Protist
Protist, Vol. 151, 253–262, October 2000 © Urban & Fischer Verlag
http://www.urbanfischer.de/journals/protist
ORIGINAL PAPER
Hyaloraphidium curvatum is not a Green Alga,
but a Lower Fungus; Amoebidium parasiticum
is not a Fungus, but a Member of the DRIPs
Iana Ustinovaa, Lothar Krienitzb, and Volker A. R. Hussa,1
a
b
Department of Botany, University of Erlangen-Nürnberg, D-91058 Erlangen, Germany
Institute of Freshwater Ecology and Inland Fisheries, D-16775 Neuglobsow, Germany
Submitted March 3, 2000; Accepted August 2, 2000
Monitoring Editor: Mitchell L. Sogin
The unicellular heterotrophic protist Hyaloraphidium is classified with a family of green algae, the
Ankistrodesmaceae. The only species that exists in pure culture and that is available for taxonomic
studies is H. curvatum. Comparative18S ribosomal RNA sequence analyses showed that H. curvatum
belongs to the fungi rather than to the algae. Within the fungi, H. curvatum preferentially clustered
with Chytridiomycetes. Unlike Chytridiomycetes, H. curvatum propagates by autosporulation, and
the presence of flagella has never been reported. Transmission electron microscopy indicated that
H. curvatum in some respects resembles Chytridiomycetes, but no elements of a flagellar apparatus
were detected. The habitus of H. curvatum is unlike that of other fungi except the trichomycete
Amoebidium parasiticum. The cell wall sugar composition of H. curvatum was unique, but to some
extent resembled that of A. parasiticum. However, H. curvatum and A. parasiticum are not closely related to each other according to 18S rRNA sequence data. Moreover, A. parasiticum clustered with
protistan animals, the Mesomycetozoa (DRIPs). Combined molecular, ultrastructural and chemical
data do not allow assignment of H. curvatum to any recognized clade of fungi. This suggests that
H. curvatum may represent an independent evolutionary lineage within the fungi.
Introduction
The lack of obvious morphological characters combined with an exclusively asexual life cycle can
cause considerable problems in the taxonomic differentiation and identification of unicellular organisms. Similarities in morphology and vegetative
propagation may be indicative of evolutionary relationships, but could also occur due to convergence,
especially when the same ecological niche is
shared. In contrast, molecular data usually circum1
Corresponding author;
fax 49-9131-8528751
e-mail [email protected]
vent such problems and provide an independent
method for drawing taxonomic conclusions. They
not only allow distinction between taxa, but also can
reveal the fundamental systematic position of dubious species. As a consequence, classifications
based on molecular data are sometimes not in
agreement with the traditional view. Examples are
not rare, where species previously assigned to fungi
turned out to be plants or protists and vice versa
(e. g. Herr et al. 1999; Huss and Sogin 1990; Ragan
et al. 1996).
The genus Hyaloraphidium Pascher et Korshikov
was first established in 1931 for some autosporine
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I. Ustinova et al.
and colourless green alga-like organisms (Korschikoff 1931). Based on similarities in morphology
and mode of reproduction, a close relationship to
the genus Ankistrodesmus Corda (Ankistrodesmaceae) was assumed. H. contortum Pasch. et
Korsh. with thin needle-like screwed cells similar to
Ankistrodesmus spiralis was described as the type
species, but H. curvatum Korsh., used in our study,
is the only species that has been isolated into pure
culture until now. It has thicker, shorter, and more or
less twisted cells compared to H. contortum, and
was isolated from a strain of the chlorophyte Hydrodictyon reticulatum (Pringsheim 1963).
The relationship of Hyaloraphidium to the family
Ankistrodesmaceae (syn. Selenastraceae) has been
the subject of many discussions. Hindák (1977) considered some species of Hyaloraphidium to be
colourless varieties of Monoraphidium KomárkováLegnerová (Ankistrodesmaceae), while Kiss (1984)
questioned its affiliation to any alga. One species, H.
moinae Korsh., has been proposed to be synonymous to Amoebidium parasiticum Cienk. (Amoebidiales), which is classified with the fungal class Trichomycetes (Hindák 1977; Komárek and Fott 1983).
Colourless algae of the genus Gloxidium Korsh., described as closely related to Hyaloraphidium and
Ankistrodesmus (Korschikoff 1931), were also proposed to belong to the Amoebidiales (Hindák 1977;
Pringsheim 1963).
