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Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences
Fungal Genetics and Biology 24, 354–363 (1998) Article No. FG981077
Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences
John Daugherty,* Timothy M. Evans,*,1 Tally Skillom,† Linda E. Watson,* and Nicholas P. Money*,2 *Department of Botany, Miami University, Oxford, Ohio 45056; and †Cincinnati Academy of Mathematics and Science, Hughes Center, 2515 Clifton Avenue, Cincinnati, Ohio 45219
Accepted for publication July 8, 1998
Daugherty, J., Evans, T. M., Skillom, T., Watson, L. E., and Money, N. P. 1998. Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence From a Phylogenetic Analysis of Internal Transcribed Spacer Sequences. Fungal Genetics and Biology 24, 354–363. Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axone1 Present address: Department of Biology, Hope College, Holland, MI 49423. 2 To whom correspondence should be addressed. E-mail: [email protected].
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mal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. r 1998 Academic Press
Index Descriptors: flagella; morphogenesis; stress response; zoospore. The different mechanisms of spore release that distinguish the water molds Saprolegnia Nees, Achlya Nees, Thraustotheca Humphrey, and Dictyuchus Leitgeib, within the Saprolegniaceae (Oomycetes), have been viewed as a transition series in which Saprolegnia is thought to exhibit the ancestral mechanism (Humphrey, 1893; Atkinson, 1909; Ho¨hnk, 1933). Sporangia of Saprolegnia release pyriform zoospores through a papilla at the tip of the sporangium (Fig. 1a). Each zoospore is equipped with a pair of flagella anchored in the spore apex by kinetosomes and associated microtubular roots (Holloway and Heath, 1977; Barr and De´saulniers, 1997). The spores swim at up to 110 µm s21, apex forward, following a counterclockwise helical path before collecting at the air–water interface, or adhering to a surface, and then encysting (Salvin, 1941). Later, the protoplast within each cyst is discharged onto the cyst surface, and in this position the initially spherical protoplast is modified into a reniform zoospore with laterally inserted flagella (Fig. 2). This type of spore, referred to as a secondary zoospore, exhibits much faster swimming (up to 260 µm s21) with a clockwise helical pitch 1087-1845/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Sporangial emptying in the Saprolegniaceae. (a) Saprolegnia ferax, primary zoospores swim after release. (b) Achlya intricata, cluster of discharged spores forms at sporangial apex. (c) Thraustotheca clavata, spores encyst and spill out through ruptured lateral wall of sporangium. (d) Dictyuchus sterile, encysted spores discharged through individual exit papillae. Bars, 50 µm.
and encysts after a much longer period of motility than the primary spore (Salvin, 1941). Encystment of the secondary zoospore can then be followed by successive phases of excystment, swimming, and encystment (Die´guezUribeondo et al., 1994); when nutrients are available, the cysts germinate and a vegetative mycelium develops. The production of two distinct types of zoospore is referred to as diplanetism, and the term polyplanetism has been used to describe the repeated pattern of encystment and excystment characteristic of Saprolegnia and other genera within the Saprolegniaceae (Webster, 1980). Achlya also releases pyriform spores from its sporangia, but flagella are not visible under the light microscope, and the spores collect and encyst in a spherical cluster or ‘‘spore ball’’ at the apex of the sporangium (Figs. 1b and 2; Money and Webster, 1987). Subsequently, secondary-type zoospores emerge from the cysts. While most studies of spore release in Achlya describe nonflagellate spores
streaming out of sporangia, some of the classical descriptions reported that the spores were flagellate like those of Saprolegnia (references in Money et al., 1987). This issue was resolved when transmission electron microscopy revealed that sporangiospores of Achlya intricata and A. flagellata possess rudimentary flagella with stunted axonemes (usually ,2 µm in length) and bear some structural features homologous to the primary-type zoospores of Saprolegnia (Money et al., 1987). The earlier accounts of active flagella, based on light microscopy, suggest that other species of Achlya develop spores with longer flagella. But in all Achlya species, the spores do not swim after discharge from the sporangium. Thraustotheca produces large cylindrical or clavate sporangia in which the spores encyst before release from the sporangium. Partial emptying of the sporangium occurs when the lateral wall ruptures and some of the cysts spill out (Figs. 1c and 2). The cysts usually adhere to one
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FIG. 2.
Diagram showing asexual phases of the life cycles of the four genera shown in Fig. 1.
another, forming a loose cluster within and around the lysed wall; after a few minutes to an hour, a secondary zoospore is released from each cyst. Spores also encyst within sporangia of Dictyuchus, but the sporangium of this genus empties when a protoplast is released from each cyst through an individual papilla that projects through the sporangial wall. On the sporangial surface the discharged protoplasts are reorganized into secondary-type zoospores. After sporangial emptying, Dictyuchus can be identified by the geodesic pattern of empty cyst walls inside the sporangium (Figs. 1d and 2). In developing a transition series to link these genera, it was assumed that the different mechanisms of sporangial emptying represented useful markers for the evolutionary longevity of one genus relative to the others (Humphrey, 1893). According to this scheme, Achlya evolved from an organism that exhibited saprolegnioid sporangial empty-
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ing, and Thraustotheca and Dictyuchus evolved from an ancestor that exhibited achlyoid sporangial emptying (terminology from Johnson, 1956, and Webster, 1980). The discovery of rudimentary flagella in Achlya (Money et al., 1987) certainly supports the idea that the achlyoid mechanism was derived from a saprolegnioid ancestor rather than vice versa. Descriptions and illustrations of these fungi in contemporary mycological textbooks are rooted in this hypothesis (Esser, 1982; Moore-Landecker, 1996). The objective of the present study was to reevaluate the hypothesis of a transition series linking these genera and their spore release mechanisms. Nucleotide sequence data from the two internal transcribed spacers (ITSs)3 of 3
Abbreviations used: CI, consistency index; ITS, internal transcribed spacer; RI, retention index.
Spore Release Mechanisms in the Saprolegniaceae
nuclear ribosomal DNA were used to construct a phylogeny. An extensive literature supports the close relationship and monophyly of the four genera (Dick, 1990; Powell and Blackwell, 1998), and the efficacy of ITS-based examinations of relationships among closely related oomycete species has been demonstrated previously (Lee and Taylor, 1992; Crawford et al., 1996). Radical differences between the responses of Achlya and Saprolegnia to osmotic stress are well documented (see references in Money, 1997) and may also reflect evolutionary divergence between these organisms. Therefore, the stress response in each genus was studied in an effort to further understand evolutionary affinities. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently.
