Eumycetozoans and molecular systematics

Eumycetozoans and molecular systematics F.W. Spiegel, S.B. Lee, and S.A. Rusk

Can. J. Bot. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/04/13 For personal use only.

Abstract: Eumycetozoans, the myxomycetes, protostelids, and dictyostelids, were first hypothesized to be a monophyletic group by L.S. Olive, who suggested that the primitive members of the group were similar to some of the extant protostelids. A review of morphological evidence supporting some aspects of this hypothesis is presented along with explicit explanations of the shortcomings of morphological data as tests of other aspects. For the hypothesis to be supported, modified, or rejected, data from other areas such as the sequences of the nuclear ribosomal small subunit genes (SSrDNA) will have to be used. Presently, sequences for this gene are known only from Physarum polycephalum and Dictyostelium discoideum. These two slime molds are treated as separate, deep clades in the grand eukaryote phylogenies derived from the sequences of SSrDNA. That is, each species represents an independent lineage that diverged early in the history of the eukaryotes. Insufficient taxon sampling may account for the molecular trees which suggest that the dictyostelids and myxomycetes are not members of a monophyletic group. We have begun to examine the SSrDNA sequence in the protostelid Protostelium mycophaga. Preliminary phylogenetic reconstructions using 11 eukaryotic outgroups suggest that the protostelids, myxomycetes, and dictyostelids are members of a single monophyletic group which may be most closely related to the Chromista. It is interesting that these results coincide with earlier phylogenetic hypotheses based on the morphological characters of these slime molds. Key words: dictyostelids, myxomycetes, protostelids, ribosomal DNA, slime molds. Resume : Les eumycCtozoens, les myxomycbtes, les prostClides et les dictyostClides ont CtC, dans une premibre hypothbse, considCrCs comme un groupe monophylCtique par L.S. Olive, qui a suggCrC que les membres primitifs de ce groupe seraient semblables ii certains protostClides actuels. L'auteur prCsente une revue des preuves morphologiques qui supportent certains aspects de cette hypothbse, ainsi que des explications des insuffisances de donnCes morphologiques pour en Cvaluer d'autres aspects. Pour supporter, modifier ou rejeter l'hypothbse, des donnCes provenant d'autres sources, telles que les sCquences des gtnes de la petite sous-unit6 de 1'ADN ribosomal nuclCique (SSrADN) devront Ctre utilistes. PrCsentement, les sCquences de ces gbnes ne sont connues que chez le Physarum polycephalum et le Dictyostelium discoideum. Ces deux myxomycbtes sont traitts comme des clades profonds et distincts dans la grande phylogCnie des eucaryotes, dCrivCe des sequences du SSrADN. Ceci signifie que chaque espbce constitue une IignCe indkpendante qui a tres t6t divergC au cours de I'histoire des eucaryotes. Un Cchantillonage insuffisant des taxons peut expliquer les dendrogrammes molCculaires qui suggtrent que les dictyostClides et les myxomycttes seraient des membres d'un m&megroupe phylogCnCtique. L'auteur a commencC ii examiner la sCquence du SSrADN du protostClide Protostelium mycophaga. Des reconstructions phylogCnCtiques prkliminaires utilisant 11 groupes d'eucaryotes distincts suggbrent que les protostClides, les myxomycbtes et les dictyostClides seraient des membres d'un seul groupe phylogCnCtique qui serait peut Stre Ctroitement liC aux chromista. I1 est intCressant que ces rCsultats coincident avec les premibres hypothbses basCes sur les caractbres morphologiques des champignons visqueux. Mots cl&s : dictyostClides, myxomycbtes, protostClides, ADN ribosomal, champignons visqueux. [Traduit par la rCdaction]

Received August 15, 1994.

F.W. Spiegel.' Department of Biological Sciences, University of Arkansas, Fayetteville, AR 72701, U.S.A. S.B. LeeZ and S.A. Rusk. Department of Biological Sciences, University of Northern Colorado, Greeley, CO 80639, U.S.A.

'

Author to whom all correspondence should be addressed. Present address: DNA Laboratory, Bureau of Forensic Services, California Department of Justice, 626 Bancrofi Way, Berkeley, CA 94701, U.S.A.

Introduction When an evolutionary lineage splits, the daughter lineages subsequently follow their own, independent histories. Hennig (1966), among others, has pointed out this fundamental but often underappreciated principle of phylogenetic systematics. When we try to determine the phylogenetic relationships between groups of organisms, we are looking for clues to their shared history prior to their divergence. HOWever, it can be the case that these clues are hidden or lost because of events in their lineages' independent histories.

