Chapter 14

Molecular Identification of AnaerobicRumen Fungi

Martin Eckart, Katerina Fliegerova, Kerstin Hoffmann, and Kerstin Voigt

Abstract Anaerobic fungi are phylogenetically unique and form a separate group,

the Neocallimastigomycota, among the chitinous fungi. Until now six genera are

described within that phylum, namely the monocentric genera Neocallimastix,Caecomyces and Piromyces as well as the polycentric genera Anaeromyces,Cyllamyces and Orpinomyces. This chapter gives a brief survey of the fascinating

world of anaerobic rumen fungi, their phylogeny, and identification. The golden

standards of molecular identification as well as promising alternatives will be

discussed.

14.1 Introduction

The physiology of the microbial community is fundamental for understanding the

processes of anaerobic decomposition of plant material, and has an economic

relevance for mankind. The distribution of organisms within the rumen is essential

for our understanding of the biochemistry of cellulose degradation (Hungate 1966).

A major part of organisms within the rumen fluid encompasses bacteria and

flagellates, but fresh and undigested plant material is rapidly colonised by anaerobic

fungi. It is now generally known that the degradation of herbal carbohydrates by

rumen fungi accelerates the digestion by downsizing the plant tissue particles.

Those particles are subsequently more easily decomposed by bacteria and protozoa.

The effectiveness of digestion is an important contributor to the health of animals in

husbandry (Wulff 2001).

M. Eckart, K. Hoffmann, and K. Voigt

Institute of Microbiology, School of Biology and Pharmacy, University of Jena, Neugasse 25,

07743 Jena, Germany

e-mail: martin.eckart@uni-jena.de

K. Fliegerova

Department of Biological Basis of Food Quality and Safety, Institute of Animal Physiology and

Genetics, Czech Academy of Sciences, v.v.i., Vıdenska 1083, 14220 Prague 4, Czech Republic

Y. Gherbawy and K. Voigt (eds.), Molecular Identification of Fungi,DOI 10.1007/978-3-642-05042-8_14, # Springer-Verlag Berlin Heidelberg 2010

297

Because of the economic and scientific interest in this topic, it is not surprising

that the first description of “flagellated organisms” living within the rumen was

given at the beginning of the twentieth century. But, astonishingly it needed more

than 60 years to discover these organisms to be fungi living without any oxygen.

The anaerobic environment is mandatory for the ecosystem rumen. It determines the

mode of life of microorganisms residing there. Besides being well-known prokar-

yotes, anaerobic fungi are important producers of short-chain fatty acids, which are

an essential source of nutrition for herbivores. Such a unique occupation of a special

ecologic niche by a group of heterotrophic, hyphal, and chitin containing eukaryotes

inevitably raises the question about the relationships of these fungi. Today, this

group is well supported by morphological and molecular data and accepted as the

Neocallimastigales (Li et al. 1993). Although the final position within the kingdom

Fungi is still unclear, it turned out to be a monophyletic group, as a basal lineage

besides or within the phylum Chytridiomycota, and is now recognised as phylum

(James et al. 2006). While a flagellated phase through the life cycle of chytridio-

mycetes is a case sui generis proved for Chytridiales, Rhizophydiales, Spizellomy-

cetales, Blastocladiales, and Neocallimastigales, the rumen fungi are characterised

by another unique attribute inside the kingdom Fungi: they live in anaerobiosis.

Until now, only several species of gut fungi have been described, probably because

of the problematic cultivation and maintenance of these organisms and high mor-

phological variability depending on growth conditions. Extensive studies of a broad

range of ruminants and application of modern methods in molecular biology will

probably bring deeper insights in microbial communities and species relationships.

This survey gives a brief overview of the historical background and of modern

trends in species recognition of this interesting fungal group.

14.2 Historical Background and the Discoveryof Rumen Fungi

Decisive for the terminology of anaerobic gut fungi was a flagellated organism

observed within the rumen of herbivores by Liebetanz (1910). This organism was

named Callimastix frontalis (Braune 1913) because of its high morphological

similarity to Callimastix cyclopis (order Blastocladiales) (Weissenberg 1912), a

flagellated parasite of Cyclops. Braune first described the multi-flagellated zoo-

spores seen in Fig. 14.1, but did not recognise them as a stage of a fungal life cycle

and misclassified this organism as parasite. The given name C. frontalis led to a

number of mis-assignments of parasitic flagellates within this genus. Ultrastructural

examinations of C. frontalis by Vavra and Joyon (1966) resulted in the establish-

ment of the new genus Neocallimastix. But unfortunately, the authors did not

recognise this organism as a fungus and still considered it as zooflagellate. Eight

years later, Whisler et al. (1974) assumed that organisms of the genus Neocalli-mastix are actually motile spores of an alternate life cycle of Coelomomycespsorophorae – a blastocladialean fungus – and declared the herbivores as

298 M. Eckart et al.

alternative hosts along with mosquitoes. Orpin (1977) first suggested that these

anaerobic organisms living in the rumen actually might be fungi and his assumption

was based on the recognition of chitin in the cell walls and on the morphological

description of the thallus of different Neocallimastix species (Orpin 1974, 1975,

1976). Orpin’s findings were in contrast to the general belief of microbiologists

that no obligate anaerobic fungi can exist and therefore fungal colonies growing

in anoxic tubes were discarded as oxygen contaminations (van der Giezen

2002). However, none of these scientists provided a taxonomic definition for

Neocallimastix. It was Heath et al. (1983) who linked Neocallimastix to the

chytridiomycetes by setting up the new family Neocallimastigaceae within the

Fig. 14.1 First description of Callimastix as a flagellate parasite. Front view, side view, and

cleavage with aequitorial layer (Braune 1913)

14 Molecular Identification of Anaerobic Rumen Fungi 299

order Spizellomycetales (phylum Chytridiomycota). The lack of multiple morpho-

logical characters has always been and still is a handicap for identifying these

organisms within the gut fungi.