MATERIALS AND METHODS Organisms and culture conditions. The following cultures were obtained from the American Type Culture Collection (ATCC; Rockville, MD): Saprolegnia ferax (Gruithuisen) Thuret (Accession No. 36051), Thraustotheca clavata (de Bary) Humphrey (34112), and Dictyuchus sterile Coker (44891). Achlya intricata Beneke, originally obtained from the culture collection at the University of Reading, United Kingdom, courtesy of Dr. Michael Dick, is now deposited at the ATCC (66594); this species was selected for the investigation because spore release has been studied more intensively in this species than in any other member of the Saprolegniaceae (Money and Webster, 1988, and references therein). Mycelia were grown in 100-ml batches of liquid PYG medium (0.1% peptone, 0.1% yeast extract, 0.3% glucose), inoculated with 10-mm-diameter plugs from PYG agar cultures, and incubated at 24°C for 2–7 days on an orbital shaker. For the osmotic stress experiments cultures were grown on PYG agar supplemented with 600 mM sucrose, sorbitol, or maltose at 22°C for up to 14 days. Colonies were photographed with standard compound and dissecting microscopes (Olympus BH-2 and SZH-10; Olympus Optical Co., Tokyo) to document the effects of these osmolytes on morphogenesis. Sequence data and phylogenetic analysis. Fresh mycelia were harvested by vacuum filtration from liquid PYG and ground in liquid nitrogen. Total DNA was extracted using the CTAB procedure (Saghai-Maroof,
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1984; Doyle and Doyle, 1987). The entire ITS region, including the 5.8S cistron, was amplified by doublestandard PCR using primers 4 and 5 of White et al. (1990). PCR products were verified on 1% agarose gels and purified using GeneClean (Bio 101, Inc., Vista, CA) prior to sequencing. The double-stranded PCR products were sequenced directly using standard dideoxy sequencing and 6% acrylamide gel electrophoresis (Sanger et al., 1977), using the snap-chill method (Winship, 1989) and Sequenase 2.0 (U.S. Biochemical, Cleveland, OH) with 35S-dATP labeling. Strands were sequenced in both directions using the four primers (ITSs 1, 2, 3, and 4) of White et al. (1990). Phytophthora megakarya (Order Peronosporales) sequences were obtained from GenBank [Accession No. S40356 (ITS1) and S50358 (ITS2)]. Sequence boundaries were determined by comparison with published sequences (White et al., 1990; Lee and Taylor, 1992; Crawford et al., 1996) and were aligned using Clustal W, Version 1.7 (Higgins et al., 1991; Thompson et al., 1994), with some manual gap adjustments made. Both ITS spacers were used in all analyses, with the 5.8S region excluded. To assess the phylogenetic signal present in the data, the g1 statistic (Hillis, 1991) was calculated. Phylogenetic analyses that included all characters, as well as an analysis that excluded 64 characters with an ambiguous alignment (positions 464–480, 509–534, and 625–653), were conducted. Sequence divergence values were estimated with PAUP 3.1 (Swofford, 1993), using the MATRIX DISTANCE option. Parsimony analyses were conducted using Fitch Parsimony (unordered, multistate characters) with the gaps treated as missing data, using PAUP 3.1 (Swofford, 1993) with the Exhaustive Search Option and Phytophthora as the outgroup. To assess the robustness of the clades, both bootstrap and decay values were calculated. Bootstrap values were calculated with PAUP 3.1 for 100 replicates and 100 random order entries of the data using the Heuristic Search Option and TBR (tree bisection reconnection) branch swapping with MULPARS on. Decay values were calculated with the software Autodecay 3.0 (Eriksson and Wikstrom, 1995), in conjunction with PAUP 3.1. Consistency index (CI) and retention index (RI) were calculated (Kluge and Farris, 1969; Farris, 1989) for all parsimony trees. In addition to the parsimony analysis, two maximum-likelihood trees that assumed transition/transversion ratios of 1.0 and 2.0 each were constructed (Felsenstein, 1989, 1993). However, because Phytophthora was highly divergent from the ingroup, a maximum-likelihood ratio test was conducted to test alternative rootings. Topologies were constructed and input as
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TABLE 1 ITS Sequence Divergence Values Calculated Using PAUP 3.1 (Swofford, 1993)
1. 2. 3. 4. 5.
Achlya Thraustotheca Saprolegnia Dictyuchus Phytophthora
1
2
3
4
5
— — — — —
0.036 — — — —
0.256 0.363 — — —
0.252 0.256 0.327 — —
0.458 0.462 0.441 0.484 —
user trees to constrain placement of Phytophthora, and likelihoods of the constrained and most parsimonious tree were compared to determine whether these alternative rootings could be rejected (Kishino and Hasegawa, 1989).
RESULTS Sequence data. Sequences were deposited with GenBank for S. ferax (Accession Nos. AF036542/AF036543 for ITS1/ITS2), A. intricata (AF036540/AF036541), T. clavata (AF036538/AF036539), and D. sterile (AF036544/ AF036545). The aligned sequence for both spacers, excluding the 5.8S coding region, was 674 base pairs (bp) in length (the aligned data matrix is available upon request from the corresponding author). The aligned length for
ITS1 was 248 bp and varied from 186 to 219 bp when excluding gaps that were inserted to achieve alignment. The aligned length of ITS2 was 375 bp and ranged from 319 to 336 bp excluding gaps. Sequence divergence estimates for the entire dataset ranged from 4 to 36% for the ingroup and 44 to 48% between the ingroup and the outgroup (Table 1). The g1 statistic was 20.58 for 10,000 random-generated trees, suggesting a relatively strong phylogenetic signal in this dataset. In the outgroup-rooted parsimony analysis, a single most parsimonious tree of 465 steps (CI 5 0.938, RI 5 0.662) was produced (Fig. 3). Saprolegnia was the most basal taxon, sister to the remaining three genera. Bootstrap and decay values indicate strong support for the sister group relationship between Achlya and Thraustotheca (100% bootstrap support, 21 decay index value) and strong support for the placement of Dictyuchus at their base (88% bootstrap support, 7 decay index value). The remaining nodes were unsupported by either measure. When 64 ambiguous characters were excluded [positions 464–480, 509–534, and 625–653; 9% (64/674) of the dataset], a single most parsimonious tree resulted, identical to the tree that included all characters. The CI (0.941) was slightly higher with these characters excluded, and the overall length was shorter (389 steps). The maximumlikelihood trees were identical to the parsimony tree, with no differences in topologies produced by either weighting scheme using transition/transversion ratios of 1.0 or 2.0.
