Species boundaries within Saprolegnia (Saprolegniales ... - Mycologia

Mycologia, 99(3), 2007, pp. 421–429. 

# 2007 by The Mycological Society of America, Lawrence, KS 66044-8897 

Species boundaries within Saprolegnia (Saprolegniales, Oomycota) based on 

morphological and DNA sequence data 

Jonathan P. Hulvey 

David E. Padgett 

J. Craig Bailey1 Department of Biology and Marine Biology, University 

of North Carolina Wilmington, 601 S. College Road, 

Wilmington, North Carolina 28403 

Abstract: Saprolegnia is a common and widespread 

genus of Oomycetes, however species identifications 

are difficult and uncertain. To test whether keys based 

on morphological characters could identify species as 

determined by molecular characters we determined 

partial DNA sequences for the 28S rRNA gene and 

the complete internal transcribed spacer (ITS) region 

for 55 isolates belonging to Saprolegnia and one 

isolate of Protoachlya hypogyna that exhibited saprolegnoid 

zoospore discharge in water culture. Phylogenetic 

analyses of the combined sequence data 

yielded 10 robustly supported clades that probably 

represent separate species. Morphological analyses of 

all isolates revealed that each DNA-based clade could 

be delimited from others by autapomorphic or 

unique combinations of morphological character 

states but not without employing several features 

previously not used at the species level. Taxonomic 

implications of these results are discussed and 

recommendations for less equivocal characterization 

of new Saprolegnia species are made. 

Key words: morphology, phylogeny, systematics, 

watermold 

INTRODUCTION 

Species of the oomycetous family Saprolegniaceae, 

commonly referred to as ‘‘watermolds’’ (history of 

term recounted in Johnson et al 2002), occur 

worldwide and can be isolated from freshwater and 

nonsaline soils with relative ease. Representatives are 

most notably characterized by the production of 

coenocytic, filamentous thalli, biflagellate, heterokont 

zoospores, and oogamous sexual structures. 

Several genera include significant pathogens of fishes 

(Willoughby 1997, Paxton and Willoughby 2000), 

crustaceans (Westman 1991, Vennerstrom et al 1998), 

midge eggs (Martin 1981), mosquito larvae (Seymour 

1984) and at least two important agricultural plants 

Accepted for publication 9 January 2006. 

1 Corresponding author. E-mail: baileyc@uncw.edu 

421 

(Wicker et al 2001, Yokosawa et al 1988, Pilet-Nayel et 

al 2002, Ramirez et al 1994, Peters and Grau 2002), 

but most included species probably are obligate 

saprotrophs. 

Kützing (1843) formally erected the Saprolegniaceae 

but considered representatives to be nonchlorophyllous 

algae. The first English language monograph 

of the family was published by Humphrey 

(1893) and revised by Coker (1923). Subsequently 

Moreau and Moreau (1935, 1938), Coker (1935), 

Dick (1969, 1972, 1978, 2001), Miller and Ristanović 

(1975), Powell and Blackwell (1998) and Padgett et al 

(2000) have made observations about watermold 

systematic concepts most of which emphasize the 

significant morphological plasticity within the family. 

Johnson et al (2002) offered an extensive systematic 

revision of the group recognizing 17 genera and 122 

species but agreed that significant species overlap 

persists because of this plasticity. 

Few investigators worldwide specialize in watermold 

systematics although several laboratories have presented 

molecular phylogenies of the family or of the 

class Peronosporomycetes to which it belongs (e.g. 

Dick 1999, Riethmueller et al 1999, Leclerc et al 2000, 

Peterson and Rosendahl 2000, Spencer et al 2002, 

Daugherty et al 1998, Hudspeth et al 2000, Inaba and 

Tokumasu 2002). To our knowledge however there 

have been no previous assessments of the degree to 

which gene sequence analyses might contribute to 

resolving the species overlap problem that has grown 

out of the use of morphological features as the sole 

basis for species circumscription. 

The present contribution focuses exclusively on 

isolates belonging to the genus Saprolegnia as determined 

by zoosporangial discharge pattern. Its sole 

objective is to determine whether all isolates of an 

inferred phylogenetic genetic clade belong to the 

same morphological species as determined by modern 

dichotomous keys ( Johnson et al 2002). If the 

trees derived from our sequence alignment are 

robustly supported, it seems logical to assume that 

all isolates belonging to the same clade should be 

recognized as a single (or ‘‘good’’) phylogenetic 

species. Thus this manuscript tests the congruency 

between modern molecular analyses and classical 

morphological identification for Saprolegnia species. 

