

40 cycles of 94°C for 30 s, 55°C for 40 s, and 72°C for 1 min (ITS). Voucher

Accepted Article

information and GenBank accession numbers of rust fungi and associated host

sequences can be found in Data S1.

Many of the specimens sampled for DNA in this study were processed as part of

a large-scale DNA barcoding project focused on generating reference sequence data

for a broad diversity of obligate plant pathogenic fungi housed in the DAOM

collection. There are challenges inherent in obtaining high-quality genomic data for

herbarium specimens collected and initially dried under unknown conditions. DNA

integrity is not necessarily related to age of the specimen (Liu & Hambleton, 2013),

meaning that success or failure at DNA barcode amplification is mostly

unpredictable. This routine initial PCR fragment for the rusts (comprising ITS2 and ~

300 bp of the adjacent 28S amplified with primers Rust2inv / ITS4Ru1) was adopted

because it can be sequenced bidirectionally with only two sequence reactions (one

forward, one reverse). Its use facilitates a rapid processing of specimens for initial

analyses and provides enough 28S signal to place the sequence near potential

relatives when the ITS2 sequence has no close matches. In analyses, this region

effectively groups specimens in OTUs that can then guide additional sequencing

efforts (eg. longer 28S), and the evaluation of other taxonomic characters.

We have observed that ITS2 is more amenable to direct sequencing than ITS1

because it possesses fewer indels and polybase regions that require cloning

approaches to resolve. In targeting the ITS2 region, we recognize that important

infraspecific variation may sometimes be difficult to interpret as compared to data

derived from the more conserved 28S region (McTaggart & Aime, 2018). Longer 28S

sequences were obtained for a select number of specimens but often only ITS2 could

be amplified and sequenced for many samples. No effort was made to sequence

alternative genes because of the lack of universal primers that work well across the

Accepted Article

diversity of rust fungi, especially for DNA from herbarium specimens and single copy

genes (Hambleton et al., 2019), and the labour-intensive effort required to design and

test specific primers for each new group being studied.

2.3 Rust and host identification

Rust identifications were based on the latest taxonomic treatments and floras

available (Savile, 1965, 1970a, 1972, 1979; Parmelee, 1967, 1969; Parmelee &

Savile, 1981; Klenke & Scholler, 2015). Preliminary phylogenetic analyses showed

that many of the species we sampled were split into two to many distinct lineages,

sometimes distantly related. In consequence, we circumscribed Operational

Taxonomic Units (OTUs) for downstream analyses using a combination of genetic,

host range and geographic criteria. OTUs were circumscribed as monophyletic

clades of ITS2-28S sequences that are consistently different from all other OTUs by

at least one substitution or indel, and occur on different telial or aecial host species,

or on a different continent from their closest relative(s). Using such a combination of

phylogenetic, sequence similarity, ecological and geographic criteria can increase the

chance that OTUs correspond to species, when compared to methods based on

sequence similarity alone (Lücking et al., 2020). All analyses used OTUs in place of

species. Samples were given a species name (New Determination/OTU in Data S1),

appended with a number for species split into two or more OTUs (e.g., “Puccinia

angustata 1”, “Puccinia angustata 2”). Host identifications were based on information

present on herbarium labels, verified using the morphology of leaf, stem and

sometimes inflorescence fragments found in rust herbarium packets, as well as by

BLAST or phylogenetic (parsimony) analysis of host rbcL, ETS or ITS barcodes.

Accepted Article

2.4 Evaluating the barcode gap and diversity of rust fungi

An ideal molecular marker for species identification and discovery would show

higher interspecific than intraspecific sequence divergence, i.e. a discrete DNA

barcode gap (Stielow et al., 2015). We assessed the utility of the ITS2-28S region for

species identification of Cyperaceae-Juncaceae rust fungi by testing for the presence

of global and local barcode gaps. Pairwise distances between every sample were

measured using the F84 substitution model in the ape v5.3 package, and an

alignment-free k-mer approach (with k=7) using the kmer v1.1.2 package in R v3.6.3

(Edgar, 2004; Wilkinson, 2018; Paradis & Schliep, 2019; R Core Team, 2020).

Distances were calculated on the central portion of the alignment which was covered

by 95% of the samples, essentially consisting of the whole ITS2 and 25bp of the

downstream 28S. There was a strong linear relationship (R²=0.86) between F84 and

k-mer distances, so we report only results obtained with k-mers, which are not

affected by alignment errors.

To detect a global barcode gap, the distribution of pairwise distances within OTUs

was plotted alongside the distribution of distances between OTUs, with the

expectation that a global gap would show as a break or minimal overlap between the

two distributions. Even in the absence of a global gap, each OTU can still form a

cluster that is distinct from all other clusters, or in other words a “local barcode gap”.

To detect a local barcode gap, we focused on OTUs represented by at least two

samples, and compared for each of these the minimum between-OTU distance to the

maximum within-OTU distance. A local barcode gap is supported for an OTU if its

minimum between-OTU distance is at least twice as large as the maximum within-

OTU distance. In other words, the length of the branches separating individuals of

two different OTUs should be at least twice as long as the length of the branches

Accepted Article

separating individuals of the same OTU for a local barcode gap to be recognized.

This is a conservative criterion compared to previous studies (e.g., Robinson et al.,

2009; Steinke et al., 2009), which considered a local barcode gap to exist when the

between-OTU distance was greater than the within-OTU distance. More sampling

within OTUs is expected to increase the maximum distance between individuals of

the same OTU. To determine whether uneven sampling of OTUs could drive

differences in the width of the local barcode gap, we ran an ordinary least-squares

analysis with number of samples per OTU as predictor, and maximum within-OTU

distance as response.

To estimate the magnitude of undescribed diversity existing in Cyperaceae-

Juncaceae rust fungi, richness accumulation curves were calculated for 11

morphologically-defined North American species (or species aggregates) where

more than five samples were available. We focused only on samples from the United

States and Canada because other countries were too poorly represented in the

dataset. A rarefaction curve was fit by treating OTUs as “species” and samples as

“individuals” in the R package vegan v2.5-6 (Oksanen et al., 2019), and checked the

presence or absence of a plateau. The Chao1 and ACE estimators of OTU richness

(O’Hara, 2005; Chiu et al., 2014) were also calculated as approximations of the total

number of OTUs (putative undescribed cryptic species) existing in each of the

studied rust species aggregates in North America north of Mexico.

2.5 Host phylogenetic data

We assembled phylogenetic datasets of monocot and dicot hosts using sequence

data available on GenBank (Data S2). For monocots, four plastid regions (rbcL,

matK, ndhF, trnL-F) and two nuclear ribosomal regions (ITS, ETS-1f) were selected

due to their good coverage across Cyperaceae and Juncaceae genera. Monocot

Accepted Article

hosts were selected based on the hosts on which rust OTUs were found, and

supplemented with host information from the literature when there was no ambiguity

about the rust species identity. In a few cases, sequence data was unavailable or

coverage was poor for a monocot host species, in which cases we selected a

representative species that is putatively closely-related based on morphology. For

these reasons, Rhynchospora capitellata was used as an examplar host for rusts on

Rhynchospora gigantea, R. rariflora, and R. glomerata; Cyperus papyrus was used

as substitute for Cyperus latifolius (both C4 Cyperus); Carex pellita for C. lasiocarpa

(both sect. Paludosae); and Carex lurida for C. frankii (both sect. Vesicariae). For

dicot hosts, sequences of two plastid barcodes (rbcL and matK) were compiled for

one examplar species of each host genus from GenBank. Voucher information and

GenBank accession numbers of host sequences used for these analyses can be

found in Data S2.

2.6 Phylogenetic analyses

Rust relationships were estimated by Bayesian analysis using MrBayes v3.2.7a

(Ronquist et al., 2012) on the Cipres Science Gateway (Miller et al., 2010). Two

GTR+G+I partitions were used: (1) 5.8S + 28S rDNA, (2) ITS2. We used a two-

exponential branch length prior with mean of 0.01 for internal and 0.1 for external

branches, following recommendations for minimizing over-estimation of posterior

probabilities (Yang & Rannala, 2005). To speed convergence, searches were started

on a tree derived from a previous short approximate-maximum likelihood analysis in

FastTree v2.1.11 (Price et al., 2010), with 50 random perturbations. Two independent

MC3 chains were run for 50 million generations sampling trees every 5000

generations, with 11 heated chains using a temperature parameter of 0.02.

Accepted Article

Branch support for rust fungi relationships were further assessed by maximum

parsimony and distance-based bootstrapping (Felsenstein, 1985). Maximum

parsimony bootstrap (PBS) searches were done in PAUP* 4.0a166 for Linux

(Swofford, 2003) using 1000 replicates, with each replicate consisting of three

random-addition sequences (RAS), retaining three trees per RAS, and using the

strict-consensus bootstrap (GRPFREQ=NO). In addition, 1000 distance-based

bootstrap (DBS) searches were done in PAUP* using a GTR+Gamma model with

empirical base frequencies and neighbor-joining (NJ) for tree estimation.

Because global-fit cophylogenetic methods work best with ultrametric trees, rust

relationships and divergence times were also jointly estimated in MrBayes v3.2.7a.

Settings were the same as above, except for the use of a birth-death (clock) branch

length prior with an exponential speciation prior of 10, flat beta(1,1) extinction prior,

and sampling probability of 5% assuming random sampling. For dating, we used an

uncorrelated gamma (IGR) clock (Lepage et al., 2007) with a lognormal clock rate of

0.0025 substitutions/site/My with a standard-deviation (SD) of 0.5 and an exponential

hyperparameter of 10. Following previously estimated divergence times of rust fungi

(Aime et al., 2018), we placed a truncated normal secondary prior on the divergence

between the Austropuccinia-Dasyspora outgroup and the Puccinia-Uromyces-

Aecidium ingroup with an average of 71 My, a SD of 8, and a minimum of 40 My.

Two independent MC3 chains were run for 120 million generations sampling trees

every 10,000 generations, with 11 heated chains using a temperature parameter of

0.02.

Monocot and dicot host relationships and divergence times were also jointly

estimated by Bayesian analysis using MrBayes v3.6.7a, to generate ultrametric trees

for cophylogenetic analysis. Fossil and secondary calibrations were placed on select

Accepted Article

nodes for dating (Smith et al., 2010; Barreda et al., 2015; Magallón et al., 2015;

Jiménez-Mejías et al., 2016; Mandel et al., 2019). Detailed methods can be found in

the supplementary material (Doc. S1).

For all Bayesian phylogenetic analyses, parameter convergence was assessed in

Tracer v1.7.1 (Rambaut et al., 2014), ten percent of runs was discarded as burnin,

and a maximum clade credibility chronogram (MCCT) was estimated using functions

of the paleotree R package (Bapst, 2012). Clade support was subjectively

characterized as poor (<0.90 pp), moderate (0.90–0.94 pp) and strong (0.95–1 pp).

2.7 Quantifying cophylogenetic signal

Cophylogenetic signal between rust fungi and their monocot and dicot hosts was

quantified on four datasets. Two “complete” datasets with all rust-monocot and rust-

dicot associations, including rust OTUs that are autoecious (with only one host type),

or where one of the alternate hosts was unknown, thus maximizing the amount of

available data. Two “reduced” datasets were also generated (rust-monocot and rust-

dicot) by pruning from phylogenies rust OTUs that are autoecious or with unknown

alternate host, thus keeping only OTUs that have known hosts for both monocots and

dicots. The “reduced” datasets are more appropriate when comparing cophylogenetic

signal between host types.

For each dataset, we ran two global-fit methods (PACo and Random TaPas), and

one event-based (Jane 4) method. Global-fit approaches work on distance matrices

(genetic or cophenetic) and are therefore robust to phylogenetic uncertainty. They

are most appropriate for the type of datasets presented here, where a single barcode

locus is used to estimate the parasite phylogeny, affording limited signal to resolve

backbone relationships. Event-based methods are less accurate when phylogenetic

Accepted Article

uncertainty is high, but they have the advantage of directly estimating the number of

cospeciation and host shift events.

PACo is based on Procrustes superimposition of Principal coordinates derived

from cophenetic matrices of host and parasite trees (Balbuena et al., 2013). Perfect

cospeciation results in host and parasite trees that are exactly identical in topology

and branch lengths, whereas other scenarios increase the distance between host

and parasite cophenetic matrices. The significance of cophylogenetic signal is tested

by random permutations of the host-parasite associations, and comparing the

observed Procrustes distance to the distribution of distances obtained by

permutations. We ran PACo analyses on all datasets using 10,000 permutations to

test for significance. While PACo is fast and relatively powerful at quantifying

cophylogenetic signal, the statistics obtained are not comparable between host-

parasite systems.

Random TaPas is a recently developed method which provides an absolute

measure of cophylogenetic signal (G*) that can be compared across different host-

parasite systems. The G* statistic ranges from G*=0 for perfect cospeciation, to

G*=2/3 for random associations, and up to G*=1 for highly uneven signal where most

clades are incongruent or random, but a few clades highly congruent (Balbuena et

al., 2020). Significant cophylogenetic signal is detected when the confidence interval

of G* does not overlap with 2/3. Random TaPas was run by calculating PACo fits on

N=10,000 random partial tanglegrams of size n=10% of the total number of

associations, which are optimal settings for quantification of cophylogenetic signal

(Balbuena et al., 2020). We obtained a point-estimate of G* on the MCCT, and

calculated 95% credibility intervals by re-running Ramdom TaPas on 500 randomly

selected host and parasite trees from the MrBayes posterior samples.

Accepted Article

Finally, we used Jane4, an event-based approach that estimates the number of

cospeciations and host-switches (Conow et al., 2010). Jane4 was run with a

population size of 200 for 20 generations using the following default costs (in steps):

cospeciation=0, duplication=1, host switch=2, loss=1, and failure to diverge=1. The

significance of the cophylogenetic signal was estimated by re-running the algorithm

on 100 randomly permuted host-parasite associations with the same settings, and

comparing the resulting cost distribution to the observed cost.

