IMA FUNGUS · 5(2): 439–448 (2014) {%"55#7|!"%V"5"!"7 Multiple barcode assessment within the Saprolegnia-Achlya clade ARTICLE (Saprolegniales, Oomycota, Straminipila) brings order in a neglected group of pathogens Mónica M. Steciow1*, Enrique Lara2CD2>2=+2 1<+H$$&DIJNI=5PIVXX%#""=+ Aires, Argentina. 2=H+Z+NI%%\]>D3*!"""IH$@ author e-mail: [email protected] C+_ Abstract: The Saprolegnia-Achlya clade comprises species of major environmental and economic importance due Key words: __[ barcoding 3` <zH which is a major obstacle to the widespread application of molecular barcoding to identify pathogenic strains with =HN\I> quarantine implications. We assessed phylogenetic relationships of major genera using three commonly used morphology <zHHHN\I>=HN\I>> Newbya H>% HHN\I> <Newbya dichotoma sp. nov., which provided the only culture water moulds <$ $ their successive reversals rendered the inference of ancestral states impossible. This highlights even more the importance of a bar-coding strategy for saprolegniacean parasite detection and monitoring. Article info:H{P">!"%V@>{!VI!"%V@{%"Z!"%V INTRODUCTION identify them quickly and reliably, in order to apply quarantine =4+!"%% Oomycetes (or water moulds) are protists with a mycelial General strategies for detecting these parasites [ Fungi, and rely on isolation and cultivation of strains isolated from that have been relocated within Heterokonta, also known [ as Straminipila, based on their molecular phylogeny. They strategy that is time-consuming and requires highly trained are ubiquitous in freshwaters, but also in soils and in marine >[ environments, where they can be found either as parasites of species and genera require the presence of reproductive or free-living. Saprolegnia-Achlya is a monophyletic clade and disseminative structures which appear sometimes of mainly freshwater species that forms a sister clade to only after weeks (Steciow 2003), urgent measures cannot Aphanomyces and includes notorious animal pathogens. be taken early enough to prevent epidemics. For this These organisms are abundant in the environment, where reason, a molecular barcoding approach could bring a [et al. valuable tool for quickly and accurately monitoring disease 2008), Daphnia (Wolinska et al.!""#$$et al. 3 !"%%&$*+$!"%%[ this approach is dependent on the existence of a reliable (Krugner-Rigby et al. 2010). As such, they are responsible [ * for millions of dollars losses to the worldwide aquaculture derived or environmental sequences to known species. industry (van West 2006), with considerable damage to < * [343%### study can provide a base for identifying known species and They can also threaten endangered wildlife, and cases of placing newly discovered strains. Unfortunately, existing local extinctions of amphibian populations due to members databases suffer major drawbacks caused by: (1) taxonomic + %#57 %#9! @ ! [ [ ; !""9 < < therefore of crucial importance to detect these organisms and therefore urgent to clarify the taxonomy within this group © 2014 International Mycological Association You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: [ Non-commercial: No derivative works: For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. VOLUME 5 · NO. 2 439 Steciow et al. by providing a reliable phylogenetic framework based DNA extraction, PCR, sequencing and phylo- _ < genetic analyses addition, it is useful to address inconsistencies that limit the \D+H* use of culture collection resources and propagate errors in I>;&+DNzI subsequent studies. Several species have been described and VKM (All-Russian Collection of Microorganisms, ARTICLE but later assigned to either Achlya or Saprolegnia. These J\&z%ZI> two genera are traditionally distinguished by the mode was extracted with a guanidine thiocyanate buffer protocol $@ Saprolegnia \ =4+!"%%D\ \ * <zHV <zH9 ; et al. 1990) (diplanetism), whereas in Achlya $ <zH ] V!& ] %V#7\ HHN \I> Z =$ et al. !""% <zHV !7H*59V\ et al. 1998, Johnson et al.!""!3 as described in Steciow et al. !"%P z D\ [ _>+<\<HJPX""ZI>>$] < + +ZTM Terminator Cycle addition, these characters can only be assessed after the H_ ] + H_ generation of sexual and asexual (dispersive) forms, which + requires the testing of many different culture conditions and Table 1. Sequences from representatives from all genera long incubation periods, a time-consuming process that can from the Saprolegnia-Achlya + be precious in the case of a disease outbreak. Therefore, the three marker genes have been surveyed and collected to development of a robust barcoding strategy to identify complete the alignments that generated the trees presented possible pathogens or implement quarantine measures here. Some sequences from the sister-clade Aphanomyces would be extremely useful. were added as outgroup. Although several diverging nom- < enclatures were used by different sequence submitters, we the morphological features used to identify species of kept original names as they were. The nomenclature used Saprolegniaceae < Z !""