Partial 28S ribosomal RNA (28S rRNA) gene 

sequences and complete ribosomal internal transcribed 

spacer (ITS) sequences were determined for

422 MYCOLOGIA 

TABLE I. Source and sequence accession numbers of isolates studied. Isolates designated as ‘sp.’ reproduced only asexually 

under the present culture conditions. Some ATCC or CBS isolates did not key to the same species as given by the source 

organization. For those the original identification is indicated by * and our identification is indicated by **. 

UNCW 

No. 

ATCC or CBS 

No. Species Collection locale 

GenBank accession No. 

28S rRNA ITS 

105 — S. megasperma Soil, New Hanover County, NC DQ393440 DQ393506 

114 — Saprolegnia sp. Fish, Pamlico River, NC DQ393441 DQ393507 

162 — S. megasperma Carolina Biological Supply DQ393442 DQ393508 

170 — Saprolegnia sp. Soil, New Hanover County, NC DQ393443 DQ393509 


176 — S. diclina Soil, New Hanover County, NC DQ393445 DQ393511 

217 — S. diclina Soil, New Hanover County, NC DQ393447 DQ393513 



250 — Saprolegnia sp. Soil, Croatan Nat. Forest, NC DQ393451 DQ393517 

251 — Saprolegnia sp. Soil, Croatan Nat. Forest, NC DQ393452 DQ393518 

253 — S. terrestris Soil, Croatan Nat. Forest, NC DQ393453 DQ393519 

254 — S. megasperma Soil, Croatan Nat. Forest, NC DQ393454 DQ393520 

257 — S. megasperma Soil, Croatan Nat. Forest, NC DQ393455 DQ393521 

258 — S. megasperma Soil, New Hanover County, NC DQ393456 DQ393522 

259 — S. richteri Soil, Great Smoky Mtns. Nat. Pk., NC DQ393457 DQ393523 

260 — S. luxurians Soil, Great Smoky Mtns. Nat. Pk., NC DQ393458 DQ393524 

263 ATCC 26116 S. ferax* S. litoralis** Pond water, California DQ393460 DQ393527 

264 ATCC 22284 S. parasitica* Saprolegnia sp.** Catfish, Virginia pond DQ393461 DQ393528 

266 ATCC 10396 S. ferax* Saprolegnia sp.** DQ393462 DQ393529 

269 ATCC 36146 S. ferax* Saprolegnia sp. ** Water, England DQ393463 DQ393530 

271 ATCC90213 S. parasitica* Saprolegnia sp. ** DQ393464 DQ393531 

273 ATCC 200013 S. parasitica* Saprolegnia sp. ** Fish, Japan DQ393466 DQ393532 

277 — Saprolegnia sp. Crayfish, Odum, GA DQ393467 DQ393533 

278 — Saprolegnia sp. Pet store fish, Wilmington, NC DQ393468 DQ393525 

280 — S. itoana Soil, Fayetteville, NC DQ393469 DQ393534 

283 — S. megasperma Creek bank, Wayne County, GA DQ393470 DQ393535 

284 — S. megasperma Water, Wayne County, GA DQ393471 DQ393536 

286 — S. megasperma Water, Wayne County, GA DQ393473 DQ393536 

288 — Saprolegnia sp. Water, Wayne County, GA DQ393474 DQ393537 

289 — Saprolegnia sp. Soil, Okefenokee Swamp, GA DQ393475 DQ393538 

290 — Saprolegnia sp. Water, Wayne County, GA DQ393476 DQ393539 



295 ATCC 36144 S. diclina* Saprolegnia sp. ** Water, England DQ393478 DQ393541 



300 — Saprolegnia sp. Soil, Italy DQ393489 DQ393551 

301 CBS 261.34 Pythiopsis cymosa United Kingdom DQ393490 DQ393552 

309 CBS 158.45 Protoachlya paradoxa* Water, Netherlands DQ393491 DQ393553 

312 — Saprolegnia sp. Soil, Richmond, VA DQ393481 DQ393544 



320 — S. megasperma Soil, Great Smoky Mtns. Nat. Pk., NC DQ393485 DQ393547 

328 — Saprolegnia sp. Pet store goldfish, Wilmington, NC DQ393486 DQ393548 

329 — Saprolegnia sp. Frog, Wilmington, NC DQ393487 DQ393549 

336 ATCC 28092 Protoachlya polysporus* Water, New Jersey DQ393492 DQ393554 

337 ATCC 44892 Protoachyla paradoxa* Pond water, Netherlands DQ393493 DQ393555 