Amoebidium parasiticum is mentioned several
times in context with Hyaloraphidium species. It resembles Hyaloraphidium in many features such as (i)
thallus morphology; (ii) sporangia with regularly arranged sporangiospores that look like autospores in
a mother cell of Hyaloraphidium. Moreover, in pure
culture A. parasiticum does not undergo an amoeboid stage in its life cycle, but reproduces only by
nonmotile sporangiospores (Whisler 1962). However, the systematic position of Amoebidium itself is
not clear. The genus Amoebidium with A. parasiticum as its type species was established by
Cienkowski in 1861 for the exocommensal epizoon
found on a variety of aquatic arthropods. It has a
colourless cigar-shaped thallus and a basal holdfast. Reproduction occurs either by nonmotile sporangiospores or by amoebae. The production of
amoebae within the sporangium was thought to be
a specific feature of this organism. Cienkowski
(1861) assigned A. parasiticum to the fungal order
Chytridiales, but others classified it with the Protozoa, usually with Sporozoa or Myxomycophyta (see
Whisler 1962). Later, Amoebidium was assigned to
the class Trichomycetes (Zygomycota) (Duboscq et
al. 1948). This class combines unique fungi that are
found in permanent association with various arthro-
pods, usually as endocommensals of the gut. Originally, the Trichomycetes consisted of four orders:
Amoebidiales, Eccrinales, Asellariales, and Harpellales (Duboscq et al. 1948, Manier 1955). Today,
Harpellales and Asellariales are proposed to belong
to the Zygomycetes, although only some members
of Harpellales produce zygospores (Lichtwardt
1986; O'Donnell et al. 1998). Combining the remaining orders, Amoebidiales and Eccrinales, in Trichomycetes is thought to be artificial (Lichtwardt
1986). This view is based on differences in morphology, ecology and development and is further supported by differences in their cell wall chemistry
(Lichtwardt 1986; Whisler 1963).
A. parasiticum was isolated into pure culture by
Whisler (1960, 1962). Its development, cell wall
chemistry, and ultrastructure were extensively studied (Trotter and Whisler 1965; Whisler1962; Whisler
1968; Whisler and Fuller 1968). Whisler (1963)
doubted the fungal origin of the Amoebidiales and
suggested their position to be “somewhere between
fungi and protozoa”. Later, based on 5S rRNA sequences, A. parasiticum was shown to be more
closely related to the protozoa than to the Zygomycota (Walker 1984). However, the data were not sufficient to allow more detailed phylogenetic conclusions.
Here we present results of phylogenetic analyses
of 18S rRNA gene sequences, electron microscopy,
and cell wall chemical analyses to discuss the relationships between Hyaloraphidium curvatum and
Ankistrodesmaceae, and between H. curvatum and
Amoebidium parasiticum.
Results
Phylogenetic Position of Hyaloraphidium
curvatum
The amplification of the 18S rRNA gene of H. curvatum resulted in a product of 1796 base pairs without
introns. Its sequence was determined and compared with the EMBL database. Unexpectedly, the
most similar sequences were found in fungi and not
in the green algae. The four highest similarities were
obtained with Spizellomyces acuminatus (Chytridiomycota; 92.1%), Basidiobolus ranarum (Zygomycota; 90.7%), the Lasioderma serricorne yeast-like
symbiont, (Ascomycota; 90.1%), and Rhizoctonia
solani (Basidiomycota; 90.1%). In order to elucidate
the phylogenetic position of H. curvatum, we constructed phylogenetic trees with the 18S rRNA gene
sequences of representatives of all major fungal
groups (Fig. 1A). In all analyses, H. curvatum was
Hyaloraphidium and Amoebidium Phylogeny
255
Figure 1. (A) Neighbor-joining tree inferred from 18S rRNA gene sequences of Hyaloraphidium curvatum, Amoebidium parasiticum, and 22 other eukaryotes. Numbers above and below the forks give the bootstrap support (100
replicates) for neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) analyses. Only bootstrap values above 50% are shown. Asterisks indicate that the topology obtained by the MP and ML analyses was
different, but statistically unsupported. (B) Maximum likelihood tree inferred from 18S rRNA sequences of A. parasiticum and members of the DRIPs (Mesomycetozoa). NJ, MP, and ML tree topologies are congruent. Numbers
above and below forks as in (A), but for 1000, 500, and 100 replicates in the NJ, MP, and ML analyses respectively.
256
I. Ustinova et al.
Hyaloraphidium and Amoebidium Phylogeny
preferentially grouped within a clade of fungi that included Chytridiomycetes and some Zygomycetes.
In spite of its tendency to group with Chytridiomycetes, the exact phylogenetic position of H. curvatum within the fungi could not be resolved, except
that a relationship with higher fungi, Ascomycetes
and Basidiomycetes, was excluded.
Transmission Electron Microscopy of
H. curvatum
The morphology and reproduction of H. curvatum
do not resemble that of any described chytridiomycete or zygomycete. Typical cells are more or
less curved with rounded ends and without any rhizoids or protrusions (Fig. 2A). “Autospores”, similar
to those of coccal green algae, are formed within the
mother cell and released by rupture of its wall (Fig.
