Molecular Phylogenetics and Evolution 35 (2005) 21–34 www.elsevier.com/locate/ympev
Eccrinales (Trichomycetes) are not fungi, but a clade of protists at the early divergence of animals and fungi
Matías J. Cafaro¤
Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, KS 66045-7534, USA
Received 22 July 2003; revised 3 September 2004 Available online 25 January 2005
Abstract
The morphologically diverse orders Eccrinales and Amoebidiales have been considered members of the fungal class Trichomyce- tes (Zygomycota) for the last 50 years. These organisms either inhabit the gut or are ectocommensals on the exoskeleton of a wide range of arthropods—Crustacea, Insecta, and Diplopoda—in varied habitats. The taxonomy of both orders is based on a few micro- morphological characters. One species, Amoebidium parasiticum, has been axenically cultured and this has permitted several bio- chemical and phylogenetic analyses. As a consequence, the order Amoebidiales has been removed from the Trichomycetes and placed in the class Mesomycetozoea. An aYnity between Eccrinales and Amoebidiales was Wrst suggested when the class Trichomy- cetes was erected by Duboscq et al. [Arch. Zool. Exp. Gen. 86 (1948) 29]. Subsequently, molecular markers have been developed to study the relationship of these orders to other groups. Ribosomal gene (18S and 28S) sequence analyses generated by this study do not support a close association of these orders to the Trichomycetes or to other fungi. Rather, Eccrinales share a common ancestry with the Amoebidiales and belong to the protist class Mesomycetozoea, placed at the animal–fungi boundary. 2004 Elsevier Inc. All rights reserved.
Keywords: Mesomycetozoea; DRIPs; Bayesian analysis; Ichthyosporea; Trichomycetes
1. Introduction holdfast. Asexual reproduction is by sporangiospores, which form basipetally from the thallus apex, a feature The Eccrinales have been placed in the class Trichomy- found only in the kingdom Fungi. Sexual reproduction cetes (Zygomycota), which are fungal associates of various has not been observed, except possibly in one species, Arthropoda. The class has traditionally included four Enteropogon sexuale, where scalariform conjugation was orders: Amoebidiales, Asellariales, Eccrinales, and Harp- reported (Hibbits, 1978). The fact that the Eccrinales share ellales. This group of symbionts inhabits a wide range of a very specialized ecological niche, the arthropod gut, with hosts—Crustacea, Insecta, and Diplopoda—in varied a group of well-known fungi, Harpellales (Trichomycetes), habitats (marine, freshwater, and terrestrial). The order in combination with the paucity of distinctive morpholog- Eccrinales is characterized by unbranched, non-septate, ical characters, have been the key criteria used in their clas- multinucleate thalli, and sporangiospore production siWcation within the Trichomycetes. The Eccrinales is the (Figs. 1A–C and F). They live attached to the cuticle lining only order of Trichomycetes with marine representatives; of the digestive tract of their hosts by a secreted, basal out of 17 genera, seven have species that inhabit crusta- ceans that live submerged in seawater. The distribution of
¤ the eccrinids ranges from tropical forests (White et al., Fax: +1 785 864 5321. Present address: Department of Bacteriolo- 2000) to hydrothermal vents (Van Dover and Lichtwardt, gy, University of Wisconsin, 420 Henry Mall, Madison, WI 53706, USA. Fax: +1 608 262 9865. 1986). They inhabit arthropods in almost all parts of the E-mail addresses: [email protected], [email protected]. world in every habitat that is suitable for their hosts.
1055-7903/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.12.019 22 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34
Fig. 1. Alacrinella limnoriae (A) sporangiospores at the thallus apex. (B) Holdfast area, lobed base. Enteromyces callianassae (C) young thalli sharing a multiple holdfast system. Paramoebidium sp. (D) swarm of released amoebae. (E) Mature thalli attached to the hindgut on a mayXy (Ephemerop- tera). Astreptonema gammari (F) sporangiospore being released with one appendage noticeable (arrow). Scale bars A, B D 25 m, C–F D 50 m.
Eccrinales are the oldest described group of Tricho- The lack of a septal pore and associated plug and the mycetes. The Wrst species, Enterobryus elegans, was presence of dictyosomes have raised some questions described from the millipede Narceus americanus (Leidy, about the true aYnity of the Eccrinales with 1849). Duboscq et al. (1948) oYcially named the class the Trichomycetes (Moss, 1999), and with fungi in Trichomycetes (meaning “hair-like” fungi) in their general. monograph referring to the Amoebidiales and Eccrinales. Lieberkühn (1856) and Schenk (1858) described Because of our inability to culture members of the amoebae-producing organisms otherwise morphologi- Eccrinales, their classiWcation has received little atten- cally similar to the Eccrinales attached to the exoskele- tion. Whisler (1963) provided early hints of some odd ton of freshwater arthropods in Europe, which characteristics for the Eccrinales; he reported a lack of Cienkowski (1861) later named Amoebidium parasiticum. chitin, a characteristic feature of fungi, in his studies of Léger and Duboscq (1929) described a second amoebae- cell-wall composition. In more recent years, there have producing genus, Paramoebidium (Figs. 1D and E), been some reports on the ultrastructure of a few species which they found in the hindgut of Nemura variegata of the group (Manier, 1979; Manier and Grizel, 1972; (Plecoptera). Together with Amoebidium, they placed MayWeld and Lichtwardt, 1980; Moss, 1975, 1999; Paramoebidium in the order Amoebidiales within the Saikawa et al., 1997; Tuzet and Manier, 1967). class Trichomycetes. M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 23