FIG. 3. Single most parsimonious tree of 465 steps, based on ITS sequence data using Phytophthora as the outgroup (CI 5 0.938, RI 5 0.062). Numbers above the line are branch lengths; percentages below the line are bootstrap values and are followed by decay index values (in parentheses).
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Spore Release Mechanisms in the Saprolegniaceae
The likelihood ratio test resulted in two alternative rootings that could not be rejected, with the first joining Phytophthora and Dictyuchus and the second placing Dictyuchus and Saprolegnia on a single branch as sister taxa to each other. Responses to osmotic stress. On unsupplemented PYG agar medium, all four species produced a richly branched coenocytic mycelium, with the proliferation of aerial hyphae and extensive invasive growth through the entire depth of the solid medium. When grown on medium supplemented with 600 mM osmolytes, the appearance of all four species was altered. The most subtle changes occurred in Saprolegnia and have been described previously (Money, 1997). Briefly, normal polarized extension was sustained on the surface of the agar medium, but invasive growth and aerial growth were inhibited by the resulting loss of turgor pressure (Fig. 4a). There was no
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polarized hyphal growth in A. intricata and the mycelium produced enlarged outgrowths that spread slowly over the agar surface (Fig. 4b); this growth form has been described previously in A. bisexualis and A. ambisexualis and was referred to as plasmodium-like (Money, 1997; Money and Hill, 1997). The stress response of Thraustotheca and Dictyuchus had not been studied previously. Polarized growth was inhibited in both Thraustotheca and Dictyuchus, and large swollen spherules (up to 200 µm in diameter) piled up on medium supplemented with sucrose, sorbitol, or maltose (Figs. 4c and 4d). Plasmodiumlike outgrowths extended from the edges of these colonies where the cells were in direct contact with the agar surface. However, in contrast to Achlya, tip-growing hyphae developed from the masses of spherules in some of the cultures after more than a week of growth at 22°C (Fig. 4d). This reversion to polarized growth occurred frequently on
FIG. 4. Appearance of Saprolegniaceae on medium supplemented with osmolytes. (a) Tip-growing hyphae of Saprolegnia on surface of PYG 1 600 mM sorbitol. (b) Plasmodium-like colony of Achlya on PYG 1 600 mM sorbitol. (c) Spherules of Thraustotheca on PYG 1 600 mM sucrose. (d) Spherules and emerging hyphae of Dictyuchus on PYG 1 600 mM sorbitol. Bar (a,d) 100 µm; (b,c) 500 µm.
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sorbitol in both Dictyuchus and Thraustotheca, but in neither case did the hyphae invade the agar. Therefore, the morphogenetic responses of Thraustotheca and Dictyuchus to osmotic stress share features in common with Achlya (depolarized growth) and with Saprolegnia (polarized). The physiological behavior underlying these morphological changes, such as changes in turgor and endoglucanase secretion (Money and Hill, 1997), was not examined.
DISCUSSION Relationships within the Saprolegniaceae. Achlya and Thraustotheca have an ITS sequence similarity of 96%, and both parsimony and maximum-likelihood analyses suggest that they are sister taxa, more closely related to each other than either is to Dictyuchus or Saprolegnia. The parsimony and maximum-likelihood analyses of the sequence data also suggest that Dictyuchus shares a more recent common ancestor with Achlya and Thraustotheca than with Saprolegnia. The trees place Dictyuchus on a separate evolutionary branch from that of Achlya and Thraustotheca, with Saprolegnia in the most basal position. The only alternative rootings that could not be rejected by the likelihood ratio test rooted Phytophthora to either Dictyuchus or the common ancestor of a Dictyuchus– Saprolegnia clade. Therefore, the interpretation of Saprolegnia as plesiomorphic is both conservative and robust. The morphogenetic response to osmotic stress exhibited by Achlya results from disruption to the normal process of cell wall assembly (Money and Hill, 1997), and the same mechanisms probably govern the behavior of Thraustotheca and Dictyuchus. The fact that wall assembly is dependent upon the expression of multiple genes improves the likelihood that these physiological comparisons may accurately reflect evolutionary relationships. This possibility is strengthened by the view that the stress responses are not adaptive mechanisms with ecological significance (and therefore subject to intense selection), but reflect constitutive differences between the mechanisms of wall assembly in each taxon (this has been demonstrated in Saprolegnia and Achlya; Money, 1997). If, as the ITS data suggest, Saprolegnia is the basal taxon of the ingroup, the maintenance of polarized hyphal extension under conditions of osmotic stress may represent an ancestral character state. It is interesting that the other three genera are united by the loss of hyphal extension under stress, which is consistent with the ITS parsimony tree that places Achlya, Thraustotheca, and Dictyuchus in a monophyletic clade.
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Daugherty et al.