2B). The number of spores per cell is 4-8, but sometimes is irregular. The ultrastructure of spores does
not differ from that of mature cells. Nuclei are chromatin-rich and often have a bulge filled with an
amorphous material associated with the nuclear envelope (Fig. 2A, 2C). This structure could be interpreted as a nucleolus or an intranuclear nucleus-associated organelle (NAO)-like structure of unknown
function. However, identification of this structure requires additional cytochemical analyses. Spores
contain only one nucleus, but mature cells with up to
four nuclei were observed (Fig. 2C). Fig. 2D shows a
nucleus in the metaphase of mitotic division. As
chromatin aligns along the metaphase plate, the
“nucleolus” or “NAO” is not visible any more, and
the spindle microtubules are seen to diverge from
pocket-like regions at the spindle poles of the intact
nuclear envelope. This kind of mitotic division resembles that shown in a chytridiomycete, Catenaria
anguillulae, except that centrioles are not present
near the “pockets” (Beckett et al. 1974). A ribosomal
core area around the nucleus was never observed in
mature cells or in autospores.
Unlike most fungi, H. curvatum possesses
stacked Golgi dictyosomes (Fig. 2E, 2F) which actively take part in the synthesis of new cell walls by
producing a large number of vesicles during sporo-
257
genesis. Mitochondria are elongated and sometimes even thread-like (Fig. 2C, 2G). Numerous
larger organelles, which are covered with only one
membrane and have a homogeneous content, may
be microbodies. They are sometimes associated
with lipid globules, mitochondria and cisternae of
the endoplasmic reticulum (Fig. 2H). To some extent, this complex resembles the microbody lipid
globule complex (MLC) of zoospores of chytridiomycetes (Barr 1990; Berger et al. 1998). The abundant vacuoles often include osmiophilic dense material or membranous structures. In some cells unusual structures were observed that are not enclosed by any membrane and have a regularly
striped or chequered pattern (Fig. 2G, 2I). These
structures resemble the rumposomes of chytridiomycetes in a face-on view (Barr 1990). However,
they are not aggregated with lipid globules, microbodies, or vacuoles, and a view that would be typical for a rumposome in cross-section was never
found. On the other hand, structures with such a
regular pattern are characteristic of phospholipids
which are often incorporated in so-called “dense
bodies”. Dense bodies are common in many fungi,
but they are surrounded by a single membrane
(Beckett et al. 1974). Sections through numerous
mature cells and spores, and even serial cross-sections through several cells gave no evidence that
centrioles, basal bodies, or flagella are present in H.
curvatum.
Cell Wall Sugar Composition of H. curvatum
The cell wall sugar composition may be an important feature at certain taxonomic levels for the systematics of fungi. In H. curvatum, the fibres of the
TFA-resistant, alkali-insoluble rigid cell wall fraction
consisted of equal amounts of galactose and mannose, while the TFA-hydrolyzable matrix was composed of three-quarters galactose and one-quarter
mannose. Glucose, glucosamine or chitin were not
found in fibre or matrix fractions. The crystallinity of
the cell wall was low, as it did not brighten in a dark
field under polarized light. This kind of cell wall is
very different from that of most other fungi. How-
Figure 2. Electron micrographs of Hyaloraphidium curvatum. (A) Typical cell appearance. The arrow shows the
bulge of the nucleus filled with an amorphous material, either nucleolus or NAO. (B) “Autospores” are released from
the mother cell. No ribosomal core area is formed around nuclei. (C) Cell with two nuclei. The filled bulge of one nucleus is indicated by the arrow. (D) Nucleus in the metaphase of mitotic division. The nucleus envelope is intact.
The microtubuli of the mitotic spindle are visible. (E–F) Dictyosomes with stacked Golgi cisternae. (G) Cross-section of the cell showing the thread-like mitochondria and a structure with “chequered” pattern (arrow). (H) MLC –
like aggregation. (I) “Chequered” structure which resembles a rumposome of Chytridiomycetes in face-on view. n –
nucleus; m – mitochondrium; mb – microbody; l – lipid globule, er – endoplasmatic reticulum. Bars indicate 1 µm
(A–C, G, H) or 0.1 µm (D–F, I).
258
I. Ustinova et al.
ever, galactan along with galactosamine are known
as main constituents in cell walls of Amoebidium
parasiticum (Trotter and Whisler 1965). For this and
other similarities between H. curvatum and A. parasiticum mentioned before, the phylogenetic relationship between the two organisms was determined.
Phylogenetic Position of Amoebidium
parasiticum
The 18S rRNA gene sequence of A. parasiticum is
1797 base pairs long and contains no introns. The
highest sequence identity (95.3%) was found with
the fish parasite Ichthyophonus hoferi which belongs to a group of protists that evolved near the animal-fungal divergence (DRIPs) (Ragan et al. 1996),
recently named Mesomycetozoa (Herr et al. 1999).
