Richards Annurev Marine 2011

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Marine Fungi: Their Ecology and Molecular Diversity
Article in Annual Review of Marine Science · January 2012

DOI: 10.1146/annurev-marine-120710-100802 · Source: PubMed
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Annu. Rev. Mar. Sci. 2012. 4:495–522 Keywords

First published online as a Review in Advance on saprotroph, osmotroph, parasite, pseudofungi, chytrid, small subunit
September 19, 2011
ribosomal DNA
The Annual Review of Marine Science is online at
marine.annualreviews.org Abstract
This article’s doi: Fungi appear to be rare in marine environments. There are relatively few
10.1146/annurev-marine-120710-100802
marine isolates in culture, and fungal small subunit ribosomal DNA (SSU
Copyright ! c 2012 by Annual Reviews. rDNA) sequences are rarely recovered in marine clone library experiments
All rights reserved
(i.e., culture-independent sequence surveys of eukaryotic microbial diversity
1941-1405/12/0115-0495$20.00 from environmental DNA samples). To explore the diversity of marine fungi,
we took a broad selection of SSU rDNA data sets and calculated a summary
phylogeny. Bringing these data together identified a diverse collection of
marine fungi, including sequences branching close to chytrids (flagellated
fungi), filamentous hypha-forming fungi, and multicellular fungi. However,
the majority of the sequences branched with ascomycete and basidiomycete
yeasts. We discuss evidence for 36 novel marine lineages, the majority and
most divergent of which branch with the chytrids. We then investigate what
these data mean for the evolutionary history of the Fungi and specifically
marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine
ecosystems.
495
INTRODUCTION
Fungi are key players in terrestrial environments (Gargas et al. 1995, James et al. 2006a, Wang
& Qiu 2006) and perform vital functions as decomposers, driving nutrient cycles in detritus
environments, and as parasites and symbionts (Webster & Weber 2007). Fungi represent a sig-
nificant proportion of the microbial diversity on Earth (Hawksworth 2001, Hibbett et al. 2007,
Mueller et al. 2007, O’Brien et al. 2005). The total diversity of the Fungi has been estimated to be
1.5–1.6 million species (Hawksworth 1991, 2001). However, much of our current understanding
of the ecology and evolutionary complexity of fungi is derived from the study of cultured fungal
isolates in the most part from terrestrial environments. Furthermore, this diversity estimate is
based on the study of fungal communities in and around plant ecosystems sampled from Western
Europe and then extrapolated to a global figure using data on global plant diversity (Hawksworth
1991, 2001). This is controversial because it assumes that plant species diversity and fungal species
diversity are correlated (May 1991, 1994; Mueller & Schmit 2007) and is likely to be too low
because it is based on counts derived from morphological observations and therefore does not
account for cryptic species diversity (Hawksworth 2001). Perhaps even more importantly, the es-
timate focuses only on fungi in a limited set of habitats and therefore does not account for diverse
and abundant fungi that are not associated with plant and soil environments, such as animal hosts,
sediment, freshwater, or marine ecosystems.

Our understanding of fungal evolutionary complexity is expanding as researchers use increas-
ingly powerful molecular methods to investigate environmental diversity (e.g., Buée et al. 2009,
Jumpponen & Jones 2009, O’Brien et al. 2005). For example, work using sequencing of inter-
nal transcribed spacer (ITS) markers from soil DNA samples has suggested a revision of global
fungal diversity from Hawksworth’s estimate of 1.5 million species to 3.5–5.1 million (O’Brien
et al. 2005). Yet current culture collections of fungi number only ∼75,000 isolates (Hawksworth
2001, Kis-Papo 2005) with updated counts suggesting 64,000 ascomycete isolates and 32,000 ba-
sidiomycete isolates (Kirk et al. 2008), not all of them representing different species. Independent
of the accuracy of these estimates, it is likely that less than 5% of the diversity of fungal species
is currently described and maintained in culture. Furthermore, the use of environmental DNA
(eDNA) methods is increasingly expanding our understanding of the diversity of fungi at the high-
est taxonomic levels with a number of discoveries of previously undescribed phylogroups ( Jones
et al. 2011, Lara et al. 2010, Porter et al. 2008, Schadt et al. 2003), suggesting that these estimates
are conservative and that culture collections are in no way representative of natural diversity.
Of the cultured species (Hawksworth 2001, Kis-Papo 2005), one count suggests there are only
467 isolates belonging to 244 genera retrieved from marine environments (Kis-Papo 2005). This
might imply that only ∼0.6% of studied fungi are derived from the marine environment, which
would be surprising as marine habitats account for 70% of the surface of the globe and have
been shown to harbor fungi from the air-surface interface to depths of kilometers. These results
have been interpreted to suggest that fungi are both nondiverse and low in abundance in marine
environments (Burgaud et al. 2009, Kis-Papo 2005, Le Calvez et al. 2009). In contrast, fungi
are thought to be a major contributor to the decomposition of woody and herbaceous substrates
and animal remains in coastal and surface marine environments (Kohlmeyer & Kohlmeyer 1979,
Mann 1988, Newell 1996). This raises questions about the significance of fungal communities in
marine environments: Are we overlooking a large diversity of fungi?
The purpose of this review is to bring together the growing body of molecular data, focusing
primarily on environmental small subunit ribosomal DNA (SSU rDNA) sequence data to summa-
rize our current knowledge of the evolutionary diversity of marine fungi. Using these data, we can
begin to investigate a number of theories and observations relating to the ecological role of fungi
496 Richards et al.

in marine ecosystems. We hope this will form an important foundation for better understanding
of the diversity and function of fungi in marine environments as metagenomic and large-scale
diversity sequencing projects gather pace.
Ascomycota:
filamentous or yeast
forms that reproduce
MOLECULAR DIVERSITY OF MARINE FUNGI sexually with internal
The kingdom Fungi was traditionally loosely classified as four major groups: (a) Ascomycota, spore maturation
(b) Basidiomycota (which together form the subkingdom Dikarya and have been the major focus within a sac-shaped
cell called an ascus;
of experimental research and genome-sequencing initiatives), (c) the zygomycetes, and (d ) the include the laboratory
chytrids (Hibbett et al. 2007, Jones et al. 2011, Webster & Weber 2007). This early model of fungal model organisms
taxonomy has been revised at a number of levels, including the placement of the microsporidia Saccharomyces cerevisiae
with (Hirt et al. 1997, 1999) and potentially within the Fungi (Adl et al. 2005, James et al. 2006a, and Neurospora crassa
Keeling 2003) and the division of the chytrids and zygomycetes into multiple interbranching Basidiomycota:
paraphyletic clades, followed by subsequent taxonomic reclassifications (see Hibbett et al. 2007; filamentous or yeast
James et al. 2006a,b; Liu et al. 2009). There still remains much uncertainty relating to the major forms that reproduce
sexually with external
divisions of the Fungi below the Dikarya. spore maturation on a
Progress in drawing the fungal tree of life has been made by studying fungi that have been iso- basidium
lated and cultured mainly from terrestrial environments (Hibbett et al. 2007; James et al. 2006a,b;
Zygomycete:
Liu et al. 2009). These approaches are limited because they preferentially sample microbes that multinucleated cell
are easily cultured or that possess larger body sizes and/or distinctive morphologies. Furthermore, that produces
these approaches bring their own specific limitations for studying fungi in marine environments: filaments and lacks a
complex fruiting body
1. The culturing of fungal isolates from marine samples has often led to the recovery of non-
fungal microbes, which are ecologically, morphologically, and trophically similar to fungi Chytrid: informal
name given to a fungus
but are not true fungi (discussed below). that produces motile
2. The ecological preferences of most fungi suggest that those in marine ecosystems are likely flagellated spores
to reside on or in host organisms or in benthic environments, including deep-sea sediments. (zoospores) during its
These habitats are difficult to examine by microscopy and in some cases pose severe sampling life cycle
difficulties. True fungi: large and
3. The majority of fungi harbor very high levels of cryptic diversity that is indistinguishable highly diverse group of
using microscopy of environmental samples and/or culturing. Further complications arise eukaryotes (mainly
because similar fungal morphotypes such as yeasts and flagellated zoospores branch in distant microbes, largely
osmotrophic and
and paraphyletic positions on the fungal tree of life ( James et al. 2006a, Liu et al. 2009), frequently with
making classifications based on observations of general morphological characters difficult chitin-rich cell walls)
and often misleading. that form a large clade
Molecular methods—specifically the polymerase chain reaction (PCR) amplification of taxo- in the opisthokont
supergroup
nomically informative gene markers from eDNA samples combined with clone library construc-
tion, sequencing, and phylogenetic analyses—have demonstrated that microbial diversity is much
more complicated than previously thought (Giovannoni et al. 1990, López-Garcı́a et al. 2001,
Moon-van der Staay et al. 2001, Olsen et al. 1986, Pace 1997). The environmental DNA, PCR,
and clone library approach has been used for both prokaryotes and eukaryotes to place many previ-
ously unrecognized branches on the tree of life, in many cases redefining our understanding of the
evolutionary complexity of the eukaryotes (Bass & Cavalier-Smith 2004, Dawson & Pace 2002,
Edgcomb et al. 2002, López-Garcı́a et al. 2001, Moon-van der Staay et al. 2001, Pace 1997, Rappé
& Giovannoni 2003, Richards & Bass 2005, Richards et al. 2005, Stoeck et al. 2006), although
these results have been the subject of much debate and revision (e.g., Berney et al. 2004, Cavalier-
Smith 2004). Molecular approaches have also demonstrated that poorly recognized groups are
important ecosystem components (Bass & Cavalier-Smith 2004, Chambouvet et al. 2008, Massana
et al. 2004b, Moreira & López-Garcia 2002). Yet most molecular surveys of microbial eukaryotic
www.annualreviews.org • Marine Fungi 497

diversity are recovered using only one primer set and sample less than 500 clones, with the result
that no eukaryote-wide study has reached sampling saturation (e.g., Edgcomb et al. 2002, Stoeck
et al. 2006). This has led to the suggestion that further sampling from the very same environments
Small subunit
ribosomal DNA would reveal more diversity and demonstrate that our understanding of the complexity of the tree
(SSU rDNA) clone of life is still very incomplete (Curtis et al. 2002, Moreira & López-Garcia 2002, Sogin et al. 2006,
library: experimental Stoeck et al. 2006).
process involving the Environmental clone library analyses specifically targeting fungi have generally sampled regions
extraction of DNA
within the ribosomal RNA (rRNA) gene array using a range of approaches and sequence targets,
from environmental
samples, PCR-targeted with some researchers focusing on the SSU rDNA sequence and others on the ITS regions. The
amplification of SSU two ITS regions are sections of DNA located between the SSU (18S) and large subunit (LSU) (28S)
rRNA (marker) genes, rRNA genes, separated by the 5.8S rRNA gene. The variable nature of the ITS regions relative to
cloning of amplicons, the flanking rRNA genes enables increased resolution and accuracy when assigning sequences to
clone sequencing, and
genus- and species-level classifications within well-sampled groups (Bruns & Gardes 1993, Gardes
phylogenetic analysis
& Bruns 1993, Horton & Bruns 2001). This process is facilitated by increasingly well-sampled
sequence databases (Buchan et al. 2002, James et al. 2006a, O’Brien et al. 2005). However, many
database sequence classifications may be erroneous (Vilgalys 2003), and variation in rates of ITS
divergence between taxonomic groups can hinder classifications using these sequences (Nilsson
et al. 2008, 2006; Vilgalys 2003). The ITS approach is useful for determining species diversity and
can be used for ecosystem comparisons when targeting well-defined taxonomic groups, but it is
of limited use for inferring higher-level phylogenetic relationships and identifying novel groups,
as ITS phylogenies demonstrate weak resolution among deeper branching relationships in the
Fungi (Horton & Bruns 2001).
Some researchers have focused on sampling the SSU rRNA gene to investigate novel fungal
diversity among higher taxonomic groups (Anderson et al. 2003, Bass et al. 2007, Jebaraj et al.
2009, Porter et al. 2008, Schadt et al. 2003, Vandenkoornhuyse et al. 2002), although this gene
cannot discriminate between closely related fungal species and is generally less intensively sampled
than the ITS regions. Therefore, others have called for the combination of the SSU and ITS
approaches (O’Brien et al. 2005), enabling multigene phylogenetic analyses encompassing the
SSU, 5.8S, and LSU sequences, which can lead to improved phylogenetic support among both
lower and higher phylogenetic nodes (e.g., Jones et al. 2011, Porter et al. 2008) and help to identify
the phylogenetic placement of many orphan environmental ITS sequences currently accumulating
in sequence databases.
Molecular Sampling in Marine Environments