372 CBS 551.62 S. eccentrica* S. richteri** Soil, England DQ393494 DQ393556 

373 — S. diclina Soil, Wilmington, NC DQ393495 DQ393557 

374 CBS 535.67 S. litoralis* Saprolegnia sp. ** Soil, England DQ393496 DQ393558 

375 CBS 213.35 S. unispora* Saprolegnia sp. ** United Kingdom DQ393497 DQ393559

TABLE I. Continued 

UNCW 

No. 

56 isolates referable to Saprolegnia. The combined 

data were analyzed yielding phylogenetic trees onto 

which we mapped sequence divergence values and 

morphological character states. Results suggest that 

the DNA-based phylogenetic hypothesis provides 

a useful framework for re-interpreting the taxonomic 

utility of vegetative and reproductive characters for 

distinguishing among Saprolegnia species. This more 

objective approach, in which both molecular and 

morphological data are employed, may provide 

a method for ultimately establishing nonoverlapping 

generic and species descriptions (and keys) for other 

watermold taxa as well. 

MATERIALS AND METHODS 

Culture propagation and morphological analyses.—Isolates 

employed in this study were derived from nonsaline 

soils collected principally from the southeastern United 

States or were purchased from the American Type 

Culture collection (ATCC) or the Centraalbureau voor 

Schimmelcultures (CBS) (TABLE I). Before morphological 

analysis or DNA extraction was attempted all isolates were 

processed to derive axenic, single spore or single hyphal tip 

cultures. 

Replicate cultures (10 for each isolate) used for morphological 

identifications were initiated by placing autoclaved, 

shelled hemp seeds on the growing edge of colonies 

propagated on Difco cornmeal agar (CMA). After a 24 h 

infestation period, single hemp seeds were transferred to 

disposable plastic Petri dishes filled with 25 mL of sterile 

distilled water. Water culture dishes were wrapped individually 

in Parafilm TM to prevent contamination during 

room temperature incubation and maintained until sacrificed 

to observe morphological characters. Wherever 

possible identifications were based on 50 replicate determinations 

of all morphological features studied ( Johnson et al 

2002). 

To preclude the possibility that individual primary 

sporangia might exhibit different patterns of sporangial 

dehiscence (Padgett and Johnson 2004), zoospore discharge 

was observed (and videotaped) from 10 separate 

HULVEY ET AL: SPECIES BOUNDARIES WITHIN SAPROLEGNIA 423 

ATCC or CBS 

No. Species Collection locale 

GenBank accession No. 

28S rRNA ITS 

377 CBS 618.97 S. polymorpha* Saprolegnia sp. ** Fish DQ393498 DQ393560 

380 CBS 542.67 S. furcata* Saprolegnia sp.** United Kingdom DQ393499 DQ393561 

381 CBS 386.52 S. megasperma * Saprolegnia sp. ** Soil, New York DQ393500 DQ393562 

382 CBS 109568 S. semi-hypogyna* S. luxurians** DQ393501 DQ393563 

383 CBS 278.52 S. subterranea* S. itoana** Mud, New York DQ393502 DQ393564 

384 CBS 305.37 S. ferax* Saprolegnia sp. ** France DQ393503 DQ393565 

345 CBS 534.67 S. ferax* Saprolegnia sp. ** Lake water, England DQ393504 DQ393566 

386 CBS 110064 S. torulosa* S. asterophora ** Soil, Japan DQ393505 DQ393567 

primary sporangia for each isolate. Discharges were 

observed on 24–48 h old, intact colonies floating in 

Parafilm-sealed culture dishes with an Olympus SZX 12 

stereo microscope equipped with a fiber-optic illuminator 

and an Hitachi CE digital camera. 