Amoebidium was the Wrst trichomycete to be cultured centrifuge tubes containing 500 l CTAB buVer (Hillis et (Whisler, 1960), which permitted more comprehensive al., 1996) for subsequent DNA extraction. Storage at studies on its biology and its phylogenetic relationships ¡20 °C from several weeks to three years yielded DNA (Trotter and Whisler, 1965; Whisler, 1962, 1968; Whisler suitable for PCR ampliWcation. and Fuller, 1968). The production of amoebae is not oth- erwise present in the kingdom Fungi; thus this character 2.2. DNA extraction alone raised many questions about the position of the Amoebidiales (Lichtwardt, 1986). Other features were First, thalli were broken by several cycles of freezing described that further caused researchers to question this and thawing. Microcentrifuge tubes were dipped in liq- relationship. Trotter and Whisler (1965) reported that A. uid nitrogen and then thawed in a heat block at 65 °C; parasiticum, like members of the Eccrinales, lacked chi- this procedure was repeated at least three times. One vol- tin in its cell wall. Ultrastructural studies indicated the ume of chloroform was added, vortexed brieXy, and cen- presence of stacked dictyosomes (Whisler and Fuller, trifuged for 10 min at maximum speed. When samples 1968), which have not been found in fungi (Beckett et al., contained large amounts of debris, the chloroform 1974; Bracker et al., 1996). Serological aYnities (Sangar extraction was repeated. After recovering the superna- et al., 1972), rRNA weight comparisons (Porter and tant, DNA was precipitated using an equal volume of Smiley, 1979), immunological studies (Peterson and isopropanol overnight or for 48 h at ¡20 °C. After centri- Lichtwardt, 1987), 5S DNA sequencing (Walker, 1984), fugation at maximum speed for 15 min, the resulting pel- and isozyme studies (Grigg and Lichtwardt, 1996) have let was washed twice with 70% ethanol and dried in a shown little aYnity between Amoebidiales and cultur- speed vac at 65 °C. The DNA was resuspended in 35 l of able members of the Trichomycetes. Recent molecular TE/10 (10 mM Tris, 0.1 mM EDTA, pH 8.1) and aliquots W sequence data have proven very useful in establishing were diluted (1:50) in ddH2O for PCR ampli cation. the true aYnity of the Amoebidiales, now a member of the Protista (Benny and O’Donnell, 2000; Mendoza et 2.3. PCR ampliWcation al., 2002; Ustinova et al., 2000). Here, I present results from phylogenetic analyses based on molecular data Universal fungal primers from White et al. (1990), for (rDNA) that include unculturable taxa of Eccrinales and nuclear ribosomal genes, were the starting point for Amoebidiales. ampliWcation procedures. Portions of the ribosomal 18S gene were ampliWed using diVerent combinations of these primers (NS1–NS8). Several Eccrinales-speciWc 2. Materials and methods primers were designed using this fragmented informa- tion and the sequence of A. parasiticum as a reference 2.1. Specimen collection (GenBank AF274051). PCR ampliWcation of the 18S rRNA gene was then performed in two separate reac- The material for this study was obtained from many tions to obtain overlapping fragments, using combina- regions and habitats (Table 1). Marine arthropods were tions of universal and speciWc primers (NS1-ECT1R and collected from mud Xats and tide pools. Freshwater crus- ECT1-NS8) (ECT1 5Ј-GAGCGTGGGCGGAGTTCG taceans and dipterans were collected in streams and GGAC-3Ј, ECT1R 5Ј-GTCCCGAACTCCGCCCACG lakes, and terrestrial millipedes and isopods were sam- CTC-3Ј), respectively. AmpliWcation of the Wrst two pled in many places. Collections were made in the Weld variable domains (D1, D2) of the 28S molecule was per- following standard procedures (Lichtwardt et al., 2001; formed either using the primers, NL1 and NL4, from White et al., 2001). Specimens were usually kept on ice in O’Donnell (1996), or a forward eccrinid-speciWc primer coolers or shaded with minimal native vegetation or (Ecc28F 5Ј-CGGACGCYTRTKTGGAYGAYGGTG- substrate to maintain specimens alive until returned to 3Ј) in combination with NL4. Primers were tested on the laboratory. Under a dissecting microscope gut fungi DNA extracted from a culture of A. parasiticum (isolate were removed from the digestive tract, with as little host FRA-1-14). The ampliWcation reactions were standard- tissue as possible, by manipulation with Wne needles. ized for a total Wnal volume of 20 l. A 2£ master mix Thalli were slide-mounted for identiWcation. Preferably, containing 0.25 mM of each primer, 0.225 mM dNTPs, wet mounts of gut fungi were photographed live using a and a 10% solution of 10£ buVer (M190A, Promega, W compound microscope or after in ltration of the Madison, WI) was used with variable MgCl2 concentra- specimens with lactophenol-cotton blue (0.05% w/v). tion (1.5–3.5 mM). A typical reaction cocktail contained Coverslips were sealed by ringing them with clear 10 l of master mix, 2 l of 5£ HiSpec Additive (Bioline Wngernail polish, rendering the slides semi-permanent. USA, Randolph, MA) and 8 l of diluted DNA sample Slide-mounted specimens were kept as vouchers. (1:50). Cycle parameters for PCR were adjusted depend- Alternatively, once the fungi had been identiWed and ing on primer pairs, but mostly followed the protocol of photographed, selected samples were preserved in micro- Gottlieb and Lichtwardt (2001). 24 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 Accession Nos. 18S 28S
b and date and , Anomura, USA California, Marin, Bay, Tomales Marsh, Creek Walker 01 Jul MJC AY336696 , Isopoda, Argentina Cruz, Santa Deseado, Puerto 98 Dec MJC AY682845 AY336695 , Anomura, False Bay, San JuanIsland, Washington, USA MJC Mar 99 AY336705 , Decapoda False Bay, San JuanIsland, Washington, USA MJC Mar 99 AY336707 , Anomura, USACalifornia, Bay, Bodega Beach, Creek Salmon 01 Jul MJC AY336694 sp., Amphipoda PineNeedle Preserve, Minnesota, USA LCF00 Mar AY336709 sp., Amphipoda Nobska Point, BarnstableMassachusetts, Co., USA MJC Mar 98 AY336704 AY336697 sp, Isopoda sp, USA Massachusetts, Hole, Woods 98 MMW Mar AY336703 sp., Diplopoda sp., Diplopoda sp., Spain Barcelona, Munt, del Llorens St. Spain Tarragona, Mora, la de Punta 02 Aug MJC AY336698 02 Aug MJC AY336700 sp., Cladocera sp., Cladocera sp., California, USA Herault, France HCW 1959 RWL Jun68 AF274051 AY336691 AY336692 Daphnia Daphnia Emerita analoga Callianassa californiensis Upogibia pugettensis Exosphaeroma studeri HostEphemeropteraLimnoria Gammarus Glomeris LocationGlomeris USA Co., Kansas, Douglas Reserve, Biological Breidenthal Diplopoda MMW 98May AY336708 USA Kansas, Douglas, Reserve, Ecological KU Tract, Woodland Rice 99 MMW Aug AY336710 Collector Hemigrapsus nudus Orchestia + + + + a A1a* FRA-1-14* CA-11-C4 KS-79-W2 CA-12-C4b WA-1-C5 ARG-D1-C15 MA-8-W4 SPA-10-C2 SPA-11-C45 MN-3-W6 WA-1-C37 sp. KS-61-W6 sp. WA-3-C3Isopoda Sphaeromatidae, USA Washington, Island, Juan San Cove, Eagle 99 Mar MJC AY336706 sp. MA-5-C17 sp.sp. sp. SPA-10-C6 SC-3-C2 Diplopoda Iulidae, MA-11-C1 Diplopoda