14.3 Traditional and Current Systematics

Their incapability of locomotion and their appearance resulted in the erroneous

classification of fungi as plants before the twentieth century. An own kingdom

Fungi was recommended only in 1969 by Whittaker (1969). The Chytridiomycetes,

besides the Oomycetes andHyphochytriomycetes, were the only group of flagellated

organisms that shared the class-characteristic cell-wall polymers (Bartnicki-Garcia

1970) and lysine synthetic pathway (Vogel 1964) of the Eumycota, comprising

Zygo-, Asco- and Basidiomycetes at that time. In the 1980s, taxonomy and phylo-

geny of Chytridiomycetes were based on the thallus development, discharge of

zoospores, the size, ultrastructural complexity, and organisation of zoospores, as

well as number and length of flagella. Furthermore, characteristics like mono- and

polycentric development as well as the release of zoospores via diffusion or via

papillae affected the taxonomy and phylogeny of these basal fungi (Barr 1978).

The anaerobic gut fungi, as a special group of the flagellated fungi, often

changed their taxonomic position within the Chytridiomycetes. Because of their

late discovery and the unusual physiological character, especially the obligate

anaerobiosis, the rumen fungi were placed into different taxonomic groups over

time, first into the subdivision Mastigomycotina (Ainsworth 1966), and later into

the division Mastigomycota (Alexopoulos and Mims 1979). Within the Mastigo-

mycotina the following zoosporic fungi were accepted: Chytridiomycetes, Hypho-

chytridiomycetes, Plasmodiophoromycetes, and Oomycetes. The basis for this

classification built the zoospore with one or two flagellae as an asexual propagative

spore. The class Chytridiomycetes traditionally contained the four orders Chytri-

diales, Harpochytriales, Blastocladiales, and Monoblepharidales. Some studies

mentioned the order Harpochytriales, now known as a synonym of the group

Chytridiales (Kirk et al. 2008). Development of molecular genetic methods such

as polymerase chain reaction (PCR), cloning, and automated sequencing enabled to

generate data for diverse analyses. Traditional phylogeny based on the short-handed

phenotypic markers such as morphology, physiology, and biochemistry is now

complemented by statistically supported evolutionary analyses, which allowed

re-evaluation and re-classification of the whole kingdom Fungi including also the

young taxonomic group covering anaerobic fungi. Molecular-biological analysis of

gut fungi performed by Li et al. (1993) resulted in the establishment of an

own order, the Neocallimastigales, with only one family: Neocallimastigaceae.

Recently, Hibbett et al. (2007) postulated a separate phylum for this group, the

Neocallimastigomycota, adapted from the paraphyletic origin of the chytridiomy-

cete fungi concluded by James et al. (2006). An informal supertree based on several

analyses showed a close relationship to the chytridiomycetes (Hibbett et al. 2007).

300 M. Eckart et al.

However, new phylogenetic approaches display the chytridiomycetes again as

monophyletic group (Ebersberger et al. 2010). Therefore, the separate phylum

Neocallimastigomycota seems to be redundant. A comparison of both classical

taxonomy, based on morphology and physiology, and modern systematic methods,

based on up-to-date molecular-genetic techniques, is shown in Table 14.1.

At present, the family Neocallimastigaceae comprises six genera1 (Adl et al.

2005): Anaeromyces (Breton et al. 1990), Caecomyces (Gold et al. 1988), Cylla-myces (Ozkose et al. 2001), Neocallimastix (Vavra and Joyon ex Heath 1983),

Orpinomyces (Barr et al. 1989), and Piromyces (Gold et al. 1988). An overview

of the six taxonomic groups within the family Neocallimastigaceae is shown in

Table 14.2.

14.4 Phylogeny

Traditional phylogenetic results supported by molecular-genetic data can redraw

evolutionary hypothesis and consequently the affinity of organisms to taxonomic

groups. Like phenotypic characterisations, molecular phylogenetics should never

be based just on one character. Comparisons or combinations of morphological and

genetic characters lead to stable and well supported evolutionary hypotheses and

with this strengths and weaknesses of genetic markers become obvious. A marker

of high diagnostic value has to be unique to a species or even to a strain and at the

Table 14.1 The systematics of the chytridiomycetes based on traditional and modern classifica-

tion schemes

Traditional system Modern system

domain Eukaryota domain Eukaryota

kingdom Fungi (Linnaeus 1753) Nees 1817 kingdom Fungi (Linnaeus 1753) Nees 1817

phylum Chytridiomycota von Arx 1967

class Chytridiomycetes (de Bary 1863)

