Fungal Diversity
Molecular taxonomy, origins and evolution of freshwater
ascomycetes
Dhanasekaran Vijaykrishna*#, Rajesh Jeewon and Kevin D. Hyde*
Centre for Research in Fungal Diversity, Department of Ecology & Biodiversity, University of
Hong Kong, Pokfulam Road, Hong Kong SAR, PR China
Vijaykrishna, D., Jeewon, R. and Hyde, K.D. (2006). Molecular taxonomy, origins and
evolution of freshwater ascomycetes. Fungal Diversity 23: 351-390.
Fungi are the most diverse and ecologically important group of eukaryotes with the majority
occurring in terrestrial habitats. Even though fewer numbers have been isolated from
freshwater habitats, fungi growing on submerged substrates exhibit great diversity, belonging
to widely differing lineages. Fungal biodiversity surveys in the tropics have resulted in a
marked increase in the numbers of fungi known from aquatic habitats. Furthermore, dominant
fungi from aquatic habitats have been isolated only from this milieu. This paper reviews
research that has been carried out on tropical lignicolous freshwater ascomycetes over the past
decade. It illustrates their diversity and discusses their role in freshwater habitats. This review
also questions, why certain ascomycetes are better adapted to freshwater habitats. Their ability
to degrade waterlogged wood and superior dispersal/ attachment strategies give freshwater
ascomycetes a competitive advantage in freshwater environments over their terrestrial
counterparts. Theories regarding the origin of freshwater ascomycetes have largely been based
on ecological findings. In this study, phylogenetic analysis is used to establish their
evolutionary origins. Phylogenetic analysis of the small subunit ribosomal DNA (18S rDNA)
sequences coupled with bayesian relaxed-clock methods are used to date the origin of
freshwater fungi and also test their relationships with their terrestrial counterparts. Phylogenies
indicate that freshwater ascomycetes have evolved from terrestrial fungi and appear to occur in
only three classes. The adaptation to populate freshwater substrates has occurred in several
lineages. The earliest possible date, when fungi became adapted to freshwater habitation is
estimated at 390 million years ago (MYA).
Key words: ascomycetes, bayesian relaxed-clock, freshwater fungi, lignicolous, molecular
dating, phylogenetics.
*
Corresponding authors: D. Vijaykrishna; email: veej@hkucc.hku.hk; K.D. Hyde; e-mail:
kdhyde@hkucc.hku.hk
#
Current address: State Key Laboratory of Emerging Infectious Diseases, Department of
Microbiology, The University of Hong Kong, Faculty of Medicine Building, Hong Kong SAR
351
Introduction
Lignicolous freshwater ascomycetes inhabit submerged woody material
in lentic (lakes, ponds, swamps, pools) and lotic (rivers, streams, creeks,
brooks) habitats (Wong et al., 1998a; Luo et al., 2004), playing an important
role in recycling organic matter in the ecosystem. This review comprises of
two sections. The first one deals with the biology of these fungi in the tropics,
bringing together the large volume of recent data available on this subject and
providing facts on their biodiversity and distribution. In the second section, the
phylogenetic relationships between freshwater taxa and their terrestrial and
marine counterparts are inferred from available rDNA sequences and their
possible routes of origins are also explored. Future research needs for these
fascinating groups of organisms are mentioned.
Lichens are associations between ascomycetes and algae, which can also
occur on river and lake margins subjected to varying water levels (e.g. Collema
dichotomum and Verrucaria rheitrophila). Lichens associated with freshwater
environments have been comprehensively reviewed by Hawksworth (2000)
and Aptroot and Seaward (2003). These will not be addressed in this paper.
The life cycle of ascomycetes are characterised by sexual (teleomorphic)
and asexual (anamorphic) states. In nature, fungi are usually encountered in
only one of these states. For a few of these ascomycetes, whose anamorphic
states are unknown from nature, teleomorphic/anamorphic connections have
been established by nucleic acid similarity and cultural studies (e.g. Huhndorf
and Fernández, 2005). Furthermore, based on nucleic acid variation, the
anamorphs have been amalgamated with the ascomycete classification, which
until recently had been segregated as Fungi Imperfecti (Taylor et al., 1999a).
What are freshwater ascomycetes?
Freshwater ascomycetes are defined as ascomycetes which have been
recorded in freshwater habitats and which complete part, or the whole of their
lifecycle within freshwater environments (Shearer, 1993; Thomas, 1996; Wong
et al., 1998a). According to this definition, in addition to species that function
in water, transient fungi present in water and terrestrial fungi that release
spores that are dispersed in water are all regarded as freshwater ascomycetes
(Luo et al., 2004). Thomas (1996), however, states that the aquatic nature of
some substrates is questionable (e.g. emerging parts of a plant), and therefore
fungi growing on these substrates cannot be classified as freshwater fungi.
Emergent macrophytes are mostly absent in rivers and streams with strong
flow rates and high riparian shading (Shearer, 2001). Therefore with the
exception of the bases of standing grasses (Van Ryckegem and Verbeken,
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Fungal Diversity
2004) and some macrophytes (Luo et al., 2004), most plant litter in an aquatic
environment is of terrestrial origin. As such, the earlier definitions of
freshwater fungi might not be appropriate, as physiological and developmental
studies are needed to support the theory that freshwater ascomycetes require
water to complete their life cycle (Shearer, 1993).
Fungi that are recorded in freshwater habitats can be indwellers or
immigrants. “Indwellers” are fully adapted to aquatic environments and can
grow and sporulate in water and are often adapted to dispersal in water, while
“immigrants” must continually immigrate from other habitats to maintain their
population in water (Park, 1972). Indwellers probably do not have unique
characters that define their aquatic existence. Wong et al. (1998a) points out
that there are numerous ascomycete species that commonly occur in freshwater
habitats and have not been found in terrestrial habitats, and only these fungi
can be confidently categorised as freshwater ascomycetes. Due to the
controversies associated with the delimitation of this ecologically distinct
group, any fungi that have been isolated from submerged plant substrates will
be considered as freshwater ascomycetes and discussed in this paper.
Biodiversity of tropical freshwater ascomycetes
Shearer (1993) listed 288 fungi that had been recorded from freshwater
habitats; this number has grown to 511 (Shearer, 2001; Cai et al, 2003a). The
number increased dramatically during the last ten years because numerous new
taxa have been recorded from submerged wood in tropical streams (e.g. Tsui et
al., 2001a; Vijaykrishna and Hyde, 2006) (see Cai et al., 2003b). Only 11 of
the 288 fungi (3.82 %) listed by Shearer (1993) were from tropical locations,
while 177 of the 511 taxa (34.6 %) have a tropical distribution (Cai et al.,
2003a). The low ratio of tropical to temperate freshwater ascomycetes had
resulted from greater research efforts in temperate regions (Hyde et al., 1997).
In similar biodiversity studies on fungi on submerged wood in temperate
(River Coln: Hyde and Goh, 1999) and tropical streams (Queensland: Hyde
and Goh, 1997a,b) it was found that tropical rivers support a greater diversity
of fungi (Ho et al., 2001; Cai et al., 2002). Of the 223 new records or species
reported from freshwater habitats since 1993, tropical ascomycetes account for
more than 75% of these new records (Cai et al., 2003a). The percentage of
tropical as compared to temperate taxa is bound to increase as more work is
carried out in the tropics (e.g. Tsui et al., 2000, Cai et al., 2002; Fryar et al.,
2004, Tsui and Hyde, 2004).
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Genera of freshwater ascomycetes
Freshwater lignicolous ascomycetes do not form a monophyletic group,
but are an ecological group of organisms. They comprise species from the three
classes, Leotiomycetes, Dothideomycetes and Sordariomycetes belonging to
the Super class Leotiomyceta, under the subphyla Pezizomycotina, represented
by 19 orders (Shearer 2001; Cai et al., 2003a) (For classification see, Eriksson
et al., 2003). The taxonomic position of a large number of these fungi is still
uncertain (see Eriksson et al., 2003). Representatives of freshwater
ascomycetes have been recorded in other habitats (terrestrial or marine).
Accordingly, the genera of freshwater lignicolous ascomycetes can be divided
in four main groups based on their occurrence. These include 1) genera that are
exclusively known from freshwater habitats, 2) genera with both freshwater
and terrestrial species, 3) genera with freshwater and marine species and 4)
genera that are found in freshwater, marine and terrestrial habitats. The most
common genera from freshwater habitats under these categories are listed in
Table 1 and each grouping is discussed below. These classifications are
probably premature, because recently, several freshwater fungi have been
discovered from terrestrial habitats. e.g. Cataractispora species have been
known only from wood submerged in freshwater (Hyde et al., 1999a; Ho et al.,
2004) however, we have recently collected a species from terrestrial grasses.