Subsequent to zoospore discharge determination, all 

replicate water cultures were individually sacrificed through 

time (over 14 d) for observing diagnostic sexual characters 

as per Johnson (1956) and Seymour (1970). These 

observations were made with an Olympus BX 41 phase 

contrast microscope and derived data recorded in specially 

designed MS Excel TM electronic spreadsheets (Ferner 

unpublished) for later analysis. Measurements of various 

quantitative features (e.g. zoospore cyst diameter, oogonial 

and oospore diameters) were made with ImagePro TM 

computer software. 

DNA isolation, PCR amplification and sequencing.—Mycelia 

for DNA extraction were propagated in glucose, yeast 

extract (GY) broth containing Difco D-glucose 

(10.0 g L 21 ) and Difco Yeast Extract (2.5 g L 21 ). Log phase 

cultures were harvested aseptically, blotted on sterile filter 

paper, transferred to sterile Whirl Paks TM and immediately 

frozen at 220 C. 

Frozen mycelia were ground with a mortar and pestle or 

disrupted via sonification in 2 3 CTAB buffer and total 

cellular DNA extracted as described in Bailey et al (1998). A 

portion of the 28S rRNA gene, including hypervariable stem 

and loop regions between helices C1 and D2 (Ben Ali et al 

1999), was amplified with primers C1 (59-ACCCGCTGATT- 

TAAGCAT-39) and D2 (59-TCCGTGTTTCAAGACGG-39) 

(Leclerc et al 2000). The entire ITS region was amplified 

with primers ITS1 (59-TCCGTAGGTGAACCTGCGG-39) and 

ITS4 (59-TCCTCCGCTTATTGATATGC-39) (White et al 

1990). The thermocycling profile included an initial 

denaturation step at 94 C for 3 min, followed by 35 cycles 

of denaturation at 94 C for 30 s, primer annealing at 50 C 

for 30 s and primer extension at 72 C for 1.5 min, a final 

extension step for 7 min at 72 C and a 4 C soak. Amplified 

products were checked for correct length and yield on 0.8% 

ethidium bromide-stained agarose gels and purified with 

the GeneClean II Kit (Qbiogene, Carlsbad, California). PCR 

products were sequenced on both strands with the 

amplification primers and BigDye Terminator cycle se-

424 MYCOLOGIA 

quencing kit (v. 2, Applied Biosystems, Foster City, 

California) according to the manufacturer’s specifications. 

Sequences were determined on an ABI3100 automated 

DNA sequencer (Applied Biosystems, Foster City, California) 

and electropherograms were edited and assembled 

with Sequence Analysis (v. 1.0.1) and Sequence Navigator 

(v. 3.4.1) programs (Applied Biosystems, Foster City, 

California). 

Phylogenetic analyses.—Sequences were aligned by eye in 

SeqApp (Gilbert 1994). The model of nucleotide substitution 

that best fit our data was determined with 

Modeltest (v. 3.06, Posada and Crandall 1998) and a general 

time reversible model with invariant sites and rates of 

substitution among sites approximated by a gamma distribution 

(5GTR + I + G) was selected. This substitution 

model and neighbor joining (NJ), parsimony (MP), and 

maximum likelihood (ML) algorithms were used to 

construct trees in PAUP (v. 4.0b10, Swofford 2001). 

Heuristic parsimony searches were conducted with TBR 

branch swapping with gaps treated as missing data and 

random orders of sequence addition (5100). The ML tree 

was inferred with the heuristic search option and random 

orders of sequence addition (510). 

Pythiopsis had been resolved as sister of Saprolegnia based 

on analyses of 28S rRNA + ITS data (Leclerc et al 2000) and 

mitochondrial cox2 gene sequences (Hudspeth et al 2000) 

and therefore was used to root the saprolegnialean trees. 

Support for nodes of the trees was assessed by calculating 

bootstrap proportion values (Felsenstein 1985). Bootstrap 

values for the NJ and MP trees were based on analyses of 

10 000 pseudoreplicate datasets whereas the ML values were 

derived from 60 replicates. 

RESULTS 

Nucleotide sequences determined in this study have 

been submitted to GenBank (TABLE I). The combined 

sequence data matrix (28S rRNA + ITS) included 1678 

characters. All saprolegnialean sequences analyzed 

differed from one another by at least one substitution; 

the maximum number of substitutions between any 

two ingroup isolates was 153. Neighbor joining 

resulted in a single tree whereas the MP analysis 

yielded 7206 equally parsimonious trees of 728 steps 

(CI 5 0.64, RI 5 0.86). Of 1678 total characters 278 

(16.6%) were found to be parsimony informative. The 

50% majority rule parsimony consensus tree, the NJ 

tree and the ML tree were consistent with one another 

differing only with respect to relationships inferred 

among some terminal taxa within clades. However the 

number of clades resolved, the composition of the 

clades and their branching order were identical in all 

three analyses; for these reasons only the ML tree is 

presented (FIG 1). 