Sparrow 1958

order Chytridiales Cohn 1879

order Spizellomycetales Barr 1980

order Blastocladiales Fitzpatrick 1930

order Monoblepharidales Sparrow 1942

phylum Chytridiomycota von Arx 1967

class Chytridiomycetes (de Bary 1863) Sparrow

1958

order Chytridiales Cohn 1879

order Spizellomycetales Barr 1980

order Rhizophydiales Letcher 2006

order Neocallimastigales Li et al. 1993

class Monoblepharidomycetes Powell 2007

order Monoblepharidales Sparrow 1942

phylum Neocallimastigomycota Powell 2007

class Neocallimastigomycetes Powell 2007

order Neocallimastigales Li et al. 1993

phylum Blastocladiomycota James et al. 2006

class Blastocladiomycetes James et al. 2006

order Blastocladiales Fitzpatrick 1930

1http://indexfungorum.org/Names/familyrecord.asp?strRecordID=81063

14 Molecular Identification of Anaerobic Rumen Fungi 301

same time ubiquitous for all taxa. The more various the set taxonomic groups is, the

more conserved the marker has to be. On the other hand, clustering on lower level,

starting with the family, requires more variable data to distinguish between species

or even strains (outlined in Fig. 14.2).

Clustering methods use differences between partitions of given data to rebuild

cladistic relationships. To get quality estimation such as bootstrap proportions (BTs

or BP), checking the robustness of a set of data (Felsenstein 1985) is required.

Highly conserved data lead to stable reconstructions in early branches caused

by low clade stability supports. An example is given in Fig. 14.2. There is no

possibility to distinguish between taxon2 and taxon3 based on identical sequence

data. This exemplary marker is not adequate for molecular diagnostics on a lower

taxonomic level such as genus or species. We demonstrate these problems with an

analysis based on real data in Fig. 14.3. The gene encoding actin is highly con-

served in eukaryotes. The coding sequence divergence between plant and non-plant

actin genes shows only 15% or less variability (Hightower and Meagher 1986).

Therefore, this marker demonstrates perfectly the relationship between anaerobic

and aerobic chytridiomycetes, with the zygomycetous order Mucorales as outgroup

(Fig. 14.3). Varieties of species level cannot be determined with actin data, as this

marker lacks molecular diagnostics possibilities.

Table 14.2 Survey of the species from the anaerobic chytrids

Genus Species Author

Neocallimastix frontalis (RA Braune) Vavra and Joyon 1966 ex IB Heath et al.

(1983)

hurleyensis Theodorou and Webb (1991)

joyonii Breton, Gaillard, Bernalier, Bonnemoy and Fonty (1988)

patriciarum Orpin and Munn (1986)

variabilis Ho and Barr (1993)

Anaeromyces elegans Ho (1993)

mucronatus Breton et al. (1990)

Caecomyces communis Gold et al. (1988)

equi Gold (1988)

sympodialis Chen, Tsai and Chien (2007)

Cyllamyces aberensis Ozkose et al. (2001)

Orpinomyces bovis Barr et al. (1989)

intercalaris Ho et al. (1994)

joyonii1 (Breton, Bernalier, Bonnemoy, Fonty, Gaillard and Gouet)

Li, Heath and Cheng (1990))

Piromyces citronii Gaillard, Breton, Dusser and Julliand (1995)

communis Gold, Heath, and Bauchop (1988)

dumbonicus Li (1990)

mae Li (1990)

minutus Ho (1993)

polycephalus Chen, Chien and Hseu (2002)

rhizinflatus Breton, Dusser, Gaillard, Guillot, Millet and Prensier (1991)

spiralis Ho (1993)1Basionym, current name: Neocallimastix joyonii Breton, Bernalier, Bonnemoy, Fonty, B. Gaillard

& Gouet 1989

302 M. Eckart et al.

Nevertheless, highly variable data could result in an unstable reconstruction of

early branches caused by “long-branch-attraction” (Bergsten 2005). Although high

variable data could help measure distances between the closest neighbours and

other taxa on lower taxonomic levels, the variability of the data could lead to false

positive congruence, such as analogy instead of homology. An example is shown in

Fig. 14.4. The high variability of the internal transcribed spacer (ITS) sequences of

chytridiomycetes allow to distinguish even between strains, but alternative ways to

cluster these data decrease the robustness of the data set. One problem is the

differentiation of homologous and paralogous markers. Homology is not a problem

if orthologous genes are involved, but paralogous genes can lead to misinterpreted

results, similar to the comparison of “apples and oranges”. One example would be

the eukaryotic translation elongation factor 1-a (EF-1a) with more than one copy

within the genomes of fungi (see fungal genomes published by the JGI at http://

genome.jgi-psf.org/). False positive results in phylogenetic analysis based on align-

ments of paralogous genes are not always obvious as shown in Fig. 14.5.

14.5 Predicted Impact of Molecular Markers on FutureIdentification and Phylogeny

Based on the theory of evolution, highly conserved genes of diverse taxonomic

groups could be amplified by the combination of PCR techniques and universal

oligonucleotides (primers). The most commonly used DNA region for molecular-

genetic phylogeny is the highly repetitive cluster of the nuclear ribosomal DNA

(rDNA). The nucleotide sequences of the nuclear small (SSU) and large (LSU)

Fig. 14.2 Schematic illustration of problems occurring during the application of single phyloge-

netic markers. The master sequence should be ATTGCTAAGCGA; the (recent) taxa show

modified sequences. Changes compared to the consensus sequences are colour-coded in red.