Genera exclusively known from freshwater habitats
Most uniquely freshwater genera are confined to the family
Annulatascaceae, which incorporates genera with some apparently exclusive
adaptations to the aquatic environment (Hyde et al., 1997, 2000a; Wong et al.,
1998b; Ho and Hyde, 2000; Tsui et al., 2002; Tsui et al., 2003; Lee et al.,
2004). Although most species share a massive apical ring, which possibly
facilitates dispersal of spores, the morphology of the ascospores is quite
diverse (Ho et al., 1999a,b; Wong et al., 1999; Lee et al., 2004). Species of
Cataractispora and Diluviocola have ascospores with uncoiling thread-like
appendages that are believed to aid in dispersal and subsequent attachment in
water (Hyde et al., 1998, 1999a; Hyde and Goh, 2003). Some species in
Annulatascus have ascospores with sticky mucilaginous sheaths, and
exosporial wall ornamentations, which are thought to aid in the attachment to
substrates (Ho et al., 1999a,b; Hyde and Wong, 2000a; Hyde and Goh, 2003).
In Fluminicola ascospores have bifurcate polar appendages (Wong et al.,
1999), while in Pseudoproboscispora ascospores have proboscis-like
appendages which uncoil (Wong and Hyde, 1999). Both appendage types are
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Fungal Diversity
Table 1. Common freshwater genera with terrestrial, marine/terrestrial or no
counterparts (modified from Hyde and Goh, 1998; Tsui et al., 2000).
Freshwater only
Freshwater/
Terrestrial
Freshwater/
Marine
Freshwater/ Marine/
Terrestrial
Sordariomycetidae
Aquaticola
Cataractispora
Pseudoproboscispora
Rivulicola
Torrentispora
Annulatascus*
Ascotaiwania*
Cercophora
Ophioceras*
Pseudohalonectria*
Aniptodera
Halosarpheia
Nais
Savoryella
Anthostomella
Phomatospora
Dothideomycetidae
Jahnula
Mamillisphaeria
Byssosphaeria
Kirschsteiniothelia
Quintaria
Lophiostoma
Massarina
Didymella
Leotiomycetidae
Vibrissea
probably useful in attachment. The unitunicate genus Ascosacculus is
characterised by deliquescent asci, with guttulate ascospores and apical
appendages and is the only genus within the Halosphaeriales found only in
freshwater habitats. Other genera of Halosphaeriales found in freshwater
habitats can also be found in marine and brackish water (Van Ryckegem and
Verbeken, 2005).
Jahnula is probably the only common ascomycete genus within the class
Dothideomycetes found only in freshwater (Table 1). The peridium of large
soft-walled cells in Jahnula and the dimorphic nature of its ascospores (one
ascospore type having bipolar mucilaginous pads) may be important freshwater
characteristics (Hawksworth, 1984; Hyde et al., 1999b). Another striking
feature, are the rhizoid-like structures which enable the ascomata to attach to
the substrate while they hang in water which may aid dispersal (Pinruan et al.,
2002). Mamillisphaeria is also known only from freshwater habitats and has
dimorphic ascospores, as in Jahnula. These characters are thought to be
important in dispersal in the freshwater environment (Hyde et al., 1996, 1999b;
Hyde and Goh, 2003).
There are other genera only known from freshwater habitats, but they
have rarely been recorded. Ascovaginospora has ascospores with remarkable
tetraradiate sheaths (Fallah et al., 1997), while in Ascominuta ascospores have
a swollen sheath (Ranghoo and Hyde, 2000). Both sheath types are probably
important in dispersal and subsequent attachment. In Ascoyunnania, a recently
described ascomycete from submerged bamboo, the hyaline ascospores
355
germinate within the asci and form dark brown to black, warted secondary
spores. These secondary dark spores bear a large hyaline, thin-walled, smooth,
non guttulate, ellipsoidal sac-like structure which are remains of the
ascospores. The condition where spores directly form secondary spores,
without formation of mycelium is known as microcyclic conidiation, where the
normal lifecycle of the fungus is bypassed (Cai et al., 2005). These spores may
help the fungus to survive dry conditions whereas the hyaline ascospores may
be adequate for aquatic dispersal.
Genera with both freshwater and terrestrial species
Common freshwater genera within the Sordariomycetes with terrestrial
representatives include Annulatascus, Ascotaiwania, Cercophora, Ophioceras
and Pseudohalonectria (Table 1). These genera probably have characteristics
that are pre-adapted for dispersal in freshwater. Species of Pseudohalonectria
were previously only known from freshwater (Hyde, 1995; Shearer, 1989a).
However, Hyde et al. (1999e) described two new species of Pseudohalonectria
from terrestrial palms and Promputtha et al. (2004) described a new species
from leaves of Magnolia liliifera. Most Ophioceras species are known from
freshwater (Tsui et al., 2001b), however Fröhlich and Hyde (2000) have
identified several species from terrestrial palms.
In most of these genera, it appears that the characters that are important
for dispersal in the terrestrial environment may also be important in the aquatic
environment. Most species in these genera have the ability to eject their
ascospores. Their ascospore appendages or sheaths may be pre-adapted for
dispersal in freshwater. Terrestrial and aquatic species of Phomatospora have
bipolar mucilaginous or hair-like appendages (Fallah and Shearer, 1998; Hyde
et al., 2000a), species of Ophioceras have sigmoid ascospores that probably
have mucilage at their tips (Goh and Hyde, 1996) and species of Saccardoella
have large mucilaginous sheaths (Tsui et al, 1998). These spore shapes, and
appendages and sheath types, have been shown to be important in the dispersal
and subsequent attachment of marine fungi (Hyde and Jones, 1989; Hyde et al.,
1993).
Bitunicate ascomycetes that occur in freshwater and terrestrial habitats
(e.g. Byssosphaeria and Kirschsteiniothelia) eject ascospores via fissitunicate
dehiscence and ascospores are often sheathed (Shearer, 1993). These
characteristics appear to be advantageous in both terrestrial and freshwater
habitats and are also observed in marine species (Hyde et al., 2000b). Some
loculoascomycetes have developed elaborate sheaths, which are massive
structures to better facilitate attachment (Shearer and Hyde, 1997), or have
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Fungal Diversity
uniquely shaped sheaths, e.g. arrow-like (Tsui et al., 1999), that arguably aid in
attachment to substrata in freshwater.
Genera within the class Leotiomycetes are predominantly terrestrial,
however a large number of these species are also found in temperate freshwater
habitats. The number of tropical isolations is very low compared to temperate
regions. However, the majority of teleomorph connections made for Ingoldian
fungi (aquatic hyphomycetes) belong to the class Leotiomycetes (Sivichai and
Jones, 2003), and several have been identified from tropical habitats. The
conidia of Ingoldian fungi are characterized with branched, sigmoid or helicoid
conidia (Gönczöl and Révay, 2004; Sakayaroj et al., 2005; Pascoal et al.,
2005), which help in the attachment of the conidia to substrates.
Genera with freshwater and marine species
The unitunicate genera Aniptodera, Halosarpheia, Nais and Savoryella
are the most commonly reported ascomycetes that occur in both freshwater and
marine habitats (Maria and Sridhar, 2003; Prasannarai and Sridhar, 2003; Tsui
and Hyde, 2004) (Table.1). These fungi may have asci that deliquesce early
(e.g. Halosarpheia species), or are persistent (e.g. Aniptodera species)
(Shearer, 1989b; Hyde et al., 1999d). In some species asci have an apical pore
(e.g. Savoryella species), but it is not clear if the pore is functional (Jones and
Hyde, 1992; Ho et al., 1997). A layer of mucilage surrounds the ascospores of
Savoryella species, which help the ascospores to adhere to the substrate they
come in contact with (Read et al., 1993). In some Aniptodera species and all
Halosarpheia species, ascospores have bipolar unfurling appendages which aid
in the attachment to substrata (Hyde et al., 1999d), but other Aniptodera
species lack appendages. Besides their occurrence in water, there are no
distinctive features that can be used to set apart these latter taxa as aquatic
fungi.
Genera found in freshwater, marine and terrestrial habitats
The fourth group is genera occurring in freshwater, marine and terrestrial
habitats and is best represented by genera within the class Dothideomycetes,
Massarina and Lophiostoma (Hyde and Aptroot, 1997; Aptroot, 1998; Liew et
al., 2002) (Table 1). Species of Massarina are frequently identified in
mangrove habitats (Hyde and Borse, 1986; Hyde et al., 1990), streams and on
terrestrial palms (Hyde and Aptroot, 1997, 1998; Aptroot et al., 2000).
Lophiostoma species are mostly terrestrial (Chesters and Bell, 1970; Holm and
Holm, 1988), but some species are also known from aquatic environments
357
(Shearer, 1993; Hyde and Aptroot, 1998; Liew et al., 2002; Hyde et al, 2002).