Saprolegnia isolates were divided among 10 clades 

each supported by robust bootstrap values (FIG. 1) 

although the branching order among clades 1, 2, 3 

and 4 could not be resolved. While the bootstrap 

value for one clade in the ML analysis was 78%, the 

remaining values were above 92% and all values for 

the 10 clades in the distance analysis were 100% 

(FIG. 1). No correlation was found between the 

number of isolates placed in a clade and the number 

of pairwise substitutions observed among those 

isolates (r 2 520.02437, p , 0.4012). The maximum 

mean number of substitutions observed among 

isolates within any of the 10 terminal clades was 10.7 

(clade 4) whereas a single substitution distinguished 

isolates SAP280 and SAP383 (clade 6). These data 

(FIG. 1) together with morphological information 

were used to justify separate recognition of clades 

7 vs. 8 as well as 9 vs. 10. For example mean 

numbers of substitutions between members of 

clades 7 and 8 (520.7) are nearly twice that observed 

among isolates placed in the most heterogeneous 

terminal clade (clade 4) (510.7). Similarly the mean 

number of substitutions between members of 

clades 9 and 10 was nearly three times that observed 

in clade 4. 

Two isolates (SAP 374 and SAP 380) did not fit 

within other bi- or multi-isolate clades thus are 

assumed to represent unique taxa. In addition unique 

(autapomorphic) morphological characters circumscribed 

clades 4, 8 and 9. 

Inspection (TABLE II) clearly reveals that, for 

almost all clades, included isolates did not all key to 

the same morphological species when identified by 

the keys presented in Johnson et al (2002). Furthermore 

in several instances the isolates did not key to 

the same species as indicated by the original culture 

source. Thus the assumption of complete congruency 

between gene sequence analysis and morphological 

identification must be rejected. Furthermore identification 

of species corresponding to each genetic 

clade using compiled morphological characters of all 

clade members does not yield different names for 

each clade (TABLE II). 

We analyzed our morphological data considering 

not only traditional sexual characters but also some 

zoosporangial characters in an effort to determine 

whether clades could be resolved from one another 

with a combination of morphological features. Results 

of this analysis are presented (FIG 2). We found that if 

we first separated species by what kind of discharge 

papilla formed on the zoosporangia we could remove 

clade 8. It is noteworthy that both members of clade 8 

exhibited long and prominently flared zoosporangial 

discharge papillae (FIG. 3) that were unique to this 

clade. The fact that resolution of numbered clades 

could not be accomplished without use of this and 

other zoosporangial characteristics (FIG. 2) represents 

a new finding for this genus.


FIG. 1. Maximum likelihood phylogram depicting phylogenetic relationships inferred among 56 saprolegnoid isolates, 

based on combined analysis of 28S rRNA and ITS DNA sequence data. The 10 terminal clades are numbered and denoted by 

dots. Bootstrap values (.70%, L5 maximum likelihood, P 5 parsimony, D 5 distance) are presented to the right of each 

numbered clade and above corresponding internal nodes.

426 MYCOLOGIA 

TABLE II. Best fit identifications of sexually reproducing clade members based on sexual morphological characteristics only 

Clade Isolate number 

DISCUSSION 

The robust bootstrap support for all 10 clades (FIG. 1) 

supports the conclusion that they represent distinct 

species. Our finding that not all members of a given 

clade keyed out to the same morphological species 

was not unexpected. The latter finding leaves no 

alternative to the conclusion that Saprolegnia species 

cannot be circumscribed solely on the basis of sexual 

features. 

Probably our most important finding was that, 

when we considered the compiled morphological 

characteristics for an entire clade, we keyed several 

different clades to the same species (TABLE II). 

Identifications based on sexual and zoosporangial 

characters that were unique to each clade did permit 

resolution to species (FIG. 1). This represents a new 

finding that should mandate use of unique features 

for circumscription not only of newly proposed 

species but also recircumscription of currently recognized 

species. 