Occurring problems are obvious: a stable backbone with statistical support is only possible with

data that are not highly diverse. However, differentiation at higher branches requires variable

sequences. To combine these datasets, several approaches like supermatrix or supertree methods

can be applied

14 Molecular Identification of Anaerobic Rumen Fungi 303

Fig.14.3

PhylogenyoftheNeocallim

astigomycota

andother

chytridiomycetesbased

onaMaxim

um

Likelihoodanalysisofactinsequencesfrom

24taxa

withatotalof903aligned

characters(unpublished

sequences).(m

ethodical

inform

ations:GAMMA

þP-Invar

model

withRAxML7.0.4

GTR-CAT(rapid

hill-clim

bingbootstrappingmethod(Stamatakis

2006,2008)),10,000rapid

bootstrap

inferencesbefore

athoroughML

search;final

ML

Optimization

Likelihood:–4676.139464)

304 M. Eckart et al.

Fig.14.4

PhylogenyoftheNeocallim

astigomycotaandother

chytridiomycetesbased

onaMaxim

umLikelihoodanalysisofITSsequencesfrom

49taxawith

atotalof1,161

aligned

characters

(unpublished

sequences).(m

ethodical

inform

ations:

GAMMA

þP-Invar

model

with

RAxML

7.0.4

GTR-CAT,

(Stamatakis2006,2008),10,000rapid

bootstrap

inferencesbefore

athoroughMLsearch.Final

MLOptimizationLikelihood:–17766.020153)

14 Molecular Identification of Anaerobic Rumen Fungi 305

Fig.14.5

PhylogenyoftheNeocallim

astigomycotaandotherchytridiomycetesbased

onaMaxim

umLikelihoodanalysisoftefsequencesfrom21taxawitha

totalof1,377aligned

characters

(unpublished

sequences).Included

paralogouscopiesdisturb

thecorrectspeciesassignments.(m

ethodical

inform

ations:

GAMMA

þP-InvarmodelwithRAxML7.0.4GTR-CAT(Stamatakis2006,2008),10,000rapidbootstrap

inferencesbeforeathoroughMLsearch.F

inalML

OptimizationLikelihood:–5723.672908)

306 M. Eckart et al.

subunits are separated by the non-coding DNA sequences of the internal transcribed

spacer (ITS) 1 and 2 and the non-transcribed intergenic spacer (IGS). Lacking a

sufficient evolutionary pressure, the non-coding regions allow the separation of

organisms down to the levels of species and strains. Using the flanking conserved

sequences of 18S (SSU) and 28S (LSU) rDNA, these regions can easily be

amplified with universal primers. Unfortunately, the ITS regions are not single

copy regions. Although the ribosomal DNA cluster follows concerted evolution

(Arnheim 1983), the intra-specific variability among organisms cannot be denied.

Usage of this region as molecular barcode marker is therefore questionable, espe-

cially if there is no reliable and supporting approach for species identification based

on e.g. morphology (Nilsson et al. 2008). Moreover, the variability of the ITS

region is sometimes not high enough to separate at the level of species as shown

for the fungal genus Penicillium (Skouboe et al. 1996, 1999). This experience

enforced the search and establishment of alternative genetic markers like the intron-

containing protein coding genes actin (act), eukaryotic translation elongation factor

1-a (tef), or beta-tubulin (btub). A profound base for this approach requires reference

strains, which need to bemorphologically and genetically well characterised, and also

the subsequent completion of the published results and sequence submissions.

First efforts to identify the anaerobic gut fungi by molecular genetic methods

were done by Dore and Stahl (1991) and Bowman et al. (1992). Their approaches

relied on partial 18S rDNA sequences for including the anaerobic fungi into the

chytridiomycetes, but the authors did not separate the species within the genera

(Dore and Stahl 1991; Bowman et al. 1992).

Trying to clarify the phylogenetic relationships within the order Neocalli-

mastigales using sequence analysis (ITS1) combined with morphological features,

ultrastructures and mitotic characters have led to seperation of the order Neocalli-

mastigales (Li et al. 1993). Isozyme analyses or DNA hybridisation has also been

used with the aim to clarify identification of anaerobic gut fungi and to increase the

level of specificity (Ho et al. 1994).

A fast and easy method for the differentiation of polycentric anaerobic fungi is

available by (RFLP) analysis of ITS spacer and/or fragments of ribosomal large

subunit (28S rDNA) digested by proper endonucleases. However, the ribosomal

small subunit (18S rDNA) turned out to be too conservative to get a well resolved

DNA polymorphism, and therefor is not very suitable for this type of analysis

(Fliegerova et al. 2006).

Methods of molecular biology are very promising, but “old-fashioned” taxon-

omy is still substantiated despite many discrepancies. The classical approach of

Neocallimastigales identification is based on their morphological characters. Thal-

lus shape (filamentous or bulbous), zoosporangial development (monocentric or

polycentric), and number of flagella per zoospore (uni- or polyflagellated) are

decisive for genus differentiations, while the ultrastructure of the zoospore is

determinative for species. (Heath et al. 1983; Orpin and Munn 1986; Munn et al.