There is now a growing trend in the use of molecular techniques to investigate microbial diversity
from marine environments. These data have given a mixed impression of the relative importance of
fungal lineages in marine environments. For example, in 2005 we conducted a meta-analysis of 13
SSU rDNA clone library studies. This analysis brought together 49 SSU rDNA environmental
clone libraries, with a total of 1,077 sequences from soils, freshwater, and marine samples. Of
these sequences, 124 (11.5%) clustered within, or close to, known fungal sequences (Richards
& Bass 2005). This analysis also suggested that although fungi are present in aquatic sediments,
low-oxygen aquatic environments, freshwater, and soils, there appeared to be very few sequences
recovered from the upper column and surface marine waters. This pattern was confirmed by a
separate, specifically marine analysis, which sampled 23 coastal water libraries (1,349 clones) and
12 open ocean libraries (826 clones) and recovered a total of only 16 fungal clones, equivalent to
0.8% of the marine SSU rDNA sequences sampled (Massana & Pedrós-Alió 2008). Consequently,
both molecular analyses and culture-based inventories (Kis-Papo 2005) have suggested that fungi
are relatively nondiverse and in low abundance in upper and surface marine ecosystems.
498 Richards et al.

Sequencing of eukaryotic SSU rDNA V9 and V4 diversity tags from marine coastal waters us-
ing 454 methods has hinted that such environments contain more fungal diversity than previously
detected using clone library methods (Stoeck et al. 2010). These diversity sequencing methods
still suggest that, compared with other eukaryotic groups, fungi appear relatively nondiverse, con-
stituting less than 5% of the operational taxonomic units recovered from marine environments.
However, these methods hint at an increased diversity in comparison with clone library methods
(Pawlowski et al. 2011, Stoeck et al. 2010). Furthermore, fungal-specific clone library analyses have
identified new fungal diversity in deep waters, anoxic marine waters, hydrothermal vent environ-
ments, and deep-sea marine sediments (Bass et al. 2007, Burgaud et al. 2009, Edgcomb et al. 2011,
Jebaraj et al. 2009, Le Calvez et al. 2009). The results of this summary and other fungal sequences
detected in marine environments are summarized in Figures 1–5. This work has made use of a
range of eDNA samples and sampling techniques and has therefore made it feasible to sample
previously inaccessible microbial communities, for example, specifically targeting communities in
deep-sea environments.
Deep-Sea and Hydrothermal Vent Environments

Using SSU rDNA clone library methods, Bass and coauthors (2007) investigated the composition
of fungal communities in deep-sea sediments and water column samples of depths of 500 m to
4,200 m, including several hydrothermal vent samples. The sequences recovered showed deep-
sea fungal communities to be dominated by ascomycete and basidiomycete forms but gener-
ally revealed a low diversity of fungi. The authors also noted that the vast majority of these se-
quences branched closest to taxonomic groups known to have a yeast morphotype, suggesting that
(a) these taxa were easier to recover or (b) yeast forms dominate these environments. Many of these
phylotypes were also shown to branch close to known pathogens, suggesting the presence of fungal
pathogens of deep-sea animals (discussed in detail below). The phylogeny reported by Bass et al.
(2007) included additional marine environmental SSU rDNA sequences recovered from general
eukaryotic PCR experiments and showed seven clusters of highly unique sequences, six of which
branched specifically within the fungal radiation, indicating the presence of unknown fungal forms
in marine environments.
A study by Le Calvez et al. (2009) using similar eDNA methods and targeting deep-sea hy-
drothermal vent ecosystems also recovered several novel fungal lineages. These lineages included
three unknown phylotypes branching within the basidiomycete radiation and two unknown phy-
lotypes branching close to chytrid sequences (Le Calvez et al. 2009). The primer set used in
this study was different from that chosen by Bass et al. (2007), but both studies demonstrate the
identification of novel fungal lineages in deep-sea marine environments.
Edgcomb et al. (2011) again used similar approaches to identify eukaryotic microbial com-
munities in deep-sea sediment cores but this time targeted both DNA- and RNA-based diversity
profiles. Although the authors specifically targeted a wide diversity of eukaryotic microbes, they
consistently recovered a high frequency of basidiomycete yeast sequences branching closely to
known Cryptococcus and Malassezia species (Edgcomb et al. 2011): 42% of the sequences recovered
from RNA-derived libraries branched with Cryptococcus sequences. This result is consistent with
another molecular analysis of eukaryotic diversity in marine sediments showing that Cryptococcus
curvatus yeasts, with a closely related genotype to those recovered by Edgcomb et al. (2011), can
dominate deep-sea microbial eukaryotic communities (Takishita et al. 2006), whereas culture sam-
pling has recovered a related Cryptococcus species (Cryptococcus surugensis) from deep-sea sediments
(Nagahama et al. 2003b) (shown in Figure 3). The recovery of these taxa in RNA-derived libraries
suggests that they are metabolically active in sedimentary ecosystems (Edgcomb et al. 2011).

Interestingly, the vast majority of the fungi sampled from deep-sea environments branch close
to, or within, clades of known terrestrial fungi. This suggests that in many cases fungi residing in
terrestrial or marine surface environments are capable of relatively easily making the transition
Saprotrophy: process
of acquiring nutrients to deep-sea habitats and is consistent with evidence that some fungi are capable of altering their
from the digestion of membrane composition to tolerate high hydrostatic pressure under short-term experimental con-
dead organic matter, ditions (Simonato et al. 2006). Taken together, these data imply that for some fungi, colonization
usually from plants or of deep-sea habitats by surface-dwelling strains is a viable ecological transition.
animals
Anoxic Marine Environments

A large fraction of the marine biosphere is anoxic or partially anoxic. In terrestrial environments,
fungi are regularly found in saprotrophic and detritus habitats that are often low in oxygen.
Fungi have been shown to possess a range of cellular and genomic adaptations to life in anoxic
environments (e.g., Embley 2006, Gojkovic et al. 2004, Hall et al. 2005). Furthermore, many fungi
have been shown to play a role in anaerobic denitrification (Shoun et al. 1992), e.g., Fusarium
oxyporum (Takaya et al. 1999, Uchimura et al. 2002). SSU rDNA sequences closely related to
F. oxyporum have been recovered from marine anaerobic environments (Figure 1; Jebaraj et al.
2009), and four marine isolates, including a Fusarium species, have been demonstrated to grow
in suboxic conditions, utilizing nitrate for respiration and accumulating nitrite, and are therefore
theoretically capable of participating in anaerobic denitrification in marine environments ( Jebaraj
& Raghukumar 2009).
A study by Jebaraj et al. (2009) examined the diversity of fungi in oxygen-depleted regions
of the Arabian Sea using clone library methods. These researchers used multiple fungal-specific
SSU rDNA primer sets and one general eukaryotic-specific primer set to amplify SSU sequences
( Jebaraj et al. 2009). Each primer set revealed an overlapping subset of fungal diversity, with
the fungal-specific primers showing a greater diversity of fungi per sampling effort than clone
libraries constructed using universal eukaryotic primers. This result demonstrates the importance
of using different primers to control for PCR biases and suggests that a significant portion of
fungal diversity is missed when using universal primer sets ( Jebaraj et al. 2009, Stoeck et al. 2006).
The phylogeny of Jebaraj et al. (2009) identified 48 distinct fungal phylotypes (clustered at
99% sequence similarity): 27 branching within the ascomycete radiation, 20 branching within the
basidiomycetes, and only 1 unique phylotype branching among the lower fungi. Several Dikarya
sequences formed highly novel branching positions in the phylogeny and clustered with additional
environmental sequences recovered from oxygen-depleted habitats ( Jebaraj et al. 2009). Indeed,
many sequences branched within a clade previously termed the hydrothermal and/or anaerobic
fungal group, which branches with the Malassezia yeasts also identified from deep-sea eukaryotic
environmental clone libraries (Bass et al. 2007, Edgcomb et al. 2011, López-Garcia et al. 2007)—
this interesting group is discussed further below. No chytrid-like sequences were identified from
this study, suggesting the possibility that these taxonomic groups have a low diversity in the marine
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 1
Subsection of a phylogenetic analysis showing the diversity of small subunit ribosomal DNA sequences recovered from marine
environmental DNA analyses. This section of the tree focuses on Pezizomycotina. The tree was calculated using PhyML (parameters
! = 0.722 and I = 0.117) with both PhyML and LogDet distance bootstraps. Marine phylotypes are highlighted in yellow. Novel
marine phylogenetic groups are marked NMFA (red, novel marine fungi ascomycetes). Gray dashed lines are false branch extensions.
Ecological information is annotated on the tree.
500 Richards et al.