Vestiges of the Saprolegnia response appear to be expressed by Thraustotheca and Dictyuchus when they revert to hyphal extension after more than a week of sustained exposure to high concentrations of osmolytes. Monophyly and algal affinities. Pringsheim (1858) was the first to recognize morphological similarities between Saprolegnia and the alga Vaucheria, but analysis of 18S rRNA gene sequences of a wide range of chromonad and xanthophycean algae and three other oomycete fungi (Achlya bisexualis, Lagenidium giganteum, and Phytophthora megasperma) showed that Vaucheria is only distantly related to the Oomycetes (Potter et al., 1997). Based on these data, Potter and colleagues questioned the oomycete nature of Saprolegnia, but did not analyze the 18S rRNA sequence of this genus. Our ITS data support the inclusion of Saprolegnia in the Oomycetes, based on comparative sequence divergence values and alignability of the ingroup taxa. While the divergence value between Saprolegnia and the remaining ingroup taxa is relatively high (25%), it is comparable to divergence values between Dictyuchus and the other genera. The only divergence value that is lower than 25% is between Achlya and Thraustotheca (4%). Evolutionary suppression of the primary-type zoospore. The relatively slow swimming velocities of primarytype zoospores (Salvin, 1941) coupled with the rarity of this spore type among the Oomycetes have led to the suggestion that the primary zoospore is more primitive than the secondary-type spore (this was most recently stated by Dick, 1990). It has been argued that suppression of the primary spore would conserve the finite quantity of food reserves (packaged during sporangial development) for the faster secondary zoospores that represent more effective vehicles for dispersal. The basal position of Saprolegnia in the ITS comparisons supports Humphrey’s century-old assertion that the diplanetic life cycle has been condensed during the evolution of the Saprolegniaceae (Humphrey, 1893), with the ultimate elimination of the primary spore stage in Dictyuchus. Likewise, the widely accepted hypothesis of flagellar suppression during the evolution of the primary spores of Achlya, rather than flagellar acquisition in Saprolegnia, is now supported by molecular phylogenetic data. Speculation on evolution of different mechanisms of sporangial emptying. Sporangial differentiation in the Saprolegniaceae follows a prescribed sequence of developmental events beginning with the cessation of hyphal elongation and ending with different patterns of wall lysis that initiate spore release (Gay and Greenwood, 1966;
Spore Release Mechanisms in the Saprolegniaceae
Armbruster, 1982a,b; Money and Brownlee, 1987). This type of rigid developmental sequence is an example of a coordinately expressed program, in which each in a succession of discrete processes occurs only upon the completion of the previous step in the program (Lawson and Poethig, 1995). For example, lysis of the sporangial wall occurs only after the completion of cleavage (in A. intricata there is a consistent lag of 8 to 10 min at 22°C between cleavage and emptying; Money and Brownlee, 1987). In Achlya, sporangial development is dependent upon the expression of many genes that are also required for vegetative growth, in addition to the transcription of a smaller suite of sporulationspecific sequences (Gwynne and Brandhorst, 1982). Synthesis of RNA and proteins continues throughout sporangial development (Griffin and Breuker, 1969), and rapid turnover of existing proteins supplies the necessary amino acids to support high levels of protein synthesis (Timberlake et al., 1973). However, after cleavage, the spore release mechanism itself may simply require the synthesis of the enzyme(s) necessary for lysis of the apical wall or papilla of the sporangium (Ross, 1979). Therefore, a small number of mutations affecting the expression of these enzymes could have profound effects on sporangial ontogeny. Since Saprolegnia is the basal genus in our comparison, it is possible that its mechanism of spore release is plesiomorphic to the other three types. In both Saprolegnia and Achlya, spores are driven out of the sporangium a
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few minutes after cleavage by osmotically generated hydrostatic pressure (Money and Webster, 1988, 1989). In Achlya, the spore ball is produced by adhesion between the spores as they are ejected from the sporangium and begin to encyst (Money and Webster, 1987). Adhesion and encystment also occur in Saprolegnia, but only after the primary zoospores have dispersed from the sporangium. Therefore, a primary difference between saprolegnioid and achlyoid emptying is a reduction in the time interval between cleavage and encystment of the spores. It seems plausible that the condensation of the life cycle observed in Achlya is a secondary consequence of mutations that suppressed axonemal elongation. Pursuing this line of inquiry, the thraustothecoid and dictyuchoid patterns of spore release can be linked to a basal saprolegnioid mechanism by coupling the same inhibition of flagellar development to changes in the temporal and spatial expression of lytic enzymes that degrade the sporangial wall (Fig. 5). Inhibition of both flagellar development and papillar lysis, accompanied by a more diffuse pattern of wall lysis, would convert an organism from a saprolegnioid to a thraustothecoid mechanism. Finally, inhibition of flagellar development and suppression of lysis of the sporangial wall, coupled with further acceleration of the encystment process (inducing encystment immediately after cleavage), could create the cyst-filled sporangium primed for dictyuchoid emptying.
FIG. 5. Hypothetical scheme showing possible developmental relationships that link four mechanisms of sporangial emptying. Minimum changes necessary to derive achlyoid, thraustothecoid, and dictyuchoid sporangial emptying from an ancestral saprolegnioid condition are mapped onto the phylogenetic topology suggested by the parsimony tree in Fig. 3.
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ACKNOWLEDGMENTS The authors acknowledge the generous support of the Miami University Committee on Faculty Research, the Miami University Summer Scholars Program, the CAMAS Scholars Program, and the National Science Foundation (Grant DEB 9596274 to L.E.W.).
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Higgins, D. G., Bleasby, A. J., and Fuchs, R. 1991. Clustal V: Improved software for multiple sequence alignment. CABIOS 8: 189–191. Hillis, D. M. 1991. Discriminating between phylogenetic signal and random noise in DNA sequences. In Phylogenetic Analysis of DNA Sequences (M. M. Miyamoto and J. Cracraft, Eds.), pp. 278–294. Oxford Univ. Press, Oxford. Ho¨hnk, W. 1933. Polyplanetism and zoo¨spore germination in Saprolegniaceae and Pythium. Amer. J. Bot. 20: 45–62. Holloway, S. A., and Heath, I. B. 1977. An ultrastructural analysis of the changes in organelle arrangement between the various spore types of Saprolegnia. Can. J. Bot. 55: 1328–1339. Humphrey, J. E. 1893. The Saprolegniaceae of the United States, with special notes on other species. Trans. Amer. Philos. Soc. (N.S.) 17: 63–148. Johnson, T. W. 1956. The Genus Achlya, Morphology and Taxonomy. Univ. of Michigan Press, Ann Arbor. Kishino, H., and Hasegawa, M. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order of the Hominoidea. J. Mol. Evol. 29: 170–179. Kluge, A. G., and Farris, J. S. 1969. Quantitative phyletics and the evolution of anurans. Syst. Zool. 18: 1–32. Lawson, E. J. R., and Poethig, R. C. 1995. Shoot development in plants: Time for a change. Trends Genet. 11: 263–268. Lee, S. B., and Taylor, J. W. 1992. Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA. Mol. Biol. Evol. 9: 636–653. Money, N. P. 1997. Wishful thinking of turgor revisited: The mechanics of fungal growth. Fungal Genet. Biol. 21: 173–187. Money, N. P., and Brownlee, C. 1987. Structural and physiological changes during sporangial development in Achlya intricata Beneke. Protoplasma 136: 199–204. Money, N. P., and Hill, T. W. 1997. Correlation between endoglucanase secretion and cell wall strength in oomycete hyphae: Implications for growth and morphogenesis. Mycologia 89: 777–785. Money, N. P., and Webster, J. 1987. Aspects of spore ball formation in Achlya. Trans. Br. Mycol. Soc. 88: 341–346. Money, N. P., and Webster, J. 1988. Cell wall permeability and its relationship to spore release in Achlya intricata. Exp. Mycol. 12: 169–179. Money, N. P., and Webster, J. 1989. The mechanism of sporangial emptying in Saprolegnia. Mycol. Res. 92: 45–49. Money, N. P., Beakes, G. W., Webster, J., and Wakeley, G. 1987. Rudimentary flagella in sporangiospores of Achlya. Trans. Br. Mycol. Soc. 89: 108–114. Moore-Landecker, E. 1996. Fundamentals of the Fungi, 4th ed. Prentice Hall, Upper Saddle River, NJ. Potter, D., Saunders, G. W., and Andersen, R. A. 1997. Phylogenetic relationships of the Raphidophyceae and Xanthophyceae as inferred from nucleotide sequences of the 18S ribosomal RNA gene. Amer. J. Bot. 84: 966–972. Powell, M. J., and Blackwell, W. H. 1998. Phentic analysis of genera of the Saprolegniaceae (Oomycetes). Mycotaxon 68: 505–516. Pringsheim, N. 1858. Beitrage zur Morphologie und Systematik der Algen. II. Die Saprolegnieen. Jahrb. Wiss. Bot. 1: 284–306. Ross, I. K. 1979. Biology of the Fungi. McGraw-Hill, New York.