There are little to no distinct differences between the species of the above
genera that occur in different habitats. Ascomata develop under a thick
pseudostroma, asci eject their ascospores during fissitunicate dehiscence, and
ascospores generally have a mucilaginous sheath (Aptroot, 1998; Liew et al.,
2002; Hyde et al., 2002), however, aquatic species of Lophiostoma have
ascospores with elaborate sheaths (Lophiostoma ingoldianum) or long
appendages (Lophiostoma frondisubmersum). These characteristics appear to
be advantageous in all the freshwater, marine and terrestrial habitats and may
account for the widespread and frequent occurrence of these species.
Species of Anthostomella, Didymella, Phomatospora and Saccardoella
also occur in all three habitats. Phomatospora is a unitunicate genus common
in terrestrial habitats (Nograsek, 1990), but has both freshwater and marine
species (Fallah and Shearer, 1998; Hyde et al., 2000b). Similarly species of the
bitunicate genus Saccardoella may occur in freshwater, marine and terrestrial
habitats (Hyde, 1992; Mathiassen, 1993, Tsui et al., 1998). The freshwater and
marine taxa of these genera do not have special adaptations, although
Phomatospora species have ascospores with bipolar mucilaginous pads of hairlike appendages (Fallah and Shearer, 1998; Hyde et al., 2000b). Didymella and
Saccardoella species have sheathed ascospores that are similar to those found
in terrestrial species of the same genera (Hyde and Aptroot, 1998; Hyde and
Wong, 2000b). The genus Vibrissea (Class Leotiomycetes), whose species
occur in all three habitats, also appear to lack any specific adaptations to the
aquatic environment (Hyde et al., 1999c).
Distribution of tropical lignicolous freshwater ascomycetes
The biogeography of freshwater ascomycetes has been discussed by
Shearer (1993), Hyde et al. (1997), Shearer (2001) and Cai et al. (2003a). Of
the 511 ascomycetes listed by Shearer (2001) and Cai et al. (2003a), 177 had a
tropical distribution with only 20 also occurring in temperate regions. This low
overlap supports the suggestion of Hyde et al. (1997) that a distinctive
freshwater ascomycete community exists in the tropics. There are numerous
taxa that have only been reported from temperate (e.g. Massarina aquatica) or
tropical regions (e.g. Aquaticola ellipsoidea). The conclusion reached for
Ingoldian fungi that many species are cosmopolitan with both tropical and
temperate distributions seems less well fitting for lignicolous ascomycetes,
which appear to have greater temperate or tropical distributions. However,
recently many ascomycetes described from tropical freshwater habitats have
been discovered in a temperate freshwater habitat (Fallah and Shearer, 2001)
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Fungal Diversity
Many freshwater ascomycetes have only been described with limited
distributions, but with the availability of more data it is apparent that many
species have much wider distributions. Annulatascus velatisporus was
previously only known from Australia (Hyde, 1992) but is now known to be
pan tropical and even occurs in temperate regions (Campbell and Shearer,
2004). These trends indicate that most freshwater ascomycetes have a pan
tropical or pan temperate distribution and some may also overlap in warm
temperate or subtropical regions.
Physiological studies of freshwater fungi indicate that the optimal growth
conditions are identical for both temperate and tropical taxa. The optimum
temperature for growth for most tropical freshwater fungi is between 20-25°C,
although several isolates exhibited optimal growth rates as low as 15°C or as
high as 30°C (Yuen et al., 1998). Temperate freshwater fungi also have similar
optimum growth rates (Zare-Maivan and Shearer, 1988a,b), showing an
optimal temperature of 25°C for most temperate taxa, although they could
grow relatively well at temperatures as low as 10°C. This would suggest that
there is no such thing as tropical or temperate taxa. Yuen et al. (1998)
however, concluded that tropical freshwater fungi do not grow well at low
temperatures, and so are absent in streams in temperate regions. On the other
hand, although temperate species grow best at 25°C, they were not able to
grow as rapidly as tropical species and this probably accounts for their absence
in tropical streams.
Probably one of the most intriguing age-old problems is how these
freshwater species have managed to colonise streams in distance parts of the
world. These aquatic fungi can have evolved in one region, so how do they
now occur in several continents. Wood-Eggenschwiler and Bärlocher (1985)
reviewed the information on the biogeography of the freshwater hyphomycetes
and found three basic trends;
1. Cosmopolitan species, although these species may be more common in the
tropics or a temperate situation.
2. Species restricted to temperate and cold tropical regions.
3. Species restricted to a very small geographical area.
Wood-Eggenschwiler and Bärlocher (1985) illustrated that aquatic
hyphomycetes are not restricted by geographical barriers and that similarities
in mycota were found between geographically distant tropical locations, as
well as in temperate locations on either side of the equator. Hyde and Goh
(2003) illustrated four possible ways by which freshwater ascomycetes appear
in different continents. Fungi might have evolved before the split of the
continents and then moved with the landmasses, or carried over to different
359
continents on plant substrates. Fungal spores might have also been dispersed
by animals (e.g. birds), or wind.
Origins of freshwater ascomycetes
Overview on the origin of ascomycetes
Ascomycetes are one of the four phyla in the Kingdom Fungi. They are
the largest group, containing more than 32,000 species, and have a variety of
associations with plants, animals, algae and cyanobacteria. Ascomycetes are
distinguished from other fungi, as they form sexual spores within a sac-like
structure called the ascus. Ascomycetes are a monophyletic group of fungi,
descended from a common ancestral species (Berbee and Taylor, 2001,
Keeling et al., 2000, Liu et al., 1999; Lumbsch, 2000) with Basidiomycetes as
sister taxa (Bruns et al., 1993; Berbee and Taylor, 1993, 2001). Information
concerning the evidence of radiation of major groups of fungi comes from
diverse sources.
Evidence on the origin of fungi has been obtained from fossil records
(Tiffney and Barghoorn, 1974, Sherwood-Pike and Gray, 1985; Hass et al.,
1994; Remy et al., 1994a,b; Taylor, 1994; Taylor et al., 1995, 1992a,b, 1999b,
2004). Based on these records, fungi are presumed to have been present in Late
Proterozoic (900-570 million years ago (MYA)). Sherwood-Pike and Gray
(1985) reported chains of asexual spores and perforate hyphae from digested
rock samples from the Silurian Period (438-408 MYA). Fossil hyphae in
association with wood decay, fossil chytrids and Glomales (arbuscular
mycorrhizae) representatives associated with plants of the Rhynie Chert
(Aberdeenshire, Scotland) are reported from the Devonian Period (408-360
MYA) (Hass et al., 1994; Remy et al., 1994a,b; Taylor et al., 1992a, 1995).
Fossil “fungi” from the Precambrian era are now considered to be filamentous
sheaths of cyanobacteria.
At present, the only oldest definite ascomycete fossils are reported from
Rynie Chert dating to the Lower Devonian Period (400 MYA) (Taylor et al.,
2004). Furthermore, interpretation of earlier fungal fossils is difficult due to
lack of diagnostic characters present in modern taxa (Berbee and Taylor,
1993). Due to the lack of interpretational fossil evidence and obscure
morphology, the dating of fungal evolution has always been a daunting task for
evolutionary biologists and mycologists. These fossil records, however, are
crucial calibration points for the molecular dating of fungal radiation (Berbee
and Taylor, 2001).
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Fungal Diversity
Nonetheless, global environmental changes have left footprints in the
DNA of living organisms from which the evolutionary history can be reliably
inferred (Brohman and Penny, 2003). With the advent of molecular techniques
several investigations have dealt with the origin of various major groups of
fungi (e.g. Bruns et al., 1993; Berbee and Taylor, 1993, 1995, 2001).
Furthermore, most information on the timescale of fungal evolution is derived
from analysis of DNA and protein sequence data with molecular clocks
(Berbee and Taylor, 2001; Heckmann et al., 2001). DNA clock estimates of
fungi have proven fruitful, particularly, in dating the evolution of Glomales
(arbuscular mycorrhizae) (Simon et al., 1993, Taylor et al., 1995). Glomales
are ubiquitous root symbionts that have evolved concurrently with land plants
and have played crucial roles in the colonization of land by plants, as
hypothesised by Pirozynsky and Malloch (1975). This importance of symbiotic
association is also suggested by evidence of arbuscular mycorrhizae in the
earliest fossil fungi (460 MYA) and in the earliest land plants (Remy et al.,
1994b; Selosse and LeTacon, 1998; Redecker et al., 2000).
