Mycoioia, 100(2), 2008, pp. 171-180. 2008 by The Mycological Society of America, Lawrence, KS 66044-8897 Issued 3 June 2008

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

Jill E. Petrisko Key words: Achiya, amphibian decline, egg, lake, Department of Biological Sciences, Idaho .SIate Leptolegnia, University, Pocatello, Idaho 83209 , Saprolegnia /irax, S. semihypo- gy na Christopher A. Pearl USGS Forest and Rangeland Ecosystem Science center, 3200 SWJe/jerson Way, Corvallis, Oregon 97331 INTRODUCTION David S. Pilliod The (, Oomycota) be- long to kingdom Chromista (sensu Aldo Leopold Wilderness Research Institute, USDA Cavalier-Smith Forest Service Rocky Mountain Research Station, 1997). These organisms often are referred to as Missoula, Montana 59801 oornycetes or "water molds" and can be found on Peter P. Sheridan decaying animal and plant debris in freshwater habitats worldwide. They can occur on adult fish and on the Charles F. Williams eggs of fish and amphibians (Hoffman 1967, Czeczuga Charles R. Peterson et al 1998). The family Saprolegniaceae has 19 genera Department oJ Biological Sciences, Ida/to State University, Pocatello, Idaho 83209 and roughly 150 (Dick 1973). Identification of Saprolegniaceae traditionally has R. Bruce Bury relied on the observation of morphological features USGS Forest and Rangeland Ecosystem Science center, (Seymour 1970, but see Hulvey et al 2007). Genera of 3200 .SWJe[Jersoii Way, Corvallis Oregon 97331 the Saprolegniaceae have been differentiated by their method of zoospore release (Seymour 1970, Daugh- erty et al 1998). Species identification has been more Abstract: We assessed the diversity and phylogeny of challenging because it has required presence of the Saprolegniaceae on amphibian eggs from the Pacific sexual structures, the oogonia and antheridia. More Northwest, with particular focus on Saroiegniaferax, recently molecular identification has been accom- a species implicated in high egg mortality. We plished with selected Saprolegniaceae using the identified isolates from eggs of six amphibians with internal transcribed spacer (ITS) and 5.8S regions the internal transcribed spacer (ITS) and 5.8S gene of ribosomal DNA (rDNA) (Molina et al 1995, Leclerc regions and BLAST of the GenBank database. We et al 2000). The most complete molecular phylogeny identified 68 sequences as Saprolegniaceae and 43 of this frsmily to date identified 10 genera and 40 sequences as true fungi from at least nine genera. Our species through analyses of ITS and the large phylogenetic analysis of the Saprolegniaceae included ribosomal subunit (LSU) (Leclerc et al 2000). isolates within the genera , and There have been relatively few published field Leptolegnia. Our phylogenvgrouped S. semihypogyna investigations of Saprolegniaceae diversity and ecolo- with Achlya rather than with the Saprolegnia reference gy. This is particularly true in the Pacific Northwest, sequences. We found only one isolate that grouped USA, which is a region where Saprolegnia closely with has been S. ferax, and this came from a hatchery- identified as a potential pathogen on amphibians that raised salmon (Idaho) that we sampled opportunisti- have experienced local declines (Kiesecker and cally. We had representatives of 7-12 species and Blaustein 1995). For example Blaustein et al (1994) three genera of Saprolegniaceae on our amphibian suggested Saprolegnia ferax was responsible for eggs. Further work on the ecological roles of different mortality of nearly 95% of western toad (Bufo boreas) species of Saprolegniaceae is needed to clarify their eggs at one site in Oregon. Despite these claims that potential importance in amphibian egg mortality and S. feraxis a pathogenic water mold of amphibian eggs. potential links to population declines. no studies have attempted to document which species of occur on the eggs of North American Accepted for publication 8 November 2007. amphibians and whether egg mortality is uniquely Co rresponding author. E-mail: [email protected] Phone: associated with S. ferax. (208) 529-8376. Current address: University of Idaho, 1776 Science Center Drive, Suite 205, Idaho Fails, ID 83402. As a result of concern over observed amphibian egg 2 Current address: USGS Forest and Rangeland Ecosystem Science mortality and the difficulty of identifying taxa Center, Snake River Field Station, 970 Lusk St., Boise, ID 83706 niicroscopicallv, we sought to ideniif- Ootfl\ccirs In