The phylogenetic tree in Fig. 1B clearly shows that
A. parasiticum is a member of the DRIPs and most
closely related to I. hoferi. This phylogeny is highly
supported with bootstrap values of 100%. The
DRIPs constitute a well-resolved clade which
groups with animals, and not with fungi (Fig. 1A). It is
split into two distinctive clusters. The first includes I.
hoferi and A. parasiticum along with Psorospermium
haeckelii, Anurofeca richardsi, and Sphaerosoma
arcticum. The second contains Dermocystidium,
Rhinosporidium seeberi, and the “rosette agent”.
In contrast to some similarities in morphology and
cell wall chemistry, no evidence was found for a
close genetic relationship of Hyaloraphidium curvatum and Amoebidium parasiticum.
Discussion
The earlier identification of Hyaloraphidium curvatum as a colorless green alga was based entirely on
its similarity to species of the green algal family
Ankistrodesmaceae in vegetative reproduction by
4–8 “autospores” and in cell morphology as observed under the light microscope. Our analyses of
18S rRNA gene sequences demonstrate that H. curvatum does not belong to the Ankistrodesmaceae,
but to lower fungi (Fig. 1A). However, the precise
systematic position of H. curvatum could not be resolved despite its preference to cluster with the
Chytridiomycetes. Within our taxon sampling, H.
curvatum constitutes the most basal branch within
Chytridiomycetes except for Blastocladiales, but the
statistical support for this topology is low. Blastocladiales, represented by Allomyces macrogynus and
Blastocladiella emersonii, group together independent of other Chytridiomycetes, and appear here to
represent the earliest lineage of fungal radiation in
concordance with an analysis based on combined
18S, 5.8S, and 28S rRNA data (Van der Auwera and
De Wachter 1996). Other Chytridiomycetes with
available 18S rRNA sequences belong to the order
Neocallimasticales and cluster with Neocallimastix
joyonii (not shown). The position of the flagella-lacking zygomycete Basidiobolus ranarum within the
Chytridiomycetes rather than the Zygomycetes has
previously been discussed by others (Jensen et al.
1998; Nagahama et al. 1995). In general, the phylogeny of lower fungi is poorly resolved and a monophyletic origin of Zygomycetes or Chytridiomycetes
is not statistically supported by ours or previous
analyses based on 18S rRNA data (Jensen et al.
1998; Nagahama et al. 1995).
The suggested relationship of H. curvatum with
the clade of Chytridiomycetes is rather surprising.
Traditionally, Chytridiomycetes are defined as fungi
that always have flagellate stages in their life cycle.
However, a flagellate stage has never been described for H. curvatum. As some Zygomycetes also
cluster with Chytridiomycetes, H. curvatum might
represent a transition form from flagellated to nonflagellated fungi. Alternatively, flagella might have
been lost during the evolution of H. curvatum, or
may be produced only at a distinctive stage of its life
cycle. In this case, the potential existence of flagella
should be recognised by the presence of basal bodies or centrioles. In contrast to Chytridiomycetes,
Zygomycetes and higher fungi do not possess centrioles but nucleus-associated organelles (NAOs).
Thus, the presence of either centrioles or NAOs
could help to resolve the systematic position of H.
curvatum at least at the class level.
The results of transmission electron microscopy
of H. curvatum do not elucidate its relationship
within fungi. On one hand, the presence of the
stacked Golgi apparatus (Fig. 2E, 2F) typical for
Chytridiomycetes but not for other fungi except Trichomycetes (Beckett et al. 1974; Weber 1993), and
a kind of microbody-lipid globule complex (MLC)
(Fig. 2H) supports the preferential clustering of H.
curvatum with Chytridiomycetes. Moreover, if the
“chequered” organelle (Fig. 2G, 2I) is a rumposome,
H. curvatum could be classified with either the
Chytridiales or the Monoblepharidales, as rumposomes are characteristic for these orders (Barr
1990; Weber 1993). On the other hand, thallus morphology and development of H. curvatum is different
from Chytridiomycetes. It does not possess a flagellar apparatus characteristic of Chytridiomycetes or
a ribosomal core area which is present in all chytridiomycetes except Spizellomycetales (Barr 1990;
Weber 1993). Moreover, H. curvatum is not a parasite (Korschikoff 1931; Pringsheim 1963), and its cell
Hyaloraphidium and Amoebidium Phylogeny
wall chemistry is different from both Chytridiomycetes and Zygomycetes, as it is composed of
equal amounts of galactose and mannose. The cell
wall of Chytridiomycetes mainly consists of chitin
and ß-glucan, while the major cell wall components
of Zygomycetes are chitosan and chitin (BartnickiGarcia 1968). However, galactose along with polygalactosamin as major cell wall constituents were
found in Amoebidium parasiticum (Trichomycetes)
(Trotter and Whisler 1965).
Where is the place of H. curvatum within fungi? A
summary of “Pros” and “Cons” for the classification
of H. curvatum with established groups of lower
fungi is given in Table 1. With the available data it is
impossible to assign H. curvatum to any of these
groups. It is obvious from molecular data, cell wall
chemistry and electron microscopy that H. curvatum
does not belong to Ascomycetes or Basidiomycetes. Neither could it be a zygomycete, as it has
a stacked Golgi apparatus and its cell wall does not
consist of chitin and chitosan.