Diplopoda Spain Munt, Barcelona, del Llorens St. USA Carolina, South University, Clemson USA Massachusetts, Essex, Point, Peach’s MJC Aug 02 AY336711 98 Oct RWL 02 Feb MJC AY336701 AY336693 exilis exilis X X *, Culture from the University of Kansas Mycological Culture Collection; +, dissection and CTAB preservation by MMW. by preservation CTAB and dissection +, Collection; Culture Mycological Kansas of University the from Culture *, Lichtwardt. W. Robert RWL, White; M. MMW, Merlin Cafaro; J. Matías MJC, Ferrington; C. Leonard LCF, Whisler; C. Howard HCW, a b Amoebidium parasiticum Amoebidium parasiticum Paramoebidium Enterobryus oxidi Enterobryus Palavascia patagonica Astreptonema Eccrinidus Eccrinidus Enterobryus halophilus Enterobryus Enterobryus Enterobryus Enteromyces callianassae Enteropogon sexuale Alacrinella limnoriae Astreptonema gammari Taeniella carcini Taeniellopsis Amoebidiales Table 1 name) genus and order by (sorted work molecular for study this in collected taxa of List Species name Source Eccrinales M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 25
2.4. Cloning and sequencing (France and USA) and Wve representatives of the Eccri- nales plus 14 outgroup sequences: Animalia: Beroe ovata Following PCR ampliWcation, if a single band was (AY026369), Leucosolenia sp. (AY026372), and M. leidyi observed on a 1% agarose gel run in 1£ TAE buVer, (AF026373); Fungi: S. culisetae (AF031072), C. stellatus products were cleaned using QIAquick PCR puriWcation (AF031073), Furculomyces boomerangus (AF031074), K. kit (Qiagen, Valencia, CA). When multiple products alabastrina (AF031064), Linderina pennispora were present, the bands were separated by electrophore- (AF031063), Coemansia reversa (AF031067), Actinomu- sis in a low melt agarose (Fisher ScientiWc, Pittsburgh, cor elegans (AF157173), Saccharomyces cerevisiae PA) gel and puriWed using QIAquick Gel extraction kit (J01355), and T. matsutake (U62964); Protista: I. hoferi (Qiagen, Valencia, CA). All products in this study were (AY026370), M. brevicollis (AY026374). The two data- cloned using the pGEM-T Easy Vector System II clon- sets were analyzed independently. The alignments were ing kit (Promega, Madison, WI). Positive bacterial inspected for the presence of ambiguously aligned clones were grown in 3 ml of LB broth + ampicillin regions caused by gap insertions. An equally weighted (10 mg/ml) overnight and their plasmids were extracted parsimony analysis was conducted on the unambigu- with QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). ously aligned regions using PAUP* 4.0 (SwoVord, 1999) Sequencing reactions were performed using Thermo via heuristic searches (MULTREES in eVect, branches Sequenase Fluorescent labeled primer cycle sequencing collapsed if maximum branch length is zero, 1000 ran- kit (Amersham–Pharmacia Biotech, Piscataway, NJ). dom stepwise additions, TBR branch swapping and Primers (IR-labeled) for the cloning vector were used in using gaps as missing characters). In a second analysis, the reactions (T7 promoter and M13Rev) by using the ambiguously aligned regions were unequivocally simultaneous bi-directional sequencing (SBS) in a LI- coded to form a new set of characters replacing these COR 4200L IR2 DNA sequencer (LI-COR, Lincoln, regions in the phylogenetic analyses. Coding of ambigu- NE). ous regions and step matrices calculations were per- formed with the program INAASE 0.2c1 (Lutzoni et al., 2.5. Data analyses 2000). Unambiguous regions were also subjected to step matrices taking into consideration the frequency of each GenBank BLAST searches were performed for raw class of possible changes (Fernández et al., 1999; Miad- sequences to determine whether the data corresponded likowska et al., 2002) with help of the program STMatrix to the desired eccrinid sample or to another gut inhabi- 2.2 (Lutzoni and Zoller, Dept. of Biology, Duke Univer- tant, food source (i.e., algae, bacteria, dinoXagellates, sity). Maximum parsimony (MP) analyses were per- etc.) or host tissue. Contaminating DNA sequences were formed using PAUP* 4.0 via heuristic searches excluded from further analysis. Sequence fragments were (MULTREES in eVect, branches collapsed if maximum combined, edited, and aligned using Sequencher 4.2 branch length is zero, 1000 random stepwise additions, (Gene Codes, Ann Arbor, MI). The resulting alignment TBR branch swapping and using gaps as Wfth character data were edited by eye using MacClade 4.06 (Maddison state). Bootstrap (BP) support was calculated for inter- and Maddison, 2003). Outgroup sequences were down- nal branches after 1000 pseudoreplicates and one ran- loaded from GenBank. Two datasets were constructed dom stepwise addition per pseudoreplicate. with 18S and 28S sequences. The small subunit (18S) Both datasets (with ambiguous regions removed) dataset contains members of several kingdoms: Anima- were analyzed under Bayesian inference using Mr. Bayes lia: Beroe cucumis (D15068), Microciona prolifera 3.0b4 (Huelsenbeck and Ronquist, 2001), which calcu- (L10825), and Mnemiopsis leidyi (AF293700); Fungi: lates posterior probabilities using a Metropolis-coupled Chytriomyces hyalinus (M59758), Smittium culisetae Markov chain Monte Carlo (MCMCMC) analysis. All (AF007540), Capniomyces stellatus (AF007531), Kickx- of the analyses employed one cold chain and three incre- ella alabastrina (AF007537), Gigaspora gigantea mentally heated chains, where the heat of the ith chain is (Z14010), Tricholoma matsutake (U62538), and Xylaria B D 1/[1 + (i ¡ 1)T] and T D 0.2, when i D 1, B D 1 corre- carpophila (Z49785); Stramenopila: Achlya bisexualis sponding to the cold chain. The general time reversible (M32705); Protista: Monosiga brevicollis (AF100940), (GTR) model was selected under a gamma distribution Diaphanoeca grandis (L10824), Sphaerothecum destru- for the among-site rate variation. Starting trees for each dens (AY267345), Rhinosporidium seeberi (AF118851), chain were generated at random; burn-in was set to 2000 Dermocystidium salmonis (U21337), Sphaeroforma arc- trees after verifying convergence to stationarity of tica (Y16260), Anurofeca richardsi (AF070445), Ichthy- parameters , lnL, and TL. The run was set to two mil- ophonus hoferi (U25637), and 12 new complete sequences lion generations with sample frequency of trees every for the Eccrinales and two for the Amoebidiales (see 100 generations. The prior distribution for the substitu- Table 1 for GenBank accession numbers). The large sub- tion rates was set to a Xat model. unit (28S) dataset contains newly generated sequences To test hypotheses that competed with the traditional for two strains of A. parasiticum from diVerent locations view that the Eccrinales are Fungi, parametric boot- 26 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 strapping (Goldman et al., 2000; Huelsenbeck et al., one new sequence to Paramoebidium sp. (Amoebidiales). 