172 MYC0L0GIA cultured from amphibian eggs from around the outgrow an bacteria in the culture. In cases where the Pacific Northwest with ITS and 5.8S rRNA gene filamentous growth was slower bacteria were evident on the sequences. We compared sequences of Saprolegnia- original PDA plate. When bacteria and filamentous growth ceae cultured from amphibian eggs to reference were present together in culture, successive hvphal transfers cultures from the American Type Culture Collection were used to obtain a pure filamentous culture. We obtained three Saprolegniaceae isolates from the (ATCC) and published sequences from GenBank. ATCC (10801 University Blvd., Manassas, Virginia 20110- The information from this survey should be of value 2209 USA): Saprolegniaferax (Grm.uthtusen) Thuret (ATCC to mycologists and amphibian biologists who are 26116), Saprolegnia parasitica Coker (ATCC 22284) and interested in the distribution of oomycetes and their Ac/i/ta americana Humphrey (ATCC 22599). We cultured ecological relationships with amphibians. and sequenced the ATCC isolates in the same manner as the egg and fish samples. MATERLLS AND METHODS DNA extraction, PR a mpizJi cation, and sequencing.—We extracted DNA from each filamentous organism using a Collection of qg samples—In 2001-2003 we collected egg procedure adapted from Griffith and Shaw (1998) for DNA samples (n = 200) of six pond-breeding amphibian species isolation from Phytophthora infestans. This process uses a from the Pacific Northwest, USA (Saprolegniaceae samples modified extraction huller (100 mM Tris-HCL, 1.4 M NaCl, TABLE 1). We coded sample numbers based on physiograph- 2% CTAB and 20 mM EDTA sodium salt pH 8.0) and a ic provinces: IS = Snake River Plain in southern Idaho; IM chloroform extraction technique. Precipitation of DNA was = Bitterroot Mountains of Idaho and western Montana; OE completed with isopropanol and centrifugation at 17000 X = Blue Mountains of eastern Oregon; OW = Cascade We confirmed the DNA was intact by running each Range and Willamette Basin of western Oregon; WA = g Cascade Range around Mount Rainier, Washington sample on 1.2% agarose gels. Samples were stored at 4 C until use in the polymerase chain reaction (PCR). (FIG. 1). The six amphibians included the long-toed salamander (Amliystoma macrodactylum), Pacific tree frog The ITS1 and ITS2 regions and the intermediate 5.8S (Pseudacris regilla), northern red-legged frog (Rana auro- ribosomal gene were amplified using the primers ITS1 ra), Cascades frog (R. cascadae), Columbia spotted frog (R. (5 TCCGTA(;(;TGAACCTGCGG 3) and ITS4 (5 luteiventris) and Oregon spotted frog (R. pretiosa). For all TCCTCCGCTTATTGATATGC 3) (White et al 1990) in a sampled amphibian egg masses, we collected equivalent 50 nmole concentration (HPLC purified) from Operon numbers of health y eggs and eggs that appeared diseased (QIAGEN). We performed PCR reactions in a 50 fIL volume (with white or gray hyphal nimbus, FIG. 2) in sterile 2 oz. using the HotStarTaq Master Mix Kit (QIAGEN) and a final polypropylene containers filled with surrounding pond I iM concentration of each primer. The final template water. Because several Saprolegnia species first were isolated DNA concentration used for each reaction was 10 ng/oL. from salmonids, we opportunistically collected three sam- We performed PCR amplifications with an Applied Biosys- ples from fish in southern Idaho: rainbow trout (Onco- tems 2400 thermocycler (Applied Biosystems). The PCR rhynchus mykiss) eggs from a hatchery and scale scrapings temperature profile was an initial activation at 95 C for from a dead rainbow trout (0. mykiss) and a hatchery-raised is min followed by 30 cycles with target temperatures at Chinook salmon (0. tshawtscha). 94 C, 54 C and 72 C each for 1 mm. A final extension at 72 C for 10 min completed the run. We analyzed PCR Culturing of osganisms from egg samples—Samples were products by electrophoresis on 1.2% agarose gels and shipped in coolers and stored at 4 C at the laboratory. stained the gels with ethidium bromide (50 isg/oL) to Because we did not want to exclude the growth of any confirm the presence of the PCR product. PCR products Saprolegniaceae species, we chose to use a nonselective were directly purified with the MinEltite PCR Purification media for our culturing procedure. We did not sterilize egg Kit (QIAGEN) for sequencing. Purified PCR products were samples because there is some indication that oomycetes sequenced with big dye terminator primers in the 3100 can grow on the outside of eggs (e.g. Czeczuga et al 1998, Genetic Analyzer (Applied Biosystems) at the Molecular Green 1999). We did not exclude any filamentous micro- Core Facility at Idaho State University at Pocatello. organisms in the culturing process so we used a nonselective media, potato-dextrose agar (PDA). PDA plates were made Alignment and phtlogenetic ana1sis.—We used PHRED from 24 g of potato dextrose (Difco) and 15 g of agar (Ewing et al 1998a) to assess the quality of DNA base call (Difco) per L. We used a sterile pipette to transfer 1-2 sequences. We considered 111 sequences to be of excellent amphibian eggs from each sample to a PDA plate. We used quality to be used for further analysis. We used PHRAP sterile forceps to place fish eggs and skin scrapings on FDA (PHRED II, Ewing et al 1998h) to build a consensus plates. We incubated cultures at 22 C for 24 h or until the sequence from forward and reverse sequences identified by growth of filamentous organisms was evident. To obtain a PHRED. pure culture of the filamentous organism we used a sterile Quality sequences were identified by BLAST (Basic Local hypodermic needle to transfer a small amount of the Alignment Search Tool, Altschul et al 1990) rising the cultured filamentous growth to a second PDA plate which National Center for Biotechnology Information (NCBI) we incubated at 22 C for 24 h. In the majority of cultures BLA.ST feature (http:/ /www.ncbi. nlm. nih .gov/ BLAST). We (75%) filamentous growth occurred quickly and was able to used the nucleotide-nucleotide search option, which uses a