On first view, H. curvatum most closely resembles
the “trichomycete” A. parasiticum. This resemblance is based on similarities in cell wall chemistry,
thallus morphology, and the observation that A. parasiticum restricts itself to a sporangiospore cycle in
pure culture (Whisler 1962), whereby unflagellated
spores are arranged in a sporangium in a manner
similar to that of H. curvatum. However, a relationship between H. curvatum and Amoebidiales was
refuted by the 18S rRNA sequence analyses (Fig.
259
1A) suggesting that these similarities occurred due
to convergence. No 18S rRNA sequences of the
second order of Trichomycetes, the Eccrinales, are
available so far. However, a relationship of H. curvatum with Eccrinales is unlikely on the basis of differences in their habits and morphology.
Despite obvious morphological differences, the
ultrastructure of H. curvatum in many aspects resembles that of Chytridiomycetes, in agreement
with the molecular phylogeny. It is commonly believed that Chytridiomycetes are the most primitive
fungal group and as such still bear flagella. If the
placement of H. curvatum within Chytridiomycetes
reflects a natural relationship than the complete loss
of flagella during the evolution of H. curvatum has to
be assumed. On the other hand, H. curvatum may
represent a link between Chytridiomycetes and Zygomycetes. However, as the cell wall composition of
H. curvatum is so different, it seems appropriate to
regard it as an independent lineage of fungal evolution. Better resolution of its position within fungi will
require further analyses of more molecular data. The
question of systematic assignment of other species
of Hyaloraphidium including the type species H.
contortum is still open.
The results of our 18S rRNA analyses clearly
show that A. parasiticum is unrelated not only to H.
curvatum but also to other fungi (Fig. 1A). Instead, it
belongs to a certain clade of protistan parasites of
fishes and crustaceans that was provisionally
named “DRIPs” after the initials of known members
Table 1. Arguments for and against assigning Hyaloraphidium curvatum to established classes of lower fungi.
Pro
Contra
Chytridiomycetes:
• Phylogenetic position
• Golgi apparatus with stacked cisternae
• Microbody-lipid globule complex
• Rumposome-like structure
Chytridiomycetes:
• Reproduction (via non-motile “autospores”) and
ecology are different from those of Chytridiomycetes
• Absence of flagella and flagellar apparatus
• Absence of ribosomal core area in spores
• Cell wall composition
Zygomycetes:
• Phylogenetic position
• Absence of flagella and flagellar apparatus
Zygomycetes:
• Thallus morphology (unicellular, fusiform, curved,
without rhizoids) and mode of propagation
• Golgi apparatus with stacked cisternae
• Cell wall composition
Trichomycetes:
• Golgi apparatus with stacked cisternae
• Thallus morphology and mode of propagation
(similar to Amoebidiales)
• Absence of flagella and flagellar apparatus
• Cell wall composition (similar to Amoebidiales)
Trichomycetes:
• Phylogenetically not related to Amoebidium
parasiticum; molecular data for Eccrinales are not
available
• Cell wall composition is different from Eccrinales
• Thallus morphology, mode of propagation and
ecology are different from Eccrinales
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I. Ustinova et al.
at that time: Dermocystidium, “rosette agent”,
Ichthyophonus, and Psorospermium (Ragan et al.
1996). Recently, the name Mesomycetozoa was
proposed (Herr et al. 1999), reflecting their relation
to both fungi and animals, as they form the most
basal branch within animals just after the point of
fungal-animal divergence (Ragan et al. 1996).
Among the DRIPs, Ichtyophonus hoferi shares the
highest 18S rRNA similarity with A. parasiticum (Fig.
1B). Some morphological and developmental features support this relation as well. Both I. hoferi and
A. parasiticum have amoeboid but no flagellate
stages in their life cycle, both possess NAOs, and
both do not contain chitin in their cell wall. However,
mitochondria of I. hoferi do not have flat but the presumptively more primitive tubulovesiculate cristae.
Although the shape of mitochondrial cristae is in
general a good indicator for the phylogenetic position, exceptions are known (see Ragan et al. 1996).
The tubulovesiculate form of cristae in I. hoferi has
been interpreted as a reversion from the flat form
typical for animals and fungi (Ragan et al. 1996). The
fact that Amoebidiales are closely related to I. hoferi
supports this hypothesis, as they possess flat mitochondrial cristae (Dang and Lichtwardt 1979;
Whisler and Fuller 1968). The number of genera assigned to the Mesomycetozoa continues to grow
(Baker et al. 1999; Herr et al. 1999; Joestensen et
al., unpublished). According to the phylogenetic tree
in Fig. 1B, Mesomycetozoa are split into two distinctive groups, one of which contains all the species
with an amoeboid life stage.