Can. J. Bot. 73(Suppl. 1): S738-S746 (1995). Printed in Canada I ImprimC au Canada


E2.6. Spiegel et al.

I

I I

These clues, the characters of the organisms in question, can become unreadable in more than one way. It is often difficult to determine if the characters of interest in the organisms are homologous. If the characters are not clearly homologous, they are relatively useless as clues. The ambiguity of the characters may be a result of convergence or parallelism between characters of the organisms being compared. Alternatively, it may be a consequence of the extreme modification of a character in one of the organisms such that its homologue in the others is unrecognizable. In other cases, characters that were present in a common ancestor can be lost in the descendants. Unless it is clear that characters have been lost, it is not possible to discern the phylogenetic significance of their absence. Finally, new sets of characters may have evolved in an ancestor, and none of these characters will have homologues outside the lineage of that organism's descendants. As one studies the phylogenetic milieux of the organisms of interest, i.e., samples as many characters in as many taxa as practicable, some of the ambiguity associated with certain characters might be expected to disappear. However, even with fairly large sample sizes, both in taxa and characters, some limits on resolution will be encountered. This is true for phylogenetic studies based upon morphological data sets, and it is likely to be true, as well, in molecular systematics. This paper will examine the systematics of the eumycetozoans with an eye to illustrate a particular case where some of the problems addressed above occur and to show how approaching the problems in this group might provide a paradigm for addressing the phylogeny of the eukaryotes as a whole. Primarily, this paper will not deal with algorithms or computer programs; rather, it will focus on the very human activity of developing a healthy skepticism about the confidence to have in the results derived from both morphological and molecular data sets.

History of the Eumycetozoans Slime molds or mycetozoans are amoeboid organisms that fruit (Olive 1975). To the mycologists who study them, there has been a tendency to behave as if all their amoebae are the same, i.e., homologous, and that all their fruiting bodies are homologous as well. L.S. Olive (1975) proposed a more skeptical approach to the question of relationships among the slime molds and effectively showed that this classical assumption of a monophyletic Mycetozoa was incorrect. Eumycetozoans, as used here, is an informal designation for a group that L.S. Olive referred to as the class Eumycetozoa in his monograph Zhe Mycetozoans (Olive 1975). He hypothesized that this is a monophyletic group of amoeboid protists that includes the myxomycetes (the classic mycetozoans), the dictyostelid cellular slime molds, and the protostelids. The eumycetozoans were only those fruiting amoebae that had pointed pseudopodia (filose pseudopodia, sensu Olive (1975)) and mitochondria with tubular cristae (Olive 1975; Dykstra 1977). While these characters are not shared derived homologies, synapomorphies, of the eumycetozoans (Spiegel 1990, 1991; Stewart and Mattox 1980; Page 1988), they could well be shared primitive homologies, symplesiomorphies, of the group, and their recognition was an important first step in the critical approach to the systematics of slime molds.