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Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A., and Allard, R. W. 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014–8018. Salvin, S. B. 1941. Comparative studies on the primary and secondary zoo¨spores of the Saprolegniaceae. I. Influence of temperature. Mycologia 33: 592–600. Sanger, F. S., Nicklen, S., and Coulson, A. R. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463–5467. Swofford, D. L. 1993. PAUP: Phylogenetic Analysis using Parsimony, Version 3.1.1. Illinois Natural History Survey, Champaign, IL. Timberlake, W. E., McDowell, L., Cheney, J., and Griffin, D. H. 1973. Protein synthesis during the differentiation of sporangia in the water mold Achlya. J. Bacteriol. 116: 67–73.
363 Thompson, J. D., Higgins, D. G., and Gibson, T. J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673–4680. Webster, J. 1980. Introduction to Fungi, 2nd ed. Cambridge Univ. Press, Cambridge. White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal genes for phylogenies. In PCR Protocols: A Guide to Methods and Applications (M. A. Innis, D. H. Gelford, J. J. Sninsky, and T. J. White, Eds.), pp. 315–322. Academic Press, San Diego. Winship, P. R. 1989. An improved method for direct sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res. 17: 1266.
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Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence from a Phylogenetic Analysis of Internal Transcribed Spacer Sequences
John Daugherty,* Timothy M. Evans,*,1 Tally Skillom,† Linda E. Watson,* and Nicholas P. Money*,2 *Department of Botany, Miami University, Oxford, Ohio 45056; and †Cincinnati Academy of Mathematics and Science, Hughes Center, 2515 Clifton Avenue, Cincinnati, Ohio 45219
Accepted for publication July 8, 1998
Daugherty, J., Evans, T. M., Skillom, T., Watson, L. E., and Money, N. P. 1998. Evolution of Spore Release Mechanisms in the Saprolegniaceae (Oomycetes): Evidence From a Phylogenetic Analysis of Internal Transcribed Spacer Sequences. Fungal Genetics and Biology 24, 354–363. Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axone1 Present address: Department of Biology, Hope College, Holland, MI 49423. 2 To whom correspondence should be addressed. E-mail: [email protected].
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mal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. r 1998 Academic Press
Index Descriptors: flagella; morphogenesis; stress response; zoospore. The different mechanisms of spore release that distinguish the water molds Saprolegnia Nees, Achlya Nees, Thraustotheca Humphrey, and Dictyuchus Leitgeib, within the Saprolegniaceae (Oomycetes), have been viewed as a transition series in which Saprolegnia is thought to exhibit the ancestral mechanism (Humphrey, 1893; Atkinson, 1909; Ho¨hnk, 1933). Sporangia of Saprolegnia release pyriform zoospores through a papilla at the tip of the sporangium (Fig. 1a). Each zoospore is equipped with a pair of flagella anchored in the spore apex by kinetosomes and associated microtubular roots (Holloway and Heath, 1977; Barr and De´saulniers, 1997). The spores swim at up to 110 µm s21, apex forward, following a counterclockwise helical path before collecting at the air–water interface, or adhering to a surface, and then encysting (Salvin, 1941). Later, the protoplast within each cyst is discharged onto the cyst surface, and in this position the initially spherical protoplast is modified into a reniform zoospore with laterally inserted flagella (Fig. 2). This type of spore, referred to as a secondary zoospore, exhibits much faster swimming (up to 260 µm s21) with a clockwise helical pitch 1087-1845/98 $25.00 Copyright r 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Sporangial emptying in the Saprolegniaceae. (a) Saprolegnia ferax, primary zoospores swim after release. (b) Achlya intricata, cluster of discharged spores forms at sporangial apex. (c) Thraustotheca clavata, spores encyst and spill out through ruptured lateral wall of sporangium. (d) Dictyuchus sterile, encysted spores discharged through individual exit papillae. Bars, 50 µm.
and encysts after a much longer period of motility than the primary spore (Salvin, 1941). Encystment of the secondary zoospore can then be followed by successive phases of excystment, swimming, and encystment (Die´guezUribeondo et al., 1994); when nutrients are available, the cysts germinate and a vegetative mycelium develops. The production of two distinct types of zoospore is referred to as diplanetism, and the term polyplanetism has been used to describe the repeated pattern of encystment and excystment characteristic of Saprolegnia and other genera within the Saprolegniaceae (Webster, 1980). Achlya also releases pyriform spores from its sporangia, but flagella are not visible under the light microscope, and the spores collect and encyst in a spherical cluster or ‘‘spore ball’’ at the apex of the sporangium (Figs. 1b and 2; Money and Webster, 1987). Subsequently, secondary-type zoospores emerge from the cysts. While most studies of spore release in Achlya describe nonflagellate spores
streaming out of sporangia, some of the classical descriptions reported that the spores were flagellate like those of Saprolegnia (references in Money et al., 1987). This issue was resolved when transmission electron microscopy revealed that sporangiospores of Achlya intricata and A. flagellata possess rudimentary flagella with stunted axonemes (usually ,2 µm in length) and bear some structural features homologous to the primary-type zoospores of Saprolegnia (Money et al., 1987). The earlier accounts of active flagella, based on light microscopy, suggest that other species of Achlya develop spores with longer flagella. But in all Achlya species, the spores do not swim after discharge from the sporangium. Thraustotheca produces large cylindrical or clavate sporangia in which the spores encyst before release from the sporangium. Partial emptying of the sporangium occurs when the lateral wall ruptures and some of the cysts spill out (Figs. 1c and 2). The cysts usually adhere to one
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FIG. 2.