Molecular clock estimates based on 18S rDNA have shown that fungi
occurred about 800 MYA and the ascomycetes and basidiomycetes diverged
about 600 MYA (Berbee and Taylor, 1993). In contradiction, an analysis using
multiple protein-coding genes, in fungi and plants, Heckman et al. (2001)
suggested that fungi originated about 1.5 billion years ago, and the
ascomycete-basiodiomycete divergence took place around 1.2 billion years
ago. However, analysis based on plastid-encoding genes, suggest that the dates
for plants proposed by Heckman et al. (2001) may be too early (Sanderson,
2003; Sanderson et al., 2004), and this when extrapolated, would also be true
for fungi (Lutzoni et al., 2004). However there has been no further analysis
involving fungi.
Ecological theories: Origin of freshwater ascomycetes
The ‘multiple’ origins of freshwater ascomycetes was first proposed by
Shearer (1993). Freshwater ascomycetes could have evolved through several
pathways. Accordingly, freshwater ascomycetes could have been present as
endophytes, pathogens or saprobes on plants, and have become adapted to
aquatic environment when these plants invaded water. Freshwater ascomycetes
could have also reached the aquatic environment via tree and shrub litter or
from the run-off of rainwater and sediments. As an example, some freshwater
ascomycetes (e.g. Didymella aptrooti, Fluminicola bipolaris) mainly occur on
bamboo (Wong et al., 1999; Hyde and Wong, 2000a; Cai et al., 2003b). In
many countries bamboo grow along the banks of rivers and may have provided
361
a direct pathway for terrestrial ascomycetes to become aquatic species (Cai et
al., 2003b).
It has also been proposed that freshwater ascomycetes are unique in
having unique adaptations, for example their capability to degrade submerged
substrates. Most freshwater lignicolous ascomycetes, which have been
repeatedly isolated from aquatic habitats, are presumed to be soft rot (a
superficial decay type restricted to surface layers of wood) fungi (Yuen et al.,
2000). These types of decay are thought to be a better adaptation to degrading
wood in water-logged conditions (Bucher et al., 2004a,b). Furthermore,
enhanced mechanisms for dispersal and subsequent attachment in freshwater
habitats, as compared to their terrestrial counterparts indicate adaptations to
freshwater lifestyles. Various in vivo and in vitro studies have been carried out
to study the ability of tropical freshwater ascomycetes to degrade wood. Yuen
et al. (1998, 2000) as part of their study on weight loss in wood caused by
freshwater fungi, found that the terrestrial fungi they tested caused greater
weight losses when incubated on agar than to those incubated in liquid
medium. The reverse was true for the freshwater fungi tested. This indicates
that freshwater fungi are better adapted to submerged conditions, than
terrestrial fungi.
The ability to produce wood degradation enzymes has been studied for
both temperate (Abdel-Raheem and Shearer, 2002) and tropical freshwater
ascomycetes (Bucher et al., 2004a). In vitro studies show that tropical
freshwater ascomycetes produce cellulases and xylanases causing soft rot
cavities and also showed that they are capable of degrading the wood surface
when not in water. The study also showed that freshwater fungi could degrade
lignin (most refractory component in wood), at least as effectively as some
terrestrial ascomycetes which are known to cause white rot decay. It has also
been shown that some freshwater fungi are competitive against or may reduce
sporulation in other fungi and this may account for the commonness of some
fungi in the freshwater milieu (Fryar et al., 2001, 2005).
Earlier studies have shown that ascomycete/basidiomycete radiation took
place after the invasion of land by plants. Based on this, it is speculated that
current day freshwater ascomycetes should have evolved from their terrestrial
counterparts. There has been no molecular study that has investigated the
origin of freshwater ascomycetes, and evolutionary theories have mainly been
based on ecological findings.
362
Fungal Diversity
Relatives of freshwater ascomycetes
The most likely candidates for the close relatives of the freshwater
ascomycetes are among the diverse group of perithecial ascomycetes. The
latter are ecologically diverse and include parasites and pathogens of plants and
animals, endophytes of grasses and trees, symbionts of arthropods, and
decomposers of a wide range of organic substrates (Kumar and Hyde, 2004;
Suryanarayanan and Thennarasan, 2004; Zhang et al., 2004; Lee et al, 2005).
There are several terrestrial and marine lineages of fungi within the perithecial
ascomycetes that display characters present among the freshwater ascomycetes.
Freshwater lignicolous ascomycetes comprise species from the classes
Leotiomycetes, Dothideomycetes and Sordariomycetes belonging to the Super
class Leotiomyceta, under the subphyla Pezizomycotina, represented by 19
orders (Shearer 2001; Cai et al., 2003a). However the majority of the species
are restricted to only seven orders, Eurotiales, Halosphaeriales, Helotiales,
Hypocreales, Pleosporales, Sordariales and Xylariales. The rest of the orders
contain less than 10 freshwater taxa (Shearer, 2001).
Molecular clock versus relaxing the clock
The basic approach for estimating molecular dates is the estimation of
genetic distance between species, and then applying calibration rates i.e. the
number of genetic changes expected per unit time (nucleotide substitution
rates), for the conversion of genetic distance into time. Since the proposal of
the molecular clock hypothesis by Zuckerkandl and Pauling (1965), which is
based on the constancy of evolutionary rate over time, several investigations
have dealt with the dating of divergence of organisms. However, there has
been constant debate over its validity (Rodriguez-Trelles et al., 2001). A
number of tests have been developed to test the constancy of evolutionary rate
across lineages (Langley and Fitch, 1974; Wu and Li, 1985), and have often
rejected the molecular clock (constancy of evolutionary rate over time) in real
datasets (see Nei and Kumar, 2000: 188).
Attempts have been made to relax the assumptions of the global
molecular clock by allowing variations of the evolutionary rate along the
phylogenetic tree (Sanderson, 1997; Rambaut and Brohman, 1998; Thorne et
al., 1998; Yoder and Yang, 2000; Aris-Brosou and Yang, 2002). Among the
newly developed methods, the bayesian relaxed clock molecular clock
approach of Thorne et al. (1998) is becoming popular. There are several
advantages of the bayesian relaxed molecular clock approach. It allows the
calculation of divergence time estimates in the presence of rate variation
363
among lineages. It also allows the incorporation of multiple paleontological
constraints as priors (Kishino et al., 2001). This method has already been
successfully used in the calibration of HIV virus (Korber et al., 2000).
Molecular clock in estimating origin of freshwater ascomycetes
Objectives
The primary goal was to test the evolutionary hypothesis that freshwater
ascomycetes have evolved from their terrestrial counterparts, based on a
phylogenetic approach. For this, 18S rDNA sequences of a number of
freshwater ascomycetes are incorporated in a phylogenetic framework
containing members of the major groups of ascomycetes and other fungi. Their
phylogenetic affinities are also discussed combining recent literature on
morphology and phylogeny pertaining to freshwater fungi. These results in turn
have been used to investigate the origin of freshwater ascomycetes, attempting
to estimate the timescale of their evolution using recently developed molecular
tools
Methods
A large number of small subunit ribosomal genes (SSU rDNA) are
available for fungi. Sequences were downloaded from GenBank and aligned
with terrestrial and marine representative using Clustal X and edited by hand
(Chenna et al., 2003). Selection of the 18S was mainly due the availability of
sequences of a wide range of taxa, and the widespread usage of the gene for
molecular clock calibrations. Sequences represented much of the taxonomic
and ecological diversity among the three classes where freshwater ascomycetes
are sampled. Our final dataset consisted of 94 species representing the major
classes of the Kingdom Fungi (Table 2). Apart from representatives from
various classes of ascomycetes, members of Chitridiomycota, Glomales
(Zygomycota) and Basidiomycota were also included in our analyses.
Outgroup taxa included 2 members of Metazoa and 1 collar flagellate.
(a) Phylogenetic analyses
Maximum likelihood was performed using the likelihood ratchet methods
(Vos, 2003). This method was implemented to search for best trees using the
computer programs PAUPRat (Sikes and Lewis, 2001) and Paup* 4b10
364
Fungal Diversity
Table 2. Sequences of fungal taxa obtained from GenBank, with codes used in
the phylogenetic trees.