Although a relationship of A. parasiticum to protozoa rather than to fungi was suggested by earlier
studies (Walker 1984), its precise assignment to any
particular group of protists could not be achieved.
This is now possible with our molecular analyses.
Moreover, our data suggest complete revision of the
systematics of the whole order Amoebidiales on a
molecular basis.
Methods
Hyaloraphidium curvatum SAG 235-1 was obtained
from the Collection of Algae, Göttingen, Germany,
and Amoebidium parasiticum ATCC 32708, from the
American Type Culture Collection, Manassas, USA.
H. curvatum was cultured in the recommended
Polytoma medium (Schlösser 1994), and A. parasiticum in an ATCC Tryptone-Glucose medium. Cells
were grown at room temperature for 4–5 days with
either aeration or shaking to prevent sedimentation.
Approximately 2 g of cells were ground in a cell mill
or treated in a French Pressure Cell (Aminco) at 106
Pa. DNA was purified by a standard phenol-chloroform procedure and precipitated with ethanol. 18S
rRNA genes were amplified by PCR and directly sequenced either manually (H. curvatum) with the T7
Sequenase PCR Product Sequencing Kit (Amersham) or with the Big Dye Terminator Cycle sequencing kit (PE Applied Biosystems) in the ABI
Prism 310 Genetic Analyser of Perkin-Elmer (A. parasiticum). PCR and sequencing primers were the
same as described previously (Huss et al. 1999).
18S rRNA sequences of H. curvatum and A. parasiticum were deposited in GenBank under the accession numbers Y17504 and Y19155 respectively.
Sequence data were compared with sequences
in the EMBL database by the use of the FASTA3 algorithm (Pearson 1990). A subalignment of the most
similar sequences found and of representatives of
all major fungal groups was obtained from the RDP
database (http://www.cme.msu.edu/rdp/html/index.
html) (Maidak et al. 1999). Sequences not included
in the RDP alignment were added manually considering the conserved secondary structure (Huss and
Sogin 1990; Neefs and DeWachter 1990). The following reference sequences were used in the
phylogenetic analyses (GenBank accession numbers are given in parentheses): Tripedalia cystophora (L10829), Anemonia sulcata (X53498), Ichthyophonus hoferi (U25637), Anurofeca richardsi
(AF070445), Sphaerosoma arcticum (Y16260),
Psorospermium haeckelii (U33180), “rosette agent”
(choanoflagellate-like sp.) (L29455), Rhinosporidium
seeberi (AF118851), Dermocystidium salmonis
(U21337), Dermocystidium sp. (U21336), Blastocladiella emersonii (M54937), Allomyces macrogynus (U23936), Sporidiobolus johnsonii (L22261),
Ustilago maydis (X62396), Saccharomyces cerevisiae (Z75578), Aspergillus fumigatus (AB008401),
Lasioderma serricorne yeast-like symbiont (D49656),
Glomus intraradices (AF004681), Acaulospora spinosa (Z14004), Endogone pisiformis (X58724),
Mortie-rella polycephala (X89436), Chytridium confervae (M59758), Basidiobolus ranarum (D29946),
Neocallimastix joyonii (M62705), Spizellomyces
acuminatus (M59759); Chlorella vulgaris (X13688),
and Ankistrodesmus stipitatus (X56100). The 18S
rRNA gene sequences of the oomycete Achlya bisexualis (M32705) and of the slime mould Dictyostelium discoideum (X00134) were used as outgroups. Highly variable regions that could not be
aligned unambiguously for all sequences were excluded, resulting in a total of 1572 (fungi) and of
1647 positions (DRIPs) that were used for the analyses. Phylogenetic trees were inferred by the neighbor-joining (NJ), the maximum parsimony (MP), and
the maximum likelihood (ML) methods using the
Hyaloraphidium and Amoebidium Phylogeny
PHYLIP program package version 3.572c (Felsenstein 1995) for NJ and MP analyses of fungal 18S
rRNA sequences (Fig. 1A), and the PAUP program
package 4.0 b3a (Swofford 2000) for all other analyses. For NJ, distance matrices were corrected with
the two-parameter method of Kimura (1980), and the
distances subsequently converted into phylogenetic
trees using the neighbor-joining method of Saitou
and Nei (1987). The addition of sequences was jumbled, and a transition/transversion ratio of 2.0 was
selected. The same ratio was used also for MP analyses. The search was fully heuristic with random addition of taxa repeated five times. The robustness of
the obtained tree topologies was estimated by the
bootstrap method (Felsenstein 1985). 100 bootstrap
resamplings were performed for each method with
the PHYLIP program and 1000 resamplings for NJ
with PAUP. For MP analyses with PAUP, characters
were weighted with rescaled consistency index and
then used as input for a bootstrap analysis (500 replications). Search settings were the same as described above. Tree-bisection-reconnection (TBR)
was chosen as a branch-swapping algorithm. For
ML analyses, the Hasegawa-Kishino-Yano model of
evolution and a transition/transversion ratio of 2.0
were selected. Settings for the full heuristic search
were the same as for MP analyses. A bootstrap analysis was performed with 100 replications.