Diagram showing asexual phases of the life cycles of the four genera shown in Fig. 1.
another, forming a loose cluster within and around the lysed wall; after a few minutes to an hour, a secondary zoospore is released from each cyst. Spores also encyst within sporangia of Dictyuchus, but the sporangium of this genus empties when a protoplast is released from each cyst through an individual papilla that projects through the sporangial wall. On the sporangial surface the discharged protoplasts are reorganized into secondary-type zoospores. After sporangial emptying, Dictyuchus can be identified by the geodesic pattern of empty cyst walls inside the sporangium (Figs. 1d and 2). In developing a transition series to link these genera, it was assumed that the different mechanisms of sporangial emptying represented useful markers for the evolutionary longevity of one genus relative to the others (Humphrey, 1893). According to this scheme, Achlya evolved from an organism that exhibited saprolegnioid sporangial empty-
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ing, and Thraustotheca and Dictyuchus evolved from an ancestor that exhibited achlyoid sporangial emptying (terminology from Johnson, 1956, and Webster, 1980). The discovery of rudimentary flagella in Achlya (Money et al., 1987) certainly supports the idea that the achlyoid mechanism was derived from a saprolegnioid ancestor rather than vice versa. Descriptions and illustrations of these fungi in contemporary mycological textbooks are rooted in this hypothesis (Esser, 1982; Moore-Landecker, 1996). The objective of the present study was to reevaluate the hypothesis of a transition series linking these genera and their spore release mechanisms. Nucleotide sequence data from the two internal transcribed spacers (ITSs)3 of 3
Abbreviations used: CI, consistency index; ITS, internal transcribed spacer; RI, retention index.
Spore Release Mechanisms in the Saprolegniaceae
nuclear ribosomal DNA were used to construct a phylogeny. An extensive literature supports the close relationship and monophyly of the four genera (Dick, 1990; Powell and Blackwell, 1998), and the efficacy of ITS-based examinations of relationships among closely related oomycete species has been demonstrated previously (Lee and Taylor, 1992; Crawford et al., 1996). Radical differences between the responses of Achlya and Saprolegnia to osmotic stress are well documented (see references in Money, 1997) and may also reflect evolutionary divergence between these organisms. Therefore, the stress response in each genus was studied in an effort to further understand evolutionary affinities. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently.
MATERIALS AND METHODS Organisms and culture conditions. The following cultures were obtained from the American Type Culture Collection (ATCC; Rockville, MD): Saprolegnia ferax (Gruithuisen) Thuret (Accession No. 36051), Thraustotheca clavata (de Bary) Humphrey (34112), and Dictyuchus sterile Coker (44891). Achlya intricata Beneke, originally obtained from the culture collection at the University of Reading, United Kingdom, courtesy of Dr. Michael Dick, is now deposited at the ATCC (66594); this species was selected for the investigation because spore release has been studied more intensively in this species than in any other member of the Saprolegniaceae (Money and Webster, 1988, and references therein). Mycelia were grown in 100-ml batches of liquid PYG medium (0.1% peptone, 0.1% yeast extract, 0.3% glucose), inoculated with 10-mm-diameter plugs from PYG agar cultures, and incubated at 24°C for 2–7 days on an orbital shaker. For the osmotic stress experiments cultures were grown on PYG agar supplemented with 600 mM sucrose, sorbitol, or maltose at 22°C for up to 14 days. Colonies were photographed with standard compound and dissecting microscopes (Olympus BH-2 and SZH-10; Olympus Optical Co., Tokyo) to document the effects of these osmolytes on morphogenesis. Sequence data and phylogenetic analysis. Fresh mycelia were harvested by vacuum filtration from liquid PYG and ground in liquid nitrogen. Total DNA was extracted using the CTAB procedure (Saghai-Maroof,
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1984; Doyle and Doyle, 1987). The entire ITS region, including the 5.8S cistron, was amplified by doublestandard PCR using primers 4 and 5 of White et al. (1990). PCR products were verified on 1% agarose gels and purified using GeneClean (Bio 101, Inc., Vista, CA) prior to sequencing. The double-stranded PCR products were sequenced directly using standard dideoxy sequencing and 6% acrylamide gel electrophoresis (Sanger et al., 1977), using the snap-chill method (Winship, 1989) and Sequenase 2.0 (U.S. Biochemical, Cleveland, OH) with 35S-dATP labeling. Strands were sequenced in both directions using the four primers (ITSs 1, 2, 3, and 4) of White et al. (1990). Phytophthora megakarya (Order Peronosporales) sequences were obtained from GenBank [Accession No. S40356 (ITS1) and S50358 (ITS2)]. Sequence boundaries were determined by comparison with published sequences (White et al., 1990; Lee and Taylor, 1992; Crawford et al., 1996) and were aligned using Clustal W, Version 1.7 (Higgins et al., 1991; Thompson et al., 1994), with some manual gap adjustments made. Both ITS spacers were used in all analyses, with the 5.8S region excluded. To assess the phylogenetic signal present in the data, the g1 statistic (Hillis, 1991) was calculated. Phylogenetic analyses that included all characters, as well as an analysis that excluded 64 characters with an ambiguous alignment (positions 464–480, 509–534, and 625–653), were conducted. Sequence divergence values were estimated with PAUP 3.1 (Swofford, 1993), using the MATRIX DISTANCE option. Parsimony analyses were conducted using Fitch Parsimony (unordered, multistate characters) with the gaps treated as missing data, using PAUP 3.1 (Swofford, 1993) with the Exhaustive Search Option and Phytophthora as the outgroup. To assess the robustness of the clades, both bootstrap and decay values were calculated. Bootstrap values were calculated with PAUP 3.1 for 100 replicates and 100 random order entries of the data using the Heuristic Search Option and TBR (tree bisection reconnection) branch swapping with MULPARS on. Decay values were calculated with the software Autodecay 3.0 (Eriksson and Wikstrom, 1995), in conjunction with PAUP 3.1. Consistency index (CI) and retention index (RI) were calculated (Kluge and Farris, 1969; Farris, 1989) for all parsimony trees. In addition to the parsimony analysis, two maximum-likelihood trees that assumed transition/transversion ratios of 1.0 and 2.0 each were constructed (Felsenstein, 1989, 1993). However, because Phytophthora was highly divergent from the ingroup, a maximum-likelihood ratio test was conducted to test alternative rootings. Topologies were constructed and input as
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TABLE 1 ITS Sequence Divergence Values Calculated Using PAUP 3.1 (Swofford, 1993)
1. 2. 3. 4. 5.