Code
Accession
number
Diaph gra
AF084234
Amoebidium parasiticum
Amoeb par
Y19155
Ichthyophonus hoferi
Chytridiomycota
Chytriomyces hyalinus
Chytridium polysiphoniae
Rhizophlyctis rosea
Spizellomyces acuminatus
Neocallimastix frontalis
Neocallimastix joyonii
Piromyces communis
Sphaeromonas communis
Zygomycota
Endogone pisiformis
Gigaspora rosea
Glomus fasciculatum
Glomus_intraradices
Basidiomycota
Hymenomycetes
Physalacria maipoensis
Pleurotus tuberregium
Limnoperdon incarnatum
Nia vibrissae
Ascomycota
Dothideomycetes
Jahnula australiensis
Jahnula bipolaris
Jahnula siamensiae
Patescospora separans
Byssothecium circinans
Cochliobolus sativus
Herpotrichia diffusa
Kirschsteiniothelia elaterascus
Kirschsteiniothelia maritima
Leptosphaeria doliolum
Ichth hof
U25637
Fungi/Metazoa incertae
sedis
Fungi/Metazoa incertae
sedis
Chytr hya
Chytr pol
Rhizo ros
Spize acu
Neoca fro
Neoca joy
Pirom com
Sphae com
M59758
AY032608
AY635829
M59759
M6270
M62705
M62706
M62707
Chytridiales
Chytridiales
Spizellomycetales
Spizellomycetales
Neocallimasticales
Neocallimasticales
Neocallimasticales
Neocallimasticales
Endog pis
Gigas ros
Glomu fas
Glomu int
X58724
X58726
Y17640
X58725
Endogonales
Glomales
Glomales
Glomales
Physa mai
Pleur tub
Limno inc
Nia vib
AYF42695
AF026595
AF42695
AF334754
Agaricales
Agaricales
Aphyllophorales
Melanogastrales
Jahnu aus
Jahnu bip
Jahnu sia
Patec sep
Bysso cir
Cochl sat
Herpo dif
Kirsc ela
Kirsc mar
Lepto dol
AF438182
AF438181
AF438180
AF438179
AY016339
U42479
U42484
AF053728
AF053726
U43457
Jahnulales
Jahnulales
Jahnulales
Jahnulales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Name
Collar flagellate
Diaphanoeca grandis
Basal animal
Group/Order
365
Table 2 continued. Sequences of fungal taxa obtained from GenBank, with
codes used in the phylogenetic trees.
Name
Lophiostoma bipolare
Lophiostoma crenatum
Massarina australiensis
Montagnula opulenta
Pleospora herbarum
Trematosphaeria hydrela
Tubeufia helicoma
Eurotiomycetes
Eupenicillium javanicum
Hamigera striata
Talaromyces flavus
Leotiomycetes
Articulospora tetracladia
Chloroscypha chloromela
Dimorphospora foliicola
Geniculospora grandis
Loramyces juncicola
Phacidium infestans
Rhabdocline parkeri
Darkera parca
Saccharomycetes
Candida albicans
Debaryomyces hansenii
Sordariomycetes
Camarops microspora
Chaetomium elatum
Aniptodera juncicola
Halosarpheia marina
Halosphaeria appendiculata
Lignincola laevis
Nimbospora effusa
Nohea umiumi
Phaeonectriella lignicola
Chaetopsina fulva
Cordyceps bifusispora
Emericellopsis minima
Hypocrea lutea
Hypocrea schweinitzii
366
Code
Lophi bip
Lophi cre
Massa aus
Monta opu
Pleos her
Trema hyd
Tubeu hel
Accession
number
AF164365
U42485
AF164364
AF164370
U43458
AF164376
AF201455
Group/Order
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Pleosporales
Eupen jav
Hamig str
Talar fla
U21298
AB003948
M83262
Eurotiales
Eurotiales
Eurotiales
Artic tet
Chlor chl
Dimor fol
Genic gra
Loram jun
Phaci inf
Rhabd par
AY357270
AF203461
AY357274
AY357276
AF203464
AF203466
AF106016
Darke par
AF203465
Helotiales
Helotiales
Helotiales
Helotiales
Helotiales
Helotiales
Heloitiales
Leotiomycetes incertae
sedis
Candi alb
Debar han
X53497
X62649
Saccharomycetales
Saccharomycetales
Camar mic
Chaet ela
Anipt jun
Halos mar
Halos app
Ligni lae
Nimbo eff
Nohea umi
Phaeo lig
Chaet ful
Cordy oph
Emeri min
Hypoc lut
Hypoc sch
AY083800
M83257
U43845
AF352082
U46872
U46873
U46877
U46878
AF050484
AB003786
AF339571
U44043
D14407
AF164357
Boliniales
Chaetomiales
Halosphaeriales
Halosphaeriales
Halosphaeriales
Halosphaeriales
Halosphaeriales
Halosphaeriales
Halosphaeriales
Hypocreales
Hypocreales
Hypocreales
Hypocreales
Hypocreales
Fungal Diversity
Table 2 continued. Sequences of fungal taxa obtained from GenBank, with
codes used in the phylogenetic trees.
Code
Melan zam
Nectr cin
Nectr lug
Nectr pse
Sphae acr
Micro cir
Petri set
Pseud boy
Ambro sur
Endom sco
Ophio que
Ophio ste
Madur myc
Lasio ovi
Sorda fim
Caini gra
Fasci pet
Hypon bux
Hypox fra
Micro niv
Xylar car
Accession
number
U78356
U32412
AY204603
AY342011
U76340
M89994
U43908
U43915
AY497509
AF267227
AY497515
M85054
AF527811
AY083799
X69851
AY083801
AY083809
AF130976
AB014046
AF548077
Z49785
Group/Order
Hypocreales
Hypocreales
Hypocreales
Hypocreales
Hypocreales
Miscroascales
Miscroascales
Miscroascales
Ophiostomatales
Ophiostomatales
Ophiostomatales
Ophiostomatales
Sordariales
Sordariales
Sordariales
Xylariales
Xylariales
Xylariales
Xylariales
Xylariales
Xylariales
Taphr wie
AY548293
Taphrinales
Savor elo
HKUCC
Savoryella longispora
Savor lon
HKUCC
Ascitendus austriaca
Ascit aus
AF24226
Magnaporthe grisea
Magna gri
AB026819
Ophioceras arcuatisporum
Ophio arc
AF050472
Pseudohalonectria falcata
Pseud fal
AF050477
Pseudohalonectria lignicola
Pseud lig
AF050478
Phomatospora arenaria
Phomatospora sp.
Phoma are
Phoma spn
CBS 372.92
HKUCC
Ascomycetes incertae
sedis
Ascomycetes incertae
sedis
Sordariomycetes
incertae sedis
Sordariomycetes
incertae sedis
Sordariomycetes
incertae sedis
Sordariomycetes
incertae sedis
Sordariomycetes
incertae sedis
Xylariales incertae sedis
Xylariales incertae sedis
Name
Melanospora zamiae
Nectria cinnabarina
Nectria lugdunensis
Nectria pseudotrichia
Sphaerodothis acrocomiae
Microascus cirrosus
Petriella setifera
Pseduallescheria boydii
Ambrosiella sulfurea
Endomyces scopilarum
Ophiostoma quercus
Ophiostoma stenoceras
Madurella mycetomatis
Lasiosphaeria ovina
Sordaria fimicola
Cainia graminis
Fasciatispora petrakii
Hyponectria buxi
Hypoxylon fragiforme
Microdochium nivale
Xylaria carpophila
Taphrinomycetes
Taphrina wiesneri
Incertae sedis
Savoryella elongata
367
(Swofford, 2000). This method is especially useful for datasets with large
number of taxa. A hierarchical maximum likelihood ratio test (LRT) with a tree
calculated under Neighbour Joining criterion under the Jukes Cantor model
(JC69) (Jukes and Cantor, 1969) was used to select an evolutionary model and
estimate all parameters needed for the ML search using the software program
MrModelTest 2 (Posada and Crandall, 1998). Gaps were treated as missing
data in all analyses.
Bayesian phylogenetic analyses were performed using the program
MrBayes 3.04b (Huelsenbeck and Ronquist, 2001) which calculates posterior
probability using a MCMC approach with sampling according to the
Metropolis-Hastings algorithm (Huelsenbeck and Ronquist, 2001). All of our
analyses employed one cold chain and three incrementally heated chains,
where the heat of the ith chain is B = 1/[1 + (i - 1)T] and T = 0.2. Joint posterior
probability distributions were obtained for the phylogeny (including branch
lengths) and the parameters of the model of sequence evolution. For the
parameters of the model of sequence evolution, the one estimated for ML
analyses through MrModeltest (Posada and Crandall, 1998) was used. Three
million generations were sampled, with every 100th generation resulting in
30000 trees. Likelihood values stabilised after 10000 to 300000 generations
(Fig. 1). To further ensure that we include trees after the chain had reached a
stable value, the burnin was fixed at 30000 generations which produced 24000
sampled trees to calculate the posterior probabilities in a consensus tree. The
analysis was repeated 3 times starting from random trees to ensure that
different tree space were being sampled.
In addition to the bayesian posterior probability, phylogenetic confidence
was also assessed using the neighbour joining bootstrap proportions with the
model settings as derived through MrModeltest.