It is clear from their morphology that both the myxomycetes, Olive's (1975) subclass Myxogastria, and the dictyostelids, subclass Dictyostelia, are each monophyletic groups. Both groups are morphologically and developmentally quite conservative. The myxomycetes all have nearly identical amoeboflagellate states, down to the details of the flagellar apparatus, and an alternation of generations with distinctive dimorphic mitosis (Martin and Alexoupoulos 1969; Martin et al. 1983; Olive 1975; Spiegel 1990, 1991). All the dictyostelids have morphologically identical amoebae, a nucleus with a peripheral nucleolus, a distinct microtubular organizing center (MTOC), and a mode of aggregation, slug formation, and fruiting that is unique (Raper 1984; Olive 1975; Bonner 1967). Although he treated the protostelids as a distinct taxon, subclass Protostelia, it is clear that Olive saw them as a paraphyletic group, and he indicated that his classification of them was artificial. This is because he believed that the myxomycetes and dictyostelids had arisen independently from within the protostelids (see Fig. 251 of Olive 1975). The protostelids are morphologically and developmentally quite diverse (Olive 1975; Spiegel 1990), and by the early 1980s there was a recognition that there was no a priori reason to accept the initial assumption that they are all closely related (Olive 1982; Whitney and Bennett 1984; Spiegel 198la, 1990). Their fruiting bodies, though all delicate and microscopic, vary with respect to stalk length and spore number (Olive 1975; Spiegel 1990). More significantly, their trophic cells, if considered independently of the rest of their life histories, would be classified in several different classes of the amoeboid protists (Spiegel and Feldman 1985; Spiegel 1990; Whitney and Bennett 1984). The validity of the concept of a monophyletic Eumycetozoa depended upon establishing the relationships, if any, among the protostelids. This would require much more detailed examinations of these organisms than had initially been the case. The order of discovery of protostelids greatly affected Olive's hypotheses about their relationships to each other and the other mycetozoans. Protostelium mycophaga Olive & Stoian. (Olive and Stoianovitch 1960), the first protostelid recognized, was considered most similar to the dictyostelid genus Acytostelium Raper because both have uninucleate, nonflagellated amoebae and make acellular stalks. Protostelium was hypothesized to be similar to the preaggregative ancestor to the dictyostelids. The next series of protostelids to be described were all species that have uninucleate amoebae with pointed pseudopodia (Olive 1962). No real attempt was made to determine whether any of the characters of the amoebae of these protostelids were homologous. Cavostelium apophysatum Olive was described next (Olive 1964), and its trophic state was interpreted incorrectly as consisting only of a uninucleate amoeboflagellate, i.e., an amoeba that can reversibly become flagellated (Spiegel 1981a; Spiegel and Feldman 1985). This type of trophic cell was thought to represent a link between those of flagellated protists and the amoebae of nonflagellated protostelids (Olive 1964). Again, no rigorous effort was made to demonstrate that any homologous characters were shared by C. apophysatum and any of the other protostelids. A number of nonflagellated protostelids with plasmodial amoeboid cells were described next (Olive and Stoianovitch 1966a, 19666; Olive 1967), and a number of ad hoc explanations of possible phylogenics were


Can. J. Bot. Vol. 73 (Suppl. I), 1995

presented to try to describe the relationships among the protostelids known at that time (Olive and Stoianovitch 1966a; 1966b; Olive 1967), again with little detailed consideration of characters. The late 1960s saw the discovery of a number of species that have amoeboflagellate stages that alternate with nonflagellated amoeboid stages (Olive 1970). Some of these species had flagellated stages that are almost identical to the swarm cells of myxomycetes in gross appearance. This and the similar life history of these species suggested that some protostelids were related to the myxomycetes. As a result of these observations, Olive (1970) presented a complex, ad hoc phylogenetic scheme including all the protostelids, the myxomycetes, and the dictyostelids as members of a monophyletic group. During the 1970s, a number of ultrastructural studies began to highlight the various differences among the trophic cells of protostelids (Furtado and Olive 1970, 1971; Hung and Olive 1973), and it was realized that there was a great deal more variability among protostelids than had been recognized. As a result, when Olive (1975) named the Eumycetozoa, he pointed out that the true relationships among them would only be discovered with much more complete examinations than had been done to date. That is, he realized that characters had to be looked at in detail and that the homologies, if any, among characters of the various mycetozoans would have to be established. By the end of the 1970s and in the early 1980s, enough ultrastructural work had been completed to suggest that the characters of the trophic cells of some protostelids were quite dissimilar to those of other species, and suggestions were made that the protostelids were a polyphyletic assemblage (Olive 1982; Whitney and Bennett 1984). These suggestions were made without carefully considering if the characters being compared among these organisms could be homologous and, therefore, phylogenetically informative. Beginning in the early 1980s, Spiegel and his co-workers began an effort to compare the morphological (including ultrastructural) characters of the cells at the various stages of the life cycles of the protostelids. Several types of life cycles are found in the group (Fig. 1). Many protostelids have an arnoeboflagellate state in the trophic portion of the life cycle (Figs. 1.1, 1.2, and 1.3). Considerable effort was made to examine these cells because the details of the flagellar apparatus could be compared among the protostelids and between the protostelids and other eukaryotes. Therefore, the characters of the flagellar apparatus could be phylogenetically informative. A series of studies of the flagellar apparatus and mitosis in the arnoeboflagellatesof protostelids (Spiegel 1981a, 1981b, 1982a, 1982b, 1990; Spiegel and Feldman 1986, 1988, 1991; Spiegel et al. 1986) led to the observation that all amoeboflagellates of protostelids and myxomycetes (Wright et al. 1979; Haskins 1978; Ishigami 1977; Aldrich 1968, 1969; Hinchee and Haskins 1980) share a number of common characters, some of which are unique to the flagellated eumycetozoans. Spiegel (198 la, 1990, 1991) has suggested that these unique characters of the amoeboflagellates are synapomorophies, which are sufficient to group the flagellated eumycetozoans, i.e., the flagellated species of protostelids and the myxomycetes, into a monophyletic group (Fig. 2, Table I). Some protostelids and the myxomycetes have a nonflagellated amoeboid state in their life cycle that develops from the