1995) was performed for both datasets. Constrained The Wnal length of the alignment was 2220 characters, of trees were imposed on the datasets under MP analysis which 637 were excluded due to ambiguous alignment. and tree length was calculated. The diVerence in tree A. bisexualis (Stramenopila) was used to root 18S trees. length between constrained and unconstrained trees is The 28S alignment was 1224 characters in length, of the parameter to be tested statistically. A constrained which 785 were excluded due to ambiguous alignment. It model tree under the maximum likelihood criterion was is also worth mentioning here that the 28S gene built in PAUP* 4.0 after establishing the model of sequences were extremely diYcult to align because of the sequence evolution using ModelTest 3.06 (Posada and variability within the D1/D2 region. M. brevicollis (Pro- Crandall, 1998). This tree and the ML model were used tista) was used to root 28S trees. to simulate evolution of DNA characters using the pro- Bayesian analysis of the 18S dataset yielded a tree gram Mesquite 1.01 (Maddison and Maddison, 2004). that is almost fully resolved except for the relationships The likelihood ratio test implemented in ModelTest 3.06 between the Mesomycetozoea, the animals, and the cho- indicated the general time reversible model with a pro- anoXagellate protists, which nonetheless collectively portion of invariant sites and a gamma distribution for form a weakly supported monophyletic group (PP 0.55, the among-site rate variation (GTR + I + ) for the 18S Fig. 2) sister to the Fungi. This analysis supports the dataset and Tamura–Nei model with a proportion of hypothesis that the Eccrinales belong to the protist class invariant sites and a gamma distribution for the among- Mesomycetozoea (PP 0.95), more speciWcally to the site rate variation (TrN + I + ) for the 28S dataset. The order Ichthyophonida (PP 1.00). Paramoebidium sp. simulation was run for 600 data matrices. The program groups, as expected, with A. parasiticum, hence forming plots the distribution of the statistic and allows to com- a well-supported Amoebidiales clade (PP 1.00) closely pare the observed valued of the test statistic (tree length) related to the Eccrinales (PP 0.97). Within the Eccrinales, to the distribution of the statistic expected under the three well-supported notable clades are present: a taxo- model as determined by the simulations (for more nomically diverse core eccrinid clade forms a strongly details, see Maddison, 2004). supported monophyletic group (PP 1.00, clade a), two Enterobryus spp. form another group (PP 1.00, clade b), and basal to both is Eccrinidus Xexilis (PP 1.00, clade c). 3. Results The Harpellales (Trichomycetes) (PP 1.00) are nested within the Zygomycota (Fungi) sharing a common 3.1. PCR ampliWcation and sequencing ancestor with the Kickxellales (PP 0.95), as previously reported (O’Donnell et al., 1998). The products ampliWed for the 18S gene varied in size Equally weighted MP analysis of the 18S dataset ranging from 1950 to 2045 bp. For the 28S gene, all matrix consisted of 2220 characters, of which 637 were products were approximately 900 bp. At least two clones excluded, 986 were constant, and 204 were variable but per ampliWed product were sequenced. It was diYcult to parsimony uninformative. This analysis of 407 parsi- obtain PCR products for most of the isolates collected in mony informative characters yielded one most parsimo- the Weld. Few samples were successfully ampliWed, with nious tree 4286.46 steps long (CI D 0.570, RI D 0.605) only 23 products out of 97 samples giving positive (Fig. 3A). This tree is very similar to the one from Bayes- results. For the small subunit, 13 almost complete ian analysis, except for the Fungi, which fail to form a sequences were obtained while there were only seven for monophyletic group. According to these data, the Eccri- the large subunit. In some cases, only partial 18S gene nales belong to the class Mesomycetozoea, more speciW- sequences were obtained due to lack of ampliWcation of cally to the order Ichthyophonida (BP 99%). The three one half of the gene and were not included in the analy- reported clades within the Eccrinales were also recov- ses (GenBank AY336699, AY336702, AY336712, and ered, but clade b is not highly supported (BP 68%). AY336713). Amoebidiales form a well-supported clade (BP 100%) closely related to the Eccrinales. Again, the relationship 3.2. Phylogenetic analyses between the Mesomycetozoea, the animals and the cho- anoXagellates is not resolved as previously reported for The 18S sequences generated in this study BLASTed the 18S gene (Ragan et al., 2003). with A. parasiticum and other Mesomycetozoea. The The MP analysis of the 18S dataset including INA- close relationship of Eccrinales to this group was unex- ASE characters resulted in a data matrix of 2234 charac- pected. To verify the hypothesis that the Eccrinales are ters after adding 14 ambiguously aligned regions recoded closely related to the Amoebidiales, phylogenetic analy- using the INAASE program. Only 456 characters were ses of representatives of several phyla of eukaryotes were used in the analysis (637 excluded, 901 constant, and 240 performed. The 18S matrix includes 33 taxa; of these 12 parsimony uninformative), which yielded one most parsi- new complete sequences belong to the Eccrinales and monious tree 5226.5 steps long (CI D 0.557, RI D 0.598) M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 27
Fig. 2. Bayesian phylogenetic tree of 33 taxa from 18S dataset after two million generations of MCMCMC chains. Values on the nodes correspond to posterior probabilities (PP). ECC, Eccrinales; AMO, Amoebidiales; DER, Dermocystidia; ICH, Ichthyophonida; CHO, ChoanoXagellates; GL, Glo- males; KI, Kickxellales; HA, Harpellales; ASC, Ascomycota; BAS, Basidiomycota; ZYG, Zygomycota; CHY, Chytridiomycota; A, Animalia; F, Fungi; S, Stramenopila; and P, Protista.