Isolate: FCAS123 [GQ120177.1] Fungal agent of coral

eDNA: GoC5_E11 [FJ153721.1] disease (Alker et al. 2001)
Isolate: Mo7 [EU887758.1]
eDNA: UI12H09 [EU446322.1]
Aspergillus sydowii [EU278600.1]
Aspergillus fumigatus [M60300.1]
eDNA: LL2_BASS [EU154986.1]
Isolate: FCAS50 [GQ120176.1]
eDNA: A1_E044 [AY046710.1] Marine fungus
eDNA: FAS_19 [GQ120115.1] >79% bootstrap support
eDNA: FAS_21 [GQ120118.1]
Penicillium verruculosum [AF510496.1] >49% bootstrap support
eDNA: FAS_20 [GQ120117.1]
eDNA: BOLA831 [AF372705.1] 0.07
eDNA: BOLA676 [AF372704.1]
eDNA: FAS_18 [GQ120114.1]
eDNA: FAS_42 [GQ120141.1] NMFA1
Chaetosartorya cremea [AB008399.1]
Penicillium chrysogenum [HQ882177.1] Isolate: MV13 [EU887738.1]
eDNA: AT2−4 [AF530541.1] Paecilomyces hepiali [HM135172.1]
eDNA: UI11B04 [EU446318.1] Isolate: MV_25C [EF638701.1]
eDNA: [AF290082.2] Isolate: MV_19C [EF638698.1]
eDNA: FAS_16 [GQ120112.1] Cordyceps sinensis [AB067701.1]
eDNA: FAS_17 [GQ120113.1] Isolate: FCAS54 [GQ120165.1]
Isolate: FCAS85 [GQ120166.1] Geosmithia putterillii [AB031390.1]
eDNA: TAGIRI-22 [AB191430.1] Isolate: FCAS132 [GQ120160.1]
Ajellomyces capsulatus [X58572.1] Tritirachium sp. [AB109761.1]
Blastomyces dermatitidis [M55624.1]

Lacazia loboi [AF255331.1] Microascus cirrosus [M89994.1]
Coccidiodes immitis [M55627.1] Pseudallescheria boydii [U43913.1]
Isolate: FCAS40 [GQ120179.1] Fungal agent of epizootic Remispora maritima [HQ111002.1]
Corollospora maritima [U46871.1]
Epicoccum nigrum [AJ295235.1] in mussels at a deep-sea Isolate: FCAS20 [GQ120173.1]
Isolate: MV_26C [EF638702.1] hydrothermal vent (Van Beauveria felina [AY261368.1]
Isolate: He5C [EF638691.1] Dover et al. 2001) Stilbocrea macrostoma [AY489693.1]
eDNA: C8 [FN263263.1] Isolate: FCAS129 [GQ120159.1]
Exophiala sp. [AJ232954.1]
Simplicillium lamellicola [AB214656.1]

Rhinocladiella atrovirens [AJ232937.1] eDNA: FAS_7 [GQ120151.1]
Nadsoniella nigra [X80706.1] Isolate: FCASAn−2 [GQ120172.1]
eDNA: Herpotrichiellaceae [DQ314803.1] eDNA: FAS_8 [GQ120152.1]
Capronia pulcherrima [AJ232944.1] eDNA: FAS_12 [GQ120108.1]
Heteroconium chaetospira [DQ521604.1] eDNA: FAS_6 [GQ120150.1]
Isolate: Mo13 [EU887764.1] Fusarium oxysporum [AB110910.1]
Isolate: GMG_C6 [FJ439580.2] Fusarium solani [AB473810]
Isolate: MV_1C [EF638692.1] Isolate: Ex7 [EU887749.1] Model fungus for
Rhinocladiella phaeophora [AJ232950.1] eDNA: FAS_11 [GQ120107.1] studying fungal
Capronia epimyces [AJ232938.1] Hypocrea jecorina [AF548103.1] denitrification
eDNA: A1_E031 [AY046698.1] Isolate: FCAS31 [GQ120170.1] under low-oxygen
eDNA: FAS_22 [GQ120119.1] eDNA: FAS_13 [GQ120109.1]
eDNA: FAS_14 [GQ120110.1] conditions (e.g.,
eDNA: FAS_23 [GQ120120.1] Takaya et al. 1999)
Cladosporium uredinicola [AY251097.2] Verticillium insectorum [AB214655]
Isolate: Mo8 [EU887759.1] Paecilomyces fumosoroseus [AB086629]
Isolate: FCAS122 [GQ120156.1] eDNA: UI11F12 [EU446307.1]
Ramularia grevilleana [GU214578.1] Humicola fuscoatra [AY706333.1]
Isolate: He2C [EF638689.1] Neurospora crassa [FJ360521]
eDNA: FAS_24 [GQ120121.1] eDNA: A2_E003 [AY046715.1]
eDNA: LEMD047 [AF372706.1] Isolate: HE7 [EU887730.1]
eDNA: 1_5F_92D12b1ab1 [GU972324.1] Isolate: Mo9 [EU887760.1]
eDNA: 1_5F_90B12b1ab1 [GU972322.1] eDNA: MV_FS1C [EF638704.1]
Coniochaeta velutina [GQ154626.1]
Isolate: Ex3 [EU887745.1]
Sporothrix schenckii [M85053.1]
Arthrinium sacchari [AB220206.1]
eDNA: He3C [EF638690.1] eDNA: T41A11 [AY882532.1]
Isolate: DIVA1 [EU887731.1] Lindra marinera [AY879000.1]
Helicodendron paradoxum [AY856945.1] Lindra thalassiae [DQ470994.1]
Bulgaria inquinans [EU107260.1] Nigrospora oryzae [AB220233.1]
Hyaloscypha vitreola [EU940080.1] Physalospora scirpi [AB220204.1]
Isolate: MV14 [EU887739.1]
eDNA: MV_FS3C [EF638705.1] eDNA: FAS_25 [GQ120122.1]
eDNA: GoC5_A12 [FJ153712.1] Westerdykella dispersa [U42488.1]
Isolate: Mo2 [EU887753.1] eDNA: FAS_27 [GQ120124.1]
eDNA: D4P07C06 [EF100327.1] eDNA: FAS_26 [GQ120123.1]
Aureobasidium pullulans [DQ278883.1] Isolate: FCAS9 [GQ120158.1]
eDNA: LC23_4EP_18 [DQ504331.1] Isolate: FCAS44 [GQ120155.1]
eDNA: D3P05F07 [EF100306.1] Preussia terricola [NG_013151.1]
eDNA: D5P09D06 [EF100360.1] Lineolata rhizophorae [GU479758]
eDNA: NB13_BASS [EU154991.1] Dendryphiella salina [EU848586.1]
Isolate: Mo5 [EU887756.1] Julella avicenniae [AF441175]
Geomyces destructans [GU999983.1] Herpotrichia juniperi [U42483.1]
eDNA: D2P04H12 [EF100257.1]
Ascocratera manglicola [GU296136]
Aigialus grandis [AF441172]
eDNA: He1C [EF638687.1] NMFA2
Sigmoidea prolifera [DQ104807.1] Dothideomycetes associated
Helicoon fuscosporum [AY856946.1] with mangrove wood (Suetrong
Isolate: Mo19 [EU887770.1]
Scolecobasidium sp. [FJ439579.2] et al. 2009)
Troposporella fumosa [AY856953.1]
Helicascus nypae [GU479755]
Massarina velataspora [GQ925841]
Massarina thalassiae [GQ925843]
Falciformispora lignatilis [GU371835]
eDNA: p15D12 [AY882537.1]
Bipolaris sorokiniana [DQ337383.1]
eDNA: 1_5F_89A12b1ab1 [GU972321.1] NMFA3
Figures 2–5

eDNA: S08H02 [AB468676.1]

eDNA: CYSGM-19 [AB275102.1]
eDNA: KD10_BASS [EU154983.1]
Metschnikoiwa sp. [FJ763557.1]
eDNA: S04H05 [AB468634.1]
eDNA: S04H01 [AB468630.1]
Metschnikoia zobellii [AB023468.1]
eDNA: D4P07A09 [EF100386.1]
Candida agrestis [AB023471.1]
eDNA: D1P02G03 [EF100222.1]
Candida akabanensis [AB013514.1]
eDNA: FAS_43 [GQ120142.1]
eDNA: FAS_1 [GQ120105.1]
eDNA: FAS_2 [GQ120116.1]
Kodamaea ohmeri [GU597327]
Saccharomyces cerevisiae [Z75578]
eDNA: LL7_BASS [EU154987.1]
Kluyveromyces nonfermentans [AB011507.1]
Pichia lachancei [EF550451.1]
eDNA: JJ15_BASS [EU154982.1]
Pichia fermentans [GQ458040.1]
eDNA: D5P09B03 [EF100409.1]
Candida abiesophila [EF550350.1]
eDNA: FAS_4 [GQ120138.1]
eDNA: FAS_3 [GQ120127.1]

Debaromyces hanseii [DQ534402.1]
eDNA: MH1_BASS [EU154990.1]
Candida parapsilosis [GQ395609]
eDNA: FAS_5 [GQ120149.1]
Schizosaccharomyces pombe [X54866]
Taphrina deformans [AJ495826.1]
eDNA: GoC5_E06 [FJ153719.1]
Marine fungus
Figure 1
>79% bootstrap support
0.07
Figures 3–5
Figure 2
environmental DNA analyses. This section of the tree mainly focuses on Saccharomycotina. The tree was calculated using PhyML
(parameters ! = 0.722 and I = 0.117) with both PhyML and LogDet distance bootstraps. Marine phylotypes are highlighted in yellow.
environments investigated or alternatively that the PCR primers or DNA sampling methodologies
used for this study were collectively biased toward the Dikarya.
Fungal-specific clone library analyses of deeper water column samples, sediments, and anoxic
environments (Bass et al. 2007, Jebaraj et al. 2009, Le Calvez et al. 2009) have significantly increased
the diversity of the fungi recovered from marine studies, especially when compared with surface
water samples (Massana & Pedrós-Alió 2008). Two of these studies ( Jebaraj et al. 2009, Le Calvez
et al. 2009) combined eDNA analysis with isolation and culture experiments, demonstrating little
overlap between the sequences recovered from isolated cultures and eDNA and suggesting the
possible presence of a more complex fungal community than identified either by eDNA or culture-
based analyses alone. Nonetheless, marine fungal-specific clone library analyses (Bass et al. 2007,
Edgcomb et al. 2011, Jebaraj et al. 2009, Le Calvez et al. 2009) reveal a simple fungal community
with relatively few phylotypes in total. This is a stark contrast to terrestrial environments, where
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 3
environmental DNA analyses. This section of the tree mainly focuses on part of the basidiomycete radiation (see Figure 4 for the
remaining basidiomycete branches). The tree was calculated using PhyML (parameters ! = 0.722 and I = 0.117) with both PhyML
and LogDet distance bootstraps. Marine phylotypes are highlighted in yellow. Novel marine phylogenetic groups are marked NMFB
(blue, novel marine fungi basidiomycetes). Ecological information is annotated on the tree.
502 Richards et al.