Fig. 1. Life history evolution among the protostelids. Cell types sharing the same hatching pattern share synapomorphic morphological characters. A, amoeboflagellate; A , , nonflagellated amoeboflagellate; B, fruiting body; C and D, nonhomologous obligate amoebae.

arnoeboflagellate and eventually gives rise to the fruiting state (Figs. 1.2 and 1.3). Comparisons of these states, which were eventually dubbed the obligate amoebae (Spiegel and Feldman 1985), showed that there are several distinct groups of protostelids with obligate amoebae and that the obligate amoebae of a given group share no unique similarities with the obligate amoebae of the other groups or with the plasmodia of the myxomycetes (Spiegel and Feldman 1985, 1986, 1988, 1991). The obligate amoebae of the different groups, therefore, were interpreted to have evolved as novelties in their respective lineages. That is, the characters they display represent the independent histories of their respective lineages, and these characters can tell us essentially nothing about a given group's shared history with other organisms. Also, the obligate amoeba of any given species shares no derived similarities with the arnoeboflagellate state from which it develops (Fig. 1). That is, no morphological synapomorphies are shared among the different trophic states of the same species. Some protostelids have no amoeboflagellate state, and


Fig. 2. Proposed phylogeny of the flagellated eumycetozoans based upon light microscopic,

ultrastructural, and developmental characters. Genera of protostelids in parentheses lack a flagellated state in the life cycle. Based upon Spiegel (1991). Oomycetes (Outgroup)

I Ceratiottiyxella (Schizoplasttrodirrm, Nettiatosteliutt~) Platioprotosteli1crr2 (Protostelircttr, s.s.) Cavostelicctti


Myxomycetes

. .

I

Clastostelircttz Protosporat~giutn Cerntionryxn

Table 1. Phylogenetically significant and phylogenetically insignificant mormphological characters for uniting eumycetozoans into a monophyletic group. Phy logenetically significant characters

Phylogenetically insignificant characters"

Trophic cells Presence of flagella Absence of flagella Structure of rootlet elements Structure of transition zone Number of flagella Mitosis of amoeboflagellate Mitosis in obligate amoebae Number of nuclei per cell Pseudopodial structure Feeding behavior Fruiting bodies

Mode of stalk elongationb

Stalk length Spore deciduous or nondeciduous Spore wall ornamentation

NOTE.For a more detailed treatment of the phylogenetically significant morphological characters, see Table 2 in Spiegel (1991). "While some of these characters may be phylogenetically significant within subgroups, they are not significant at uniting the whole group. "The characters associated with stalk elongation may be synapomorphic for the group (see text). their nonflagellated trophic states may have arisen either from amoeboflagellates of ancestors that lost the ability to produce flagella (Figs. 1.4 and 2) as is clearly the case with the genus Protosteliurn, sensu stricto, (Spiegel 1982a, 1984, 1990; Spiegel et al. 1994; Best 1984), or from ancestors with obligate amoebae that lost the amoeboflagellate state and retained only the obligate amoeba (Figs. 1.5, 1.6, and 2), as is the case with the genera Schizoplasrnodiurn Olive & Stoian. and Nernatosteliurn Olive & Stoian. (Whitney and Bennett 1984; Spiegel 1990; Spiegel and Feldman 1986). Such organisms can be easily recognized as members of the monophyletic flagellated eumycetozoans that have lost the ability to produce flagellated cells. The suggestion by Whitney and Bennett (1984) that protostelids are a polyphyletic assemblage was based on the comparison of yeast-ingesting characters of the obligate amoebae of the genera Ceratiornyxella, Schizoplasrnodiurn,