(Fig. 3B). This tree is fully resolved with the Fungi form- lost throughout the tree. Although the posterior proba- ing a well-supported monophyletic group (BP 85%). The bilities are not comparable to the bootstrap values Eccrinales are not monophyletic, but the core eccrinid (Alfaro et al., 2003), all the analyses support the place- clade is well-supported (clade a, BP 100%). Amoebidiales ment of the Eccrinales within the Mesomycetozoea. fail to form a monophyletic group, though they are still Bayesian analysis of the 28S dataset produced a tree closely related to other members of the Ichthyophonida. (Fig. 4) that is in general concordance with the results Though it is a resolved tree, major relationship between obtained with the 18S gene, i.e., Eccrinales are part of the animals, protists, and fungi are not supported. The addi- class Mesomycetozoea (PP 1.00). Although the Eccri- tion of recoded regions with INAASE into the analysis nales appear as a monophyletic group there is low sup- increases the resolution of the tree, but general support is port (PP 0.53). This result might be due to poor taxon 28 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34
Fig. 3. (A) Phylogram derived from an equally weighted MP analysis with bootstrap values indicated at the nodes. (B) Phylogram derived from a MP analysis including recoded characters from INAASE program. For both trees, only MP BP > 50% are shown. ECC, Eccrinales; AMO, Amoebidiales; CHO, ChoanoXagellates; A, Animalia; F, Fungi; and P, Protista. sampling in both the ingroup and outgroup. The paucity steps long (CI D 0.562, RI D0.590) (Fig. 4). This tree has of sequences for the 28S gene for the Mesomycetozoea in the same topology as the tree from Bayesian analysis GenBank made it diYcult to add an adequate represen- except for the position of A. elegans. The Eccrinales form tation of the class. According to these data, Meso- a well-supported monophyletic group (BP 98%) within mycetozoea is more closely related to the fungi than to the Mesomycetozoea (BP 79%). The fungi appear as a the animals (PP 0.98) and A. parasiticum forms a mono- weakly supported monophyletic group (BP 57%) most phyletic group with I. hoferi (PP 0.81). Again, within the closely related to the Mesomycetozoea (BP 88%). fungi, the Harpellales and Kickxellales group together Tree length diVerences between constrained and (PP 0.98). unconstrained analyses placing the Eccrinales with the The MP analysis of the 28S data included seven fungi, rather than with the Mesomycetozoea, were sig- ambiguously aligned regions recoded using INAASE. Of niWcant for both datasets. DiVerences of 1042.28 and the 446 included characters 140 were constant and 62 var- 40.14 steps for 18S and 28S datasets, respectively, were iable, but parsimony uninformative. The analysis of 249 recorded. The null hypothesis that the Eccrinales form a characters yielded one most parsimonious tree 2522.96 monophyletic group with the fungi was rejected at M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 29
Fig. 3. (continued)
P < 0.01 in both datasets. Monophyly for Eccrinales + monophyletic group following the addition of Paramoe- Amoebidiales + Ichthyophonida with fungi was also bidium sp. Amoebidiales and Eccrinales were identiWed as rejected (P < 0.01) for the 18S data. closely related within the order Ichthyophonida. Duboscq et al. (1948) recognized this relationship when they erected the Trichomycetes and established the Eccrinides 4. Discussion for the Eccrinales and Amoebidiales. These authors based their grouping on rather unusual morphological charac- 4.1. Phylogenetic relationships ters that provided few clues concerning the identity of their relatives at that time. Cavalier-Smith (1998) placed The results of the present phylogenetic study strongly the Eccrinales and Amoebidiales together in the class support the aYliation of the Eccrinales with the Enteromycetes based on morphology, ecology, and ultra- class Mesomycetozoea. Monophyly of the group was structure (presence of dictyosomes). Lichtwardt (1986) maintained in almost all the analyses, though not well considered the Eccrinales to be the most derived order of supported in some cases. The Amoebidiales remained a the Trichomycetes, whereas Moss (1999) suggested that 30 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34
Fig. 4. Bayesian phylogenetic tree from 28S dataset after two million generations of MCMCMC chains. Values to the left and right of the slash cor- respond to posterior probabilities (PP) and parsimony bootstrap values (BP) derived from a separate analysis, respectively. Only maximum parsi- mony BP values >50% are shown; if support is lower, a dash is indicated. The asterisk indicates the only diVerence found in the MP tree (the relative position of Actinomucor elegans). ECC, Eccrinales; AMO, Amoebidiales; CHO, ChoanoXagellates; ASC, Ascomycota; BAS, Basidiomycota; ZYG, Zygomycota; KI, Kickxellales; HA, Harpellales; A, Animalia; F, Fungi; and P, Protista. there is no evidence, beyond similarity in habitat, to Eccrinales are form during reproduction, and their consider them part of this group. In the Eccrinales, the function within a coenocytic thallus, preventing loss of presence of dictyosomes, lack of chitin in the cell wall cytoplasmic content upon release of sporangiospores, is and the formation of septa without a plug support not equivalent to those in the other trichomycete orders. Moss’ hypothesis. The septum that forms between spo- Asellariales and Harpellales have thalli that are septate rangia is the only one present in the otherwise coeno- throughout, with a deWned septal pore structure that cytic thallus. The septum is initially perforated but allows cellular continuity across the hyphae. The sep- occluded with wall material deposited at maturity, as tum in Eccrinales is a complete barrier; no cellular con- described for Astreptonema gammari (Moss, 1999). The tinuity or migration of organelles occurs after its septa between sporangia do not appear to be homolo- formation. Simple organization of the thallus, sporan- gous to the septa in Harpellales and Asellariales giospores, and common association with arthropods are because of functional diVerences. These structures in the characters shared by the four traditional orders of M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 31