3 Results

3.1 Barcoding results

ITS2 barcodes were obtained for 157 of the 254 specimens sampled for this study

(62% sequencing success), including one specimen (DAOM 181219) yielding ITS2

barcodes for two different rust species from separate DNA extractions of aecial and

uredinial pustules. These 158 sequences were combined with previously sequenced

and unpublished ITS2 barcodes for 94 additional DAOM specimens. Sequencing of

the rust failed for 84 newly sampled specimens with collection dates between 1883

and 2013, and sequences were too short or difficult to interpret for another 13. As

well as the ITS2 spacer, the 28S was also successfully sequenced for a short length

(97–346 bp) in 211 sequences (83%), and for a longer length (421–1174 bp) in 37

sequences (15%). Good amplicons were obtained for specimens that ranged in age

from <1 to 136 years (oldest specimen collected in 1880), with the majority of

sequences from specimens collected before 1962. Basic sequence statistics can be

found in Table 1. Rust sequences were obtained to represent all the diversity of

Cyperaceae hosts, including 12 genera representing 11 tribes, although sampling

was biased towards North America and Europe. Additional sequences were obtained

on related Juncaceae and Iridaceae rusts, and on alternate dicot (aecial) hosts,

Accepted Article

including samples from Adoxaceae, Celastraceae, Elaeagnaceae, Grossulariaceae,

Lamiaceae, Orobanchaceae, Primulaceae, Urticaceae, and 37 genera and 9 tribes of

Asteraceae.

We obtained rbcL host plant barcodes for 130 of the 158 (82%) specimens

successfully sequenced for the rust in this study, and included previously generated

rbcL barcodes for 70 of the other 94 specimens. We also selectively generated ETS-

1f (21) or ITS (1) barcodes for 22 of those specimens, and ETS-1f (2) or ITS (3) for

five specimens for which rbcL sequencing failed. The rbcL host plant barcodes

revealed two host genus misidentifications; the 23 ETS-1f barcodes revealed three

host genus misidentifications and three host species misidentifications, and the four

ITS barcodes revealed a single host genus misidentification. Overall, host genus

misidentification rate was around 2.9% (6 misidentifications out of 205 specimens

with host barcodes). Host misidentification is difficult to assess at the species level,

because of the poor resolution of rbcL barcodes and limited number of more

informative ETS-1f and ITS barcodes.

3.2 Barcode gap analysis

Barcode gap analyses were done on the final rust alignment including a total of

296 sequences (252 sequences published here + 44 sequences from Genbank). The

distribution of within-OTU pairwise k-mer distances overlapped and was completely

included within the distribution of distances between OTUs, rejecting the presence of

a global barcode gap (Fig. 1). However, 56 out of the 72 OTUs (78%) represented by

more than one sample showed a local barcode gap, because the distance to the

nearest OTU was more than twice as large as the maximum within-OTU distance

(Fig. 1; supplementary Fig. S1), supporting the status of most OTUs as distinct

entities (differentiated populations or species). The maximum within-OTU distance,

Accepted Article

which is used to identify the width of the local barcode gap, was only weakly and

non-significantly associated with the number of individuals sampled per OTU (least

squares p=0.067, R2=0.047). A t-test did not detect a significant difference (p=0.37)

in sampling depth between OTUs that showed a local barcode gap (averaging 3.3

samples per OTU) and those that did not (averaging 2.9 samples per OTU). Depth of

sampling within OTUs is therefore unlikely to have influenced the detection of the

local barcode gap.

3.3 Rarefaction and richness analysis

Eleven rust species or aggregates were represented by enough North American

samples to be used in rarefaction and richness analyses. Of those, Puccinia

mcclatchieana Dietel & Holw. was the only taxon where all samples formed a clade

with little molecular variation. All others showed sample paraphyly, polyphyly, or

sufficient molecular variation for multiple OTUs (possible cryptic species) to be

recognized. The chao1 and ACE statistics estimated between two and four OTUs for

most rust aggregates, including the Puccinia asteris Duby agg., P. eriophori Thüm.

agg., P. lagenophorae Cooke agg., Uromyces junci Tul. agg., and U. silphii (Syd. &

P. Syd.) Arthur agg.

A few aggregates stood out by their high number of observed and estimated

OTUs. The rarefaction curve for the P. angustata Peck agg. was nearly flat at 25

samples, thus chao1 and ACE estimated the same number of OTUs as observed: 6

OTUs in total (Fig. 2). The situation was similar for the P. urticata DC. agg. with 14

North American samples included and 5 OTUs observed and estimated. The

rarefaction curve was still growing fast for the P. obtecta Peck agg. with only five

sampled specimens and ca. 3 OTUs observed, so that chao1 and ACE estimated

respectively 4 and 6.7 OTUs. The rarefaction curve for the P. caricina DC. agg. was

Accepted Article

also still growing fast at 28 included North American samples, so that 20 and 29.8

total OTUs were estimated, much more than the 13 observed OTUs. The rarefaction

curve for the highly paraphyletic Puccinia dioicae Magnus agg. also showed no sign

of leveling off at 12 samples, and chao1 and ACE were consequently high,

respectively estimating a total of 19.5 and 26.8 OTUs for this aggregate in North

America.

3.4 Phylogenetic results

Austropuccinia psidii and Dasyspora amazonica were sister to a strongly-

supported Puccinia + Uromyces + Aecidium clade (1.00 pp; 99% PBS; 93% DBS).

Within the latter, rusts of Poaceae [Puccinia coronata Corda, P. graminis Pers., P.

heterospora Berk. & M.A. Curtis, P. kuehnii (W. Krüger) E.J. Butler], P. malvacearum

Bertero ex Mont., and P. myrsiphylli (Thüm.) G. Winter formed a grade of lineages

(outgroups, not shown in Figs. 3–4) leading to a nested clade comprising all

Cyperaceae-Juncaceae rusts and related microcyclic species (Figs. 3–4). The rusts

on Cyperaceae-Juncaceae (ingroup) were monophyletic in all analyses, although

support was low (<0.50 pp; <50% PBS; 74% DBS; Figs. 3–4). However, a single

Carex rust, Puccinia microsora Körn., was placed with low support as part of the

early-diverged grade of Poaceae rusts that was included in the outgroup. Within the

clade of Cyperaceae-Juncaceae rusts, backbone relationships also received poor

support, but 143 OTUs were determined, including 43 identified at the species level,

and 100 corresponding best to 50 species in 27 species aggregates (Data S1).

Several moderately to strongly-supported clades emerged from a highly paraphyletic

group of microcyclic Asteraceae rusts, and samples identified as part of the Puccinia

dioicae agg. This aggregate accommodates nearly all rusts alternating between

Carex and Asteraceae. The 12 sampled specimens of the P. dioicae agg. formed 9

Accepted Article

distinct lineages placed throughout the phylogeny. Rusts on Juncaceae (Juncus L.

and Luzula DC.) formed seven lineages scattered throughout the phylogeny and

often associated with specimens of the P. dioicae agg. The crown age of

Cyperaceae-Juncaceae rusts was estimated to be Late Eocene (ca. 35 Mya), and

most major rust clades appeared between 34 and 12 Mya, coincident with the crown

age of Carex (34–37 Mya) and the divergence of its major clades (23–18 Mya;

Martín-Bravo et al., 2019; Figs. 3–4).

Rusts of Scirpeae formed several distantly related clades throughout the

phylogeny. A strongly-supported P. angustata agg. (1.00 pp, 99% DBS, 98% PBS;

Fig. 3) comprised rusts with telia on Scirpus Tourn. ex L. or Eriophorum L.

(Scirpeae), and aecia on Mentha L. or Lycopus L. (Lamiaceae tribe Mentheae).

Within the P. angustata agg., five subclades received high support (>0.95 pp), with

one of these subclades comprising rusts of Eriophorum originally identified as “P.

eriophori”. Other samples of P. eriophori formed two distantly related clades. One

comprising accessions of Puccinia eriophori var. apargidii Savile, including the

holotype, formed a strongly-supported clade (1.00 pp, <50% DBS, 96% PBS; Fig. 3)

that was distantly related to other accessions of “P. eriophori 1” (1.00 pp, 83% DBS,

97% PBS; Fig. 4). Puccinia mcclatchieana, a species on Scirpus without known

aecial host, formed a strongly supported subclade (1.00 pp, 95% DBS, 95% PBS;

Fig. 3) within the P. urticata Clade, which mostly comprises rusts of Carex. Puccinia

mcclatchieana showed no important sequence variation, although two samples

originally identified as “P. mcclatchieana” on Scirpus from Oregon and California

(DAOM 70596 and DAOM 134101) were found to be distantly related to other P.

mcclatchieana accessions. They clustered instead with samples of P. urticata, and

were accordingly re-identified as “P. urticata agg. 2”.

Accepted Article

A moderately supported P. eriophori-alpini Allesch.–P. dulichii P. Syd. & Syd.

clade consisted of species that alternate between Solidago L. (Asteraceae tribe

Astereae) and members of the Scirpo-Caricoid Clade: Trichophorum, Carex, and

Dulichium Pers. (0.93 pp, 98% DBS, 70% PBS; Fig. 3). Samples from Solidago

confirmed this aecial host for P. dulichii and one P. dioicae agg. species, but no

samples were available to confirm the aecial host of P. eriophori-alpini, which is

known from a single record on Solidago in Europe (Jørstad, 1942).

A moderately supported “Uromyces lineolatus Clade” (0.94 pp, <50% DBS and

PBS; Fig. 3) comprised all rusts of Fuireneae s.lat. with unicellular teliospores and

aecia on Apiaceae: Uromyces lineolatus (Desm.) Schröt. subsp. lineolatus, U.

lineolatus subsp. neoarcticus Savile, and U. americanus Speg. Within this clade, U.

lineolatus subsp. lineolatus, an Old World rust on Bolboschoenus (Asch.) Palla, was

separated by a long branch from a poorly-supported New World clade consisting of

U. lineolatus subsp. neoarcticus and U. americanus, a rust that uses Schoenoplectus

(Rchb.) Palla as a host.

Sister to the U. lineolatus Clade was a strongly-supported “Puccinia urticata

Clade” (1.00 pp, <50% DBS, 51% PBS; Fig. 3), comprising all rusts of Carex with 3+

pores on the urediniospores, and with aecia usually on Urtica L. (Urticaceae).

Samples identified as “P. urticata” formed five distinct lineages separated by long

branches. One sample of a segregate species recognized in Europe, P. urticae-

acutae Kleb., was not positioned with the other samples of this species. Nested

within the P. urticata clade were a few species with different host alternations: P.

iridis Wallr. alternating between Iris L. (Iridaceae) and Valeriana L. (Valerianaceae);

P. paludosa Plowr. between Carex and Pedicularis L. (Orobanchaceae); and P.

minutissima Arthur between Carex and Decodon J.F.Gmel. (Lythraceae).

Accepted Article

A strongly-supported “Puccinia caricina Clade” included all samples of P. caricina

and segregate species (1.00 pp, 66% DBS, 70% PBS; Fig. 4). These heteroecious

rusts alternate between Carex and Ribes L., Lysimachia L. or Parnassia L. The

autoecious derivative species P. parkerae Dietel & Holw. and P. ribis-japonici Henn.

of Ribes were nested in this clade. Species with distinctive characters such as single-

pored urediniospores (P. uniporula Orton) or aecia on genera other than Ribes (P.

karelica Tranzschel, P. limosae Magnus, P. uliginosa Juel) were nested within a

highly paraphyletic “P. caricina agg.” that formed subclades without obvious structure

in host ranges, but usually restricted either to samples from North America or from

Europe.

A strongly supported “Puccinia obtecta Clade” (1.00 pp, 83% DBS, 97% PBS; Fig.

4) consisted of all heteroecious rusts of Schoenoplectus and Cyperus L. with telia in

locules or surrounded by fused paraphyses, and aecia on Asteraceae, as well as a

few autoecious rusts on related genera of Asteraceae. Within this clade, a strongly-

supported subclade contained intermixed samples of P. obtecta and P. osoyoosensis

Savile (1.00 pp, 66% DBS, 98% PBS; Fig. 4), two species infecting Schoenoplectus

and alternating either on Bidens L. or Xanthium L. (Asteraceae tribe Heliantheae)

that differ by several morphological characters. Sister to this subclade was a

strongly-supported subclade including P. canaliculata (Schwein.) Lagerh. 2 (0.95 pp,

83% DBS, 68% PBS; Fig. 4), a rust of Cyperus also thought to alternate on

Xanthium, and autoecious derivatives on Xanthium and various other Heliantheae,

including accessions identified as P. xanthii Schwein. and P. melampodii Dietel &

Holw. that were not monophyletic.

Accepted Article

3.5 Cophylogenetic signal

Significant cophylogenetic signal was detected by global-fit (PACo, Random

TaPas) and event-based (Jane4) methods for complete and reduced datasets, and

for dicot and monocot hosts (Table 2). Yet, the event-based approach, Jane4,

estimated comparatively few cospeciations and a large number of host-switches and

duplications, suggesting that the significant cophylogenetic signal is not driven by

strict cospeciation.

The Random TaPas G* coefficient was >2/3 for all datasets, indicating that the

cophylogenetic signal was split between highly congruent clades (blue colours in Fig.

5), and highly discordant clades (red colours in Fig. 5). In monocots, high

cophylogenetic signal was visible in the deeper nodes of the Carex phylogeny

(Cariceae), and in the Scirpus-Eriophorum clade (Scirpeae; Fig. 5A). Highly

discordant signal comes from genera of Fuireneae s.lat., Eleocharis R.Br.

(Eleocharideae) and Cyperus (Cypereae), as well as Juncaceae. In dicots, the clade

comprising Grossulariaceae, Lythraceae, Celastraceae, Elaeagnaceae and

Urticaceae (Rosids) showed particularly discordant cophylogenetic signal as they

were hosts to several unrelated rust lineages (Fig. 5B). No other dicot lineage

showed particularly strong cophylogenetic signal.

In the reduced datasets, Jane4 estimated slightly more cospeciations and fewer

host switches in dicots compared to monocots, suggesting higher cophylogenetic

signal between rusts and dicot hosts. However, the confidence interval of the G*

coefficients of monocots and dicots overlapped in the reduced datasets (Table 2),

and there is thus no significant difference in cophylogenetic signal between monocot

and dicot hosts.