eDNA: 10_1F_26b1ab1 [GU972474.1]

eDNA: 10132 [GU972195.1] NMFB1
eDNA: 10180 [GU972227.1]
eDNA: 10_1F_63b1ab1 [GU972505.1]
eDNA: 1H3cG10 [GU972030.1]
Marine fungus eDNA: 1H3cD8 [GU972011.1]
>79% bootstrap support eDNA: AB3F14RJ10F01 [GU823324.1]
eDNA: 10194 [GU972235.1]
>49% bootstrap support eDNA: PAT6_EK5_11 [DQ504360.1]
eDNA: KM10_BASS [EU154984.1]
0.07 eDNA: AT9−6 [AF530542.1]
eDNA: 10109 [GU972179.1]
eDNA: [FN690504.1]
eDNA: RU12192007A11 [HQ427485.1]
eDNA: [FN690505.1]
eDNA: UI14F08 [EU446360.1]
Malassezia restricta [EU192367.1]
eDNA: FAS_41 [GQ120140.1]
eDNA: LC23_5EP_14 [DQ504335.1]
eDNA: BAQA52 [AF372708.1]
eDNA: FAS_40 [GQ120139.1]
eDNA: MV5E1_EF15 [EF638647.1]
eDNA: A93F14RM1E05 [GU823227.1]
eDNA: 5H2dA11 [GU972109.1]
eDNA: CK2_BASS [EU154972.1]
eDNA: MV5E1_EF2 [EF638638.1]
eDNA: FAS_15 [GQ120111.1]
eDNA: BOLA39 [AF372707.1] NMFB2
eDNA: 10137 [GU972199.1] NMFB3
eDNA: 10158 [GU972212.1]
Malassezia furfur [AY083223.1]

eDNA: 1H3dC09 [GU971936.1]
eDNA: BOH3_EK2_20 [DQ504358.1]
eDNA: 1H3dB07 [GU971924.1]
eDNA: 9_1F_90B12b1ab1 [GU972448.1]
eDNA: CE2_BASS [EU154971.1]
Malassezia sympodialis [EU192369.1]
Malassezia pachydermatis [EU192366.1]
Malassezia globosa [EU192364.1]
eDNA: 10_1F_31b1ab1 [GU972479.1]
eDNA: 10148 [GU972207.1] NMFB4

eDNA: 10_1F_19b1ab1 [GU972468.1]
Ustilago maydis [X62396]
eDNA: FAS_37 [GQ120135.1]
Moesziomyces bullatus [DQ363307]
eDNA: FAS_38 [GQ120136.1] NMFB5
eDNA: FAS_39 [GQ120137.1]
Thecaphora spilanthis [DQ832242]
eDNA: FAS_51 [GU072550.1]
eDNA: FAS_30 [GQ120128.1]
Graphiola cylindrica [D63929.1]
eDNA: UI12B09 [EU446331.1]
eDNA: cLA11E07 [EU446368.1]
eDNA: LKM33[AJ130854.1]
Exobasidium rhododendri [AJ271381.1]
Doassansia hygrophilae [DQ198788]
Rhamphospora nymphaeae [DQ831033.1]
Tilletiopsis ashingtonensis [AJ271382.1]
Isolate: HE6 [EU887729.1]
Tilletiopsis derxii [AB045704]
eDNA: FAS_45 [GQ120144.1]
eDNA: UI13D02 [EU446343.1]
Coprinus cinereus [M92991]
Exidia uvapsassa [NG_013159.1]
eDNA: MV5E1_EF3 [EF638639.1]
Athelia epiphylla [GU187613.1]
eDNA: MV5E1_EF5 [EF638641.1] Form multicellular bodies
eDNA: FAS_48 [GQ120147.1] in terrestrial environments
eDNA: FAS_46 [GQ120145.1]
eDNA: FAS_47 [GQ120146.1]
eDNA: NB16_BASS [EU158830.1]
Antrodia variiformis [AY336782.1]
Cystofilobasidium infirmo-miniatum [AB072226.1]
eDNA: MD13_BASS [EU154988.1]
eDNA: 1H2cA11 [GU971833.1]
eDNA: 1H2cG5 [GU971887.1]
Cryptococcus pseudolongus [AB051047.1]
eDNA: 1H2cD8_1 [GU971902.1]
eDNA: 1H2cD6 [GU971865.1] NMFB8
eDNA: 1H2cA7 [GU971897.1]
Trichosporon aquatile [AB001730]
eDNA: DSGM-62 [AB275062.1] Dominant microbial eukaryote in sediment at the Kuroshima
eDNA: KUROS1 [AB234889.1] Knoll methane seep (Takishita et al. 2006)
Cryptococcus curvatus [AB032626]
Cryptococcus surugaensis [AB100440.1]
eDNA: BL00122143 [AY426889.1] Yeast species isolated from deep-sea floor sediment,
eDNA: CH1_S1_56 [AY821990.1] Suruga Bay, Japan (Nagahama et al. 2003b)
eDNA: HC8_BASS [EU154978.1]
Cryptococcus carnescens [AB105437.1]
Cryptococcus gattii [HQ659559]
Filobasidium elegans [AB075545.1]
eDNA: H18E12_5 [EF638517.1]
Filobasidium elegans [AB075545.1]
eDNA:[AB505508.1]
Cryptococcus albidus [HQ231895]
eDNA: MV2E1_40 [EF638491.1] NMFB9
eDNA: MV2E1_7 [EF638470.1]
Figure 4
Figures 1, 2, and 5

eDNA: UI11B06 [EU446299.1]

eDNA: B42 [FJ914435.1]
Rhodotorula glutinis [DQ832194.1] Red yeasts isolated from
eDNA: MD7_BASS [EU154989.1] deep-sea sediments,
eDNA: UI12D05 [EU446334.1] Pacific Ocean
Rhodotorula mucilaginosa [GQ433375.1] (Nagahama et al. 2001)
eDNA: FAS_29 [GQ120126.1]
eDNA: FAS_28 [GQ120125.1] Example of red yeast isolated from
Rhodotorula benthica [AB126647.1] a deep-sea tubeworm, Pacific Ocean
Rhodotorula nymphaeae [AB055189.1] (Nagahama et al. 2003a)
Rhodotorula aurantiaca [AB030354.1]
Kurtzmanomyces nectairei [D64122.1]
Sporobolomyces xanthus [D64118.1]
Sporobolomyces pyrrosiae [AB126045.2]
Bensingtonia naganoensis [D38366.1]
eDNA: GoC4_A07 [FJ153692.1]
Kondoa malvinella [D13776.1]
Mycogloea macrospora [U41848.1]
Tritirachium egenum [JF263567.1]
Platygloea disciformis [DQ234563.1]
Figure 3
Marine fungus
Figures 1, 2, and 5 >49% bootstrap support

0.07
Figure 4
Subsection of a phylogenetic analysis demonstrating the diversity of small subunit ribosomal (SSU) DNA
sequences recovered from marine environmental DNA analyses. This section of the tree focuses on the other
subsection of the basidiomycete phylogeny (see Figure 3 for the remaining basidiomycete branches). The
tree was calculated using PhyML (parameters ! = 0.722 and I = 0.117) with both PhyML and LogDet
distance bootstraps. Marine phylotypes are highlighted in yellow. Ecological information is annotated on the
tree. Three Rhodotorula sequences are not highlighted in yellow, because actual SSU sequences were not
recovered from a marine environment; marine provenance here is based on comparison of internal
transcribed spacer (ITS) sequences in the papers referenced.
fungal communities appear to be much more complex (e.g., Buée et al. 2009, Jumpponen &
Jones 2009, O’Brien et al. 2005), clearly demonstrated by the comparison of species accumulation
curves from marine environments (Bass et al. 2007, Jebaraj et al. 2009) with the results of clone
library analyses of nonmarine habitats. Based on currently available data, it is difficult to describe
the difference in the diversity and relative abundance of fungal forms between terrestrial and
marine environments. Yet the molecular data seem to be consistent with the results of culturing
efforts, although different diversity profiles are revealed by these two methods, and suggest that
marine fungi are relatively nondiverse and lower in abundance. Therefore, some fundamental
questions remain to be addressed: (a) Is this apparent difference in diversity and abundance due
to an asymmetric sampling effort between marine and terrestrial environments? (b) Is there a
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→
Figure 5
environmental DNA analyses. This section of the tree mainly focuses on the lower fungi (i.e., chytrids and zygomycetes). The tree was
calculated using PhyML (parameters ! = 0.722 and I = 0.117) with both PhyML and LogDet distance bootstraps. Marine phylotypes
are highlighted in yellow. Novel marine phylogenetic groups are marked NMF (orange, novel marine fungi). Ecological information is
annotated on the tree.
504 Richards et al.

Figures 1–4
Rhopalomyces elegans [NG_017191.1]

eDNA: D1P01D10 [EF100208.1] NMF1
Syncephalis depressa [AB016011.1]
Thamnocephalis sphaerospora [AB016013.1]
Scutellospora dipurpurescens [FM212931.1]
Glomus mosseae [NG_017178]
Paraglomus occultum [NG_017179]
Endogone lactiflua [DQ536471.1]
eDNA: D5P09E04 [EF100388.1] Marine fungus
eDNA: KD12_BASS [EU154985.1] NMF2 >79% bootstrap support
Chytridiales sp. AF011 [EF432819.2]
eDNA: NPK97_82 [EU371354.1] NMF3 >49% bootstrap support
eDNA: T37A10 [AY882534.1]
eDNA: p15H03 [AY882540.2] NMF4 0.07
eDNA: CCW64 [AY180029.1]
Chytridiales sp. [EF443138]
Lobulomyces angularis [AF164253.2]
Chytriomyces poculatus [EF443135.1]
eDNA:MV5E2_90 [EF638685.1]
eDNA: MV2E2_B7R [EF638571.1] NMF5
eDNA: D2P03H04 [EF100399.1] NMF6
Chytridium polysiphoniae [AY032608.1]
eDNA: D2P03H05 [EF100242.1] NMF7 Parasite of brown
Nowakowskiella sp. [AY635835] macroalgae
Cladochytrium replicatum [NG_017169] (Küpper et al. 2006)
Neocallimastix frontalis [EF014370.1]

Piromyces sp. [HQ585899]
Cyllamyces aberensis [DQ536481.1]
Triparticalcar arcticum [DQ536480.1]
Entophlyctis confervae-glomeratae [EF014367.1]
Catenomyces sp. [AY635830.1]
eDNA: DB39_BASS [EU154976.1]
eDNA: AI3F14RJ1H10 [GU823836] NMF8
Entophlyctis sp. [AY635824.1]
eDNA: PA2009A3 [HQ191313.1]

eDNA:T5P2AeD02 [GQ995333.1]
Rhizophlyctis rosea [NG_017175.1]
Basidiobolus ranarum [NG_017184]
Basidiobolus haptosporus [AF368504.1]
Rhizophydium sphaerotheca [AY635823]
Batrachochytrium dendrobatidis (Broad genome project)
eDNA: D3P06C06 [EF100403.1] NMF9
Kappamyces laurelensis [DQ536478.1]
Rhizophydium sp. [AY635821.1]
eDNA: T5P2AeG04 [GQ995437.1] NMF10
eDNA: BAQA128 [AF372721.1] NMF11
eDNA: BL01032036 [AY426915.1] NMF12
Rhizophlyctis harderi [AF164272.2]
Rhizophydium elyensis [DQ536479.1]
eDNA: M1_18C05 [DQ103818.1] NMF13
Rhizophydium sp. [DQ536492.1]
Karlingiomyces sp. [AF164278]
Polychytrium aggregatum [NG_017168]
Mortierella olfii [AF113425.1]
Mortierella chlamydospora [AF157143.1]
Blastocladiella emersonii [AY635842]
Allomyces arbuscula [NG_017166]
eDNA: TAGIRI-24 [AB191432.1] NMF14
Rhizoclosmatium sp. [AY601709.1]
Chytriomyces hyalinus [DQ536487]
Hyaloraphidium curvatum [NG_017172]
Monoblepharella sp. [AY546682]
eDNA: I34 [EU910604]
eDNA: LKM15 [AJ130850]
eDNA: IAFDv7 [AY835675.2] NMF16
eDNA: NAMAKO-36 AB252776
Rozella sp. [AY601707]
Rozella allomycis [AY635838]
eDNA: DSGM-64 [AB275064.1]
eDNA: TAGIRI-23 [AB191431.1] NMF17
eDNA: NAMAKO-37 [AB252777]
eDNA: NAMAKO-35 [AB252775.1]
eDNA: CCW48 [AY180024.1] NMF18
eDNA: CCW24 [AY230211.1] NMF19
eDNA: LKM46 [AJ130857]
eDNA: BL01032033 [AY426912.1] NMF20
eDNA: EMPE7 [AF372715.1]
eDNA: LS_CM2 [FJ687267.1]
eDNA: BRKC111 [AF372714.1]
eDNA: GoC6_F01 [FJ153737.1] NMF21
eDNA: WS_CM1 [FJ687268]
eDNA: IAFDv110 [AY835696.2] NMF22
eDNA: LKM11: [AJ130849] Cryptomycota
eDNA: BAQA254 [AF372713.1] (Jones et al. 2011)
eDNA: KD14_BASS [EU154992.1]
eDNA: [AB468638.1]
eDNA: [AB275063.1] NMF24
eDNA: SS1_E_01_42 [EU050974.1]
eDNA: SS1_E_01_10 [EU050973.1]
Physoderma dulichii [DQ536472]
Physoderma maculare [DQ536489]
Amoebidium parasiticum [Y19155]
eDNA: NW61702 [DQ060525.1]
eDNA: FAS_49 [GQ120148.1]
Nuclearia simplex Outgroup
Capsaspora owczarzaki
Suberites ficus [AJ627184]
Aurelia sp. [EU276014]

real ecological barrier that has prevented marine fungi from diversifying to the same degree as
terrestrial fungi? (c) Is the broad success and evolutionary complexity of the fungal radiation so
intimately linked with the radiation of land plants and the colonization of terrestrial environments
that these forms dominate the diversity of the fungal kingdom? (d ) Are marine niches dominated
by a different set of microbes performing the equivalent roles in the ecosystem as fungi in terrestrial
environments, so as to outcompete fungi in marine habitats? To begin to address these questions,
it is important to investigate which fungi have been detected in marine environments.
PHYLOGENETIC SUMMARY OF THE SMALL SUBUNIT RIBOSOMAL