1988; Webb and Theodorou 1991). Unfortunately, characters observable by light

microscopy vary with culture conditions and are highly pleomorphic (Brookman

et al. 2000). Moreover, the cultures often fail to produce important structures

14 Molecular Identification of Anaerobic Rumen Fungi 307

(sporangia and zoospores) making identification even more problematic. Also the

differentiation of species using ultrastructural features of the zoospores is question-

able, because ultrastructure depends not only on the age of microorganisms but also

on the method and quality of their preparation (Ho and Barr 1995).

14.6 Molecular Identification and DNA Barcoding

One of the important characteristics of anaerobic fungi is their flagellated stage in life

cycle. However, flagellated zoospores, can be found also in other aquatic fungi, like

the Blastocladiomycota and the Chytridiomycota sensu stricto and also in some

protists, e.g., the stramenopiles and among those the oomycetes, which are derived

brown algae. The flagellae of these organisms caused the mis-applications of taxo-

nomic and phylogenetic assignments as it happened to Braune with Neocallimastix(1913). The elucidation of morphological characters is valuable and indispensable,

but has to be supported by techniques of molecular genetics because the pleomorphic

shape of fungi leads to complications in their identification. Therefore, molecular

information becomes more and more important as a primary source for species

recognition. Now, 90 years after the discovery of the anaerobic rumen fungi, molec-

ular phylogenetic studies confirmed their relationship to the kingdom Fungi (Forster

et al. 1990; Bowman et al. 1992). The choice of molecular genetic markers in the

kingdom Fungi, respectively the phylum Chytridiomycota, is clearly arranged.

Today, state of the art comprises seven markers for fungal phylogeny that provides

data over the complete spectrum of the kingdom: 18S rDNA, 28S rDNA, ITS1 and

ITS2 including the 5.8S rDNA, rpb1, rpb2, tef, and beta-tubulin. In special cases

like pathogenic species or organisms of industrial importance, some additional

markers exhibiting a higher specificity were developed. Such markers encompass

not only genes encoding calmodulin, Mcm7 (MS456), and Tsr1 (MS277) (Aguileta

et al. 2008; Schmitt et al. 2009) but also physiological properties such as toxins or

extrolite profiles, which are well established, for example, the ascomycetous genus

Penicillium (Samson and Frisvad 2004).

To find the most useful marker for “tagging” all forms of life is the aim of many

current projects involved in “DNA barcoding”. DNA barcoding is an approach to

identify any organism based on sequence analysis of selected genomic regions.

Access to these regions should be as universal as possible, comparable, reproduc-

ible, and relatively easy to accomplish. Barcoding is thought to serve not only the

identification or verification of known specimens but also to contribute in the

discovery of new, undescribed species. Although DNA barcoding already proved

to be a very useful tool for the discovery of cryptic species, which are by definition

not differentiable by morphological features (Hebert et al. 2004), barcoding is

nevertheless error-prone. Depending on the method used, DNA barcoding turned

out to be not always sufficient for species recognition (Brower 2006; DeSalle

et al. 2005; Whitworth et al. 2007). One of the major problems in all barcoding

308 M. Eckart et al.

approaches is still the question which molecular tool should be used, since every

further step in species identification is based on it.

In animal systems, the mitochondrial cox1 is widely applied (Hebert et al. 2003),

although its sufficiency is already questioned (Goetze 2003). With a slower evolu-

tionary rate of this cytochrome c oxidase, this marker is not applicable for flowering

plants (Kress et al. 2005).

One of the most discussed marker in fungal taxonomy and phylogenetics is still

the ITS regionwith all its aforementioned advantages and disadvantages (see Sect. 5).

Phylum CoSAscomycota 1,124,132

Basidiomycota 197,974environmental samples 41,955Glomeromycota 11,698Microsporidia 5,676unclassified fungi 5,107Fungi incertae sedis 5,092

Zoopagomycotina 32Entomophthoromycotina 439Kickxellomycotina 276Mucoromycotina 3,948unkown 397

Chytridiomycota 1,683Neocallimastigomycota 465Blastocladiomycota 134

a

b

Fig. 14.6 Schematic illustration and number of nucleotide sequences provided by the Interna-

tional Nucleotide Sequence Database Collaboration. (e.g., Genbank). (a) Percentages of the

number of fungal sequences provided in GenBank. The graph shows the total number of submitted

sequences within the kingdom Fungi. The subparts describe the single phyla based on the

taxonomy provided by the TaxBrowser at NCBI. The group Dikarya is represented by approxi-

mately 96% of all available sequences. The number of sequences that were generated of environ-

mental samples is higher than that of all other phyla with the exception of the Dikarya. The

Neocallimastigomycota represent with 465 sequences the second smallest group of fungal organ-

isms represented as nucleotide sequences within GenBank. (b) Survey of the nucleotide sequencesprovided by the International Nucleotide Sequence Database Collaboration (as of May 1st, 2009)

14 Molecular Identification of Anaerobic Rumen Fungi 309

Nevertheless, provided sequence data usable for species identification for anaerobic

gut fungi are restricted, e.g., only 163 “ITS” tagged sequences are assigned to the

order Neocallimastigales (465 nucleotide sequences in summary, compare Fig. 14.6),

with only 17 sequences assigned to full taxon names. Because of missing mitochon-

dria in anaerobic fungi (hydrogenosomes instead), mitochondrial based barcode

markers are out of question (Bullerwell and Lang 2005). The need for a complete bar-

coding database, as always demanded (Ekrem 2007), is obvious. But another major

drawback is the data deposited in such a barcode database. An adequate number

of well-defined reference specimens are a prerequisite for species identification

and especially for species discovery. Such references should encompass all possi-

ble variances within defined species boundaries, e.g., geographically based varia-

tions (DeSalle et al. 2005; Meyer and Paulay 2005).