PETRIsI\o ET \I: SAPROLEGNLCEAE AND AMPHIBIANS 73

FIG. 1. Sample sites in the Pacific Northwest, USA. Site numbers correspond to TABLE I. Physiographic regions are: Idaho- Montana (IM), Idaho-South (IS), Oregon-East (OF) antI Oregon-West (OW), Washington (WA). heuristic algorithm to search for sequence homology cultured and sequenced in our lab (S. ferax [Gruithuisen] (Altschul et a! 1990). Thuret, S. Parasztzca Coker and A. americana Humphrey); To see the phvlogenetic relationships among our (iii) 16 Saprolegnia, Ac/iia, or Leptolegnia (Leclerc et al Saprolegniaceae sequences, we used Geneious Pro® (ver- 2000) that we downloaded from the GenBank NCBI sion 2.5.2) to produce a multiple global alignment of 88 database (TABLE II); and (iv) the non-Saprolegniaceae sequences. The sequences in our phylogeny included: (i) all outgroup Ihytophthora botiyosa (ITS sequence GenBank samples identified by BLAST as Saprolegnia, Aclilya, or AF266784, Cooke et al 2000). We saved the alignment in the Leptolegnia (n = 68); (ii) three ATCC type strains that were Nexus format. The sequence alignment was analyzed with both a y analysis using PAUP (version 4.0 S... distance and parsimon beta; Swofford 2003). We performed the distance analysis I .. I - using the jukes-Cantor option with equal rates for variable i •k.; i. :. .••) sites (Jukes and Cantor 1969). A bootstrap analysis of the c.: . . neighbor joining tree was done with a neighbor joining search with 1000 replicates. Groups with 70% or greater frequency were retained. The parsimony analysis was done using the heuristic search option. The maximum number of trees saved was set to 2000. The parsimony consensus tree was computed with 70% majority rule. A bootstrap analysis of the parsimony consensus tree was done using a fast heuristic search with 1000 replicates.