Achlya Thraustotheca Saprolegnia Dictyuchus Phytophthora
1
2
3
4
5
— — — — —
0.036 — — — —
0.256 0.363 — — —
0.252 0.256 0.327 — —
0.458 0.462 0.441 0.484 —
user trees to constrain placement of Phytophthora, and likelihoods of the constrained and most parsimonious tree were compared to determine whether these alternative rootings could be rejected (Kishino and Hasegawa, 1989).
RESULTS Sequence data. Sequences were deposited with GenBank for S. ferax (Accession Nos. AF036542/AF036543 for ITS1/ITS2), A. intricata (AF036540/AF036541), T. clavata (AF036538/AF036539), and D. sterile (AF036544/ AF036545). The aligned sequence for both spacers, excluding the 5.8S coding region, was 674 base pairs (bp) in length (the aligned data matrix is available upon request from the corresponding author). The aligned length for
ITS1 was 248 bp and varied from 186 to 219 bp when excluding gaps that were inserted to achieve alignment. The aligned length of ITS2 was 375 bp and ranged from 319 to 336 bp excluding gaps. Sequence divergence estimates for the entire dataset ranged from 4 to 36% for the ingroup and 44 to 48% between the ingroup and the outgroup (Table 1). The g1 statistic was 20.58 for 10,000 random-generated trees, suggesting a relatively strong phylogenetic signal in this dataset. In the outgroup-rooted parsimony analysis, a single most parsimonious tree of 465 steps (CI 5 0.938, RI 5 0.662) was produced (Fig. 3). Saprolegnia was the most basal taxon, sister to the remaining three genera. Bootstrap and decay values indicate strong support for the sister group relationship between Achlya and Thraustotheca (100% bootstrap support, 21 decay index value) and strong support for the placement of Dictyuchus at their base (88% bootstrap support, 7 decay index value). The remaining nodes were unsupported by either measure. When 64 ambiguous characters were excluded [positions 464–480, 509–534, and 625–653; 9% (64/674) of the dataset], a single most parsimonious tree resulted, identical to the tree that included all characters. The CI (0.941) was slightly higher with these characters excluded, and the overall length was shorter (389 steps). The maximumlikelihood trees were identical to the parsimony tree, with no differences in topologies produced by either weighting scheme using transition/transversion ratios of 1.0 or 2.0.
FIG. 3. Single most parsimonious tree of 465 steps, based on ITS sequence data using Phytophthora as the outgroup (CI 5 0.938, RI 5 0.062). Numbers above the line are branch lengths; percentages below the line are bootstrap values and are followed by decay index values (in parentheses).
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Spore Release Mechanisms in the Saprolegniaceae
The likelihood ratio test resulted in two alternative rootings that could not be rejected, with the first joining Phytophthora and Dictyuchus and the second placing Dictyuchus and Saprolegnia on a single branch as sister taxa to each other. Responses to osmotic stress. On unsupplemented PYG agar medium, all four species produced a richly branched coenocytic mycelium, with the proliferation of aerial hyphae and extensive invasive growth through the entire depth of the solid medium. When grown on medium supplemented with 600 mM osmolytes, the appearance of all four species was altered. The most subtle changes occurred in Saprolegnia and have been described previously (Money, 1997). Briefly, normal polarized extension was sustained on the surface of the agar medium, but invasive growth and aerial growth were inhibited by the resulting loss of turgor pressure (Fig. 4a). There was no
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polarized hyphal growth in A. intricata and the mycelium produced enlarged outgrowths that spread slowly over the agar surface (Fig. 4b); this growth form has been described previously in A. bisexualis and A. ambisexualis and was referred to as plasmodium-like (Money, 1997; Money and Hill, 1997). The stress response of Thraustotheca and Dictyuchus had not been studied previously. Polarized growth was inhibited in both Thraustotheca and Dictyuchus, and large swollen spherules (up to 200 µm in diameter) piled up on medium supplemented with sucrose, sorbitol, or maltose (Figs. 4c and 4d). Plasmodiumlike outgrowths extended from the edges of these colonies where the cells were in direct contact with the agar surface. However, in contrast to Achlya, tip-growing hyphae developed from the masses of spherules in some of the cultures after more than a week of growth at 22°C (Fig. 4d). This reversion to polarized growth occurred frequently on
FIG. 4. Appearance of Saprolegniaceae on medium supplemented with osmolytes. (a) Tip-growing hyphae of Saprolegnia on surface of PYG 1 600 mM sorbitol. (b) Plasmodium-like colony of Achlya on PYG 1 600 mM sorbitol. (c) Spherules of Thraustotheca on PYG 1 600 mM sucrose. (d) Spherules and emerging hyphae of Dictyuchus on PYG 1 600 mM sorbitol. Bar (a,d) 100 µm; (b,c) 500 µm.
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sorbitol in both Dictyuchus and Thraustotheca, but in neither case did the hyphae invade the agar. Therefore, the morphogenetic responses of Thraustotheca and Dictyuchus to osmotic stress share features in common with Achlya (depolarized growth) and with Saprolegnia (polarized). The physiological behavior underlying these morphological changes, such as changes in turgor and endoglucanase secretion (Money and Hill, 1997), was not examined.
DISCUSSION Relationships within the Saprolegniaceae. Achlya and Thraustotheca have an ITS sequence similarity of 96%, and both parsimony and maximum-likelihood analyses suggest that they are sister taxa, more closely related to each other than either is to Dictyuchus or Saprolegnia. The parsimony and maximum-likelihood analyses of the sequence data also suggest that Dictyuchus shares a more recent common ancestor with Achlya and Thraustotheca than with Saprolegnia. The trees place Dictyuchus on a separate evolutionary branch from that of Achlya and Thraustotheca, with Saprolegnia in the most basal position. The only alternative rootings that could not be rejected by the likelihood ratio test rooted Phytophthora to either Dictyuchus or the common ancestor of a Dictyuchus– Saprolegnia clade. Therefore, the interpretation of Saprolegnia as plesiomorphic is both conservative and robust. The morphogenetic response to osmotic stress exhibited by Achlya results from disruption to the normal process of cell wall assembly (Money and Hill, 1997), and the same mechanisms probably govern the behavior of Thraustotheca and Dictyuchus. The fact that wall assembly is dependent upon the expression of multiple genes improves the likelihood that these physiological comparisons may accurately reflect evolutionary relationships. This possibility is strengthened by the view that the stress responses are not adaptive mechanisms with ecological significance (and therefore subject to intense selection), but reflect constitutive differences between the mechanisms of wall assembly in each taxon (this has been demonstrated in Saprolegnia and Achlya; Money, 1997). If, as the ITS data suggest, Saprolegnia is the basal taxon of the ingroup, the maintenance of polarized hyphal extension under conditions of osmotic stress may represent an ancestral character state. It is interesting that the other three genera are united by the loss of hyphal extension under stress, which is consistent with the ITS parsimony tree that places Achlya, Thraustotheca, and Dictyuchus in a monophyletic clade.