DNA DIVERSITY OF MARINE FUNGI
To bring together the growing body of data investigating the molecular diversity of fungi in
marine environments, we have calculated a summary phylogenetic tree of all unique fungal SSU
rDNA sequences detected from marine eDNA samples. Sampling was conducted by a literature
review followed by phylogenetic tree construction to obtain a preliminary census of the molecular
diversity of fungal SSU sequences. Using this preliminary tree, we conducted BLASTn searches
(Altschul et al. 1990) of the NCBI nonredundant DNA database (GenBank) to identify additional
marine fungal eDNA SSU sequences. We focused on SSU, rather than ITS, sequences to generate
as robust a phylogeny as possible, allowing us to construct a comprehensive picture of the diversity

and identify any novel or distinct phylogenetic groups. Therefore, many marine culture-derived
sequences are not included in our analysis, as ITS rDNA is often the marker of choice in culture-
based studies (Horton & Bruns 2001).
Our tree (Figures 1–5) was constructed in an iterative fashion with many highly similar se-
quences from the same environment sample and/or publication removed and thus represents a
summary of the diversity of marine fungal SSU sequences available in GenBank. Therefore, this
phylogenetic tree should be viewed not as an exhaustive census, but rather as a summary of our
understanding of the molecular diversity of marine fungi.
We also note that the nucleic acid extraction methods used in many of the published envi-
ronmental clone library studies often do not include chemical/physical preparations specifically
tailored to the lysis of fungal cells. It is therefore possible that DNA from cells enclosed within
robust chitin-rich cell walls typical of many fungi may have been missed by these studies. Fur-
thermore, the PCR primers used convey differential PCR bias toward or against particular fungal
groups. Therefore, our summary tree is likely to be incomplete and is affected by systematic biases
in the sampling methods used.
The sequences in Figures 1–5 are derived from a range of methods, including both fungal-
specific clone library analyses and general eukaryotic analyses, and the sequences used were gener-
ated by a number of different primer combinations. Therefore, the number of DNA positions that
could be reliably sampled across all the data sets analyzed was limited. Nonetheless, we were able
to sample a masked alignment region of 656 characters surrounding the variable SSU V4 region
(Wuyts et al. 2000). We then conducted preliminary phylogenetic analysis to remove redundant
and highly similar SSU sequences. The alignment was calculated using MUSCLE (Edgar 2004),
extensively corrected by eye, and was masked using SEAVIEW (Galtier et al. 1996). Phylogenetic
analysis was conducted using PHYML (Guindon & Gascuel 2003) with 100 PHYML bootstraps
and 1,000 LOG-DET distance bootstraps (Lockhart et al. 1994).
The Dikarya Majority on Land and at Sea

Our phylogeny (Figures 1–5) shows a diverse collection of environmental marine fungal SSU
rDNA sequences, with the vast majority branching within the basidiomycetes and ascomycetes
506 Richards et al.

(Figures 1–4). This dominance of Dikarya in marine habitats is consistent with previous analyses
(Bass et al. 2007, Edgcomb et al. 2011, Jebaraj et al. 2009, Le Calvez et al. 2009). Many Dikarya
sequences sampled during our preliminary phylogenetic analysis were highly similar to each other;
much of this redundancy was removed during the tree processing steps described above and is
not shown in Figures 1–4. Consequently, this phylogenetic analysis actually underestimates the
relative dominance of Dikarya (Figures 1–4) compared to lower fungi (Figure 5).
Our phylogenetic analysis suggests that filamentous fungi are more diverse in marine habitats
than previous studies have suggested (Bass et al. 2007). Examples include lineages within the
Pezizomycotina ascomycete fungi, Neurospora, Aspergillus, Cordyceps, and Fusarium (Figure 1),
and some basidiomycetes, e.g., Ustilago-like lineages (Figure 3). The phylogeny also identified
several marine sequences branching close to taxa that form multicellular structures in terrestrial
environments, e.g., Coprinus, Antrodia, and Exidia (Figure 3). We had previously suggested that
yeast forms dominate fungal diversity in the deep water column and marine sediment environments
(Bass et al. 2007). The summary phylogeny partially supports this analysis, with a large proportion
of the sequence diversity recovered clustering with known yeast taxa [e.g., Candida, Metschnikowia,
Pichia, Taphrina, Malassezia, Cryptococcus, and Rhodotorula (Figures 2–4)], but suggests that marine
fungal communities comprise a wider diversity of fungal phenotypes.
We also note that a large proportion of the filamentous Pezizomycotina diversity shown in
Figure 1 was recovered from cultured isolates (Burgaud et al. 2009, Jebaraj et al. 2009, Le Calvez
et al. 2009), suggesting that eDNA studies miss these usually filamentous fungi. This may be
explained by primer bias or by the DNA extraction process failing to recover DNA from these
microbes, owing to their robust cell walls reducing DNA recovery from these groups. Alternatively,
these Pezizomycotina microbes may be at a very low level of abundance, or present as cysts, and
are consequently often missed during eDNA sampling. Yet filamentous forms seem to be readily
recovered during culturing experiments, perhaps because this sampling method can preferentially
select for such fungal forms (e.g., Le Calvez et al. 2009). In conclusion, our phylogenetic results
suggest that Dikarya are the most readily recovered marine fungal lineages, and the majority of
sequences recovered from eDNA analyses cluster on the phylogeny with known yeast groups,
although culturing efforts suggest the presence of a higher proportion of filamentous forms.
Novel Marine Fungal Phylotypes and the Marine-Terrestrial Transition

Environmental clone library sequencing has identified a number of novel and phylogenetically
distinct clades that branch with the fungi but seem to represent highly unique groups ( Jones et al.
2011, Lara et al. 2010, Porter et al. 2008, Schadt et al. 2003). Our summary phylogeny shows 36
such groups (Figures 1, 3, and 5). We defined these novel groups on the basis of a minimum
of 3% nucleotide character difference, identified using BLASTn analysis, to all sequences in the
GenBank nonredundant database that were not marine eDNA sequences. Interestingly, although
the majority of marine fungal sequences recovered for this analysis branched within the Dikarya
(Figures 1–4), 24 of the 36 novel groups branched among the lower fungi (Figure 5). Figure 6
plots the percentage difference of all 36 novel fungal groups to cultured and described fungi versus
the percentage difference of the novel groups to all fungal SSU sequences recovered from terrestrial
environments (including both eDNA and cultured isolates). This graph clearly demonstrates that
the novel marine lower fungi generally comprise more divergent SSU types compared to novel
groups within the Dikarya. Furthermore, the novel marine lower fungi seem to be particularly
distantly related to cultured and described fungi. Taken together, these data suggest that marine
environments host a significant number of highly novel groups, with the majority of these novel
groups branching below the Dikarya radiation, close to chytrid branches, implying that they may

NMF
NMFB (Dikarya)
15 NMFA (Dikarya)
Line of best fit for novel
14 marine Dikarya groups
(n = 12, NMFB + NMFA)
13 Line of best fit for novel
marine lower-fungi groups
terrestrial fungi (either cultured isolate or eDNA)

12 (n = 24)
Percent difference among SSU sequences from

11
10
8
6
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Percent difference among SSU sequences of all
cultured and described fungal isolates
Figure 6
Graph comparing the percentage difference of novel marine fungi to terrestrial fungi verses the percentage
difference of the novel fungi to all isolated and described fungi (either terrestrial or marine). This graph
demonstrates that among the marine fungi, the novel marine groups branch mainly among the lower fungi
(Figure 5) and that these novel lower fungal branches (shown in orange) have higher levels of small subunit
(SSU) ribosomal DNA sequence difference to cultured and described fungi (x axis) than fungi detected in
terrestrial environments ( y axis). This demonstrates that novel marine fungi (NMF) tend to have closer
relatives detected by sequencing of terrestrial environments than they have represented in culture
collections, suggesting that isolation and description of marine fungi are lagging behind. Abbreviations:
eDNA, environmental DNA; n, number of novel phylotypes identified in the tree analysis (see Figures 1–5);
NMFA, novel marine fungi ascomycetes; NMFB, novel marine fungi basidiomycetes.
form flagellated zoospores (Figure 5). These results are consistent with the conclusion of Le
Calvez et al. (2009), who suggest that the marine environment hosts numerous, unclassified deep-
branching fungal forms, reflecting an ancient transition from marine environments to terrestrial
environments. The lower fungi may be significantly more diverse and numerous in marine habitats
than Figure 5 suggests if the PCR process (including primer choice/design) used to generate most
marine clone libraries (whether targeting fungi or microbial eukaryotes in general) is biased toward
Dikarya fungi.
The majority of fungal environmental SSU rDNA sequences and isolated cultures are from
terrestrial environments, leading to the hypothesis that fungi are a mainly terrestrial group (e.g.,
508 Richards et al.