Trichomycetes, including Amoebidiales. The data pre- (Lichtwardt, 1986). The phylogenetic analyses do not sented here indicate that the Trichomycetes, as tradi- support this relationship (Figs. 2–4). tionally established, are polyphyletic. Eccrinales present a great range of host types and habitats. Several families of Diplopoda consistently have 4.2. Morphology, biology, and evolution eccrinids in their guts. Other terrestrial hosts include iso- pods and beetles. Crustaceans that bear Eccrinales The Eccrinales are a morphologically diverse group, inhabit terrestrial, freshwater, and marine environments in part owing to the wide range of its hosts (Diplopoda, from intertidal zones to deep oceans (Van Dover and Crustacea, and Insecta) and the varied habitats of these Lichtwardt, 1986). Members of the Amoebidiales, in hosts. The main characteristics of Eccrinales are that contrast, are limited to freshwater habitats and larval they are unbranched, unicellular, multinucleate thalli aquatic insects and small crustaceans. Results of the that produce sporangiospores by basipetal septation present study do not indicate any particular pattern from the thallus apex (Figs. 1A–C and F). The Amoebid- associated with host type. It is possible that the Eccri- iales morphology is less diverse, but possesses the basic nales radiated very early in their association with arthro- cylindrical shape like the Eccrinales. The two genera pods, thus resulting in a wide variety of hosts. Amoebidium and Paramoebidium have coenocytic, multi- Paleontological evidence supports an origin of arthro- nucleate thalli that produce amoebae (Fig. 1D), which pods in the Upper Proterozoic as suggested by Ediaca- typically encyst and produce cystospores. Paramoebid- ran rocks containing jointed-legged animals (Margulis ium diVers in its larger size and location in the host—it and Schwartz, 2001). Recent fossil discoveries and attaches to the hindgut lining—while Amoebidium spp. molecular clock data suggest that arthropod diversiWca- are attached to the exoskeleton. Another important tion began in the Precambrian (Brusca, 2000). An “eccri- intergeneric diVerence is that Amoebidium produces spo- nid-like” organism has been documented from siliciWed rangiospores; hence the whole thallus is considered a peat deposits collected in the Antarctica formation of sporangium. Fremouw from the Middle Triassic (White and Taylor, Members of the Eccrinales produce two types of spo- 1989). The authors compared the organism to Enter- rangiospores, a primary infestation type which is typi- obryus, pointing out the presence of holdfast, aseptate cally uninucleate, oval to ellipsoidal and thick-walled thalli, spores, and septal plugs. However, septal plugs are that can act as a resistant spore or dissemination unit, absent in extant eccrinids. This absence and the lack of and a secondary infestation type, which is multinucleate, an arthropod host associated with this fossil raise the elongate and thin-walled. Secondary infestation spores question about the true aYnity of this fossil organism. It are believed to germinate within the host gut thus serv- is more probable that the common ancestor of Amoe- ing to increase infestation levels within the same individ- bidiales and Eccrinales was present in superWcial—and ual. There is considerable variation in shape and size of probably facultative—associations with arthropods. these sporangiospores, ranging from oval to ellipsoidal Under this hypothesis, the Amoebidiales may be a very to allantoid. Most of the eccrinids live in hosts that are old group with members that attached to the exterior gregarious, exhibit parental care of the young, or live in exoskeleton of arthropods. This interpretation would lentic waters; all of these factors favor the possibility of view colonization of the gut as an independent event in ingestion of released spores by new hosts. their evolutionary history. Lichtwardt (1986) presented a In marine and lotic freshwater environments, sporan- scenario in which Amoebidium evolved by Wrst attaching giospores have evolved appendages that probably externally and promiscuously to a wide range of diVer- increase their ability to Xoat and remain near the host ent aquatic hosts because it does not appear to have spe- population that gave origin to the spore, increasing the cial nutritional requirements. It is presumed that chances of being ingested. Four genera of Eccrinales continual ingestion by some hosts of resistant spores produce appendaged spores (Arundinula, Astreptonema, from Amoebidium resulted in spore germination in the Palavascia, and Taeniella). The appendages are formed gut, and thus Paramoebidium -like organisms adapted to within the sporangium and only become apparent fol- gut habitation. Given this scenario, the eccrinids could lowing spore release. The appendages of A. gammari have been derived from this Paramoebidium-like ances- (Fig. 1F) are the only ones that have been studied in tor before losing the sporangiospore phase or by re- detail (Moss, 1975, 1979, 1999). They are extensions of acquiring it. Further studies and data are needed to an outer, mucilaginous, sporangiospore wall which is address these hypotheses. formed early in spore ontogeny by the extracellular deposition of material derived from dictyosome vesicles. 4.3. ClassiWcation and position of Amoebidiales and Appendages have not been reported for any species of Eccrinales within Mesomycetozoea Amoebidiales. The presence of appendages in the Eccri- nales and Harpellales (Trichomycetes) was used as a The class Mesomycetozoea was established by shared character to indicate common ancestry Mendoza et al. (2001) to accommodate a group of Wsh 32 M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 and shellWsh parasites, human and anuran pathogens, amoebae at higher pH (Okamoto et al., 1995). The sym- and A. parasiticum. The group was previously named biotic life style and basic morphology of Ichthyophonus, DRIPs, an acronym for Dermocystidium, Rhinosporid- Amoebidiales and Eccrinales suggest that they are ium, Ichthyophonus, and Psorospermium, by Ragan et al. closely related. This hypothesis is supported by the phy- (1996), who recognized its monophyly based on 18S logenies recovered in this study. Biochemical characters sequence data. The clade was positioned near the ani- might help clarify this relationship further; for example, mal–fungal divergence based on molecular data (Ragan the reported lack of chitin in the cell wall of Amoebidi- et al., 1996; Spanggaard et al., 1996). Cavalier-Smith ales and Eccrinales (Whisler, 1963) is apparently not the (1998) proposed the class Ichthyosporea and divided the case in Ichthyophonus (Spanggaard et al., 1995). This group into two orders, Ichthyophonida and Dermocysti- important character suggests that the Mesomycetozoea dia. Mendoza et