Preparation of H. curvatum for transmission electron microscopy was done as described by Gaffal
(1987) and by Krienitz et al. (1983). After removing
the culture medium with 50 mM collidine buffer, cells
were fixed in collidine-buffered 2% glutaraldehyde
containing 5 mM CaCl2 for 1 h at room temperature
and postfixed in 2% OsO4 and O.8% K3Fe3(CN)6 for
1 h at room temperature. Cells were then rinsed 3
times with H2O and treated for 2 h with 2% uranyl
acetate. After standard dehydration, embedding,
and sectioning, samples were stained with lead citrate. Observations were made under a Zeiss EM-10
electron microscope.
The rigid cell wall fraction of H. curvatum cells
was isolated and analysed as described by Takeda
(1991) and Krienitz et al. (1999). Cell walls of H. curvatum were first refined from soluble material,
treated with boiling ethanol and a boiling ether/acetone mixture (v/v), and then successively hydrolysed
with trifluoracetic (TFA), sulfuric, and hydrochloric
acid. The composition of neutral- and amino sugars
was determined by gas-liquid chromotography and
by high-voltage paper electrophoresis respectively.
Neutral sugars were analysed by TFA and sulfuric
acid hydrolyses, and amino sugars by HCl hydrolysis. Anisotropy of isolated cell walls was examined
under a polarizing microscope.
261
Acknowledgements
We thank K. P. Gaffal and G. Friedrichs for supervision and help in transmission electron microscopy,
and G. Steingräber for excellent technical assistance. H. Takeda (Hokkaido University School of
Medicine, Sapporo, Japan) kindly performed the cell
wall sugar composition analyses. We also thank E.
Schnepf, J. Longcore, B. F. Lang, and H. C. Whisler
for helpful discussions and advice. This work was
supported by the Deutsche Forschungsgemeinschaft Grant Hu 410/6 to V.A.R.H.
References
Baker GC, Beebee TJ, Ragan MA (1999) Prototheca
richardsi, a pathogen of anuran larvae, is related to a
clade of protistan parasites near the animal-fungal divergence. Microbiology UK 145: 1777–1784
Barr DJS (1990) Phylum Chytridiomycota. In Margulis
L, Corliss JO, Melkonian M, Chapman DJ (eds) Handbook of Protoctista. Jones and Bartlett Publ, Boston,
pp 454–466
Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu Rev Microbiol 22:
87–105
Beckett A, Heath IB, McLaughlin DJ (1974) An Atlas
of Fungal Ultrastructure. Longman, London
Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R, Ragan MA, Hyatt
AD, McDonald KR, Hines HB, Lips KR, Marantelli G,
Parkes H (1998) Chytridiomycosis causes amphibian
mortality associated with population declines in the rain
forests of Australia and Central America. Proc Natl
Acad Sci USA 95: 9031–9036
Cienkowski L (1861) Ueber parasitische Schläuche auf
Crustaceen. Botan Zeitung 19: 167–174
Dang S, Lichtwardt RW (1979) Fine structure of
Paramoebidium (Trichomycetes) and a new species
with viruslike particles. Am J Bot 66: 1093–1104
Duboscq O, Leger L, Tuzet O (1948) Contribution a la
connaissance des Eccrinides: les Trichomycetes. Arch
Zool Exp Gen 86: 29–144
Felsenstein J (1985) Confidence limits on phylogenies:
an approach using the bootstrap. Evolution 39: 783–791
Felsenstein J (1995) PHYLIP (Phylogeny Inference
Package) Version 3.572c. Department of Genetics, University of Washington, Seattle, USA
Gaffal KP (1987) Mitosis-specific oscillations of mitochondrial morphology in Chlamydomonas reinhardtii.
Endocyt Cell Res 4: 41–62
Herr RA, Ajello L, Taylor JW, Arseculeratne SN,
Mendoza L (1999) Phylogenetic analysis of Rhinosporidium seeberi's 18S small-subunit ribosomal DNA