(b) Molecular dating
The same original dataset consisting of 94 taxa for the small subunit
ribosomal DNA (18S rDNA) was used for dating. As the relaxed molecular
clock relies on a topology to infer divergence times, we used the bayesian tree
topology (Fig. 1) as previously identified, which conforms to the current views
on ascomycete/basidiomycete relationships.
In the bayesian relaxed molecular clock approach (Thorne et al., 1998), it
is important to use prior constraints on independent calibration points dispersed
across the tree in order to reduce potential regional effects (Kishino et al.,
2001). To provide comparability among studies, we used the calibrations
according to Berbee and Taylor (2001) that are comparable with our taxon
sampling. Among the four calibration points defining the priors constraints;
368
Fungal Diversity
Fig1. Maximum likelihood tree with Bayesian posterior probability (%) at internodes.
369
three were minimum age constraints and one was a maximum age constraint.
In all subsequent bayesian analyses these six prior constraints on calibration
were simultaneously used to derive posterior estimates of divergence ages
(Kishino et al., 2001).
The analysis of divergence time estimation was conducted combining the
two different approaches: the likelihood ratio test (LRT: Felsenstein, 1981) and
the bayesian approach (Kishino et al., 2001) as described in a step-by-step
manual by Rutchmann (2004). In brief, the dataset was subject to maximum
likelihood analyses using the modules BASEML in the PAML 3.12 program
package (Yang, 1997), using the tree shown in Fig. 1. This was done for the
estimation of model parameters such as, unequal nucleotide frequencies,
transition/transversion rate ratio (parameter: κ) and the rate heterogeneity
among sites (Shape parameter: α including a discrete γ model of rates among
sites). The bayesian relaxed molecular clock was applied using the software
package MULTIDIVTIME. The program ESTBRANCHES was first used to
compute branch lengths of the constrained topology. The program
MULTIDIVTIME used the variance-covariance matrices produced by
ESTBRANCHES to run a MCMC for estimating mean posterior divergence
times on nodes with associated standard deviation (SD) and 95% credibility
interval. The following settings were used: numstamp (10,000), burnin
(100,000), and samplefreq (100). The following priors were used for the
expected number of time units between tip and root if there has been no
constraint on node times, and 1000 MYA for the minimum possible number of
time units between tip and root. Other priors for gamma distribution of the rate
at root node and the Brownian motion constant describing the rate variation
(i.e. the degree of rate autocorrelation along the descending branches of the
tree), were derived from the median branch length for each data set as advised
by Thorne et al. (1998).
Results and Discussion
Dataset
The final alignment of the 18S rDNA sequences for the 94 taxa was 1641
characters long. The general time reversible model including invariant sites and
assuming a discrete gamma distribution (GTR + γ + I) was the best-fit model
estimated through MrModeltest 2. The settings for the GTR + γ + I were as
follows: R-matrix = (1.1370, 2.9815, 1.4865, 0.7903, 4.9487, 1.0000; base
frequencies = (A=0.2471, C=0.2105, G=0.2660; T=0.2764); proportion of
invariant sites = 0.3675; and shape of the gamma distribution = 0.6303. This
370
Fungal Diversity
model of evolution was used in the Maximum likelihood analysis using the
likelihood ratchet method. To assess clade stability, in addition to bayesian
posterior probability, which is known to show high support for wrong
topology, we also assessed clade stability based on a neighbour joining
bootstrap proportion. Figure 1 shows the maximum likelihood tree with
bayesian posterior probabilities at the internodes.
Phylogeny
Phylogeny shows significant bayesian support for most of the fungal
groups and subgroups (Fig. 1). Among the four major clades within fungi, both
Ascomycota and Basidiomycota were highly supported, by 100% bayesian
posterior probability (BPP), while the neighbour joining bootstrap proportions
(NJBP) were 100% and 97 % respectively. Chytrids were moderately
supported (Bpp = 99%, NJBP = 83), while, the Zygomycota comprising
Endogone and the Glomales was supported with 100% BPP, but, did not
receive clade support for the NJBP. However, the Glomales received high
support (BPP = 100, NJBP = 98). The major divisions and relationships are
similar to those found previously with analyses of fungal genome sequences
(e.g. Lutzoni et al., 2004).
Test for molecular clock
The molecular clock hypothesis is rejected for the SSU rDNA data set by
a likelihood ratio test under the F84 model (Felsenstein, 1984). This reveals
that extensive rate variation occurs among different lineages, with fast evolving
taxa and slow evolving ones distributed all over the tree. This could also be due
to the uneven taxon sampling, with high density of taxa among certain
lineages. Thus, the use of a relaxed molecular clock approach designed to
accommodate rate variation was preferable for estimating divergence ages with
this dataset.
Estimation of divergence time
Times of divergence were estimated for all nodes in the phylogenies as
derived through the consensus of 24000 generations of the bayesian analysis,
using the 4 calibrations points described in Table 3.
The chronogram obtained on the 18S rDNA data set is presented in
Figure 2. Based on this data, the mean posterior age for the basal animal-fungal
group, is estimated to be 1150 ± 220 MYA (95% credibility interval: 870371
Table 3. Calibration points for evolutionary analyses.
Divergence points
Basal animals –
Fungi
Endogonales - rest of
Fungi
Ascomceyetes basidiomycetes
Stomach chitrids free living chytrids
Paleontological data
Radiation of animals and
fungi (minimum age
constraint)
Evolution of terrestrial
taxa (minimum age
constraint)
Identification of fossil
clamp connections
(minimum age constraint)
Stomach chitrids
evolution after the
evolution of marsupials
(maximum age
constraint)
Age (Myr)
References
965
Doolittle et al., 1996
800
Berbee & Taylor,
2001
600
Berbee & Taylor,
2001
150-200
Berbee & Taylor,
2001
1670). This early split is followed by the divergence between the Chytrids and
the rest of fungi 1020 ± 140 MYA (820 – 149). Accordingly, the ancestral
marine fungi, should have their ancestors, in the Precambrian era (Fig. 2). The
evolution of terrestrial fungi, is seen at the divergence between Endogone and
the rest of fungi at 890 ± 160 MYA (800-1400) and the AscomycetesBasidiomycetes divergence at 780 ± 160 MYA (520-1100).
The earliest divergence of freshwater species is seen in the Jahnulales at
380 ± 100 MYA (220-660), however most of the freshwater lineages appear to
have diverged during the Mesozoic period (66-245 MYA).
The evolution of fungi on land has been one of the most intriguing
questions in mycology. Some of the few studies which have attempted to date
the evolution of fungi on land, have varied greatly (600 MYA and 1200 MYA)
(Berbee and Taylor., 2001; Heckmann et al., 2001). The estimated dates in our
study based on a 18S rDNA dataset, is at 890 MYA. Our results are much later
than the dates estimated by Heckmann et al. (2001). Sanderson (2003), also
showed that the dates proposed by Heckman et al. (2001) to be much earlier,
and suggested this might be due to calibration errors and the assumptions of a
molecular clock. Our divergence estimates are also in odds with those
estimated by Berbee and Taylor (2001), who used the same 18S rDNA gene,
yet their dates were around 600 MYA. A possible explanation for the differing
conclusions is that the earlier studies were based on the molecular clock
hypothesis (Zuckerlandl and Pauling, 1965), which did not hold good for out
dataset. It has been suggested that violation of the clock could have drastic
effects on date estimation (Brohman and Penny, 2000; Yoder and Yang, 2000).
372
Fungal Diversity
Fig 2. A timescale of fungal evolution. Thick blue lines indicating calibration points used.
373
The ascomycetes, basidiomycetes and Glomales, which diverged after
the initial colonization of land, are mainly saprobic on vascular plants. The
estimation of their divergence time at 890 MYA is almost two folds older than
the most recent dates obtained from the evolution of land plants (483-490
MYA, Sanderson, 2003). With the absence of land plants at 890 MYA, which
are the main sources of food for fungi that have colonised land, how did these
fungi survive? Berbee and Taylor (2001) suggested that the actual origin of
land plants could be much older than their fossil records, or that the fungi were
initially associated with the ancestors of the vascular land plants, following
their hosts onto land.
The only fossil record which clearly indicates the earliest possible
presence of perithecial ascomycetes is at Rhynie Chert deposits dating 400
MYA (Taylor et al., 2004). Our dates suggest that the filamentous ascomycetes
originated during the Cambrian era (550 MYA) about 150 MY earlier than the
fossil record.
Multiple origins of freshwater ascomycetes
The topology of the tree obtained through the maximum likelihood and
bayesian analyses accords well with the classification system of fungi derived
from other phylogenetic studies (Lutzoni et al., 2004) and was assumed to be
true for the date estimates. Phylogeny clearly shows that freshwater taxa have
evolved independently through several lineages (Fig. 1). Furthermore, it is also
evident that freshwater taxa have evolved directly from terrestrial species.