RESULTS

Of the 203 samples we cultured, 138 filamentous cultures grew. BLAST was able to identify sequences FIG. 2. Oregon spotted frog (Rana pretlosa) egg mass from 111 isolates from 28 collection sites. We with high mortality and microorganisms on embryos identified 68 sequences as Saprolegniaceae (TABLE I). (center) surrounded by healthy developing egg masses The remaining 43 sequences were true ftlngi: from Oregon. Trichoderina (n = 27), Mucor (n = 6), Verticillium

Nl\ .( )I,O( .l\ n

FABLE I. Site and host species for successful cultures of Saprolegniaceae (n = 68). Sample codes are iii format Region-Site- Egg Sample. Amphibians are AMMA (Arnbystona macrodactylum); PSRE (Pseudacris regilla), RAAU (Rana aurora), RACA (Rana cascadae), RALU (Rana lutei vent ris) , RAPR (Rana pretiosa). Fish are Oneorhynchus tshawytschsa (Chinook Salmon) and 0. myhiss (Rainbow Trout)

Site Site Name State Sample code Species

I Lost Horse Creek MT IM-1-1 AMMA 2 LRC Frog Pond MT IM-2-1 RALU 3 Lost Horse Pond MT IM-3-6, IM-3-7 AMMA IM-3-5 1SRE IM-3-1, IM-3-2, IM-3-3, IM-3-4 RALU 4 Kramis Pond MT IM-4-8 AMMA JM-4-1, IM-4-2, IM-4-3, IM-4-4, IM-4--5 PSRE IM-4-6, IM-4--7 RAI,U ) Jones Pond MT IM-5-1, IM-5-2, IM-5-3, TM-5-4 AMMA 6 Duffy Pond East MT IM-6-1, IM-6-2, IM-6-3, IM-6-4, IM-6-5, IM-6-6 RALU 7 Little Rock Lake MT IM-7-1, IM-7-2, IM-7-3 RALU 8 Granite Lake ID IM-8-1, TM-8-2, IM-8-3 RALU 9 Frog Pond Lake ID IM-9-1, IM-9-2 RALU 10 North Walton Lake ID IM-10-1 RALU 11 Eagle Fish Health ID IS-11-1 (fish) Oncorhynchus Laboratory tshawytschsa 12 Fish Hatchery ID IS-12-1 (fish eggs) 0. mykiss 13 Gibson Jack Stream ID IS-13-1 (fish) 0. mykiss 14 Marjorie Lake Pond WA W-14-1 RACA 15 Snow Lake WA WA-15-1, WA-15-2, WA-15-3, WA-15-4, WA-15-5 RACA 16 Upper Deadwood Lake WA WA-16-1 RACA 17 Ethel Lake WA-i 7-I RACA 18 Bench Lake WA WA-18-1, WA-18-2 RACA 19 Jack Creek OR OW-1 9-1 RAPR 20 Half Moon Pond OR OW-20-1 RAAU 21 Dugout Pond OR OW-21-1 PSRE OW-21-2, OW-21-3 RAAU

22 Black Mountain Lower OR OE-22-1, OE-22-2, OE-22-3, OE-22-4 RALU Pond

23 Penn Lake OR OW-23-1 RACA OW-23-2, OW-23-3 RAPR 24 Muskrat Lake OR OW-24-1 PSRE 25 China Lake Small Pond OR OE-25-1 RALU 26 Camp Creek Lowest Pond OR OE-26-1, OE-26-2 RALU 27 Black Mountain Middle OR OE-27-1 AMMA Pond OE-27-2 RALU 28 Big Marsh OR OW-28-1 RAPR