Originally thought to be a fast, cheap, and easy-to-access method for the

assignment of “unknown” to “known” specimens, molecular barcoding should be

used with caution. On the one hand, supplementing a barcode marker with addi-

tional information about e.g., morphology, biogeography, or even more molecular

data will miss the aim of a single easy-to-use marker for species assignment. But on

the other hand, supplementing data is necessary as a specimen cannot be identified

or described with certainty by one molecular attribute (Brower 2006; Will et al.

2005). Storing new data in a database is always tied with responsibility of the

submitter. Open-access to such databases is necessary but at the same time prone to

errors and losing its value as shown by GenBank at the NCBI (Bridge et al. 2003).

14.7 Conclusion and Future Line of Research

According to the efforts of Aguileta et al. (2008) and Schmitt et al. (2009) more

alterantive barcoding markers need to be established and validated in order to get a

reliable identification which is in concordance with morphological and ultrastructural

characters. The increase of the complexity of research on anaerobic rumen fungi in

their composite ecosystems requires a common platform for strain and data shared

among the scientific community. It is necessary to gain a certain homogeneity and

common use of reference and type strains including reference sequences of barcode

markers and other characters suitable for a reliable identification of anaerobic rumen

fungi. This is a fundamental for cultivation-independent detection in the natural

ecosystems and habitats of anaerobic fungi as performed by Fliegerova et al. (2010).

Acknowledgments The authors express their gratitude to L. Jay Yanke (Agriculture and Agri-

Food Canada, Lethbridge Research Centre, Lethbridge, AB, Canada) for providing Neocallimastixfrontalis strain SR4. This project was a component of the institutional research plan (AV OZ 5045

0515) of the Institute of Animal Physiology and Genetics, Academy of Sciences of the CzechRepublic in Prague. The czech team was supported by The National Agency for AgricultureResearch (project no. QI92A286/2008), The german team was supported by the Deutsche For-schungsgemeinschaft (project no. Vo 772/7-1), which is part of a bilateral grant between the CzechScience Foundation and the Deutsche Forschungsgemeinschaft.

310 M. Eckart et al.

References

Adl SM, Simpson AGB, Farmer MA, Andersen RA, Anderson OR, Barta JR, Bowser SS,

Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE,

Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup O, Mozley-

Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MFJR (2005)

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

J Eukaryot Microbiol 52:399–451

Aguileta G, Marthey S, Chiapello H, Lebrun MH, Rodolphe F, Fournier E, Gendrault-Jacquemard A,

Giraud T (2008) Assessing the performance of single-copy genes for recovering robust phylo-

genies. Syst Biol 57:613–627

Ainsworth GC (1966) A general purpose classification of the fungi. Bibl System Mycol 4:1–4

Alexopoulos CJ, Mims CW (1979) Introductory Mycology 38:1–613

Arnheim N (1983) Concerted evolution of multigene families. In: Nei M, Koehn RK (eds)

Evolution of genes and proteins. Sinauer Associated, Sunderland, MA, pp 38–61

Barr DJS (1978) Taxonomy and phylogeny of chytrids. Biosystems 10:153–165

Barr DJS, Kudo H, Jakober KD, Cheng KJ (1989) Morphology and development of rumen fungi:

Neocallimastix sp., Piromyces communis, and Orpinomyces bovis gen.nov., sp.nov. Can J Bot

67:2815–2824

Bartnicki-Garcia S (1970) Cell wall composition and other biochemical markers in fungal

phylogeny. In: Harborne JB (ed) Phytochemical phylogeny. Academic Press, New York,

pp 81–103

Bergsten J (2005) A review of long-branch attraction. Cladistics 21(2):163–193

Bowman BH, Taylor JW, Brownlee AG, Lee J, Lu S, White TJ (1992) Molecular evolution of the

fungi: relationship of the basidiomycetes, ascomycetes, and chytridiomycetes. Mol Biol Evol

9:285–296

Braune R (1913) Untersuchungen uber die im Wiederkauermagen vorkommenden Protozoen.

Arch Protistenk 32:111–170

Breton A, Bernalier A, Dusser M, Fonty G, Gaillard-Martinie B, Guillot J (1990) Anaeromycesmucronatus nov. gen., nov. sp. A new strictly anaerobic rumen fungus with polycentric thallus.