Le Calvez et al. 2009). However, all terrestrial life-forms, including freshwater forms, must trace
their ancestry back to marine-dwelling ancestors. It has therefore been inferred that fungi arose
in the oceans and then colonized terrestrial environments (Le Calvez et al. 2009). The transition
from marine to terrestrial environments involves a radical change in biological capacities, and
therefore it is widely suggested that symbioses between fungi and photosynthetic microbes facil-
itated multiple terrestrial colonization events, including the phototrophic microbial ancestor of
the land plants (Pirozynski & Malloch 1975, Selosse & Le Tacon 1998, Simon et al. 1993). Fossil
evidence has suggested that several fungal subgroups [e.g., vesicular-arbuscular (VA) mycorrhizae,
ascomycetes, blastocladiomycetes] were established in the Devonian 400 Mya, suggesting the fun-
gal lineage is much older than this (Berbee & Taylor 2010) and therefore that the fungal kingdom
is much older than the colonization of land by both plants and fungi. These results imply a long
phase of fungal evolution in aquatic/marine environments before fungi colonized the land (i.e., an
ancient marine fungal divergence) (Le Calvez et al. 2009). Evidence of divergent deep-branching
marine fungal SSU sequences (e.g., Figures 5 and 6) has been used to argue that these lower fun-
gal marine groups are derived from an ancient phase of fungal evolution in marine environments
prior to the colonization of terrestrial environments (Le Calvez et al. 2009). However, environ-
mental sequencing of marine DNA samples also shows that marine lineages frequently form short
branches interdispersed between terrestrial lineages (Figures 1–5). One interpretation is that the
evolutionary diversification of the Fungi has involved multiple marine-terrestrial transitions in

both directions.
In contradiction to the ancient marine fungal divergence theory, studies of freshwater environ-
ments have recovered novel deep-branching fungal sequences at a much higher frequency than in
the marine environment. For example, freshwater studies frequently report that a high proportion
of all the clones recovered from general eukaryotic clone libraries are fungal sequences [e.g., 19%
(Lef èvre et al. 2008), 23% (Lef èvre et al. 2007), 33% (Berney et al. 2004), or 25% (Lepère et al.
2006)]. Furthermore, in contrast to the majority of fungal sequences recovered from marine envi-
ronments, which are Dikarya (e.g., Figures 1–5; Bass et al. 2007, Edgcomb et al. 2011, Jebaraj et al.
2009, Le Calvez et al. 2009), a much larger proportion of the freshwater fungal sequences branch
among the lower fungi (Figure 7). These results point to a large and underexplored diversity of
lower fungal groups in freshwater (Lef èvre et al. 2007, 2008; Lefranc et al. 2005; Slapeta et al.
2005) compared to marine environments. With freshwater environments being part of the terres-
trial biome, this emerging pattern therefore seems to be inconsistent with the idea that divergent
deep-branching novel fungal forms are derived directly from marine ancestors, separate from the
terrestrial branch(es) (Le Calvez et al. 2009). A more realistic model might be a terrestrial origin or
very early primary terrestrial radiation of fungi followed by multiple marine-terrestrial transitions
in both directions. Indeed, phylogenetic analysis of marine ascomycetes has identified multiple
terrestrial-to-marine transitions (Spatafora et al. 1998, Suetrong et al. 2009), whereas data support-
ing multiple losses of flagella within the fungal radiation ( James et al. 2006a,b; Liu et al. 2009) show
a similarly complex evolutionary pattern involving multiple aquatic-to-nonaquatic transitions.
Cryptomycota: A New Basal Branch in the Fungi?

Interestingly, freshwater studies have also identified a large and complex clade of environmental
sequences that form one of the deepest branches in the Fungi (e.g., Berney et al. 2004; Lef èvre
et al. 2007, 2008; van Hannen et al. 1999). Van Hannen and coauthors (1999) first identified this
group from sequencing DNA recovered from a freshwater-derived experimental detritus system,
and it has now been recovered from numerous environment types ( Jones et al. 2011, Lara et al.
2010). Of the four fungal-like sequences recovered by van Hannen et al. (1999), three formed this

Edgcomb et al. (2011)

deep-sea sediment
with a nested PCR step
Le Calvez et al. (2009) Marine fungi identifed using

hydrothermal vents seven different primer sets
(five fungal specific)
Bass et al. (2007)
deep sea
Jebaraj et al. (2009)

oxygen-depleted Arabian
sea environments
Lefèvre et al. (2008)

lake water
picoeukaryotes 2
Lefèvre et al. (2007)
lake water Freshwater fungi identified

picoeukaryotes 1 using three different general
eukaryotic primer sets
Lefranc et al. (2005)
lake water columns
Berney et al. (2004)

stream sediment
10 20 30 40 50
Phylotypes
Dikarya Lower fungi
Figure 7
Comparison of small subunit ribosomal clone library analyses from marine and freshwater environmental
DNAs, demonstrating that freshwater sampling identifies many more lower fungi phylotypes than similar
marine analyses, whereas marine analyses tend to detect Dikarya fungi. Note that the freshwater studies
summarized used general eukaryotic primers, whereas the marine studies summarized used both general
eukaryotic primers and fungal-specific primers.
deep-branching clade. Subsequent clone library experiments have considerably expanded the di-
versity of this clade in soils and freshwater environments (e.g., Jones et al. 2011; Lef èvre et al.
2007, 2008; Lefranc et al. 2005); sequencing of marine libraries has also recovered representatives
Phagotrophy:
process of acquiring of this group (Figure 5; also see Massana et al. 2004a; Takishita et al. 2005, 2007). Using terminal
nutrients by engulfing restriction fragment length polymorphism analyses, Lepère et al. (2006) found that representatives
large particles or prey of this group are highly abundant in freshwater environments and that population abundance is
cells in a phagosome linked to an abundance of algal populations.
(cell vacuole), followed
Further phylogenetic analysis with additional environmental rDNA sampling suggests that
by digestion and
intracellular this group branches with the intracellular parasitic genus Rozella, thought to be the first branch
absorption of nutrients in the fungal radiation ( James et al. 2006a, Jones et al. 2011, Lara et al. 2010). This is an inter-
Osmotrophy: mode esting relationship because the genus Rozella comprises intracellular parasites that do not possess
of nutrition involving a chitin/cellulose cell wall for the stages of their life cycle identified and furthermore appear to
secretion of enzymes be capable of phagotrophy (Held 1981). These characteristics represent distinct differences from
to break down complex the fundamental fungal phenotype: a rigid chitin wall, the use of osmotrophy, and an inability
biological polymers
to perform phagotrophy. Thus it is questionable whether the Rozella genus and this wider clade
followed by uptake of
simplified molecules of environmental sequences should be classified within the Fungi or whether they represent an
intermediate form (Lara et al. 2010, Jones et al. 2011).
510 Richards et al.

Jones et al. (2011) used fluorescence in situ hybridization to investigate the ecology and cell
biology of this novel group and showed that it possesses at least three distinct life-cycle phases:
(a) a flagellated zoospore, (b) single cells attached to other (often algal) cells, and (c) cysts or
Cryptomycota:
resting cells. Using a combination of cell wall markers, the authors could not identify a fungal highly diverse group of
chitin/cellulose cell wall, suggesting that similar to Rozella, these microbes live without this cellular microbes, mainly
trait for much of their life cycle. In anticipation of formal classification, and because this clade known from
groups with true fungi, Jones et al. (2011) named this group cryptomycota (hidden fungi). environmental DNA
techniques; includes
Interestingly, both the cryptomycota and chytrid-like sequences recovered from freshwater
Rozella
environments branch most closely to fungi known to parasitize algae, other fungi, or protists (Held
1981; Lef èvre et al. 2007, 2008; Lefranc et al. 2005). These observations have led some authors
to re-evaluate the functional role of fungal parasites in freshwater environments, suggesting that
they play a significant role in the microbial loop of freshwater ecosystems (Lef èvre et al. 2008).
ECOLOGICAL ROLE OF FUNGI IN MARINE ENVIRONMENTS

Fungi are considered as saprotrophs, parasites, or symbionts, partly because of the bias of research
interests, but mainly as a consequence of the evolution of fungal cell biology and feeding strategies.
In contrast to the example of the cryptomycota discussed above ( Jones et al. 2011), fungi generally
possess robust chitin-rich cell walls and obtain nutrients exclusively by feeding osmotrophically.
This process involves the secretion of depolymerizing enzymes followed by the transportation of
nutrients, usually as digested monomers, back into the cell.
These traits underpin the ecological success of the Fungi and are mechanistically linked;
the chitin/cellulose reinforces the fungal cell and enables it to resist (a) the substantial osmotic
pressure produced during osmotrophic feeding, (b) structural strains during growth (usually as
polarized cells in the form of hyphae or rhizoids), and (c) the diverse and heterogeneous envi-
ronments within which fungal filaments grow. These adaptations drive the high metabolic rate,
fast growth, and ecological success of fungi (Bartnicki-Garcia 1987). However, as a consequence
of this lifestyle, many fungi have lost the ability to perform phagocytosis and therefore can-
not engulf and digest prey cells in the same way as many other eukaryotes. This reliance on
osmotrophy determines the ecology of fungi: They thrive in nutritionally rich environments
such as plant and animal host organisms, soils, sediments, and detritus environments, where they
can attach to substrates, secrete enzymes, break down complex biological polymers, and take up
nutrients.
These ecological characteristics in part may explain why fungi have been considered both
nondiverse and of low abundance in many upper and surface marine water column samples
(Kis-Papo 2005, Massana & Pedrós-Alió 2008, Richards & Bass 2005). Many pelagic and surface
water environments are often low in nutrients, and the microbial eukaryotic component of the
planktonic food chain is predominately that of free-floating or swimming single-celled organisms
performing primary production and/or phagotrophic grazing. Such environments are unlikely to
favor organisms that feed primarily by attachment to larger physical substrates and osmotrophy,
partly because secreted enzymes and target nutrients are likely to be lost by rapid diffusion in the
liquid environment, but also because of key differences in the relationship between photosynthesis
and biomass accumulation on land and at sea. Fixed carbon on land is largely invested in the
construction of large and complex plant tissues rich in energy and nutrients. These complex
structures are difficult to digest, driving the evolution of the osmotrophic lifestyle, specialized
plant/fungi associations, and the subsequent diversification of fungi. Yet in the open ocean,
primary producers do not extend much beyond the scale of small single-celled organisms (∼3 µm
in diameter) and do not make complex energy- and nutrient-rich structures like land plants.

As such, the very niches that favor fungal diversification and osmotrophic feeding on land are
largely absent in the open ocean. Therefore, this trophic relationship is essentially closed off to
the microbial community, with only single-celled primary producers and phagotrophic grazers
supported in the upper oceanic water column.
There are, of course, exceptions to this model: (a) In the lower water column, below the pho-
tozone, as particulate matter descends and enters the sediment, logically there must be increased
niche availability for saprotrophs (potentially fungi) driving the principal steps in food webs, which
at these lower depths are born out of detritus processing. Observations that fungi are the dominant
active eukaryotic microbes in these environments are consistent with this hypothesis (Edgcomb
et al. 2011, Takishita et al. 2006). Detrital processing represents a largely understudied area of
marine ecology but represents an important gap in our understanding of how nutrients, including
carbon, are processed. (b) Coastal systems harboring large multicellular algae or mangroves must
provide numerous niches for saprotrophs and endophytes, again providing potential ecosystems
for marine fungi (discussed below). (c) A large fraction of marine fungi must also reside as parasitic
forms in marine animals.
Fungi as Parasitic Agents in the Marine Environment