and Nernatosteliurn with the yeast-ingesting characters of the amoeboflagellates and amoebae of the genera Planoprotosteliurn Olive & Stoian and Protosteliurn, sensu stricto. Because these stages of the life cycle share no history in a common ancestor, these characters are useless to evaluate relationships. Olive (1982) expressed skepticism about the common origin of the protostelids that was based on a vague disquiet about the range of morphologies found in the trophic cells of protostelids, but particular characters were not discussed. Some nonflagellated protostelids have trophic cells that are not obviously similar to either the amoeboflagellates or the obligate amoebae of any of the flagellated lineages. These are mostly species which were originally assigned to the genera Protosteliurn (Olive 1962, 1975; Olive and Stoianovitch 1969, 1981) and Schizoplasrnodiopsis Olive (Olive 1967, 1975; Olive and Stoianovitch 1975; Olive and Whitney 1982). Work by Best (1984), Olive et al. (1984), and Spiegel and co-workers (Spiegel 1990; Spiegel et al. 1994; F.W. Spiegel and J. Feldman, unpublished) and Bennett (1986a, 19866) has shown that three genera should be segregated from Protosteliurn, sensu stricto, and Bortnick (1993) has shown that Schizoplasrnodiopsis should be split into at least two genera. None of the segregates from Protosteliurn nor either of the genera of the original Schizoplasrnodiopsis has any characters of its trophic cells that are unequivocally synapomorphic with the characters of the trophic cells of any of the flagellated lineages (Table I). One of the segregates from Protosteliurn, the genus Endosteliurn Olive (Olive et al. 1984; Bennett 1986a; Spiegel 1990; F.W. Spiegel and J. Feldman, unpublished), appears not to be related to any of the other eumycetozoans at all. None of the characters of the amoebae of dictyostelids is clearly synapomorphic with the characters of any of the flagellated eumycetozoans (Spiegel 1990). The ultrastructure of fruiting bodies has been studied to varying extents in several protostelids (Dykstra 1978; Spiegel et al. 1979; Olive and Whitney 1982; Whitney 1984, 1985; Blanton and Chanzy 1984; Smith 1987; F.W. Spiegel and J. Feldman 1993, unpublished), and Spiegel (1990, 1991; Spiegel and Feldman 1993) has suggested that the characters of fruiting in most protostelids other than Endosteliurn spp. (Olive et al. 1984) appear to be sy napomorphic for the group, although this needs further, in-depth analysis. Spiegel et al. (1979) also suggested that there are similarities

Can. J. Bot. Vol. 73 (Suppl. I ) , 1995

among the stalk tube synthesizing prestalk cells of dictyostelids (George et al. 1972; Hohl et al. 1968), which might be indicative of a relationship between protostelids and dictyostelids.


Present status of the eumycetozoans and its significance Our present understanding of the relationships among Olive's Eumycetozoa, based on the morphological data that have been gathered since 1975, is that there is a monophyletic group-of flagellated eumycetozoans (Fig. 2) and a number of nonflagellated groups that are of uncertain status. The flagellated group includes the myxomycetes and the flagellated protostelids (Spiegel 1991). Some of the lineages of flagellated protostelids include species that lack the flagellated state (Fig. 2) but share clearly synapomorphic characters with their flagellated sister groups. These characters include (i) fruiting body development (Olive 1975; Spiegel et al. 1994); (ii) feeding behavior (Whitney and Bennett 1984) of obligate amoebae; and (iii) mitosis and detailed morphology of amoebae (Spiegel 1982a, 1982b, 1984, 1990; Spiegel and Feldman 1985, 1986; Spiegel et al. 1994). Most of the nonflagellated protostelids have fruiting bodies that are similar to-those of the flagellated protostelids, but they have dissimilar trophic cells (Spiegel 1990; Spiegel et al. 1994; Best 1984; Bortnick 1993). This dissimilarity could be the result of (i) independent origins from other protist~,as seems the case with Endostelium spp. (Olive et al. 1984; Bennett 1986~);(ii) it could result from the possibility that some protostelids have trophic cells that are derived from the obligate amoebae of lineages for which the flagellated sibling groups are extinct or undiscovered (Spiegel et al. 1994; Bortnick 1993); or (iii) it could be that the characters of the trophic cells of these protostelids have diverged so greatly from those of their sibling taxa that homologies are unrecognizable. In any case, it is difficult to use morphological characters to determine the relationshi~sof these nonflagellated protostelids with the flagellated eumycetozoans. The morphological case for including the dictyostelids in a monophyletic Eumycetozoa is highly equivocal at best. After considering all that is known of the morphology of the known eumycetozoans, sensu lato, it is clear that morphology alone cannot provide the information necessary to resolve unambiguously the relationships among all these organisms. Because of the way that life cycles appear to have evolved in the protostelids (Fig. I), some nonflagellated protostelids with obligate amoebae as their sole trophic states (Figs. 1.5 and 1.6) may share no morphological synapomorphies even though they are closely related. That is, close relationships might exist between groups, but they are impossible-toresolve because whole sets of shared characters have been lost during the independent evolution of each group. If the dictyostelids share a common ancestry with other eumycetozoans, it cannot be resolved with morphology alone for essentially the same reason that not all protostelids can be clearly recognized as related. In a general sense, this is the downfall of using morphological characters to determine the relationships among some groups of eukaryotes. Protists and fungi do not lack morphological character sets, as is the often-heard lament. Rather, it u