FEMS Microbiol Lett 70:177–182

Bridge PD, Roberts PJ, Spooner BM, Panchal G (2003) On the unreliability of published DNA

sequences. New Phytol 160:43–48

Brookman JL, Mennim G, Trinci AP, Theodorou MK, Tuckwell DS (2000) Identification and

characterization of anaerobic gut fungi using molecular methodologies based on ribosomal

ITS1 and 18S rRNA. Microbiology 146:393–403

Brower AVZ (2006) Problems with DNA barcodes for species delimitation: ‘ten species’ of

Astraptes fulgerator reassessed (Lepidoptera: Hesperiidae). Syst Biodivers 4:127–132

Bullerwell CE, Lang BF (2005) Fungal evolution: the case of the vanishing mitochondrion. Curr

Opin Microbiol 8:362–369

DeSalle R, Egan MG, Siddall M (2005) The unholy trinity: taxonomy, species delimtiation and

DNA barcoding. Philos Trans R Soc Lond B Biol Sci 360:1905–1916

Dore J, Stahl DA (1991) Phylogeny of anaerobic rumen Chytridiomycetes inferred from small

subunit ribosomal RNA sequence comparisons. Can J Bot 69:1964–1971

Ebersberger I, Strauss S, Kupczok A, Kothe E, von Haeseler A, Gube M, Eckart M, Voigt K (2010)

A stable backbone for the fungi. in revision

Ekrem T, Willassen E, Stur E (2007) A comprehensive DNA sequence library is essential for

identification with DNA barcodes. Mol Phylogenet Evol 43:530–542

Felsenstein J (1985) Confidence limits of phylogenies: an approach using the bootstrap. Evolution

39:783–791

Fliegerova K, Mrazek J, Voigt K (2006) Differentiation of anaerobic polycentric fungi by rDNA

PCR-RFLP. Folia Microbiol (Praha) 51:273–277

14 Molecular Identification of Anaerobic Rumen Fungi 311

Fliegerova K, Mrazek J, Hoffmann K, Zabranska J, Voigt K (2010) Diversity of anaerobic fungi

within cow manure determined by ITS1 analysis. Folia Microbiol (Praha): in press

Forster H, Coffey MD, Elwood H, Sogin ML (1990) Sequence analysis of the small subunit

ribosomal RNAs of three zoosporic fungi and implications for fungal evolution. Mycologia

82:306–312

Goetze E (2003) Cryptic speciation on the high seas; global phylogenetics of the copepod family

Eucalanidae. Proc R Soc B 270:2321–2331

Gold JJ, Heath IB, Bauchop T (1988) Ultrastructural description of a new chytrid genus of caecum

anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae. BioSys-

tems 21:403–415

Heath IB, Bauchop T, Skipp RA (1983) Assignment of the rumen anaerobe Neocallimastixfrontalis to the Spizellomycetales (Chytridiomycetes) on the basis of its polyflagellate zoo-

spore ultrastructure. Can J Bot 61:295–307

Hebert PDN, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cytochrome c oxidase

subunit 1 divergences among closely related species. Proc R Soc B 270(Suppl):96–99

Hebert PD, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one: DNA

barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. ProcNatl Acad Sci USA 101:14812–14817

Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, Eriksson OE, Huhndorf S, James T,

Kirk PM, Lucking R, Lumbs HT, Lutzoni F, Matheny PB, McLaughlin DJ, Powell MJ,

Redhead S, Schoch CL, Spatafora JW, Stalpers JA, Vilgalys R, Aime MC, Aptroot A,

Bauer R, Begerow D, Benny GL, Castlebury LA, Crous PW, Dai YC, Gams W, Geiser DM,

Griffith GW, Gueidan C, Hawksworth DL, Hestmark G, Hosaka K, Humber RA, Hyde KD,

Ironside JE, Koljalg U, Kurtzman CP, Larsson KH, Lichtwardt R, Longcore J, Miadlikowska J,

Miller A, Moncalvo JM, Mozley-Standridge S, Oberwinkler F, Parmasto E, Reeb V,

Rogers JD, Roux C, Ryvarden L, Sampaio JP, Schussler A, Sugiyama J, Thorn RG, Tibell L,

Untereiner WA,Walker C, Wang Z,Weir A,Weiss M,White MM,Winka K, Yao YJ, Zhang N

(2007) A higher-level phylogenetic classification of the fungi. Mycol Res 111:509–547

Hightower RC, Meagher RB (1986) The molecular evolution of actin. Genetics 114:315–332

Ho Y, Barr D (1995) Classification of anaerobic gut fungi from herbivores with emphasis on

rumen fungi from malaysia. Mycologia 87:655–677

Ho YW, Khoo IY, Tan SG, Abdullah N, Jalaludin S, Kudo H (1994) Isozyme analysis of anaerobic

rumen fungi and their relationship to aerobic chytrids. Microbiology 140:1495–1504

Hungate RE (1966) The rumen and its microbes. Academic Press, New York

James TY, Letcher PM, Longcore JE, Mozley-Standridge SE, Porter D, Powell MJ, Griffith GW,

Vilgalys R (2006) A molecular phylogeny of the flagellated fungi (Chytridiomycota) and a

proposal for a new phylum (Blastocladiomycota). Mycologia 98:860–871

Kirk PM, Cannon PF, Minter DW, Stalpers JA (2008) Dictionary of the fungi, 10th edn. CABI