Accepted Article

4 Discussion

4.1 Cophylogenetic signal without cospeciation in Cyperaceae-Juncaceae rusts

Using the largest barcoding dataset of rust fungi to date (Feau et al., 2011;

Beenken et al., 2017), we demonstrated the utility of ITS2-28S barcodes for species

identification and discovery in North American Cyperaceae-Juncaceae rust fungi.

Perhaps surprisingly, our analyses suggest large numbers of host jumps and a

relatively minor role for cospeciation, in spite of the significant cophylogenetic signal

detected by both global-fit and event-based methods. A strict cospeciation scenario

can confidently be rejected, but what then explains the significant correlation we

observed between host and parasite phylogenies?

An emerging consensus is that strict cospeciation is exceedingly rare in nature,

and that other processes are responsible for the cophylogenetic signal observed in

most host-parasite associations (Nylin et al., 2018). The most recent review of the

empirical literature on the subject found only seven convincing examples of systems

dominated by cospeciation out of 103 cophylogenetic studies (de Vienne et al.,

2013). All conclusive cases were between invertebrates and internal mutualistic

prokaryotes that are transmitted internally from parent to offspring, limiting the

possibility of dispersal to new hosts (Bright & Bulgheresi, 2010). In external,

horizontally-transmitted parasites such as smut and rust fungi (Refrégier et al., 2008),

host jumps were the dominant mode of evolution. This could be due to the fact that

chance encounters with new hosts are more likely in external parasites. Indeed, rust

spores are mostly wind dispersed, and are therefore frequently deposited on non-

host species (Savile, 1976), providing constant opportunities to colonize new

species. Jumps may be further favored when related plant species live in close

sympatry, as is often the case in Cyperaceae (Elliott et al., 2016). Jumps may also be

Accepted Article

common due to the fact that it can rescue parasites from extinction in the

coevolutionary arms race with their hosts (Thines, 2019).

One way to explain the significant cophylogenetic signal we detected in

Cyperaceae-Juncaceae rusts, even in the absence of cospeciation, would be to

assume that host jumps occur most often between closely related hosts (Charleston

& Robertson, 2002). Such a model of “preferential host jumps” is supported by

inoculation experiments that showed fungal pathogens better able to infect novel host

plants when they are closely related to their normal hosts (Gilbert & Webb, 2007; de

Vienne et al., 2009). The same pattern was demonstrated in RNA viruses, with

probability of host jumps following a sigmoidal relationship with phylogenetic distance

between hosts, indicating that jumps to closely related hosts are easy, whereas

moderately and distantly related hosts become quickly unreachable (Cuthill &

Charleston, 2013). Preferential host jumps have now been documented in a large

variety of plant and animal systems, and appear to be the rule in most systems (de

Vienne et al., 2013). Preferential host jumps were also suggested to be the main

driver behind cophylogenetic congruence of Anthracoidea smuts and their Carex

hosts (Hendrichs et al., 2005; Escudero, 2015), a classic example of host-parasite

correlation in Cyperaceae (Savile, 1952; Savile & Calder, 1953; Kukkonen &

Timonen, 1979). The rust fungi of Cyperaceae and Juncaceae appear to be yet

another example of cophylogenetic signal driven by preferential host jumps.

4.2 Cophylogenetic signal does not differ significantly between aecial and telial hosts

A previous study found that correlations between rusts and host phylogenies are

stronger on aecial than on telial hosts (Aime et al., 2018), a pattern we call here

“differential host conservatism”. Differential host conservatism could be explained by

the fact that sexual reproduction occurs on dicot aecial hosts, therefore making

Accepted Article

infection of novel aecial hosts more disadvantageous than infection of novel telial

hosts. Indeed, a novel aecial host reduces the rust’s outcrossing opportunities,

whereas a novel telial hosts does not affect any component of the rust reproductive

cycle.

We find indications of differential host conservatism in the complete datasets,

when cophylogenetic signal is compared in dicot (mostly aecial) and monocot (telial)

hosts. However, analyses on reduced datasets where both aecial and telial hosts are

known for all terminals finds no significant difference in cophylogenetic signal. Thus,

the differential host conservatism detected on the complete datasets is likely caused

by the different size of the datasets, with the complete monocot dataset containing a

larger number of terminals and hence higher chance of catching instances of

incongruent signal. In consequence, the lack of a significant difference in

cophylogenetic signal between telial and aecial hosts in Cyperaceae-Juncaceae

rusts contrasts with the much stronger differential host conservatism reported by

Aime et al. (2018) at higher taxonomic levels in rust fungi.

Our results would fit a pattern where differential host conservatism is stronger in

more ancient rust lineages, such as between rust families (Aime et al., 2018), but

weaker in recent lineages such as within rust genera (present results). Such a decay

of differential host conservatism rejects the explanation that aecial hosts are

conserved due to reduced mating opportunities on novel aecial hosts, because this

phenomenon should act relatively fast in creating differences in host jump

probabilities. Hence, if this was true, it should be equally detectable at all taxonomic

levels. Consequently, the decay we observed points to a role for slowly-acting

evolutionary forces that would only create differences at longer evolutionary

timescales. Such forces could include differential speed of the coevolutionary arms

Accepted Article

race in aecial and telial hosts, or the effect of one of the many biological

characteristics that differ between spore stages, such as ploidy (Ono, 2002) or

virulence (Rice and Westoby, 1982). More studies are needed to determine the

extent of differential host conservatism and to identify the causal factors behind the

pattern.

4.3 Utility of rust fungi relationships in Cyperaceae-Juncaceae classification

The fact that cophylogenetic signal is driven by preferential host jumps in the

Cyperaceae-Juncaceae rusts does not exclude the possibility that rust relationships

might corroborate proposed host relationships. In some of his most influential

studies, Savile (1972, 1979) hypothesized a close affinity between several groups of

Cyperaceae genera that shared the same rust lineages. For instance, he proposed

that Carex, Trichophorum, Scirpus, Eriophorum and Dulichium were closely related

based on the occurrence of rusts classified within the “dioicae-hieracii lineage”

infecting all these genera (Savile, 1970a). Such a close relationship among these

temperate sedge genera had never been proposed before, but molecular

phylogenetic studies have since placed them all in a strongly-supported “Scirpo-

Caricoid Clade” (Léveillé-Bourret et al., 2014, 2015, 2018a; Semmouri et al., 2019;

Léveillé-Bourret & Starr, 2019). Nevertheless, molecular analysis of the rusts

invalidates Savile’s hypothesis because members of his “dioicae-hieracii lineage”,

such as the Puccinia dioicae agg., P. dulichii, P. eriophori and U. perigynius Halst.

are scattered throughout the rust phylogeny, as in previous analyses (van der Merwe

et al., 2007, 2008). It therefore seems probable that characters used to delimit

Savile’s “dioicae-hieracii lineage”, such as bizonate aeciospores, flattened

urediniospores with two super-equatorial pores, and telia without paraphyses, are

Accepted Article

plesiomorphic or convergent in Cyperaceae-Juncaceae rusts (Puccinia–Uromyces

clade I sensu Maier et al., 2007).

The close relationship of Bulbostylis Kunth and Eleocharis, supported by all

phylogenetic analyses of Cyperaceae (e.g., Muasya et al., 2009; Hinchliff & Roalson,

2013; Semmouri et al., 2019), was also thought to be supported by rust fungi (Savile,

1979). However, the rusts of Bulbostylis and Eleocharis are here shown to form three

separate and seemingly distant clades. Likewise, the fact that all rusts infecting

Fuireneae s.lat. genera (Bolboschoenus, Schoenoplectus, Schoenoplectiella Lye,

Fuirena Rottb.) and Eleocharis possess urediniospores with 3+ equatorial pores and

loculate telia has been suggested to reflect correlated evolution of rusts and their

hosts (Savile, 1979; Goetghebeur, 1998). However, urediniospores with 3+ pores

and loculate telia are here shown to have evolved in four lineages from 2-pored, non-

loculate ancestors: (1) in the Uromyces lineolatus Clade; (2) in Puccinia liberta F.

Kern; (3) in P. canaliculata 1; (4) and in the P. obtecta Clade. Repeated evolution of

loculate telia might be better understood as another example of parallel adaptation in

rusts subject to similar ecological pressures (Savile, 1976, 1978), as most Fuireneae

s.lat. and Eleocharideae produce emergent, leafless culms in open marshes. Their

culms offer a substrate subject to intense sunlight and heat that may have favored

rust species able to protect their resting spores by enclosing them within locules.

Stronger cophylogenetic signal is observed at lower taxonomic levels. Notably,

subclades within the Puccinia angustata agg. are each restricted to different

subclades of Scirpus and Eriophorum species, suggesting greater host specificity

than previously recognized (Scirpeae; Léveillé-Bourret et al., 2014, 2015). Rusts on

genera of Fuireneae s.lat. (Bolboschoenus, Schoenoplectus), a Cyperaceae tribe

long suspected to be unnatural (Shiels et al., 2014; Glon et al., 2017; Semmouri et

Accepted Article

al., 2019), formed two distantly related clades that diverged in the Mid-Miocene (ca.

13 Mya), despite sharing highly distinctive loculate telia. These two rust clades differ

in important morphological and ecological features (Savile, 1972; Klenke & Scholler,

2015), and their divergence supports the recognition of separate tribes for the genera

Bolboschoenus and Schoenoplectus (Bolboschoeneae and Schoenoplecteae; see

Starr et al., 2021 in this issue). A more detailed discussion of cophylogenetic patterns

and their taxonomic implications can be found in the supplementary material (Doc.

S2).

4.4 Challenges and prospects of ITS2-28S for the discovery and identification of rust

species

The ITS2-28S provides good resolution for the identification of rust fungi, and

enough variation to serve as a first step in the discovery of new species, as

demonstrated here and in previous studies (e.g., McTaggart & Aime, 2018). The

absence of a global barcode gap is in line with previous studies on this locus (Stielow

et al., 2015), but is compensated by the presence of a local gap in most species or

OTUs (Robinson et al., 2009; Steinke et al., 2009). Combined with host and

distribution information, we expect ITS2-28S to provide good resolution for rust

identification, even in the most species-rich lineages. The large number of indels in

ITS2, and the short length of the 28S sequence usually obtained with our primers

provided only limited phylogenetic signal, but its relative ease of amplification and

specificity enables the rapid and extensive study of hundreds of taxa, even when old

specimens (>100 years) must be used. This is an important feature for barcoding a

group of fungi that is very diverse, but also severely under-collected in recent years

(Liu & Hambleton, 2010). In the future, increasingly sensitive genome sequencing

techniques and an acceleration in the availability of a diversity of rust genomes will

Accepted Article

lead to the recognition of optimal regions for molecular phylogenetic analyses and

facilitate the development of new robust PCR primers for the rusts.

Our barcoding approach highlights the need for more in-depth studies of the

taxonomy of Cyperaceae-Juncaceae rusts, by demonstrating that the broad species

limits still in use in North America are in many case unnatural (high levels of sample

paraphyly or polyphyly). We estimate that between 5 and 24 potential cryptic species

(OTUs) could exist within each of the four most common and abundant North

American species aggregates on Carex, Scirpus and Eriophorum, the Puccinia

angustata s.lat., P. caricina agg., P. dioicae agg. and P. urticata agg. This leads us to

believe that the total number of rust species on Cyperaceae-Juncaceae recognized

in North America will more than double when detailed taxonomic studies combining

molecular and ecological data are made. In fact, if every OTU delimited here

corresponds to one rust species and the richness estimates are reliable, a minimum

of 90 rust species of Cyperaceae and 10 of Juncaceae would be expected in North

America north of Mexico. This would be roughly one rust species for every ten

Cyperaceae-Juncaceae species (Ball et al., 2002), which is well below the ratio of

one rust species for every two to three Cyperaceae species reported from Germany

(Klenke & Scholler, 2015). Such numbers are unsurprising given that similarly large

numbers of cryptic species are being discovered in many other groups of fungal plant

parasites (Roy et al., 1998; Pažoutová et al., 2015; Riess et al., 2019; Shoukouhi et

al., 2019; Kemler et al., 2020; Liu et al., 2020). Moreover, taxonomists have long

recognized the likelihood of cryptic speciation in several of the rust complexes we

studied, but were unable to discern robust diagnostic features due to the limited

number of characters that can be studied with traditional morphological approaches

(Savile, 1972, 1973, 1984; Parmelee, 1989).

Accepted Article

In Europe, narrowly circumscribed species have been in use for a long time in the

notoriously difficult heteroecious rusts of Carex, giving greater weight to host range

and ecology compared to the broader “morphological species” recognized in North

America (Banz & Zwetko, 2018). This was made possible by abundant cross-

inoculation studies and careful observations in the field, data sources that are more

limited in North America. Our molecular results suggest that the narrower species

limits used in Europe may be more appropriate, although future studies will need to

confirm that the OTUs delimited here correspond to species, rather than intraspecific

variants. DNA barcoding promises to play an important role in rust species

delimitation by replacing labour-intensive cross-inoculations and field observations

with easily-obtained molecular data when linking life stages on telial (monocot) and

aecial (dicot) hosts. Combined with other modern techniques such as multi-locus

approaches to species delimitation (Rintoul et al., 2012), and the re-examination of

morphological features using electron microscopy and morphometric analysis

(Zwetko & Blanz, 2012; Liu & Hambleton, 2013), the tools are now available to

document the diversity of undescribed microfungi that occur on Cyperaceae and

Juncaceae in North America. A detailed discussion of the taxonomic implications of

the phylogenetic results for a few important rust species aggregates and their

correlated microcyclic or autoecious relatives can be found in the supplementary

material (Doc. S2).