is likely that many lineages have lost the characters that are the clues to their shared histories with other organisms and express only characters that illustrate their lineages' independent histories. Though this phenomenon is rarely acknowledged, understanding it makes it clear that morphological systematists can and should state which morphological characters of their organisms are phylogenetically informative and which are not. When the uselessness of morphological characters can be determined in a given situation, other characters sets, such as DNA sequences, must be used if relationships are to be resolved. However, the characters of molecular systematics are as likely as morphological characters to be uninformative in certain situations (Hillis 1991). Therefore, a first test of molecular systematics in a group should be made using organisms for which there is a good morphological understanding of relationships. In the particular case of the eumycetozoans, a molecular tree should show members of the myxomycetes and the flagellated protostelids in the same clade exclusive of other groups of eukaryotes. Failure to resolve such relationships would indicate either that the sequence of choice is uninformative and useless or that the morphological data have been misinterpreted. Where there is a preponderance of detailed morphological data to support a relationship, the sequence data should be interpreted as useless.

The history of the molecular systematics of eumycetozoans The most widely available sequences used in molecular systematics are the sequences of the small subunit ribosomal RNA gene (SSrDNA) (Sogin et al. 1989; Forster et al. 1990; Schlegel 1991; Wolters 1991; Bruns et al. 1991, 1992; Li and Heath 1992). The only eumycetozoans for which this gene has been sequenced are the widely studied Physarum polycephalum Schw. (Johansen et al. 1988) and Dictyostelium discoideum Raper (McCarroll et al. 1983). Both species are interpreted as being members of lineages that diverged early in the history of the eukaryotes, but they have never been reported as the only members of the same lineage. It is interesting that no publications have focused on the molecular systematics of the eumcyetozoans as a group, and little comment has been made concerning the observation that these two species do not group together on either distance matrix or varsimonv trees. one or the other of these species is often used as an outgroup in phylogenetic studies based on SsrDNA sequences, but they are rarely both included. In those cases where both s~ecieshave been included in trees with outgroups that diverge earlier than they do, P. polycephalum either diverges from the rest of the eukaryotes with the subsequent branching event separating D. discoideum from the higher eukaryotes (Forster et al. 1990; Bruns et al. 1991; Li and Heath 1992), or P. polycephalum is included in a trichotomy with the heterolobosean amoebae and the rest of the eukaryotes with D. discoideum as the next of the eukaryotes to diverge (Schlegel 1991; Wolters 1991). Gene sequences can tell us nothing about the morphology of ancestral organisms. However, the branching pattern of the molecular trees is consistent with the interpretation that, with the origin of the myxomycetes followed by the origin of


Fig. 3. Partial nuclear SSrDNA sequence from Protostelium mycophaga. Positions correspond with those of Physarum polycephalum.


CCCCGTGGCG AGGGGCCTGA GCAGGCGCGC AGTGACAAGA AAAGGAATTG GCTAATTTGA ATAGTAAGGA ATGGGTGGTG

REGION BETWEEN #305-454 ACGGGTAACG GACAAGGGTC GAAATGGCCC CGCTTCTAAG AAATTACCCA ATCTCCAATT AATAGTAATG CCTTGGCTCA REGION BETWEEN #1132-1291 ACGGAAGGNC ACACACAGGA CTCAACACGG GAAACTTACC TTGACAGATT GAGAGCTCTT GTGCATGGGT TCTTAGTTGG

GATTCCGGAG GAAGGCAGCA CGAGGGAGGT

Fig. 4. Single most parsimonious tree from analysis of 310 bp of sequence of Protostelium rnycophaga and 14 outgroups. Length = 377 steps, C.I. = 0.71 1. The number above the eumycetozoan branch and the Vairimorpha-heterolobosean branch is the bootstrap value for 1000 repetitions. The number below is the decay value. All other branches had a bootstrap value of