262
I. Ustinova et al.
groups this pathogen among members of the protoctistan Mesomycetozoa clade. J Clin Microbiol 37:
2750–2754
Hindák F (1977) Studies on the chlorococcal algae
(Chlorophyceae).I. Biol Pr 23: 1–190
Huss VAR, Sogin ML (1990) Phylogenetic position of
some Chlorella species based upon complete smallsubunit ribosomal RNA sequences. J Mol Evol 31:
432–442
Huss VAR, Frank C, Hartmann EC, Hirmer M,
Kloboucek A, Seidel BM, Wenzeler P, Kessler E
(1999) Biochemical taxonomy and molecular phylogeny
of the genus Chlorella sensu lato (Chlorophyta). J Phycol 35: 587–598
Jensen AB, Gargas A, Eilenberg J, Rosendahl S
(1998) Relationships of the insect-pathogenic order Entomophthorales (Zygomycota, Fungi) based on phylogenetic analyses of nuclear small subunit ribosomal
DNA sequences (SSU rDNA). Fungal Genet Biol 24:
325–334
Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative
studies of nucleotide sequences. J Mol Evol 16: 111–120
Kiss KT (1984) Special problems in studying phytoplankton associations (occurrence and ecological evaluation of Hyaloraphidium contortum). Acta Bot Hung
30: 269–276
Neefs J-M, DeWachter R (1990) A proposal for the
secondary structure of a variable area of eukaryotic
small ribosomal subunit RNA involving the existence of
pseudoknot. Nucleic Acids Res 18: 5695–5704
O'Donnell K, Cigelnik E, Benny GL (1998) Phylogenetic relationships among the Harpellales and Kickxellales. Mycologia 90: 624–639
Pearson WR (1990) Rapid and sensitive sequence
comparison with FASTP and FASTA. Methods Enzymol
183: 63–98
Pringsheim EG (1963) Farblose Algen. G. Fischer-Verlag, Stuttgart
Ragan MA, Goggin CL, Cawthorn RJ, Cerenius L,
Jamieson AV, Plourde SM, Rand TG, Soderhall K,
Gutell RR (1996) A novel clade of protistan parasites
near the animal-fungal divergence. Proc Natl Acad Sci
USA 93: 11907–11912
Saitou N, Nei M (1987) The neighbor-joining method: a
new method for reconstructing phylogenetic trees. Mol
Biol Evol 4: 406–425
Schlösser UG (1994) SAG – Sammlung von Algenkulturen at the University of Göttingen. Catalogue of
strains 1994. Bot Acta 107: 113–186
Swofford DL (2000) PAUP*. Phylogenetic Analysis
Using Parsimony (*and other methods). Sinauer Associates, Sunderland, Massachusetts
Komárek J, Fott B (1983) Das Phytoplankton des
Suesswassers 7,1. Chlorophyceae (Gruenalgen). Ordnung: Chlorococcales. Schweizerbart, Stuttgart
Takeda H (1991) Sugar composition of the cell wall and
the taxonomy of Chlorella (Chlorophyceae). J Phycol
27: 224–232
Korschikoff AA (1931) Notizen über einige neue apochlorotische Algen. Arch Protistenkd 74: 249–258
Trotter MJ, Whisler HC (1965) Chemical composition
of the cell wall of Amoebidium parasiticum. Can J Bot
43: 869–876
Krienitz L, Klein G, Heynig H, Böhm H (1983) Morphologie und Ultrastruktur einiger Arten der Gattung
Monoraphidium (Chlorellales) I. Monoraphidium griffithii, M. neglectum und M. tortile. Arch Hydrobiol Suppl
Algol Stud 33: 401–417
Van der Auwera G, De Wachter R (1996) Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii, and implications for the evolution of
zoosporic fungi. J Mol Evol 43: 476–483
Krienitz L, Takeda H, Hepperle D (1999) Ultrastructure, cell wall composition, and phylogenetic position of
Pseudodictyosphaerium jurisii (Chlorococcales, Chlorophyta) including a comparison with other picoplanktonic green algae. Phycologia 38: 100–107
Walker WF (1984) 5S ribosomal RNA sequences from
Zygomycotina and evolutionary implications. System
Appl Microbiol 5: 448–456
Lichtwardt RW (1986) The Trichomycetes, Fungal Associates of Arthropods. Springer-Verlag, New York
Whisler HC (1960) Pure culture of the Trichomycete,
Amoebidium parasiticum. Nature 186: 732–733
Maidak BL, Cole JR, Parker CT Jr, Garrity GM, Larsen N, Li B, Lilburn TG, McCaughey MJ, Olsen GJ,
Overbeek R, Pramanik S, Schmidt TM, Tiedje JM,
Woese CR (1999) A new version of the RDP (Ribosomal
Database Project). Nucleic Acids Res 27: 171–173
Manier JF (1955) Classification et nomenclature des
Trichomycetes. Ann Sci Nat Zool 17: 395–397
Nagahama T, Sato H, Shimazu M., Sugiyama J (1995)
Phylogenetic divergence of the Entomophthoralean
fungi: evidence from nuclear 18S ribosomal RNA gene
sequence. Mycologia 87: 203–209
Weber H (1993) Allgemeine Mykologie. G. Fischer-Verlag, Jena, Stuttgart
Whisler HC (1962) Culture and nutrition of Amoebidium
parasiticum. Am J Bot 49: 193–199
Whisler HC (1963) Observations on some new and
unusual enterophilous Phycomycetes. Can J Bot 41:
887–900
Whisler HC (1968) Developmental control of Amoebidium parasiticum. Devel Biol 17: 562–570
Whisler HC, Fuller MS (1968) Preliminary observations
on the holdfast of Amoebidium parasiticum. Mycologia
60: 1068–1079