For the convenience of discussing evidence for their origin based on
morphological and molecular characters from previously derived phylogenies,
freshwater fungi that are present in three classes are discussed into five
morphological groups based on characteristics of their asci and ascospores.
Class: Sordariomycetes
Annulatascaceae
The Annulatascaceae was introduced by Wong et al. (1998b) to
accommodate the freshwater genus Annulatascus and related genera (Ho and
Hyde, 2000). Species in this family have asci with massive apical rings that
probably facilitate strong ejection of ascospores in air and under water, and
may be adapted for dispersal in freshwater, or in the air when washed to the
banks of rivers (Hyde and Goh, 2003). The apical ring comprises of a thick
electron dense upper region and an extensively elongated lower region that is
374
Fungal Diversity
filled with compact electron-dense granules. Only the inner wall layer is
responsible for the ring formation, the outer ascus wall layer disintegrates at
the apex (Ho and Hyde, 2000). The ascospores in most species are equipped
with sheaths or unique appendages (Goh and Hyde, 1996, 1999; Hyde et al.,
1997; Ho and Hyde, 2000; Ho et al., 2004). Appendages in marine fungi are
known to be important in the dispersal and attachment of ascospores to
substrata (Hyde and Jones, 1989; Hyde et al., 2000b). The species can also
degrade submerged wood (Yuen et al., 1998). It is likely that these three
factors are responsible for the success of these fungi in freshwater ecosystems
(Hyde et al., 1997). The Annulatascaceae were originally thought to be unique
to freshwater (Wong et al., 1998a). However, Fröhlich and Hyde (2000) have
discovered two new terrestrial species from palms and two terrestrial species
are known from bamboo (Dalisay, 1998) that has taxonomic affinities in this
family. There has been no record of any marine genera with morphological
similarities to the Annulatascaceae.
Annulatascaceae now consists of 14 genera. Recent molecular sequence
data have been used to shed light on the phylogeny of Annulatascaceae. 28S
rDNA sequences have been widely used to analyse the phylogenetic affinities
of the Annulatascaceae and various views regarding their phylogeny have been
reported. Ranghoo et al. (1999) analysed partial sequences from the 28S rDNA
to determine the taxonomic placement of Ascotaiwania and Ascolacicola at the
familial level. Two Annulatascus species and seven other annulatascaceous
taxa were also sequenced as part of this study. In the resulting trees these taxa
clustered with two sordariaceous species, indicating a closer phylogenetic
relationship to the Sordariaceae than other pyrenomycete families. The
Sordariales comprise mostly terrestrial taxa and provide evidence that the
Annulatascaceae have affinities with terrestrial fungi.
Réblova and Wong (2001) found only Clohesia corticola had affinities
with the Sordariales, while the principal genus Annulatascus clustered with
Aquaticola
and
Rhamphoria
representing
the
Annulatascaceae/
Trichosphaeriaceae clade. The Trichosphaeriaceae, mainly contains terrestrial
saprobic species. Recently, Huhndorf et al. (2004) found Annulatascus, the
principle genus of the Annulatascaceae shared phylogenetic affinities to the
Ophiostomatales, which mainly consists of plant pathogens. Due to the lack of
bootstrap support, the affinities of Annulatascaceae to either one of the two
groups (Trichosphaeriales or Ophiostomatales) could not be confirmed.
However, recently, Annulatascaceae was found to form a monophyletic clade
with members of both the orders with low bootstrap support with the inclusion
of additional freshwater taxa (Vijaykrishna et al., 2005).
375
Annulatascaceae was not included in our analysis of the 18S rDNA
analysis, because of the absence of data in the GenBank. Phylogenetic studies
based on the 28S rDNA show a relationship of Annultascaceae to the
Ophiostomatales (Vijaykrishna et al., 2005) indicating a terrestrial origin of the
family. Extrapolating the results obtained from our divergence estimates,
Annulatascaceae members should have evolved between 200 to 300 MYA
(Fig. 2).
Terrestrial unitunicate ascomycete genera with aquatic species
Several genera previously known from terrestrial habitats have also been
shown to have common freshwater representatives, e.g. Phomatospora,
Saccardoella, Zopfiella (Shearer, 1993). Many of the species in these genera
occur in terrestrial habitats. Species of Chaetomium, Coniochaeta and Nectria
have infrequently been recorded in freshwater (Shearer, 1993). As species in
these genera are usually found in terrestrial habitats, the aquatic species are
probably not truly aquatic taxa. Species of Ophioceras and Pseudohalonectria
are more frequently identified in freshwater habitats (Chen et al., 1995) than in
terrestrial habitats (Hyde et al., 1999e; Fröhlich and Hyde, 2000).
Phylogenetic ordinal placement of the terrestrial unitunicate genera with
aquatic species clearly shows the multiple origins of freshwater ascomycetes.
The above species can be seen in three distinct clades within monophyletic
pyrenomycetes clade. Ophioceras, Phomatospora and Pseudohalonectria
showing a closer relationship to the Magnaporthaceae. Whilst Chaetomium
and Coniochaeta are closer to the Sordariales, Nectria falls within the
Hypocreales, providing further evidence that these aquatic taxa had terrestrial
predecessors.
Time estimates of the evolution of terrestrial and freshwater species of
Ophioceras, Phomatospora and Pseudohalonectria show a fairly recent
evolutionary divergence (approx 40 – 190 MYA).
Halosphaeriales
The Halosphaeriales are rather unique amongst freshwater fungi in not
having terrestrial representatives, but in being well represented by marine
species. The Halosphaeriaceae contains two distinct morphological groups: 1)
those with appendaged ascospores and early deliquescing asci, and 2) those
with persistent asci, often with an apical apparatus (it is not known if this is
functional), and mostly with ascospores with polar filamentous unfurling
appendages (Hyde et al., 1999d, 2000b). The first group is not common in
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Fungal Diversity
freshwater and only represented by three species of Fluviatispora and one
species of Ayria (Fryar and Hyde, 2004). Species with early deliquescing asci
are rare in freshwater fungi and this character may not be a good adaptation in
this milieu. The second group, are however, common in marine and freshwater
habitats (Hyde et al., 1999d). The possible ability of their asci to eject
ascospores, and the presence of ascospores with unfurling filamentous
appendages, may be important for dispersal and subsequent attachment in
freshwater habitats (Goh and Hyde, 1996).
Spatafora et al. (1998) analysed partial sequences from the large and
small subunit rDNA from 15 marine representatives of the Halosphaeriaceae
and integrated this with a data set of homologous sequences from terrestrial
ascomycetes. In their analyses, a group of Halosphaeriales (e.g. Aniptodera,
Ceriosporopsis, Corollospora, Halosarpheia, Nimbospora) clustered with
other terrestrial Microascales. Representatives of Lindra and Lulworthia,
which are marine, formed a well-supported clade that was isolated among the
perithecial ascomycetes and these genera have recently been accommodated in
Lulworthiales a new fungal order (Kohlmeyer et al., 2000). Spatafora et al.
(1998) therefore concluded that Halosphaeriales and the separate
Lulworthiales (Lulworthia/Lindra clade) were independently derived from
terrestrial ancestors. Members of the Microascales are characterised by
evanescent asci with passively discharged ascospores, which are dispersed by
insects. The origin of evanescent asci may have evolved on several occasions
being adapted from insect dispersal and dispersal in the marine environment.
The Halosphaeriales have been shown to have terrestrial ancestors that have
evolved morphologically to adapt to marine environment (Spatafora et al.,
1998).
In a similar study, partial sequences of the LSU rDNA from
representatives of freshwater and marine Halosphaeriales clustered into two
separate groups (Ranghoo et al., 2000). One group contained representatives of
Aniptodera and Savoryella, while the second group contained representatives
of the marine Halosphaeriales (e.g. Corollospora, Halosphaeriopsis,
Halosarpheia, Nimbospora). However, the marine and freshwater
representatives of the genera Aniptodera and Savoryella grouped together in
their individual genera, rather than separating according to their habitats. Kang
et al. (2000) analysed the SSU rDNA of four Halosarpheia species, Lignincola
laevis and Nais inornata and found that these marine species formed a
subclade with taxa from the Microascales, indicating a terrestrial origin. Okada
et al. (1997) also sequenced 19 marine fungi and showed that the Microascales
and Halosphaeriales are phylogenetically related.