4.5 Diversity begets diversity in the rusts of Carex

The genus Carex (>2000 species) is larger than 92% of all flowering plant

families. With a distribution that spans all the continents except Antarctica, its species

can be found in habitats as varied as tropical rain forests, deserts, prairies and arctic

tundra (Starr et al., 2015; Global Carex Group, 2015; Martín-Bravo et al., 2019). This

Accepted Article

broad diversity is probably the driving factor behind the importance of Carex as telial

host to such a phylogenetically and taxonomically diverse collection of rusts. Our

results provide evidence that Carex diversity has promoted the diversification of their

rust parasites because (1) the timing of origin of Carex and its major clades appears

to coincide roughly with the origin and diversification of the Cyperaceae-Juncaceae

rusts (Puccinia–Uromyces clade I sensu Maier et al., 2007), and (2) several major

rust lineages such as the P. urticata Clade and the P. caricina Clade are mostly

restricted to a single small aecial host genus (respectively Urtica and Ribes), and yet

they appear to comprise numerous cryptic rust species or races that have probably

evolved by specialization on one or a few Carex hosts. This is not entirely

unexpected as studies on other parasitic associations have shown that the

diversification of plant hosts drives the diversification of their insect associates (Janz

et al., 2006; Cruaud et al., 2012), and that taxonomically diverse host communities

increase parasite diversity (e.g., in amphibians, Johnson et al., 2016; birds,

Hechinger and Lafferty, 2005; and insect protective symbionts (Hafer & Vorburger,

2019). Simulations also suggest that rapidly diversifying host clades promote parasite

diversification in the presence of preferential host shifts (Engelstädter & Fortuna,

2019).

The high rate of ecological niche evolution observed in rapidly radiating lineages

of Carex (Pender, 2015; Spalink et al., 2016; Pender et al., 2021 in this issue) may

also have played a role in the diversification of their rust fungi, by facilitating the

evolution of new aecial host associations or by promoting ecological specialization.

As more phylogenetic data accumulates on rust fungi and as taxonomic studies

clarify species limits, diversity and host ranges, it should soon be possible to explicitly

test for correlations between rust and host diversification rates. We suspect that such

Accepted Article

studies will find that the amazing diversity of rust fungi on Carex is more than mere

coincidence.

Acknowledgements

Development of molecular data for DAOM specimens was supported by funding

from the Genomics Research and Development Initiative (GRDI-QIS, Project ID

2679) of the Government of Canada (https://grdi.canada.ca/en). For assistance in

locating herbarium specimens and permitting DNA work, we thank Jennifer Wilkinson

and Scott Redhead (Canadian National Mycological Herbarium, Ottawa, Canada).

We also thank Sylvia Wilson and the Molecular Technologies Laboratory (MTL) at

the Ottawa Research & Development Centre, specifically Julie T. Chapados, Kasia

Dadej, Wayne McCormick and Lisa Koziol, for technical assistance. Finally, we thank

Julie Carey for preparing all the GenBank submissions. Part of this research was

conducted while the first author was a PhD student at the University of Ottawa

(UofO) with support from an Alexander-Graham-Bell NSERC Research Scholarship,

a FRQNT Doctoral Scholarship, and an UofO Excellence Scholarship. This work was

also supported by National Science and Engineering Research Council of Canada

(NSERC) Discovery Grants to Julian R. Starr (RGPINs 342278-2013 and 2018-

04115). Two anonymous reviewers provided helpful comments on an early version of

this manuscript.

References

Aime MC. 2006. Toward resolving family-level relationships in rust fungi

(Uredinales). Mycoscience 47, 112–122. https://doi.org/10.1007/S10267-006-

0281-0

Aime MC, Bell CD, Wilson AW. 2018. Deconstructing the evolutionary complexity

between rust fungi (Pucciniales) and their plant hosts. Studies in Mycology 89,

143–152.

Accepted Article

Aime MC, Toome M, McLaughlin DJ. 2014. Pucciniomycotina. In: McLaughlin DJ,

Spatafora JW eds. The Mycota, volume 7A: systematics and evolution.

Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 271–294.

https://doi.org/10.1007/978-3-642-55318-9_10

Arthur JC. 1934. Manual of the rusts in United States and Canada. Purdue Research

Foundation, Lafayette, Indiana (United-States).

Balbuena JA, Míguez-Lozano R, Blasco-Costa I. 2013. PACo: a novel procrustes

application to cophylogenetic analysis. PLoS ONE 8, e61048.

https://doi.org/10.1371/journal.pone.0061048

Balbuena JA, Pérez-Escobar ÓA, Llopis-Belenguer C, Blasco-Costa I. 2020. Random

Tanglegram Partitions (Random TaPas): an alexandrian approach to the

cophylogenetic gordian knot. Systematic Biology 69: 1212–1230.

Ball PW, Reznicek AA, Murray DF 2002. Cyperaceae Jussieu. In: Flora of North

America Editorial Committee eds. Flora of North America north of Mexico,

vol. 23. Oxford University Press, Oxford.

Brooks RE, Clemants SE. 2000. Juncaceae A. L. Jussieu. In: Flora of North America

Editorial Committee eds. Flora of North America north of Mexico, vol. 22.

Oxford University Press, Oxford.

Banz P, Zwetko P. 2018. Remarks on species concepts in European Florae of rust

fungi. In: Banz P ed. Biodiversity and ecology of fungi, lichens, and mosses:

Kerner von Marilaun Workshop 2015 in Memory of Josef Poelt. Verlag der

Österreichischen Akademie der Wissenschaften, Wien, pp. 289–310.

Bapst DW. 2012. paleotree: an R package for paleontological and phylogenetic

analyses of evolution. Methods in Ecology and Evolution 3: 803–807.

https://doi.org/10.1111/j.2041-210X.2012.00223.x

Barnes CW, Kinkel LL, Groth JV. 2005. Spatial and temporal dynamics of Puccinia

andropogonis on Comandra umbellata and Andropogon gerardii in a native

prairie. Canadian Journal of Botany 83, 1159–1173.

https://doi.org/10.1139/b05-087

Barreda VD, Palazzesi L, Tellería MC, Olivero EB, Raine JI, Forest F. 2015. Early

evolution of the angiosperm clade Asteraceae in the Cretaceous of Antarctica.

Proceedings of the National Academy of Sciences 112, 10989–10994.

https://doi.org/10.1073/pnas.1423653112

Beenken L, Lutz M, Scholler M. 2017. DNA barcoding and phylogenetic analyses of

the genus Coleosporium (Pucciniales) reveal that the North American

goldenrod rust C. solidaginis is a neomycete on introduced and native

Solidago species in Europe. Mycological Progress 16, 1073–1085.

https://doi.org/10.1007/s11557-017-1357-2

Accepted Article

Blackwell M. 2011. The Fungi: 1, 2, 3 … 5.1 million species? American Journal of

Botany 98, 426–438. https://doi.org/10.3732/ajb.1000298

Blackwell M, Vega FE. 2018. Lives within lives: hidden fungal biodiversity and the

importance of conservation. Fungal Ecology 35, 127–134.

https://doi.org/10.1016/j.funeco.2018.05.011

Bright M, Bulgheresi S. 2010. A complex journey: transmission of microbial

symbionts. Nature Reviews Microbiology 8, 218–230.

https://doi.org/10.1038/nrmicro2262

Brodie JF, Aslan CE, Rogers HS, Redford KH, Maron JL, Bronstein JL, Groves CR.

2014. Secondary extinctions of biodiversity. Trends in Ecology & Evolution

29, 664–672. https://doi.org/10.1016/j.tree.2014.09.012

Bruhl JJ, 1995. Sedge genera of the world: relationships and a new classification of

the Cyperaceae. Australian Systematic Botany 8, 125–305.

Caro T. 2010. Conservation by proxy: indicator, umbrella, keystone, flagship, and

other surrogate species, 2nd ed. Island Press.

Charleston MA, Robertson DL. 2002. Preferential host switching by primate

Lentiviruses can account for phylogenetic similarity with the primate

phylogeny. Systematic Biology 51, 528–535.

https://doi.org/10.1080/10635150290069940

Cheng T, Xu C, Lei L, Li C, Zhang Y, Zhou S. 2016. Barcoding the kingdom Plantae:

new PCR primers for ITS regions of plants with improved universality and

specificity. Molecular Ecology Resources 16, 138–149.

https://doi.org/10.1111/1755-0998.12438

Chiu CH, Wang YT, Walther BA, Chao A. 2014. An improved nonparametric lower

bound of species richness via a modified good-turing frequency formula.

Biometrics 70, 671–682. https://doi.org/10.1111/biom.12200

Conow C, Fielder D, Ovadia Y, Libeskind-Hadas R. 2010. Jane: a new tool for the

cophylogeny reconstruction problem. Algorithms for Molecular Biology 5,

Article 16. https://doi.org/10.1186/1748-7188-5-16

Cruaud A, Rønsted N, Chantarasuwan B, Chou LS, Clement WL, Couloux A,

Cousins B, Genson G, Harrison RD, Hanson PE, Hossaert-Mckey M, Jabbour-

Zahab R, Jousselin E, Kerdelhué C, Kjellberg F, Lopez-Vaamonde C, Peebles

J, Peng YQ, Pereira RAS, Schramm T, Ubaidillah R, van Noort S, Weiblen

GD, Yang DR, Yodpinyanee A, Libeskind-Hadas R, Cook JM, Rasplus JY,

Savolainen V. 2012. An extreme case of plant–insect codiversification: figs

and fig-pollinating wasps. Systematic Biology 61, 1029–1047.

https://doi.org/10.1093/sysbio/sys068

Accepted Article

Cummins GB, Hiratsuka Y. 2003. Illustrated genera of rust fungi, 3rd ed. American

Phytopathological Society (APS), St. Paul, Minnesota (United States).

Cuthill JH, Charleston MA. 2013. A simple model explains the dynamics of

preferential host switching among mammal RNA viruses. Evolution 67, 980–

990. https://doi.org/10.1111/evo.12064

Dahlgren R. 1983. General aspects of angiosperm evolution and macrosystematics.

Nordic Journal of Botany 3, 119–149. https://doi.org/10.1111/j.1756-

1051.1983.tb01448.x

de Vienne DM, Hood ME, Giraud T. 2009. Phylogenetic determinants of potential

host shifts in fungal pathogens. Journal of Evolutionary Biology 22, 2532–

2541. https://doi.org/10.1111/j.1420-9101.2009.01878.x

de Vienne DM, Refrégier G, López-Villavicencio M, Tellier A, Hood ME, Giraud T.

2013. Cospeciation vs host-shift speciation: methods for testing, evidence

from natural associations and relation to coevolution. New Phytologist 198,

347–385. https://doi.org/10.1111/nph.12150

Demers JE, Liu M, Hambleton S, Castlebury LA. 2017. Rust fungi on Panicum.

Mycologia 109, 1–17. https://doi.org/10.1080/00275514.2016.1262656

Edgar RC. 2004. Local homology recognition and distance measures in linear time

using compressed amino acid alphabets. Nucleic Acids Research 32, 380–385.

https://doi.org/10.1093/nar/gkh180

Eichler W. 1948. XLI.--Some rules in ectoparasitism. The Annals and Magazine of

Natural History Series 12 1, 588–598.

Elliott TL, Waterway MJ, Davies TJ. 2016. Contrasting lineage-specific patterns

conceal community phylogenetic structure in larger clades. Journal of

Vegetation Science 27, 69–79. https://doi.org/10.1111/jvs.12345

Engelstädter J, Fortuna NZ. 2019. The dynamics of preferential host switching: host

phylogeny as a key predictor of parasite distribution. Evolution 73: 1330–

1340. https://doi.org/10.1111/evo.13716

Escudero M. 2015. Phylogenetic congruence of parasitic smut fungi (Anthracoidea,

Anthracoideaceae) and their host plants (Carex, Cyperaceae): cospeciation or

host-shift speciation? American Journal of Botany 102, 1108–1114.

https://doi.org/10.3732/ajb.1500130

Fahrenholz H. 1913. Ectoparasiten und abstammungslehre. Zoologischer Anzeiger 41,

371–374.

Accepted Article

Feau N, Vialle A, Allaire M, Maier W, Hamelin RC. 2011. DNA barcoding in the rust

genus Chrysomyxa and its implications for the phylogeny of the genus.

Mycologia 103, 1250–1266. https://doi.org/10.3852/10-426

Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the

bootstrap. Evolution 39, 783–791. https://doi.org/10.2307/2408678

Fernald ML. 1905. The North American species of Eriophorum. Part II. Notes on the

preceeding synopsis. Rhodora 7, 129–136.

Frodin DG. 2004. History and concepts of big plant genera. Taxon 53, 753–776.

Geils BW, Hummer KE, Hunt RS. 2010. White pines, Ribes, and blister rust: a review

and synthesis. Forest Pathology 40, 147–185. https://doi.org/10.1111/j.1439-

0329.2010.00654.x

Gilbert GS, Webb CO. 2007. Phylogenetic signal in plant pathogen-host range.

Proceedings of the National Academy of Sciences 104, 4979–4983.

https://doi.org/10.1073/pnas.0607968104

Gilmour CN, Starr JR, Naczi RFC. 2013. Calliscirpus, a new genus for two narrow

endemics of the California Floristic Province, C. criniger and C. brachythrix

sp. nov. (Cyperaceae). Kew Bulletin 68, 84–105.

https://doi.org/10.1007/s12225-012-9420-2

Global Carex Group, 2015. Making Carex monophyletic (Cyperaceae, tribe

Cariceae): a new broader circumscription. Botanical Journal of the Linnean

Society 179, 1–42. https://doi.org/10.1111/boj.12298

Glon HE, Shiels DR, Linton E, Starr JR, Shorkey AL, Fleming S, Lichtenwald SK,

Schick ER, Pozo D, Monfils AK. 2017. A five gene phylogenetic study of

Fuireneae (Cyperaceae) with a revision of Isolepis humillima. Systematic

Botany 42, 26–36. https://doi.org/10.1600/036364417X694601

Goetghebeur P. 1998. Cyperaceae. In: Kubitzki K ed. Flowering Plants,

Monocotyledons: Alismatanae and Commelinanae (except Gramineae), The

Families and Genera of Vascular Plants. Springer, New York (United-States),

pp. 141–190.

Govaerts R, Simpson DA, Bruhl JJ, Egorova, T., Goetghebeur P, Wilson K. 2007.

World checklist of Cyperaceae. Kew Publishing, Kew (England).