(n = 3), Penicillium (n = 1), Cordyceps (n = 1), duced identical groupings of Leptolegnia, Saprolegnia, Engyodontium (n = 1), Gibberella (n = 1), Guignardia Achlya and S. semihypogyna. While the groupings were (n = I.), Leptosphaeria (n = 1) and one unidentified identical, the bootstrap support was higher in the fungus. Because we did not sterilize our egg samples, branch groupings of the neighbor joining than the it is possible that these true fungi were environmental parsimony consensus tree. We chose to show the Contaminants that were present in the egg samples. neighbor joining (FIG. 3) rather than the parsimony While we considered it important to report all the consensus tree to see the amount of evolutionary organisms cultured from our amphibian eggs, we change and therefore determine how closely isolates focused on the Saprolegniaceae for this study. were related within each grouping of Saprolegnia, Of 884 total characters, 312 were parsimony Leptolegnia, Achlya or S. semihypogyna. informative, 411 were constant and 1611 variable Our distance analysis resolved the Leptolegnia and characters were parsimony uninformative. The neigh- Saprolegnia groups from the rest of the tree with bor joining and maximum parsimony analysis pro- bootstrap support of 75. The Achlya and S. semihypo- Pt. IRISKO r i i Siioi i;NI\ :it. VND AMPHIBIANS

TABLE II. Reference sequences and cultures used for phylogenetic analysis Reference Cultures Source and accession number

Achlya amen cana GenBank AF2 18145 Achlya amen cana Humphrey ATCC 22599 our laboratory Achlya aquatica GenBank AF218 150 Achlya colorata GenBank AF218159 Achlya intricata GenBank AF2 18148 Achlya oligacantha GenBank AF218162 Achlya papillosa GenBank AF218161 Achlya racemosa GenBank AF2 18158 Leptolegnia CBS 177.86 (;eiBaik AY310502 Phytophthora botiyoca GenBank AF266784 Saprolegnia anomalies GenBauk DQ322632 Saprolegn ia bulbosa (;eisBank AY26701 1 Saprolegnia diclina Humphrey ATCC 90215 GenBank AY455775 Saprolegnia frrax (Cruithuisen) Thuret ATCC 26116 our laboratory Saprolegnia hypogna (;enBaik AY6471 88 SaprOleg7iia longicaulis (;erB2Ink AY270032 Saprolegnia oliviae GenBank AY270031 Saprolegnia parasitica Coker ATCC 22284 our laboratory Saprolegnia salmon is Geuhank AY647 193 Saprolegnia semzhvpogvna GenBank AY647194

na groups are resolved with bootstrap support of Idaho-Montana cluster. A sample from a wild trout 100. The Saprolegnia group is resolved from Lepto- scraping (IS-13-1) also might he associated with the legnia with bootstrap support of 91. The consensus group including S. parasitica and S. diclina. The parsimony tree has lower bootstrap support of 73 for group containing S. salmonis, S. hypogyna and S. ferax the resolution of the Sap rolegnia and Leplolegnia ATCC 26116 included one field sample from a groups from the tree and lower bootstrap support Chinook salmon scraping (IS-11-1). A grouping of S. (86) for the resolution of the Achlya and S. bulbosa, S. oliviae, S. ion gicaulis and S. anomalies semihypoyna groupings. The Saprolegnia group is included 13 isolates from amphibian eggs and one resolved from Leptolegnia with bootstrap support of isolate from eggs of hatchery rainbow trout (IS-12-1). 71 instead of the 91 seen in the neighbor joining tree. With the exception of the hatchery trout, all samples Our neighbor joining distance tree (FIG.) re- iii this group were collected in Oregon and Washing- solved field and reference samples into two major ton. groups outside the Phytophthora outgroup. The first The second major group also was resolved into two major group (bootstrap value 75) comprised two subgroups. The Achlya group included all eight subgroups that further resolved into a total of six Achlya reference sequences and was resolved further groupings. Thirteen isolates from amphibian eggs into five groupings. Our finding of low bootstrap grouped strongly with the Leptolegnia reference support (60) for the Achlya group concurs with other sequence (bootstrap 100), and two egg samples studies that imply the is likely to be polyphyletic separated from that group with strong support (Leclerc et al 2000). Outside the three groups of (bootstrap 100). A group including all Saprolegnia Achlya reference samples were two groups of amphib- reference sequences except S. seinihypogyna had ian egg samples that were not directly affiliated with strong support (bootstrap 91). The separation be- reference species. The first group (including IM-1-I, tween the group including S. parasitica and S. diclina OW-20-1, OE-22-2, OW-21-1 and OE-22-3) was most and the group including the other seven reference closely related to the grouping of A. colorata and A. Saproleg7iia had weaker support (bootstrap <70). racemosa. The second group of egg samples (OW-23- Isolates from two Idaho-Montana amphibian egg 3, WA-15-5 and WA-15-4) also was distinct from all our samples grouped with S. parasitica ATCC 22284 other Achlya sequences (bootstrap 100). Separate (bootstrap 93) which was near but distinct from 14 from the main Achlya group was a second subgroup amphibian samples that grouped strongly with S. including one amphibian egg sample with S. semi- diclinaATCC 90215 (bootstrap 100). Thirteen of the hypogyna (bootstrap 99) and a distinct cluster of 12 14 isolates in the S. diclina grouping were from the amphibian egg isolates (bootstrap 100). All these