It is striking that many fungi that cause or are associated with disease in marine habitats are
closely related in phylogenetic analyses to well-characterized nonmarine taxa. Figures 1–3 show
several examples, with marine sequences branching closely to terrestrial parasitic fungi, e.g.,
Cordyceps/Paecilomyces (Figure 1), Geomyces (Figure 1), Taphrina (Figure 2), Candida (Figure 2),
Malassezia (Figure 3), and Ustilago (Figure 3). Furthermore, fungi are often isolated from marine
animals, suggesting a range of additional and unstudied parasitic associations. For example, new
species of the basidiomycete yeast Rhodotorula have been isolated from deep-sea tubeworms and
clams by Nagahama et al. (2003a) and have also been recovered from a range of eDNA and cul-
turing studies derived from deep-sea samples (Nagahama et al. 2001, 2003a) (Figure 4). Although
they have not been directly shown to be parasitic agents, they appear to be closely associated with
certain marine invertebrates (Nagahama et al. 2003a). Similarly, black yeast (Herpotrichiellaceae
of the order Chaetothyriales) has been proposed as a causal agent in disease of the deep-sea mussel
Bathymodiolus brevior in the Fiji Basin (Van Dover et al. 2007). Other known fungal pathogens
closely related to terrestrial fungi are particularly prevalent in marine mammals, including As-
pergillus (aspergillosis), Blastomyces (blastomycosis), Coccidioides (coccidioidomycosis), Cryptococcus
(cryptococcosis), Candida (candidiasis), Fusarium (fusariomycosis), Histoplasma, Sporothrix, der-
matitis caused by Malassezia, and a diverse group of disease-causing zygomycetes (Higgins 2000).
Many of these groups are frequently recovered by eDNA analysis (e.g., Bass et al. 2007, Edgcomb
et al. 2002, Jebaraj et al. 2009), confirming their widespread presence in marine environments
(Figures 1–4).
Fungi generally have been shown to associate with coral and are suggested to represent
opportunistic pathogens of corals weakened by environmental stress (Le Campion-Alsumard
et al. 1995). For example, Aspergillus sydowii (shown in Figure 1) is closely related to terrestrial
Aspergillus species and has been identified as a pathogen of sea fan corals (Alker et al. 2001, Shinn
et al. 2000). Such corals are known to release defensive chemicals during infection by endoliths
such as A. sydowii (Alker et al. 2001).
Cawthorn (2011) identified the ascomycete fungus Fusarium as a disease-causing agent in
American lobsters (Fusarium is also represented in multiple marine eDNA analyses; see Figure 1),
noting its opportunistic nature and propensity to invade through damaged or dead tissues
(Cawthorn 2011). In a review of emerging diseases and their links to climate and anthropogenic
512 Richards et al.

factors, Harvell and coauthors (1999) found that three of 34 mass mortalities in marine environ-
ments are attributed to fungi.
Wang & Johnson (2009) pointed out that studies of fungal pathogens of marine algae mostly
focus on seaweed or macroalgae and also rely on cultivation-based methods, which can give a
false impression of natural diversity. This means that culture-independent molecular probing of
potentially infected tissue could reveal important new information about organismal interactions of
marine fungi. Several dozen ascomycete fungi are already known to be pathogens of marine algae
(Kohlmeyer & Kohlmeyer 1979), while chytrids (e.g., Chytridium polysiphoniae) are also known
to parasitize macroalgae (Küpper et al. 2006). Seaweeds have been shown to respond to fungal
attack, by the production of antifungal lobophorolide and tolytoxin by the brown alga Lobophora
variegata against the parasitic ascomycetes Lindra thalassiae and Dendryphiella salina (Kubanek et al.
2003). Many ascomycetes appear to be specific to red, green, or brown algae, although some are
generalists. Some cause galls on seaweeds, whereas others are involved in symbioses with seaweeds,
outside of which neither partner appears able to grow. For example, the ascomycete Mycophycias
ascophylli is involved in a three-way symbiosis with the phaeophyte Ascophyllum nodosum and its red
algal epiphyte Polysiphonia lanosa. The fungus interacts with both, possibly transferring nutrients
between them (Spooner & Roberts 2005).
Another relatively well-studied set of interactions is between sponges and fungi. A culture-
based study by Li & Wang (2009) revealed a large diversity of fungi associated with sponge species
(e.g., Mycosphearellales, Eurotialies, Dothideales, Hypocreales, Diapothales, Xylariales, Pleospo-
rales, Aphyllophorales, and Saccharomycetales). They were able to classify their isolates into three
categories: those found in all sponge species, those in more than one but not all, and specialists
found only in a single sponge species. Cladosporium, Penicillium, Aspergillus, and Eupenicillium were
recovered from the majority of sponges investigated, suggesting that fungi from these groups are
regularly associated with sponges. Again, these phylogenetic groups are consistently recovered
in marine eDNA analyses, and many of the lineages are closely related to nonmarine species
(Figure 1).
The isolation-independent denaturing gradient gel electrophoresis approach used by Gao et al.
(2008) also showed a high diversity of fungi associated with sponges, and demonstrated that fungal
communities differed between sponge species and between sponges and the surrounding seawater.
They found a high incidence and microdiversity of Malassezia lineages. Malassezia are ubiquitous
lipophilic yeasts and are found in soils, sediments, and deep-sea habitats and on terrestrial metazoa.
Gao et al. (2008) suggested that their analysis of sponges had detected the highest diversity to date
of Malassezia lineages from a single host. It is currently unclear whether the diversity of sponge-
associated fungi represents symbionts and/or parasites.
Our phylogenetic analysis also demonstrated a large number of Malassezia-like sequences re-
covered from a number of marine environments, including deep-sea water column and sediment
samples. These sequences formed a large clade displaying complex microvariation, suggesting a
radiation of marine Malassezia lineages and suggesting that this represents an important and di-
verse group of fungi in marine environments (Figure 3; also see Bass et al. 2007, Edgcomb et al.
2011, Jebaraj et al. 2009, López-Garcia et al. 2007).
Fungi as Saprotrophic Agents in the Marine Environment

In terrestrial ecosystems, fungi perform critical roles as saprotrophs in detrital environments,
breaking down complex biopolymers and recycling nutrients. This process is important because it
underpins the wider ecosystem, but it is much less clear which organisms perform equivalent roles
in marine environments. Marine yeasts are generally associated with nutrient concentrations [e.g.,

pollution, plankton blooms, and macroalgae (Kohlmeyer & Kohlmeyer 1979)], suggesting that
fungi are also important saprotrophs in marine environments. Furthermore, saprotrophy and life in
detritus are often associated with anoxic or partially anoxic conditions, and fungi have been shown
to possess a range of cellular and genomic adaptations to anoxic environments (discussed briefly
above). Therefore, although marine fungal diversity appears to be limited compared to terrestrial
environments (Kis-Papo 2005, Massana & Pedrós-Alió 2008, Richards & Bass 2005), fungi may
still play a critical role in detritus processing in marine ecosystems (Mann 1988, Raghukumar
2004), thus providing essential nutrients to the wider food web, such as the amino acids lysine
and methionine, various vitamins, polyunsaturated fatty acids, and sterols (an important precursor
for the manufacture of cholesterols in marine animals) (Phillips 1984). These pathways, through
which organic matter re-enters the food web, are vital for the survival of detritivorous animals,
which are unable to synthesize such compounds by themselves (Raghukumar 2004). For example,
Crustacea require the polyunsaturated fatty acid docosahexaenoic acid for growth (Harrison 1990),
which is provided to benthic food webs by detrital microbes (Raghukumar 2004), although it is
not clear whether true fungi mediate this process. In addition, fungi are thought to play a role in
the degradation of tucinin, an animal cellulose, which occurs in the test of tunicates (Kohlmeyer &
Kohlmeyer 1979). Indeed, phylogenetic analysis of environmental marine fungal sequences shows
a number of sequences branching closely to known saprotrophic fungi, including, for example,
Aspergillus (Figure 1), Fusarium (Figure 1), Coprinus (Figure 3), and Exidia (Figure 3).
One relatively strong research focus regarding marine fungal interactions has been the break-
down of calcareous structures by fungi, whether of living or dead organisms. Marine fungi are
known to degrade structures such as mollusc shells, burrow linings, and barnacle shells (Hyde
et al. 1998). For example, the ascomycete genera Arenariomyces, Corollospora, and Lindra degrade
foraminiferan tests and, with related genera including Remispora, attack other calcareous struc-
tures, for example, those of barnacles and shipworms (Spooner & Roberts 2005). Other endolithic
fungi (the general term for those living inside rocks and boring into calcareous substances) associ-
ated with many coral genera include basidiomycetes, many ascomycetes (e.g., A. sydowii ), and the
chytrids Dodgella priscus and Conchyliastrum enderi. Ostracoblabe and Lithopythium are often cited as
endolithic fungi (Golubic et al. 2005, Kendrick et al. 1982), although sequence data for all four of
these genera are currently unavailable in GenBank.
Fungi may also be important in degrading lignocellulose in marine environments, as they are
in terrestrial ones. They are able to tolerate reduced oxygen concentrations (discussed above)
and could potentially be the main degrader of these compounds in low-oxygen and anoxic ma-
rine sediments (Hyde et al. 1998) and of plant material in the oceans in general, especially in
mangrove ecosystems. Evidence for lignocellulose degradation by marine fungi (through the pro-
duction of endoglucanase enzymes, allowing growth on a carboxymethylcellulose substrate) has
been found in over 30 strains of phylogenetically diverse fungi isolated from marine environments
(Hyde et al. 1998). Mangrove leaves are rich in lignocellulosic structural polymers, soluble or-
ganics, phenolics, and tannins, making them highly resistant to degradation from many microbes.
However, fungi such as Pestalotiopsis and Cladosporium—common primary saprotrophs—are of-
ten recovered from mangrove leaves (Raghukumar 2004). The initial production of degrading
enzymes by fungal colonizers therefore likely plays a vital role in the breakdown of robust plant
materials.
Fungal strains isolated from leaves of Spartina alterniflora have proven capable of cellulose,
cellobiose, pectin, lipid, tannic acid, starch, and xylan degradation (Gessner 1980). Suetrong et al.
(2009) reported a diversity of intertidal marine Dothideomycetes mainly associated with mangrove
habitats in tropical/subtropical environments, noting that these species are well adapted to marine
environments with active discharge of ascospores and with a mucilaginous sheath. A representative
514 Richards et al.

diversity of species associated with mangrove plants is summarized in Figure 1, showing the wide
diversity of Dothideomycetes fungi associated with this marine environment (Suetrong et al.
2009). Dothideomycetes are frequently found as saprobes on decaying woody material in marine
Pseudofungi: group
environments. They are also parasites or symbionts of seagrasses or marine algae. Suetrong et al. of stramenopiles
(2009) therefore posed the question of whether this pattern represents a radiation, or multiple (heterokonts)
radiations, of Dothideomycetes specifically in mangrove marine habitats. unrelated to true fungi
but that feed and grow
using analogous
Filling the Fungal Niche in the Marine Ecosystems: Fungal Analogs methods to true fungi
Labyrinthulids and
The relative contribution of fungi to disease load and saprotrophic operations within marine
thraustochytrids:
ecosystems has been obscured by the high incidence of and taxonomic confusion caused by organ- stramenopile
isms that have fungal-like characteristics but are not true fungi (Cavalier-Smith 1987, Cavalier- (heterokont) protists
Smith & Chao 2006), i.e., those that do not branch within the fungal clade within the opisthokont with partially similar
supergroup (Adl et al. 2005, Simpson & Roger 2004). These fungi-like organisms also feed by morphologies and
analogous life cycles to