Hafer N, Vorburger C. 2019. Diversity begets diversity: do parasites promote

variation in protective symbionts? Current Opinion in Insect Science 32, 8–14.

https://doi.org/10.1016/j.cois.2018.08.008

Accepted Article

Hambleton S, Liu M, Eggertson Q, Wilson S, Carey J, Anikster Y, Kolmer JA. 2019.

Crown rust fungi with short lifecycles – the Puccinia mesnieriana species

complex. Sydowia 71: 47–63.

Hawksworth DL, Rossman AY. 1997. Where are all the undescribed fungi?

Phytopathology 87, 888–891. https://doi.org/10.1094/PHYTO.1997.87.9.888

Hechinger RF, Lafferty KD. 2005. Host diversity begets parasite diversity: bird final

hosts and trematodes in snail intermediate hosts. Proceedings of the Royal

Society B: Biological Sciences 272, 1059–1066.

https://doi.org/10.1098/rspb.2005.3070

Henderson DM. 2004. The rust fungi of the British Isles: a guide to identification by

their host plant. British Mycological Society, Kew (England).

Hendrichs M, Begerow D, Bauer R, Oberwinkler F. 2005. The genus Anthracoidea

(Basidiomycota, Ustilaginales): a molecular phylogenetic approach using LSU

rDNA sequences. Mycological Research 109, 31–40.

Henk DA, Farr DF, Aime MC, 2011. Mycodiplosis (Diptera) infestation of rust fungi

is frequent, wide spread and possibly host specific. Fungal Ecology 4, 284–

289. https://doi.org/10.1016/j.funeco.2011.03.006

Hinchliff CE, Roalson EH, 2013. Using supermatrices for phylogenetic inquiry: an

example using the sedges. Systematic Biology 62, 205–219.

https://doi.org/10.1093/sysbio/sys088

Hiratsuka N. 1933. Inoculation experiments with heteroecious species of the Japanese

rust fungi. The Botanical Magazine, Tokyo 47, 710–714.

Ito S. 1934. Cultures of Japanese Uredinales I. The Botanical Magazine, Tokyo 48,

531–539. https://doi.org/10.15281/jplantres1887.48.531

Jackson HS. 1931. Present evolutionary tendencies and the origin of the life cycles in

the Uredinales. Memoirs of the Torrey Botanical Club 18, 1–108.

Janz N, Nylin S, Wahlberg N. 2006. Diversity begets diversity: host expansions and

the diversification of plant-feeding insects. BMC Evolutionary Biolology 6, 4.

https://doi.org/10.1186/1471-2148-6-4

Jiménez-Mejías P, Martinetto E, Momohara A, Popova S, Smith SY, Roalson EH.

2016. A commented synopsis of the pre-Pleistocene fossil record of Carex

(Cyperaceae). The Botanical Review 82, 258–345.

https://doi.org/10.1007/s12229-016-9169-7

Johnson PTJ, Wood CL, Joseph MB, Preston DL, Haas SE, Springer YP. 2016.

Habitat heterogeneity drives the host-diversity-begets-parasite-diversity

Accepted Article

relationship: evidence from experimental and field studies. Ecology Letters 19,

752–761. https://doi.org/10.1111/ele.12609

Jørstad I. 1942. The aecidial stage of Puccinia confinis Sydow. Nyt Magazin for

Naturvidenskaberne 83, 100–103.

Kemler M, Denchev TT, Denchev CM, Begerow D, Piątek M, Lutz M. 2020. Host

preference and sorus location correlate with parasite phylogeny in the smut

fungal genus Microbotryum (Basidiomycota, Microbotryales). Mycological

Progress 19, 481–493. https://doi.org/10.1007/s11557-020-01571-x

Kirschner J. 2002. Juncaceae 1: Rostkovia to Luzula, in: Orchard AE, Bleyerveen JE,

Wilson AJG, Kuchlmayer B eds. Species Plantarum: Flora of the World.

Australian Biological Ressources Study, Canberra, p. 122.

Klassen GJ. 1992. Coevolution: a history of the macroevolutionary approach to

studying host-parasite associations. Journal of Parasitology 78, 573–587.

Klenke F, Scholler M. 2015. Pflanzenparasitische Kleinpilze. Springer Berlin

Heidelberg, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-46162-4

Koh LP. 2004. Species coextinctions and the biodiversity crisis. Science 305, 1632–

1634. https://doi.org/10.1126/science.1101101

Koyama T. 1958. Taxonomic study of the genus Scirpus Linné. Journal of the Faculty

of Science, University of Tokyo, Section 3. Botany 7, 271–366.

Kukkonen I, Timonen T. 1979. Species of Ustilaginales, especially of the genus

Anthracoidea, as tools in plant taxonomy. Symbolae botanicae Upsalienses

22, 166–176.

Larsen BB, Miller EC, Rhodes MK, Wiens JJ. 2017. Inordinate fondness multiplied

and redistributed: the number of species on Earth and the new pie of life. The

Quaternary Review of Biology 92, 229–265. https://doi.org/10.1086/693564

Lepage T, Bryant D, Philippe H, Lartillot N. 2007. A general comparison of

molecular clock models. Molecular Biology and Evolution 24, 2669–2680.

https://doi.org/10.1093/molbev/msm193

Léveillé-Bourret É, Donadío S, Gilmour CN, Starr JR, 2015. Rhodoscirpus

(Cyperaceae: Scirpeae), a new South American sedge genus supported by

molecular, morphological, anatomical and embryological data. Taxon 64, 931–

944. http://dx.doi.org/10.12705/645.4

Léveillé-Bourret É, Gilmour CN, Starr JR, Naczi RFC, Spalink D, Sytsma KJ. 2014.

Searching for the sister to sedges (Carex): resolving relationships in the

Cariceae-Dulichieae-Scirpeae clade (Cyperaceae). Botanical Journal of the

Linnean Society 176, 1–21. https://doi.org/10.1111/boj.12193

Accepted Article

Léveillé-Bourret É, Starr JR. 2019. Molecular and morphological data reveal three

new tribes within the Scirpo-Caricoid Clade (Cyperoideae, Cyperaceae).

Taxon 68, 218–245. https://doi.org/10.1002/tax.12055

Léveillé-Bourret É, Starr JR, Ford BA. 2018a. Why are there so many sedges?

Sumatroscirpeae, a missing piece in the evolutionary puzzle of the giant genus

Carex (Cyperaceae). Molecular Phylogenetics Evolution 119, 93–104.

https://doi.org/10.1016/j.ympev.2017.10.025

Léveillé-Bourret É, Starr JR, Ford BA, Lemmon, E.M., Lemmon, A.R., 2018b.

Resolving rapid radiations within angiosperm families using anchored

phylogenomics. Systematic Biology 67, 94–112.

https://doi.org/10.1093/sysbio/syx050

Linnaeus C. 1753. Species plantarum, 1st ed. Printed by Lawrence Salvius,

Stockholm (Sweden).

Liu M, Hambleton S. 2013. Laying the foundation for a taxonomic review of Puccinia

coronata s.l. in a phylogenetic context. Mycological Progress 12, 63–89.

Liu M, Hambleton S. 2012. Puccinia chunjii, a close relative of the cereal stem rusts

revealed by molecular phylogeny and morphological study. Mycologia 104,

1056–1067. https://doi.org/10.3852/11-251

Liu M, Hambleton S. 2010. Taxonomic study of stripe rust, Puccinia striiformis sensu

lato, based on molecular and morphological evidence. Fungal Biology 114,

881–899. https://doi.org/10.1016/j.funbio.2010.08.005

Liu M, Overy DP, Cayouette J, Shoukouhi P, Hicks C, Bisson K, Sproule A, Wyka

SA, Broders K, Popovic Z, Menzies JG. 2020. Four phylogenetic species of

ergot from Canada and their characteristics in morphology, alkaloid

production, and pathogenicity. Mycologia 1–15.

https://doi.org/10.1080/00275514.2020.1797372

Lücking R, Aime MC, Robbertse B, Miller AN, Ariyawansa HA, Aoki T, Cardinali

G, Crous PW, Druzhinina IS, Geiser DM, Hawksworth DL, Hyde KD, Irinyi

L, Jeewon R, Johnston PR, Kirk PM, Malosso E, May TW, Meyer W, Öpik

M, Robert V, Stadler M, Thines M, Vu D, Yurkov AM, Zhang N, Schoch CL.

2020. Unambiguous identification of fungi: where do we stand and how

accurate and precise is fungal DNA barcoding? IMA Fungus 11: 14.

https://doi.org/10.1186/s43008-020-00033-z

Lutz M, Bauer R, Begerow D, Oberwinkler F. 2004a. Tuberculina —

Thanatophytum/Rhizoctonia crocorum — Helicobasidium: a unique

mycoparasitic-phytoparasitic life strategy. Mycological Research 108, 227–

238. https://doi.org/10.1017/S0953756204009359

Accepted Article

Lutz M, Bauer R, Begerow D, Oberwinkler F, Triebel, D. 2004b. Tuberculina: rust

relatives attack rusts. Mycologia 96, 614–626.

https://doi.org/10.1080/15572536.2005.11832957

Magallón, S., Gómez-Acevedo, S., Sánchez-Reyes, L.L., Hernández-Hernández, T.,

2015. A metacalibrated time-tree documents the early rise of flowering plant

phylogenetic diversity. New Phytologist 207, 437–453.

https://doi.org/10.1111/nph.13264

Maier, W., Begerow D, Weiß M, Oberwinkler F. 2003. Phylogeny of the rust fungi:

an approach using nuclear large subunit ribosomal DNA sequences. Canadian

Journal of Botany 81, 12–23. https://doi.org/10.1139/B02-113

Maier W, Wingfield BD, Mennicken M, Wingfield MJ. 2007. Polyphyly and two

emerging lineages in the rust genera Puccinia and Uromyces. Mycological

Research 111, 176–185.

Mandel JR, Dikow RB, Siniscalchi CM, Thapa R, Watson LE, Funk VA. 2019. A

fully resolved backbone phylogeny reveals numerous dispersals and explosive

diversifications throughout the history of Asteraceae. Proceedings of the

National Academy of Sciences 116, 14083–14088.

https://doi.org/10.1073/pnas.1903871116

Martín‐Bravo S, Jiménez‐Mejías P, Villaverde T, Escudero M, Hahn M, Spalink D,

Roalson EH, Hipp AL, the Global Carex Group, Benítez‐Benítez C, P.

Bruederle L, Fitzek E, A. Ford B, A. Ford K, Garner M, Gebauer S, H.

Hoffmann M, Jin X, Larridon I, Léveillé‐Bourret É, Lu Y, Luceño M,

Maguilla E, Márquez‐Corro JI, Míguez M, Naczi R, A. Reznicek A, R. Starr J.

2019. A tale of worldwide success: behind the scenes of Carex (Cyperaceae)

biogeography and diversification. Journal of Systematics and Evolution 57:

695–718.

McTaggart AR, Aime MC. 2018. The species of Coleosporium (Pucciniales) on

Solidago in North America. Fungal Biology 122, 800–809.

https://doi.org/10.1016/j.funbio.2018.04.007

Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for

inference of large phylogenetic trees, in: Proceedings of the Gateway

Computing Environments Workshop (GCE). Presented at the Gateway

Computing Environments Workshop (GCE), New Orleans (United-States), pp.

1–8.

Muasya AM, Simpson DA, Verboom GA, Goetghebeur P, Naczi RFC, Chase MW,

Smets E. 2009. Phylogeny of Cyperaceae based on DNA sequence data:

current progress and future prospects. The Botanical Review 75, 2–21.

https://doi.org/10.1007/s12229-008-9019-3

Accepted Article

Nelsen DJ. 2010. A phylogenetic analysis of species diversity, specificity, and

distribution of Mycodiplosis on rust fungi (M.Sc.). Minnesota State

University.

Nischwitz C, Newcombe G, Anderson CL. 2005. Host specialization of the

mycoparasite Eudarluca caricis and its evolutionary relationship to

Ampelomyces. Mycological Research 109, 421–428.

https://doi.org/10.1017/S0953756205002431

Nylin S, Agosta S, Bensch S, Boeger WA, Braga MP, Brooks DR, Forister ML,

Hambäck PA, Hoberg EP, Nyman T, Schäpers A, Stigall AL, Wheat CW,

Österling M, Janz N. 2018. Embracing colonizations: a new paradigm for

species association dynamics. Trends in Ecology & Evolution 33: 4–14.

Ødegaard F. 2000. How many species of arthropods? Erwin’s estimate revised.

Biological Journal of the Linnean Society 71, 583–597.

https://doi.org/10.1111/j.1095-8312.2000.tb01279.x

O’Hara RB. 2005. Species richness estimators: how many species can dance on the

head of a pin? Journal of Animal Ecology 74, 375–386.

https://doi.org/10.1111/j.1365-2656.2005.00940.x

Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR,

O’Hara RB, Simpson GL, Solymos P, Stevens HH, Szoecs E, Wagner H.

2019. vegan: community ecology package. R package version 2.5-6. 2019.

Ono Y. 2002. The diversity of nuclear cycle in microcyclic rust fungi (Uredinales)

and its ecological and evolutionary implications. Mycoscience 43, 421–439.

https://doi.org/10.1007/S102670200062

Palla E. 1896. Zur Systematic der Gattung Eriophorum. Botanische Zeitung 54, 141–

158.

Paradis E, Schliep K. 2019. ape 5.0: an environment for modern phylogenetics and

evolutionary analyses in R. Bioinformatics 35, 526–528.

https://doi.org/10.1093/bioinformatics/bty633

Parmelee JA. 1989. The rusts (Uredinales) of arctic Canada. Canadian Journal of

Botany 67, 3315–3365. https://doi.org/10.1139/b89-407

Parmelee JA. 1967. The autoecious species of Puccinia on Heliantheae in North

America. Canadian Journal of Botany 45, 2267–2327.

https://doi.org/10.1139/b67-248

Parmelee JA. 1969. The autoecious species of Puccinia on Heliantheae

[’Ambrosiaceae’] in North America. Canadian Journal of Botany 47, 1391–

1402. https://doi.org/10.1139/b69-199

Accepted Article

Parmelee JA, Savile DBO. 1981. Autoecious species of Puccinia on Cichorieae in

North America. Canadian Journal of Botany 59, 1078–1101.

https://doi.org/10.1139/b81-147

Pažoutová S, Pešicová K, Chudíčková M, Šrůtka P, Kolařík M. 2015. Delimitation of

cryptic species inside Claviceps purpurea. Fungal Biology 119, 7–26.

https://doi.org/10.1016/j.funbio.2014.10.003

Pender JE. 2015. Climatic niche estimation, trait evolution and species richness in

North American Carex (Cyperaceae) (M.Sc.). University of Ottawa, Ottawa,

Ontario (Canada).