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Daugherty et al.
Vestiges of the Saprolegnia response appear to be expressed by Thraustotheca and Dictyuchus when they revert to hyphal extension after more than a week of sustained exposure to high concentrations of osmolytes. Monophyly and algal affinities. Pringsheim (1858) was the first to recognize morphological similarities between Saprolegnia and the alga Vaucheria, but analysis of 18S rRNA gene sequences of a wide range of chromonad and xanthophycean algae and three other oomycete fungi (Achlya bisexualis, Lagenidium giganteum, and Phytophthora megasperma) showed that Vaucheria is only distantly related to the Oomycetes (Potter et al., 1997). Based on these data, Potter and colleagues questioned the oomycete nature of Saprolegnia, but did not analyze the 18S rRNA sequence of this genus. Our ITS data support the inclusion of Saprolegnia in the Oomycetes, based on comparative sequence divergence values and alignability of the ingroup taxa. While the divergence value between Saprolegnia and the remaining ingroup taxa is relatively high (25%), it is comparable to divergence values between Dictyuchus and the other genera. The only divergence value that is lower than 25% is between Achlya and Thraustotheca (4%). Evolutionary suppression of the primary-type zoospore. The relatively slow swimming velocities of primarytype zoospores (Salvin, 1941) coupled with the rarity of this spore type among the Oomycetes have led to the suggestion that the primary zoospore is more primitive than the secondary-type spore (this was most recently stated by Dick, 1990). It has been argued that suppression of the primary spore would conserve the finite quantity of food reserves (packaged during sporangial development) for the faster secondary zoospores that represent more effective vehicles for dispersal. The basal position of Saprolegnia in the ITS comparisons supports Humphrey’s century-old assertion that the diplanetic life cycle has been condensed during the evolution of the Saprolegniaceae (Humphrey, 1893), with the ultimate elimination of the primary spore stage in Dictyuchus. Likewise, the widely accepted hypothesis of flagellar suppression during the evolution of the primary spores of Achlya, rather than flagellar acquisition in Saprolegnia, is now supported by molecular phylogenetic data. Speculation on evolution of different mechanisms of sporangial emptying. Sporangial differentiation in the Saprolegniaceae follows a prescribed sequence of developmental events beginning with the cessation of hyphal elongation and ending with different patterns of wall lysis that initiate spore release (Gay and Greenwood, 1966;
Spore Release Mechanisms in the Saprolegniaceae
Armbruster, 1982a,b; Money and Brownlee, 1987). This type of rigid developmental sequence is an example of a coordinately expressed program, in which each in a succession of discrete processes occurs only upon the completion of the previous step in the program (Lawson and Poethig, 1995). For example, lysis of the sporangial wall occurs only after the completion of cleavage (in A. intricata there is a consistent lag of 8 to 10 min at 22°C between cleavage and emptying; Money and Brownlee, 1987). In Achlya, sporangial development is dependent upon the expression of many genes that are also required for vegetative growth, in addition to the transcription of a smaller suite of sporulationspecific sequences (Gwynne and Brandhorst, 1982). Synthesis of RNA and proteins continues throughout sporangial development (Griffin and Breuker, 1969), and rapid turnover of existing proteins supplies the necessary amino acids to support high levels of protein synthesis (Timberlake et al., 1973). However, after cleavage, the spore release mechanism itself may simply require the synthesis of the enzyme(s) necessary for lysis of the apical wall or papilla of the sporangium (Ross, 1979). Therefore, a small number of mutations affecting the expression of these enzymes could have profound effects on sporangial ontogeny. Since Saprolegnia is the basal genus in our comparison, it is possible that its mechanism of spore release is plesiomorphic to the other three types. In both Saprolegnia and Achlya, spores are driven out of the sporangium a
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few minutes after cleavage by osmotically generated hydrostatic pressure (Money and Webster, 1988, 1989). In Achlya, the spore ball is produced by adhesion between the spores as they are ejected from the sporangium and begin to encyst (Money and Webster, 1987). Adhesion and encystment also occur in Saprolegnia, but only after the primary zoospores have dispersed from the sporangium. Therefore, a primary difference between saprolegnioid and achlyoid emptying is a reduction in the time interval between cleavage and encystment of the spores. It seems plausible that the condensation of the life cycle observed in Achlya is a secondary consequence of mutations that suppressed axonemal elongation. Pursuing this line of inquiry, the thraustothecoid and dictyuchoid patterns of spore release can be linked to a basal saprolegnioid mechanism by coupling the same inhibition of flagellar development to changes in the temporal and spatial expression of lytic enzymes that degrade the sporangial wall (Fig. 5). Inhibition of both flagellar development and papillar lysis, accompanied by a more diffuse pattern of wall lysis, would convert an organism from a saprolegnioid to a thraustothecoid mechanism. Finally, inhibition of flagellar development and suppression of lysis of the sporangial wall, coupled with further acceleration of the encystment process (inducing encystment immediately after cleavage), could create the cyst-filled sporangium primed for dictyuchoid emptying.
FIG. 5. Hypothetical scheme showing possible developmental relationships that link four mechanisms of sporangial emptying. Minimum changes necessary to derive achlyoid, thraustothecoid, and dictyuchoid sporangial emptying from an ancestral saprolegnioid condition are mapped onto the phylogenetic topology suggested by the parsimony tree in Fig. 3.
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ACKNOWLEDGMENTS The authors acknowledge the generous support of the Miami University Committee on Faculty Research, the Miami University Summer Scholars Program, the CAMAS Scholars Program, and the National Science Foundation (Grant DEB 9596274 to L.E.W.).
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