This conclusion, that species can be identified only 

of isolate 

Best fit morphological identification 

Based on compiled characteristics of 

all clade isolates 

1 263 S. litoralis S. megasperma 

373 S. diclina 

2 283 S. megasperma S. ferax 

295 S. asterophora 

253 S. terrestris S. megasperma 

3 269 S. diclina 

284 S. megasperma 





176 S. diclina 


4 320 S. megasperma S. megasperma 

5 217 S. diclina S. diclina 

6 280 S. itoana S. itoana 

383 S. itoana 

257 S. megasperma S. megasperma 

7 258 S. megasperma 

386 S. asterophora 

8 260 S. luxurians S. richteri 

259 S. richteri 

9 372 S. richteri S. richteri 

382 S. luxurians 

292 S. megasperma S. megasperma 

289 ? 

10 291 S. subeccentrica 


using a combination of unique sexual and zoosporangial 

characters, agrees with Dick’s (1973) contention 

that a revised multivariate analytical approach 

likely would be needed for meaningful systematic 

revision of the watermolds. Indeed he argued that 

knowledge of the family could advance only after this 

is done. 

To aid this daunting task of systematic revision we 

offer these suggestions: (i) Species circumscription 

for Saprolegnia can be done best using the compiled 

sexual and zoosporangial features for all members of 

a clade. This can be done however only after clade 

membership is determined on the basis of comparative 

sequence analysis (i.e. DNA data for new isolates 

first must be compared with those previously deposited 

in GenBank [e.g. TABLE I]). This conclusion 

necessarily implies that the type species of Saprolegnia 

taxa should be redefined and reference to DNA 

sequence data should be included in the new 

diagnoses (Bailey et al 1998, Deane et al 1998, 

Guillou et al 1999, Andersen et al 2002, Moro et al 

2002, Hoef-Emden and Melkonian 2003). With this

FIG. 2. Key based on morphological characters to 

phylogenetic clades within Saprolegnia. 

method any new or previously studied isolate can be 

described or identified to species regardless of 

whether that particular isolate produces characteristic 

zoosporangial or sexual features (Lange et al 2002, 

Hoef-Emden and Melkonian 2003, Marin et al 2003). 

(ii) Whereas replicate character state determinations 

for sexual features of Saprolegnia isolates often are 

not normally distributed (our unpublished data), 

sample sizes must be sufficiently large for each 

taxonomically significant character (we suggest n 5 

50) to render dependable isolate characterization 

before new taxa are described. (iii) Whereas historically 

descriptions of Saprolegnia species have employed 

ambiguous terms such as ‘‘predominant’, 

‘‘frequent’’, ‘‘occasional’’ and ‘‘rare’’, we propose 

that all listing of sexual and zoosporangial features of 

a proposed taxon be made using percentage occurrence 

values for each morphological feature based on 

a minimum of n 5 50 replicate determinations. (iv) 

Our analyses of other watermold genera (unpublished) 

indicate morphological plasticity equal to or 

greater than that presented here for Saprolegnia. We 

suggest therefore that these guidelines be used for 

revising all genera of the family Saprolegniaceae. 

Results (TABLE II) suggest to us that assignment of 

unequivocal specific epithets to phylogenetic clades at 


FIG. 3. Micrographs of typical sporangial discharge 

papillae. a, b. Prominent, long and flared papillae (arrows) 

unique to clade 8. c, d. Typical papillae of other clades.

428 MYCOLOGIA 

this date would be unwise. The extreme divergence of 

our findings from classically accepted systematic 

criteria for Saprolegnia suggests that taxonomic 

criteria for this genus, and probably all others 

belonging to the Saprolegniaceae, must be comprehensively 

reevaluated. For these reasons we have 

elected not to devise any temporary scheme of 

classification at this time. 

ACKNOWLEDGMENTS 

We gratefully acknowledge financial support provided by 

the National Science Foundation grant DEB 0328316 

(PEET program). Raymond R. Stone, Maris Durako and 

Zhuoer Lin provided assistance with morphological characterizations, 

and Melissa Smith assisted with preparation of 

FIG. 3. Dr T.W. Johnson Jr. (Duke Univ., retired) kindly 

provided advice on the morphological identifications, and 

critical comments on the manuscript. 

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Species boundaries within Saprolegnia (Saprolegniales ... - Mycologia

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