377
Based on SSU and LSU rDNA sequences, Campbell et al. (2003),
reported that the presence or absence of the unfurling thread like appendages is
not a good phylogenetic marker within the aquatic genus Halosarpheia. They
also found that species occurring in freshwater habitats are phylogenetically
distinct from those occurring only in marine habitats. Furthermore, at the
morphological level, significant differences can be found between marine and
freshwater representatives of the Halosphaeriales. Species of Corollospora,
Halosphaeriopsis, Halosphaeria and Nimbospora are characteristic of the
Halosphaeriales, having deliquescing asci which lack an apical ring, and
ascospores with different kinds of appendages (Jones, 1995). Species of
Aniptodera and Savoryella (marine and freshwater), however, have persistent
asci with definite apical rings (Hyde et al., 1999d), but in freshwater
representatives of Halosarpheia the asci deliquesce much earlier than marine
representatives of Halosarpheia (Campbell et al., 2003).
Ranghoo (1998) indicated that marine and freshwater representatives of
the Halosphaeriales are closely related to each other. Spatafora et al. (1998)
had already shown that marine Halosphaeriales were related to terrestrial
Microascales and could have originated from them. Accordingly we can infer
that freshwater representatives of the Halosphaeriales could have terrestrial
ancestors.
The data of Spatafora et al. (1998), Ranghoo (1998) and Campbell et al.
(2003) provide support to the hypothesis that the Halosphaeriales are
secondary marine ascomycetes, having derived from terrestrial predecessors
and do not support the hypothesis of Kohlmeyer and Kohlmeyer (1979a,b), that
the Halosphaeriales are primary marine fungi. Taking into account the
freshwater representatives, the Halosphaeriales are therefore secondary aquatic
ascomycetes as a whole. Another intriguing question is: could freshwater
habitats be a pathway for certain genera to have become adapted to marine
environments? To understand this, a wider species sampling among the
Halosphaeriales, can be used to trace ancestral character states.
Class: Dothideomycetes
The Loculoascomycetes have bitunicate asci, which are effective
discharge structures (Shearer, 1993). Many terrestrial genera, e.g. Didymella,
Lophiostoma, Massarina, contain species that occur in freshwater and marine
habitats, while there are a few genera that are restricted to either marine or
freshwater habitats, e.g. Manglicola and Jahnula respectively (Kohlmeyer and
Kohlmeyer, 1979 b; Hyde and Wong, 1999). The discharge abilities of the asci
in these taxa are probably important characters for the survival in aquatic
378
Fungal Diversity
habitats, as the ascospores need to be ejected through air or possibly in water.
Most loculoascomycetes have ascospores with mucilaginous sheaths, and Hyde
and Jones (1989) have shown these to be important in dispersal in marine
environments. It therefore appears that Loculoascomycete spores may be preadapted for dispersal in water and that many genera, e.g. Leptosphaeria,
Massarina, Trematosphaeria, have evolved species capable of an aquatic
lifestyle.
Comparative analyses based on molecular phylogenetic information have
shown the relatedness of marine, freshwater and terrestrial taxa supporting the
terrestrial origin of aquatic loculoascomycetes (Spatafora et al., 1995; Liew et
al., 2000). Marine members of Melanommatales and Dothideales clustered
with the terrestrial Dothideales and Pleosporales (Spatafora et al., 1995). Liew
et al. (2000) included two aquatic fungi (i.e. Massarina australiensis, M.
bipolaris) in their analysis of Loculoascomycetes with pseudoparaphyses.
These taxa clustered within the groups comprising terrestrial Dothideales and
Pleosporales indicating a terrestrial origin.
Nucleotide sequence analyses show that freshwater Dothideomycete taxa
have evolved in clearly three distinct lineages. Members of Jahnula belonging
to the order Jahnulales, form a well-supported clade separate from the other
members of the Dothideomycetes. The order Jahnulales was erected to
accommodate a very unique group of freshwater ascomycetes, with stalks
subtending the ascomata and containing dimorphic ascospores (Pang et al.,
2002). The Jahnulales show the earliest divergence times for freshwater
ascomycetes in our estimates (390MYA). Other freshwater taxa from the class
Dothideomycetes are dispersed among terrestrial species. The third group
Tubeufiaceae, better represented in aquatic habitats, in their anamorphic form
(e.g. Helicosporium, Helicoma). Therefore, molecular systematics and
morphological evidence provide strong evidence that loculoascomycetes have
repeatedly gained the ability to inhabit freshwater habitats.
Class: Leotiomycetes
Fungi that produce cup-shaped fruiting structures (apothecial
ascomycetes) are commonly known as cup fungi or Discomycetes. Fungi with
apotheicial ascomata are found in three classes, viz. Pezizomycetes,
Leotiomycetes and Leconaromycetes. Freshwater genera, however, are found
only in the Class Leotiomycetes.
Freshwater Discomycetes are not common in tropical freshwater habitats,
but are frequently isolated from submerged grasses and twigs in the temperate
region (Shearer, 1993). Many discomycete genera found in the aquatic habitats
379
e.g. Hymenoscyphus, Lophodermium, Mollisia, are better represented in
terrestrial habitats. Species of Apostemidium, Niptera and Vibrissea are mostly
confined to freshwater environments. The sigmoid or filiform ascospores in
species of these genera appear to be adapted for dispersal and attachment in
aquatic habitats (Webster and Davey, 1984; Shearer, 1993). Aquadiscula and
Loramyces are genera described to accommodate freshwater species. In
Loramyces the ascospores are filiform and have a large mucilaginous head and
filiform tail and seem to be well adapted for dispersal and subsequent
attachment in water (Goh and Hyde, 1996). In Aquadiscula, ascospores have a
mucilaginous appendage (Shearer and Crane, 1985). The freshwater
discomycetes appear to have no other special features, besides the sigmoid and
filiform ascospores, adapted to the aquatic habitat. However, the filiform
ascospores are also found in terrestrial discomycetes.
A number of Ingoldian fungi have their sexual stages within the
Leotiomycetes. These aquatic (Ingoldian) hyphomycetes grow in aquatic
habitats and release a large number of conidia, with branched, sigmoid or
helicoid conidia, which become trapped in foam (Sakayaroj et al., 2005). The
foam is however temporary inoculum of conidia, with high viability, which
eventually get attached to suitable substrata (Sridhar and Barlocher, 1994;
Sakayaroj et al., 2005). Therefore, their existence in freshwater habitats in their
asexual form is itself an adaptation for their continued occurrence in
freshwater.
Conclusion
The major finding of this study is that freshwater fungi, like marine fungi
(Spatafora et al., 1995), have evolved from terrestrial ancestors. Molecular data
show that freshwater ascomycetes have evolved separately through different
lineages, which supports the hypothesis proposed by Shearer (1993). Unlike,
freshwater hyphomycetes, lignicolous fungi appears appear to have few special
adaptations to survive in freshwater habitats. The majority of the characters
that appear to be adaptations to freshwater habitats can also be seen in
terrestrial fungi (eg. ascospore sheath, and presence of active discharge
mechanisms). The lack of exclusive adaptations further indicates, that
freshwater ascomycetes share a common ancestor with terrestrial ascomycetes.
Morphological and molecular data indicate that the freshwater and marine
Halosphaeriales have terrestrial ancestors. One of the major findings of
Spatafora et al. (1998) is that some of the major lineages of marine fungi were
derived from terrestrial predecessors that possessed evanescent asci. Whether
the marine representatives of these genera evolved from terrestrial or
380
Fungal Diversity
freshwater ancestors remains unknown. It would be interesting to select species
from these different habitats for molecular studies to investigate the ancestral
states of freshwater and marine lineages.
The appearance of freshwater ascomycetes in distant parts of the world
has always been intriguing and several possible mechanisms of dispersal have
been proposed by Hyde and Goh (2003). Our estimates show the earliest
possible origins of freshwater taxa at 390 MYA. This is much earlier than the
separation of Pangea into several continents, indicating that the dispersal may
have occurred with separation of continents. Evidence which refutes this
however, are the freshwater fungal communities on newly formed volcanic
islands (e.g. Hawaii), which are also similar to those on older continents
(Eldredge and Miller, 1995), indicating that fungi must have arrived in these
islands by various dispersal mechanisms (Hyde and Goh, 2003). Studies are
needed to understand the biogeography, of commonly found freshwater
ascomycetes from different parts of the world. Studies on rDNA sequences
have provided valuable insights in the biogeography and evolution of several
fungi (Isikhuemhen, 1999; Moncalvo, 2000, 2002). The methods used should
be extended to understand the biogeography of widely distributed freshwater
fungi, which might provide an answer for the appearance of freshwater fungi in
newly formed islands.
Acknowledgements
We thank Dr. Jean-Marc Moncalvo for ideas regarding the molecular work presented in
this review. The University of Hong Kong is acknowledged for supporting this research by
providing D.V with a postgraduate studentship. The Hong Kong Research Grants Council
(HKU 7370/02M, HKU 7322/04M) is thanked for providing R.J with a postdoctoral
fellowship. Helen Leung and Heidi Kong are thanked for technical assistance. Justin Bahl and
Lei Cai are thanked for their valuable comments and discussion.
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