osmotrophy, in many cases forming polarized cells analogous to hyphae and rhizoids of true fungi,
chytrid fungi; produce
and grow on standard fungal growth media; hence they are often recovered during culture-based a network of filaments
analyses of environmental diversity and have historically been considered to be fungi. However, for feeding and
these fungal-like organisms have evolved this lifestyle independently and possess cellular char- movement
acteristics that separate them from true fungi. For example, true fungi generally form a robust Endomyxa: a
exoskeleton containing chitin during part of their life cycle however, the exoskeleton of the nondiverse group of filose
fungal analogs does not contain chitin (Bartnicki-Garcia 1987). amoebae, parasites,
and uncharacterized
Fungal analogs include oomycetes; Pirsonia; hyphochytriomycetes [collectively forming the
environmental
pseudofungi (Cavalier-Smith & Chao 2006)]; labyrinthulids and thraustochytrids (which are stra- sequences currently
menopiles); ichthyosporeans (e.g., Ichthyophonus), which also branch within the opisthokonts but included within the
separately from true fungi; and endomyxan Cercozoa [ascetosporeans (e.g., Paradinium and Hap- phylum Cercozoa
losporidium) and phytomyxids (plasmodiophorids and phagomyxids)]. All these groups have at some (supergroup Rhizaria),
comprising the sister
stage been classified as true fungi. Ascetosporea are well known for causing MSX disease of oysters
group to core
and other diseases of marine invertebrates; plasmodiophorids and their sister group phagomyxids Cercozoa
are important marine parasites of seagrasses, diatoms, and phaeophycean algae (e.g., Parodi et al.
Marine snow:
2010). organic detritus falling
Indeed, general surveys of marine fungi, both recent and from earlier in the twentieth century, from the upper water
report more fungal analogs than true fungi, thereby implying that true fungi are relatively unimpor- column layers, often
tant in marine habitats. For example, a comprehensive review of marine fungi from Woods Hole by comprising mucus
secreted by
Sparrow (1936) described mostly oomycetes, along with labyrinthulids, a thraustochytrid, Proto-
phytoplankton, most
myxa (an unclassified large branched amoeba, probably endomyxan), and the chytrid Chytridium— prolifically diatoms
the only true fungus listed (Figure 5; also see Sparrow 1936). A similarly comprehensive review (which form
of zoosporic fungal parasites of marine biota (Raghukumar 1988) described many pseudofungal aggregations in the
groups, including 11 genera of oomycetes infecting diatoms, green, red, and brown algae, and water column)
crustaceans, and also hyphochytriomycetes, including members of the genus Anisolpidium, para-
sitizing filamentous brown algae, and Hyphochytridium peniliae, causing mycosis of the planktonic
cladoceran Penilia avirostris.
Labyrinthulids and thraustochytrids (e.g., Schizochytrium, Ullkenia, Labyrinthula, Labyrinthu-
loides) are generally considered exclusively marine, although more recently labyrinthulids have been
found in nonmarine habitats (Douhan et al. 2009). These stramenopiles parasitize a wide range
of marine organisms, including diatoms, octopus, squid, seagrasses, shellfish, and fish. Thraus-
tochytrids have been detected on marine substrates such as salp fecal pellets and marine snow.
They reproduce through the production of motile zoospores, which swim to a food source and
colonize it (in an analogous manner to fungal chytrids). There is also evidence of saprotrophic

function among fungal analogs, and studies within mangrove swamps reveal the colonization of
fallen leaves by the oomycete Halophytophthora sp. of red mangrove plants within two hours of
submergence (Raghukumar 2004).
SUMMARY POINTS
1. Currently known fungal diversity in marine environments represents a tiny fraction of
that from terrestrial environments.
2. Fungal sequences detected in marine environments span a large diversity of forms and
lineages, including chytrids, filamentous hyphal forms, and multicellular forms.
3. Dikarya yeast forms appear to dominate the known diversity of marine fungi.
4. Marine environments, like freshwater environments, harbor a number of highly diver-

gent, deep-branching, and uncultured fungi, of which future study will greatly improve
our understanding of fungal cell diversity and evolution.
5. There appears to have been frequent marine-terrestrial and terrestrial-marine coloniza-
tion events during the radiation of the Fungi.

6. Fungi play diverse ecological roles in marine ecosystems and have frequently been asso-
ciated with parasitism of marine animals, plants, and algae.
7. Many ecological niches inhabited by true fungi in terrestrial and freshwater habitats are
occupied by diverse fungal analogs in marine habitats.
8. Fungal-specific molecular studies in marine environments are so far relatively few, yet
many reveal a marine fungal diversity that is significantly higher than other methods
suggest but much less diverse than terrestrial environments.
FUTURE ISSUES
1. What are the main drivers of detrital and saprotrophic processes in marine environments?
2. What is the diversity of true fungal parasites in marine environments, and what dangers
do they represent for marine conservation?
3. What is the true diversity of lower fungi in marine environments, and how do they relate
to terrestrial taxa and the fungal tree of life?
4. What are the evolutionary and physiological changes between truly marine fungi and
their close terrestrial relatives?
5. Do fungal analogs perform the majority of saprotrophic functions in marine environ-
ments?
6. Do fungal analogs actually exclude true fungi from colonizing apparently suitable marine
habitats?
7. By what evolutionary mechanisms have fungal analogs been able to occupy fungal-like
niches in marine habitats?
516 Richards et al.

DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
This work was primarily supported by the European Funding Agencies from the ERA-net program
BiodivERsA, under the BioMarKs project. The authors are supported by research grants from the
Biotechnology and Biological Sciences Research Council (BBSRC), Department for Environment,
Food and Rural Affairs (DEFRA), and Natural Environment Research Council (NERC).
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MA04-FrontMatter ARI 16 November 2011 9:36
Annual Review of
Marine Science
Volume 4, 2012 Contents
A Conversation with Karl K. Turekian

Karl K. Turekian and J. Kirk Cochran ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1
Climate Change Impacts on Marine Ecosystems

Scott C. Doney, Mary Ruckelshaus, J. Emmett Duffy, James P. Barry, Francis Chan,
Chad A. English, Heather M. Galindo, Jacqueline M. Grebmeier, Anne B. Hollowed,
Nancy Knowlton, Jeffrey Polovina, Nancy N. Rabalais, William J. Sydeman,

and Lynne D. Talley ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !11
The Physiology of Global Change: Linking Patterns to Mechanisms
George N. Somero ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !39
Shifting Patterns of Life in the Pacific Arctic and Sub-Arctic Seas
Jacqueline M. Grebmeier ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !63
Understanding Continental Margin Biodiversity: A New Imperative
Lisa A. Levin and Myriam Sibuet ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !79
Nutrient Ratios as a Tracer and Driver of Ocean Biogeochemistry
Curtis Deutsch and Thomas Weber ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 113
Progress in Understanding Harmful Algal Blooms: Paradigm Shifts
and New Technologies for Research, Monitoring, and Management
Donald M. Anderson, Allan D. Cembella, and Gustaaf M. Hallegraeff ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 143
Thin Phytoplankton Layers: Characteristics, Mechanisms,
and Consequences
William M. Durham and Roman Stocker ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 177
Jellyfish and Ctenophore Blooms Coincide with Human Proliferations
and Environmental Perturbations
Jennifer E. Purcell ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 209
Benthic Foraminiferal Biogeography: Controls on Global Distribution
Patterns in Deep-Water Settings
Andrew J. Gooday and Frans J. Jorissen ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 237
vi
MA04-FrontMatter ARI 16 November 2011 9:36
Plankton and Particle Size and Packaging: From Determining Optical

Properties to Driving the Biological Pump
L. Stemmann and E. Boss ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 263
Overturning in the North Atlantic
M. Susan Lozier ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 291
The Wind- and Wave-Driven Inner-Shelf Circulation
Steven J. Lentz and Melanie R. Fewings ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 317
Serpentinite Mud Volcanism: Observations, Processes,
and Implications
Patricia Fryer ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 345
Marine Microgels
Pedro Verdugo ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 375
The Fate of Terrestrial Organic Carbon in the Marine Environment

Neal E. Blair and Robert C. Aller ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 401
Marine Viruses: Truth or Dare

Mya Breitbart ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 425
The Rare Bacterial Biosphere
Carlos Pedrós-Alió ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 449
Marine Protistan Diversity
David A. Caron, Peter D. Countway, Adriane C. Jones, Diane Y. Kim,
and Astrid Schnetzer ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 467
Marine Fungi: Their Ecology and Molecular Diversity
Thomas A. Richards, Meredith D.M. Jones, Guy Leonard, and David Bass ! ! ! ! ! ! ! ! ! ! ! ! 495
Genomic Insights into Bacterial DMSP Transformations
Mary Ann Moran, Chris R. Reisch, Ronald P. Kiene, and William B. Whitman ! ! ! ! ! ! 523
Errata
An online log of corrections to Annual Review of Marine Science articles may be found at
http://marine.annualreviews.org/errata.shtml
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Marine Fungi: Their Ecology and Molecular Diversity

Article in Annual Review of Marine Science · January 2012

Meredith Jones Guy Leonard

Hyphochytrium catenoides Genome Project View project

Soil protist initiative View project

The user has requested enhancement of the downloaded file.

• Our comprehensive search

Annu. Rev. Mar. Sci. 2012. 4:495–522 Keywords

sediment, freshwater, or marine ecosystems.

496 Richards et al.

www.annualreviews.org • Marine Fungi 497

Molecular Sampling in Marine Environments

498 Richards et al.

Deep-Sea and Hydrothermal Vent Environments

www.annualreviews.org • Marine Fungi 499

Anoxic Marine Environments

500 Richards et al.

Isolate: FCAS123 [GQ120177.1] Fungal agent of coral

Blastomyces dermatitidis [M55624.1]

Simplicillium lamellicola [AB214656.1]

www.annualreviews.org • Marine Fungi 501

eDNA: S08H02 [AB468676.1]

eDNA: FAS_3 [GQ120127.1]

502 Richards et al.

eDNA: 10_1F_26b1ab1 [GU972474.1]

Malassezia furfur [AY083223.1]

eDNA: 10148 [GU972207.1] NMFB4

www.annualreviews.org • Marine Fungi 503

eDNA: UI11B06 [EU446299.1]

Figures 1, 2, and 5 >49% bootstrap support

504 Richards et al.

Rhopalomyces elegans [NG_017191.1]

Neocallimastix frontalis [EF014370.1]

eDNA: PA2009A3 [HQ191313.1]

www.annualreviews.org • Marine Fungi 505

PHYLOGENETIC SUMMARY OF THE SMALL SUBUNIT RIBOSOMAL

as robust a phylogeny as possible, allowing us to construct a comprehensive picture of the diversity

The Dikarya Majority on Land and at Sea

506 Richards et al.

Novel Marine Fungal Phylotypes and the Marine-Terrestrial Transition

www.annualreviews.org • Marine Fungi 507

terrestrial fungi (either cultured isolate or eDNA)

Percent difference among SSU sequences from

508 Richards et al.

evolutionary diversification of the Fungi has involved multiple marine-terrestrial transitions in

Cryptomycota: A New Basal Branch in the Fungi?

www.annualreviews.org • Marine Fungi 509

Edgcomb et al. (2011)

Le Calvez et al. (2009) Marine fungi identifed using

Jebaraj et al. (2009)

Lefèvre et al. (2008)

lake water Freshwater fungi identified

Berney et al. (2004)

Dikarya Lower fungi

510 Richards et al.

ECOLOGICAL ROLE OF FUNGI IN MARINE ENVIRONMENTS

www.annualreviews.org • Marine Fungi 511

forms in marine animals.

Fungi as Parasitic Agents in the Marine Environment

512 Richards et al.

Fungi as Saprotrophic Agents in the Marine Environment

www.annualreviews.org • Marine Fungi 513