Pender JE, Starr JR. 2021. Trait evolution rates shape species richness on a

continental scale in sedges (Carex, Cyperaceae). Journal of Systematics and

Evolution manuscript number: JSE-2020-10-239

Petersen RH. 1974. The rust fungus life cycle. The Botanical Review 40, 453–513.

https://doi.org/10.1007/BF02860021

Piepenbring M, Begerow D, Oberwinkler F, 1999. Molecular sequence data assess the

value of morphological characteristics for a phylogenetic classification of

Cintractia. Mycologia 91, 485–498.

Price MN, Dehal PS, Arkin AP. 2010. FastTree 2 – Approximately maximum-

likelihood trees for large alignments. PLoS ONE 5, e9490.

https://doi.org/10.1371/journal.pone.0009490

Price PW, Westoby M, Rice B. 1988. Parasite-mediated competition: some

predictions and tests. The American Naturalist 131, 544–555.

https://doi.org/10.1086/284805

R Core Team, 2020. R: a language and environment for statistical computing. R

Foundation for Statistical Computing, Vienna (Austria).

Rambaut A, Shuchard MA, Xie D, Drummond AJ. 2014. Tracer version 1.6.

Refrégier G, Gac ML, Jabbour F, Widmer A, Shykoff JA, Yockteng R, Hood ME,

Giraud T. 2008. Cophylogeny of the anther smut fungi and their

caryophyllaceous hosts: prevalence of host shifts and importance of delimiting

parasite species for inferring cospeciation. BMC Evolutionary Biology 8: 100.

https://doi.org/10.1186/1471-2148-8-100

Rice B, Westoby M. 1982. Heteroecious rusts as agents of interference competition.

Evolutionary Theory 6, 43–52.

Riess K, Schön ME, Ziegler R, Lutz M, Shivas RG, Piątek M, Garnica S. 2019. The

origin and diversification of the Entorrhizales: deep evolutionary roots but

recent speciation with a phylogenetic and phenotypic split between associates

Accepted Article

of the Cyperaceae and Juncaceae. Organisms Diversity & Evolution 19: 13–

30. https://doi.org/10.1007/s13127-018-0384-4

Rintoul TL, Eggertson QA, Lévesque CA. 2012. Multigene phylogenetic analyses to

delimit new species in fungal plant pathogens. In: Bolton MD, Thomma BPHJ

eds. Plant Fungal Pathogens. Springer, New York, pp. 549–569.

Rioux S, Mimee B, Gagnon AÈ, Hambleton S. 2015. First report of stripe rust

(Puccinia striiformis f. sp. tritici) on wheat in Quebec, Canada.

Phytoprotection 95, 7–9. https://doi.org/10.7202/1028400ar

Robinson E, Blagoev G, Hebert P, Adamowicz S. 2009. Prospects for using DNA

barcoding to identify spiders in species-rich genera. ZooKeys 16, 27–46.

https://doi.org/10.3897/zookeys.16.239

Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B,

Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient bayesian

phylogenetic inference and model choice across a large model space.

Systematic Biology 61, 539–542. https://doi.org/10.1093/sysbio/sys029

Roy BA, Vogler DR, Bruns TD, Szaro TM. 1998. Cryptic species in the Puccinia

monoica complex. Mycologia 90, 846. https://doi.org/10.2307/3761326

Saari EE, Prescott JM. 1985. World distribution in relation to economic losses. In:

Roelfs AP, Buschnell WR eds. The cereal rusts: diseases, distribution,

epidemiology, and control. Elsevier, 259–298. https://doi.org/10.1016/B978-0-

12-148402-6.50017-1

Savile DBO. 1990. Relationships of Poaceae, Cyperaceae, and Juncaceae reflected by

their fungal parasites. Canadian Journal of Botany 68, 731–734.

Savile DBO. 1984. Taxonomy of the cereal rust fungi, in: Bushnell, W.R., Roelfs,

A.P. (Eds.), The Cereal Rusts: Origins, Specificity, Structure, and Physiology.

Academic Press, Montreal (Canada), pp. 79–112.

Savile DBO. 1979. Fungi as aids in higher plant classification. The Botanical Review

45, 377–503.

Savile DBO. 1978. Paleoecology and convergent evolution in rust fungi (Uredinales).

Biosystems 10, 31–36.

Savile DBO. 1976. Evolution of the rust fungi (Uredinales) as reflected by their

ecological problems, in: Hecht MK, Steere WC, Wallace B eds. Evolutionary

Biology. Plenum Press, New York, NY (United States), pp. 137–207.

Savile DBO. 1973. Aeciospore types in Puccinia and Uromyces attacking

Cyperaceae, Juncaceae and Poaceae. Reports of the Tottori Mycological

Institute 10, 225–241.

Accepted Article

Savile DBO. 1972. Some rusts of Scirpus and allied genera. Canadian Journal of

Botany 50, 2579–2596. https://doi.org/10.1139/b72-331

Savile DBO. 1970a. Some Eurasian Puccinia species attacking Cardueae. Canadian

Journal of Botany 48, 1553–1566.

Savile DBO. 1965. Puccinia karelica and species delimitation in the Uredinales.

Canadian Journal of Botany 43, 231–238. https://doi.org/10.1139/b65-027

Savile DBO. 1952. A study of the species of Cintractia on Carex, Kobresia, and

Scirpus in North America. Canadian Journal of Botany 30, 410–435.

Savile DBO, Calder JA. 1953. Phylogeny of Carex in the light of parasitism by the

smut fungi. Canadian Journal of Botany 31, 164–174.

Schmit JP, Mueller GM. 2007. An estimate of the lower limit of global fungal

diversity. Biodiversity and Conservation 16, 99–111.

https://doi.org/10.1007/s10531-006-9129-3

Semmouri I, Bauters K, Léveillé-Bourret É, Starr JR, Goetghebeur P, Larridon I.

2019. Phylogeny and systematics of Cyperaceae, the evolution and importance

of embryo morphology. The Botanical Review 85, 1–39.

https://doi.org/10.1007/s12229-018-9202-0

Shiels DR, Hurlbut DL, Lichtenwald SK, Monfils AK. 2014. Monophyly and

phylogeny of Schoenoplectus and Schoenoplectiella (Cyperaceae): evidence

from chloroplast and nuclear DNA sequences. Systematic Botany 39, 132–144.

Shoukouhi P, Hicks C, Menzies JG, Popovic Z, Chen W, Seifert KA, Assabgui R, Liu

M. 2019. Phylogeny of Canadian ergot fungi and a detection assay by real-

time polymerase chain reaction. Mycologia 111, 493–505.

https://doi.org/10.1080/00275514.2019.1581018

Small E, Cayouette J. 2016. 50. Sedges – the key sustainable resource for Arctic

biodiversity. Biodiversity 17, 60–69.

https://doi.org/10.1080/14888386.2016.1164624

Smith SY, Collinson ME, Rudall PJ, Simpson DA. 2010. The Cretaceous and

Paleogene fossil record of Poales: review and current research. In: Seberg O,

Petersen G, Barfod AS, Davis JI eds. Diversity, Phylogeny, and Evolution in

the Monocotyledons: Proceedings of the Fourth International Conference on

the Comparative Biology of the Monocotyledons and the Fifth International

Symposium on Grass Systematics and Evolution. Aarhus University Press,

Denmark, pp. 333–356.

Spalink D, Drew BT, Pace MC, Zaborsky JG, Li P, Cameron KM, Givnish TJ,

Sytsma KJ. 2016. Evolution of geographical place and niche space: patterns of

diversification in the North American sedge (Cyperaceae) flora. Molecular

Accepted Article

Phylogenetics Evolution 95: 183–195.

https://doi.org/10.1016/j.ympev.2015.09.028

Starr JR, Harris SA, Simpson DA. 2003. Potential of the 5′ and 3′ ends of the

intergenic spacer (IGS) of rDNA in the Cyperaceae: new sequences for lower‐

level phylogenies in sedges with an example from Uncinia Pers. International

Journal of Plant Sciences 164, 213–227. https://doi.org/10.1086/346168

Starr JR, Janzen FH, Ford BA. 2015. Three new, early diverging Carex (Cariceae,

Cyperaceae) lineages from East and Southeast Asia with important

evolutionary and biogeographic implications. Molecular Phylogenetics

Evolution 88, 105–120. https://doi.org/doi:10.1016/j.ympev.2015.04.001

Starr JR, Jiménez-Mejías P, Zuntini AR, Semmouri I, Muasya AM, Baker WJ,

Brewer GE, Epitawalage N, Fairlie I, Forest F, Léveillé-Bourret É, Pokorny L,

Larridon I. 2021. Targeted sequencing supports morphology and embryo

features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Journal of Systematics and Evolution Early View.

https://doi.org/10.1111/jse.12721

Starr JR, Léveillé-Bourret É, Vũ Anh T, Nguyễn Thị KT, Ford BA. 2019. The

rediscovery of the rare Vietnamese endemic Eriophorum scabriculme

redefines generic limits in the Scirpo-Caricoid Clade (Cyperaceae). PeerJ 7,

e7538.

Steinke D, Zemlak TS, Hebert PDN. 2009. Barcoding Nemo: DNA-based

identifications for the ornamental fish trade. PLoS ONE 4, e6300.

https://doi.org/10.1371/journal.pone.0006300

Stielow JB, Lévesque CA, Seifert KA, Meyer W, Irinyi L, Smits D, Renfurm R,

Verkley GJM, Groenewald M, Chaduli D, Lomascolo A, Welti S, Lesage-

Meessen L, Favel A, Al-Hatmi AMS, Damm U, Yilmaz N, Houbraken J,

Lombard L, Quaedvlieg W, Binder M, Vaas LAI, Vu D, Yurkov A, Begerow

D, Roehl O, Guerreiro M, Fonseca A, Samerpitak K, van Diepeningen AD,

Dolatabadi S, Moreno LF, Casaregola S, Mallet S, Jacques N, Roscini L, Egidi

E, Bizet C, Garcia-Hermoso D, Martín MP, Deng S, Groenewald JZ,

Boekhout T, de Beer ZW, Barnes I, Duong TA, Wingfield MJ, de Hoog GS,

Crous PW, Lewis CT, Hambleton S, Moussa TAA, Al-Zahrani HS,

Almaghrabi OA, Louis-Seize G, Assabgui R, McCormick W, Omer G, Dukik

K, Cardinali G, Eberhardt U, de Vries M, Robert V. 2015. One fungus, which

genes? Development and assessment of universal primers for potential

secondary fungal DNA barcodes. Persoonia - Molecular Phylogeny and

Evolution of Fungi 35: 242–263. https://doi.org/10.3767/003158515X689135

Takhtajan AL. 1980. Outline of the classification of flowering plants

(Magnoliophyta). The Botanical Review 46, 225–359.

Accepted Article

Thines M. 2019. An evolutionary framework for host shifts – jumping ships for

survival. New Phytologist 224, 605–617. https://doi.org/10.1111/nph.16092

Thorne RF. 2000. The classification and geography of the flowering plants:

Dicotyledons of the class Angiospermae: Subclasses Magnoliidae,

Ranunculidae, Caryophyllidae, Dilleniidae, Rosidae, Asteridae, and Lamiidae.

The Botanical Review 66, 441–647. https://doi.org/10.1007/BF02869011

Trakunyingcharoen T, Lombard L, Groenewald JZ, Cheewangkoon R, Toanun C,

Crous PW. 2014. Mycoparasitic species of Sphaerellopsis, and allied

lichenicolous and other genera. IMA Fungus 5, 391–414.

https://doi.org/10.5598/imafungus.2014.05.02.05

van der Merwe M, Ericson L, Walker J, Thrall PH, Burdon JJ. 2007. Evolutionary

relationships among species of Puccinia and Uromyces (Pucciniaceae,

Uredinales) inferred from partial protein coding gene phylogenies.

Mycological Research 111, 163–175.

https://doi.org/10.1016/j.mycres.2006.09.015

van der Merwe MM, Walker J, Ericson L, Burdon J. 2008. Coevolution with higher

taxonomic host groups within the Puccinia/Uromyces rust lineage obscured by

host jumps. Mycological Research 112, 1387–1408.

Vánky K. 2012. Smut fungi of the world. APS Press, United-States.

Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of

enzymatically amplified ribosomal DNA from several Cryptococcus species.

Journal of Bacteriology 172, 4238–4246.

https://doi.org/10.1128/JB.172.8.4238-4246.1990

White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of

fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH,

Sninsky JJ, White TJ eds. PCR protocols: a guide to methods and

applications. Academic Press, New York, pp. 315–322.

Wilkinson SP. 2018. kmer: an R package for fast alignment-free clustering of

biological sequences. R package version 1.1.2.

Yang Z, Rannala B. 2005. Branch-length prior influences Bayesian posterior

probability of phylogeny. Systematic Biology 54, 455–470.

https://doi.org/10.1080/10635150590945313

Yano O, Ikeda H, Watson MF, Rajbhandari K, Jin XF, Hoshino T, Muasya AM, Ohba

H. 2012. Phylogenetic position of the Himalayan genus Erioscirpus

(Cyperaceae) inferred from DNA sequence data. Botanical Journal of the

Linnean Society 170, 1–11.

Accepted Article

Zhou D, Hyde KD. 2001. Host-specificity, host-exclusivity, and host-recurrence in

saprobic fungi. Mycological Research 105, 1449–1457.

https://doi.org/10.1017/S0953756201004713

Zwetko P, Blanz P. 2012. Aeciospore types in rusts on Ranunculus and allied genera.