176 MYCOLOGIA r 100 IM-7-3 IM -7-1 WA-18-2 100 OF -27 -1 WA -17-1 I Leptolegnia CBS 17786 Leptolegnia

IM -9- 1 M-3-3 IM -4-4 IM -4-8 IM -7-2 -4-2 I M-4-1M OW -19-1 IM -4-7 [1 IM -4-6 0(1 [l

M -4-3 IM -4-5 IM -3-4 S. thdi,rn ATCC 90215 Saprolegnia IM-5-2 93 ______IM -5-4 S po.rasi€k Al CC 27284 IS-13 -1 (fish) 06 -22-1 IM -5-3 S. wrnmafies S. 1oegkdLv IS -12-1 (fish eggs) 91 OF -27-2 OF -22 -4 IM -5-1 72 OW -21 -2 OW -28-1 06 -26-1 OW -24-1 WA - 16-1 S. siirie S. b,dJse 05 -26 -2 OF -25-1 OW -21 -3 -S. efrnonjs S.Jère-c ATCC 26116 100 IS -11 -1 (fish) S I IM-1-1 I OW -20-1 06 -22 -2 OW-2i-i -i91 - ISO 4. r"emosa Achlya A. e,kern ATCC 22599 A. aqualzea A. ppillose A. eli,sth,, 100 OW -23-3 ttt I WA -15-5 1 WA -15-4 WA -18-1 I— IM -6-2 100 IM-6-1 IM-2-1 IM -6-5 WA -15-2 WA -14-1 M -6-3 I M -10-1 IM -6-4 9 WA -15-3 IM -6-6 WA -15-1 S. semihypogyna S. seihypogyna U P. bo8-ya