al. (2001) emended the class by chang- may be more closely related to Fungi than to Metazoa. ing the name to Mesomycetozoea and recently, Recently, Ragan et al. (2003) concluded that Meso- Mendoza et al. (2002) revised the current classiWcation. mycetozoea are members of the clade containing ani- The group has a long history of misidentiWcations and mals, fungi, and choanoXagellates based on elongation unclassiWed descriptions for animal parasites and sapro- factor 1 sequences. Lang et al. (2002) sequenced the trophic microbes that comprise the class. The organisms entire mitochondrial genomes of representatives of the included in the Mesomycetozoea have a diverse array of choanoXagellates (M. brevicollis) and the Meso- shared characters that suggest a common origin, such as mycetozoea (A. parasiticum). The authors’ results Xat mitochondrial cristae, symbiotic habit, formation of showed that the choanoXagellates are a sister taxon to some kind of endospore, cyst or spherical resistant struc- the Metazoa and that the Mesomycetozoea is sister to ture, and unicellular thalli. However, the strongest sup- these taxa. They placed them all together in a new group port for the Mesomycetozoea as a monophyletic group named Holozoa. The relationship of the Meso- comes from molecular data (Medina et al., 2001; Ragan mycetozoea to animals and fungi is inconsistent in anal- et al., 2003, 1996). The present study increases that sup- yses incorporating one or few loci. A multiple nuclear port for the Mesomycetozoea by adding more members protein-coding gene analysis may be needed to address to the group and by showing the same monophyletic this deep–node relationship (Baldauf et al., 2000). clade obtained with sequence data from the 28S ribo- Currently, the Eccrinales are divided into three fami- somal gene (Fig. 4). The Eccrinales are placed as sister lies: Eccrinaceae, Palavasciaceae, and Parataeniellaceae. taxon of the Amoebidiales in the order Ichthyophonida. Target loci from members of the Parataeniellaceae failed This result will require a re-description of the class Mes- to amplify and were not included in this study. There- omycetozoea in the future to include these symbionts of fore, it is clear that a wider range of taxon sampling is various arthropods. both possible and needed to address the internal organi- The type of symbiotic relationship of the Eccrinales zation of the group. New or understudied groups of and Amoebidiales to their hosts has always been a sub- organisms, like the Eccrinales, might be found and ject of discussion (Lichtwardt, 1986; Moss, 1999). Tradi- added to the Mesomycetozoea in the future. Detailed tionally, as Trichomycetes, they have been regarded as comparisons of members of these groups are needed to commensalistic in the absence of evidence to indicate deWne their internal relationships. In the case of the that these organisms acted as parasites or mutualists. Eccrinales, it is necessary to gather sequences from more The lack of axenic cultures (except for the ectocommen- taxa and from other genes to establish if there is a pat- sal A. parasiticum) made it almost impossible to study tern in their relationships regarding host, habitat, and/or the relationship between arthropod host and symbiont. taxonomic arrangements. Few studies have been attempted to answer this question in the Eccrinales (Charmantier and Manier, 1981). Recently, Kimura et al. (2002) studied the relationship Acknowledgments between Enteromyces callianassae and the mud shrimp Nihonotrypaea harmandi in two populations one with I am indebted to Robert W. Lichtwardt for giving me and one without infestation. The authors concluded that the opportunity to study Trichomycetes under his super- a parasitic situation is unlikely but that the relationship vision. I thank C. HauXer, C. Martin, C. Currie, M. could be either commensalistic, when nutrients are abun- White, K. Peterson, T. James, L. Mendoza, and R. Licht- dant, or mutualistic, when nutrient supply drops below wardt for reading and commenting on the manuscript. an unknown threshold. Ernest Estevez supplied samples from Florida and Todd Ichthyophonus hoferi is the closest relatives of Amoe- Haney samples from South Carolina. Gratefully, I want bidiales and Eccrinales within the order Ichthyophonida to mention all the people who assisted me with Weld col- (Figs. 2 and 4). Like A. parasiticum, I. hoferi has the abil- lection all over the world. I had the opportunity to meet ity to develop hyphal forms. Interestingly, I. hoferi pro- with many biologists: J-F. Manier, S. Santamaria, L. duces hyphae at a low pH (Baker et al., 1999) and Guardia Valle, H. Whisler, J. K. Misra, C-Y. Chien, M.J. Cafaro / Molecular Phylogenetics and Evolution 35 (2005) 21–34 33
H. Sato, and A. Rizzo. National Science Foundation Hillis, D., Mable, B., Larson, A., Davis, S., Zimmer, A., 1996. Nucleic PEET award DEB-9521811 supported most of this acids IV: sequencing and cloning. In: Hillis, D., Moritz, C., Mable, study (R.W. Lichtwardt—PI), NSF PEET award DEB- B. (Eds.), Molecular Systematics. Sinauer Associates Inc., Publish- ers, Sunderland, Massachusetts, pp. 291–321. 9521649 (R.C. Brusca—PI). Other sources include: Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference Department of Ecology and Evolutionary Biology, Uni- of phylogeny. Bioinformatics 17, 754–755. versity of Kansas, Tinker Field Research Grant, Center Huelsenbeck, J.P., Hillis, D.M., Jones, R., 1995. Parametric bootstrap- for Latin American Studies, Asociación Latinoameri- ping in molecular phylogenetics: applications and performance. In: cana de Micología, NSF PEET Program, Deep Hypha Ferraris, J.D., Palumbi, S.R. (Eds.), Molecular zoology: Advances, Strategies, and Protocols. Symposium held during Annual Meeting (NSF RCN Program) and NSF Assembling the Fungal of the American Society of Zoologists, St. Louis, Missouri, USA, Tree of Life (AFTOL). Indirectly, providing space and January 5–8, 1995. Wiley-Liss, New York, pp. 19–45. other supplies, Friday Harbor Marine Laboratories, Kimura, H., Harada, K., Hara, K., Tamaki, A., 2002. Enzymatic WA, Bodega Bay Marine Laboratory, UC Davis, CA, approach to fungal association with arthropod guts: a case study Ifremer, Séte, France, Departament de Biologia Animal, for the crustacean host, Nihonotrypaea harmandi, and its foregut fungus, Enteromyces callianassae. Mar. Ecol. 23, 157–183. Biologia Vegetal i Ecologia, Universidad Autónoma de Lang, B.F., O’Kelly, C., Nerad, T., Gray, M.W., Burger, G., 2002. The Barcelona, Spain. closest unicellular relatives of animals. Curr. Biol. 12, 1773–1778. Léger, L., Duboscq, O., 1929. L’évolution des Paramoebidium, nouveau genre d’Eccrinides, parasite des larves aquatiques d’Insectes. C.R. Acad. Sci. Paris 189, 75–77. References Leidy, J., 1849. Enterobrus, a new genus of Confervaceae. Proc. Acad. Natl. Sci. Phila. 