Stapfia 96, 105–121.

Figure 1. Barcode gap in the ITS2-28S dataset. Histogram of within-OTU (blue) and

between-OTU (orange, hatched) k-mer distances between samples, showing important

overlap and suggesting the absence of a global barcode gap. Inset shows violin and dot

plots of the ratio between minimum between-OTU and maximum within-OTU k-mer

distances (log scale). A local barcode gap is present in OTUs with a ratio above 2.

Figure 2. Rarefaction curves of the eleven North American rust species and

aggregates included in rarefaction and richness analyses. The four aggregates where

>10 individuals were sequenced are identified by name.

Accepted Article

Figure 3. Bayesian Maximum clade credibility chronogram of rust fungi (Aecidium,

Puccinia and Uromyces) parasites of Cyperaceae-Juncaceae, based on ITS2-

28S barcodes. Branch support indicated as Bayesian posterior probabilities/NJ

bootstrap/ML bootstrap. Support values <50% bootstrap or 0.50 posterior

probability indicated by a hyphen (-) or left blank. Wider branch lines indicate

≥0.95 Bayesian posterior probabilities. Bold names indicate rust species with at

least one life stage on Cyperaceae or Juncaceae (all other rusts with all stages

on dicots, except for Puccinia iridis on Iridaceae). Species that alternate between

Carex and Asteraceae, or autoecious on Asteraceae, are not highlighted with

colors. All other clades or lineages highlighted with colors, and with telial host(s)

labelled beside the clade preceded by “ex” (Latin “from”). Ages corresponding to

the origins of Carex and of its major clades (Martín-Bravo et al., 2019), and

Pleistocene glaciations (2.58 Mya) are indicated with blue and yellow shaded

boxes. Outgroup species are not shown.

Accepted Article

Figure 4. Continued from Figure 3.

Accepted Article

Figure 5. Tanglegrams showing associations between rust fungi and their monocot

(A) or dicot (B) hosts. Links colored in dark blue are highly congruent, those in

dark red are highly discordant, and those in light grey are purely random. The

amount of cophylogenetic signal is also expressed at branch nodes using the

same color codes. A pure cospeciation scenario would be visualized as

tanglegrams without any overlapping lines, and dark blue colors throughout.

These tanglegrams show uneven cophylogenetic signal (both dark blue and dark

red are present).

Accepted Article

Table 1. Sequence statistics for the ITS2-28S dataset.

Dataset

Aligned

length

Terminals

missing

Gaps,

missing, &

ambiguous

characters

Variable

characters

Parsimony

informative

characters

Consistency

index (CI)

Retention

index (RI)

5.8S

159

10 (3.7%)

48%

22 (14%)

15 (9.4%)

0.585

0.877

ITS2

360

57%

257 (71%)

199 (55%)

0.234

0.761

28S

1244

9 (3.0%)

71%

321 (26%)

176 (14%)

0.529

0.671

Full

alignment

1763

72%

600 (34%)

390 (22%)

0.304

0.737

Table 2. Cophylogenetic signal and estimated coevolutionary events between rust

fungi and their monocot and dicot hosts. An asterisk (*) indicates a statistically

significant value at alpha=0.05.

Statistics

Complete datasets

Reduced datasets

Monocot hosts

Dicot hosts

Monocot hosts

Dicot hosts

PACo

p-value

<0.0001 *

Random TaPas

G* coefficient

0.706

[0.704–0.732] *

0.747

[0.733–0.760] *

0.735

[0.708–0.744] *

0.741

[0.709–0.763] *

Accepted Article

Statistics

Complete datasets

Reduced datasets

Jane 4

p-value

<0.01 *

cost

284

211

209

105

events

240

176

191

cospeciations

14 (5.8%)

14 (8.0%)

10 (5.2%)

6 (6.7%)

duplications

18 (7.5%)

42 (24%)

19 (10%)

30 (33%)

host switches

58 (24%)

52 (30%)

28 (15%)

21 (23%)

losses/sorting

127 (53%)

49 (28%)

113 (59%)

21 (23%)

failures to diverge

23 (9.6%)

16 (9.2%)

21 (11%)

12 (13%)

Genetic time traveling: sequencing old herbarium specimens, including the oldest herbarium specimen sequenced from kingdom Fungi, reveals the population structure of an agriculturally significant rust

Article

Full-text available

Dec 2022
NEW PHYTOL

Sequencing herbarium specimens can be instrumental in answering ecological, evolutionary, and taxonomic inquiries. We developed a protocol for sequencing herbarium specimens of rust fungi (Pucciniales) and proceeded to sequence specimens ranging from 4 to 211 yr old from five different genera. We then obtained sequences from an economically important biological control agent, Puccinia suaveolens, to highlight the potential of sequencing herbarium specimens in an ecological sense and to evaluate the following hypotheses: (1) The population structure of a plant pathogen changes over time, and (2) introduced pathogens are more diverse in their native range. Our efforts resulted in sequences from 87 herbarium specimens that revealed a high level of diversity with a population structure that exhibited spatial–temporal patterns. The specimens sequenced from Europe showed more diversity than the ones from North America, uncovering an invasion pattern likely related to its European native host in North America. Additionally, to the best of our knowledge, the specimen from France collected in c. 1811 is the oldest herbarium specimen sequenced from kingdom Fungi. In conclusion, sequencing old herbarium specimens is an important tool that can be extrapolated to better understand plant–microbe evolution and to evaluate old type specimens to solidify the taxonomy of plant pathogenic fungi.

Strong phylogenetic congruence between Tulasnella fungi and their associated Drakaeinae orchids

Article

Full-text available

Oct 2022

The study of congruency between phylogenies of interacting species can provide a powerful approach for understanding the evolutionary history of symbiotic associations. Orchid mycorrhizal fungi can survive independently of orchids making cospeciation unlikely, leading us to predict that any congruence would arise from host‐switches to closely related fungal species. The Australasian orchid subtribe Drakaeinae is an iconic group of sexually deceptive orchids that consists of approximately 66 species. In this study, we investigated the evolutionary relationships between representatives of all six Drakaeinae orchid genera (39 species) and their mycorrhizal fungi. We used an exome capture dataset to generate the first well‐resolved phylogeny of the Drakaeinae genera. A total of 10 closely related Tulasnella Operational Taxonomic Units (OTUs) and previously described species were associated with the Drakaeinae orchids. Three of them were shared among orchid genera, with each genus associating with 1–6 Tulasnella lineages. Cophylogenetic analyses show Drakaeinae orchids and their Tulasnella associates exhibit significant congruence (p < 0.001) in the topology of their phylogenetic trees. An event‐based method also revealed significant congruence in Drakaeinae–Tulasnella relationships, with duplications (35), losses (25), and failure to diverge (9) the most frequent events, with minimal evidence for cospeciation (1) and host‐switches (2). The high number of duplications suggests that the orchids speciate independently from the fungi, and the fungal species association of the ancestral orchid species is typically maintained in the daughter species. For the Drakaeinae–Tulasnella interaction, a pattern of phylogenetic niche conservatism rather than coevolution likely explains the observed phylogenetic congruency in orchid and fungal phylogenies. Given that many orchid genera are characterized by sharing of fungal species between closely related orchid species, we predict that these findings may apply to a wide range of orchid lineages. Representatives of five genera of Drakaeinae orchids, from top left to bottom right: Arthrochilus latipes, Chiloglottis trilabra, Caleana major, Paracaleana minor, Drakaea livida.

Rtapas: An R Package to Assess Cophylogenetic Signal between Two Evolutionary Histories

Article

Mar 2023
SYST BIOL

Cophylogeny represents a framework to understand how ecological and evolutionary process influence lineage diversification. The recently developed algorithm Random Tanglegram Partitions provides a directly interpretable statistic to quantify the strength of cophylogenetic signal and incorporates phylogenetic uncertainty into its estimation, and maps onto a tanglegram the contribution to cophylogenetic signal of individual host-symbiont associations. We introduce Rtapas, an R package to perform Random Tanglegram Partitions. Rtapas applies a given global-fit method to random partial tanglegrams of a fixed size to identify the associations, terminals, and internal nodes that maximize phylogenetic congruence. This new package extends the original implementation with a new algorithm that examines the contribution to phylogenetic incongruence of each host-symbiont association and adds ParaFit, a method designed to test for topological congruence between two phylogenies, to the list of global-fit methods than can be applied. Rtapas facilitates and speeds up cophylogenetic analysis, as it can handle large phylogenies (100+ terminals) in affordable computational time as illustrated with two real-world examples. Rtapas can particularly cater for the need for causal inference in cophylogeny in two domains: (i) Analysis of complex and intricate host-symbiont evolutionary histories and (ii) assessment of topological (in)congruence between phylogenies produced with different DNA markers and specifically identify subsets of loci for phylogenetic analysis that are most likely to reflect gene-tree evolutionary histories.

DNA barcode sequencing discovered the aecial host of Puccinia chunjiei

Article

Full-text available

Dec 2022

The telial stage of Puccinia chunjiei was described in 2012 from a single specimen collected in China (DAOM 240982). This species is the closest relative of P. graminis but differs in telial morphology on their grass hosts and in DNA sequences. As part of a DNA-barcoding project for rust herbarium specimens, collections of the aecial stage of P. graminis on Berberis were processed. For one specimen, BPI 1103856, the ITS sequence matched that of P. chunjiei and the aecial morphology differed from P. graminis. An expanded description of P. chunjiei is presented with photographs of the aecial stages of both species.

Molecular-based diversity studies and field surveys are not mutually exclusive: On the importance of integrated methodologies in mycological research. FRONTIERS IN FUNGAL BIOLOGY (2022) 3:860777

Article

Full-text available

Mar 2022

Understanding and describing the diversity of living organisms is a great challenge. Fungi have for a long time been, and unfortunately still are, underestimated when it comes to taxonomic research. The foundations were laid by the first mycologists through field observations. These important fundamental works have been and remain vital reference works. Nevertheless, a non-negligible part of the studied funga escaped their attention. Thanks to modern developments in molecular techniques, the study of fungal diversity has been revolutionized in terms of tools and knowledge. Despite a number of disadvantages inherent to these techniques, traditional field-based inventory work has been increasingly superseded and neglected. This perspective aims to demonstrate the central importance of field-based research in fungal diversity studies, and encourages researchers not to be blinded by the sole use of molecular methods.

Delimiting species in Basidiomycota: a review

Article

Full-text available

Sep 2021

Species delimitation is one of the most fundamental processes in biology. Biodiversity undertakings, for instance, require explicit species concepts and criteria for species delimitation in order to be relevant and translatable. However, a perfect species concept does not exist for Fungi. Here, we review the species concepts commonly used in Basidiomycota, the second largest phylum of Fungi that contains some of the best known species of mushrooms, rusts, smuts, and jelly fungi. In general, best practice is to delimitate species, publish new taxa, and conduct taxonomic revisions based on as many independent lines of evidence as possible, that is, by applying a so-called unifying (or integrative) conceptual framework. However, the types of data used vary considerably from group to group. For this reason we discuss the different classes of Basidiomycota, and for each provide: (i) a general introduction with difficulties faced in species recognition, (ii) species concepts and methods for species delimitation, and (iii) community recommendations and conclusions.

What is a species in fungal plant pathogens?

Article

Aug 2021

Scientific names are crucial for communicating knowledge concerning fungi and fungus-like organisms. In plant pathology, they link information regarding biology, host range, distribution and potential risk to agriculture and food security. In the past, delimitation among pathogenic taxa was primarily based on morphological characteristics. Due to distinct species sharing overlapping characteristics, the morphological identification of species is often neither straightforward nor reliable. Hence, the phylogenetic species concept based on molecular phylogenetic reconstructions gained importance. The present opinion discusses what a fungal species is and how identification of species in plant pathology has changed over the past decades. In this context, host-specialization and species complexes are discussed. Furthermore, species concepts in plant pathology are examined using case studies from Bipolaris, Colletotrichum, Curvularia, Diaporthe, Diplodia, Meliola, Plasmopara, rust fungi and Trichoderma. Each entry contains a brief introduction to the genus, concepts used in species identification so far and the problems in describing a species followed by recommendations. The importance of correctly naming and identifying a species is addressed in the context of recent introductions, and we also discuss whether the introduction of new species in pathogenic genera has been overestimated. We also provide guidelines to be considered when introducing a new species in a plant pathogenic genus.

Four new species of Puccinia from herbaceous plants in China

Article

Full-text available

Jan 2024
MYCOLOGIA

Members of Puccinia (Pucciniaceae, Pucciniales) are known as plant pathogens worldwide, which are characterized by their morphology, host association, and molecular data of various genes. In the present study, 10 specimens of Puccinia were collected from four herbaceous plants (Anaphalis hancockii, Anthriscus sylvestris, Halenia elliptica, and Pilea pumila) in China and identified based on morphology and phylogeny. As a result, 10 samples represent four undescribed species of Puccinia, viz., P. apdensia, P. decidua, P. dermatis, and P. lianchengensis, spp. nov. P. apdensia is characterized by its smooth teliospores with thickened apex. P. decidua represents the first Puccinia species inhabiting the host Anaphalis hancockii and is distinguished from the other Puccinia species by its telia and uredinia surrounded by the epidermis. P. dermatis from Halenia elliptica differs from the other Puccinia species on the host genus Halenia by the telia that have epidermis and teliospores with sparsely irregular granulated protrusions. P. lianchengensis is characterized by its teliospore surface with fishnet ornamentation and urediniospores without prominent caps. All of the new species are described and illustrated in this study.