0005 subs5tu0osiss

FIG. 3. Jukes-Cantor neighbor joining distance tree for Saprolegniaceae isolates from amphibian eggs (n = 65), fish scrapings or eggs (n = 3) and reference samples (n 20). Bootstrap values <70 are not shown. Pi:i R1sk1) i:! At,: S\p l.oIi(\L\( 1\t \\I) \\lII IIRI \.S samples came from our northern sites in Washington, pathogenic on mosquito larvae (Bisht et at 1996, Montana and Idaho. The generic and specific Scholte et at 2004). Although not sampled specifical- identities of these 12 isolates are unclear. ly, mosquitoes were abundant at many of our sites. Our neighhorjoining distance analysis also suggests a distinct division between most of our Saprolegnia DISCUSSION samples and members of the genus Ach1a. This To our knowledge this is the first broad survey of generally agrees with 28S rDNA data (Riethmuller et oornycetes on amphibian eggs outside work in at 1999) and the cladistic analysis of LSU rDNA data Poland. This also appears to be one of the first reported by Leclerc et at (2000). In contrast a studies of filamentous microorganisms on amphibian neighbor joining distance tree (Leclerc et at 2000) eggs that uses molecular phylogenetic techniques to placed some Ach1a (including A. racemosa, A. circumvent some of the difficulties of oomycete colorata, A. oligacantha and A. papjllosa) closer to identification based on unstable reproductive struc- Saprolegnia than with the main Achiya dade (includ- tures. The BLAST analysis of the NCBI database was ing A. aquatica, A. americana and A. intricata). Both able to identify most of our isolates to the generic our phylogeny and analysis by Leclerc et at (2000) of level. The GenBank and ATCC reference species the LSU identify the same three Achiya subgroups: (i) served as the foundation for our phylogeny. Our A. americana/A. aquatca/A. iniricata, (ii) A. oliga- neighbor joining distance analysis of ITS regions cantha/A. paplilosa and (iii) A. racemosa/A. colorata. successfully resolved the known Saprolegnza, Achlya These Achlya subgroups are consistent with divisions and Leptolegnia sequences into distinct genera and based on oospore morphology outlined by Dick species groups and helped us to see the phylogenetic (1969). None of our eight isolates from field samples relationships among these three groups. directly matched sequences of our reference Achiya Our sequence identification and phylogenetic species, so we are unsure of their species-level analysis confirm that a diversity of Saprolegniaceae . Given the paucity of field sampling for occur on amphibian eggs in the Pacific Northwest. We Saprolegniaceae in western North America, it is estimate that we had representatives of at least 7-12 possible this lineage represents a new species. species and three genera of Saprolegniaceae on out- Czeczuga Ct at (1998) reported 15 Achlya species amphibian eggs. Czeczuga et at (1998) also reported a (including A. colorata) from amphibian eggs in diversity of taxa based on morphological identifica- Poland, but little else is published on Achlya on tion, including 33 species of Saprolcgniaceae (14 amphibians. A variety of Achlya (including A. Achlya, 10 Leptoleg7iia, nine Saprolegnia) and 18 other americana, A. colorata, and A. raceniosa) have been zoosporic fungi from Polish amphibians raised in five reported on fish eggs (Czeczuga and Musz yiiska 1997, different water sources. 1999). Our phylogenetic analysis is in general agreement Most phylogcnetic work suggests that the genus with recent molecular work on the Saprolegniaceae Achlya is not a monophyletic unit in its current (e.g. Dick et at 1999, Riethmuller et at 1999, Leclerc configuration (e.g. Green and Dick 1972, Dick et at et at 2000). Most (78%) of our oomycete samples (68) 1999, Riethmuller et at 1999, Leclerc et at 2000). grouped within Saprolegnia and Achlya but we also Similarly Leclerc et at (2000) indicated that Sapro- identified a subset of our amphibian egg cultures as legnia might not be monophyletic, and difficulties probable members of Leptolegnia (n = 13) or a remain in distinguishing generic affiliations of some related but separable group (bootstrap support of Achlya and Saprolegnia species. Our phylogeny 100; n = 2). Few Leptolegnia species are documented grouped reference sequences of Saprolegnia (boot- in GenBank, so it is possible that our samples could strap 91) and Achlla together in relatively cohesive be further resolved with more reference species. The respective groups. However our S. semihpogyna and separation of the Leptolegnia group from our primary an associated group of field samples resolved more Saprolegnia group is strongly supported (bootstrap closely with Achlya than to the main group of 100) and generally concurs with phylogenies based on Saprolegnia. Type specimens of S. semihypo,gyna were 18S rDNA (Dick et at 1999) and LSU rDNA data described from Japan (Inaba and Tokumasu 2002), (Leclerc et at 2000). Our Leptolegnia samples came and ITS and 28 LSU sequences are in GenBank. We from four amphibians (A. macrodactylum, R. cascadae, did not inspect the morphology of any of our samples, R. luteiventris, R. pretiosa) and from all four states. but if our phylogeny is correct the generic attribution Czeczuga et at (1998) reported L. caudata de Bary of S. semihypogyna may merit reconsideration. The from eggs of five amphibians when they were reared identity of the tightly clustered group adjoining S. in water from five different wetlands. Leptolegnia semihypogyna (bootstrap 99) and its relationship to caudata and other members of the genus are other Achlya and Saprolegnia is unclear.