% H analyses of the group based on the three most frequently et al. (2002). Sequences were aligned manually using <zHHHN=HN\I> +] 3 %### z respective resolution powers. We also inferred a consensus reconstructed using a Maximum Likelihood approach as tree and inferred possible scenarios for trait evolution. And, implemented in MEGA v. 6 (Tamura et al. 2013) using the [* following parameters: general time reversible model with Newbya dichotoma \ invariant sites and four categories among site variation, and [ 1000 bootstraps to evaluate node robustness. Alignments 55# <zH %9PPHHN\I>X75=HN MATERIALS AND METHODS \I>z_ Aphanomyces species and relatives, as these organisms are Isolation of Newbya dichotoma the closest known relatives of the Saprolegnia-Achlya clade Floating organic matter (twigs, leaves) were collected from =4+!"%%+_ D \ I_ > from different gene markers did not derive from the same taken to the laboratory in separate sterile polyethylene bags. organisms, we could not concatenate the three markers The methods for collection and isolation of mycelium-forming and proceed to a joint phylogenetic analysis. Therefore, we \D%#!P inferred a consensus tree on the basis of keeping every node %#59H%#9"H4&%#7X that was supported robustly by at least one marker and not +$ contradicted by another. dishes containing several halves of hemp seeds (Cannabis sativa%5M!"D; mycelial growth was observed on the seeds, a single hypha RESULTS was isolated and transferred to cornmeal-agar medium (CMA) `>PMV Gene trees and consensus tree $ z<zH H for the monophyly of all genera except Achlya, with bootstrap $ [ supports ranging between 0.89 and 1 (Fig. 1). The genus $ Leptolegnia appears paraphyletic if Geolegnia is accepted as `$ a separate genus; likewise, Thraustotheca appears nested sexual organs (structure of the oogonium and antheridium). within Achlya Z Observations and measurements were made with an Olympus branchings (i.e. relationships between genera) are generally + V" z _ `H>%Leptolegnia/Geolegnia Z "#X Aplanes/Aplanopsis/Newbya species was repeatedly isolated from the same locality in May (bootstrap value = 0.99). This tree placed Newbya, at the and June 2009, and May 2010. base of the genera Aplanes and Aplanopsis. This branching 440 IMA FUNGUS Barcode assessment within the Saprolegnia-Achyla clade Table 1.=$D+H+ sequenced. ARTICLE Species VKM, LPSC and CBS GenBank accession numbers accession numbers ITS SSU LSU Achlya debaryana J&*%#"V "#7P5! "#7PX% "#7P55 Achlya sparrowii J&*!!%X "#7PVV "#7P7" "#7P59 Aplanopsis spinosa D+H5X99X "#7PVX "#7P95 "#7P5# Aplanes treleaseanus VKM F-2129 >+!%#PXP "#7P9P "#7P9" Aplanopsis spinosa D+H5XX9X "#7PV9 "#7P99 "#7P57 Leptolegnia caudata D+H%%PVP% "#7PV% "#7P9# IZ1 Leptolegnia caudata D+H97"9# "#7PV" "#7PX" "#7P5X Leptolegnia sp. D+HP#!7% "#7PV! "#7P9X IZ1 Protoachlya paradoxa D+H!9%P7 "#7P5% "#7PX5 IZ1 Isoachlya humphreyana D+H%%""5X "#7PVP "#7PX9 IZ1 Pythiopsis intermedia D+HP"VP5 "#7PV7 "#7PXX IZ1 Pythiopsis terrestris D+H%%""57 "#7P5" "#7PX7 "#7P9! Pythiopsis terrestris D+H%%""5# "#7PV# "#7PX# IZ1 Thraustotheca clavata D+HPVPPP "#7P5V "#7PX! 3995!%P Leptolegnia caudata D+HP5#P5 "#7PP7 "#7P97 IZ1 Thraustotheca clavata D+H55X9X "#7P5P "#7PXP 3995!97 Newbya dichotoma =HD7XX "#7PV5 "#7P9V "#7P9% 1IZI Table 2.JH*>$ \ Genus Discharge mode Sporangia Flagellated zoospores Saprolegnia saprolegnoid I dimorphic (primary+secondary) Pythiopsis saprolegnoid I monomorphic (primary) basipetalous fashion Protoachlya protoachloid H$ dimorphic (primary +secondary) cluster Isoachlya saprolegnoid I dimorphic (primary+ secondary) proliferation Scoliolegnia saprolegnoid I dimorphic (primary +secondary) branching or basipetal succession Newbya achlyoid H monomorphic (secondary) Aplanes aplanoid or achlyoid Sporangia cylindrical or fusiform; spores germinate in situ, "\$ inside sporangium Calyptralegnia thraustothecoid or Sporangia fusiform, cylindrical or clavate; renewed sympodially, monomorphic (secondary) calyptralegnoid or in a basipetalous or cymose fashion