4, 225–233. Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstrap? A simu- Lichtwardt, R.W., 1986. The Trichomycetes: Fungal Associates of lation study comparing the performance of Bayesian Markov chain Arthropods. Springer-Verlag, New York pp. 343. Monte Carlo sampling and bootstrapping in assessing phylogenetic Lichtwardt, R.W., Cafaro, M.J., White, M.M., 2001. The Trichomyce- conWdence. Mol. Biol. Evol. 20, 255–266. tes: fungal associates of Arthropods. Revised Edition. Available Baker, G.C., Beebee, T.J.C., Ragan, M.A., 1999. Prototheca richardsi, a from: <(http://www.nhm.ku.edu/~fungi)>. pathogen of anuran larvae, is related to a clade of protistan para- Lieberkühn, N., 1856. Ueber parasitische Schläuche auf einigen Insec- sites near the animal–fungal divergence. Microbiology 145, 1777– tenlarven. Arch. Anat. Physl. Wiss. Med. 25, 494–495. 1784. Lutzoni, F., Wagner, P., Reeb, V., Zoller, S., 2000. Integrating ambigu- Baldauf, S.L., Roger, A.J., Wenk-Siefert, I., Doolittle, W.F., 2000. A ously aligned regions of DNA sequences in phylogenetic analyses kingdom-level phylogeny of eukaryotes based on combined protein without violating positional homology. Sys. Biol. 49, 628–651. data. Science 290, 972–977. Maddison, D.R., 2004. Testing monophyly of a group of beetles. Study Beckett, A., Heath, I.B., McLaughlin, D.J., 1974. An Atlas of Fungal 1. In: Mesquite: A Modular System for Evolutionary Analysis, Ver- Ultrastructure. Longman Group, London pp. 221. sion 1.01, Available from:
Moss, S.T., 1975. Septal structure in the Trichomycetes with special ref- hoferi, and fungi, choanoXagellates and the rosette agent. Mar. Biol. erence to Astreptomena gammari (Eccrinales). Trans. Br. Mycol. 126, 109–115. Soc. 65, 115–127. SwoVord, D.L., 1999. PAUP* 4.0. Phylogenetic Analysis Using Parsi- Moss, S.T., 1979. Commensalism of the Trichomycetes. In: Batra, L.R. mony (* and Other Methods). Version 4.0. Sinauer Associates, Sun- (Ed.), Insect–Fungus Symbiosis: Nutrition, Mutualism, and Com- derland, Massachusetts. mensalism. Allanheld, Osmun, Montclair, pp. 175–227. Trotter, M.J., Whisler, H.C., 1965. Chemical composition of the cell Moss, S.T., 1999. Astreptonema gammari: an eccrinid with appendaged wall of Amoebidium parasiticum. Can. J. Bot. 43, 869–876. spores. Kew Bull. 54, 637–650. Tuzet, O., Manier, J.-F., 1967. Enterobryus oxidi Lichtwardt, Trich- O’Donnell, K.O., 1996. Progress towards a phylogenetic classiWcation omycète Eccrinale parasite du Myriapode Diplopode Oxidus graci- of Fusarium. Sydowia 48, 57–70. lis (Koch) (cycle, ultrastructure). Protistologica 3, 413–421. O’Donnell, K., Cigelnick, E., Benny, G.L., 1998. Phylogenetic relation- Ustinova, I., Krienitz, L., Huss, V.A.R., 2000. Hyaloraphidium curva- ships among the Harpellales and Kickxellales. Mycologia 90, 624–639. tum is not a green alga, but a lower fungus; Amoebidium parasiti- Okamoto, N., Nakase, K., Suzuki, H., Nakai, Y., Fujii, K., Sano, T., cum is not a fungus, but a member of the DRIPS. Protist 151, 1995. Life history and morphology of Ichthyophonus hoferi in vitro. 253–262. Fish Pathol. 20, 273–285. Van Dover, C.L., Lichtwardt, R.W., 1986. A new trichomycete com- Peterson, S.W., Lichtwardt, R.W., 1987. Antigenic variation within and mensal with a galatheid squat lobster from deep-sea hydrothermal between populations of three genera of Harpellales (Trichomyce- vents. Biol. Bull. 171, 461–468. tes). Trans. Br. Mycol. Soc. 88, 189–197. Walker, W.F., 1984. 5S ribosomal RNA sequences from Zygomycotina Porter, D., Smiley, R., 1979. Ribosomal RNA molecular weights of and evolutionary implications. Syst. Appl. Microbiol. 5, 448–456. Trichomycetes and Zygomycetes. Exp. Mycol. 3, 188–193. Whisler, H.C., 1960. Pure culture of the Trichomycete, Amoebidium Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA parasiticum. Nature 186, 732–733. substitution. Bioinformatics 14, 817–818. Whisler, H.C., 1962. Culture and nutrition of Amoebidium parasiticum. Ragan, M.A., Murphy, C.A., Rand, T.G., 2003. Are Ichthyosporea ani- Am. J. Bot. 49, 193–199. mals or fungi? Bayesian phylogenetic analysis of elongation factor Whisler, H.C., 1963. Observations on some new and unusual enteroph- 1 of Ichthyophonus irregularis. Mol. Phyl. Evol. 29, 550–562. ilous Phycomycetes. Can. J. Bot. 41, 887–900. Ragan, M.A., Goggin, C.L., Cawthorn, R.J., Cerenius, L., Jamieson, Whisler, H.C., 1968. Developmental control of Amoebidium parasiti- A.V.C., Plourde, S.M., Rand, T.G., Söderhäll, K., Gutell, R.R., 1996. cum. Dev. Biol. 17, 562–570. A novel clade of protistan parasites near the animal–fungal diver- Whisler, H.C., Fuller, M.S., 1968. Preliminary observation on the hold- gence. Proc. Natl. Acad. Sci. USA 93, 11907–11912. fast of Amoebidium parasiticum. Mycologia 60, 1068–1079. Saikawa, M., Sugiura, K., Sato, H., 1997. Electron microscopy of two White Jr., J.F., Taylor, T.N., 1989. A trichomycete-like fossil from the trichomycetous fungi attached to the hindgut lining of pill bugs. Triassic of Antarctica. Mycologia 81, 643–646. Can. J. Bot. 75, 1479–1484. White, M.M., Cafaro, M.J., Gottlieb, A.M., 2001. Taxonomy and sys- Sangar, V.K., Lichtwardt, R.W., Kirsch, J.A.W., Lester, R.N., 1972. tematics of Trichomycetes-past, present and future. In: Misra, J.K., Immunological studies on the fungal genus Smittium (Trichomyce- Horn, B.W. (Eds.), Trichomycetes and Other Fungal Groups. Sci- tes). Mycologia 64, 342–358. ence Publishers, EnWeld, New Hampshire, USA, pp. 27–37. Schenk, A., 1858. Ueber parasitische Schläuche auf Crustaceen. In: White, M.M., Cafaro, M.J., Lichtwardt, R.W., 2000. Arthropod gut Physikalisch-Medizinische Gesellschaft, Vol. 8, Würzburg, pp. 252– fungi from Puerto Rico and summary of tropical Trichomycetes 259. worldwide. Caribb. J. Sci. 36, 210–220. Spanggaard, B., Huss, H.H., Brescianni, J., 1995. Morphology of Ich- White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. AmpliWcation and thyophonus hoferi assessed by light and scanning electron micros- direct sequencing of fungal ribosomal RNA genes for phylogenet- copy. J. Fish Dis. 18, 567–577. ics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), Spanggaard, B., Skouboe, P., Rossen, L., Taylor, J.W., 1996. Phyloge- PCR Protocols: A Guide to Methods and Applications. Academic netic relationships of the intercellular Wsh pathogen Ichthyophonus Press, New York, pp. 315–322.