178 MYcoI.0GIA

The distribution of samples among Sap rolegnia cultured Saprolegniaceae from amphibian eggs that species in our main group was somewhat unexpected. were developing normally. Many pond-breeding Fourteen isolates grouped strongly with S. dielina amphibians deposit eggs among herbaceous vegeta- ATCC 90215. The strong support for our S. diclina tion in shallow margins, and these littoral zones can group (bootstrap 100) and our S. parasitica group support the highest density and diversity of oomycete (bootstrap 93; n = 2 egg isolates) concurs with the propagules in ponds or lakes (OSullivan 1965; conclusion by Molina et al (1995) that S. diclina and Dick 1971, 1976). Several amphibians with this S. paraMtica are closely related but separate species. oviposition habit have adaptations to reduce egg Our S. diclina group was the most geographically losses to aquatic microorganisms, such as thickened homogeneous of our groups with larger sample sizes: egg capsules, separating egg masses to reduce direct 13 of 14 samples came from the Bitterroot Mountain hyphal invasion or accelerating development in region of Idaho-Montana. Also consistent with Molina presence of oomycete hyphae (Kiesecker and Blaus- et al (1995) was our finding of a close relationship tein 1997, Green 1999, Gomez-Mestre et al 2006, between S. ferax and S. hypogyna. Touchon et a! 2006). Similar to saprolegniosis in Although others (Blaustein et al 1994, Kiesecker fish, it is unclear how frequently different members of and Blaustein 1995) report S. frrax associated with the Saprolegniaceae act as pathogens on healthy amphibian egg mortality in the Pacific Northwest, amphibian eggs, colonize dead eggs (Robinson et al none of our isolates from amphibian eggs were 2003) or take advantage of another stressor or grouped closely with this species. Many of our isolates infection (Kiesecker and Blaustein 1995, Lefcort et from amphibians were grouped closely with S. diclina al 1997). and other Saprolegnia spp. (S. buibosa,S. oliviae, S. To better understand the threat posed by Sapro- anomalies and S. longicaulis). The single sample that legniaceae to pond-breeding amphibians afield, we aligned near our reference S. ferax came from a recommend additional work to clarify the taxonomy Chinook fish scraping (IS-il-i). Our two other fish of the colonizing microorganisms, controlled tests of samples were grouped with S. parasitica (sample IS- pathogenicity on individual amphibian species and 13-1) and S. anomalies/Iongicaulis (15-12-1). The S. experimental study of mechanisms by which stressors diclina/ S. parasitica complex is commonly associated increase susceptibility of embryos. with saprolegniosis in fish around the world (e.g. Beakes and Ford 1983). Of note S. diclina colonized ACKNOWLEDGMENTS dead or unfertilized perch eggs in lab trials but did not invade adjacent live perch eggs (Paxton and This work was supported by the Idaho National Engineering Willoughby 2000). and Environmental Laboratory (now INL) Education Our lack of S. ftrax seems noteworthy because it is Outreach BBW Bechtel grant and a grant from the US among the most widespread and abundant taxa of its Geological Survey Amphibian Research and Monitoring genus (Seymour 1970, Dick 1971, Czeczuga et al 1998, Initiative (ARMI). We thank V. Winston for his contribu- tions throughout this project and G. Carroll for his Johnson et al 2002). A recent study that combined thoughtful review of an earlier version of this manuscript. genetic and morphological data (Hulvey et al 2007) Samples were gathered under scientific collecting permits showed that species designation of Saprolegnia based from the National Park Service, Washington Department of solely upon morphological characteristics is not Fish and Wildlife, Oregon Department of Fish and Wildlife, sufficient. Because we used sequence rather than Idaho Department of Fish and Game and Montana morphology for identification, we should not have Department of Fish and Wildlife. We thank R. Hoffman encountered this problem to the same degree that (USGS FRESC), B. Santora (Mount Rainier National Park), earlier observational studies may have experienced. P. Murphy (Idaho Department of Fish and Game) and B. This might explain why we found a large diversity of Maxell (Montana National Heritage Program) for help Saprolegnia species on our amphibian eggs. We identifying sampling sites. We thank S. Galvan for assem- bling the map and B. Cummings, J . Jones, J. Kittrell, B. suggest that future identification of field-collected McCreary, J. Oertley and C. Rombough for assistance in the Saprolegnia with sequence and morphological data field. will contribute to a new understanding of distribution and ecological function of this diverse group in aquatic systems. LITERATURE CITED The effects of colonization by Saprolegniaceae on Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. amphibian eggs and populations remain unclear. Basic local alignment search tool. J Mol Biol 215:403- Most of the amphibian populations we sampled do 410. not appear to be in decline (CAP and DSP unpub- Beakes GW, Ford H. 1983. Esterase isoenzyme variation in lished data). 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