Publishing, Wallingford, UK

Kress WJ, Wurdack KJ, Zimmer EA, Weigt LA, Janzen DH (2005) Use of DNA barcodes to

identify flowering plants. Proc Natl Acad Sci USA 102:8369–8374

Li J, Heath IB, Packer L (1993) The phylogenetic relationships of the anaerobic chytridiomycetous

gut fungi (Neocallimasticaceae) and the Chytridiomycota II. Cladistic analysis of structural

data and description of Neocallimasticales ord. nov. Can J Bot 71:393–407

Liebetanz E (1910) Die parasitischen Protozoen des Wiederkauermagens. Arch Protistenk

19:19–90

Meyer CP, Paulay G (2005) DNA barcoding: error rates based on comprehensive sampling. PLoS

Biol 3:e422

Munn EA, Orpin CG, Greenwood CA (1988) The ultrastructure and possible relationships of four

obligate anaerobic chytridiomycete fungi from the rumen of sheep. Biosystems 22:67–81

Nilsson RH, Kristiansson E, Ryberg M, Hallenberg N, Larsson K (2008) Intraspecific ITS

variability in the kingdom fungi as expressed in the international sequence databases and its

implications for molecular species identification. Evol Bioinform 4:193–201

312 M. Eckart et al.

Orpin CG (1974) The rumen flagellate Callimastix frontalis: does sequestration occur? J Gen

Microbiol 84:395–398

Orpin CG (1975) Studies on the rumen flagellate Neocallimastix frontalis. J Gen Microbiol

91:249–262

Orpin CG (1976) Studies on the rumen flagellate Sphaeromonas communis. J Gen Microbiol.

94:270–280

Orpin CG (1977) Invasion of plant tissue in the rumen by the flagellate Neocallimastix frontalis.J Gen Microbiol 98:423–430

Orpin C, Munn E (1986) Neocallimastix patriciarum: a new member of the Neocallimasticaceae

inhabiting the sheep rumen. Trans Br Mycol Soc 86:178–181

Ozkose E, Thomas BJ, Davies DR, Griffith GW, Theodorou MK (2001) Cyllamyces aberensis gen.nov sp.nov., a new anaerobic gut fungus with branched sporangiophores isolated from cattle.

Can J Bot 79:666–673

Samson RA, Frisvad JC (eds) (2004) Penicillium subgenus Penicillium: new taxonomic schemes,

mycotoxins and other extrolites. Stud Mycol 49:1–251

Schmitt I, Crespo A, Divakar PK, Fankhauser JD, Herman-Sackett E, Kalb K, Nelsen MP,

Nelson NA, Rivas-Plata E, Shimp AD, Widhelm T, Lumbsch HT (2009) New primers for

promising single copy genes in fungal phylogenetics and systematics. Persoonia 23:35–40

Skouboe P, Boysen M, Pedersen LH, Frisvad JC, Rossen L (1996) Identification of Penicillium

species using the internal transcribed spacer (ITS) regions. In: Rossen L, Rubio V, DawsonMT,

Frisvad JC (eds) Fungal identification techniques. European Commission, Brussels, Belgium,

pp 160–164

Skouboe P, Frisvad JC, Taylor JW, Lauritsen D, Boysen M, Rossen L (1999) Phylogenetic

analysis of nucleotide sequences from the ITS region of terverticillate Penicillium species.

Mycol Res 103:873–881

Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with

thousands of taxa and mixed models. Bioinformatics 22(21):2688–2690

Stamatakis A, Hoover P, Rougemont J (2008) A rapid bootstrap algorithm for the RAxML web-

servers. Systematic Biology 75(5):758–771

van der Giezen M (2002) Strange fungi with even stranger insides. Mycologist 16:129–131

Vavra J, Joyon L (1966) Etude sur la morphologie, le cycle evolutif et la position systematique de

Callimastix cyclopis Weissenberg, 1912. Protistologica 2:5–16

Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implications. Am Nat

98:435

Webb J, Theodorou MK (1991) Neocallimastix hurleyensis sp.nov., an anaerobic fungus from the

ovine rumen. Can J Bot 69:1220–1224

Weissenberg S (1912) Sitzungsbericht der Gesellschaft naturfoschender Freunde zu Berlin 5:299

Whisler HC, Zebold SL, Shemanchuk JA (1974) Alternate host for mosquito parasite Coelomo-myces. Nature 251:715–716

Whittaker RH (1969) New concepts of kingdoms or organisms. Evolutionary relations are better

represented by new classifications than by the traditional two kingdoms. Science 163:150–160

Whitworth TL, Dawson RD, Magalon H, Baudry E (2007) DNA barcoding cannot reliably

identify species of the blowfly genus Protocalliphora (Diptera: Calliphoridae) Proc Bio Sci

274:1731–1739

Will KW, Mishler BD, Wheeler QD (2005) The perils of DNA barcoding and the need for

integrative taxonomy. Syst Biol 54:844–851

Wulff C (2001) Untersuchungen zum Thiamin- und Thiaminderivatgehalt im Pansensaft des

Rindes nach Verfutterung von mit Mucor racemosus Fresenius verpilztem Heu (in vitro),

Thesis, Tierarztliche Hochschule Hannover, Hannover

14 Molecular Identification of Anaerobic Rumen Fungi 313