First report of Puccinia gibberosa on blue oatgrass (Helictotrichon sempervirens) in Canada, with taxonomic revision of the rust species

Article

Oct 2023

Abstract: Helictotrichon sempervirens(blue oatgrass) is a perennial grass widely grown for ornamental use in Canada. Rusted blue oatgrass plants with diseased leaves covered with uredinia were collected from two locations at metropolitan Vancouver, British Columbia (BC), in summer 2022. The rust fungus was identified as Puccinia gibberosa (= P. coronata var. gibberosa) based on combined results of morphological characteristics and molecular analyses. The internal transcribed spacer two (ITS2) and partial large subunit ribosomal DNA were amplified and sequenced. The sequence obtained for the rust fungus showed 99.9% identity with the previously reported P. coronata var. gibberosa from USA. This is the first report of rust caused by P. gibberosa on blue oatgrass in Canada. Evidence from phylogenetic analyses supports the recognition of this rust at the species level. A revised description for P. gibberosa is provided, including synonyms, host range and distribution across the globe, and incorporating comprehensive molecular and morphological results for several North American and European specimens. In addition, a lectotype and an epitype are designated here for the rust species. Keywords: British Columbia, fungal disease, Poaceae, Pucciniales, rust fungi. Résumé : Helictotrichon sempervirens (avoine sempervirente) est une graminée vivace cultivée couramment à des fins ornementales au Canada. À l’été2022, des plants d’avoine sempervirente rouillée dont les feuilles infectées étaient couvertes d’urédosores ont été collectés à deux endroits de la région métropolitaine de Vancouver, en Colombie-Britannique. Le champignon de la rouille a été identifié en tant que Puccinia gibberosa (= P. coronata var. gibberosa), en se basant sur les résultats combinés des analyses des caractéristiques morphologiques et moléculaires. L’espaceur transcrit interne2 (ITS2) et la grande sous-unité partielle de l’ADN ribosomique ont été amplifiés et séquencés. La séquence obtenue pour le champignon de la rouille était identique M Abbasi et al. First report of Puccinia gibberosa in Canada à 99,9 % à P. coronata var. gibberosa préalablement rapporté aux États-Unis. Il s’agit de la première mention de rouille causée par P. gibberosa chez l’avoine sempervirente au Canada. L’indice obtenu des analyses phylogénétiques soutient la reconnaissance de cette rouille au niveau de l’espèce. Une description révisée de P. gibberosa est fournie, y compris les synonymes, la gamme d’hôtes ainsi que l’aire de distribution mondiale, et comprend les résultats détaillés des analyses moléculaires et morphologiques pour plusieurs spécimens nord-américains et européens. En outre, un lectotype et un épitype ont été sélectionnés pour l’espèce de rouille. Mots clés: champignons de la rouille, Colombie-Britannique, maladie fongique, poacées, Pucciniales

Genomic biosurveillance to protect the world’s forest resources

Chapter

Jan 2023

Rc Hamelin

We live at an unprecedented time when the planet’s biomes are being constantly challenged by invasions and climate change. This threatens our precious natural resources, an essential component of the bioeconomy, as well as the numerous ecosystem services that are provided by our forests. The best way to protect terrestrial ecosystems, halt biodiversity loss, and sustainably manage forests is by improving our ability to prevent forest disease and pest outbreaks before they can cause damage by capitalizing on the remarkable advances in genomic technologies. The implementation of global biosurveillance of forests requires the development of tools and their adoption by partners around the world and the sharing and communication of data in real time. This chapter will explore the technological advances as well as the challenges that we face in implementing such approaches.

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Article

Full-text available

Jan 2021

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing in order to: (1) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae and Cypereae; (2) define the limits of Fuireneae s.s., and (3) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognise as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr.nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub‐Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognised tribes and genera are provided. This article is protected by copyright. All rights reserved.

Unambiguous identification of fungi: Where do we stand and how accurate and precise is fungal DNA barcoding?

Article

Full-text available

Jul 2020

True fungi (Fungi) and fungus-like organisms (e.g. Mycetozoa, Oomycota) constitute the second largest group of organisms based on global richness estimates, with around 3 million predicted species. Compared to plants and animals, fungi have simple body plans with often morphologically and ecologically obscure structures. This poses challenges for accurate and precise identifications. Here we provide a conceptual framework for the identification of fungi, encouraging the approach of integrative (polyphasic) taxonomy for species delimitation, i.e. the combination of genealogy (phylogeny), phenotype (including autecology), and reproductive biology (when feasible). This allows objective evaluation of diagnostic characters, either phenotypic or molecular or both. Verification of identifications is crucial but often neglected. Because of clade-specific evolutionary histories, there is currently no single tool for the identification of fungi, although DNA barcoding using the internal transcribed spacer (ITS) remains a first diagnosis, particularly in metabarcoding studies. Secondary DNA barcodes are increasingly implemented for groups where ITS does not provide sufficient precision. Issues of pairwise sequence similarity-based identifications and OTU clustering are discussed, and multiple sequence alignment-based phylogenetic approaches with subsequent verification are recommended as more accurate alternatives. In metabarcoding approaches, the trade-off between speed and accuracy and precision of molecular identifications must be carefully considered. Intragenomic variation of the ITS and other barcoding markers should be properly documented, as phylotype diversity is not necessarily a proxy of species richness. Important strategies to improve molecular identification of fungi are: (1) broadly document intraspecific and intragenomic variation of barcoding markers; (2) substantially expand sequence repositories, focusing on undersampled clades and missing taxa; (3) improve curation of sequence labels in primary repositories and substantially increase the number of sequences based on verified material; (4) link sequence data to digital information of voucher specimens including imagery. In parallel, technological improvements to genome sequencing offer promising alternatives to DNA barcoding in the future. Despite the prevalence of DNA-based fungal taxonomy, phenotype-based approaches remain an important strategy to catalog the global diversity of fungi and establish initial species hypotheses.

Host preference and sorus location correlate with parasite phylogeny in the smut fungal genus Microbotryum (Basidiomycota, Microbotryales)

Article

Full-text available

Apr 2020

The smut fungal genus Microbotryum (Microbotryales, Pucciniomycotina) contains species that parasitize plants from many different lineages of euasterids, with host specificity of individual parasite species in general being exceptionally high. Additionally, it has been shown that the location of spore production in some species is related to spore dispersal. In this phylogenetic study based on ITS and LSU rDNA data of 57 Microbotryum spp., host spectra and sorus location are mapped on the phylogeny of Microbotryum species in order to understand the macroevolutionary patterns of these two traits.We find that monophyletic parasite clades correspond well with monophyletic host clades and also that monophyletic parasite groups in general produce their spores in the same plant organ. Ancestral state reconstruction inferred the most probable ancestral trait for sorus location being leaves and the most probable ancestral host family for the genus Microbotryum as being the Polygonaceae. According to molecular analyses, a newly sequenced specimen of Ustilago ducellieri, a seed parasite on Arenaria leptoclados, previously treated as synonym of Microbotryum duriaeanum, belongs to a lineage distinct from specimens of M. duriaeanum. A new combination, Microbotryum ducellieri, is accordingly proposed. Taxonomic implications of the presented analyses for the genera Bauhinus and Haradaea are briefly discussed.

Random Tanglegram Partitions (Random TaPas): An Alexandrian Approach to the Cophylogenetic Gordian Knot

Article

Full-text available

Apr 2020
SYST BIOL

Symbiosis is a key driver of evolutionary novelty and ecological diversity, but our understanding of how macroevolutionary processes originate extant symbiotic associations is still very incomplete. Cophylogenetic tools are used to assess the congruence between the phylogenies of two groups of organisms related by extant associations. If phylogenetic congruence is higher than expected by chance, we conclude that there is cophylogenetic signal in the system under study. However, how to quantify cophylogenetic signal is still an open issue. We present a novel approach, Random Tanglegram Partitions (Random TaPas) that applies a given global-fit method to random partial tanglegrams of a fixed size to identify the associations, terminals and nodes that maximize phylogenetic congruence. By means of simulations, we show that the output value produced is inversely proportional to the number and proportion of cospeciation events employed to build simulated tanglegrams. In addition, with time-calibrated trees, Random TaPas can also distinguish cospeciation from pseudocospeciation. Random TaPas can handle large tanglegrams in affordable computational time and incorporates phylogenetic uncertainty in the analyses. We demonstrate its application with two real examples: Passerine birds and their feather mites, and orchids and bee pollinators. In both systems, Random TaPas revealed low cophylogenetic signal, but mapping its variation onto the tanglegram pointed to two different coevolutionary processes. We suggest that the recursive partitioning of the tanglegram buffers the effect of phylogenetic nonindependence occurring in current global-fit methods and therefore Random TaPas is more reliable than regular global-fit methods to identify host-symbiont associations that contribute most to cophylogenetic signal. Random TaPas can be implemented in the public-domain statistical software R with scripts provided herein. A User's Guide is also available at GitHub.

A tale of worldwide success: Behind the scenes of Carex (Cyperaceae) biogeography and diversification

Article

Full-text available

Nov 2019

The megadiverse genus Carex (c. 2000 species, Cyperaceae) has a nearly cosmopolitan distribution, displaying an inverted latitudinal richness gradient with higher species diversity in cold‐temperate areas of the Northern Hemisphere. Despite great expansion in our knowledge of the phylogenetic history of the genus and many molecular studies focusing on the biogeography of particular groups during the last few decades, a global analysis of Carex biogeography and diversification is still lacking. For this purpose, we built the hitherto most comprehensive Carex‐dated phylogeny based on three markers (ETS–ITS–matK), using a previous phylogenomic Hyb‐Seq framework, and a sampling of two‐thirds of its species and all recognized sections. Ancestral area reconstruction, biogeographic stochastic mapping, and diversification rate analyses were conducted to elucidate macroevolutionary biogeographic and diversification patterns. Our results reveal that Carex originated in the late Eocene in E Asia, where it probably remained until the synchronous diversification of its main subgeneric lineages during the late Oligocene. E Asia is supported as the cradle of Carex diversification, as well as a “museum” of extant species diversity. Subsequent “out‐of‐Asia” colonization patterns feature multiple asymmetric dispersals clustered toward present times among the Northern Hemisphere regions, with major regions acting both as source and sink (especially Asia and North America), as well as several independent colonization events of the Southern Hemisphere. We detected 13 notable diversification rate shifts during the last 10My, including remarkable radiations in North America and New Zealand, which occurred concurrently with the late Neogene global cooling, which suggests that diversification involved the colonization of new areas and expansion into novel areas of niche space.

The rediscovery of the rare Vietnamese endemic Eriophorum scabriculme redefines generic limits in the Scirpo-Caricoid Clade (Cyperaceae)

Article

Full-text available

Sep 2019

For those familiar with boreal bogs and wet tundra, species of Eriophorum (“the cotton grasses”) will undoubtedly represent some of the most striking and memorable taxa they have encountered. This small genus of 20 Holarctic sedge species (Cyperaceae) is remarkable because its inflorescences produce large, brilliantly white to rusty-red cottony masses when its flowers develop a perianth of highly elongated bristles after anthesis. In this study, we document the rediscovery of Eriophorum scabriculme , a narrow Vietnamese endemic known from only two collections made approximately 7 km apart near Sa Pa in Lào Cai Province over 75 years ago. Using plastid DNA sequences ( matK , ndhF ), embryology, and morphology, we test whether E. scabriculme is aligned within the Scirpo-Caricoid Clade (genus Khaosokia and tribes Cariceae, Dulichieae, Scirpeae, and Sumatroscirpeae) or the Ficinia Clade (Cypereae), and we determine whether its unique character combinations (≥10 elongated bristles, reduced sheathing basal leaves, 1–4 spikelets) could be evidence for a new genus or simply mark it as an unusual species within currently recognised genera. In addition, we document the discovery of seven new populations, and we extend its range westward to Lai Châu Province and southward in Lào Cai Province by more than 47 km. Our results demonstrate that Eriophorum scabriculme is best treated in the genus Trichophorum , thus re-circumscribing both genera and their limits with Scirpus s.str. In addition, we emend the description of Trichophorum scabriculme (Beetle) J.R.Starr, Lév.-Bourret & B.A. Ford, provide the first pictures and accurate illustration of the species, and assess its conservation status in Vietnam (VU, Vulnerable). Our study corroborates the fact that in such a diverse and taxonomically difficult family like the sedges, conspicuous characters like highly elongated bristles may be useful for dividing diversity, but they are no guarantee that the groups they mark are natural.

Trait evolution rates shape continental patterns of species richness in North America’s most diverse angiosperm genus (Carex, Cyperaceae)

Article

Mar 2021

Ecological opportunity has been associated with increases in diversification rates across the tree of life. Under an ecological diversification model, the emergence of novel environments promotes morpho‐ and ecospace evolution. Whether this model holds at the clade‐level within the most species‐rich angiosperm genus found in North America (Carex, Cyperaceae) has yet to be tested. Recent work demonstrates a temporal coupling of climate cooling and widespread colonization of Carex in North America, implicating ecological diversification. Additionally, research has consistently found asymmetric patterns of lineage‐level diversification in the genus. Why does variation in clade sizes exist in the genus? Is ecological diversification involved? In this study, we tested whether rates of morphological and ecological trait evolution are correlated with clade‐level species richness in Carex of North America north of Mexico. We constructed a phylogeny of 477 species—an almost complete regional sample. We estimated rates of evolution of morphological traits, habitat, and climatic niche and assessed whether differences in rates of evolution correlate with species richness differences in replicate non‐nested sister clades. Our work demonstrates significant positive correlations between climatic niche rates, habitat and reproductive morphological evolution, and species richness. This coupling of trait and niche evolution and species richness in a diverse, continental clade‐sample strongly suggests that the ability of clades to explore niche and functional space has shaped disparities in richness and functional diversity across the North American flora region. Our findings highlight the importance of the evolutionary history of trait and niche evolution in shaping continental and regional floras. This article is protected by copyright. All rights reserved.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity

Article

Sep 2020

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

3. Taxonomy of the Cereal Rust Fungi

Chapter

Dec 1984

D.B.O. Savile

Crown rust fungi with short lifecycles – the Puccinia mesnieriana species complex

Article

Jun 2019
SYDOWIA

Cryptic diversity and significant cophylogenetic signal detected by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae‐Juncaceae

Abstract and Figures

Recommended publications

Phylogeny and Systematics of Cyperaceae, the Evolution and Importance of Embryo Morphology

Molecular and morphological data reveal three new tribes within the Scirpo‐Caricoid Clade (Cyperoide...

Why are there so many sedges? Sumatroscirpeae, a missing piece in the evolutionary puzzle of the gia...

The rediscovery of the rare Vietnamese endemic Eriophorum scabriculme redefines generic limits in th...